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Sample records for regulating microtubule structure

  1. 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.

  2. Structural insights into microtubule doublet interactions inaxonemes

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    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.

  3. 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.

  4. 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.

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

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    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.

  6. 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)

  7. 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.

  8. 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.

  9. 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

  10. 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.

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

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    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.

  12. 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.

  13. 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

  14. 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.

  15. 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

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

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    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.

  17. 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.

  18. 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.

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

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    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

  20. 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

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

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    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  3. 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

  4. 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

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

  8. 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.

  9. 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.

  10. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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

  17. 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.

  18. 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.

  19. 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

  20. 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...

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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...

  6. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  8. 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

  9. 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.

  10. 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...

  11. 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.

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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.

  17. 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.

  18. 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.

  19. 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

  20. 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.

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

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    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. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. 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

  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. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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).

  20. Structure of Dynamic, Taxol-Stabilized, and GMPPCP-Stabilized Microtubule.

    Science.gov (United States)

    Ginsburg, Avi; Shemesh, Asaf; Millgram, Abigail; Dharan, Raviv; Levi-Kalisman, Yael; Ringel, Israel; Raviv, Uri

    2017-09-14

    Microtubule (MT) is made of αβ-tubulin heterodimers that dynamically assemble into a hollow nanotube composed of straight protofilaments. MT dynamics is facilitated by hydrolysis of guanosine-5'-triphosphate (GTP) and can be inhibited by either anticancer agents like taxol or the nonhydrolyzable GTP analogues like GMPPCP. Using high-resolution synchrotron X-ray scattering, we have measured and analyzed the scattering curves from solutions of dynamic MT (in other words, in the presence of excess GTP and free of dynamic-inhibiting agents) and examined the effect of two MT stabilizers: taxol and GMPPCP. Previously, we have analyzed the structure of dynamic MT by docking the atomic model of tubulin dimer onto a 3-start left handed helical lattice, derived from the PDB ID 3J6F . 3J6F corresponds to a MT with 14 protofilaments. In this paper, we took into account the possibility of having MT structures containing between 12 and 15 protofilaments. MTs with 12 protofilaments were never observed. We determined the radii, the pitch, and the distribution of protofilament number that best fit the scattering data from dynamic MT or stabilized MT by taxol or GMPPCP. We found that the protofilament number distribution shifted when the MT was stabilized. Taxol increased the mass fraction of MT with 13 protofilaments and decreased the mass fraction of MT with 14 protofilaments. GMPPCP reduced the mass fraction of MT with 15 protofilaments and increased the mass fraction of MT with 14 protofilaments. The pitch, however, remained unchanged regardless of whether the MT was dynamic or stabilized. Higher tubulin concentrations increased the fraction of dynamic MT with 14 protofilaments.

  1. 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

  2. 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

  3. 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

  4. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  6. 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".

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. Structural analysis of the role of TPX2 in branching microtubule nucleation

    Science.gov (United States)

    Thawani, Akanksha

    2017-01-01

    The mitotic spindle consists of microtubules (MTs), which are nucleated by the γ-tubulin ring complex (γ-TuRC). How the γ-TuRC gets activated at the right time and location remains elusive. Recently, it was uncovered that MTs nucleate from preexisting MTs within the mitotic spindle, which requires the protein TPX2, but the mechanism basis for TPX2 action is unknown. Here, we investigate the role of TPX2 in branching MT nucleation. We establish the domain organization of Xenopus laevis TPX2 and define the minimal TPX2 version that stimulates branching MT nucleation, which we find is unrelated to TPX2’s ability to nucleate MTs in vitro. Several domains of TPX2 contribute to its MT-binding and bundling activities. However, the property necessary for TPX2 to induce branching MT nucleation is contained within newly identified γ-TuRC nucleation activator motifs. Separation-of-function mutations leave the binding of TPX2 to γ-TuRC intact, whereas branching MT nucleation is abolished, suggesting that TPX2 may activate γ-TuRC to promote branching MT nucleation. PMID:28264915

  12. 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.

  13. 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

  14. 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.

  15. Determining the structure-mechanics relationships of dense microtubule networks with confocal microscopy and magnetic tweezers-based microrheology.

    Science.gov (United States)

    Yang, Yali; Valentine, Megan T

    2013-01-01

    The microtubule (MT) cytoskeleton is essential in maintaining the shape, strength, and organization of cells. Its spatiotemporal organization is fundamental for numerous dynamic biological processes, and mechanical stress within the MT cytoskeleton provides an important signaling mechanism in mitosis and neural development. This raises important questions about the relationships between structure and mechanics in complex MT structures. In vitro, reconstituted cytoskeletal networks provide a minimal model of cell mechanics while also providing a testing ground for the fundamental polymer physics of stiff polymer gels. Here, we describe our development and implementation of a broad tool kit to study structure-mechanics relationships in reconstituted MT networks, including protocols for the assembly of entangled and cross-linked MT networks, fluorescence imaging, microstructure characterization, construction and calibration of magnetic tweezers devices, and mechanical data collection and analysis. In particular, we present the design and assembly of three neodymium iron boron (NdFeB)-based magnetic tweezers devices optimized for use with MT networks: (1) high-force magnetic tweezers devices that enable the application of nano-Newton forces and possible meso- to macroscale materials characterization; (2) ring-shaped NdFeB-based magnetic tweezers devices that enable oscillatory microrheology measurements; and (3) portable magnetic tweezers devices that enable direct visualization of microscale deformation in soft materials under applied force. Copyright © 2013 Elsevier Inc. All rights reserved.

  16. 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.

  17. 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.

  18. 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...

  19. The structure of tubulin-binding cofactor A from Leishmania major infers a mode of association during the early stages of microtubule assembly

    Energy Technology Data Exchange (ETDEWEB)

    Barrack, Keri L.; Fyfe, Paul K.; Hunter, William N., E-mail: w.n.hunter@dundee.ac.uk [University of Dundee, Dow Street, Dundee DD1 5EH, Scotland (United Kingdom)

    2015-04-21

    The structure of a tubulin-binding cofactor from L. major is reported and compared with yeast, plant and human orthologues. Tubulin-binding cofactor A (TBCA) participates in microtubule formation, a key process in eukaryotic biology to create the cytoskeleton. There is little information on how TBCA might interact with β-tubulin en route to microtubule biogenesis. To address this, the protozoan Leishmania major was targeted as a model system. The crystal structure of TBCA and comparisons with three orthologous proteins are presented. The presence of conserved features infers that electrostatic interactions that are likely to involve the C-terminal tail of β-tubulin are key to association. This study provides a reagent and template to support further work in this area.

  20. 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.

  1. 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."

  2. 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.

  3. 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.

  4. BICD2, dynactin, and LIS1 cooperate in regulating dynein recruitment to cellular structures

    Science.gov (United States)

    Splinter, Daniël; Razafsky, David S.; Schlager, Max A.; Serra-Marques, Andrea; Grigoriev, Ilya; Demmers, Jeroen; Keijzer, Nanda; Jiang, Kai; Poser, Ina; Hyman, Anthony A.; Hoogenraad, Casper C.; King, Stephen J.; Akhmanova, Anna

    2012-01-01

    Cytoplasmic dynein is the major microtubule minus-end–directed cellular motor. Most dynein activities require dynactin, but the mechanisms regulating cargo-dependent dynein–dynactin interaction are poorly understood. In this study, we focus on dynein–dynactin recruitment to cargo by the conserved motor adaptor Bicaudal D2 (BICD2). We show that dynein and dynactin depend on each other for BICD2-mediated targeting to cargo and that BICD2 N-terminus (BICD2-N) strongly promotes stable interaction between dynein and dynactin both in vitro and in vivo. Direct visualization of dynein in live cells indicates that by itself the triple BICD2-N–dynein–dynactin complex is unable to interact with either cargo or microtubules. However, tethering of BICD2-N to different membranes promotes their microtubule minus-end–directed motility. We further show that LIS1 is required for dynein-mediated transport induced by membrane tethering of BICD2-N and that LIS1 contributes to dynein accumulation at microtubule plus ends and BICD2-positive cellular structures. Our results demonstrate that dynein recruitment to cargo requires concerted action of multiple dynein cofactors. PMID:22956769

  5. 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.

  6. Using cell structures to develop functional nanomaterials and nanostructures--case studies of actin filaments and microtubules.

    Science.gov (United States)

    Wu, Kevin Chia-Wen; Yang, Chung-Yao; Cheng, Chao-Min

    2014-04-25

    This article is based on the continued development of biologically relevant elements (i.e., actin filaments and microtubules in living cells) as building blocks to create functional nanomaterials and nanostructures that can then be used to manufacture nature-inspired small-scale devices or systems. Here, we summarize current progress in the field and focus specifically on processes characterized by (1) robustness and ease of use, (2) inexpensiveness, and (3) potential expandability to mass production. This article, we believe, will provide scientists and engineers with a more comprehensive understanding of how to mine biological materials and natural design features to construct functional materials and devices.

  7. Scoulerine affects microtubule structure, inhibits proliferation, arrests cell cycle and thus culminates in the apoptotic death of cancer cells.

    Science.gov (United States)

    Habartova, Klara; Havelek, Radim; Seifrtova, Martina; Kralovec, Karel; Cahlikova, Lucie; Chlebek, Jakub; Cermakova, Eva; Mazankova, Nadezda; Marikova, Jana; Kunes, Jiri; Novakova, Lucie; Rezacova, Martina

    2018-03-19

    Scoulerine is an isoquinoline alkaloid, which indicated promising suppression of cancer cells growth. However, the mode of action (MOA) remained unclear. Cytotoxic and antiproliferative properties were determined in this study. Scoulerine reduces the mitochondrial dehydrogenases activity of the evaluated leukemic cells with IC 50 values ranging from 2.7 to 6.5 µM. The xCELLigence system revealed that scoulerine exerted potent antiproliferative activity in lung, ovarian and breast carcinoma cell lines. Jurkat and MOLT-4 leukemic cells treated with scoulerine were decreased in proliferation and viability. Scoulerine acted to inhibit proliferation through inducing G2 or M-phase cell cycle arrest, which correlates well with the observed breakdown of the microtubule network, increased Chk1 Ser345, Chk2 Thr68 and mitotic H3 Ser10 phosphorylation. Scoulerine was able to activate apoptosis, as determined by p53 upregulation, increase caspase activity, Annexin V and TUNEL labeling. Results highlight the potent antiproliferative and proapoptotic function of scoulerine in cancer cells caused by its ability to interfere with the microtubule elements of the cytoskeleton, checkpoint kinase signaling and p53 proteins. This is the first study of the mechanism of scoulerine at cellular and molecular level. Scoulerine is a potent antimitotic compound and that it merits further investigation as an anticancer drug.

  8. 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.

  9. 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

  10. Taxol crystals can masquerade as stabilized microtubules.

    Directory of Open Access Journals (Sweden)

    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.

  11. 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

  12. 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...

  13. 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

  14. Microtubules: A network for solitary waves

    Directory of Open Access Journals (Sweden)

    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

  15. 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.

  16. 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

  17. 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).

  18. 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

  19. 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.

  20. 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.

  1. 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.

  2. 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....

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. SAS-4 is recruited to a dynamic structure in newly forming centrioles that is stabilized by the gamma-tubulin-mediated addition of centriolar microtubules.

    Science.gov (United States)

    Dammermann, Alexander; Maddox, Paul S; Desai, Arshad; Oegema, Karen

    2008-02-25

    Centrioles are surrounded by pericentriolar material (PCM), which is proposed to promote new centriole assembly by concentrating gamma-tubulin. Here, we quantitatively monitor new centriole assembly in living Caenorhabditis elegans embryos, focusing on the conserved components SAS-4 and SAS-6. We show that SAS-4 and SAS-6 are coordinately recruited to the site of new centriole assembly and reach their maximum levels during S phase. Centriolar SAS-6 is subsequently reduced by a mechanism intrinsic to the early assembly pathway that does not require progression into mitosis. Centriolar SAS-4 remains in dynamic equilibrium with the cytoplasmic pool until late prophase, when it is stably incorporated in a step that requires gamma-tubulin and microtubule assembly. These results indicate that gamma-tubulin in the PCM stabilizes the nascent daughter centriole by promoting microtubule addition to its outer wall. Such a mechanism may help restrict new centriole assembly to the vicinity of preexisting parent centrioles that recruit PCM.

  14. 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.

  15. 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.

  16. 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.

  17. 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...

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. 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.

  4. BICD2, dynactin, and LIS1 cooperate in regulating dynein recruitment to cellular structures

    NARCIS (Netherlands)

    D. Splinter (Daniël); D.S. Razafsky (David); M.A. Schlager (Max); A. Serra-Marques (Andrea); I. Grigoriev (Ilya); J.A.A. Demmers (Jeroen); N. Keijzer (Nanda); K. Jiang (Kai); S. Poser; A. Hyman (Anthony); C.C. Hoogenraad (Casper); S.J. King (Stephen); A.S. Akhmanova (Anna)

    2012-01-01

    textabstractCytoplasmic dynein is the major microtubule minus-end-directed cellular motor. Most dynein activities require dynactin, but the mechanisms regulating cargo-dependent dynein-dynactin interaction are poorly understood. In this study, we focus on dynein-dynactin recruitment to cargo by the

  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. 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.

  7. 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

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

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    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

  9. 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.

  10. Microtubule-Targeting Agents Enter the Central Nervous System (CNS: Double-edged Swords for Treating CNS Injury and Disease

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    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.

  11. 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.

  12. 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

  13. Structural biases in prudential regulation of banks

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    Mario Tonveronachi

    2001-12-01

    Full Text Available According to Mario Tonveronachi, the Basle rules produce distortions that are no less serious than those attributed to the former structural regulation. Excessive competition is no less harmful than low competition, the level playing field approach helps the large dimension and “too big to fail” results, capital crunches produce serious effects on the economy while the regulatory costs go on absorbing important resources in small banks. It is a matter for further research to verify if the new approach to regulation has also fostered an increase in the part of GDP absorbed by the financial system without bringing about a better distribution of risks and a proportionate increase in what James Tobin (1984 termed full-insurance efficiency.

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

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    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.

  15. 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

  16. Adaptive compliant structures for flow regulation

    Science.gov (United States)

    Brinkmeyer, Alex; Theunissen, Raf; M. Weaver, Paul; Pirrera, Alberto

    2017-01-01

    This paper introduces conceptual design principles for a novel class of adaptive structures that provide both flow regulation and control. While of general applicability, these design principles, which revolve around the idea of using the instabilities and elastically nonlinear behaviour of post-buckled panels, are exemplified through a case study: the design of a shape-adaptive air inlet. The inlet comprises a deformable post-buckled member that changes shape depending on the pressure field applied by the surrounding fluid, thereby regulating the inlet aperture. By tailoring the stress field in the post-buckled state and the geometry of the initial, stress-free configuration, the deformable section can snap through to close or open the inlet completely. Owing to its inherent ability to change shape in response to external stimuli—i.e. the aerodynamic loads imposed by different operating conditions—the inlet does not have to rely on linkages and mechanisms for actuation, unlike conventional flow-controlling devices. PMID:28878567

  17. 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).

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

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    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.

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

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    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.

  20. 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.

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

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    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.

  2. Dendritic Actin Cytoskeleton: Structure, Functions, and Regulations

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    Anja Konietzny

    2017-05-01

    Full Text Available Actin is a versatile and ubiquitous cytoskeletal protein that plays a major role in both the establishment and the maintenance of neuronal polarity. For a long time, the most prominent roles that were attributed to actin in neurons were the movement of growth cones, polarized cargo sorting at the axon initial segment, and the dynamic plasticity of dendritic spines, since those compartments contain large accumulations of actin filaments (F-actin that can be readily visualized using electron- and fluorescence microscopy. With the development of super-resolution microscopy in the past few years, previously unknown structures of the actin cytoskeleton have been uncovered: a periodic lattice consisting of actin and spectrin seems to pervade not only the whole axon, but also dendrites and even the necks of dendritic spines. Apart from that striking feature, patches of F-actin and deep actin filament bundles have been described along the lengths of neurites. So far, research has been focused on the specific roles of actin in the axon, while it is becoming more and more apparent that in the dendrite, actin is not only confined to dendritic spines, but serves many additional and important functions. In this review, we focus on recent developments regarding the role of actin in dendrite morphology, the regulation of actin dynamics by internal and external factors, and the role of F-actin in dendritic protein trafficking.

  3. Microtubular stability affects pVHL-mediated regulation of HIF-1alpha via the p38/MAPK pathway in hypoxic cardiomyocytes.

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    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.

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

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    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

  5. 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

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

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    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.

  7. 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.

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

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    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.

  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. 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.

  11. Analytical review of structure and regulation of hemopoiesis

    Energy Technology Data Exchange (ETDEWEB)

    Cronkite, E.P.

    1987-01-01

    The development of knowledge on the structure of hemopoiesis and its regulation can be divided into four broad areas: descriptive morphology, kinetics of cell proliferation, regulation of rates of cell proliferation through interaction of molecular regulators and their cell surface receptors, and clinical applications. 60 refs., 6 figs.

  12. Analytical review of structure and regulation of hemopoiesis

    International Nuclear Information System (INIS)

    Cronkite, E.P.

    1987-01-01

    The development of knowledge on the structure of hemopoiesis and its regulation can be divided into four broad areas: descriptive morphology, kinetics of cell proliferation, regulation of rates of cell proliferation through interaction of molecular regulators and their cell surface receptors, and clinical applications. 60 refs., 6 figs

  13. 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

  14. 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

  15. 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

  16. 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.

  17. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  19. Foreign Banking in Ukraine: Development Trends and Ownership Structure Regulation

    Directory of Open Access Journals (Sweden)

    Serhij Reverchuk

    2015-10-01

    Full Text Available The purpose of the article is to research the theoretical and practical aspects of foreign participation ownership structure of banks in Ukraine; to analyze the tendencies and challenges of structure regulation of bank ownership; to provide recommendations related to the role of enhancing the transparency of banking business. This research was conducted by way of review of the data on bank ownership and the regulation of the ownership structure of the banking sector in Ukraine.

  20. 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.

  1. 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

  2. Spatial Structures and Regulation in Biological Systems

    DEFF Research Database (Denmark)

    Yde, Pernille

    , and the other is the spatial regulation of biological systems, here related to different aspects of the inflammatory response. All systems are studied using computational modelling and mathematical analysis. The first part of the thesis explores different protein aggregation scenarios. In Chapter 1, we consider...... a previously studied and very general aggregation model describing frangible linear filaments. This model is especially relevant for the growth of amyloid fibres, that have been related to a number of serious human diseases, and which are known to grow in an accelerated self-enhanced manner.We derive...... model of the tissue and show how coupled cells are able to function as an excitable medium and propagate waves of high cytokine concentration through the tissue. If the internal regulation in the cells is over-productive, the model predicts a continuous amplification of cytokines, which spans the entire...

  3. Ofd1, a human disease gene, regulates the length and distal structure of centrioles.

    Science.gov (United States)

    Singla, Veena; Romaguera-Ros, Miriam; Garcia-Verdugo, Jose Manuel; Reiter, Jeremy F

    2010-03-16

    Centrosomes and their component centrioles represent the principal microtubule organizing centers of animal cells. Here, we show that the gene underlying orofaciodigital syndrome 1, Ofd1, is a component of the distal centriole that controls centriole length. In the absence of Ofd1, distal regions of centrioles, but not procentrioles, elongate abnormally. These long centrioles are structurally similar to normal centrioles but contain destabilized microtubules with abnormal posttranslational modifications. Ofd1 is also important for centriole distal appendage formation and centriolar recruitment of the intraflagellar transport protein Ift88. To model OFD1 syndrome in embryonic stem cells, we replaced the Ofd1 gene with missense alleles from human OFD1 patients. Distinct disease-associated mutations cause different degrees of excessive or decreased centriole elongation, all of which are associated with diminished ciliogenesis. Our results indicate that Ofd1 acts at the distal centriole to build distal appendages, recruit Ift88, and stabilize centriolar microtubules at a defined length. Copyright 2010 Elsevier Inc. All rights reserved.

  4. 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.

  5. 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.

  6. 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.

  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. 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).

  9. 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.

  10. GAR22β regulates cell migration, sperm motility, and axoneme structure.

    Science.gov (United States)

    Gamper, Ivonne; Fleck, David; Barlin, Meltem; Spehr, Marc; El Sayad, Sara; Kleine, Henning; Maxeiner, Sebastian; Schalla, Carmen; Aydin, Gülcan; Hoss, Mareike; Litchfield, David W; Lüscher, Bernhard; Zenke, Martin; Sechi, Antonio

    2016-01-15

    Spatiotemporal cytoskeleton remodeling is pivotal for cell adhesion and migration. Here we investigated the function of Gas2-related protein on chromosome 22 (GAR22β), a poorly characterized protein that interacts with actin and microtubules. Primary and immortalized GAR22β(-/-) Sertoli cells moved faster than wild-type cells. In addition, GAR22β(-/-) cells showed a more prominent focal adhesion turnover. GAR22β overexpression or its reexpression in GAR22β(-/-) cells reduced cell motility and focal adhesion turnover. GAR22β-actin interaction was stronger than GAR22β-microtubule interaction, resulting in GAR22β localization and dynamics that mirrored those of the actin cytoskeleton. Mechanistically, GAR22β interacted with the regulator of microtubule dynamics end-binding protein 1 (EB1) via a novel noncanonical amino acid sequence, and this GAR22β-EB1 interaction was required for the ability of GAR22β to modulate cell motility. We found that GAR22β is highly expressed in mouse testes, and its absence resulted in reduced spermatozoa generation, lower actin levels in testes, and impaired motility and ultrastructural disorganization of spermatozoa. Collectively our findings identify GAR22β as a novel regulator of cell adhesion and migration and provide a foundation for understanding the molecular basis of diverse cytoskeleton-dependent processes. © 2016 Gamper 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).

  11. 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.

  12. 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

  13. 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.

  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. 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.

  17. 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.

  18. 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.

  19. 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

  20. Entry Regulations, Product Differentiation and Determinants of Market Structure

    OpenAIRE

    Maican, Florin; Orth, ´Matilda

    2013-01-01

    We use a dynamic oligopoly model of entry and exit to evaluate how entry regulations affect profitability and market structure in retail. The model incorporates demand and store-level heterogeneity. Based on unique data for all retail food stores in Sweden, we find that the average entry costs for small and large stores are 10 and 18 percent lower, respectively, in markets with liberal compared with restrictive regulations. Counterfactual simulations show that lower entry costs in restrictive...

  1. Cultural regulation of emotion: Individual, relational, and structural sources

    Directory of Open Access Journals (Sweden)

    Jozefien eDe Leersnyder

    2013-02-01

    Full Text Available The most prevalent and intense emotional experiences differ across cultures. These differences in emotional experience can be understood as the outcomes of emotion regulation, because emotions that fit the valued relationships within a culture tend to be most common and intense. We review evidence suggesting that emotion regulation underlying cultural differences in emotional experience often takes place at the point of emotion elicitation through the promotion of situations and appraisals that are consistent with culturally valued relationships. These regulatory processes depend on individual tendencies, but are also co-regulated within relationships—close others shape people’s environment and help them appraise events in culturally valued ways—and are afforded by structural conditions—people’s daily lives limit the opportunities for emotion, and afford certain appraisals. The combined evidence suggests that cultural differences in emotion regulation go well beyond the effortful regulation based on display rules.

  2. Cultural regulation of emotion: individual, relational, and structural sources.

    Science.gov (United States)

    De Leersnyder, Jozefien; Boiger, Michael; Mesquita, Batja

    2013-01-01

    The most prevalent and intense emotional experiences differ across cultures. These differences in emotional experience can be understood as the outcomes of emotion regulation, because emotions that fit the valued relationships within a culture tend to be most common and intense. We review evidence suggesting that emotion regulation underlying cultural differences in emotional experience often takes place at the point of emotion elicitation through the promotion of situations and appraisals that are consistent with culturally valued relationships. These regulatory processes depend on individual tendencies, but are also co-regulated within relationships-close others shape people's environment and help them appraise events in culturally valued ways-and are afforded by structural conditions-people's daily lives "limit" the opportunities for emotion, and afford certain appraisals. The combined evidence suggests that cultural differences in emotion regulation go well beyond the effortful regulation based on display rules.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. Structural remedies in merger regulation in a Cournot framework

    Czech Academy of Sciences Publication Activity Database

    Medvedev, Andrei

    -, 2004-006 (2004), s. 1-21 ISSN 1572-4042 Institutional research plan: CEZ:AV0Z7085904 Keywords : merger regulation * structural remedies * auction Subject RIV: AH - Economics http://www.tilburguniversity.nl/tilec/publications/discussionpapers/2004-006.pdf

  9. Cellular regulation of the structure and function of aortic valves

    Directory of Open Access Journals (Sweden)

    Ismail El-Hamamsy

    2010-01-01

    Full Text Available The aortic valve was long considered a passive structure that opens and closes in response to changes in transvalvular pressure. Recent evidence suggests that the aortic valve performs highly sophisticated functions as a result of its unique microscopic structure. These functions allow it to adapt to its hemodynamic and mechanical environment. Understanding the cellular and molecular mechanisms involved in normal valve physiology is essential to elucidate the mechanisms behind valve disease. We here review the structure and developmental biology of aortic valves; we examine the role of its cellular parts in regulating its function and describe potential pathophysiological and clinical implications.

  10. Approaches to link RNA secondary structures with splicing regulation

    DEFF Research Database (Denmark)

    Plass, Mireya; Eyras, Eduardo

    2014-01-01

    In higher eukaryotes, alternative splicing is usually regulated by protein factors, which bind to the pre-mRNA and affect the recognition of splicing signals. There is recent evidence that the secondary structure of the pre-mRNA may also play an important role in this process, either by facilitat...... describes the steps in the analysis of the secondary structure of the pre-mRNA and its possible relation to splicing. As a working example, we use the case of yeast and the problem of the recognition of the 3' splice site (3'ss).......In higher eukaryotes, alternative splicing is usually regulated by protein factors, which bind to the pre-mRNA and affect the recognition of splicing signals. There is recent evidence that the secondary structure of the pre-mRNA may also play an important role in this process, either...

  11. 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

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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

  18. The adventitia: essential regulator of vascular wall structure and function.

    Science.gov (United States)

    Stenmark, Kurt R; Yeager, Michael E; El Kasmi, Karim C; Nozik-Grayck, Eva; Gerasimovskaya, Evgenia V; Li, Min; Riddle, Suzette R; Frid, Maria G

    2013-01-01

    The vascular adventitia acts as a biological processing center for the retrieval, integration, storage, and release of key regulators of vessel wall function. It is the most complex compartment of the vessel wall and is composed of a variety of cells, including fibroblasts, immunomodulatory cells (dendritic cells and macrophages), progenitor cells, vasa vasorum endothelial cells and pericytes, and adrenergic nerves. In response to vascular stress or injury, resident adventitial cells are often the first to be activated and reprogrammed to influence the tone and structure of the vessel wall; to initiate and perpetuate chronic vascular inflammation; and to stimulate expansion of the vasa vasorum, which can act as a conduit for continued inflammatory and progenitor cell delivery to the vessel wall. This review presents the current evidence demonstrating that the adventitia acts as a key regulator of vascular wall function and structure from the outside in.

  19. 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.

  20. Soil structural quality assessment for soil protection regulation

    Science.gov (United States)

    Johannes, Alice; Boivin, Pascal

    2017-04-01

    Soil quality assessment is rapidly developing worldwide, though mostly focused on the monitoring of arable land and soil fertility. Soil protection regulations assess soil quality differently, focusing on priority pollutants and threshold values. The soil physical properties are weakly considered, due to lack of consensus and experimental difficulties faced with characterization. Non-disputable, easy to perform and inexpensive methods should be available for environmental regulation to be applied, which is unfortunately not the case. As a consequence, quantitative soil physical protection regulation is not applied, and inexpensive soil physical quality indicators for arable soil management are not available. Overcoming these limitations was the objective of a research project funded by the Swiss federal office for environment (FOEN). The main results and the perspectives of application are given in this presentation. A first step of the research was to characterize soils in a good structural state (reference soils) under different land use. The structural quality was assessed with field expertise and Visual Evaluation of the Soil Structure (VESS), and the physical properties were assessed with Shrinkage analysis. The relationships between the physical properties and the soil constituents were linear and highly determined. They represent the reference properties of the corresponding soils. In a second step, the properties of physically degraded soils were analysed and compared to the reference properties. This allowed defining the most discriminant parameters departing the different structure qualities and their threshold limits. Equivalent properties corresponding to these parameters but inexpensive and easy to determine were defined and tested. More than 90% of the samples were correctly classed with this method, which meets, therefore, the requirements for practical application in regulation. Moreover, result-oriented agri-environmental schemes for soil quality

  1. Entry regulations, welfare and determinants of market structure

    OpenAIRE

    Maican, Florin; Orth, Matilda

    2015-01-01

    We use a dynamic oligopoly model of entry and exit with store-type differentiation to evaluate how entry regulations affect profitability, market structure and welfare. Based on unique data for all retail food stores in Sweden, we estimate demand, recover variable profits, and estimate entry costs and fixed costs by store type. Counterfactual policy experiments show that welfare increases when competition is enhanced by lower entry costs. Protecting small stores by imposing licensing fees on ...

  2. 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.

  3. 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.

  4. 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).

  5. 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

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. Structural Fine-Tuning of MIT-Interacting Motif 2 (MIM2) and Allosteric Regulation of ESCRT-III by Vps4 in Yeast.

    Science.gov (United States)

    Kojima, Rieko; Obita, Takayuki; Onoue, Kousuke; Mizuguchi, Mineyuki

    2016-06-05

    The endosomal sorting complex required for transport (ESCRT) facilitates roles in membrane remodeling, such as multivesicular body biogenesis, enveloped virus budding and cell division. In yeast, Vps4 plays a crucial role in intraluminal vesicle formation by disassembling ESCRT proteins. Vps4 is recruited by ESCRT-III proteins to the endosomal membrane through the interaction between the microtubule interacting and trafficking (MIT) domain of Vps4 and the C-terminal MIT-interacting motif (MIM) of ESCRT-III proteins. Here, we have determined the crystal structure of Vps4-MIT in a complex with Vps20, a member of ESCRT-III, and revealed that Vps20 adopts a unique MIM2 conformation. Based on structural comparisons with other known MIM2s, we have refined the consensus sequence of MIM2. We have shown that another ESCRT-III protein, Ist1, binds to Vps4-MIT via its C-terminal MIM1 with higher affinity than Vps2, but lacks MIM2 by surface plasmon resonance. Surprisingly, the Ist1 MIM1 competed with the MIM2 of Vfa1, a regulator of Vps4, for binding to Vps4-MIT, even though these MIMs bind in non-overlapping sites on the MIT. These findings provide insight into the allosteric recognition of MIMs of ESCRT-III by Vps4 and also the regulation of ESCRT machinery at the last step of membrane remodeling. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. 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

  12. 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

  13. 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

  14. Structure, inhibition, and regulation of essential lipid A enzymes.

    Science.gov (United States)

    Zhou, Pei; Zhao, Jinshi

    2017-11-01

    The Raetz pathway of lipid A biosynthesis plays a vital role in the survival and fitness of Gram-negative bacteria. Research efforts in the past three decades have identified individual enzymes of the pathway and have provided a mechanistic understanding of the action and regulation of these enzymes at the molecular level. This article reviews the discovery, biochemical and structural characterization, and regulation of the essential lipid A enzymes, as well as continued efforts to develop novel antibiotics against Gram-negative pathogens by targeting lipid A biosynthesis. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. 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

  16. 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

  17. 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.

  18. 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

  19. 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.

  20. Structural insights into central hypertension regulation by human aminopeptidase A.

    Science.gov (United States)

    Yang, Yang; Liu, Chang; Lin, Yi-Lun; Li, Fang

    2013-08-30

    Hypertension is regulated through both the central and systemic renin-angiotensin systems. In the central renin-angiotensin system, zinc-dependent aminopeptidase A (APA) up-regulates blood pressure by specifically cleaving the N-terminal aspartate, but not the adjacent arginine, from angiotensin II, a process facilitated by calcium. Here, we determined the crystal structures of human APA and its complexes with different ligands and identified a calcium-binding site in the S1 pocket of APA. Without calcium, the S1 pocket can bind both acidic and basic residues through formation of salt bridges with the charged side chains. In the presence of calcium, the binding of acidic residues is enhanced as they ligate the cation, whereas the binding of basic residues is no longer favorable due to charge repulsion. Of the peptidomimetic inhibitors of APA, amastatin has higher potency than bestatin by fitting better in the S1 pocket and interacting additionally with the S3' subsite. These results explain the calcium-modulated substrate specificity of APA in central hypertension regulation and can guide the design and development of brain-targeting antihypertensive APA inhibitors.

  1. Structural Insights into Central Hypertension Regulation by Human Aminopeptidase A*

    Science.gov (United States)

    Yang, Yang; Liu, Chang; Lin, Yi-Lun; Li, Fang

    2013-01-01

    Hypertension is regulated through both the central and systemic renin-angiotensin systems. In the central renin-angiotensin system, zinc-dependent aminopeptidase A (APA) up-regulates blood pressure by specifically cleaving the N-terminal aspartate, but not the adjacent arginine, from angiotensin II, a process facilitated by calcium. Here, we determined the crystal structures of human APA and its complexes with different ligands and identified a calcium-binding site in the S1 pocket of APA. Without calcium, the S1 pocket can bind both acidic and basic residues through formation of salt bridges with the charged side chains. In the presence of calcium, the binding of acidic residues is enhanced as they ligate the cation, whereas the binding of basic residues is no longer favorable due to charge repulsion. Of the peptidomimetic inhibitors of APA, amastatin has higher potency than bestatin by fitting better in the S1 pocket and interacting additionally with the S3′ subsite. These results explain the calcium-modulated substrate specificity of APA in central hypertension regulation and can guide the design and development of brain-targeting antihypertensive APA inhibitors. PMID:23888046

  2. 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

  3. 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

  4. 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.

  5. 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...

  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

    Full Text Available Abstract Background Cucurbitacin B, an oxygenated tetracyclic triterpenoid compound extracted from the Thai medicinal plant Trichosanthes cucumerina L., has been reported to have several biological activities including anti-inflammatory, antimicrobial and anticancer. Cucurbitacin B is great of interest because of its biological activity. This agent inhibits growth of various types of human cancer cells lines. Methods In this study, we explored the novel molecular response of cucurbitacin B in human breast cancer cells, MCF-7 and MDA-MB-231. The growth inhibitory effect of cucurbitacin B on breast cancer cells was assessed by MTT assay. The effects of cucurbitacin B on microtubules morphological structure and tubulin polymerization were analyzed using immunofluorescence technique and tubulin polymerization assay kit, respectively. Proteomic analysis was used to identify the target-specific proteins that involved in cucurbitacin B treatment. Some of the differentially expressed genes and protein products were validated by real-time RT-PCR and western blot analysis. Cell cycle distributions and apoptosis were investigated using flow cytometry. Results Cucurbitacin B exhibited strong antiproliferative effects against breast cancer cells in a dose-dependent manner. We show that cucurbitacin B prominently alters the cytoskeletal network of breast cancer cells, inducing rapid morphologic changes and improper polymerization of the microtubule network. Moreover, the results of 2D-PAGE, real-time RT-PCR, and western blot analysis revealed that the expression of nucleophosmin/B23 and c-Myc decreased markedly after cucurbitacin B treatment. Immunofluorescence microscopy showed that cucurbitacin B induced translocation of nucleophosmin/B23 from the nucleolus to nucleoplasm. Treatment with cucurbitacin B resulted in cell cycle arrest at G2/M phase and the enhancement of apoptosis. Conclusions Our findings suggest that cucurbitacin B may inhibit the

  7. 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.

  8. RNA polymerase III transcription - regulated by chromatin structure and regulator of nuclear chromatin organization.

    Science.gov (United States)

    Pascali, Chiara; Teichmann, Martin

    2013-01-01

    RNA polymerase III (Pol III) transcription is regulated by modifications of the chromatin. DNA methylation and post-translational modifications of histones, such as acetylation, phosphorylation and methylation have been linked to Pol III transcriptional activity. In addition to being regulated by modifications of DNA and histones, Pol III genes and its transcription factors have been implicated in the organization of nuclear chromatin in several organisms. In yeast, the ability of the Pol III transcription system to contribute to nuclear organization seems to be dependent on direct interactions of Pol III genes and/or its transcription factors TFIIIC and TFIIIB with the structural maintenance of chromatin (SMC) protein-containing complexes cohesin and condensin. In human cells, Pol III genes and transcription factors have also been shown to colocalize with cohesin and the transcription regulator and genome organizer CCCTC-binding factor (CTCF). Furthermore, chromosomal sites have been identified in yeast and humans that are bound by partial Pol III machineries (extra TFIIIC sites - ETC; chromosome organizing clamps - COC). These ETCs/COC as well as Pol III genes possess the ability to act as boundary elements that restrict spreading of heterochromatin.

  9. 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.

  10. 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.

  11. Regulation of chromatin structure by poly(ADP-ribosylation

    Directory of Open Access Journals (Sweden)

    Sascha eBeneke

    2012-09-01

    Full Text Available The interaction of DNA with proteins in the context of chromatin has to be tightly regulated to achieve so different tasks as packaging, transcription, replication and repair. The very rapid and transient post-translational modification of proteins by poly(ADP-ribose has been shown to take part in all four. Originally identified as immediate cellular answer to a variety of genotoxic stresses, already early data indicated the ability of this highly charged nucleic acid-like polymer to modulate nucleosome structure, the basic unit of chromatin. At the same time the enzyme responsible for synthesizing poly(ADP-ribose, the zinc-finger protein poly(ADP-ribose polymerase-1 (PARP1, was shown to control transcription initiation as basic factor TFIIC within the RNA-polymerase II machinery. Later research focused more on PARP-mediated regulation of DNA repair and cell death, but in the last few years, transcription as well as chromatin modulation has re-appeared on the scene. This review will discuss the impact of PARP1 on transcription and transcription factors, its implication in chromatin remodeling for DNA repair and probably also replication, and its role in controlling epigenetic events such as DNA methylation and the functionality of the insulator protein CCCTC-binding factor.

  12. 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.

  13. 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.

  14. 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.

  15. 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

  16. Structure, Function and Regulation of the Hsp90 Machinery

    Directory of Open Access Journals (Sweden)

    Jing Li

    2013-06-01

    Full Text Available Heat shock protein 90 (Hsp90 is an ATP-dependent molecular chaperone which is essential in eukaryotes. It is required for the activation and stabilization of a wide variety of client proteins and many of them are involved in important cellular pathways. Since Hsp90 affects numerous physiological processes such as signal transduction, intracellular transport, and protein degradation, it became an interesting target for cancer therapy. Structurally, Hsp90 is a flexible dimeric protein composed of three different domains which adopt structurally distinct conformations. ATP binding triggers directionality in these conformational changes and leads to a more compact state. To achieve its function, Hsp90 works together with a large group of cofactors, termed co-chaperones. Co-chaperones form defined binary or ternary complexes with Hsp90, which facilitate the maturation of client proteins. In addition, posttranslational modifications of Hsp90, such as phosphorylation and acetylation, provide another level of regulation. They influence the conformational cycle, co-chaperone interaction, and inter-domain communications. In this review, we discuss the recent progress made in understanding the Hsp90 machinery.

  17. Self-regulating star formation and disk structure

    International Nuclear Information System (INIS)

    Dopita, M.A.

    1987-01-01

    Star formation processes determine the disk structure of galaxies. Stars heavier than about 1 solar mass determine the chemical evolution of the system and are produced at a rate which maintains (by the momentum input of the stars) the phase structure, pressure, and vertical velocity dispersion of the gas. Low mass stars are produced quiescently within molecular clouds, and their associated T-Tauri winds maintain the support of molecular clouds and regulate the star formation rate. Inefficient cooling suppresses this mode of star formation at low metallicity. Applied to the solar neighborhood, such a model can account for age/metallicity relationships, the increase in the O/Fe ratio at low metallicity, the paucity of metal-poor G and K dwarf stars, the missing mass in the disk and, possibly, the existence of a metal-poor thick disk. For other galaxies, it accounts for constant w-velocity dispersion of the gas, the relationship between gas content and specific rates of star formation, the surface brightness/metallicity relationship and for the shallow radial gradients in both star formation rates and HI content. 71 references

  18. 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.

  19. 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)

  20. 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...

  1. Electricity supply industry. Structure, ownership and regulation in OECD countries

    International Nuclear Information System (INIS)

    1994-01-01

    This study surveys developments and implications in the electricity supply industries in OECD countries. Chapter 1 introduces the issues. (Competition or electricity supply for everybody?) Electricity markets are dynamic and the participants are restructuring and repositioning themselves in order to benefit from new opportunities or policy initiatives. These changes are described in chapter 2. Privatisation is being pursued by some governments, not only for reasons of economic efficiency. Arguments for and against privatisation and different ways of introducing it are discussed in chapter 3. Fair trade and competition legislation, as it applies to all corporate entities, creates the institutional framework within which the utility has to operate. Various approaches to regulation and recent developments are described in chapter 4; the implications of regulatory changes are analysed in chapter 5. Having surveyed recent developments and their direct consequences, this study then goes on to look at their broader implications for the achievement of a range of energy policy objectives. Chapter 6 looks at fuel choice and investment decisions. Chapter 7 considers the issue of security of electricity supply, which has many special characteristics for both suppliers and regulators. OECD countries use different approaches for ensuring security of supply. Chapter 8 looks at environmental protection. Chapter 9 looks at energy efficiency. Chapter 10 discusses pricing. The introduction of competition has significant effects: it tends to reduce costs, remove cross subsidies, and bring prices more closely in line with the structure of costs. But there is no clear evidence at this stage as to whether, in the long run, competition produces lower overall prices. Finally chapter 11 analyses risk. The electricity business, like every other business, is faced with a variety of risks that cover every financial and technical facet of electricity production, transport, and supply. (N.C.)

  2. 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 ...

  3. 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.

  4. 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

  5. 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.

  6. 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

  7. 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

  8. 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.

  9. 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.

  10. 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.

  11. Tetraspanin 7 regulates sealing zone formation and the bone-resorbing activity of osteoclasts

    Energy Technology Data Exchange (ETDEWEB)

    Kwon, Jun-Oh; Lee, Yong Deok; Kim, Haemin; Kim, Min Kyung; Song, Min-Kyoung; Lee, Zang Hee; Kim, Hong-Hee, E-mail: hhbkim@snu.ac.kr

    2016-09-02

    Tetraspanin family proteins regulate morphology, motility, fusion, and signaling in various cell types. We investigated the role of the tetraspanin 7 (Tspan7) isoform in the differentiation and function of osteoclasts. Tspan7 was up-regulated during osteoclastogenesis. When Tspan7 expression was reduced in primary precursor cells by siRNA-mediated gene knock-down, the generation of multinuclear osteoclasts was not affected. However, a striking cytoskeletal abnormality was observed: the formation of the podosome belt structure was inhibited and the microtubular network were disrupted by Tspan7 knock-down. Decreases in acetylated microtubules and levels of phosphorylated Src and Pyk2 in Tspan7 knock-down cells supported the involvement of Tspan7 in cytoskeletal rearrangement signaling in osteoclasts. This cytoskeletal defect interfered with sealing zone formation and subsequently the bone-resorbing activity of mature osteoclasts on dentin surfaces. Our results suggest that Tspan7 plays an important role in cytoskeletal organization required for the bone-resorbing function of osteoclasts by regulating signaling to Src, Pyk2, and microtubules. - Highlights: • Tspan7 expression is up-regulated during osteoclastogenesis. • Tspan7 regulates podosome belt organization in osteoclasts. • Tspan7 is crucial for sealing zone formation and bone-resorption by osteoclasts. • Src and Pyk2 phosphorylation and microtubule acetylation mediate Tspan7 function.

  12. 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.

  13. 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.

  14. The plant microtubule-associated protein AtMAP65-3/PLE is essential for cytokinetic phragmoplast function.

    Science.gov (United States)

    Müller, Sabine; Smertenko, Andrei; Wagner, Vera; Heinrich, Maria; Hussey, Patrick J; Hauser, Marie-Theres

    2004-03-09

    Directional cell expansion in interphase and nuclear and cell division in M-phase are mediated by four microtubule arrays, three of which are unique to plants: the interphase array, the preprophase band, and the phragmoplast. The plant microtubule-associated protein MAP65 has been identified as a key structural component in these arrays. The Arabidopsis genome has nine MAP65 genes, and here we show that one, AtMAP65-3/PLE, locates only to the mitotic arrays and is essential for cytokinesis. The Arabidopsis pleiade (ple) alleles are single recessive mutations, and we show that these mutations are in the AtMAP65-3 gene. Moreover, these mutations cause C-terminal truncations that abolish microtubule binding. In the ple mutants the anaphase spindle is normal, and the cytokinetic phragmoplast can form but is distorted; not only is it wider, but the midline, the region where oppositely oriented microtubules overlap, is unusually expanded. Here we present data that demonstrate an essential role for AtMAP65-3/PLE in cytokinesis in plant cells.

  15. Aggregation of SND1 in Stress Granules is Associated with the Microtubule Cytoskeleton During Heat Shock Stimulus.

    Science.gov (United States)

    Shao, Jie; Gao, Fei; Zhang, Bingbing; Zhao, Meng; Zhou, Yunli; He, Jinyan; Ren, Li; Yao, Zhi; Yang, Jie; Su, Chao; Gao, Xingjie

    2017-12-01

    Stress granules (SGs) are dynamic dense structures in the cytoplasm that form in response to a variety of environmental stress stimuli. Staphylococcal nuclease and Tudor domain containing 1 (SND1) is a type of RNA-binding protein and has been identified as a transcriptional co-activator. Our previous studies have shown that SND1 is a component of the stress granule, which forms under stress conditions. Here, we observed that SND1 granules were often surrounded by ɑ-tubulin-microtubules in 45°C-treated HeLa cells at 15 min or colocalized with microtubules at 30 or 45 min. Furthermore, Nocodazole-mediated microtubule depolymerization could significantly affect the efficient recruitment of SND1 proteins to the SGs during heat shock stress. In addition, the 45°C heat shock mediated the enhancement of eIF2α phosphorylation, which was not affected by treatment with Nocodazole, an agent that disrupts the cytoskeleton. The intact microtubule cytoskeletal tracks are important for the efficient assembly of SND1 granules under heat shock stress and may facilitate SND1 shuttling between cytoplasmic RNA foci. Anat Rec, 300:2192-2199, 2017. © 2017 The Authors The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists. Copyright © 2017 The Authors The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.

  16. 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.

  17. 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

  18. Structural Basis for Bc12-Regulated Mitochondrion-Dependent Apoptosis

    National Research Council Canada - National Science Library

    Marassi, Francesca M

    2005-01-01

    ...; by dimerization with other Bcl-2 family members; by binding to other non-homologous proteins; and by formation of membrane pores that are believed to regulate apoptosis by perturbing mitochondrial physiology...

  19. Operator/regulator interface: Organizational structure and responsibilities

    Energy Technology Data Exchange (ETDEWEB)

    Wiebe, J S [American Electric Power Service Corp. (United States)

    1997-09-01

    In the context of promotion of safety culture for the nuclear power plants the following aspects are briefly described: operator/regulator interface regulatory organization; policy making body; operating organization; regulatory interface.

  20. Cultural regulation of emotion: Individual, relational, and structural sources

    OpenAIRE

    De Leersnyder, Jozefien; Boiger, Michael; Mesquita, Batja

    2013-01-01

    The most prevalent and intense emotional experiences differ across cultures. These differences in emotional experience can be understood as the outcomes of emotion regulation, because emotions that fit the valued relationships within a culture tend to be most common and intense. We review evidence suggesting that emotion regulation underlying cultural differences in emotional experience often takes place at the point of emotion elicitation through the promotion of situations and appraisals th...

  1. 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

  2. A structural self-regulation of functioning macromolecules

    International Nuclear Information System (INIS)

    Khristoforov, L.N.

    1998-01-01

    An approach to describing the functional structural changes of macromolecules processing the flows of low-mass agents is formulated. The latter appear as a source of a discrete noise whose defining parameters depend on structural variables. We derive a forward evolution equation and then, by adiabatic elimination, effective Fokker-Planck's equation for the structural modes. Within the dichotomous case, we discuss noise-induced nonequilibrium phase transitions reflecting the regulatory role of the structural subsystem

  3. 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

  4. 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...

  5. 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...

  6. 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.

  7. Structure and function of the cystic fibrosis transmembrane conductance regulator

    Directory of Open Access Journals (Sweden)

    M.M. Morales

    1999-08-01

    Full Text Available Cystic fibrosis (CF is a lethal autosomal recessive genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR. Mutations in the CFTR gene may result in a defective processing of its protein and alter the function and regulation of this channel. Mutations are associated with different symptoms, including pancreatic insufficiency, bile duct obstruction, infertility in males, high sweat Cl-, intestinal obstruction, nasal polyp formation, chronic sinusitis, mucus dehydration, and chronic Pseudomonas aeruginosa and Staphylococcus aureus lung infection, responsible for 90% of the mortality of CF patients. The gene responsible for the cellular defect in CF was cloned in 1989 and its protein product CFTR is activated by an increase of intracellular cAMP. The CFTR contains two membrane domains, each with six transmembrane domain segments, two nucleotide-binding domains (NBDs, and a cytoplasmic domain. In this review we discuss the studies that have correlated the role of each CFTR domain in the protein function as a chloride channel and as a regulator of the outwardly rectifying Cl- channels (ORCCs.

  8. 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.

  9. 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

  10. 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.

  11. 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

  12. HDAC up-regulation in early colon field carcinogenesis is involved in cell tumorigenicity through regulation of chromatin structure.

    Directory of Open Access Journals (Sweden)

    Yolanda Stypula-Cyrus

    Full Text Available Normal cell function is dependent on the proper maintenance of chromatin structure. Regulation of chromatin structure is controlled by histone modifications that directly influence chromatin architecture and genome function. Specifically, the histone deacetylase (HDAC family of proteins modulate chromatin compaction and are commonly dysregulated in many tumors, including colorectal cancer (CRC. However, the role of HDAC proteins in early colorectal carcinogenesis has not been previously reported. We found HDAC1, HDAC2, HDAC3, HDAC5, and HDAC7 all to be up-regulated in the field of human CRC. Furthermore, we observed that HDAC2 up-regulation is one of the earliest events in CRC carcinogenesis and observed this in human field carcinogenesis, the azoxymethane-treated rat model, and in more aggressive colon cancer cell lines. The universality of HDAC2 up-regulation suggests that HDAC2 up-regulation is a novel and important early event in CRC, which may serve as a biomarker. HDAC inhibitors (HDACIs interfere with tumorigenic HDAC activity; however, the precise mechanisms involved in this process remain to be elucidated. We confirmed that HDAC inhibition by valproic acid (VPA targeted the more aggressive cell line. Using nuclease digestion assays and transmission electron microscopy imaging, we observed that VPA treatment induced greater changes in chromatin structure in the more aggressive cell line. Furthermore, we used the novel imaging technique partial wave spectroscopy (PWS to quantify nanoscale alterations in chromatin. We noted that the PWS results are consistent with the biological assays, indicating a greater effect of VPA treatment in the more aggressive cell type. Together, these results demonstrate the importance of HDAC activity in early carcinogenic events and the unique role of higher-order chromatin structure in determining cell tumorigenicity.

  13. Structural interaction and functional regulation of polycystin-2 by filamin.

    Directory of Open Access Journals (Sweden)

    Qian Wang

    Full Text Available Filamins are important actin cross-linking proteins implicated in scaffolding, membrane stabilization and signal transduction, through interaction with ion channels, receptors and signaling proteins. Here we report the physical and functional interaction between filamins and polycystin-2, a TRP-type cation channel mutated in 10-15% patients with autosomal dominant polycystic kidney disease. Yeast two-hybrid and GST pull-down experiments demonstrated that the C-termini of filamin isoforms A, B and C directly bind to both the intracellular N- and C-termini of polycystin-2. Reciprocal co-immunoprecipitation experiments showed that endogenous polycystin-2 and filamins are in the same complexes in renal epithelial cells and human melanoma A7 cells. We then examined the effect of filamin on polycystin-2 channel function by electrophysiology studies with a lipid bilayer reconstitution system and found that filamin-A substantially inhibits polycystin-2 channel activity. Our study indicates that filamins are important regulators of polycystin-2 channel function, and further links actin cytoskeletal dynamics to the regulation of this channel protein.

  14. 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.

  15. Vfa1 Binds to the N-terminal Microtubule-interacting and Trafficking (MIT) Domain of Vps4 and Stimulates Its ATPase Activity*

    Science.gov (United States)

    Vild, Cody J.; Xu, Zhaohui

    2014-01-01

    The endosomal sorting complexes required for transport (ESCRT) are responsible for multivesicular body biogenesis, membrane abscission during cytokinesis, and retroviral budding. They function as transiently assembled molecular complexes on the membrane, and their disassembly requires the action of the AAA-ATPase Vps4. Vps4 is regulated by a multitude of ESCRT and ESCRT-related proteins. Binding of these proteins to Vps4 is often mediated via the microtubule-interacting and trafficking (MIT) domain of Vps4. Recently, a new Vps4-binding protein Vfa1 was identified in a yeast genetic screen, where overexpression of Vfa1 caused defects in vacuolar morphology. However, the function of Vfa1 and its role in vacuolar biology were largely unknown. Here, we provide the first detailed biochemical and biophysical study of Vps4-Vfa1 interaction. The MIT domain of Vps4 binds to the C-terminal 17 residues of Vfa1. This interaction is of high affinity and greatly stimulates the ATPase activity of Vps4. The crystal structure of the Vps4-Vfa1 complex shows that Vfa1 adopts a canonical MIT-interacting motif 2 structure that has been observed previously in other Vps4-ESCRT interactions. These findings suggest that Vfa1 is a novel positive regulator of Vps4 function. PMID:24567329

  16. Vfa1 binds to the N-terminal microtubule-interacting and trafficking (MIT) domain of Vps4 and stimulates its ATPase activity.

    Science.gov (United States)

    Vild, Cody J; Xu, Zhaohui

    2014-04-11

    The endosomal sorting complexes required for transport (ESCRT) are responsible for multivesicular body biogenesis, membrane abscission during cytokinesis, and retroviral budding. They function as transiently assembled molecular complexes on the membrane, and their disassembly requires the action of the AAA-ATPase Vps4. Vps4 is regulated by a multitude of ESCRT and ESCRT-related proteins. Binding of these proteins to Vps4 is often mediated via the microtubule-interacting and trafficking (MIT) domain of Vps4. Recently, a new Vps4-binding protein Vfa1 was identified in a yeast genetic screen, where overexpression of Vfa1 caused defects in vacuolar morphology. However, the function of Vfa1 and its role in vacuolar biology were largely unknown. Here, we provide the first detailed biochemical and biophysical study of Vps4-Vfa1 interaction. The MIT domain of Vps4 binds to the C-terminal 17 residues of Vfa1. This interaction is of high affinity and greatly stimulates the ATPase activity of Vps4. The crystal structure of the Vps4-Vfa1 complex shows that Vfa1 adopts a canonical MIT-interacting motif 2 structure that has been observed previously in other Vps4-ESCRT interactions. These findings suggest that Vfa1 is a novel positive regulator of Vps4 function.

  17. Regulation and the Ownership Structure of European Listed Firms

    DEFF Research Database (Denmark)

    Rapp, Marc Steffen; Trinchera, Oliver

    2017-01-01

    In this paper, we explore an extensive panel data set covering more than 4,000 listed firms in 16 European countries to study the effects of shareholder protection on ownership structure and firm performance. We document a negative firm-level correlation between shareholder protection and ownersh...

  18. 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.

  19. 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.

  20. 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.

  1. SLC6 Neurotransmitter Transporters: Structure, Function, and Regulation

    DEFF Research Database (Denmark)

    Kristensen, Anders S; Andersen, Jacob; Jørgensen, Trine N

    2011-01-01

    The neurotransmitter transporters (NTTs) belonging to the solute carrier 6 (SLC6) gene family (also referred to as the neurotransmitter-sodium-symporter family or Na(+)/Cl(-)-dependent transporters) comprise a group of nine sodium- and chloride-dependent plasma membrane transporters...... for the monoamine neurotransmitters serotonin (5-hydroxytryptamine), dopamine, and norepinephrine, and the amino acid neurotransmitters GABA and glycine. The SLC6 NTTs are widely expressed in the mammalian brain and play an essential role in regulating neurotransmitter signaling and homeostasis by mediating uptake...... of released neurotransmitters from the extracellular space into neurons and glial cells. The transporters are targets for a wide range of therapeutic drugs used in treatment of psychiatric diseases, including major depression, anxiety disorders, attention deficit hyperactivity disorder and epilepsy...

  2. Local chromatin structure of heterochromatin regulates repeated DNA stability, nucleolus structure, and genome integrity

    Energy Technology Data Exchange (ETDEWEB)

    Peng, Jamy C. [Univ. of California, Berkeley, CA (United States)

    2007-01-01

    Heterochromatin constitutes a significant portion of the genome in higher eukaryotes; approximately 30% in Drosophila and human. Heterochromatin contains a high repeat DNA content and a low density of protein-encoding genes. In contrast, euchromatin is composed mostly of unique sequences and contains the majority of single-copy genes. Genetic and cytological studies demonstrated that heterochromatin exhibits regulatory roles in chromosome organization, centromere function and telomere protection. As an epigenetically regulated structure, heterochromatin formation is not defined by any DNA sequence consensus. Heterochromatin is characterized by its association with nucleosomes containing methylated-lysine 9 of histone H3 (H3K9me), heterochromatin protein 1 (HP1) that binds H3K9me, and Su(var)3-9, which methylates H3K9 and binds HP1. Heterochromatin formation and functions are influenced by HP1, Su(var)3-9, and the RNA interference (RNAi) pathway. My thesis project investigates how heterochromatin formation and function impact nuclear architecture, repeated DNA organization, and genome stability in Drosophila melanogaster. H3K9me-based chromatin reduces extrachromosomal DNA formation; most likely by restricting the access of repair machineries to repeated DNAs. Reducing extrachromosomal ribosomal DNA stabilizes rDNA repeats and the nucleolus structure. H3K9me-based chromatin also inhibits DNA damage in heterochromatin. Cells with compromised heterochromatin structure, due to Su(var)3-9 or dcr-2 (a component of the RNAi pathway) mutations, display severe DNA damage in heterochromatin compared to wild type. In these mutant cells, accumulated DNA damage leads to chromosomal defects such as translocations, defective DNA repair response, and activation of the G2-M DNA repair and mitotic checkpoints that ensure cellular and animal viability. My thesis research suggests that DNA replication, repair, and recombination mechanisms in heterochromatin differ from those in

  3. Structural Elements Regulating AAA+ Protein Quality Control Machines.

    Science.gov (United States)

    Chang, Chiung-Wen; Lee, Sukyeong; Tsai, Francis T F

    2017-01-01

    Members of the ATPases Associated with various cellular Activities (AAA+) superfamily participate in essential and diverse cellular pathways in all kingdoms of life by harnessing the energy of ATP binding and hydrolysis to drive their biological functions. Although most AAA+ proteins share a ring-shaped architecture, AAA+ proteins have evolved distinct structural elements that are fine-tuned to their specific functions. A central question in the field is how ATP binding and hydrolysis are coupled to substrate translocation through the central channel of ring-forming AAA+ proteins. In this mini-review, we will discuss structural elements present in AAA+ proteins involved in protein quality control, drawing similarities to their known role in substrate interaction by AAA+ proteins involved in DNA translocation. Elements to be discussed include the pore loop-1, the Inter-Subunit Signaling (ISS) motif, and the Pre-Sensor I insert (PS-I) motif. Lastly, we will summarize our current understanding on the inter-relationship of those structural elements and propose a model how ATP binding and hydrolysis might be coupled to polypeptide translocation in protein quality control machines.

  4. 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.

  5. 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

  6. Nature and regulation of the insulin receptor: structure and function

    International Nuclear Information System (INIS)

    Czech, M.P.

    1985-01-01

    Native, cell-surface insulin receptor consists of two glycoprotein subunit types with apparent masses of about 125,000 daltons (alpha subunit) and 90,000 daltons (beta subunit). The alpha and beta insulin-receptor subunits seem to have distinct functions such that alpha appears to bind hormone whereas beta appears to possess intrinsic tyrosine kinase activity. In detergent extracts, insulin activates receptor autophosphorylation of tyrosine residues on its beta subunit, whereas in the presence of reductant, the alpha subunit is also phosphorylated. In intact cells, insulin activates serine/threonine phosphorylation of insulin receptor beta subunit as well as tyrosine phosphorylation. The biological role of the receptor-associated tyrosine kinase is not known. The insulin receptor kinase is regulated by beta-adrenergic agonists and other agents that elevate cAMP in adipocytes, presumably via the cAMP-dependent protein kinase. Such agents decrease receptor affinity for insulin and partially uncouple receptor tyrosine kinase activity from activation by insulin. These effects appear to contribute to the biological antagonism between insulin and beta-agonists. These data suggest the hypothesis that a complex network of tyrosine and serine/threonine phosphorylations on the insulin receptor modulate its binding and kinase activities in an antagonistic manner

  7. Bank regulation and financial fragility in developing countries: Does bank structure matter?

    Directory of Open Access Journals (Sweden)

    Jeroen Klomp

    2015-12-01

    Full Text Available Using data for 1238 banks located in 94 developing and emerging countries, we explore whether the impact of bank regulation and supervision on banking risk (measured by the banks’ Z-scores depends on bank structure. Our findings suggest that stricter regulation and supervision increases the banks’ Z-scores. Notably capital requirements and supervisory control diminish banking risk. However, the effectiveness of other dimensions of regulation and supervision depends on the organizational structure of banks. Notably activity restrictions reduce risk of large and foreign owned banks, while liquidity restrictions have most effect on the Z-scores of unlisted and commercial banks.

  8. Renewable resource regulation and uncertain prices: The role of financial structure and bankruptcy

    International Nuclear Information System (INIS)

    Damania, Richard; Bulte, Erwin H.

    2006-01-01

    We analyze the interaction between regulatory policies and the financial structure of a fishery and show that firms with debts may respond differently to regulations than firms that have not accumulated debts. There are conditions where more stringent regulation is counterproductive, providing a perverse incentive to increase harvesting effort. We show that optimal regulation depends on the sector's financial structure, and demonstrate that there are cases when intervention is counterproductive, or too costly to implement. There are also cases where successful regulatory intervention can only be implemented when accompanied by a sufficiently large subsidy. (author)

  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. DMPD: Structure, function and regulation of the Toll/IL-1 receptor adaptor proteins. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 17667936 Structure, function and regulation of the Toll/IL-1 receptor adaptor prote... (.svg) (.html) (.csml) Show Structure, function and regulation of the Toll/IL-1 receptor adaptor proteins. ...PubmedID 17667936 Title Structure, function and regulation of the Toll/IL-1 recep

  11. Orchestrated structure evolution: modeling growth-regulated nanomanufacturing

    Energy Technology Data Exchange (ETDEWEB)

    Abbasi, Shaghayegh; Boehringer, Karl F [Department of Electrical Engineering, University of Washington, Seattle, WA 98195-2500 (United States); Kitayaporn, Sathana; Schwartz, Daniel T, E-mail: karlb@washington.edu [Department of Chemical Engineering, University of Washington, Seattle, WA 98195-2500 (United States)

    2011-04-22

    Orchestrated structure evolution (OSE) is a scalable manufacturing method that combines the advantages of top-down (tool-directed) and bottom-up (self-propagating) approaches. The method consists of a seed patterning step that defines where material nucleates, followed by a growth step that merges seeded islands into the final patterned thin film. We develop a model to predict the completed pattern based on a computationally efficient approximate Green's function solution of the diffusion equation plus a Voronoi diagram based approach that defines the final grain boundary structure. Experimental results rely on electron beam lithography to pattern the seeds, followed by the mass transfer limited growth of copper via electrodeposition. The seed growth model is compared with experimental results to quantify nearest neighbor seed-to-seed interactions as well as how seeds interact with the pattern boundary to impact the local growth rate. Seed-to-seed and seed-to-pattern interactions are shown to result in overgrowth of seeds on edges and corners of the shape, where seeds have fewer neighbors. We explore how local changes to the seed location can be used to improve the patterning quality without increasing the manufacturing cost. OSE is shown to enable a unique set of trade-offs between the cost, time, and quality of thin film patterning.

  12. Surface topography regulates wnt signaling through control of primary cilia structure in mesenchymal stem cells

    Science.gov (United States)

    McMurray, R. J.; Wann, A. K. T.; Thompson, C. L.; Connelly, J. T.; Knight, M. M.

    2013-01-01

    The primary cilium regulates cellular signalling including influencing wnt sensitivity by sequestering β-catenin within the ciliary compartment. Topographic regulation of intracellular actin-myosin tension can control stem cell fate of which wnt is an important mediator. We hypothesized that topography influences mesenchymal stem cell (MSC) wnt signaling through the regulation of primary cilia structure and function. MSCs cultured on grooves expressed elongated primary cilia, through reduced actin organization. siRNA inhibition of anterograde intraflagellar transport (IFT88) reduced cilia length and increased active nuclear β-catenin. Conversely, increased primary cilia assembly in MSCs cultured on the grooves was associated with decreased levels of nuclear active β-catenin, axin-2 induction and proliferation, in response to wnt3a. This negative regulation, on grooved topography, was reversed by siRNA to IFT88. This indicates that subtle regulation of IFT and associated cilia structure, tunes the wnt response controlling stem cell differentiation. PMID:24346024

  13. Hormonal regulation of alveolarization: structure-function correlation

    Directory of Open Access Journals (Sweden)

    Godinez Marye H

    2006-03-01

    Full Text Available Abstract Background Dexamethasone (Dex limits and all-trans-retinoic acid (RA promotes alveolarization. While structural changes resulting from such hormonal exposures are known, their functional consequences are unclear. Methods Neonatal rats were treated with Dex and/or RA during the first two weeks of life or were given RA after previous exposure to Dex. Morphology was assessed by light microscopy and radial alveolar counts. Function was evaluated by plethysmography at d13, pressure volume curves at d30, and exercise swim testing and arterial blood gases at both d15 and d30. Results Dex-treated animals had simplified lung architecture without secondary septation. Animals given RA alone had smaller, more numerous alveoli. Concomitant treatment with Dex + RA prevented the Dex-induced changes in septation. While the results of exposure to Dex + RA were sustained, the effects of RA alone were reversed two weeks after treatment was stopped. At d13, Dex-treated animals had increased lung volume, respiratory rate, tidal volume, and minute ventilation. On d15, both RA- and Dex-treated animals had hypercarbia and low arterial pH. By d30, the RA-treated animals resolved this respiratory acidosis, but Dex-treated animals continued to demonstrate blood gas and lung volume abnormalities. Concomitant RA treatment improved respiratory acidosis, but failed to normalize Dex-induced changes in pulmonary function and lung volumes. No differences in exercise tolerance were noted at either d15 or d30. RA treatment after the period of alveolarization also corrected the effects of earlier Dex exposure, but the structural changes due to RA alone were again lost two weeks after treatment. Conclusion We conclude that both RA- and corticosteroid-treatments are associated with respiratory acidosis at d15. While RA alone-induced changes in structure andrespiratory function are reversed, Dex-treated animals continue to demonstrate increased respiratory rate, minute

  14. Modulation of microtubule assembly by the HIV-1 Tat protein is strongly dependent on zinc binding to Tat

    Directory of Open Access Journals (Sweden)

    Muller Sylviane

    2008-07-01

    Full Text Available Abstract Background During HIV-1 infection, the Tat protein plays a key role by transactivating the transcription of the HIV-1 proviral DNA. In addition, Tat induces apoptosis of non-infected T lymphocytes, leading to a massive loss of immune competence. This apoptosis is notably mediated by the interaction of Tat with microtubules, which are dynamic components essential for cell structure and division. Tat binds two Zn2+ ions through its conserved cysteine-rich region in vitro, but the role of zinc in the structure and properties of Tat is still controversial. Results To investigate the role of zinc, we first characterized Tat apo- and holo-forms by fluorescence correlation spectroscopy and time-resolved fluorescence spectroscopy. Both of the Tat forms are monomeric and poorly folded but differ by local conformational changes in the vicinity of the cysteine-rich region. The interaction of the two Tat forms with tubulin dimers and microtubules was monitored by analytical ultracentrifugation, turbidity measurements and electron microscopy. At 20°C, both of the Tat forms bind tubulin dimers, but only the holo-Tat was found to form discrete complexes. At 37°C, both forms promoted the nucleation and increased the elongation rates of tubulin assembly. However, only the holo-Tat increased the amount of microtubules, decreased the tubulin critical concentration, and stabilized the microtubules. In contrast, apo-Tat induced a large amount of tubulin aggregates. Conclusion Our data suggest that holo-Tat corresponds to the active form, responsible for the Tat-mediated apoptosis.

  15. 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.

  16. Structural elements regulating amyloidogenesis: a cholinesterase model system.

    Directory of Open Access Journals (Sweden)

    Létitia Jean

    2008-03-01

    Full Text Available Polymerization into amyloid fibrils is a crucial step in the pathogenesis of neurodegenerative syndromes. Amyloid assembly is governed by properties of the sequence backbone and specific side-chain interactions, since fibrils from unrelated sequences possess similar structures and morphologies. Therefore, characterization of the structural determinants driving amyloid aggregation is of fundamental importance. We investigated the forces involved in the amyloid assembly of a model peptide derived from the oligomerization domain of acetylcholinesterase (AChE, AChE(586-599, through the effect of single point mutations on beta-sheet propensity, conformation, fibrilization, surfactant activity, oligomerization and fibril morphology. AChE(586-599 was chosen due to its fibrilization tractability and AChE involvement in Alzheimer's disease. The results revealed how specific regions and residues can control AChE(586-599 assembly. Hydrophobic and/or aromatic residues were crucial for maintaining a high beta-strand propensity, for the conformational transition to beta-sheet, and for the first stage of aggregation. We also demonstrated that positively charged side-chains might be involved in electrostatic interactions, which could control the transition to beta-sheet, the oligomerization and assembly stability. Further interactions were also found to participate in the assembly. We showed that some residues were important for AChE(586-599 surfactant activity and that amyloid assembly might preferentially occur at an air-water interface. Consistently with the experimental observations and assembly models for other amyloid systems, we propose a model for AChE(586-599 assembly in which a steric-zipper formed through specific interactions (hydrophobic, electrostatic, cation-pi, SH-aromatic, metal chelation and polar-polar would maintain the beta-sheets together. We also propose that the stacking between the strands in the beta-sheets along the fiber axis could

  17. Src protein-tyrosine kinase structure and regulation

    International Nuclear Information System (INIS)

    Roskoski, Robert

    2004-01-01

    Src and Src-family protein kinases are proto-oncogenes that play key roles in cell morphology, motility, proliferation, and survival. v-Src (a viral protein) is encoded by the chicken oncogene of Rous sarcoma virus, and Src (the cellular homologue) is encoded by a physiological gene, the first of the proto-oncogenes. From the N- to C-terminus, Src contains an N-terminal 14-carbon myristoyl group, a unique segment, an SH3 domain, an SH2 domain, a protein-tyrosine kinase domain, and a C-terminal regulatory tail. The chief phosphorylation sites of Src include tyrosine 416 that results in activation from autophosphorylation and tyrosine 527 that results in inhibition from phosphorylation by C-terminal Src kinase. In the restrained state, the SH2 domain forms a salt bridge with phosphotyrosine 527, and the SH3 domain binds to the kinase domain via a polyproline type II left-handed helix. The SH2 and SH3 domains occur on the backside of the kinase domain away from the active site where they stabilize a dormant enzyme conformation. Protein-tyrosine phosphatases such as PTPα displace phosphotyrosine 527 from the Src SH2 domain and mediate its dephosphorylation leading to Src kinase activation. C-terminal Src kinase consists of an SH3, SH2, and kinase domain; it lacks an N-terminal myristoyl group and a C-terminal regulatory tail. Its X-ray structure has been determined, and the SH2 lobe occupies a position that is entirely different from that of Src. Unlike Src, the C-terminal Src kinase SH2 and SH3 domains stabilize an active enzyme conformation. Amino acid residues in the αD helix near the catalytic loop in the large lobe of C-terminal Src kinase serve as a docking site for the physiological substrate (Src) but not for an artificial substrate (polyGlu 4 Tyr)

  18. 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....

  19. 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

  20. 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.

  1. Tubulation of Class II MHC Compartments Is Microtubule Dependent and Involves Multiple Endolysosomal Membrane Proteins in Primary Dendritic Cells1

    Science.gov (United States)

    Vyas, Jatin M.; Kim, You-Me; Artavanis-Tsakonas, Katerina; Love, J. Christopher; Van der Veen, Annemarthe G.; Ploegh, Hidde L.

    2009-01-01

    Immature dendritic cells (DCs) capture exogenous Ags in the periphery for eventual processing in endolysosomes. Upon maturation by TLR agonists, DCs deliver peptide-loaded class II MHC molecules from these compartments to the cell surface via long tubular structures (endolysosomal tubules). The nature and rules that govern the movement of these DC compartments are unknown. In this study, we demonstrate that the tubules contain multiple proteins including the class II MHC molecules and LAMP1, a lysosomal resident protein, as well as CD63 and CD82, members of the tetraspanin family. Endolysosomal tubules can be stained with acidotropic dyes, indicating that they are extensions of lysosomes. However, the proper trafficking of class II MHC molecules themselves is not necessary for endolysosomal tubule formation. DCs lacking MyD88 can also form endolysosomal tubules, demonstrating that MyD88-dependent TLR activation is not necessary for the formation of this compartment. Endolysosomal tubules in DCs exhibit dynamic and saltatory movement, including bidirectional travel. Measured velocities are consistent with motor-based movement along microtubules. Indeed, nocodazole causes the collapse of endolysosomal tubules. In addition to its association with microtubules, endolysosomal tubules follow the plus ends of microtubules as visualized in primary DCs expressing end binding protein 1 (EB1)-enhanced GFP. PMID:17513769

  2. Tubulation of class II MHC compartments is microtubule dependent and involves multiple endolysosomal membrane proteins in primary dendritic cells.

    Science.gov (United States)

    Vyas, Jatin M; Kim, You-Me; Artavanis-Tsakonas, Katerina; Love, J Christopher; Van der Veen, Annemarthe G; Ploegh, Hidde L

    2007-06-01

    Immature dendritic cells (DCs) capture exogenous Ags in the periphery for eventual processing in endolysosomes. Upon maturation by TLR agonists, DCs deliver peptide-loaded class II MHC molecules from these compartments to the cell surface via long tubular structures (endolysosomal tubules). The nature and rules that govern the movement of these DC compartments are unknown. In this study, we demonstrate that the tubules contain multiple proteins including the class II MHC molecules and LAMP1, a lysosomal resident protein, as well as CD63 and CD82, members of the tetraspanin family. Endolysosomal tubules can be stained with acidotropic dyes, indicating that they are extensions of lysosomes. However, the proper trafficking of class II MHC molecules themselves is not necessary for endolysosomal tubule formation. DCs lacking MyD88 can also form endolysosomal tubules, demonstrating that MyD88-dependent TLR activation is not necessary for the formation of this compartment. Endolysosomal tubules in DCs exhibit dynamic and saltatory movement, including bidirectional travel. Measured velocities are consistent with motor-based movement along microtubules. Indeed, nocodazole causes the collapse of endolysosomal tubules. In addition to its association with microtubules, endolysosomal tubules follow the plus ends of microtubules as visualized in primary DCs expressing end binding protein 1 (EB1)-enhanced GFP.

  3. The free energy profile of tubulin straight-bent conformational changes, with implications for microtubule assembly and drug discovery.

    Directory of Open Access Journals (Sweden)

    Lili X Peng

    2014-02-01

    Full Text Available αβ-tubulin dimers need to convert between a 'bent' conformation observed for free dimers in solution and a 'straight' conformation required for incorporation into the microtubule lattice. Here, we investigate the free energy landscape of αβ-tubulin using molecular dynamics simulations, emphasizing implications for models of assembly, and modulation of the conformational landscape by colchicine, a tubulin-binding drug that inhibits microtubule polymerization. Specifically, we performed molecular dynamics, potential-of-mean force simulations to obtain the free energy profile for unpolymerized GDP-bound tubulin as a function of the ∼12° intradimer rotation differentiating the straight and bent conformers. Our results predict that the unassembled GDP-tubulin heterodimer exists in a continuum of conformations ranging between straight and bent, but, in agreement with existing structural data, suggests that an intermediate bent state has a lower free energy (by ∼1 kcal/mol and thus dominates in solution. In agreement with predictions of the lattice model of microtubule assembly, lateral binding of two αβ-tubulins strongly shifts the conformational equilibrium towards the straight state, which is then ∼1 kcal/mol lower in free energy than the bent state. Finally, calculations of colchicine binding to a single αβ-tubulin dimer strongly shifts the equilibrium toward the bent states, and disfavors the straight state to the extent that it is no longer thermodynamically populated.

  4. Cellular effects of the microtubule-targeting agent peloruside A in hypoxia-conditioned colorectal carcinoma cells.

    Science.gov (United States)

    Řehulka, Jiří; Annadurai, Narendran; Frydrych, Ivo; Znojek, Pawel; Džubák, Petr; Northcote, Peter; Miller, John H; Hajdúch, Marián; Das, Viswanath

    2017-07-01

    Hypoxia is a prominent feature of solid tumors, dramatically remodeling microtubule structures and cellular pathways and contributing to paclitaxel resistance. Peloruside A (PLA), a microtubule-targeting agent, has shown promising anti-tumor effects in preclinical studies. Although it has a similar mode of action to paclitaxel, it binds to a distinct site on β-tubulin that differs from the classical taxane site. In this study, we examined the unexplored effects of PLA in hypoxia-conditioned colorectal HCT116 cancer cells. Cytotoxicity of PLA was determined by cell proliferation assay. The effects of a pre-exposure to hypoxia on PLA-induced cell cycle alterations and apoptosis were examined by flow cytometry, time-lapse imaging, and western blot analysis of selected markers. The hypoxia effect on stabilization of microtubules by PLA was monitored by an intracellular tubulin polymerization assay. Our findings show that the cytotoxicity of PLA is not altered in hypoxia-conditioned cells compared to paclitaxel and vincristine. Furthermore, hypoxia does not alter PLA-induced microtubule stabilization nor the multinucleation of cells. PLA causes cyclin B1 and G2/M accumulation followed by apoptosis. The cellular and molecular effects of PLA have been determined in normoxic conditions, but there are no reports of PLA effects in hypoxic cells. Our findings reveal that hypoxia preconditioning does not alter the sensitivity of HCT116 to PLA. These data report on the cellular and molecular effects of PLA in hypoxia-conditioned cells for the first time, and will encourage further exploration of PLA as a promising anti-tumor agent. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. 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

  6. 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.

  7. 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.

  8. Automatic control of negative emotions: evidence that structured practice increases the efficiency of emotion regulation.

    Science.gov (United States)

    Christou-Champi, Spyros; Farrow, Tom F D; Webb, Thomas L

    2015-01-01

    Emotion regulation (ER) is vital to everyday functioning. However, the effortful nature of many forms of ER may lead to regulation being inefficient and potentially ineffective. The present research examined whether structured practice could increase the efficiency of ER. During three training sessions, comprising a total of 150 training trials, participants were presented with negatively valenced images and asked either to "attend" (control condition) or "reappraise" (ER condition). A further group of participants did not participate in training but only completed follow-up measures. Practice increased the efficiency of ER as indexed by decreased time required to regulate emotions and increased heart rate variability (HRV). Furthermore, participants in the ER condition spontaneously regulated their negative emotions two weeks later and reported being more habitual in their use of ER. These findings indicate that structured practice can facilitate the automatic control of negative emotions and that these effects persist beyond training.

  9. 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.

  10. The structure, regulation, and performance of pension funds in nine industrial countries

    OpenAIRE

    Davis, E.P.; DEC

    1993-01-01

    The author offers an overview of issues relating to the development of funded pension schemes in industrial countries. The analysis applies the economic theory of pension regulation to experience with the structure, regulation, and performance of funds in nine countries - Canada, Denmark, Germany, Japan, Netherlands, Sweden, Switzerland, the United Kingdom, and the United States - seeking to shed light on the finance of old age security in developing countries and the reform of pension funds ...

  11. Cytoskeleton Molecular Motors: Structures and Their Functions in Neuron.

    Science.gov (United States)

    Xiao, Qingpin; Hu, Xiaohui; Wei, Zhiyi; Tam, Kin Yip

    2016-01-01

    Cells make use of molecular motors to transport small molecules, macromolecules and cellular organelles to target region to execute biological functions, which is utmost important for polarized cells, such as neurons. In particular, cytoskeleton motors play fundamental roles in neuron polarization, extension, shape and neurotransmission. Cytoskeleton motors comprise of myosin, kinesin and cytoplasmic dynein. F-actin filaments act as myosin track, while kinesin and cytoplasmic dynein move on microtubules. Cytoskeleton motors work together to build a highly polarized and regulated system in neuronal cells via different molecular mechanisms and functional regulations. This review discusses the structures and working mechanisms of the cytoskeleton motors in neurons.

  12. 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 ...

  13. 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.

  14. 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....

  15. Breakup and then makeup: a predictive model of how cilia self-regulate hardness for posture control.

    Science.gov (United States)

    Bandyopadhyay, Promode R; Hansen, Joshua C

    2013-01-01

    Functioning as sensors and propulsors, cilia are evolutionarily conserved organelles having a highly organized internal structure. How a paramecium's cilium produces off-propulsion-plane curvature during its return stroke for symmetry breaking and drag reduction is not known. We explain these cilium deformations by developing a torsional pendulum model of beat frequency dependence on viscosity and an olivo-cerebellar model of self-regulation of posture control. The phase dependence of cilia torsion is determined, and a bio-physical model of hardness control with predictive features is offered. Crossbridge links between the central microtubule pair harden the cilium during the power stroke; this stroke's end is a critical phase during which ATP molecules soften the crossbridge-microtubule attachment at the cilium inflection point where torsion is at its maximum. A precipitous reduction in hardness ensues, signaling the start of ATP hydrolysis that re-hardens the cilium. The cilium attractor basin could be used as reference for perturbation sensing.

  16. Resolution 8.069/12. It approve the regulations for the large size structures installation, destined for wind power generation

    International Nuclear Information System (INIS)

    2012-01-01

    This resolution approve the regulations for the large size structures installation, destined to wind power generation. The objective of this rule is to regulate the urban conditions of the facilities and the environmental guarantees, safety and inhabitants wholesomeness

  17. 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.

  18. Structure-mechanism-based engineering of chemical regulators targeting distinct pathological factors in Alzheimer's disease.

    Science.gov (United States)

    Beck, Michael W; Derrick, Jeffrey S; Kerr, Richard A; Oh, Shin Bi; Cho, Woo Jong; Lee, Shin Jung C; Ji, Yonghwan; Han, Jiyeon; Tehrani, Zahra Aliakbar; Suh, Nayoung; Kim, Sujeong; Larsen, Scott D; Kim, Kwang S; Lee, Joo-Yong; Ruotolo, Brandon T; Lim, Mi Hee

    2016-10-13

    The absence of effective therapeutics against Alzheimer's disease (AD) is a result of the limited understanding of its multifaceted aetiology. Because of the lack of chemical tools to identify pathological factors, investigations into AD pathogenesis have also been insubstantial. Here we report chemical regulators that demonstrate distinct specificity towards targets linked to AD pathology, including metals, amyloid-β (Aβ), metal-Aβ, reactive oxygen species, and free organic radicals. We obtained these chemical regulators through a rational structure-mechanism-based design strategy. We performed structural variations of small molecules for fine-tuning their electronic properties, such as ionization potentials and mechanistic pathways for reactivity towards different targets. We established in vitro and/or in vivo efficacies of the regulators for modulating their targets' reactivities, ameliorating toxicity, reducing amyloid pathology, and improving cognitive deficits. Our chemical tools show promise for deciphering AD pathogenesis and discovering effective drugs.

  19. Structure, function, and regulation of enzymes involved in amino acid metabolism of bacteria and archaea.

    Science.gov (United States)

    Tomita, Takeo

    2017-11-01

    Amino acids are essential components in all organisms because they are building blocks of proteins. They are also produced industrially and used for various purposes. For example, L-glutamate is used as the component of "umami" taste and lysine has been used as livestock feed. Recently, many kinds of amino acids have attracted attention as biological regulators and are used for a healthy life. Thus, to clarify the mechanism of how amino acids are biosynthesized and how they work as biological regulators will lead to further effective utilization of them. Here, I review the leucine-induced-allosteric activation of glutamate dehydrogenase (GDH) from Thermus thermophilus and the relationship with the allosteric regulation of GDH from mammals. Next, I describe structural insights into the efficient production of L-glutamate by GDH from an excellent L-glutamate producer, Corynebacterium glutamicum. Finally, I review the structural biology of lysine biosynthesis of thermophilic bacterium and archaea.

  20. Structural basis of dual Ca2+/pH regulation of the endolysosomal TRPML1 channel

    Energy Technology Data Exchange (ETDEWEB)

    Li, Minghui; Zhang, Wei K.; Benvin, Nicole M.; Zhou, Xiaoyuan; Su, Deyuan; Li, Huan; Wang, Shu; Michailidis, Ioannis E.; Tong, Liang; Li, Xueming; Yang, Jian

    2017-01-23

    The activities of organellar ion channels are often regulated by Ca2+ and H+, which are present in high concentrations in many organelles. Here we report a structural element critical for dual Ca2+/pH regulation of TRPML1, a Ca2+-release channel crucial for endolysosomal function. TRPML1 mutations cause mucolipidosis type IV (MLIV), a severe lysosomal storage disorder characterized by neurodegeneration, mental retardation and blindness. We obtained crystal structures of the 213-residue luminal domain of human TRPML1 containing three missense MLIV-causing mutations. This domain forms a tetramer with a highly electronegative central pore formed by a novel luminal pore loop. Cysteine cross-linking and cryo-EM analyses confirmed that this architecture occurs in the full-length channel. Structure–function studies demonstrated that Ca2+ and H+ interact with the luminal pore and exert physiologically important regulation. The MLIV-causing mutations disrupt the luminal-domain structure and cause TRPML1 mislocalization. Our study reveals the structural underpinnings of TRPML1's regulation, assembly and pathogenesis.

  1. Abnormalities in the structural covariance of emotion regulation networks in major depressive disorder.

    Science.gov (United States)

    Wu, Huawang; Sun, Hui; Wang, Chao; Yu, Lin; Li, Yilan; Peng, Hongjun; Lu, Xiaobing; Hu, Qingmao; Ning, Yuping; Jiang, Tianzi; Xu, Jinping; Wang, Jiaojian

    2017-01-01

    Major depressive disorder (MDD) is a common psychiatric disorder that is characterized by cognitive deficits and affective symptoms. To date, an increasing number of neuroimaging studies have focused on emotion regulation and have consistently shown that emotion dysregulation is one of the central features and underlying mechanisms of MDD. Although gray matter morphological abnormalities in regions within emotion regulation networks have been identified in MDD, the interactions and relationships between these gray matter structures remain largely unknown. Thus, in this study, we adopted a structural covariance method based on gray matter volume to investigate the brain morphological abnormalities within the emotion regulation networks in a large cohort of 65 MDD patients and 65 age- and gender-matched healthy controls. A permutation test with p covariance connectivity strengths between MDD patients and healthy controls. The structural covariance analysis revealed an increased correlation strength of gray matter volume between the left angular gyrus and the left amygdala and between the right angular gyrus and the right amygdala, as well as a decreased correlation strength of the gray matter volume between the right angular gyrus and the posterior cingulate cortex in MDD. Our findings support the notion that emotion dysregulation is an underlying mechanism of MDD by revealing disrupted structural covariance patterns in the emotion regulation network. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. 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

  3. GntR family of regulators in Mycobacterium smegmatis: a sequence and structure based characterization

    Directory of Open Access Journals (Sweden)

    Ranjan Akash

    2007-08-01

    Full Text Available Abstract Background Mycobacterium smegmatis is fast growing non-pathogenic mycobacteria. This organism has been widely used as a model organism to study the biology of other virulent and extremely slow growing species like Mycobacterium tuberculosis. Based on the homology of the N-terminal DNA binding domain, the recently sequenced genome of M. smegmatis has been shown to possess several putative GntR regulators. A striking characteristic feature of this family of regulators is that they possess a conserved N-terminal DNA binding domain and a diverse C-terminal domain involved in the effector binding and/or oligomerization. Since the physiological role of these regulators is critically dependent upon effector binding and operator sites, we have analysed and classified these regulators into their specific subfamilies and identified their potential binding sites. Results The sequence analysis of M. smegmatis putative GntRs has revealed that FadR, HutC, MocR and the YtrA-like regulators are encoded by 45, 8, 8 and 1 genes respectively. Further out of 45 FadR-like regulators, 19 were classified into the FadR group and 26 into the VanR group. All these proteins showed similar secondary structural elements specific to their respective subfamilies except MSMEG_3959, which showed additional secondary structural elements. Using the reciprocal BLAST searches, we further identified the orthologs of these regulators in Bacillus subtilis and other mycobacteria. Since the expression of many regulators is auto-regulatory, we have identified potential operator sites for a number of these GntR regulators by analyzing the upstream sequences. Conclusion This study helps in extending the annotation of M. smegmatis GntR proteins. It identifies the GntR regulators of M. smegmatis that could serve as a model for studying orthologous regulators from virulent as well as other saprophytic mycobacteria. This study also sheds some light on the nucleotide preferences in the

  4. Duplication and Nuclear Envelope Insertion of the Yeast Microtubule Organizing Centre, the Spindle Pole Body

    Directory of Open Access Journals (Sweden)

    Diana Rüthnick

    2018-05-01

    Full Text Available The main microtubule organizing centre in the unicellular model organisms Saccharomyces cerevisiae and Schizosaccharomyces pompe is the spindle pole body (SPB. The SPB is a multilayer structure, which duplicates exactly once per cell cycle. Unlike higher eukaryotic cells, both yeast model organisms undergo mitosis without breakdown of the nuclear envelope (NE, a so-called closed mitosis. Therefore, in order to simultaneously nucleate nuclear and cytoplasmic MTs, it is vital to embed the SPB into the NE at least during mitosis, similarly to the nuclear pore complex (NPC. This review aims to embrace the current knowledge of the SPB duplication cycle with special emphasis on the critical step of the insertion of the new SPB into the NE.

  5. 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.

  6. 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 ...

  7. Functional 5' UTR mRNA structures in eukaryotic translation regulation and how to find them.

    Science.gov (United States)

    Leppek, Kathrin; Das, Rhiju; Barna, Maria

    2018-03-01

    RNA molecules can fold into intricate shapes that can provide an additional layer of control of gene expression beyond that of their sequence. In this Review, we discuss the current mechanistic understanding of structures in 5' untranslated regions (UTRs) of eukaryotic mRNAs and the emerging methodologies used to explore them. These structures may regulate cap-dependent translation initiation through helicase-mediated remodelling of RNA structures and higher-order RNA interactions, as well as cap-independent translation initiation through internal ribosome entry sites (IRESs), mRNA modifications and other specialized translation pathways. We discuss known 5' UTR RNA structures and how new structure probing technologies coupled with prospective validation, particularly compensatory mutagenesis, are likely to identify classes of structured RNA elements that shape post-transcriptional control of gene expression and the development of multicellular organisms.

  8. Mms1 is an assistant for regulating G-quadruplex DNA structures.

    Science.gov (United States)

    Schwindt, Eike; Paeschke, Katrin

    2017-11-02

    The preservation of genome stability is fundamental for every cell. Genomic integrity is constantly challenged. Among those challenges are also non-canonical nucleic acid structures. In recent years, scientists became aware of the impact of G-quadruplex (G4) structures on genome stability. It has been shown that folded G4-DNA structures cause changes in the cell, such as transcriptional up/down-regulation, replication stalling, or enhanced genome instability. Multiple helicases have been identified to regulate G4 structures and by this preserve genome stability. Interestingly, although these helicases are mostly ubiquitous expressed, they show specificity for G4 regulation in certain cellular processes (e.g., DNA replication). To this date, it is not clear how this process and target specificity of helicases are achieved. Recently, Mms1, an ubiquitin ligase complex protein, was identified as a novel G4-DNA-binding protein that supports genome stability by aiding Pif1 helicase binding to these regions. In this perspective review, we discuss the question if G4-DNA interacting proteins are fundamental for helicase function and specificity at G4-DNA structures.

  9. A DNA Origami Mechanical Device for the Regulation of Microcosmic Structural Rigidity.

    Science.gov (United States)

    Wan, Neng; Hong, Zhouping; Wang, Huading; Fu, Xin; Zhang, Ziyue; Li, Chao; Xia, Han; Fang, Yan; Li, Maoteng; Zhan, Yi; Yang, Xiangliang

    2017-11-01

    DNA origami makes it feasible to fabricate a tremendous number of DNA nanostructures with various geometries, dimensions, and functionalities. Moreover, an increasing amount of research on DNA nanostructures is focused on biological and biomedical applications. Here, the reversible regulation of microcosmic structural rigidity is accomplished using a DNA origami device in vitro. The designed DNA origami monomer is composed of an internal central axis and an external sliding tube. Due to the external tube sliding, the device transforms between flexible and rigid states. By transporting the device into the liposome, the conformational change of the origami device induces a structural change in the liposome. The results obtained demonstrate that the programmed DNA origami device can be applied to regulate the microcosmic structural rigidity of liposomes. Because microcosmic structural rigidity is important to cell proliferation and function, the results obtained potentially provide a foundation for the regulation of cell microcosmic structural rigidity using DNA nanostructures. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Phosphorylation Regulates the Bound Structure of an Intrinsically Disordered Protein: The p53-TAZ2 Case.

    Directory of Open Access Journals (Sweden)

    Raúl Esteban Ithuralde

    Full Text Available Disordered regions and Intrinsically Disordered Proteins (IDPs are involved in critical cellular processes and may acquire a stable three-dimensional structure only upon binding to their partners. IDPs may follow a folding-after-binding process, known as induced folding, or a folding-before-binding process, known as conformational selection. The transcription factor p53 is involved in the regulation of cellular events that arise upon stress or DNA damage. The p53 domain structure is composed of an N-terminal transactivation domain (p53TAD, a DNA Binding Domain and a tetramerization domain. The activity of TAD is tightly regulated by interactions with cofactors, inhibitors and phosphorylation. To initiate transcription, p53TAD binds to the TAZ2 domain of CBP, a co-transcription factor, and undergoes a folding and binding process, as revealed by the recent NMR structure of the complex. The activity of p53 is regulated by phosphorylation at multiple sites on the TAD domain and recent studies have shown that modifications at three residues affect the binding towards TAZ2. However, we still do not know how these phosphorylations affect the structure of the bound state and, therefore, how they regulate the p53 function. In this work, we have used computational simulations to understand how phosphorylation affects the structure of the p53TAD:TAZ2 complex and regulates the recognition mechanism. Phosphorylation has been proposed to enhance binding by direct interaction with the folded protein or by changing the unbound conformation of IDPs, for example by pre-folding the protein favoring the recognition mechanism. Here, we show an interesting turn in the p53 case: phosphorylation mainly affects the bound structure of p53TAD, highlighting the complexity of IDP protein-protein interactions. Our results are in agreement with previous experimental studies, allowing a clear picture of how p53 is regulated by phosphorylation and giving new insights into how

  11. Template-free fabrication and morphology regulation of Ag@carbon composite structure

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Wenyan, E-mail: zhangwenyan8531@gmail.com [College of Material Engineering, Jinling Institute of Technology, Nanjing (China); Hao, Lingyun; Lin, Qin [College of Material Engineering, Jinling Institute of Technology, Nanjing (China); Lu, Chunhua; Xu, Zhongzi [College of Materials Science and Engineering, Nanjing Technology University, Nanjing (China); Chen, Xiaoyu [College of Material Engineering, Jinling Institute of Technology, Nanjing (China)

    2014-12-15

    Graphical abstract: - Highlights: • A simple and low-cost method to prepare Ag@C composite material. • AgNO{sub 3} plays an important role in tuning size and functional groups of products. • HTC reaction time is also a key factor for regulating the Ag@C structure. - Abstract: Ag–carbon composite materials were prepared without any template by hydrothermal carbonization of solvable starch. The composite materials are composed of Ag cores and carbonaceous shell to form a core–shell (Ag@carbon) structure. During the hydrothermal carbonization process, the aromatization and carbonization of solvable starch endowed the Ag@carbon composite structure with abundant aromatic, hydroxyl and carbonyl groups. The AgNO{sub 3} concentration and HTC reaction time are two important factors for regulating the size, morphology and functional groups of the composite material. With the increasing of AgNO{sub 3} concentration, morphologies of the composite material turned from spheres to wires.

  12. 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

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. Live visualizations of single isolated tubulin protein self-assembly via tunneling current: effect of electromagnetic pumping during spontaneous growth of microtubule.

    Science.gov (United States)

    Sahu, Satyajit; Ghosh, Subrata; Fujita, Daisuke; Bandyopadhyay, Anirban

    2014-12-03

    As we bring tubulin protein molecules one by one into the vicinity, they self-assemble and entire event we capture live via quantum tunneling. We observe how these molecules form a linear chain and then chains self-assemble into 2D sheet, an essential for microtubule, --fundamental nano-tube in a cellular life form. Even without using GTP, or any chemical reaction, but applying particular ac signal using specially designed antenna around atomic sharp tip we could carry out the self-assembly, however, if there is no electromagnetic pumping, no self-assembly is observed. In order to verify this atomic scale observation, we have built an artificial cell-like environment with nano-scale engineering and repeated spontaneous growth of tubulin protein to its complex with and without electromagnetic signal. We used 64 combinations of plant, animal and fungi tubulins and several doping molecules used as drug, and repeatedly observed that the long reported common frequency region where protein folds mechanically and its structures vibrate electromagnetically. Under pumping, the growth process exhibits a unique organized behavior unprecedented otherwise. Thus, "common frequency point" is proposed as a tool to regulate protein complex related diseases in the future.

  18. The structure of common emotion regulation strategies: A meta-analytic examination.

    Science.gov (United States)

    Naragon-Gainey, Kristin; McMahon, Tierney P; Chacko, Thomas P

    2017-04-01

    Emotion regulation has been examined extensively with regard to important outcomes, including psychological and physical health. However, the literature includes many different emotion regulation strategies but little examination of how they relate to one another, making it difficult to interpret and synthesize findings. The goal of this meta-analysis was to examine the underlying structure of common emotion regulation strategies (i.e., acceptance, behavioral avoidance, distraction, experiential avoidance, expressive suppression, mindfulness, problem solving, reappraisal, rumination, worry), and to evaluate this structure in light of theoretical models of emotion regulation. We also examined how distress tolerance-an important emotion regulation ability -relates to strategy use. We conducted meta-analyses estimating the correlations between emotion regulation strategies (based on 331 samples and 670 effect sizes), as well as between distress tolerance and strategies. The resulting meta-analytic correlation matrix was submitted to confirmatory and exploratory factor analyses. None of the confirmatory models, based on prior theory, was an acceptable fit to the data. Exploratory factor analysis suggested that 3 underlying factors best characterized these data. Two factors-labeled Disengagement and Aversive Cognitive Perseveration-emerged as strongly correlated but distinct factors, with the latter consisting of putatively maladaptive strategies. The third factor, Adaptive Engagement, was a less unified factor and weakly related to the other 2 factors. Distress tolerance was most closely associated with low levels of repetitive negative thought and experiential avoidance, and high levels of acceptance and mindfulness. We discuss the theoretical implications of these findings and applications to emotion regulation assessment. (PsycINFO Database Record (c) 2017 APA, all rights reserved).

  19. 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 ...

  20. Structural Basis for DNA Recognition by the Two-Component Response Regulator RcsB.

    Science.gov (United States)

    Filippova, Ekaterina V; Zemaitaitis, Bozena; Aung, Theint; Wolfe, Alan J; Anderson, Wayne F

    2018-02-27

    RcsB is a highly conserved transcription regulator of the Rcs phosphorelay system, a complex two-component signal transduction system (N. Majdalani and S. Gottesman, Annu Rev Microbiol 59:379-405, 2005; A. J. Wolfe, Curr Opin Microbiol 13:204-209, 2010, https://doi.org/10.1016/j.mib.2010.01.002; D. J. Clarke, Future Microbiol 5:1173-1184, 2010, https://doi.org/10.2217/fmb.10.83). RcsB plays an important role in virulence and pathogenicity in human hosts by regulating biofilm formation. RcsB can regulate transcription alone or together with its auxiliary transcription regulators by forming heterodimers. This complexity allows RcsB to regulate transcription of more than 600 bacterial genes in response to different stresses (D. Wang et al., Mol Plant Microbe Interact 25:6-17, 2012, https://doi.org/10.1094/MPMI-08-11-0207). Despite increasing knowledge of RcsB importance, molecular mechanisms that drive the ability of RcsB to control transcription of a large number of genes remain unclear. Here, we present crystal structures of unphosphorylated RcsB in complex with the consensus DNA-binding sequence of 22-mer (DNA22) and 18-mer (DNA18) of the flhDC operon from Escherichia coli determined at 3.15- and 3.37-Å resolution, respectively. The results of our structural analysis combined with the results of in vitro binding assays provide valuable insights to the protein regulatory mechanism, demonstrate how RcsB recognizes target DNA sequences, and reveal a unique oligomeric state that allows RcsB to form homo- and heterodimers. This information will help us understand the complex mechanisms of transcriptional regulation by RcsB in bacteria. IMPORTANCE RcsB is a well-studied two-component response regulator of the Rcs phosphorelay system, conserved within the family Enterobacteriaceae , which includes many pathogens. It is a global regulator, controlling more than 5% of bacterial genes associated with capsule biosynthesis, flagellar biogenesis, cell wall biosynthesis

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. Crystal Structure of a Putative HTH-Type Transcriptional Regulator yxaF from Bacillus subtilis

    International Nuclear Information System (INIS)

    Seetharaman, J.; Kumaran, D.; Bonanno, J.; Burley, S.; Swaminathan, S.

    2006-01-01

    The New York Structural GenomiX Research Consortium (NYSGXRC) has selected the protein coded by yxaF gene from Bacillus subtilis as a target for structure determination. The yxaF protein has 191 residues with a molecular mass of 21 kDa and had no sequence homology to any structure in the Protein Data Bank (PDB) at the time of target selection. We aimed to elucidate the three-dimensional structure for the putative protein yxaF to better understand the relationship between protein sequence, structure, and function. This protein is annotated as a putative helix-turn-helix (HTH) type transcriptional regulator. Many transcriptional regulators like TetR and QacR use a structurally well-defined DNA-binding HTH motif to recognize the target DNA sequences. DNA-HTH motif interactions have been extensively studied. As the HTH motif is structurally conserved in many regulatory proteins, these DNA-protein complexes show some similarity in DNA recognition patterns. Many such regulatory proteins have a ligand-binding domain in addition to the DNA-binding domain. Structural studies on ligand-binding regulatory proteins provide a wealth of information on ligand-, and possibly drug-, binding mechanisms. Understanding the ligand-binding mechanism may help overcome problems with drug resistance, which represent increasing challenges in medicine. The protein encoded by yxaF, hereafter called T1414, shows fold similar to QacR repressor and TetR/CamR repressor and possesses putative DNA and ligand-binding domains. Here, we report the crystal structure of T1414 and compare it with structurally similar drug and DNA-binding proteins

  6. The structure of brain glycogen phosphorylase-from allosteric regulation mechanisms to clinical perspectives.

    Science.gov (United States)

    Mathieu, Cécile; Dupret, Jean-Marie; Rodrigues Lima, Fernando

    2017-02-01

    Glycogen phosphorylase (GP) is the key enzyme that regulates glycogen mobilization in cells. GP is a complex allosteric enzyme that comprises a family of three isozymes: muscle GP (mGP), liver GP (lGP), and brain GP (bGP). Although the three isozymes display high similarity and catalyze the same reaction, they differ in their sensitivity to the allosteric activator adenosine monophosphate (AMP). Moreover, inactivating mutations in mGP and lGP have been known to be associated with glycogen storage diseases (McArdle and Hers disease, respectively). The determination, decades ago, of the structure of mGP and lGP have allowed to better understand the allosteric regulation of these two isoforms and the development of specific inhibitors. Despite its important role in brain glycogen metabolism, the structure of the brain GP had remained elusive. Here, we provide an overview of the human brain GP structure and its relationship with the two other members of this key family of the metabolic enzymes. We also summarize how this structure provides valuable information to understand the regulation of bGP and to design specific ligands of potential pharmacological interest. © 2016 Federation of European Biochemical Societies.

  7. Structure of noncoding RNA is a determinant of function of RNA binding proteins in transcriptional regulation

    Directory of Open Access Journals (Sweden)

    Oyoshi Takanori

    2012-01-01

    Full Text Available Abstract The majority of the noncoding regions of mammalian genomes have been found to be transcribed to generate noncoding RNAs (ncRNAs, resulting in intense interest in their biological roles. During the past decade, numerous ncRNAs and aptamers have been identified as regulators of transcription. 6S RNA, first described as a ncRNA in E. coli, mimics an open promoter structure, which has a large bulge with two hairpin/stalk structures that regulate transcription through interactions with RNA polymerase. B2 RNA, which has stem-loops and unstructured single-stranded regions, represses transcription of mRNA in response to various stresses, including heat shock in mouse cells. The interaction of TLS (translocated in liposarcoma with CBP/p300 was induced by ncRNAs that bind to TLS, and this in turn results in inhibition of CBP/p300 histone acetyltransferase (HAT activity in human cells. Transcription regulator EWS (Ewing's sarcoma, which is highly related to TLS, and TLS specifically bind to G-quadruplex structures in vitro. The carboxy terminus containing the Arg-Gly-Gly (RGG repeat domains in these proteins are necessary for cis-repression of transcription activation and HAT activity by the N-terminal glutamine-rich domain. Especially, the RGG domain in the carboxy terminus of EWS is important for the G-quadruplex specific binding. Together, these data suggest that functions of EWS and TLS are modulated by specific structures of ncRNAs.

  8. 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.

  9. Structural and functional organization of growing tips of Neurospora crassa Hyphae.

    Science.gov (United States)

    Potapova, T V

    2014-07-01

    Data are presented on a variety of intracellular structures of the vegetative hyphae of the filamentous fungus Neurospora crassa and the involvement of these structures in the tip growth of the hyphae. Current ideas on the molecular and genetic mechanisms of tip growth and regulation of this process are considered. On the basis of comparison of data on behaviors of mitochondria and microtubules and data on the electrical heterogeneity of the hyphal apex, a hypothesis is proposed about a possible supervisory role of the longitudinal electric field in the structural and functional organization of growing tips of the N. crassa hyphae.

  10. Recovery of microtubules on the blepharoplast of Ceratopteris spermatogenous cells after oryzalin treatment.

    Science.gov (United States)

    Vaughn, Kevin C; Bowling, Andrew J

    2008-11-01

    Most land plants have ill-defined microtubule-organizing centers (MTOCs), consisting of sites on the nuclear envelope or even along microtubules (MTs). In contrast, the spermatogenous cells of the pteridophyte Ceratopteris richardii have a well-defined MTOC, the blepharoplast, which organizes MTs through the last two division cycles. This allows a rare opportunity to study the organization and workings of a structurally well-defined plant MTOC. In this study, antheridial plants were treated with levels of oryzalin that cause complete MT loss from the cells containing blepharoplasts. The oryzalin was then washed out and plants were allowed to recover for varying amounts of time. If the spermatogenous cells were fixed prior to washing out, the blepharoplasts had an unusual appearance. In the matrix (pericentriolar) material where MT ends are normally found, clear areas of about the diameter of MTs were seen embedded in a much deeper matrix, made more obvious in stereo pairs. Occasionally, the matrix material was highly distended, although the basal body template cylinder morphology appeared to be unaltered. The blepharoplasts often occurred as clusters of 2 or 4, indicating that blepharoplast reproduction is not affected by the lack of MTs, but that their movement to the poles is. Gamma (gamma) tubulin antibodies labeled the edge of the blepharoplast in areas where the pits are located, indicating that these might be sites for MT nucleation. After wash out, the new MTs always re-appeared on the blepharoplast and the recovery occurred within an hour of washout. MT lengths increased with increasing washout time and were indistinguishable from untreated blepharoplasts after 24 h of recovery. After washout, arrays formed in new sperm cells such as the spline and basal bodies were often malformed or present in multiple copies, as were the blepharoplasts in these cells prior to wash out. These data indicate that the blepharoplast serves as the site of MT nucleation and

  11. Proposal of guidelines for structuring an independent regulation body for the Brazilian nuclear sector

    International Nuclear Information System (INIS)

    Nicoll Junior, Ricardo

    2016-01-01

    Regulatory bodies are responsible for regulation in various sectors of society. In Brazil, they work in various areas for the development of the country and have as main objective the social, economic and national development. The progress of new technologies in the nuclear field and their commercialization underscores the need for regulation according to international safety standards. The present research searches through an extensive review of the literature identify the international guidelines for regulatory bodies and make a comparative analysis between Brazil and five countries that have independent regulatory bodies in the nuclear sector. The purpose of the work is to contribute to the Brazilian public sectors, with an evaluation of the country's regulation in the perception of specialists and propose guidelines for the structuring of an independent regulatory body, respecting international agreements and the legislation in force in the country. (author)

  12. Structural Characterization of Lignin in Wild-Type versus COMT Down-Regulated Switchgrass

    Energy Technology Data Exchange (ETDEWEB)

    Samuel, Reichel [School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA (United States); BioEnergy Science Center, Oak Ridge, TN (United States); Pu, Yunqiao, E-mail: yunqiao.pu@ipst.gatech.edu [BioEnergy Science Center, Oak Ridge, TN (United States); Institute of Paper Science and Technology, Georgia Institute of Technology, Atlanta, GA (United States); Jiang, Nan [School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA (United States); BioEnergy Science Center, Oak Ridge, TN (United States); Fu, Chunxiang [Forage Improvement Division, The Samuel Roberts Noble Foundation, Ardmore, OK (United States); Wang, Zeng-Yu [BioEnergy Science Center, Oak Ridge, TN (United States); Forage Improvement Division, The Samuel Roberts Noble Foundation, Ardmore, OK (United States); Ragauskas, Arthur, E-mail: yunqiao.pu@ipst.gatech.edu [School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA (United States); BioEnergy Science Center, Oak Ridge, TN (United States)

    2014-01-20

    This study examined the chemical structural characteristics of cellulolytic enzyme lignin isolated from switchgrass focusing on comparisons between wild-type control and caffeic acid 3-O-methyltransferase (COMT) down-regulated transgenic line. Nuclear magnetic resonance techniques including {sup 13}C, {sup 31}P, and two-dimensional {sup 13}C-{sup 1}H heteronuclear single quantum coherence as well as gel permeation chromatography were employed. Compared to the wild-type, the COMT down-regulated transgenic switchgrass lignin demonstrated a decrease in syringyl (S):guaiacyl (G) ratio and p-coumarate:ferulate ratio, an increase in relative abundance of phenylcoumaran unit, and a comparable content of total free phenolic OH groups along with formation of benzodioxane unit. In addition, COMT down-regulation had no significant effects on the lignin molecular weights during its biosynthesis process.

  13. Structure of Rot, a global regulator of virulence genes in Staphylococcus aureus.

    Science.gov (United States)

    Zhu, Yuwei; Fan, Xiaojiao; Zhang, Xu; Jiang, Xuguang; Niu, Liwen; Teng, Maikun; Li, Xu

    2014-09-01

    Staphylococcus aureus is a highly versatile pathogen that can infect human tissue by producing a large arsenal of virulence factors that are tightly regulated by a complex regulatory network. Rot, which shares sequence similarity with SarA homologues, is a global regulator that regulates numerous virulence genes. However, the recognition model of Rot for the promoter region of target genes and the putative regulation mechanism remain elusive. In this study, the 1.77 Å resolution X-ray crystal structure of Rot is reported. The structure reveals that two Rot molecules form a compact homodimer, each of which contains a typical helix-turn-helix module and a β-hairpin motif connected by a flexible loop. Fluorescence polarization results indicate that Rot preferentially recognizes AT-rich dsDNA with ~30-base-pair nucleotides and that the conserved positively charged residues on the winged-helix motif are vital for binding to the AT-rich dsDNA. It is proposed that the DNA-recognition model of Rot may be similar to that of SarA, SarR and SarS, in which the helix-turn-helix motifs of each monomer interact with the major grooves of target dsDNA and the winged motifs contact the minor grooves. Interestingly, the structure shows that Rot adopts a novel dimerization model that differs from that of other SarA homologues. As expected, perturbation of the dimer interface abolishes the dsDNA-binding ability of Rot, suggesting that Rot functions as a dimer. In addition, the results have been further confirmed in vivo by measuring the transcriptional regulation of α-toxin, a major virulence factor produced by most S. aureus strains.

  14. Structure and Chromosomal Organization of Yeast Genes Regulated by Topoisomerase II.

    Science.gov (United States)

    Joshi, Ricky S; Nikolaou, Christoforos; Roca, Joaquim

    2018-01-03

    Cellular DNA topoisomerases (topo I and topo II) are highly conserved enzymes that regulate the topology of DNA during normal genome transactions, such as DNA transcription and replication. In budding yeast, topo I is dispensable whereas topo II is essential, suggesting fundamental and exclusive roles for topo II, which might include the functions of the topo IIa and topo IIb isoforms found in mammalian cells. In this review, we discuss major findings of the structure and chromosomal organization of genes regulated by topo II in budding yeast. Experimental data was derived from short (10 min) and long term (120 min) responses to topo II inactivation in top-2 ts mutants. First, we discuss how short term responses reveal a subset of yeast genes that are regulated by topo II depending on their promoter architecture. These short term responses also uncovered topo II regulation of transcription across multi-gene clusters, plausibly by common DNA topology management. Finally, we examine the effects of deactivated topo II on the elongation of RNA transcripts. Each study provides an insight into the particular chromatin structure that interacts with the activity of topo II. These findings are of notable clinical interest as numerous anti-cancer therapies interfere with topo II activity.

  15. Control of 2D Flexible Structures by Confinement of Vibrations and Regulation of Their Energy Flow

    Directory of Open Access Journals (Sweden)

    Fakhreddine Landolsi

    2009-01-01

    Full Text Available In this paper, we investigate the control of 2D flexible structures by vibration confinement and the regulation of their energy flow along prespecified spatial paths. A discretized-model-based feedback strategy, aiming at confining and suppressing simultaneously the vibration, is proposed. It is assumed that the structure consists of parts that are sensitive to vibrations. The control design introduces a new pseudo-modal matrix derived from the computed eigenvectors of the discretized model. Simulations are presented to show the efficacy of the proposed control law. A parametric study is carried out to examine the effects of the different control parameters on the simultaneous confinement and suppression of vibrations. In addition, we conducted a set of simulations to investigate the flow control of vibrational energy during the confinement-suppression process. We found that the energy flow can be regulated via a set of control parameters for different confinement configurations.

  16. Structural insights into GDP-mediated regulation of a bacterial acyl-CoA thioesterase.

    Science.gov (United States)

    Khandokar, Yogesh B; Srivastava, Parul; Cowieson, Nathan; Sarker, Subir; Aragao, David; Das, Shubagata; Smith, Kate M; Raidal, Shane R; Forwood, Jade K

    2017-12-15

    Thioesterases catalyze the cleavage of thioester bonds within many activated fatty acids and acyl-CoA substrates. They are expressed ubiquitously in both prokaryotes and eukaryotes and are subdivided into 25 thioesterase families according to their catalytic active site, protein oligomerization, and substrate specificity. Although many of these enzyme families are well-characterized in terms of function and substrate specificity, regulation across most thioesterase families is poorly understood. Here, we characterized a TE6 thioesterase from the bacterium Neisseria meningitidis Structural analysis with X-ray crystallographic diffraction data to 2.0-Å revealed that each protein subunit harbors a hot dog-fold and that the TE6 enzyme forms a hexamer with D3 symmetry. An assessment of thioesterase activity against a range of acyl-CoA substrates revealed the greatest activity against acetyl-CoA, and structure-guided mutagenesis of putative active site residues identified Asn 24 and Asp 39 as being essential for activity. Our structural analysis revealed that six GDP nucleotides bound the enzyme in close proximity to an intersubunit disulfide bond interactions that covalently link thioesterase domains in a double hot dog dimer. Structure-guided mutagenesis of residues within the GDP-binding pocket identified Arg 93 as playing a key role in the nucleotide interaction and revealed that GDP is required for activity. All mutations were confirmed to be specific and not to have resulted from structural perturbations by X-ray crystallography. This is the first report of a bacterial GDP-regulated thioesterase and of covalent linkage of thioesterase domains through a disulfide bond, revealing structural similarities with ADP regulation in the human ACOT12 thioesterase. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. A Subset of Autism-Associated Genes Regulate the Structural Stability of Neurons

    Science.gov (United States)

    Lin, Yu-Chih; Frei, Jeannine A.; Kilander, Michaela B. C.; Shen, Wenjuan; Blatt, Gene J.

    2016-01-01

    Autism spectrum disorder (ASD) comprises a range of neurological conditions that affect individuals’ ability to communicate and interact with others. People with ASD often exhibit marked qualitative difficulties in social interaction, communication, and behavior. Alterations in neurite arborization and dendritic spine morphology, including size, shape, and number, are hallmarks of almost all neurological conditions, including ASD. As experimental evidence emerges in recent years, it becomes clear that although there is broad heterogeneity of identified autism risk genes, many of them converge into similar cellular pathways, including those regulating neurite outgrowth, synapse formation and spine stability, and synaptic plasticity. These mechanisms together regulate the structural stability of neurons and are vulnerable targets in ASD. In this review, we discuss the current understanding of those autism risk genes that affect the structural connectivity of neurons. We sub-categorize them into (1) cytoskeletal regulators, e.g., motors and small RhoGTPase regulators; (2) adhesion molecules, e.g., cadherins, NCAM, and neurexin superfamily; (3) cell surface receptors, e.g., glutamatergic receptors and receptor tyrosine kinases; (4) signaling molecules, e.g., protein kinases and phosphatases; and (5) synaptic proteins, e.g., vesicle and scaffolding proteins. Although the roles of some of these genes in maintaining neuronal structural stability are well studied, how mutations contribute to the autism phenotype is still largely unknown. Investigating whether and how the neuronal structure and function are affected when these genes are mutated will provide insights toward developing effective interventions aimed at improving the lives of people with autism and their families. PMID:27909399

  18. Structure analysis of the global metabolic regulator Crc from Pseudomonas aeruginosa.

    Science.gov (United States)

    Wei, Yong; Zhang, Heng; Gao, Zeng-Qiang; Xu, Jian-Hua; Liu, Quan-Sheng; Dong, Yu-Hui

    2013-01-01

    The global metabolic regulator catabolite repression control (Crc) has recently been found to modulate the susceptibility to antibiotics and virulence in the opportunistic pathogen Pseudomonas aeruginosa and been suggested as a nonlethal target for novel antimicrobials. In P. aeruginosa, Crc couples with the CA motifs from the small RNA CrcZ to form a post-transcriptional regulator system and is removed from the 5'-end of the target mRNAs. In this study, we first reported the crystal structure of Crc from P. aeruginosa refined to 2.20 Å. The structure showed that it consists of two halves with similar overall topology and there are 11 β strands surrounded by 13 helices, forming a four-layered α/β-sandwich. The circular dichroism spectroscopy revealed that it is thermostable in solution and shares similar characteristics to that in crystal. Comprehensive structural analysis and comparison with the homologies of Crc showed high similarity with several known nucleases and consequently may be classified into a member exodeoxyribonuclease III. However, it shows distinct substrate specificity (RNA as the preferred substrate) compared to these DNA endonucleases. Structural comparisons also revealed potential RNA recognition and binding region mainly consisting of five flexible loops. Our structure study provided the basis for the future application of Crc as a target to develop new antibiotics. Copyright © 2012 International Union of Biochemistry and Molecular Biology, Inc.

  19. Regulation of Silk Material Structure by Temperature-Controlled Water Vapor Annealing

    Science.gov (United States)

    Hu, Xiao; Shmelev, Karen; Sun, Lin; Gil, Eun-Seok; Park, Sang-Hyug; Cebe, Peggy; Kaplan, David L.

    2011-01-01

    We present a simple and effective method to obtain refined control of the molecular structure of silk biomaterials through physical temperature-controlled water vapor annealing (TCWVA). The silk materials can be prepared with control of crystallinity, from a low content using conditions at 4°C (alpha-helix dominated silk I structure), to highest content of ~60% crystallinity at 100°C (beta-sheet dominated silk II structure). This new physical approach covers the range of structures previously reported to govern crystallization during the fabrication of silk materials, yet offers a simpler, green chemistry, approach with tight control of reproducibility. The transition kinetics, thermal, mechanical, and biodegradation properties of the silk films prepared at different temperatures were investigated and compared by Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), uniaxial tensile studies, and enzymatic degradation studies. The results revealed that this new physical processing method accurately controls structure, in turn providing control of mechanical properties, thermal stability, enzyme degradation rate, and human mesenchymal stem cell interactions. The mechanistic basis for the control is through the temperature controlled regulation of water vapor, to control crystallization. Control of silk structure via TCWVA represents a significant improvement in the fabrication of silk-based biomaterials, where control of structure-property relationships is key to regulating material properties. This new approach to control crystallization also provides an entirely new green approach, avoiding common methods which use organic solvents (methanol, ethanol) or organic acids. The method described here for silk proteins would also be universal for many other structural proteins (and likely other biopolymers), where water controls chain interactions related to material properties. PMID:21425769

  20. Hypergravity signal transduction in HeLa cells with concomitant phosphorylation of proteins immunoprecipitated with anti-microtubule-associated protein antibodies

    Science.gov (United States)

    Kumei, Yasuhiro; Whitson, Peggy A.; Sato, Atsushige; Cintron, Nitza M.

    1991-01-01

    It is shown that hypergravity (35g) stimulates the production of inositol 1,4,5-trisphosphate (IP3) and decreases adenosine 3-prime,5-prime-cyclic monophosphate (cAMP) levels in HeLa cells. It is proposed that IP3 and cAMP may act as second messengers in hypergravity signal transduction. Phosphorylation of microtubule-associated proteins in both the detergent-soluble and -insoluble fractions suggests that cytoskeletal structures may be influenced by gravity.

  1. 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.

  2. 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

  3. 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.

  4. 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

  5. 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

  6. 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.

  7. Crystal structures of two transcriptional regulators from Bacillus cereus define the conserved structural features of a PadR subfamily.

    Directory of Open Access Journals (Sweden)

    Guntur Fibriansah

    Full Text Available PadR-like transcriptional regulators form a structurally-related family of proteins that control the expression of genes associated with detoxification, virulence and multi-drug resistance in bacteria. Only a few members of this family have been studied by genetic, biochemical and biophysical methods, and their structure/function relationships are still largely undefined. Here, we report the crystal structures of two PadR-like proteins from Bacillus cereus, which we named bcPadR1 and bcPadR2 (products of gene loci BC4206 and BCE3449 in strains ATCC 14579 and ATCC 10987, respectively. BC4206, together with its neighboring gene BC4207, was previously shown to become significantly upregulated in presence of the bacteriocin AS-48. DNA mobility shift assays reveal that bcPadR1 binds to a 250 bp intergenic region containing the putative BC4206-BC4207 promoter sequence, while in-situ expression of bcPadR1 decreases bacteriocin tolerance, together suggesting a role for bcPadR1 as repressor of BC4206-BC4207 transcription. The function of bcPadR2 (48% identical in sequence to bcPadR1 is unknown, but the location of its gene just upstream from genes encoding a putative antibiotic ABC efflux pump, suggests a role in regulating antibiotic resistance. The bcPadR proteins are structurally similar to LmrR, a PadR-like transcription regulator in Lactococcus lactis that controls expression of a multidrug ABC transporter via a mechanism of multidrug binding and induction. Together these proteins define a subfamily of conserved, relatively small PadR proteins characterized by a single C-terminal helix for dimerization. Unlike LmrR, bcPadR1 and bcPadR2 lack a central pore for ligand binding, making it unclear whether the transcriptional regulatory roles of bcPadR1 and bcPadR2 involve direct ligand recognition and induction.

  8. The solution structure of ChaB, a putative membrane ion antiporter regulator from Escherichia coli

    Directory of Open Access Journals (Sweden)

    Iannuzzi Pietro

    2004-08-01

    Full Text Available Abstract Background ChaB is a putative regulator of ChaA, a Na+/H+ antiporter that also has Ca+/H+ activity in E. coli. ChaB contains a conserved 60-residue region of unknown function found in other bacteria, archaeabacteria and a series of baculoviral proteins. As part of a structural genomics project, the structure of ChaB was elucidated by NMR spectroscopy. Results The structure of ChaB is composed of 3 α-helices and a small sheet that pack tightly to form a fold that is found in the cyclin-box family of proteins. Conclusion ChaB is distinguished from its putative DNA binding sequence homologues by a highly charged flexible loop region that has weak affinity to Mg2+ and Ca2+ divalent metal ions.

  9. High-resolution structure of TBP with TAF1 reveals anchoring patterns in transcriptional regulation.

    Science.gov (United States)

    Anandapadamanaban, Madhanagopal; Andresen, Cecilia; Helander, Sara; Ohyama, Yoshifumi; Siponen, Marina I; Lundström, Patrik; Kokubo, Tetsuro; Ikura, Mitsuhiko; Moche, Martin; Sunnerhagen, Maria

    2013-08-01

    The general transcription factor TFIID provides a regulatory platform for transcription initiation. Here we present the crystal structure (1.97 Å) and NMR analysis of yeast TAF1 N-terminal domains TAND1 and TAND2 bound to yeast TBP, together with mutational data. We find that yeast TAF1-TAND1, which in itself acts as a transcriptional activator, binds TBP's concave DNA-binding surface by presenting similar anchor residues to TBP as does Mot1 but from a distinct structural scaffold. Furthermore, we show how TAF1-TAND2 uses an aromatic and acidic anchoring pattern to bind a conserved TBP surface groove traversing the basic helix region, and we find highly similar TBP-binding motifs also presented by the structurally distinct TFIIA, Mot1 and Brf1 proteins. Our identification of these anchoring patterns, which can be easily disrupted or enhanced, provides insight into the competitive multiprotein TBP interplay critical to transcriptional regulation.

  10. Chloroplast Translation: Structural and Functional Organization, Operational Control, and Regulation[OPEN

    Science.gov (United States)

    2018-01-01

    Chloroplast translation is essential for cellular viability and plant development. Its positioning at the intersection of organellar RNA and protein metabolism makes it a unique point for the regulation of gene expression in response to internal and external cues. Recently obtained high-resolution structures of plastid ribosomes, the development of approaches allowing genome-wide analyses of chloroplast translation (i.e., ribosome profiling), and the discovery of RNA binding proteins involved in the control of translational activity have greatly increased our understanding of the chloroplast translation process and its regulation. In this review, we provide an overview of the current knowledge of the chloroplast translation machinery, its structure, organization, and function. In addition, we summarize the techniques that are currently available to study chloroplast translation and describe how translational activity is controlled and which cis-elements and trans-factors are involved. Finally, we discuss how translational control contributes to the regulation of chloroplast gene expression in response to developmental, environmental, and physiological cues. We also illustrate the commonalities and the differences between the chloroplast and bacterial translation machineries and the mechanisms of protein biosynthesis in these two prokaryotic systems. PMID:29610211

  11. Distorsioni strutturali della regolamentazione prudenziale delle banche (Structural distortions in prudential regulation of banks

    Directory of Open Access Journals (Sweden)

    Mario Tonveronachi

    2002-03-01

    Full Text Available At the national level, financial regulation based on prohibitions, the so-called structural rules, was accused of producing significant distortion. The degree of competition it was limited, encouraging inefficiencies of all types; the culture of risk it was weakened; wide discretionary powers were used by national authorities to distort market mechanisms and public ownership distorted competition and fostered cronyism.A new common regulative culture is therefore emerged, based on internal free competition within the banking sector and the financial system as a whole. It requires the elimination of strict limits to banking, the abandonment of the principle of specialization between commercial banking and financial asset bank, the affirmation of private property, the liability of the banks in a stronger market discipline, even with regard to their corporate governance.The Basel rules distorts no less serious than those attributed to the previous structural rules. Excessive competition is no less harmful than a low degree of competition, setting the “level playing field” helps large dimension and practicas “too big to fail”, the “capital crunches” produce serious effects on the economy, while the regulatory costs absorb important share resources of smaller banks. It is a matter of further research whether the new approach to regulation has also favored an increase in the share of national income absorbed by the financial system without having produced a better distribution of risk and a proportional increase in what James Tobin (1984 has defined efficiency of full insurance.

  12. Functional Role of Cyclin-Dependent Kinase 5 in the Regulation of Melanogenesis and Epidermal Structure.

    Science.gov (United States)

    Dong, Changsheng; Yang, Shanshan; Fan, Ruiwen; Ji, Kaiyuan; Zhang, Junzhen; Liu, Xuexian; Hu, Shuaipeng; Xie, Jianshan; Liu, Yu; Gao, Wenjun; Wang, Haidong; Yao, Jianbo; Smith, George W; Herrid, Muren

    2017-10-23

    The mammalian integumentary system plays important roles in body homeostasis, and dysfunction of melanogenesis or epidermal development may lead to a variety of skin diseases, including melanoma. Skin pigmentation in humans and coat color in fleece-producing animals are regulated by many genes. Among them, microphthalmia-associated transcription factor (MITF) and paired-box 3 (PAX3) are at the top of the cascade and regulate activities of many important melanogenic enzymes. Here, we report for the first time that cyclin-dependent kinase 5 (Cdk5) is an essential regulator of MITF and PAX3. Cdk5 knockdown in mice causes a lightened coat color, a polarized distribution of melanin and hyperproliferation of basal keratinocytes. Reduced expression of Keratin 10 (K10) resulting from Cdk5 knockdown may be responsible for an abnormal epidermal structure. In contrast, overexpression of Cdk5 in sheep (Ovis aries) only produces brown patches on a white background, with no other observable abnormalities. Collectively, our findings show that Cdk5 has an important functional role in the regulation of melanin production and transportation and in normal development of the integumentary system.

  13. Factor structure of the Self-Regulation Questionnaire (SRQ) at Spanish universities.

    Science.gov (United States)

    Pichardo, Carmen; Justicia, Fernando; de la Fuente, Jesús; Martínez-Vicente, José Manuel; Berbén, Ana B G

    2014-08-04

    The Self-Regulation Questionnaire (SRQ) has been used in psychology research during the last decade. The instrument has been used in a variety of life domains: psychological well-being, dispositional happiness, depressive symptoms and career adaptability. This investigation studies the factor structure and internal consistency of the SRQ, extracting a short version in the Spanish context and examining its relation to academic variables (self-regulated learning and grades). The analysis started from a version with 63 items, representing seven conceptual dimensions. This version was administered to a sample of 834 students from Education and Psychology. The data from the above-mentioned sample were randomly divided into two sets, each containing 50% of the students (n = 417): exploratory and confirmatory. In the exploratory sample, exploratory factor analysis findings suggested a more parsimonious measurement model, with 17 items and 4 first-order factors. The confirmatory sample was used in the confirmatory factor analysis. The results show evidence for the internal consistency of the Short Self-Regulation Questionnaire (SSRQ) in the Spanish context, with indices greater than .90 and errors around .05. Regarding academic variables, both versions are related to self-regulated learning (r = .40, p < .01) and students' grades (r = .15, p < .01). Differences from other studies done in North America are discussed, as well as similarities to a study from North-West University (in South Africa).

  14. Specific chlamydial inclusion membrane proteins associate with active Src family kinases in microdomains that interact with the host microtubule network.

    Science.gov (United States)

    Mital, Jeffrey; Miller, Natalie J; Fischer, Elizabeth R; Hackstadt, Ted

    2010-09-01

    Chlamydiae are Gram-negative obligate intracellular bacteria that cause diseases with significant medical and economic impact. Chlamydia trachomatis replicates within a vacuole termed an inclusion, which is extensively modified by the insertion of a number of bacterial effector proteins known as inclusion membrane proteins (Incs). Once modified, the inclusion is trafficked in a dynein-dependent manner to the microtubule-organizing centre (MTOC), where it associates with host centrosomes. Here we describe a novel structure on the inclusion membrane comprised of both host and bacterial proteins. Members of the Src family of kinases are recruited to the chlamydial inclusion in an active form. These kinases display a distinct, localized punctate microdomain-like staining pattern on the inclusion membrane that colocalizes with four chlamydial inclusion membrane proteins (Incs) and is enriched in cholesterol. Biochemical studies show that at least two of these Incs stably interact with one another. Furthermore, host centrosomes associate with these microdomain proteins in C. trachomatis-infected cells and in uninfected cells exogenously expressing one of the chlamydial effectors. Together, the data suggest that a specific structure on the C. trachomatis inclusion membrane may be responsible for the known interactions of chlamydiae with the microtubule network and resultant effects on centrosome stability.

  15. 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.

  16. 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.

  17. Non-bilayer structures in mitochondrial membranes regulate ATP synthase activity.

    Science.gov (United States)

    Gasanov, Sardar E; Kim, Aleksandr A; Yaguzhinsky, Lev S; Dagda, Ruben K

    2018-02-01

    Cardiolipin (CL) is an anionic phospholipid at the inner mitochondrial membrane (IMM) that facilitates the formation of transient non-bilayer (non-lamellar) structures to maintain mitochondrial integrity. CL modulates mitochondrial functions including ATP synthesis. However, the biophysical mechanisms by which CL generates non-lamellar structures and the extent to which these structures contribute to ATP synthesis remain unknown. We hypothesized that CL and ATP synthase facilitate the formation of non-bilayer structures at the IMM to stimulate ATP synthesis. By using 1 H NMR and 31 P NMR techniques, we observed that increasing the temperature (8°C to 37°C), lowering the pH (3.0), or incubating intact mitochondria with CTII - an IMM-targeted toxin that increases the formation of immobilized non-bilayer structures - elevated the formation of non-bilayer structures to stimulate ATP synthesis. The F 0 sector of the ATP synthase complex can facilitate the formation of non-bilayer structures as incubating model membranes enriched with IMM-specific phospholipids with exogenous DCCD-binding protein of the F 0 sector (DCCD-BPF) elevated the formation of immobilized non-bilayer structures to a similar manner as CTII. Native PAGE assays revealed that CL, but not other anionic phospholipids, specifically binds to DCCD-BPF to promote the formation of stable lipid-protein complexes. Mechanistically, molecular docking studies identified two lipid binding sites for CL in DCCD-BPF. We propose a new model of ATP synthase regulation in which CL mediates the formation of non-bilayer structures that serve to cluster protons and ATP synthase complexes as a mechanism to enhance proton translocation to the F 0 sector, and thereby increase ATP synthesis. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Photoionization-regulated star formation and the structure of molecular clouds

    Science.gov (United States)

    Mckee, Christopher F.

    1989-01-01

    A model for the rate of low-mass star formation in Galactic molecular clouds and for the influence of this star formation on the structure and evolution of the clouds is presented. The rate of energy injection by newly formed stars is estimated, and the effect of this energy injection on the size of the cloud is determined. It is shown that the observed rate of star formation appears adequate to support the observed clouds against gravitational collapse. The rate of photoionization-regulated star formation is estimated and it is shown to be in agreement with estimates of the observed rate of star formation if the observed molecular cloud parameters are used. The mean cloud extinction and the Galactic star formation rate per unit mass of molecular gas are predicted theoretically from the condition that photionization-regulated star formation be in equilibrium. A simple model for the evolution of isolated molecular clouds is developed.

  19. Neuronal process structure and growth proteins are targets of heavy PTM regulation during brain development

    DEFF Research Database (Denmark)

    Edwards, Alistair V G; Schwämmle, Veit; Larsen, Martin Røssel

    2014-01-01

    UNLABELLED: Brain development is a process requiring precise control of many different cell types. One method to achieve this is through specific and temporally regulated modification of proteins in order to alter structure and function. Post-translational modification (PTM) of proteins is known...... on protein-level events, this study also provides significant insight into detailed roles for individual modified proteins in the developing brain, helping to advance the understanding of the complex protein-driven processes that underlie development. Finally, the use of a novel bioinformatic analytical tool...... provides one of the most comprehensive sets of individual PTM site regulation data for mammalian brain tissue. This will provide a valuable resource for those wishing to perform comparisons or meta-analyses of large scale PTMomic data, as are becoming increasingly common. Furthermore, being focussed...

  20. Fluctuation-driven mechanotransduction regulates mitochondrial-network structure and function

    Science.gov (United States)

    Bartolák-Suki, Erzsébet; Imsirovic, Jasmin; Parameswaran, Harikrishnan; Wellman, Tyler J.; Martinez, Nuria; Allen, Philip G.; Frey, Urs; Suki, Béla

    2015-10-01

    Cells can be exposed to irregular mechanical fluctuations, such as those arising from changes in blood pressure. Here, we report that ATP production, assessed through changes in mitochondrial membrane potential, is downregulated in vascular smooth muscle cells in culture exposed to monotonous stretch cycles when compared with cells exposed to a variable cyclic stretch that incorporates physiological levels of cycle-by-cycle variability in stretch amplitude. Variable stretch enhances ATP production by increasing the expression of ATP synthase’s catalytic domain, cytochrome c oxidase and its tyrosine phosphorylation, mitofusins and PGC-1α. Such a fluctuation-driven mechanotransduction mechanism is mediated by motor proteins and by the enhancement of microtubule-, actin- and mitochondrial-network complexity. We also show that, in aorta rings isolated from rats, monotonous stretch downregulates--whereas variable stretch maintains--physiological vessel-wall contractility through mitochondrial ATP production. Our results have implications for ATP-dependent and mechanosensitive intracellular processes.

  1. 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.)

  2. Reelin exerts structural, biochemical and transcriptional regulation over presynaptic and postsynaptic elements in the adult hippocampus

    Directory of Open Access Journals (Sweden)

    Carles eBosch

    2016-05-01

    Full Text Available Reelin regulates neuronal positioning and synaptogenesis in the developing brain, and adult brain plasticity. Here we used transgenic mice overexpressing Reelin (Reelin-OE mice to perform a comprehensive dissection of the effects of this protein on the structural and biochemical features of dendritic spines and axon terminals in the adult hippocampus. Electron microscopy (EM revealed both higher density of synapses and structural complexity of both pre- and postsynaptic elements in transgenic mice than in WT mice. Dendritic spines had larger spine apparatuses, which correlated with a redistribution of Synaptopodin. Most of the changes observed in Reelin-OE mice were reversible after blockade of transgene expression, thus supporting the specificity of the observed phenotypes. Western blot and transcriptional analyses did not show major changes in the expression of pre- or postsynaptic proteins, including SNARE proteins, glutamate receptors, and scaffolding and signaling proteins. However, EM immunogold assays revealed that the NMDA receptor subunits NR2a and NR2b, and p-Cofilin showed a redistribution from synaptic to extrasynaptic pools. Taken together with previous studies, the present results suggest that Reelin regulates the structural and biochemical properties of adult hippocampal synapses by increasing their density and morphological complexity and by modifying the distribution and trafficking of major glutamatergic components.

  3. Structural basis of SUFU–GLI interaction in human Hedgehog signalling regulation

    International Nuclear Information System (INIS)

    Cherry, Amy L.; Finta, Csaba; Karlström, Mikael; Jin, Qianren; Schwend, Thomas; Astorga-Wells, Juan; Zubarev, Roman A.; Del Campo, Mark; Criswell, Angela R.; Sanctis, Daniele de; Jovine, Luca; Toftgård, Rune

    2013-01-01

    Crystal and small-angle X-ray scattering structures of full-length human SUFU alone and in complex with the conserved SYGHL motif from GLI transcription factors show major conformational changes associated with binding and reveal an intrinsically disordered region crucial for pathway activation. Hedgehog signalling plays a fundamental role in the control of metazoan development, cell proliferation and differentiation, as highlighted by the fact that its deregulation is associated with the development of many human tumours. SUFU is an essential intracellular negative regulator of mammalian Hedgehog signalling and acts by binding and modulating the activity of GLI transcription factors. Despite its central importance, little is known about SUFU regulation and the nature of SUFU–GLI interaction. Here, the crystal and small-angle X-ray scattering structures of full-length human SUFU and its complex with the key SYGHL motif conserved in all GLIs are reported. It is demonstrated that GLI binding is associated with major conformational changes in SUFU, including an intrinsically disordered loop that is also crucial for pathway activation. These findings reveal the structure of the SUFU–GLI interface and suggest a mechanism for an essential regulatory step in Hedgehog signalling, offering possibilities for the development of novel pathway modulators and therapeutics

  4. DNA breaks and chromatin structural changes enhance the transcription of autoimmune regulator target genes.

    Science.gov (United States)

    Guha, Mithu; Saare, Mario; Maslovskaja, Julia; Kisand, Kai; Liiv, Ingrid; Haljasorg, Uku; Tasa, Tõnis; Metspalu, Andres; Milani, Lili; Peterson, Pärt

    2017-04-21

    The autoimmune regulator (AIRE) protein is the key factor in thymic negative selection of autoreactive T cells by promoting the ectopic expression of tissue-specific genes in the thymic medullary epithelium. Mutations in AIRE cause a monogenic autoimmune disease called autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy. AIRE has been shown to promote DNA breaks via its interaction with topoisomerase 2 (TOP2). In this study, we investigated topoisomerase-induced DNA breaks and chromatin structural alterations in conjunction with AIRE-dependent gene expression. Using RNA sequencing, we found that inhibition of TOP2 religation activity by etoposide in AIRE-expressing cells had a synergistic effect on genes with low expression levels. AIRE-mediated transcription was not only enhanced by TOP2 inhibition but also by the TOP1 inhibitor camptothecin. The transcriptional activation was associated with structural rearrangements in chromatin, notably the accumulation of γH2AX and the exchange of histone H1 with HMGB1 at AIRE target gene promoters. In addition, we found the transcriptional up-regulation to co-occur with the chromatin structural changes within the genomic cluster of carcinoembryonic antigen-like cellular adhesion molecule genes. Overall, our results suggest that the presence of AIRE can trigger molecular events leading to an altered chromatin landscape and the enhanced transcription of low-expressed genes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Getting in (and out of) the loop: regulating higher order telomere structures.

    Science.gov (United States)

    Luke-Glaser, Sarah; Poschke, Heiko; Luke, Brian

    2012-01-01

    The DNA at the ends of linear chromosomes (the telomere) folds back onto itself and forms an intramolecular lariat-like structure. Although the telomere loop has been implicated in the protection of chromosome ends from nuclease-mediated resection and unscheduled DNA repair activities, it potentially poses an obstacle to the DNA replication machinery during S-phase. Therefore, the coordinated regulation of telomere loop formation, maintenance, and resolution is required in order to establish a balance between protecting the chromosome ends and promoting their duplication prior to cell division. Until recently, the only factor known to influence telomere looping in human cells was TRF2, a component of the shelterin complex. Recent work in yeast and mouse cells has uncovered additional regulatory factors that affect the loop structure at telomeres. In the following "perspective" we outline what is known about telomere looping and highlight the latest results regarding the regulation of this chromosome end structure. We speculate about how the manipulation of the telomere loop may have therapeutic implications in terms of diseases associated with telomere dysfunction and uncontrolled proliferation.

  6. Getting in (and out of the loop: regulating higher order telomere structures

    Directory of Open Access Journals (Sweden)

    Sarah eLuke-Glaser

    2012-11-01

    Full Text Available The DNA at the ends of linear chromosomes (the telomere folds back onto itself and forms an intramolecular lariat-like structure. Although the telomere loop has been implicated in the protection of chromosome ends from nuclease-mediated resection and unscheduled DNA repair activities, it potentially poses an obstacle to the DNA replication machinery during S phase. Therefore, the coordinated regulation of telomere loop formation, maintenance and resolution is required in order to establish a balance between protecting the chromosome ends and promoting their duplication prior to cell division. Until recently, the only factor know to influence telomere looping in human cells was TRF2, a component of the shelterin complex. Recent work in yeast and mouse cells has uncovered additional regulatory factors that affect the loop structure at telomeres. In the following perspective we will outline what is known about telomere looping and highlight the latest results regarding the regulation of this chromosome end structure. We will speculate about how the manipulation of the telomere loop may have therapeutic implications in terms of diseases associated with telomere dysfunction and uncontrolled proliferation.

  7. Structural basis of SUFU–GLI interaction in human Hedgehog signalling regulation

    Energy Technology Data Exchange (ETDEWEB)

    Cherry, Amy L.; Finta, Csaba; Karlström, Mikael; Jin, Qianren; Schwend, Thomas [Karolinska Institutet, Novum, Hälsovägen 7, SE-141 83 Huddinge (Sweden); Astorga-Wells, Juan [Karolinska Institutet, Scheeles väg 2, SE-171 77 Stockholm (Sweden); Biomotif AB, Enhagsvägen 7, SE-182 12 Danderyd (Sweden); Zubarev, Roman A. [Karolinska Institutet, Scheeles väg 2, SE-171 77 Stockholm (Sweden); Del Campo, Mark; Criswell, Angela R. [Rigaku Americas Corporation, 9009 New Trails Drive, The Woodlands, TX 77381 (United States); Sanctis, Daniele de [European Synchrotron Radiation Facility, 6 Rue Jules Horowitz, 38043 Grenoble (France); Jovine, Luca, E-mail: luca.jovine@ki.se; Toftgård, Rune, E-mail: luca.jovine@ki.se [Karolinska Institutet, Novum, Hälsovägen 7, SE-141 83 Huddinge (Sweden)

    2013-12-01

    Crystal and small-angle X-ray scattering structures of full-length human SUFU alone and in complex with the conserved SYGHL motif from GLI transcription factors show major conformational changes associated with binding and reveal an intrinsically disordered region crucial for pathway activation. Hedgehog signalling plays a fundamental role in the control of metazoan development, cell proliferation and differentiation, as highlighted by the fact that its deregulation is associated with the development of many human tumours. SUFU is an essential intracellular negative regulator of mammalian Hedgehog signalling and acts by binding and modulating the activity of GLI transcription factors. Despite its central importance, little is known about SUFU regulation and the nature of SUFU–GLI interaction. Here, the crystal and small-angle X-ray scattering structures of full-length human SUFU and its complex with the key SYGHL motif conserved in all GLIs are reported. It is demonstrated that GLI binding is associated with major conformational changes in SUFU, including an intrinsically disordered loop that is also crucial for pathway activation. These findings reveal the structure of the SUFU–GLI interface and suggest a mechanism for an essential regulatory step in Hedgehog signalling, offering possibilities for the development of novel pathway modulators and therapeutics.

  8. Alternative oxidase in the branched mitochondrial respiratory network: an overview on structure, function, regulation, and role

    Directory of Open Access Journals (Sweden)

    Sluse F.E.

    1998-01-01

    Full Text Available Plants and some other organisms including protists possess a complex branched respiratory network in their mitochondria. Some pathways of this network are not energy-conserving and allow sites of energy conservation to be bypassed, leading to a decrease of the energy yield in the cells. It is a challenge to understand the regulation of the partitioning of electrons between the various energy-dissipating and -conserving pathways. This review is focused on the oxidase side of the respiratory chain that presents a cyanide-resistant energy-dissipating alternative oxidase (AOX besides the cytochrome pathway. The known structural properties of AOX are described including transmembrane topology, dimerization, and active sites. Regulation of the alternative oxidase activity is presented in detail because of its complexity. The alternative oxidase activity is dependent on substrate availability: total ubiquinone concentration and its redox state in the membrane and O2 concentration in the cell. The alternative oxidase activity can be long-term regulated (gene expression or short-term (post-translational modification, allosteric activation regulated. Electron distribution (partitioning between the alternative and cytochrome pathways during steady-state respiration is a crucial measurement to quantitatively analyze the effects of the various levels of regulation of the alternative oxidase. Three approaches are described with their specific domain of application and limitations: kinetic approach, oxygen isotope differential discrimination, and ADP/O method (thermokinetic approach. Lastly, the role of the alternative oxidase in non-thermogenic tissues is discussed in relation to the energy metabolism balance of the cell (supply in reducing equivalents/demand in energy and carbon and with harmful reactive oxygen species formation.

  9. Structure, signaling mechanism and regulation of the natriuretic peptide receptor guanylate cyclase.

    Energy Technology Data Exchange (ETDEWEB)

    Misono, K. S.; Philo, J. S.; Arakawa, T.; Ogata, C. M.; Qiu, Y.; Ogawa, H.; Young, H. S. (Biosciences Division); (Univ. of Nevada); (Alliance Protein Labs.)

    2011-06-01

    Atrial natriuretic peptide (ANP) and the homologous B-type natriuretic peptide are cardiac hormones that dilate blood vessels and stimulate natriuresis and diuresis, thereby lowering blood pressure and blood volume. ANP and B-type natriuretic peptide counterbalance the actions of the renin-angiotensin-aldosterone and neurohormonal systems, and play a central role in cardiovascular regulation. These activities are mediated by natriuretic peptide receptor-A (NPRA), a single transmembrane segment, guanylyl cyclase (GC)-linked receptor that occurs as a homodimer. Here, we present an overview of the structure, possible chloride-mediated regulation and signaling mechanism of NPRA and other receptor GCs. Earlier, we determined the crystal structures of the NPRA extracellular domain with and without bound ANP. Their structural comparison has revealed a novel ANP-induced rotation mechanism occurring in the juxtamembrane region that apparently triggers transmembrane signal transduction. More recently, the crystal structures of the dimerized catalytic domain of green algae GC Cyg12 and that of cyanobacterium GC Cya2 have been reported. These structures closely resemble that of the adenylyl cyclase catalytic domain, consisting of a C1 and C2 subdomain heterodimer. Adenylyl cyclase is activated by binding of G{sub s}{alpha} to C2 and the ensuing 7{sup o} rotation of C1 around an axis parallel to the central cleft, thereby inducing the heterodimer to adopt a catalytically active conformation. We speculate that, in NPRA, the ANP-induced rotation of the juxtamembrane domains, transmitted across the transmembrane helices, may induce a similar rotation in each of the dimerized GC catalytic domains, leading to the stimulation of the GC catalytic activity.

  10. Aspects of gene structure and functional regulation of the isozymes of Na,K-ATPase

    DEFF Research Database (Denmark)

    Jorgensen, P.L.

    2001-01-01

    genomes, the genes of four alpha-subunit and at least three beta-subunit isoforms of Na,K-ATPase are identified and two gamma-subunits are expressed in kidney. The isoforms combine in a number of Na,K-ATPase isozymes that are expressed in a tissue and cell specific manner. Models of the molecular...... mechanism of regulation of these isozymes have become more reliable due to progress in understanding the three-dimensional protein structure and conformational transitions mediating transfer of energy from the P-domain to intramembrane Na+ and K+ binding sites....

  11. 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.

  12. 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.

  13. 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.

  14. 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)

  15. 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).

  16. Codon usage regulates protein structure and function by affecting translation elongation speed in Drosophila cells.

    Science.gov (United States)

    Zhao, Fangzhou; Yu, Chien-Hung; Liu, Yi

    2017-08-21

    Codon usage biases are found in all eukaryotic and prokaryotic genomes and have been proposed to regulate different aspects of translation process. Codon optimality has been shown to regulate translation elongation speed in fungal systems, but its effect on translation elongation speed in animal systems is not clear. In this study, we used a Drosophila cell-free translation system to directly compare the velocity of mRNA translation elongation. Our results demonstrate that optimal synonymous codons speed up translation elongation while non-optimal codons slow down translation. In addition, codon usage regulates ribosome movement and stalling on mRNA during translation. Finally, we show that codon usage affects protein structure and function in vitro and in Drosophila cells. Together, these results suggest that the effect of codon usage on translation elongation speed is a conserved mechanism from fungi to animals that can affect protein folding in eukaryotic organisms. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  17. Structural Insights into RNA Recognition by the Alternate-Splicing Regulator CUG-Binding Protein 1

    Energy Technology Data Exchange (ETDEWEB)

    M Teplova; J Song; H Gaw; A Teplov; D Patel

    2011-12-31

    CUG-binding protein 1 (CUGBP1) regulates multiple aspects of nuclear and cytoplasmic mRNA processing, with implications for onset of myotonic dystrophy. CUGBP1 harbors three RRM domains and preferentially targets UGU-rich mRNA elements. We describe crystal structures of CUGBP1 RRM1 and tandem RRM1/2 domains bound to RNAs containing tandem UGU(U/G) elements. Both RRM1 in RRM1-RNA and RRM2 in RRM1/2-RNA complexes use similar principles to target UGU(U/G) elements, with recognition mediated by face-to-edge stacking and water-mediated hydrogen-bonding networks. The UG step adopts a left-handed Z-RNA conformation, with the syn guanine recognized through Hoogsteen edge-protein backbone hydrogen-bonding interactions. NMR studies on the RRM1/2-RNA complex establish that both RRM domains target tandem UGUU motifs in solution, whereas filter-binding assays identify a preference for recognition of GU over AU or GC steps. We discuss the implications of CUGBP1-mediated targeting and sequestration of UGU(U/G) elements on pre-mRNA alternative-splicing regulation, translational regulation, and mRNA decay.

  18. Structural Basis for Regulated Proteolysis by the α-Secretase ADAM10.

    Science.gov (United States)

    Seegar, Tom C M; Killingsworth, Lauren B; Saha, Nayanendu; Meyer, Peter A; Patra, Dhabaleswar; Zimmerman, Brandon; Janes, Peter W; Rubinstein, Eric; Nikolov, Dimitar B; Skiniotis, Georgios; Kruse, Andrew C; Blacklow, Stephen C

    2017-12-14

    Cleavage of membrane-anchored proteins by ADAM (a disintegrin and metalloproteinase) endopeptidases plays a key role in a wide variety of biological signal transduction and protein turnover processes. Among ADAM family members, ADAM10 stands out as particularly important because it is both responsible for regulated proteolysis of Notch receptors and catalyzes the non-amyloidogenic α-secretase cleavage of the Alzheimer's precursor protein (APP). We present here the X-ray crystal structure of the ADAM10 ectodomain, which, together with biochemical and cellular studies, reveals how access to the enzyme active site is regulated. The enzyme adopts an unanticipated architecture in which the C-terminal cysteine-rich domain partially occludes the enzyme active site, preventing unfettered substrate access. Binding of a modulatory antibody to the cysteine-rich domain liberates the catalytic domain from autoinhibition, enhancing enzymatic activity toward a peptide substrate. Together, these studies reveal a mechanism for regulation of ADAM activity and offer a roadmap for its modulation. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Pulsed flows, tributary inputs, and food web structure in a highly regulated river

    Science.gov (United States)

    Sabo, John; Caron, Melanie; Doucett, Richard R.; Dibble, Kimberly L.; Ruhi, Albert; Marks, Jane; Hungate, Bruce; Kennedy, Theodore A.

    2018-01-01

    1.Dams disrupt the river continuum, altering hydrology, biodiversity, and energy flow. Although research indicates that tributary inputs have the potential to dilute these effects, knowledge at the food web level is still scarce.2.Here we examined the riverine food web structure of the Colorado River below Glen Canyon Dam, focusing on organic matter sources, trophic diversity, and food chain length. We asked how these components respond to pulsed flows from tributaries following monsoon thunderstorms that seasonally increase streamflow in the American Southwest.3.Tributaries increased the relative importance of terrestrial organic matter, particularly during the wet season below junctures of key tributaries. This contrasted with the algal-based food web present immediately below Glen Canyon Dam.4.Tributary inputs during the monsoon also increased trophic diversity and food chain length: food chain length peaked below the confluence with the largest tributary (by discharge) in Grand Canyon, increasing by >1 trophic level over a 4-5 kilometre reach possibly due to aquatic prey being flushed into the mainstem during heavy rain events.5.Our results illustrate that large tributaries can create seasonal discontinuities, influencing riverine food web structure in terms of allochthony, food web diversity, and food chain length.6.Synthesis and applications. Pulsed flows from unregulated tributaries following seasonal monsoon rains increase the importance of terrestrially-derived organic matter in large, regulated river food webs, increasing food chain length and trophic diversity downstream of tributary inputs. Protecting unregulated tributaries within hydropower cascades may be important if we are to mitigate food web structure alteration due to flow regulation by large dams. This is critical in the light of global hydropower development, especially in megadiverse, developing countries where dam placement (including completed and planned structures) is in tributaries.

  20. Developing a Structural Model on the Relationship among Motivational Beliefs, Self-Regulated Learning Strategies, and Achievement in Mathematics

    Science.gov (United States)

    Fadlelmula, Fatma Kayan; Cakiroglu, Erdinc; Sungur, Semra

    2015-01-01

    This study examines the interrelationships among students' motivational beliefs (i.e. achievement goal orientations, perception of classroom goal structure, and self-efficacy), use of self-regulated learning strategies (i.e. elaboration, organization, and metacognitive self-regulation strategies), and achievement in mathematics, by proposing and…

  1. Detangling the Interrelationships between Self- Regulation and Ill-Structured Problem Solving in Problem-Based Learning

    Science.gov (United States)

    Ge, Xun; Law, Victor; Huang, Kun

    2016-01-01

    One of the goals for problem-based learning (PBL) is to promote self-regulation. Although self-regulation has been studied extensively, its interrelationships with ill-structured problem solving have been unclear. In order to clarify the interrelationships, this article proposes a conceptual framework illustrating the iterative processes among…

  2. KIAA0100 Modulates Cancer Cell Aggression Behavior of MDA-MB-231 through Microtubule and Heat Shock Proteins

    Directory of Open Access Journals (Sweden)

    Zhenyu Zhong

    2018-06-01

    Full Text Available The KIAA0100 gene was identified in the human immature myeloid cell line cDNA library. Recent studies have shown that its expression is elevated in breast cancer and associated with more aggressive cancer types as well as poor outcomes. However, its cellular and molecular function is yet to be understood. Here we show that silencing KIAA0100 by siRNA in the breast cancer cell line MDA-MB-231 significantly reduced the cancer cells’ aggressive behavior, including cell aggregation, reattachment, cell metastasis and invasion. Most importantly, silencing the expression of KIAA0100 particularly sensitized the quiescent cancer cells in suspension culture to anoikis. Immunoprecipitation, mass spectrometry and immunofluorescence analysis revealed that KIAA0100 may play multiple roles in the cancer cells, including stabilizing microtubule structure as a microtubule binding protein, and contributing to MDA-MB-231 cells Anoikis resistance by the interaction with stress protein HSPA1A. Our study also implies that the interaction between KIAA0100 and HSPA1A may be targeted for new drug development to specifically induce anoikis cell death in the cancer cell.

  3. 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.

  4. 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

  5. Microtubule-targeting drugs rescue axonal swellings in cortical neurons from spastin knockout mice

    Directory of Open Access Journals (Sweden)

    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.

  6. 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.

  7. Ionic wave propagation and collision in an excitable circuit model of microtubules

    Science.gov (United States)

    Guemkam Ghomsi, P.; Tameh Berinyoh, J. T.; Moukam Kakmeni, F. M.

    2018-02-01

    In this paper, we report the propensity to excitability of the internal structure of cellular microtubules, modelled as a relatively large one-dimensional spatial array of electrical units with nonlinear resistive features. We propose a model mimicking the dynamics of a large set of such intracellular dynamical entities as an excitable medium. We show that the behavior of such lattices can be described by a complex Ginzburg-Landau equation, which admits several wave solutions, including the plane waves paradigm. A stability analysis of the plane waves solutions of our dynamical system is conducted both analytically and numerically. It is observed that perturbed plane waves will always evolve toward promoting the generation of localized periodic waves trains. These modes include both stationary and travelling spatial excitations. They encompass, on one hand, localized structures such as solitary waves embracing bright solitons, dark solitons, and bisolitonic impulses with head-on collisions phenomena, and on the other hand, the appearance of both spatially homogeneous and spatially inhomogeneous stationary patterns. This ability exhibited by our array of proteinic elements to display several states of excitability exposes their stunning biological and physical complexity and is of high relevance in the description of the developmental and informative processes occurring on the subcellular scale.

  8. 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)

  9. Preliminary structural studies of the transcriptional regulator CmeR from Campylobacter jejuni

    Energy Technology Data Exchange (ETDEWEB)

    Su, Chih-Chia [Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011 (United States); Shi, Feng [Department of Veterinary Microbiology, College of Veterinary Medicine, Iowa State University, Ames, IA 50011 (United States); Gu, Ruoyu; Li, Ming [Department of Physics and Astronomy, Iowa State University, Ames, IA 50011 (United States); McDermott, Gerry [Department of Anatomy, School of Medicine, University of California, San Francisco, CA 94143 (United States); Yu, Edward W., E-mail: ewyu@iastate.edu [Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011 (United States); Department of Physics and Astronomy, Iowa State University, Ames, IA 50011 (United States); Zhang, Qijing [Department of Veterinary Microbiology, College of Veterinary Medicine, Iowa State University, Ames, IA 50011 (United States); Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011 (United States)

    2007-01-01

    The transcriptional regulator CmeR from C. jejuni has been purified and crystallized and X-ray diffraction data have been collected to a resolution of 2.2 Å. In Campylobacter jejuni, a Gram-negative bacterial pathogen causing gastroenteritis in humans, the CmeR regulatory protein controls transcription of the multidrug transporter gene operon cmeABC. CmeR belongs to the TetR family of transcriptional regulators. The 210-residue CmeR consists of two functional motifs: an N-terminal DNA-binding domain and a C-terminal ligand-binding domain. It is predicted that the DNA-binding domain interacts directly with target promoters, while the C-terminal motif interacts with inducing ligands (such as bile salts). As an initial step towards confirming this structural model, recombinant CmeR protein containing a 6×His tag at the N-terminus was crystallized. Crystals of ligand-free CmeR belonged to space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 37.4, b = 57.6, c = 93.3 Å. Diffraction was observed to at least 2.2 Å at 100 K. Analysis of the detailed CmeR structure is currently in progress.

  10. Modulation of chromatin structure by the FACT histone chaperone complex regulates HIV-1 integration.

    Science.gov (United States)

    Matysiak, Julien; Lesbats, Paul; Mauro, Eric; Lapaillerie, Delphine; Dupuy, Jean-William; Lopez, Angelica P; Benleulmi, Mohamed Salah; Calmels, Christina; Andreola, Marie-Line; Ruff, Marc; Llano, Manuel; Delelis, Olivier; Lavigne, Marc; Parissi, Vincent

    2017-07-28

    Insertion of retroviral genome DNA occurs in the chromatin of the host cell. This step is modulated by chromatin structure as nucleosomes compaction was shown to prevent HIV-1 integration and chromatin remodeling has been reported to affect integration efficiency. LEDGF/p75-mediated targeting of the integration complex toward RNA polymerase II (polII) transcribed regions ensures optimal access to dynamic regions that are suitable for integration. Consequently, we have investigated the involvement of polII-associated factors in the regulation of HIV-1 integration. Using a pull down approach coupled with mass spectrometry, we have selected the FACT (FAcilitates Chromatin Transcription) complex as a new potential cofactor of HIV-1 integration. FACT is a histone chaperone complex associated with the polII transcription machinery and recently shown to bind LEDGF/p75. We report here that a tripartite complex can be formed between HIV-1 integrase, LEDGF/p75 and FACT in vitro and in cells. Biochemical analyzes show that FACT-dependent nucleosome disassembly promotes HIV-1 integration into chromatinized templates, and generates highly favored nucleosomal structures in vitro. This effect was found to be amplified by LEDGF/p75. Promotion of this FACT-mediated chromatin remodeling in cells both increases chromatin accessibility and stimulates HIV-1 infectivity and integration. Altogether, our data indicate that FACT regulates HIV-1 integration by inducing local nucleosomes dissociation that modulates the functional association between the incoming intasome and the targeted nucleosome.

  11. A symphony of regulations centered on p63 to control development of ectoderm-derived structures.

    Science.gov (United States)

    Guerrini, Luisa; Costanzo, Antonio; Merlo, Giorgio R

    2011-01-01

    The p53-related transcription factor p63 is critically important for basic cellular functions during development of the ectoderm and derived structure and tissues, including skin, limb, palate, and hair. On the one side, p63 is required to sustain the proliferation of keratinocyte progenitors, while on the other side it is required for cell stratification, commitment to differentiate, cell adhesion, and epithelial-mesenchymal signaling. Molecules that are components or regulators of the p63 pathway(s) are rapidly being identified, and it comes with no surprise that alterations in the p63 pathway lead to congenital conditions in which the skin and other ectoderm-derived structures are affected. In this paper, we summarize the current knowledge of the molecular and cellular regulations centered on p63, derived from the comprehension of p63-linked human diseases and the corresponding animal models, as well as from cellular models and high-throughput molecular approaches. We point out common themes and features, that allow to speculate on the possible role of p63 downstream events and their potential exploitation in future attempts to correct the congenital defect in preclinical studies.

  12. Structural determinants of PIP(2) regulation of inward rectifier K(ATP) channels.

    Science.gov (United States)

    Shyng, S L; Cukras, C A; Harwood, J; Nichols, C G

    2000-11-01

    Phosphatidylinositol 4,5-bisphosphate (PIP(2)) activates K(ATP) and other inward rectifier (Kir) channels. To determine residues important for PIP(2) regulation, we have systematically mutated each positive charge in the COOH terminus of Kir6.2 to alanine. The effects of these mutations on channel function were examined using (86)Rb efflux assays on intact cells and inside-out patch-clamp methods. Both methods identify essentially the same basic residues in two narrow regions (176-222 and 301-314) in the COOH terminus that are important for the maintenance of channel function and interaction with PIP(2). Only one residue (R201A) simultaneously affected ATP and PIP(2) sensitivity, which is consistent with the notion that these ligands, while functionally competitive, are unlikely to bind to identical sites. Strikingly, none of 13 basic residues in the terminal portion (residues 315-390) of the COOH terminus affected channel function when neutralized. The data help to define the structural requirements for PIP(2) sensitivity of K(ATP) channels. Moreover, the regions and residues defined in this study parallel those uncovered in recent studies of PIP(2) sensitivity in other inward rectifier channels, indicating a common structural basis for PIP(2) regulation.

  13. Recapitulating the Structural Evolution of Redox Regulation in Adenosine 5'-Phosphosulfate Kinase from Cyanobacteria to Plants.

    Science.gov (United States)

    Herrmann, Jonathan; Nathin, David; Lee, Soon Goo; Sun, Tony; Jez, Joseph M

    2015-10-09

    In plants, adenosine 5'-phosphosulfate (APS) kinase (APSK) is required for reproductive viability and the production of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as a sulfur donor in specialized metabolism. Previous studies of the APSK from Arabidopsis thaliana (AtAPSK) identified a regulatory disulfide bond formed between the N-terminal domain (NTD) and a cysteine on the core scaffold. This thiol switch is unique to mosses, gymnosperms, and angiosperms. To understand the structural evolution of redox control of APSK, we investigated the redox-insensitive APSK from the cyanobacterium Synechocystis sp. PCC 6803 (SynAPSK). Crystallographic analysis of SynAPSK in complex with either APS and a non-hydrolyzable ATP analog or APS and sulfate revealed the overall structure of the enzyme, which lacks the NTD found in homologs from mosses and plants. A series of engineered SynAPSK variants reconstructed the structural evolution of the plant APSK. Biochemical analyses of SynAPSK, SynAPSK H23C mutant, SynAPSK fused to the AtAPSK NTD, and the fusion protein with the H23C mutation showed that the addition of the NTD and cysteines recapitulated thiol-based regulation. These results reveal the molecular basis for structural changes leading to the evolution of redox control of APSK in the green lineage from cyanobacteria to plants. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. A study for the structural and functional regulation of chaperon protein by radiation

    International Nuclear Information System (INIS)

    Lee, Seung Sik; Chung, Byung Yeoup; Kim, Jin Hong

    2011-01-01

    The purpose of the this project provides new application areas for radiation technology for improvement of protein activities using radiation through the structural changes and functional regulations of molecular chaperon. Research scope includes 1) isolation of molecular chaperon proteins related radiation response from Psedomonads and purification of recombinant protein from E. coli., 2) the establishment of effective irradiation dose for the structural changes of chaperon protein, 3) analysis of the structural and functional changes of molecular chaperon by gamma irradiation. Main results are as follow: the chaperon activities of 2-Cys peroxiredxin show the maximum (about 3 times) at 15-30 kGy of gamma irradiation, but they were reduced greater than 30 kGy of gamma rays: the peroxidase activities show a tendency to decrease with increasing gamma irradiation: the structural change of peroxiredoxin (PP1084 and PA3529) by gamma irradiation (the formation of low molecular weight complexes or fragmentation of peroxiredoxin by gamma irradiation, the increase of beta-sheet and random coil by gamma irradiation and the decrease of alpha-helix and turn by gamma irradiation, and increased chaperon activity is related with increased hydrophobicity)

  15. The Ndc80 internal loop is required for recruitment of the Ska complex to establish end-on microtubule attachment to kinetochores.

    Science.gov (United States)

    Zhang, Gang; Kelstrup, Christian D; Hu, Xiao-Wen; Kaas Hansen, Mathilde J; Singleton, Martin R; Olsen, Jesper V; Nilsson, Jakob

    2012-07-01

    The Ndc80 complex establishes end-on attachment of kinetochores to microtubules, which is 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 that generates a kink in the structure disrupts the long coiled-coil region but the exact function of this loop, has until now, not been clear. Here we show that this loop region is essential for end-on attachment of kinetochores to microtubules in human cells. Cells expressing loop mutants of Ndc80 are unable to align the chromosomes, and stable kinetochore fibers are absent. Through quantitative mass spectrometry and immunofluorescence we found that the binding of the spindle and kinetochore associated (Ska) complex depends on the loop region, explaining why end-on attachment is defective. This underscores the importance of the Ndc80 loop region in coordinating chromosome segregation through the recruitment of specific proteins to the kinetochore.

  16. 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)

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. The structure of ions and zwitterionic lipids regulates the charge of dipolar membranes.

    Science.gov (United States)

    Szekely, Or; Steiner, Ariel; Szekely, Pablo; Amit, Einav; Asor, Roi; Tamburu, Carmen; Raviv, Uri

    2011-06-21

    In pure water, zwitterionic lipids form lamellar phases with an equilibrium water gap on the order of 2 to 3 nm as a result of the dominating van der Waals attraction between dipolar bilayers. Monovalent ions can swell those neutral lamellae by a small amount. Divalent ions can adsorb onto dipolar membranes and charge them. Using solution X-ray scattering, we studied how the structure of ions and zwitterionic lipids regulates the charge of dipolar membranes. We found that unlike monovalent ions that weakly interact with all of the examined dipolar membranes, divalent and trivalent ions adsorb onto membranes containing lipids with saturated tails, with an association constant on the order of ∼10 M(-1). One double bond in the lipid tail is sufficient to prevent divalent ion adsorption. We suggest that this behavior is due to the relatively loose packing of lipids with unsaturated tails that increases the area per lipid headgroup, enabling their free rotation. Divalent ion adsorption links two lipids and limits their free rotation. The ion-dipole interaction gained by the adsorption of the ions onto unsaturated membranes is insufficient to compensate for the loss of headgroup free-rotational entropy. The ion-dipole interaction is stronger for cations with a higher valence. Nevertheless, polyamines behave as monovalent ions near dipolar interfaces in the sense that they interact weakly with the membrane surface, whereas in the bulk their behavior is similar to that of multivalent cations. Advanced data analysis and comparison with theory provide insight into the structure and interactions between ion-induced regulated charged interfaces. This study models biologically relevant interactions between cell membranes and various ions and the manner in which the lipid structure governs those interactions. The ability to monitor these interactions creates a tool for probing systems that are more complex and forms the basis for controlling the interactions between dipolar

  2. 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

  3. 'Elaioplasts' identified as lipotubuloids in Althaea rosea, Funkia sieboldiana and Vanilla planifolia contain lipid bodies connected with microtubules

    Directory of Open Access Journals (Sweden)

    Maria Kwiatkowska

    2011-09-01

    Full Text Available "Elaioplasts" observed in Vanilla planifolia, Funkia Sieboldiana and Althaea rosea exhibit all the features characteristic of lipotubuloids earlier described in Ornithogalum umbellatum. They are cytoplasmic domains containing aggregates of lipid bodies connected with microtubules. The immunogold technique confirmed the presence of tubulin in this domain. These structures do not have their own membranes but they are surrounded by a tonoplast at the side of a vacuole since they invaginate into it. In cytoplasm of this domain among lipid bodies there are numerous ribosomes, ER cisternae and vesicles as well as few mitochondria, Golgi structures and microbodies while at older developmental stages there are also autolytic vacuoles. The fact that they are so similar to O. umbellatum lipotubuloids suggest that "elaioplasts" of V. planifolia, F. Sieboldiana and A. rosea can also be named lipotubuloids.

  4. The Legal Structure of Commercial Banks and Financial Regulation : does organizational form matter for the design of bank regulation?

    NARCIS (Netherlands)

    V.L.E. Cedeno-Brea (Enmanuel)

    2017-01-01

    markdownabstractDo the different ways that commercial banks are legally organized matter for the design of financial regulation? It is often assumed that most commercial banks are setup as investor owned business corporations. However, this is not always the case In many jurisdictions, banks are

  5. 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...

  6. The structure of Plasmodium falciparum serine hydroxymethyltransferase reveals a novel redox switch that regulates its activities

    Energy Technology Data Exchange (ETDEWEB)

    Chitnumsub, Penchit; Ittarat, Wanwipa; Jaruwat, Aritsara; Noytanom, Krittikar [National Center for Genetic Engineering and Biotechnology, 113 Thailand Science Park, Paholyothin Road, Klong 1, Klong Luang, Pathumthani 12120 (Thailand); Amornwatcharapong, Watcharee [Mahidol University, Bangkok (Thailand); Pornthanakasem, Wichai [National Center for Genetic Engineering and Biotechnology, 113 Thailand Science Park, Paholyothin Road, Klong 1, Klong Luang, Pathumthani 12120 (Thailand); Chaiyen, Pimchai [Mahidol University, Bangkok (Thailand); Yuthavong, Yongyuth; Leartsakulpanich, Ubolsree [National Center for Genetic Engineering and Biotechnology, 113 Thailand Science Park, Paholyothin Road, Klong 1, Klong Luang, Pathumthani 12120 (Thailand)

    2014-06-01

    The crystal structure of P. falciparum SHMT revealed snapshots of an intriguing disulfide/sulfhydryl switch controlling the functional activity. Plasmodium falciparum serine hydroxymethyltransferase (PfSHMT), an enzyme in the dTMP synthesis cycle, is an antimalarial target because inhibition of its expression or function has been shown to be lethal to the parasite. As the wild-type enzyme could not be crystallized, protein engineering of residues on the surface was carried out. The surface-engineered mutant PfSHMT-F292E was successfully crystallized and its structure was determined at 3 Å resolution. The PfSHMT-F292E structure is a good representation of PfSHMT as this variant revealed biochemical properties similar to those of the wild type. Although the overall structure of PfSHMT is similar to those of other SHMTs, unique features including the presence of two loops and a distinctive cysteine pair formed by Cys125 and Cys364 in the tetrahydrofolate (THF) substrate binding pocket were identified. These structural characteristics have never been reported in other SHMTs. Biochemical characterization and mutation analysis of these two residues confirm that they act as a disulfide/sulfhydryl switch to regulate the THF-dependent catalytic function of the enzyme. This redox switch is not present in the human enzyme, in which the cysteine pair is absent. The data reported here can be further exploited as a new strategy to specifically disrupt the activity of the parasite enzyme without interfering with the function of the human enzyme.

  7. The structure of Plasmodium falciparum serine hydroxymethyltransferase reveals a novel redox switch that regulates its activities

    International Nuclear Information System (INIS)

    Chitnumsub, Penchit; Ittarat, Wanwipa; Jaruwat, Aritsara; Noytanom, Krittikar; Amornwatcharapong, Watcharee; Pornthanakasem, Wichai; Chaiyen, Pimchai; Yuthavong, Yongyuth; Leartsakulpanich, Ubolsree

    2014-01-01

    The crystal structure of P. falciparum SHMT revealed snapshots of an intriguing disulfide/sulfhydryl switch controlling the functional activity. Plasmodium falciparum serine hydroxymethyltransferase (PfSHMT), an enzyme in the dTMP synthesis cycle, is an antimalarial target because inhibition of its expression or function has been shown to be lethal to the parasite. As the wild-type enzyme could not be crystallized, protein engineering of residues on the surface was carried out. The surface-engineered mutant PfSHMT-F292E was successfully crystallized and its structure was determined at 3 Å resolution. The PfSHMT-F292E structure is a good representation of PfSHMT as this variant revealed biochemical properties similar to those of the wild type. Although the overall structure of PfSHMT is similar to those of other SHMTs, unique features including the presence of two loops and a distinctive cysteine pair formed by Cys125 and Cys364 in the tetrahydrofolate (THF) substrate binding pocket were identified. These structural characteristics have never been reported in other SHMTs. Biochemical characterization and mutation analysis of these two residues confirm that they act as a disulfide/sulfhydryl switch to regulate the THF-dependent catalytic function of the enzyme. This redox switch is not present in the human enzyme, in which the cysteine pair is absent. The data reported here can be further exploited as a new strategy to specifically disrupt the activity of the parasite enzyme without interfering with the function of the human enzyme

  8. Receptor for advanced glycation endproducts (RAGE maintains pulmonary structure and regulates the response to cigarette smoke.

    Directory of Open Access Journals (Sweden)

    Lisa Wolf

    Full Text Available The receptor for advanced glycation endproducts (RAGE is highly expressed in the lung but its physiological functions in this organ is still not completely understood. To determine the contribution of RAGE to physiological functions of the lung, we analyzed pulmonary mechanics and structure of wildtype and RAGE deficient (RAGE-/- mice. RAGE deficiency spontaneously resulted in a loss of lung structure shown by an increased mean chord length, increased respiratory system compliance, decreased respiratory system elastance and increased concentrations of serum protein albumin in bronchoalveolar lavage fluids. Pulmonary expression of RAGE was mainly localized on alveolar epithelial cells and alveolar macrophages. Primary murine alveolar epithelial cells isolated from RAGE-/- mice revealed an altered differentiation and defective barrier formation under in vitro conditions. Stimulation of interferone-y (IFNy-activated alveolar macrophages deficient for RAGE with Toll-like receptor (TLR ligands resulted in significantly decreased release of proinflammatory cytokines and chemokines. Exposure to chronic cigarette smoke did not affect emphysema-like changes in lung parenchyma in RAGE-/- mice. Acute cigarette smoke exposure revealed a modified inflammatory response in RAGE-/- mice that was characterized by an influx of macrophages and a decreased keratinocyte-derived chemokine (KC release. Our data suggest that RAGE regulates the differentiation of alveolar epithelial cells and impacts on the development and maintenance of pulmonary structure. In cigarette smoke-induced lung pathology, RAGE mediates inflammation that contributes to lung damage.

  9. Structural basis for the regulation mechanism of the tyrosine kinase CapB from Staphylococcus aureus.

    Directory of Open Access Journals (Sweden)

    Vanesa Olivares-Illana

    2008-06-01

    Full Text Available Bacteria were thought to be devoid of tyrosine-phosphorylating enzymes. However, several tyrosine kinases without similarity to their eukaryotic counterparts have recently been identified in bacteria. They are involved in many physiological processes, but their accurate functions remain poorly understood due to slow progress in their structural characterization. They have been best characterized as copolymerases involved in the synthesis and export of extracellular polysaccharides. These compounds play critical roles in the virulence of pathogenic bacteria, and bacterial tyrosine kinases can thus be considered as potential therapeutic targets. Here, we present the crystal structures of the phosphorylated and unphosphorylated states of the tyrosine kinase CapB from the human pathogen Staphylococcus aureus together with the activator domain of its cognate transmembrane modulator CapA. This first high-resolution structure of a bacterial tyrosine kinase reveals a 230-kDa ring-shaped octamer that dissociates upon intermolecular autophosphorylation. These observations provide a molecular basis for the regulation mechanism of the bacterial tyrosine kinases and give insights into their copolymerase function.

  10. Regulating temporospatial dynamics of morphogen for structure formation of the lacrimal gland by chitosan biomaterials.

    Science.gov (United States)

    Hsiao, Ya-Chuan; Yang, Tsung-Lin

    2017-01-01

    The lacrimal gland is an important organ responsible for regulating tear synthesis and secretion. The major work of lacrimal gland (LG) is to lubricate the ocular surface and maintain the health of eyes. Functional deterioration of the lacrimal gland happens because of aging, diseases, or therapeutic complications, but without effective treatments till now. The LG originates from the epithelium of ocular surface and develops by branching morphogenesis. To regenerate functional LGs, it is required to explore the way of recapitulating and facilitating the organ to establish the intricate and ramified structure. In this study, we proposed an approach using chitosan biomaterials to create a biomimetic environment beneficial to the branching structure formation of developing LG. The morphogenetic effect of chitosan was specific and optimized to promote LG branching. With chitosan, increase in temporal expression and local concentration of endogenous HGF-related molecules creates an environment around the emerging tip of LG epithelia. By efficiently enhancing downstream signaling of HGF pathways, the cellular activities and behaviors were activated to contribute to LG branching morphogenesis. The morphogenetic effect of chitosan was abolished by either ligand or receptor deprivation, or inhibition of downstream signaling transduction. Our results elucidated the underlying mechanism accounting for chitosan morphogenetic effects on LG, and also proposed promising approaches with chitosan to assist tissue structure formation of the LG. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Structural Insight on the Mechanism of Regulation of the MarR Family of Proteins: High-Resolution Crystal Structure of a Transcriptional Repressor from Methanobacterium thermoautotrophicum

    Energy Technology Data Exchange (ETDEWEB)

    Saridakis, Vivian; Shahinas, Dea; Xu, Xiaohui; Christendat, Dinesh (York); (Toronto); (CG)

    2008-03-31

    Transcriptional regulators belonging to the MarR family are characterized by a winged-helix DNA binding domain. These transcriptional regulators regulate the efflux and influx of phenolic agents in bacteria and archaea. In Escherichia coli, MarR regulates the multiple antibiotic resistance operon and its inactivation produces a multiple antibiotic resistance phenotype. In some organisms, active efflux of drug compounds will produce a drug resistance phenotype, whereas in other organisms, active influx of chlorinated hydrocarbons results in their rapid degradation. Although proteins in the MarR family are regulators of important biological processes, their mechanism of action is not well understood and structural information about how phenolic agents regulate the activity of these proteins is lacking. This article presents the three-dimensional structure of a protein of the MarR family, MTH313, in its apo form and in complex with salicylate, a known inactivator. A comparison of these two structures indicates that the mechanism of regulation involves a large conformational change in the DNA binding lobe. Electrophoretic mobility shift assay and biophysical analyses further suggest that salicylate inactivates MTH313 and prevents it from binding to its promoter region.

  12. Non-critical string theory formulation of microtubule dynamics and quantum aspects of brain function

    CERN Document Server

    Mavromatos, Nikolaos E

    1995-01-01

    Microtubule (MT) networks, subneural paracrystalline cytosceletal structures, seem to play a fundamental role in the neurons. We cast here the complicated MT dynamics in the form of a 1+1-dimensional non-critical string theory, thus enabling us to provide a consistent quantum treatment of MTs, including enviromental {\\em friction} effects. We suggest, thus, that the MTs are the microsites, in the brain, for the emergence of stable, macroscopic quantum coherent states, identifiable with the {\\em preconscious states}. Quantum space-time effects, as described by non-critical string theory, trigger then an {\\em organized collapse} of the coherent states down to a specific or {\\em conscious state}. The whole process we estimate to take {\\cal O}(1\\,{\\rm sec}), in excellent agreement with a plethora of experimental/observational findings. The {\\em microscopic arrow of time}, endemic in non-critical string theory, and apparent here in the self-collapse process, provides a satisfactory and simple resolution to the age...

  13. Plant community structure regulates responses of prairie soil respiration to decadal experimental warming.

    Science.gov (United States)

    Xu, Xia; Shi, Zheng; Li, Dejun; Zhou, Xuhui; Sherry, Rebecca A; Luo, Yiqi

    2015-10-01

    Soil respiration is recognized to be influenced by temperature, moisture, and ecosystem production. However, little is known about how plant community structure regulates responses of soil respiration to climate change. Here, we used a 13-year field warming experiment to explore the mechanisms underlying plant community regulation on feedbacks of soil respiration to climate change in a tallgrass prairie in Oklahoma, USA. Infrared heaters were used to elevate temperature about 2 °C since November 1999. Annual clipping was used to mimic hay harvest. Our results showed that experimental warming significantly increased soil respiration approximately from 10% in the first 7 years (2000-2006) to 30% in the next 6 years (2007-2012). The two-stage warming stimulation of soil respiration was closely related to warming-induced increases in ecosystem production over the years. Moreover, we found that across the 13 years, warming-induced increases in soil respiration were positively affected by the proportion of aboveground net primary production (ANPP) contributed by C3 forbs. Functional composition of the plant community regulated warming-induced increases in soil respiration through the quantity and quality of organic matter inputs to soil and the amount of photosynthetic carbon (C) allocated belowground. Clipping, the interaction of clipping with warming, and warming-induced changes in soil temperature and moisture all had little effect on soil respiration over the years (all P > 0.05). Our results suggest that climate warming may drive an increase in soil respiration through altering composition of plant communities in grassland ecosystems. © 2015 John Wiley & Sons Ltd.

  14. Australia's private health insurance industry: structure, competition, regulation and role in a less than 'ideal world'.

    Science.gov (United States)

    Shamsullah, Ardel

    2011-02-01

    Australia's private health insurance funds have been prominent participants in the nation's health system for 60 years. Yet there is relatively little public awareness of the distinctive origins of the health funds, the uncharacteristic organisational nature of these commercial enterprises and the peculiarly regulated nature of their industry. The conventional corporate responsibility to shareholders was, until recently, completely irrelevant, and remains marginal to the sector. However, their purported answerability to contributors, styled as 'members', was always doubtful for most health funds. After a long period of remarkable stability in the sector, despite significant shifts in health funding policy, recent years have brought notable changes, with mergers, acquisitions and exits from the industry. The research is based on the detailed study of the private health funds, covering their history, organisational character and industry structure. It argues that the funds have always been divorced from the disciplines of the competitive market and generally have operated complacently within a system of comprehensive regulation and generous subsidy. The prospect of the private health funds enjoying an expanded role under a form of 'social insurance', as suggested by the National Health and Hospitals Reform Commission, is not supported.

  15. Motivation, emotion regulation, and the latent structure of psychopathology: An integrative and convergent historical perspective.

    Science.gov (United States)

    Beauchaine, Theodore P; Zisner, Aimee

    2017-09-01

    Motivational models of psychopathology have long been advanced by psychophysiologists, and have provided key insights into neurobiological mechanisms of a wide range of psychiatric disorders. These accounts emphasize individual differences in activity and reactivity of bottom-up, subcortical neural systems of approach and avoidance in affecting behavior. Largely independent literatures emphasize the roles of top-down, cortical deficits in emotion regulation and executive function in conferring vulnerability to psychopathology. To date however, few models effectively integrate functions performed by bottom-up emotion generation system with those performed by top-down emotion regulation systems in accounting for alternative expressions of psychopathology. In this article, we present such a model, and describe how it accommodates the well replicated bifactor structure of psychopathology. We describe how excessive approach motivation maps directly into externalizing liability, how excessive passive avoidance motivation maps directly into internalizing liability, and how emotion dysregulation and executive function map onto general liability. This approach is consistent with the Research Domain Criteria initiative, which assumes that a limited number of brain systems interact to confer vulnerability to many if not most forms of psychopathology. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Structural analysis of DNA–protein complexes regulating the restriction–modification system Esp1396I

    International Nuclear Information System (INIS)

    Martin, Richard N. A.; McGeehan, John E.; Ball, Neil J.; Streeter, Simon D.; Thresh, Sarah-Jane; Kneale, G. G.

    2013-01-01

    Comparison of bound and unbound DNA in protein–DNA co-crystal complexes reveals insights into controller-protein binding and DNA distortion in transcriptional regulation. The controller protein of the type II restriction–modification (RM) system Esp1396I binds to three distinct DNA operator sequences upstream of the methyltransferase and endonuclease genes in order to regulate their expression. Previous biophysical and crystallographic studies have shown molecular details of how the controller protein binds to the operator sites with very different affinities. Here, two protein–DNA co-crystal structures containing portions of unbound DNA from native operator sites are reported. The DNA in both complexes shows significant distortion in the region between the conserved symmetric sequences, similar to that of a DNA duplex when bound by the controller protein (C-protein), indicating that the naked DNA has an intrinsic tendency to bend when not bound to the C-protein. Moreover, the width of the major groove of the DNA adjacent to a bound C-protein dimer is observed to be significantly increased, supporting the idea that this DNA distortion contributes to the substantial cooperativity found when a second C-protein dimer binds to the operator to form the tetrameric repression complex

  17. Structure of the transcriptional regulator LmrR and its mechanism of multidrug recognition.

    Science.gov (United States)

    Madoori, Pramod Kumar; Agustiandari, Herfita; Driessen, Arnold J M; Thunnissen, Andy-Mark W H

    2009-01-21

    LmrR is a PadR-related transcriptional repressor that regulates the production of LmrCD, a major multidrug ABC transporter in Lactococcus lactis. Transcriptional regulation is presumed to follow a drug-sensitive induction mechanism involving the direct binding of transporter ligands to LmrR. Here, we present crystal structures of LmrR in an apo state and in two drug-bound states complexed with Hoechst 33342 and daunomycin. LmrR shows a common topology containing a typical beta-winged helix-turn-helix domain with an additional C-terminal helix involved in dimerization. Its dimeric organization is highly unusual with a flat-shaped hydrophobic pore at the dimer centre serving as a multidrug-binding site. The drugs bind in a similar manner with their aromatic rings sandwiched in between the indole groups of two dimer-related tryptophan residues. Multidrug recognition is facilitated by conformational plasticity and the absence of drug-specific hydrogen bonds. Combined analyses using site-directed mutagenesis, fluorescence-based drug binding and protein-DNA gel shift assays reveal an allosteric coupling between the multidrug- and DNA-binding sites of LmrR that most likely has a function in the induction mechanism.

  18. Modeling of a New Structure of Precision Air Conditioning System Using Secondary Condenser for Rh Regulation

    Directory of Open Access Journals (Sweden)

    Aries Subiantoro

    2012-05-01

    Full Text Available A dynamic mathematical model for a new structure of precision air conditioning (PAC has been developed. The proposed PAC uses an additional secondary condenser for relative humidity regulation compared to a basic refrigeration system. The work mechanism for this system and a vapour-compression cycle process of the system are illustrated using psychrometric chart and pressure-enthalpy diagram. A non-linear system model is derived based on the conservation of mass and energy balance principles and then linearized at steady state operating point for developing a 8th-order state space model suited for multivariable controller design. The quality of linearized model is analyzed in terms of transient response, controllability, observability, and interaction between input-output variables. The developed model is verified through simulation showing its ability for imitating the nonlinear behavior and the interaction of input-output variables.

  19. A framework to assess landscape structural capacity to provide regulating ecosystem services in West Africa.

    Science.gov (United States)

    Inkoom, Justice Nana; Frank, Susanne; Greve, Klaus; Fürst, Christine

    2018-03-01

    The Sudanian savanna landscapes of West Africa are amongst the world's most vulnerable areas to climate change impacts. Inappropriate land use and agriculture management practices continuously impede the capacity of agricultural landscapes to provide ecosystem services (ES). Given the absence of practical assessment techniques to evaluate the landscape's capacity to provide regulating ES in this region, the goal of this paper is to propose an integrative assessment framework which combines remote sensing, geographic information systems, expert weighting and landscape metrics-based assessment. We utilized Analytical Hierarchical Process and Likert scale for the expert weighting of landscape capacity. In total, 56 experts from several land use and landscape management related departments participated in the assessment. Further, we adapted the hemeroby concept to define areas of naturalness while landscape metrics including Patch Density, Shannon's Diversity, and Shape Index were utilized for structural assessment. Lastly, we tested the reliability of expert weighting using certainty measurement rated by experts themselves. Our study focused on four regulating ES including flood control, pest and disease control, climate control, and wind erosion control. Our assessment framework was tested on four selected sites in the Vea catchment area of Ghana. The outcome of our study revealed that highly heterogeneous landscapes have a higher capacity to provide pest and disease control, while less heterogeneous landscapes have a higher potential to provide climate control. Further, we could show that the potential capacities to provide ecosystem services are underestimated by 15% if landscape structural aspects assessed through landscape metrics are not considered. We conclude that the combination of adapted land use and an optimized land use pattern could contribute considerably to lower climate change impacts in West African agricultural landscapes. Copyright © 2017 Elsevier

  20. Structure, control and regulation of the formal market for medicinal plants' products in Nigeria.

    Science.gov (United States)

    Oguntade, Adegboyega E; Oluwalana, Isaac B

    2011-01-01

    There are informal and formal markets for medicinal plants' products in Nigeria. The formal market is subject to the national regulatory framework for Food and Drug Administration and Control. It is relatively new and underdeveloped. This study was designed to appraise this market with special emphasis on the market participants, market structure, marketing functions performed, conduct of sellers in the market and; standards and regulations to which the market is subject. Information used for this study was collected through personal interviews and interactions with key participants in the market; especially the officials of regulatory agency. The market structure was analysed in terms of the share of market controlled by participants and product types. Concentration Ratios (CR2 and CR4) were used to assess the market share. Marketing functions being performed were described in terms of the exchange, physical and facilitating functions while the conduct was described in terms of pricing and promotional strategies. The regulatory framework under which the market operates was appraised. The market was highly concentrated with a CR2 and CR4 of 58.5% and 80.8 %; respectively. Imported products accounted for only 12.3% of the market. The predominant modes of presentation of the product were capsule (41.6%) and liquid (36.2%). About 20.77% of the products were classified as multivitamins, 13.85% were antibiotics while 10.77% addressed sexual dysfunctional problems. These products were regulated under the Food and Drug Administration and Control (NAFDAC) decrees, 1993-1999. Only 2.3% of the products have received full registration status while the others were only listed.

  1. The Legal Structure of Commercial Banks and Financial Regulation : does organizational form matter for the design of bank regulation?

    OpenAIRE

    Cedeno-Brea, Enmanuel

    2017-01-01

    markdownabstractDo the different ways that commercial banks are legally organized matter for the design of financial regulation? It is often assumed that most commercial banks are setup as investor owned business corporations. However, this is not always the case In many jurisdictions, banks are legally organized using a plethora of organizational forms, which include: co-operatives, mutual associations and even nonprofit entities. Thus, some of the distinctive legal attributes and features o...

  2. The Relationship between Structure-Related Food Parenting Practices and Children's Heightened Levels of Self-Regulation in Eating.

    Science.gov (United States)

    Frankel, Leslie A; Powell, Elisabeth; Jansen, Elena

    Food parenting practices influence children's eating behaviors and weight status. Food parenting practices also influence children's self-regulatory abilities around eating, which has important implications for children's eating behaviors. The purpose of the following study is to examine use of structure-related food parenting practices and the potential impact on children's ability to self-regulate energy intake. Parents (n = 379) of preschool age children (M = 4.10 years, SD = 0.92) were mostly mothers (68.6%), Non-White (54.5%), and overweight/obese (50.1%). Hierarchical Multiple Regression was conducted to predict child self-regulation in eating from structure-related food parenting practices (structured meal setting, structured meal timing, family meal setting), while accounting for child weight status, parent age, gender, BMI, race, and yearly income. Hierarchical Multiple Regression results indicated that structure-related feeding practices (structured meal setting and family meal setting, but not structured meal timing) are associated with children's heightened levels of self-regulation in eating. Models examining the relationship within children who were normal weight and overweight/obese indicated the following: a relationship between structured meal setting and heightened self-regulation in eating for normal-weight children and a relationship between family meal setting and heightened self-regulation in eating for overweight/obese children. Researchers should further investigate these potentially modifiable parent feeding behaviors as a protective parenting technique, which possibly contributes to a healthy weight development by enhancing self-regulation in eating.

  3. Molecular biology of C4 phosphoenolpyruvate carboxylase: Structure, regulation and genetic engineering.

    Science.gov (United States)

    Rajagopalan, A V; Devi, M T; Raghavendra, A S

    1994-02-01

    photoactivation of PEPC-PK and PEPC in leaves, extensive use of site-directed mutagenesis to precisely identify other key amino acid residues, changes in quarternary structure of PEPC in vivo, a high-resolution crystal structure, and hormonal regulation of PEPC expression.

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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.

  10. 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

  11. 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

  12. 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.

  13. Dna2 nuclease-helicase structure, mechanism and regulation by Rpa.

    Science.gov (United States)

    Zhou, Chun; Pourmal, Sergei; Pavletich, Nikola P

    2015-11-02

    The Dna2 nuclease-helicase maintains genomic integrity by processing DNA double-strand breaks, Okazaki fragments and stalled replication forks. Dna2 requires ssDNA ends, and is dependent on the ssDNA-binding protein Rpa, which controls cleavage polarity. Here we present the 2.3 Å structure of intact mouse Dna2 bound to a 15-nucleotide ssDNA. The nuclease active site is embedded in a long, narrow tunnel through which the DNA has to thread. The helicase domain is required for DNA binding but not threading. We also present the structure of a flexibly-tethered Dna2-Rpa interaction that recruits Dna2 to Rpa-coated DNA. We establish that a second Dna2-Rpa interaction is mutually exclusive with Rpa-DNA interactions and mediates the displacement of Rpa from ssDNA. This interaction occurs at the nuclease tunnel entrance and the 5' end of the Rpa-DNA complex. Hence, it only displaces Rpa from the 5' but not 3' end, explaining how Rpa