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Sample records for regulates microtubule densities

  1. Tubulin cofactor B regulates microtubule densities during microglia transition to the reactive states

    International Nuclear Information System (INIS)

    Fanarraga, M.L.; Villegas, J.C.; Carranza, G.; Castano, R.; Zabala, J.C.

    2009-01-01

    Microglia are highly dynamic cells of the CNS that continuously survey the welfare of the neural parenchyma and play key roles modulating neurogenesis and neuronal cell death. In response to injury or pathogen invasion parenchymal microglia transforms into a more active cell that proliferates, migrates and behaves as a macrophage. The acquisition of these extra skills implicates enormous modifications of the microtubule and actin cytoskeletons. Here we show that tubulin cofactor B (TBCB), which has been found to contribute to various aspects of microtubule dynamics in vivo, is also implicated in microglial cytoskeletal changes. We find that TBCB is upregulated in post-lesion reactive parenchymal microglia/macrophages, in interferon treated BV-2 microglial cells, and in neonate amoeboid microglia where the microtubule densities are remarkably low. Our data demonstrate that upon TBCB downregulation both, after microglia differentiation to the ramified phenotype in vivo and in vitro, or after TBCB gene silencing, microtubule densities are restored in these cells. Taken together these observations support the view that TBCB functions as a microtubule density regulator in microglia during activation, and provide an insight into the understanding of the complex mechanisms controlling microtubule reorganization during microglial transition between the amoeboid, ramified, and reactive phenotypes

  2. EML proteins in microtubule regulation and human disease.

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    Fry, Andrew M; O'Regan, Laura; Montgomery, Jessica; Adib, Rozita; Bayliss, Richard

    2016-10-15

    The EMLs are a conserved family of microtubule-associated proteins (MAPs). The founding member was discovered in sea urchins as a 77-kDa polypeptide that co-purified with microtubules. This protein, termed EMAP for echinoderm MAP, was the major non-tubulin component present in purified microtubule preparations made from unfertilized sea urchin eggs [J. Cell Sci. (1993) 104: , 445-450; J. Cell Sci. (1987) 87: (Pt 1), 71-84]. Orthologues of EMAP were subsequently identified in other echinoderms, such as starfish and sand dollar, and then in more distant eukaryotes, including flies, worms and vertebrates, where the name of ELP or EML (both for EMAP-like protein) has been adopted [BMC Dev. Biol. (2008) 8: , 110; Dev. Genes Evol. (2000) 210: , 2-10]. The common property of these proteins is their ability to decorate microtubules. However, whether they are associated with particular microtubule populations or exercise specific functions in different microtubule-dependent processes remains unknown. Furthermore, although there is limited evidence that they regulate microtubule dynamics, the biochemical mechanisms of their molecular activity have yet to be explored. Nevertheless, interest in these proteins has grown substantially because of the identification of EML mutations in neuronal disorders and oncogenic fusions in human cancers. Here, we summarize our current knowledge of the expression, localization and structure of what is proving to be an interesting and important class of MAPs. We also speculate about their function in microtubule regulation and highlight how the studies of EMLs in human diseases may open up novel avenues for patient therapy. © 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  3. EWSR1 regulates mitosis by dynamically influencing microtubule acetylation.

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    Wang, Yi-Long; Chen, Hui; Zhan, Yi-Qun; Yin, Rong-Hua; Li, Chang-Yan; Ge, Chang-Hui; Yu, Miao; Yang, Xiao-Ming

    2016-08-17

    EWSR1, participating in transcription and splicing, has been identified as a translocation partner for various transcription factors, resulting in translocation, which in turn plays crucial roles in tumorigenesis. Recent studies have investigated the role of EWSR1 in mitosis. However, the effect of EWSR1 on mitosis is poorly understood. Here, we observed that depletion of EWSR1 resulted in cell cycle arrest in the mitotic phase, mainly due to an increase in the time from nuclear envelope breakdown to metaphase, resulting in a high percentage of unaligned chromosomes and multipolar spindles. We also demonstrated that EWSR1 is a spindle-associated protein that interacts with α-tubulin during mitosis. EWSR1 depletion increased the cold-sensitivity of spindle microtubules, and decreased the rate of spindle assembly. EWSR1 regulated the level of microtubule acetylation in the mitotic spindle; microtubule acetylation was rescued in EWSR1-depleted mitotic cells following suppression of HDAC6 activity by its specific inhibitor or siRNA treatment. In summary, these results suggest that EWSR1 regulates the acetylation of microtubules in a cell cycle-dependent manner through its dynamic location on spindle MTs, and may be a novel regulator for mitosis progress independent of its translocation.

  4. Spatiotemporal Regulation of Nuclear Transport Machinery and Microtubule Organization

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    Okada, Naoyuki; Sato, Masamitsu

    2015-01-01

    Spindle microtubules capture and segregate chromosomes and, therefore, their assembly is an essential event in mitosis. To carry out their mission, many key players for microtubule formation need to be strictly orchestrated. Particularly, proteins that assemble the spindle need to be translocated at appropriate sites during mitosis. A small GTPase (hydrolase enzyme of guanosine triphosphate), Ran, controls this translocation. Ran plays many roles in many cellular events: nucleocytoplasmic shuttling through the nuclear envelope, assembly of the mitotic spindle, and reorganization of the nuclear envelope at the mitotic exit. Although these events are seemingly distinct, recent studies demonstrate that the mechanisms underlying these phenomena are substantially the same as explained by molecular interplay of the master regulator Ran, the transport factor importin, and its cargo proteins. Our review focuses on how the transport machinery regulates mitotic progression of cells. We summarize translocation mechanisms governed by Ran and its regulatory proteins, and particularly focus on Ran-GTP targets in fission yeast that promote spindle formation. We also discuss the coordination of the spatial and temporal regulation of proteins from the viewpoint of transport machinery. We propose that the transport machinery is an essential key that couples the spatial and temporal events in cells. PMID:26308057

  5. Kindlin1 regulates microtubule function to ensure normal mitosis.

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    Patel, Hitesh; Stavrou, Ifigeneia; Shrestha, Roshan L; Draviam, Viji; Frame, Margaret C; Brunton, Valerie G

    2016-08-01

    Loss of Kindlin 1 (Kin1) results in the skin blistering disorder Kindler Syndrome (KS), whose symptoms also include skin atrophy and reduced keratinocyte proliferation. Kin1 binds to integrins to modulate their activation and more recently it has been shown to regulate mitotic spindles and cell survival in a Plk1-dependent manner. Here we report that short-term Kin1 deletion in mouse skin results in impaired mitosis, which is associated with reduced acetylated tubulin (ac-tub) levels and cell proliferation. In cells, impaired mitosis and reduced ac-tub levels are also accompanied by reduced microtubule stability, all of which are rescued by HDAC6 inhibition. The ability of Kin1 to regulate HDAC6-dependent cellular ac-tub levels is dependent on its phosphorylation by Plk1. Taken together, these data define a novel role for Kin1 in microtubule acetylation and stability and offer a mechanistic insight into how certain KS phenotypes, such as skin atrophy and reduced cell proliferation, arise. © The Author (2016). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS.

  6. Regulation of developmental and environmental signaling by interaction between microtubules and membranes in plant cells

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    Qun Zhang

    2015-12-01

    Full Text Available ABSTRACT Cell division and expansion require the ordered arrangement of microtubules, which are subject to spatial and temporal modifications by developmental and environmental factors. Understanding how signals translate to changes in cortical microtubule organization is of fundamental importance. A defining feature of the cortical microtubule array is its association with the plasma membrane; modules of the plasma membrane are thought to play important roles in the mediation of microtubule organization. In this review, we highlight advances in research on the regulation of cortical microtubule organization by membrane-associated and membrane-tethered proteins and lipids in response to phytohormones and stress. The transmembrane kinase receptor Rho-like guanosine triphosphatase, phospholipase D, phosphatidic acid, and phosphoinositides are discussed with a focus on their roles in microtubule organization.

  7. Regulation of microtubule-based transport by MAP4

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    Semenova, Irina; Ikeda, Kazuho; Resaul, Karim; Kraikivski, Pavel; Aguiar, Mike; Gygi, Steven; Zaliapin, Ilya; Cowan, Ann; Rodionov, Vladimir

    2014-01-01

    Microtubule (MT)-based transport of organelles driven by the opposing MT motors kinesins and dynein is tightly regulated in cells, but the underlying molecular mechanisms remain largely unknown. Here we tested the regulation of MT transport by the ubiquitous protein MAP4 using Xenopus melanophores as an experimental system. In these cells, pigment granules (melanosomes) move along MTs to the cell center (aggregation) or to the periphery (dispersion) by means of cytoplasmic dynein and kinesin-2, respectively. We found that aggregation signals induced phosphorylation of threonine residues in the MT-binding domain of the Xenopus MAP4 (XMAP4), thus decreasing binding of this protein to MTs. Overexpression of XMAP4 inhibited pigment aggregation by shortening dynein-dependent MT runs of melanosomes, whereas removal of XMAP4 from MTs reduced the length of kinesin-2–dependent runs and suppressed pigment dispersion. We hypothesize that binding of XMAP4 to MTs negatively regulates dynein-dependent movement of melanosomes and positively regulates kinesin-2–based movement. Phosphorylation during pigment aggregation reduces binding of XMAP4 to MTs, thus increasing dynein-dependent and decreasing kinesin-2–dependent motility of melanosomes, which stimulates their accumulation in the cell center, whereas dephosphorylation of XMAP4 during dispersion has an opposite effect. PMID:25143402

  8. GIT1/βPIX signaling proteins and PAK1 kinase regulate microtubule nucleation.

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    Černohorská, Markéta; Sulimenko, Vadym; Hájková, Zuzana; Sulimenko, Tetyana; Sládková, Vladimíra; Vinopal, Stanislav; Dráberová, Eduarda; Dráber, Pavel

    2016-06-01

    Microtubule nucleation from γ-tubulin complexes, located at the centrosome, is an essential step in the formation of the microtubule cytoskeleton. However, the signaling mechanisms that regulate microtubule nucleation in interphase cells are largely unknown. In this study, we report that γ-tubulin is in complexes containing G protein-coupled receptor kinase-interacting protein 1 (GIT1), p21-activated kinase interacting exchange factor (βPIX), and p21 protein (Cdc42/Rac)-activated kinase 1 (PAK1) in various cell lines. Immunofluorescence microscopy revealed association of GIT1, βPIX and activated PAK1 with centrosomes. Microtubule regrowth experiments showed that depletion of βPIX stimulated microtubule nucleation, while depletion of GIT1 or PAK1 resulted in decreased nucleation in the interphase cells. These data were confirmed for GIT1 and βPIX by phenotypic rescue experiments, and counting of new microtubules emanating from centrosomes during the microtubule regrowth. The importance of PAK1 for microtubule nucleation was corroborated by the inhibition of its kinase activity with IPA-3 inhibitor. GIT1 with PAK1 thus represent positive regulators, and βPIX is a negative regulator of microtubule nucleation from the interphase centrosomes. The regulatory roles of GIT1, βPIX and PAK1 in microtubule nucleation correlated with recruitment of γ-tubulin to the centrosome. Furthermore, in vitro kinase assays showed that GIT1 and βPIX, but not γ-tubulin, serve as substrates for PAK1. Finally, direct interaction of γ-tubulin with the C-terminal domain of βPIX and the N-terminal domain of GIT1, which targets this protein to the centrosome, was determined by pull-down experiments. We propose that GIT1/βPIX signaling proteins with PAK1 kinase represent a novel regulatory mechanism of microtubule nucleation in interphase cells. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. A mutation of the fission yeast EB1 overcomes negative regulation by phosphorylation and stabilizes microtubules

    International Nuclear Information System (INIS)

    Iimori, Makoto; Ozaki, Kanako; Chikashige, Yuji; Habu, Toshiyuki; Hiraoka, Yasushi; Maki, Takahisa; Hayashi, Ikuko; Obuse, Chikashi; Matsumoto, Tomohiro

    2012-01-01

    Mal3 is a fission yeast homolog of EB1, a plus-end tracking protein (+ TIP). We have generated a mutation (89R) replacing glutamine with arginine in the calponin homology (CH) domain of Mal3. Analysis of the 89R mutant in vitro has revealed that the mutation confers a higher affinity to microtubules and enhances the intrinsic activity to promote the microtubule-assembly. The mutant Mal3 is no longer a + TIP, but binds strongly the microtubule lattice. Live cell imaging has revealed that while the wild type Mal3 proteins dissociate from the tip of the growing microtubules before the onset of shrinkage, the mutant Mal3 proteins persist on microtubules and reduces a rate of shrinkage after a longer pausing period. Consequently, the mutant Mal3 proteins cause abnormal elongation of microtubules composing the spindle and aster. Mal3 is phosphorylated at a cluster of serine/threonine residues in the linker connecting the CH and EB1-like C-terminal motif domains. The phosphorylation occurs in a microtubule-dependent manner and reduces the affinity of Mal3 to microtubules. We propose that because the 89R mutation is resistant to the effect of phosphorylation, it can associate persistently with microtubules and confers a stronger stability of microtubules likely by reinforcing the cylindrical structure. -- Highlights: ► We characterize a mutation (mal3-89R) in fission yeast homolog of EB1. ► The mutation enhances the activity to assemble microtubules. ► Mal3 is phosphorylated in a microtubule-dependent manner. ► The phosphorylation negatively regulates the Mal3 activity.

  10. A mutation of the fission yeast EB1 overcomes negative regulation by phosphorylation and stabilizes microtubules

    Energy Technology Data Exchange (ETDEWEB)

    Iimori, Makoto; Ozaki, Kanako [Graduate School of Biostudies, Kyoto University, Kitashirakawa-Oiwake cho, Sakyo ku, Kyoto, 606-8502 (Japan); Chikashige, Yuji [Kobe Advanced ICT Research Center, National Institute of Information and Communications Technology, Kobe, 651-2492 (Japan); Habu, Toshiyuki [Graduate School of Biostudies, Kyoto University, Kitashirakawa-Oiwake cho, Sakyo ku, Kyoto, 606-8502 (Japan); Radiation Biology Center, Kyoto University, Yoshida-Konoe cho, Sakyo ku, Kyoto, 606-8501 (Japan); Hiraoka, Yasushi [Kobe Advanced ICT Research Center, National Institute of Information and Communications Technology, Kobe, 651-2492 (Japan); Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, 565-0871 (Japan); Maki, Takahisa; Hayashi, Ikuko [Graduate School of Nanobioscience, Yokohama City University, Tsurumi, Yokohama, 230-0045 (Japan); Obuse, Chikashi [Graduate School of Life Science, Hokkaido University, Sapporo 001-0021 (Japan); Matsumoto, Tomohiro, E-mail: tmatsumo@house.rbc.kyoto-u.ac.jp [Graduate School of Biostudies, Kyoto University, Kitashirakawa-Oiwake cho, Sakyo ku, Kyoto, 606-8502 (Japan); Radiation Biology Center, Kyoto University, Yoshida-Konoe cho, Sakyo ku, Kyoto, 606-8501 (Japan)

    2012-02-01

    Mal3 is a fission yeast homolog of EB1, a plus-end tracking protein (+ TIP). We have generated a mutation (89R) replacing glutamine with arginine in the calponin homology (CH) domain of Mal3. Analysis of the 89R mutant in vitro has revealed that the mutation confers a higher affinity to microtubules and enhances the intrinsic activity to promote the microtubule-assembly. The mutant Mal3 is no longer a + TIP, but binds strongly the microtubule lattice. Live cell imaging has revealed that while the wild type Mal3 proteins dissociate from the tip of the growing microtubules before the onset of shrinkage, the mutant Mal3 proteins persist on microtubules and reduces a rate of shrinkage after a longer pausing period. Consequently, the mutant Mal3 proteins cause abnormal elongation of microtubules composing the spindle and aster. Mal3 is phosphorylated at a cluster of serine/threonine residues in the linker connecting the CH and EB1-like C-terminal motif domains. The phosphorylation occurs in a microtubule-dependent manner and reduces the affinity of Mal3 to microtubules. We propose that because the 89R mutation is resistant to the effect of phosphorylation, it can associate persistently with microtubules and confers a stronger stability of microtubules likely by reinforcing the cylindrical structure. -- Highlights: Black-Right-Pointing-Pointer We characterize a mutation (mal3-89R) in fission yeast homolog of EB1. Black-Right-Pointing-Pointer The mutation enhances the activity to assemble microtubules. Black-Right-Pointing-Pointer Mal3 is phosphorylated in a microtubule-dependent manner. Black-Right-Pointing-Pointer The phosphorylation negatively regulates the Mal3 activity.

  11. Measuring and modeling polymer concentration profiles near spindle boundaries argues that spindle microtubules regulate their own nucleation

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    Kaye, Bryan; Stiehl, Olivia; Foster, Peter J.; Shelley, Michael J.; Needleman, Daniel J.; Fürthauer, Sebastian

    2018-05-01

    Spindles are self-organized microtubule-based structures that segregate chromosomes during cell division. The mass of the spindle is controlled by the balance between microtubule turnover and nucleation. The mechanisms that control the spatial regulation of microtubule nucleation remain poorly understood. While previous work found that microtubule nucleators bind to pre-existing microtubules in the spindle, it is still unclear whether this binding regulates the activity of those nucleators. Here we use a combination of experiments and mathematical modeling to investigate this issue. We measured the concentration of microtubules and soluble tubulin in and around the spindle. We found a very sharp decay in the concentration of microtubules at the spindle interface. This is inconsistent with a model in which the activity of nucleators is independent of their association with microtubules but consistent with a model in which microtubule nucleators are only active when bound to pre-existing microtubules. This argues that the activity of microtubule nucleators is greatly enhanced when bound to pre-existing microtubules. Thus, microtubule nucleators are both localized and activated by the microtubules they generate.

  12. Brassinosteroids regulate pavement cell growth by mediating BIN2-induced microtubule stabilization.

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    Liu, Xiaolei; Yang, Qin; Wang, Yuan; Wang, Linhai; Fu, Ying; Wang, Xuelu

    2018-02-23

    Brassinosteroids (BRs), a group of plant steroid hormones, play important roles in regulating plant development. The cytoskeleton also affects key developmental processes and a deficiency in BR biosynthesis or signaling leads to abnormal phenotypes similar to those of microtubule-defective mutants. However, how BRs regulate microtubule and cell morphology remains unknown. Here, using liquid chromatography-tandem mass spectrometry, we identified tubulin proteins that interact with Arabidopsis BRASSINOSTEROID INSENSITIVE2 (BIN2), a negative regulator of BR responses in plants. In vitro and in vivo pull-down assays confirmed that BIN2 interacts with tubulin proteins. High-speed co-sedimentation assays demonstrated that BIN2 also binds microtubules. The Arabidopsis genome also encodes two BIN2 homologs, BIN2-LIKE 1 (BIL1) and BIL2, which function redundantly with BIN2. In the bin2-3 bil1 bil2 triple mutant, cortical microtubules were more sensitive to treatment with the microtubule-disrupting drug oryzalin than in wild-type, whereas in the BIN2 gain-of-function mutant bin2-1, cortical microtubules were insensitive to oryzalin treatment. These results provide important insight into how BR regulates plant pavement cell and leaf growth by mediating the stabilization of microtubules by BIN2.

  13. Ibuprofen regulation of microtubule dynamics in cystic fibrosis epithelial cells.

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    Rymut, Sharon M; Kampman, Claire M; Corey, Deborah A; Endres, Tori; Cotton, Calvin U; Kelley, Thomas J

    2016-08-01

    High-dose ibuprofen, an effective anti-inflammatory therapy for the treatment of cystic fibrosis (CF), has been shown to preserve lung function in a pediatric population. Despite its efficacy, few patients receive ibuprofen treatment due to potential renal and gastrointestinal toxicity. The mechanism of ibuprofen efficacy is also unclear. We have previously demonstrated that CF microtubules are slower to reform after depolymerization compared with respective wild-type controls. Slower microtubule dynamics in CF cells are responsible for impaired intracellular transport and are related to inflammatory signaling. Here, it is identified that high-dose ibuprofen treatment in both CF cell models and primary CF nasal epithelial cells restores microtubule reformation rates to wild-type levels, as well as induce extension of microtubules to the cell periphery. Ibuprofen treatment also restores microtubule-dependent intracellular transport monitored by measuring intracellular cholesterol transport. These effects are specific to ibuprofen as other cyclooxygenase inhibitors have no effect on these measures. Effects of ibuprofen are mimicked by stimulation of AMPK and blocked by the AMPK inhibitor compound C. We conclude that high-dose ibuprofen treatment enhances microtubule formation in CF cells likely through an AMPK-related pathway. These findings define a potential mechanism to explain the efficacy of ibuprofen therapy in CF. Copyright © 2016 the American Physiological Society.

  14. TONNEAU2/FASS Regulates the Geometry of Microtubule Nucleation and Cortical Array Organization in Interphase Arabidopsis Cells[C][W

    Science.gov (United States)

    Kirik, Angela; Ehrhardt, David W.; Kirik, Viktor

    2012-01-01

    Organization of microtubules into ordered arrays involves spatial and temporal regulation of microtubule nucleation. Here, we show that acentrosomal microtubule nucleation in plant cells involves a previously unknown regulatory step that determines the geometry of microtubule nucleation. Dynamic imaging of interphase cortical microtubules revealed that the ratio of branching to in-bundle microtubule nucleation on cortical microtubules is regulated by the Arabidopsis thaliana B′′ subunit of protein phosphatase 2A, which is encoded by the TONNEAU2/FASS (TON2) gene. The probability of nucleation from γ-tubulin complexes localized at the cell cortex was not affected by a loss of TON2 function, suggesting a specific role of TON2 in regulating the nucleation geometry. Both loss of TON2 function and ectopic targeting of TON2 to the plasma membrane resulted in defects in cell shape, suggesting the importance of TON2-mediated regulation of the microtubule cytoskeleton in cell morphogenesis. Loss of TON2 function also resulted in an inability for cortical arrays to reorient in response to light stimulus, suggesting an essential role for TON2 and microtubule branching nucleation in reorganization of microtubule arrays. Our data establish TON2 as a regulator of interphase microtubule nucleation and provide experimental evidence for a novel regulatory step in the process of microtubule-dependent nucleation. PMID:22395485

  15. Regulation of microtubule nucleation mediated by gamma-tubulin complexes

    Czech Academy of Sciences Publication Activity Database

    Sulimenko, Vadym; Hájková, Zuzana; Klebanovych, Anastasiya; Dráber, Pavel

    2017-01-01

    Roč. 254, č. 3 (2017), s. 1187-1199 ISSN 0033-183X R&D Projects: GA MŠk(CZ) LD13015 Institutional support: RVO:68378050 Keywords : mitotic spindle formation * ring complex * fission yeast * organizing centers * protein complex * golgi-complex * cell-cycle * pole body * augmin * centrosome * Centrosomes * Microtubule nucleation * Microtubule-organizing centers * Non-centrosomal nucleation sites * Spindle pole bodies * gamma-Tubulin complexes Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Cell biology Impact factor: 2.870, year: 2016

  16. Cep169, a Novel Microtubule Plus-End-Tracking Centrosomal Protein, Binds to CDK5RAP2 and Regulates Microtubule Stability.

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    Yusuke Mori

    Full Text Available The centrosomal protein, CDK5RAP2, is a microcephaly protein that regulates centrosomal maturation by recruitment of a γ-tubulin ring complex (γ-TuRC onto centrosomes. In this report, we identified a novel human centrosomal protein, Cep169, as a binding partner of CDK5RAP2, a member of microtubule plus-end-tracking proteins (+TIPs. Cep169 interacts directly with CDK5RAP2 through CM1, an evolutionarily conserved domain, and colocalizes at the pericentriolar matrix (PCM around centrioles with CDK5RAP2. In addition, Cep169 interacts with EB1 through SxIP-motif responsible for EB1 binding, and colocalizes with CDK5RAP2 at the microtubule plus-end. EB1-binding-deficient Cep169 abolishes EB1 interaction and microtubule plus-end attachment, indicating Cep169 as a novel member of +TIPs. We further show that ectopic expression of either Cep169 or CDK5RAP2 induces microtubule bundling and acetylation in U2OS cells, and depletion of Cep169 induces microtubule depolymerization in HeLa cells, although Cep169 is not required for assembly of γ-tubulin onto centrosome by CDK5RAP2. These results show that Cep169 targets microtubule tips and regulates stability of microtubules with CDK5RAP2.

  17. Complementary activities of TPX2 and chTOG constitute an efficient importin-regulated microtubule nucleation module.

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    Roostalu, Johanna; Cade, Nicholas I; Surrey, Thomas

    2015-11-01

    Spindle assembly and function require precise control of microtubule nucleation and dynamics. The chromatin-driven spindle assembly pathway exerts such control locally in the vicinity of chromosomes. One of the key targets of this pathway is TPX2. The molecular mechanism of how TPX2 stimulates microtubule nucleation is not understood. Using microscopy-based dynamic in vitro reconstitution assays with purified proteins, we find that human TPX2 directly stabilizes growing microtubule ends and stimulates microtubule nucleation by stabilizing early microtubule nucleation intermediates. Human microtubule polymerase chTOG (XMAP215/Msps/Stu2p/Dis1/Alp14 homologue) only weakly promotes nucleation, but acts synergistically with TPX2. Hence, a combination of distinct and complementary activities is sufficient for efficient microtubule formation in vitro. Importins control the efficiency of the microtubule nucleation by selectively blocking the interaction of TPX2 with microtubule nucleation intermediates. This in vitro reconstitution reveals the molecular mechanism of regulated microtubule formation by a minimal nucleation module essential for chromatin-dependent microtubule nucleation in cells.

  18. SDF1 Reduces Interneuron Leading Process Branching through Dual Regulation of Actin and Microtubules

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    Lysko, Daniel E.; Putt, Mary

    2014-01-01

    Normal cerebral cortical function requires a highly ordered balance between projection neurons and interneurons. During development these two neuronal populations migrate from distinct progenitor zones to form the cerebral cortex, with interneurons originating in the more distant ganglionic eminences. Moreover, deficits in interneurons have been linked to a variety of neurodevelopmental disorders underscoring the importance of understanding interneuron development and function. We, and others, have identified SDF1 signaling as one important modulator of interneuron migration speed and leading process branching behavior in mice, although how SDF1 signaling impacts these behaviors remains unknown. We previously found SDF1 inhibited leading process branching while increasing the rate of migration. We have now mechanistically linked SDF1 modulation of leading process branching behavior to a dual regulation of both actin and microtubule organization. We find SDF1 consolidates actin at the leading process tip by de-repressing calpain protease and increasing proteolysis of branched-actin-supporting cortactin. Additionally, SDF1 stabilizes the microtubule array in the leading process through activation of the microtubule-associated protein doublecortin (DCX). DCX stabilizes the microtubule array by bundling microtubules within the leading process, reducing branching. These data provide mechanistic insight into the regulation of interneuron leading process dynamics during neuronal migration in mice and provides insight into how cortactin and DCX, a known human neuronal migration disorder gene, participate in this process. PMID:24695713

  19. SDF1 reduces interneuron leading process branching through dual regulation of actin and microtubules.

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    Lysko, Daniel E; Putt, Mary; Golden, Jeffrey A

    2014-04-02

    Normal cerebral cortical function requires a highly ordered balance between projection neurons and interneurons. During development these two neuronal populations migrate from distinct progenitor zones to form the cerebral cortex, with interneurons originating in the more distant ganglionic eminences. Moreover, deficits in interneurons have been linked to a variety of neurodevelopmental disorders underscoring the importance of understanding interneuron development and function. We, and others, have identified SDF1 signaling as one important modulator of interneuron migration speed and leading process branching behavior in mice, although how SDF1 signaling impacts these behaviors remains unknown. We previously found SDF1 inhibited leading process branching while increasing the rate of migration. We have now mechanistically linked SDF1 modulation of leading process branching behavior to a dual regulation of both actin and microtubule organization. We find SDF1 consolidates actin at the leading process tip by de-repressing calpain protease and increasing proteolysis of branched-actin-supporting cortactin. Additionally, SDF1 stabilizes the microtubule array in the leading process through activation of the microtubule-associated protein doublecortin (DCX). DCX stabilizes the microtubule array by bundling microtubules within the leading process, reducing branching. These data provide mechanistic insight into the regulation of interneuron leading process dynamics during neuronal migration in mice and provides insight into how cortactin and DCX, a known human neuronal migration disorder gene, participate in this process.

  20. Calcium regulates ATP-sensitive microtubule binding by Chlamydomonas outer arm dynein.

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    Sakato, Miho; King, Stephen M

    2003-10-31

    The Chlamydomonas outer dynein arm contains three distinct heavy chains (alpha, beta, and gamma) that exhibit different motor properties. The LC4 protein, which binds 1-2 Ca2+ with KCa = 3 x 10-5 m, is associated with the gamma heavy chain and has been proposed to act as a sensor to regulate dynein motor function in response to alterations in intraflagellar Ca2+ levels. Here we genetically dissect the outer arm to yield subparticles containing different motor unit combinations and assess the microtubule-binding properties of these complexes both prior to and following preincubation with tubulin and ATP, which was used to inhibit ATP-insensitive (structural) microtubule binding. We observed that the alpha heavy chain exhibits a dominant Ca2+-independent ATP-sensitive MT binding activity in vitro that is inhibited by attachment of tubulin to the structural microtubule-binding domain. Furthermore, we show that ATP-sensitive microtubule binding by a dynein subparticle containing only the beta and gamma heavy chains does not occur at Ca2+ concentrations below pCa 6 but is maximally activated above pCa 5. This activity was not observed in mutant dyneins containing small deletions in the microtubule-binding region of the beta heavy chain or in dyneins that lack both the alpha heavy chain and the motor domain of the beta heavy chain. These findings strongly suggest that Ca2+ binding directly to a component of the dynein complex regulates ATP-sensitive interactions between the beta heavy chain and microtubules and lead to a model for how individual motor units are controlled within the outer dynein arm.

  1. KATNAL1 regulation of sertoli cell microtubule dynamics is essential for spermiogenesis and male fertility.

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    Lee B Smith

    Full Text Available Spermatogenesis is a complex process reliant upon interactions between germ cells (GC and supporting somatic cells. Testicular Sertoli cells (SC support GCs during maturation through physical attachment, the provision of nutrients, and protection from immunological attack. This role is facilitated by an active cytoskeleton of parallel microtubule arrays that permit transport of nutrients to GCs, as well as translocation of spermatids through the seminiferous epithelium during maturation. It is well established that chemical perturbation of SC microtubule remodelling leads to premature GC exfoliation demonstrating that microtubule remodelling is an essential component of male fertility, yet the genes responsible for this process remain unknown. Using a random ENU mutagenesis approach, we have identified a novel mouse line displaying male-specific infertility, due to a point mutation in the highly conserved ATPase domain of the novel KATANIN p60-related microtubule severing protein Katanin p60 subunit A-like1 (KATNAL1. We demonstrate that Katnal1 is expressed in testicular Sertoli cells (SC from 15.5 days post-coitum (dpc and that, consistent with chemical disruption models, loss of function of KATNAL1 leads to male-specific infertility through disruption of SC microtubule dynamics and premature exfoliation of spermatids from the seminiferous epithelium. The identification of KATNAL1 as an essential regulator of male fertility provides a significant novel entry point into advancing our understanding of how SC microtubule dynamics promotes male fertility. Such information will have resonance both for future treatment of male fertility and the development of non-hormonal male contraceptives.

  2. Microtubule-Mediated Inositol Lipid Signaling Plays Critical Roles in Regulation of Blebbing.

    Directory of Open Access Journals (Sweden)

    Tatsuroh Sugiyama

    Full Text Available Cells migrate by extending pseudopods such as lamellipodia and blebs. Although the signals leading to lamellipodia extension have been extensively investigated, those for bleb extension remain unclear. Here, we investigated signals for blebbing in Dictyostelium cells using a newly developed assay to induce blebbing. When cells were cut into two pieces with a microneedle, the anucleate fragments vigorously extended blebs. This assay enabled us to induce blebbing reproducibly, and analyses of knockout mutants and specific inhibitors identified candidate molecules that regulate blebbing. Blebs were also induced in anucleate fragments of leukocytes, indicating that this assay is generally applicable to animal cells. After cutting, microtubules in the anucleate fragments promptly depolymerized, followed by the extension of blebs. Furthermore, when intact cells were treated with a microtubule inhibitor, they frequently extended blebs. The depolymerization of microtubules induced the delocalization of inositol lipid phosphatidylinositol 3,4,5-trisphosphate from the cell membrane. PI3 kinase-null cells frequently extended blebs, whereas PTEN-null cells extended fewer blebs. From these observations, we propose a model in which microtubules play a critical role in bleb regulation via inositol lipid metabolism.

  3. Optimization of microtubule affinity regulating kinase (MARK) inhibitors with improved physical properties

    Energy Technology Data Exchange (ETDEWEB)

    Sloman, David L.; Noucti, Njamkou; Altman, Michael D.; Chen, Dapeng; Mislak, Andrea C.; Szewczak, Alexander; Hayashi, Mansuo; Warren, Lee; Dellovade, Tammy; Wu, Zhenhua; Marcus, Jacob; Walker, Deborah; Su, Hua-Poo; Edavettal, Suzanne C.; Munshi, Sanjeev; Hutton, Michael; Nuthall, Hugh; Stanton, Matthew G. (Merck)

    2016-09-01

    Inhibition of microtubule affinity regulating kinase (MARK) represents a potentially attractive means of arresting neurofibrillary tangle pathology in Alzheimer’s disease. This manuscript outlines efforts to optimize a pyrazolopyrimidine series of MARK inhibitors by focusing on improvements in potency, physical properties and attributes amenable to CNS penetration. A unique cylcyclohexyldiamine scaffold was identified that led to remarkable improvements in potency, opening up opportunities to reduce MW, Pgp efflux and improve pharmacokinetic properties while also conferring improved solubility.

  4. A Genome-wide RNAi Screen for Microtubule Bundle Formation and Lysosome Motility Regulation in Drosophila S2 Cells

    Directory of Open Access Journals (Sweden)

    Amber L. Jolly

    2016-01-01

    Full Text Available Long-distance intracellular transport of organelles, mRNA, and proteins (“cargo” occurs along the microtubule cytoskeleton by the action of kinesin and dynein motor proteins, but the vast network of factors involved in regulating intracellular cargo transport are still unknown. We capitalize on the Drosophila melanogaster S2 model cell system to monitor lysosome transport along microtubule bundles, which require enzymatically active kinesin-1 motor protein for their formation. We use an automated tracking program and a naive Bayesian classifier for the multivariate motility data to analyze 15,683 gene phenotypes and find 98 proteins involved in regulating lysosome motility along microtubules and 48 involved in the formation of microtubule filled processes in S2 cells. We identify innate immunity genes, ion channels, and signaling proteins having a role in lysosome motility regulation and find an unexpected relationship between the dynein motor, Rab7a, and lysosome motility regulation.

  5. Shaping the tracks : Regulation of microtubule dynamics by kinesins KIF21A and KIF21B

    NARCIS (Netherlands)

    van Riel, W.E.|info:eu-repo/dai/nl/338772634

    2016-01-01

    Control of microtubule dynamics is important for cell morphogenesis. Kinesins, motor proteins known to function in cargo transport, were recently also implicated in altering the microtubule network. Several kinesins are described to cause microtubule network reorganization or stabilization, either

  6. Microtubule Regulation of Kv7 Channels Orchestrates cAMP-Mediated Vasorelaxations in Rat Arterial Smooth Muscle

    DEFF Research Database (Denmark)

    Lindman, Johanna; Khammy, Makhala M; Lundegaard, Pia R

    2018-01-01

    Microtubules can regulate GPCR (G protein-coupled receptor) signaling in various cell types. In vascular smooth muscle, activation of the β-adrenoceptor leads to production of cAMP to mediate a vasorelaxation. Little is known about the role of microtubules in smooth muscle, and given the importance...... of renal and mesenteric arteries that the microtubule stabilizer, paclitaxel, prevented. Sharp microelectrode experiments showed that colchicine treatment caused increased hyperpolarization of mesenteric artery segments in response to isoprenaline. Application of the Kv7 channel blocker, XE991, attenuated...

  7. Microtubule Protofilament Number Is Modulated in a Step-Wise Fashion By the Charge of Density of An Enveloping Layer

    International Nuclear Information System (INIS)

    Raviv, U.; Nguyen, T.; Ghafouri, R.; Needleman, D.J.; Li, Y.; Miller, H.P.; Wilson, L.; Bruinsma, R.F.; Safinya, C.R.; UC, Santa Barbara; UCLA

    2007-01-01

    Microtubules are able to adjust their protofilament (PF) number and, as a consequence, their dynamics and function, to the assembly conditions and presence of cofactors. However, the principle behind such variations is poorly understood. Using synchrotron x-ray scattering and transmission electron microscopy, we studied how charged membranes, which under certain conditions can envelop preassembled MTs, regulate the PF number of those MTs. We show that the mean PF number, , is modulated primarily by the charge density of the membranes. decreases in a stepwise fashion with increasing membrane charge density. does not depend on the membrane-protein stoichiometry or the solution ionic strength. We studied the effect of taxol and found that increases logarithmically with taxol/tubulin stoichiometry. We present a theoretical model, which by balancing the electrostatic and elastic interactions in the system accounts for the trends in our findings and reveals an effective MT bending stiffness of order 10-100 k B T/nm, associated with the observed changes in PF number

  8. GTSE1 is a microtubule plus-end tracking protein that regulates EB1-dependent cell migration.

    Directory of Open Access Journals (Sweden)

    Massimilano Scolz

    Full Text Available The regulation of cell migration is a highly complex process that is often compromised when cancer cells become metastatic. The microtubule cytoskeleton is necessary for cell migration, but how microtubules and microtubule-associated proteins regulate multiple pathways promoting cell migration remains unclear. Microtubule plus-end binding proteins (+TIPs are emerging as important players in many cellular functions, including cell migration. Here we identify a +TIP, GTSE1, that promotes cell migration. GTSE1 accumulates at growing microtubule plus ends through interaction with the EB1+TIP. The EB1-dependent +TIP activity of GTSE1 is required for cell migration, as well as for microtubule-dependent disassembly of focal adhesions. GTSE1 protein levels determine the migratory capacity of both nontransformed and breast cancer cell lines. In breast cancers, increased GTSE1 expression correlates with invasive potential, tumor stage, and time to distant metastasis, suggesting that misregulation of GTSE1 expression could be associated with increased invasive potential.

  9. TCS1, a Microtubule-Binding Protein, Interacts with KCBP/ZWICHEL to Regulate Trichome Cell Shape in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Liangliang Chen

    2016-10-01

    Full Text Available How cell shape is controlled is a fundamental question in developmental biology, but the genetic and molecular mechanisms that determine cell shape are largely unknown. Arabidopsis trichomes have been used as a good model system to investigate cell shape at the single-cell level. Here we describe the trichome cell shape 1 (tcs1 mutants with the reduced trichome branch number in Arabidopsis. TCS1 encodes a coiled-coil domain-containing protein. Pharmacological analyses and observations of microtubule dynamics show that TCS1 influences the stability of microtubules. Biochemical analyses and live-cell imaging indicate that TCS1 binds to microtubules and promotes the assembly of microtubules. Further results reveal that TCS1 physically associates with KCBP/ZWICHEL, a microtubule motor involved in the regulation of trichome branch number. Genetic analyses indicate that kcbp/zwi is epistatic to tcs1 with respect to trichome branch number. Thus, our findings define a novel genetic and molecular mechanism by which TCS1 interacts with KCBP to regulate trichome cell shape by influencing the stability of microtubules.

  10. Integrins Regulate Apical Constriction via Microtubule Stabilization in the Drosophila Eye Disc Epithelium

    Directory of Open Access Journals (Sweden)

    Vilaiwan M. Fernandes

    2014-12-01

    Full Text Available During morphogenesis, extracellular signals trigger actomyosin contractility in subpopulations of cells to coordinate changes in cell shape. To illuminate the link between signaling-mediated tissue patterning and cytoskeletal remodeling, we study the progression of the morphogenetic furrow (MF, the wave of apical constriction that traverses the Drosophila eye imaginal disc preceding photoreceptor neurogenesis. Apical constriction depends on actomyosin contractility downstream of the Hedgehog (Hh and bone morphogenetic protein (BMP pathways. We identify a role for integrin adhesion receptors in MF progression. We show that Hh and BMP regulate integrin expression, the loss of which disrupts apical constriction and slows furrow progression; conversely, elevated integrins accelerate furrow progression. We present evidence that integrins regulate MF progression by promoting microtubule stabilization, since reducing microtubule stability rescues integrin-mediated furrow acceleration. Thus, integrins act as a genetic link between tissue-level signaling events and morphological change at the cellular level, leading to morphogenesis and neurogenesis in the eye.

  11. Microtubule affinity-regulating kinases are potential druggable targets for Alzheimer's disease.

    Science.gov (United States)

    Annadurai, Narendran; Agrawal, Khushboo; Džubák, Petr; Hajdúch, Marián; Das, Viswanath

    2017-11-01

    Alzheimer's disease (AD) is a progressive neurodegenerative disorder that affects normal functions of the brain. Currently, AD is one of the leading causes of death in developed countries and the only one of the top ten diseases without a means to prevent, cure, or significantly slow down its progression. Therefore, newer therapeutic concepts are urgently needed to improve survival and the quality of life of AD patients. Microtubule affinity-regulating kinases (MARKs) regulate tau-microtubule binding and play a crucial role in neurons. However, their role in hyperphosphorylation of tau makes them potential druggable target for AD therapy. Despite the relevance of MARKs in AD pathogenesis, only a few small molecules are known to have anti-MARK activity and not much has been done to progress these compounds into therapeutic candidates. But given the diverse role of MARKs, the specificity of novel inhibitors is imperative for their successful translation from bench to bedside. In this regard, a recent co-crystal structure of MARK4 in association with a pyrazolopyrimidine-based inhibitor offers a potential scaffold for the development of more specific MARK inhibitors. In this manuscript, we review the biological role of MARKs in health and disease, and draw attention to the largely unexplored area of MARK inhibitors for AD.

  12. Phosphorylation of the yeast γ-tubulin Tub4 regulates microtubule function

    DEFF Research Database (Denmark)

    Lin, Tien-chen; Gombos, Linda; Neuner, Annett

    2011-01-01

    The yeast ¿-tubulin Tub4 is assembled with Spc97 and Spc98 into the small Tub4 complex. The Tub4 complex binds via the receptor proteins Spc72 and Spc110 to the spindle pole body (SPB), the functional equivalent of the mammalian centrosome, where the Tub4 complex organizes cytoplasmic and nuclear...... microtubules. Little is known about the regulation of the Tub4 complex. Here, we isolated the Tub4 complex with the bound receptors from yeast cells. Analysis of the purified Tub4 complex by mass spectrometry identified more than 50 phosphorylation sites in Spc72, Spc97, Spc98, Spc110 and Tub4. To examine...... the functional relevance of the phosphorylation sites, phospho-mimicking and non-phosphorylatable mutations in Tub4, Spc97 and Spc98 were analyzed. Three phosphorylation sites in Tub4 were found to be critical for Tub4 stability and microtubule organization. One of the sites is highly conserved in ¿-tubulins...

  13. ATX-2, the C. elegans Ortholog of Human Ataxin-2, Regulates Centrosome Size and Microtubule Dynamics.

    Directory of Open Access Journals (Sweden)

    Michael D Stubenvoll

    2016-09-01

    Full Text Available Centrosomes are critical sites for orchestrating microtubule dynamics, and exhibit dynamic changes in size during the cell cycle. As cells progress to mitosis, centrosomes recruit more microtubules (MT to form mitotic bipolar spindles that ensure proper chromosome segregation. We report a new role for ATX-2, a C. elegans ortholog of Human Ataxin-2, in regulating centrosome size and MT dynamics. ATX-2, an RNA-binding protein, forms a complex with SZY-20 in an RNA-independent fashion. Depleting ATX-2 results in embryonic lethality and cytokinesis failure, and restores centrosome duplication to zyg-1 mutants. In this pathway, SZY-20 promotes ATX-2 abundance, which inversely correlates with centrosome size. Centrosomes depleted of ATX-2 exhibit elevated levels of centrosome factors (ZYG-1, SPD-5, γ-Tubulin, increasing MT nucleating activity but impeding MT growth. We show that ATX-2 influences MT behavior through γ-Tubulin at the centrosome. Our data suggest that RNA-binding proteins play an active role in controlling MT dynamics and provide insight into the control of proper centrosome size and MT dynamics.

  14. Differential regulation of microtubule severing by APC underlies distinct patterns of projection neuron and interneuron migration

    Science.gov (United States)

    Eom, Tae-Yeon; Stanco, Amelia; Guo, Jiami; Wilkins, Gary; Deslauriers, Danielle; Yan, Jessica; Monckton, Chase; Blair, Josh; Oon, Eesim; Perez, Abby; Salas, Eduardo; Oh, Adrianna; Ghukasyan, Vladimir; Snider, William D.; Rubenstein, John L. R.; Anton, E. S.

    2014-01-01

    Coordinated migration of distinct classes of neurons to appropriate positions leads to the formation of functional neuronal circuitry in the cerebral cortex. Two major classes of cortical neurons, interneurons and projection neurons, utilize distinctly different modes (radial vs. tangential) and routes of migration to arrive at their final positions in the cerebral cortex. Here, we show that adenomatous polyposis coli (APC) modulates microtubule (MT) severing in interneurons to facilitate tangential mode of interneuron migration, but not the glial-guided, radial migration of projection neurons. APC regulates the stability and activity of the MT severing protein p60-katanin in interneurons to promote the rapid remodeling of neuronal processes necessary for interneuron migration. These findings reveal how severing and restructuring of MTs facilitate distinct modes of neuronal migration necessary for laminar organization of neurons in the developing cerebral cortex. PMID:25535916

  15. Glucose regulated proteins 78 and 75 bind to the receptor for hyaluronan mediated motility in interphase microtubules

    International Nuclear Information System (INIS)

    Kuwabara, Hiroko; Yoneda, Masahiko; Hayasaki, Hana; Nakamura, Toshiya; Mori, Hiroshi

    2006-01-01

    The receptor for hyaluronan mediated motility (RHAMM), which is a hyaluronan-binding protein, is a centrosomal and microtubal protein. Here, we have identified two RHAMM-binding proteins, glucose regulated protein (GRP) 78 and GRP75, using co-immunoprecipitation analysis. These two proteins directly bound to glutathione-S-transferase-RHAMM fusion proteins. By double immunostaining, GRP78 and GRP75 colocalized with RHAMM in interphase microtubules, but were separated in mitotic spindles. Prevention of microtubule polymerization by TN-16 and vincristine sulfate induced RHAMM overexpression without a significant change in GRP78/75. Taken together, GRP78/75 and RHAMM complexes may stabilize microtubules in the interphase, associated with a downregulation of RHAMM. These results reveal a new biochemical activity of RHAMM

  16. The Kinesin Adaptor Calsyntenin-1 Organizes Microtubule Polarity and Regulates Dynamics during Sensory Axon Arbor Development

    Directory of Open Access Journals (Sweden)

    Mary C. Halloran

    2017-04-01

    Full Text Available Axon growth and branching, and development of neuronal polarity are critically dependent on proper organization and dynamics of the microtubule (MT cytoskeleton. MTs must organize with correct polarity for delivery of diverse cargos to appropriate subcellular locations, yet the molecular mechanisms regulating MT polarity remain poorly understood. Moreover, how an actively branching axon reorganizes MTs to direct their plus ends distally at branch points is unknown. We used high-speed, in vivo imaging of polymerizing MT plus ends to characterize MT dynamics in developing sensory axon arbors in zebrafish embryos. We find that axonal MTs are highly dynamic throughout development, and that the peripheral and central axons of sensory neurons show differences in MT behaviors. Furthermore, we show that Calsyntenin-1 (Clstn-1, a kinesin adaptor required for sensory axon branching, also regulates MT polarity in developing axon arbors. In wild type neurons the vast majority of MTs are directed in the correct plus-end-distal orientation from early stages of development. Loss of Clstn-1 causes an increase in MTs polymerizing in the retrograde direction. These misoriented MTs most often are found near growth cones and branch points, suggesting Clstn-1 is particularly important for organizing MT polarity at these locations. Together, our results suggest that Clstn-1, in addition to regulating kinesin-mediated cargo transport, also organizes the underlying MT highway during axon arbor development.

  17. PP2A regulates kinetochore-microtubule attachment during meiosis I in oocyte.

    Science.gov (United States)

    Tang, An; Shi, Peiliang; Song, Anying; Zou, Dayuan; Zhou, Yue; Gu, Pengyu; Huang, Zan; Wang, Qinghua; Lin, Zhaoyu; Gao, Xiang

    2016-06-02

    Studies using in vitro cultured oocytes have indicated that the protein phosphatase 2A (PP2A), a major serine/threonine protein phosphatase, participates in multiple steps of meiosis. Details of oocyte maturation regulation by PP2A remain unclear and an in vivo model can provide more convincing information. Here, we inactivated PP2A by mutating genes encoding for its catalytic subunits (PP2Acs) in mouse oocytes. We found that eliminating both PP2Acs caused female infertility. Oocytes lacking PP2Acs failed to complete 1(st) meiotic division due to chromosome misalignment and abnormal spindle assembly. In mitosis, PP2A counteracts Aurora kinase B/C (AurkB/C) to facilitate correct kinetochore-microtubule (KT-MT) attachment. In meiosis I in oocyte, we found that PP2Ac deficiency destabilized KT-MT attachments. Chemical inhibition of AurkB/C in PP2Ac-null oocytes partly restored the formation of lateral/merotelic KT-MT attachments but not correct KT-MT attachments. Taken together, our findings demonstrate that PP2Acs are essential for chromosome alignments and regulate the formation of correct KT-MT attachments in meiosis I in oocytes.

  18. GSK3 controls axon growth via CLASP-mediated regulation of growth cone microtubules

    Science.gov (United States)

    Hur, Eun-Mi; Saijilafu; Lee, Byoung Dae; Kim, Seong-Jin; Xu, Wen-Lin; Zhou, Feng-Quan

    2011-01-01

    Suppression of glycogen synthase kinase 3 (GSK3) activity in neurons yields pleiotropic outcomes, causing both axon growth promotion and inhibition. Previous studies have suggested that specific GSK3 substrates, such as adenomatous polyposis coli (APC) and collapsin response mediator protein 2 (CRMP2), support axon growth by regulating the stability of axonal microtubules (MTs), but the substrate(s) and mechanisms conveying axon growth inhibition remain elusive. Here we show that CLIP (cytoplasmic linker protein)-associated protein (CLASP), originally identified as a MT plus end-binding protein, displays both plus end-binding and lattice-binding activities in nerve growth cones, and reveal that the two MT-binding activities regulate axon growth in an opposing manner: The lattice-binding activity mediates axon growth inhibition induced by suppression of GSK3 activity via preventing MT protrusion into the growth cone periphery, whereas the plus end-binding property supports axon extension via stabilizing the growing ends of axonal MTs. We propose a model in which CLASP transduces GSK3 activity levels to differentially control axon growth by coordinating the stability and configuration of growth cone MTs. PMID:21937714

  19. Coupling of kinesin ATP turnover to translocation and microtubule regulation: one engine, many machines.

    Science.gov (United States)

    Friel, Claire T; Howard, Jonathon

    2012-12-01

    The cycle of ATP turnover is integral to the action of motor proteins. Here we discuss how variation in this cycle leads to variation of function observed amongst members of the kinesin superfamily of microtubule associated motor proteins. Variation in the ATP turnover cycle among superfamily members can tune the characteristic kinesin motor to one of the range of microtubule-based functions performed by kinesins. The speed at which ATP is hydrolysed affects the speed of translocation. The ratio of rate constants of ATP turnover in relation to association and dissociation from the microtubule influence the processivity of translocation. Variation in the rate-limiting step of the cycle can reverse the way in which the motor domain interacts with the microtubule producing non-motile kinesins. Because the ATP turnover cycle is not fully understood for the majority of kinesins, much work remains to show how the kinesin engine functions in such a wide variety of molecular machines.

  20. The Monopolin Complex Crosslinks Kinetochore Components to Regulate Chromosome-Microtubule Attachments

    Energy Technology Data Exchange (ETDEWEB)

    Corbett, Kevin D.; Yip, Calvin K.; Ee, Ly-Sha; Walz, Thomas; Amon, Angelika; Harrison, Stephen C. (Harvard-Med); (MIT)

    2010-09-27

    The monopolin complex regulates different types of kinetochore-microtubule attachments in fungi, ensuring sister chromatid co-orientation in Saccharomyces cerevisiae meiosis I and inhibiting merotelic attachment in Schizosaccharomyces pombe mitosis. In addition, the monopolin complex maintains the integrity and silencing of ribosomal DNA (rDNA) repeats in the nucleolus. We show here that the S. cerevisiae Csm1/Lrs4 monopolin subcomplex has a distinctive V-shaped structure, with two pairs of protein-protein interaction domains positioned {approx}10 nm apart. Csm1 presents a conserved hydrophobic surface patch that binds two kinetochore proteins: Dsn1, a subunit of the outer-kinetochore MIND/Mis12 complex, and Mif2/CENP-C. Csm1 point-mutations that disrupt kinetochore-subunit binding also disrupt sister chromatid co-orientation in S. cerevisiae meiosis I. We further show that the same Csm1 point-mutations affect rDNA silencing, probably by disrupting binding to the rDNA-associated protein Tof2. We propose that Csm1/Lrs4 functions as a molecular clamp, crosslinking kinetochore components to enforce sister chromatid co-orientation in S. cerevisiae meiosis I and to suppress merotelic attachment in S. pombe mitosis, and crosslinking rDNA repeats to aid rDNA silencing.

  1. Active Erk Regulates Microtubule Stability in H-ras-Transformed Cells

    Directory of Open Access Journals (Sweden)

    Rene E. Harrison

    2001-01-01

    Full Text Available Increasing evidence suggests that activated erk regulates cell functions, at least in part, by mechanisms that do not require gene transcription. Here we show that the map kinase, erk, decorates microtubules (MTs and mitotic spindles in both parental and mutant active rastransfected 10T1 /2 fibroblasts and MCF10A breast epithelial cells. Approximately 20% of total cellular erk decorated MTs in both cell lines. A greater proportion of activated erk was associated with MTs in the presence of mutant active H-ras than in parental cells. Activation of erk by the ras pathway coincided with a decrease in the stability of MT, as detected by a stability marker. The MKK1 inhibitor, PD98059 and transfection of a dominant negative MKK1 blocked ras-induced instability of MTs but did not modify the association of erk with MTs or affect MT stability of the parental cells. These results indicate that the subset of active erk kinase that associates with MTs contributes to their instability in the presence of a mutant active ras. The MT-associated subset of active erk likely contributes to the enhanced invasive and proliferative abilities of cells containing mutant active H-ras.

  2. Hypothesis: NDL proteins function in stress responses by regulating microtubule organization.

    Science.gov (United States)

    Khatri, Nisha; Mudgil, Yashwanti

    2015-01-01

    N-MYC DOWNREGULATED-LIKE proteins (NDL), members of the alpha/beta hydrolase superfamily were recently rediscovered as interactors of G-protein signaling in Arabidopsis thaliana. Although the precise molecular function of NDL proteins is still elusive, in animals these proteins play protective role in hypoxia and expression is induced by hypoxia and nickel, indicating role in stress. Homology of NDL1 with animal counterpart N-MYC DOWNREGULATED GENE (NDRG) suggests similar functions in animals and plants. It is well established that stress responses leads to the microtubule depolymerization and reorganization which is crucial for stress tolerance. NDRG is a microtubule-associated protein which mediates the microtubule organization in animals by causing acetylation and increases the stability of α-tubulin. As NDL1 is highly homologous to NDRG, involvement of NDL1 in the microtubule organization during plant stress can also be expected. Discovery of interaction of NDL with protein kinesin light chain- related 1, enodomembrane family protein 70, syntaxin-23, tubulin alpha-2 chain, as a part of G protein interactome initiative encourages us to postulate microtubule stabilizing functions for NDL family in plants. Our search for NDL interactors in G protein interactome also predicts the role of NDL proteins in abiotic stress tolerance management. Based on published report in animals and predicted interacting partners for NDL in G protein interactome lead us to hypothesize involvement of NDL in the microtubule organization during abiotic stress management in plants.

  3. Calcium-dependent depletion zones in the cortical microtubule array coincide with sites of, but do not regulate, wall ingrowth papillae deposition in epidermal transfer cells

    Science.gov (United States)

    Zhang, Hui-ming; Talbot, Mark J.; McCurdy, David W.; Patrick, John W.; Offler, Christina E.

    2015-01-01

    Trans-differentiation to a transfer-cell morphology is characterized by the localized deposition of wall ingrowth papillae that protrude into the cytosol. Whether the cortical microtubule array directs wall ingrowth papillae formation was investigated using a Vicia faba cotyledon culture system in which their adaxial epidermal cells were spontaneously induced to trans-differentiate to transfer cells. During deposition of wall ingrowth papillae, the aligned cortical microtubule arrays in precursor epidermal cells were reorganized into a randomized array characterized by circular depletion zones. Concurrence of the temporal appearance, spatial pattern, and size of depletion zones and wall ingrowth papillae was consistent with each papilla occupying a depletion zone. Surprisingly, microtubules appeared not to regulate construction of wall ingrowth papillae, as neither depolymerization nor stabilization of cortical microtubules changed their deposition pattern or morphology. Moreover, the size and spatial pattern of depletion zones was unaltered when the formation of wall ingrowth papillae was blocked by inhibiting cellulose biosynthesis. In contrast, the depletion zones were absent when the cytosolic calcium plumes, responsible for directing wall ingrowth papillae formation, were blocked or dissipated. Thus, we conclude that the depletion zones within the cortical microtubule array result from localized depolymerization of microtubules initiated by elevated cytosolic Ca2+ levels at loci where wall ingrowth papillae are deposited. The physiological significance of the depletion zones as a mechanism to accommodate the construction of wall ingrowth papillae without compromising maintenance of the plasma membrane–microtubule inter-relationship is discussed. PMID:26136268

  4. Plasma membrane factor XIIIA transglutaminase activity regulates osteoblast matrix secretion and deposition by affecting microtubule dynamics.

    Directory of Open Access Journals (Sweden)

    Hadil F Al-Jallad

    2011-01-01

    Full Text Available Transglutaminase activity, arising potentially from transglutaminase 2 (TG2 and Factor XIIIA (FXIIIA, has been linked to osteoblast differentiation where it is required for type I collagen and fibronectin matrix deposition. In this study we have used an irreversible TG-inhibitor to 'block -and-track' enzyme(s targeted during osteoblast differentiation. We show that the irreversible TG-inhibitor is highly potent in inhibiting osteoblast differentiation and mineralization and reduces secretion of both fibronectin and type I collagen and their release from the cell surface. Tracking of the dansyl probe by Western blotting and immunofluorescence microscopy demonstrated that the inhibitor targets plasma membrane-associated FXIIIA. TG2 appears not to contribute to crosslinking activity on the osteoblast surface. Inhibition of FXIIIA with NC9 resulted in defective secretory vesicle delivery to the plasma membrane which was attributable to a disorganized microtubule network and decreased microtubule association with the plasma membrane. NC9 inhibition of FXIIIA resulted in destabilization of microtubules as assessed by cellular Glu-tubulin levels. Furthermore, NC9 blocked modification of Glu-tubulin into 150 kDa high-molecular weight Glu-tubulin form which was specifically localized to the plasma membrane. FXIIIA enzyme and its crosslinking activity were colocalized with plasma membrane-associated tubulin, and thus, it appears that FXIIIA crosslinking activity is directed towards stabilizing the interaction of microtubules with the plasma membrane. Our work provides the first mechanistic cues as to how transglutaminase activity could affect protein secretion and matrix deposition in osteoblasts and suggests a novel function for plasma membrane FXIIIA in microtubule dynamics.

  5. TRESK background K(+ channel is inhibited by PAR-1/MARK microtubule affinity-regulating kinases in Xenopus oocytes.

    Directory of Open Access Journals (Sweden)

    Gabriella Braun

    Full Text Available TRESK (TWIK-related spinal cord K(+ channel, KCNK18 is a major background K(+ channel of sensory neurons. Dominant-negative mutation of TRESK is linked to familial migraine. This important two-pore domain K(+ channel is uniquely activated by calcineurin. The calcium/calmodulin-dependent protein phosphatase directly binds to the channel and activates TRESK current several-fold in Xenopus oocytes and HEK293 cells. We have recently shown that the kinase, which is responsible for the basal inhibition of the K(+ current, is sensitive to the adaptor protein 14-3-3. Therefore we have examined the effect of the 14-3-3-inhibited PAR-1/MARK, microtubule-associated-protein/microtubule affinity-regulating kinase on TRESK in the Xenopus oocyte expression system. MARK1, MARK2 and MARK3 accelerated the return of TRESK current to the resting state after the calcium-dependent activation. Several other serine-threonine kinase types, generally involved in the modulation of other ion channels, failed to influence TRESK current recovery. MARK2 phosphorylated the primary determinant of regulation, the cluster of three adjacent serine residues (S274, 276 and 279 in the intracellular loop of mouse TRESK. In contrast, serine 264, the 14-3-3-binding site of TRESK, was not phosphorylated by the kinase. Thus MARK2 selectively inhibits TRESK activity via the S274/276/279 cluster, but does not affect the direct recruitment of 14-3-3 to the channel. TRESK is the first example of an ion channel phosphorylated by the dynamically membrane-localized MARK kinases, also known as general determinants of cellular polarity. These results raise the possibility that microtubule dynamics is coupled to the regulation of excitability in the neurons, which express TRESK background potassium channel.

  6. Wood cell-wall structure requires local 2D-microtubule disassembly by a novel plasma membrane-anchored protein.

    Science.gov (United States)

    Oda, Yoshihisa; Iida, Yuki; Kondo, Yuki; Fukuda, Hiroo

    2010-07-13

    Plant cells have evolved cortical microtubules, in a two-dimensional space beneath the plasma membrane, that regulate patterning of cellulose deposition. Although recent studies have revealed that several microtubule-associated proteins facilitate self-organization of transverse cortical microtubules, it is still unknown how diverse patterns of cortical microtubules are organized in different xylem cells, which are the major components of wood. Using our newly established in vitro xylem cell differentiation system, we found that a novel microtubule end-tracking protein, microtubule depletion domain 1 (MIDD1), was anchored to distinct plasma membrane domains and promoted local microtubule disassembly, resulting in pits on xylem cell walls. The introduction of RNA interference for MIDD1 resulted in the failure of local microtubule depletion and the formation of secondary walls without pits. Conversely, the overexpression of MIDD1 reduced microtubule density. MIDD1 has two coiled-coil domains for the binding to microtubules and for the anchorage to plasma membrane domains, respectively. Combination of the two coils caused end tracking of microtubules during shrinkage and suppressed their rescue events. Our results indicate that MIDD1 integrates spatial information in the plasma membrane with cortical microtubule dynamics for determining xylem cell wall pattern. Copyright 2010 Elsevier Ltd. All rights reserved.

  7. GIT1/beta PIX signaling proteins and PAK1 kinase regulate microtubule nucleation

    Czech Academy of Sciences Publication Activity Database

    Černohorská, Markéta; Sulimenko, Vadym; Hájková, Zuzana; Sulimenko, Tetyana; Sládková, Vladimíra; Vinopal, Stanislav; Dráberová, Eduarda; Dráber, Pavel

    2016-01-01

    Roč. 1863, č. 6 (2016), s. 1282-1297 ISSN 0167-4889 R&D Projects: GA ČR GAP302/12/1673; GA ČR GA15-22194S; GA MŠk LH12050; GA MZd NT14467; GA ČR GA16-23702S Institutional support: RVO:68378050 Keywords : Centrosome * Microtubule nucleation * gamma-tubulin * GIT1/beta PIX signaling proteins * PAK1 kinase Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.521, year: 2016

  8. A Novel Plasma Membrane-Anchored Protein Regulates Xylem Cell-Wall Deposition through Microtubule-Dependent Lateral Inhibition of Rho GTPase Domains.

    Science.gov (United States)

    Sugiyama, Yuki; Wakazaki, Mayumi; Toyooka, Kiminori; Fukuda, Hiroo; Oda, Yoshihisa

    2017-08-21

    Spatial control of cell-wall deposition is essential for determining plant cell shape [1]. Rho-type GTPases, together with the cortical cytoskeleton, play central roles in regulating cell-wall patterning [2]. In metaxylem vessel cells, which are the major components of xylem tissues, active ROP11 Rho GTPases form oval plasma membrane domains that locally disrupt cortical microtubules, thereby directing the formation of oval pits in secondary cell walls [3-5]. However, the regulatory mechanism that determines the planar shape of active Rho of Plants (ROP) domains is still unknown. Here we show that IQD13 associates with cortical microtubules and the plasma membrane to laterally restrict the localization of ROP GTPase domains, thereby directing the formation of oval secondary cell-wall pits. Loss and overexpression of IQD13 led to the formation of abnormally round and narrow secondary cell-wall pits, respectively. Ectopically expressed IQD13 increased the presence of parallel cortical microtubules by promoting microtubule rescue. A reconstructive approach revealed that IQD13 confines the area of active ROP domains within the lattice of the cortical microtubules, causing narrow ROP domains to form. This activity required the interaction of IQD13 with the plasma membrane. These findings suggest that IQD13 positively regulates microtubule dynamics as well as their linkage to the plasma membrane, which synergistically confines the area of active ROP domains, leading to the formation of oval secondary cell-wall pits. This finding sheds light on the role of microtubule-plasma membrane linkage as a lateral fence that determines the planar shape of Rho GTPase domains. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Regulation of retinoschisin secretion in Weri-Rb1 cells by the F-actin and microtubule cytoskeleton.

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    Eiko Kitamura

    Full Text Available Retinoschisin is encoded by the gene responsible for X-linked retinoschisis (XLRS, an early onset macular degeneration that results in a splitting of the inner layers of the retina and severe loss in vision. Retinoschisin is predominantly expressed and secreted from photoreceptor cells as a homo-oligomer protein; it then associates with the surface of retinal cells and maintains the retina cellular architecture. Many missense mutations in the XLRS1 gene are known to cause intracellular retention of retinoschisin, indicating that the secretion process of the protein is a critical step for its normal function in the retina. However, the molecular mechanisms underlying retinoschisin's secretion remain to be fully elucidated. In this study, we investigated the role of the F-actin cytoskeleton in the secretion of retinoschisin by treating Weri-Rb1 cells, which are known to secrete retinoschisin, with cytochalasin D, jasplakinolide, Y-27632, and dibutyryl cGMP. Our results show that cytochalasin D and jasplakinolide inhibit retinoschisin secretion, whereas Y-27632 and dibutyryl cGMP enhance secretion causing F-actin alterations. We also demonstrate that high concentrations of taxol, which hyperpolymerizes microtubules, inhibit retinoschisin secretion. Our data suggest that retinoschisin secretion is regulated by the F-actin cytoskeleton, that cGMP or inhibition of ROCK alters F-actin structure enhancing the secretion, and that the microtubule cytoskeleton is also involved in this process.

  10. Regulation of Retinoschisin Secretion in Weri-Rb1 Cells by the F-Actin and Microtubule Cytoskeleton

    Science.gov (United States)

    Kitamura, Eiko; Gribanova, Yekaterina E.; Farber, Debora B.

    2011-01-01

    Retinoschisin is encoded by the gene responsible for X-linked retinoschisis (XLRS), an early onset macular degeneration that results in a splitting of the inner layers of the retina and severe loss in vision. Retinoschisin is predominantly expressed and secreted from photoreceptor cells as a homo-oligomer protein; it then associates with the surface of retinal cells and maintains the retina cellular architecture. Many missense mutations in the XLRS1 gene are known to cause intracellular retention of retinoschisin, indicating that the secretion process of the protein is a critical step for its normal function in the retina. However, the molecular mechanisms underlying retinoschisin's secretion remain to be fully elucidated. In this study, we investigated the role of the F-actin cytoskeleton in the secretion of retinoschisin by treating Weri-Rb1 cells, which are known to secrete retinoschisin, with cytochalasin D, jasplakinolide, Y-27632, and dibutyryl cGMP. Our results show that cytochalasin D and jasplakinolide inhibit retinoschisin secretion, whereas Y-27632 and dibutyryl cGMP enhance secretion causing F-actin alterations. We also demonstrate that high concentrations of taxol, which hyperpolymerizes microtubules, inhibit retinoschisin secretion. Our data suggest that retinoschisin secretion is regulated by the F-actin cytoskeleton, that cGMP or inhibition of ROCK alters F-actin structure enhancing the secretion, and that the microtubule cytoskeleton is also involved in this process. PMID:21738583

  11. T1R3 homomeric sweet taste receptor regulates adipogenesis through Gαs-mediated microtubules disassembly and Rho activation in 3T3-L1 cells.

    Directory of Open Access Journals (Sweden)

    Yosuke Masubuchi

    Full Text Available We previously reported that 3T3-L1 cells express a functional sweet taste receptor possibly as a T1R3 homomer that is coupled to Gs and negatively regulates adipogenesis by a Gαs-mediated but cAMP-independent mechanism. Here, we show that stimulation of this receptor with sucralose or saccharin induced disassembly of the microtubules in 3T3-L1 preadipocytes, which was attenuated by overexpression of the dominant-negative mutant of Gαs (Gαs-G226A. In contrast, overexpression of the constitutively active mutant of Gαs (Gαs-Q227L as well as treatment with cholera toxin or isoproterenol but not with forskolin caused disassembly of the microtubules. Sweetener-induced microtubule disassembly was accompanied by activation of RhoA and Rho-associated kinase (ROCK. This was attenuated with by knockdown of GEF-H1, a microtubule-localized guanine nucleotide exchange factor for Rho GTPase. Furthermore, overexpression of the dominant-negative mutant of RhoA (RhoA-T19N blocked sweetener-induced dephosphorylation of Akt and repression of PPARγ and C/EBPα in the early phase of adipogenic differentiation. These results suggest that the T1R3 homomeric sweet taste receptor negatively regulates adipogenesis through Gαs-mediated microtubule disassembly and consequent activation of the Rho/ROCK pathway.

  12. Calcium-dependent depletion zones in the cortical microtubule array coincide with sites of, but do not regulate, wall ingrowth papillae deposition in epidermal transfer cells.

    Science.gov (United States)

    Zhang, Hui-ming; Talbot, Mark J; McCurdy, David W; Patrick, John W; Offler, Christina E

    2015-09-01

    Trans-differentiation to a transfer-cell morphology is characterized by the localized deposition of wall ingrowth papillae that protrude into the cytosol. Whether the cortical microtubule array directs wall ingrowth papillae formation was investigated using a Vicia faba cotyledon culture system in which their adaxial epidermal cells were spontaneously induced to trans-differentiate to transfer cells. During deposition of wall ingrowth papillae, the aligned cortical microtubule arrays in precursor epidermal cells were reorganized into a randomized array characterized by circular depletion zones. Concurrence of the temporal appearance, spatial pattern, and size of depletion zones and wall ingrowth papillae was consistent with each papilla occupying a depletion zone. Surprisingly, microtubules appeared not to regulate construction of wall ingrowth papillae, as neither depolymerization nor stabilization of cortical microtubules changed their deposition pattern or morphology. Moreover, the size and spatial pattern of depletion zones was unaltered when the formation of wall ingrowth papillae was blocked by inhibiting cellulose biosynthesis. In contrast, the depletion zones were absent when the cytosolic calcium plumes, responsible for directing wall ingrowth papillae formation, were blocked or dissipated. Thus, we conclude that the depletion zones within the cortical microtubule array result from localized depolymerization of microtubules initiated by elevated cytosolic Ca(2+) levels at loci where wall ingrowth papillae are deposited. The physiological significance of the depletion zones as a mechanism to accommodate the construction of wall ingrowth papillae without compromising maintenance of the plasma membrane-microtubule inter-relationship is discussed. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  13. Nerve growth factor stimulates axon outgrowth through negative regulation of growth cone actomyosin restraint of microtubule advance.

    Science.gov (United States)

    Turney, Stephen G; Ahmed, Mostafa; Chandrasekar, Indra; Wysolmerski, Robert B; Goeckeler, Zoe M; Rioux, Robert M; Whitesides, George M; Bridgman, Paul C

    2016-02-01

    Nerve growth factor (NGF) promotes growth, differentiation, and survival of sensory neurons in the mammalian nervous system. Little is known about how NGF elicits faster axon outgrowth or how growth cones integrate and transform signal input to motor output. Using cultured mouse dorsal root ganglion neurons, we found that myosin II (MII) is required for NGF to stimulate faster axon outgrowth. From experiments inducing loss or gain of function of MII, specific MII isoforms, and vinculin-dependent adhesion-cytoskeletal coupling, we determined that NGF causes decreased vinculin-dependent actomyosin restraint of microtubule advance. Inhibition of MII blocked NGF stimulation, indicating the central role of restraint in directed outgrowth. The restraint consists of myosin IIB- and IIA-dependent processes: retrograde actin network flow and transverse actin bundling, respectively. The processes differentially contribute on laminin-1 and fibronectin due to selective actin tethering to adhesions. On laminin-1, NGF induced greater vinculin-dependent adhesion-cytoskeletal coupling, which slowed retrograde actin network flow (i.e., it regulated the molecular clutch). On fibronectin, NGF caused inactivation of myosin IIA, which negatively regulated actin bundling. On both substrates, the result was the same: NGF-induced weakening of MII-dependent restraint led to dynamic microtubules entering the actin-rich periphery more frequently, giving rise to faster elongation. © 2016 Turney et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  14. Nonstructural protein 5A is incorporated into hepatitis C virus low-density particle through interaction with core protein and microtubules during intracellular transport.

    Directory of Open Access Journals (Sweden)

    Chao-Kuen Lai

    Full Text Available Nonstructural protein 5A (NS5A of hepatitis C virus (HCV serves dual functions in viral RNA replication and virus assembly. Here, we demonstrate that HCV replication complex along with NS5A and Core protein was transported to the lipid droplet (LD through microtubules, and NS5A-Core complexes were then transported from LD through early-to-late endosomes to the plasma membrane via microtubules. Further studies by cofractionation analysis and immunoelectron microscopy of the released particles showed that NS5A-Core complexes, but not NS4B, were present in the low-density fractions, but not in the high-density fractions, of the HCV RNA-containing virions and associated with the internal virion core. Furthermore, exosomal markers CD63 and CD81 were also detected in the low-density fractions, but not in the high-density fractions. Overall, our results suggest that HCV NS5A is associated with the core of the low-density virus particles which exit the cell through a preexisting endosome/exosome pathway and may contribute to HCV natural infection.

  15. NAT10, a nucleolar protein, localizes to the midbody and regulates cytokinesis and acetylation of microtubules

    International Nuclear Information System (INIS)

    Shen, Qi; Zheng, Xingzheng; McNutt, Michael A.; Guang, Lizhao; Sun, Ying; Wang, Jiaochen; Gong, Yilei; Hou, Lin; Zhang, Bo

    2009-01-01

    The midbody is a structural organelle formed in late phase mitosis which is responsible for completion of cytokinesis. Although various kinds of proteins have been found to distribute or immigrate to this organelle, their functions have still not been completely worked out. In this study, we demonstrated that NAT10 (N-acetyltransferase 10, NAT10) is not only predominantly distributed in the nucleolus in interphase, but is also concentrated in the mitotic midbody during telophase. The domain in N-terminal residues 549-834 of NAT10 specifically mediated its subcellular localization. Treatment with genotoxic agents or irradiation increased concentration of NAT10 in both the nucleolus and midbody. Moreover, DNA damage induced increase of NAT10 in the midbody apparently accompanied by in situ elevation of the level of acetylated α-tubulin, suggesting that it plays a role in maintaining or enhancing stability of α-tubulin. The depletion of NAT10 induced defects in nucleolar assembly, cytokinesis and decreased acetylated α-tubulin, leading to G2/M cell cycle arrest or delay of mitotic exit. In addition, over-expression of NAT10 was found in a variety of soft tissue sarcomas, and correlated with tumor histological grading. These results indicate that NAT10 may play an important role in cell division through facilitating reformation of the nucleolus and midbody in the late phase of cell mitosis, and stabilization of microtubules.

  16. NAT10, a nucleolar protein, localizes to the midbody and regulates cytokinesis and acetylation of microtubules

    Energy Technology Data Exchange (ETDEWEB)

    Shen, Qi; Zheng, Xingzheng; McNutt, Michael A.; Guang, Lizhao; Sun, Ying; Wang, Jiaochen; Gong, Yilei; Hou, Lin [Department of Pathology, Health Science Center of Peking University, 38 Xueyuan Road, Haidian District, Beijing 100191 (China); Zhang, Bo, E-mail: zhangbo@bjmu.edu.cn [Department of Pathology, Health Science Center of Peking University, 38 Xueyuan Road, Haidian District, Beijing 100191 (China)

    2009-06-10

    The midbody is a structural organelle formed in late phase mitosis which is responsible for completion of cytokinesis. Although various kinds of proteins have been found to distribute or immigrate to this organelle, their functions have still not been completely worked out. In this study, we demonstrated that NAT10 (N-acetyltransferase 10, NAT10) is not only predominantly distributed in the nucleolus in interphase, but is also concentrated in the mitotic midbody during telophase. The domain in N-terminal residues 549-834 of NAT10 specifically mediated its subcellular localization. Treatment with genotoxic agents or irradiation increased concentration of NAT10 in both the nucleolus and midbody. Moreover, DNA damage induced increase of NAT10 in the midbody apparently accompanied by in situ elevation of the level of acetylated {alpha}-tubulin, suggesting that it plays a role in maintaining or enhancing stability of {alpha}-tubulin. The depletion of NAT10 induced defects in nucleolar assembly, cytokinesis and decreased acetylated {alpha}-tubulin, leading to G2/M cell cycle arrest or delay of mitotic exit. In addition, over-expression of NAT10 was found in a variety of soft tissue sarcomas, and correlated with tumor histological grading. These results indicate that NAT10 may play an important role in cell division through facilitating reformation of the nucleolus and midbody in the late phase of cell mitosis, and stabilization of microtubules.

  17. Down-Regulation of Desmosomes in Cultured Cells: The Roles of PKC, Microtubules and Lysosomal/Proteasomal Degradation

    Science.gov (United States)

    McHarg, Selina; Hopkins, Gemma; Lim, Lusiana; Garrod, David

    2014-01-01

    Desmosomes are intercellular adhesive junctions of major importance for tissue integrity. To allow cell motility and migration they are down-regulated in epidermal wound healing. Electron microscopy indicates that whole desmosomes are internalised by cells in tissues, but the mechanism of down-regulation is unclear. In this paper we provide an overview of the internalisation of half-desmosomes by cultured cells induced by calcium chelation. Our results show that: (i) half desmosome internalisation is dependent on conventional PKC isoforms; (ii) microtubules transport internalised half desmosomes to the region of the centrosome by a kinesin-dependent mechanism; (iii) desmosomal proteins remain colocalised after internalisation and are not recycled to the cell surface; (iv) internalised desmosomes are degraded by the combined action of lysosomes and proteasomes. We also confirm that half desmosome internalisation is dependent upon the actin cytoskeleton. These results suggest that half desmosomes are not disassembled and recycled during or after internalisation but instead are transported to the centrosomal region where they are degraded. These findings may have significance for the down-regulation of desmosomes in wounds. PMID:25291180

  18. The Vip1 inositol polyphosphate kinase family regulates polarized growth and modulates the microtubule cytoskeleton in fungi.

    Directory of Open Access Journals (Sweden)

    Jennifer Pöhlmann

    2014-09-01

    Full Text Available Microtubules (MTs are pivotal for numerous eukaryotic processes ranging from cellular morphogenesis, chromosome segregation to intracellular transport. Execution of these tasks requires intricate regulation of MT dynamics. Here, we identify a new regulator of the Schizosaccharomyces pombe MT cytoskeleton: Asp1, a member of the highly conserved Vip1 inositol polyphosphate kinase family. Inositol pyrophosphates generated by Asp1 modulate MT dynamic parameters independent of the central +TIP EB1 and in a dose-dependent and cellular-context-dependent manner. Importantly, our analysis of the in vitro kinase activities of various S. pombe Asp1 variants demonstrated that the C-terminal phosphatase-like domain of the dual domain Vip1 protein negatively affects the inositol pyrophosphate output of the N-terminal kinase domain. These data suggest that the former domain has phosphatase activity. Remarkably, Vip1 regulation of the MT cytoskeleton is a conserved feature, as Vip1-like proteins of the filamentous ascomycete Aspergillus nidulans and the distantly related pathogenic basidiomycete Ustilago maydis also affect the MT cytoskeleton in these organisms. Consistent with the role of interphase MTs in growth zone selection/maintenance, all 3 fungal systems show aspects of aberrant cell morphogenesis. Thus, for the first time we have identified a conserved biological process for inositol pyrophosphates.

  19. Dynamic microtubule organization and mitochondrial transport are regulated by distinct Kinesin-1 pathways

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    Anna Melkov

    2015-12-01

    Full Text Available The microtubule (MT plus-end motor kinesin heavy chain (Khc is well known for its role in long distance cargo transport. Recent evidence showed that Khc is also required for the organization of the cellular MT network by mediating MT sliding. We found that mutations in Khc and the gene of its adaptor protein, kinesin light chain (Klc resulted in identical bristle morphology defects, with the upper part of the bristle being thinner and flatter than normal and failing to taper towards the bristle tip. We demonstrate that bristle mitochondria transport requires Khc but not Klc as a competing force to dynein heavy chain (Dhc. Surprisingly, we demonstrate for the first time that Dhc is the primary motor for both anterograde and retrograde fast mitochondria transport. We found that the upper part of Khc and Klc mutant bristles lacked stable MTs. When following dynamic MT polymerization via the use of GFP-tagged end-binding protein 1 (EB1, it was noted that at Khc and Klc mutant bristle tips, dynamic MTs significantly deviated from the bristle parallel growth axis, relative to wild-type bristles. We also observed that GFP-EB1 failed to concentrate as a focus at the tip of Khc and Klc mutant bristles. We propose that the failure of bristle tapering is due to defects in directing dynamic MTs at the growing tip. Thus, we reveal a new function for Khc and Klc in directing dynamic MTs during polarized cell growth. Moreover, we also demonstrate a novel mode of coordination in mitochondrial transport between Khc and Dhc.

  20. Microtubule plus end-tracking proteins play critical roles in directional growth of hyphae by regulating the dynamics of cytoplasmic microtubules in Aspergillus nidulans.

    Science.gov (United States)

    Zeng, Cui J Tracy; Kim, Hye-Ryun; Vargas Arispuro, Irasema; Kim, Jung-Mi; Huang, An-Chi; Liu, Bo

    2014-11-01

    Cytoplasmic microtubules (MTs) serve as a rate-limiting factor for hyphal tip growth in the filamentous fungus Aspergillus nidulans. We hypothesized that this function depended on the MT plus end-tracking proteins (+TIPs) including the EB1 family protein EBA that decorated the MT plus ends undergoing polymerization. The ebAΔ mutation reduced colony growth and the mutant hyphae appeared in an undulating pattern instead of exhibiting unidirectional growth in the control. These phenotypes were enhanced by a mutation in another +TIP gene clipA. EBA was required for plus end-tracking of CLIPA, the Kinesin-7 motor KipA, and the XMAP215 homologue AlpA. In addition, cytoplasmic dynein also depended on EBA to track on most polymerizing MT plus ends, but not for its conspicuous appearance at the MT ends near the hyphal apex. The loss of EBA reduced the number of cytoplasmic MTs and prolonged dwelling times for MTs after reaching the hyphal apex. Finally, we found that colonies were formed in the absence of EBA, CLIPA, and NUDA together, suggesting that they were dispensable for fundamental functions of MTs. This study provided a comprehensive delineation of the relationship among different +TIPs and their contributions to MT dynamics and unidirectional hyphal expansion in filamentous fungi. © 2014 John Wiley & Sons Ltd.

  1. A novel mechanism important for the alignment of microtubules.

    Science.gov (United States)

    Wightman, Raymond; Turner, Simon R

    2008-04-01

    Using a live-cell imaging approach to study individual micro-tubules, we have compared microtubule behavior between net-like and aligned cortical arrays. In contrast to previous studies, a steep angled collision between the growing end of a microtubule and a preexisting microtubule was found to favor crossover. Frequencies of microtubule crossovers, bundling and catastrophes are similar regardless of whether the cell exhibited a net-like or aligned microtubule array. In the predominantly aligned array of petiole cells, severing occurs at the sites of microtubule crossovers and serves to remove unaligned microtubules and to increase microtubule density. Severing was observed to be rare in net-like arrays. Microtubule severing is carried out by the katanin enzyme. In this addendum, we present new insights into the possible mechanism of crossing over and preliminary data looking at organization of the array in a katanin mutant.

  2. Recruitment of EB1, a Master Regulator of Microtubule Dynamics, to the Surface of the Theileria annulata Schizont

    KAUST Repository

    Woods, Kerry L.

    2013-05-09

    The apicomplexan parasite Theileria annulata transforms infected host cells, inducing uncontrolled proliferation and clonal expansion of the parasitized cell population. Shortly after sporozoite entry into the target cell, the surrounding host cell membrane is dissolved and an array of host cell microtubules (MTs) surrounds the parasite, which develops into the transforming schizont. The latter does not egress to invade and transform other cells. Instead, it remains tethered to host cell MTs and, during mitosis and cytokinesis, engages the cell\\'s astral and central spindle MTs to secure its distribution between the two daughter cells. The molecular mechanism by which the schizont recruits and stabilizes host cell MTs is not known. MT minus ends are mostly anchored in the MT organizing center, while the plus ends explore the cellular space, switching constantly between phases of growth and shrinkage (called dynamic instability). Assuming the plus ends of growing MTs provide the first point of contact with the parasite, we focused on the complex protein machinery associated with these structures. We now report how the schizont recruits end-binding protein 1 (EB1), a central component of the MT plus end protein interaction network and key regulator of host cell MT dynamics. Using a range of in vitro experiments, we demonstrate that T. annulata p104, a polymorphic antigen expressed on the schizont surface, functions as a genuine EB1-binding protein and can recruit EB1 in the absence of any other parasite proteins. Binding strictly depends on a consensus SxIP motif located in a highly disordered C-terminal region of p104. We further show that parasite interaction with host cell EB1 is cell cycle regulated. This is the first description of a pathogen-encoded protein to interact with EB1 via a bona-fide SxIP motif. Our findings provide important new insight into the mode of interaction between Theileria and the host cell cytoskeleton. 2013 Woods et al.

  3. Recruitment of EB1, a master regulator of microtubule dynamics, to the surface of the Theileria annulata schizont.

    Directory of Open Access Journals (Sweden)

    Kerry L Woods

    2013-05-01

    Full Text Available The apicomplexan parasite Theileria annulata transforms infected host cells, inducing uncontrolled proliferation and clonal expansion of the parasitized cell population. Shortly after sporozoite entry into the target cell, the surrounding host cell membrane is dissolved and an array of host cell microtubules (MTs surrounds the parasite, which develops into the transforming schizont. The latter does not egress to invade and transform other cells. Instead, it remains tethered to host cell MTs and, during mitosis and cytokinesis, engages the cell's astral and central spindle MTs to secure its distribution between the two daughter cells. The molecular mechanism by which the schizont recruits and stabilizes host cell MTs is not known. MT minus ends are mostly anchored in the MT organizing center, while the plus ends explore the cellular space, switching constantly between phases of growth and shrinkage (called dynamic instability. Assuming the plus ends of growing MTs provide the first point of contact with the parasite, we focused on the complex protein machinery associated with these structures. We now report how the schizont recruits end-binding protein 1 (EB1, a central component of the MT plus end protein interaction network and key regulator of host cell MT dynamics. Using a range of in vitro experiments, we demonstrate that T. annulata p104, a polymorphic antigen expressed on the schizont surface, functions as a genuine EB1-binding protein and can recruit EB1 in the absence of any other parasite proteins. Binding strictly depends on a consensus SxIP motif located in a highly disordered C-terminal region of p104. We further show that parasite interaction with host cell EB1 is cell cycle regulated. This is the first description of a pathogen-encoded protein to interact with EB1 via a bona-fide SxIP motif. Our findings provide important new insight into the mode of interaction between Theileria and the host cell cytoskeleton.

  4. Manipulation and quantification of microtubule lattice integrity

    Directory of Open Access Journals (Sweden)

    Taylor A. Reid

    2017-08-01

    Full Text Available Microtubules are structural polymers that participate in a wide range of cellular functions. The addition and loss of tubulin subunits allows the microtubule to grow and shorten, as well as to develop and repair defects and gaps in its cylindrical lattice. These lattice defects act to modulate the interactions of microtubules with molecular motors and other microtubule-associated proteins. Therefore, tools to control and measure microtubule lattice structure will be invaluable for developing a quantitative understanding of how the structural state of the microtubule lattice may regulate its interactions with other proteins. In this work, we manipulated the lattice integrity of in vitro microtubules to create pools of microtubules with common nucleotide states, but with variations in structural states. We then developed a series of novel semi-automated analysis tools for both fluorescence and electron microscopy experiments to quantify the type and severity of alterations in microtubule lattice integrity. These techniques will enable new investigations that explore the role of microtubule lattice structure in interactions with microtubule-associated proteins.

  5. Microtubule catastrophe and rescue.

    Science.gov (United States)

    Gardner, Melissa K; Zanic, Marija; Howard, Jonathon

    2013-02-01

    Microtubules are long cylindrical polymers composed of tubulin subunits. In cells, microtubules play an essential role in architecture and motility. For example, microtubules give shape to cells, serve as intracellular transport tracks, and act as key elements in important cellular structures such as axonemes and mitotic spindles. To accomplish these varied functions, networks of microtubules in cells are very dynamic, continuously remodeling through stochastic length fluctuations at the ends of individual microtubules. The dynamic behavior at the end of an individual microtubule is termed 'dynamic instability'. This behavior manifests itself by periods of persistent microtubule growth interrupted by occasional switching to rapid shrinkage (called microtubule 'catastrophe'), and then by switching back from shrinkage to growth (called microtubule 'rescue'). In this review, we summarize recent findings which provide new insights into the mechanisms of microtubule catastrophe and rescue, and discuss the impact of these findings in regards to the role of microtubule dynamics inside of cells. Copyright © 2012 Elsevier Ltd. All rights reserved.

  6. Microtubule nucleation and organization in dendrites

    Science.gov (United States)

    Delandre, Caroline; Amikura, Reiko; Moore, Adrian W.

    2016-01-01

    ABSTRACT Dendrite branching is an essential process for building complex nervous systems. It determines the number, distribution and integration of inputs into a neuron, and is regulated to create the diverse dendrite arbor branching patterns characteristic of different neuron types. The microtubule cytoskeleton is critical to provide structure and exert force during dendrite branching. It also supports the functional requirements of dendrites, reflected by differential microtubule architectural organization between neuron types, illustrated here for sensory neurons. Both anterograde and retrograde microtubule polymerization occur within growing dendrites, and recent studies indicate that branching is enhanced by anterograde microtubule polymerization events in nascent branches. The polarities of microtubule polymerization events are regulated by the position and orientation of microtubule nucleation events in the dendrite arbor. Golgi outposts are a primary microtubule nucleation center in dendrites and share common nucleation machinery with the centrosome. In addition, pre-existing dendrite microtubules may act as nucleation sites. We discuss how balancing the activities of distinct nucleation machineries within the growing dendrite can alter microtubule polymerization polarity and dendrite branching, and how regulating this balance can generate neuron type-specific morphologies. PMID:27097122

  7. Microtubule bundling plays a role in ethylene-mediated cortical microtubule reorientation in etiolated Arabidopsis hypocotyls.

    Science.gov (United States)

    Ma, Qianqian; Sun, Jingbo; Mao, Tonglin

    2016-05-15

    The gaseous hormone ethylene is known to regulate plant growth under etiolated conditions (the 'triple response'). Although organization of cortical microtubules is essential for cell elongation, the underlying mechanisms that regulate microtubule organization by hormone signaling, including ethylene, are ambiguous. In the present study, we demonstrate that ethylene signaling participates in regulation of cortical microtubule reorientation. In particular, regulation of microtubule bundling is important for this process in etiolated hypocotyls. Time-lapse analysis indicated that selective stabilization of microtubule-bundling structures formed in various arrays is related to ethylene-mediated microtubule orientation. Bundling events and bundle growth lifetimes were significantly increased in oblique and longitudinal arrays, but decreased in transverse arrays in wild-type cells in response to ethylene. However, the effects of ethylene on microtubule bundling were partially suppressed in a microtubule-bundling protein WDL5 knockout mutant (wdl5-1). This study suggests that modulation of microtubule bundles that have formed in certain orientations plays a role in reorienting microtubule arrays in response to ethylene-mediated etiolated hypocotyl cell elongation. © 2016. Published by The Company of Biologists Ltd.

  8. Rho-associated coiled-coil kinase (ROCK) protein controls microtubule dynamics in a novel signaling pathway that regulates cell migration.

    Science.gov (United States)

    Schofield, Alice V; Steel, Rohan; Bernard, Ora

    2012-12-21

    The two members of the Rho-associated coiled-coil kinase (ROCK1 and 2) family are established regulators of actin dynamics that are involved in the regulation of the cell cycle as well as cell motility and invasion. Here, we discovered a novel signaling pathway whereby ROCK regulates microtubule (MT) acetylation via phosphorylation of the tubulin polymerization promoting protein 1 (TPPP1/p25). We show that ROCK phosphorylation of TPPP1 inhibits the interaction between TPPP1 and histone deacetylase 6 (HDAC6), which in turn results in increased HDAC6 activity followed by a decrease in MT acetylation. As a consequence, we show that TPPP1 phosphorylation by ROCK increases cell migration and invasion via modulation of cellular acetyl MT levels. We establish here that the ROCK-TPPP1-HDAC6 signaling pathway is important for the regulation of cell migration and invasion.

  9. Biological Information Processing in Single Microtubules

    Science.gov (United States)

    2014-03-05

    generated by synchronized oscillations of microtubules, centrosomes and chromosomes regulate the dynamics of mitosis and meiosis, Yue Zhao and Qimin...of frequency by a single microtubule. Green arrows depict the peaks that appear in absorption and disappear in transmission. Purple arrows show

  10. Assembly and control of large microtubule complexes

    Science.gov (United States)

    Korolev, Kirill; Ishihara, Keisuke; Mitchison, Timothy

    Motility, division, and other cellular processes require rapid assembly and disassembly of microtubule structures. We report a new mechanism for the formation of asters, radial microtubule complexes found in very large cells. The standard model of aster growth assumes elongation of a fixed number of microtubules originating from the centrosomes. However, aster morphology in this model does not scale with cell size, and we found evidence for microtubule nucleation away from centrosomes. By combining polymerization dynamics and auto-catalytic nucleation of microtubules, we developed a new biophysical model of aster growth. The model predicts an explosive transition from an aster with a steady-state radius to one that expands as a travelling wave. At the transition, microtubule density increases continuously, but aster growth rate discontinuously jumps to a nonzero value. We tested our model with biochemical perturbations in egg extract and confirmed main theoretical predictions including the jump in the growth rate. Our results show that asters can grow even though individual microtubules are short and unstable. The dynamic balance between microtubule collapse and nucleation could be a general framework for the assembly and control of large microtubule complexes. NIH GM39565; Simons Foundation 409704; Honjo International 486 Scholarship Foundation.

  11. Microtubule-Organizing Centers.

    Science.gov (United States)

    Wu, Jingchao; Akhmanova, Anna

    2017-10-06

    The organization of microtubule networks is crucial for controlling chromosome segregation during cell division, for positioning and transport of different organelles, and for cell polarity and morphogenesis. The geometry of microtubule arrays strongly depends on the localization and activity of the sites where microtubules are nucleated and where their minus ends are anchored. Such sites are often clustered into structures known as microtubule-organizing centers, which include the centrosomes in animals and spindle pole bodies in fungi. In addition, other microtubules, as well as membrane compartments such as the cell nucleus, the Golgi apparatus, and the cell cortex, can nucleate, stabilize, and tether microtubule minus ends. These activities depend on microtubule-nucleating factors, such as γ-tubulin-containing complexes and their activators and receptors, and microtubule minus end-stabilizing proteins with their binding partners. Here, we provide an overview of the current knowledge on how such factors work together to control microtubule organization in different systems.

  12. Microtubule Catastrophe and Rescue

    OpenAIRE

    Gardner, Melissa K.; Zanic, Marija; Howard, Jonathon

    2012-01-01

    Microtubules are long cylindrical polymers composed of tubulin subunits. In cells, microtubules play an essential role in architecture and motility. For example, microtubules give shape to cells, serve as intracellular transport tracks, and act as key elements in important cellular structures such as axonemes and mitotic spindles. To accomplish these varied functions, networks of microtubules in cells are very dynamic, continuously remodeling through stochastic length fluctuations at the ends...

  13. Drosophila homologue of Diaphanous 1 (DIAPH1) controls the metastatic potential of colon cancer cells by regulating microtubule-dependent adhesion.

    Science.gov (United States)

    Lin, Yuan-Na; Bhuwania, Ridhirama; Gromova, Kira; Failla, Antonio Virgilio; Lange, Tobias; Riecken, Kristoffer; Linder, Stefan; Kneussel, Matthias; Izbicki, Jakob R; Windhorst, Sabine

    2015-07-30

    Drosophila homologue of Diaphanous 1 (DIAPH1) regulates actin polymerization and microtubule (MT) stabilization upon stimulation with lysophosphatidic acid (LPA). Recently, we showed strongly reduced lung metastasis of DIAPH1-depleted colon cancer cells but we found accumulations of DIAPH1-depleted cells in bone marrow. Here, we analyzed possible organ- or tissue-specific metastasis of DIAPH1-depleted HCT-116 cells. Our data confirmed that depletion of DIAPH1 strongly inhibited lung metastasis and revealed that, in contrast to control cells, DIAPH1-depleted cells did not form metastases in further organs. Detailed mechanistic analysis on cells that were not stimulated with LPA to activate the cytoskeleton-modulating activity of DIAPH1, revealed that even under basal conditions DIAPH1 was essential for cellular adhesion to collagen. In non-stimulated cells DIAPH1 did not control actin dynamics but, interestingly, was essential for stabilization of microtubules (MTs). Additionally, DIAPH1 controlled directed vesicle trafficking and with this, local clustering of the adhesion protein integrin-β1 at the plasma membrane. Therefore, we conclude that under non-stimulating conditions DIAPH1 controls cellular adhesion by stabilizing MTs required for local clustering of integrin-β1 at the plasma membrane. Thus, blockade of DIAPH1-tubulin interaction may be a promising approach to inhibit one of the earliest steps in the metastatic cascade of colon cancer.

  14. The Arabidopsis microtubule-associated protein MAP65-3 supports infection by filamentous biotrophic pathogens by down-regulating salicylic acid-dependent defenses.

    Science.gov (United States)

    Quentin, Michaël; Baurès, Isabelle; Hoefle, Caroline; Caillaud, Marie-Cécile; Allasia, Valérie; Panabières, Franck; Abad, Pierre; Hückelhoven, Ralph; Keller, Harald; Favery, Bruno

    2016-03-01

    The oomycete Hyaloperonospora arabidopsidis and the ascomycete Erysiphe cruciferarum are obligate biotrophic pathogens causing downy mildew and powdery mildew, respectively, on Arabidopsis. Upon infection, the filamentous pathogens induce the formation of intracellular bulbous structures called haustoria, which are required for the biotrophic lifestyle. We previously showed that the microtubule-associated protein AtMAP65-3 plays a critical role in organizing cytoskeleton microtubule arrays during mitosis and cytokinesis. This renders the protein essential for the development of giant cells, which are the feeding sites induced by root knot nematodes. Here, we show that AtMAP65-3 expression is also induced in leaves upon infection by the downy mildew oomycete and the powdery mildew fungus. Loss of AtMAP65-3 function in the map65-3 mutant dramatically reduced infection by both pathogens, predominantly at the stages of leaf penetration. Whole-transcriptome analysis showed an over-represented, constitutive activation of genes involved in salicylic acid (SA) biosynthesis, signaling, and defense execution in map65-3, whereas jasmonic acid (JA)-mediated signaling was down-regulated. Preventing SA synthesis and accumulation in map65-3 rescued plant susceptibility to pathogens, but not the developmental phenotype caused by cytoskeleton defaults. AtMAP65-3 thus has a dual role. It positively regulates cytokinesis, thus plant growth and development, and negatively interferes with plant defense against filamentous biotrophs. Our data suggest that downy mildew and powdery mildew stimulate AtMAP65-3 expression to down-regulate SA signaling for infection. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  15. YB-1 promotes microtubule assembly in vitro through interaction with tubulin and microtubules

    Directory of Open Access Journals (Sweden)

    Baconnais Sonia

    2008-09-01

    Full Text Available Abstract Background YB-1 is a major regulator of gene expression in eukaryotic cells. In addition to its role in transcription, YB-1 plays a key role in translation and stabilization of mRNAs. Results We show here that YB-1 interacts with tubulin and microtubules and stimulates microtubule assembly in vitro. High resolution imaging via electron and atomic force microscopy revealed that microtubules assembled in the presence of YB-1 exhibited a normal single wall ultrastructure and indicated that YB-1 most probably coats the outer microtubule wall. Furthermore, we found that YB-1 also promotes the assembly of MAPs-tubulin and subtilisin-treated tubulin. Finally, we demonstrated that tubulin interferes with RNA:YB-1 complexes. Conclusion These results suggest that YB-1 may regulate microtubule assembly in vivo and that its interaction with tubulin may contribute to the control of mRNA translation.

  16. Acetylcholine receptor (AChR) clustering is regulated both by glycogen synthase kinase 3β (GSK3β)-dependent phosphorylation and the level of CLIP-associated protein 2 (CLASP2) mediating the capture of microtubule plus-ends.

    Science.gov (United States)

    Basu, Sreya; Sladecek, Stefan; Pemble, Hayley; Wittmann, Torsten; Slotman, Johan A; van Cappellen, Wiggert; Brenner, Hans-Rudolf; Galjart, Niels

    2014-10-31

    The postsynaptic apparatus of the neuromuscular junction (NMJ) traps and anchors acetylcholine receptors (AChRs) at high density at the synapse. We have previously shown that microtubule (MT) capture by CLASP2, a MT plus-end-tracking protein (+TIP), increases the size and receptor density of AChR clusters at the NMJ through the delivery of AChRs and that this is regulated by a pathway involving neuronal agrin and several postsynaptic kinases, including GSK3. Phosphorylation by GSK3 has been shown to cause CLASP2 dissociation from MT ends, and nine potential phosphorylation sites for GSK3 have been mapped on CLASP2. How CLASP2 phosphorylation regulates MT capture at the NMJ and how this controls the size of AChR clusters are not yet understood. To examine this, we used myotubes cultured on agrin patches that induce AChR clustering in a two-dimensional manner. We show that expression of a CLASP2 mutant, in which the nine GSK3 target serines are mutated to alanine (CLASP2-9XS/9XA) and are resistant to GSK3β-dependent phosphorylation, promotes MT capture at clusters and increases AChR cluster size, compared with myotubes that express similar levels of wild type CLASP2 or that are noninfected. Conversely, myotubes expressing a phosphomimetic form of CLASP2 (CLASP2-8XS/D) show enrichment of immobile mutant CLASP2 in clusters, but MT capture and AChR cluster size are reduced. Taken together, our data suggest that both GSK3β-dependent phosphorylation and the level of CLASP2 play a role in the maintenance of AChR cluster size through the regulated capture and release of MT plus-ends. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Neuronal plasticity in hibernation and the proposed role of the microtubule-associated protein tau as a "master switch" regulating synaptic gain in neuronal networks.

    Science.gov (United States)

    Arendt, Thomas; Bullmann, Torsten

    2013-09-01

    The present paper provides an overview of adaptive changes in brain structure and learning abilities during hibernation as a behavioral strategy used by several mammalian species to minimize energy expenditure under current or anticipated inhospitable environmental conditions. One cellular mechanism that contributes to the regulated suppression of metabolism and thermogenesis during hibernation is reversible phosphorylation of enzymes and proteins, which limits rates of flux through metabolic pathways. Reversible phosphorylation during hibernation also affects synaptic membrane proteins, a process known to be involved in synaptic plasticity. This mechanism of reversible protein phosphorylation also affects the microtubule-associated protein tau, thereby generating a condition that in the adult human brain is associated with aggregation of tau protein to paired helical filaments (PHFs), as observed in Alzheimer's disease. Here, we put forward the concept that phosphorylation of tau is a neuroprotective mechanism to escape NMDA-mediated hyperexcitability of neurons that would otherwise occur during slow gradual cooling of the brain. Phosphorylation of tau and its subsequent targeting to subsynaptic sites might, thus, work as a kind of "master switch," regulating NMDA receptor-mediated synaptic gain in a wide array of neuronal networks, thereby enabling entry into torpor. If this condition lasts too long, however, it may eventually turn into a pathological trigger, driving a cascade of events leading to neurodegeneration, as in Alzheimer's disease or other "tauopathies".

  18. Autocatalytic microtubule nucleation determines the size and mass of Xenopus laevis egg extract spindles.

    Science.gov (United States)

    Decker, Franziska; Oriola, David; Dalton, Benjamin; Brugués, Jan

    2018-01-11

    Regulation of size and growth is a fundamental problem in biology. A prominent example is the formation of the mitotic spindle, where protein concentration gradients around chromosomes are thought to regulate spindle growth by controlling microtubule nucleation. Previous evidence suggests that microtubules nucleate throughout the spindle structure. However, the mechanisms underlying microtubule nucleation and its spatial regulation are still unclear. Here, we developed an assay based on laser ablation to directly probe microtubule nucleation events in Xenopus laevis egg extracts. Combining this method with theory and quantitative microscopy, we show that the size of a spindle is controlled by autocatalytic growth of microtubules, driven by microtubule-stimulated microtubule nucleation. The autocatalytic activity of this nucleation system is spatially regulated by the limiting amounts of active microtubule nucleators, which decrease with distance from the chromosomes. This mechanism provides an upper limit to spindle size even when resources are not limiting. © 2018, Decker et al.

  19. Microtubules Growth Rate Alteration in Human Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Irina B. Alieva

    2010-01-01

    Full Text Available To understand how microtubules contribute to the dynamic reorganization of the endothelial cell (EC cytoskeleton, we established an EC model expressing EB3-GFP, a protein that marks microtubule plus-ends. Using this model, we were able to measure microtubule growth rate at the centrosome region and near the cell periphery of a single human EC and in the EC monolayer. We demonstrate that the majority of microtubules in EC are dynamic, the growth rate of their plus-ends is highest in the internal cytoplasm, in the region of the centrosome. Growth rate of microtubule plus-ends decreases from the cell center toward the periphery. Our data suggest the existing mechanism(s of local regulation of microtubule plus-ends growth in EC. Microtubule growth rate in the internal cytoplasm of EC in the monolayer is lower than that of single EC suggesting the regulatory effect of cell-cell contacts. Centrosomal microtubule growth rate distribution in single EC indicated the presence of two subpopulations of microtubules with “normal” (similar to those in monolayer EC and “fast” (three times as much growth rates. Our results indicate functional interactions between cell-cell contacts and microtubules.

  20. Short Linear Sequence Motif LxxPTPh Targets Diverse Proteins to Growing Microtubule Ends

    NARCIS (Netherlands)

    Kumar, Anil; Manatschal, Cristina; Rai, Ankit; Grigoriev, Ilya; Degen, Miriam Steiner; Jaussi, Rolf; Kretzschmar, Ines; Prota, Andrea E; Volkmer, Rudolf; Kammerer, Richard A.; Akhmanova, Anna; Steinmetz, Michel O.

    2017-01-01

    Microtubule plus-end tracking proteins (+TIPs) are involved in virtually all microtubule-based processes. End-binding (EB) proteins are considered master regulators of +TIP interaction networks, since they autonomously track growing microtubule ends and recruit a plethora of proteins to this

  1. Microtubules move the nucleus to quiescence.

    Science.gov (United States)

    Laporte, Damien; Sagot, Isabelle

    2014-01-01

    The nucleus is a cellular compartment that hosts several macro-molecular machines displaying a highly complex spatial organization. This tight architectural orchestration determines not only DNA replication and repair but also regulates gene expression. In budding yeast microtubules play a key role in structuring the nucleus since they condition the Rabl arrangement in G1 and chromosome partitioning during mitosis through their attachment to centromeres via the kinetochore proteins. Recently, we have shown that upon quiescence entry, intranuclear microtubules emanating from the spindle pole body elongate to form a highly stable bundle that spans the entire nucleus. Here, we examine some molecular mechanisms that may underlie the formation of this structure. As the intranuclear microtubule bundle causes a profound re-organization of the yeast nucleus and is required for cell survival during quiescence, we discuss the possibility that the assembly of such a structure participates in quiescence establishment.

  2. KIF7 Controls the Proliferation of Cells of the Respiratory Airway through Distinct Microtubule Dependent Mechanisms.

    Directory of Open Access Journals (Sweden)

    Garry L Coles

    2015-10-01

    Full Text Available The cell cycle must be tightly coordinated for proper control of embryonic development and for the long-term maintenance of organs such as the lung. There is emerging evidence that Kinesin family member 7 (Kif7 promotes Hedgehog (Hh signaling during embryonic development, and its misregulation contributes to diseases such as ciliopathies and cancer. Kif7 encodes a microtubule interacting protein that controls Hh signaling through regulation of microtubule dynamics within the primary cilium. However, whether Kif7 has a function in nonciliated cells remains largely unknown. The role Kif7 plays in basic cell biological processes like cell proliferation or cell cycle progression also remains to be elucidated. Here, we show that Kif7 is required for coordination of the cell cycle, and inactivation of this gene leads to increased cell proliferation in vivo and in vitro. Immunostaining and transmission electron microscopy experiments show that Kif7dda/dda mutant lungs are hyperproliferative and exhibit reduced alveolar epithelial cell differentiation. KIF7 depleted C3H10T1/2 fibroblasts and Kif7dda/dda mutant mouse embryonic fibroblasts have increased growth rates at high cellular densities, suggesting that Kif7 may function as a general regulator of cellular proliferation. We ascertained that in G1, Kif7 and microtubule dynamics regulate the expression and activity of several components of the cell cycle machinery known to control entry into S phase. Our data suggest that Kif7 may function to regulate the maintenance of the respiratory airway architecture by controlling cellular density, cell proliferation, and cycle exit through its role as a microtubule associated protein.

  3. Electrodynamic effects on microtubules

    Czech Academy of Sciences Publication Activity Database

    Kučera, Ondřej; Havelka, Daniel; Deriu, M.A.; Cifra, Michal

    2015-01-01

    Roč. 44, Jul (2015), s. 169-169 ISSN 0175-7571. [10th European-Biophysical-Societies-Association (EBSA) European Biophysics Congress. 18.07.2015-22.07.2015, Dresden] R&D Projects: GA ČR(CZ) GA15-17102S Institutional support: RVO:67985882 Keywords : Microtubules * Electric al polarity Subject RIV: JA - Electronics ; Optoelectronics, Electric al Engineering

  4. Microtubule's conformational cap

    DEFF Research Database (Denmark)

    Flyvbjerg, H.

    1999-01-01

    The molecular mechanisms that allow elongation of the unstable microtubule lattice remain unclear. It is usually thought that the GDP-liganded tubulin lattice is capped by a small layer of GTP- or GDP-P(i)-liganded molecules, the so called "GTP-cap". Here, we point-out that the elastic properties...

  5. The Microtubule Regulatory Protein Stathmin Is Required to Maintain the Integrity of Axonal Microtubules in Drosophila

    Science.gov (United States)

    Duncan, Jason E.; Lytle, Nikki K.; Zuniga, Alfredo; Goldstein, Lawrence S. B.

    2013-01-01

    Axonal transport, a form of long-distance, bi-directional intracellular transport that occurs between the cell body and synaptic terminal, is critical in maintaining the function and viability of neurons. We have identified a requirement for the stathmin (stai) gene in the maintenance of axonal microtubules and regulation of axonal transport in Drosophila . The stai gene encodes a cytosolic phosphoprotein that regulates microtubule dynamics by partitioning tubulin dimers between pools of soluble tubulin and polymerized microtubules, and by directly binding to microtubules and promoting depolymerization. Analysis of stai function in Drosophila , which has a single stai gene, circumvents potential complications with studies performed in vertebrate systems in which mutant phenotypes may be compensated by genetic redundancy of other members of the stai gene family. This has allowed us to identify an essential function for stai in the maintenance of the integrity of axonal microtubules. In addition to the severe disruption in the abundance and architecture of microtubules in the axons of stai mutant Drosophila , we also observe additional neurological phenotypes associated with loss of stai function including a posterior paralysis and tail-flip phenotype in third instar larvae, aberrant accumulation of transported membranous organelles in stai deficient axons, a progressive bang-sensitive response to mechanical stimulation reminiscent of the class of Drosophila mutants used to model human epileptic seizures, and a reduced adult lifespan. Reductions in the levels of Kinesin-1, the primary anterograde motor in axonal transport, enhance these phenotypes. Collectively, our results indicate that stai has an important role in neuronal function, likely through the maintenance of microtubule integrity in the axons of nerves of the peripheral nervous system necessary to support and sustain long-distance axonal transport. PMID:23840848

  6. S. pombe kinesins-8 promote both nucleation and catastrophe of microtubules.

    Directory of Open Access Journals (Sweden)

    Muriel Erent

    Full Text Available The kinesins-8 were originally thought to be microtubule depolymerases, but are now emerging as more versatile catalysts of microtubule dynamics. We show here that S. pombe Klp5-436 and Klp6-440 are non-processive plus-end-directed motors whose in vitro velocities on S. pombe microtubules at 7 and 23 nm s(-1 are too slow to keep pace with the growing tips of dynamic interphase microtubules in living S. pombe. In vitro, Klp5 and 6 dimers exhibit a hitherto-undescribed combination of strong enhancement of microtubule nucleation with no effect on growth rate or catastrophe frequency. By contrast in vivo, both Klp5 and Klp6 promote microtubule catastrophe at cell ends whilst Klp6 also increases the number of interphase microtubule arrays (IMAs. Our data support a model in which Klp5/6 bind tightly to free tubulin heterodimers, strongly promoting the nucleation of new microtubules, and then continue to land as a tubulin-motor complex on the tips of growing microtubules, with the motors then dissociating after a few seconds residence on the lattice. In vivo, we predict that only at cell ends, when growing microtubule tips become lodged and their growth slows down, will Klp5/6 motor activity succeed in tracking growing microtubule tips. This mechanism would allow Klp5/6 to detect the arrival of microtubule tips at cells ends and to amplify the intrinsic tendency for microtubules to catastrophise in compression at cell ends. Our evidence identifies Klp5 and 6 as spatial regulators of microtubule dynamics that enhance both microtubule nucleation at the cell centre and microtubule catastrophe at the cell ends.

  7. ARPP-16 Is a Striatal-Enriched Inhibitor of Protein Phosphatase 2A Regulated by Microtubule-Associated Serine/Threonine Kinase 3 (Mast 3 Kinase).

    Science.gov (United States)

    Andrade, Erika C; Musante, Veronica; Horiuchi, Atsuko; Matsuzaki, Hideo; Brody, A Harrison; Wu, Terence; Greengard, Paul; Taylor, Jane R; Nairn, Angus C

    2017-03-08

    ARPP-16 (cAMP-regulated phospho-protein of molecular weight 16 kDa) is one of several small acid-soluble proteins highly expressed in medium spiny neurons of striatum that are phosphorylated in response to dopamine acting via D1 receptor/protein kinase A (PKA) signaling. We show here that ARPP-16 is also phosphorylated in vitro and in vivo by microtubule-associated serine/threonine kinase 3 (MAST3 kinase), an enzyme of previously unknown function that is enriched in striatum. We find that ARPP-16 interacts directly with the scaffolding A subunit of the serine/threonine protein phosphatase, PP2A, and that phosphorylation of ARPP-16 at Ser46 by MAST3 kinase converts the protein into a selective inhibitor of B55α- and B56δ-containing heterotrimeric forms of PP2A. Ser46 of ARPP-16 is phosphorylated to a high basal stoichiometry in striatum, suggestive of basal inhibition of PP2A in striatal neurons. In support of this hypothesis, conditional knock-out of ARPP-16 in CaMKIIα::cre/floxed ARPP-16/19 mice results in dephosphorylation of a subset of PP2A substrates including phospho-Thr75-DARPP-32, phospho-T308-Akt, and phospho-T202/Y204-ERK. Conditional knock-out of ARPP-16/19 is associated with increased motivation measured on a progressive ratio schedule of food reinforcement, yet an attenuated locomotor response to acute cocaine. Our previous studies have shown that ARPP-16 is phosphorylated at Ser88 by PKA. Activation of PKA in striatal slices leads to phosphorylation of Ser88, and this is accompanied by marked dephosphorylation of Ser46. Together, these studies suggest that phospho-Ser46-ARPP-16 acts to basally control PP2A in striatal medium spiny neurons but that dopamine acting via PKA inactivates ARPP-16 leading to selective potentiation of PP2A signaling. SIGNIFICANCE STATEMENT We describe a novel mechanism of signal transduction enriched in medium spiny neurons of striatum that likely mediates effects of the neurotransmitter dopamine acting on these cells. We

  8. Optical Tweezers-Based Measurements of Forces and Dynamics at Microtubule Ends.

    Science.gov (United States)

    Baclayon, Marian; Kalisch, Svenja-Marei; Hendel, Ed; Laan, Liedewij; Husson, Julien; Munteanu, E Laura; Dogterom, Marileen

    2017-01-01

    Microtubules are dynamic cytoskeletal polymers that polymerize and depolymerize while interacting with different proteins and structures within the cell. The highly regulated dynamic properties as well as the pushing and pulling forces generated by dynamic microtubule ends play important roles in processes such as in cell division. For instance, microtubule end-binding proteins are known to affect dramatically the dynamic properties of microtubules, and cortical dyneins are known to mediate pulling forces on microtubule ends. We discuss in this chapter our efforts to reconstitute these systems in vitro and mimic their interactions with structures within the cell using micro-fabricated barriers. Using an optical tweezers setup, we investigate the dynamics and forces of microtubules growing against functionalized barriers in the absence and presence of end-binding proteins and barrier-attached motor proteins. This setup allows high-speed as well as nanometer and piconewton resolution measurements on dynamic microtubules.

  9. Microtubule nucleation in mouse bone marrow-derived mast cells is regulated by the concerted action of GIT1/βPIX proteins and calcium

    Czech Academy of Sciences Publication Activity Database

    Sulimenko, Vadym; Hájková, Zuzana; Černohorská, Markéta; Sulimenko, Tetyana; Sládková, Vladimíra; Dráberová, Lubica; Vinopal, Stanislav; Dráberová, Eduarda; Dráber, Pavel

    2015-01-01

    Roč. 194, č. 9 (2015), s. 4099-4111 ISSN 0022-1767 R&D Projects: GA ČR GAP302/12/1673; GA ČR GPP302/11/P709; GA ČR(CZ) GA14-09807S; GA ČR GA15-22194S; GA MŠk(CZ) LD13015; GA MŠk LH12050; GA MZd NT14467 Institutional support: RVO:68378050 Keywords : Bone Marrow-Derived Mast Cells * Microtubule Nucleation * GIT1/beta PIX Proteins * Calcium Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.985, year: 2015

  10. Nonlinear dynamics of C-terminal tails in cellular microtubules

    Science.gov (United States)

    Sekulic, Dalibor L.; Sataric, Bogdan M.; Zdravkovic, Slobodan; Bugay, Aleksandr N.; Sataric, Miljko V.

    2016-07-01

    The mechanical and electrical properties, and information processing capabilities of microtubules are the permanent subject of interest for carrying out experiments in vitro and in silico, as well as for theoretical attempts to elucidate the underlying processes. In this paper, we developed a new model of the mechano-electrical waves elicited in the rows of very flexible C-terminal tails which decorate the outer surface of each microtubule. The fact that C-terminal tails play very diverse roles in many cellular functions, such as recruitment of motor proteins and microtubule-associated proteins, motivated us to consider their collective dynamics as the source of localized waves aimed for communication between microtubule and associated proteins. Our approach is based on the ferroelectric liquid crystal model and it leads to the effective asymmetric double-well potential which brings about the conditions for the appearance of kink-waves conducted by intrinsic electric fields embedded in microtubules. These kinks can serve as the signals for control and regulation of intracellular traffic along microtubules performed by processive motions of motor proteins, primarly from kinesin and dynein families. On the other hand, they can be precursors for initiation of dynamical instability of microtubules by recruiting the proper proteins responsible for the depolymerization process.

  11. Slingshot mechanism for clusters: Gas density regulates star density in the Orion Nebula Cluster (M42)

    Science.gov (United States)

    Stutz, Amelia M.

    2018-02-01

    We characterize the stellar and gas volume density, potential, and gravitational field profiles in the central ∼0.5 pc of the Orion Nebula Cluster (ONC), the nearest embedded star cluster (or rather, protocluster) hosting massive star formation available for detailed observational scrutiny. We find that the stellar volume density is well characterized by a Plummer profile ρstars(r) = 5755 M⊙ pc- 3 (1 + (r/a)2)- 5/2, where a = 0.36 pc. The gas density follows a cylindrical power law ρgas(R) = 25.9 M⊙ pc- 3 (R/pc)- 1.775. The stellar density profile dominates over the gas density profile inside r ∼ 1 pc. The gravitational field is gas-dominated at all radii, but the contribution to the total field by the stars is nearly equal to that of the gas at r ∼ a. This fact alone demonstrates that the protocluster cannot be considered a gas-free system or a virialized system dominated by its own gravity. The stellar protocluster core is dynamically young, with an age of ∼2-3 Myr, a 1D velocity dispersion of σobs = 2.6 km s-1, and a crossing time of ∼0.55 Myr. This time-scale is almost identical to the gas filament oscillation time-scale estimated recently by Stutz & Gould. This provides strong evidence that the protocluster structure is regulated by the gas filament. The protocluster structure may be set by tidal forces due to the oscillating filamentary gas potential. Such forces could naturally suppress low density stellar structures on scales ≳ a. The analysis presented here leads to a new suggestion that clusters form by an analogue of the 'slingshot mechanism' previously proposed for stars.

  12. Synchronous Oscillations in Microtubule Polymerization

    Science.gov (United States)

    Carlier, M. F.; Melki, R.; Pantaloni, D.; Hill, T. L.; Chen, Y.

    1987-08-01

    Under conditions where microtubule nucleation and growth are fast (i.e., high magnesium ion and tubulin concentrations and absence of glycerol), microtubule assembly in vitro exhibits an oscillatory regime preceding the establishment of steady state. The amplitude of the oscillations can represent >50% of the maximum turbidity change and oscillations persist for up to 20 periods of 80 s each. Oscillations are accompanied by extensive length redistribution of microtubules. Preliminary work suggests that the oscillatory kinetics can be simulated using a model in which many microtubules undergo synchronous transitions between growing and rapidly depolymerizing phases, complicated by the kinetically limiting rate of nucleotide exchange on free tubulin.

  13. Forms of density regulation and (quasi-) stationary distributions of population sizes in birds

    DEFF Research Database (Denmark)

    Sæther, Bernt-Erik; Engen, Steinar; Grøtan, Vidar

    2008-01-01

    The theta-logistic model of density regulation is an especially flexible class of density regulation models where different forms of non-linear density regulation can be expressed by only one parameter, u. Estimating the parameters of the thetalogistic model is, however, challenging. This is main...

  14. Modeling microtubule oscillations

    DEFF Research Database (Denmark)

    Jobs, E.; Wolf, D.E.; Flyvbjerg, H.

    1997-01-01

    Synchronization of molecular reactions in a macroscopic volume may cause the volume's physical properties to change dynamically and thus reveal much about the reactions. As an example, experimental time series for so-called microtubule oscillations are analyzed in terms of a minimal model...... for this complex polymerization-depolymerization cycle. The model reproduces well the qualitatively different time series that result from different experimental conditions, and illuminates the role and importance of individual processes in the cycle. Simple experiments are suggested that can further test...... and define the model and the polymer's reaction cycle....

  15. NAD+ and SIRT3 control microtubule dynamics and reduce susceptibility to antimicrotubule agents

    Science.gov (United States)

    Harkcom, William T.; Ghosh, Ananda K.; Sung, Matthew S.; Matov, Alexandre; Brown, Kevin D.; Giannakakou, Paraskevi; Jaffrey, Samie R.

    2014-01-01

    Nicotinamide adenine dinucleotide (NAD+) is an endogenous enzyme cofactor and cosubstrate that has effects on diverse cellular and physiologic processes, including reactive oxygen species generation, mitochondrial function, apoptosis, and axonal degeneration. A major goal is to identify the NAD+-regulated cellular pathways that may mediate these effects. Here we show that the dynamic assembly and disassembly of microtubules is markedly altered by NAD+. Furthermore, we show that the disassembly of microtubule polymers elicited by microtubule depolymerizing agents is blocked by increasing intracellular NAD+ levels. We find that these effects of NAD+ are mediated by the activation of the mitochondrial sirtuin sirtuin-3 (SIRT3). Overexpression of SIRT3 prevents microtubule disassembly and apoptosis elicited by antimicrotubule agents and knockdown of SIRT3 prevents the protective effects of NAD+ on microtubule polymers. Taken together, these data demonstrate that NAD+ and SIRT3 regulate microtubule polymerization and the efficacy of antimicrotubule agents. PMID:24889606

  16. Microtubules as mechanical force sensors.

    Science.gov (United States)

    Karafyllidis, Ioannis G; Lagoudas, Dimitris C

    2007-03-01

    Microtubules are polymers of tubulin subunits (dimers) arranged on a hexagonal lattice. Each tubulin dimer comprises two monomers, the alpha-tubulin and beta-tubulin, and can be found in two states. In the first state a mobile negative charge is located into the alpha-tubulin monomer and in the second into the beta-tubulin monomer. Each tubulin dimer is modeled as an electrical dipole coupled to its neighbors by electrostatic forces. The location of the mobile charge in each dimer depends on the location of the charges in the dimer's neighborhood. Mechanical forces that act on the microtubule affect the distances between the dimers and alter the electrostatic potential. Changes in this potential affect the mobile negative charge location in each dimer and the charge distribution in the microtubule. The net effect is that mechanical forces affect the charge distribution in microtubules. We propose to exploit this effect and use microtubules as mechanical force sensors. We model each dimer as a two-state quantum system and, following the quantum computation paradigm, we use discrete quantum random walk on the hexagonal microtubule lattice to determine the charge distribution. Different forces applied on the microtubule are modeled as different coin biases leading to different probability distributions of the quantum walker location, which are directly connected to different charge distributions. Simulation results show that there is a strong indication that microtubules can be used as mechanical force sensors and that they can also detect the force directions and magnitudes.

  17. Nonlinear dynamics of C–terminal tails in cellular microtubules

    Energy Technology Data Exchange (ETDEWEB)

    Sekulic, Dalibor L., E-mail: dalsek@uns.ac.rs; Sataric, Bogdan M.; Sataric, Miljko V. [University of Novi Sad, Faculty of Technical Sciences, Novi Sad (Serbia); Zdravkovic, Slobodan [University of Belgrade, Institute of Nuclear Sciences Vinca, Belgrade (Serbia); Bugay, Aleksandr N. [Laboratory of Radiation Biology, Joint Institute for Nuclear Research, Dubna (Russian Federation)

    2016-07-15

    The mechanical and electrical properties, and information processing capabilities of microtubules are the permanent subject of interest for carrying out experiments in vitro and in silico, as well as for theoretical attempts to elucidate the underlying processes. In this paper, we developed a new model of the mechano–electrical waves elicited in the rows of very flexible C–terminal tails which decorate the outer surface of each microtubule. The fact that C–terminal tails play very diverse roles in many cellular functions, such as recruitment of motor proteins and microtubule–associated proteins, motivated us to consider their collective dynamics as the source of localized waves aimed for communication between microtubule and associated proteins. Our approach is based on the ferroelectric liquid crystal model and it leads to the effective asymmetric double-well potential which brings about the conditions for the appearance of kink–waves conducted by intrinsic electric fields embedded in microtubules. These kinks can serve as the signals for control and regulation of intracellular traffic along microtubules performed by processive motions of motor proteins, primarly from kinesin and dynein families. On the other hand, they can be precursors for initiation of dynamical instability of microtubules by recruiting the proper proteins responsible for the depolymerization process.

  18. Nonlinear dynamics of C–terminal tails in cellular microtubules

    International Nuclear Information System (INIS)

    Sekulic, Dalibor L.; Sataric, Bogdan M.; Sataric, Miljko V.; Zdravkovic, Slobodan; Bugay, Aleksandr N.

    2016-01-01

    The mechanical and electrical properties, and information processing capabilities of microtubules are the permanent subject of interest for carrying out experiments in vitro and in silico, as well as for theoretical attempts to elucidate the underlying processes. In this paper, we developed a new model of the mechano–electrical waves elicited in the rows of very flexible C–terminal tails which decorate the outer surface of each microtubule. The fact that C–terminal tails play very diverse roles in many cellular functions, such as recruitment of motor proteins and microtubule–associated proteins, motivated us to consider their collective dynamics as the source of localized waves aimed for communication between microtubule and associated proteins. Our approach is based on the ferroelectric liquid crystal model and it leads to the effective asymmetric double-well potential which brings about the conditions for the appearance of kink–waves conducted by intrinsic electric fields embedded in microtubules. These kinks can serve as the signals for control and regulation of intracellular traffic along microtubules performed by processive motions of motor proteins, primarly from kinesin and dynein families. On the other hand, they can be precursors for initiation of dynamical instability of microtubules by recruiting the proper proteins responsible for the depolymerization process.

  19. Density regulation in Northeast Atlantic fish populations: Density dependence is stronger in recruitment than in somatic growth.

    Science.gov (United States)

    Zimmermann, Fabian; Ricard, Daniel; Heino, Mikko

    2018-05-01

    Population regulation is a central concept in ecology, yet in many cases its presence and the underlying mechanisms are difficult to demonstrate. The current paradigm maintains that marine fish populations are predominantly regulated by density-dependent recruitment. While it is known that density-dependent somatic growth can be present too, its general importance remains unknown and most practical applications neglect it. This study aimed to close this gap by for the first time quantifying and comparing density dependence in growth and recruitment over a large set of fish populations. We fitted density-dependent models to time-series data on population size, recruitment and age-specific weight from commercially exploited fish populations in the Northeast Atlantic Ocean and the Baltic Sea. Data were standardized to enable a direct comparison within and among populations, and estimated parameters were used to quantify the impact of density regulation on population biomass. Statistically significant density dependence in recruitment was detected in a large proportion of populations (70%), whereas for density dependence in somatic growth the prevalence of density dependence depended heavily on the method (26% and 69%). Despite age-dependent variability, the density dependence in recruitment was consistently stronger among age groups and between alternative approaches that use weight-at-age or weight increments to assess growth. Estimates of density-dependent reduction in biomass underlined these results: 97% of populations with statistically significant parameters for growth and recruitment showed a larger impact of density-dependent recruitment on population biomass. The results reaffirm the importance of density-dependent recruitment in marine fishes, yet they also show that density dependence in somatic growth is not uncommon. Furthermore, the results are important from an applied perspective because density dependence in somatic growth affects productivity and

  20. Linking cortical microtubule attachment and exocytosis [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Ivar Noordstra

    2017-04-01

    Full Text Available Exocytosis is a fundamental cellular process whereby secreted molecules are packaged into vesicles that move along cytoskeletal filaments and fuse with the plasma membrane. To function optimally, cells are strongly dependent on precisely controlled delivery of exocytotic cargo. In mammalian cells, microtubules serve as major tracks for vesicle transport by motor proteins, and thus microtubule organization is important for targeted delivery of secretory carriers. Over the years, multiple microtubule-associated and cortical proteins have been discovered that facilitate the interaction between the microtubule plus ends and the cell cortex. In this review, we focus on mammalian protein complexes that have been shown to participate in both cortical microtubule capture and exocytosis, thereby regulating the spatial organization of secretion. These complexes include microtubule plus-end tracking proteins, scaffolding factors, actin-binding proteins, and components of vesicle docking machinery, which together allow efficient coordination of cargo transport and release.

  1. The dynamic interplay of plasma membrane domains and cortical microtubules in secondary cell wall patterning

    Directory of Open Access Journals (Sweden)

    Yoshihisa eOda

    2013-12-01

    Full Text Available Patterning of the cellulosic cell wall underlies the shape and function of plant cells. The cortical microtubule array plays a central role in the regulation of cell wall patterns. However, the regulatory mechanisms by which secondary cell wall patterns are established through cortical microtubules remain to be fully determined. Our recent study in xylem vessel cells revealed that a mutual inhibitory interaction between cortical microtubules and distinct plasma membrane domains leads to distinctive patterning in secondary cell walls. Our research revealed that the recycling of active and inactive ROP proteins by a specific GAP and GEF pair establishes distinct de novo plasma membrane domains. Active ROP recruits a plant-specific microtubule-associated protein, MIDD1, which mediates the mutual interaction between cortical microtubules and plasma membrane domains. In this mini review, we summarize recent research regarding secondary wall patterning, with a focus on the emerging interplay between plasma membrane domains and cortical microtubules through MIDD1 and ROP.

  2. A structural model for microtubule minus-end recognition and protection by CAMSAP proteins

    NARCIS (Netherlands)

    Atherton, Joseph; Jiang, Kai; Stangier, Marcel M.; Luo, Yanzhang; Hua, Shasha; Houben, Klaartje; Van Hooff, Jolien J.E.; Joseph, Agnel Praveen; Scarabelli, Guido; Grant, Barry J.; Roberts, Anthony J.; Topf, Maya; Steinmetz, Michel O.; Baldus, Marc; Moores, Carolyn A.; Akhmanova, Anna

    2017-01-01

    CAMSAP and Patronin family members regulate microtubule minus-end stability and localization and thus organize noncentrosomal microtubule networks, which are essential for cell division, polarization and differentiation. Here, we found that the CAMSAP C-terminal CKK domain is widely present among

  3. Oscillatory fluid flow influences primary cilia and microtubule mechanics.

    Science.gov (United States)

    Espinha, Lina C; Hoey, David A; Fernandes, Paulo R; Rodrigues, Hélder C; Jacobs, Christopher R

    2014-07-01

    Many tissues are sensitive to mechanical stimuli; however, the mechanotransduction mechanism used by cells remains unknown in many cases. The primary cilium is a solitary, immotile microtubule-based extension present on nearly every mammalian cell which extends from the basal body. The cilium is a mechanosensitive organelle and has been shown to transduce fluid flow-induced shear stress in tissues, such as the kidney and bone. The majority of microtubules assemble from the mother centriole (basal body), contributing significantly to the anchoring of the primary cilium. Several studies have attempted to quantify the number of microtubules emanating from the basal body and the results vary depending on the cell type. It has also been shown that cellular response to shear stress depends on microtubular integrity. This study hypothesizes that changing the microtubule attachment of primary cilia in response to a mechanical stimulus could change primary cilia mechanics and, possibly, mechanosensitivity. Oscillatory fluid flow was applied to two different cell types and the microtubule attachment to the ciliary base was quantified. For the first time, an increase in microtubules around primary cilia both with time and shear rate in response to oscillatory fluid flow stimulation was demonstrated. Moreover, it is presented that the primary cilium is required for this loading-induced cellular response. This study has demonstrated a new role for the cilium in regulating alterations in the cytoplasmic microtubule network in response to mechanical stimulation, and therefore provides a new insight into how cilia may regulate its mechanics and thus the cells mechanosensitivity. Copyright © 2014 Wiley Periodicals, Inc.

  4. Polyamine sharing between tubulin dimers favours microtubule nucleation and elongation via facilitated diffusion.

    Directory of Open Access Journals (Sweden)

    Alain Mechulam

    2009-01-01

    Full Text Available We suggest for the first time that the action of multivalent cations on microtubule dynamics can result from facilitated diffusion of GTP-tubulin to the microtubule ends. Facilitated diffusion can promote microtubule assembly, because, upon encountering a growing nucleus or the microtubule wall, random GTP-tubulin sliding on their surfaces will increase the probability of association to the target sites (nucleation sites or MT ends. This is an original explanation for understanding the apparent discrepancy between the high rate of microtubule elongation and the low rate of tubulin association at the microtubule ends in the viscous cytoplasm. The mechanism of facilitated diffusion requires an attraction force between two tubulins, which can result from the sharing of multivalent counterions. Natural polyamines (putrescine, spermidine, and spermine are present in all living cells and are potent agents to trigger tubulin self-attraction. By using an analytical model, we analyze the implication of facilitated diffusion mediated by polyamines on nucleation and elongation of microtubules. In vitro experiments using pure tubulin indicate that the promotion of microtubule assembly by polyamines is typical of facilitated diffusion. The results presented here show that polyamines can be of particular importance for the regulation of the microtubule network in vivo and provide the basis for further investigations into the effects of facilitated diffusion on cytoskeleton dynamics.

  5. Centriolar CPAP/SAS-4 Imparts Slow Processive Microtubule Growth

    NARCIS (Netherlands)

    Sharma, Ashwani; Aher, Amol; Dynes, Nicola J; Frey, Daniel; Katrukha, Eugene A; Jaussi, Rolf; Grigoriev, Ilya; Croisier, Marie; Kammerer, Richard A; Akhmanova, Anna; Gönczy, Pierre; Steinmetz, Michel O

    2016-01-01

    Centrioles are fundamental and evolutionarily conserved microtubule-based organelles whose assembly is characterized by microtubule growth rates that are orders of magnitude slower than those of cytoplasmic microtubules. Several centriolar proteins can interact with tubulin or microtubules, but how

  6. Dietary flavonoid fisetin binds to β-tubulin and disrupts microtubule dynamics in prostate cancer cells.

    Science.gov (United States)

    Mukhtar, Eiman; Adhami, Vaqar Mustafa; Sechi, Mario; Mukhtar, Hasan

    2015-10-28

    Microtubule targeting based therapies have revolutionized cancer treatment; however, resistance and side effects remain a major limitation. Therefore, novel strategies that can overcome these limitations are urgently needed. We made a novel discovery that fisetin, a hydroxyflavone, is a microtubule stabilizing agent. Fisetin binds to tubulin and stabilizes microtubules with binding characteristics far superior than paclitaxel. Surface plasmon resonance and computational docking studies suggested that fisetin binds to β-tubulin with superior affinity compared to paclitaxel. Fisetin treatment of human prostate cancer cells resulted in robust up-regulation of microtubule associated proteins (MAP)-2 and -4. In addition, fisetin treated cells were enriched in α-tubulin acetylation, an indication of stabilization of microtubules. Fisetin significantly inhibited PCa cell proliferation, migration, and invasion. Nudc, a protein associated with microtubule motor dynein/dynactin complex that regulates microtubule dynamics, was inhibited with fisetin treatment. Further, fisetin treatment of a P-glycoprotein overexpressing multidrug-resistant cancer cell line NCI/ADR-RES inhibited the viability and colony formation. Our results offer in vitro proof-of-concept for fisetin as a microtubule targeting agent. We suggest that fisetin could be developed as an adjuvant for treatment of prostate and other cancer types. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  7. The growth speed of microtubules with XMAP215-coated beads coupled to their ends is increased by tensile force

    Science.gov (United States)

    Trushko, Anastasiya; Schäffer, Erik; Howard, Jonathon

    2013-01-01

    The generation of pulling and pushing forces is one of the important functions of microtubules, which are dynamic and polarized structures. The ends of dynamic microtubules are able to form relatively stable links to cellular structures, so that when a microtubule grows it can exert a pushing force and when it shrinks it can exert a pulling force. Microtubule growth and shrinkage are tightly regulated by microtubule-associated proteins (MAPs) that bind to microtubule ends. Given their localization, MAPs may be exposed to compressive and tensile forces. The effect of such forces on MAP function, however, is poorly understood. Here we show that beads coated with the microtubule polymerizing protein XMAP215, the Xenopus homolog of Dis1 and chTOG, are able to link stably to the plus ends of microtubules, even when the ends are growing or shrinking; at growing ends, the beads increase the polymerization rate. Using optical tweezers, we found that tensile force further increased the microtubule polymerization rate. These results show that physical forces can regulate the activity of MAPs. Furthermore, our results show that XMAP215 can be used as a handle to sense and mechanically manipulate the dynamics of the microtubule tip. PMID:23964126

  8. A ROP2-RIC1 pathway fine-tunes microtubule reorganization for salt tolerance in Arabidopsis.

    Science.gov (United States)

    Li, Changjiang; Lu, Hanmei; Li, Wei; Yuan, Ming; Fu, Ying

    2017-07-01

    The reorganization of microtubules induced by salt stress is required for Arabidopsis survival under high salinity conditions. RIC1 is an effector of Rho-related GTPase from plants (ROPs) and a known microtubule-associated protein. In this study, we demonstrated that RIC1 expression decreased with long-term NaCl treatment, and ric1-1 seedlings exhibited a higher survival rate under salt stress. We found that RIC1 reduced the frequency of microtubule transition from shortening to growing status and knockout of RIC1 improved the reassembly of depolymerized microtubules caused by either oryzalin treatment or salt stress. Further investigation showed that constitutively active ROP2 promoted the reassembly of microtubules and the survival of seedlings under salt stress. A rop2-1 ric1-1 double mutant rescued the salt-sensitive phenotype of rop2-1, indicating that ROP2 functions in salt tolerance through RIC1. Although ROP2 did not regulate RIC1 expression upon salt stress, a quick but mild increase of ROP2 activity was induced, led to reduction of RIC1 on microtubules. Collectively, our study reveals an ROP2-RIC1 pathway that fine-tunes microtubule dynamics in response to salt stress in Arabidopsis. This finding not only reveals a new regulatory mechanism for microtubule reorganization under salt stress but also the importance of ROP signalling for salinity tolerance. © 2017 John Wiley & Sons Ltd.

  9. Stable and dynamic microtubules coordinately shape the myosin activation zone during cytokinetic furrow formation

    Science.gov (United States)

    Foe, Victoria E.; von Dassow, George

    2008-01-01

    The cytokinetic furrow arises from spatial and temporal regulation of cortical contractility. To test the role microtubules play in furrow specification, we studied myosin II activation in echinoderm zygotes by assessing serine19-phosphorylated regulatory light chain (pRLC) localization after precisely timed drug treatments. Cortical pRLC was globally depressed before cytokinesis, then elevated only at the equator. We implicated cell cycle biochemistry (not microtubules) in pRLC depression, and differential microtubule stability in localizing the subsequent myosin activation. With no microtubules, pRLC accumulation occurred globally instead of equatorially, and loss of just dynamic microtubules increased equatorial pRLC recruitment. Nocodazole treatment revealed a population of stable astral microtubules that formed during anaphase; among these, those aimed toward the equator grew longer, and their tips coincided with cortical pRLC accumulation. Shrinking the mitotic apparatus with colchicine revealed pRLC suppression near dynamic microtubule arrays. We conclude that opposite effects of stable versus dynamic microtubules focuses myosin activation to the cell equator during cytokinesis. PMID:18955555

  10. Four-stranded mini microtubules formed by Prosthecobacter BtubAB show dynamic instability.

    Science.gov (United States)

    Deng, Xian; Fink, Gero; Bharat, Tanmay A M; He, Shaoda; Kureisaite-Ciziene, Danguole; Löwe, Jan

    2017-07-18

    Microtubules, the dynamic, yet stiff hollow tubes built from αβ-tubulin protein heterodimers, are thought to be present only in eukaryotic cells. Here, we report a 3.6-Å helical reconstruction electron cryomicroscopy structure of four-stranded mini microtubules formed by bacterial tubulin-like Prosthecobacter dejongeii BtubAB proteins. Despite their much smaller diameter, mini microtubules share many key structural features with eukaryotic microtubules, such as an M-loop, alternating subunits, and a seam that breaks overall helical symmetry. Using in vitro total internal reflection fluorescence microscopy, we show that bacterial mini microtubules treadmill and display dynamic instability, another hallmark of eukaryotic microtubules. The third protein in the btub gene cluster, BtubC, previously known as "bacterial kinesin light chain," binds along protofilaments every 8 nm, inhibits BtubAB mini microtubule catastrophe, and increases rescue. Our work reveals that some bacteria contain regulated and dynamic cytomotive microtubule systems that were once thought to be only useful in much larger and sophisticated eukaryotic cells.

  11. Interactive domains in the molecular chaperone human alphaB crystallin modulate microtubule assembly and disassembly.

    Directory of Open Access Journals (Sweden)

    Joy G Ghosh

    2007-06-01

    Full Text Available Small heat shock proteins regulate microtubule assembly during cell proliferation and in response to stress through interactions that are poorly understood.Novel functions for five interactive sequences in the small heat shock protein and molecular chaperone, human alphaB crystallin, were investigated in the assembly/disassembly of microtubules and aggregation of tubulin using synthetic peptides and mutants of human alphaB crystallin.The interactive sequence (113FISREFHR(120 exposed on the surface of alphaB crystallin decreased microtubule assembly by approximately 45%. In contrast, the interactive sequences, (131LTITSSLSSDGV(142 and (156ERTIPITRE(164, corresponding to the beta8 strand and the C-terminal extension respectively, which are involved in complex formation, increased microtubule assembly by approximately 34-45%. The alphaB crystallin peptides, (113FISREFHR(120 and (156ERTIPITRE(164, inhibited microtubule disassembly by approximately 26-36%, and the peptides (113FISREFHR(120 and (131LTITSSLSSDGV(142 decreased the thermal aggregation of tubulin by approximately 42-44%. The (131LTITSSLSSDGV(142 and (156ERTIPITRE(164 peptides were more effective than the widely used anti-cancer drug, Paclitaxel, in modulating tubulinmicrotubule dynamics. Mutagenesis of these interactive sequences in wt human alphaB crystallin confirmed the effects of the alphaB crystallin peptides on microtubule assembly/disassembly and tubulin aggregation. The regulation of microtubule assembly by alphaB crystallin varied over a narrow range of concentrations. The assembly of microtubules was maximal at alphaB crystallin to tubulin molar ratios between 1:4 and 2:1, while molar ratios >2:1 inhibited microtubule assembly.Interactive sequences on the surface of human alphaB crystallin collectively modulate microtubule assembly through a dynamic subunit exchange mechanism that depends on the concentration and ratio of alphaB crystallin to tubulin. These are the first

  12. Structural insights into microtubule doublet interactions inaxonemes

    Energy Technology Data Exchange (ETDEWEB)

    Downing, Kenneth H.; Sui, Haixin

    2007-06-06

    Coordinated sliding of microtubule doublets, driven by dynein motors, produces periodic beating of the axoneme. Recent structural studies of the axoneme have used cryo-electron tomography to reveal new details of the interactions among some of the multitude of proteins that form the axoneme and regulate its movement. Connections among the several sets of dyneins, in particular, suggest ways in which their actions may be coordinated. Study of the molecular architecture of isolated doublets has provided a structural basis for understanding the doublet's mechanical properties that are related to the bending of the axoneme, and has also offered insight into its potential role in the mechanism of dynein activity regulation.

  13. Dynamic release of nuclear RanGTP triggers TPX2-dependent microtubule assembly during the apoptotic execution phase.

    Science.gov (United States)

    Moss, David K; Wilde, Andrew; Lane, Jon D

    2009-03-01

    During apoptosis, the interphase microtubule network is dismantled then later replaced by a novel, non-centrosomal microtubule array. These microtubules assist in the peripheral redistribution of nuclear fragments in the apoptotic cell; however, the regulation of apoptotic microtubule assembly is not understood. Here, we demonstrate that microtubule assembly depends upon the release of nuclear RanGTP into the apoptotic cytoplasm because this process is blocked in apoptotic cells overexpressing dominant-negative GDP-locked Ran (T24N). Actin-myosin-II contractility provides the impetus for Ran release and, consequently, microtubule assembly is blocked in blebbistatin- and Y27632-treated apoptotic cells. Importantly, the spindle-assembly factor TPX2 (targeting protein for Xklp2), colocalises with apoptotic microtubules, and siRNA silencing of TPX2, but not of the microtubule motors Mklp1 and Kid, abrogates apoptotic microtubule assembly. These data provide a molecular explanation for the assembly of the apoptotic microtubule network, and suggest important similarities with the process of RanGTP- and TPX2-mediated mitotic spindle formation.

  14. Surface regulated arsenenes as Dirac materials: From density functional calculations

    International Nuclear Information System (INIS)

    Yuan, Junhui; Xie, Qingxing; Yu, Niannian; Wang, Jiafu

    2017-01-01

    Highlights: • The presence of Dirac cones in chemically decorated buckled arsenene AsX (X = CN, NC, NCO, NCS, and NCSe) has been revealed. • First-principles calculations show that all these chemically decorated arsenenes are kinetically stable in defending thermal fluctuations in room temperature. - Abstract: Using first principle calculations based on density functional theory (DFT), we have systematically investigated the structure stability and electronic properties of chemically decorated arsenenes, AsX (X = CN, NC, NCO, NCS and NCSe). Phonon dispersion and formation energy analysis reveal that all the five chemically decorated buckled arsenenes are energetically favorable and could be synthesized. Our study shows that wide-bandgap arsenene would turn into Dirac materials when functionalized by -X (X = CN, NC, NCO, NCS and NCSe) groups, rendering new promises in next generation high-performance electronic devices.

  15. Explaining density-dependent regulation in earthworm populations using life-history analysis

    NARCIS (Netherlands)

    Kammenga, J.E.; Spurgeon, D.J.; Svendsen, C.; Weeks, J.M.

    2003-01-01

    At present there is little knowledge about how density regulates population growth rate and to what extent this is determined by life-history patterns. We compared density dependent population consequences in the Nicholsonian sense based oil experimental observations and life-history modeling for

  16. Katanin: A Sword Cutting Microtubules for Cellular, Developmental, and Physiological Purposes

    Directory of Open Access Journals (Sweden)

    Ivan Luptovčiak

    2017-11-01

    Full Text Available KATANIN is a well-studied microtubule severing protein affecting microtubule organization and dynamic properties in higher plants. By regulating mitotic and cytokinetic and cortical microtubule arrays it is involved in the progression of cell division and cell division plane orientation. KATANIN is also involved in cell elongation and morphogenesis during plant growth. In this way KATANIN plays critical roles in diverse plant developmental processes including the development of pollen, embryo, seed, meristem, root, hypocotyl, cotyledon, leaf, shoot, and silique. KATANIN-dependent microtubule regulation seems to be under the control of plant hormones. This minireview provides an overview on available KATANIN mutants and discusses advances in our understanding of KATANIN biological roles in plants.

  17. Microtubule heterogeneity of Ornithogalum umbellatum ovary epidermal cells: non-stable cortical microtubules and stable lipotubuloid microtubules.

    Science.gov (United States)

    Kwiatkowska, Maria; Stępiński, Dariusz; Polit, Justyna T; Popłońska, Katarzyna; Wojtczak, Agnieszka

    2011-01-01

    Lipotubuloids, structures containing lipid bodies and microtubules, are described in ovary epidermal cells of Ornithogalum umbellatum. Microtubules of lipotubuloids can be fixed in electron microscope fixative containing only buffered OsO(4) or in glutaraldehyde with OsO(4) post-fixation, or in a mixture of OsO(4) and glutaraldehyde. None of these substances fixes cortical microtubules of ovary epidermis of this plant which is characterized by dynamic longitudinal growth. However, cortical microtubules can be fixed with cold methanol according immunocytological methods with the use of β-tubulin antibodies and fluorescein. The existence of cortical microtubules has also been evidenced by EM observations solely after the use of taxol, microtubule stabilizer, and fixation in a glutaraldehyde/OsO(4) mixture. These microtubules mostly lie transversely, sometimes obliquely, and rarely parallel to the cell axis. Staining, using Ruthenium Red and silver hexamine, has revealed that lipotubuloid microtubules surface is covered with polysaccharides. The presumption has been made that the presence of a polysaccharide layer enhances the stability of lipotubuloid microtubules.

  18. Taking directions: the role of microtubule-bound nucleation in the self-organization of the plant cortical array

    International Nuclear Information System (INIS)

    Deinum, Eva E; Tindemans, Simon H; Mulder, Bela M

    2011-01-01

    The highly aligned cortical microtubule array of interphase plant cells is a key regulator of anisotropic cell expansion. Recent computational and analytical work has shown that the non-equilibrium self-organization of this structure can be understood on the basis of experimentally observed collisional interactions between dynamic microtubules attached to the plasma membrane. Most of these approaches assumed that new microtubules are homogeneously and isotropically nucleated on the cortical surface. Experimental evidence, however, shows that nucleation mostly occurs from other microtubules and under specific relative angles. Here, we investigate the impact of directed microtubule-bound nucleations on the alignment process using computer simulations. The results show that microtubule-bound nucleations can increase the degree of alignment achieved, decrease the timescale of the ordering process and widen the regime of dynamic parameters for which the system can self-organize. We establish that the major determinant of this effect is the degree of co-alignment of the nucleations with the parent microtubule. The specific role of sideways branching nucleations appears to allow stronger alignment while maintaining a measure of overall spatial homogeneity. Finally, we investigate the suggestion that observed persistent rotation of microtubule domains can be explained through a handedness bias in microtubule-bound nucleations, showing that this is possible only for an extreme bias and over a limited range of parameters

  19. Taking directions: the role of microtubule-bound nucleation in the self-organization of the plant cortical array

    Science.gov (United States)

    Deinum, Eva E.; Tindemans, Simon H.; Mulder, Bela M.

    2011-10-01

    The highly aligned cortical microtubule array of interphase plant cells is a key regulator of anisotropic cell expansion. Recent computational and analytical work has shown that the non-equilibrium self-organization of this structure can be understood on the basis of experimentally observed collisional interactions between dynamic microtubules attached to the plasma membrane. Most of these approaches assumed that new microtubules are homogeneously and isotropically nucleated on the cortical surface. Experimental evidence, however, shows that nucleation mostly occurs from other microtubules and under specific relative angles. Here, we investigate the impact of directed microtubule-bound nucleations on the alignment process using computer simulations. The results show that microtubule-bound nucleations can increase the degree of alignment achieved, decrease the timescale of the ordering process and widen the regime of dynamic parameters for which the system can self-organize. We establish that the major determinant of this effect is the degree of co-alignment of the nucleations with the parent microtubule. The specific role of sideways branching nucleations appears to allow stronger alignment while maintaining a measure of overall spatial homogeneity. Finally, we investigate the suggestion that observed persistent rotation of microtubule domains can be explained through a handedness bias in microtubule-bound nucleations, showing that this is possible only for an extreme bias and over a limited range of parameters.

  20. Regulation of bacterioplankton density and biomass in tropical shallow coastal lagoons

    Directory of Open Access Journals (Sweden)

    Fabiana MacCord

    Full Text Available AIM: Estimating bacterioplankton density and biomass and their regulating factors is important in order to evaluate aquatic systems' carrying capacity, regarding bacterial growth and the stock of matter in the bacterial community, which can be consumed by higher trophic levels. We aim to evaluate the limnological factors which regulate - in space and time - the bacterioplankton dynamics (abundance and biomass in five tropical coastal lagoons in the state of Rio de Janeiro, Brazil. METHOD: The current study was carried out at the following lagoons: Imboassica, Cabiúnas, Comprida, Carapebus and Garças. They differ in morphology and in their main limnological factors. The limnological variables as well as bacterioplankton abundance and biomass were monthly sampled for 14 months. Model selection analyses were performed in order to evaluate the main variables regulating the bacterioplankton's dynamics in these lagoons. RESULT: The salt concentration and the "space" factor (i.e. different lagoons explained great part of the bacterial density and biomass variance in the studied tropical coastal lagoons. When the lagoons were analyzed separately, salinity still explained great part of the variation of bacterial density and biomass in the Imboassica and Garças lagoons. On the other hand, phosphorus concentration was the main factor explaining the variance of bacterial density and biomass in the distrophic Cabiúnas, Comprida and Carapebus lagoons. There was a strong correlation between bacterial density and biomass (r² = 0.70, p < 0.05, indicating that bacterial biomass variations are highly dependent on bacterial density variations. CONCLUSION: (i Different limnological variables regulate the bacterial density and biomass in the studied coastal lagoons, (ii salt and phosphorus concentrations greatly explained the variation of bacterial density and biomass in the saline and distrophic lagoons, respectively, and (iii N-nitrate and chlorophyll

  1. Effects of Larval Density on Gene Regulation in Caenorhabditis elegans During Routine L1 Synchronization.

    Science.gov (United States)

    Chan, Io Long; Rando, Oliver J; Conine, Colin C

    2018-05-04

    Bleaching gravid C. elegans followed by a short period of starvation of the L1 larvae is a routine method performed by worm researchers for generating synchronous populations for experiments. During the process of investigating dietary effects on gene regulation in L1 stage worms by single-worm RNA-Seq, we found that the density of resuspended L1 larvae affects expression of many mRNAs. Specifically, a number of genes related to metabolism and signaling are highly expressed in worms arrested at low density, but are repressed at higher arrest densities. We generated a GFP reporter strain based on one of the most density-dependent genes in our dataset - lips-15 - and confirmed that this reporter was expressed specifically in worms arrested at relatively low density. Finally, we show that conditioned media from high density L1 cultures was able to downregulate lips-15 even in L1 animals arrested at low density, and experiments using daf-22 mutant animals demonstrated that this effect is not mediated by the ascaroside family of signaling pheromones. Together, our data implicate a soluble signaling molecule in density sensing by L1 stage C. elegans , and provide guidance for design of experiments focused on early developmental gene regulation. Copyright © 2018 Chan et al.

  2. An ELMO2-RhoG-ILK network modulates microtubule dynamics.

    Science.gov (United States)

    Jackson, Bradley C; Ivanova, Iordanka A; Dagnino, Lina

    2015-07-15

    ELMO2 belongs to a family of scaffold proteins involved in phagocytosis and cell motility. ELMO2 can simultaneously bind integrin-linked kinase (ILK) and RhoG, forming tripartite ERI complexes. These complexes are involved in promoting β1 integrin-dependent directional migration in undifferentiated epidermal keratinocytes. ELMO2 and ILK have also separately been implicated in microtubule regulation at integrin-containing focal adhesions. During differentiation, epidermal keratinocytes cease to express integrins, but ERI complexes persist. Here we show an integrin-independent role of ERI complexes in modulation of microtubule dynamics in differentiated keratinocytes. Depletion of ERI complexes by inactivating the Ilk gene in these cells reduces microtubule growth and increases the frequency of catastrophe. Reciprocally, exogenous expression of ELMO2 or RhoG stabilizes microtubules, but only if ILK is also present. Mechanistically, activation of Rac1 downstream from ERI complexes mediates their effects on microtubule stability. In this pathway, Rac1 serves as a hub to modulate microtubule dynamics through two different routes: 1) phosphorylation and inactivation of the microtubule-destabilizing protein stathmin and 2) phosphorylation and inactivation of GSK-3β, which leads to the activation of CRMP2, promoting microtubule growth. At the cellular level, the absence of ERI species impairs Ca(2+)-mediated formation of adherens junctions, critical to maintaining mechanical integrity in the epidermis. Our findings support a key role for ERI species in integrin-independent stabilization of the microtubule network in differentiated keratinocytes. © 2015 Jackson et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  3. Dissecting the nanoscale distributions and functions of microtubule-end-binding proteins EB1 and ch-TOG in interphase HeLa cells.

    Directory of Open Access Journals (Sweden)

    Satoko Nakamura

    Full Text Available Recently, the EB1 and XMAP215/TOG families of microtubule binding proteins have been demonstrated to bind autonomously to the growing plus ends of microtubules and regulate their behaviour in in vitro systems. However, their functional redundancy or difference in cells remains obscure. Here, we compared the nanoscale distributions of EB1 and ch-TOG along microtubules using high-resolution microscopy techniques, and also their roles in microtubule organisation in interphase HeLa cells. The ch-TOG accumulation sites protruded ∼100 nm from the EB1 comets. Overexpression experiments showed that ch-TOG and EB1 did not interfere with each other's localisation, confirming that they recognise distinct regions at the ends of microtubules. While both EB1 and ch-TOG showed similar effects on microtubule plus end dynamics and additively increased microtubule dynamicity, only EB1 exhibited microtubule-cell cortex attachment activity. These observations indicate that EB1 and ch-TOG regulate microtubule organisation differently via distinct regions in the plus ends of microtubules.

  4. The demography of climate-driven and density-regulated population dynamics in a perennial plant

    DEFF Research Database (Denmark)

    Dahlgren, Johan; Bengstsson, Karin; Ehrlén, Johan

    2016-01-01

    Identifying the internal and external drivers of population dynamics is a key objective in ecology, currently accentuated by the need to forecast the effects of climate change on species distributions and abundances. The interplay between environmental and density effects is one particularly...... important aspect of such forecasts. We examined the simultaneous impact of climate and intraspecific density on vital rates of the dwarf shrub Fumana procumbens over 20 yr, using generalized additive mixed models. We then analyzed effects on population dynamics using integral projection models...... to be driven solely by the environment can overestimate extinction risks if there is density dependence. We conclude that density regulation can dampen effects of climate change on Fumana population size, and discuss the need to quantify density dependence in predictions of population responses...

  5. Microtubule dynamics: Caps, catastrophes, and coupled hydrolysis

    DEFF Research Database (Denmark)

    Flyvbjerg, H.; Holy, T.E.; Leibler, S.

    1996-01-01

    An effective theory is formulated for the dynamics of the guanosine triphosphate (GTP) cap believed to stabilize growing microtubules. The theory provides a ''coarse-grained'' description of the cap's dynamics. ''Microscopic'' details, such as the microtubule lattice structure and the fate of its...

  6. Mechanics of microtubules: effects of protofilament orientation.

    Science.gov (United States)

    Donhauser, Zachary J; Jobs, William B; Binka, Edem C

    2010-09-08

    Microtubules are hollow cylindrical polymers of the protein tubulin that play a number of important dynamic and structural roles in eukaryotic cells. Both in vivo and in vitro microtubules can exist in several possible configurations, differing in the number of protofilaments, helical rise of tubulin dimers, and protofilament skew angle with respect to the main tube axis. Here, finite element modeling is applied to examine the mechanical response of several known microtubule types when subjected to radial deformation. The data presented here provide an important insight into microtubule stiffness and reveal that protofilament orientation does not affect radial stiffness. Rather, stiffness is primarily dependent on the effective Young's modulus of the polymerized material and the effective radius of the microtubule. These results are also directly correlated to atomic force microscopy nanoindentation measurements to allow a more detailed interpretation of previous experiments. When combined with experimental data that show a significant difference between microtubules stabilized with a slowly hydrolyzable GTP analog and microtubules stabilized with paclitaxel, the finite element data suggest that paclitaxel increases the overall radial flexibility of the microtubule wall. Copyright 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  7. C-terminal region of MAP7 domain containing protein 3 (MAP7D3 promotes microtubule polymerization by binding at the C-terminal tail of tubulin.

    Directory of Open Access Journals (Sweden)

    Saroj Yadav

    Full Text Available MAP7 domain containing protein 3 (MAP7D3, a newly identified microtubule associated protein, has been shown to promote microtubule assembly and stability. Its microtubule binding region has been reported to consist of two coiled coil motifs located at the N-terminus. It possesses a MAP7 domain near the C-terminus and belongs to the microtubule associated protein 7 (MAP7 family. The MAP7 domain of MAP7 protein has been shown to bind to kinesin-1; however, the role of MAP7 domain in MAP7D3 remains unknown. Based on the bioinformatics analysis of MAP7D3, we hypothesized that the MAP7 domain of MAP7D3 may have microtubule binding activity. Indeed, we found that MAP7 domain of MAP7D3 bound to microtubules as well as enhanced the assembly of microtubules in vitro. Interestingly, a longer fragment MDCT that contained the MAP7 domain (MD with the C-terminal tail (CT of the protein promoted microtubule polymerization to a greater extent than MD and CT individually. MDCT stabilized microtubules against dilution induced disassembly. MDCT bound to reconstituted microtubules with an apparent dissociation constant of 3.0 ± 0.5 µM. An immunostaining experiment showed that MDCT localized along the length of the preassembled microtubules. Competition experiments with tau indicated that MDCT shares its binding site on microtubules with tau. Further, we present evidence indicating that MDCT binds to the C-terminal tail of tubulin. In addition, MDCT could bind to tubulin in HeLa cell extract. Here, we report a microtubule binding region in the C-terminal region of MAP7D3 that may have a role in regulating microtubule assembly dynamics.

  8. Density-dependent intraspecific aggression regulates survival in northern Yellowstone wolves (Canis lupus).

    Science.gov (United States)

    Cubaynes, Sarah; MacNulty, Daniel R; Stahler, Daniel R; Quimby, Kira A; Smith, Douglas W; Coulson, Tim

    2014-11-01

    Understanding the population dynamics of top-predators is essential to assess their impact on ecosystems and to guide their management. Key to this understanding is identifying the mechanisms regulating vital rates. Determining the influence of density on survival is necessary to understand the extent to which human-caused mortality is compensatory or additive. In wolves (Canis lupus), empirical evidence for density-dependent survival is lacking. Dispersal is considered the principal way in which wolves adjust their numbers to prey supply or compensate for human exploitation. However, studies to date have primarily focused on exploited wolf populations, in which density-dependent mechanisms are likely weak due to artificially low wolf densities. Using 13 years of data on 280 collared wolves in Yellowstone National Park, we assessed the effect of wolf density, prey abundance and population structure, as well as winter severity, on age-specific survival in two areas (prey-rich vs. prey-poor) of the national park. We further analysed cause-specific mortality and explored the factors driving intraspecific aggression in the prey-rich northern area of the park. Overall, survival rates decreased during the study. In northern Yellowstone, density dependence regulated adult survival through an increase in intraspecific aggression, independent of prey availability. In the interior of the park, adult survival was less variable and density-independent, despite reduced prey availability. There was no effect of prey population structure in northern Yellowstone, or of winter severity in either area. Survival was similar among yearlings and adults, but lower for adults older than 6 years. Our results indicate that density-dependent intraspecific aggression is a major driver of adult wolf survival in northern Yellowstone, suggesting intrinsic density-dependent mechanisms have the potential to regulate wolf populations at high ungulate densities. When low prey availability or high

  9. Local density regulates migratory songbird reproductive success through effects on double-brooding and nest predation.

    Science.gov (United States)

    Woodworth, Bradley K; Wheelwright, Nathaniel T; Newman, Amy E M; Norris, D Ryan

    2017-08-01

    Knowledge of the density-dependent processes that regulate animal populations is key to understanding, predicting, and conserving populations. In migratory birds, density-dependence is most often studied during the breeding season, yet we still lack a robust understanding of the reproductive traits through which density influences individual reproductive success. We used 27-yr of detailed, individual-level productivity data from an island-breeding population of Savannah sparrows Passerculus sandwichensis to evaluate effects of local and total annual population density on female reproductive success. Local density (number of neighbors within 50 m of a female's nest) had stronger effects on the number of young fledged than did total annual population density. Females nesting in areas of high local density were more likely to suffer nest predation and less likely to initiate and fledge a second clutch, which led to fewer young fledged in a season. Fledging fewer young subsequently decreased the likelihood of a female recruiting offspring into the breeding population in a subsequent year. Collectively, these results provide insight into the scale and reproductive mechanisms mediating density-dependent reproductive success and fitness in songbirds. © 2017 by the Ecological Society of America.

  10. Survivin counteracts the therapeutic effect of microtubule de-stabilizers by stabilizing tubulin polymers

    Directory of Open Access Journals (Sweden)

    Hsieh Hsing-Pang

    2009-07-01

    Full Text Available Abstract Background Survivin is a dual function protein. It inhibits the apoptosis of cells by inhibiting caspases, and also promotes cell growth by stabilizing microtubules during mitosis. Over-expression of survivin has been demonstrated to induce drug-resistance to various chemo-therapeutic agents such as cisplatin (DNA damaging agent and paclitaxel (microtubule stabilizer in cancers. However, survivin-induced resistance to microtubule de-stabilizers such as Vinca alkaloids and Combretastatin A-4 (CA-4-related compounds were seldom demonstrated in the past. Furthermore, the question remains as to whether survivin plays a dominant role in processing cytokinesis or inhibiting caspases activity in cells treated with anti-mitotic compounds. The purpose of this study is to evaluate the effect of survivin on the resistance and susceptibility of human cancer cells to microtubule de-stabilizer-induced cell death. Results BPR0L075 is a CA-4 analog that induces microtubule de-polymerization and subsequent caspase-dependent apoptosis. To study the relationship between the expression of survivin and the resistance to microtubule de-stabilizers, a KB-derived BPR0L075-resistant cancer cell line, KB-L30, was generated for this study. Here, we found that survivin was over-expressed in the KB-L30 cells. Down-regulation of survivin by siRNA induced hyper-sensitivity to BPR0L075 in KB cells and partially re-stored sensitivity to BPR0L075 in KB-L30 cells. Western blot analysis revealed that down-regulation of survivin induced microtubule de-stabilization in both KB and KB-L30 cells. However, the same treatment did not enhance the down-stream caspase-3/-7 activities in BPR0L075-treated KB cells. Translocation of a caspase-independent apoptosis-related molecule, apoptosis-inducing factor (AIF, from cytoplasm to the nucleus was observed in survivin-targeted KB cells under BPR0L075 treatment. Conclusion In this study, survivin plays an important role in the

  11. Learning-induced and stathmin-dependent changes in microtubule stability are critical for memory and disrupted in ageing

    Science.gov (United States)

    Uchida, Shusaku; Martel, Guillaume; Pavlowsky, Alice; Takizawa, Shuichi; Hevi, Charles; Watanabe, Yoshifumi; Kandel, Eric R.; Alarcon, Juan Marcos; Shumyatsky, Gleb P.

    2014-01-01

    Changes in the stability of microtubules regulate many biological processes, but their role in memory remains unclear. Here we show that learning causes biphasic changes in the microtubule-associated network in the hippocampus. In the early phase, stathmin is dephosphorylated, enhancing its microtubule-destabilizing activity by promoting stathmin-tubulin binding, whereas in the late phase these processes are reversed leading to an increase in microtubule/KIF5-mediated localization of the GluA2 subunit of AMPA receptors at synaptic sites. A microtubule stabilizer paclitaxel decreases or increases memory when applied at the early or late phases, respectively. Stathmin mutations disrupt changes in microtubule stability, GluA2 localization, synaptic plasticity and memory. Aged wild-type mice show impairments in stathmin levels, changes in microtubule stability, and GluA2 localization. Blocking GluA2 endocytosis rescues memory deficits in stathmin mutant and aged wild-type mice. These findings demonstrate a role for microtubules in memory in young adult and aged individuals. PMID:25007915

  12. Disruption of microtubule network rescues aberrant actin comets in dynamin2-depleted cells.

    Directory of Open Access Journals (Sweden)

    Yuji Henmi

    Full Text Available A large GTPase dynamin, which is required for endocytic vesicle formation, regulates the actin cytoskeleton through its interaction with cortactin. Dynamin2 mutants impair the formation of actin comets, which are induced by Listeria monocytogenes or phosphatidylinositol-4-phosphate 5-kinase. However, the role of dynamin2 in the regulation of the actin comet is still unclear. Here we show that aberrant actin comets in dynamin2-depleted cells were rescued by disrupting of microtubule networks. Depletion of dynamin2, but not cortactin, significantly reduced the length and the speed of actin comets induced by Listeria. This implies that dynamin2 may regulate the actin comet in a cortactin-independent manner. As dynamin regulates microtubules, we investigated whether perturbation of microtubules would rescue actin comet formation in dynamin2-depleted cells. Treatment with taxol or colchicine created a microtubule-free space in the cytoplasm, and made no difference between control and dynamin2 siRNA cells. This suggests that the alteration of microtubules by dynamin2 depletion reduced the length and the speed of the actin comet.

  13. Magnolol Inhibits the Growth of Non-Small Cell Lung Cancer via Inhibiting Microtubule Polymerization

    Directory of Open Access Journals (Sweden)

    Jia Shen

    2017-07-01

    Full Text Available Background: The tubulin/microtubule system, which is an integral component of the cytoskeleton, plays an essential role in mitosis. Targeting mitotic progression by disturbing microtubule dynamics is a rational strategy for cancer treatment. Methods: Microtubule polymerization assay was performed to examine the effect of Magnolol (a novel natural phenolic compound isolated from Magnolia obovata on cellular microtubule polymerization in human non-small cell lung cancer (NSCLC cells. Cell cycle analysis, mitotic index assay, cell proliferation assay, colony formation assay, western blotting analysis of cell cycle regulators, Annexin V-FITC/PI staining, and live/dead viability staining were carried out to investigate the Magnolol’s inhibitory effect on proliferation and viability of NSCLS cells in vitro. Xenograft model of human A549 NSCLC tumor was used to determine the Magnolol’s efficacy in vivo. Results: Magnolol treatment effectively inhibited cell proliferation and colony formation of NSCLC cells. Further study proved that Magnolol induced the mitotic phase arrest and inhibited G2/M progression in a dose-dependent manner, which were mechanistically associated with expression alteration of a series of cell cycle regulators. Furthermore, Magnolol treatment disrupted the cellular microtubule organization via inhibiting the polymerization of microtubule. We also found treatment with NSCLC cells with Magnolol resulted in apoptosis activation through a p53-independent pathway, and autophgy induction via down-regulation of the Akt/mTOR pathway. Finally, Magnolol treatment significantly suppressed the NSCLC tumor growth in mouse xenograft model in vivo. Conclusion: These findings identify Magnolol as a promising candidate with anti-microtubule polymerization activity for NSCLC treatment.

  14. Three-dimensional fine structure of the organization of microtubules in neurite varicosities by ultra-high voltage electron microscope tomography.

    Science.gov (United States)

    Nishida, Tomoki; Yoshimura, Ryoichi; Endo, Yasuhisa

    2017-09-01

    Neurite varicosities are highly specialized compartments that are involved in neurotransmitter/ neuromodulator release and provide a physiological platform for neural functions. However, it remains unclear how microtubule organization contributes to the form of varicosity. Here, we examine the three-dimensional structure of microtubules in varicosities of a differentiated PC12 neural cell line using ultra-high voltage electron microscope tomography. Three-dimensional imaging showed that a part of the varicosities contained an accumulation of organelles that were separated from parallel microtubule arrays. Further detailed analysis using serial sections and whole-mount tomography revealed microtubules running in a spindle shape of swelling in some other types of varicosities. These electron tomographic results showed that the structural diversity and heterogeneity of microtubule organization supported the form of varicosities, suggesting that a different distribution pattern of microtubules in varicosities is crucial to the regulation of varicosities development.

  15. The plant formin AtFH4 interacts with both actin and microtubules, and contains a newly identified microtubule-binding domain

    Czech Academy of Sciences Publication Activity Database

    Deeks, M.J.; Fendrych, Matyáš; Smertenko, A.; Bell, K.S.; Oparka, K.; Cvrčková, F.; Žárský, Viktor; Hussey, P.J.

    2010-01-01

    Roč. 123, č. 8 (2010), s. 1209-1215 ISSN 0021-9533 R&D Projects: GA MŠk(CZ) LC06004; GA ČR GAP305/10/0433 Institutional research plan: CEZ:AV0Z50380511 Keywords : Actin regulating proteins * Membrane * Microtubule Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 6.290, year: 2010

  16. Flagellum density regulates Proteus mirabilis swarmer cell motility in viscous environments.

    Science.gov (United States)

    Tuson, Hannah H; Copeland, Matthew F; Carey, Sonia; Sacotte, Ryan; Weibel, Douglas B

    2013-01-01

    Proteus mirabilis is an opportunistic pathogen that is frequently associated with urinary tract infections. In the lab, P. mirabilis cells become long and multinucleate and increase their number of flagella as they colonize agar surfaces during swarming. Swarming has been implicated in pathogenesis; however, it is unclear how energetically costly changes in P. mirabilis cell morphology translate into an advantage for adapting to environmental changes. We investigated two morphological changes that occur during swarming--increases in cell length and flagellum density--and discovered that an increase in the surface density of flagella enabled cells to translate rapidly through fluids of increasing viscosity; in contrast, cell length had a small effect on motility. We found that swarm cells had a surface density of flagella that was ∼5 times larger than that of vegetative cells and were motile in fluids with a viscosity that inhibits vegetative cell motility. To test the relationship between flagellum density and velocity, we overexpressed FlhD(4)C(2), the master regulator of the flagellar operon, in vegetative cells of P. mirabilis and found that increased flagellum density produced an increase in cell velocity. Our results establish a relationship between P. mirabilis flagellum density and cell motility in viscous environments that may be relevant to its adaptation during the infection of mammalian urinary tracts and movement in contact with indwelling catheters.

  17. Microtubule array reorientation in response to hormones does not involve changes in microtubule nucleation modes at the periclinal cell surface

    Science.gov (United States)

    Atkinson, Samantha; Kirik, Angela; Kirik, Viktor

    2014-01-01

    Aligned microtubule arrays spatially organize cell division, trafficking, and determine the direction of cell expansion in plant cells. In response to changes in environmental and developmental signals, cells reorganize their microtubule arrays into new configurations. Here, we tested the role of microtubule nucleation during hormone-induced microtubule array reorientation. We have found that in the process of microtubule array reorientation the ratios between branching, parallel, and de-novo nucleations remained constant, suggesting that the microtubule reorientation mechanism does not involve changes in nucleation modes. In the ton2/fass mutant, which has reduced microtubule branching nucleation frequency and decreased nucleation activity of the γ-tubulin complexes, microtubule arrays were able to reorient. Presented data suggest that reorientation of microtubules into transverse arrays in response to hormones does not involve changes in microtubule nucleation at the periclinal cell surface PMID:25135522

  18. Spatial distribution of limited resources and local density regulation in juvenile Atlantic salmon.

    Science.gov (United States)

    Finstad, Anders G; Einum, Sigurd; Ugedal, Ola; Forseth, Torbjørn

    2009-01-01

    1. Spatial heterogeneity of resources may influence competition among individuals and thus have a fundamental role in shaping population dynamics and carrying capacity. In the present study, we identify shelter opportunities as a limiting resource for juvenile Atlantic salmon (Salmo salar L.). Experimental and field studies are combined in order to demonstrate how the spatial distribution of shelters may influence population dynamics on both within and among population scales. 2. In closed experimental streams, fish performance scaled negatively with decreasing shelter availability and increasing densities. In contrast, the fish in open stream channels dispersed according to shelter availability and performance of fish remaining in the streams did not depend on initial density or shelters. 3. The field study confirmed that spatial variation in densities of 1-year-old juveniles was governed both by initial recruit density and shelter availability. Strength of density-dependent population regulation, measured as carrying capacity, increased with decreasing number of shelters. 4. Nine rivers were surveyed for spatial variation in shelter availability and increased shelter heterogeneity tended to decrease maximum observed population size (measured using catch statistics of adult salmon as a proxy). 5. Our studies highlight the importance of small-scale within-population spatial structure in population dynamics and demonstrate that not only the absolute amount of limiting resources but also their spatial arrangement can be an important factor influencing population carrying capacity.

  19. Mto2 multisite phosphorylation inactivates non-spindle microtubule nucleation complexes during mitosis

    Science.gov (United States)

    Borek, Weronika E.; Groocock, Lynda M.; Samejima, Itaru; Zou, Juan; de Lima Alves, Flavia; Rappsilber, Juri; Sawin, Kenneth E.

    2015-01-01

    Microtubule nucleation is highly regulated during the eukaryotic cell cycle, but the underlying molecular mechanisms are largely unknown. During mitosis in fission yeast Schizosaccharomyces pombe, cytoplasmic microtubule nucleation ceases simultaneously with intranuclear mitotic spindle assembly. Cytoplasmic nucleation depends on the Mto1/2 complex, which binds and activates the γ-tubulin complex and also recruits the γ-tubulin complex to both centrosomal (spindle pole body) and non-centrosomal sites. Here we show that the Mto1/2 complex disassembles during mitosis, coincident with hyperphosphorylation of Mto2 protein. By mapping and mutating multiple Mto2 phosphorylation sites, we generate mto2-phosphomutant strains with enhanced Mto1/2 complex stability, interaction with the γ-tubulin complex and microtubule nucleation activity. A mutant with 24 phosphorylation sites mutated to alanine, mto2[24A], retains interphase-like behaviour even in mitotic cells. This provides a molecular-level understanding of how phosphorylation ‘switches off' microtubule nucleation complexes during the cell cycle and, more broadly, illuminates mechanisms regulating non-centrosomal microtubule nucleation. PMID:26243668

  20. Microtubules self-repair in response to mechanical stress

    Science.gov (United States)

    Schaedel, Laura; John, Karin; Gaillard, Jérémie; Nachury, Maxence V.; Blanchoin, Laurent; Théry, Manuel

    2015-11-01

    Microtubules--which define the shape of axons, cilia and flagella, and provide tracks for intracellular transport--can be highly bent by intracellular forces, and microtubule structure and stiffness are thought to be affected by physical constraints. Yet how microtubules tolerate the vast forces exerted on them remains unknown. Here, by using a microfluidic device, we show that microtubule stiffness decreases incrementally with each cycle of bending and release. Similar to other cases of material fatigue, the concentration of mechanical stresses on pre-existing defects in the microtubule lattice is responsible for the generation of more extensive damage, which further decreases microtubule stiffness. Strikingly, damaged microtubules were able to incorporate new tubulin dimers into their lattice and recover their initial stiffness. Our findings demonstrate that microtubules are ductile materials with self-healing properties, that their dynamics does not exclusively occur at their ends, and that their lattice plasticity enables the microtubules' adaptation to mechanical stresses.

  1. Insulin-like growth factor-II receptors in cultured rat hepatocytes: regulation by cell density

    International Nuclear Information System (INIS)

    Scott, C.D.; Baxter, R.C.

    1987-01-01

    Insulin-like growth factor-II (IGF-II) receptors in primary cultures of adult rat hepatocytes were characterized and their regulation by cell density examined. In hepatocytes cultured at 5 X 10(5) cells per 3.8 cm2 plate [ 125 I]IGF-II bound to specific, high affinity receptors (Ka = 4.4 +/- 0.5 X 10(9) l/mol). Less than 1% cross-reactivity by IGF-I and no cross-reactivity by insulin were observed. IGF-II binding increased when cells were permeabilized with 0.01% digitonin, suggesting the presence of an intracellular receptor pool. Determined by Scatchard analysis and by polyacrylamide gel electrophoresis after affinity labeling, the higher binding was due solely to an increase in binding sites present on 220 kDa type II IGF receptors. In hepatocytes cultured at low densities, the number of cell surface receptors increased markedly, from 10-20,000 receptors per cell at a culture density of 6 X 10(5) cells/well to 70-80,000 receptors per cell at 0.38 X 10(5) cells/well. The increase was not due simply to the exposure of receptors from the intracellular pool, as a density-related increase in receptors was also seen in cells permeabilized with digitonin. There was no evidence that IGF binding proteins, either secreted by hepatocytes or present in fetal calf serum, had any effect on the measurement of receptor concentration or affinity. We conclude that rat hepatocytes in primary culture contain specific IGF-II receptors and that both cell surface and intracellular receptors are regulated by cell density

  2. GMC Collisions as Triggers of Star Formation. III. Density and Magnetically Regulated Star Formation

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Benjamin [National Astronomical Observatory of Japan, Mitaka, Tokyo 181-8588 (Japan); Tan, Jonathan C. [Department of Physics, University of Florida, Gainesville, FL 32611 (United States); Christie, Duncan [Department of Astronomy, University of Florida, Gainesville, FL 32611 (United States); Nakamura, Fumitaka [National Astronomical Observatory, Mitaka, Tokyo 181-8588 (Japan); Van Loo, Sven [School of Physics and Astronomy, University of Leeds, Leeds LS2 9JT (United Kingdom); Collins, David, E-mail: ben.wu@nao.ac.jp [Department of Physics, Florida State University, Tallahassee, FL 32306-4350 (United States)

    2017-06-01

    We study giant molecular cloud (GMC) collisions and their ability to trigger star cluster formation. We further develop our three-dimensional magnetized, turbulent, colliding GMC simulations by implementing star formation subgrid models. Two such models are explored: (1) “Density-Regulated,” i.e., fixed efficiency per free-fall time above a set density threshold and (2) “Magnetically Regulated,” i.e., fixed efficiency per free-fall time in regions that are magnetically supercritical. Variations of parameters associated with these models are also explored. In the non-colliding simulations, the overall level of star formation is sensitive to model parameter choices that relate to effective density thresholds. In the GMC collision simulations, the final star formation rates and efficiencies are relatively independent of these parameters. Between the non-colliding and colliding cases, we compare the morphologies of the resulting star clusters, properties of star-forming gas, time evolution of the star formation rate (SFR), spatial clustering of the stars, and resulting kinematics of the stars in comparison to the natal gas. We find that typical collisions, by creating larger amounts of dense gas, trigger earlier and enhanced star formation, resulting in 10 times higher SFRs and efficiencies. The star clusters formed from GMC collisions show greater spatial substructure and more disturbed kinematics.

  3. Cep192 controls the balance of centrosome and non-centrosomal microtubules during interphase.

    Directory of Open Access Journals (Sweden)

    Brian P O'Rourke

    Full Text Available Cep192 is a centrosomal protein that contributes to the formation and function of the mitotic spindle in mammalian cells. Cep192's mitotic activities stem largely from its role in the recruitment to the centrosome of numerous additional proteins such as gamma-tubulin and Pericentrin. Here, we examine Cep192's function in interphase cells. Our data indicate that, as in mitosis, Cep192 stimulates the nucleation of centrosomal microtubules thereby regulating the morphology of interphase microtubule arrays. Interestingly, however, cells lacking Cep192 remain capable of generating normal levels of MTs as the loss of centrosomal microtubules is augmented by MT nucleation from other sites, most notably the Golgi apparatus. The depletion of Cep192 results in a significant decrease in the level of centrosome-associated gamma-tubulin, likely explaining its impact on centrosome microtubule nucleation. However, in stark contrast to mitosis, Cep192 appears to maintain an antagonistic relationship with Pericentrin at interphase centrosomes. Interphase cells depleted of Cep192 display significantly higher levels of centrosome-associated Pericentrin while overexpression of Cep192 reduces the levels of centrosomal Pericentrin. Conversely, depletion of Pericentrin results in elevated levels of centrosomal Cep192 and enhances microtubule nucleation at centrosomes, at least during interphase. Finally, we show that depletion of Cep192 negatively impacts cell motility and alters normal cell polarization. Our current working hypothesis is that the microtubule nucleating capacity of the interphase centrosome is determined by an antagonistic balance of Cep192, which promotes nucleation, and Pericentrin, which inhibits nucleation. This in turn determines the relative abundance of centrosomal and non-centrosomal microtubules that tune cell movement and shape.

  4. Pruning for crop regulation in high density guava (Psidium guajava L.) plantation

    Energy Technology Data Exchange (ETDEWEB)

    Thakre, M.; Lal, S.; Uniyal, S.; Goswami, A.K. Prakash. P.

    2016-11-01

    High density management and crop regulation are two important aspects in guava (Psidium guajava L.) production. Therefore, to find out the economic way of managing high density planting and crop regulation, the present work was carried out on 6-year-old guava trees of cv. Pant Prabhat under double-hedge row system of planting during 2009-10 and 2010-11. Seven different forms of pruning [FBT: flower bud thinning by hand, FBTT: flower bud thinning by hand followed by removal of terminal one leaf pair, RLFO: removal of leaves and flower buds by hand, retaining one leaf pair at the top, RLF: removal of all leaves and flowers by hand, OLPS: one leaf pair shoot pruning, FSP: full shoot pruning, OLPF: one leaf pair pruning of fruited shoots only] were studied along with control (C).Minimum annual increase in tree volume (6.764 m3) was recorded with the treatment OLPF, which was 2.31 times less than the control (15.682 m3). Highest yield during winter season (55.30 kg/tree) and total yield (59.87 kg/tree) was obtained from treatment OLPF. One leaf pair pruning of fruited shoots only (OLPF) was also found profitable among other treatments by recording cost:benefit ratio of 1:2.96. This treatment also recorded the highest return distributed in rainy as well as in winter season. On the basis of findings it can be concluded that one leaf pair pruning of fruited shoots only is suitable for profitable high density management as well as crop regulation of guava in farmer friendly manner. (Author)

  5. Pruning for crop regulation in high density guava (Psidium guajava L. plantation

    Directory of Open Access Journals (Sweden)

    Madhubala Thakre

    2016-06-01

    Full Text Available High density management and crop regulation are two important aspects in guava (Psidium guajava L. production. Therefore, to find out the economic way of managing high density planting and crop regulation, the present work was carried out on 6-year-old guava trees of cv. Pant Prabhat under double-hedge row system of planting during 2009-10 and 2010-11. Seven different forms of pruning [FBT: flower bud thinning by hand, FBTT: flower bud thinning by hand followed by removal of terminal one leaf pair, RLFO: removal of leaves and flower buds by hand, retaining one leaf pair at the top, RLF: removal of all leaves and flowers by hand, OLPS: one leaf pair shoot pruning, FSP: full shoot pruning, OLPF: one leaf pair pruning of fruited shoots only] were studied along with control (C.Minimum annual increase in tree volume (6.764 m3 was recorded with the treatment OLPF, which was 2.31 times less than the control (15.682 m3. Highest yield during winter season (55.30 kg/tree and total yield (59.87 kg/tree was obtained from treatment OLPF. One leaf pair pruning of fruited shoots only (OLPF was also found profitable among other treatments by recording cost:benefit ratio of 1:2.96. This treatment also recorded the highest return distributed in rainy as well as in winter season. On the basis of findings it can be concluded that one leaf pair pruning of fruited shoots only is suitable for profitable high density management as well as crop regulation of guava in farmer friendly manner.

  6. Mechanisms regulating amphipod population density within macroalgal communities with low predator impact

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    Hartvig Christie

    2004-04-01

    Full Text Available In eight mesocosms (land based basins macroalgae communities with associated fauna were transplanted from the sea and established during two years. Then, different doses of nutrients (N and P were added to the basins throughout the following three years. During the period of nutrient addition, macroinvertebrate grazers showed seasonal fluctuations with densities usually between 500,000 and 1 million individuals per mesocosm during summer and to a level of about 100,000 during winter. The macroinvertebrate grazers mainly consisted of about 10 species of amphipods and isopods, among which the amphipod Gammarus locusta dominated strongly by biomass. Although the number of predators was very low, the grazer populations never reached a density where considerable grazing impact could be found on the macroalgae. No increase in grazer density was found in the basins with improved nutrient conditions. Thus food quality may be insufficient for further population growth, or density dependant regulation mechanisms may have prevented the grazers from flourishing and overgrazing the system. In aquarium experiments we showed that G. locusta could grow and reproduce on Fucus serratus, Ulva lactuca, periphyton and detritus, and that cannibalism by adult G. locusta on juveniles may have great impact on the population growth. The basins were run with a water flow through system. Nets were placed in front of the inflow and outflow tubes to measure immigration and emigration. Only few individuals (and no Gammarus sp. were recorded in the inflowing water, while high numbers of both amphipods and isopods were found in the outflowing water. Emigration reached peak values during night-time, and it was then two to three times as high as during day-time. Emigration of mobile grazers from the basins amounted to 1-2% of the standing stock daily. These mechanisms that regulate grazers do contribute to maintenance of the seaweed dominance and thus the stability of the seaweed

  7. Ferritin associates with marginal band microtubules

    International Nuclear Information System (INIS)

    Infante, Anthony A.; Infante, Dzintra; Chan, M.-C.; How, P.-C.; Kutschera, Waltraud; Linhartova, Irena; Muellner, Ernst W.; Wiche, Gerhard; Propst, Friedrich

    2007-01-01

    We characterized chicken erythrocyte and human platelet ferritin by biochemical studies and immunofluorescence. Erythrocyte ferritin was found to be a homopolymer of H-ferritin subunits, resistant to proteinase K digestion, heat stable, and contained iron. In mature chicken erythrocytes and human platelets, ferritin was localized at the marginal band, a ring-shaped peripheral microtubule bundle, and displayed properties of bona fide microtubule-associated proteins such as tau. Red blood cell ferritin association with the marginal band was confirmed by temperature-induced disassembly-reassembly of microtubules. During erythrocyte differentiation, ferritin co-localized with coalescing microtubules during marginal band formation. In addition, ferritin was found in the nuclei of mature erythrocytes, but was not detectable in those of bone marrow erythrocyte precursors. These results suggest that ferritin has a function in marginal band formation and possibly in protection of the marginal band from damaging effects of reactive oxygen species by sequestering iron in the mature erythrocyte. Moreover, our data suggest that ferritin and syncolin, a previously identified erythrocyte microtubule-associated protein, are identical. Nuclear ferritin might contribute to transcriptional silencing or, alternatively, constitute a ferritin reservoir

  8. Taxol crystals can masquerade as stabilized microtubules.

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    Margit Foss

    Full Text Available Taxol is a potent anti-mitotic drug used in chemotherapy, angioplastic stents, and cell biology research. By binding and stabilizing microtubules, Taxol inhibits their dynamics, crucial for cell division, motility, and survival. The drug has also been reported to induce formation of asters and bundles composed of stabilized microtubules. Surprisingly, at commonly used concentrations, Taxol forms crystals that rapidly bind fluorescent tubulin subunits, generating structures with an uncanny resemblance to microtubule asters and bundles. Kinetic and topological considerations suggest that tubulin subunits, rather than microtubules, bind the crystals. This sequestration of tubulin from the subunit pool would be expected to shift the equilibrium of free to polymerized tubulin to disfavor assembly. Our results imply that some previously reported Taxol-induced asters or bundles could include or be composed of tubulin-decorated Taxol crystals. Thus, reevaluation of certain morphological, chemical, and physical properties of Taxol-treated microtubules may be necessary. Moreover, our findings suggest a novel mechanism for chemotherapy-induced cytotoxicity in non-dividing cells, with far-reaching medical implications.

  9. Stabilization of microtubules by inorganic phosphate and its structural analogues, the fluoride complexes of aluminum and beryllium

    International Nuclear Information System (INIS)

    Carlier, M.F.; Didry, D.; Melki, R.; Chabre, M.; Pantaloni, D.

    1988-01-01

    In order to elucidate how the elementary reactions of GTP cleavage and subsequent inorganic phosphate (P/sub i/) release, which accompany microtubule assembly, regulate microtubule dynamics, the effect of P/sub i/ and of its structural analogues AlF 4 - and BeF 3 - on the stability of GDP-microtubules has been investigated. Inorganic phosphate binds to microtubules with a low affinity (K/sub D/ = 25 mM) and slows down the rate of GDP-subunit dissociation by about 2 orders of magnitude. AlF 4 - and BeF 3 - exhibit phosphate-like effects with 1000-fold higher affinity. Evidence has been obtained for direct binding of BeF 3 - to microtubules with a stoichiometry of 1 mol of BeF 3 - per mole of GDP-subunit and an equilibrium dissociation constant of 12-15 μM. AlF 4 - and P/sub i/ compete for this site. Phosphate analogues abolish oscillatory polymerization kinetics and slow down microtubule turnover at steady state. In view of these results, the authors propose that P/sub i/ and its structural analogues bind to the site of the γ-phosphate of GTP in the E site and reconstitute a GDP-P/sub i/-microtubule, from which tubulin subunits dissociate very slowly. They therefore understand that, following GTP cleavage on microtubules, P/sub i/ release in the medium is accompanied by a structural change resulting in a large destabilization of the polymer. A cap of slowly dissociating GDP-P/sub i/-subunits prevents depolymerization of the microtubule GDP-core at steady state. The similarity with the actin system is studied

  10. Energy Density, Energy Intake, and Body Weight Regulation in Adults12345

    Science.gov (United States)

    Karl, J. Philip; Roberts, Susan B.

    2014-01-01

    The role of dietary energy density (ED) in the regulation of energy intake (EI) is controversial. Methodologically, there is also debate about whether beverages should be included in dietary ED calculations. To address these issues, studies examining the effects of ED on EI or body weight in nonelderly adults were reviewed. Different approaches to calculating dietary ED do not appear to alter the direction of reported relations between ED and body weight. Evidence that lowering dietary ED reduces EI in short-term studies is convincing, but there are currently insufficient data to determine long-term effectiveness for weight loss. The review also identified key barriers to progress in understanding the role of ED in energy regulation, in particular the absence of a standard definition of ED, and the lack of data from multiple long-term clinical trials examining the effectiveness of low-ED diet recommendations for preventing both primary weight gain and weight regain in nonobese individuals. Long-term clinical trials designed to examine the impact of dietary ED on energy regulation, and including multiple ED calculation methods within the same study, are still needed to determine the importance of ED in the regulation of EI and body weight. PMID:25398750

  11. The imidazopyridine derivative JK184 reveals dual roles for microtubules in Hedgehog signaling.

    Science.gov (United States)

    Cupido, Tommaso; Rack, Paul G; Firestone, Ari J; Hyman, Joel M; Han, Kyuho; Sinha, Surajit; Ocasio, Cory A; Chen, James K

    2009-01-01

    Eradicating hedgehogs: The title molecule has been previously identified as a potent inhibitor of the Hedgehog signaling pathway, which gives embryonic cells information needed to develop properly. This molecule is shown to modulate Hedgehog target gene expression by depolymerizing microtubules, thus revealing dual roles of the cytoskeleton in pathway regulation (see figure).

  12. Xyloglucan Deficiency Disrupts Microtubule Stability and Cellulose Biosynthesis in Arabidopsis, Altering Cell Growth and Morphogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Xiao, Chaowen; Zhang, Tian; Zheng, Yunzhen; Cosgrove, Daniel J.; Anderson, Charles T.

    2015-11-02

    Xyloglucan constitutes most of the hemicellulose in eudicot primary cell walls and functions in cell wall structure and mechanics. Although Arabidopsis (Arabidopsis thaliana) xxt1 xxt2 mutants lacking detectable xyloglucan are viable, they display growth defects that are suggestive of alterations in wall integrity. To probe the mechanisms underlying these defects, we analyzed cellulose arrangement, microtubule patterning and dynamics, microtubule- and wall-integrity-related gene expression, and cellulose biosynthesis in xxt1 xxt2 plants. We found that cellulose is highly aligned in xxt1 xxt2 cell walls, that its three-dimensional distribution is altered, and that microtubule patterning and stability are aberrant in etiolated xxt1 xxt2 hypocotyls. We also found that the expression levels of microtubule-associated genes, such as MAP70-5 and CLASP, and receptor genes, such as HERK1 and WAK1, were changed in xxt1 xxt2 plants and that cellulose synthase motility is reduced in xxt1 xxt2 cells, corresponding with a reduction in cellulose content. Our results indicate that loss of xyloglucan affects both the stability of the microtubule cytoskeleton and the production and patterning of cellulose in primary cell walls. These findings establish, to our knowledge, new links between wall integrity, cytoskeletal dynamics, and wall synthesis in the regulation of plant morphogenesis.

  13. TIPsy tour guides: How microtubule plus-end tracking proteins (+TIPs facilitate axon guidance

    Directory of Open Access Journals (Sweden)

    Elizabeth A Bearce

    2015-06-01

    Full Text Available The growth cone is a dynamic cytoskeletal vehicle, which drives the end of a developing axon. It serves to interpret and navigate through the complex landscape and guidance cues of the early nervous system. The growth cone’s distinctive cytoskeletal organization offers a fascinating platform to study how extracellular cues can be translated into mechanical outgrowth and turning behaviors. While many studies of cell motility highlight the importance of actin networks in signaling, adhesion, and propulsion, both seminal and emerging works in the field have highlighted a unique and necessary role for microtubules in growth cone navigation. Here, we focus on the role of singular pioneer microtubules, which extend into the growth cone periphery and are regulated by a diverse family of microtubule plus-end tracking proteins (+TIPs. These +TIPs accumulate at the dynamic ends of microtubules, where they are well-positioned to encounter and respond to key signaling events downstream of guidance receptors, catalyzing immediate changes in microtubule stability and actin cross-talk, that facilitate both axonal outgrowth and turning events.

  14. Potential mechanisms of resistance to microtubule inhibitors.

    Science.gov (United States)

    Kavallaris, Maria; Annereau, Jean-Philippe; Barret, Jean-Marc

    2008-06-01

    Antimitotic drugs targeting the microtubules, such as the taxanes and vinca alkaloids, are widely used in the treatment of neoplastic diseases. Development of drug resistance over time, however, limits the efficacy of these agents and poses a clinical challenge to long-term improvement of patient outcomes. Understanding the mechanism(s) of drug resistance becomes paramount to allowing for alternative, if not improved, therapeutic options that might circumvent this challenge. Vinflunine, a novel microtubule inhibitor, has shown superior preclinical antitumor activity, and displays a different pattern of resistance, compared with other agents in the vinca alkaloid class.

  15. Microtubules: A network for solitary waves

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    Zdravković Slobodan

    2017-01-01

    Full Text Available In the present paper we deal with nonlinear dynamics of microtubules. The structure and role of microtubules in cells are explained as well as one of models explaining their dynamics. Solutions of the crucial nonlinear differential equation depend on used mathematical methods. Two commonly used procedures, continuum and semi-discrete approximations, are explained. These solutions are solitary waves usually called as kink solitons, breathers and bell-type solitons. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. III45010

  16. Maize yield and quality in response to plant density and application of a novel plant growth regulator

    NARCIS (Netherlands)

    Zhang, Q.; Zhang, L.; Evers, J.B.; Werf, van der W.; Zhang, W.; Duan, L.

    2014-01-01

    Farmers in China have gradually increased plant density in maize to achieve higher yields, but this has increased risk of lodging due to taller and weaker stems at higher plant densities. Plant growth regulators can be used to reduce lodging risk. In this study, for the first time, the performance

  17. Crop growth, light utilization and yield of relay intercropped cotton as affected by plant density and a plant growth regulator

    NARCIS (Netherlands)

    Mao, L.; Zhang, L.; Zhao, X.; Liu, S.; Werf, van der W.; Zhang, S.; Spiertz, J.H.J.; Li, Z.

    2014-01-01

    Modern cotton cultivation requires high plant densities and compact plants. Here we study planting density and growth regulator effects on plant structure and production of cotton when the cotton is grown in a relay intercrop with wheat, a cultivation system that is widespread in China. Field

  18. GDP-tubulin incorporation into growing microtubules modulates polymer stability.

    Science.gov (United States)

    Valiron, Odile; Arnal, Isabelle; Caudron, Nicolas; Job, Didier

    2010-06-04

    Microtubule growth proceeds through the endwise addition of nucleotide-bound tubulin dimers. The microtubule wall is composed of GDP-tubulin subunits, which are thought to come exclusively from the incorporation of GTP-tubulin complexes at microtubule ends followed by GTP hydrolysis within the polymer. The possibility of a direct GDP-tubulin incorporation into growing polymers is regarded as hardly compatible with recent structural data. Here, we have examined GTP-tubulin and GDP-tubulin incorporation into polymerizing microtubules using a minimal assembly system comprised of nucleotide-bound tubulin dimers, in the absence of free nucleotide. We find that GDP-tubulin complexes can efficiently co-polymerize with GTP-tubulin complexes during microtubule assembly. GDP-tubulin incorporation into microtubules occurs with similar efficiency during bulk microtubule assembly as during microtubule growth from seeds or centrosomes. Microtubules formed from GTP-tubulin/GDP-tubulin mixtures display altered microtubule dynamics, in particular a decreased shrinkage rate, apparently due to intrinsic modifications of the polymer disassembly properties. Thus, although microtubules polymerized from GTP-tubulin/GDP-tubulin mixtures or from homogeneous GTP-tubulin solutions are both composed of GDP-tubulin subunits, they have different dynamic properties, and this may reveal a novel form of microtubule "structural plasticity."

  19. Regulating Hypothalamus Gene Expression in Food Intake: Dietary Composition or Calorie Density?

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    Mi Jang

    2017-01-01

    Full Text Available BackgroundThe proportion of saturated fatty acids/unsaturated fatty acids in the diet seems to act as a physiological regulation on obesity, cardiovascular diseases, and diabetes. Differently composed fatty acid diets may induce satiety of the hypothalamus in different ways. However, the direct effect of the different fatty acid diets on satiety in the hypothalamus is not clear.MethodsThree experiments in mice were conducted to determine whether: different compositions of fatty acids affects gene mRNA expression of the hypothalamus over time; different types of fatty acids administered into the stomach directly affect gene mRNA expression of the hypothalamus; and fat composition changes in the diet affects gene mRNA expression of the hypothalamus.ResultsThe type of fat in cases of purified fatty acid administration directly into the stomach may cause changes of gene expressions in the hypothalamus. Gene expression by dietary fat may be regulated by calorie amount ingested rather than weight amount or type of fat.ConclusionTherefore, the calorie density factor of the diet in regulating hypothalamic gene in food intake may be detrimental, although the possibility of type of fat cannot be ruled out.

  20. Cationic membranes complexed with oppositely charged microtubules: hierarchical self-assembly leading to bio-nanotubes

    International Nuclear Information System (INIS)

    Raviv, Uri; Needleman, Daniel J; Safinya, Cyrus R

    2006-01-01

    The self-assembly of microtubules and charged membranes has been studied, using x-ray diffraction and electron microscopy. Polyelectrolyte lipid complexes usually form structures templated by the lipid phase, when the polyelectrolyte curvature is much larger than the membrane spontaneous curvature. When the polyelectrolyte curvature approaches the membrane spontaneous curvature, as in microtubules, two types of new structures emerge. Depending on the conditions, vesicles either adsorb onto the microtubule, forming a 'beads on a rod' structure, or coat the microtubule, which now forms the template. Tubulin oligomers then coat the external lipid layer, forming a lipid protein nanotube. The tubulin oligomer coverage at the external layer is determined by the membrane charge density. The energy barrier between the beads on a rod and the lipid-protein nanotube states depends on the membrane bending rigidity and membrane charge density. By controlling the lipid/tubulin stoichiometry we can switch between lipid-protein nanotubes with open ends to lipid-protein nanotubes with closed end with lipid cups. This forms the basis for controlled drug encapsulation and release

  1. Colchicine Depolymerizes Microtubules, Increases Junctophilin-2, and Improves Right Ventricular Function in Experimental Pulmonary Arterial Hypertension.

    Science.gov (United States)

    Prins, Kurt W; Tian, Lian; Wu, Danchen; Thenappan, Thenappan; Metzger, Joseph M; Archer, Stephen L

    2017-05-31

    Pulmonary arterial hypertension (PAH) is a lethal disease characterized by obstructive pulmonary vascular remodeling and right ventricular (RV) dysfunction. Although RV function predicts outcomes in PAH, mechanisms of RV dysfunction are poorly understood, and RV-targeted therapies are lacking. We hypothesized that in PAH, abnormal microtubular structure in RV cardiomyocytes impairs RV function by reducing junctophilin-2 (JPH2) expression, resulting in t-tubule derangements. Conversely, we assessed whether colchicine, a microtubule-depolymerizing agent, could increase JPH2 expression and enhance RV function in monocrotaline-induced PAH. Immunoblots, confocal microscopy, echocardiography, cardiac catheterization, and treadmill testing were used to examine colchicine's (0.5 mg/kg 3 times/week) effects on pulmonary hemodynamics, RV function, and functional capacity. Rats were treated with saline (n=28) or colchicine (n=24) for 3 weeks, beginning 1 week after monocrotaline (60 mg/kg, subcutaneous). In the monocrotaline RV, but not the left ventricle, microtubule density is increased, and JPH2 expression is reduced, with loss of t-tubule localization and t-tubule disarray. Colchicine reduces microtubule density, increases JPH2 expression, and improves t-tubule morphology in RV cardiomyocytes. Colchicine therapy diminishes RV hypertrophy, improves RV function, and enhances RV-pulmonary artery coupling. Colchicine reduces small pulmonary arteriolar thickness and improves pulmonary hemodynamics. Finally, colchicine increases exercise capacity. Monocrotaline-induced PAH causes RV-specific derangement of microtubules marked by reduction in JPH2 and t-tubule disarray. Colchicine reduces microtubule density, increases JPH2 expression, and improves both t-tubule architecture and RV function. Colchicine also reduces adverse pulmonary vascular remodeling. These results provide biological plausibility for a clinical trial to repurpose colchicine as a RV-directed therapy for PAH

  2. Fatherhood contributes to increased hippocampal spine density and anxiety regulation in California mice.

    Science.gov (United States)

    Glasper, Erica R; Hyer, Molly M; Katakam, Jhansi; Harper, Robyn; Ameri, Cyrus; Wolz, Thomas

    2016-01-01

    Parenting alters the hippocampus, an area of the brain that undergoes significant experience-induced plasticity and contributes to emotional regulation. While the relationship between maternal care and hippocampal neuroplasticity has been characterized, the extent to which fatherhood alters the structure and function of the hippocampus is far less understood. Here, we investigated to what extent fatherhood altered anxiety regulation and dendritic morphology of the hippocampus using the highly paternal California mouse (Peromyscus californicus). Fathers spent significantly more time on the open arms of the elevated plus maze, compared to non-fathers. Total distance traveled in the EPM was not changed by paternal experience, which suggests that the increased time spent on the open arms of the maze indicates decreased anxiety-like behavior. Fatherhood also increased dendritic spine density of granule cells in the dentate gyrus and basal dendrites of pyramidal cells in area CA1 of the hippocampus. These findings parallel those observed in maternal rodents, suggesting that the hippocampus of fathers and mothers respond similarly to offspring.

  3. Involvement of microtubules in lipoprotein degradation and utilization for steroidogenesis in cultured rat luteal cells

    International Nuclear Information System (INIS)

    Rajan, V.P.; Menon, K.M.

    1985-01-01

    Cells isolated from superovulated rat ovaries metabolize low density lipoprotein (LDL) and high density lipoprotein (HDL) of human or rat origin and use the lipoprotein-derived cholesterol as a precursor for progesterone production. Under in vitro conditions, both lipoproteins are internalized and degraded in the lysosomes, although degradation of HDL is of lower magnitude than that of LDL. In this report we have examined the role of cellular microtubules in the internalization and degradation of human LDL and HDL in cultured rat luteal cells. The microtubule depolymerizing agents colchicine, podophyllotoxin, vinblastine, and nocodazole as well as taxol, deuterium oxide, and dimethyl sulfoxide, which are known to rapidly polymerize cellular tubulin into microtubules, were used to block the function of microtubules. When these antimicrotubule agents were included in the incubations, degradation of the apolipoproteins of [ 125 I]iodo-LDL and [ 125 I]iodo-HDL by the luteal cells was inhibited by 50-85% compared to untreated control values. Maximum inhibitory effects were observed when the cells were preincubated with the inhibitor for at least 4 h at 37 C before treatment with the labeled lipoprotein. Lipoprotein-stimulated progesterone production by luteal cells was also inhibited by 50% or more in the presence of antimicrotubule agents. However, basal and hCG-stimulated progesterone production were unaffected by these inhibitors. The binding of [ 125 I]iodo-LDL and [ 125 I]iodo-HDL to luteal cell plasma membrane receptors was not affected by the microtubule inhibitors. Although binding was unaffected and degradation was impaired in the presence of the inhibitors, there was no detectable accumulation of undegraded lipoprotein within the cells during the 24 h of study

  4. Measurement of in vitro microtubule polymerization by turbidity and fluorescence.

    Science.gov (United States)

    Mirigian, Matthew; Mukherjee, Kamalika; Bane, Susan L; Sackett, Dan L

    2013-01-01

    Tubulin polymerization may be conveniently monitored by the increase in turbidity (optical density, or OD) or by the increase in fluorescence intensity of diamidino-phenylindole. The resulting data can be a quantitative measure of microtubule (MT) assembly, but some care is needed in interpretation, especially of OD data. Buffer formulations used for the assembly reaction significantly influence the polymerization, both by altering the critical concentration for polymerization and by altering the exact polymer produced-for example, by increasing the production of sheet polymers in addition to MT. Both the turbidity and the fluorescence methods are useful for demonstrating the effect of MT-stabilizing or -destabilizing additives. 2013 Published by Elsevier Inc.

  5. The HO-1/CO system regulates mitochondrial-capillary density relationships in human skeletal muscle.

    Science.gov (United States)

    Pecorella, Shelly R H; Potter, Jennifer V F; Cherry, Anne D; Peacher, Dionne F; Welty-Wolf, Karen E; Moon, Richard E; Piantadosi, Claude A; Suliman, Hagir B

    2015-10-15

    The heme oxygenase-1 (HO-1)/carbon monoxide (CO) system induces mitochondrial biogenesis, but its biological impact in human skeletal muscle is uncertain. The enzyme system generates CO, which stimulates mitochondrial proliferation in normal muscle. Here we examined whether CO breathing can be used to produce a coordinated metabolic and vascular response in human skeletal muscle. In 19 healthy subjects, we performed vastus lateralis muscle biopsies and tested one-legged maximal O2 uptake (V̇o2max) before and after breathing air or CO (200 ppm) for 1 h daily for 5 days. In response to CO, there was robust HO-1 induction along with increased mRNA levels for nuclear-encoded mitochondrial transcription factor A (Tfam), cytochrome c, cytochrome oxidase subunit IV (COX IV), and mitochondrial-encoded COX I and NADH dehydrogenase subunit 1 (NDI). CO breathing did not increase V̇o2max (1.96 ± 0.51 pre-CO, 1.87 ± 0.50 post-CO l/min; P = not significant) but did increase muscle citrate synthase, mitochondrial density (139.0 ± 34.9 pre-CO, 219.0 ± 36.2 post-CO; no. of mitochondrial profiles/field), myoglobin content and glucose transporter (GLUT4) protein level and led to GLUT4 localization to the myocyte membrane, all consistent with expansion of the tissue O2 transport system. These responses were attended by increased cluster of differentiation 31 (CD31)-positive muscle capillaries (1.78 ± 0.16 pre-CO, 2.37 ± 0.59 post-CO; capillaries/muscle fiber), implying the enrichment of microvascular O2 reserve. The findings support that induction of the HO-1/CO system by CO not only improves muscle mitochondrial density, but regulates myoglobin content, GLUT4 localization, and capillarity in accordance with current concepts of skeletal muscle plasticity. Copyright © 2015 the American Physiological Society.

  6. Tracking of plus-ends reveals microtubule functional diversity in different cell types

    Science.gov (United States)

    Shaebani, M. Reza; Pasula, Aravind; Ott, Albrecht; Santen, Ludger

    2016-07-01

    Many cellular processes are tightly connected to the dynamics of microtubules (MTs). While in neuronal axons MTs mainly regulate intracellular trafficking, they participate in cytoskeleton reorganization in many other eukaryotic cells, enabling the cell to efficiently adapt to changes in the environment. We show that the functional differences of MTs in different cell types and regions is reflected in the dynamic properties of MT tips. Using plus-end tracking proteins EB1 to monitor growing MT plus-ends, we show that MT dynamics and life cycle in axons of human neurons significantly differ from that of fibroblast cells. The density of plus-ends, as well as the rescue and catastrophe frequencies increase while the growth rate decreases toward the fibroblast cell margin. This results in a rather stable filamentous network structure and maintains the connection between nucleus and membrane. In contrast, plus-ends are uniformly distributed along the axons and exhibit diverse polymerization run times and spatially homogeneous rescue and catastrophe frequencies, leading to MT segments of various lengths. The probability distributions of the excursion length of polymerization and the MT length both follow nearly exponential tails, in agreement with the analytical predictions of a two-state model of MT dynamics.

  7. Dinitroaniline herbicide resistance and the microtubule cytoskeleton.

    Science.gov (United States)

    Anthony; Hussey

    1999-03-01

    Dinitroaniline herbicides have been used for pre-emergence weed control for the past 25 years in cotton, soybean, wheat and oilseed crops. Considering their long persistence and extensive use, resistance to dinitroanilines is fairly rare. However, the most widespread dinitroaniline-resistant weeds, the highly resistant (R) and the intermediate (I) biotypes of the invasive goosegrass Eleusine indica, are now infesting more than 1000 cotton fields in the southern states of the USA. The molecular basis of this resistance has been identified, and found to be a point mutation in a major microtubule cytoskeletal protein, alpha-tubulin. These studies have served both to explain the establishment of resistance and to reveal fundamental properties of tubulin gene expression and microtubule structure.

  8. HSPB1 facilitates the formation of non-centrosomal microtubules.

    Directory of Open Access Journals (Sweden)

    Leonardo Almeida-Souza

    Full Text Available The remodeling capacity of microtubules (MT is essential for their proper function. In mammals, MTs are predominantly formed at the centrosome, but can also originate from non-centrosomal sites, a process that is still poorly understood. We here show that the small heat shock protein HSPB1 plays a role in the control of non-centrosomal MT formation. The HSPB1 expression level regulates the balance between centrosomal and non-centrosomal MTs. The HSPB1 protein can be detected specifically at sites of de novo forming non-centrosomal MTs, while it is absent from the centrosomes. In addition, we show that HSPB1 binds preferentially to the lattice of newly formed MTs in vitro, suggesting that its function occurs by stabilizing MT seeds. Our findings open new avenues for the understanding of the role of HSPB1 in the development, maintenance and protection of cells with specialized non-centrosomal MT arrays.

  9. Near-atomic model of microtubule-tau interactions.

    Science.gov (United States)

    Kellogg, Elizabeth H; Hejab, Nisreen M A; Poepsel, Simon; Downing, Kenneth H; DiMaio, Frank; Nogales, Eva

    2018-06-15

    Tau is a developmentally regulated axonal protein that stabilizes and bundles microtubules (MTs). Its hyperphosphorylation is thought to cause detachment from MTs and subsequent aggregation into fibrils implicated in Alzheimer's disease. It is unclear which tau residues are crucial for tau-MT interactions, where tau binds on MTs, and how it stabilizes them. We used cryo-electron microscopy to visualize different tau constructs on MTs and computational approaches to generate atomic models of tau-tubulin interactions. The conserved tubulin-binding repeats within tau adopt similar extended structures along the crest of the protofilament, stabilizing the interface between tubulin dimers. Our structures explain the effect of phosphorylation on MT affinity and lead to a model of tau repeats binding in tandem along protofilaments, tethering together tubulin dimers and stabilizing polymerization interfaces. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  10. In vivo FRET imaging revealed a regulatory role of RanGTP in kinetochore-microtubule attachments via Aurora B kinase.

    Directory of Open Access Journals (Sweden)

    Yoke-Peng Lee

    Full Text Available Under the fluctuating circumstances provided by the innate dynamics of microtubules and opposing tensions resulted from microtubule-associated motors, it is vital to ensure stable kinetochore-microtubule attachments for accurate segregation. However, a comprehensive understanding of how this regulation is mechanistically achieved remains elusive. Using our newly designed live cell FRET time-lapse imaging, we found that post-metaphase RanGTP is crucial in the maintenance of stable kinetochore-microtubule attachments by regulating Aurora B kinase via the NES-bearing Mst1. More importantly, our study demonstrates that by ensuring stable alignment of metaphase chromosomes prior to segregation, RanGTP is indispensible in governing the genomic integrity and the fidelity of cell cycle progression. Our findings suggest an additional role of RanGTP beyond its known function in mitotic spindle assembly during the prometaphase-metaphase transition.

  11. The nucleoporin MEL-28 promotes RanGTP-dependent γ-tubulin recruitment and microtubule nucleation in mitotic spindle formation.

    Science.gov (United States)

    Yokoyama, Hideki; Koch, Birgit; Walczak, Rudolf; Ciray-Duygu, Fulya; González-Sánchez, Juan Carlos; Devos, Damien P; Mattaj, Iain W; Gruss, Oliver J

    2014-01-01

    The GTP-bound form of the Ran GTPase (RanGTP), produced around chromosomes, drives nuclear envelope and nuclear pore complex (NPC) re-assembly after mitosis. The nucleoporin MEL-28/ELYS binds chromatin in a RanGTP-regulated manner and acts to seed NPC assembly. Here we show that, upon mitotic NPC disassembly, MEL-28 dissociates from chromatin and re-localizes to spindle microtubules and kinetochores. MEL-28 directly binds microtubules in a RanGTP-regulated way via its C-terminal chromatin-binding domain. Using Xenopus egg extracts, we demonstrate that MEL-28 is essential for RanGTP-dependent microtubule nucleation and spindle assembly, independent of its function in NPC assembly. Specifically, MEL-28 interacts with the γ-tubulin ring complex and recruits it to microtubule nucleation sites. Our data identify MEL-28 as a RanGTP target that functions throughout the cell cycle. Its cell cycle-dependent binding to chromatin or microtubules discriminates MEL-28 functions in interphase and mitosis, and ensures that spindle assembly occurs only after NPC breakdown.

  12. The Role of Molecular Microtubule Motors and the Microtubule Cytoskeleton in Stress Granule Dynamics

    Directory of Open Access Journals (Sweden)

    Kristen M. Bartoli

    2011-01-01

    Full Text Available Stress granules (SGs are cytoplasmic foci that appear in cells exposed to stress-induced translational inhibition. SGs function as a triage center, where mRNAs are sorted for storage, degradation, and translation reinitiation. The underlying mechanisms of SGs dynamics are still being characterized, although many key players have been identified. The main components of SGs are stalled 48S preinitiation complexes. To date, many other proteins have also been found to localize in SGs and are hypothesized to function in SG dynamics. Most recently, the microtubule cytoskeleton and associated motor proteins have been demonstrated to function in SG dynamics. In this paper, we will discuss current literature examining the function of microtubules and the molecular microtubule motors in SG assembly, coalescence, movement, composition, organization, and disassembly.

  13. Microtubules are organized independently of the centrosome in Drosophila neurons

    Directory of Open Access Journals (Sweden)

    Nguyen Michelle M

    2011-12-01

    Full Text Available Abstract Background The best-studied arrangement of microtubules is that organized by the centrosome, a cloud of microtubule nucleating and anchoring proteins is clustered around centrioles. However, noncentrosomal microtubule arrays are common in many differentiated cells, including neurons. Although microtubules are not anchored at neuronal centrosomes, it remains unclear whether the centrosome plays a role in organizing neuronal microtubules. We use Drosophila as a model system to determine whether centrosomal microtubule nucleation is important in mature neurons. Results In developing and mature neurons, centrioles were not surrounded by the core nucleation protein γ-tubulin. This suggests that the centrioles do not organize functional centrosomes in Drosophila neurons in vivo. Consistent with this idea, centriole position was not correlated with a specific region of the cell body in neurons, and growing microtubules did not cluster around the centriole, even after axon severing when the number of growing plus ends is dramatically increased. To determine whether the centrosome was required for microtubule organization in mature neurons, we used two approaches. First, we used DSas-4 centriole duplication mutants. In these mutants, centrioles were present in many larval sensory neurons, but they were not fully functional. Despite reduced centriole function, microtubule orientation was normal in axons and dendrites. Second, we used laser ablation to eliminate the centriole, and again found that microtubule polarity in axons and dendrites was normal, even 3 days after treatment. Conclusion We conclude that the centrosome is not a major site of microtubule nucleation in Drosophila neurons, and is not required for maintenance of neuronal microtubule organization in these cells.

  14. Spindle pole body-anchored Kar3 drives the nucleus along microtubules from another nucleus in preparation for nuclear fusion during yeast karyogamy.

    Science.gov (United States)

    Gibeaux, Romain; Politi, Antonio Z; Nédélec, François; Antony, Claude; Knop, Michael

    2013-02-01

    Nuclear migration during yeast karyogamy, termed nuclear congression, is required to initiate nuclear fusion. Congression involves a specific regulation of the microtubule minus end-directed kinesin-14 motor Kar3 and a rearrangement of the cytoplasmic microtubule attachment sites at the spindle pole bodies (SPBs). However, how these elements interact to produce the forces necessary for nuclear migration is less clear. We used electron tomography, molecular genetics, quantitative imaging, and first principles modeling to investigate how cytoplasmic microtubules are organized during nuclear congression. We found that Kar3, with the help of its light chain, Cik1, is anchored during mating to the SPB component Spc72 that also serves as a nucleator and anchor for microtubules via their minus ends. Moreover, we show that no direct microtubule-microtubule interactions are required for nuclear migration. Instead, SPB-anchored Kar3 exerts the necessary pulling forces laterally on microtubules emanating from the SPB of the mating partner nucleus. Therefore, a twofold symmetrical application of the core principle that drives nuclear migration in higher cells is used in yeast to drive nuclei toward each other before nuclear fusion.

  15. Effect of radiation on microtubule structure in cancer cells

    International Nuclear Information System (INIS)

    Tripath, Shambhoo Sharan; Panda, Dulal; Jayakumar, S.; Maikho, Thoh; Sandur, Santosh Kumar

    2017-01-01

    Microtubules (MT) are dynamic structural cellular components. In proliferating cells, they are essential components in cell division through the formation of the mitotic spindle. Radiotherapy is an integral part of cancer treatment for most of the solid cancers. Scanty data exists in the literature related to how ionizing radiation affects microtubule reorganization in tumor cells. In the present study, breast cancer cell line (MCF-7 cells) was exposed to different doses of radiation (2-10Gy). Cells were cultured for 24 h, fixed and stained with antitubulin antibody and subjected to immunofluorescence microscopy. In another experiment, cells were subjected to cold treatment for 5 min or 30 min for studying the disassembly of microtubules after 24 h of irradiation. Further, these cells were incubated at 37°C for 20 min for studying the reassembly of microtubules. Acetylation of microtubule was also examined after exposure of cells to radiation. Experiments were also performed by combining radiation with low concentration of CXI-Benzo 84 (MT destabilizing agent 1 and 2.5 uM). Exposure of MCF-7 cells to radiation lead to destabilization of microtubules. Interestingly, destabilization of microtubule was faster upon cold treatment in irradiated group as compared to control group. These cells failed to re-stabilize at 37°C. Radiation also reduced the acetylation level of microtubule. Combination treatment of CXI-Benzo 84 with radiation exhibited additive effect in terms of depolymerization of MT. Our results suggest that ionizing radiation indeed modulates microtubule dynamics. (author)

  16. Producing Conditional Mutants for Studying Plant Microtubule Function

    Energy Technology Data Exchange (ETDEWEB)

    Richard Cyr

    2009-09-29

    The cytoskeleton, and in particular its microtubule component, participates in several processes that directly affect growth and development in higher plants. Normal cytoskeletal function requires the precise and orderly arrangement of microtubules into several cell cycle and developmentally specific arrays. One of these, the cortical array, is notable for its role in directing the deposition of cellulose (the most prominent polymer in the biosphere). An understanding of how these arrays form, and the molecular interactions that contribute to their function, is incomplete. To gain a better understanding of how microtubules work, we have been working to characterize mutants in critical cytoskeletal genes. This characterization is being carried out at the subcellular level using vital microtubule gene constructs. In the last year of funding colleagues have discovered that gamma-tubulin complexes form along the lengths of cortical microtubules where they act to spawn new microtubules at a characteristic 40 deg angle. This finding complements nicely the finding from our lab (which was funded by the DOE) showing that microtubule encounters are angle dependent; high angles encounters results in catastrophic collisions while low angle encounters result in favorable zippering. The finding of a 40 deg spawn of new microtubules from extant microtubule, together with aforementioned rules of encounters, insures favorable co-alignment in the array. I was invited to write a New and Views essay on this topic and a PDF is attached (News and Views policy does not permit funding acknowledgments and so I was not allowed to acknowledge support from the DOE).

  17. Effects of ultraviolet radiation on microtubule organisation and morphogenesis in plants

    International Nuclear Information System (INIS)

    Staxen, I.

    1994-09-01

    The involvement of the cytoskeleton in the development of somatic embryos was studied in Larix x eurolepis. Protoplasts were isolated from both somatic embryo-regenerating and non-generating cultures and fractionated on a discontinuous Percoll density gradient, whereby a highly embryogenic protoplast fraction could be enriched. Protoplasts of two cell lines of Larix eurolepis, one with regenerating potential and one lacking this potential, were compared. In contrast to the non-regenerating line were a protoplast-like organisation of the cortical microtubules was maintained, re-organisation of this microtubular network occurred in the regenerable line after only three days of culture, indicating that organised growth was occurring. However, this early organisation of cortical microtubules may not always be a valid marker for regenerable and non-regenerable material. In order to investigate the effect of ultraviolet-B (UV-B, 280-320 nm) radiation on the microtubule cytoskeleton, protoplasts were isolated from leaves of Petunia hybrida and subjected to four different doses of UV-B radiation. The organisation of the microtubules and the progression of the cells through the cell cycle was observed at 0, 24, 48 and 72 h after irradiation. UV-B induced breaks in the cortical microtubules resulting in shorter fragments with increasing amounts of radiation. Also, the division of the protoplasts was delayed, which was related to the absence of an microtubule network. Whole Petunia plants were grown in growth chambers in the presence and absence of UV-B. The plants responded to UV-B with increased rates of CO 2 assimilation, a 60% increase in UV-screening compounds and the changes in the morphology of the leaves that were reflected in a 70-100% increase in leaf area and 20% decrease in leaf thickness. The microtubules of the epidermal cells was not affected by UV-B, nor was the number of epidermal cells (per unit area). The increase in leaf area in the UV-treated plants

  18. Identification of a lysosome membrane protein which could mediate ATP-dependent stable association of lysosomes to microtubules

    International Nuclear Information System (INIS)

    Mithieux, G.; Rousset, B.

    1989-01-01

    We have previously reported that purified thyroid lysosomes bind to reconstituted microtubules to form stable complexes, a process which is inhibited by ATP. Among detergent-solubilized lysosomal membrane protein, we identified a 50-kDa molecular component which binds to preassembled microtubules. The binding of this polypeptide to microtubules was decreased in the presence of ATP. We purified this 50-kDa protein by affinity chromatography on immobilized ATP. The 50-kDa protein bound to the ATP column was eluted by 1 mM ATP. The purified protein, labeled with 125I, exhibited the ability of interacting with microtubules. The binding process was inhibited by increasing concentrations of ATP, the half-maximal inhibitory effect being obtained at an ATP concentration of 0.35 mM. The interaction of the 50-kDa protein with microtubules is a saturable phenomenon since the binding of the 125I-labeled 50-kDa protein was inhibited by unlabeled solubilized lysosomal membrane protein containing the 50-kDa polypeptide but not by the same protein fraction from which the 50-kDa polypeptide had been removed by the ATP affinity chromatography procedure. The 50-kDa protein has the property to bind to pure tubulin coupled to an insoluble matrix. The 50-kDa protein was eluted from the tubulin affinity column by ATP. These findings support the conclusion that a protein inserted into the lysosomal membrane is able to bind directly to microtubules in a process which can be regulated by ATP. We propose that this protein could account for the association of lysosomes to microtubules demonstrated both in vitro and in intact cells

  19. The polarity protein Par6 is coupled to the microtubule network during molluscan early embryogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Homma, Taihei [Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Shimizu, Miho [Kuroda Chiromorphology Team, ERATO-SORST, JST, Komaba, Meguro-ku, Tokyo 153-8902 (Japan); Kuroda, Reiko, E-mail: ckuroda@mail.ecc.u-tokyo.ac.jp [Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Kuroda Chiromorphology Team, ERATO-SORST, JST, Komaba, Meguro-ku, Tokyo 153-8902 (Japan); Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Komaba, Meguro-ku, Tokyo 153-8902 (Japan)

    2011-01-07

    Research highlights: {yields} The cDNAs encoding Par6 and aPKC homologues were cloned from the snail Lymnaea stagnalis. {yields} L. stagnalis Par6 directly interacts with tubulin and microtubules and localizes to the microtubule cytoskeleton during the early embryogenesis. {yields} Identical sequence and localization of LsPar6 for the dextral and the sinistral snails exclude the possibility of the gene being the primary determinant of body handedness. -- Abstract: Cell polarity, which directs the orientation of asymmetric cell division and segregation of fate determinants, is a fundamental feature of development and differentiation. Regulators of polarity have been extensively studied, and the critical importance of the Par (partitioning-defective) complex as the polarity machinery is now recognized in a wide range of eukaryotic systems. The Par polarity module is evolutionarily conserved, but its mechanism and cooperating factors vary among different systems. Here we describe the cloning and characterization of a pond snail Lymnaea stagnalis homologue of partitioning-defective 6 (Lspar6). The protein product LsPar6 shows high affinity for microtubules and localizes to the mitotic apparatus during embryonic cell division. In vitro assays revealed direct binding of LsPar6 to tubulin and microtubules, which is the first evidence of the direct interaction between the two proteins. The interaction is mediated by two distinct regions of LsPar6 both located in the N-terminal half. Atypical PKC, a functional partner of Par6, was also found to localize to the mitotic spindle. These results suggest that the L. stagnalis Par complex employs the microtubule network in cell polarity processes during the early embryogenesis. Identical sequence and localization of LsPar6 for the dextral and the sinistral snails exclude the possibility of the gene being the primary determinant of handedness.

  20. The polarity protein Par6 is coupled to the microtubule network during molluscan early embryogenesis

    International Nuclear Information System (INIS)

    Homma, Taihei; Shimizu, Miho; Kuroda, Reiko

    2011-01-01

    Research highlights: → The cDNAs encoding Par6 and aPKC homologues were cloned from the snail Lymnaea stagnalis. → L. stagnalis Par6 directly interacts with tubulin and microtubules and localizes to the microtubule cytoskeleton during the early embryogenesis. → Identical sequence and localization of LsPar6 for the dextral and the sinistral snails exclude the possibility of the gene being the primary determinant of body handedness. -- Abstract: Cell polarity, which directs the orientation of asymmetric cell division and segregation of fate determinants, is a fundamental feature of development and differentiation. Regulators of polarity have been extensively studied, and the critical importance of the Par (partitioning-defective) complex as the polarity machinery is now recognized in a wide range of eukaryotic systems. The Par polarity module is evolutionarily conserved, but its mechanism and cooperating factors vary among different systems. Here we describe the cloning and characterization of a pond snail Lymnaea stagnalis homologue of partitioning-defective 6 (Lspar6). The protein product LsPar6 shows high affinity for microtubules and localizes to the mitotic apparatus during embryonic cell division. In vitro assays revealed direct binding of LsPar6 to tubulin and microtubules, which is the first evidence of the direct interaction between the two proteins. The interaction is mediated by two distinct regions of LsPar6 both located in the N-terminal half. Atypical PKC, a functional partner of Par6, was also found to localize to the mitotic spindle. These results suggest that the L. stagnalis Par complex employs the microtubule network in cell polarity processes during the early embryogenesis. Identical sequence and localization of LsPar6 for the dextral and the sinistral snails exclude the possibility of the gene being the primary determinant of handedness.

  1. Dynamics and regulation at the tip : a high resolution view on microtubele assembly

    NARCIS (Netherlands)

    Munteanu, Laura

    2008-01-01

    Microtubules are highly dynamic protein polymers that and are essential for intracellular organization and fundamental processes like transport and cell division. In cells, a wide family of microtubule-associated proteins (MAPs) tightly regulates microtubule dynamics. The work presented in this

  2. Optomechanical proposal for monitoring microtubule mechanical vibrations

    Czech Academy of Sciences Publication Activity Database

    Barzanjeh, Sh.; Salari, V.; Tuszynski, J. A.; Cifra, Michal; Simon, C.

    2017-01-01

    Roč. 96, č. 1 (2017), č. článku 012404. ISSN 2470-0045 R&D Projects: GA ČR(CZ) GA15-17102S Grant - others:AV ČR(CZ) SAV-15-22 Program:Bilaterální spolupráce Institutional support: RVO:67985882 Keywords : Vibrational modes * Microtubule * Resonance frequencies Subject RIV: BH - Optics, Masers, Lasers OBOR OECD: Optics (including laser optics and quantum optics) Impact factor: 2.366, year: 2016

  3. Emerging microtubule targets in glioma therapy

    Czech Academy of Sciences Publication Activity Database

    Katsetos, C.D.; Reginato, M.J.; Baas, P.W.; D'Agostino, L.; Legido, A.; Tuszynski, J. A.; Dráberová, Eduarda; Dráber, Pavel

    2015-01-01

    Roč. 22, č. 1 (2015), s. 49-72 ISSN 1071-9091 R&D Projects: GA MŠk LH12050; GA MZd NT14467 Grant - others:GA AV ČR M200521203PIPP; NIH(US) R01 NS028785; Philadelphia Health Education Corporation (PHEC)–St. Christopher’s Hospital for Children Reunified Endowment (C.D.K.)(US) 323256 Institutional support: RVO:68378050 Keywords : glioma tumorigenesis * glioblastoma * tubulin * microtubules Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.303, year: 2015

  4. Changes in microtubule overlap length regulate kinesin-14-driven microtubule sliding

    Czech Academy of Sciences Publication Activity Database

    Braun, Marcus; Lánský, Zdeněk; Szuba, A.; Schwarz, F. W.; Mitra, A.; Gao, M.; Luedecke, A.; ten Wolde, P.R.; Diez, S.

    2017-01-01

    Roč. 13, č. 12 (2017), s. 1245-1252 ISSN 1552-4450 R&D Projects: GA ČR(CZ) GA15-17488S; GA ČR(CZ) GA17-12496Y; GA ČR(CZ) GJ17-12496Y; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:86652036 Keywords : SPINDLE ELONGATION * MITOTIC SPINDLE * KINESIN-5 CIN8 * CROSS-LINKERS Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Biophysics Impact factor: 15.066, year: 2016

  5. Quantitative analysis of microtubule orientation in interdigitated leaf pavement cells.

    Science.gov (United States)

    Akita, Kae; Higaki, Takumi; Kutsuna, Natsumaro; Hasezawa, Seiichiro

    2015-01-01

    Leaf pavement cells are shaped like a jigsaw puzzle in most dicotyledon species. Molecular genetic studies have identified several genes required for pavement cells morphogenesis and proposed that microtubules play crucial roles in the interdigitation of pavement cells. In this study, we performed quantitative analysis of cortical microtubule orientation in leaf pavement cells in Arabidopsis thaliana. We captured confocal images of cortical microtubules in cotyledon leaf epidermis expressing GFP-tubulinβ and quantitatively evaluated the microtubule orientations relative to the pavement cell growth axis using original image processing techniques. Our results showed that microtubules kept parallel orientations to the growth axis during pavement cell growth. In addition, we showed that immersion treatment of seed cotyledons in solutions containing tubulin polymerization and depolymerization inhibitors decreased pavement cell complexity. Treatment with oryzalin and colchicine inhibited the symmetric division of guard mother cells.

  6. Stochastic seasonality and nonlinear density-dependent factors regulate population size in an African rodent

    DEFF Research Database (Denmark)

    Leirs, Herwig; Steneth, Nils Chr.; Nichols, James D.

    1997-01-01

    , but clear examples of both processes acting in the same population are rare(7,8). Key-factor analysis (regression of population changes on possible causal factors) and time-series analysis are often used to investigate the presence of density dependence, but such approaches may be biased and provide...... no information on actual demographic rates(9,10). Here we report on both density-dependent and density-independent effects in a murid rodent pest species, the multimammute rat Mastomys natalensis (Smith, 1834), using statistical capture-recapture models, Both effects occur simultaneously, but we also demonstrate...

  7. Role of membrane sterols and cortical microtubules in gravity resistance in plants

    Science.gov (United States)

    Hoson, T.; Koizumi, T.; Matsumoto, S.; Kumasaki, S.; Soga, K.; Wakabayashi, K.; Sakaki, T.

    Resistance to the gravitational force is a principal graviresponse in plants comparable to gravitropism Nevertheless only limited information has been obtained for this graviresponse We have examined mechanisms of signal perception transformation and transduction of the perceived signal and response to the transduced signal in gravity resistance using hypergravity conditions produced by centrifugation In Arabidopsis hypocotyls hypergravity treatment greatly increased the expression level of 3-hydroxy-3-methylglutaryl-Coenzyme A reductase HMGR which catalyzes a reaction producing mevalonic acid a key precursor of terpenoids such as membrane sterols Geranyl diphosphate synthase gene was also up-regulated by hypergravity whereas the expression of other genes involved in membrane lipid metabolism was not influenced Hypergravity caused an increase in sterol content in azuki bean epicotyls but not in phospholipid glycolipid or fatty acid content Also hypergravity did not influence fatty acid composition in any lipid class Thus the effect of hypergravity on membrane lipid metabolism was specific for sterol synthesis On the other hand alpha- and beta-tubulin genes were up-regulated by hypergravity treatment in Arabidopsis hypocotyls Hypergravity also induced reorientation of cortical microtubules in azuki epicotyls the percentage of epidermal cells with transverse microtubles was decreased whereas that with longitudinal microtubules was increased Inhibitors of HMGR action and microtubule-disrupting agents completely prevented the gravity resistance

  8. Branching microtubule nucleation in Xenopus egg extracts mediated by augmin and TPX2

    Science.gov (United States)

    Petry, Sabine; Groen, Aaron C.; Ishihara, Keisuke; Mitchison, Timothy J.; Vale, Ronald D.

    2013-01-01

    Summary The microtubules that comprise mitotic spindles in animal cells are nucleated at centrosomes and by spindle assembly factors that are activated in the vicinity of chromatin. Indirect evidence also has suggested that microtubules might be nucleated from pre-existing microtubules throughout the spindle, but this process has not been observed directly. Here, we demonstrate microtubule nucleation from the sides of existing microtubules in meiotic Xenopus egg extracts. Daughter microtubules grow at a low branch angle and with the same polarity as mother filaments. Branching microtubule nucleation requires gamma-tubulin and augmin and is stimulated by GTP-bound Ran and its effector TPX2, factors previously implicated in chromatin-stimulated nucleation. Because of the rapid amplification of microtubule numbers and the preservation of microtubule polarity, microtubule-dependent microtubule nucleation is well suited for spindle assembly and maintenance. PMID:23415226

  9. Daple Coordinates Planar Polarized Microtubule Dynamics in Ependymal Cells and Contributes to Hydrocephalus

    Directory of Open Access Journals (Sweden)

    Maki Takagishi

    2017-07-01

    Full Text Available Motile cilia in ependymal cells, which line the cerebral ventricles, exhibit a coordinated beating motion that drives directional cerebrospinal fluid (CSF flow and guides neuroblast migration. At the apical cortex of these multi-ciliated cells, asymmetric localization of planar cell polarity (PCP proteins is required for the planar polarization of microtubule dynamics, which coordinates cilia orientation. Daple is a disheveled-associating protein that controls the non-canonical Wnt signaling pathway and cell motility. Here, we show that Daple-deficient mice present hydrocephalus and their ependymal cilia lack coordinated orientation. Daple regulates microtubule dynamics at the anterior side of ependymal cells, which in turn orients the cilial basal bodies required for the directional cerebrospinal fluid flow. These results demonstrate an important role for Daple in planar polarity in motile cilia and provide a framework for understanding the mechanisms and functions of planar polarization in the ependymal cells.

  10. The Drosophila Microtubule-Associated Protein Mars Stabilizes Mitotic Spindles by Crosslinking Microtubules through Its N-Terminal Region

    DEFF Research Database (Denmark)

    Zhang, Gang; Beati, Hamze; Nilsson, Jakob

    2013-01-01

    Correct segregation of genetic material relies on proper assembly and maintenance of the mitotic spindle. How the highly dynamic microtubules (MTs) are maintained in stable mitotic spindles is a key question to be answered. Motor and non-motor microtubule associated proteins (MAPs) have been...

  11. Drosophila Regulate Yeast Density and Increase Yeast Community Similarity in a Natural Substrate

    OpenAIRE

    Stamps, Judy A.; Yang, Louie H.; Morales, Vanessa M.; Boundy-Mills, Kyria L.

    2012-01-01

    Drosophila melanogaster adults and larvae, but especially larvae, had profound effects on the densities and community structure of yeasts that developed in banana fruits. Pieces of fruit exposed to adult female flies previously fed fly-conditioned bananas developed higher yeast densities than pieces of the same fruits that were not exposed to flies, supporting previous suggestions that adult Drosophila vector yeasts to new substrates. However, larvae alone had dramatic effects on yeast densit...

  12. Ion channel density regulates switches between regular and fast spiking in soma but not in axons.

    Directory of Open Access Journals (Sweden)

    Hugo Zeberg

    2010-04-01

    Full Text Available The threshold firing frequency of a neuron is a characterizing feature of its dynamical behaviour, in turn determining its role in the oscillatory activity of the brain. Two main types of dynamics have been identified in brain neurons. Type 1 dynamics (regular spiking shows a continuous relationship between frequency and stimulation current (f-I(stim and, thus, an arbitrarily low frequency at threshold current; Type 2 (fast spiking shows a discontinuous f-I(stim relationship and a minimum threshold frequency. In a previous study of a hippocampal neuron model, we demonstrated that its dynamics could be of both Type 1 and Type 2, depending on ion channel density. In the present study we analyse the effect of varying channel density on threshold firing frequency on two well-studied axon membranes, namely the frog myelinated axon and the squid giant axon. Moreover, we analyse the hippocampal neuron model in more detail. The models are all based on voltage-clamp studies, thus comprising experimentally measurable parameters. The choice of analysing effects of channel density modifications is due to their physiological and pharmacological relevance. We show, using bifurcation analysis, that both axon models display exclusively Type 2 dynamics, independently of ion channel density. Nevertheless, both models have a region in the channel-density plane characterized by an N-shaped steady-state current-voltage relationship (a prerequisite for Type 1 dynamics and associated with this type of dynamics in the hippocampal model. In summary, our results suggest that the hippocampal soma and the two axon membranes represent two distinct kinds of membranes; membranes with a channel-density dependent switching between Type 1 and 2 dynamics, and membranes with a channel-density independent dynamics. The difference between the two membrane types suggests functional differences, compatible with a more flexible role of the soma membrane than that of the axon membrane.

  13. Effects of ultraviolet radiation on microtubule organisation and morphogenesis in plants

    Energy Technology Data Exchange (ETDEWEB)

    Staxen, I.

    1994-09-01

    The involvement of the cytoskeleton in the development of somatic embryos was studied in Larix x eurolepis. Protoplasts were isolated from both somatic embryo-regenerating and non-generating cultures and fractionated on a discontinuous Percoll density gradient. Protoplasts of two cell lines of Larix eurolepis, one with regenerating potential and one lacking this potential, were compared. In contrast to the non-regenerating line were a protoplast-like organisation of the cortical microtubules was maintained, re-organisation of this microtubular network occurred in the regenerable line after only three days of culture, indicating that organised growth was occurring. However, this early organisation of cortical microtubules may not always be a valid marker for regenerable and non-regenerable material. In order to investigate the effect of ultraviolet-B (UV-B, 280-320 nm) radiation on the microtubule cytoskeleton, protoplasts were isolated from leaves of Petunia hybrida and subjected to four different doses of UV-B radiation. The organisation of the microtubules and the progression of the cells through the cell cycle was observed at 0, 24, 48 and 72 h after irradiation. UV-B induced breaks in the cortical microtubules resulting in shorter fragments with increasing amounts of radiation. Also, the division of the protoplasts was delayed. Whole Petunia plants were grown in growth chambers in the presence and absence of UV-B. The plants responded to UV-B with increased rates of CO{sub 2} assimilation, a 60% increase in UV-screening compounds and the changes in the morphology of the leaves that were reflected in a 70-100% increase in leaf area and 20% decrease in leaf thickness. The microtubules of the epidermal cells was not affected by UV-B, nor was the number of epidermal cells (per unit area). The increase in leaf area in the UV-treated plants appeared due to stimulation of cell division in the leaf meristems. 111 refs, 5 figs, 2 tabs.

  14. Multiscale modeling and simulation of microtubule-motor-protein assemblies

    Science.gov (United States)

    Gao, Tong; Blackwell, Robert; Glaser, Matthew A.; Betterton, M. D.; Shelley, Michael J.

    2015-12-01

    Microtubules and motor proteins self-organize into biologically important assemblies including the mitotic spindle and the centrosomal microtubule array. Outside of cells, microtubule-motor mixtures can form novel active liquid-crystalline materials driven out of equilibrium by adenosine triphosphate-consuming motor proteins. Microscopic motor activity causes polarity-dependent interactions between motor proteins and microtubules, but how these interactions yield larger-scale dynamical behavior such as complex flows and defect dynamics is not well understood. We develop a multiscale theory for microtubule-motor systems in which Brownian dynamics simulations of polar microtubules driven by motors are used to study microscopic organization and stresses created by motor-mediated microtubule interactions. We identify polarity-sorting and crosslink tether relaxation as two polar-specific sources of active destabilizing stress. We then develop a continuum Doi-Onsager model that captures polarity sorting and the hydrodynamic flows generated by these polar-specific active stresses. In simulations of active nematic flows on immersed surfaces, the active stresses drive turbulent flow dynamics and continuous generation and annihilation of disclination defects. The dynamics follow from two instabilities, and accounting for the immersed nature of the experiment yields unambiguous characteristic length and time scales. When turning off the hydrodynamics in the Doi-Onsager model, we capture formation of polar lanes as observed in the Brownian dynamics simulation.

  15. Multiscale modeling and simulation of microtubule-motor-protein assemblies.

    Science.gov (United States)

    Gao, Tong; Blackwell, Robert; Glaser, Matthew A; Betterton, M D; Shelley, Michael J

    2015-01-01

    Microtubules and motor proteins self-organize into biologically important assemblies including the mitotic spindle and the centrosomal microtubule array. Outside of cells, microtubule-motor mixtures can form novel active liquid-crystalline materials driven out of equilibrium by adenosine triphosphate-consuming motor proteins. Microscopic motor activity causes polarity-dependent interactions between motor proteins and microtubules, but how these interactions yield larger-scale dynamical behavior such as complex flows and defect dynamics is not well understood. We develop a multiscale theory for microtubule-motor systems in which Brownian dynamics simulations of polar microtubules driven by motors are used to study microscopic organization and stresses created by motor-mediated microtubule interactions. We identify polarity-sorting and crosslink tether relaxation as two polar-specific sources of active destabilizing stress. We then develop a continuum Doi-Onsager model that captures polarity sorting and the hydrodynamic flows generated by these polar-specific active stresses. In simulations of active nematic flows on immersed surfaces, the active stresses drive turbulent flow dynamics and continuous generation and annihilation of disclination defects. The dynamics follow from two instabilities, and accounting for the immersed nature of the experiment yields unambiguous characteristic length and time scales. When turning off the hydrodynamics in the Doi-Onsager model, we capture formation of polar lanes as observed in the Brownian dynamics simulation.

  16. Molecular architecture of axonemal microtubule doublets revealedby cryo-electron tomography

    Energy Technology Data Exchange (ETDEWEB)

    Sui, Haixin; Downing, Kenneth H.

    2006-05-22

    The axoneme, which forms the core of eukaryotic flagella and cilia, is one of the largest macromolecular machines with a structure that is largely conserved from protists to mammals. Microtubule doublets are structural components of axonemes containing a number of proteins besides tubulin, and are usually found in arrays of nine doublets arranged around two singlet microtubules. Coordinated sliding of adjacent doublets, which involves a host of other proteins in the axoneme, produces periodic beating movements of the axoneme. We have obtained a 3D density map of intact microtubule doublets using cryo-electron tomography and image averaging. Our map, with a resolution of about 3 nm, provides insights into locations of particular proteins within the doublets and the structural features of the doublets that define their mechanical properties. We identify likely candidates for several of these non-tubulin components of the doublets. This work offers novel insight on how tubulin protofilaments and accessory proteins attach together to form the doublets and provides a structural basis for understanding doublet function in axonemes.

  17. Centrosome and microtubule instability in aging Drosophila cells

    Science.gov (United States)

    Schatten, H.; Chakrabarti, A.; Hedrick, J.

    1999-01-01

    Several cytoskeletal changes are associated with aging which includes alterations in muscle structure leading to muscular atrophy, and weakening of the microtubule network which affects cellular secretion and maintenance of cell shape. Weakening of the microtubule network during meiosis in aging oocytes can result in aneuploidy or trisomic zygotes with increasing maternal age. Imbalances of cytoskeletal organization can lead to disease such as Alzheimer's, muscular disorders, and cancer. Because many cytoskeletal diseases are related to age we investigated the effects of aging on microtubule organization in cell cultures of the Drosophila cell model system (Schneider S-1 and Kc23 cell lines). This cell model is increasingly being used as an alternative system to mammalian cell cultures. Drosophila cells are amenable to genetic manipulations and can be used to identify and manipulate genes which are involved in the aging processes. Immunofluorescence, scanning, and transmission electron microscopy were employed for the analysis of microtubule organizing centers (centrosomes) and microtubules at various times after subculturing cells in fresh medium. Our results reveal that centrosomes and the microtubule network becomes significantly affected in aging cells after 5 days of subculture. At 5-14 days of subculture, 1% abnormal out of 3% mitoses were noted which were clearly distinguishable from freshly subcultured control cells in which 3% of cells undergo normal mitosis with bipolar configurations. Microtubules are also affected in the midbody during cell division. The midbody in aging cells becomes up to 10 times longer when compared with midbodies in freshly subcultured cells. During interphase, microtubules are often disrupted and disorganized, which may indicate improper function related to transport of cell organelles along microtubules. These results are likely to help explain some cytoskeletal disorders and diseases related to aging.

  18. Birefringence of single and bundled microtubules.

    Science.gov (United States)

    Oldenbourg, R; Salmon, E D; Tran, P T

    1998-01-01

    We have measured the birefringence of microtubules (MTs) and of MT-based macromolecular assemblies in vitro and in living cells by using the new Pol-Scope. A single microtubule in aqueous suspension and imaged with a numerical aperture of 1.4 had a peak retardance of 0.07 nm. The peak retardance of a small bundle increased linearly with the number of MTs in the bundle. Axonemes (prepared from sea urchin sperm) had a peak retardance 20 times higher than that of single MTs, in accordance with the nine doublets and two singlets arrangement of parallel MTs in the axoneme. Measured filament retardance decreased when the filament was defocused or the numerical aperture of the imaging system was decreased. However, the retardance "area," which we defined as the image retardance integrated along a line perpendicular to the filament axis, proved to be independent of focus and of numerical aperture. These results are in good agreement with a theory that we developed for measuring retardances with imaging optics. Our theoretical concept is based on Wiener's theory of mixed dielectrics, which is well established for nonimaging applications. We extend its use to imaging systems by considering the coherence region defined by the optical set-up. Light scattered from within that region interferes coherently in the image point. The presence of a filament in the coherence region leads to a polarization dependent scattering cross section and to a finite retardance measured in the image point. Similar to resolution measurements, the linear dimension of the coherence region for retardance measurements is on the order lambda/(2 NA), where lambda is the wavelength of light and NA is the numerical aperture of the illumination and imaging lenses.

  19. Spatiotemporal relationships between growth and microtubule orientation as revealed in living root cells of Arabidopsis thaliana transformed with green-fluorescent-protein gene construct GFP-MBD

    Science.gov (United States)

    Granger, C. L.; Cyr, R. J.

    2001-01-01

    Arabidopsis thaliana plants were transformed with GFP-MBD (J. Marc et al., Plant Cell 10: 1927-1939, 1998) under the control of a constitutive (35S) or copper-inducible promoter. GFP-specific fluorescence distributions, levels, and persistence were determined and found to vary with age, tissue type, transgenic line, and individual plant. With the exception of an increased frequency of abnormal roots of 35S GFP-MBD plants grown on kanamycin-containing media, expression of GFP-MBD does not appear to affect plant phenotype. The number of leaves, branches, bolts, and siliques as well as overall height, leaf size, and seed set are similar between wild-type and transgenic plants as is the rate of root growth. Thus, we conclude that the transgenic plants can serve as a living model system in which the dynamic behavior of microtubules can be visualized. Confocal microscopy was used to simultaneously monitor growth and microtubule behavior within individual cells as they passed through the elongation zone of the Arabidopsis root. Generally, microtubules reoriented from transverse to oblique or longitudinal orientations as growth declined. Microtubule reorientation initiated at the ends of the cell did not necessarily occur simultaneously in adjacent neighboring cells and did not involve complete disintegration and repolymerization of microtubule arrays. Although growth rates correlated with microtubule reorientation, the two processes were not tightly coupled in terms of their temporal relationships, suggesting that other factor(s) may be involved in regulating both events. Additionally, microtubule orientation was more defined in cells whose growth was accelerating and less stringent in cells whose growth was decelerating, indicating that microtubule-orienting factor(s) may be sensitive to growth acceleration, rather than growth per se.

  20. Cell density and N-cadherin interactions regulate cell proliferation in the sensory epithelia of the inner ear.

    Science.gov (United States)

    Warchol, Mark E

    2002-04-01

    Sensory hair cells in the inner ears of nonmammalian vertebrates can regenerate after injury. In many species, replacement hair cells are produced by the proliferation of epithelial supporting cells. Thus, the ability of supporting cells to undergo renewed proliferation is a key determinant of regenerative ability. The present study used cultures of isolated inner ear sensory epithelia to identify cellular signals that regulate supporting cell proliferation. Small pieces of sensory epithelia from the chicken utricle were cultured in glass microwells. Under those conditions, cell proliferation was inversely related to local cell density. The signaling molecules N-cadherin, beta-catenin, and focal adhesion kinase were immunolocalized in the cultured epithelial cells, and high levels of phosphotyrosine immunoreactivity were present at cell-cell junctions and focal contacts of proliferating cells. Binding of microbeads coated with a function-blocking antibody to N-cadherin inhibited ongoing proliferation. The growth of epithelial cells was also affected by the density of extracellular matrix molecules. The results suggest that cell density, cell-cell contact, and the composition of the extracellular matrix may be critical influences on the regulation of sensory regeneration in the inner ear.

  1. Microtubule and Cell Contact Dependency of ER-bound PTP1B Localization in Growth Cones

    Science.gov (United States)

    Fuentes, Federico

    2009-01-01

    PTP1B is an ER-bound protein tyrosine phosphatase implied in the regulation of cell adhesion. Here we investigated mechanisms involved in the positioning and dynamics of PTP1B in axonal growth cones and evaluated the role of this enzyme in axons. In growth cones, PTP1B consistently localizes in the central domain, and occasionally at the peripheral region and filopodia. Live imaging of GFP-PTP1B reveals dynamic excursions of fingerlike processes within the peripheral region and filopodia. PTP1B and GFP-PTP1B colocalize with ER markers and coalign with microtubules at the peripheral region and redistribute to the base of the growth cone after treatment with nocodazole, a condition that is reversible. Growth cone contact with cellular targets is accompanied by invasion of PTP1B and stable microtubules in the peripheral region aligned with the contact axis. Functional impairment of PTP1B causes retardation of axon elongation, as well as reduction of growth cone filopodia lifetime and Src activity. Our results highlight the role of microtubules and cell contacts in the positioning of ER-bound PTP1B to the peripheral region of growth cones, which may be required for the positive role of PTP1B in axon elongation, filopodia stabilization, and Src activity. PMID:19158394

  2. Changes in microtubule-associated protein tau during peripheral nerve injury and regeneration

    Directory of Open Access Journals (Sweden)

    Guang-bin Zha

    2016-01-01

    Full Text Available Tau, a primary component of microtubule-associated protein, promotes microtubule assembly and/or disassembly and maintains the stability of the microtubule structure. Although the importance of tau in neurodegenerative diseases has been well demonstrated, whether tau is involved in peripheral nerve regeneration remains unknown. In the current study, we obtained sciatic nerve tissue from adult rats 0, 1, 4, 7, and 14 days after sciatic nerve crush and examined tau mRNA and protein expression levels and the location of tau in the sciatic nerve following peripheral nerve injury. The results from our quantitative reverse transcription polymerase chain reaction analysis showed that compared with the uninjured control sciatic nerve, mRNA expression levels for both tau and tau tubulin kinase 1, a serine/threonine kinase that regulates tau phosphorylation, were decreased following peripheral nerve injury. Our western blot assay results suggested that the protein expression levels of tau and phosphorylated tau initially decreased 1 day post nerve injury but then gradually increased. The results of our immunohistochemical labeling showed that the location of tau protein was not altered by nerve injury. Thus, these results showed that the expression of tau was changed following sciatic nerve crush, suggesting that tau may be involved in peripheral nerve repair and regeneration.

  3. Effects of the KIF2C neck peptide on microtubules: lateral disintegration of microtubules and β-structure formation.

    Science.gov (United States)

    Shimizu, Youské; Shimizu, Takashi; Nara, Masayuki; Kikumoto, Mahito; Kojima, Hiroaki; Morii, Hisayuki

    2013-04-01

    Members of the kinesin-13 sub-family, including KIF2C, depolymerize microtubules. The positive charge-rich 'neck' region extending from the N-terminus of the catalytic head is considered to be important in the depolymerization activity. Chemically synthesized peptides, covering the basic region (A182-E200), induced a sigmoidal increase in the turbidity of a microtubule suspension. The increase was suppressed by salt addition or by reduction of basicity by amino acid substitutions. Electron microscopic observations revealed ring structures surrounding the microtubules at high peptide concentrations. Using the peptide A182-D218, we also detected free thin straight filaments, probably protofilaments disintegrated from microtubules. Therefore, the neck region, even without the catalytic head domain, may induce lateral disintegration of microtubules. With microtubules lacking anion-rich C-termini as a result of subtilisin treatment, addition of the peptide induced only a moderate increase in turbidity, and rings and protofilaments were rarely detected, while aggregations, also thought to be caused by lateral disintegration, were often observed in electron micrographs. Thus, the C-termini are not crucial for the action of the peptides in lateral disintegration but contribute to structural stabilization of the protofilaments. Previous structural studies indicated that the neck region of KIF2C is flexible, but our IR analysis suggests that the cation-rich region (K190-A204) forms β-structure in the presence of microtubules, which may be of significance with regard to the action of the neck region. Therefore, the neck region of KIF2C is sufficient to cause disintegration of microtubules into protofilaments, and this may contribute to the ability of KIF2C to cause depolymerization of microtubules. © 2013 The Authors Journal compilation © 2013 FEBS.

  4. Lysosomes are associated with microtubules and not with intermediate filaments in cultured fibroblasts.

    OpenAIRE

    Collot, M; Louvard, D; Singer, S J

    1984-01-01

    Double immunofluorescent labeling experiments for lysosomes and either microtubules or vimentin intermediate filaments in cultured well-spread fibroblasts show a remarkable degree of superposition of the lysosomes and the microtubules. Under two different sets of conditions where the microtubules and intermediate filaments are well segregated from one another, the lysosomes remain codistributed with the microtubules. It is suggested that this specific association of lysosomes with microtubule...

  5. and its allicin on microtubule and cancer cell lines

    African Journals Online (AJOL)

    STORAGESEVER

    2009-10-05

    Oct 5, 2009 ... microtubule protein polymer that treated by A. hirtifolium. (A), and allicin (B) in .... with a chromogenic thiol: reaction of 4-mercaptopyridine with ... transformed tumor growth in vivo by diallyl disulfide is associated with inhibition ...

  6. APC functions at the centrosome to stimulate microtubule growth.

    Science.gov (United States)

    Lui, Christina; Ashton, Cahora; Sharma, Manisha; Brocardo, Mariana G; Henderson, Beric R

    2016-01-01

    The adenomatous polyposis coli (APC) tumor suppressor is multi-functional. APC is known to localize at the centrosome, and in mitotic cells contributes to formation of the mitotic spindle. To test whether APC contributes to nascent microtubule (MT) growth at interphase centrosomes, we employed MT regrowth assays in U2OS cells to measure MT assembly before and after nocodazole treatment and release. We showed that siRNA knockdown of full-length APC delayed both initial MT aster formation and MT elongation/regrowth. In contrast, APC-mutant SW480 cancer cells displayed a defect in MT regrowth that was unaffected by APC knockdown, but which was rescued by reconstitution of full-length APC. Our findings identify APC as a positive regulator of centrosome MT initial assembly and suggest that this process is disrupted by cancer mutations. We confirmed that full-length APC associates with the MT-nucleation factor γ-tubulin, and found that the APC cancer-truncated form (1-1309) also bound to γ-tubulin through APC amino acids 1-453. While binding to γ-tubulin may help target APC to the site of MT nucleation complexes, additional C-terminal sequences of APC are required to stimulate and stabilize MT growth. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. Microtubules Enable the Planar Cell Polarity of Airway Cilia

    Science.gov (United States)

    Vladar, Eszter K.; Bayly, Roy D.; Sangoram, Ashvin; Scott, Matthew P.; Axelrod, Jeffrey D.

    2012-01-01

    Summary Background Airway cilia must be physically oriented along the longitudinal tissue axis for concerted, directional motility that is essential for proper mucociliary clearance. Results We show that Planar Cell Polarity (PCP) signaling specifies directionality and orients respiratory cilia. Within all airway epithelial cells a conserved set of PCP proteins shows interdependent, asymmetric junctional localization; non-autonomous signaling coordinates polarization between cells; and a polarized microtubule (MT) network is likely required for asymmetric PCP protein localization. We find that basal bodies dock after polarity of PCP proteins is established, are polarized nearly simultaneously, and refinement of basal body/cilium orientation continues during airway epithelial development. Unique to mature multiciliated cells, we identify PCP-regulated, planar polarized MTs that originate from basal bodies and interact, via their plus ends, with membrane domains associated with the PCP proteins Frizzled and Dishevelled. Disruption of MTs leads to misoriented cilia. Conclusions A conserved PCP pathway orients airway cilia by communicating polarity information from asymmetric membrane domains at the apical junctions, through MTs, to orient the MT and actin based network of ciliary basal bodies below the apical surface. PMID:23122850

  8. High-Density and Very-Low-Density Lipoprotein Have Opposing Roles in Regulating Tumor-Initiating Cells and Sensitivity to Radiation in Inflammatory Breast Cancer

    Energy Technology Data Exchange (ETDEWEB)

    Wolfe, Adam R. [Department of Radiation Oncology, University of Texas MD Anderson Cancer Center, Houston, Texas (United States); Morgan Welch Inflammatory Breast Cancer Research Program and Clinic, University of Texas MD Anderson Cancer Center, Houston, Texas (United States); Atkinson, Rachel L. [Morgan Welch Inflammatory Breast Cancer Research Program and Clinic, University of Texas MD Anderson Cancer Center, Houston, Texas (United States); Reddy, Jay P. [Department of Radiation Oncology, University of Texas MD Anderson Cancer Center, Houston, Texas (United States); Debeb, Bisrat G.; Larson, Richard; Li, Li [Department of Radiation Oncology, University of Texas MD Anderson Cancer Center, Houston, Texas (United States); Department of Clinical Cancer Prevention, University of Texas MD Anderson Cancer Center, Houston, Texas (United States); Masuda, Hiroko; Brewer, Takae [Department of Clinical Cancer Prevention, University of Texas MD Anderson Cancer Center, Houston, Texas (United States); Atkinson, Bradley J. [Department of Clinical Pharmacy Services, University of Texas MD Anderson Cancer Center, Houston, Texas (United States); Brewster, Abeena [Morgan Welch Inflammatory Breast Cancer Research Program and Clinic, University of Texas MD Anderson Cancer Center, Houston, Texas (United States); Ueno, Naoto T. [Department of Clinical Cancer Prevention, University of Texas MD Anderson Cancer Center, Houston, Texas (United States); Woodward, Wendy A., E-mail: wwoodward@mdanderson.org [Department of Radiation Oncology, University of Texas MD Anderson Cancer Center, Houston, Texas (United States); Department of Clinical Cancer Prevention, University of Texas MD Anderson Cancer Center, Houston, Texas (United States)

    2015-04-01

    Purpose: We previously demonstrated that cholesterol-lowering agents regulate radiation sensitivity of inflammatory breast cancer (IBC) cell lines in vitro and are associated with less radiation resistance among IBC patients who undergo postmastectomy radiation. We hypothesized that decreasing IBC cellular cholesterol induced by treatment with lipoproteins would increase radiation sensitivity. Here, we examined the impact of specific transporters of cholesterol (ie lipoproteins) on the responses of IBC cells to self-renewal and to radiation in vitro and on clinical outcomes in IBC patients. Methods and Materials: Two patient-derived IBC cell lines, SUM 149 and KPL4, were incubated with low-density lipoproteins (LDL), very-low-density lipoproteins (VLDL), or high-density lipoproteins (HDL) for 24 hours prior to irradiation (0-6 Gy) and mammosphere formation assay. Cholesterol panels were examined in a cohort of patients with primary IBC diagnosed between 1995 and 2011 at MD Anderson Cancer Center. Lipoprotein levels were then correlated to patient outcome, using the log rank statistical model, and examined in multivariate analysis using Cox regression. Results: VLDL increased and HDL decreased mammosphere formation compared to untreated SUM 149 and KPL4 cells. Survival curves showed enhancement of survival in both of the IBC cell lines when pretreated with VLDL and, conversely, radiation sensitization in all cell lines when pretreated with HDL. In IBC patients, higher VLDL values (>30 mg/dL) predicted a lower 5-year overall survival rate than normal values (hazard ratio [HR] = 1.9 [95% confidence interval [CI]: 1.05-3.45], P=.035). Lower-than-normal patient HDL values (<60 mg/dL) predicted a lower 5-year overall survival rate than values higher than 60 mg/dL (HR = 3.21 [95% CI: 1.25-8.27], P=.015). Conclusions: This study discovered a relationship among the plasma levels of lipoproteins, overall patient response, and radiation resistance in IBC patients

  9. High-Density and Very-Low-Density Lipoprotein Have Opposing Roles in Regulating Tumor-Initiating Cells and Sensitivity to Radiation in Inflammatory Breast Cancer

    International Nuclear Information System (INIS)

    Wolfe, Adam R.; Atkinson, Rachel L.; Reddy, Jay P.; Debeb, Bisrat G.; Larson, Richard; Li, Li; Masuda, Hiroko; Brewer, Takae; Atkinson, Bradley J.; Brewster, Abeena; Ueno, Naoto T.; Woodward, Wendy A.

    2015-01-01

    Purpose: We previously demonstrated that cholesterol-lowering agents regulate radiation sensitivity of inflammatory breast cancer (IBC) cell lines in vitro and are associated with less radiation resistance among IBC patients who undergo postmastectomy radiation. We hypothesized that decreasing IBC cellular cholesterol induced by treatment with lipoproteins would increase radiation sensitivity. Here, we examined the impact of specific transporters of cholesterol (ie lipoproteins) on the responses of IBC cells to self-renewal and to radiation in vitro and on clinical outcomes in IBC patients. Methods and Materials: Two patient-derived IBC cell lines, SUM 149 and KPL4, were incubated with low-density lipoproteins (LDL), very-low-density lipoproteins (VLDL), or high-density lipoproteins (HDL) for 24 hours prior to irradiation (0-6 Gy) and mammosphere formation assay. Cholesterol panels were examined in a cohort of patients with primary IBC diagnosed between 1995 and 2011 at MD Anderson Cancer Center. Lipoprotein levels were then correlated to patient outcome, using the log rank statistical model, and examined in multivariate analysis using Cox regression. Results: VLDL increased and HDL decreased mammosphere formation compared to untreated SUM 149 and KPL4 cells. Survival curves showed enhancement of survival in both of the IBC cell lines when pretreated with VLDL and, conversely, radiation sensitization in all cell lines when pretreated with HDL. In IBC patients, higher VLDL values (>30 mg/dL) predicted a lower 5-year overall survival rate than normal values (hazard ratio [HR] = 1.9 [95% confidence interval [CI]: 1.05-3.45], P=.035). Lower-than-normal patient HDL values (<60 mg/dL) predicted a lower 5-year overall survival rate than values higher than 60 mg/dL (HR = 3.21 [95% CI: 1.25-8.27], P=.015). Conclusions: This study discovered a relationship among the plasma levels of lipoproteins, overall patient response, and radiation resistance in IBC patients

  10. Optical properties of template synthesized nanowalled ZnS microtubules

    Science.gov (United States)

    Kumar, Rajesh; Chakarvarti, S. K.

    2007-12-01

    Electrodeposition is a versatile technique combining low processing cost with ambient conditions that can be used to prepare metallic, polymeric and semiconducting nano/micro structures. In the present work, track-etch membranes (TEMs) of makrofol (KG) have been used as templates for synthesis of ZnS nanowalled microtubules using electrodeposition technique. The morphology of the microtubules was characterized by scanning electron microscopy. Size effects on the band gap of tubules have also been studied by UV-visible spectrophotometer.

  11. Microtubules become more dynamic but not shorter during preprophase band formation: A possible "search-and-capture" mechanism for microtubule translocation

    NARCIS (Netherlands)

    Vos, J.W.; Dogterom, M.; Emons, A.M.C.

    2004-01-01

    The dynamic behavior of the microtubule cytoskeleton plays a crucial role in cellular organization, but the physical mechanisms underlying microtubule (re)organization in plant cells are poorly understood. We investigated microtubule dynamics in tobacco BY-2 suspension cells during interphase and

  12. The Microtubule Plus-End Tracking Protein CLASP2 Is Required for Hematopoiesis and Hematopoietic Stem Cell Maintenance

    Directory of Open Access Journals (Sweden)

    Ksenija Drabek

    2012-10-01

    Full Text Available Mammalian CLASPs are microtubule plus-end tracking proteins whose essential function as regulators of microtubule behavior has been studied mainly in cultured cells. We show here that absence of murine CLASP2 in vivo results in thrombocytopenia, progressive anemia, and pancytopenia, due to defects in megakaryopoiesis, in erythropoiesis, and in the maintenance of hematopoietic stem cell activity. Furthermore, microtubule stability and organization are affected upon attachment of Clasp2 knockout hematopoietic stem-cell-enriched populations, and these cells do not home efficiently toward their bone marrow niche. Strikingly, CLASP2-deficient hematopoietic stem cells contain severely reduced mRNA levels of c-Mpl, which encodes the thrombopoietin receptor, an essential factor for megakaryopoiesis and hematopoietic stem cell maintenance. Our data suggest that thrombopoietin signaling is impaired in Clasp2 knockout mice. We propose that the CLASP2-mediated stabilization of microtubules is required for proper attachment, homing, and maintenance of hematopoietic stem cells and that this is necessary to sustain c-Mpl transcription.

  13. Reduced density and altered regulation of rat atrial L-type Ca2+ current in heart failure.

    Science.gov (United States)

    Bond, Richard C; Bryant, Simon M; Watson, Judy J; Hancox, Jules C; Orchard, Clive H; James, Andrew F

    2017-03-01

    Constitutive regulation by PKA has recently been shown to contribute to L-type Ca 2+ current ( I CaL ) at the ventricular t-tubule in heart failure. Conversely, reduction in constitutive regulation by PKA has been proposed to underlie the downregulation of atrial I CaL in heart failure. The hypothesis that downregulation of atrial I CaL in heart failure involves reduced channel phosphorylation was examined. Anesthetized adult male Wistar rats underwent surgical coronary artery ligation (CAL, N =10) or equivalent sham-operation (Sham, N =12). Left atrial myocytes were isolated ~18 wk postsurgery and whole cell currents recorded (holding potential=-80 mV). I CaL activated by depolarizing pulses to voltages from -40 to +50 mV were normalized to cell capacitance and current density-voltage relations plotted. CAL cell capacitances were ~1.67-fold greater than Sham ( P ≤ 0.0001). Maximal I CaL conductance ( G max ) was downregulated more than 2-fold in CAL vs. Sham myocytes ( P 50% more effectively in CAL than in Sham so that differences in I CaL density were abolished. Differences between CAL and Sham G max were not abolished by calyculin A (100 nmol/l), suggesting that increased protein dephosphorylation did not account for I CaL downregulation. Treatment with either H-89 (10 μmol/l) or AIP (5 μmol/l) had no effect on basal currents in Sham or CAL myocytes, indicating that, in contrast to ventricular myocytes, neither PKA nor CaMKII regulated basal I CaL Expression of the L-type α 1C -subunit, protein phosphatases 1 and 2A, and inhibitor-1 proteins was unchanged. In conclusion, reduction in PKA-dependent regulation did not contribute to downregulation of atrial I CaL in heart failure. NEW & NOTEWORTHY Whole cell recording of L-type Ca 2+ currents in atrial myocytes from rat hearts subjected to coronary artery ligation compared with those from sham-operated controls reveals marked reduction in current density in heart failure without change in channel subunit

  14. Association of TCTP with Centrosome and Microtubules

    Directory of Open Access Journals (Sweden)

    Mariusz K. Jaglarz

    2012-01-01

    Full Text Available Translationally Controlled Tumour Protein (TCTP associates with microtubules (MT, however, the details of this association are unknown. Here we analyze the relationship of TCTP with MTs and centrosomes in Xenopus laevis and mammalian cells using immunofluorescence, tagged TCTP expression and immunoelectron microscopy. We show that TCTP associates both with MTs and centrosomes at spindle poles when detected by species-specific antibodies and by Myc-XlTCTP expression in Xenopus and mammalian cells. However, when the antibodies against XlTCTP were used in mammalian cells, TCTP was detected exclusively in the centrosomes. These results suggest that a distinct pool of TCTP may be specific for, and associate with, the centrosomes. Double labelling for TCTP and γ-tubulin with immuno-gold electron microscopy in Xenopus laevis oogonia shows localization of TCTP at the periphery of the γ-tubulin-containing pericentriolar material (PCM enveloping the centriole. TCTP localizes in the close vicinity of, but not directly on the MTs in Xenopus ovary suggesting that this association requires unidentified linker proteins. Thus, we show for the first time: (1 the association of TCTP with centrosomes, (2 peripheral localization of TCTP in relation to the centriole and the γ-tubulin-containing PCM within the centrosome, and (3 the indirect association of TCTP with MTs.

  15. Identification of miR-185 as a regulator of de novo cholesterol biosynthesis and low density lipoprotein uptake

    Science.gov (United States)

    Yang, Muhua; Liu, Weidong; Pellicane, Christina; Sahyoun, Christine; Joseph, Biny K.; Gallo-Ebert, Christina; Donigan, Melissa; Pandya, Devanshi; Giordano, Caroline; Bata, Adam; Nickels, Joseph T.

    2014-01-01

    Dysregulation of cholesterol homeostasis is associated with various metabolic diseases, including atherosclerosis and type 2 diabetes. The sterol response element binding protein (SREBP)-2 transcription factor induces the expression of genes involved in de novo cholesterol biosynthesis and low density lipoprotein (LDL) uptake, thus it plays a crucial role in maintaining cholesterol homeostasis. Here, we found that overexpressing microRNA (miR)-185 in HepG2 cells repressed SREBP-2 expression and protein level. miR-185-directed inhibition caused decreased SREBP-2-dependent gene expression, LDL uptake, and HMG-CoA reductase activity. In addition, we found that miR-185 expression was tightly regulated by SREBP-1c, through its binding to a single sterol response element in the miR-185 promoter. Moreover, we found that miR-185 expression levels were elevated in mice fed a high-fat diet, and this increase correlated with an increase in total cholesterol level and a decrease in SREBP-2 expression and protein. Finally, we found that individuals with high cholesterol had a 5-fold increase in serum miR-185 expression compared with control individuals. Thus, miR-185 controls cholesterol homeostasis through regulating SREBP-2 expression and activity. In turn, SREBP-1c regulates miR-185 expression through a complex cholesterol-responsive feedback loop. Thus, a novel axis regulating cholesterol homeostasis exists that exploits miR-185-dependent regulation of SREBP-2 and requires SREBP-1c for function. PMID:24296663

  16. Effect of Porphyromonas gingivalis infection on post-transcriptional regulation of the low-density lipoprotein receptor in mice

    Directory of Open Access Journals (Sweden)

    Miyazawa Haruna

    2012-09-01

    Full Text Available Abstract Background Periodontal disease is suggested to increase the risk of atherothrombotic disease by inducing dyslipidemia. Recently, we demonstrated that proprotein convertase subtilisin/kexin type 9 (PCSK9, which is known to play a critical role in the regulation of circulating low-density lipoprotein (LDL cholesterol levels, is elevated in periodontitis patients. However, the underlying mechanisms of elevation of PCSK9 in periodontitis patients are largely unknown. Here, we explored whether Porphyromonas gingivalis, a representative periodontopathic bacterium, -induced inflammatory response regulates serum PCSK9 and cholesterol levels using animal models. Methods We infected C57BL/6 mice intraperitoneally with Porphyromonas gingivalis, a representative strain of periodontopathic bacteria, and evaluated serum PCSK9 levels and the serum lipid profile. PCSK9 and LDL receptor (LDLR gene and protein expression, as well as liver X receptors (Lxrs, inducible degrader of the LDLR (Idol, and sterol regulatory element binding transcription factor (Srebf2 gene expression, were examined in the liver. Results P. gingivalis infection induced a significant elevation of serum PCSK9 levels and a concomitant elevation of total and LDL cholesterol compared with sham-infected mice. The LDL cholesterol levels were significantly correlated with PCSK9 levels. Expression of the Pcsk9, Ldlr, and Srebf2 genes was upregulated in the livers of the P. gingivalis-infected mice compared with the sham-infected mice. Although Pcsk9 gene expression is known to be positively regulated by sterol regulatory element binding protein (SREBP2 (human homologue of Srebf2, whereas Srebf2 is negatively regulated by cholesterol, the elevated expression of Srebf2 found in the infected mice is thought to be mediated by P. gingivalis infection. Conclusions P. gingivalis infection upregulates PCSK9 production via upregulation of Srebf2, independent of cholesterol levels. Further studies

  17. Circulating levels of IGF-1 directly regulate bone growth and density

    Science.gov (United States)

    Yakar, Shoshana; Rosen, Clifford J.; Beamer, Wesley G.; Ackert-Bicknell, Cheryl L.; Wu, Yiping; Liu, Jun-Li; Ooi, Guck T.; Setser, Jennifer; Frystyk, Jan; Boisclair, Yves R.; LeRoith, Derek

    2002-01-01

    IGF-1 is a growth-promoting polypeptide that is essential for normal growth and development. In serum, the majority of the IGFs exist in a 150-kDa complex including the IGF molecule, IGF binding protein 3 (IGFBP-3), and the acid labile subunit (ALS). This complex prolongs the half-life of serum IGFs and facilitates their endocrine actions. Liver IGF-1–deficient (LID) mice and ALS knockout (ALSKO) mice exhibited relatively normal growth and development, despite having 75% and 65% reductions in serum IGF-1 levels, respectively. Double gene disrupted mice were generated by crossing LID+ALSKO mice. These mice exhibited further reductions in serum IGF-1 levels and a significant reduction in linear growth. The proximal growth plates of the tibiae of LID+ALSKO mice were smaller in total height as well as in the height of the proliferative and hypertrophic zones of chondrocytes. There was also a 10% decrease in bone mineral density and a greater than 35% decrease in periosteal circumference and cortical thickness in these mice. IGF-1 treatment for 4 weeks restored the total height of the proximal growth plate of the tibia. Thus, the double gene disruption LID+ALSKO mouse model demonstrates that a threshold concentration of circulating IGF-1 is necessary for normal bone growth and suggests that IGF-1, IGFBP-3, and ALS play a prominent role in the pathophysiology of osteoporosis. PMID:12235108

  18. CREB regulates spine density of lateral amygdala neurons: implications for memory allocation

    Directory of Open Access Journals (Sweden)

    Derya eSargin

    2013-12-01

    Full Text Available Neurons may compete against one another for integration into a memory trace. Specifically, neurons in the lateral nucleus of the amygdala with relatively higher levels of CREB seem to be preferentially allocated to a fear memory trace, while neurons with relatively decreased CREB function seem to be excluded from a fear memory trace. CREB is a ubiquitous transcription factor that modulates many diverse cellular processes, raising the question as to which of these CREB-mediated processes underlie memory allocation. CREB is implicated in modulating dendritic spine number and morphology. As dendritic spines are intimately involved in memory formation, we investigated whether manipulations of CREB function alter spine number or morphology of neurons at the time of fear conditioning. We used viral vectors to manipulate CREB function in the lateral amygdala principal neurons in mice maintained in their homecages. At the time that fear conditioning normally occurs, we observed that neurons with high levels of CREB had more dendritic spines, while neurons with low CREB function had relatively fewer spines compared to control neurons. These results suggest that the modulation of spine density provides a potential mechanism for preferential allocation of a subset of neurons to the memory trace.

  19. Regulation of low density lipoprotein receptor function in a human hepatoma cell line

    International Nuclear Information System (INIS)

    Leichtner, A.M.; Krieger, M.; Schwartz, A.L.

    1984-01-01

    Low density lipoprotein (LDL) processing was investigated in a human hepatoma-derived cell line, Hep G2. Hep G2 cells bound, internalized and degraded LDL via a saturable, high affinity pathway similar to that present in other mammalian cells. Although 80% of the uptake and degradation of 125 I-LDL was inhibited by 40-fold excess native LDL, the same concentration of methylated LDL, which cannot bind to LDL receptors, had virtually no effect on processing. When added at low concentrations, the lysosomotropic agent, chloroquine, inhibited degradation without affecting the rate of lipoprotein internalization. Receptor activity was decreased 60% by preincubation of the cells in medium containing a source of cholesterol (LDL or unesterified cholesterol) and increased 1.7-fold by preincubation with compactin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase. The Hep G2 cell line may prove a useful system both for the further study of hepatic lipoprotein metabolism and for the evaluation of new antihypercholesterolemic agents

  20. MiR-130a regulates neurite outgrowth and dendritic spine density by targeting MeCP2

    Directory of Open Access Journals (Sweden)

    Yunjia Zhang

    2016-06-01

    Full Text Available ABSTRACT MicroRNAs (miRNAs are critical for both development and function of the central nervous system. Significant evidence suggests that abnormal expression of miRNAs is associated with neurodevelopmental disorders. MeCP2 protein is an epigenetic regulator repressing or activating gene transcription by binding to methylated DNA. Both loss-of-function and gain-of-function mutations in the MECP2 gene lead to neurodevelopmental disorders such as Rett syndrome, autism and MECP2 duplication syndrome. In this study, we demonstrate that miR-130a inhibits neurite outgrowth and reduces dendritic spine density as well as dendritic complexity. Bioinformatics analyses, cell cultures and biochemical experiments indicate that miR-130a targets MECP2 and down-regulates MeCP2 protein expression. Furthermore, expression of the wild-type MeCP2, but not a loss-of-function mutant, rescues the miR-130a-induced phenotype. Our study uncovers the MECP2 gene as a previous unknown target for miR-130a, supporting that miR-130a may play a role in neurodevelopment by regulating MeCP2. Together with data from other groups, our work suggests that a feedback regulatory mechanism involving both miR-130a and MeCP2 may serve to ensure their appropriate expression and function in neural development.

  1. A microtubule inhibitor, ABT-751, induces autophagy and delays apoptosis in Huh-7 cells

    Energy Technology Data Exchange (ETDEWEB)

    Wei, Ren-Jie [Institute of Biomedical Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan (China); Department of Pathology, Kaohsiung Armed Forces General Hospital, Kaohsiung, Taiwan (China); Lin, Su-Shuan [Department of Pathology, Kaohsiung Armed Forces General Hospital, Kaohsiung, Taiwan (China); Wu, Wen-Ren [Institute of Biomedical Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan (China); Chen, Lih-Ren [Department of Biotechnology, Southern Taiwan University of Science and Technology, Tainan, Taiwan (China); Division of Physiology, Livestock Research Institute, Council of Agriculture, Taiwan (China); Li, Chien-Feng [Department of Biotechnology, Southern Taiwan University of Science and Technology, Tainan, Taiwan (China); Department of Pathology, Chi-Mei Medical Center, Tainan, Taiwan (China); National Institute of Cancer Research, National Health Research Institute, Tainan, Taiwan (China); Institute of Clinical Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan (China); Chen, Han-De; Chou, Chien-Ting; Chen, Ya-Chun [Institute of Biomedical Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan (China); Liang, Shih-Shin [Institute of Biomedical Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan (China); Department of Biotechnology, Kaohsiung Medical University, Kaohsiung, Taiwan (China); Chien, Shang-Tao [Department of Pathology, Kaohsiung Armed Forces General Hospital, Kaohsiung, Taiwan (China); Shiue, Yow-Ling, E-mail: ylshiue@mail.nsysu.edu.tw [Institute of Biomedical Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan (China); Department of Biological Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan (China); Doctoral Degree Program in Marine Biotechnology, National Sun Yat-sen University, Kaohsiung, Taiwan (China)

    2016-11-15

    The objective was to investigate the upstream mechanisms of apoptosis which were triggered by a novel anti-microtubule drug, ABT-751, in hepatocellular carcinoma-derived Huh-7 cells. Effects of ABT-751 were evaluated by immunocytochemistry, flow cytometric, alkaline comet, soft agar, immunoblotting, CytoID, green fluorescent protein-microtubule associated protein 1 light chain 3 beta detection, plasmid transfection, nuclear/cytosol fractionation, coimmunoprecipitation, quantitative reverse transcription-polymerase chain reaction, small-hairpin RNA interference and mitochondria/cytosol fractionation assays. Results showed that ABT-751 caused dysregulation of microtubule, collapse of mitochondrial membrane potential, generation of reactive oxygen species (ROS), DNA damage, G{sub 2}/M cell cycle arrest, inhibition of anchorage-independent cell growth and apoptosis in Huh-7 cells. ABT-751 also induced early autophagy via upregulation of nuclear TP53 and downregulation of the AKT serine/threonine kinase (AKT)/mechanistic target of rapamycin (MTOR) pathway. Through modulation of the expression levels of DNA damage checkpoint proteins and G{sub 2}/M cell cycle regulators, ABT-751 induced G{sub 2}/M cell cycle arrest. Subsequently, ABT-751 triggered apoptosis with marked downregulation of B-cell CLL/lymphoma 2, upregulation of mitochondrial BCL2 antagonist/killer 1 and BCL2 like 11 protein levels, and cleavages of caspase 8 (CASP8), CASP9, CASP3 and DNA fragmentation factor subunit alpha proteins. Suppression of ROS significantly decreased ABT-751-induced autophagic and apoptotic cells. Pharmacological inhibition of autophagy significantly increased the percentages of ABT-751-induced apoptotic cells. The autophagy induced by ABT-751 plays a protective role to postpone apoptosis by exerting adaptive responses following microtubule damage, ROS and/or impaired mitochondria. - Highlights: • An anti-microtubule agent, ABT-751, induces autophagy and apoptosis in Huh-7 cells.

  2. The hydrometeorological implications of zoning laws: Can land use regulations of urban density and sprawl improve a city's resilience?

    Science.gov (United States)

    Bou-Zeid, E.; Ryu, Y. H.; Smith, J. A.; Newburn, D. A.

    2015-12-01

    The intensification of heat waves and of the hydrological cycle due to global climate change pose particularly high risks to urban residents. Cities are already hotter than their surroundings due to the urban heat island effect and are known to result in local intensification of rainfall and flooding due to their coupled impacts on the surface and the lower atmosphere. These interacting local and global changes can adversely affect the health and well being of urban residents, and city administrators are increasing efforts to mitigate and adapt to the potential disruptions though various infrastructure and preparedness programs. However, as cities worldwide continue to expand, a key decision is how to manage that urban sprawl and regulate its spatial features to aid in the mitigation and adaptation effort. This study assesses whether alternative zoning regulations that modify the density and extent of a metropolitan region, but have a minimal impact on total population and demographic growth, have an appreciable impact on its response to extreme weather events, and as such, whether they can be used to increase urban resilience. We consider Baltimore (the city and its surrounding suburbs), which in 1967 adopted one of the first urban growth boundaries (UGBs) in the United States, as our test case. Departing from the urban extent circa 1900, we create alternative land use patterns that, compared to the actual current land use baseline, would have resulted from drastically different policy scenarios and approaches to zoning that the city would have undertaken. We consider various alternatives where the city is smaller and denser, due to stricter regulation, versus larger and less dense than the actual baseline, while maintaining the same total population. Our findings indicate that lower densities have significant benefits: compared to the current landscape and to denser patterns, they reduce both extreme temperatures during heat waves and spatio-temporal rainfall

  3. Melanophores for microtubule dynamics and motility assays.

    Science.gov (United States)

    Ikeda, Kazuho; Semenova, Irina; Zhapparova, Olga; Rodionov, Vladimir

    2010-01-01

    Microtubules (MTs) are cytoskeletal structures essential for cell division, locomotion, intracellular transport, and spatial organization of the cytoplasm. In most interphase cells, MTs are organized into a polarized radial array with minus-ends clustered at the centrosome and plus-ends extended to the cell periphery. This array directs transport of organelles driven by MT-based motor proteins that specifically move either to plus- or to minus-ends. Along with using MTs as tracks for cargo, motor proteins can organize MTs into a radial array in the absence of the centrosome. Transport of organelles and motor-dependent radial organization of MTs require MT dynamics, continuous addition and loss of tubulin subunits at minus- and plus-ends. A unique experimental system for studying the role of MT dynamics in these processes is the melanophore, which provides a useful tool for imaging of both dynamic MTs and moving membrane organelles. Melanophores are filled with pigment granules that are synchronously transported by motor proteins in response to hormonal stimuli. The flat shape of the cell and the radial organization of MTs facilitate imaging of dynamic MT plus-ends and monitoring of their interaction with membrane organelles. Microsurgically produced cytoplasmic fragments of melanophores are used to study the centrosome-independent rearrangement of MTs into a radial array. Here we describe the experimental approaches to study the role of MT dynamics in intracellular transport and centrosome-independent MT organization in melanophores. We focus on the preparation of cell cultures, microsurgery and microinjection, fluorescence labeling, and live imaging of MTs. 2010 Elsevier Inc. All rights reserved.

  4. Quantitative Analysis of Tau-Microtubule Interaction Using FRET

    Directory of Open Access Journals (Sweden)

    Isabelle L. Di Maïo

    2014-08-01

    Full Text Available The interaction between the microtubule associated protein, tau and the microtubules is investigated. A fluorescence resonance energy transfer (FRET assay was used to determine the distance separating tau to the microtubule wall, as well as the binding parameters of the interaction. By using microtubules stabilized with Flutax-2 as donor and tau labeled with rhodamine as acceptor, a donor-to-acceptor distance of 54 ± 1 Å was found. A molecular model is proposed in which Flutax-2 is directly accessible to tau-rhodamine molecules for energy transfer. By titration, we calculated the stoichiometric dissociation constant to be equal to 1.0 ± 0.5 µM. The influence of the C-terminal tails of αβ-tubulin on the tau-microtubule interaction is presented once a procedure to form homogeneous solution of cleaved tubulin has been determined. The results indicate that the C-terminal tails of α- and β-tubulin by electrostatic effects and of recruitment seem to be involved in the binding mechanism of tau.

  5. Kinesin expands and stabilizes the GDP-microtubule lattice

    Science.gov (United States)

    Peet, Daniel R.; Burroughs, Nigel J.; Cross, Robert A.

    2018-05-01

    Kinesin-1 is a nanoscale molecular motor that walks towards the fast-growing (plus) ends of microtubules, hauling molecular cargo to specific reaction sites in cells. Kinesin-driven transport is central to the self-organization of eukaryotic cells and shows great promise as a tool for nano-engineering1. Recent work hints that kinesin may also play a role in modulating the stability of its microtubule track, both in vitro2,3 and in vivo4, but the results are conflicting5-7 and the mechanisms are unclear. Here, we report a new dimension to the kinesin-microtubule interaction, whereby strong-binding state (adenosine triphosphate (ATP)-bound and apo) kinesin-1 motor domains inhibit the shrinkage of guanosine diphosphate (GDP) microtubules by up to two orders of magnitude and expand their lattice spacing by 1.6%. Our data reveal an unexpected mechanism by which the mechanochemical cycles of kinesin and tubulin interlock, and so allow motile kinesins to influence the structure, stability and mechanics of their microtubule track.

  6. APC and Smad7 link TGFβ type I receptors to the microtubule system to promote cell migration

    Science.gov (United States)

    Ekman, Maria; Mu, Yabing; Lee, So Young; Edlund, Sofia; Kozakai, Takaharu; Thakur, Noopur; Tran, Hoanh; Qian, Jiang; Groeden, Joanna; Heldin, Carl-Henrik; Landström, Maréne

    2012-01-01

    Cell migration occurs by activation of complex regulatory pathways that are spatially and temporally integrated in response to extracellular cues. Binding of adenomatous polyposis coli (APC) to the microtubule plus ends in polarized cells is regulated by glycogen synthase kinase 3β (GSK-3β). This event is crucial for establishment of cell polarity during directional migration. However, the role of APC for cellular extension in response to extracellular signals is less clear. Smad7 is a direct target gene for transforming growth factor-β (TGFβ) and is known to inhibit various TGFβ-induced responses. Here we report a new function for Smad7. We show that Smad7 and p38 mitogen–activated protein kinase together regulate the expression of APC and cell migration in prostate cancer cells in response to TGFβ stimulation. In addition, Smad7 forms a complex with APC and acts as an adaptor protein for p38 and GSK-3β kinases to facilitate local TGFβ/p38–dependent inactivation of GSK-3β, accumulation of β-catenin, and recruitment of APC to the microtubule plus end in the leading edge of migrating prostate cancer cells. Moreover, the Smad7–APC complex links the TGFβ type I receptor to the microtubule system to regulate directed cellular extension and migratory responses evoked by TGFβ. PMID:22496417

  7. Microtubule–microtubule sliding by kinesin-1 is essential for normal cytoplasmic streaming in Drosophila oocytes

    Science.gov (United States)

    Lu, Wen; Winding, Michael; Lakonishok, Margot; Wildonger, Jill

    2016-01-01

    Cytoplasmic streaming in Drosophila oocytes is a microtubule-based bulk cytoplasmic movement. Streaming efficiently circulates and localizes mRNAs and proteins deposited by the nurse cells across the oocyte. This movement is driven by kinesin-1, a major microtubule motor. Recently, we have shown that kinesin-1 heavy chain (KHC) can transport one microtubule on another microtubule, thus driving microtubule–microtubule sliding in multiple cell types. To study the role of microtubule sliding in oocyte cytoplasmic streaming, we used a Khc mutant that is deficient in microtubule sliding but able to transport a majority of cargoes. We demonstrated that streaming is reduced by genomic replacement of wild-type Khc with this sliding-deficient mutant. Streaming can be fully rescued by wild-type KHC and partially rescued by a chimeric motor that cannot move organelles but is active in microtubule sliding. Consistent with these data, we identified two populations of microtubules in fast-streaming oocytes: a network of stable microtubules anchored to the actin cortex and free cytoplasmic microtubules that moved in the ooplasm. We further demonstrated that the reduced streaming in sliding-deficient oocytes resulted in posterior determination defects. Together, we propose that kinesin-1 slides free cytoplasmic microtubules against cortically immobilized microtubules, generating forces that contribute to cytoplasmic streaming and are essential for the refinement of posterior determinants. PMID:27512034

  8. Mutations in Human Tubulin Proximal to the Kinesin-Binding Site Alter Dynamic Instability at Microtubule Plus- and Minus-Ends

    Energy Technology Data Exchange (ETDEWEB)

    Ti, Shih-Chieh; Pamula, Melissa C.; Howes, Stuart C.; Duellberg, Christian; Cade, Nicholas I.; Kleiner, Ralph E.; Forth, Scott; Surrey, Thomas; Nogales, Eva; Kapoor, Tarun M.

    2016-04-01

    The assembly of microtubule-based cellular structures depends on regulated tubulin polymerization and directional transport. In this research, we have purified and characterized tubulin heterodimers that have human β-tubulin isotype III (TUBB3), as well as heterodimers with one of two β-tubulin mutations (D417H or R262H). Both point mutations are proximal to the kinesin-binding site and have been linked to an ocular motility disorder in humans. Compared to wild-type, microtubules with these mutations have decreased catastrophe frequencies and increased average lifetimes of plus- and minus-end-stabilizing caps. Importantly, the D417H mutation does not alter microtubule lattice structure or Mal3 binding to growing filaments. Instead, this mutation reduces the affinity of tubulin for TOG domains and colchicine, suggesting that the distribution of tubulin heterodimer conformations is changed. Together, our findings reveal how residues on the surface of microtubules, distal from the GTP-hydrolysis site and inter-subunit contacts, can alter polymerization dynamics at the plus- and minus-ends of microtubules.

  9. Mouse genome-wide association and systems genetics identify Asxl2 as a regulator of bone mineral density and osteoclastogenesis.

    Directory of Open Access Journals (Sweden)

    Charles R Farber

    2011-04-01

    Full Text Available Significant advances have been made in the discovery of genes affecting bone mineral density (BMD; however, our understanding of its genetic basis remains incomplete. In the current study, genome-wide association (GWA and co-expression network analysis were used in the recently described Hybrid Mouse Diversity Panel (HMDP to identify and functionally characterize novel BMD genes. In the HMDP, a GWA of total body, spinal, and femoral BMD revealed four significant associations (-log10P>5.39 affecting at least one BMD trait on chromosomes (Chrs. 7, 11, 12, and 17. The associations implicated a total of 163 genes with each association harboring between 14 and 112 genes. This list was reduced to 26 functional candidates by identifying those genes that were regulated by local eQTL in bone or harbored potentially functional non-synonymous (NS SNPs. This analysis revealed that the most significant BMD SNP on Chr. 12 was a NS SNP in the additional sex combs like-2 (Asxl2 gene that was predicted to be functional. The involvement of Asxl2 in the regulation of bone mass was confirmed by the observation that Asxl2 knockout mice had reduced BMD. To begin to unravel the mechanism through which Asxl2 influenced BMD, a gene co-expression network was created using cortical bone gene expression microarray data from the HMDP strains. Asxl2 was identified as a member of a co-expression module enriched for genes involved in the differentiation of myeloid cells. In bone, osteoclasts are bone-resorbing cells of myeloid origin, suggesting that Asxl2 may play a role in osteoclast differentiation. In agreement, the knockdown of Asxl2 in bone marrow macrophages impaired their ability to form osteoclasts. This study identifies a new regulator of BMD and osteoclastogenesis and highlights the power of GWA and systems genetics in the mouse for dissecting complex genetic traits.

  10. CENTROSOMES AND MICROTUBULES DURING MEIOSIS IN THE MUSHROOM BOLETUS RUBINELLUS

    Science.gov (United States)

    McLaughlin, David J.

    1971-01-01

    The double centrosome in the basidium of Boletus rubinellus has been observed in three planes with the electron microscope at interphase preceding nuclear fusion, at prophase I, and at interphase I. It is composed of two components connected by a band-shaped middle part. At anaphase I a single, enlarged centrosome is found at the spindle pole, which is attached to the cell membrane. Microtubules mainly oriented parallel to the longitudinal axis of the basidium are present at prefusion, prophase I and interphase I. Cytoplasmic microtubules are absent when the spindle is present. The relationship of the centrosome in B. rubinellus to that in other organisms and the role of the cytoplasmic microtubules are discussed. PMID:4329156

  11. The engine of microtubule dynamics comes into focus.

    Science.gov (United States)

    Mitchison, T J

    2014-05-22

    In this issue, Alushin et al. report high-resolution structures of three states of the microtubule lattice: GTP-bound, which is stable to depolymerization; unstable GDP-bound; and stable Taxol and GDP-bound. By comparing these structures at near-atomic resolution, they are able to propose a detailed model for how GTP hydrolysis destabilizes the microtubule and thus powers dynamic instability and chromosome movement. Destabilization of cytoskeleton filaments by nucleotide hydrolysis is an important general principle in cell dynamics, and this work represents a major step forward on a problem with a long history. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. Tau can switch microtubule network organizations: from random networks to dynamic and stable bundles.

    Science.gov (United States)

    Prezel, Elea; Elie, Auréliane; Delaroche, Julie; Stoppin-Mellet, Virginie; Bosc, Christophe; Serre, Laurence; Fourest-Lieuvin, Anne; Andrieux, Annie; Vantard, Marylin; Arnal, Isabelle

    2018-01-15

    In neurons, microtubule networks alternate between single filaments and bundled arrays under the influence of effectors controlling their dynamics and organization. Tau is a microtubule bundler that stabilizes microtubules by stimulating growth and inhibiting shrinkage. The mechanisms by which tau organizes microtubule networks remain poorly understood. Here, we studied the self-organization of microtubules growing in the presence of tau isoforms and mutants. The results show that tau's ability to induce stable microtubule bundles requires two hexapeptides located in its microtubule-binding domain and is modulated by its projection domain. Site-specific pseudophosphorylation of tau promotes distinct microtubule organizations: stable single microtubules, stable bundles, or dynamic bundles. Disease-related tau mutations increase the formation of highly dynamic bundles. Finally, cryo-electron microscopy experiments indicate that tau and its variants similarly change the microtubule lattice structure by increasing both the protofilament number and lattice defects. Overall, our results uncover novel phosphodependent mechanisms governing tau's ability to trigger microtubule organization and reveal that disease-related modifications of tau promote specific microtubule organizations that may have a deleterious impact during neurodegeneration. © 2018 Prezel, Elie, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  13. Dietary flavonoid fisetin binds to β-tubulin and disrupts microtubule dynamics in prostate cancer cells

    OpenAIRE

    Mukhtar, Eiman; Adhami, Vaqar Mustafa; Sechi, Mario; Mukhtar, Hasan

    2015-01-01

    Microtubule targeting based therapies have revolutionized cancer treatment; however, resistance and side effects remain a major limitation. Therefore, novel strategies that can overcome these limitations are urgently needed. We made a novel discovery that fisetin, a hydroxyflavone, is a microtubule stabilizing agent. Fisetin binds to tubulin and stabilizes microtubules with binding characteristics far superior than paclitaxel. Surface plasmon resonance and computational docking studies sugges...

  14. Microtubule protein ADP-ribosylation in vitro leads to assembly inhibition and rapid depolymerization

    Energy Technology Data Exchange (ETDEWEB)

    Scaife, R.M. (Fred Hutchinson Cancer Research Center, Seattle, WA (United States)); Wilson, L. (Univ. of California, Santa Barbara (United States)); Purich, D.L. (Univ. of Florida, Gainesville (United States))

    1992-01-14

    Bovine brain microtubule protein, containing both tubulin and microtubule-associated proteins, undergoes ADP-ribosylation in the presence of ({sup 14}C)NAD{sup +} and a turkey erythrocyte mono-ADP-ribosyltransferase in vitro. The modification reaction could be demonstrated in crude brain tissue extracts where selective ADP-ribosylation of both the {alpha} and {beta} chains of tubulin and of the high molecular weight microtubule-associated protein MAP-2 occurred. In experiments with purified microtubule protein, tubulin dimer, the high molecular weight microtubule-associated protein MAP-2, and another high molecular weight microtubule-associated protein which may be a MAP-1 species were heavily labeled. Tubulin and MAP-2 incorporated ({sup 14}C)ADP-ribose to an average extent of approximately 2.4 and 30 mol of ADP-ribose/mol of protein, respectively. Assembly of microtubule protein into microtubules in vitro was inhibited by ADP-ribosylation, and incubation of assembled steady-state microtubules with ADP-ribosyltransferase and NAD{sup +} resulted in rapid depolymerization of the microtubules. Thus, the eukaryotic enzyme can ADP-ribosylate tubulin and microtubule-associated proteins to much greater extents than previously observed with cholera and pertussis toxins, and the modification can significantly modulate microtubule assembly and disassembly.

  15. TCTEX1D4, a novel protein phosphatase 1 interactor: connecting the phosphatase to the microtubule network

    Science.gov (United States)

    Korrodi-Gregório, Luís; Vieira, Sandra I.; Esteves, Sara L. C.; Silva, Joana V.; Freitas, Maria João; Brauns, Ann-Kristin; Luers, Georg; Abrantes, Joana; Esteves, Pedro J.; da Cruz e Silva, Odete A. B.; Fardilha, Margarida; da Cruz e Silva, Edgar F.

    2013-01-01

    Summary Reversible phosphorylation plays an important role as a mechanism of intracellular control in eukaryotes. PPP1, a major eukaryotic Ser/Thr-protein phosphatase, acquires its specificity by interacting with different protein regulators, also known as PPP1 interacting proteins (PIPs). In the present work we characterized a physiologically relevant PIP in testis. Using a yeast two-hybrid screen with a human testis cDNA library, we identified a novel PIP of PPP1CC2 isoform, the T-complex testis expressed protein 1 domain containing 4 (TCTEX1D4) that has recently been described as a Tctex1 dynein light chain family member. The overlay assays confirm that TCTEX1D4 interacts with the different spliced isoforms of PPP1CC. Also, the binding domain occurs in the N-terminus, where a consensus PPP1 binding motif (PPP1BM) RVSF is present. The distribution of TCTEX1D4 in testis suggests its involvement in distinct functions, such as TGFβ signaling at the blood–testis barrier and acrosome cap formation. Immunofluorescence in human ejaculated sperm shows that TCTEX1D4 is present in the flagellum and in the acrosome region of the head. Moreover, TCTEX1D4 and PPP1 co-localize in the microtubule organizing center (MTOC) and microtubules in cell cultures. Importantly, the TCTEX1D4 PPP1BM seems to be relevant for complex formation, for PPP1 retention in the MTOC and movement along microtubules. These novel results open new avenues to possible roles of this dynein, together with PPP1. In essence TCTEX1D4/PPP1C complex appears to be involved in microtubule dynamics, sperm motility, acrosome reaction and in the regulation of the blood–testis barrier. PMID:23789093

  16. Inhibition of glycogen synthase kinase-3 reduces extension of the axonal leading process by destabilizing microtubules in cerebellar granule neurons.

    Science.gov (United States)

    Inami, Yoshihiro; Omura, Mitsuru; Kubota, Kenta; Konishi, Yoshiyuki

    2018-07-01

    Recent studies have uncovered various molecules that play key roles in neuronal morphogenesis. Nevertheless, the mechanisms underlying the neuron-type-dependent regulation of morphogenesis remain unknown. We have previously reported that inhibition of glycogen synthase kinase-3 (GSK3) markedly reduced axonal length of cerebellar granule neurons (CGNs) in a neuron-type-dependent manner. In the present study, we investigated the mechanisms by which the growth of CGN axons was severely suppressed upon GSK3 inhibition. Using time-lapse imaging of cultured CGNs at early morphogenesis, we found that extension of the leading process was severely inhibited by the pharmacological inhibition of GSK3. The rate of somal migration was also reduced with a GSK3 inhibitor in dissociated culture as well as in microexplant culture. In addition, CGNs ectopically expressed with a catalytically inactive mutant of GSK3 exhibited a migration defect in vivo. In axonal leading processes of CGNs, detyrosination and acetylation of α-tubulin, which are known to correlate with microtubule stability, were decreased by GSK3 inhibition. A photoconversion analysis found that inhibition of GSK3 increases the turnover of microtubules. Furthermore, in the presence of paclitaxel, a microtubule-stabilizing reagent, inhibition of GSK3 recovered the axonal leading process extension that was reduced by paclitaxel. Our results suggest that GSK3 supports the extension of axonal processes by stabilizing microtubules, contrary to its function in other neuron-types, lending mechanical insight into neuron-type-dependent morphological regulation. Copyright © 2018 Elsevier B.V. All rights reserved.

  17. How biological microtubules may avoid decoherence

    International Nuclear Information System (INIS)

    Hameroff, S.

    2005-01-01

    Full text: Entangled superpositions persisting for hundreds of milliseconds in protein assemblies such as microtubules (MTs) are proposed in biological functions, e.g. quantum computation relevant to consciousness in the Penrose-Hameroff 'Orch OR' model. Cylindrical polymers of the protein tubulin, MTs organize cell activities. The obvious question is how biological quantum states could avoid decoherence, e.g. in the brain at 37.6 degrees centigrade. Screening/sheelding: tubulin protein states/functions are governed by van der Waals London forces, quantum interactions among clouds of delocalizable electrons in nonpolar 'hydrophobic' intra-protein pockets screened from external van der Waals thermal interactions. Such pockets include amino acid resonance structures benzene and indole rings. (Anesthetic gases erase consciousness solely by interfering with London forces in hydrophobic pockets in various brain proteins). Hence tubulin states may act as superpositioned qubits also shielded at the MT level by counter-ion Debye plasma layers (due to charged C-termini tails on tubulin) and by water-ordering actin gels which embed MTs in a quasi-solid. Biological systems may also exploit thermodynamic gradients to give extremely low effective temperatures. Decoherence free subspaces: paradoxically, a system coupled strongly to its environment through certain degrees of freedom can effectively 'freeze' other degrees of freedom (quantum Zeno effect), enabling coherent superpositions and entanglement to persist. Metabolic energy supplied to MT collective dynamics (e.g. Froehlich coherence) can cause Bose-Einstein condenzation and counter decoherence as lasers avoid decoherence at room temperature. Topological quantum error correction: MT lattice structure reveals various helical winding paths through adjacent tubulins which follow the Fibonacci series. Propagation/interactions of quasi-particles along these paths may process information. As proposed by Kitaev (1997), various

  18. Prion protein inhibits microtubule assembly by inducing tubulin oligomerization

    International Nuclear Information System (INIS)

    Nieznanski, Krzysztof; Podlubnaya, Zoya A.; Nieznanska, Hanna

    2006-01-01

    A growing body of evidence points to an association of prion protein (PrP) with microtubular cytoskeleton. Recently, direct binding of PrP to tubulin has also been found. In this work, using standard light scattering measurements, sedimentation experiments, and electron microscopy, we show for First time the effect of a direct interaction between these proteins on tubulin polymerization. We demonstrate that full-length recombinant PrP induces a rapid increase in the turbidity of tubulin diluted below the critical concentration for microtubule assembly. This effect requires magnesium ions and is weakened by NaCl. Moreover, the PrP-induced light scattering structures of tubulin are cold-stable. In preparations of diluted tubulin incubated with PrP, electron microscopy revealed the presence of ∼50 nm disc-shaped structures not reported so far. These unique tubulin oligomers may form large aggregates. The effect of PrP is more pronounced under the conditions promoting microtubule formation. In these tubulin samples, PrP induces formation of the above oligomers associated with short protofilaments and sheets of protofilaments into aggregates. Noticeably, this is accompanied by a significant reduction of the number and length of microtubules. Hence, we postulate that prion protein may act as an inhibitor of microtubule assembly by inducing formation of stable tubulin oligomers

  19. Neuronal microtubule organization: from minus end to plus end

    NARCIS (Netherlands)

    Yau, K.W.

    2016-01-01

    Neurons are highly polarized cells consisting of a dendritic part and axonal part. Dendrites receive signals from other cells while axons transmit signals to other cells. In this thesis, mostly hippocampal neurons from rat embryos are used to study fundamental aspects of the microtubule organization

  20. Microtubules in cell migration, morphogenesis and metabolism: Making the connections

    NARCIS (Netherlands)

    Noordstra, I.

    2017-01-01

    Cell polarity refers to a fundamental property of eukaryotic cells, in which cellular components and structures are organized in an asymmetric fashion. In order to control their polarity, cells make use of microtubules, hollow polymers that extend throughout the cytoplasm. Due to the asymmetry of

  1. Capture of microtubule plus-ends at the actin cortex promotes axophilic neuronal migration by enhancing microtubule tension in the leading process.

    Science.gov (United States)

    Hutchins, B Ian; Wray, Susan

    2014-01-01

    Microtubules are a critical part of neuronal polarity and leading process extension, thus microtubule movement plays an important role in neuronal migration. However, the dynamics of microtubules during the forward movement of the nucleus into the leading process (nucleokinesis) is unclear and may be dependent on the cell type and mode of migration used. In particular, little is known about cytoskeletal changes during axophilic migration, commonly used in anteroposterior neuronal migration. We recently showed that leading process actin flow in migrating GnRH neurons is controlled by a signaling cascade involving IP3 receptors, CaMKK, AMPK, and RhoA. In the present study, microtubule dynamics were examined in GnRH neurons. Failure of the migration of these cells leads to the neuroendocrine disorder Kallmann Syndrome. Microtubules translocated forward along the leading process shaft during migration, but reversed direction and moved toward the nucleus when migration stalled. Blocking calcium release through IP3 receptors halted migration and induced the same reversal of microtubule translocation, while blocking cortical actin flow prevented microtubules from translocating toward the distal leading process. Super-resolution imaging revealed that microtubule plus-end tips are captured at the actin cortex through calcium-dependent mechanisms. This work shows that cortical actin flow draws the microtubule network forward through calcium-dependent capture in order to promote nucleokinesis, revealing a novel mechanism engaged by migrating neurons to facilitate movement.

  2. Microtubule depolymerization normalizes in vivo myocardial contractile function in dogs with pressure-overload left ventricular hypertrophy

    Science.gov (United States)

    Koide, M.; Hamawaki, M.; Narishige, T.; Sato, H.; Nemoto, S.; DeFreyte, G.; Zile, M. R.; Cooper G, I. V.; Carabello, B. A.

    2000-01-01

    BACKGROUND: Because initially compensatory myocardial hypertrophy in response to pressure overloading may eventually decompensate to myocardial failure, mechanisms responsible for this transition have long been sought. One such mechanism established in vitro is densification of the cellular microtubule network, which imposes a viscous load that inhibits cardiocyte contraction. METHODS AND RESULTS: In the present study, we extended this in vitro finding to the in vivo level and tested the hypothesis that this cytoskeletal abnormality is important in the in vivo contractile dysfunction that occurs in experimental aortic stenosis in the adult dog. In 8 dogs in which gradual stenosis of the ascending aorta had caused severe left ventricular (LV) pressure overloading (gradient, 152+/-16 mm Hg) with contractile dysfunction, LV function was measured at baseline and 1 hour after the intravenous administration of colchicine. Cardiocytes obtained by biopsy before and after in vivo colchicine administration were examined in tandem. Microtubule depolymerization restored LV contractile function both in vivo and in vitro. CONCLUSIONS: These and additional corroborative data show that increased cardiocyte microtubule network density is an important mechanism for the ventricular contractile dysfunction that develops in large mammals with adult-onset pressure-overload-induced cardiac hypertrophy.

  3. Cdk1-Cyclin B1-mediated Phosphorylation of Tumor-associated Microtubule-associated Protein/Cytoskeleton-associated Protein 2 in Mitosis*

    OpenAIRE

    Uk Hong, Kyung; Kim, Hyun-Jun; Kim, Hyo-Sil; Seong, Yeon-Sun; Hong, Kyeong-Man; Bae, Chang-Dae; Park, Joobae

    2009-01-01

    During mitosis, establishment of structurally and functionally sound bipolar spindles is necessary for maintaining the fidelity of chromosome segregation. Tumor-associated microtubule-associated protein (TMAP), also known as cytoskeleton-associated protein 2 (CKAP2), is a mitotic spindle-associated protein whose level is frequently up-regulated in various malignancies. Previous reports have suggested that TMAP is a potential regulator of mitotic spindle assembly and dynamics and that it is re...

  4. Coin Tossing Explains the Activity of Opposing Microtubule Motors on Phagosomes.

    Science.gov (United States)

    Sanghavi, Paulomi; D'Souza, Ashwin; Rai, Ashim; Rai, Arpan; Padinhatheeri, Ranjith; Mallik, Roop

    2018-05-07

    How the opposing activity of kinesin and dynein motors generates polarized distribution of organelles inside cells is poorly understood and hotly debated [1, 2]. Possible explanations include stochastic mechanical competition [3, 4], coordinated regulation by motor-associated proteins [5-7], mechanical activation of motors [8], and lipid-induced organization [9]. Here, we address this question by using phagocytosed latex beads to generate early phagosomes (EPs) that move bidirectionally along microtubules (MTs) in an in vitro assay [9]. Dynein/kinesin activity on individual EPs is recorded as real-time force generation of the motors against an optical trap. Activity of one class of motors frequently coincides with, or is rapidly followed by opposite motors. This leads to frequent and rapid reversals of EPs in the trap. Remarkably, the choice between dynein and kinesin can be explained by the tossing of a coin. Opposing motors therefore appear to function stochastically and independently of each other, as also confirmed by observing no effect on kinesin function when dynein is inhibited on the EPs. A simple binomial probability calculation based on the geometry of EP-microtubule contact explains the observed activity of dynein and kinesin on phagosomes. This understanding of intracellular transport in terms of a hypothetical coin, if it holds true for other cargoes, provides a conceptual framework to explain the polarized localization of organelles inside cells. Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  5. Inhibition of the Ras-Net (Elk-3) pathway by a novel pyrazole that affects microtubules.

    Science.gov (United States)

    Wasylyk, Christine; Zheng, Hong; Castell, Christelle; Debussche, Laurent; Multon, Marie-Christine; Wasylyk, Bohdan

    2008-03-01

    Net (Elk-3/SAP-2/Erp) is a transcription factor that is phosphorylated and activated by the Ras-extracellular signal-regulated kinase (Erk) signaling pathway and is involved in wound healing, angiogenesis, and tumor growth. In a cell-based screen for small molecule inhibitors of Ras activation of Net transcriptional activity, we identified a novel pyrazole, XRP44X. XRP44X inhibits fibroblast growth factor 2 (FGF-2)-induced Net phosphorylation by the Ras-Erk signaling upstream from Ras. It also binds to the colchicine-binding site of tubulin, depolymerizes microtubules, stimulates cell membrane blebbing, and affects the morphology of the actin skeleton. Interestingly, Combretastin-A4, which produces similar effects on the cytoskeleton, also inhibits FGF-2 Ras-Net signaling. This differs from other classes of agents that target microtubules, which have either little effect (vincristine) or no effect (docetaxel and nocodazole) on the Ras-Net pathway. XRP44X inhibits various cellular properties, including cell growth, cell cycle progression, and aortal sprouting, similar to other molecules that bind to the tubulin colchicine site. XRP44X has the potentially interesting property of connecting two important pathways involved in cell transformation and may thereby represent an interesting class of molecules that could be developed for cancer treatment.

  6. Heuristic consequences of a load of oxygen in microtubules.

    Science.gov (United States)

    Denis, Pierre A

    2014-04-01

    The current cell oxygen paradigm shows some major gaps that have not yet been resolved. Something seems to be lacking for the comprehensive statement of the oxygen distribution in the cell, especially the low cytoplasmic oxygen level. The entrapment of oxygen in microtubules (MTs) resolves the latter observation, as well as the occurrence of an extensive cytoplasmic foam formation. It leads to a novel oxygen paradigm for cells. During the steady-state treadmilling, the mobile cavity would absorb oxygenated cytoplasm forward, entrap gas nuclei and concentrate them. A fluorescence method is described to confirm the in vitro load of oxygen in MTs during their periodic growths and shrinkages. The latter operating mechanism is called the gas dynamic instability (GDI) of MTs. Several known biosystems could rest on the GDI. (1) The GTP-cap is linked with the gas meniscus encountered in a tube filled with gas. The GTP hydrolysis is linked to the conformational change of the GTPase domain according to the bubble pressure, and to the shaking of protofilaments with gas particles (soliton-like waves). (2) The GDI provides a free energy water pump because water molecules have to escape from MT pores when foam concentrates within the MT. Beside ATP hydrolysis in motor proteins, the GDI provides an additional driving force in intracellular transport of cargo. The water streams flowing from the MT through slits organize themselves as water layers between the cargo and the MT surface, and break ionic bridges. It makes the cargo glide over a water rail. (3) The GDI provides a universal motor for chromosome segregation because the depolymerization of kinetochorial MTs is expected to generate a strong cytoplasmic foam. Chromosomes are sucked up according to the pressure difference (or density difference) applied to opposite sides of the kinetochore, which is in agreement with Archimedes' principle of buoyancy. Non-kinetochorial MTs reabsorb foam during GDI. Last, the mitotic spindle

  7. The neurosteroid pregnenolone reverts microtubule derangement induced by the loss of a functional CDKL5-IQGAP1 complex.

    Science.gov (United States)

    Barbiero, Isabella; Peroni, Diana; Tramarin, Marco; Chandola, Chetan; Rusconi, Laura; Landsberger, Nicoletta; Kilstrup-Nielsen, Charlotte

    2017-09-15

    CDKL5 is a protein kinase that plays a key role for neuronal functions as testified by the onset of complex neuronal dysfunctions in patients with genetic lesions in CDKL5. Here we identify a novel interactor of CDKL5, IQGAP1, a fundamental regulator of cell migration and polarity. In accordance with a functional role of this interaction, depletion of CDKL5 impairs cell migration and impedes the localization of IQGAP1 at the leading edge. Moreover, we demonstrate that CDKL5 is required for IQGAP1 to form a functional complex with its effectors, Rac1 and the microtubule plus end tracking protein CLIP170. These defects eventually impact on the microtubule association of CLIP170, thus deranging their dynamics. CLIP170 is a cellular target of the neurosteroid pregnenolone; by blocking CLIP170 in its active conformation, pregnenolone is capable of restoring the microtubule association of CLIP170 in CDKL5 deficient cells and rescuing morphological defects in neurons devoid of CDKL5. These findings provide novel insights into CDKL5 functions and pave the way for target-specific therapeutic strategies for individuals affected with CDKL5-disorder. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  8. The nucleation of microtubules in Aspergillus nidulans germlings

    Directory of Open Access Journals (Sweden)

    Cristina de Andrade-Monteiro

    1999-09-01

    Full Text Available Microtubules are filaments composed of dimers of alpha- and beta-tubulins, which have a variety of functions in living cells. In fungi, the spindle pole bodies usually have been considered to be microtubule-organizing centers. We used the antimicrotubule drug Benomyl in block/release experiments to depolymerize and repolymerize microtubules in Aspergillus nidulans germlings to learn more about the microtubule nucleation process in this filamentous fungus. Twenty seconds after release from Benomyl short microtubules were formed from several bright (immunofluorescent dots distributed along the germlings, suggesting that microtubule nucleation is randomly distributed in A. nidulans germlings. Since nuclear movement is dependent on microtubules in A. nidulans we analyzed whether mutants defective in nuclear distribution along the growing hyphae (nud mutants have some obvious microtubule defect. Cytoplasmic, astral and spindle microtubules were present and appeared to be normal in all nud mutants. However, significant changes in the percentage of short versus long mitotic spindles were observed in nud mutants. This suggests that some of the nuclei of nud mutants do not reach the late stage of cell division at normal temperatures.Microtúbulos são filamentos compostos por dímeros das tubulinas a e b e têm uma variedade de funções nas células vivas. Em fungos, os corpúsculos polares dos fusos são geralmente considerados os centros organizadores dos microtúbulos. Com o objetivo de contribuir para uma melhor compreensão dos processos de nucleação dos microtúbulos no fungo filamentoso A. nidulans, nós utilizamos a droga antimicrotúbulo Benomil em experimentos de bloqueio e liberação para depolimerizar e repolimerizar os microtúbulos. Após 20 segundos de reincubação em meio sem Benomil, pequenos microtúbulos foram formados a partir de pontos distribuídos pela célula, sugerindo que os pontos de nucleação de microtúbulos s

  9. PMA synergistically enhances apicularen A-induced cytotoxicity by disrupting microtubule networks in HeLa cells

    International Nuclear Information System (INIS)

    Seo, Kang-Sik; Hwang, Byung-Doo; Kim, Jong-Seok; Park, Ji-Hoon; Song, Kyoung-Sub; Yun, Eun-Jin; Park, Jong-Il; Kweon, Gi Ryang; Yoon, Wan-Hee; Lim, Kyu

    2014-01-01

    Combination therapy is key to improving cancer treatment efficacy. Phorbol 12-myristate 13-acetate (PMA), a well-known PKC activator, increases the cytotoxicity of several anticancer drugs. Apicularen A induces cytotoxicity in tumor cells through disrupting microtubule networks by tubulin down-regulation. In this study, we examined whether PMA increases apicularen A-induced cytotoxicity in HeLa cells. Cell viability was examined by thiazolyl blue tetrazolium (MTT) assays. To investigate apoptotic potential of apicularen A, DNA fragmentation assays were performed followed by extracting genomic DNA, and caspase-3 activity assays were performed by fluorescence assays using fluorogenic substrate. The cell cycle distribution induced by combination with PMA and apicularen A was examined by flow cytometry after staining with propidium iodide (PI). The expression levels of target proteins were measured by Western blotting analysis using specific antibodies, and α-tubulin mRNA levels were assessed by reverse transcription polymerase chain reaction (RT-PCR). To examine the effect of combination of PMA and apicularen A on the microtubule architecture, α-tubulin protein and nuclei were visualized by immunofluorescence staining using an anti-α-tubulin antibody and PI, respectively. We found that apicularen A induced caspase-dependent apoptosis in HeLa cells. PMA synergistically increased cytotoxicity and apoptotic sub-G 1 population induced by apicularen A. These effects were completely blocked by the PKC inhibitors Ro31-8220 and Go6983, while caspase inhibition by Z-VAD-fmk did not prevent cytotoxicity. RNA interference using siRNA against PKCα, but not PKCβ and PKCγ, inhibited cytotoxicity induced by combination PMA and apicularen A. PMA increased the apicularen A-induced disruption of microtubule networks by further decreasing α- and β-tubulin protein levels in a PKC-dependent manner. These results suggest that the synergy between PMA and apicularen A is involved by

  10. SOCS-1 localizes to the microtubule organizing complex-associated 20S proteasome.

    Science.gov (United States)

    Vuong, Bao Q; Arenzana, Teresita L; Showalter, Brian M; Losman, Julie; Chen, X Peter; Mostecki, Justin; Banks, Alexander S; Limnander, Andre; Fernandez, Neil; Rothman, Paul B

    2004-10-01

    The regulation of cytokine signaling is critical for controlling cellular proliferation and activation during an immune response. SOCS-1 is a potent inhibitor of Jak kinase activity and of signaling initiated by several cytokines. SOCS-1 protein levels are tightly regulated, and recent data suggest that SOCS-1 may regulate the protein levels of some signaling proteins by the ubiquitin proteasome pathway; however, the cellular mechanism by which SOCS-1 directs proteins for degradation is unknown. In this report, SOCS-1 is found to colocalize and biochemically copurify with the microtubule organizing complex (MTOC) and its associated 20S proteasome. The SOCS-1 SH2 domain is required for the localization of SOCS-1 to the MTOC. Overexpression of SOCS-1 targets Jak1 in an SH2-dependent manner to a perinuclear distribution resembling the MTOC-associated 20S proteasome. Analysis of MTOCs fractionated from SOCS-1-deficient cells demonstrates that SOCS-1 may function redundantly to regulate the localization of Jak1 to the MTOC. Nocodazole inhibits the protein turnover of SOCS-1, demonstrating that the minus-end transport of SOCS-1 to the MTOC-associated 20S proteasome is required to regulate SOCS-1 protein levels. These data link SOCS-1 directly with the proteasome pathway and suggest another function for the SH2 domain of SOCS-1 in the regulation of Jak/STAT signaling.

  11. Mechanical splitting of microtubules into protofilament bundles by surface-bound kinesin-1.

    Science.gov (United States)

    VanDelinder, Virginia; Adams, Peter G; Bachand, George D

    2016-12-21

    The fundamental biophysics of gliding microtubule (MT) motility by surface-tethered kinesin-1 motor proteins has been widely studied, as well as applied to capture and transport analytes in bioanalytical microdevices. In these systems, phenomena such as molecular wear and fracture into shorter MTs have been reported due the mechanical forces applied on the MT during transport. In the present work, we show that MTs can be split longitudinally into protofilament bundles (PFBs) by the work performed by surface-bound kinesin motors. We examine the properties of these PFBs using several techniques (e.g., fluorescence microscopy, SEM, AFM), and show that the PFBs continue to be mobile on the surface and display very high curvature compared to MT. Further, higher surface density of kinesin motors and shorter kinesin-surface tethers promote PFB formation, whereas modifying MT with GMPCPP or higher paclitaxel concentrations did not affect PFB formation.

  12. TgICMAP1 is a novel microtubule binding protein in Toxoplasma gondii.

    Directory of Open Access Journals (Sweden)

    Aoife T Heaslip

    Full Text Available The microtubule cytoskeleton provides essential structural support for all eukaryotic cells and can be assembled into various higher order structures that perform drastically different functions. Understanding how microtubule-containing assemblies are built in a spatially and temporally controlled manner is therefore fundamental to understanding cell physiology. Toxoplasma gondii, a protozoan parasite, contains at least five distinct tubulin-containing structures, the spindle pole, centrioles, cortical microtubules, the conoid, and the intra-conoid microtubules. How these five structurally and functionally distinct sets of tubulin containing structures are constructed and maintained in the same cell is an intriguing problem. Previously, we performed a proteomic analysis of the T. gondii apical complex, a cytoskeletal complex located at the apical end of the parasite that is composed of the conoid, three ring-like structures, and the two short intra-conoid microtubules. Here we report the characterization of one of the proteins identified in that analysis, TgICMAP1. We show that TgICMAP1 is a novel microtubule binding protein that can directly bind to microtubules in vitro and stabilizes microtubules when ectopically expressed in mammalian cells. Interestingly, in T. gondii, TgICMAP1 preferentially binds to the intra-conoid microtubules, providing us the first molecular tool to investigate the intra-conoid microtubule assembly process during daughter construction.

  13. Stabilizing versus Destabilizing the Microtubules: A Double-Edge Sword for an Effective Cancer Treatment Option?

    Directory of Open Access Journals (Sweden)

    Daniele Fanale

    2015-01-01

    Full Text Available Microtubules are dynamic and structural cellular components involved in several cell functions, including cell shape, motility, and intracellular trafficking. In proliferating cells, they are essential components in the division process through the formation of the mitotic spindle. As a result of these functions, tubulin and microtubules are targets for anticancer agents. Microtubule-targeting agents can be divided into two groups: microtubule-stabilizing, and microtubule-destabilizing agents. The former bind to the tubulin polymer and stabilize microtubules, while the latter bind to the tubulin dimers and destabilize microtubules. Alteration of tubulin-microtubule equilibrium determines the disruption of the mitotic spindle, halting the cell cycle at the metaphase-anaphase transition and, eventually, resulting in cell death. Clinical application of earlier microtubule inhibitors, however, unfortunately showed several limits, such as neurological and bone marrow toxicity and the emergence of drug-resistant tumor cells. Here we review several natural and synthetic microtubule-targeting agents, which showed antitumor activity and increased efficacy in comparison to traditional drugs in various preclinical and clinical studies. Cryptophycins, combretastatins, ombrabulin, soblidotin, D-24851, epothilones and discodermolide were used in clinical trials. Some of them showed antiangiogenic and antivascular activity and others showed the ability to overcome multidrug resistance, supporting their possible use in chemotherapy.

  14. Microtubule dynamics. II. Kinetics of self-assembly

    DEFF Research Database (Denmark)

    Flyvbjerg, H.; Jobs, E.

    1997-01-01

    Inverse scattering theory describes the conditions necessary and sufficient to determine an unknown potential from known scattering data. No similar theory exists for when and how one may deduce the kinetics of an unknown chemical reaction from quantitative information about its final state and i...... to analyze the self-assembly of microtubules from tubulin are general, and many other reactions and processes may be studied as inverse problems with these methods when enough experimental data are available....

  15. Vibrations of microtubules: Physics that has not met biology yet

    Czech Academy of Sciences Publication Activity Database

    Kučera, Ondřej; Havelka, Daniel; Cifra, Michal

    2017-01-01

    Roč. 72, 1 July (2017), s. 13-22 ISSN 0165-2125 R&D Projects: GA ČR(CZ) GA15-17102S Grant - others:AV ČR(CZ) SAV-15-22 Program:Bilaterální spolupráce Institutional support: RVO:67985882 Keywords : Models * Vibrations * Microtubules Subject RIV: BO - Biophysics OBOR OECD: Biophysics Impact factor: 1.575, year: 2016

  16. Dictyoceratidan poisons: Defined mark on microtubule-tubulin dynamics.

    Science.gov (United States)

    Gnanambal K, Mary Elizabeth; Lakshmipathy, Shailaja Vommi

    2016-03-01

    Tubulin/microtubule assembly and disassembly is characterized as one of the chief processes during cell growth and division. Hence drugs those perturb these process are considered to be effective in killing fast multiplying cancer cells. There is a collection of natural compounds which disturb microtubule/tubulin dis/assemblage and there have been a lot of efforts concerted in the marine realm too, to surveying such killer molecules. Close to half the natural compounds shooting out from marine invertebrates are generally with no traceable definite mechanisms of action though may be tough anti-cancerous hits at nanogram levels, hence fatefully those discoveries conclude therein without a capacity of translation from laboratory to pharmacy. Astoundingly at least 50% of natural compounds which have definite mechanisms of action causing disorders in tubulin/microtubule kinetics have an isolation history from sponges belonging to the Phylum: Porifera. Poriferans have always been a wonder worker to treat cancers with a choice of, yet precise targets on cancerous tissues. There is a specific order: Dictyoceratida within this Phylum which has contributed to yielding at least 50% of effective compounds possessing this unique mechanism of action mentioned above. However, not much notice is driven to Dictyoceratidans alongside the order: Demospongiae thus dictating the need to know its select microtubule/tubulin irritants since the unearthing of avarol in the year 1974 till date. Hence this review selectively pinpoints all the compounds, noteworthy derivatives and analogs stemming from order: Dictyoceratida focusing on the past, present and future. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. GIT1 enhances neurite outgrowth by stimulating microtubule assembly

    Directory of Open Access Journals (Sweden)

    Yi-sheng Li

    2016-01-01

    Full Text Available GIT1, a G-protein-coupled receptor kinase interacting protein, has been reported to be involved in neurite outgrowth. However, the neurobiological functions of the protein remain unclear. In this study, we found that GIT1 was highly expressed in the nervous system, and its expression was maintained throughout all stages of neuritogenesis in the brain. In primary cultured mouse hippocampal neurons from GIT1 knockout mice, there was a significant reduction in total neurite length per neuron, as well as in the average length of axon-like structures, which could not be prevented by nerve growth factor treatment. Overexpression of GIT1 significantly promoted axon growth and fully rescued the axon outgrowth defect in the primary hippocampal neuron cultures from GIT1 knockout mice. The GIT1 N terminal region, including the ADP ribosylation factor-GTPase activating protein domain, the ankyrin domains and the Spa2 homology domain, were sufficient to enhance axonal extension. Importantly, GIT1 bound to many tubulin proteins and microtubule-associated proteins, and it accelerated microtubule assembly in vitro. Collectively, our findings suggest that GIT1 promotes neurite outgrowth, at least partially by stimulating microtubule assembly. This study provides new insight into the cellular and molecular pathogenesis of GIT1-associated neurological diseases.

  18. Adult trees cause density-dependent mortality in conspecific seedlings by regulating the frequency of pathogenic soil fungi.

    Science.gov (United States)

    Liang, Minxia; Liu, Xubing; Gilbert, Gregory S; Zheng, Yi; Luo, Shan; Huang, Fengmin; Yu, Shixiao

    2016-12-01

    Negative density-dependent seedling mortality has been widely detected in tropical, subtropical and temperate forests, with soil pathogens as a major driver. Here we investigated how host density affects the composition of soil pathogen communities and consequently influences the strength of plant-soil feedbacks. In field censuses of six 1-ha permanent plots, we found that survival was much lower for newly germinated seedlings that were surrounded by more conspecific adults. The relative abundance of pathogenic fungi in soil increased with increasing conspecific tree density for five of nine tree species; more soil pathogens accumulated around roots where adult tree density was higher, and this greater pathogen frequency was associated with lower seedling survival. Our findings show how tree density influences populations of soil pathogens, which creates plant-soil feedbacks that contribute to community-level and population-level compensatory trends in seedling survival. © 2016 John Wiley & Sons Ltd/CNRS.

  19. The Drosophila Microtubule-Associated Protein Mars Stabilizes Mitotic Spindles by Crosslinking Microtubules through Its N-Terminal Region

    Science.gov (United States)

    Zhang, Gang; Beati, Hamze; Nilsson, Jakob; Wodarz, Andreas

    2013-01-01

    Correct segregation of genetic material relies on proper assembly and maintenance of the mitotic spindle. How the highly dynamic microtubules (MTs) are maintained in stable mitotic spindles is a key question to be answered. Motor and non-motor microtubule associated proteins (MAPs) have been reported to stabilize the dynamic spindle through crosslinking adjacent MTs. Mars, a novel MAP, is essential for the early development of Drosophila embryos. Previous studies showed that Mars is required for maintaining an intact mitotic spindle but did not provide a molecular mechanism for this function. Here we show that Mars is able to stabilize the mitotic spindle in vivo. Both in vivo and in vitro data reveal that the N-terminal region of Mars functions in the stabilization of the mitotic spindle by crosslinking adjacent MTs. PMID:23593258

  20. The Drosophila microtubule-associated protein mars stabilizes mitotic spindles by crosslinking microtubules through its N-terminal region.

    Directory of Open Access Journals (Sweden)

    Gang Zhang

    Full Text Available Correct segregation of genetic material relies on proper assembly and maintenance of the mitotic spindle. How the highly dynamic microtubules (MTs are maintained in stable mitotic spindles is a key question to be answered. Motor and non-motor microtubule associated proteins (MAPs have been reported to stabilize the dynamic spindle through crosslinking adjacent MTs. Mars, a novel MAP, is essential for the early development of Drosophila embryos. Previous studies showed that Mars is required for maintaining an intact mitotic spindle but did not provide a molecular mechanism for this function. Here we show that Mars is able to stabilize the mitotic spindle in vivo. Both in vivo and in vitro data reveal that the N-terminal region of Mars functions in the stabilization of the mitotic spindle by crosslinking adjacent MTs.

  1. The Drosophila microtubule-associated protein mars stabilizes mitotic spindles by crosslinking microtubules through its N-terminal region.

    Science.gov (United States)

    Zhang, Gang; Beati, Hamze; Nilsson, Jakob; Wodarz, Andreas

    2013-01-01

    Correct segregation of genetic material relies on proper assembly and maintenance of the mitotic spindle. How the highly dynamic microtubules (MTs) are maintained in stable mitotic spindles is a key question to be answered. Motor and non-motor microtubule associated proteins (MAPs) have been reported to stabilize the dynamic spindle through crosslinking adjacent MTs. Mars, a novel MAP, is essential for the early development of Drosophila embryos. Previous studies showed that Mars is required for maintaining an intact mitotic spindle but did not provide a molecular mechanism for this function. Here we show that Mars is able to stabilize the mitotic spindle in vivo. Both in vivo and in vitro data reveal that the N-terminal region of Mars functions in the stabilization of the mitotic spindle by crosslinking adjacent MTs.

  2. Simultaneous 3D tracking of passive tracers and microtubule bundles in an active gel

    Science.gov (United States)

    Fan, Yi; Breuer, Kenneth S.; Fluids Team

    Kinesin-driven microtubule bundles generate a spontaneous flow in unconfined geometries. They exhibit properties of active matter, including the emergence of collective motion, reduction of apparent viscosity and consumption of local energy. Here we present results from 3D tracking of passive tracers (using Airy rings and 3D scanning) synchronized with 3D measurement of the microtubule bundles motion. This technique is applied to measure viscosity variation and collective flow in a confined geometry with particular attention paid to the self-pumping system recently reported by Wu et al. (2016). Results show that the viscosity in an equilibrium microtubule network is around half that of the isotropic unbundled microtubule solution. Cross-correlations of the active microtubule network and passive tracers define a neighborhood around microtubule bundles in which passive tracers are effectively transported. MRSEC NSF.

  3. Ase1p Organizes Antiparallel Microtubule Arrays during Interphase and Mitosis in Fission YeastV⃞

    OpenAIRE

    Loïodice, Isabelle; Staub, Jayme; Setty, Thanuja Gangi; Nguyen, Nam-Phuong T.; Paoletti, Anne; Tran, P. T.

    2005-01-01

    Proper microtubule organization is essential for cellular processes such as organelle positioning during interphase and spindle formation during mitosis. The fission yeast Schizosaccharomyces pombe presents a good model for understanding microtubule organization. We identify fission yeast ase1p, a member of the conserved ASE1/PRC1/MAP65 family of microtubule bundling proteins, which functions in organizing the spindle midzone during mitosis. Using fluorescence live cell imaging, we show that ...

  4. Epidermal growth factor receptor mediated proliferation depends on increased lipid droplet density regulated via a negative regulatory loop with FOXO3/Sirtuin6

    International Nuclear Information System (INIS)

    Penrose, Harrison; Heller, Sandra; Cable, Chloe; Makboul, Rania; Chadalawada, Gita; Chen, Ying; Crawford, Susan E.; Savkovic, Suzana D.

    2016-01-01

    The proliferation of colon cancer cells is mediated in part by epidermal growth factor receptor (EGFR) signaling and requires sustained levels of cellular energy to meet its high metabolic needs. Intracellular lipid droplets (LDs) are a source of energy used for various cellular functions and they are elevated in density in human cancer, yet their regulation and function are not well understood. Here, in human colon cancer cells, EGF stimulates increases in LD density, which depends on EGFR expression and activation as well as the individual cellular capacity for lipid synthesis. Increases in LDs are blockaded by inhibition of PI3K/mTOR and PGE2 synthesis, supporting their dependency on select upstream pathways. In colon cancer cells, silencing of the FOXO3 transcription factor leads to down regulation of SIRT6, a negative regulator of lipid synthesis, and consequent increases in the LD coat protein PLIN2, revealing that increases in LDs depend on loss of FOXO3/SIRT6. Moreover, EGF stimulates loss of FOXO3/SIRT6, which is blockaded by the inhibition of upstream pathways as well as lipid synthesis, revealing existence of a negative regulatory loop between LDs and FOXO3/SIRT6. Elevated LDs are utilized by EGF treatment and their depletion through the inhibition of lipid synthesis or silencing of PLIN2 significantly attenuates proliferation. This novel mechanism of proliferative EGFR signaling leading to elevated LD density in colon cancer cells could potentially be therapeutically targeted for the treatment of tumor progression. - Highlights: • In colon cancer cells, EGFR activation leads to increases in LD density. • EGFR signaling includes PI3K/mTOR and PGE2 leading to lipid synthesis. • Increases in LDs are controlled by a negative regulatory loop with FOXO3/SIRT6. • EGFR mediated colon cancer cell proliferation depends on increased LD density.

  5. Epidermal growth factor receptor mediated proliferation depends on increased lipid droplet density regulated via a negative regulatory loop with FOXO3/Sirtuin6

    Energy Technology Data Exchange (ETDEWEB)

    Penrose, Harrison; Heller, Sandra; Cable, Chloe [Department of Pathology and Laboratory Medicine, Tulane University School of Medicine, 1430 Tulane Ave SL-79, New Orleans, LA 70112 (United States); Makboul, Rania [Department of Pathology and Laboratory Medicine, Tulane University School of Medicine, 1430 Tulane Ave SL-79, New Orleans, LA 70112 (United States); Pathology Department, Assiut University, Assiut (Egypt); Chadalawada, Gita; Chen, Ying [Department of Pathology and Laboratory Medicine, Tulane University School of Medicine, 1430 Tulane Ave SL-79, New Orleans, LA 70112 (United States); Crawford, Susan E. [Department of Pathology, Saint Louis University School of Medicine, 1402 South Grand Blvd, Saint Louis, MO 63104 (United States); Savkovic, Suzana D., E-mail: ssavkovi@tulane.edu [Department of Pathology and Laboratory Medicine, Tulane University School of Medicine, 1430 Tulane Ave SL-79, New Orleans, LA 70112 (United States)

    2016-01-15

    The proliferation of colon cancer cells is mediated in part by epidermal growth factor receptor (EGFR) signaling and requires sustained levels of cellular energy to meet its high metabolic needs. Intracellular lipid droplets (LDs) are a source of energy used for various cellular functions and they are elevated in density in human cancer, yet their regulation and function are not well understood. Here, in human colon cancer cells, EGF stimulates increases in LD density, which depends on EGFR expression and activation as well as the individual cellular capacity for lipid synthesis. Increases in LDs are blockaded by inhibition of PI3K/mTOR and PGE2 synthesis, supporting their dependency on select upstream pathways. In colon cancer cells, silencing of the FOXO3 transcription factor leads to down regulation of SIRT6, a negative regulator of lipid synthesis, and consequent increases in the LD coat protein PLIN2, revealing that increases in LDs depend on loss of FOXO3/SIRT6. Moreover, EGF stimulates loss of FOXO3/SIRT6, which is blockaded by the inhibition of upstream pathways as well as lipid synthesis, revealing existence of a negative regulatory loop between LDs and FOXO3/SIRT6. Elevated LDs are utilized by EGF treatment and their depletion through the inhibition of lipid synthesis or silencing of PLIN2 significantly attenuates proliferation. This novel mechanism of proliferative EGFR signaling leading to elevated LD density in colon cancer cells could potentially be therapeutically targeted for the treatment of tumor progression. - Highlights: • In colon cancer cells, EGFR activation leads to increases in LD density. • EGFR signaling includes PI3K/mTOR and PGE2 leading to lipid synthesis. • Increases in LDs are controlled by a negative regulatory loop with FOXO3/SIRT6. • EGFR mediated colon cancer cell proliferation depends on increased LD density.

  6. Combing and self-assembly phenomena in dry films of Taxol-stabilized microtubules

    Directory of Open Access Journals (Sweden)

    Rose Franck

    2007-01-01

    Full Text Available AbstractMicrotubules are filamentous proteins that act as a substrate for the translocation of motor proteins. As such, they may be envisioned as a scaffold for the self-assembly of functional materials and devices. Physisorption, self-assembly and combing are here investigated as a potential prelude to microtubule-templated self-assembly. Dense films of self-assembled microtubules were successfully produced, as well as patterns of both dendritic and non-dendritic bundles of microtubules. They are presented in the present paper and the mechanism of their formation is discussed.

  7. Msd1/SSX2IP-dependent microtubule anchorage ensures spindle orientation and primary cilia formation

    OpenAIRE

    Hori, Akiko; Ikebe, Chiho; Tada, Masazumi; Toda, Takashi

    2014-01-01

    Anchoring microtubules to the centrosome is critical for cell geometry and polarity, yet the molecular mechanism remains unknown. Here we show that the conserved human Msd1/SSX2IP is required for microtubule anchoring. hMsd1/SSX2IP is delivered to the centrosome in a centriolar satellite-dependent manner and binds the microtubule-nucleator ?-tubulin complex. hMsd1/SSX2IP depletion leads to disorganised interphase microtubules and misoriented mitotic spindles with reduced length and intensity....

  8. CYP Suppression in Human Hepatocytes by Monomethyl Auristatin E, the Payload in Brentuximab Vedotin (Adcetris®), is Associated with Microtubule Disruption.

    Science.gov (United States)

    Wolenski, Francis S; Xia, Cindy Q; Ma, Bingli; Han, Tae H; Shyu, Wen C; Balani, Suresh K

    2018-06-01

    Monomethyl auristatin E (MMAE), the toxin linked to CD30-specific monoclonal antibody of Adcetris ® (brentuximab vedotin), is a potent anti-microtubule agent. Brentuximab vedotin has been approved for the treatment of relapsed or refractory Hodgkin lymphoma and anaplastic large cell lymphoma. Cytochrome P450 (CYP) induction assessment of MMAE was conducted in human hepatocytes to assess DDI potentials and its translation to clinic. MMAE was incubated at 1-1000 nM with cultured primary human hepatocytes for 72 h, and CYP1A2, CYP2B6, and CYP3A4 mRNA expression was assessed by quantitative reverse transcription-polymerase chain reaction and CYP-specific probe substrate by liquid chromatography coupled with mass spectrometry, along with microtubule disruption by immunofluorescence staining using anti-β-tubulin antibody and imaging. MMAE up to 10 nM had no significant effect on CYP1A2, CYP2B6, and CYP3A4 mRNA expression and activity, whereas at higher concentrations of 100- and 1000-nM MMAE, the CYP mRNA expression and activity were diminished substantially. Further investigation showed that the degree of CYP suppression was paralleled by that of microtubule disruption by MMAE, as measured by increase in the number of β-tubulin-positive aggregates. At the clinical dose, the concentration of MMAE was 7 nM which did not show any significant CYP suppression or microtubule disruption in hepatocytes. MMAE was not a CYP inducer in human hepatocytes. However, it caused a concentration-dependent CYP mRNA suppression and activity. The CYP suppression was associated with microtubule disruption, supporting the reports that intact microtubule architecture is required for CYP regulations. The absence of CYP suppression and microtubule disruption in vitro at the clinical plasma concentrations of MMAE (< 10 nM) explains the lack of pharmacokinetic drug interaction between brentuximab vedotin and midazolam, a sensitive CYP3A substrate, reported in patients.

  9. The actin-binding protein capulet genetically interacts with the microtubule motor kinesin to maintain neuronal dendrite homeostasis.

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    Paul M B Medina

    Full Text Available BACKGROUND: Neurons require precise cytoskeletal regulation within neurites, containing microtubule tracks for cargo transport in axons and dendrites or within synapses containing organized actin. Due to the unique architecture and specialized function of neurons, neurons are particularly susceptible to perturbation of the cytoskeleton. Numerous actin-binding proteins help maintain proper cytoskeletal regulation. METHODOLOGY/PRINCIPAL FINDINGS: From a Drosophila forward genetic screen, we identified a mutation in capulet--encoding a conserved actin-binding protein--that causes abnormal aggregates of actin within dendrites. Through interaction studies, we demonstrate that simultaneous genetic inactivation of capulet and kinesin heavy chain, a microtubule motor protein, produces elongate cofilin-actin rods within dendrites but not axons. These rods resemble actin-rich structures induced in both mammalian neurodegenerative and Drosophila Alzheimer's models, but have not previously been identified by loss of function mutations in vivo. We further demonstrate that mitochondria, which are transported by Kinesin, have impaired distribution along dendrites in a capulet mutant. While Capulet and Cofilin may biochemically cooperate in certain circumstances, in neuronal dendrites they genetically antagonize each other. CONCLUSIONS/SIGNIFICANCE: The present study is the first molecularly defined loss of function demonstration of actin-cofilin rods in vivo. This study suggests that simultaneous, seemingly minor perturbations in neuronal dendrites can synergize producing severe abnormalities affecting actin, microtubules and mitochondria/energy availability in dendrites. Additionally, as >90% of Alzheimer's and Parkinson's cases are sporadic this study suggests mechanisms by which multiple mutations together may contribute to neurodegeneration instead of reliance on single mutations to produce disease.

  10. On the nature and shape of tubulin trails: implications on microtubule self-organization.

    Science.gov (United States)

    Glade, Nicolas

    2012-06-01

    Microtubules, major elements of the cell skeleton are, most of the time, well organized in vivo, but they can also show self-organizing behaviors in time and/or space in purified solutions in vitro. Theoretical studies and models based on the concepts of collective dynamics in complex systems, reaction-diffusion processes and emergent phenomena were proposed to explain some of these behaviors. In the particular case of microtubule spatial self-organization, it has been advanced that microtubules could behave like ants, self-organizing by 'talking to each other' by way of hypothetic (because never observed) concentrated chemical trails of tubulin that are expected to be released by their disassembling ends. Deterministic models based on this idea yielded indeed like-looking spatio-temporal self-organizing behaviors. Nevertheless the question remains of whether microscopic tubulin trails produced by individual or bundles of several microtubules are intense enough to allow microtubule self-organization at a macroscopic level. In the present work, by simulating the diffusion of tubulin in microtubule solutions at the microscopic scale, we measure the shape and intensity of tubulin trails and discuss about the assumption of microtubule self-organization due to the production of chemical trails by disassembling microtubules. We show that the tubulin trails produced by individual microtubules or small microtubule arrays are very weak and not elongated even at very high reactive rates. Although the variations of concentration due to such trails are not significant compared to natural fluctuations of the concentration of tubuline in the chemical environment, the study shows that heterogeneities of biochemical composition can form due to microtubule disassembly. They could become significant when produced by numerous microtubule ends located in the same place. Their possible formation could play a role in certain conditions of reaction. In particular, it gives a mesoscopic

  11. Disruption of the mouse Jhy gene causes abnormal ciliary microtubule patterning and juvenile hydrocephalus

    Science.gov (United States)

    Appelbe, Oliver K.; Bollman, Bryan; Attarwala, Ali; Triebes, Lindy A.; Muniz-Talavera, Hilmarie; Curry, Daniel J.; Schmidt, Jennifer V.

    2013-01-01

    SUMMARY Congenital hydrocephalus, the accumulation of excess cerebrospinal fluid (CSF) in the ventricles of the brain, affects one of every 1,000 children born today, making it one of the most common human developmental disorders. Genetic causes of hydrocephalus are poorly understood in humans, but animal models suggest a broad genetic program underlying the regulation of CSF balance. In this study, the random integration of a transgene into the mouse genome led to the development of an early onset and rapidly progressive hydrocephalus. Juvenile hydrocephalus transgenic mice (JhylacZ) inherit communicating hydrocephalus in an autosomal recessive fashion with dilation of the lateral ventricles observed as early as postnatal day 1.5. Ventricular dilation increases in severity over time, becoming fatal at 4-8 weeks of age. The ependymal cilia lining the lateral ventricles are morphologically abnormal and reduced in number in JhylacZ/lacZ brains, and ultrastructural analysis revealed disorganization of the expected 9+2 microtubule pattern. Rather, the majority of JhylacZ/lacZ cilia develop axonemes with 9+0 or 8+2 microtubule structures. Disruption of an unstudied gene, 4931429I11Rik (now named Jhy) appears to underlie the hydrocephalus of JhylacZ/lacZ mice, and the Jhy transcript and protein are decreased in JhylacZ/lacZ mice. Partial phenotypic rescue was achieved in JhylacZ/lacZ mice by the introduction of a bacterial artificial chromosome (BAC) carrying 60-70% of the JHY protein coding sequence. Jhy is evolutionarily conserved from humans to basal vertebrates, but the predicted JHY protein lacks identifiable functional domains. Ongoing studies are directed at uncovering the physiological function of JHY and its role in CSF homeostasis. PMID:23906841

  12. Katanin localization requires triplet microtubules in Chlamydomonas reinhardtii.

    Directory of Open Access Journals (Sweden)

    Jessica M Esparza

    Full Text Available Centrioles and basal bodies are essential for a variety of cellular processes that include the recruitment of proteins to these structures for both centrosomal and ciliary function. This recruitment is compromised when centriole/basal body assembly is defective. Mutations that cause basal body assembly defects confer supersensitivity to Taxol. These include bld2, bld10, bld12, uni3, vfl1, vfl2, and vfl3. Flagellar motility mutants do not confer sensitivity with the exception of mutations in the p60 (pf19 and p80 (pf15 subunits of the microtubule severing protein katanin. We have identified additional pf15 and bld2 (ε-tubulin alleles in screens for Taxol sensitivity. Null pf15 and bld2 alleles are viable and are not essential genes in Chlamydomonas. Analysis of double mutant strains with the pf15-3 and bld2-6 null alleles suggests that basal bodies in Chlamydomonas may recruit additional proteins beyond katanin that affect spindle microtubule stability. The bld2-5 allele is a hypomorphic allele and its phenotype is modulated by nutritional cues. Basal bodies in bld2-5 cells are missing proximal ends. The basal body mutants show aberrant localization of an epitope-tagged p80 subunit of katanin. Unlike IFT proteins, katanin p80 does not localize to the transition fibers of the basal bodies based on an analysis of the uni1 mutant as well as the lack of colocalization of katanin p80 with IFT74. We suggest that the triplet microtubules are likely to play a key role in katanin p80 recruitment to the basal body of Chlamydomonas rather than the transition fibers that are needed for IFT localization.

  13. Stocking density affects the growth performance and metabolism of Amur sturgeon by regulating expression of genes in the GH/IGF axis

    Science.gov (United States)

    Ren, Yuanyuan; Wen, Haishen; Li, Yun; Li, Jifang

    2017-07-01

    The effects of stocking density on the growth and metabolism of Amur sturgeon were assessed. Amur sturgeon were grown for 70 days at three different stocking densities (low stocking density, LSD: 5.5 kg/m3; medium stocking density, MSD: 8.0 kg/m3; and high stocking density, HSD: 11.0 kg/m3), and the biometric index, muscle composition, and serum biochemical parameters were evaluated. In addition, pituitary, liver, and muscle samples were collected for gene cloning and expression analyses. After 70 days of growth, the fish maintained at HSD had significantly lower final body weight and specific growth rate, and a higher feed conversion ratio than those of the fish in the MSD and LSD groups. The HSD group had the lowest lipid and protein concentrations in serum and muscle. The serum cortisol concentration increased significantly in the HSD group, indicating that the stress-response system was activated in these fish. There was no change in the concentration of serum insulin-like growth factor 2 (IGF-2), while the concentrations of serum growth hormone (GH) and insulin-like growth factor 1 (IGF-1) decreased in the HSD group. The full-length cDNAs of GH and IGF-2 genes (995-bp and 1 207-bp long, respectively), were cloned and analyzed. In the HSD group, the expressions of GH in the pituitary and growth hormone receptor (GHR) and IGF-1 in the liver were down-regulated at the end of the 70-day experiment. In the HSD group, the transcript level of IGF-2 significantly decreased in the liver, but did not change in muscle. Overall, our results indicated that a HSD negatively affects the growth performance and leads to changes in lipid and protein metabolism in Amur sturgeon. The down-regulated expression of genes related to the GH/IGF axis may be responsible for the poor growth performance of Amur sturgeon under crowding stress.

  14. Interaction of microtubules with active principles of Xanthium strumarium.

    Science.gov (United States)

    Menon, G S; Kuchroo, K; Dasgupta, D

    2001-01-01

    Indigenous variety of Xanthium strumarium (X. strumarium) was screened for its antimitotic activity using the microtubule-tubulin system isolated from mammalian tissue. A preliminary phytochemical screening of the whole extracts of the plant was carried out followed by partial purification of the whole extract of X.strumarium. The separated fractions obtained were identified and used for in vitro polymerization studies. The whole as well as partially separated chemical constituents of X. strumarium showed effective inhibition of tubulin polymerization. The results thus suggest that X. strumarium may possess antimitotic components.

  15. Genetic analysis of a Drosophila microtubule-associated protein

    OpenAIRE

    1992-01-01

    The 205-kD microtubule-associated protein (205K MAP) is one of the principal MAPs in Drosophila. 205K MAP is similar to the HeLa 210K/MAP4 family of MAPs since it shares the following biochemical properties: it is present in several isoforms, has a molecular mass of approximately 200 kD, and is thermostable. Furthermore, immuno-crossreactivity has been observed between mouse MAP4, HeLa 210K, and Drosophila 205K MAP. Currently, there is little information concerning the biological function of ...

  16. Polysaccharide charge density regulating protein adsorption to air/water interfaces by protein/polysaccharide complex formation

    NARCIS (Netherlands)

    Ganzevles, R.A.; Kosters, H.; Vliet, T. van; Stuart, M.A.C.; Jongh, H.H.J. de

    2007-01-01

    Because the formation of protein/polysaccharide complexes is dominated by electrostatic interaction, polysaccharide charge density is expected to play a major role in the adsorption behavior of the complexes. In this study, pullulan (a non-charged polysaccharide) carboxylated to four different

  17. Biallelic Mutations in TBCD, Encoding the Tubulin Folding Cofactor D, Perturb Microtubule Dynamics and Cause Early-Onset Encephalopathy

    NARCIS (Netherlands)

    Flex, Elisabetta; Niceta, Marcello; Cecchetti, Serena; Thiffault, Isabelle; Au, Margaret G.; Capuano, Alessandro; Piermarini, Emanuela; Ivanova, Anna A.; Francis, Joshua W.; Chillemi, Giovanni; Chandramouli, Balasubramanian; Carpentieri, Giovanna; Haaxma, Charlotte A.; Ciolfi, Andrea; Pizzi, Simone; Douglas, Ganka V.; Levine, Kara; Sferra, Antonella; Dentici, Maria Lisa; Pfundt, Rolph R.; Le Pichon, Jean-Baptiste; Farrow, Emily; Baas, Frank; Piemonte, Fiorella; Dallapiccola, Bruno; Graham, John M.; Saunders, Carol J.; Bertini, Enrico; Kahn, Richard A.; Koolen, David A.; Tartaglia, Marco

    2016-01-01

    Microtubules are dynamic cytoskeletal elements coordinating and supporting a variety of neuronal processes, including cell division, migration, polarity, intracellular trafficking, and signal transduction. Mutations in genes encoding tubulins and microtubule-associated proteins are known to cause

  18. Microtubule reorganization in tobacco BY-2 cells stably expressing GFP-MBD

    Science.gov (United States)

    Granger, C. L.; Cyr, R. J.

    2000-01-01

    Microtubule organization plays an important role in plant morphogenesis; however, little is known about how microtubule arrays transit from one organized state to another. The use of a genetically incorporated fluorescent marker would allow long-term observation of microtubule behavior in living cells. Here, we have characterized a Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) cell line that had been stably transformed with a gfp-mbd construct previously demonstrated to label microtubules (J. Marc et al., 1998, Plant Cell 10: 1927-1939). Fluorescence levels were low, but interphase and mitotic microtubule arrays, as well as the transitions between these arrays, could be observed in individual gfp-mbd-transformed cells. By comparing several attributes of transformed and untransformed cells it was concluded that the transgenic cells are not adversely affected by low-level expression of the transgene and that these cells will serve as a useful and accurate model system for observing microtubule reorganization in vivo. Indeed, some initial observations were made that are consistent with the involvement of motor proteins in the transition between the spindle and phragmoplast arrays. Our observations also support the role of the perinuclear region in nucleating microtubules at the end of cell division with a progressive shift of these microtubules and/or nucleating activity to the cortex to form the interphase cortical array.

  19. Synthesis and biological evaluation of structurally simplified noscapine analogues as microtubule binding agents

    Czech Academy of Sciences Publication Activity Database

    Ghaly, P.E.; Churchill, C.D.M.; Abou El-Magd, R.M.; Hájková, Zuzana; Dráber, Pavel; West, F.G.; Tuszyński, J.A.

    2017-01-01

    Roč. 95, č. 6 (2017), s. 649-655 ISSN 0008-4042 R&D Projects: GA ČR GA15-22194S Institutional support: RVO:68378050 Keywords : noscapine * microtubule * tubulin * cytotoxicity * microtubule dynamics * docking Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Biochemistry and molecular biology Impact factor: 1.080, year: 2016

  20. Feeding cells induced by phytoparasitic nematodes require γ-tubulin ring complex for microtubule reorganization.

    Directory of Open Access Journals (Sweden)

    Mohamed Youssef Banora

    2011-12-01

    Full Text Available Reorganization of the microtubule network is important for the fast isodiametric expansion of giant-feeding cells induced by root-knot nematodes. The efficiency of microtubule reorganization depends on the nucleation of new microtubules, their elongation rate and activity of microtubule severing factors. New microtubules in plants are nucleated by cytoplasmic or microtubule-bound γ-tubulin ring complexes. Here we investigate the requirement of γ-tubulin complexes for giant feeding cells development using the interaction between Arabidopsis and Meloidogyne spp. as a model system. Immunocytochemical analyses demonstrate that γ-tubulin localizes to both cortical cytoplasm and mitotic microtubule arrays of the giant cells where it can associate with microtubules. The transcripts of two Arabidopsis γ-tubulin (TUBG1 and TUBG2 and two γ-tubulin complex proteins genes (GCP3 and GCP4 are upregulated in galls. Electron microscopy demonstrates association of GCP3 and γ-tubulin as part of a complex in the cytoplasm of giant cells. Knockout of either or both γ-tubulin genes results in the gene dose-dependent alteration of the morphology of feeding site and failure of nematode life cycle completion. We conclude that the γ-tubulin complex is essential for the control of microtubular network remodelling in the course of initiation and development of giant-feeding cells, and for the successful reproduction of nematodes in their plant hosts.

  1. Zwint-1 is required for spindle assembly checkpoint function and kinetochore-microtubule attachment during oocyte meiosis.

    Science.gov (United States)

    Woo Seo, Dong; Yeop You, Seung; Chung, Woo-Jae; Cho, Dong-Hyung; Kim, Jae-Sung; Su Oh, Jeong

    2015-10-21

    The key step for faithful chromosome segregation during meiosis is kinetochore assembly. Defects in this process result in aneuploidy, leading to miscarriages, infertility and various birth defects. However, the roles of kinetochores in homologous chromosome segregation during meiosis are ill-defined. Here we found that Zwint-1 is required for homologous chromosome segregation during meiosis. Knockdown of Zwint-1 accelerated the first meiosis by abrogating the kinetochore recruitment of Mad2, leading to chromosome misalignment and a high incidence of aneuploidy. Although Zwint-1 knockdown did not affect Aurora C kinase activity, the meiotic defects following Zwint-1 knockdown were similar to those observed with ZM447439 treatment. Importantly, the chromosome misalignment following Aurora C kinase inhibition was not restored after removing the inhibitor in Zwint-1-knockdown oocytes, whereas the defect was rescued after the inhibitor washout in the control oocytes. These results suggest that Aurora C kinase-mediated correction of erroneous kinetochore-microtubule attachment is primarily regulated by Zwint-1. Our results provide the first evidence that Zwint-1 is required to correct erroneous kinetochore-microtubule attachment and regulate spindle checkpoint function during meiosis.

  2. JMJD5 (Jumonji Domain-containing 5) Associates with Spindle Microtubules and Is Required for Proper Mitosis.

    Science.gov (United States)

    He, Zhimin; Wu, Junyu; Su, Xiaonan; Zhang, Ye; Pan, Lixia; Wei, Huimin; Fang, Qiang; Li, Haitao; Wang, Da-Liang; Sun, Fang-Lin

    2016-02-26

    Precise mitotic spindle assembly is a guarantee of proper chromosome segregation during mitosis. Chromosome instability caused by disturbed mitosis is one of the major features of various types of cancer. JMJD5 has been reported to be involved in epigenetic regulation of gene expression in the nucleus, but little is known about its function in mitotic process. Here we report the unexpected localization and function of JMJD5 in mitotic progression. JMJD5 partially accumulates on mitotic spindles during mitosis, and depletion of JMJD5 results in significant mitotic arrest, spindle assembly defects, and sustained activation of the spindle assembly checkpoint (SAC). Inactivating SAC can efficiently reverse the mitotic arrest caused by JMJD5 depletion. Moreover, JMJD5 is found to interact with tubulin proteins and associate with microtubules during mitosis. JMJD5-depleted cells show a significant reduction of α-tubulin acetylation level on mitotic spindles and fail to generate enough interkinetochore tension to satisfy the SAC. Further, JMJD5 depletion also increases the susceptibility of HeLa cells to the antimicrotubule agent. Taken together, these results suggest that JMJD5 plays an important role in regulating mitotic progression, probably by modulating the stability of spindle microtubules. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. BORC Functions Upstream of Kinesins 1 and 3 to Coordinate Regional Movement of Lysosomes along Different Microtubule Tracks.

    Science.gov (United States)

    Guardia, Carlos M; Farías, Ginny G; Jia, Rui; Pu, Jing; Bonifacino, Juan S

    2016-11-15

    The multiple functions of lysosomes are critically dependent on their ability to undergo bidirectional movement along microtubules between the center and the periphery of the cell. Centrifugal and centripetal movement of lysosomes is mediated by kinesin and dynein motors, respectively. We recently described a multi-subunit complex named BORC that recruits the small GTPase Arl8 to lysosomes to promote their kinesin-dependent movement toward the cell periphery. Here, we show that BORC and Arl8 function upstream of two structurally distinct kinesin types: kinesin-1 (KIF5B) and kinesin-3 (KIF1Bβ and KIF1A). Remarkably, KIF5B preferentially moves lysosomes on perinuclear tracks enriched in acetylated α-tubulin, whereas KIF1Bβ and KIF1A drive lysosome movement on more rectilinear, peripheral tracks enriched in tyrosinated α-tubulin. These findings establish BORC as a master regulator of lysosome positioning through coupling to different kinesins and microtubule tracks. Common regulation by BORC enables coordinate control of lysosome movement in different regions of the cell. Published by Elsevier Inc.

  4. Expansion and Polarity Sorting in Microtubule-Dynein Bundles(WHAT IS LIFE? THE NEXT 100 YEARS OF YUKAWA'S DREAM)

    OpenAIRE

    Assaf, ZEMEL; Alex, MOGILNER; Department of Neurobiology, Physiology and Behavior, University of California; Department of Neurobiology, Physiology and Behavior, University of California

    2008-01-01

    Interactions of multiple molecular motors with dynamic polymers, such as actin and microtubules, form the basis for many processes in the cell cytoskeleton. One example is the active 'sorting' of microtubule bundles by dynein molecular motors into aster-like arrays of microtubules; in these bundles dynein motors cross-link and slide neighboring microtubules apart. A number of models have been suggested to quantify the active dynamics of cross-linked bundles of polar filaments. In the case of ...

  5. Hepatocyte cotransport of taurocholate and bilirubin glucuronides: Role of microtubules

    International Nuclear Information System (INIS)

    Crawford, J.M.; Gollan, J.L.

    1988-01-01

    Modulation of bile pigment excretion by bile salts has been attributed to modification of canalicular membrane transport or a physical interaction in bile. Based on the observation that a microtubule-dependent pathway is involved in the hepatocellular transport of bile salts, the authors investigated the possibility that bilirubin glucuronides are associated with bile salts during intracellular transport. Experiments were conducted in intact rats (basal) or after overnight biliary diversion and intravenous reinfusion of taurocholate (depleted/reinfused). All rats were pretreated with intravenous low-dose colchicine or its inactive isomer lumicolchicine. Biliary excretion of radiolabeled bilirubin glucuronides derived from tracer [ 14 C]bilirubin-[ 3 H]bilirubin monoglucuronide (coinjected iv) was unchanged in basal rats but was consistently delayed in depleted/reinfused rats. This was accompanied by a significant shift toward bilirubin diglucuronide formation from both substrates. In basal Gunn rats, with deficient bilirubin glucuronidation, biliary excretion of intravenous [ 14 C]bilirubin monoglucuronide-[ 3 H]bilirubin diglucuronide was unaffected by colchicine but was retarded in depleted/reinfused Gunn rats. Colchicine had no effect on the rate of bilirubin glucuronidation in vitro in rat liver microsomes. They conclude that a portion of the bilirubin glucuronides generated endogenously in hepatocytes or taken up directly from plasma may be cotransported with bile salts to the bile canalicular membrane via a microtubule-dependent mechanism

  6. Endoplasmic-reticulum-mediated microtubule alignment governs cytoplasmic streaming.

    Science.gov (United States)

    Kimura, Kenji; Mamane, Alexandre; Sasaki, Tohru; Sato, Kohta; Takagi, Jun; Niwayama, Ritsuya; Hufnagel, Lars; Shimamoto, Yuta; Joanny, Jean-François; Uchida, Seiichi; Kimura, Akatsuki

    2017-04-01

    Cytoplasmic streaming refers to a collective movement of cytoplasm observed in many cell types. The mechanism of meiotic cytoplasmic streaming (MeiCS) in Caenorhabditis elegans zygotes is puzzling as the direction of the flow is not predefined by cell polarity and occasionally reverses. Here, we demonstrate that the endoplasmic reticulum (ER) network structure is required for the collective flow. Using a combination of RNAi, microscopy and image processing of C. elegans zygotes, we devise a theoretical model, which reproduces and predicts the emergence and reversal of the flow. We propose a positive-feedback mechanism, where a local flow generated along a microtubule is transmitted to neighbouring regions through the ER. This, in turn, aligns microtubules over a broader area to self-organize the collective flow. The proposed model could be applicable to various cytoplasmic streaming phenomena in the absence of predefined polarity. The increased mobility of cortical granules by MeiCS correlates with the efficient exocytosis of the granules to protect the zygotes from osmotic and mechanical stresses.

  7. Microtubules Nonlinear Models Dynamics Investigations through the exp(−Φ(ξ-Expansion Method Implementation

    Directory of Open Access Journals (Sweden)

    Nur Alam

    2016-02-01

    Full Text Available In this research article, we present exact solutions with parameters for two nonlinear model partial differential equations(PDEs describing microtubules, by implementing the exp(−Φ(ξ-Expansion Method. The considered models, describing highly nonlinear dynamics of microtubules, can be reduced to nonlinear ordinary differential equations. While the first PDE describes the longitudinal model of nonlinear dynamics of microtubules, the second one describes the nonlinear model of dynamics of radial dislocations in microtubules. The acquired solutions are then graphically presented, and their distinct properties are enumerated in respect to the corresponding dynamic behavior of the microtubules they model. Various patterns, including but not limited to regular, singular kink-like, as well as periodicity exhibiting ones, are detected. Being the method of choice herein, the exp(−Φ(ξ-Expansion Method not disappointing in the least, is found and declared highly efficient.

  8. Centriole triplet microtubules are required for stable centriole formation and inheritance in human cells

    Science.gov (United States)

    Wang, Jennifer T; Kong, Dong; Hoerner, Christian R; Loncarek, Jadranka

    2017-01-01

    Centrioles are composed of long-lived microtubules arranged in nine triplets. However, the contribution of triplet microtubules to mammalian centriole formation and stability is unknown. Little is known of the mechanism of triplet microtubule formation, but experiments in unicellular eukaryotes indicate that delta-tubulin and epsilon-tubulin, two less-studied tubulin family members, are required. Here, we report that centrioles in delta-tubulin and epsilon-tubulin null mutant human cells lack triplet microtubules and fail to undergo centriole maturation. These aberrant centrioles are formed de novo each cell cycle, but are unstable and do not persist to the next cell cycle, leading to a futile cycle of centriole formation and disintegration. Disintegration can be suppressed by paclitaxel treatment. Delta-tubulin and epsilon-tubulin physically interact, indicating that these tubulins act together to maintain triplet microtubules and that these are necessary for inheritance of centrioles from one cell cycle to the next. PMID:28906251

  9. Centriole triplet microtubules are required for stable centriole formation and inheritance in human cells.

    Science.gov (United States)

    Wang, Jennifer T; Kong, Dong; Hoerner, Christian R; Loncarek, Jadranka; Stearns, Tim

    2017-09-14

    Centrioles are composed of long-lived microtubules arranged in nine triplets. However, the contribution of triplet microtubules to mammalian centriole formation and stability is unknown. Little is known of the mechanism of triplet microtubule formation, but experiments in unicellular eukaryotes indicate that delta-tubulin and epsilon-tubulin, two less-studied tubulin family members, are required. Here, we report that centrioles in delta-tubulin and epsilon-tubulin null mutant human cells lack triplet microtubules and fail to undergo centriole maturation. These aberrant centrioles are formed de novo each cell cycle, but are unstable and do not persist to the next cell cycle, leading to a futile cycle of centriole formation and disintegration. Disintegration can be suppressed by paclitaxel treatment. Delta-tubulin and epsilon-tubulin physically interact, indicating that these tubulins act together to maintain triplet microtubules and that these are necessary for inheritance of centrioles from one cell cycle to the next.

  10. Vorinostat positively regulates synaptic plasticity genes expression and spine density in HIV infected neurons: role of nicotine in progression of HIV-associated neurocognitive disorder

    Science.gov (United States)

    2014-01-01

    Background HIV-associated neurocognitive disorder (HAND) is characterized by development of cognitive, behavioral and motor abnormalities, and occurs in approximately 50% of HIV infected individuals. In the United States, the prevalence of cigarette smoking ranges from 35-70% in HIV-infected individuals compared to 20% in general population. Cognitive impairment in heavy cigarette smokers has been well reported. However, the synergistic effects of nicotine and HIV infection and the underlying mechanisms in the development of HAND are unknown. Results In this study, we explored the role of nicotine in the progression of HAND using SK-N-MC, a neuronal cell line. SK-N-MC cells were infected with HIV-1 in the presence or absence of nicotine for 7 days. We observed significant increase in HIV infectivity in SK-N-MC treated with nicotine compared to untreated HIV-infected neuronal cells. HIV and nicotine synergize to significantly dysregulate the expression of synaptic plasticity genes and spine density; with a concomitant increase of HDAC2 levels in SK-N-MC cells. In addition, inhibition of HDAC2 up-regulation with the use of vorinostat resulted in HIV latency breakdown and recovery of synaptic plasticity genes expression and spine density in nicotine/HIV alone and in co-treated SK-N-MC cells. Furthermore, increased eIF2 alpha phosphorylation, which negatively regulates eukaryotic translational process, was observed in HIV alone and in co-treatment with nicotine compared to untreated control and nicotine alone treated SK-N-MC cells. Conclusions These results suggest that nicotine and HIV synergize to negatively regulate the synaptic plasticity gene expression and spine density and this may contribute to the increased risk of HAND in HIV infected smokers. Apart from disrupting latency, vorinostat may be a useful therapeutic to inhibit the negative regulatory effects on synaptic plasticity in HIV infected nicotine abusers. PMID:24886748

  11. Modelling the role of intrinsic electric fields in microtubules as an additional control mechanism of bi-directional intracellular transport.

    Science.gov (United States)

    Sataric, M V; Budinski-Petkovic, L; Loncarevic, I; Tuszynski, J A

    2008-01-01

    Active transport is essential for cellular function, while impaired transport has been linked to diseases such as neuronal degeneration. Much long distance transport in cells uses opposite polarity molecular motors of the kinesin and dynein families to move cargos along microtubules. It is clear that many types of cargo are moved by both sets of motors, and frequently in a reverse direction. The general question of how the direction of transport is regulated is still open. The mechanism of the cell's differential control of diverse cargos within the same cytoplasmic background is still unclear as is the answer to the question how endosomes and mitochondria move to different locations within the same cell. To answer these questions we postulate the existence of a local signaling mechanism used by the cell to specifically control different cargos. In particular, we propose an additional physical mechanism that works through the use of constant and alternating intrinsic (endogenous) electric fields as a means of controlling the speed and direction of microtubule-based transport. A specific model is proposed and analyzed in this paper. The model involves the rotational degrees of freedom of the C-termini of tubulin, their interactions and the coupling between elastic and dielectric degrees of freedom. Viscosity of the solution is also included and the resultant equation of motion is found as a nonlinear elliptic equation with dissipation. A particular analytical solution of this equation is obtained in the form of a kink whose properties are analyzed. It is concluded that this solution can be modulated by the presence of electric fields and hence may correspond to the observed behavior of motor protein transport along microtubules.

  12. Plus- and minus-end directed microtubule motors bind simultaneously to herpes simplex virus capsids using different inner tegument structures.

    Directory of Open Access Journals (Sweden)

    Kerstin Radtke

    2010-07-01

    Full Text Available Many viruses depend on host microtubule motors to reach their destined intracellular location. Viral particles of neurotropic alphaherpesviruses such as herpes simplex virus 1 (HSV1 show bidirectional transport towards the cell center as well as the periphery, indicating that they utilize microtubule motors of opposing directionality. To understand the mechanisms of specific motor recruitment, it is necessary to characterize the molecular composition of such motile viral structures. We have generated HSV1 capsids with different surface features without impairing their overall architecture, and show that in a mammalian cell-free system the microtubule motors dynein and kinesin-1 and the dynein cofactor dynactin could interact directly with capsids independent of other host factors. The capsid composition and surface was analyzed with respect to 23 structural proteins that are potentially exposed to the cytosol during virus assembly or cell entry. Many of these proteins belong to the tegument, the hallmark of all herpesviruses located between the capsid and the viral envelope. Using immunoblots, quantitative mass spectrometry and quantitative immunoelectron microscopy, we show that capsids exposing inner tegument proteins such as pUS3, pUL36, pUL37, ICP0, pUL14, pUL16, and pUL21 recruited dynein, dynactin, kinesin-1 and kinesin-2. In contrast, neither untegumented capsids exposing VP5, VP26, pUL17 and pUL25 nor capsids covered by outer tegument proteins such as vhs, pUL11, ICP4, ICP34.5, VP11/12, VP13/14, VP16, VP22 or pUS11 bound microtubule motors. Our data suggest that HSV1 uses different structural features of the inner tegument to recruit dynein or kinesin-1. Individual capsids simultaneously accommodated motors of opposing directionality as well as several copies of the same motor. Thus, these associated motors either engage in a tug-of-war or their activities are coordinately regulated to achieve net transport either to the nucleus during

  13. Rad GTPase is essential for the regulation of bone density and bone marrow adipose tissue in mice.

    Science.gov (United States)

    Withers, Catherine N; Brown, Drew M; Byiringiro, Innocent; Allen, Matthew R; Condon, Keith W; Satin, Jonathan; Andres, Douglas A

    2017-10-01

    The small GTP-binding protein Rad (RRAD, Ras associated with diabetes) is the founding member of the RGK (Rad, Rem, Rem2, and Gem/Kir) family that regulates cardiac voltage-gated Ca 2+ channel function. However, its cellular and physiological functions outside of the heart remain to be elucidated. Here we report that Rad GTPase function is required for normal bone homeostasis in mice, as Rad deletion results in significantly lower bone mass and higher bone marrow adipose tissue (BMAT) levels. Dynamic histomorphometry in vivo and primary calvarial osteoblast assays in vitro demonstrate that bone formation and osteoblast mineralization rates are depressed, while in vitro osteoclast differentiation is increased, in the absence of Rad. Microarray analysis revealed that canonical osteogenic gene expression (Runx2, osterix, etc.) is not altered in Rad -/- calvarial osteoblasts; instead robust up-regulation of matrix Gla protein (MGP, +11-fold), an inhibitor of extracellular matrix mineralization and a protein secreted during adipocyte differentiation, was observed. Strikingly, Rad deficiency also resulted in significantly higher marrow adipose tissue levels in vivo and promoted spontaneous in vitro adipogenesis of primary calvarial osteoblasts. Adipogenic differentiation of wildtype calvarial osteoblasts resulted in the loss of endogenous Rad protein, further supporting a role for Rad in the control of BMAT levels. These findings reveal a novel in vivo function for Rad and establish a role for Rad signaling in the complex physiological control of skeletal homeostasis and bone marrow adiposity. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Clostridium difficile toxin CDT induces formation of microtubule-based protrusions and increases adherence of bacteria.

    Directory of Open Access Journals (Sweden)

    Carsten Schwan

    2009-10-01

    Full Text Available Clostridium difficile causes antibiotic-associated diarrhea and pseudomembranous colitis by production of the Rho GTPase-glucosylating toxins A and B. Recently emerging hypervirulent Clostridium difficile strains additionally produce the binary ADP-ribosyltransferase toxin CDT (Clostridium difficile transferase, which ADP-ribosylates actin and inhibits actin polymerization. Thus far, the role of CDT as a virulence factor is not understood. Here we report by using time-lapse- and immunofluorescence microscopy that CDT and other binary actin-ADP-ribosylating toxins, including Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin, induce redistribution of microtubules and formation of long (up to >150 microm microtubule-based protrusions at the surface of intestinal epithelial cells. The toxins increase the length of decoration of microtubule plus-ends by EB1/3, CLIP-170 and CLIP-115 proteins and cause redistribution of the capture proteins CLASP2 and ACF7 from microtubules at the cell cortex into the cell interior. The CDT-induced microtubule protrusions form a dense meshwork at the cell surface, which wrap and embed bacterial cells, thereby largely increasing the adherence of Clostridia. The study describes a novel type of microtubule structure caused by less efficient microtubule capture and offers a new perspective for the pathogenetic role of CDT and other binary actin-ADP-ribosylating toxins in host-pathogen interactions.

  15. Structural differences between yeast and mammalian microtubules revealed by cryo-EM

    Energy Technology Data Exchange (ETDEWEB)

    Howes, Stuart C. [Univ. of California, Berkeley, CA (United States). Biophysics Graduate Group; Geyer, Elisabeth A. [Univ. of Texas Southwestern Medical Center, Dallas, TX (United States). Dept. of Biophysics; Univ. of Texas Southwestern Medical Center, Dallas, TX (United States). Dept. of Biochemistry; LaFrance, Benjamin [Univ. of California, Berkeley, CA (United States). Molecular and Cell Biology Graduate Program; Zhang, Rui [Univ. of California, Berkeley, CA (United States). Howard Hughes Medical Inst.; Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Molecular Biophysics and Integrated Bioimaging Division; Kellogg, Elizabeth H. [Univ. of California, Berkeley, CA (United States). Howard Hughes Medical Inst.; Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Molecular Biophysics and Integrated Bioimaging Division; Westermann, Stefan [Univ. of Duisburg-Essen, Essen (Germany). Dept. of Molecular Genetics, Center for Medical Biotechnology; Rice, Luke M. [Univ. of Texas Southwestern Medical Center, Dallas, TX (United States). Dept. of Biophysics; Univ. of Texas Southwestern Medical Center, Dallas, TX (United States). Dept. of Biochemistry; Nogales, Eva [Univ. of California, Berkeley, CA (United States). Howard Hughes Medical Inst.; Univ. of California, Berkeley, CA (United States). Dept. of Molecular Biology and California Inst. for Quantitative Biosciences; Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Molecular Biophysics and Integrated Bioimaging Division

    2017-06-26

    Microtubules are polymers of αβ-tubulin heterodimers essential for all eukaryotes. Despite sequence conservation, there are significant structural differences between microtubules assembled in vitro from mammalian or budding yeast tubulin. Yeast MTs were not observed to undergo compaction at the interdimer interface as seen for mammalian microtubules upon GTP hydrolysis. Lack of compaction might reflect slower GTP hydrolysis or a different degree of allosteric coupling in the lattice. The microtubule plus end–tracking protein Bim1 binds yeast microtubules both between αβ-tubulin heterodimers, as seen for other organisms, and within tubulin dimers, but binds mammalian tubulin only at interdimer contacts. At the concentrations used in cryo-electron microscopy, Bim1 causes the compaction of yeast microtubules and induces their rapid disassembly. In conclusion, our studies demonstrate structural differences between yeast and mammalian microtubules that likely underlie their differing polymerization dynamics. These differences may reflect adaptations to the demands of different cell size or range of physiological growth temperatures.

  16. Buckling analysis of orthotropic protein microtubules under axial and radial compression based on couple stress theory.

    Science.gov (United States)

    Beni, Yaghoub Tadi; Zeverdejani, M Karimi; Mehralian, Fahimeh

    2017-10-01

    Protein microtubules (MTs) are one of the important intercellular components and have a vital role in the stability and strength of the cells. Due to applied external loads, protein microtubules may be involved buckling phenomenon. Due to impact of protein microtubules in cell reactions, it is important to determine their critical buckling load. Considering nature of protein microtubules, various parameters are effective on microtubules buckling. The small size of microtubules and also lack of uniformity of MTs properties in different directions caused the necessity of accuracy in the analysis of these bio-structure. In fact, microtubules must be considered as a size dependent cylinder, which behave as an orthotropic material. Hence, in the present work using first-order shear deformation model (FSDT), the buckling equations of anisotropic MTs are derived based on new modified couple stress theory (NMCST). After solving the stability equations, the influences of various parameters are measured on the MTs critical buckling load. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Model for the orientational ordering of the plant microtubule cortical array

    Science.gov (United States)

    Hawkins, Rhoda J.; Tindemans, Simon H.; Mulder, Bela M.

    2010-07-01

    The plant microtubule cortical array is a striking feature of all growing plant cells. It consists of a more or less homogeneously distributed array of highly aligned microtubules connected to the inner side of the plasma membrane and oriented transversely to the cell growth axis. Here, we formulate a continuum model to describe the origin of orientational order in such confined arrays of dynamical microtubules. The model is based on recent experimental observations that show that a growing cortical microtubule can interact through angle dependent collisions with pre-existing microtubules that can lead either to co-alignment of the growth, retraction through catastrophe induction or crossing over the encountered microtubule. We identify a single control parameter, which is fully determined by the nucleation rate and intrinsic dynamics of individual microtubules. We solve the model analytically in the stationary isotropic phase, discuss the limits of stability of this isotropic phase, and explicitly solve for the ordered stationary states in a simplified version of the model.

  18. The putative bZIP transcription factor BzpN slows proliferation and functions in the regulation of cell density by autocrine signals in Dictyostelium.

    Directory of Open Access Journals (Sweden)

    Jonathan E Phillips

    Full Text Available The secreted proteins AprA and CfaD function as autocrine signals that inhibit cell proliferation in Dictyostelium discoideum, thereby regulating cell numbers by a negative feedback mechanism. We report here that the putative basic leucine zipper transcription factor BzpN plays a role in the inhibition of proliferation by AprA and CfaD. Cells lacking BzpN proliferate more rapidly than wild-type cells but do not reach a higher stationary density. Recombinant AprA inhibits wild-type cell proliferation but does not inhibit the proliferation of cells lacking BzpN. Recombinant CfaD also inhibits wild-type cell proliferation, but promotes the proliferation of cells lacking BzpN. Overexpression of BzpN results in a reduced cell density at stationary phase, and this phenotype requires AprA, CfaD, and the kinase QkgA. Conditioned media from high-density cells stops the proliferation of wild-type but not bzpN(- cells and induces a nuclear localization of a BzpN-GFP fusion protein, though this localization does not require AprA or CfaD. Together, the data suggest that BzpN is necessary for some but not all of the effects of AprA and CfaD, and that BzpN may function downstream of AprA and CfaD in a signal transduction pathway that inhibits proliferation.

  19. The Putative bZIP Transcripton Factor BzpN Slows Proliferation and Functions in the Regulation of Cell Density by Autocrine Signals in Dictyostelium

    Science.gov (United States)

    Phillips, Jonathan E.; Huang, Eryong; Shaulsky, Gad; Gomer, Richard H.

    2011-01-01

    The secreted proteins AprA and CfaD function as autocrine signals that inhibit cell proliferation in Dictyostelium discoideum, thereby regulating cell numbers by a negative feedback mechanism. We report here that the putative basic leucine zipper transcription factor BzpN plays a role in the inhibition of proliferation by AprA and CfaD. Cells lacking BzpN proliferate more rapidly than wild-type cells but do not reach a higher stationary density. Recombinant AprA inhibits wild-type cell proliferation but does not inhibit the proliferation of cells lacking BzpN. Recombinant CfaD also inhibits wild-type cell proliferation, but promotes the proliferation of cells lacking BzpN. Overexpression of BzpN results in a reduced cell density at stationary phase, and this phenotype requires AprA, CfaD, and the kinase QkgA. Conditioned media from high-density cells stops the proliferation of wild-type but not bzpN− cells and induces a nuclear localization of a BzpN-GFP fusion protein, though this localization does not require AprA or CfaD. Together, the data suggest that BzpN is necessary for some but not all of the effects of AprA and CfaD, and that BzpN may function downstream of AprA and CfaD in a signal transduction pathway that inhibits proliferation. PMID:21760904

  20. The putative bZIP transcription factor BzpN slows proliferation and functions in the regulation of cell density by autocrine signals in Dictyostelium.

    Science.gov (United States)

    Phillips, Jonathan E; Huang, Eryong; Shaulsky, Gad; Gomer, Richard H

    2011-01-01

    The secreted proteins AprA and CfaD function as autocrine signals that inhibit cell proliferation in Dictyostelium discoideum, thereby regulating cell numbers by a negative feedback mechanism. We report here that the putative basic leucine zipper transcription factor BzpN plays a role in the inhibition of proliferation by AprA and CfaD. Cells lacking BzpN proliferate more rapidly than wild-type cells but do not reach a higher stationary density. Recombinant AprA inhibits wild-type cell proliferation but does not inhibit the proliferation of cells lacking BzpN. Recombinant CfaD also inhibits wild-type cell proliferation, but promotes the proliferation of cells lacking BzpN. Overexpression of BzpN results in a reduced cell density at stationary phase, and this phenotype requires AprA, CfaD, and the kinase QkgA. Conditioned media from high-density cells stops the proliferation of wild-type but not bzpN(-) cells and induces a nuclear localization of a BzpN-GFP fusion protein, though this localization does not require AprA or CfaD. Together, the data suggest that BzpN is necessary for some but not all of the effects of AprA and CfaD, and that BzpN may function downstream of AprA and CfaD in a signal transduction pathway that inhibits proliferation.

  1. Control of cell volume in the J774 macrophage by microtubule disassembly and cyclic AMP

    Science.gov (United States)

    Melmed, RN; Karanian, PJ; Berlin, RD

    1981-01-01

    We have explored the possibilities that cell volume is regulated by the status of microtubule assembly and cyclic AMP metabolism and may be coordinated with shape change. Treatment of J774.2 mouse macrophages with colchicine caused rapid microtubule disassembly and was associated with a striking increase (from 15-20 to more than 90 percent) in the proportion of cells with a large protuberance at one pole. This provided a simple experimental system in which shape changes occurred in virtually an entire cell population in suspension. Parallel changes in cell volume could then be quantified by isotope dilution techniques. We found that the shape change caused by colchicine was accompanied by a decrease in cell volume of approximately 20 percent. Nocodozole, but not lumicolchicine, caused identical changes in both cell shape and cell volume. The volume loss was not due to cell lysis nor to inhibition of pinocytosis. The mechanism of volume loss was also examined. Colchicine induced a small but reproducible increase in activity of the ouabain-sensitive Na(+), K(+)-dependent ATPase. However, inhibition of this enzyme/transport system by ouabain did not change cell volume nor did it block the colchicines-induced decrease in volume. One the other hand, SITS (4’acetamido, 4-isothiocyano 2,2’ disulfonic acid stilbene), an inhibitor of anion transport, inhibited the effects of colchicines, thus suggesting a role for an anion transport system in cell volume regulation. Because colchicine is known to activate adenylate cyclase in several systems and because cell shape changes are often induced by hormones that elevate cyclic AMP, we also examined the effects of cyclic AMP on cell volume. Agents that act to increase syclic AMP (cholera toxin, which activates adenylate cyclase; IBMX, and inhibitor of phosphodiesterase; and dibutyryl cyclic AMP) all caused a volume decrease comparable to that of colchicine. To define the effective metabolic pathway, we studied two mutants of J

  2. STIM1-Directed Reorganization of Microtubules in Activated Mast Cells

    Czech Academy of Sciences Publication Activity Database

    Hájková, Zuzana; Bugajev, Viktor; Dráberová, Eduarda; Vinopal, Stanislav; Dráberová, Lubica; Janáček, Jiří; Dráber, Petr; Dráber, Pavel

    2011-01-01

    Roč. 186, č. 2 (2011), s. 913-923 ISSN 0022-1767 R&D Projects: GA ČR(CZ) GD204/09/H084; GA ČR GA204/09/1777; GA ČR GA301/09/1826; GA ČR GAP302/10/1759; GA MŠk LC545; GA MŠk(CZ) LC06063; GA AV ČR KAN200520701 Institutional research plan: CEZ:AV0Z50520514; CEZ:AV0Z50110509 Keywords : STIM1 * bone marrow-derived mast cells * microtubules Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.788, year: 2011

  3. A Mechanism for Cytoplasmic Streaming: Kinesin-Driven Alignment of Microtubules and Fast Fluid Flows.

    Science.gov (United States)

    Monteith, Corey E; Brunner, Matthew E; Djagaeva, Inna; Bielecki, Anthony M; Deutsch, Joshua M; Saxton, William M

    2016-05-10

    The transport of cytoplasmic components can be profoundly affected by hydrodynamics. Cytoplasmic streaming in Drosophila oocytes offers a striking example. Forces on fluid from kinesin-1 are initially directed by a disordered meshwork of microtubules, generating minor slow cytoplasmic flows. Subsequently, to mix incoming nurse cell cytoplasm with ooplasm, a subcortical layer of microtubules forms parallel arrays that support long-range, fast flows. To analyze the streaming mechanism, we combined observations of microtubule and organelle motions with detailed mathematical modeling. In the fast state, microtubules tethered to the cortex form a thin subcortical layer and undergo correlated sinusoidal bending. Organelles moving in flows along the arrays show velocities that are slow near the cortex and fast on the inward side of the subcortical microtubule layer. Starting with fundamental physical principles suggested by qualitative hypotheses, and with published values for microtubule stiffness, kinesin velocity, and cytoplasmic viscosity, we developed a quantitative coupled hydrodynamic model for streaming. The fully detailed mathematical model and its simulations identify key variables that can shift the system between disordered (slow) and ordered (fast) states. Measurements of array curvature, wave period, and the effects of diminished kinesin velocity on flow rates, as well as prior observations on f-actin perturbation, support the model. This establishes a concrete mechanistic framework for the ooplasmic streaming process. The self-organizing fast phase is a result of viscous drag on kinesin-driven cargoes that mediates equal and opposite forces on cytoplasmic fluid and on microtubules whose minus ends are tethered to the cortex. Fluid moves toward plus ends and microtubules are forced backward toward their minus ends, resulting in buckling. Under certain conditions, the buckling microtubules self-organize into parallel bending arrays, guiding varying directions

  4. Iron oxide nanoparticles induce human microvascular endothelial cell permeability through reactive oxygen species production and microtubule remodeling

    Directory of Open Access Journals (Sweden)

    Shi Xianglin

    2009-01-01

    Full Text Available Abstract Background Engineered iron nanoparticles are being explored for the development of biomedical applications and many other industry purposes. However, to date little is known concerning the precise mechanisms of translocation of iron nanoparticles into targeted tissues and organs from blood circulation, as well as the underlying implications of potential harmful health effects in human. Results The confocal microscopy imaging analysis demonstrates that exposure to engineered iron nanoparticles induces an increase in cell permeability in human microvascular endothelial cells. Our studies further reveal iron nanoparticles enhance the permeability through the production of reactive oxygen species (ROS and the stabilization of microtubules. We also showed Akt/GSK-3β signaling pathways are involved in iron nanoparticle-induced cell permeability. The inhibition of ROS demonstrate ROS play a major role in regulating Akt/GSK-3β – mediated cell permeability upon iron nanoparticle exposure. These results provide new insights into the bioreactivity of engineered iron nanoparticles which can inform potential applications in medical imaging or drug delivery. Conclusion Our results indicate that exposure to iron nanoparticles induces an increase in endothelial cell permeability through ROS oxidative stress-modulated microtubule remodeling. The findings from this study provide new understandings on the effects of nanoparticles on vascular transport of macromolecules and drugs.

  5. Inhibitory neurons modulate spontaneous signaling in cultured cortical neurons: density-dependent regulation of excitatory neuronal signaling

    International Nuclear Information System (INIS)

    Serra, Michael; Guaraldi, Mary; Shea, Thomas B

    2010-01-01

    Cortical neuronal activity depends on a balance between excitatory and inhibitory influences. Culturing of neurons on multi-electrode arrays (MEAs) has provided insight into the development and maintenance of neuronal networks. Herein, we seeded MEAs with murine embryonic cortical/hippocampal neurons at different densities ( 1000 cells mm −2 ) and monitored resultant spontaneous signaling. Sparsely seeded cultures displayed a large number of bipolar, rapid, high-amplitude individual signals with no apparent temporal regularity. By contrast, densely seeded cultures instead displayed clusters of signals at regular intervals. These patterns were observed even within thinner and thicker areas of the same culture. GABAergic neurons (25% of total neurons in our cultures) mediated the differential signal patterns observed above, since addition of the inhibitory antagonist bicuculline to dense cultures and hippocampal slice cultures induced the signal pattern characteristic of sparse cultures. Sparsely seeded cultures likely lacked sufficient inhibitory neurons to modulate excitatory activity. Differential seeding of MEAs can provide a unique model for analyses of pertubation in the interaction between excitatory and inhibitory function during aging and neuropathological conditions where dysregulation of GABAergic neurons is a significant component

  6. Control of density-dependent, cell state-specific signal transduction by the cell adhesion molecule CEACAM1, and its influence on cell cycle regulation

    International Nuclear Information System (INIS)

    Scheffrahn, Inka; Singer, Bernhard B.; Sigmundsson, Kristmundur; Lucka, Lothar; Oebrink, Bjoern

    2005-01-01

    Growth factor receptors, extracellular matrix receptors, and cell-cell adhesion molecules co-operate in regulating the activities of intracellular signaling pathways. Here, we demonstrate that the cell adhesion molecule CEACAM1 co-regulates growth-factor-induced DNA synthesis in NBT-II epithelial cells in a cell-density-dependent manner. CEACAM1 exerted its effects by regulating the activity of the Erk 1/2 MAP kinase pathway and the expression levels of the cyclin-dependent kinase inhibitor p27 Kip1 . Interestingly, both inhibitory and stimulatory effects were observed. Confluent cells continuously exposed to fetal calf serum showed little Erk activity and DNA synthesis compared with sparse cells. Under these conditions, anti-CEACAM1 antibodies strongly stimulated Erk activation, decreased p27 expression, and induced DNA synthesis. In serum-starved confluent cells, re-addition of 10% fetal calf serum activated the Erk pathway, decreased p27 expression, and stimulated DNA synthesis to the same levels as in sparse cells. Under these conditions anti-CEACAM1 antibodies de-activated Erk, restored the level of p27, and inhibited DNA synthesis. These data indicate that CEACAM1 mediates contact inhibition of proliferation in cells that are constantly exposed to growth factors, but co-activates growth-factor-induced proliferation in cells that have been starved for growth factors; exposure to extracellular CEACAM1 ligands reverts these responses

  7. Dietary fatty acids regulate hepatic low density lipoprotein (LDL) transport by altering LDL receptor protein and mRNA levels.

    Science.gov (United States)

    Horton, J D; Cuthbert, J A; Spady, D K

    1993-01-01

    The concentration of LDL in plasma is strongly influenced by the amount and the type of lipid in the diet. Recent studies in the hamster have shown that dietary fatty acids differentially affect circulating LDL levels primarily by altering receptor-dependent LDL uptake in the liver. To investigate the mechanistic basis of this effect, rates of receptor-dependent LDL transport in the liver were correlated with LDL receptor protein and mRNA levels in hamsters fed safflower oil or coconut oil and varying amounts of cholesterol. Hepatic LDL receptor activity was significantly lower in animals fed coconut oil than in animals fed safflower oil at all levels of cholesterol intake (26, 53, and 61% lower at cholesterol intakes of 0, 0.06, and 0.12%, respectively). These fatty acid-induced changes in hepatic LDL receptor activity were accompanied by parallel changes in hepatic LDL receptor protein and mRNA levels, suggesting that dietary fatty acids regulate the LDL receptor pathway largely at the mRNA level. Images PMID:8349814

  8. MVL-PLA2, a snake venom phospholipase A2, inhibits angiogenesis through an increase in microtubule dynamics and disorganization of focal adhesions.

    Directory of Open Access Journals (Sweden)

    Amine Bazaa

    Full Text Available Integrins are essential protagonists of the complex multi-step process of angiogenesis that has now become a major target for the development of anticancer therapies. We recently reported and characterized that MVL-PLA2, a novel phospholipase A2 from Macrovipera lebetina venom, exhibited anti-integrin activity. In this study, we show that MVL-PLA2 also displays potent anti-angiogenic properties. This phospholipase A2 inhibited adhesion and migration of human microvascular-endothelial cells (HMEC-1 in a dose-dependent manner without being cytotoxic. Using Matrigel and chick chorioallantoic membrane assays, we demonstrated that MVL-PLA2, as well as its catalytically inactivated form, significantly inhibited angiogenesis both in vitro and in vivo. We have also found that the actin cytoskeleton and the distribution of alphav beta3 integrin, a critical regulator of angiogenesis and a major component of focal adhesions, were disturbed after MVL-PLA2 treatment. In order to further investigate the mechanism of action of this protein on endothelial cells, we analyzed the dynamic instability behavior of microtubules in living endothelial cells. Interestingly, we showed that MVL-PLA2 significantly increased microtubule dynamicity in HMEC-1 cells by 40%. We propose that the enhancement of microtubule dynamics may explain the alterations in the formation of focal adhesions, leading to inhibition of cell adhesion and migration.

  9. Quantitative cell polarity imaging defines leader-to-follower transitions during collective migration and the key role of microtubule-dependent adherens junction formation.

    Science.gov (United States)

    Revenu, Céline; Streichan, Sebastian; Donà, Erika; Lecaudey, Virginie; Hufnagel, Lars; Gilmour, Darren

    2014-03-01

    The directed migration of cell collectives drives the formation of complex organ systems. A characteristic feature of many migrating collectives is a 'tissue-scale' polarity, whereby 'leader' cells at the edge of the tissue guide trailing 'followers' that become assembled into polarised epithelial tissues en route. Here, we combine quantitative imaging and perturbation approaches to investigate epithelial cell state transitions during collective migration and organogenesis, using the zebrafish lateral line primordium as an in vivo model. A readout of three-dimensional cell polarity, based on centrosomal-nucleus axes, allows the transition from migrating leaders to assembled followers to be quantitatively resolved for the first time in vivo. Using live reporters and a novel fluorescent protein timer approach, we investigate changes in cell-cell adhesion underlying this transition by monitoring cadherin receptor localisation and stability. This reveals that while cadherin 2 is expressed across the entire tissue, functional apical junctions are first assembled in the transition zone and become progressively more stable across the leader-follower axis of the tissue. Perturbation experiments demonstrate that the formation of these apical adherens junctions requires dynamic microtubules. However, once stabilised, adherens junction maintenance is microtubule independent. Combined, these data identify a mechanism for regulating leader-to-follower transitions within migrating collectives, based on the relocation and stabilisation of cadherins, and reveal a key role for dynamic microtubules in this process.

  10. Enhancement of the efficiency of photodynamic therapy by combination with the microtubule inhibitor vincristine

    Science.gov (United States)

    Ma, Li Wei; Berg, Kristian; Danielsen, Havard E.; Iani, Vladimir; Moan, Johan

    1996-01-01

    Combination effects of photodynamic therapy (PDT) with meso-tetra (di-adjacent- sulfonatophenyl) porphine (TPPS2a) and the microtubule (MT) inhibitor, vincristine (VCR), were studied in the CaD2 mouse tumor model in mice. A synergistic effect was found when VCR, at an almost nontoxic dose (1 mg/kg), was injected i.p. into the mice 6 hr before PDT. The data on mitotic index show a 4 - 5 fold accumulation of the cells in mitosis 6 hr after injection of VCR into the mice. Cell cycle and ploidy distributions in tumor tissues were determined by means of image analysis with measurement of integrated optical density after Feulgen reaction on monolayers. Ploidy distribution of the tumors was not significantly changed 6 and 12 hr after administration of VCR only, while an increasing aneuploidy was observed 24 and 48 hr after VCR treatment. No prominent changes of the cell cycle and ploidy distributions were found in the tumor tissues after PDT or PDT combined with VCR.

  11. Collective behavior of minus-ended motors in mitotic microtubule asters gliding toward DNA

    International Nuclear Information System (INIS)

    Athale, Chaitanya A; Dinarina, Ana; Nedelec, Francois; Karsenti, Eric

    2014-01-01

    Microtubules (MTs) nucleated by centrosomes form star-shaped structures referred to as asters. Aster motility and dynamics is vital for genome stability, cell division, polarization and differentiation. Asters move either toward the cell center or away from it. Here, we focus on the centering mechanism in a membrane independent system of Xenopus cytoplasmic egg extracts. Using live microscopy and single particle tracking, we find that asters move toward chromatinized DNA structures. The velocity and directionality profiles suggest a random-walk with drift directed toward DNA. We have developed a theoretical model that can explain this movement as a result of a gradient of MT length dynamics and MT gliding on immobilized dynein motors. In simulations, the antagonistic action of the motor species on the radial array of MTs leads to a tug-of-war purely due to geometric considerations and aster motility resembles a directed random-walk. Additionally, our model predicts that aster velocities do not change greatly with varying initial distance from DNA. The movement of asymmetric asters becomes increasingly super-diffusive with increasing motor density, but for symmetric asters it becomes less super-diffusive. The transition of symmetric asters from superdiffusive to diffusive mobility is the result of number fluctuations in bound motors in the tug-of-war. Overall, our model is in good agreement with experimental data in Xenopus cytoplasmic extracts and predicts novel features of the collective effects of motor-MT interactions. (paper)

  12. Regulation

    International Nuclear Information System (INIS)

    Ballereau, P.

    1999-01-01

    The different regulations relative to nuclear energy since the first of January 1999 are given here. Two points deserve to be noticed: the decree of the third august 1999 authorizing the national Agency for the radioactive waste management to install and exploit on the commune of Bures (Meuse) an underground laboratory destined to study the deep geological formations where could be stored the radioactive waste. The second point is about the uranium residues and the waste notion. The judgment of the administrative tribunal of Limoges ( 9. july 1998) forbidding the exploitation of a storage installation of depleted uranium considered as final waste and qualifying it as an industrial waste storage facility has been annulled bu the Court of Appeal. It stipulated that, according to the law number 75663 of the 15. july 1965, no criteria below can be applied to depleted uranium: production residue (possibility of an ulterior enrichment), abandonment of a personal property or simple intention to do it ( future use aimed in the authorization request made in the Prefecture). This judgment has devoted the primacy of the waste notion on this one of final waste. (N.C.)

  13. Polo-like kinase phosphorylation determines Caenorhabditis elegans centrosome size and density by biasing SPD-5 toward an assembly-competent conformation

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    Oliver Wueseke

    2016-10-01

    Full Text Available Centrosomes are major microtubule-organizing centers composed of centrioles surrounded by an extensive proteinacious layer called the pericentriolar material (PCM. In Caenorhabditis elegans embryos, the mitotic PCM expands by Polo-like kinase 1 (PLK-1 phosphorylation-accelerated assembly of SPD-5 molecules into supramolecular scaffolds. However, how PLK-1 phosphorylation regulates SPD-5 assembly is not known. We found that a mutant version of SPD-5 that is insensitive to PLK-1 phosphorylation (SPD-54A could localize to PCM but was unable to rescue the reduction in PCM size and density when wild-type SPD-5 levels were decreased. In vitro, purified SPD-54A self-assembled into functional supramolecular scaffolds over long time scales, suggesting that phosphorylation only controls the rate of SPD-5 scaffold assembly. Furthermore, the SPD-5 scaffold, once assembled, remained intact and supported microtubule nucleation in the absence of PLK-1 activity in vivo. We conclude that PLK-1 is required for rapid assembly of the PCM scaffold but not for scaffold maintenance or function. Based on this idea, we developed a theoretical model that adequately predicted PCM growth rates in different mutant conditions in vivo. We propose that PLK-1 phosphorylation-dependent conversion of SPD-5 into an assembly-competent form underlies PCM formation in vivo and that the rate of this conversion determines final PCM size and density.

  14. Cdk1-cyclin B1-mediated phosphorylation of tumor-associated microtubule-associated protein/cytoskeleton-associated protein 2 in mitosis.

    Science.gov (United States)

    Hong, Kyung Uk; Kim, Hyun-Jun; Kim, Hyo-Sil; Seong, Yeon-Sun; Hong, Kyeong-Man; Bae, Chang-Dae; Park, Joobae

    2009-06-12

    During mitosis, establishment of structurally and functionally sound bipolar spindles is necessary for maintaining the fidelity of chromosome segregation. Tumor-associated microtubule-associated protein (TMAP), also known as cytoskeleton-associated protein 2 (CKAP2), is a mitotic spindle-associated protein whose level is frequently up-regulated in various malignancies. Previous reports have suggested that TMAP is a potential regulator of mitotic spindle assembly and dynamics and that it is required for chromosome segregation to occur properly. So far, there have been no reports on how its mitosis-related functions are regulated. Here, we report that TMAP is hyper-phosphorylated at the C terminus specifically during mitosis. At least four different residues (Thr-578, Thr-596, Thr-622, and Ser-627) were responsible for the mitosis-specific phosphorylation of TMAP. Among these, Thr-622 was specifically phosphorylated by Cdk1-cyclin B1 both in vitro and in vivo. Interestingly, compared with the wild type, a phosphorylation-deficient mutant form of TMAP, in which Thr-622 had been replaced with an alanine (T622A), induced a significant increase in the frequency of metaphase cells with abnormal bipolar spindles, which often displayed disorganized, asymmetrical, or narrow and elongated morphologies. Formation of these abnormal bipolar spindles subsequently resulted in misalignment of metaphase chromosomes and ultimately caused a delay in the entry into anaphase. Moreover, such defects resulting from the T622A mutation were associated with a decrease in the rate of protein turnover at spindle microtubules. These findings suggest that Cdk1-cyclin B1-mediated phosphorylation of TMAP is important for and contributes to proper regulation of microtubule dynamics and establishment of functional bipolar spindles during mitosis.

  15. Cdk1-Cyclin B1-mediated Phosphorylation of Tumor-associated Microtubule-associated Protein/Cytoskeleton-associated Protein 2 in Mitosis*

    Science.gov (United States)

    Uk Hong, Kyung; Kim, Hyun-Jun; Kim, Hyo-Sil; Seong, Yeon-Sun; Hong, Kyeong-Man; Bae, Chang-Dae; Park, Joobae

    2009-01-01

    During mitosis, establishment of structurally and functionally sound bipolar spindles is necessary for maintaining the fidelity of chromosome segregation. Tumor-associated microtubule-associated protein (TMAP), also known as cytoskeleton-associated protein 2 (CKAP2), is a mitotic spindle-associated protein whose level is frequently up-regulated in various malignancies. Previous reports have suggested that TMAP is a potential regulator of mitotic spindle assembly and dynamics and that it is required for chromosome segregation to occur properly. So far, there have been no reports on how its mitosis-related functions are regulated. Here, we report that TMAP is hyper-phosphorylated at the C terminus specifically during mitosis. At least four different residues (Thr-578, Thr-596, Thr-622, and Ser-627) were responsible for the mitosis-specific phosphorylation of TMAP. Among these, Thr-622 was specifically phosphorylated by Cdk1-cyclin B1 both in vitro and in vivo. Interestingly, compared with the wild type, a phosphorylation-deficient mutant form of TMAP, in which Thr-622 had been replaced with an alanine (T622A), induced a significant increase in the frequency of metaphase cells with abnormal bipolar spindles, which often displayed disorganized, asymmetrical, or narrow and elongated morphologies. Formation of these abnormal bipolar spindles subsequently resulted in misalignment of metaphase chromosomes and ultimately caused a delay in the entry into anaphase. Moreover, such defects resulting from the T622A mutation were associated with a decrease in the rate of protein turnover at spindle microtubules. These findings suggest that Cdk1-cyclin B1-mediated phosphorylation of TMAP is important for and contributes to proper regulation of microtubule dynamics and establishment of functional bipolar spindles during mitosis. PMID:19369249

  16. Protein friction limits diffusive and directed movements of kinesin motors on microtubules.

    Science.gov (United States)

    Bormuth, Volker; Varga, Vladimir; Howard, Jonathon; Schäffer, Erik

    2009-08-14

    Friction limits the operation of macroscopic engines and is critical to the performance of micromechanical devices. We report measurements of friction in a biological nanomachine. Using optical tweezers, we characterized the frictional drag force of individual kinesin-8 motor proteins interacting with their microtubule tracks. At low speeds and with no energy source, the frictional drag was related to the diffusion coefficient by the Einstein relation. At higher speeds, the frictional drag force increased nonlinearly, consistent with the motor jumping 8 nanometers between adjacent tubulin dimers along the microtubule, and was asymmetric, reflecting the structural polarity of the microtubule. We argue that these frictional forces arise from breaking bonds between the motor domains and the microtubule, and they limit the speed and efficiency of kinesin.

  17. [Genetic transformation of flax (Linum usitatissimum L.) with chimeric GFP-TUA6 gene for visualisation of microtubules].

    Science.gov (United States)

    Shisha, E N; Korkhovoĭ, V I; Baer, G Ia; Guzenko, E V; Lemesh, V A; Kartel', N A; Emets, A I; Blium, Ia B

    2013-01-01

    The data of Agrobacterium-mediated transformation of some Linum usitatissimum cultivars zoned on the territories of Belarus and Ukraine with the plasmid carrying chimeric GFP-TUA6 gene and nptII gene as selectable marker conferring resistance to kanamycin are presented in this study. Transformation was affected by a number of factors including optical density (OD600), time of inoculation of explants with Agrobacterium and co-culture conditions. Transgenic nature of obtained lines was confirmed by PCR analysis. Expression of GFP-TUA6 gene was detected with confocal laser scanning microscopy. The obtained transgenic lines can be used for further functional studies the role of microtubules in the processes of building the flax fibres and resistance to wind.

  18. In vivo control mechanisms of motor-cargo movement on microtubules

    Science.gov (United States)

    Gunawardena, Shermali

    2014-03-01

    Within axons, molecular motors transport essential components required for neuronal growth and viability. Although many levels of regulation must exist for proper anterograde and retrograde transport of vital proteins, little is known about these mechanisms. Previous work suggested that the amyloid precursor protein (APP) functions as a kinesin-1 receptor during transport. However, how APP vesicle motility is regulated is unclear. Using genetics and in vivo imaging in Drosophila we showed that reduction of presenilin (PS) substantially increased anterograde and retrograde APP vesicle velocities. Strikingly, PS deficiency had no effect on an unrelated cargo vesicle containing synaptotagmin, which is powered by a different kinesin motor. Increased PS-mediated velocities required functional kinesin-1 and dynein motors. We also found that these PS-mediated effects on motor protein function were mediated via a pathway that involves glycogen synthase kinase-3 β (GSK-3 β) . PS genetically interacted with GSK-3 β in an activity dependent manner. Excess of active GSK-3 β perturbed transport by causing axonal blockages, which were enhanced by reduction of kinesin-1 or dynein, while excess of non-functional GSK-3 β had no effect. Strikingly, GSK-3 β-activity dependent transport defects were enhanced by reduction of PS. Collectively, our findings suggest that PS and GSK-3 β are required for normal motor protein function, and we propose a model in which PS likely regulates GSK-3 β activity during transport. These findings have important implications for our understanding of the complex regulatory machinery that must exist in vivo and how this system is coordinated during vesicle motility on microtubules.

  19. Bacillus anthracis TIR Domain-Containing Protein Localises to Cellular Microtubule Structures and Induces Autophagy.

    Science.gov (United States)

    Carlsson, Emil; Thwaite, Joanne E; Jenner, Dominic C; Spear, Abigail M; Flick-Smith, Helen; Atkins, Helen S; Byrne, Bernadette; Ding, Jeak Ling

    2016-01-01

    Toll-like receptors (TLRs) recognise invading pathogens and mediate downstream immune signalling via Toll/IL-1 receptor (TIR) domains. TIR domain proteins (Tdps) have been identified in multiple pathogenic bacteria and have recently been implicated as negative regulators of host innate immune activation. A Tdp has been identified in Bacillus anthracis, the causative agent of anthrax. Here we present the first study of this protein, designated BaTdp. Recombinantly expressed and purified BaTdp TIR domain interacted with several human TIR domains, including that of the key TLR adaptor MyD88, although BaTdp expression in cultured HEK293 cells had no effect on TLR4- or TLR2- mediated immune activation. During expression in mammalian cells, BaTdp localised to microtubular networks and caused an increase in lipidated cytosolic microtubule-associated protein 1A/1B-light chain 3 (LC3), indicative of autophagosome formation. In vivo intra-nasal infection experiments in mice showed that a BaTdp knockout strain colonised host tissue faster with higher bacterial load within 4 days post-infection compared to the wild type B. anthracis. Taken together, these findings indicate that BaTdp does not play an immune suppressive role, but rather, its absence increases virulence. BaTdp present in wild type B. anthracis plausibly interact with the infected host cell, which undergoes autophagy in self-defence.

  20. Deficiency of RITA results in multiple mitotic defects by affecting microtubule dynamics.

    Science.gov (United States)

    Steinhäuser, K; Klöble, P; Kreis, N-N; Ritter, A; Friemel, A; Roth, S; Reichel, J M; Michaelis, J; Rieger, M A; Louwen, F; Oswald, F; Yuan, J

    2017-04-01

    Deregulation of mitotic microtubule (MT) dynamics results in defective spindle assembly and chromosome missegregation, leading further to chromosome instability, a hallmark of tumor cells. RBP-J interacting and tubulin-associated protein (RITA) has been identified as a negative regulator of the Notch signaling pathway. Intriguingly, deregulated RITA is involved in primary hepatocellular carcinoma and other malignant entities. We were interested in the potential molecular mechanisms behind its involvement. We show here that RITA binds to tubulin and localizes to various mitotic MT structures. RITA coats MTs and affects their structures in vitro as well as in vivo. Tumor cell lines deficient of RITA display increased acetylated α-tubulin, enhanced MT stability and reduced MT dynamics, accompanied by multiple mitotic defects, including chromosome misalignment and segregation errors. Re-expression of wild-type RITA, but not RITA Δtub ineffectively binding to tubulin, restores the phenotypes, suggesting that the role of RITA in MT modulation is mediated via its interaction with tubulin. Mechanistically, RITA interacts with tubulin/histone deacetylase 6 (HDAC6) and its suppression decreases the binding of the deacetylase HDAC6 to tubulin/MTs. Furthermore, the mitotic defects and increased MT stability are also observed in RITA -/- mouse embryonic fibroblasts. RITA has thus a novel role in modulating MT dynamics and its deregulation results in erroneous chromosome segregation, one of the major reasons for chromosome instability in tumor cells.

  1. Bug22 influences cilium morphology and the post-translational modification of ciliary microtubules

    Directory of Open Access Journals (Sweden)

    Teresa Mendes Maia

    2014-01-01

    Cilia and flagella are organelles essential for motility and sensing of environmental stimuli. Depending on the cell type, cilia acquire a defined set of functions and, accordingly, are built with an appropriate length and molecular composition. Several ciliary proteins display a high degree of conservation throughout evolution and mutations in ciliary genes are associated with various diseases such as ciliopathies and infertility. Here, we describe the role of the highly conserved ciliary protein, Bug22, in Drosophila. Previous studies in unicellular organisms have shown that Bug22 is required for proper cilia function, but its exact role in ciliogenesis has not been investigated yet. Null Bug22 mutant flies display cilia-associated phenotypes and nervous system defects. Furthermore, sperm differentiation is blocked at the individualization stage, due to impaired migration of the individualization machinery. Tubulin post-translational modifications (PTMs such as polyglycylation, polyglutamylation or acetylation, are determinants of microtubule (MT functions and stability in centrioles, cilia and neurons. We found defects in the timely incorporation of polyglycylation in sperm axonemal MTs of Bug22 mutants. In addition, we found that depletion of human Bug22 in RPE1 cells resulted in the appearance of longer cilia and reduced axonemal polyglutamylation. Our work identifies Bug22 as a protein that plays a conserved role in the regulation of PTMs of the ciliary axoneme.

  2. The microtubule associated protein END BINDING 1 represses root responses to mechanical cues.

    Science.gov (United States)

    Gleeson, Laura; Squires, Shannon; Bisgrove, Sherryl R

    2012-05-01

    The ability of roots to navigate around rocks and other debris as they grow through the soil requires a mechanism for detecting and responding to input from both touch and gravity sensing systems. The microtubule associated protein END BINDING 1b (EB1b) is involved in this process as mutants have defects responding to combinations of touch and gravity cues. This study investigates the role of EB1b in root responses to mechanical cues. We find that eb1b-1 mutant roots exhibit an increase over wild type in their response to touch and that the expression of EB1b genes in transgenic mutants restores the response to wild type levels, indicating that EB1b is an inhibitor of the response. Mutant roots are also hypersensitive to increased levels of mechanical stimulation, revealing the presence of another process that activates the response. These findings are supported by analyses of double mutants between eb1b-1 and seedlings carrying mutations in PHOSPHOGLUCOMUTASE (PGM), ALTERED RESPONSE TO GRAVITY1 (ARG1), or TOUCH3 (TCH3), genes that encode proteins involved in gravity sensing, signaling, or touch responses, respectively. A model is proposed in which root responses to mechanical cues are modulated by at least two competing regulatory processes, one that promotes touch-mediated growth and another, regulated by EB1b, which dampens root responses to touch and enhances gravitropism. © 2012. Published by Elsevier Ireland Ltd. All rights reserved.

  3. Biophysics of filament length regulation by molecular motors

    International Nuclear Information System (INIS)

    Kuan, Hui-Shun; Betterton, M D

    2013-01-01

    Regulating physical size is an essential problem that biological organisms must solve from the subcellular to the organismal scales, but it is not well understood what physical principles and mechanisms organisms use to sense and regulate their size. Any biophysical size-regulation scheme operates in a noisy environment and must be robust to other cellular dynamics and fluctuations. This work develops theory of filament length regulation inspired by recent experiments on kinesin-8 motor proteins, which move with directional bias on microtubule filaments and alter microtubule dynamics. Purified kinesin-8 motors can depolymerize chemically-stabilized microtubules. In the length-dependent depolymerization model, the rate of depolymerization tends to increase with filament length, because long filaments accumulate more motors at their tips and therefore shorten more quickly. When balanced with a constant filament growth rate, this mechanism can lead to a fixed polymer length. However, the mechanism by which kinesin-8 motors affect the length of dynamic microtubules in cells is less clear. We study the more biologically realistic problem of microtubule dynamic instability modulated by a motor-dependent increase in the filament catastrophe frequency. This leads to a significant decrease in the mean filament length and a narrowing of the filament length distribution. The results improve our understanding of the biophysics of length regulation in cells. (paper)

  4. Cellular effects of curcumin on Plasmodium falciparum include disruption of microtubules.

    Directory of Open Access Journals (Sweden)

    Rimi Chakrabarti

    Full Text Available Curcumin has been widely investigated for its myriad cellular effects resulting in reduced proliferation of various eukaryotic cells including cancer cells and the human malaria parasite Plasmodium falciparum. Studies with human cancer cell lines HT-29, Caco-2, and MCF-7 suggest that curcumin can bind to tubulin and induce alterations in microtubule structure. Based on this finding, we investigated whether curcumin has any effect on P. falciparum microtubules, considering that mammalian and parasite tubulin are 83% identical. IC50 of curcumin was found to be 5 µM as compared to 20 µM reported before. Immunofluorescence images of parasites treated with 5 or 20 µM curcumin showed a concentration-dependent effect on parasite microtubules resulting in diffuse staining contrasting with the discrete hemispindles and subpellicular microtubules observed in untreated parasites. The effect on P. falciparum microtubules was evident only in the second cycle for both concentrations tested. This diffuse pattern of tubulin fluorescence in curcumin treated parasites was similar to the effect of a microtubule destabilizing drug vinblastine on P. falciparum. Molecular docking predicted the binding site of curcumin at the interface of alpha and beta tubulin, similar to another destabilizing drug colchicine. Data from predicted drug binding is supported by results from drug combination assays showing antagonistic interactions between curcumin and colchicine, sharing a similar binding site, and additive/synergistic interactions of curcumin with paclitaxel and vinblastine, having different binding sites. This evidence suggests that cellular effects of curcumin are at least, in part, due to its perturbing effect on P. falciparum microtubules. The action of curcumin, both direct and indirect, on P. falciparum microtubules is discussed.

  5. GDP-to-GTP exchange on the microtubule end can contribute to the frequency of catastrophe.

    Science.gov (United States)

    Piedra, Felipe-Andrés; Kim, Tae; Garza, Emily S; Geyer, Elisabeth A; Burns, Alexander; Ye, Xuecheng; Rice, Luke M

    2016-11-07

    Microtubules are dynamic polymers of αβ-tubulin that have essential roles in chromosome segregation and organization of the cytoplasm. Catastrophe-the switch from growing to shrinking-occurs when a microtubule loses its stabilizing GTP cap. Recent evidence indicates that the nucleotide on the microtubule end controls how tightly an incoming subunit will be bound (trans-acting GTP), but most current models do not incorporate this information. We implemented trans-acting GTP into a computational model for microtubule dynamics. In simulations, growing microtubules often exposed terminal GDP-bound subunits without undergoing catastrophe. Transient GDP exposure on the growing plus end slowed elongation by reducing the number of favorable binding sites on the microtubule end. Slower elongation led to erosion of the GTP cap and an increase in the frequency of catastrophe. Allowing GDP-to-GTP exchange on terminal subunits in simulations mitigated these effects. Using mutant αβ-tubulin or modified GTP, we showed experimentally that a more readily exchangeable nucleotide led to less frequent catastrophe. Current models for microtubule dynamics do not account for GDP-to-GTP exchange on the growing microtubule end, so our findings provide a new way of thinking about the molecular events that initiate catastrophe. © 2016 Piedra et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  6. Essential oil of Pinus koraiensis leaves exerts antihyperlipidemic effects via up-regulation of low-density lipoprotein receptor and inhibition of acyl-coenzyme A: cholesterol acyltransferase.

    Science.gov (United States)

    Kim, Ji-Hyun; Lee, Hyo-Jung; Jeong, Soo-Jin; Lee, Min-Ho; Kim, Sung-Hoon

    2012-09-01

    Hyperlipidemia is an important factor to induce metabolic syndrome such as obesity, diabetes and cardiovascular diseases. Recently, some antihyperlipidemic agents from herbal medicines have been in the spotlight in the medical science field. Thus, the present study evaluated the antihyperlipidemic activities of the essential oil from the leaves of Pinus koraiensis SIEB (EOPK) that has been used as a folk remedy for heart disease. The reverse transcription polymerase chain reaction (RT-PCR) revealed that EOPK up-regulated low density lipoprotein receptor (LDLR) at the mRNA level as well as negatively suppressed the expression of sterol regulatory element-binding protein (SREBP)-1c, SREBP-2, 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR), fatty acid synthase (FAS) and glycerol-3-phosphate acyltransferase (GPAT) involved in lipid metabolism in HepG2 cells. Also, western blotting showed that EOPK activated LDLR and attenuated the expression of FAS at the protein level in the cells. Consistently, EOPK significantly inhibited the level of human acylcoenzyme A: cholesterol acyltransferase (hACAT)1 and 2 and reduced the low-density lipoprotein (LDL) oxidation activity. Furthermore, chromatography-mass spectrometry (GC-MS) analysis showed that EOPK, an essential oil mixture, contained camphene (21.11%), d-limonene (21.01%), α-pinene (16.74%) and borneol (11.52%). Overall, the findings suggest that EOPK can be a potent pharmaceutical agent for the prevention and treatment of hyperlipidemia. Copyright © 2012 John Wiley & Sons, Ltd.

  7. Interplay between microtubule bundling and sorting factors ensures acentriolar spindle stability during C. elegans oocyte meiosis.

    Directory of Open Access Journals (Sweden)

    Timothy J Mullen

    2017-09-01

    Full Text Available In many species, oocyte meiosis is carried out in the absence of centrioles. As a result, microtubule organization, spindle assembly, and chromosome segregation proceed by unique mechanisms. Here, we report insights into the principles underlying this specialized form of cell division, through studies of C. elegans KLP-15 and KLP-16, two highly homologous members of the kinesin-14 family of minus-end-directed kinesins. These proteins localize to the acentriolar oocyte spindle and promote microtubule bundling during spindle assembly; following KLP-15/16 depletion, microtubule bundles form but then collapse into a disorganized array. Surprisingly, despite this defect we found that during anaphase, microtubules are able to reorganize into a bundled array that facilitates chromosome segregation. This phenotype therefore enabled us to identify factors promoting microtubule organization during anaphase, whose contributions are normally undetectable in wild-type worms; we found that SPD-1 (PRC1 bundles microtubules and KLP-18 (kinesin-12 likely sorts those bundles into a functional orientation capable of mediating chromosome segregation. Therefore, our studies have revealed an interplay between distinct mechanisms that together promote spindle formation and chromosome segregation in the absence of structural cues such as centrioles.

  8. Emerging roles for microtubules in angiosperm pollen tube growth highlight new research cues

    Directory of Open Access Journals (Sweden)

    Alessandra eMoscatelli

    2015-02-01

    Full Text Available In plants, actin filaments have an important role in organelle movement and cytoplasmic streaming. Otherwise microtubules have a role in restricting organelles to specific areas of the cell and in maintaining organelle morphology. In somatic plant cells, microtubules also participate in cell division and morphogenesis, allowing cells to take their definitive shape in order to perform specific functions. In the latter case, microtubules influence assembly of the cell wall, controlling the delivery of enzymes involved in cellulose synthesis and of wall modulation material to the proper sites.In angiosperm pollen tubes, organelle movement is generally attributed to the acto-myosin system, the main role of which is in distributing organelles in the cytoplasm and in carrying secretory vesicles to the apex for polarized growth. Recent data on membrane trafficking suggests a role of microtubules in fine delivery and repositioning of vesicles to sustain pollen tube growth. This review examines the role of microtubules in secretion and endocytosis, highlighting new research cues regarding cell wall construction and pollen tube-pistil crosstalk, that help unravel the role of microtubules in polarized growth.

  9. ATPase Cycle of the Nonmotile Kinesin NOD Allows Microtubule End Tracking and Drives Chromosome Movement

    Energy Technology Data Exchange (ETDEWEB)

    Cochran, J.; Sindelar, C; Mulko, N; Collins, K; Kong, S; Hawley, R; Kull, F

    2009-01-01

    Segregation of nonexchange chromosomes during Drosophila melanogaster meiosis requires the proper function of NOD, a nonmotile kinesin-10. We have determined the X-ray crystal structure of the NOD catalytic domain in the ADP- and AMPPNP-bound states. These structures reveal an alternate conformation of the microtubule binding region as well as a nucleotide-sensitive relay of hydrogen bonds at the active site. Additionally, a cryo-electron microscopy reconstruction of the nucleotide-free microtubule-NOD complex shows an atypical binding orientation. Thermodynamic studies show that NOD binds tightly to microtubules in the nucleotide-free state, yet other nucleotide states, including AMPPNP, are weakened. Our pre-steady-state kinetic analysis demonstrates that NOD interaction with microtubules occurs slowly with weak activation of ADP product release. Upon rapid substrate binding, NOD detaches from the microtubule prior to the rate-limiting step of ATP hydrolysis, which is also atypical for a kinesin. We propose a model for NOD's microtubule plus-end tracking that drives chromosome movement.

  10. Direct evidence for GTP and GDP-Pi intermediates in microtubule assembly

    International Nuclear Information System (INIS)

    Melki, R.; Carlier, M.F.; Pantaloni, D.

    1990-01-01

    Identification of the kinetic intermediates in GTP hydrolysis on microtubules and characterization of their assembly properties is essential in understanding microtubule dynamics. By using an improved glass filter assay that selectively traps microtubules with a dead time of 2 s and monitoring taxol-induced rapid assembly of microtubules from [γ- 32 P, 3 H]GTP-tubulin 1:1 complex, direct evidence has been obtained for GTP- and GDP-P i -microtubule transient states in the early stages of the polymerization process. A simple kinetic analysis of GTP hydrolysis on microtubules within two sequential pseudo-first-order processes led to apparent first-order rate constants of 0.065 s -1 for the cleavage of the γ-phosphate and 0.02 s -1 for the liberation of P i , assuming a simple random model. Apparent rate constants for GTP hydrolysis and P i release were independent of the composition of the buffer used to polymerize tubulin. The significance of these values with respect to those derived from previous studies from this and other laboratories and the possibility of a vectorial model for GTP hydrolysis are discussed

  11. Microtubule-targeting drugs rescue axonal swellings in cortical neurons from spastin knockout mice

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    Coralie Fassier

    2013-01-01

    Mutations in SPG4, encoding the microtubule-severing protein spastin, are responsible for the most frequent form of hereditary spastic paraplegia (HSP, a heterogeneous group of genetic diseases characterized by degeneration of the corticospinal tracts. We previously reported that mice harboring a deletion in Spg4, generating a premature stop codon, develop progressive axonal degeneration characterized by focal axonal swellings associated with impaired axonal transport. To further characterize the molecular and cellular mechanisms underlying this mutant phenotype, we have assessed microtubule dynamics and axonal transport in primary cultures of cortical neurons from spastin-mutant mice. We show an early and marked impairment of microtubule dynamics all along the axons of spastin-deficient cortical neurons, which is likely to be responsible for the occurrence of axonal swellings and cargo stalling. Our analysis also reveals that a modulation of microtubule dynamics by microtubule-targeting drugs rescues the mutant phenotype of cortical neurons. Together, these results contribute to a better understanding of the pathogenesis of SPG4-linked HSP and ascertain the influence of microtubule-targeted drugs on the early axonal phenotype in a mouse model of the disease.

  12. Actin and microtubule networks contribute differently to cell response for small and large strains

    Science.gov (United States)

    Kubitschke, H.; Schnauss, J.; Nnetu, K. D.; Warmt, E.; Stange, R.; Kaes, J.

    2017-09-01

    Cytoskeletal filaments provide cells with mechanical stability and organization. The main key players are actin filaments and microtubules governing a cell’s response to mechanical stimuli. We investigated the specific influences of these crucial components by deforming MCF-7 epithelial cells at small (≤5% deformation) and large strains (>5% deformation). To understand specific contributions of actin filaments and microtubules, we systematically studied cellular responses after treatment with cytoskeleton influencing drugs. Quantification with the microfluidic optical stretcher allowed capturing the relative deformation and relaxation of cells under different conditions. We separated distinctive deformational and relaxational contributions to cell mechanics for actin and microtubule networks for two orders of magnitude of drug dosages. Disrupting actin filaments via latrunculin A, for instance, revealed a strain-independent softening. Stabilizing these filaments by treatment with jasplakinolide yielded cell softening for small strains but showed no significant change at large strains. In contrast, cells treated with nocodazole to disrupt microtubules displayed a softening at large strains but remained unchanged at small strains. Stabilizing microtubules within the cells via paclitaxel revealed no significant changes for deformations at small strains, but concentration-dependent impact at large strains. This suggests that for suspended cells, the actin cortex is probed at small strains, while at larger strains; the whole cell is probed with a significant contribution from the microtubules.

  13. L-Cysteine-induced up-regulation of the low-density lipoprotein receptor is mediated via a transforming growth factor-alpha signalling pathway.

    Science.gov (United States)

    Tanaka, Yuma; Shimada, Masaya; Nagaoka, Satoshi

    2014-02-14

    Sulphur-containing amino acids regulate plasma cholesterol levels in animals and humans. However, their mechanism of action remains unclear. Low-density lipoprotein receptor (LDLR) plays an important role in cholesterol metabolism. We therefore investigated the effects of sulphur-containing amino acids on the expression of LDLR in hepatocytes. HepG2 cells were cultured in Dulbecco's Modified Eagle's Medium with or without sulphur-containing amino acids and cysteine-containing compounds. We found that L-cysteine increased LDLR mRNA and enhanced LDLR gene promoter activity through the extracellular-signal-related kinase and p38 mitogen-activated protein kinase signalling pathways in HepG2 cells. Moreover, we observed that L-cysteine stimulated the release of transforming growth factor-alpha (TGF-α) and that TGF-α increased the LDLR mRNA levels. This study provides a report of the L-cysteine mediated up-regulation of the LDLR expression via TGF-α signalling pathway. Our findings provide insights into cholesterol homeostasis and amino acid signalling. Copyright © 2014 Elsevier Inc. All rights reserved.

  14. Effects of microtubule mechanics on hydrolysis and catastrophes

    International Nuclear Information System (INIS)

    Müller, N; Kierfeld, J

    2014-01-01

    We introduce a model for microtubule (MT) mechanics containing lateral bonds between dimers in neighboring protofilaments, bending rigidity of dimers, and repulsive interactions between protofilaments modeling steric constraints to investigate the influence of mechanical forces on hydrolysis and catastrophes. We use the allosteric dimer model, where tubulin dimers are characterized by an equilibrium bending angle, which changes from 0 ∘ to 22 ∘ by hydrolysis of a dimer. This also affects the lateral interaction and bending energies and, thus, the mechanical equilibrium state of the MT. As hydrolysis gives rise to conformational changes in dimers, mechanical forces also influence the hydrolysis rates by mechanical energy changes modulating the hydrolysis rate. The interaction via the MT mechanics then gives rise to correlation effects in the hydrolysis dynamics, which have not been taken into account before. Assuming a dominant influence of mechanical energies on hydrolysis rates, we investigate the most probable hydrolysis pathways both for vectorial and random hydrolysis. Investigating the stability with respect to lateral bond rupture, we identify initiation configurations for catastrophes along the hydrolysis pathways and values for a lateral bond rupture force. If we allow for rupturing of lateral bonds between dimers in neighboring protofilaments above this threshold force, our model exhibits avalanche-like catastrophe events. (papers)

  15. Calmodulin immunolocalization to cortical microtubules is calcium independent

    Energy Technology Data Exchange (ETDEWEB)

    Fisher, D.D.; Cyr, R.J.

    1992-12-31

    Calcium affects the stability of cortical microtubules (MTs) in lysed protoplasts. This calmodulin (CaM)-mediated interaction may provide a mechanism that serves to integrate cellular behavior with MT function. To test the hypothesis that CaM associates with these MTs, monoclonal antibodies were produced against CaM, and one (designated mAb1D10), was selected for its suitability as an immunocytochemical reagent. It is shown that CaM associates with the cortical Mats of cultured carrot (Daucus carota L.) and tobacco (Nicotiana tobacum L.) cells. Inasmuch as CaM interacts with calcium and affects the behavior of these Mats, we hypothesized that calcium would alter this association. To test this, protoplasts containing taxol-stabilized Mats were lysed in the presence of various concentrations of calcium and examined for the association of Cam with cortical Mats. At 1 {mu}M calcium, many protoplasts did not have CaM in association with the cortical Mats, while at 3.6 {mu}M calcium, this association was completely abolished. The results are discussed in terms of a model in which CaM associates with Mats via two types of interactions; one calcium dependent and one independent.

  16. Calmodulin immunolocalization to cortical microtubules is calcium independent

    Energy Technology Data Exchange (ETDEWEB)

    Fisher, D.D.; Cyr, R.J.

    1992-01-01

    Calcium affects the stability of cortical microtubules (MTs) in lysed protoplasts. This calmodulin (CaM)-mediated interaction may provide a mechanism that serves to integrate cellular behavior with MT function. To test the hypothesis that CaM associates with these MTs, monoclonal antibodies were produced against CaM, and one (designated mAb1D10), was selected for its suitability as an immunocytochemical reagent. It is shown that CaM associates with the cortical Mats of cultured carrot (Daucus carota L.) and tobacco (Nicotiana tobacum L.) cells. Inasmuch as CaM interacts with calcium and affects the behavior of these Mats, we hypothesized that calcium would alter this association. To test this, protoplasts containing taxol-stabilized Mats were lysed in the presence of various concentrations of calcium and examined for the association of Cam with cortical Mats. At 1 [mu]M calcium, many protoplasts did not have CaM in association with the cortical Mats, while at 3.6 [mu]M calcium, this association was completely abolished. The results are discussed in terms of a model in which CaM associates with Mats via two types of interactions; one calcium dependent and one independent.

  17. Characterization of gold nanoparticle binding to microtubule filaments

    International Nuclear Information System (INIS)

    Zhou, Jing C.; Wang Xianghuai; Xue Mei; Xu Zheng; Hamasaki, Toshikazu; Yang, Yang; Wang Kang; Dunn, Bruce

    2010-01-01

    Microtubule (MT) protein filaments were used as templates for fabricating Au nanowires as a bottom-up approach for fabricating building blocks for future integrated circuits. Photochemical reduction methods were employed to form Au nanoparticles which bind and uniformly cover the MT filaments. Synthesis of the MT-templated Au nanowires was characterized using UV/vis spectroscopy and transmission electron microscopy (TEM). In addition, binding between the MT filaments and Au nanoparticles was investigated using surface enhanced Raman spectroscopy (SERS) and X-ray photoelectron spectroscopy (XPS) to establish the nature of the binding sites. A variety of functional groups were identified by SERS to interact with the Au including imidazole, sulfur, aromatic rings, amine, and carboxylate. The imidazole ring in the histidine is the most prominent functional group for Au binding. The results from these studies provide better understanding of the binding between Au and the biotemplate and give insight concerning methods to improve Au coverage for MT-templated Au nanowires.

  18. Halogenated auxins affect microtubules and root elongation in Lactuca sativa

    Science.gov (United States)

    Zhang, N.; Hasenstein, K. H.

    2000-01-01

    We studied the effect of 4,4,4-trifluoro-3-(indole-3-)butyric acid (TFIBA), a recently described root growth stimulator, and 5,6-dichloro-indole-3-acetic acid (DCIAA) on growth and microtubule (MT) organization in roots of Lactuca sativa L. DCIAA and indole-3-butyric acid (IBA) inhibited root elongation and depolymerized MTs in the cortex of the elongation zone, inhibited the elongation of stele cells, and promoted xylem maturation. Both auxins caused the plane of cell division to shift from anticlinal to periclinal. In contrast, TFIBA (100 micromolar) promoted elongation of primary roots by 40% and stimulated the elongation of lateral roots, even in the presence of IBA, the microtubular inhibitors oryzalin and taxol, or the auxin transport inhibitor naphthylphthalamic acid. However, TFIBA inhibited the formation of lateral root primordia. Immunostaining showed that TFIBA stabilized MTs orientation perpendicular to the root axis, doubled the cortical cell length, but delayed xylem maturation. The data indicate that the auxin-induced inhibition of elongation and swelling of roots results from reoriented phragmoplasts, the destabilization of MTs in elongating cells, and promotion of vessel formation. In contrast, TFIBA induced promotion of root elongation by enhancing cell length, prolonging transverse MT orientation, delaying cell and xylem maturation.

  19. The effects of 60Co γ-ray irradiation on cytoplasmic microtubules of mouse macrophages and lymphocytes

    International Nuclear Information System (INIS)

    Li Qianqian; Mao Zijun; Yin Zhiwei; Hu Yumin

    1989-05-01

    The effects of 60 Co γ-ray irradiation on cytoplasmic microtubules of mouse macrophages and lymphocytes were investigated by immunofluorescence microscopy and scanning electron microscope. The results indicated. (1) microtubule organization of the irradiated cells remarkably differed from that of the control since the doses over 4 Gy; (2) 144 hours after irradiation the alterations of microtubules have been shown to be basically r epaired ; (3) the cytoplasmic microtubules and centrioles disappeared under transmission electron microscope, the membranes irradiated and microvilli showed changes under scanning electron microscope too. From these observations and those of other workers who studied the radiation effect on extracted microtubule proteins in vitro, the authors support that 60 Co γ-ray irradiation can inhabits cytoplasmic microtubule assembling

  20. Microtubule-Targeting Agents Enter the Central Nervous System (CNS): Double-edged Swords for Treating CNS Injury and Disease.

    Science.gov (United States)

    Hur, Eun-Mi; Lee, Byoung Dae

    2014-12-01

    Microtubules have been among the most successful targets in anticancer therapy and a large number of microtubule-targeting agents (MTAs) are in various stages of clinical development for the treatment of several malignancies. Given that injury and diseases in the central nervous system (CNS) are accompanied by acute or chronic disruption of the structural integrity of neurons and that microtubules provide structural support for the nervous system at cellular and intracellular levels, microtubules are emerging as potential therapeutic targets for treating CNS disorders. It has been postulated that exogenous application of MTAs might prevent the breakdown or degradation of microtubules after injury or during neurodegeneration, which will thereby aid in preserving the structural integrity and function of the nervous system. Here we review recent evidence that supports this notion and also discuss potential risks of targeting microtubules as a therapy for treating nerve injury and neurodegenerative diseases.

  1. Microtubule-Targeting Agents Enter the Central Nervous System (CNS: Double-edged Swords for Treating CNS Injury and Disease

    Directory of Open Access Journals (Sweden)

    Eun-Mi Hur

    2014-12-01

    Full Text Available Microtubules have been among the most successful targets in anticancer therapy and a large number of microtubule-targeting agents (MTAs are in various stages of clinical development for the treatment of several malignancies. Given that injury and diseases in the central nervous system (CNS are accompanied by acute or chronic disruption of the structural integrity of neurons and that microtubules provide structural support for the nervous system at cellular and intracellular levels, microtubules are emerging as potential therapeutic targets for treating CNS disorders. It has been postulated that exogenous application of MTAs might prevent the breakdown or degradation of microtubules after injury or during neurodegeneration, which will thereby aid in preserving the structural integrity and function of the nervous system. Here we review recent evidence that supports this notion and also discuss potential risks of targeting microtubules as a therapy for treating nerve injury and neurodegenerative diseases.

  2. The role of Cra in regulating acetate excretion and osmotic tolerance in E. coli K-12 and E. coli B at high density growth.

    Science.gov (United States)

    Son, Young-Jin; Phue, Je-Nie; Trinh, Loc B; Lee, Sang Jun; Shiloach, Joseph

    2011-06-30

    E. coli B (BL21), unlike E.coli K-12 (JM109) is insensitive to glucose concentration and, therefore, grows faster and produces less acetate than E. coli K-12, especially when growing to high cell densities at high glucose concentration. By performing genomic analysis, it was demonstrated that the cause of this difference in sensitivity to the glucose concentration is the result of the differences in the central carbon metabolism activity. We hypothesized that the global transcription regulator Cra (FruR) is constitutively expressed in E. coli B and may be responsible for the different behaviour of the two strains. To investigate this possibility and better understand the function of Cra in the two strains, cra - negative E. coli B (BL21) and E. coli K-12 (JM109) were prepared and their growth behaviour and gene expression at high glucose were evaluated using microarray and real-time PCR. The deletion of the cra gene in E. coli B (BL21) minimally affected the growth and maximal acetate accumulation, while the deletion of the same gene in E.coli K-12 (JM109) caused the cells to stop growing as soon as acetate concentration reached 6.6 g/L and the media conductivity reached 21 mS/cm. ppsA (gluconeogenesis gene), aceBA (the glyoxylate shunt genes) and poxB (the acetate producing gene) were down-regulated in both strains, while acs (acetate uptake gene) was down-regulated only in E.coli B (BL21). These transcriptional differences had little effect on acetate and pyruvate production. Additionally, it was found that the lower growth of E. coli K-12 (JM109) strain was the result of transcription inhibition of the osmoprotectant producing bet operon (betABT). The transcriptional changes caused by the deletion of cra gene did not affect the activity of the central carbon metabolism, suggesting that Cra does not act alone; rather it interacts with other pleiotropic regulators to create a network of metabolic effects. An unexpected outcome of this work is the finding that cra

  3. Transient phosphorylation of tumor associated microtubule associated protein (TMAP)/cytoskeleton associated protein 2 (CKAP2) at Thr-596 during early phases of mitosis

    OpenAIRE

    Hong, Kyung Uk; Choi, Yong-Bock; Lee, Jung-Hwa; Kim, Hyun-Jun; Kwon, Hye-Rim; Seong, Yeon-Sun; Kim, Heung Tae; Park, Joobae; Bae, Chang-Dae; Hong, Kyeong-Man

    2008-01-01

    Tumor associated microtubule associated protein (TMAP), also known as cytoskeleton associated protein 2 (CKAP2) is a mitotic spindle-associated protein whose expression is cell cycle-regulated and also frequently deregulated in cancer cells. Two monoclonal antibodies (mAbs) against TMAP/CKAP2 were produced: B-1-13 and D-12-3. Interestingly, the reactivity of mAb D-12-3 to TMAP/CKAP2 was markedly decreased specifically in mitotic cell lysate. The epitope mapping study showed that mAb D-12-3 re...

  4. Chlorpyrifos, chlorpyrifos-oxon, and diisopropylfluorophosphate inhibit kinesin-dependent microtubule motility

    International Nuclear Information System (INIS)

    Gearhart, Debra A.; Sickles, Dale W.; Buccafusco, Jerry J.; Prendergast, Mark A.; Terry, Alvin V.

    2007-01-01

    Diisopropylfluorophosphate, originally developed as a chemical warfare agent, is structurally similar to nerve agents, and chlorpyrifos has extensive worldwide use as an agricultural pesticide. While inhibition of cholinesterases underlies the acute toxicity of these organophosphates, we previously reported impaired axonal transport in the sciatic nerves from rats treated chronically with subthreshold doses of chlorpyrifos. Those data indicate that chlorpyrifos (and/or its active metabolite, chlorpyrifos-oxon) might directly affect the function of kinesin and/or microtubules-the principal proteins that mediate anterograde axonal transport. The current report describes in vitro assays to assess the concentration-dependent effects of chlorpyrifos (0-10 μM), chlorpyrifos-oxon (0-10 μM), and diisopropylfluorophosphate (0-0.59 nM) on kinesin-dependent microtubule motility. Preincubating bovine brain microtubules with the organophosphates did not alter kinesin-mediated microtubule motility. In contrast, preincubation of bovine brain kinesin with diisopropylfluorophosphate, chlorpyrifos, or chlorpyrifos-oxon produced a concentration-dependent increase in the number of locomoting microtubules that detached from the kinesin-coated glass cover slip. Our data suggest that the organophosphates-chlorpyrifos-oxon, chlorpyrifos, and diisopropylfluorophosphate-directly affect kinesin, thereby disrupting kinesin-dependent transport on microtubules. Kinesin-dependent movement of vesicles, organelles, and other cellular components along microtubules is fundamental to the organization of all eukaryotic cells, especially in neurons where organelles and proteins synthesized in the cell body must move down long axons to pre-synaptic sites in nerve terminals. We postulate that disruption of kinesin-dependent intracellular transport could account for some of the long-term effects of organophosphates on the peripheral and central nervous system

  5. Moonlighting microtubule-associated proteins: regulatory functions by day and pathological functions at night.

    Science.gov (United States)

    Oláh, J; Tőkési, N; Lehotzky, A; Orosz, F; Ovádi, J

    2013-11-01

    The sensing, integrating, and coordinating features of the eukaryotic cells are achieved by the complex ultrastructural arrays and multifarious functions of the cytoskeletal network. Cytoskeleton comprises fibrous protein networks of microtubules, actin, and intermediate filaments. These filamentous polymer structures are highly dynamic and undergo constant and rapid reorganization during cellular processes. The microtubular system plays a crucial role in the brain, as it is involved in an enormous number of cellular events including cell differentiation and pathological inclusion formation. These multifarious functions of microtubules can be achieved by their decoration with proteins/enzymes that exert specific effects on the dynamics and organization of the cytoskeleton and mediate distinct functions due to their moonlighting features. This mini-review focuses on two aspects of the microtubule cytoskeleton. On the one hand, we describe the heteroassociation of tubulin/microtubules with metabolic enzymes, which in addition to their catalytic activities stabilize microtubule structures via their cross-linking functions. On the other hand, we focus on the recently identified moonlighting tubulin polymerization promoting protein, TPPP/p25. TPPP/p25 is a microtubule-associated protein and it displays distinct physiological or pathological (aberrant) functions; thus it is a prototype of Neomorphic Moonlighting Proteins. The expression of TPPP/p25 is finely controlled in the human brain; this protein is indispensable for the development of projections of oligodendrocytes that are responsible for the ensheathment of axons. The nonphysiological, higher or lower TPPP/p25 level leads to distinct CNS diseases. Mechanisms contributing to the control of microtubule stability and dynamics by metabolic enzymes and TPPP/p25 will be discussed. Copyright © 2013 Wiley Periodicals, Inc.

  6. Cucurbitacin B inhibits human breast cancer cell proliferation through disruption of microtubule polymerization and nucleophosmin/B23 translocation

    Directory of Open Access Journals (Sweden)

    Duangmano Suwit

    2012-10-01

    proliferation of human breast cancer cells through disruption of the microtubule network and down-regulation of c-Myc and nucleophosmin/B23 as well as the perturbation in nucleophosmin/B23 trafficking from the nucleolus to nucleoplasm, resulting in G2/M arrest.

  7. α-Tubulin Tyrosination and CLIP-170 Phosphorylation Regulate the Initiation of Dynein-Driven Transport in Neurons

    Directory of Open Access Journals (Sweden)

    Jeffrey J. Nirschl

    2016-03-01

    Full Text Available Motor-cargo recruitment to microtubules is often the rate-limiting step of intracellular transport, and defects in this recruitment can cause neurodegenerative disease. Here, we use in vitro reconstitution assays with single-molecule resolution, live-cell transport assays in primary neurons, computational image analysis, and computer simulations to investigate the factors regulating retrograde transport initiation in the distal axon. We find that phosphorylation of the cytoskeletal-organelle linker protein CLIP-170 and post-translational modifications of the microtubule track combine to precisely control the initiation of retrograde transport. Computer simulations of organelle dynamics in the distal axon indicate that while CLIP-170 primarily regulates the time to microtubule encounter, the tyrosination state of the microtubule lattice regulates the likelihood of binding. These mechanisms interact to control transport initiation in the axon in a manner sensitive to the specialized cytoskeletal architecture of the neuron.

  8. TBCD links centriologenesis, spindle microtubule dynamics, and midbody abscission in human cells.

    Directory of Open Access Journals (Sweden)

    Mónica López Fanarraga

    2010-01-01

    Full Text Available Microtubule-organizing centers recruit alpha- and beta-tubulin polypeptides for microtubule nucleation. Tubulin synthesis is complex, requiring five specific cofactors, designated tubulin cofactors (TBCs A-E, which contribute to various aspects of microtubule dynamics in vivo. Here, we show that tubulin cofactor D (TBCD is concentrated at the centrosome and midbody, where it participates in centriologenesis, spindle organization, and cell abscission. TBCD exhibits a cell-cycle-specific pattern, localizing on the daughter centriole at G1 and on procentrioles by S, and disappearing from older centrioles at telophase as the protein is recruited to the midbody. Our data show that TBCD overexpression results in microtubule release from the centrosome and G1 arrest, whereas its depletion produces mitotic aberrations and incomplete microtubule retraction at the midbody during cytokinesis. TBCD is recruited to the centriole replication site at the onset of the centrosome duplication cycle. A role in centriologenesis is further supported in differentiating ciliated cells, where TBCD is organized into "centriolar rosettes". These data suggest that TBCD participates in both canonical and de novo centriolar assembly pathways.

  9. Kinesin-8 effects on mitotic microtubule dynamics contribute to spindle function in fission yeast

    Science.gov (United States)

    Gergely, Zachary R.; Crapo, Ammon; Hough, Loren E.; McIntosh, J. Richard; Betterton, Meredith D.

    2016-01-01

    Kinesin-8 motor proteins destabilize microtubules. Their absence during cell division is associated with disorganized mitotic chromosome movements and chromosome loss. Despite recent work studying effects of kinesin-8s on microtubule dynamics, it remains unclear whether the kinesin-8 mitotic phenotypes are consequences of their effect on microtubule dynamics, their well-established motor activity, or additional, unknown functions. To better understand the role of kinesin-8 proteins in mitosis, we studied the effects of deletion of the fission yeast kinesin-8 proteins Klp5 and Klp6 on chromosome movements and spindle length dynamics. Aberrant microtubule-driven kinetochore pushing movements and tripolar mitotic spindles occurred in cells lacking Klp5 but not Klp6. Kinesin-8–deletion strains showed large fluctuations in metaphase spindle length, suggesting a disruption of spindle length stabilization. Comparison of our results from light microscopy with a mathematical model suggests that kinesin-8–induced effects on microtubule dynamics, kinetochore attachment stability, and sliding force in the spindle can explain the aberrant chromosome movements and spindle length fluctuations seen. PMID:27146110

  10. Identification and characterization of SSE15206, a microtubule depolymerizing agent that overcomes multidrug resistance

    KAUST Repository

    Manzoor, Safia

    2018-02-13

    Microtubules are highly dynamic structures that form spindle fibres during mitosis and are one of the most validated cancer targets. The success of drugs targeting microtubules, however, is often limited by the development of multidrug resistance. Here we describe the discovery and characterization of SSE15206, a pyrazolinethioamide derivative [3-phenyl-5-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole-1-carbothioamide] that has potent antiproliferative activities in cancer cell lines of different origins and overcomes resistance to microtubule-targeting agents. Treatment of cells with SSE15206 causes aberrant mitosis resulting in G2/M arrest due to incomplete spindle formation, a phenotype often associated with drugs that interfere with microtubule dynamics. SSE15206 inhibits microtubule polymerization both in biochemical and cellular assays by binding to colchicine site in tubulin as shown by docking and competition studies. Prolonged treatment of cells with the compound results in apoptotic cell death [increased Poly (ADP-ribose) polymerase cleavage and Annexin V/PI staining] accompanied by p53 induction. More importantly, we demonstrate that SSE15206 is able to overcome resistance to chemotherapeutic drugs in different cancer cell lines including multidrug-resistant KB-V1 and A2780-Pac-Res cell lines overexpressing MDR-1, making it a promising hit for the lead optimization studies to target multidrug resistance.

  11. Microtubule dynamics of the centrosome-like polar organizers from the basal land plant Marchantia polymorpha.

    Science.gov (United States)

    Buschmann, Henrik; Holtmannspötter, Michael; Borchers, Agnes; O'Donoghue, Martin-Timothy; Zachgo, Sabine

    2016-02-01

    The liverwort Marchantia employs both modern and ancestral devices during cell division: it forms preprophase bands and in addition it shows centrosome-like polar organizers. We investigated whether polar organizers and preprophase bands cooperate to set up the division plane. To this end, two novel green fluorescent protein-based microtubule markers for dividing cells of Marchantia were developed. Cells of the apical notch formed polar organizers first and subsequently assembled preprophase bands. Polar organizers were formed de novo from multiple mobile microtubule foci localizing to the nuclear envelope. The foci then became concentrated by bipolar aggregation. We determined the comet production rate of polar organizers and show that microtubule plus ends of astral microtubules polymerize faster than those found on cortical microtubules. Importantly, it was observed that conditions increasing polar organizer numbers interfere with preprophase band formation. The data show that polar organizers have much in common with centrosomes, but that they also have specialized features. The results suggest that polar organizers contribute to preprophase band formation and in this way are involved in controlling the division plane. Our analyses of the basal land plant Marchantia shed new light on the evolution of plant cell division. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

  12. Self-Sustained Oscillatory Sliding Movement of Doublet Microtubules and Flagellar Bend Formation.

    Directory of Open Access Journals (Sweden)

    Sumio Ishijima

    Full Text Available It is well established that the basis for flagellar and ciliary movements is ATP-dependent sliding between adjacent doublet microtubules. However, the mechanism for converting microtubule sliding into flagellar and ciliary movements has long remained unresolved. The author has developed new sperm models that use bull spermatozoa divested of their plasma membrane and midpiece mitochondrial sheath by Triton X-100 and dithiothreitol. These models enable the observation of both the oscillatory sliding movement of activated doublet microtubules and flagellar bend formation in the presence of ATP. A long fiber of doublet microtubules extruded by synchronous sliding of the sperm flagella and a short fiber of doublet microtubules extruded by metachronal sliding exhibited spontaneous oscillatory movements and constructed a one beat cycle of flagellar bending by alternately actuating. The small sliding displacement generated by metachronal sliding formed helical bends, whereas the large displacement by synchronous sliding formed planar bends. Therefore, the resultant waveform is a half-funnel shape, which is similar to ciliary movements.

  13. Fission yeast cells undergo nuclear division in the absence of spindle microtubules.

    Directory of Open Access Journals (Sweden)

    Stefania Castagnetti

    2010-10-01

    Full Text Available Mitosis in eukaryotic cells employs spindle microtubules to drive accurate chromosome segregation at cell division. Cells lacking spindle microtubules arrest in mitosis due to a spindle checkpoint that delays mitotic progression until all chromosomes have achieved stable bipolar attachment to spindle microtubules. In fission yeast, mitosis occurs within an intact nuclear membrane with the mitotic spindle elongating between the spindle pole bodies. We show here that in fission yeast interference with mitotic spindle formation delays mitosis only briefly and cells proceed to an unusual nuclear division process we term nuclear fission, during which cells perform some chromosome segregation and efficiently enter S-phase of the next cell cycle. Nuclear fission is blocked if spindle pole body maturation or sister chromatid separation cannot take place or if actin polymerization is inhibited. We suggest that this process exhibits vestiges of a primitive nuclear division process independent of spindle microtubules, possibly reflecting an evolutionary intermediate state between bacterial and Archeal chromosome segregation where the nucleoid divides without a spindle and a microtubule spindle-based eukaryotic mitosis.

  14. Direct incorporation of guanosine 5'-diphosphate into microtubules without guanosine 5'-triphosphate hydrolysis

    International Nuclear Information System (INIS)

    Hamel, E.; Batra, J.K.; Lin, C.M.

    1986-01-01

    Using highly purified calf brain tubulin bearing [8- 14 C]guanosine 5'-diphosphate (GDP) in the exchangeable nucleotide site and heat-treated microtubule-associated proteins, the authors have found that a significant proportion of exchangeable-site GDP in microtubules can be incorporated directly during guanosine 5'-triphosphate (GTP) dependent polymerization of tubulin, without an initial exchange of GDP for GTP and subsequent GTP hydrolysis during assembly. The precise amount of GDP incorporated directly into microtubules is highly dependent on specific reaction conditions, being favored by high tubulin concentrations, low GTP and Mg 2+ concentrations, and exogenous GDP in the reaction mixture. Minimum effects were observed with changes in reaction pH or temperature, changes in concentration of microtubule-associated proteins, alteration of the sulfonate buffer, or the presence of a calcium chelator in the reaction mixture. Under conditions most favorable for direct GDP incorporation, about one-third of the GDP in microtubules is incorporated directly (without GTP hydrolysis) and two-thirds is incorporated hydrolytically (as a consequence of GTP hydrolysis). Direct incorporation of GDP occurs in a constant proportion throughout elongation, and the amount of direct incorporation probably reflects the rapid equilibration of GDP and GTP at the exchangeable site that occurs before the onset of assembly

  15. Stabilization, not polymerization, of microtubules inhibits the nuclear translocation of STATs in adipocytes

    International Nuclear Information System (INIS)

    Gleason, Evanna L.; Hogan, Jessica C.; Stephens, Jacqueline M.

    2004-01-01

    Signal transducers and activators of transcriptions (STATs) are a family of latent transcription factors which are activated by a variety of growth factors and cytokines in many cell types. However, the mechanism by which these transcription factors translocate to the nucleus is poorly understood. The goal of this study was to determine the requirement of microfilaments and microtubules for cytokine induced STAT activation in cultured adipocytes. We used seven different actin-specific and microtubule-specific agents that are well-established effectors of these cytoskeletal networks. Our results clearly demonstrate that inhibition of microfilaments or the prevention of microtubule polymerization has no effect on the ability of STATs to be tyrosine phosphorylated or to translocate to the nucleus. However, we observed that paclitaxel, a microtubule stabilizer, resulted in a significant decrease in the nuclear translocation of STATs without affecting the cytosolic tyrosine phosphorylation of these transcription factors. In summary, our results demonstrate that the dynamic instability, but not the polymerization, of microtubules contributes to nuclear translocation of STAT proteins in adipocytes

  16. Prickle isoforms control the direction of tissue polarity by microtubule independent and dependent mechanisms

    Directory of Open Access Journals (Sweden)

    Katherine A. Sharp

    2016-03-01

    Full Text Available Planar cell polarity signaling directs the polarization of cells within the plane of many epithelia. While these tissues exhibit asymmetric localization of a set of core module proteins, in Drosophila, more than one mechanism links the direction of core module polarization to the tissue axes. One signaling system establishes a polarity bias in the parallel, apical microtubules upon which vesicles containing core proteins traffic. Swapping expression of the differentially expressed Prickle isoforms, Prickle and Spiny-legs, reverses the direction of core module polarization. Studies in the proximal wing and the anterior abdomen indicated that this results from their differential control of microtubule polarity. Prickle and Spiny-legs also control the direction of polarization in the distal wing (D-wing and the posterior abdomen (P-abd. We report here that this occurs without affecting microtubule polarity in these tissues. The direction of polarity in the D-wing is therefore likely determined by a novel mechanism independent of microtubule polarity. In the P-abd, Prickle and Spiny-legs interpret at least two directional cues through a microtubule-polarity-independent mechanism.

  17. Nonrandom γ-TuNA-dependent spatial pattern of microtubule nucleation at the Golgi.

    Science.gov (United States)

    Sanders, Anna A W M; Chang, Kevin; Zhu, Xiaodong; Thoppil, Roslin J; Holmes, William R; Kaverina, Irina

    2017-11-07

    Noncentrosomal microtubule (MT) nucleation at the Golgi generates MT network asymmetry in motile vertebrate cells. Investigating the Golgi-derived MT (GDMT) distribution, we find that MT asymmetry arises from nonrandom nucleation sites at the Golgi (hotspots). Using computational simulations, we propose two plausible mechanistic models of GDMT nucleation leading to this phenotype. In the "cooperativity" model, formation of a single GDMT promotes further nucleation at the same site. In the "heterogeneous Golgi" model, MT nucleation is dramatically up-regulated at discrete and sparse locations within the Golgi. While MT clustering in hotspots is equally well described by both models, simulating MT length distributions within the cooperativity model fits the data better. Investigating the molecular mechanism underlying hotspot formation, we have found that hotspots are significantly smaller than a Golgi subdomain positive for scaffolding protein AKAP450, which is thought to recruit GDMT nucleation factors. We have further probed potential roles of known GDMT-promoting molecules, including γ-TuRC-mediated nucleation activator (γ-TuNA) domain-containing proteins and MT stabilizer CLASPs. While both γ-TuNA inhibition and lack of CLASPs resulted in drastically decreased GDMT nucleation, computational modeling revealed that only γ-TuNA inhibition suppressed hotspot formation. We conclude that hotspots require γ-TuNA activity, which facilitates clustered GDMT nucleation at distinct Golgi sites. © 2017 Sanders et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  18. The XMAP215 Ortholog Alp14 Promotes Microtubule Nucleation in Fission Yeast.

    Science.gov (United States)

    Flor-Parra, Ignacio; Iglesias-Romero, Ana Belén; Chang, Fred

    2018-06-04

    The organization and number of microtubules (MTs) in a cell depend on the proper regulation of MT nucleation. Currently, the mechanism of nucleation is the most poorly understood aspect of MT dynamics. XMAP215/chTOG/Alp14/Stu2 proteins are MT polymerases that stimulate MT polymerization at MT plus ends by binding and releasing tubulin dimers. Although these proteins also localize to MT organizing centers and have nucleating activity in vitro, it is not yet clear whether these proteins participate in MT nucleation in vivo. Here, we demonstrate that in the fission yeast Schizosaccharomyces pombe, the XMAP215 ortholog Alp14 is critical for efficient MT nucleation in vivo. In multiple assays, loss of Alp14 function led to reduced nucleation rate and numbers of interphase MT bundles. Conversely, activation of Alp14 led to increased nucleation frequency. Alp14 associated with Mto1 and γ-tubulin complex components, and artificially targeting Alp14 to the γ-tubulin ring complexes (γ-TuRCs) stimulated nucleation. In imaging individual nucleation events, we found that Alp14 transiently associated with a γ-tubulin particle shortly before the appearance of a new MT. The transforming acidic coiled-coil (TACC) ortholog Alp7 mediated the localization of Alp14 at nucleation sites but not plus ends, and was required for efficient nucleation but not for MT polymerization. Our findings provide the strongest evidence to date that Alp14 serves as a critical MT nucleation factor in vivo. We suggest a model in which Alp14 associates with the γ-tubulin complex in an Alp7-dependent manner to facilitate the assembly or stabilization of the nascent MT. Copyright © 2018 Elsevier Ltd. All rights reserved.

  19. Katanin spiral and ring structures shed light on power stroke for microtubule severing

    Energy Technology Data Exchange (ETDEWEB)

    Zehr, Elena; Szyk, Agnieszka; Piszczek, Grzegorz; Szczesna, Ewa; Zuo, Xiaobing; Roll-Mecak, Antonina

    2017-08-07

    Microtubule-severing enzymes katanin, spastin and fidgetin are AAA ATPases critical for the biogenesis and maintenance of complex microtubule arrays in axons, spindles and cilia. Because of a lack of 3D structures, their mechanism has remained poorly understood. We report the first X-ray structure of the monomeric AAA katanin module and cryo-EM reconstructions of the hexamer in two conformations. These reveal an unexpected asymmetric arrangement of the AAA domains mediated by structural elements unique to severing enzymes and critical for their function. Our reconstructions show that katanin cycles between open spiral and closed ring conformations, depending on the ATP occupancy of a gating protomer that tenses or relaxes inter-protomer interfaces. Cycling of the hexamer between these conformations would provide the power stroke for microtubule severing.

  20. Template-free electrosynthesis of aligned poly(p-phenylene) microtubules

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Poly(p-phenylene) (PPP) microtubules with diameters of 0.2-0.8μm and lengths of~10 (m have been synthesized by direct oxidation of benzene in the mixed electrolyte of boron trifluoride diethyl etherate (BFEE) and trifluoroacetic acid (TFA) (BFEE:TFA= 2:1, by volume), containing a certain amount of sodium dodecylbenzene- sulfonate (SDBS) as surfactant. The microtubules were grown vertically on the working electrode surface. The tubular morphology has been confirmed by scanning and transmission electron microscopies and the chain structure of the skin of the tubules has been characterized by Raman spectroscopy. The electrode property, monomer/surfactant molar ratio and the value of applied potential have strong effects on the morphology of the microtubules.

  1. Reassessing the Roles of PIN Proteins and Anticlinal Microtubules during Pavement Cell Morphogenesis1[OPEN

    Science.gov (United States)

    Sawchuk, Megan G.; Scarpella, Enrico

    2018-01-01

    The leaf epidermis is a biomechanical shell that influences the size and shape of the organ. Its morphogenesis is a multiscale process in which nanometer-scale cytoskeletal protein complexes, individual cells, and groups of cells pattern growth and define macroscopic leaf traits. Interdigitated growth of neighboring cells is an evolutionarily conserved developmental strategy. Understanding how signaling pathways and cytoskeletal proteins pattern cell walls during this form of tissue morphogenesis is an important research challenge. The cellular and molecular control of a lobed cell morphology is currently thought to involve PIN-FORMED (PIN)-type plasma membrane efflux carriers that generate subcellular auxin gradients. Auxin gradients were proposed to function across cell boundaries to encode stable offset patterns of cortical microtubules and actin filaments between adjacent cells. Many models suggest that long-lived microtubules along the anticlinal cell wall generate local cell wall heterogeneities that restrict local growth and specify the timing and location of lobe formation. Here, we used Arabidopsis (Arabidopsis thaliana) reverse genetics and multivariate long-term time-lapse imaging to test current cell shape control models. We found that neither PIN proteins nor long-lived microtubules along the anticlinal wall predict the patterns of lobe formation. In fields of lobing cells, anticlinal microtubules are not correlated with cell shape and are unstable at the time scales of cell expansion. Our analyses indicate that anticlinal microtubules have multiple functions in pavement cells and that lobe initiation is likely controlled by complex interactions among cell geometry, cell wall stress patterns, and transient microtubule networks that span the anticlinal and periclinal walls. PMID:29192026

  2. A computational framework for cortical microtubule dynamics in realistically shaped plant cells.

    Directory of Open Access Journals (Sweden)

    Bandan Chakrabortty

    2018-02-01

    Full Text Available Plant morphogenesis is strongly dependent on the directional growth and the subsequent oriented division of individual cells. It has been shown that the plant cortical microtubule array plays a key role in controlling both these processes. This ordered structure emerges as the collective result of stochastic interactions between large numbers of dynamic microtubules. To elucidate this complex self-organization process a number of analytical and computational approaches to study the dynamics of cortical microtubules have been proposed. To date, however, these models have been restricted to two dimensional planes or geometrically simple surfaces in three dimensions, which strongly limits their applicability as plant cells display a wide variety of shapes. This limitation is even more acute, as both local as well as global geometrical features of cells are expected to influence the overall organization of the array. Here we describe a framework for efficiently simulating microtubule dynamics on triangulated approximations of arbitrary three dimensional surfaces. This allows the study of microtubule array organization on realistic cell surfaces obtained by segmentation of microscopic images. We validate the framework against expected or known results for the spherical and cubical geometry. We then use it to systematically study the individual contributions of global geometry, cell-edge induced catastrophes and cell-face induced stability to array organization in a cuboidal geometry. Finally, we apply our framework to analyze the highly non-trivial geometry of leaf pavement cells of Arabidopsis thaliana, Nicotiana benthamiana and Hedera helix. We show that our simulations can predict multiple features of the microtubule array structure in these cells, revealing, among others, strong constraints on the orientation of division planes.

  3. Reassessing the Roles of PIN Proteins and Anticlinal Microtubules during Pavement Cell Morphogenesis.

    Science.gov (United States)

    Belteton, Samuel A; Sawchuk, Megan G; Donohoe, Bryon S; Scarpella, Enrico; Szymanski, Daniel B

    2018-01-01

    The leaf epidermis is a biomechanical shell that influences the size and shape of the organ. Its morphogenesis is a multiscale process in which nanometer-scale cytoskeletal protein complexes, individual cells, and groups of cells pattern growth and define macroscopic leaf traits. Interdigitated growth of neighboring cells is an evolutionarily conserved developmental strategy. Understanding how signaling pathways and cytoskeletal proteins pattern cell walls during this form of tissue morphogenesis is an important research challenge. The cellular and molecular control of a lobed cell morphology is currently thought to involve PIN-FORMED (PIN)-type plasma membrane efflux carriers that generate subcellular auxin gradients. Auxin gradients were proposed to function across cell boundaries to encode stable offset patterns of cortical microtubules and actin filaments between adjacent cells. Many models suggest that long-lived microtubules along the anticlinal cell wall generate local cell wall heterogeneities that restrict local growth and specify the timing and location of lobe formation. Here, we used Arabidopsis ( Arabidopsis thaliana ) reverse genetics and multivariate long-term time-lapse imaging to test current cell shape control models. We found that neither PIN proteins nor long-lived microtubules along the anticlinal wall predict the patterns of lobe formation. In fields of lobing cells, anticlinal microtubules are not correlated with cell shape and are unstable at the time scales of cell expansion. Our analyses indicate that anticlinal microtubules have multiple functions in pavement cells and that lobe initiation is likely controlled by complex interactions among cell geometry, cell wall stress patterns, and transient microtubule networks that span the anticlinal and periclinal walls. © 2018 American Society of Plant Biologists. All Rights Reserved.

  4. A computational framework for cortical microtubule dynamics in realistically shaped plant cells

    KAUST Repository

    Chakrabortty, Bandan; Blilou, Ikram; Scheres, Ben; Mulder, Bela M.

    2018-01-01

    Plant morphogenesis is strongly dependent on the directional growth and the subsequent oriented division of individual cells. It has been shown that the plant cortical microtubule array plays a key role in controlling both these processes. This ordered structure emerges as the collective result of stochastic interactions between large numbers of dynamic microtubules. To elucidate this complex self-organization process a number of analytical and computational approaches to study the dynamics of cortical microtubules have been proposed. To date, however, these models have been restricted to two dimensional planes or geometrically simple surfaces in three dimensions, which strongly limits their applicability as plant cells display a wide variety of shapes. This limitation is even more acute, as both local as well as global geometrical features of cells are expected to influence the overall organization of the array. Here we describe a framework for efficiently simulating microtubule dynamics on triangulated approximations of arbitrary three dimensional surfaces. This allows the study of microtubule array organization on realistic cell surfaces obtained by segmentation of microscopic images. We validate the framework against expected or known results for the spherical and cubical geometry. We then use it to systematically study the individual contributions of global geometry, cell-edge induced catastrophes and cell-face induced stability to array organization in a cuboidal geometry. Finally, we apply our framework to analyze the highly non-trivial geometry of leaf pavement cells of Arabidopsis thaliana, Nicotiana benthamiana and Hedera helix. We show that our simulations can predict multiple features of the microtubule array structure in these cells, revealing, among others, strong constraints on the orientation of division planes.

  5. Reassessing the roles of PIN proteins and anticlinal microtubules during pavement cell morphogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Belteton, Samuel; Sawchuk, Megan G.; Donohoe, Bryon S.; Scarpella, Enrico; Szymanski, Daniel B.

    2017-11-30

    The leaf epidermis is a biomechanical shell that influences the size and shape of the organ. Its morphogenesis is a multiscale process in which nanometer-scale cytoskeletal protein complexes, individual cells, and groups of cells pattern growth and define macroscopic leaf traits. Interdigitated growth of neighboring cells is an evolutionarily conserved developmental strategy. Understanding how signaling pathways and cytoskeletal proteins pattern cell walls during this form of tissue morphogenesis is an important research challenge. The cellular and molecular control of a lobed cell morphology is currently thought to involve PIN-FORMED (PIN)-type plasma membrane efflux carriers that generate subcellular auxin gradients. Auxin gradients were proposed to function across cell boundaries to encode stable offset patterns of cortical microtubules and actin filaments between adjacent cells. Many models suggest that long-lived microtubules along the anticlinal cell wall generate local cell wall heterogeneities that restrict local growth and specify the timing and location of lobe formation. Here we used Arabidopsis reverse genetics and multivariate long-term time-lapse imaging to test current cell shape control models. We found that neither PIN proteins nor microtubules along the anticlinal wall predict the patterns of lobe formation. In fields of lobing cells, anticlinal microtubules are not correlated with cell shape and are unstable at the time scales of cell expansion. Our analyses indicate that anticlinal microtubules have multiple functions in pavement cells, and that lobe initiation is likely controlled by complex interactions among cell geometry, cell wall stress patterns, and transient microtubule networks that span the anticlinal and periclinal walls.

  6. A computational framework for cortical microtubule dynamics in realistically shaped plant cells

    KAUST Repository

    Chakrabortty, Bandan

    2018-02-02

    Plant morphogenesis is strongly dependent on the directional growth and the subsequent oriented division of individual cells. It has been shown that the plant cortical microtubule array plays a key role in controlling both these processes. This ordered structure emerges as the collective result of stochastic interactions between large numbers of dynamic microtubules. To elucidate this complex self-organization process a number of analytical and computational approaches to study the dynamics of cortical microtubules have been proposed. To date, however, these models have been restricted to two dimensional planes or geometrically simple surfaces in three dimensions, which strongly limits their applicability as plant cells display a wide variety of shapes. This limitation is even more acute, as both local as well as global geometrical features of cells are expected to influence the overall organization of the array. Here we describe a framework for efficiently simulating microtubule dynamics on triangulated approximations of arbitrary three dimensional surfaces. This allows the study of microtubule array organization on realistic cell surfaces obtained by segmentation of microscopic images. We validate the framework against expected or known results for the spherical and cubical geometry. We then use it to systematically study the individual contributions of global geometry, cell-edge induced catastrophes and cell-face induced stability to array organization in a cuboidal geometry. Finally, we apply our framework to analyze the highly non-trivial geometry of leaf pavement cells of Arabidopsis thaliana, Nicotiana benthamiana and Hedera helix. We show that our simulations can predict multiple features of the microtubule array structure in these cells, revealing, among others, strong constraints on the orientation of division planes.

  7. Arabidopsis cortical microtubules position cellulose synthase delivery to the plasma membrane and interact with cellulose synthase trafficking compartments.

    NARCIS (Netherlands)

    Gutierrez, R.; Lindeboom, J.J.; Paredez, A.R.; Emons, A.M.C.; Ehrhardt, D.W.

    2009-01-01

    Plant cell morphogenesis relies on the organization and function of two polymer arrays separated by the plasma membrane: the cortical microtubule cytoskeleton and cellulose microfibrils in the cell wall. Studies using in vivo markers confirmed that one function of the cortical microtubule array is

  8. Capu and Spire Assemble a Cytoplasmic Actin~Mesh that Maintains Microtubule Organization in the Drosophila Oocyte

    DEFF Research Database (Denmark)

    Dahlgaard, K.; Raposo, A.A.S.F.; Niccoli, T.

    2007-01-01

    Mutants in the actin nucleators Cappuccino and Spire disrupt the polarized microtubule network in the Drosophila oocyte that defines the anterior-posterior axis, suggesting that microtubule organization depends on actin. Here, we show that Cappuccino and Spire organize an isotropic mesh of actin...

  9. Microtubule-dependent targeting of the exocyst complex is necessary for xylem development in Arabidopsis

    Czech Academy of Sciences Publication Activity Database

    Vukašinović, Nemanja; Oda, Y.; Pejchar, Přemysl; Synek, Lukáš; Pečenková, Tamara; Rawat, Anamika; Sekereš, Juraj; Potocký, Martin; Žárský, Viktor

    2017-01-01

    Roč. 213, č. 3 (2017), s. 1052-1067 ISSN 0028-646X R&D Projects: GA ČR(CZ) GA15-14886S Grant - others:GA MŠk(CZ) LO1417 Institutional support: RVO:61389030 Keywords : secondary cell-wall * tracheary element differentiation * cortical microtubules * plasma-membrane * vesicle trafficking * secretory pathways * auxin transport * exocytosis * deposition * thaliana * conserved oligomeric Golgi (COG) complex * exocyst * microtubules * secondary cell wall * tracheary elements * xylem Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Cell biology Impact factor: 7.330, year: 2016

  10. TRX-ASK1-JNK signaling regulation of cell density-dependent cytotoxicity in cigarette smoke-exposed human bronchial epithelial cells.

    Science.gov (United States)

    Lee, Yong Chan; Chuang, Chun-Yu; Lee, Pak-Kei; Lee, Jin-Soo; Harper, Richart W; Buckpitt, Alan B; Wu, Reen; Oslund, Karen

    2008-05-01

    Cigarette smoke is a major environmental air pollutant that injures airway epithelium and incites subsequent diseases including chronic obstructive pulmonary disease. The lesion that smoke induces in airway epithelium is still incompletely understood. Using a LIVE/DEAD cytotoxicity assay, we observed that subconfluent cultures of bronchial epithelial cells derived from both human and monkey airway tissues and an immortalized normal human bronchial epithelial cell line (HBE1) were more susceptible to injury by cigarette smoke extract (CSE) and by direct cigarette smoke exposure than cells in confluent cultures. Scraping confluent cultures also caused an enhanced cell injury predominately in the leading edge of the scraped confluent cultures by CSE. Cellular ATP levels in both subconfluent and confluent cultures were drastically reduced after CSE exposure. In contrast, GSH levels were significantly reduced only in subconfluent cultures exposed to smoke and not in confluent cultures. Western blot analysis demonstrated ERK activation in both confluent and subconfluent cultures after CSE. However, activation of apoptosis signal-regulating kinase 1 (ASK1), JNK, and p38 were demonstrated only in subconfluent cultures and not in confluent cultures after CSE. Using short interfering RNA (siRNA) to JNK1 and JNK2 and a JNK inhibitor, we attenuated CSE-mediated cell death in subconfluent cultures but not with an inhibitor of the p38 pathway. Using the tetracycline (Tet)-on inducible approach, overexpression of thioredoxin (TRX) attenuated CSE-mediated cell death and JNK activation in subconfluent cultures. These results suggest that the TRX-ASK1-JNK pathway may play a critical role in mediating cell density-dependent CSE cytotoxicity.

  11. Regulation of cellulose synthesis in response to stress.

    Science.gov (United States)

    Kesten, Christopher; Menna, Alexandra; Sánchez-Rodríguez, Clara

    2017-12-01

    The cell wall is a complex polysaccharide network that provides stability and protection to the plant and is one of the first layers of biotic and abiotic stimuli perception. A controlled remodeling of the primary cell wall is essential for the plant to adapt its growth to environmental stresses. Cellulose, the main component of plant cell walls is synthesized by plasma membrane-localized cellulose synthases moving along cortical microtubule tracks. Recent advancements demonstrate a tight regulation of cellulose synthesis at the primary cell wall by phytohormone networks. Stress-induced perturbations at the cell wall that modify cellulose synthesis and microtubule arrangement activate similar phytohormone-based stress response pathways. The integration of stress perception at the primary cell wall and downstream responses are likely to be tightly regulated by phytohormone signaling pathways in the context of cellulose synthesis and microtubule arrangement. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  12. Challenges and opportunities in the high-resolution cryo-EM visualization of microtubules and their binding partners.

    Science.gov (United States)

    Nogales, Eva; Kellogg, Elizabeth H

    2017-10-01

    As non-crystallizable polymers, microtubules have been the target of cryo-electron microscopy (cryo-EM) studies since the technique was first established. Over the years, image processing strategies have been developed that take care of the unique, pseudo-helical symmetry of the microtubule. With recent progress in data quality and data processing, cryo-EM reconstructions are now reaching resolutions that allow the generation of atomic models of microtubules and the factors that bind them. These include cellular partners that contribute to microtubule cellular functions, or small ligands that interfere with those functions in the treatment of cancer. The stage is set to generate a family portrait for all identified microtubule interacting proteins and to use cryo-EM as a drug development tool in the targeting of tubulin. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  13. Cell proliferation, cell shape, and microtubule and cellulose microfibril organization of tobacco BY-2 cells are not altered by exposure to near weightlessness in space

    NARCIS (Netherlands)

    Sieberer, B.; Kieft, H.; Franssen-Verheijen, M.A.W.; Emons, A.M.C.; Vos, J.W.

    2009-01-01

    The microtubule cytoskeleton and the cell wall both play key roles in plant cell growth and division, determining the plant’s final stature. At near weightlessness, tubulin polymerizes into microtubules in vitro, but these microtubules do not self-organize in the ordered patterns observed at 1g.

  14. Microtubule organization in three-dimensional confined geometries: Evaluating the role of elasticity through a combined in vitro and modeling approach

    NARCIS (Netherlands)

    Cosentino Lagomarsino, M.; Tanase, C.; Vos, J.W.; Emons, A.M.C.; Mulder, B.; Dogterom, M.

    2007-01-01

    Microtubules or microtubule bundles in cells often grow longer than the size of the cell, which causes their shape and organization to adapt to constraints imposed by the cell geometry. We test the reciprocal role of elasticity and confinement in the organization of growing microtubules in a

  15. Postsynaptic density protein 95-regulated NR2B tyrosine phosphorylation and interactions of Fyn with NR2B in levodopa-induced dyskinesia rat models

    Directory of Open Access Journals (Sweden)

    Ba M

    2014-12-01

    Full Text Available Maowen Ba,1,* Min Kong,2,* Guozhao Ma3 1Department of Neurology, Yuhuangding Hospital, Yantai City, Shandong, People’s Republic of China; 2Department of Neurology, Yantaishan Hospital, Yantai City, Shandong, People’s Republic of China; 3Department of Neurology, Shandong Provincial Qianfoshan Hospital, Shandong University, Shandong, People’s Republic of China *These authors contributed equally to this work Context: Abnormality in interactions between N-methyl-d-aspartate (NMDA receptor and its signaling molecules occurs in the lesioned striatum in Parkinson’s disease (PD and levodopa-induced dyskinesia (LID. It was reported that Fyn-mediated NR2B tyrosine phosphorylation, can enhance NMDA receptor function. Postsynaptic density protein 95 (PSD-95, one of the synapse-associated proteins, regulates interactions between receptor and downstream-signaling molecules. In light of the relationship between PSD-95, NR2B, and Fyn kinases, does PSD-95 contribute to the overactivity of NMDA receptor function induced by dopaminergic treatment? To further prove the possibility, the effects of regulating the PSD-95 expression on the augmented NR2B tyrosine phosphorylation and on the interactions of Fyn and NR2B in LID rat models were evaluated.Methods: In the present study, parkinsonian rat models were established by injecting 6-hydroxydopamine. Subsequently, valid PD rats were treated with levodopa (50 mg/kg/day with benserazide 12.5 mg/kg/day, twice daily intraperitoneally for 22 days to create LID rat models. Then, the effect of pretreatment with an intrastriatal injection of the PSD-95mRNA antisense oligonucleotides (PSD-95 ASO on the rotational response to levodopa challenge was assessed. The effects of pretreatment with an intrastriatal injection of PSD-95 ASO on the augmented NR2B tyrosine phosphorylation and interactions of Fyn with NR2B in the LID rat models were detected by immunoblotting and immunoprecipitation. Results: Levodopa

  16. Biallelic Mutations in TBCD, Encoding the Tubulin Folding Cofactor D, Perturb Microtubule Dynamics and Cause Early-Onset Encephalopathy.

    Science.gov (United States)

    Flex, Elisabetta; Niceta, Marcello; Cecchetti, Serena; Thiffault, Isabelle; Au, Margaret G; Capuano, Alessandro; Piermarini, Emanuela; Ivanova, Anna A; Francis, Joshua W; Chillemi, Giovanni; Chandramouli, Balasubramanian; Carpentieri, Giovanna; Haaxma, Charlotte A; Ciolfi, Andrea; Pizzi, Simone; Douglas, Ganka V; Levine, Kara; Sferra, Antonella; Dentici, Maria Lisa; Pfundt, Rolph R; Le Pichon, Jean-Baptiste; Farrow, Emily; Baas, Frank; Piemonte, Fiorella; Dallapiccola, Bruno; Graham, John M; Saunders, Carol J; Bertini, Enrico; Kahn, Richard A; Koolen, David A; Tartaglia, Marco

    2016-10-06

    Microtubules are dynamic cytoskeletal elements coordinating and supporting a variety of neuronal processes, including cell division, migration, polarity, intracellular trafficking, and signal transduction. Mutations in genes encoding tubulins and microtubule-associated proteins are known to cause neurodevelopmental and neurodegenerative disorders. Growing evidence suggests that altered microtubule dynamics may also underlie or contribute to neurodevelopmental disorders and neurodegeneration. We report that biallelic mutations in TBCD, encoding one of the five co-chaperones required for assembly and disassembly of the αβ-tubulin heterodimer, the structural unit of microtubules, cause a disease with neurodevelopmental and neurodegenerative features characterized by early-onset cortical atrophy, secondary hypomyelination, microcephaly, thin corpus callosum, developmental delay, intellectual disability, seizures, optic atrophy, and spastic quadriplegia. Molecular dynamics simulations predicted long-range and/or local structural perturbations associated with the disease-causing mutations. Biochemical analyses documented variably reduced levels of TBCD, indicating relative instability of mutant proteins, and defective β-tubulin binding in a subset of the tested mutants. Reduced or defective TBCD function resulted in decreased soluble α/β-tubulin levels and accelerated microtubule polymerization in fibroblasts from affected subjects, demonstrating an overall shift toward a more rapidly growing and stable microtubule population. These cells displayed an aberrant mitotic spindle with disorganized, tangle-shaped microtubules and reduced aster formation, which however did not alter appreciably the rate of cell proliferation. Our findings establish that defective TBCD function underlies a recognizable encephalopathy and drives accelerated microtubule polymerization and enhanced microtubule stability, underscoring an additional cause of altered microtubule dynamics with

  17. CEP295 interacts with microtubules and is required for centriole elongation.

    Science.gov (United States)

    Chang, Ching-Wen; Hsu, Wen-Bin; Tsai, Jhih-Jie; Tang, Chieh-Ju C; Tang, Tang K

    2016-07-01

    Centriole duplication is a tightly ordered process during which procentrioles are assembled in G1-S and elongate during S and G2. Here, we show that human CEP295 (Drosophila Ana1) is not essential for initial cartwheel assembly, but is required to build distal half centrioles during S and G2. Using super-resolution and immunogold electron microscopy, we demonstrate that CEP295 is recruited to the proximal end of procentrioles in early S phase, when it is also localized at the centriolar microtubule wall that surrounds the human SAS6 cartwheel hub. Interestingly, depletion of CEP295 not only inhibits the recruitments of POC5 and POC1B to the distal half centrioles in G2, resulting in shorter centrioles, it also blocks the post-translational modification of centriolar microtubules (e.g. acetylation and glutamylation). Importantly, our results indicate that CEP295 directly interacts with microtubules, and that excess CEP295 could induce the assembly of overly long centrioles. Furthermore, exogenous expression of the N-terminal domain of CEP295 exerts a dominant-negative effect on centriole elongation. Collectively, these findings suggest that CEP295 is essential for building the distal half centrioles and for post-translational modification of centriolar microtubules. © 2016. Published by The Company of Biologists Ltd.

  18. Dissecting the function and assembly of acentriolar microtubule organizing centers in Drosophila cells in vivo.

    Directory of Open Access Journals (Sweden)

    Janina Baumbach

    2015-05-01

    Full Text Available Acentriolar microtubule organizing centers (aMTOCs are formed during meiosis and mitosis in several cell types, but their function and assembly mechanism is unclear. Importantly, aMTOCs can be overactive in cancer cells, enhancing multipolar spindle formation, merotelic kinetochore attachment and aneuploidy. Here we show that aMTOCs can form in acentriolar Drosophila somatic cells in vivo via an assembly pathway that depends on Asl, Cnn and, to a lesser extent, Spd-2--the same proteins that appear to drive mitotic centrosome assembly in flies. This finding enabled us to ablate aMTOC formation in acentriolar cells, and so perform a detailed genetic analysis of the contribution of aMTOCs to acentriolar mitotic spindle formation. Here we show that although aMTOCs can nucleate microtubules, they do not detectably increase the efficiency of acentriolar spindle assembly in somatic fly cells. We find that they are required, however, for robust microtubule array assembly in cells without centrioles that also lack microtubule nucleation from around the chromatin. Importantly, aMTOCs are also essential for dynein-dependent acentriolar spindle pole focusing and for robust cell proliferation in the absence of centrioles and HSET/Ncd (a kinesin essential for acentriolar spindle pole focusing in many systems. We propose an updated model for acentriolar spindle pole coalescence by the molecular motors Ncd/HSET and dynein in conjunction with aMTOCs.

  19. The current of a particle along a microtubule in microscopic plasma

    International Nuclear Information System (INIS)

    Li Wei; Chen Junfang; Wang Teng; Lai Xiuqiong

    2008-01-01

    Transport of a particle along the axis of a microtubule in a plasma-enhanced chemical vapor deposition (PECVD) system is investigated. The current, respectively, as a function of the temperature, the magnetic field and the external force is obtained. The value and direction of the current may be controlled by changing the above parameters

  20. Phospholipase D family interactions with the cytoskeleton: isoform delta promotes plasma membrane anchoring of cortical microtubules

    Czech Academy of Sciences Publication Activity Database

    Andreeva, Z.; Ho, A. Y. Y.; Barthet, M. M.; Potocký, Martin; Bezvoda, R.; Žárský, Viktor; Marc, J.

    2009-01-01

    Roč. 36, č. 7 (2009), s. 600-612 ISSN 1445-4408 R&D Projects: GA AV ČR IAA601110916 Institutional research plan: CEZ:AV0Z50380511 Keywords : Allium * Arabidopsis * F-actin-microtubule interactions Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.678, year: 2009

  1. Microtubule Abnormalities Underlying Gulf War Illness in Neurons from Human-Induced Pluripotent Cells

    Science.gov (United States)

    2016-09-01

    cells derived from human induced pluripotent stem cells (hiPSCs), originating from GW...AWARD NUMBER: W81XWH-15-1-0433 TITLE: Microtubule Abnormalities Underlying Gulf War Illness in Neurons from Human- Induced Pluripotent Cells ...A simple blood sample is taken from the soldier, and then transduced, using reliable established methods , to make the cells pluripotent .

  2. Wolbachia utilizes host microtubules and Dynein for anterior localization in the Drosophila oocyte.

    Directory of Open Access Journals (Sweden)

    Patrick M Ferree

    2005-10-01

    Full Text Available To investigate the role of the host cytoskeleton in the maternal transmission of the endoparasitic bacteria Wolbachia, we have characterized their distribution in the female germ line of Drosophila melanogaster. In the germarium, Wolbachia are distributed to all germ cells of the cyst, establishing an early infection in the cell destined to become the oocyte. During mid-oogenesis, Wolbachia exhibit a distinct concentration between the anterior cortex and the nucleus in the oocyte, where many bacteria appear to contact the nuclear envelope. Following programmed rearrangement of the microtubule network, Wolbachia dissociate from this anterior position and become dispersed throughout the oocyte. This localization pattern is distinct from mitochondria and all known axis determinants. Manipulation of microtubules and cytoplasmic Dynein and Dynactin, but not Kinesin-1, disrupts anterior bacterial localization in the oocyte. In live egg chambers, Wolbachia exhibit movement in nurse cells but not in the oocyte, suggesting that the bacteria are anchored by host factors. In addition, we identify mid-oogenesis as a period in the life cycle of Wolbachia in which bacterial replication occurs. Total bacterial counts show that Wolbachia increase at a significantly higher rate in the oocyte than in the average nurse cell, and that normal Wolbachia levels in the oocyte depend on microtubules. These findings demonstrate that Wolbachia utilize the host microtubule network and associated proteins for their subcellular localization in the Drosophila oocyte. These interactions may also play a role in bacterial motility and replication, ultimately leading to the bacteria's efficient maternal transmission.

  3. Novel mitochondrial extensions provide evidence for a link between microtubule-directed movement and mitochondrial fission

    International Nuclear Information System (INIS)

    Bowes, Timothy; Gupta, Radhey S.

    2008-01-01

    Mitochondrial dynamics play an important role in a large number of cellular processes. Previously, we reported that treatment of mammalian cells with the cysteine-alkylators, N-ethylmaleimide and ethacrynic acid, induced rapid mitochondrial fusion forming a large reticulum approximately 30 min after treatment. Here, we further investigated this phenomenon using a number of techniques including live-cell confocal microscopy. In live cells, drug-induced fusion coincided with a cessation of fast mitochondrial movement which was dependent on microtubules. During this loss of movement, thin mitochondrial tubules extending from mitochondria were also observed, which we refer to as 'mitochondrial extensions'. The formation of these mitochondrial extensions, which were not observed in untreated cells, depended on microtubules and was abolished by pretreatment with nocodazole. In this study, we provide evidence that these extensions result from of a block in mitochondrial fission combined with continued application of motile force by microtubule-dependent motor complexes. Our observations strongly suggest the existence of a link between microtubule-based mitochondrial trafficking and mitochondrial fission

  4. Deformation pattern in vibrating microtubule: Structural mechanics study based on an atomistic approach

    Czech Academy of Sciences Publication Activity Database

    Havelka, Daniel; Deriu, M.A.; Cifra, Michal; Kučera, Ondřej

    2017-01-01

    Roč. 7, č. 1 (2017), č. článku 4227. ISSN 2045-2322 R&D Projects: GA ČR(CZ) GA15-17102S Institutional support: RVO:67985882 Keywords : Continuum model * Protein microtubules * Molecular-dymamics Subject RIV: BO - Biophysics OBOR OECD: Biophysics Impact factor: 4.259, year: 2016

  5. CLASP2 interacts with p120-catenin and governs microtubule dynamics at adherens junctions

    DEFF Research Database (Denmark)

    Shahbazi, Marta N; Megias, Diego; Epifano, Carolina

    2013-01-01

    Classical cadherins and their connections with microtubules (MTs) are emerging as important determinants of cell adhesion. However, the functional relevance of such interactions and the molecular players that contribute to tissue architecture are still emerging. In this paper, we report that the ...

  6. Proteomics of cancer cell lines resistant to microtubule-stabilizing agents

    DEFF Research Database (Denmark)

    Albrethsen, Jakob; Angeletti, Ruth H; Horwitz, Susan Band

    2014-01-01

    Despite the clinical success of microtubule-interacting agents (MIA), a significant challenge for oncologists is the inability to predict the response of individual patients with cancer to these drugs. In the present study, six cell lines were compared by 2D DIGE proteomics to investigate cellula...

  7. Application of quasi-steady state methods to molecular motor transport on microtubules in fungal hyphae.

    Science.gov (United States)

    Dauvergne, Duncan; Edelstein-Keshet, Leah

    2015-08-21

    We consider bidirectional transport of cargo by molecular motors dynein and kinesin that walk along microtubules, and/or diffuse in the cell. The motors compete to transport cargo in opposite directions with respect to microtubule polarity (towards the plus or minus end of the microtubule). In recent work, Gou et al. (2014) used a hierarchical set of models, each consisting of continuum transport equations to track the evolution of motors and their cargo (early endosomes) in the specific case of the fungus Ustilago maydis. We complement their work using a framework of quasi-steady state analysis developed by Newby and Bressloff (2010) and Bressloff and Newby (2013) to reduce the models to an approximating steady state Fokker-Plank equation. This analysis allows us to find analytic approximations to the steady state solutions in many cases where the full models are not easily solved. Consequently, we can make predictions about parameter dependence of the resulting spatial distributions. We also characterize the overall rates of bulk transport and diffusion, and how these are related to state transition parameters, motor speeds, microtubule polarity distribution, and specific assumptions made. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Tubular lysosome morphology and distribution within macrophages depend on the integrity of cytoplasmic microtubules

    International Nuclear Information System (INIS)

    Swanson, J.; Bushnell, A.; Silverstein, S.C.

    1987-01-01

    Pinocytosis of the fluorescent dye lucifer yellow labels elongated, membrane-bound tubular organelles in several cell types, including cultured human monocytes, thioglycolate-elicited mouse peritoneal macrophages, and the macrophage-like cell line J774.2. These tubular structures can be identified as lysosomes by acid phosphatase histochemistry and immunofluorescence localization of cathepsin L. The abundance of tubular lysosomes is markedly increased by treatment with phorbol 12-myristate 13-acetate. When labeled by pinocytosis of microperoxidase and examined by electron microscopic histochemistry, the tubular lysosomes have an outside diameter of ≅ 75 nm and a length of several micrometers; they radiate from the cell's centrosphere in alignment with cytoplasmic microtubules and intermediate filaments. Incubation of phorbol myristate acetate-treated macrophages at 4 0 C or in medium containing 5 μM colchicine or nocodazole at 37 0 C leads to disassembly of microtubules and fragmentation of the tubular lysosomes. Return of the cultures to 37 0 C or removal of nocodazole from the medium leads to reassembly of microtubules and the reappearance of tubular lysosomes within 10-20 min. The authors conclude that microtubules are essential for the maintenance of tubular lysosome morphology and that, in macrophages, a significant proportion of the lysosomal compartment is contained within these tubular structures

  9. Vault mobility depends in part on microtubules and vaults can be recruited to the nuclear envelope

    International Nuclear Information System (INIS)

    Zon, Arend van; Mossink, Marieke H.; Houtsmuller, Adriaan B.; Schoester, Martijn; Scheffer, George L.; Scheper, Rik J.; Sonneveld, Pieter; Wiemer, Erik A.C.

    2006-01-01

    Vaults are ribonucleoproteins that may function in intracellular transport processes. We investigated the intracellular distribution and dynamics of vaults in non-small cell lung cancer cells in which vaults are labeled with the green fluorescent protein. Immunofluorescence experiments showed that vaults are dispersed throughout the cytoplasm; a small fraction is found in close proximity to microtubules. Immunoprecipitation experiments corroborated these results showing co-precipitation of MVP and β-tubulin. Using quantitative fluorescence-recovery after photobleaching (FRAP), we demonstrated that vault mobility over longer distances in part depends on intact microtubules; vaults moving slower when microtubules are depolymerized by nocodazole. Biochemical fractionation indicated a small fraction of MVP associated with the nucleus, however, no GFP-tagged vaults could be observed inside the nucleus. We observed an accumulation of vaults at the nuclear envelope upon treatment of cells with the protein synthesis inhibitor cycloheximide. Analysis of nucleo-cytoplasmic transport using a fluorescent substrate containing a classical NLS and NES expressed in MVP +/+ and MVP -/- mouse embryonic fibroblasts indicated no differences in nuclear import/export kinetics, suggesting no role for vaults in these processes. We hypothesize that a subset of vaults moves directionally via microtubules, possibly towards the nucleus

  10. Aluminum ions inhibit phospholipase D in a microtubule-dependent manner

    Czech Academy of Sciences Publication Activity Database

    Pejchar, Přemysl; Pleskot, R.; Schwarzerová, K.; Martinec, Jan; Valentová, O.; Novotná, Z.

    2008-01-01

    Roč. 32, č. 5 (2008), s. 554-556 ISSN 1065-6995 R&D Projects: GA ČR GA522/05/0340 Institutional research plan: CEZ:AV0Z50380511 Keywords : Aluminum toxicity * Phospholipase D * Microtubules Subject RIV: ED - Physiology Impact factor: 1.619, year: 2008

  11. Clustering of Nuclei in Multinucleated Hyphae Is Prevented by Dynein-Driven Bidirectional Nuclear Movements and Microtubule Growth Control in Ashbya gossypii ▿ †

    Science.gov (United States)

    Grava, Sandrine; Keller, Miyako; Voegeli, Sylvia; Seger, Shanon; Lang, Claudia; Philippsen, Peter

    2011-01-01

    During filamentous fungus development, multinucleated hyphae employ a system for long-range nuclear migration to maintain an equal nuclear density. A decade ago the microtubule motor dynein was shown to play a central role in this process. Previous studies with Ashbya gossypii revealed extensive bidirectional movements and bypassings of nuclei, an autonomous cytoplasmic microtubule (cMT) cytoskeleton emanating from each nucleus, and pulling of nuclei by sliding of cMTs along the cortex. Here, we show that dynein is the sole motor for bidirectional movements and bypassing because these movements are concomitantly decreased in mutants carrying truncations of the dynein heavy-chain DYN1 promoter. The dynactin component Jnm1, the accessory proteins Dyn2 and Ndl1, and the potential dynein cortical anchor Num1 are also involved in the dynamic distribution of nuclei. In their absence, nuclei aggregate to different degrees, whereby the mutants with dense nuclear clusters grow extremely long cMTs. As in budding yeast, we found that dynein is delivered to cMT plus ends, and its activity or processivity is probably controlled by dynactin and Num1. Together with its role in powering nuclear movements, we propose that dynein also plays (directly or indirectly) a role in the control of cMT length. Those combined dynein actions prevent nuclear clustering in A. gossypii and thus reveal a novel cellular role for dynein. PMID:21642510

  12. The use of compressive sensing and peak detection in the reconstruction of microtubules length time series in the process of dynamic instability.

    Science.gov (United States)

    Mahrooghy, Majid; Yarahmadian, Shantia; Menon, Vineetha; Rezania, Vahid; Tuszynski, Jack A

    2015-10-01

    Microtubules (MTs) are intra-cellular cylindrical protein filaments. They exhibit a unique phenomenon of stochastic growth and shrinkage, called dynamic instability. In this paper, we introduce a theoretical framework for applying Compressive Sensing (CS) to the sampled data of the microtubule length in the process of dynamic instability. To reduce data density and reconstruct the original signal with relatively low sampling rates, we have applied CS to experimental MT lament length time series modeled as a Dichotomous Markov Noise (DMN). The results show that using CS along with the wavelet transform significantly reduces the recovery errors comparing in the absence of wavelet transform, especially in the low and the medium sampling rates. In a sampling rate ranging from 0.2 to 0.5, the Root-Mean-Squared Error (RMSE) decreases by approximately 3 times and between 0.5 and 1, RMSE is small. We also apply a peak detection technique to the wavelet coefficients to detect and closely approximate the growth and shrinkage of MTs for computing the essential dynamic instability parameters, i.e., transition frequencies and specially growth and shrinkage rates. The results show that using compressed sensing along with the peak detection technique and wavelet transform in sampling rates reduces the recovery errors for the parameters. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Regulation of Population Densities of Heterodera cajani and Other Plant-Parasitic Nematodes by Crop Rotations on Vertisols, in Semi-Arid Tropical Production Systems in India

    Science.gov (United States)

    Sharma, S. B.; Rego, T. J.; Mohiuddin, M.; Rao, V. N.

    1996-01-01

    The significance of double crop (intercrop and sequential crop), single crop (rainy season crop fallow from June to September), and rotations on densities of Heterodera cajani, Helicotylenchus retusus, and Rotylenchulus reniformis was studied on Vertisol (Typic Pellusterts) between 1987 and 1993. Cowpea (Vigna sinensis), mungbean (Phaseolus aureus), and pigeonpea (Cajanus cajan) greatly increased the population densities of H. cajani and suppressed the population densities of other plant-parasitic nematodes. Mean population densities of H. cajani were about 8 times lower in single crop systems than in double crop systems, with pigeonpea as a component intercrop. Plots planted to sorghum, safflower, and chickpea in the preceding year contained fewer H. cajani eggs and juveniles than did plots previously planted to pigeonpea, cowpea, or mungbean. Continuous cropping of sorghum in the rainy season and safflower in the post-rainy season markedly reduced the population density of H. cajani. Sorghum, safflower, and chickpea favored increased population densities of H. retusus. Adding cowpea to the system resulted in a significant increase in the densities of R. reniformis. Mean densities of total plant-parasitic nematodes were three times greater in double crop systems, with pigeonpea as a component intercrop than in single crop systems with rainy season fallow component. Cropping systems had a regulatory effect on the nematode populations and could be an effective nematode management tactic. Intercropping of sorghum with H. cajani tolerant pigeonpea could be effective in increasing the productivity of traditional production systems in H. cajani infested regions. PMID:19277141

  14. INDUCTION OF LOW-DENSITY AND UP-REGULATION OF CD11B EXPRESSION OF NEUTROPHILS AND EOSINOPHILS BY DEXTRAN SEDIMENTATION AND CENTRIFUGATION

    NARCIS (Netherlands)

    DIJKHUIZEN, B; DEMONCHY, JGR; GERRITSEN, J; KAUFFMAN, HF

    1994-01-01

    Neutrophils and eosinophils circulating in an activated state are of low density. However, purification procedures such as dextran sedimentation and centrifugation may influence the density and function of cells. In the present study we have evaluated the effect of dextran sedimentation and

  15. Hypothesis: NDL proteins function in stress responses by regulating microtubule organization

    OpenAIRE

    Khatri, Nisha; Mudgil, Yashwanti

    2015-01-01

    N-MYC DOWNREGULATED-LIKE proteins (NDL), members of the alpha/beta hydrolase superfamily were recently rediscovered as interactors of G-protein signaling in Arabidopsis thaliana. Although the precise molecular function of NDL proteins is still elusive, in animals these proteins play protective role in hypoxia and expression is induced by hypoxia and nickel, indicating role in stress. Homology of NDL1 with animal counterpart N-MYC DOWNREGULATED GENE (NDRG) suggests similar functions in animals...

  16. Luminal localization of α-tubulin K40 acetylation by cryo-EM analysis of fab-labeled microtubules.

    Directory of Open Access Journals (Sweden)

    Virupakshi Soppina

    Full Text Available The αβ-tubulin subunits of microtubules can undergo a variety of evolutionarily-conserved post-translational modifications (PTMs that provide functional specialization to subsets of cellular microtubules. Acetylation of α-tubulin residue Lysine-40 (K40 has been correlated with increased microtubule stability, intracellular transport, and ciliary assembly, yet a mechanistic understanding of how acetylation influences these events is lacking. Using the anti-acetylated tubulin antibody 6-11B-1 and electron cryo-microscopy, we demonstrate that the K40 acetylation site is located inside the microtubule lumen and thus cannot directly influence events on the microtubule surface, including kinesin-1 binding. Surprisingly, the monoclonal 6-11B-1 antibody recognizes both acetylated and deacetylated microtubules. These results suggest that acetylation induces structural changes in the K40-containing loop that could have important functional consequences on microtubule stability, bending, and subunit interactions. This work has important implications for acetylation and deacetylation reaction mechanisms as well as for interpreting experiments based on 6-11B-1 labeling.

  17. Interaction of the Tobacco mosaic virus movement protein with microtubules during the cell cycle in tobacco BY-2 cells.

    Science.gov (United States)

    Boutant, Emmanuel; Fitterer, Chantal; Ritzenthaler, Christophe; Heinlein, Manfred

    2009-10-01

    Cell-to-cell movement of Tobacco mosaic virus (TMV) involves the interaction of virus-encoded 30-kDa movement protein (MP) with microtubules. In cells behind the infection front that accumulate high levels of MP, this activity is reflected by the formation of stabilized MP/microtubule complexes. The ability of MP to bind along and stabilize microtubules is conserved upon expression in mammalian cells. In mammalian cells, the protein also leads to inhibition of mitosis and cell division through a microtubule-independent process correlated with the loss of centrosomal gamma-tubulin and of centrosomal microtubule-nucleation activity. Since MP has the capacity to interact with plant factors involved in microtubule nucleation and dynamics, we used inducible expression in BY-2 cells to test whether MP expression inhibits mitosis and cell division also in plants. We demonstrate that MP:GFP associates with all plant microtubule arrays and, unlike in mammalian cells, does not interfere with mitosis. Thus, MP function and the interaction of MP with factors of the cytoskeleton do not entail an inhibition of mitosis in plants. We also report that the protein targets primary plasmodesmata in BY-2 cells immediately upon or during cytokinesis and that the accumulation of MP in plasmodesmata occurs in the presence of inhibitors of the cytoskeleton and the secretory pathway.

  18. Influence of carbon nanotubes on the buckling of microtubule bundles in viscoelastic cytoplasm using nonlocal strain gradient theory

    Directory of Open Access Journals (Sweden)

    A. Farajpour

    Full Text Available Carbon nanotubes are a new class of microtubule-stabilizing agents since they interact with protein microtubules in living cells, interfering with cell division and inducing apoptosis. In the present work, a modified beam model is developed to investigate the effect of carbon nanotubes on the buckling of microtubule bundles in living cell. A realistic interaction model is employed using recent experimental data on the carbon nanotube-stabilized microtubules. Small scale and surface effects are taken into account applying the nonlocal strain gradient theory and surface elasticity theory. Pasternak model is used to describe the normal and shearing effects of enclosing filament matrix on the buckling behavior of the system. An exact solution is obtained for the buckling growth rates of the mixed bundle in viscoelastic surrounding cytoplasm. The present results are compared with those reported in the open literature for single microtubules and an excellent agreement is found. Finally, the effects of different parameters such as the size, chirality, position and surface energy of carbon nanotubes on the buckling growth rates of microtubule bundles are studied. It is found that the buckling growth rate may increase or decrease by adding carbon nanotubes, depending on the diameter and chirality of carbon nanotubes. Keywords: Microtubules, Carbon nanotubes, Buckling, Size effects

  19. Finding the Cell Center by a Balance of Dynein and Myosin Pulling and Microtubule Pushing: A Computational Study

    Science.gov (United States)

    Zhu, Jie; Burakov, Anton; Rodionov, Vladimir

    2010-01-01

    The centrosome position in many types of interphase cells is actively maintained in the cell center. Our previous work indicated that the centrosome is kept at the center by pulling force generated by dynein and actin flow produced by myosin contraction and that an unidentified factor that depends on microtubule dynamics destabilizes position of the centrosome. Here, we use modeling to simulate the centrosome positioning based on the idea that the balance of three forces—dyneins pulling along microtubule length, myosin-powered centripetal drag, and microtubules pushing on organelles—is responsible for the centrosome displacement. By comparing numerical predictions with centrosome behavior in wild-type and perturbed interphase cells, we rule out several plausible hypotheses about the nature of the microtubule-based force. We conclude that strong dynein- and weaker myosin-generated forces pull the microtubules inward competing with microtubule plus-ends pushing the microtubule aster outward and that the balance of these forces positions the centrosome at the cell center. The model also predicts that kinesin action could be another outward-pushing force. Simulations demonstrate that the force-balance centering mechanism is robust yet versatile. We use the experimental observations to reverse engineer the characteristic forces and centrosome mobility. PMID:20980619

  20. Atomic-resolution structure of the CAP-Gly domain of dynactin on polymeric microtubules determined by magic angle spinning NMR spectroscopy.

    Science.gov (United States)

    Yan, Si; Guo, Changmiao; Hou, Guangjin; Zhang, Huilan; Lu, Xingyu; Williams, John Charles; Polenova, Tatyana

    2015-11-24

    Microtubules and their associated proteins perform a broad array of essential physiological functions, including mitosis, polarization and differentiation, cell migration, and vesicle and organelle transport. As such, they have been extensively studied at multiple levels of resolution (e.g., from structural biology to cell biology). Despite these efforts, there remain significant gaps in our knowledge concerning how microtubule-binding proteins bind to microtubules, how dynamics connect different conformational states, and how these interactions and dynamics affect cellular processes. Structures of microtubule-associated proteins assembled on polymeric microtubules are not known at atomic resolution. Here, we report a structure of the cytoskeleton-associated protein glycine-rich (CAP-Gly) domain of dynactin motor on polymeric microtubules, solved by magic angle spinning NMR spectroscopy. We present the intermolecular interface of CAP-Gly with microtubules, derived by recording direct dipolar contacts between CAP-Gly and tubulin using double rotational echo double resonance (dREDOR)-filtered experiments. Our results indicate that the structure adopted by CAP-Gly varies, particularly around its loop regions, permitting its interaction with multiple binding partners and with the microtubules. To our knowledge, this study reports the first atomic-resolution structure of a microtubule-associated protein on polymeric microtubules. Our approach lays the foundation for atomic-resolution structural analysis of other microtubule-associated motors.

  1. Lateral motion and bending of microtubules studied with a new single-filament tracking routine in living cells.

    Science.gov (United States)

    Pallavicini, Carla; Levi, Valeria; Wetzler, Diana E; Angiolini, Juan F; Benseñor, Lorena; Despósito, Marcelo A; Bruno, Luciana

    2014-06-17

    The cytoskeleton is involved in numerous cellular processes such as migration, division, and contraction and provides the tracks for transport driven by molecular motors. Therefore, it is very important to quantify the mechanical behavior of the cytoskeletal filaments to get a better insight into cell mechanics and organization. It has been demonstrated that relevant mechanical properties of microtubules can be extracted from the analysis of their motion and shape fluctuations. However, tracking individual filaments in living cells is extremely complex due, for example, to the high and heterogeneous background. We introduce a believed new tracking algorithm that allows recovering the coordinates of fluorescent microtubules with ∼9 nm precision in in vitro conditions. To illustrate potential applications of this algorithm, we studied the curvature distributions of fluorescent microtubules in living cells. By performing a Fourier analysis of the microtubule shapes, we found that the curvatures followed a thermal-like distribution as previously reported with an effective persistence length of ∼20 μm, a value significantly smaller than that measured in vitro. We also verified that the microtubule-associated protein XTP or the depolymerization of the actin network do not affect this value; however, the disruption of intermediate filaments decreased the persistence length. Also, we recovered trajectories of microtubule segments in actin or intermediate filament-depleted cells, and observed a significant increase of their motion with respect to untreated cells showing that these filaments contribute to the overall organization of the microtubule network. Moreover, the analysis of trajectories of microtubule segments in untreated cells showed that these filaments presented a slower but more directional motion in the cortex with respect to the perinuclear region, and suggests that the tracking routine would allow mapping the microtubule dynamical organization in cells

  2. Erucin, the major isothiocyanate in arugula (Eruca sativa, inhibits proliferation of MCF7 tumor cells by suppressing microtubule dynamics.

    Directory of Open Access Journals (Sweden)

    Olga Azarenko

    Full Text Available Consumption of cruciferous vegetables is associated with reduced risk of various types of cancer. Isothiocyanates including sulforaphane and erucin are believed to be responsible for this activity. Erucin [1-isothiocyanato-4-(methylthiobutane], which is metabolically and structurally related to sulforaphane, is present in large quantities in arugula (Eruca sativa, Mill., kohlrabi and Chinese cabbage. However, its cancer preventive mechanisms remain poorly understood. We found that erucin inhibits proliferation of MCF7 breast cancer cells (IC50 = 28 µM in parallel with cell cycle arrest at mitosis (IC50 = 13 µM and apoptosis, by a mechanism consistent with impairment of microtubule dynamics. Concentrations of 5-15 µM erucin suppressed the dynamic instability of microtubules during interphase in the cells. Most dynamic instability parameters were inhibited, including the rates and extents of growing and shortening, the switching frequencies between growing and shortening, and the overall dynamicity. Much higher erucin concentrations were required to reduce the microtubule polymer mass. In addition, erucin suppressed dynamic instability of microtubules reassembled from purified tubulin in similar fashion. The effects of erucin on microtubule dynamics, like those of sulforaphane, are similar qualitatively to those of much more powerful clinically-used microtubule-targeting anticancer drugs, including taxanes and the vinca alkaloids. The results suggest that suppression of microtubule dynamics by erucin and the resulting impairment of critically important microtubule-dependent cell functions such as mitosis, cell migration and microtubule-based transport may be important in its cancer preventive activities.

  3. Mechanical unloading reduces microtubule actin crosslinking factor 1 expression to inhibit β-catenin signaling and osteoblast proliferation.

    Science.gov (United States)

    Yin, Chong; Zhang, Yan; Hu, Lifang; Tian, Ye; Chen, Zhihao; Li, Dijie; Zhao, Fan; Su, Peihong; Ma, Xiaoli; Zhang, Ge; Miao, Zhiping; Wang, Liping; Qian, Airong; Xian, Cory J

    2018-07-01

    Mechanical unloading was considered a major threat to bone homeostasis, and has been shown to decrease osteoblast proliferation although the underlying mechanism is unclear. Microtubule actin crosslinking factor 1 (MACF1) is a cytoskeletal protein that regulates cellular processes and Wnt/β-catenin pathway, an essential signaling pathway for osteoblasts. However, the relationship between MACF1 expression and mechanical unloading, and the function and the associated mechanisms of MACF1 in regulating osteoblast proliferation are unclear. This study investigated effects of mechanical unloading on MACF1 expression levels in cultured MC3T3-E1 osteoblastic cells and in femurs of mice with hind limb unloading; and it also examined the role and potential action mechanisms of MACF1 in osteoblast proliferation in MACF1-knockdown, overexpressed or control MC3T3-E1 cells treated with or without the mechanical unloading condition. Results showed that the mechanical unloading condition inhibited osteoblast proliferation and MACF1 expression in MC3T3-E1 osteoblastic cells and mouse femurs. MACF1 knockdown decreased osteoblast proliferation, while MACF1 overexpression increased it. The inhibitory effect of mechanical unloading on osteoblast proliferation also changed with MACF1 expression levels. Furthermore, MACF1 was found to enhance β-catenin expression and activity, and mechanical unloading decreased β-catenin expression through MACF1. Moreover, β-catenin was found an important regulator of osteoblast proliferation, as its preservation by treatment with its agonist lithium attenuated the inhibitory effects of MACF1-knockdown or mechanical unloading on osteoblast proliferation. Taken together, mechanical unloading decreases MACF1 expression, and MACF1 up-regulates osteoblast proliferation through enhancing β-catenin signaling. This study has thus provided a mechanism for mechanical unloading-induced inhibited osteoblast proliferation. © 2017 Wiley Periodicals, Inc.

  4. Phenotypic Dissection of Bone Mineral Density Reveals Skeletal Site Specificity and Facilitates the Identification of Novel Loci in the Genetic Regulation of Bone Mass Attainment

    NARCIS (Netherlands)

    J.P. Kemp (John); M.C. Medina-Gomez (Carolina); K. Estrada Gil (Karol); B. St Pourcain (Beate); D.H.M. Heppe (Denise); N.M. Warrington (Nicole); L. Oei (Ling); S.M. Ring (Susan); C.J. Kruithof (Claudia); N.J. Timpson (Nicholas); L.E. Wolber (Lisa); S. Reppe (Sjur); K.M. Gautvik (Kaare); E. Grundberg (Elin); B. Ge (Bing); B.C.J. van der Eerden (Bram); J. van de Peppel (Jeroen); M.A. Hibbs (Matthew); C.L. Ackert-Bicknell (Cheryl); K. Choi (Kunho); D.L. Koller (Daniel); M.J. Econs (Michael); F.M. Williams (Frances); T. Foroud (Tatiana); M.C. Zillikens (Carola); C. Ohlsson (Claes); A. Hofman (Albert); A.G. Uitterlinden (André); G. Davey-Smith (George); V.W.V. Jaddoe (Vincent); J.H. Tobias (Jon); F. Rivadeneira Ramirez (Fernando); D.M. Evans (David)

    2014-01-01

    textabstractHeritability of bone mineral density (BMD) varies across skeletal sites, reflecting different relative contributions of genetic and environmental influences. To quantify the degree to which common genetic variants tag and environmental factors influence BMD, at different sites, we

  5. Npas4 Regulates Mdm2 and thus Dcx in Experience-Dependent Dendritic Spine Development of Newborn Olfactory Bulb Interneurons

    Directory of Open Access Journals (Sweden)

    Sei-ichi Yoshihara

    2014-08-01

    Full Text Available Sensory experience regulates the development of various brain structures, including the cortex, hippocampus, and olfactory bulb (OB. Little is known about how sensory experience regulates the dendritic spine development of OB interneurons, such as granule cells (GCs, although it is well studied in mitral/tufted cells. Here, we identify a transcription factor, Npas4, which is expressed in OB GCs immediately after sensory input and is required for dendritic spine formation. Npas4 overexpression in OB GCs increases dendritic spine density, even under sensory deprivation, and rescues reduction of dendrite spine density in the Npas4 knockout OB. Furthermore, loss of Npas4 upregulates expression of the E3-ubiquitin ligase Mdm2, which ubiquitinates a microtubule-associated protein Dcx. This leads to reduction in the dendritic spine density of OB GCs. Together, these findings suggest that Npas4 regulates Mdm2 expression to ubiquitinate and degrade Dcx during dendritic spine development in newborn OB GCs after sensory experience.

  6. Microtubular stability affects pVHL-mediated regulation of HIF-1alpha via the p38/MAPK pathway in hypoxic cardiomyocytes.

    Directory of Open Access Journals (Sweden)

    Miao Teng

    Full Text Available BACKGROUND: Our previous research found that structural changes of the microtubule network influence glycolysis in cardiomyocytes by regulating the hypoxia-inducible factor (HIF-1α during the early stages of hypoxia. However, little is known about the underlying regulatory mechanism of the changes of HIF-1α caused by microtubule network alternation. The von Hippel-Lindau tumor suppressor protein (pVHL, as a ubiquitin ligase, is best understood as a negative regulator of HIF-1α. METHODOLOGY/PRINCIPAL FINDINGS: In primary rat cardiomyocytes and H9c2 cardiac cells, microtubule-stabilization was achieved by pretreating with paclitaxel or transfection of microtubule-associated protein 4 (MAP4 overexpression plasmids and microtubule-depolymerization was achieved by pretreating with colchicine or transfection of MAP4 siRNA before hypoxia treatment. Recombinant adenovirus vectors for overexpressing pVHL or silencing of pVHL expression were constructed and transfected in primary rat cardiomyocytes and H9c2 cells. With different microtubule-stabilizing and -depolymerizing treaments, we demonstrated that the protein levels of HIF-1α were down-regulated through overexpression of pVHL and were up-regulated through knockdown of pVHL in hypoxic cardiomyocytes. Importantly, microtubular structure breakdown activated p38/MAPK pathway, accompanied with the upregulation of pVHL. In coincidence, we found that SB203580, a p38/MAPK inhibitor decreased pVHL while MKK6 (Glu overexpression increased pVHL in the microtubule network altered-hypoxic cardiomyocytes and H9c2 cells. CONCLUSIONS/SIGNIFICANCE: This study suggests that pVHL plays an important role in the regulation of HIF-1α caused by the changes of microtubular structure and the p38/MAPK pathway participates in the process of pVHL change following microtubule network alteration in hypoxic cardiomyocytes.

  7. Microtubule Destabilizer KIF2A Undergoes Distinct Site-Specific Phosphorylation Cascades that Differentially Affect Neuronal Morphogenesis

    Directory of Open Access Journals (Sweden)

    Tadayuki Ogawa

    2015-09-01

    Full Text Available Neurons exhibit dynamic structural changes in response to extracellular stimuli. Microtubules (MTs provide rapid and dramatic cytoskeletal changes within the structural framework. However, the molecular mechanisms and signaling networks underlying MT dynamics remain unknown. Here, we have applied a comprehensive and quantitative phospho-analysis of the MT destabilizer KIF2A to elucidate the regulatory mechanisms of MT dynamics within neurons in response to extracellular signals. Interestingly, we identified two different sets of KIF2A phosphorylation profiles that accelerate (A-type and brake (B-type the MT depolymerization activity of KIF2A. Brain-derived neurotrophic factor (BDNF stimulates PAK1 and CDK5 kinases, which decrease the MT depolymerizing activity of KIF2A through B-type phosphorylation, resulting in enhanced outgrowth of neural processes. In contrast, lysophosphatidic acid (LPA induces ROCK2 kinase, which suppresses neurite outgrowth from round cells via A-type phosphorylation. We propose that these two mutually exclusive forms of KIF2A phosphorylation differentially regulate neuronal morphogenesis during development.

  8. Proteasome Failure Promotes Positioning of Lysosomes around the Aggresome via Local Block of Microtubule-Dependent Transport

    Science.gov (United States)

    Zaarur, Nava; Meriin, Anatoli B.; Bejarano, Eloy; Xu, Xiaobin; Gabai, Vladimir L.; Cuervo, Ana Maria

    2014-01-01

    Ubiquitinated proteins aggregate upon proteasome failure, and the aggregates are transported to the aggresome. In aggresomes, protein aggregates are actively degraded by the autophagy-lysosome pathway, but why targeting the aggresome promotes degradation of aggregated species is currently unknown. Here we report that the important factor in this process is clustering of lysosomes around the aggresome via a novel mechanism. Proteasome inhibition causes formation of a zone around the centrosome where microtubular transport of lysosomes is suppressed, resulting in their entrapment and accumulation. Microtubule-dependent transport of other organelles, including autophagosomes, mitochondria, and endosomes, is also blocked in this entrapment zone (E-zone), while movement of organelles at the cell periphery remains unaffected. Following the whole-genome small interfering RNA (siRNA) screen for proteins involved in aggresome formation, we defined the pathway that regulates formation of the E-zone, including the Stk11 protein kinase, the Usp9x deubiquitinating enzyme, and their substrate kinase MARK4. Therefore, upon proteasome failure, targeting of aggregated proteins of the aggresome is coordinated with lysosome positioning around this body to facilitate degradation of the abnormal species. PMID:24469403

  9. Road density

    Data.gov (United States)

    U.S. Environmental Protection Agency — Road density is generally highly correlated with amount of developed land cover. High road densities usually indicate high levels of ecological disturbance. More...

  10. Non-equilibrium assembly of microtubules: from molecules to autonomous chemical robots.

    Science.gov (United States)

    Hess, H; Ross, Jennifer L

    2017-09-18

    Biological systems have evolved to harness non-equilibrium processes from the molecular to the macro scale. It is currently a grand challenge of chemistry, materials science, and engineering to understand and mimic biological systems that have the ability to autonomously sense stimuli, process these inputs, and respond by performing mechanical work. New chemical systems are responding to the challenge and form the basis for future responsive, adaptive, and active materials. In this article, we describe a particular biochemical-biomechanical network based on the microtubule cytoskeletal filament - itself a non-equilibrium chemical system. We trace the non-equilibrium aspects of the system from molecules to networks and describe how the cell uses this system to perform active work in essential processes. Finally, we discuss how microtubule-based engineered systems can serve as testbeds for autonomous chemical robots composed of biological and synthetic components.

  11. Explaining the Microtubule Energy Balance: Contributions Due to Dipole Moments, Charges, van der Waals and Solvation Energy

    Directory of Open Access Journals (Sweden)

    Ahmed Taha Ayoub

    2017-09-01

    Full Text Available Microtubules are the main components of mitotic spindles, and are the pillars of the cellular cytoskeleton. They perform most of their cellular functions by virtue of their unique dynamic instability processes which alternate between polymerization and depolymerization phases. This in turn is driven by a precise balance between attraction and repulsion forces between the constituents of microtubules (MTs—tubulin dimers. Therefore, it is critically important to know what contributions result in a balance of the interaction energy among tubulin dimers that make up microtubules and what interactions may tip this balance toward or away from a stable polymerized state of tubulin. In this paper, we calculate the dipole–dipole interaction energy between tubulin dimers in a microtubule as part of the various contributions to the energy balance. We also compare the remaining contributions to the interaction energies between tubulin dimers and establish a balance between stabilizing and destabilizing components, including the van der Waals, electrostatic, and solvent-accessible surface area energies. The energy balance shows that the GTP-capped tip of the seam at the plus end of microtubules is stabilized only by − 9 kcal/mol, which can be completely reversed by the hydrolysis of a single GTP molecule, which releases + 14 kcal/mol and destabilizes the seam by an excess of + 5 kcal/mol. This triggers the breakdown of microtubules and initiates a disassembly phase which is aptly called a catastrophe.

  12. Spatio-temporal dependence of the signaling response in immune-receptor trafficking networks regulated by cell density: a theoretical model.

    Directory of Open Access Journals (Sweden)

    Pilar García-Peñarrubia

    Full Text Available Cell signaling processes involve receptor trafficking through highly connected networks of interacting components. The binding of surface receptors to their specific ligands is a key factor for the control and triggering of signaling pathways. In most experimental systems, ligand concentration and cell density vary within a wide range of values. Dependence of the signal response on cell density is related with the extracellular volume available per cell. This dependence has previously been studied using non-spatial models which assume that signaling components are well mixed and uniformly distributed in a single compartment. In this paper, a mathematical model that shows the influence exerted by cell density on the spatio-temporal evolution of ligands, cell surface receptors, and intracellular signaling molecules is developed. To this end, partial differential equations were used to model ligand and receptor trafficking dynamics through the different domains of the whole system. This enabled us to analyze several interesting features involved with these systems, namely: a how the perturbation caused by the signaling response propagates through the system; b receptor internalization dynamics and how cell density affects the robustness of dose-response curves upon variation of the binding affinity; and c that enhanced correlations between ligand input and system response are obtained under conditions that result in larger perturbations of the equilibrium ligand + surface receptor [Please see text] ligand - receptor complex. Finally, the results are compared with those obtained by considering that the above components are well mixed in a single compartment.

  13. Central nervous system neuropeptide Y regulates mediators of hepatic phospholipid remodeling and very low-density lipoprotein triglyceride secretion via sympathetic innervation

    NARCIS (Netherlands)

    Rojas, Jennifer M; Bruinstroop, E.; Printz, Richard L; Alijagic-Boers, Aldijana; Foppen, E.; Turney, Maxine K; George, Leena; Beck-Sickinger, Annette G; Kalsbeek, A.; Niswender, Kevin D

    OBJECTIVE: Elevated very low-density lipoprotein (VLDL)-triglyceride (TG) secretion from the liver contributes to an atherogenic dyslipidemia that is associated with obesity, diabetes and the metabolic syndrome. Numerous models of obesity and diabetes are characterized by increased central nervous

  14. Central nervous system neuropeptide Y regulates mediators of hepatic phospholipid remodeling and very low-density lipoprotein triglyceride secretion via sympathetic innervation

    NARCIS (Netherlands)

    Rojas, Jennifer M.; Bruinstroop, Eveline; Printz, Richard L.; Alijagic-Boers, Aldijana; Foppen, Ewout; Turney, Maxine K.; George, Leena; Beck-Sickinger, Annette G.; Kalsbeek, Andries; Niswender, Kevin D.

    2015-01-01

    Objective: Elevated very low-density lipoprotein (VLDL)-triglyceride (TG) secretion from the liver contributes to an atherogenic dyslipidemia that is associated with obesity, diabetes and the metabolic syndrome. Numerous models of obesity and diabetes are characterized by increased central nervous

  15. In vivo regulation of scavenger receptor BI and the selective uptake of high density lipoprotein cholesteryl esters in rat liver parenchymal and Kupffer cells

    NARCIS (Netherlands)

    Fluiter, K.; van der Westhuijzen, D. R.; van Berkel, T. J.

    1998-01-01

    High density lipoprotein cholesteryl esters (HDL-CE) are selectively taken up by liver parenchymal cells without parallel apolipoprotein uptake. This selective uptake route forms an important step in the so-called reverse cholesterol transport. Scavenger receptor BI (SR-BI) is the only known HDL

  16. Brain regions implicated in inhibitory control and appetite regulation are activated in response to food portion size and energy density in children

    NARCIS (Netherlands)

    English, L.K.; Fearnbach, S.N.; Lasschuijt, M.; Schlegel, A.; Anderson, K.; Harris, S.; Fisher, J.O.; Savage, J.S.; Rolls, B.J.; Keller, K.L.

    2016-01-01

    Objective:Large portions of energy-dense foods drive energy intake but the brain mechanisms underlying this effect are not clear. Our main objective was to investigate brain function in response to food images varied by portion size (PS) and energy density (ED) in children using functional

  17. The centrosomal linker and microtubules provide dual levels of spatial coordination of centrosomes.

    Directory of Open Access Journals (Sweden)

    Marko Panic

    2015-05-01

    Full Text Available The centrosome is the principal microtubule organizing center in most animal cells. It consists of a pair of centrioles surrounded by pericentriolar material. The centrosome, like DNA, duplicates exactly once per cell cycle. During interphase duplicated centrosomes remain closely linked by a proteinaceous linker. This centrosomal linker is composed of rootletin filaments that are anchored to the centrioles via the protein C-Nap1. At the onset of mitosis the linker is dissolved by Nek2A kinase to support the formation of the bipolar mitotic spindle. The importance of the centrosomal linker for cell function during interphase awaits characterization. Here we assessed the phenotype of human RPE1 C-Nap1 knockout (KO cells. The absence of the linker led to a modest increase in the average centrosome separation from 1 to 2.5 μm. This small impact on the degree of separation is indicative of a second level of spatial organization of centrosomes. Microtubule depolymerisation or stabilization in C-Nap1 KO cells dramatically increased the inter-centrosomal separation (> 8 μm. Thus, microtubules position centrosomes relatively close to one another in the absence of linker function. C-Nap1 KO cells had a Golgi organization defect with a two-fold expansion of the area occupied by the Golgi. When the centrosomes of C-Nap1 KO cells showed considerable separation, two spatially distinct Golgi stacks could be observed. Furthermore, migration of C-Nap1 KO cells was slower than their wild type RPE1 counterparts. These data show that the spatial organization of centrosomes is modulated by a combination of centrosomal cohesion and microtubule forces. Furthermore a modest increase in centrosome separation has major impact on Golgi organization and cell migration.

  18. Characterization of mitosis-specific phosphorylation of tumor-associated microtubule-associated protein

    OpenAIRE

    Hong, Kyung Uk; Kim, Hyun-Jun; Bae, Chang-Dae; Park, Joobae

    2009-01-01

    Tumor-associated microtubule-associated protein (TMAP), also known as cytoskeleton associated protein 2 (CKAP2), has been recently shown to be involved in the assembly and maintenance of mitotic spindle and also plays an essential role in maintaining the fidelity of chromosome segregation during mitosis. We have previously reported that TMAP is phosphorylated at multiple residues specifically during mitosis, and characterized the mechanism and functional importance of phosphorylation at one o...

  19. Identification of interphase functions for the NIMA kinase involving microtubules and the ESCRT pathway.

    Directory of Open Access Journals (Sweden)

    Meera Govindaraghavan

    2014-03-01

    Full Text Available The Never in Mitosis A (NIMA kinase (the founding member of the Nek family of kinases has been considered a mitotic specific kinase with nuclear restricted roles in the model fungus Aspergillus nidulans. By extending to A. nidulans the results of a synthetic lethal screen performed in Saccharomyces cerevisiae using the NIMA ortholog KIN3, we identified a conserved genetic interaction between nimA and genes encoding proteins of the Endosomal Sorting Complex Required for Transport (ESCRT pathway. Absence of ESCRT pathway functions in combination with partial NIMA function causes enhanced cell growth defects, including an inability to maintain a single polarized dominant cell tip. These genetic insights suggest NIMA potentially has interphase functions in addition to its established mitotic functions at nuclei. We therefore generated endogenously GFP-tagged NIMA (NIMA-GFP which was fully functional to follow its interphase locations using live cell spinning disc 4D confocal microscopy. During interphase some NIMA-GFP locates to the tips of rapidly growing cells and, when expressed ectopically, also locates to the tips of cytoplasmic microtubules, suggestive of non-nuclear interphase functions. In support of this, perturbation of NIMA function either by ectopic overexpression or through partial inactivation results in marked cell tip growth defects with excess NIMA-GFP promoting multiple growing cell tips. Ectopic NIMA-GFP was found to locate to the plus ends of microtubules in an EB1 dependent manner, while impairing NIMA function altered the dynamic localization of EB1 and the cytoplasmic microtubule network. Together, our genetic and cell biological analyses reveal novel non-nuclear interphase functions for NIMA involving microtubules and the ESCRT pathway for normal polarized fungal cell tip growth. These insights extend the roles of NIMA both spatially and temporally and indicate that this conserved protein kinase could help integrate cell

  20. Molecular recognition of epothilones by microtubules and tubulin dimers revealed by biochemical and NMR approaches.

    Science.gov (United States)

    Canales, Angeles; Nieto, Lidia; Rodríguez-Salarichs, Javier; Sánchez-Murcia, Pedro A; Coderch, Claire; Cortés-Cabrera, Alvaro; Paterson, Ian; Carlomagno, Teresa; Gago, Federico; Andreu, José M; Altmann, Karl-Heinz; Jiménez-Barbero, Jesús; Díaz, J Fernando

    2014-04-18

    The binding of epothilones to dimeric tubulin and to microtubules has been studied by means of biochemical and NMR techniques. We have determined the binding constants of epothilone A (EpoA) and B (EpoB) to dimeric tubulin, which are 4 orders of magnitude lower than those for microtubules, and we have elucidated the conformation and binding epitopes of EpoA and EpoB when bound to tubulin dimers and microtubules in solution. The determined conformation of epothilones when bound to dimeric tubulin is similar to that found by X-ray crystallographic techniques for the binding of EpoA to the Tubulin/RB3/TTL complex; it is markedly different from that reported for EpoA bound to zinc-induced sheets obtained by electron crystallography. Likewise, only the X-ray structure of EpoA bound to the Tubulin/RB3/TTL complex at the luminal site, but not the electron crystallography structure, is compatible with the results obtained by STD on the binding epitope of EpoA bound to dimeric tubulin, thus confirming that the allosteric change (structuring of the M-loop) is the biochemical mechanism of induction of tubulin assembly by epothilones. TR-NOESY signals of EpoA bound to microtubules have been obtained, supporting the interaction with a transient binding site with a fast exchange rate (pore site), consistent with the notion that epothilones access the luminal site through the pore site, as has also been observed for taxanes. Finally, the differences in the tubulin binding affinities of a series of epothilone analogues has been quantitatively explained using the newly determined binding pose and the COMBINE methodology.

  1. Phosphatase PP2A and microtubule-mediated pulling forces disassemble centrosomes during mitotic exit

    Directory of Open Access Journals (Sweden)

    Stephen J. Enos

    2018-01-01

    Full Text Available Centrosomes are microtubule-nucleating organelles that facilitate chromosome segregation and cell division in metazoans. Centrosomes comprise centrioles that organize a micron-scale mass of protein called pericentriolar material (PCM from which microtubules nucleate. During each cell cycle, PCM accumulates around centrioles through phosphorylation-mediated assembly of PCM scaffold proteins. During mitotic exit, PCM swiftly disassembles by an unknown mechanism. Here, we used Caenorhabditis elegans embryos to determine the mechanism and importance of PCM disassembly in dividing cells. We found that the phosphatase PP2A and its regulatory subunit SUR-6 (PP2ASUR-6, together with cortically directed microtubule pulling forces, actively disassemble PCM. In embryos depleted of these activities, ∼25% of PCM persisted from one cell cycle into the next. Purified PP2ASUR-6 could dephosphorylate the major PCM scaffold protein SPD-5 in vitro. Our data suggest that PCM disassembly occurs through a combination of dephosphorylation of PCM components and force-driven fragmentation of the PCM scaffold.

  2. SAS-6 engineering reveals interdependence between cartwheel and microtubules in determining centriole architecture.

    Science.gov (United States)

    Hilbert, Manuel; Noga, Akira; Frey, Daniel; Hamel, Virginie; Guichard, Paul; Kraatz, Sebastian H W; Pfreundschuh, Moritz; Hosner, Sarah; Flückiger, Isabelle; Jaussi, Rolf; Wieser, Mara M; Thieltges, Katherine M; Deupi, Xavier; Müller, Daniel J; Kammerer, Richard A; Gönczy, Pierre; Hirono, Masafumi; Steinmetz, Michel O

    2016-04-01

    Centrioles are critical for the formation of centrosomes, cilia and flagella in eukaryotes. They are thought to assemble around a nine-fold symmetric cartwheel structure established by SAS-6 proteins. Here, we have engineered Chlamydomonas reinhardtii SAS-6-based oligomers with symmetries ranging from five- to ten-fold. Expression of a SAS-6 mutant that forms six-fold symmetric cartwheel structures in vitro resulted in cartwheels and centrioles with eight- or nine-fold symmetries in vivo. In combination with Bld10 mutants that weaken cartwheel-microtubule interactions, this SAS-6 mutant produced six- to eight-fold symmetric cartwheels. Concurrently, the microtubule wall maintained eight- and nine-fold symmetries. Expressing SAS-6 with analogous mutations in human cells resulted in nine-fold symmetric centrioles that exhibited impaired length and organization. Together, our data suggest that the self-assembly properties of SAS-6 instruct cartwheel symmetry, and lead us to propose a model in which the cartwheel and the microtubule wall assemble in an interdependent manner to establish the native architecture of centrioles.

  3. Torsion of the central pair microtubules in eukaryotic flagella due to bending-driven lateral buckling

    International Nuclear Information System (INIS)

    Li, C.; Ru, C.Q.; Mioduchowski, A.

    2006-01-01

    Inspired by recent interest in torsion of the central pair microtubules in eukaryotic flagella, a novel thin-walled elastic beam model is suggested to study critical condition under which uniform bending of a flagellum will cause lateral/torsional buckling of the central pair. The model is directed to the central pair itself and the role of all surrounding cross-linkings inside the flagellum is modeled as an equivalent surrounding elastic medium. The model predicts that bending-driven torsion of the central pair does occur when the radius of curvature of the bent flagellum reduces to a moderate critical value typically of tens of microns. In particular, this critical value is almost independent of the flagellum length, and more sensitive to the parameters defining the surrounding elastic medium than the shear modulus of microtubules. The predicted wavelengths of the torsional buckling mode are insensitive to the flagellum length and comparable to some known related experimental data. These results indicate that torsion of the central pair microtubules in flagella is inevitable as a result of bending-driven lateral buckling. This offers an entirely new insight into the ongoing research on the mechanism of the central pair torsion

  4. Noscapine alters microtubule dynamics in living cells and inhibits the progression of melanoma.

    Science.gov (United States)

    Landen, Jaren W; Lang, Roland; McMahon, Steve J; Rusan, Nasser M; Yvon, Anne-Marie; Adams, Ashley W; Sorcinelli, Mia D; Campbell, Ross; Bonaccorsi, Paola; Ansel, John C; Archer, David R; Wadsworth, Patricia; Armstrong, Cheryl A; Joshi, Harish C

    2002-07-15

    Cellular microtubules, polymers of tubulin, alternate relentlessly between phases of growth and shortening. We now show that noscapine, a tubulin-binding agent, increases the time that cellular microtubules spend idle in a paused state. As a result, most mammalian cell types observed arrest in mitosis in the presence of noscapine. We demonstrate that noscapine-treated murine melanoma B16LS9 cells do not arrest in mitosis but rather become polyploid followed by cell death, whereas primary melanocytes reversibly arrest in mitosis and resume a normal cell cycle after noscapine removal. Furthermore, in a syngeneic murine model of established s.c. melanoma, noscapine treatment resulted in an 85% inhibition of tumor volume on day 17 when delivered by gavage compared with untreated animals (P microtubule dynamics, with no detected toxicity to the host. Consequently, noscapine could be a valuable chemotherapeutic agent, alone or in combination, for the treatment of advanced melanoma.

  5. Curcumin alters the cytoskeleton and microtubule organization on trophozoites of Giardia lamblia.

    Science.gov (United States)

    Gutiérrez-Gutiérrez, Filiberto; Palomo-Ligas, Lissethe; Hernández-Hernández, José Manuel; Pérez-Rangel, Armando; Aguayo-Ortiz, Rodrigo; Hernández-Campos, Alicia; Castillo, Rafael; González-Pozos, Sirenia; Cortés-Zárate, Rafael; Ramírez-Herrera, Mario Alberto; Mendoza-Magaña, María Luisa; Castillo-Romero, Araceli

    2017-08-01

    Giardia lamblia is a worldwide protozoan responsible for a significant number of intestinal infections. There are several drugs for the treatment of giardiasis, but they often cause side effects. Curcumin, a component of turmeric, has antigiardial activity; however, the molecular target and mechanism of antiproliferative activity are not clear. The effects of curcumin on cellular microtubules have been widely investigated. Since tubulin is the most abundant protein in the cytoskeleton of Giardia, to elucidate whether curcumin has activity against the microtubules of this parasite, we treated trophozoites with curcumin and the cells were analyzed by scanning electron microscopy and confocal microscopy. Curcumin inhibited Giardia proliferation and adhesion in a time-concentration-dependent mode. The higher inhibitory concentrations of curcumin (3 and 15μM) disrupted the cytoskeletal structures of trophozoites; the damage was evident on the ventral disk, flagella and in the caudal region, also the membrane was affected. The immunofluorescence images showed altered distribution of tubulin staining on ventral disk and flagella. Additionally, we found that curcumin caused a clear reduction of tubulin expression. By docking analysis and molecular dynamics we showed that curcumin has a high probability to bind at the interface of the tubulin dimer close to the vinblastine binding site. All the data presented indicate that curcumin may inhibit Giardia proliferation by perturbing microtubules. Copyright © 2017. Published by Elsevier B.V.

  6. Effects of colchicine treatment on the microtubule cytoskeleton and total protein during microsporogenesis in ginkgo biloba

    International Nuclear Information System (INIS)

    Cao, Q.J.; Mei, F.F.

    2015-01-01

    The purpose of this study was to examine the effects of colchicine treatment on the microtubule cytoskeleton and the expression of proteins during microsporogenesis in G. biloba, as observed by immunofluorescence and 2-DE analysis in microsporangia treated with colchicine. The results showed the microtubule structures were affected by the colchicine in Ginkgo biloba, but the treatment effect of the colchicine had certain limitation in G. biloba. The percentage of microsporocytes whose microtubule structures were affected by the colchicine treatment was less than that observed in other plant species, not higher than 10 %. It was also found that the expression level of several endogenous proteins were changed in G. biloba when the microsporangia were treated with colchicine. Although we only tested colchicines was only tested in the present study, G. biloba appeared to possess factors that restricted the effect of such chemical agents. Our observations led us to speculate that the endogenous proteins are possibly responsible for the reduced effects of colchicine treatment in G. biloba. (author)

  7. Heterotrimeric Kinesin II Is the Microtubule Motor Protein Responsible for Pigment Dispersion in Xenopus Melanophores

    Science.gov (United States)

    Tuma, M. Carolina; Zill, Andrew; Le Bot, Nathalie; Vernos, Isabelle; Gelfand, Vladimir

    1998-01-01

    Melanophores move pigment organelles (melanosomes) from the cell center to the periphery and vice-versa. These bidirectional movements require cytoplasmic microtubules and microfilaments and depend on the function of microtubule motors and a myosin. Earlier we found that melanosomes purified from Xenopus melanophores contain the plus end microtubule motor kinesin II, indicating that it may be involved in dispersion (Rogers, S.L., I.S. Tint, P.C. Fanapour, and V.I. Gelfand. 1997. Proc. Natl. Acad. Sci. USA. 94: 3720–3725). Here, we generated a dominant-negative construct encoding green fluorescent protein fused to the stalk-tail region of Xenopus kinesin-like protein 3 (Xklp3), the 95-kD motor subunit of Xenopus kinesin II, and introduced it into melanophores. Overexpression of the fusion protein inhibited pigment dispersion but had no effect on aggregation. To control for the specificity of this effect, we studied the kinesin-dependent movement of lysosomes. Neither dispersion of lysosomes in acidic conditions nor their clustering under alkaline conditions was affected by the mutant Xklp3. Furthermore, microinjection of melanophores with SUK4, a function-blocking kinesin antibody, inhibited dispersion of lysosomes but had no effect on melanosome transport. We conclude that melanosome dispersion is powered by kinesin II and not by conventional kinesin. This paper demonstrates that kinesin II moves membrane-bound organelles. PMID:9852150

  8. Image-based compound profiling reveals a dual inhibitor of tyrosine kinase and microtubule polymerization.

    Science.gov (United States)

    Tanabe, Kenji

    2016-04-27

    Small-molecule compounds are widely used as biological research tools and therapeutic drugs. Therefore, uncovering novel targets of these compounds should provide insights that are valuable in both basic and clinical studies. I developed a method for image-based compound profiling by quantitating the effects of compounds on signal transduction and vesicle trafficking of epidermal growth factor receptor (EGFR). Using six signal transduction molecules and two markers of vesicle trafficking, 570 image features were obtained and subjected to multivariate analysis. Fourteen compounds that affected EGFR or its pathways were classified into four clusters, based on their phenotypic features. Surprisingly, one EGFR inhibitor (CAS 879127-07-8) was classified into the same cluster as nocodazole, a microtubule depolymerizer. In fact, this compound directly depolymerized microtubules. These results indicate that CAS 879127-07-8 could be used as a chemical probe to investigate both the EGFR pathway and microtubule dynamics. The image-based multivariate analysis developed herein has potential as a powerful tool for discovering unexpected drug properties.

  9. Transcription co-activator Arabidopsis ANGUSTIFOLIA3 (AN3) regulates water-use efficiency and drought tolerance by modulating stomatal density and improving root architecture by the transrepression of YODA (YDA).

    Science.gov (United States)

    Meng, Lai-Sheng; Yao, Shun-Qiao

    2015-09-01

    One goal of modern agriculture is the improvement of plant drought tolerance and water-use efficiency (WUE). Although stomatal density has been linked to WUE, the causal molecular mechanisms and engineered alternations of this relationship are not yet fully understood. Moreover, YODA (YDA), which is a MAPKK kinase gene, negatively regulates stomatal development. BR-INSENSITIVE 2 interacts with phosphorylates and inhibits YDA. However, whether YDA is modulated in the transcriptional level is still unclear. Plants lacking ANGUSTIFOLIA3 (AN3) activity have high drought stress tolerance because of low stomatal densities and improved root architecture. Such plants also exhibit enhanced WUE through declining transpiration without a demonstrable reduction in biomass accumulation. AN3 negatively regulated YDA expression at the transcriptional level by target-gene analysis. Chromatin immunoprecipitation analysis indicated that AN3 was associated with a region of the YDA promoter in vivo. YDA mutation significantly decreased the stomatal density and root length of an3 mutant, thus proving the participation of YDA in an3 drought tolerance and WUE enhancement. These components form an AN3-YDA complex, which allows the integration of water deficit stress signalling into the production or spacing of stomata and cell proliferation, thus leading to drought tolerance and enhanced WUE. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  10. Functional connectivity analysis of the neural bases of emotion regulation: A comparison of independent component method with density-based k-means clustering method.

    Science.gov (United States)

    Zou, Ling; Guo, Qian; Xu, Yi; Yang, Biao; Jiao, Zhuqing; Xiang, Jianbo

    2016-04-29

    Functional magnetic resonance imaging (fMRI) is an important tool in neuroscience for assessing connectivity and interactions between distant areas of the brain. To find and characterize the coherent patterns of brain activity as a means of identifying brain systems for the cognitive reappraisal of the emotion task, both density-based k-means clustering and independent component analysis (ICA) methods can be applied to characterize the interactions between brain regions involved in cognitive reappraisal of emotion. Our results reveal that compared with the ICA method, the density-based k-means clustering method provides a higher sensitivity of polymerization. In addition, it is more sensitive to those relatively weak functional connection regions. Thus, the study concludes that in the process of receiving emotional stimuli, the relatively obvious activation areas are mainly distributed in the frontal lobe, cingulum and near the hypothalamus. Furthermore, density-based k-means clustering method creates a more reliable method for follow-up studies of brain functional connectivity.

  11. Epothilones as lead structures for the synthesis-based discovery of new chemotypes for microtubule stabilization.

    Science.gov (United States)

    Feyen, Fabian; Cachoux, Frédéric; Gertsch, Jürg; Wartmann, Markus; Altmann, Karl-Heinz

    2008-01-01

    Epothilones are macrocyclic bacterial natural products with potent microtubule-stabilizing and antiproliferative activity. They have served as successful lead structures for the development of several clinical candidates for anticancer therapy. However, the structural diversity of this group of clinical compounds is rather limited, as their structures show little divergence from the original natural product leads. Our own research has explored the question of whether epothilones can serve as a basis for the development of new structural scaffolds, or chemotypes, for microtubule stabilization that might serve as a basis for the discovery of new generations of anticancer drugs. We have elaborated a series of epothilone-derived macrolactones whose overall structural features significantly deviate from those of the natural epothilone scaffold and thus define new structural families of microtubule-stabilizing agents. Key elements of our hypermodification strategy are the change of the natural epoxide geometry from cis to trans, the incorporation of a conformationally constrained side chain, the removal of the C3-hydroxyl group, and the replacement of C12 with nitrogen. So far, this approach has yielded analogs 30 and 40 that are the most advanced, the most rigorously modified, structures, both of which are potent antiproliferative agents with low nanomolar activity against several human cancer cell lines in vitro. The synthesis was achieved through a macrolactone-based strategy or a high-yielding RCM reaction. The 12-aza-epothilone ("azathilone" 40) may be considered a "non-natural" natural product that still retains most of the overall structural characteristics of a true natural product but is structurally unique, because it lies outside of the general scope of Nature's biosynthetic machinery for polyketide synthesis. Like natural epothilones, both 30 and 40 promote tubulin polymerization in vitro and at the cellular level induce cell cycle arrest in mitosis. These

  12. Lung density

    DEFF Research Database (Denmark)

    Garnett, E S; Webber, C E; Coates, G

    1977-01-01

    The density of a defined volume of the human lung can be measured in vivo by a new noninvasive technique. A beam of gamma-rays is directed at the lung and, by measuring the scattered gamma-rays, lung density is calculated. The density in the lower lobe of the right lung in normal man during quiet...... breathing in the sitting position ranged from 0.25 to 0.37 g.cm-3. Subnormal values were found in patients with emphsema. In patients with pulmonary congestion and edema, lung density values ranged from 0.33 to 0.93 g.cm-3. The lung density measurement correlated well with the findings in chest radiographs...... but the lung density values were more sensitive indices. This was particularly evident in serial observations of individual patients....

  13. Replicate high-density rat genome oligonucleotide microarrays reveal hundreds of regulated genes in the dorsal root ganglion after peripheral nerve injury.

    Directory of Open Access Journals (Sweden)

    Mannion James W

    2002-10-01

    Full Text Available Abstract Background Rat oligonucleotide microarrays were used to detect changes in gene expression in the dorsal root ganglion (DRG 3 days following sciatic nerve transection (axotomy. Two comparisons were made using two sets of triplicate microarrays, naïve versus naïve and naïve versus axotomy. Results Microarray variability was assessed using the naïve versus naïve comparison. These results support use of a P 1.5-fold expression change and P 1.5-fold and P in situ hybridization verified the expression of 24 transcripts. These data showed an 83% concordance rate with the arrays; most mismatches represent genes with low expression levels reflecting limits of array sensitivity. A significant correlation was found between actual mRNA differences and relative changes between microarrays (r2 = 0.8567. Temporal patterns of individual genes regulation varied. Conclusions We identify parameters for microarray analysis which reduce error while identifying many putatively regulated genes. Functional classification of these genes suggest reorganization of cell structural components, activation of genes expressed by immune and inflammatory cells and down-regulation of genes involved in neurotransmission.

  14. Two Types of Genetic Interaction Implicate the Whirligig Gene of Drosophila Melanogaster in Microtubule Organization in the Flagellar Axoneme

    Science.gov (United States)

    Green, L. L.; Wolf, N.; McDonald, K. L.; Fuller, M. T.

    1990-01-01

    The mutant nc4 allele of whirligig (3-54.4) of Drosophila melanogaster fails to complement mutations in an α-tubulin locus, α1t, mutations in a β-tubulin locus, B2t, or a mutation in the haywire locus. However, wrl fails to map to any of the known α- or β-tubulin genes. The extragenic failure to complement could indicate that the wrl product participates in structural interactions with microtubule proteins. The whirligig locus appears to be haploinsufficient for male fertility. Both a deficiency of wrl and possible loss of function alleles obtained by reverting the failure to complement between wrl(nc4) and B2t(n) are dominant male sterile in a genetic background wild type for tubulin. The dominant male sterility of the revertant alleles is suppressed if the flies are also heterozygous for B2t(n), for a deficiency of α1t, or for the hay(nc2) allele. These results suggest that it is not the absolute level of wrl gene product but its level relative to tubulin or microtubule function that is important for normal spermatogenesis. The phenotype of homozygous wrl mutants suggests that the whirligig product plays a role in postmeiotic spermatid differentiation, possibly in organizing the microtubules of the sperm flagellar axoneme. Flies homozygous for either wrl(nc4) or revertant alleles are viable and female fertile but male sterile. Premeiotic and meiotic stages of spermatogenesis appear normal. However, in post-meiotic stages, flagellar axonemes show loss of the accessory microtubule on the B-subfiber of outer doublet microtubules, outer triplet instead of outer doublet microtubules, and missing central pair microtubules. PMID:2127579

  15. Destiny from density

    OpenAIRE

    Seewaldt, Victoria L.

    2012-01-01

    The identification of a signalling protein that regulates the accumulation of fat and connective tissue in breasts may help to explain why high mammographic density is linked to breast-cancer risk and may provide a marker for predicting this risk.

  16. Microtubule-Associated Proteins in Mesial Temporal Lobe Epilepsy with and without Psychiatric Comorbidities and Their Relation with Granular Cell Layer Dispersion

    Directory of Open Access Journals (Sweden)

    Ludmyla Kandratavicius

    2013-01-01

    Full Text Available Background. Despite strong association between epilepsy and psychiatric comorbidities, biological substrates are unknown. We have previously reported decreased mossy fiber sprouting in mesial temporal lobe epilepsy (MTLE patients with psychosis and increased in those with major depression. Microtubule associated proteins (MAPs are essentially involved in dendritic and synaptic sprouting. Methods. MTLE hippocampi of subjects without psychiatric history, MTLE + major depression, and MTLE + interictal psychosis derived from epilepsy surgery and control necropsies were investigated for neuronal density, granular layer dispersion, and MAP2 and tau immunohistochemistry. Results. Altered MAP2 and tau expression in MTLE and decreased tau expression in MTLE with psychosis were found. Granular layer dispersion correlated inversely with verbal memory scores, and with MAP2 and tau expression in the entorhinal cortex. Patients taking fluoxetine showed increased neuronal density in the granular layer and those taking haloperidol decreased neuronal density in CA3 and subiculum. Conclusions. Our results indicate relations between MAPs, granular layer dispersion, and memory that have not been previously investigated. Differential MAPs expression in human MTLE hippocampi with and without psychiatric comorbidities suggests that psychopathological states in MTLE rely on differential morphological and possibly neurochemical backgrounds. This clinical study was approved by our institution’s Research Ethics Board (HC-FMRP no. 1270/2008 and is registered under the Brazilian National System of Information on Ethics in Human Research (SISNEP no. 0423.0.004.000-07.

  17. Regulation of low-density lipoprotein receptor and 3-hydroxy-3-methylglutaryl coenzyme A reductase expression by Zingiber officinale in the liver of high-fat diet-fed rats.

    Science.gov (United States)

    Nammi, Srinivas; Kim, Moon S; Gavande, Navnath S; Li, George Q; Roufogalis, Basil D

    2010-05-01

    Zingiber officinale has been used to control lipid disorders and reported to possess remarkable cholesterol-lowering activity in experimental hyperlipidaemia. In the present study, the effect of a characterized and standardized extract of Zingiber officinale on the hepatic lipid levels as well as on the hepatic mRNA and protein expression of low-density lipoprotein (LDL) receptor and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase was investigated in a high-fat diet-fed rat model. Rats were treated with an ethanol extract of Zingiber officinale (400 mg/kg) extract along with a high-fat diet for 6 weeks. The extract of Zingiber officinale significantly decreased hepatic triglyceride and tended to decrease hepatic cholesterol levels when administered over 6 weeks to the rats fed a high-fat diet. We found that in parallel, the extract up-regulated both LDL receptor mRNA and protein level and down-regulated HMG-CoA reductase protein expression in the liver of these rats. The metabolic control of body lipid homeostasis is in part due to enhanced cholesterol biosynthesis and reduced expression of LDL receptor sites following long-term consumption of high-fat diets. The present results show restoration of transcriptional and post-transcriptional changes in low-density lipoprotein and HMG CoA reductase by Zingiber officinale administration with a high-fat diet and provide a rational explanation for the effect of ginger in the treatment of hyperlipidaemia.

  18. Auxin-dependent microtubule responses and seedling development are affected in a rice mutant resistant to EPC

    International Nuclear Information System (INIS)

    Nick, P.; Yatou, O.; Furuya, M.; Lambert, A.M.

    1994-01-01

    Mutants in rice (Oryza sativa L. cv. japonica) were used to study the role of the cytoskeleton in signal-dependent morphogenesis. Mutants obtained by gamma ray irradiation were selected that failed to show inhibition of coleoptile elongation by the anti microtubular drug ethyl-N-phenylcarbamate (EPC). The mutation EPC-Resistant 31 (ER31), isolated from such a screen, caused lethality in putatively homozygous embryos. Heterozygotes exhibited drug resistance, impaired development of crown roots, and characteristic changes in the pattern of cell elongation: cell elongation was enhanced in mesocotyls and leaf sheaths, but inhibited in coleoptiles. The orientation of cortical microtubules changed correspondingly: for etiolated seedlings, compared with the wild-type, they were more transverse with respect to the long cell axis in mesocotyls and leaf sheaths, but more longitudinal in coleoptiles. In mutant coleoptiles, in contrast to wild-type, microtubules did not reorient in response to auxin, and their response to microtubule-eliminating and microtubule-stabilizing drugs was conspicuously reduced. In contrast, they responded normally to other stimuli such as gibberellins or red light. Auxin sensitivity as assayed by the dose-response for callus induction did not show any significant differences between wild-type and mutant. The mutant phenotype is interpreted in terms of an interrupted link between auxin-triggered signal transduction and microtubule reorientation. (author)

  19. An agent-based model contrasts opposite effects of dynamic and stable microtubules on cleavage furrow positioning.

    Science.gov (United States)

    Odell, Garrett M; Foe, Victoria E

    2008-11-03

    From experiments by Foe and von Dassow (Foe, V.E., and G. von Dassow. 2008. J. Cell Biol. 183:457-470) and others, we infer a molecular mechanism for positioning the cleavage furrow during cytokinesis. Computer simulations reveal how this mechanism depends on quantitative motor-behavior details and explore how robustly this mechanism succeeds across a range of cell sizes. The mechanism involves the MKLP1 (kinesin-6) component of centralspindlin binding to and walking along microtubules to stimulate cortical contractility where the centralspindlin complex concentrates. The majority of astral microtubules are dynamically unstable. They bind most MKLP1 and suppress cortical Rho/myosin II activation because the tips of unstable microtubules usually depolymerize before MKLP1s reach the cortex. A subset of astral microtubules stabilizes during anaphase, becoming effective rails along which MKLP1 can actually reach the cortex. Because stabilized microtubules aim statistically at the equatorial spindle midplane, that is where centralspindlin accumulates to stimulate furrow formation.

  20. One-dimensional Brownian motion of charged nanoparticles along microtubules: a model system for weak binding interactions.

    Science.gov (United States)

    Minoura, Itsushi; Katayama, Eisaku; Sekimoto, Ken; Muto, Etsuko

    2010-04-21

    Various proteins are known to exhibit one-dimensional Brownian motion along charged rodlike polymers, such as microtubules (MTs), actin, and DNA. The electrostatic interaction between the proteins and the rodlike polymers appears to be crucial for one-dimensional Brownian motion, although the underlying mechanism has not been fully clarified. We examined the interactions of positively-charged nanoparticles composed of polyacrylamide gels with MTs. These hydrophilic nanoparticles bound to MTs and displayed one-dimensional Brownian motion in a charge-dependent manner, which indicates that nonspecific electrostatic interaction is sufficient for one-dimensional Brownian motion. The diffusion coefficient decreased exponentially with an increasing particle charge (with the exponent being 0.10 kBT per charge), whereas the duration of the interaction increased exponentially (exponent of 0.22 kBT per charge). These results can be explained semiquantitatively if one assumes that a particle repeats a cycle of binding to and movement along an MT until it finally dissociates from the MT. During the movement, a particle is still electrostatically constrained in the potential valley surrounding the MT. This entire process can be described by a three-state model analogous to the Michaelis-Menten scheme, in which the two parameters of the equilibrium constant between binding and movement, and the rate of dissociation from the MT, are derived as a function of the particle charge density. This study highlights the possibility that the weak binding interactions between proteins and rodlike polymers, e.g., MTs, are mediated by a similar, nonspecific charge-dependent mechanism. Copyright 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  1. Microtubule-dependent changes in morphology and localization of chloride transport proteins in gill mitochondria-rich cells of the tilapia, Oreochromis mossambicus.

    Science.gov (United States)

    Yang, Wen-Kai; Wu, Yu-Ching; Tang, Cheng-Hao; Lee, Tsung-Han

    2016-08-01

    The tilapia (Oreochromis mossambicus) is a euryhaline fish exhibiting adaptive changes in cell size, phenotype, and ionoregulatory functions upon salinity challenge. Na(+) /Cl(-) cotransporter (NCC) and Na(+) /K(+) /2Cl(-) cotransporter (NKCC) are localized in the apical and basolateral membranes of mitochondria-rich (MR) cells of the gills. These cells are responsible for chloride absorption (NCC) and secretion (NKCC), respectively, thus, the switch of gill NCC and NKCC expression is a crucial regulatory mechanism for salinity adaptation in tilapia. However, little is known about the interaction of cytoskeleton and these adaptive changes. In this study, we examined the time-course of changes in the localization of NKCC/NCC in the gills of tilapia transferred from fresh water (FW) to brackish water (20‰) and from seawater (SW; 35‰) to FW. The results showed that basolateral NKCC disappeared and NCC was expressed in the apical membrane of MR cells. To further clarify the process of these adaptive changes, colchicine, a specific inhibitor of microtubule-dependent cellular regulating processes was used. SW-acclimated tilapia were transferred to SW, FW, and FW with colchicine (colchicine-FW) for 96 h. Compared with the FW-treatment group, in the MR cells of colchicine-FW-treatment group, (1) the average size was significantly larger, (2) only wavy-convex-subtype apical surfaces were found, and (3) the basolateral (cytoplasmic) NKCC signals were still exhibited. Taken together, our results suggest that changes in size, phenotype, as well as the expression of NCC and NKCC cotransporters of MR cells in the tilapia are microtubule-dependent. J. Morphol. 277:1113-1122, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  2. Activation of Ran GTPase by a Legionella effector promotes microtubule polymerization, pathogen vacuole motility and infection.

    Directory of Open Access Journals (Sweden)

    Eva Rothmeier

    2013-09-01

    Full Text Available The causative agent of Legionnaires' disease, Legionella pneumophila, uses the Icm/Dot type IV secretion system (T4SS to form in phagocytes a distinct "Legionella-containing vacuole" (LCV, which intercepts endosomal and secretory vesicle trafficking. Proteomics revealed the presence of the small GTPase Ran and its effector RanBP1 on purified LCVs. Here we validate that Ran and RanBP1 localize to LCVs and promote intracellular growth of L. pneumophila. Moreover, the L. pneumophila protein LegG1, which contains putative RCC1 Ran guanine nucleotide exchange factor (GEF domains, accumulates on LCVs in an Icm/Dot-dependent manner. L. pneumophila wild-type bacteria, but not strains lacking LegG1 or a functional Icm/Dot T4SS, activate Ran on LCVs, while purified LegG1 produces active Ran(GTP in cell lysates. L. pneumophila lacking legG1 is compromised for intracellular growth in macrophages and amoebae, yet is as cytotoxic as the wild-type strain. A downstream effect of LegG1 is to stabilize microtubules, as revealed by conventional and stimulated emission depletion (STED fluorescence microscopy, subcellular fractionation and Western blot, or by microbial microinjection through the T3SS of a Yersinia strain lacking endogenous effectors. Real-time fluorescence imaging indicates that LCVs harboring wild-type L. pneumophila rapidly move along microtubules, while LCVs harboring ΔlegG1 mutant bacteria are stalled. Together, our results demonstrate that Ran activation and RanBP1 promote LCV formation, and the Icm/Dot substrate LegG1 functions as a bacterial Ran activator, which localizes to LCVs and promotes microtubule stabilization, LCV motility as well as intracellular replication of L. pneumophila.

  3. Activation of Ran GTPase by a Legionella Effector Promotes Microtubule Polymerization, Pathogen Vacuole Motility and Infection

    Science.gov (United States)

    Rothmeier, Eva; Pfaffinger, Gudrun; Hoffmann, Christine; Harrison, Christopher F.; Grabmayr, Heinrich; Repnik, Urska; Hannemann, Mandy; Wölke, Stefan; Bausch, Andreas; Griffiths, Gareth; Müller-Taubenberger, Annette; Itzen, Aymelt; Hilbi, Hubert

    2013-01-01

    The causative agent of Legionnaires' disease, Legionella pneumophila, uses the Icm/Dot type IV secretion system (T4SS) to form in phagocytes a distinct “Legionella-containing vacuole” (LCV), which intercepts endosomal and secretory vesicle trafficking. Proteomics revealed the presence of the small GTPase Ran and its effector RanBP1 on purified LCVs. Here we validate that Ran and RanBP1 localize to LCVs and promote intracellular growth of L. pneumophila. Moreover, the L. pneumophila protein LegG1, which contains putative RCC1 Ran guanine nucleotide exchange factor (GEF) domains, accumulates on LCVs in an Icm/Dot-dependent manner. L. pneumophila wild-type bacteria, but not strains lacking LegG1 or a functional Icm/Dot T4SS, activate Ran on LCVs, while purified LegG1 produces active Ran(GTP) in cell lysates. L. pneumophila lacking legG1 is compromised for intracellular growth in macrophages and amoebae, yet is as cytotoxic as the wild-type strain. A downstream effect of LegG1 is to stabilize microtubules, as revealed by conventional and stimulated emission depletion (STED) fluorescence microscopy, subcellular fractionation and Western blot, or by microbial microinjection through the T3SS of a Yersinia strain lacking endogenous effectors. Real-time fluorescence imaging indicates that LCVs harboring wild-type L. pneumophila rapidly move along microtubules, while LCVs harboring ΔlegG1 mutant bacteria are stalled. Together, our results demonstrate that Ran activation and RanBP1 promote LCV formation, and the Icm/Dot substrate LegG1 functions as a bacterial Ran activator, which localizes to LCVs and promotes microtubule stabilization, LCV motility as well as intracellular replication of L. pneumophila. PMID:24068924

  4. Electron tomography of the microtubule cytoskeleton in multinucleated hyphae of Ashbya gossypii.

    Science.gov (United States)

    Gibeaux, Romain; Lang, Claudia; Politi, Antonio Z; Jaspersen, Sue L; Philippsen, Peter; Antony, Claude

    2012-12-01

    We report the mechanistic basis guiding the migration pattern of multiple nuclei in hyphae of Ashbya gossypii. Using electron tomography, we reconstructed the cytoplasmic microtubule (cMT) cytoskeleton in three tip regions with a total of 13 nuclei and also the spindle microtubules of four mitotic nuclei. Each spindle pole body (SPB) nucleates three cMTs and most cMTs above a certain length grow according to their plus-end structure. Long cMTs closely align for several microns along the cortex, presumably marking regions where dynein generates pulling forces on nuclei. Close proximity between cMTs emanating from adjacent nuclei was not observed. The majority of nuclei carry duplicated side-by-side SPBs, which together emanate an average of six cMTs, in most cases in opposite orientation with respect to the hyphal growth axis. Such cMT arrays explain why many nuclei undergo short-range back and forth movements. Only occasionally do all six cMTs orient in one direction, a precondition for long-range nuclear bypassing. Following mitosis, daughter nuclei carry a single SPB with three cMTs. The increased probability that all three cMTs orient in one direction explains the high rate of nuclear bypassing observed in these nuclei. The A. gossypii mitotic spindle was found to be structurally similar to that of Saccharomyces cerevisiae in terms of nuclear microtubule (nMT) number, length distribution and three-dimensional organization even though the two organisms differ significantly in chromosome number. Our results suggest that two nMTs attach to each kinetochore in A. gossypii and not only one nMT like in S. cerevisiae.

  5. Gravity resistance, another graviresponse in plants - role of microtubule-membrane-cell wall continuum

    Science.gov (United States)

    Hoson, T.; Saito, Y.; Usui, S.; Soga, K.; Wakabayashi, K.

    Resistance to the gravitational force has been a serious problem for plants to survive on land, after they first went ashore more than 400 million years ago. Thus, gravity resistance is the principal graviresponse in plants comparable to gravitropism. Nevertheless, only limited information has been obtained for this second gravity response. We have examined the mechanism of gravity resistance using hypergravity conditions produced by centrifugation. The results led a hypothesis on the mechanism of plant resistance to the gravitational force that the plant constructs a tough body by increasing the cell wall rigidity, which are brought about by modification of the cell wall metabolism and cell wall environment, especially pH. The hypothesis was further supported by space experiments during the Space Shuttle STS-95 mission. On the other hand, we have shown that gravity signal may be perceived by mechanoreceptors (mechanosensitive ion channels) on the plasma membrane and amyloplast sedimentation in statocytes is not involved in gravity resistance. Moreover, hypergravity treatment increased the expression levels of genes encoding alpha-tubulin, a component of microtubules and 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (HMGR), which catalyzes a reaction producing mevalonic acid, a key precursor of terpenoids such as membrane sterols. The expression of HMGR and alpha- and beta-tubulin genes increased within several hours after hypergravity treatment, depending on the magnitude of gravity. The determination of levels of gene products as well as the analysis with knockout mutants of these genes by T-DNA insertions in Arabidopsis supports the involvement of both membrane sterols and microtubules in gravity resistance. These results suggest that structural or physiological continuum of microtubule-cell membrane-cell wall is responsible for plant resistance to the gravitational force.

  6. Quantum walks in brain microtubules--a biomolecular basis for quantum cognition?

    Science.gov (United States)

    Hameroff, Stuart

    2014-01-01

    Cognitive decisions are best described by quantum mathematics. Do quantum information devices operate in the brain? What would they look like? Fuss and Navarro () describe quantum lattice registers in which quantum superpositioned pathways interact (compute/integrate) as 'quantum walks' akin to Feynman's path integral in a lattice (e.g. the 'Feynman quantum chessboard'). Simultaneous alternate pathways eventually reduce (collapse), selecting one particular pathway in a cognitive decision, or choice. This paper describes how quantum walks in a Feynman chessboard are conceptually identical to 'topological qubits' in brain neuronal microtubules, as described in the Penrose-Hameroff 'Orch OR' theory of consciousness. Copyright © 2013 Cognitive Science Society, Inc.

  7. XTACC3-XMAP215 association reveals an asymmetric interaction promoting microtubule elongation

    DEFF Research Database (Denmark)

    Mortuza, Gulnahar B; Cavazza, Tommaso; Garcia-Mayoral, Maria Flor

    2014-01-01

    215 (chTOG), dissecting the mechanism by which their interaction promotes microtubule elongation during spindle assembly. Using SAXS, we show that the TACC domain (TD) is an elongated structure that mediates the interaction with the C terminus of XMAP215. Our data suggest that one TD and two XMAP215...... molecules associate to form a four-helix coiled-coil complex. A hybrid methods approach was used to define the precise regions of the TACC heptad repeat and the XMAP215 C terminus required for assembly and functioning of the complex. We show that XTACC3 can induce the recruitment of larger amounts of XMAP...

  8. Antagonism between the dynein and Ndc80 complexes at kinetochores controls the stability of kinetochore-microtubule attachments during mitosis.

    Science.gov (United States)

    Amin, Mohammed A; McKenney, Richard J; Varma, Dileep

    2018-04-20

    Chromosome alignment and segregation during mitosis require kinetochore-microtubule (kMT) attachments that are mediated by the molecular motor dynein and the kMT-binding complex Ndc80. The Rod-ZW10-Zwilch (RZZ) complex is central to this coordination as it has an important role in dynein recruitment and has recently been reported to have a key function in the regulation of stable kMT attachments in Caenorhabditis elegans besides its role in activating the spindle assembly checkpoint (SAC). However, the mechanism by which these protein complexes control kMT attachments to drive chromosome motility during early mitosis is still unclear. Here, using in vitro total internal reflection fluorescence microscopy, we observed that higher concentrations of Ndc80 inhibited dynein binding to MTs, providing evidence that Ndc80 and dynein antagonize each other's function. High-resolution microscopy and siRNA-mediated functional disruption revealed that severe defects in chromosome alignment induced by depletion of dynein or the dynein adapter Spindly are rescued by codepletion of the RZZ component Rod in human cells. Interestingly, rescue of the chromosome alignment defects was independent of Rod function in SAC activation and was accompanied by a remarkable restoration of stable kMT attachments. Furthermore, the chromosome alignment rescue depended on the plus-end-directed motility of centromere protein E (CENP-E) because cells codepleted of CENP-E, Rod, and dynein could not establish stable kMT attachments or align their chromosomes properly. Our findings support the idea that dynein may control the function of the Ndc80 complex in stabilizing kMT attachments directly by interfering with Ndc80-MT binding or indirectly by controlling the Rod-mediated inhibition of Ndc80. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. A novel dual inhibitor of microtubule and Bruton's tyrosine kinase inhibits survival of multiple myeloma and osteoclastogenesis.

    Science.gov (United States)

    Pandey, Manoj K; Gowda, Krishne; Sung, Shen-Shu; Abraham, Thomas; Budak-Alpdogan, Tulin; Talamo, Giampolo; Dovat, Sinisa; Amin, Shantu

    2017-09-01

    Bruton's tyrosine kinase (BTK) regulates many vital signaling pathways and plays a critical role in cell proliferation, survival, migration, and resistance. Previously, we reported that a small molecule, KS99, is an inhibitor of tubulin polymerization. In the present study, we explored whether KS99 is a dual inhibitor of BTK and tubulin polymerization. Although it is known that BTK is required for clonogenic growth and resistance, and microtubules are essential for cancer cell growth, dual targeting of these two components has not been explored previously. Through docking studies, we predicted that KS99 interacts directly with the catalytic domain of BTK and inhibits phosphorylation at the Y223 residue and kinase activities. Treatment of KS99 reduces the cell viability of multiple myeloma (MM) and CD138 + cells, with an IC 50 of between 0.5 and 1.0 μmol/L. We found that KS99 is able to induce apoptosis in MM cells in a caspase-dependent manner. KS99 suppressed the receptor activator of NF-κB ligand (RANKL)-induced differentiation of macrophages to osteoclasts in a dose-dependent manner and, importantly, inhibited the expression of cytokines associated with bone loss. Finally, we found that KS99 inhibits the in vivo tumor growth of MM cells through the inhibition of BTK and tubulin. Overall, our results show that dual inhibition of BTK and tubulin polymerization by KS99 is a viable option in MM treatment, particularly in the inhibition of refraction and relapse. Copyright © 2017 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

  10. Olfactory receptor signaling is regulated by the post-synaptic density 95, Drosophila discs large, zona-occludens 1 (PDZ) scaffold multi-PDZ domain protein 1.

    LENUS (Irish Health Repository)

    Dooley, Ruth

    2009-12-01

    The unique ability of mammals to detect and discriminate between thousands of different odorant molecules is governed by the diverse array of olfactory receptors expressed by olfactory sensory neurons in the nasal epithelium. Olfactory receptors consist of seven transmembrane domain G protein-coupled receptors and comprise the largest gene superfamily in the mammalian genome. We found that approximately 30% of olfactory receptors possess a classical post-synaptic density 95, Drosophila discs large, zona-occludens 1 (PDZ) domain binding motif in their C-termini. PDZ domains have been established as sites for protein-protein interaction and play a central role in organizing diverse cell signaling assemblies. In the present study, we show that multi-PDZ domain protein 1 (MUPP1) is expressed in the apical compartment of olfactory sensory neurons. Furthermore, on heterologous co-expression with olfactory sensory neurons, MUPP1 was shown to translocate to the plasma membrane. We found direct interaction of PDZ domains 1 + 2 of MUPP1 with the C-terminus of olfactory receptors in vitro. Moreover, the odorant-elicited calcium response of OR2AG1 showed a prolonged decay in MUPP1 small interfering RNA-treated cells. We have therefore elucidated the first building blocks of the putative \\'olfactosome\\

  11. The low-density lipoprotein receptor-related protein 10 is a negative regulator of the canonical Wnt/β-catenin signaling pathway

    International Nuclear Information System (INIS)

    Jeong, Young-Hee; Sekiya, Manami; Hirata, Michiko; Ye, Mingjuan; Yamagishi, Azumi; Lee, Sang-Mi; Kang, Man-Jong; Hosoda, Akemi; Fukumura, Tomoe; Kim, Dong-Ho; Saeki, Shigeru

    2010-01-01

    Wnt signaling pathways play fundamental roles in the differentiation, proliferation and functions of many cells as well as developmental, growth, and homeostatic processes in animals. Low-density lipoprotein receptor (LDLR)-related protein (LRP) 5 and LRP6 serve as coreceptors of Wnt proteins together with Frizzled receptors, triggering activation of canonical Wnt/β-catenin signaling. Here, we found that LRP10, a new member of the LDLR gene family, inhibits the canonical Wnt/β-catenin signaling pathway. The β-catenin/T cell factor (TCF) transcriptional activity in HEK293 cells was activated by transfection with Wnt3a or LRP6, which was then inhibited by co-transfection with LRP10. Deletion of the extracellular domain of LRP10 negated its inhibitory effect. The inhibitory effect of LRP10 was consistently conserved in HEK293 cells even when GSK3β phosphorylation was inhibited by incubation with lithium chloride and co-transfection with constitutively active S33Y-mutated β-catenin. Nuclear β-catenin accumulation was unaffected by LRP10. The present studies suggest that LRP10 may interfere with the formation of the β-catenin/TCF complex and/or its binding to target DNA in the nucleus, and that the extracellular domain of LRP10 is critical for inhibition of the canonical Wnt/β-catenin signaling pathway.

  12. Coordinated regulation of nitrogen supply mode and initial cell density for energy storage compounds production with economized nitrogen utilization in a marine microalga Isochrysis zhangjiangensis.

    Science.gov (United States)

    Chi, Lei; Yao, Changhong; Cao, Xupeng; Xue, Song

    2016-01-01

    Lipids and carbohydrates are main energy storage compounds (ESC) of microalgae under stressed conditions and they are potential feedstock for biofuel production. Yet, the sustainable and commercially successful production of ESC in microalgae needs to consider nitrogen utilization efficiency. Here the impact of different initial cell densities (ICDs) on ESC accumulation in Isochrysis zhangjiangensis under two nitrogen supply modes (an initially equal concentration of nitrogen per-cell in the medium (N1) and an equal total concentration of nitrogen in the culture system (N2)) were investigated. The results demonstrated that the highest ESC yield (1.36gL(-1)) at N1, which included a maximal nitrogen supply in the cultivation system, and the highest ESC content (66.5%) and ESC productivity per mass of nitrogen (3.28gg(-1) (N) day(-1)) at N2, were all obtained under a high ICD of 8.0×10(6)cellsmL(-1). Therefore I. zhangjiangensis qualifies for ESC-enriched biomass production with economized nitrogen utilization. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Low Bone Density

    Science.gov (United States)

    ... Density Exam/Testing › Low Bone Density Low Bone Density Low bone density is when your bone density ... people with normal bone density. Detecting Low Bone Density A bone density test will determine whether you ...

  14. Microtubule self-organisation by reaction-diffusion processes causes collective transport and organisation of cellular particles

    Directory of Open Access Journals (Sweden)

    Demongeot Jacques

    2004-06-01

    Full Text Available Abstract Background The transport of intra-cellular particles by microtubules is a major biological function. Under appropriate in vitro conditions, microtubule preparations behave as a 'complex' system and show 'emergent' phenomena. In particular, they form dissipative structures that self-organise over macroscopic distances by a combination of reaction and diffusion. Results Here, we show that self-organisation also gives rise to a collective transport of colloidal particles along a specific direction. Particles, such as polystyrene beads, chromosomes, nuclei, and vesicles are carried at speeds of several microns per minute. The process also results in the macroscopic self-organisation of these particles. After self-organisation is completed, they show the same pattern of organisation as the microtubules. Numerical simulations of a population of growing and shrinking microtubules, incorporating experimentally realistic reaction dynamics, predict self-organisation. They forecast that during self-organisation, macroscopic parallel arrays of oriented microtubules form which cross the reaction space in successive waves. Such travelling waves are capable of transporting colloidal particles. The fact that in the simulations, the aligned arrays move along the same direction and at the same speed as the particles move, suggest that this process forms the underlying mechanism for the observed transport properties. Conclusions This process constitutes a novel physical chemical mechanism by which chemical energy is converted into collective transport of colloidal particles along a given direction. Self-organisation of this type provides a new mechanism by which intra cellular particles such as chromosomes and vesicles can be displaced and simultaneously organised by microtubules. It is plausible that processes of this type occur in vivo.

  15. Microtubule self-organisation by reaction-diffusion processes causes collective transport and organisation of cellular particles

    Science.gov (United States)

    Glade, Nicolas; Demongeot, Jacques; Tabony, James

    2004-01-01

    Background The transport of intra-cellular particles by microtubules is a major biological function. Under appropriate in vitro conditions, microtubule preparations behave as a 'complex' system and show 'emergent' phenomena. In particular, they form dissipative structures that self-organise over macroscopic distances by a combination of reaction and diffusion. Results Here, we show that self-organisation also gives rise to a collective transport of colloidal particles along a specific direction. Particles, such as polystyrene beads, chromosomes, nuclei, and vesicles are carried at speeds of several microns per minute. The process also results in the macroscopic self-organisation of these particles. After self-organisation is completed, they show the same pattern of organisation as the microtubules. Numerical simulations of a population of growing and shrinking microtubules, incorporating experimentally realistic reaction dynamics, predict self-organisation. They forecast that during self-organisation, macroscopic parallel arrays of oriented microtubules form which cross the reaction space in successive waves. Such travelling waves are capable of transporting colloidal particles. The fact that in the simulations, the aligned arrays move along the same direction and at the same speed as the particles move, suggest that this process forms the underlying mechanism for the observed transport properties. Conclusions This process constitutes a novel physical chemical mechanism by which chemical energy is converted into collective transport of colloidal particles along a given direction. Self-organisation of this type provides a new mechanism by which intra cellular particles such as chromosomes and vesicles can be displaced and simultaneously organised by microtubules. It is plausible that processes of this type occur in vivo. PMID:15176973

  16. Brain regions implicated in inhibitory control and appetite regulation are activated in response to food portion size and energy density in children.

    Science.gov (United States)

    English, L K; Fearnbach, S N; Lasschuijt, M; Schlegel, A; Anderson, K; Harris, S; Wilson, S J; Fisher, J O; Savage, J S; Rolls, B J; Keller, K L

    2016-10-01

    Large portions of energy-dense foods drive energy intake but the brain mechanisms underlying this effect are not clear. Our main objective was to investigate brain function in response to food images varied by portion size (PS) and energy density (ED) in children using functional magnetic resonance imaging (fMRI). Blood-oxygen-level-dependent (BOLD) fMRI was completed in 36 children (ages 7-10 years) after a 2-h fast while viewing food images at two levels of PS (Large PS, Small PS) and two levels of ED (High ED, Low ED). Children rated perceived fullness pre- and post-fMRI, as well as liking of images on visual analog scales post-fMRI. Anthropometrics were completed 4 weeks before the fMRI. Large PS vs Small PS and High ED vs Low ED were compared with region-of-interest analyses using Brain Voyager v 2.8. Region-of-interest analyses revealed that activation in the right inferior frontal gyrus (P=0.03) was greater for Large PS vs Small PS. Activation was reduced for High ED vs Low ED in the left hypothalamus (P=0.03). Main effects were no longer significant after adjustment for pre-fMRI fullness and liking ratings (PS, P=0.92; ED, P=0.58). This is the first fMRI study to report increased activation to large portions in a brain region that is involved in inhibitory control. These findings may contribute to understanding why some children overeat when presented with large portions of palatable food.

  17. The Ndc80 internal loop is required for recruitment of the Ska complex to establish end-on microtubule attachment to kinetochores

    DEFF Research Database (Denmark)

    Zhang, Gang; Kelstrup, Christian D; Hu, Xiao-Wen

    2012-01-01

    The Ndc80 complex establishes end-on attachment of kinetochores to microtubules essential for chromosome segregation. The Ndc80 subunit is characterized by an N-terminal region, that binds directly to microtubules, and a long coiled-coil region that interacts with Nuf2. A loop region in Ndc80 tha...... chromosome segregation through the recruitment of specific proteins to the kinetochore....

  18. An antitubulin agent BCFMT inhibits proliferation of cancer cells and induces cell death by inhibiting microtubule dynamics.

    Directory of Open Access Journals (Sweden)

    Ankit Rai

    Full Text Available Using cell based screening assay, we identified a novel anti-tubulin agent (Z-5-((5-(4-bromo-3-chlorophenylfuran-2-ylmethylene-2-thioxothiazolidin-4-one (BCFMT that inhibited proliferation of human cervical carcinoma (HeLa (IC(50, 7.2 ± 1.8 µM, human breast adenocarcinoma (MCF-7 (IC(50, 10.0 ± 0.5 µM, highly metastatic breast adenocarcinoma (MDA-MB-231 (IC(50, 6.0 ± 1 µM, cisplatin-resistant human ovarian carcinoma (A2780-cis (IC(50, 5.8 ± 0.3 µM and multi-drug resistant mouse mammary tumor (EMT6/AR1 (IC(50, 6.5 ± 1 µM cells. Using several complimentary strategies, BCFMT was found to inhibit cancer cell proliferation at G2/M phase of the cell cycle apparently by targeting microtubules. In addition, BCFMT strongly suppressed the dynamics of individual microtubules in live MCF-7 cells. At its half maximal proliferation inhibitory concentration (10 µM, BCFMT reduced the rates of growing and shortening phases of microtubules in MCF-7 cells by 37 and 40%, respectively. Further, it increased the time microtubules spent in the pause (neither growing nor shortening detectably state by 135% and reduced the dynamicity (dimer exchange per unit time of microtubules by 70%. In vitro, BCFMT bound to tubulin with a dissociation constant of 8.3 ± 1.8 µM, inhibited tubulin assembly and suppressed GTPase activity of microtubules. BCFMT competitively inhibited the binding of BODIPY FL-vinblastine to tubulin with an inhibitory concentration (K(i of 5.2 ± 1.5 µM suggesting that it binds to tubulin at the vinblastine site. In cultured cells, BCFMT-treatment depolymerized interphase microtubules, perturbed the spindle organization and accumulated checkpoint proteins (BubR1 and Mad2 at the kinetochores. BCFMT-treated MCF-7 cells showed enhanced nuclear accumulation of p53 and its downstream p21, which consequently activated apoptosis in these cells. The results suggested that BCFMT inhibits proliferation of several types of cancer cells including drug

  19. Duplication in the microtubule-actin cross-linking factor 1 gene causes a novel neuromuscular condition

    DEFF Research Database (Denmark)

    Jørgensen, Louise H; Mosbech, Mai-Britt; Færgeman, Nils J

    2014-01-01

    Spectrins and plakins are important communicators linking cytoskeletal components to each other and to cellular junctions. Microtubule-actin cross-linking factor 1 (MACF1) belongs to the spectraplakin family and is involved in control of microtubule dynamics. Complete knock out of MACF1 in mice...... muscles and diminished motor skills, with heterogeneous presentation among the affected family members. To corroborate these findings we used RNA interference to knock down the VAB-10 locus containing the MACF1 homologue in C. elegans, and we could show that this also causes movement disturbances...

  20. Functional regulation of Q by microRNA172 and transcriptional co-repressor TOPLESS in controlling bread wheat spikelet density.

    Science.gov (United States)

    Liu, Pan; Liu, Jie; Dong, Huixue; Sun, Jiaqiang

    2018-02-01

    Bread wheat (Triticum aestivum) spike architecture is an important agronomic trait. The Q gene plays a key role in the domestication of bread wheat spike architecture. However, the regulatory mechanisms of Q expression and transcriptional activity remain largely unknown. In this study, we show that overexpression of bread wheat tae-miR172 caused a speltoid-like spike phenotype, reminiscent of that in wheat plants with the q gene. The reduction in Q transcript levels in the tae-miR172 overexpression transgenic bread wheat lines suggests that the Q expression can be suppressed by tae-miR172 in bread wheat. Indeed, our RACE analyses confirmed that the Q mRNA is targeted by tae-miR172 for cleavage. According to our analyses, the Q protein is localized in nucleus and confers transcriptional repression activity. Meanwhile, the Q protein could physically interact with the bread wheat transcriptional co-repressor TOPLESS (TaTPL). Specifically, the N-terminal ethylene-responsive element binding factor-associated amphiphilic repression (EAR) (LDLNVE) motif but not the C-terminal EAR (LDLDLR) motif of Q protein mediates its interaction with the CTLH motif of TaTPL. Moreover, we show that the N-terminal EAR motif of Q protein is also essentially required for the transcriptional repression activity of Q protein. Taken together, we reveal the functional regulation of Q protein by tae-miR172 and transcriptional co-repressor TaTPL in controlling the bread wheat spike architecture. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  1. Microtubule-dependent association of AKAP350A and CCAR1 with RNA stress granules

    International Nuclear Information System (INIS)

    Kolobova, Elena; Efimov, Andrey; Kaverina, Irina; Rishi, Arun K.; Schrader, John W.; Ham, Amy-Joan; Larocca, M. Cecilia; Goldenring, James R.

    2009-01-01

    Recent investigations have highlighted the importance of subcellular localization of mRNAs to cell function. While AKAP350A, a multifunctional scaffolding protein, localizes to the Golgi apparatus and centrosomes, we have now identified a cytosolic pool of AKAP350A. Analysis of AKAP350A scaffolded complexes revealed two novel interacting proteins, CCAR1 and caprin-1. CCAR1, caprin-1 and AKAP350A along with G3BP, a stress granule marker, relocate to RNA stress granules after arsenite treatment. Stress also caused loss of AKAP350 from the Golgi and fragmentation of the Golgi apparatus. Disruption of microtubules with nocodazole altered stress granule formation and changed their morphology by preventing fusion of stress granules. In the presence of nocodazole, arsenite induced smaller granules with the vast majority of AKAP350A and CCAR1 separated from G3BP-containing granules. Similar to nocodazole treatment, reduction of AKAP350A or CCAR1 expression also altered the size and number of G3BP-containing stress granules induced by arsenite treatment. A limited set of 69 mRNA transcripts was immunoisolated with AKAP350A even in the absence of stress, suggesting the association of AKAP350A with mRNA transcripts. These results provide the first evidence for the microtubule dependent association of AKAP350A and CCAR1 with RNA stress granules

  2. Statistical mechanics provides novel insights into microtubule stability and mechanism of shrinkage.

    Directory of Open Access Journals (Sweden)

    Ishutesh Jain

    2015-02-01

    Full Text Available Microtubules are nano-machines that grow and shrink stochastically, making use of the coupling between chemical kinetics and mechanics of its constituent protofilaments (PFs. We investigate the stability and shrinkage of microtubules taking into account inter-protofilament interactions and bending interactions of intrinsically curved PFs. Computing the free energy as a function of PF tip position, we show that the competition between curvature energy, inter-PF interaction energy and entropy leads to a rich landscape with a series of minima that repeat over a length-scale determined by the intrinsic curvature. Computing Langevin dynamics of the tip through the landscape and accounting for depolymerization, we calculate the average unzippering and shrinkage velocities of GDP protofilaments and compare them with the experimentally known results. Our analysis predicts that the strength of the inter-PF interaction (E(s(m has to be comparable to the strength of the curvature energy (E(b(m such that E(s(m - E(b(m ≈ 1kBT, and questions the prevalent notion that unzippering results from the domination of bending energy of curved GDP PFs. Our work demonstrates how the shape of the free energy landscape is crucial in explaining the mechanism of MT shrinkage where the unzippered PFs will fluctuate in a set of partially peeled off states and subunit dissociation will reduce the length.

  3. Synthesis and biological evaluation of indolyl-pyridinyl-propenones having either methuosis or microtubule disruption activity.

    Science.gov (United States)

    Trabbic, Christopher J; Overmeyer, Jean H; Alexander, Evan M; Crissman, Emily J; Kvale, Heather M; Smith, Marcie A; Erhardt, Paul W; Maltese, William A

    2015-03-12

    Methuosis is a form of nonapoptotic cell death characterized by an accumulation of macropinosome-derived vacuoles with eventual loss of membrane integrity. Small molecules inducing methuosis could offer significant advantages compared to more traditional anticancer drug therapies that typically rely on apoptosis. Herein we further define the effects of chemical substitutions at the 2- and 5-indolyl positions on our lead compound 3-(5-methoxy-2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propene-1-one (MOMIPP). We have identified a number of compounds that induce methuosis at similar potencies, including an interesting analogue having a hydroxypropyl substituent at the 2-position. In addition, we have discovered that certain substitutions on the 2-indolyl position redirect the mode of cytotoxicity from methuosis to microtubule disruption. This switch in activity is associated with an increase in potency as large as 2 orders of magnitude. These compounds appear to represent a new class of potent microtubule-active anticancer agents.

  4. Disruption of Microtubules Post-Virus Entry Enhances Adeno-Associated Virus Vector Transduction

    Science.gov (United States)

    Xiao, Ping-Jie; Mitchell, Angela M.; Huang, Lu; Li, Chengwen; Samulski, R. Jude

    2016-01-01

    Perinuclear retention of viral particles is a poorly understood phenomenon observed during many virus infections. In this study, we investigated whether perinuclear accumulation acts as a barrier to limit recombinant adeno-associated virus (rAAV) transduction. After nocodazole treatment to disrupt microtubules at microtubule-organization center (MT-MTOC) after virus entry, we observed higher rAAV transduction. To elucidate the role of MT-MTOC in rAAV infection and study its underlying mechanisms, we demonstrated that rAAV's perinuclear localization was retained by MT-MTOC with fluorescent analysis, and enhanced rAAV transduction from MT-MTOC disruption was dependent on the rAAV capsid's nuclear import signals. Interestingly, after knocking down RhoA or inhibiting its downstream effectors (ROCK and Actin), MT-MTOC disruption failed to increase rAAV transduction or nuclear entry. These data suggest that enhancement of rAAV transduction is the result of increased trafficking to the nucleus via the RhoA-ROCK-Actin pathway. Ten-fold higher rAAV transduction was also observed by disrupting MT-MTOC in brain, liver, and tumor in vivo. In summary, this study indicates that virus perinuclear accumulation at MT-MTOC is a barrier-limiting parameter for effective rAAV transduction and defines a novel defense mechanism by which host cells restrain viral invasion. PMID:26942476

  5. Development of other microtubule-stabilizer families: the epothilones and their derivatives.

    Science.gov (United States)

    Brogdon, Cynthia F; Lee, Francis Y; Canetta, Renzo M

    2014-05-01

    Chemotherapy is the mainstay of treatment for numerous cancer types, but resistance to chemotherapy remains a major clinical issue and is one of the driving influences underlying the development of new anticancer medications. One of the most important classes of chemotherapy agents is the taxanes, which target the cytoskeleton and spindle apparatus of tumor cells by binding to the microtubules, thereby disrupting key cellular mechanisms, including mitosis. Taxane resistance, however, limits treatment options and creates a major challenge for clinicians. Ongoing research has identified several newer classes of microtubule-targeting chemotherapies that may retain activity despite clinical resistance to taxanes. Among these classes, the epothilones have been studied most extensively in the clinical setting. Like taxanes, epothilones stabilize microtubulin turnover, and they have properties favoring their development as anticancer agents. The most clinically advanced epothilone analog is ixabepilone, which is currently the only approved epothilone derivative. Ixabepilone is indicated for the treatment of metastatic or locally advanced breast cancer in combination with capecitabine after failure of an anthracycline and a taxane, or as monotherapy after failure of an anthracycline, a taxane, and capecitabine. In phase II and III trials, ixabepilone showed efficacy in several patient subgroups and in various stages of breast cancer. Common adverse reactions include peripheral sensory neuropathy and asthenia. This paper will discuss the preclinical and clinical development of epothilones and their derivatives across a variety of cancer types.

  6. A 31-residue peptide induces aggregation of tau's microtubule-binding region in cells

    Science.gov (United States)

    Stöhr, Jan; Wu, Haifan; Nick, Mimi; Wu, Yibing; Bhate, Manasi; Condello, Carlo; Johnson, Noah; Rodgers, Jeffrey; Lemmin, Thomas; Acharya, Srabasti; Becker, Julia; Robinson, Kathleen; Kelly, Mark J. S.; Gai, Feng; Stubbs, Gerald; Prusiner, Stanley B.; Degrado, William F.

    2017-09-01

    The self-propagation of misfolded conformations of tau underlies neurodegenerative diseases, including Alzheimer's. There is considerable interest in discovering the minimal sequence and active conformational nucleus that defines this self-propagating event. The microtubule-binding region, spanning residues 244-372, reproduces much of the aggregation behaviour of tau in cells and animal models. Further dissection of the amyloid-forming region to a hexapeptide from the third microtubule-binding repeat resulted in a peptide that rapidly forms fibrils in vitro. We show that this peptide lacks the ability to seed aggregation of tau244-372 in cells. However, as the hexapeptide is gradually extended to 31 residues, the peptides aggregate more slowly and gain potent activity to induce aggregation of tau244-372 in cells. X-ray fibre diffraction, hydrogen-deuterium exchange and solid-state NMR studies map the beta-forming region to a 25-residue sequence. Thus, the nucleus for self-propagating aggregation of tau244-372 in cells is packaged in a remarkably small peptide.

  7. A novel spiroindoline targets cell cycle and migration via modulation of microtubule cytoskeleton.

    Science.gov (United States)

    Kumar, Naveen; Hati, Santanu; Munshi, Parthapratim; Sen, Subhabrata; Sehrawat, Seema; Singh, Shailja

    2017-05-01

    Natural product-inspired libraries of molecules with diverse architectures have evolved as one of the most useful tools for discovering lead molecules for drug discovery. In comparison to conventional combinatorial libraries, these molecules have been inferred to perform better in phenotypic screening against complicated targets. Diversity-oriented synthesis (DOS) is a forward directional strategy to access such multifaceted library of molecules. From a successful DOS campaign of a natural product-inspired library, recently a small molecule with spiroindoline motif was identified as a potent anti-breast cancer compound. Herein we report the subcellular studies performed for this molecule on breast cancer cells. Our investigation revealed that it repositions microtubule cytoskeleton and displaces AKAP9 located at the microtubule organization centre. DNA ladder assay and cell cycle experiments further established the molecule as an apoptotic agent. This work further substantiated the amalgamation of DOS-phenotypic screening-sub-cellular studies as a consolidated blueprint for the discovery of potential pharmaceutical drug candidates.

  8. Phenotypic dissection of bone mineral density reveals skeletal site specificity and facilitates the identification of novel loci in the genetic regulation of bone mass attainment.

    Directory of Open Access Journals (Sweden)

    John P Kemp

    2014-06-01

    Full Text Available Heritability of bone mineral density (BMD varies across skeletal sites, reflecting different relative contributions of genetic and environmental influences. To quantify the degree to which common genetic variants tag and environmental factors influence BMD, at different sites, we estimated the genetic (rg and residual (re correlations between BMD measured at the upper limbs (UL-BMD, lower limbs (LL-BMD and skull (SK-BMD, using total-body DXA scans of ∼ 4,890 participants recruited by the Avon Longitudinal Study of Parents and their Children (ALSPAC. Point estimates of rg indicated that appendicular sites have a greater proportion of shared genetic architecture (LL-/UL-BMD rg = 0.78 between them, than with the skull (UL-/SK-BMD rg = 0.58 and LL-/SK-BMD rg = 0.43. Likewise, the residual correlation between BMD at appendicular sites (r(e = 0.55 was higher than the residual correlation between SK-BMD and BMD at appendicular sites (r(e = 0.20-0.24. To explore the basis for the observed differences in rg and re, genome-wide association meta-analyses were performed (n ∼ 9,395, combining data from ALSPAC and the Generation R Study identifying 15 independent signals from 13 loci associated at genome-wide significant level across different skeletal regions. Results suggested that previously identified BMD-associated variants may exert site-specific effects (i.e. differ in the strength of their association and magnitude of effect across different skeletal sites. In particular, variants at CPED1 exerted a larger influence on SK-BMD and UL-BMD when compared to LL-BMD (P = 2.01 × 10(-37, whilst variants at WNT16 influenced UL-BMD to a greater degree when compared to SK- and LL-BMD (P = 2.31 × 10(-14. In addition, we report a novel association between RIN3 (previously associated with Paget's disease and LL-BMD (rs754388: β = 0.13, SE = 0.02, P = 1.4 × 10(-10. Our results suggest that BMD at different skeletal sites is under a mixture of shared and

  9. Phenotypic dissection of bone mineral density reveals skeletal site specificity and facilitates the identification of novel loci in the genetic regulation of bone mass attainment.

    Science.gov (United States)

    Kemp, John P; Medina-Gomez, Carolina; Estrada, Karol; St Pourcain, Beate; Heppe, Denise H M; Warrington, Nicole M; Oei, Ling; Ring, Susan M; Kruithof, Claudia J; Timpson, Nicholas J; Wolber, Lisa E; Reppe, Sjur; Gautvik, Kaare; Grundberg, Elin; Ge, Bing; van der Eerden, Bram; van de Peppel, Jeroen; Hibbs, Matthew A; Ackert-Bicknell, Cheryl L; Choi, Kwangbom; Koller, Daniel L; Econs, Michael J; Williams, Frances M K; Foroud, Tatiana; Zillikens, M Carola; Ohlsson, Claes; Hofman, Albert; Uitterlinden, André G; Davey Smith, George; Jaddoe, Vincent W V; Tobias, Jonathan H; Rivadeneira, Fernando; Evans, David M

    2014-06-01

    Heritability of bone mineral density (BMD) varies across skeletal sites, reflecting different relative contributions of genetic and environmental influences. To quantify the degree to which common genetic variants tag and environmental factors influence BMD, at different sites, we estimated the genetic (rg) and residual (re) correlations between BMD measured at the upper limbs (UL-BMD), lower limbs (LL-BMD) and skull (SK-BMD), using total-body DXA scans of ∼ 4,890 participants recruited by the Avon Longitudinal Study of Parents and their Children (ALSPAC). Point estimates of rg indicated that appendicular sites have a greater proportion of shared genetic architecture (LL-/UL-BMD rg = 0.78) between them, than with the skull (UL-/SK-BMD rg = 0.58 and LL-/SK-BMD rg = 0.43). Likewise, the residual correlation between BMD at appendicular sites (r(e) = 0.55) was higher than the residual correlation between SK-BMD and BMD at appendicular sites (r(e) = 0.20-0.24). To explore the basis for the observed differences in rg and re, genome-wide association meta-analyses were performed (n ∼ 9,395), combining data from ALSPAC and the Generation R Study identifying 15 independent signals from 13 loci associated at genome-wide significant level across different skeletal regions. Results suggested that previously identified BMD-associated variants may exert site-specific effects (i.e. differ in the strength of their association and magnitude of effect across different skeletal sites). In particular, variants at CPED1 exerted a larger influence on SK-BMD and UL-BMD when compared to LL-BMD (P = 2.01 × 10(-37)), whilst variants at WNT16 influenced UL-BMD to a greater degree when compared to SK- and LL-BMD (P = 2.31 × 10(-14)). In addition, we report a novel association between RIN3 (previously associated with Paget's disease) and LL-BMD (rs754388: β = 0.13, SE = 0.02, P = 1.4 × 10(-10)). Our results suggest that BMD at different skeletal sites is under a mixture of shared and

  10. Central nervous system neuropeptide Y regulates mediators of hepatic phospholipid remodeling and very low-density lipoprotein triglyceride secretion via sympathetic innervation

    Science.gov (United States)

    Rojas, Jennifer M.; Bruinstroop, Eveline; Printz, Richard L.; Alijagic-Boers, Aldijana; Foppen, Ewout; Turney, Maxine K.; George, Leena; Beck-Sickinger, Annette G.; Kalsbeek, Andries; Niswender, Kevin D.

    2015-01-01

    Objective Elevated very low-density lipoprotein (VLDL)-triglyceride (TG) secretion from the liver contributes to an atherogenic dyslipidemia that is associated with obesity, diabetes and the metabolic syndrome. Numerous models of obesity and diabetes are characterized by increased central nervous system (CNS) neuropeptide Y (NPY); in fact, a single intracerebroventricular (icv) administration of NPY in lean fasted rats elevates hepatic VLDL-TG secretion and does so, in large part, via signaling through the CNS NPY Y1 receptor. Thus, our overarching hypothesis is that elevated CNS NPY action contributes to dyslipidemia by activating central circuits that modulate liver lipid metabolism. Methods Chow-fed Zucker fatty (ZF) rats were pair-fed by matching their caloric intake to that of lean controls and effects on body weight, plasma TG, and liver content of TG and phospholipid (PL) were compared to ad-libitum (ad-lib) fed ZF rats. Additionally, lean 4-h fasted rats with intact or disrupted hepatic sympathetic innervation were treated with icv NPY or NPY Y1 receptor agonist to identify novel hepatic mechanisms by which NPY promotes VLDL particle maturation and secretion. Results Manipulation of plasma TG levels in obese ZF rats, through pair-feeding had no effect on liver TG content; however, hepatic PL content was substantially reduced and was tightly correlated with plasma TG levels. Treatment with icv NPY or a selective NPY Y1 receptor agonist in lean fasted rats robustly activated key hepatic regulatory proteins, stearoyl-CoA desaturase-1 (SCD-1), ADP-ribosylation factor-1 (ARF-1), and lipin-1, known to be involved in remodeling liver PL into TG for VLDL maturation and secretion. Lastly, we show that the effects of CNS NPY on key liporegulatory proteins are attenuated by hepatic sympathetic denervation. Conclusions These data support a model in which CNS NPY modulate