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Sample records for microtubules coordinately shape

  1. Microtubules contribute to maintain nucleus shape in epithelial cell monolayer

    Science.gov (United States)

    Tremblay, Dominique; Andrzejewski, Lukasz; Pelling, Andrew

    2013-03-01

    INTRODUCTION: Tissue strains can result in significant nuclear deformations and may regulate gene expression. However, the precise role of the cytoskeleton in regulating nuclear mechanics remains poorly understood. Here, we investigate the nuclear deformability of Madin-Darky canine kidney cells (MDCK) under various stretching conditions to clarify the role of the microtubules and actin network on the mechanical behavior of the nucleus. METHODS: A custom-built cell-stretching device allowing for real time imaging of MDCK nuclei was used. Cells were seeded on a silicone membrane coated with rat-tail collagen I. A nuclear stain, Hoechst-33342, was used to image nuclei during stretching. We exposed cells to a compressive and non-compressive stretching strain field of 25%. Nocodazole and cytochalasin-D were used to depolymerize the microtubules and actin network. RESULTS: Nuclei in control cells stretched more along their minor axis than major axis with a deformation of 5% and 2% respectively. This anisotropy vanished completely in microtubule-deprived cells and these cells showed a very high nuclear deformability along the minor axis when exposed to a compressive stretching strain field. CONCLUSIONS: The microtubules drive the anisotropic deformability of MDCK nuclei in a monolayer and maintain nuclear shape when exposed to compressive strain. Such intrinsic mechanical behavior indicates that microtubules are essential to maintain nuclear shape and may prevent down regulation of gene expression.

  2. ErbB2-dependent chemotaxis requires microtubule capture and stabilization coordinated by distinct signaling pathways.

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    Khedidja Benseddik

    Full Text Available Activation of the ErbB2 receptor tyrosine kinase stimulates breast cancer cell migration. Cell migration is a complex process that requires the synchronized reorganization of numerous subcellular structures including cell-to-matrix adhesions, the actin cytoskeleton and microtubules. How the multiple signaling pathways triggered by ErbB2 coordinate, in time and space, the various processes involved in cell motility, is poorly defined. We investigated the mechanism whereby ErbB2 controls microtubules and chemotaxis. We report that activation of ErbB2 increased both cell velocity and directed migration. Impairment of the Cdc42 and RhoA GTPases, but not of Rac1, prevented the chemotactic response. RhoA is a key component of the Memo/ACF7 pathway whereby ErbB2 controls microtubule capture at the leading edge. Upon Memo or ACF7 depletion, microtubules failed to reach the leading edge and cells lost their ability to follow the chemotactic gradient. Constitutive ACF7 targeting to the membrane in Memo-depleted cells reestablished directed migration. ErbB2-mediated activation of phospholipase C gamma (PLCγ also contributed to cell guidance. We further showed that PLCγ signaling, via classical protein kinases C, and Memo signaling converged towards a single pathway controlling the microtubule capture complex. Finally, inhibiting the PI3K/Akt pathway did not affect microtubule capture, but disturbed microtubule stability, which also resulted in defective chemotaxis. PI3K/Akt-dependent stabilization of microtubules involved repression of GSK3 activity on the one hand and inhibition of the microtubule destabilizing protein, Stathmin, on the other hand. Thus, ErbB2 triggers distinct and complementary pathways that tightly coordinate microtubule capture and microtubule stability to control chemotaxis.

  3. Energy shaping for coordinating internally actuated vehicles

    Institute of Scientific and Technical Information of China (English)

    2011-01-01

    This paper considers the stable coordination problem of two vehicles equipping with internal moving mass actuators.The coordinating and stabilizing control are derived by energy shaping. The proposed method is physically motivated and avoids cancelation or domination of nonlinearities.

  4. Microtubules as key coordinators of nuclear envelope and endoplasmic reticulum dynamics during mitosis.

    Science.gov (United States)

    Schlaitz, Anne-Lore

    2014-07-01

    During mitosis, cells comprehensively restructure their interior to promote the faithful inheritance of DNA and cytoplasmic contents. In metazoans, this restructuring entails disassembly of the nuclear envelope, redistribution of its components into the endoplasmic reticulum (ER) and eventually nuclear envelope reassembly around the segregated chromosomes. The microtubule cytoskeleton has recently emerged as a critical regulator of mitotic nuclear envelope and ER dynamics. Microtubules and associated molecular motors tear open the nuclear envelope in prophase and remove nuclear envelope remnants from chromatin. Additionally, two distinct mechanisms of microtubule-based regulation of ER dynamics operate later in mitosis. First, association of the ER with microtubules is reduced, preventing invasion of ER into the spindle area, and second, organelle membrane is actively cleared from metaphase chromosomes. However, we are only beginning to understand the role of microtubules in shaping and distributing ER and other organelles during mitosis. © 2014 WILEY Periodicals, Inc.

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

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    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.

  6. Collective coordinates, shape transitions and shape coexistence a microscopic approach

    CERN Document Server

    Nakatsukasa, T; Nakatsukasa, Takashi; Walet, Niels R.

    1998-01-01

    We investigate a description of shape-mixing and shape-transitions using collective coordinates. To that end we apply a theory of adiabatic large-amplitude motion to a simplified nuclear shell-model, where the approximate results can be contrasted with exact diagonalisations. We find excellent agreement for different regimes, and contrast the results with those from a more standard calculation using a quadrupole constraint. We show that the method employed in this work selects diabatic (crossing) potential energy curves where these are appropriate, and discuss the implications for a microscopic study of shape coexistence.

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

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    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.

  8. Microtubules as key cytoskeletal elements in cellular transport and shape changes: their expected responses to space environments

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    Conrad, G. W.; Conrad, A. H.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    Application of reference standard reagents to alternatively depolymerize or stabilize microtubules in a cell that undergoes very regular cytoskeleton-dependent shape changes provides a model system in which some expected components of the environments of spacecraft and space can be tested on Earth for their effects on the cytoskeleton. The fertilized eggs of Ilyanassa obsoleta undergo polar lobe formation by repeated, dramatic, constriction and relaxation of a microfilamentous band localized in the cortical cytoplasm and activated by microtubules.

  9. Morphological transformation of liposomes caused by assembly of encapsulated tubulin and determination of shape by microtubule-associated proteins (MAPs).

    Science.gov (United States)

    Kaneko, T; Itoh, T J; Hotani, H

    1998-12-18

    To examine the role of cytoskeletons in cellular morphogenesis, we generated liposomes encapsulating tubulin, with or without microtubule-associated proteins (MAPs), and observed their transformation using dark-field microscopy. When tubulin was polymerized with MAPs in liposomes, liposomes were transformed into a "bipolar" shape with a central sphere and two tubular membrane protrusions that aligned in a straight line. On the other hand, when pure tubulin was polymerized in liposomes without MAPs, they initially transformed into a bipolar shape but subsequently re-transformed into a "monopolar" shape, i.e. a sphere with only one straight tubular portion. This re-transformation occurred in two ways: first, by shortening of one of the tubular portions due to microtubule disassembly; or second, by fluctuation of the central sphere toward one of the ends without shortening of the tube portion. MAPs prevented this re-transformation, and their role in stabilizing the shape of transformed liposomes was studied by the co-sedimentation method. The results show that MAPs, particularly MAP1 and MAP2, mediate binding between microtubules and the liposomal membrane. However, MAP2 by itself did not bind to liposomes, but was able to stabilize bipolar liposomes. This stabilization is caused not only by direct links between microtubules and liposomes, but also by prevention of Brownian motion of microtubules through an increase in friction. Copyright 1998 Academic Press

  10. Hoxb1b controls oriented cell division, cell shape and microtubule dynamics in neural tube morphogenesis.

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    Zigman, Mihaela; Laumann-Lipp, Nico; Titus, Tom; Postlethwait, John; Moens, Cecilia B

    2014-02-01

    Hox genes are classically ascribed to function in patterning the anterior-posterior axis of bilaterian animals; however, their role in directing molecular mechanisms underlying morphogenesis at the cellular level remains largely unstudied. We unveil a non-classical role for the zebrafish hoxb1b gene, which shares ancestral functions with mammalian Hoxa1, in controlling progenitor cell shape and oriented cell division during zebrafish anterior hindbrain neural tube morphogenesis. This is likely distinct from its role in cell fate acquisition and segment boundary formation. We show that, without affecting major components of apico-basal or planar cell polarity, Hoxb1b regulates mitotic spindle rotation during the oriented neural keel symmetric mitoses that are required for normal neural tube lumen formation in the zebrafish. This function correlates with a non-cell-autonomous requirement for Hoxb1b in regulating microtubule plus-end dynamics in progenitor cells in interphase. We propose that Hox genes can influence global tissue morphogenesis by control of microtubule dynamics in individual cells in vivo.

  11. Depolymerizing kinesins Kip3 and MCAK shape cellular microtubule architecture by differential control of catastrophe.

    Science.gov (United States)

    Gardner, Melissa K; Zanic, Marija; Gell, Christopher; Bormuth, Volker; Howard, Jonathon

    2011-11-23

    Microtubules are dynamic filaments whose ends alternate between periods of slow growth and rapid shortening as they explore intracellular space and move organelles. A key question is how regulatory proteins modulate catastrophe, the conversion from growth to shortening. To study this process, we reconstituted microtubule dynamics in the absence and presence of the kinesin-8 Kip3 and the kinesin-13 MCAK. Surprisingly, we found that, even in the absence of the kinesins, the microtubule catastrophe frequency depends on the age of the microtubule, indicating that catastrophe is a multistep process. Kip3 slowed microtubule growth in a length-dependent manner and increased the rate of aging. In contrast, MCAK eliminated the aging process. Thus, both kinesins are catastrophe factors; Kip3 mediates fine control of microtubule length by narrowing the distribution of maximum lengths prior to catastrophe, whereas MCAK promotes rapid restructuring of the microtubule cytoskeleton by making catastrophe a first-order random process.

  12. Coordination of microtubule and microfilament dynamics by Drosophila Rho1, Spire and Cappuccino.

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    Rosales-Nieves, Alicia E; Johndrow, James E; Keller, Lani C; Magie, Craig R; Pinto-Santini, Delia M; Parkhurst, Susan M

    2006-04-01

    The actin-nucleation factors Spire and Cappuccino (Capu) regulate the onset of ooplasmic streaming in Drosophila melanogaster. Although this streaming event is microtubule-based, actin assembly is required for its timing. It is not understood how the interaction of microtubules and microfilaments is mediated in this context. Here, we demonstrate that Capu and Spire have microtubule and microfilament crosslinking activity. The spire locus encodes several distinct protein isoforms (SpireA, SpireC and SpireD). SpireD was recently shown to nucleate actin, but the activity of the other isoforms has not been addressed. We find that SpireD does not have crosslinking activity, whereas SpireC is a potent crosslinker. We show that SpireD binds to Capu and inhibits F-actin/microtubule crosslinking, and activated Rho1 abolishes this inhibition, establishing a mechanistic basis for the regulation of Capu and Spire activity. We propose that Rho1, cappuccino and spire are elements of a conserved developmental cassette that is capable of directly mediating crosstalk between microtubules and microfilaments.

  13. The coordination of centromere replication, spindle formation, and kinetochore-microtubule interaction in budding yeast.

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    Hong Liu

    2008-11-01

    Full Text Available The kinetochore is a protein complex that assembles on centromeric DNA to mediate chromosome-microtubule interaction. Most eukaryotic cells form the spindle and establish kinetochore-microtubule interaction during mitosis, but budding yeast cells finish these processes in S-phase. It has long been noticed that the S-phase spindle in budding yeast is shorter than that in metaphase, but the biological significance of this short S-phase spindle structure remains unclear. We addressed this issue by using ask1-3, a temperature-sensitive kinetochore mutant that exhibits partially elongated spindles at permissive temperature in the presence of hydroxyurea (HU, a DNA synthesis inhibitor. After exposure to and removal of HU, ask1-3 cells show a delayed anaphase entry. This delay depends on the spindle checkpoint, which monitors kinetochore-microtubule interaction defects. Overproduction of microtubule-associated protein Ase1 or Cin8 also induces spindle elongation in HU-arrested cells. The spindle checkpoint-dependent anaphase entry delay is also observed after ASE1 or CIN8 overexpression in HU-arrested cells. Therefore, the shorter spindle in S-phase cells is likely to facilitate proper chromosome-microtubule interaction.

  14. On the Nature and Shape of Tubulin Trails: Implications on Microtubule Self-Organization

    CERN Document Server

    Glade, Nicolas

    2012-01-01

    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 macr...

  15. A unique ball-shaped Golgi apparatus in the rat pituitary gonadotrope: its functional implications in relation to the arrangement of the microtubule network.

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    Watanabe, Tsuyoshi; Sakai, Yuko; Koga, Daisuke; Bochimoto, Hiroki; Hira, Yoshiki; Hosaka, Masahiro; Ushiki, Tatsuo

    2012-08-01

    In polarized exocrine cells, the Golgi apparatus is cup-shaped and its convex and concave surfaces are designated as cis and trans faces, functionally confronting the rough endoplasmic reticulum and the cell surface, respectively. To clarify the morphological characteristics of the Golgi apparatus in non-polarized endocrine cells, the investigators immunocytochemically examined its precise architecture in pituitary gonadotropes, especially in relation to the arrangement of the intracellular microtubule network. The Golgi apparatus in the gonadotropes was not cup-shaped but ball-shaped or spherical, and its outer and inner surfaces were the cis and trans faces, respectively. Centrioles were situated at the center of the Golgi apparatus, from which radiating microtubules isotropically extended to the cell periphery through the gaps in the spherical wall of the Golgi stack. The shape of the Golgi apparatus and the arrangement of microtubules demonstrated in the present study could explain the microtubule-dependent movements of tubulovesicular carriers and granules within the gonadotropes. Furthermore, the spherical shape of the Golgi apparatus possibly reflects the highly symmetrical arrangement of microtubule arrays, as well as the poor polarity in the cell surface of pituitary gonadotropes.

  16. Stars advantages vs parallel coordinates: shape perception as visualization reserve

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    Grishin, Vladimir; Kovalerchuk, Boris

    2013-12-01

    Although shape perception is the main information channel for brain, it has been poor used by recent visualization techniques. The difficulties of its modeling are key obstacles for visualization theory and application. Known experimental estimates of shape perception capabilities have been made for low data dimension, and they were usually not connected with data structures. More applied approach for certain data structures detection by means of shape displays are considered by the example of analytical and experimental comparison of popular now Parallel Coordinates (PCs), i.e. 2D Cartesian displays of data vectors, with polar displays known as stars. Advantages of stars vs. PCs by Gestalt Laws are shown. About twice faster feature selection and classification with stars than PCs are showed by psychological experiments for hyper-tubes structures detection in data space with dimension up to 100-200 and its subspaces. This demonstrates great reserves of visualization enhancement in comparison with many recent techniques usually focused on few data attributes analysis.

  17. Coordinate transformations make perfect invisibility cloaks with arbitrary shape

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    Yan Wei; Yan Min; Ruan Zhichao; Qiu Min [Laboratory of Optics, Photonics and Quantum Electronics, Department of Microelectronics and Applied Physics, Royal Institute of Technology, 164 40 Kista (Sweden)], E-mail: min@kth.se

    2008-04-15

    By investigating wave properties at cloak boundaries, invisibility cloaks with arbitrary shape constructed by general coordinate transformations are confirmed to be perfectly invisible to the external incident wave. The differences between line transformed cloaks and point transformed cloaks are discussed. The fields in the cloak medium are found analytically to be related to the fields in the original space via coordinate transformation functions. At the exterior boundary of the cloak, it is shown that no reflection is excited even though the permittivity and permeability do not always have a perfectly matched layer form, whereas at the inner boundary, no reflection is excited either, and in particular no field can penetrate into the cloaked region. However, for the inner boundary of any line transformed cloak, the permittivity and permeability in a specific tangential direction are always required to be infinitely large. Furthermore, the field discontinuity at the inner boundary always exists; the surface current is induced to make this discontinuity self-consistent. A point transformed cloak does not experience such problems. The tangential fields at the inner boundary are all zero, implying that no field discontinuity exists.

  18. Polymeric microtubules that breathe: CO2 -driven polymer controlled-self-assembly and shape transformation.

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    Yan, Qiang; Zhao, Yue

    2013-09-16

    Tubular breathing motion: Polymer tubules self-assembled from a gas-sensitive triblock copolymer can undergo shape evolution. A sequence from microtubes through submicroscopic vesicles to nanosized spherical micelles is modulated by CO2 stimulation levels.

  19. Coordination of multi-agent systems via energy-shaping : Networking improves robustness

    NARCIS (Netherlands)

    Nuno, Emmanuel; Ortega, Romeo; Jayawardhana, Bayu; Basanez, Luis

    2012-01-01

    In this paper the problem of robust coordination of multi–agent systems via energy–shaping is studied. The agents are nonidentical, Euler–Lagrange systems with uncertain parameters. The control objective is to drive all agents states to the same constant equilibrium— which is achieved shaping their

  20. Cep70 promotes microtubule assembly in vitro by increasing microtubule elongation

    Institute of Scientific and Technical Information of China (English)

    Xingjuan Shi; Jun Wang; Yunfan Yang; Yuan Ren; Jun Zhou; Dengwen Li

    2012-01-01

    Microtubules are dynamic cytoskeletal polymers present in all eukaryotic cells,In animal cells,they are organized by the centrosome,the major microtubule-organizing center.Many centrosomal proteins act coordinately to modulate microtubule assembly and organization.Our previous work has shown that Cep70,a novel centrosomal protein regulates microtubule assembly and organization in mammalian cells.However,the molecular details remain to be investigated,in this study,we investigated the molecular mechanism of how Cep70 regulates microtubule assembly using purified proteins.Our data showed that Cep70 increased the microtubule length without affecting the microtubule number in the purified system.These results demonstrate that Cep70 could directly regulate microtubule assembly by promoting microtubule elongation instead of microtubule nucleation.

  1. Intersegmental dynamics shape joint coordination during catching in typically developing children but not in children with developmental coordination disorder.

    Science.gov (United States)

    Asmussen, Michael J; Przysucha, Eryk P; Dounskaia, Natalia

    2014-04-01

    Factors shaping joint coordination during multijoint movements were studied using a one-handed ball-catching task. Typically developing (TD) boys between 9 and 12 yr of age, at which catching becomes consistently successful, and boys with developmental coordination disorder (DCD) of the same age participated in the study. The arm was initially stretched down. Catching was performed by flexing the shoulder and elbow and extending the wrist in the parasagittal plane. Catching success rate was substantially lower in children with DCD. Amplitudes and directions of joint motions were similar in both groups. Group differences were found in shoulder and elbow coordination patterns. TD children performed the movement predominantly by actively accelerating into flexion, one joint at a time-first the elbow and then the shoulder-and allowing passive interaction torque (IT) to accelerate the other joint into extension. Children with DCD tended to accelerate both joints into flexion simultaneously, suppressing IT. The results suggest that the TD joint coordination was shaped by the tendency to minimize active control of IT despite the complexity of the emergent joint kinematics. The inefficient control of IT in children with DCD points to deficiency of the internal model of intersegmental dynamics. Together, the findings advocate that joint coordination throughout a multijoint movement is a by-product of the control strategy that benefits from movement dynamics by actively accelerating a single joint and using IT for rotation of the other joint. Reduction of control-dependent noise is discussed as a possible advantage of this control strategy.

  2. P-glycoprotein (ABCB1) inhibited network of mitochondrion transport along microtubule and BMP signal-induced cell shape in chimpanzee left cerebrum by systems-theoretical analysis.

    Science.gov (United States)

    Lin, Hong; Wang, Lin; Jiang, Minghu; Huang, Juxiang; Qi, Lianxiu

    2012-10-01

    We constructed the significant low-expression P-glycoprotein (ABCB1) inhibited transport and signal network in chimpanzee compared with high-expression (fold change ≥2) the human left cerebrum in GEO data set, by using integration of gene regulatory activated and inhibited network inference method with gene ontology (GO) analysis. Our result showed that ABCB1 transport and signal upstream network RAB2A inhibited ABCB1, and downstream ABCB1-inhibited SMAD1_2, NCK2, SLC25A46, GDF10, RASGRP1, EGFR, LRPPRC, RASSF2, RASA4, CA2, CBLB, UBR5, SLC25A16, ITGB3BP, DDIT4, PDPN, RAB2A in chimpanzee left cerebrum. We obtained that the different biological processes of ABCB1 inhibited transport and signal network repressed carbon dioxide transport, ER to Golgi vesicle-mediated transport, folic acid transport, mitochondrion transport along microtubule, water transport, BMP signaling pathway, Ras protein signal transduction, transforming growth factor beta receptor signaling pathway in chimpanzee compared with the inhibited network of the human left cerebrum, as a result of inducing inhibition of mitochondrion transport along microtubule and BMP signal-induced cell shape in chimpanzee left cerebrum. Our hypothesis was verified by the same and different biological processes of ABCB1 inhibited transport and signal network of chimpanzee compared with the corresponding activated network of chimpanzee and the human left cerebrum, respectively. Copyright © 2012 John Wiley & Sons, Ltd.

  3. Microtubule binding by the formin Cappuccino and its implications for Drosophila oogenesis

    OpenAIRE

    Roth-Johnson, Elizabeth Anne

    2014-01-01

    Coordination of actin and microtubule cytoskeletal networks is required for a number of fundamental cellular processes. Formin family actin nucleators are emerging coordinators of the actin and microtubule cytoskeletons, as they can both nucleate actin filaments and bind microtubules in vitro. To gain a more detailed mechanistic understanding of formin-microtubule interactions and formin-mediated actin-microtubule crosstalk, we studied microtubule binding by Cappuccino (Capu), a formin involv...

  4. Visualization of microtubule growth in living platelets reveals a dynamic marginal band with multiple microtubules

    NARCIS (Netherlands)

    S. Patel-Hett (Sunita); J.L. Richardson (Jennifer); H. Schulze (Harald); K. Drabek (Ksenija); N.A. Isaac (Natasha); K. Hoffmeister (Karin); R.A. Shivdasani (Ramesh); J.C. Bulinski (J. Chloë); N.J. Galjart (Niels); J.H. Hartwig (John); J. Italiano (Joseph)

    2008-01-01

    textabstractThe marginal band of microtubules maintains the discoid shape of resting blood platelets. Although studies of platelet microtubule coil structure conclude that it is composed of a single microtubule, no investigations of its dynamics exist. In contrast to previous studies, permeabilized

  5. Bending nanofibers into nanospirals: coordination chemistry as a tool for shaping hydrophobic assemblies.

    Science.gov (United States)

    Kossoy, Elizaveta; Weissman, Haim; Rybtchinski, Boris

    2015-01-01

    In the current work, we demonstrate how coordination chemistry can be employed to direct self-assembly based on strong hydrophobic interactions. To investigate the influence of coordination sphere geometry on aqueous self-assembly, we synthesized complexes of the amphiphilic perylene diimide terpyridine ligand with the first-row transition-metal centers (zinc, cobalt, and nickel). In aqueous medium, aggregation of these complexes is induced by hydrophobic interactions between the ligands. However, the final shapes of the resulting assemblies depend on the preferred geometry of the coordination spheres typical for the particular metal center. The self-assembly process was characterized by UV/Vis spectroscopy, zeta potential measurements, and cryogenic transmission electron microscopy (cryo-TEM). Coordination of zinc(II) and cobalt(II) leads to the formation of unique nanospiral assemblies, whereas complexation of nickel(II) leads to the formation of straight nanofibers. Notably, coordination bonds are utilized not as connectors between elementary building blocks, but as directing interactions, enabling control over supramolecular geometry.

  6. Coordinated cell-shape changes control epithelial movement in zebrafish and Drosophila.

    Science.gov (United States)

    Köppen, Mathias; Fernández, Beatriz García; Carvalho, Lara; Jacinto, Antonio; Heisenberg, Carl-Philipp

    2006-07-01

    Epithelial morphogenesis depends on coordinated changes in cell shape, a process that is still poorly understood. During zebrafish epiboly and Drosophila dorsal closure, cell-shape changes at the epithelial margin are of critical importance. Here evidence is provided for a conserved mechanism of local actin and myosin 2 recruitment during theses events. It was found that during epiboly of the zebrafish embryo, the movement of the outer epithelium (enveloping layer) over the yolk cell surface involves the constriction of marginal cells. This process depends on the recruitment of actin and myosin 2 within the yolk cytoplasm along the margin of the enveloping layer. Actin and myosin 2 recruitment within the yolk cytoplasm requires the Ste20-like kinase Msn1, an orthologue of Drosophila Misshapen. Similarly, in Drosophila, actin and myosin 2 localization and cell constriction at the margin of the epidermis mediate dorsal closure and are controlled by Misshapen. Thus, this study has characterized a conserved mechanism underlying coordinated cell-shape changes during epithelial morphogenesis.

  7. Water-Energy Nexus in Shared River Basins: How Hydropower Shapes Cooperation and Coordination

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    Kouangpalath Phimthong

    2015-01-01

    Full Text Available The construction of hydropower plants on transboundary rivers is seldom done with equal benefits to all riparians, and therefore presents coordination and cooperation challenges. Without a supra-national authority in charge of transboundary river basins, coordination between sectors (water, energy and environment and cooperation between countries largely depends on willingness of the individual nation states and the power relations between these countries. This paper discusses how the interests and relative power positions of actors in transboundary water management shape the outcomes, and what roles are played by River Basin Organisations and foreign investors (especially in hydropower development. These issues are illustrated with examples from the Mekong river in Southeast Asia (Laos, Thailand, Cambodia and Vietnam, the Euphrates-Tigris (Turkey, Syria, Iraq, Iran and Kuwait and the Çoruh in Turkey and Georgia.

  8. Shape-memory nanopores induced in coordination frameworks by crystal downsizing.

    Science.gov (United States)

    Sakata, Yoko; Furukawa, Shuhei; Kondo, Mio; Hirai, Kenji; Horike, Nao; Takashima, Yohei; Uehara, Hiromitsu; Louvain, Nicolas; Meilikhov, Mikhail; Tsuruoka, Takaaki; Isoda, Seiji; Kosaka, Wataru; Sakata, Osami; Kitagawa, Susumu

    2013-01-11

    Flexible porous coordination polymers change their structure in response to molecular incorporation but recover their original configuration after the guest has been removed. We demonstrated that the crystal downsizing of twofold interpenetrated frameworks of [Cu(2)(dicarboxylate)(2)(amine)](n) regulates the structural flexibility and induces a shape-memory effect in the coordination frameworks. In addition to the two structures that contribute to the sorption process (that is, a nonporous closed phase and a guest-included open phase), we isolated an unusual, metastable open dried phase when downsizing the crystals to the mesoscale, and the closed phase was recovered by thermal treatment. Crystal downsizing suppressed the structural mobility and stabilized the open dried phase. The successful isolation of two interconvertible empty phases, the closed phase and the open dried phase, provided switchable sorption properties with or without gate-opening behavior.

  9. Interpersonal coordination tendencies shape 1-vs-1 sub-phase performance outcomes in youth soccer.

    Science.gov (United States)

    Duarte, Ricardo; Araújo, Duarte; Davids, Keith; Travassos, Bruno; Gazimba, Vítor; Sampaio, Jaime

    2012-05-01

    This study investigated the influence of interpersonal coordination tendencies on performance outcomes of 1-vs-1 sub-phases in youth soccer. Eight male developing soccer players (age: 11.8 ± 0.4 years; training experience: 3.6 ± 1.1 years) performed an in situ simulation of a 1-vs-1 sub-phase of soccer. Data from 82 trials were obtained with motion-analysis techniques, and relative phase used to measure the space-time coordination tendencies of attacker-defender dyads. Approximate entropy (ApEn) was then used to quantify the unpredictability of interpersonal interactions over trials. Results revealed how different modes of interpersonal coordination emerging from attacker-defender dyads influenced the 1-vs-1 performance outcomes. High levels of space-time synchronisation (47%) and unpredictability in interpersonal coordination processes (ApEn: 0.91 ± 0.34) were identified as key features of an attacking player's success. A lead-lag relation attributed to a defending player (34% around -30° values) and a more predictable coordination mode (ApEn: 0.65 ± 0.27, P success of defending players in 1-vs-1 sub-phases. These findings revealed how the mutual influence of each player on the behaviour of dyadic systems shaped emergent performance outcomes. More specifically, the findings showed that attacking players should be constrained to exploit the space-time synchrony with defenders in an unpredictable and creative way, while defenders should be encouraged to adopt postures and behaviours that actively constrain the attacker's actions.

  10. Persistence Length of Stable Microtubules

    Science.gov (United States)

    Hawkins, Taviare; Mirigian, Matthew; Yasar, M. Selcuk; Ross, Jennifer

    2011-03-01

    Microtubules are a vital component of the cytoskeleton. As the most rigid of the cytoskeleton filaments, they give shape and support to the cell. They are also essential for intracellular traffic by providing the roadways onto which organelles are transported, and they are required to reorganize during cellular division. To perform its function in the cell, the microtubule must be rigid yet dynamic. We are interested in how the mechanical properties of stable microtubules change over time. Some ``stable'' microtubules of the cell are recycled after days, such as in the axons of neurons or the cilia and flagella. We measured the persistence length of freely fluctuating taxol-stabilized microtubules over the span of a week and analyzed them via Fourier decomposition. As measured on a daily basis, the persistence length is independent of the contour length. Although measured over the span of the week, the accuracy of the measurement and the persistence length varies. We also studied how fluorescently-labeling the microtubule affects the persistence length and observed that a higher labeling ratio corresponded to greater flexibility. National Science Foundation Grant No: 0928540 to JLR.

  11. Preparation of Novel Banana-Shaped Triple Helical Liquid Crystals by Metal Coordination

    Directory of Open Access Journals (Sweden)

    Jon A. Preece

    2009-03-01

    Full Text Available The synthesis of a series of banana-shaped structures has been carried out, in which the bend unit is formed by a 4,4’-methylenedianiline or 3,3’-methylenedianiline core bearing two symmetric pyridylimine linkages to di- and tri- alkoxyphenylester moieties on the side arms. The molecules, in addition to providing an elongated aromatic central core associated with liquid crystal (LC molecules, also provide binding sites for metals. The methylenedianiline spacer incorporates phenylene groups that sterically prevent the two binding sites from co-ordinating to a single metal centre and the central methylene unit introduces enhanced flexibility into the ligand backbone. Furthermore, complexes have been formed by the co-ordination between 3, 3’-methylenedianiline containing ligands and Cu (I ions [Cu2(3a-c2][PF6]2. Electrospray Mass Spectrometry (ESMS and Fast Atom Bombardment Mass Spectrometry (FABMS showed the formation of dimeric species; [Cu (L2][PF6]2. Finally,thermal analysis of the ligands (1a-d, 2a-d, 3a-c and 4a-d andCu complexes [Cu2(3a-c2][PF6]2 has been carried out in order to investigate the phase properties of these materials. None of the banana-shaped ligands and the metal complexes [Cu2(3a-c2][PF6]2 showed any mesophases.

  12. Revisiting Vertical Models To Simulate the Line Shape of Electronic Spectra Adopting Cartesian and Internal Coordinates.

    Science.gov (United States)

    Cerezo, Javier; Santoro, Fabrizio

    2016-10-11

    Vertical models for the simulation of spectroscopic line shapes expand the potential energy surface (PES) of the final state around the equilibrium geometry of the initial state. These models provide, in principle, a better approximation of the region of the band maximum. At variance, adiabatic models expand each PES around its own minimum. In the harmonic approximation, when the minimum energy structures of the two electronic states are connected by large structural displacements, adiabatic models can breakdown and are outperformed by vertical models. However, the practical application of vertical models faces the issues related to the necessity to perform a frequency analysis at a nonstationary point. In this contribution we revisit vertical models in harmonic approximation adopting both Cartesian (x) and valence internal curvilinear coordinates (s). We show that when x coordinates are used, the vibrational analysis at nonstationary points leads to a deficient description of low-frequency modes, for which spurious imaginary frequencies may even appear. This issue is solved when s coordinates are adopted. It is however necessary to account for the second derivative of s with respect to x, which here we compute analytically. We compare the performance of the vertical model in the s-frame with respect to adiabatic models and previously proposed vertical models in x- or Q1-frame, where Q1 are the normal coordinates of the initial state computed as combination of Cartesian coordinates. We show that for rigid molecules the vertical approach in the s-frame provides a description of the final state very close to the adiabatic picture. For sizable displacements it is a solid alternative to adiabatic models, and it is not affected by the issues of vertical models in x- and Q1-frames, which mainly arise when temperature effects are included. In principle the G matrix depends on s, and this creates nonorthogonality problems of the Duschinsky matrix connecting the normal

  13. Supersymmetry and Shape Invariance of Hartmann Potential and Ring-Shaped Oscillator Potential in the γ and θ Dimensions of Spherical Polar Coordinates

    Institute of Scientific and Technical Information of China (English)

    QIAN Shang-Wu; HUANG Bo-Wen; WANG De-Yun; GU Zhi-Yu

    2002-01-01

    This article shows that in spherical polar coordinates, some noncentral separable potentials have super-symmetry and shape invariance in the r and θ dimensions, we choose Hartmann potential and ring-shaped oscillator astwo important examples, thus in principle the energy eigenvalues and energy eigenfunctions of such the potentials in ther and θ dimensions can be obtained by the method of supersymmetric quantum mechanics. Here we use an alternativemethod to get the required results.

  14. Physical Basis of Large Microtubule Aster Growth

    CERN Document Server

    Ishihara, Keisuke; Mitchison, Timothy J

    2016-01-01

    Microtubule asters - radial arrays of microtubules organized by centrosomes - play a fundamental role in the spatial coordination of animal cells. The standard model of aster growth assumes a fixed number of microtubules originating from the centrosomes. However, aster morphology in this model does not scale with cell size, and we recently found evidence for non-centrosomal microtubule nucleation. Here, we combine autocatalytic nucleation and polymerization dynamics to develop a biophysical model of aster growth. Our model predicts that asters expand as traveling waves and recapitulates all major aspects of aster growth. As the nucleation rate increases, the model predicts an explosive transition from stationary to growing asters with a discontinuous jump of the growth velocity to a nonzero value. Experiments in frog egg extract confirm the main theoretical predictions. Our results suggest that asters observed in large frog and amphibian eggs are a meshwork of short, unstable microtubules maintained by autoca...

  15. New 3-D coordination polymers based on semi-rigid V-shape tetracarboxylates

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Jing-Jing; Xu, Wei; Wang, Yan-Ning [College of Chemistry, Jilin University, Jiefang Road 2519, Changchun 130023, Jilin (China); State Key Laboratory of Inorganic Synthesis and Preparative Chemistry, Jilin University, Qianjin Road 2699, Changchun 130012, Jilin (China); Yu, Jie-Hui, E-mail: jhyu@jlu.edu.cn [College of Chemistry, Jilin University, Jiefang Road 2519, Changchun 130023, Jilin (China); State Key Laboratory of Inorganic Synthesis and Preparative Chemistry, Jilin University, Qianjin Road 2699, Changchun 130012, Jilin (China); Zhang, Ping, E-mail: zhangping@jlu.edu.cn [College of Chemistry, Jilin University, Jiefang Road 2519, Changchun 130023, Jilin (China); Xu, Ji-Qing [College of Chemistry, Jilin University, Jiefang Road 2519, Changchun 130023, Jilin (China); State Key Laboratory of Inorganic Synthesis and Preparative Chemistry, Jilin University, Qianjin Road 2699, Changchun 130012, Jilin (China)

    2015-03-15

    Under the hydrothermal conditions, the reactions of transition-metal salts, tetracarboxylic acids and N,N′-donor ligands yielded three new coordination polymers as [Cu{sub 4}(fph){sub 2}(bpe){sub 3}(H{sub 2}O){sub 2}]·2H{sub 2}O (fph=4,4′-(hexafluoroisopropylidene)diphthalate, bpe=1,2-bis(pyridyl)ethylene) 1, [Co{sub 2}(fph)(bpa){sub 2}(H{sub 2}O){sub 2}]·3H{sub 2}O (bpa=1,2-bis(pyridyl)ethylane) 2, and [Ni(H{sub 2}O)(H{sub 2}oph)(bpa)] (oph=4,4′-oxydiphthalate) 3. X-ray single-crystal diffraction analysis revealed that the title three compounds all possess the three-dimensional (3-D) network structures. For compound 1, the fph molecules first link the Cu{sup 2+} ions into a two-dimensional (2-D) wave-like layer with a (4,4) topology. The bpe molecules act as the second linkers, extending the 2-D layers into a 3-D network. For compound 2, the fph molecules still serve as the first connectors, linking the Co{sup 2+} ions into a one-dimensional (1-D) tube-like chain. Then the bpa molecules propagate the chains into a 3-D (4,4,4)-connected network. In the formation of the 3-D network of compound 3, the oph molecule does not play a role. The bpa molecules as well as the water molecules act as a mixed bridge. Only a kind of 4-connected metal node is observed in compound 3. The magnetic properties of compounds 1–3 were investigated and all exhibit the predominant antiferromegnetic magnetic behaviors. - Graphical abstract: Structures of three semi-rigid V-shape tetracarboxylate-based coordination polymers were reported, and their magnetic properties were investigated. - Highlights: • Structures of three tetracarboxylate-based coordination polymers were reported. • Role of organic bases in metal–tetracarboxylate compounds was discussed. • Characters of V-shape and semi-rigidity for tetracarboxylate play a key role in crystal growth. • Their magnetic properties were investigated.

  16. beta-Dystroglycan modulates the interplay between actin and microtubules in human-adhered platelets.

    Science.gov (United States)

    Cerecedo, Doris; Cisneros, Bulmaro; Suárez-Sánchez, Rocío; Hernández-González, Enrique; Galván, Iván

    2008-05-01

    To maintain the continuity of an injured blood vessel, platelets change shape, secrete granule contents, adhere, aggregate, and retract in a haemostatic plug. Ordered arrays of microtubules, microfilaments, and associated proteins are responsible for these platelet responses. In full-spread platelets, microfilament bundles in association with other cytoskeleton proteins are anchored in focal contacts. Recent studies in migrating cells suggest that co-ordination and direct physical interaction of microtubules and actin network modulate adhesion development. In platelets, we have proposed a feasible association between these two cytoskeletal systems, as well as the participation of the dystrophin-associated protein complex, as part of the focal adhesion complex. The present study analysed the participation of microtubules and actin during the platelet adhesion process. Confocal microscopy, fluorescence resonance transfer energy and immunoprecipitation assays were used to provide evidence of a cross-talk between these two cytoskeletal systems. Interestingly, beta-dystroglycan was found to act as an interplay protein between actin and microtubules and an additional communication between these two cytoskeleton networks was maintained through proteins of focal adhesion complex. Altogether our data are indicative of a dynamic co-participation of actin filaments and microtubules in modulating focal contacts to achieve platelet function.

  17. Microtubule-driven nuclear movements and linear elements as meiosis-specific characteristics of the fission yeasts Schizosaccharomyces versatilis and Schizosaccharomyces pombe.

    Science.gov (United States)

    Svoboda, A; Bähler, J; Kohli, J

    1995-11-01

    Meiotic prophase in Schizosaccharomyces pombe is characterized by striking nuclear movements and the formation of linear elements along chromosomes instead of tripartite synaptonemal complexes. We analysed the organization of nuclei and microtubules in cells of fission yeasts undergoing sexual differentiation. S. japonicus var. versatilis and S. pombe cells were studied in parallel, taking advantage of the better cytology in S. versatilis. During conjugation, microtubules were directed towards the mating projection. These microtubules seem to lead the haploid nuclei together in the zygote by interaction with the spindle pole bodies at the nuclear periphery. After karyogamy, arrays of microtubules emanating from the spindle pole body of the diploid nucleus extended to both cell poles. The same differentiated microtubule configuration was elaborated upon induction of azygotic meiosis in S. pombe. The cyclic movements of the elongated nuclei between the cell poles is reflected by a dynamic and coordinated shortening and lengthening of the two microtubule arrays. When the nucleus was at a cell end, one array was short while the other bridged the whole cell length. Experiments with inhibitors showed that microtubules are required for karyogamy and for the elongated shape and movement of nuclei during meiotic prophase. In both fission yeasts the SPBs and nucleoli are at the leading ends of the moving nuclei. Astral and cytoplasmic microtubules were also prominent during meiotic divisions and sporulation. We further show that in S. versatilis the linear elements formed during meiotic prophase are similar to those in S. pombe. Tripartite synaptonemal complexes were never detected. Taken together, these findings suggest that S. pombe and S. versatilis share basic characteristics in the organization of microtubules and the structure and behaviour of nuclei during their meiotic cell cycle. The prominent differentiations of microtubules and nuclei may be involved in the

  18. Microtubules, Tubulins and Associated Proteins.

    Science.gov (United States)

    Raxworthy, Michael J.

    1988-01-01

    Reviews much of what is known about microtubules, which are biopolymers consisting predominantly of subunits of the globular protein, tubulin. Describes the functions of microtubules, their structure and assembly, microtube associated proteins, and microtubule-disrupting agents. (TW)

  19. An Improved Surface Simplification Method for Facial Expression Animation Based on Homogeneous Coordinate Transformation Matrix and Maximum Shape Operator

    Directory of Open Access Journals (Sweden)

    Juin-Ling Tseng

    2016-01-01

    Full Text Available Facial animation is one of the most popular 3D animation topics researched in recent years. However, when using facial animation, a 3D facial animation model has to be stored. This 3D facial animation model requires many triangles to accurately describe and demonstrate facial expression animation because the face often presents a number of different expressions. Consequently, the costs associated with facial animation have increased rapidly. In an effort to reduce storage costs, researchers have sought to simplify 3D animation models using techniques such as Deformation Sensitive Decimation and Feature Edge Quadric. The studies conducted have examined the problems in the homogeneity of the local coordinate system between different expression models and in the retainment of simplified model characteristics. This paper proposes a method that applies Homogeneous Coordinate Transformation Matrix to solve the problem of homogeneity of the local coordinate system and Maximum Shape Operator to detect shape changes in facial animation so as to properly preserve the features of facial expressions. Further, root mean square error and perceived quality error are used to compare the errors generated by different simplification methods in experiments. Experimental results show that, compared with Deformation Sensitive Decimation and Feature Edge Quadric, our method can not only reduce the errors caused by simplification of facial animation, but also retain more facial features.

  20. General theory for the mechanics of confined microtubule asters

    Science.gov (United States)

    Ma, Rui; Laan, Liedewij; Dogterom, Marileen; Pavin, Nenad; Jülicher, Frank

    2014-01-01

    In cells, dynamic microtubules organize into asters or spindles to assist positioning of organelles. Two types of forces are suggested to contribute to the positioning process: (i) microtubule-growth based pushing forces; and (ii) motor protein mediated pulling forces. In this paper, we present a general theory to account for aster positioning in a confinement of arbitrary shape. The theory takes account of microtubule nucleation, growth, catastrophe, slipping, as well as interaction with cortical force generators. We calculate microtubule distributions and forces acting on microtubule organizing centers in a sphere and in an ellipsoid. Positioning mechanisms based on both pushing forces and pulling forces can be distinguished in our theory for different parameter regimes or in different geometries. In addition, we investigate positioning of microtubule asters in the case of asymmetric distribution of motors. This analysis enables us to characterize situations relevant for Caenorrhabditis elegans embryos.

  1. Synthesis and crystal structure of a wheel-shaped supramolecular coordination complex

    Indian Academy of Sciences (India)

    Deepak Gupta; Palanisamy Rajakannu; Bhaskaran Shankar; Firasat Hussain; Malaichamy Sathiyendiran

    2014-09-01

    Supramolecular coordination complex (SCC) possessing spatially arranged three anthraquinone dimers in a slipped-cofacial orientation was achieved by the treatment of Re2(CO)10, 2-hydroxymethylanthraquinone and tritopic N-donor via fac-Re(CO)3-directed one pot approach. The off-set -stacking and C≡O…H bonding interactions stabilize the ring structure.

  2. Development of a coordinated controller for robot-assisted shape memory alloy actuated needle for prostate brachytherapy.

    Science.gov (United States)

    Joseph, Felix Orlando Maria; Franz, Karly; Luan, Yaxin; Zhao, Yan-Jiang; Datla, Naresh; Hutapea, Parsaoran; Dicker, Adam; Yu, Yan; Podder, Tarun

    2014-01-01

    This paper deals with the development of a coordinated control system for a robot and robot-driven shape memory alloy (SMA) actuated needle to follow a curvilinear path for percutaneous intervention. The robot driving the needle is considered as the outer loop and the non-linear SMA actuated flexible needle system forms the inner loop. The two feedback control loops are coordinated in such a way that the robot drives the needle considering the needle's actual deflection so that the needle tip reaches the target location with an acceptable accuracy. Simulation results are presented to verify the efficacy of the controller for tracking the overall desired trajectory which includes the combined trajectory of the robot and the needle.

  3. Dirichlet boundary conditions for arbitrary-shaped boundaries in stellarator-like magnetic fields for the Flux-Coordinate Independent method

    CERN Document Server

    Hill, Peter; Dudson, Ben

    2016-01-01

    We present a technique for handling Dirichlet boundary conditions with the Flux Coordinate Independent (FCI) parallel derivative operator with arbitrary-shaped material geometry in general 3D magnetic fields. The FCI method constructs a finite difference scheme for $\

  4. DETERMINED MODEL FOR COORDINATED REGULATION OF MOTOR TRANSPORT MOVEMENT ON HIGHWAY WITH T-SHAPE CROSSROADS

    Directory of Open Access Journals (Sweden)

    V. N. Shut

    2009-01-01

    Full Text Available The paper examines variants of higher control efficiency in respect of road traffic by creating coordinated regulation  with the help of a determined module. Model application conditions have been determined for specific traffic situations with due account of transport-pedestrian load. The paper contains proposals for the model optimization directed on reduction of  motor vehicle delay in front of the in-traffic light  stop-line along the main highway direction.

  5. Shape assisted fabrication of fluorescent cages of squarate based metal-organic coordination frameworks.

    Science.gov (United States)

    Jayaramulu, Kolleboyina; Krishna, Katla Sai; George, Subi J; Eswaramoorthy, Muthuswamy; Maji, Tapas Kumar

    2013-05-11

    Micronic cage structures of squarate based metal-organic coordination frameworks (MOCFs) have been fabricated for the first time by specific anion selective etching of metal squarate cubes. Time and stoichiometry dependent synthesis and the corresponding microscopic studies have provided mechanistic insight into the cage formation. Furthermore, a non-covalent post-synthetic strategy has been adopted to functionalize the micronic cubes or cages with chromophores rendering the resulting hybrids green fluorescent.

  6. Microtubule doublets are double-track railways for intraflagellar transport trains.

    Science.gov (United States)

    Stepanek, Ludek; Pigino, Gaia

    2016-05-06

    The cilium is a large macromolecular machine that is vital for motility, signaling, and sensing in most eukaryotic cells. Its conserved core structure, the axoneme, contains nine microtubule doublets, each comprising a full A-microtubule and an incomplete B-microtubule. However, thus far, the function of this doublet geometry has not been understood. We developed a time-resolved correlative fluorescence and three-dimensional electron microscopy approach to investigate the dynamics of intraflagellar transport (IFT) trains, which carry ciliary building blocks along microtubules during the assembly and disassembly of the cilium. Using this method, we showed that each microtubule doublet is used as a bidirectional double-track railway: Anterograde IFT trains move along B-microtubules, and retrograde trains move along A-microtubules. Thus, the microtubule doublet geometry provides direction-specific rails to coordinate bidirectional transport of ciliary components.

  7. Interaction between microtubules and the Drosophila formin Cappuccino and its effect on actin assembly.

    Science.gov (United States)

    Roth-Johnson, Elizabeth A; Vizcarra, Christina L; Bois, Justin S; Quinlan, Margot E

    2014-02-14

    Formin family actin nucleators are potential coordinators of the actin and microtubule cytoskeletons, as they can both nucleate actin filaments and bind microtubules in vitro. To gain a more detailed mechanistic understanding of formin-microtubule interactions and formin-mediated actin-microtubule cross-talk, we studied microtubule binding by Cappuccino (Capu), a formin involved in regulating actin and microtubule organization during Drosophila oogenesis. We found that two distinct domains within Capu, FH2 and tail, work together to promote high-affinity microtubule binding. The tail domain appears to bind microtubules through nonspecific charge-based interactions. In contrast, distinct residues within the FH2 domain are important for microtubule binding. We also report the first visualization of a formin polymerizing actin filaments in the presence of microtubules. Interestingly, microtubules are potent inhibitors of the actin nucleation activity of Capu but appear to have little effect on Capu once it is bound to the barbed end of an elongating filament. Because Capu does not simultaneously bind microtubules and assemble actin filaments in vitro, its actin assembly and microtubule binding activities likely require spatial and/or temporal regulation within the Drosophila oocyte.

  8. Interaction between Microtubules and the Drosophila Formin Cappuccino and Its Effect on Actin Assembly*

    Science.gov (United States)

    Roth-Johnson, Elizabeth A.; Vizcarra, Christina L.; Bois, Justin S.; Quinlan, Margot E.

    2014-01-01

    Formin family actin nucleators are potential coordinators of the actin and microtubule cytoskeletons, as they can both nucleate actin filaments and bind microtubules in vitro. To gain a more detailed mechanistic understanding of formin-microtubule interactions and formin-mediated actin-microtubule cross-talk, we studied microtubule binding by Cappuccino (Capu), a formin involved in regulating actin and microtubule organization during Drosophila oogenesis. We found that two distinct domains within Capu, FH2 and tail, work together to promote high-affinity microtubule binding. The tail domain appears to bind microtubules through nonspecific charge-based interactions. In contrast, distinct residues within the FH2 domain are important for microtubule binding. We also report the first visualization of a formin polymerizing actin filaments in the presence of microtubules. Interestingly, microtubules are potent inhibitors of the actin nucleation activity of Capu but appear to have little effect on Capu once it is bound to the barbed end of an elongating filament. Because Capu does not simultaneously bind microtubules and assemble actin filaments in vitro, its actin assembly and microtubule binding activities likely require spatial and/or temporal regulation within the Drosophila oocyte. PMID:24362037

  9. Dynamics of Microtubule Instabilities

    CERN Document Server

    Antal, T; Redner, S

    2007-01-01

    We investigate the dynamics of an idealized model of microtubule growth that evolves by: (i) attachment of guanosine triphosphate (GTP) at rate lambda, (ii) conversion of GTP to guanosine diphosphate (GDP) at rate 1, and (iii) detachment of GDP at rate mu. As a function of these rates, a microtubule can grow steadily or its length can fluctuate wildly. For mu=0, we find the exact tubule and GTP cap length distributions, and power-law length distributions of GTP and GDP islands. For mu=infinity, we argue that the time between catastrophes, where the microtubule shrinks to zero length, scales as exp(lambda). We also find the phase boundary between a growing and shrinking microtubule.

  10. The ability to induce microtubule acetylation is a general feature of formin proteins.

    Directory of Open Access Journals (Sweden)

    Susan F Thurston

    Full Text Available Cytoplasmic microtubules exist as distinct dynamic and stable populations within the cell. Stable microtubules direct and maintain cell polarity and it is thought that their stabilization is dependent on coordinative organization between the microtubule network and the actin cytoskeleton. A growing body of work suggests that some members of the formin family of actin remodeling proteins also regulate microtubule organization and stability. For example, we showed previously that expression of the novel formin INF1 is sufficient to induce microtubule stabilization and tubulin acetylation, but not tubulin detyrosination. An important issue with respect to the relationship between formins and microtubules is the determination of which formin domains mediate microtubule stabilization. INF1 has a distinct microtubule-binding domain at its C-terminus and the endogenous INF1 protein is associated with the microtubule network. Surprisingly, the INF1 microtubule-binding domain is not essential for INF1-induced microtubule acetylation. We show here that expression of the isolated FH1 + FH2 functional unit of INF1 is sufficient to induce microtubule acetylation independent of the INF1 microtubule-binding domain. It is not yet clear whether or not microtubule stabilization is a general property of all mammalian formins; therefore we expressed constitutively active derivatives of thirteen of the fifteen mammalian formin proteins in HeLa and NIH3T3 cells and measured their effects on stress fiber formation, MT organization and MT acetylation. We found that expression of the FH1 + FH2 unit of the majority of mammalian formins is sufficient to induce microtubule acetylation. Our results suggest that the regulation of microtubule acetylation is likely a general formin activity and that the FH2 should be thought of as a dual-function domain capable of regulating both actin and microtubule networks.

  11. Alignment of Microtubule Imagery

    OpenAIRE

    Yu, Feiyang; Oerlemans, Ard; Bakker, Erwin M.

    2011-01-01

    This work discusses preliminary work aimed at simulating and visualizing the growth process of a tiny structure inside the cell---the microtubule. Difficulty of recording the process lies in the fact that the tissue preparation method for electronic microscopes is highly destructive to live cells. Here in this paper, our approach is to take pictures of microtubules at different time slots and then appropriately combine these images into a coherent video. Experimental results are given on real...

  12. Alignment of Microtubule Imagery

    CERN Document Server

    Yu, Feiyang; Bakker, Erwin M

    2011-01-01

    This work discusses preliminary work aimed at simulating and visualizing the growth process of a tiny structure inside the cell---the microtubule. Difficulty of recording the process lies in the fact that the tissue preparation method for electronic microscopes is highly destructive to live cells. Here in this paper, our approach is to take pictures of microtubules at different time slots and then appropriately combine these images into a coherent video. Experimental results are given on real data.

  13. 2-D boundary-fitted coordinate grid generation for casting with complex shape

    Directory of Open Access Journals (Sweden)

    Wang Ye

    2013-05-01

    Full Text Available In order to overcome the zigzag grids generated by conventional finite difference method on complicated casting boundaries in the simulation of casting process, the generation program for 2-D boundary-fitted coordinate grid has been developed by solving a set of partial differential equations (PDE numerically. The STL format files were treated as input data for 2-D physical regions. The equipartition method for boundary points was used to improve the self-adaptability of grid according to the characteristic of the STL format files. The program was demonstrated through some examples. The comparison between the conventional finite difference method and the proposed method shows that this program is effective and flexible for generation of boundary-fitted grid in any arbitrary 2-D complex domain, and the grid is in accordance with the variety of boundary curvature finely. The program also provides two types of boundary-fitted grids for double-connected region, O-type and C-type. The limitation of the step-like boundary with the rectangle grid could be avoided effectively. Therefore, the computational accuracy and efficiency would be improved and the computational time would be saved significantly by the application of boundary-fitted grids.

  14. Mechanical operation and intersubunit coordination of ring-shaped molecular motors: insights from single-molecule studies.

    Science.gov (United States)

    Liu, Shixin; Chistol, Gheorghe; Bustamante, Carlos

    2014-05-06

    Ring NTPases represent a large and diverse group of proteins that couple their nucleotide hydrolysis activity to a mechanical task involving force generation and some type of transport process in the cell. Because of their shape, these enzymes often operate as gates that separate distinct cellular compartments to control and regulate the passage of chemical species across them. In this manner, ions and small molecules are moved across membranes, biopolymer substrates are segregated between cells or moved into confined spaces, double-stranded nucleic acids are separated into single strands to provide access to the genetic information, and polypeptides are unfolded and processed for recycling. Here we review the recent advances in the characterization of these motors using single-molecule manipulation and detection approaches. We describe the various mechanisms by which ring motors convert chemical energy to mechanical force or torque and coordinate the activities of individual subunits that constitute the ring. We also examine how single-molecule studies have contributed to a better understanding of the structural elements involved in motor-substrate interaction, mechanochemical coupling, and intersubunit coordination. Finally, we discuss how these molecular motors tailor their operation-often through regulation by other cofactors-to suit their unique biological functions.

  15. Zn(II) coordination polymers with flexible V-shaped dicarboxylate ligand: Syntheses, helical structures and properties

    Energy Technology Data Exchange (ETDEWEB)

    Li, Lin [School of Environment and Chemical Engineering, Nanchang Hangkong University, Nanchang 330063 (China); Liu, Chong-Bo, E-mail: cbliu@nchu.edu.cn [School of Environment and Chemical Engineering, Nanchang Hangkong University, Nanchang 330063 (China); Yang, Gao-Shan [School of Environment and Chemical Engineering, Nanchang Hangkong University, Nanchang 330063 (China); Xiong, Zhi-Qiang [Center for Analysis and Testing, Nanchang Hangkong University, Nanchang 330063 (China); Liu, Hong [School of Environment and Chemical Engineering, Nanchang Hangkong University, Nanchang 330063 (China); Wen, Hui-Liang [State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047 (China)

    2015-11-15

    Hydrothermal reactions of 2,2′-[hexafluoroisopropylidenebis(p-phenyleneoxy)]diacetic acid (H{sub 2}L) and zinc ions in the presence of N-donor ancillary ligands afford four novel coordination polymers, namely, [Zn{sub 2}(μ{sub 2}-OH)(μ{sub 4}-O){sub 0.5}(L)]·0.5H{sub 2}O (1), [Zn(L)(2,2′-bipy)(H{sub 2}O)] (2), [Zn{sub 3}(L){sub 3}(phen){sub 2}]·H{sub 2}O (3) and [Zn{sub 2}(L){sub 2}(4,4′-bipy)] (4) (2,2′-bipy=2,2′-bipyridine; 4,4′-bipy=4,4′-bipyridine; phen=1,10-phenanthroline). Their structures have been determined by single-crystal X-ray diffraction analyses, elemental analyses, IR spectra, powder X-ray diffraction (PXRD), and thermogravimetric (TG) analyses. Complex 1 shows a 3-D clover framework consisting of [Zn{sub 4}(µ{sub 4}-O)(µ{sub 2}-OH){sub 2}]{sup 4+} clusters, and exhibits a novel (3,8)-connected topological net with the Schläfli symbol of {3·4·5}{sub 2}{3"4·4"4·5"2·6"6·7"1"0·8"2}, and contains double-stranded and two kinds of meso-helices. 2 displays a helical chain structure, which is further extended via hydrogen bonds into a 3-D supramolecular structure with meso-helix chains. 3 displays a 2-D {4"4·6"2} parallelogram structure, which is further extended via hydrogen bonds into a 3-D supramolecular structure with single-stranded helical chains. 4 shows a 2-D {4"4·6"2} square structure with left- and right-handed helical chains. Moreover, the luminescent properties of 1–4 have been investigated. - Graphical abstract: Four new Zn(II) coordination polymers with helical structures based on flexible V-shaped dicarboxylate ligand have been synthesized and structurally characterized. Photoluminescent properties have been investigated. - Highlights: • Four novel Zn(II) coordination polymers with V-shaped ligand were characterized. • Complexes 1–4 show diverse intriguing helical characters. • Fluorescence properties of complexes 1–4 were investigated.

  16. Do prokaryotes contain microtubules?

    Science.gov (United States)

    Bermudes, D.; Hinkle, G.; Margulis, L.

    1994-01-01

    In eukaryotic cells, microtubules are 24-nm-diameter tubular structures composed of a class of conserved proteins called tubulin. They are involved in numerous cell functions including ciliary motility, nerve cell elongation, pigment migration, centrosome formation, and chromosome movement. Although cytoplasmic tubules and fibers have been observed in bacteria, some with diameters similar to those of eukaryotes, no homologies to eukaryotic microtubules have been established. Certain groups of bacteria including azotobacters, cyanobacteria, enteric bacteria, and spirochetes have been frequently observed to possess microtubule-like structures, and others, including archaebacteria, have been shown to be sensitive to drugs that inhibit the polymerization of microtubules. Although little biochemical or molecular biological information is available, the differences observed among these prokaryotic structures suggest that their composition generally differs among themselves as well as from that of eukaryotes. We review the distribution of cytoplasmic tubules in prokaryotes, even though, in all cases, their functions remain unknown. At least some tend to occur in cells that are large, elongate, and motile, suggesting that they may be involved in cytoskeletal functions, intracellular motility, or transport activities comparable to those performed by eukaryotic microtubules. In Escherichia coli, the FtsZ protein is associated with the formation of a ring in the division zone between the newly forming offspring cells. Like tubulin, FtsZ is a GTPase and shares with tubulin a 7-amino-acid motif, making it a promising candidate in which to seek the origin of tubulins.

  17. Microtubule as a Transmission Line for Ionic Currents

    Institute of Scientific and Technical Information of China (English)

    ILI(C) D.I.; SATARI(C) M.V.; RALEVI(C) N.

    2009-01-01

    We establish a new model for ionic waves along microtubules based on polyelectrolyte features of cylindrical biopolymers. The nonlinear transmission line described by a nonlinear differential equation is obtained with stable kink solution pertinent to the shape of the front of accompanying potential. The localized ionic wave could be used to explain the behavior of microtubules as biomolecular transistors capable of amplifying electrical information in neurons.

  18. Roles for microtubule and microfilament cytoskeletons in animal cell cytokinesis

    Institute of Scientific and Technical Information of China (English)

    CHEN Zhongcai; CAI Shang; JIANG Qing; ZHANG Chuanmao; TANG Xiaowei

    2005-01-01

    Microtubule and microfilament cytoskeletons play key roles in the whole process of cytokinesis. Although a number of hypotheses have been proposed to elucidate the mechanism of cytokinesis by microtubule and actin filament cytoskeletons, many reports are conflicting. In our study, combining the cytoskeletons drug treatments with the time-lapse video technology, we retested the key roles of microtubule and actin filament in cytokinesis. The results showed that depolymerization of microtubules by Nocodazole after the initiation of furrowing would not inhibit the furrow ingression, but obviously decrease the stiffness of daughter cells. Depolymerizing actin filaments by Cytochalasin B before metaphase would inhibit the initiation of furrowing but not chromosome segregation, resulting in the formation of binucleate cells; however, depolymerizing actin filaments during anaphase would prevent furrowing and lead to the regress of established furrow, also resulting in the formation of binucleate cells. Further, depolymerizing microtubules and actin filaments simultaneously after metaphase would cause the quick regress of the furrow and the formation of binucleate cells. From these results we propose that a successful cytokinesis requires functions and coordination of both the microtubule and actin filament cytoskeletons. Microtubule cytoskeleton may function in the positioning and initiation of cleavage furrow, and the actin filament cytoskeleton may play key roles in the initiation and ingression of the furrow.

  19. Dissecting the molecular mechanism underlying the intimate relationship between cellulose microfibrils and cortical microtubules

    Directory of Open Access Journals (Sweden)

    Lei eLei

    2014-03-01

    Full Text Available A central question in plant cell development is how the cell wall determines directional cell expansion and therefore the final shape of the cell. As the major load-bearing component of the cell wall, cellulose microfibrils are laid down transversely to the axis of elongation, thus forming a spring-like structure that reinforces the cell laterally and while favoring longitudinal expansion in most growing cells. Mounting evidence suggests that cortical microtubules organize the deposition of cellulose microfibrils, but the precise molecular mechanisms linking microtubules to cellulose organization have remained unclear until the recent discovery of CSI1, a linker protein between the cortical microtubules and the cellulose biosynthesizing machinery. In this review, we will focus on the intimate relationship between cellulose microfibrils and cortical microtubules, in particular, we will discuss microtubule arrangement and cell wall architecture, the linkage between cellulose synthase complexes and microtubules, and the feedback mechanisms between cell wall and microtubules.

  20. Microtubule's conformational cap

    DEFF Research Database (Denmark)

    Chretien, D.; Janosi, I.; Taveau, J.C.

    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...

  1. A series of coordination polymers based on a V-shaped multicarboxylate and bisimidazole ligands: Synthesis, characterization and luminescent properties

    Science.gov (United States)

    Guo, Huadong; Yan, Yongnian; Guo, Xianmin; Wang, Nan; Qi, Yanjuan

    2016-03-01

    Based on a V-shaped multicarboxylic acid and various bisimidazole ligands, six new coordination polymers, namely, [Zn4(otba) (1,4-bix)4]·3H2O (1), [Ni4(otba)2(1,4-bix)3(H2O)2]·2H2O (2), [Zn2(H2otba)2(bib) (H2O)2] (3), [Cd2(H2otba)2(bib)]·2H2O (4), [Zn3(otba)2(bidpe)2(H2O)2] (5), [Ni2(H2otba)2(bidpe)3(H2O)2]·H2O (6) (H4otba = 3,5,3‧,5‧-oxytetrabenzoic acid, 1,4-bix = 1,2-bis(imidazol-1-ylmethyl)benzene, bib = 1,4-bis(imidazol-1-yl)benzene and bidpe = 4,4'-bis(imidazol-1-yl)diphenyl ether) have been hydrothermally synthesized and structurally characterized. Compound 1 displays an uncommon hexanodal 3D 4-connected network. Compound 2 features a trinodal 3D (4, 4, 6)-connected framework. Compound 3 shows an interesting polythreaded 1D→3D species. Compound 4 exhibits a binodal 3D (3, 8)-connected network. Compound 5 displays a three-fold interpenetration of trinodal (3, 3, 4)-connected network. Compound 6 shows a five-fold interpenetration of five-connected bilayer network. The luminescent properties of compounds 1, 3, 4 and 5 were also measured.

  2. Shape-, size-, and functional group-selective binding of small organic guests in a paramagnetic coordination cage.

    Science.gov (United States)

    Turega, Simon; Whitehead, Martina; Hall, Benjamin R; Meijer, Anthony J H M; Hunter, Christopher A; Ward, Michael D

    2013-01-18

    The host-guest chemistry of the octanuclear cubic coordination cage [Co(8)L(12)](16+) (where L is a bridging ligand containing two chelating pyrazolyl-pyridine units connected to a central naphthalene-1,5-diyl spacer via methylene "hinges") has been investigated in detail by (1)H NMR spectroscopy. The cage encloses a cavity of volume of ca. 400 Å(3), which is accessible through 4 Å diameter portals in the centers of the cube faces. The paramagnetism of the cage eliminates overlap of NMR signals by dispersing them over a range of ca. 200 ppm, making changes of specific signals easy to observe, and also results in large complexation-induced shifts of bound guests. The cage, in CD(3)CN solution, acts as a remarkably size- and shape-selective host for small organic guests such as coumarin (K = 78 M(-1)) and other bicyclic molecules of comparable size and shape such as isoquinoline-N-oxide (K = 2100 M(-1)). Binding arises from two independent recognition elements, which have been separately quantified. These are (i) a polar component arising from interaction of the H-bond accepting O atom of the guest with a convergent group of CH protons inside the cavity that lie close to a fac tris-chelate metal center and are therefore in a region of high electrostatic potential; and (ii) an additional component arising from the second aromatic ring (aromatic/van der Waals interactions with the interior surface of the cage and/or solvophobic interactions). The strength of the first component varies linearly with the H-bond-accepting ability of the guest; the second component is fixed at approximately 10 kJ mol(-1). We have also used (1)H-(1)H exchange spectroscopy (EXSY) experiments to analyze semiquantitatively two distinct dynamic processes, viz. movement of the guest into and out of the cavity and tumbling of the guest inside the host cavity. Depending on the size of the guest and the position of substituents, the rates of these processes can vary substantially, and the rates

  3. 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.

  4. 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.

  5. Cell Biology: Microtubule Collisions to the Rescue.

    Science.gov (United States)

    Gardner, Melissa K

    2016-12-19

    The proper regulation of microtubule lengths is fundamental to their cellular function. New work now reports that the collision of a growing microtubule end with another object, such as a microtubule, can contribute to the regulation of microtubule lengths by leaving behind damage that ultimately acts to stabilize the microtubule network. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. TPX2 phosphorylation maintains metaphase spindle length by regulating microtubule flux

    Science.gov (United States)

    Fu, Jingyan; Bian, Minglei; Xin, Guangwei; Deng, Zhaoxuan; Luo, Jia; Guo, Xiao; Chen, Hao; Wang, Yao; Jiang, Qing

    2015-01-01

    A steady-state metaphase spindle maintains constant length, although the microtubules undergo intensive dynamics. Tubulin dimers are incorporated at plus ends of spindle microtubules while they are removed from the minus ends, resulting in poleward movement. Such microtubule flux is regulated by the microtubule rescue factors CLASPs at kinetochores and depolymerizing protein Kif2a at the poles, along with other regulators of microtubule dynamics. How microtubule polymerization and depolymerization are coordinated remains unclear. Here we show that TPX2, a microtubule-bundling protein and activator of Aurora A, plays an important role. TPX2 was phosphorylated by Aurora A during mitosis. Its phospho-null mutant caused short metaphase spindles coupled with low microtubule flux rate. Interestingly, phosphorylation of TPX2 regulated its interaction with CLASP1 but not Kif2a. The effect of its mutant in shortening the spindle could be rescued by codepletion of CLASP1 and Kif2a that abolished microtubule flux. Together we propose that Aurora A–dependent TPX2 phosphorylation controls mitotic spindle length through regulating microtubule flux. PMID:26240182

  7. Stabilizing versus destabilizing the microtubules: a double-edge sword for an effective cancer treatment option?

    Science.gov (United States)

    Fanale, Daniele; Bronte, Giuseppe; Passiglia, Francesco; Calò, Valentina; Castiglia, Marta; Di Piazza, Florinda; Barraco, Nadia; Cangemi, Antonina; Catarella, Maria Teresa; Insalaco, Lavinia; Listì, Angela; Maragliano, Rossella; Massihnia, Daniela; Perez, Alessandro; Toia, Francesca; Cicero, Giuseppe; Bazan, Viviana

    2015-01-01

    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.

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

    Science.gov (United States)

    Fanale, Daniele; Bronte, Giuseppe; Passiglia, Francesco; Calò, Valentina; Castiglia, Marta; Di Piazza, Florinda; Barraco, Nadia; Cangemi, Antonina; Catarella, Maria Teresa; Insalaco, Lavinia; Listì, Angela; Maragliano, Rossella; Massihnia, Daniela; Perez, Alessandro; Toia, Francesca; Cicero, Giuseppe; Bazan, Viviana

    2015-01-01

    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. PMID:26484003

  9. 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.

  10. Microtubule organization in three-dimensional confined geometries: Evaluating the role of elasticity through a combined in vitro and modeling approach

    NARCIS (Netherlands)

    Cosentino Lagomarsino, M.; Tanase, C.; Vos, J.W.; Emons, A.M.C.; Mulder, B.; Dogterom, M.

    2007-01-01

    Microtubules or microtubule bundles in cells often grow longer than the size of the cell, which causes their shape and organization to adapt to constraints imposed by the cell geometry. We test the reciprocal role of elasticity and confinement in the organization of growing microtubules in a

  11. 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....

  12. Folding of the Tau Protein on Microtubules.

    Science.gov (United States)

    Kadavath, Harindranath; Jaremko, Mariusz; Jaremko, Łukasz; Biernat, Jacek; Mandelkow, Eckhard; Zweckstetter, Markus

    2015-08-24

    Microtubules are regulated by microtubule-associated proteins. However, little is known about the structure of microtubule-associated proteins in complex with microtubules. Herein we show that the microtubule-associated protein Tau, which is intrinsically disordered in solution, locally folds into a stable structure upon binding to microtubules. While Tau is highly flexible in solution and adopts a β-sheet structure in amyloid fibrils, in complex with microtubules the conserved hexapeptides at the beginning of the Tau repeats two and three convert into a hairpin conformation. Thus, binding to microtubules stabilizes a unique conformation in Tau. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Mechanical modulation of cardiac microtubules.

    Science.gov (United States)

    White, Ed

    2011-07-01

    Microtubules are a major component of the cardiac myocyte cytoskeleton. Interventions that alter it may influence cardiac mechanical and electrical activity by disrupting the trafficking of proteins to and from the surface membrane by molecular motors such as dynein, which use microtubules as tracks to step along. Free tubulin dimers may transfer GTP to the α-subunits of G-proteins, thus an increase in free tubulin could increase the activity of G-proteins; evidence for and against such a role exists. There is more general agreement that microtubules act as compression-resisting structures within myocytes, influencing visco-elasticity of myocytes and increasing resistance to shortening when proliferated and resisting deformation from longitudinal shear stress. In response to pressure overload, there can be post-translational modifications resulting in more stable microtubules and an increase in microtubule density. This is accompanied by contractile dysfunction of myocytes which can be reversed by microtubule disruption. There are reports of mechanically induced changes in electrical activity that are dependent upon microtubules, but at present, a consensus is lacking on whether disruption or proliferation would be beneficial in the prevention of arrhythmias. Microtubules certainly play a role in the response of cardiac myocytes to mechanical stimulation, the exact nature and significance of this role is still to be fully determined.

  14. Aging of dynamically stabilized microtubules

    CERN Document Server

    Ebbinghaus, M

    2009-01-01

    The microtubule network, an important part of the cytoskeleton, is constantly remodeled by alternating phases of growth and shrinkage of individual filaments. Plus-end tracking proteins (+TIPs) interact with the microtubule and in many cases alter its dynamics. While it is established that the prototypal CLIP-170 enhances microtubule stability by increasing rescues, the plus-end tracking mechanism is still under debate. We present a model for microtubule dynamics in which a rescue factor is dynamically added to the filament while growing. As a consequence, the filament shows aging behavior which should be experimentally accessible and thus allow one to exclude some hypothesized models of the inclusion of rescue factors at the microtubule plus end. Additionally, we show the strong influence of the cell geometry on the quantitative results.

  15. Microtubule organization : from the centrosome to the Golgi apparatus

    NARCIS (Netherlands)

    Wu, J.

    2017-01-01

    Similar to the skeleton of a human body, every cell possesses the so-called cytoskeleton, a system of filaments that support cell shape and enable cells to divide and move. One of the major types of cytoskeletal fibers are microtubules, microscopic tubes that cells use as rails to transport their co

  16. Structural insights into microtubule doublet interactions inaxonemes

    Energy Technology Data Exchange (ETDEWEB)

    Downing, Kenneth H.; Sui, Haixin

    2007-06-06

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

  17. Variations of structures and solid-state conductivity of isomeric silver(I) coordination polymers having linear and V-shaped thiophene-centered ditriazole ligands

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Bin [State Key Laboratory of Coordination Chemistry, Nanjing National Laboratory of Microstructures, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093 (China); School of Environmental and Chemical Engineering, Nanchang Hangkong University, Nanchang, 330063 (China); Geng, Jiao; Zhang, Lie [State Key Laboratory of Coordination Chemistry, Nanjing National Laboratory of Microstructures, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093 (China); Huang, Wei, E-mail: whuang@nju.edu.cn [State Key Laboratory of Coordination Chemistry, Nanjing National Laboratory of Microstructures, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093 (China)

    2014-07-01

    A pair of new linear and V-shaped acceptor–donor–acceptor (A−D−A) thiophene-centered ditriazole structural isomers, i.e., 2,5-di(1H-1,2,4-triazol-1-yl)thiophene (L{sup 1}) and 3,4-di(1H-1,2,4-triazol-1-yl)thiophene (L{sup 2}), has been synthesized and characterized. They are used as μ{sub 2}-bridging ligands to prepare a pair of silver(I) coordination polymers formulated as [Ag(L{sup 1})(NO{sub 3})]{sub n} (1) and [Ag(L{sup 2})(NO{sub 3})]{sub n} (2), which are also structural isomers at the supramolecular level. X-ray single-crystal diffraction analyses for 1 and 2 reveal that they exhibit the same one-dimensional (1D) coordination polymers but different structural architectures because of the distinguishable shape and configuration of isomeric ligands (L{sup 1} and L{sup 2}) and the alterations of the coordination numbers. More interestingly, compared with the free ligands, 1D silver(I) polymeric isomers 1 and 2 show significant enhancement of solid-state conductivity to different extents (1.42×10{sup 4} and 2.17×10{sup 3} times), where 6.96 times' enhancement of solid-state conductivity from 1 to 2 has been observed. The formation of Ag–N coordinative bonds and the configurational discrepancy of L{sup 1} and L{sup 2} are believed to play important roles in facilitating the electron transport between molecules, which can also be supported by Density Function Theory calculations of their band gaps. - Graphical abstract: A pair of linear and V-shaped isomeric thiophene-centered ditriazole ligands (L{sup 1}) and L{sup 2} are used to prepare a pair of silver(I) polymeric isomers (1 and 2), where significant enhancement of solid-state conductivity to different extents are observed originating from the distinguishable shape and configuration of isomeric ligands. - Highlights: • A pair of linear and V-shaped thiophene-centered ditriazole structural isomers is prepared. • They are used as µ{sub 2}-bridging ligands to prepare a pair of silver

  18. The development and geometry of shape change in Arabidopsis thaliana cotyledon pavement cells

    Directory of Open Access Journals (Sweden)

    Halsey Leah E

    2011-02-01

    Full Text Available Abstract Background The leaf epidermis is an important architectural control element that influences the growth properties of underlying tissues and the overall form of the organ. In dicots, interdigitated pavement cells are the building blocks of the tissue, and their morphogenesis includes the assembly of specialized cell walls that surround the apical, basal, and lateral (anticlinal cell surfaces. The microtubule and actin cytoskeletons are highly polarized along the cortex of the anticlinal wall; however, the relationships between these arrays and cell morphogenesis are unclear. Results We developed new quantitative tools to compare population-level growth statistics with time-lapse imaging of cotyledon pavement cells in an intact tissue. The analysis revealed alternating waves of lobe initiation and a phase of lateral isotropic expansion that persisted for days. During lateral isotropic diffuse growth, microtubule organization varied greatly between cell surfaces. Parallel microtubule bundles were distributed unevenly along the anticlinal surface, with subsets marking stable cortical domains at cell indentations and others clearly populating the cortex within convex cell protrusions. Conclusions Pavement cell morphogenesis is discontinuous, and includes punctuated phases of lobe initiation and lateral isotropic expansion. In the epidermis, lateral isotropic growth is independent of pavement cell size and shape. Cortical microtubules along the upper cell surface and stable cortical patches of anticlinal microtubules may coordinate the growth behaviors of orthogonal cell walls. This work illustrates the importance of directly linking protein localization data to the growth behavior of leaf epidermal cells.

  19. 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.

  20. Self-assembly of iron coordination polymer of bowl-shaped N-ligand and dodecamolybdosilicate anion

    Science.gov (United States)

    Tang, Qun; Chen, Ya-Guang

    2014-06-01

    [FeII2(ttmb)2(H2O)6][SiMo12O40]·4H2O (1) (ttmb = 1,3,5-tris(triazol-1-ylmethyl) benzene) was synthesized under hydrothermal conditions and characterized by IR spectroscopy, TG analysis and X-ray diffraction method. In 1 the bowl-like tridentate ligand, ttmb, bridges Fe2+ ions forming a coordination polymeric layer, [FeII2(ttmb)2(H2O)6]n4n+, with a large dimensional grid of 7.00 × 7.11 Å, in the presence of Keggin-type polyoxoanion as a template. The coordination polymeric layers and the polyoxoanions are fused into an inorganic-organic hybrid by hydrogen bonds. The photoluminescent spectrum of 1 in solid state at room temperature shows that the coordination of ttmb to Fe2+ ion results in a quench of the ttmb intramolecular fluorescence and a weak O-Mo LMCT fluorescent emission was observed at 520 nm.

  1. Motility and microtubule depolymerization mechanisms of the Kinesin-8 motor, KIF19A

    Science.gov (United States)

    Wang, Doudou; Nitta, Ryo; Morikawa, Manatsu; Yajima, Hiroaki; Inoue, Shigeyuki; Shigematsu, Hideki; Kikkawa, Masahide; Hirokawa, Nobutaka

    2016-01-01

    The kinesin-8 motor, KIF19A, accumulates at cilia tips and controls cilium length. Defective KIF19A leads to hydrocephalus and female infertility because of abnormally elongated cilia. Uniquely among kinesins, KIF19A possesses the dual functions of motility along ciliary microtubules and depolymerization of microtubules. To elucidate the molecular mechanisms of these functions we solved the crystal structure of its motor domain and determined its cryo-electron microscopy structure complexed with a microtubule. The features of KIF19A that enable its dual function are clustered on its microtubule-binding side. Unexpectedly, a destabilized switch II coordinates with a destabilized L8 to enable KIF19A to adjust to both straight and curved microtubule protofilaments. The basic clusters of L2 and L12 tether the microtubule. The long L2 with a characteristic acidic-hydrophobic-basic sequence effectively stabilizes the curved conformation of microtubule ends. Hence, KIF19A utilizes multiple strategies to accomplish the dual functions of motility and microtubule depolymerization by ATP hydrolysis. DOI: http://dx.doi.org/10.7554/eLife.18101.001 PMID:27690357

  2. Cell wall matrix polysaccharide distribution and cortical microtubule organization: two factors controlling mesophyll cell morphogenesis in land plants.

    Science.gov (United States)

    Sotiriou, P; Giannoutsou, E; Panteris, E; Apostolakos, P; Galatis, B

    2016-03-01

    This work investigates the involvement of local differentiation of cell wall matrix polysaccharides and the role of microtubules in the morphogenesis of mesophyll cells (MCs) of three types (lobed, branched and palisade) in the dicotyledon Vigna sinensis and the fern Asplenium nidus. Homogalacturonan (HGA) epitopes recognized by the 2F4, JIM5 and JIM7 antibodies and callose were immunolocalized in hand-made leaf sections. Callose was also stained with aniline blue. We studied microtubule organization by tubulin immunofluorescence and transmission electron microscopy. In both plants, the matrix cell wall polysaccharide distribution underwent definite changes during MC differentiation. Callose constantly defined the sites of MC contacts. The 2F4 HGA epitope in V. sinensis first appeared in MC contacts but gradually moved towards the cell wall regions facing the intercellular spaces, while in A. nidus it was initially localized at the cell walls delimiting the intercellular spaces, but finally shifted to MC contacts. In V. sinensis, the JIM5 and JIM7 HGA epitopes initially marked the cell walls delimiting the intercellular spaces and gradually shifted in MC contacts, while in A. nidus they constantly enriched MC contacts. In all MC types examined, the cortical microtubules played a crucial role in their morphogenesis. In particular, in palisade MCs, cortical microtubule helices, by controlling cellulose microfibril orientation, forced these MCs to acquire a truncated cone-like shape. Unexpectedly in V. sinensis, the differentiation of colchicine-affected MCs deviated completely, since they developed a cell wall ingrowth labyrinth, becoming transfer-like cells. The results of this work and previous studies on Zea mays (Giannoutsou et al., Annals of Botany 2013; 112: : 1067-1081) revealed highly controlled local cell wall matrix differentiation in MCs of species belonging to different plant groups. This, in coordination with microtubule-dependent cellulose microfibril

  3. Microtubule affinity-regulating kinase 4: structure, function, and regulation.

    Science.gov (United States)

    Naz, Farha; Anjum, Farah; Islam, Asimul; Ahmad, Faizan; Hassan, Md Imtaiyaz

    2013-11-01

    MAP/Microtubule affinity-regulating kinase 4 (MARK4) belongs to the family of serine/threonine kinases that phosphorylate the microtubule-associated proteins (MAP) causing their detachment from the microtubules thereby increasing microtubule dynamics and facilitating cell division, cell cycle control, cell polarity determination, cell shape alterations, etc. The MARK4 gene encodes two alternatively spliced isoforms, L and S that differ in their C-terminal region. These isoforms are differentially regulated in human tissues including central nervous system. MARK4L is a 752-residue-long polypeptide that is divided into three distinct domains: (1) protein kinase domain (59-314), (2) ubiquitin-associated domain (322-369), and (3) kinase-associated domain (703-752) plus 54 residues (649-703) involved in the proper folding and function of the enzyme. In addition, residues 65-73 are considered to be the ATP-binding domain and Lys88 is considered as ATP-binding site. Asp181 has been proposed to be the active site of MARK4 that is activated by phosphorylation of Thr214 side chain. The isoform MARK4S is highly expressed in the normal brain and is presumably involved in neuronal differentiation. On the other hand, the isoform MARK4L is upregulated in hepatocarcinoma cells and gliomas suggesting its involvement in cell cycle. Several biological functions are also associated with MARK4 including microtubule bundle formation, nervous system development, and positive regulation of programmed cell death. Therefore, MARK4 is considered as the most suitable target for structure-based rational drug design. Our sequence, structure- and function-based analysis should be helpful for better understanding of mechanisms of regulation of microtubule dynamics and MARK4 associated diseases.

  4. Competitive coordination aggregation for V-shaped [Co3] and disc-like [Co7] complexes: synthesis, magnetic properties and catechol oxidase activity.

    Science.gov (United States)

    Singha Mahapatra, Tufan; Basak, Dipmalya; Chand, Santanu; Lengyel, Jeff; Shatruk, Michael; Bertolasi, Valerio; Ray, Debashis

    2016-09-14

    Unique dependence on the nature of metal salt and reaction conditions for coordination assembly reactions of varying architecture and nuclearity have been identified in V-shaped [Co3L4] and planar disc-like [Co7L6] compounds: [CoL2(μ-L)2(μ-OH2)2(CF3CO2)2] (1) and [Co(μ-L)6(μ-OMe)6]Cl2 (2) (HL = 2-{(3-ethoxypropylimino)methyl}-6-methoxyphenol). At room temperature varying reaction conditions, cobalt-ligand ratios and use of different bases allowed unique types of coordination self-assembly. The synthetic marvel lies in the nature of aggregation with respect to the two unrelated cores in 1 and 2. Complex 1 assumes a V-shaped arrangement bound to L(-), water and a trifluoroacetate anion, while 2 grows around a central Co(II) ion surrounded by a {Co} hexagon bound to methoxide and L(-). Magnetic measurements revealed that the intermetallic interactions are antiferromagnetic in nature in the case of complex 1 and ferromagnetic in the case of 2 involving high spin cobalt(ii) ions with stabilization of the high-spin ground state in the latter case. In MeCN solutions complexes 1 and 2 showed catalytic oxidation of 3,5-di-tert-butylcatechol (3,5-DTBCH2) to 3,5-di-tert-butylbenzoquinone (3,5-DTBQ) in air. The kinetic study in MeCN revealed that with respect to the catalytic turnover number (kcat) 2 is more effective than 1 for oxidation of 3,5-DTBCH2.

  5. Cytoskeletal architecture of isolated mitotic spindle with special reference to microtubule-associated proteins and cytoplasmic dynein.

    Science.gov (United States)

    Hirokawa, N; Takemura, R; Hisanaga, S

    1985-11-01

    We have studied cytoskeletal architectures of isolated mitotic apparatus from sea urchin eggs using quick-freeze, deep-etch electron microscopy. This method revealed the existence of an extensive three-dimensional network of straight and branching crossbridges between spindle microtubules. The surface of the spindle microtubules was almost entirely covered with hexagonally packed, small, round button-like structures which were very uniform in shape and size (approximately 8 nm in diameter), and these microtubule buttons frequently provided bases for crossbridges between adjacent microtubules. These structures were removed from the surface of microtubules by high salt (0.6 M NaCl) extraction. Microtubule-associated proteins (MAPs) and microtubules isolated from mitotic spindles which were mainly composed of a large amount of 75-kD protein and some high molecular mass (250 kD, 245 kD) proteins were polymerized in vitro and examined by quick-freeze, deep-etch electron microscopy. The surfaces of microtubules were entirely covered with the same hexagonally packed round buttons, the arrangement of which is intimately related to that of tubulin dimers. Short crossbridges and some longer crossbridges were also observed. High salt treatment (0.6 M NaCl) extracted both 75-kD protein and high molecular weight proteins and removed microtubule buttons and most of crossbridges from the surface of microtubules. Considering the relatively high amount of 75-kD protein among MAPs isolated from mitotic spindles, it is concluded that these microtubule buttons probably consist of 75-kD MAP and that some of the crossbridges in vivo could belong to MAPs. Another kind of granule, larger in size (11-26 nm in diameter), was also on occasion associated with the surface of microtubules of mitotic spindles. A fine sidearm sometimes connected the larger granule to adjacent microtubules. Localization of cytoplasmic dynein ATPase in the mitotic spindle was investigated by electron microscopic

  6. Poisson Coordinates.

    Science.gov (United States)

    Li, Xian-Ying; Hu, Shi-Min

    2013-02-01

    Harmonic functions are the critical points of a Dirichlet energy functional, the linear projections of conformal maps. They play an important role in computer graphics, particularly for gradient-domain image processing and shape-preserving geometric computation. We propose Poisson coordinates, a novel transfinite interpolation scheme based on the Poisson integral formula, as a rapid way to estimate a harmonic function on a certain domain with desired boundary values. Poisson coordinates are an extension of the Mean Value coordinates (MVCs) which inherit their linear precision, smoothness, and kernel positivity. We give explicit formulas for Poisson coordinates in both continuous and 2D discrete forms. Superior to MVCs, Poisson coordinates are proved to be pseudoharmonic (i.e., they reproduce harmonic functions on n-dimensional balls). Our experimental results show that Poisson coordinates have lower Dirichlet energies than MVCs on a number of typical 2D domains (particularly convex domains). As well as presenting a formula, our approach provides useful insights for further studies on coordinates-based interpolation and fast estimation of harmonic functions.

  7. A stochastic model for microtubule motors describes the in vivo cytoplasmic transport of human adenovirus.

    Directory of Open Access Journals (Sweden)

    Mattia Gazzola

    2009-12-01

    Full Text Available Cytoplasmic transport of organelles, nucleic acids and proteins on microtubules is usually bidirectional with dynein and kinesin motors mediating the delivery of cargoes in the cytoplasm. Here we combine live cell microscopy, single virus tracking and trajectory segmentation to systematically identify the parameters of a stochastic computational model of cargo transport by molecular motors on microtubules. The model parameters are identified using an evolutionary optimization algorithm to minimize the Kullback-Leibler divergence between the in silico and the in vivo run length and velocity distributions of the viruses on microtubules. The present stochastic model suggests that bidirectional transport of human adenoviruses can be explained without explicit motor coordination. The model enables the prediction of the number of motors active on the viral cargo during microtubule-dependent motions as well as the number of motor binding sites, with the protein hexon as the binding site for the motors.

  8. A stochastic model for microtubule motors describes the in vivo cytoplasmic transport of human adenovirus.

    Science.gov (United States)

    Gazzola, Mattia; Burckhardt, Christoph J; Bayati, Basil; Engelke, Martin; Greber, Urs F; Koumoutsakos, Petros

    2009-12-01

    Cytoplasmic transport of organelles, nucleic acids and proteins on microtubules is usually bidirectional with dynein and kinesin motors mediating the delivery of cargoes in the cytoplasm. Here we combine live cell microscopy, single virus tracking and trajectory segmentation to systematically identify the parameters of a stochastic computational model of cargo transport by molecular motors on microtubules. The model parameters are identified using an evolutionary optimization algorithm to minimize the Kullback-Leibler divergence between the in silico and the in vivo run length and velocity distributions of the viruses on microtubules. The present stochastic model suggests that bidirectional transport of human adenoviruses can be explained without explicit motor coordination. The model enables the prediction of the number of motors active on the viral cargo during microtubule-dependent motions as well as the number of motor binding sites, with the protein hexon as the binding site for the motors.

  9. Microtubule depolymerization induces traction force increase through two distinct pathways.

    Science.gov (United States)

    Rape, Andrew; Guo, Wei-hui; Wang, Yu-li

    2011-12-15

    Traction forces increase after microtubule depolymerization; however, the signaling mechanisms underlying this, in particular the dependence upon myosin II, remain unclear. We investigated the mechanism of traction force increase after nocodazole-induced microtubule depolymerization by applying traction force microscopy to cells cultured on micropatterned polyacrylamide hydrogels to obtain samples of homogeneous shape and size. Control cells and cells treated with a focal adhesion kinase (FAK) inhibitor showed similar increases in traction forces, indicating that the response is independent of FAK. Surprisingly, pharmacological inhibition of myosin II did not prevent the increase of residual traction forces upon nocodazole treatment. This increase was abolished upon pharmacological inhibition of FAK. These results suggest two distinct pathways for the regulation of traction forces. First, microtubule depolymerization activates a myosin-II-dependent mechanism through a FAK-independent pathway. Second, microtubule depolymerization also enhances traction forces through a myosin-II-independent, FAK-regulated pathway. Traction forces are therefore regulated by a complex network of complementary signals and force-generating mechanisms.

  10. 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.

  11. Optomechanical proposal for monitoring microtubule mechanical vibrations

    Science.gov (United States)

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

    2017-07-01

    Microtubules provide the mechanical force required for chromosome separation during mitosis. However, little is known about the dynamic (high-frequency) mechanical properties of microtubules. Here, we theoretically propose to control the vibrations of a doubly clamped microtubule by tip electrodes and to detect its motion via the optomechanical coupling between the vibrational modes of the microtubule and an optical cavity. In the presence of a red-detuned strong pump laser, this coupling leads to optomechanical-induced transparency of an optical probe field, which can be detected with state-of-the art technology. The center frequency and line width of the transparency peak give the resonance frequency and damping rate of the microtubule, respectively, while the height of the peak reveals information about the microtubule-cavity field coupling. Our method opens the new possibilities to gain information about the physical properties of microtubules, which will enhance our capability to design physical cancer treatment protocols as alternatives to chemotherapeutic drugs.

  12. Microtubules, polarity and vertebrate neural tube morphogenesis.

    Science.gov (United States)

    Cearns, Michael D; Escuin, Sarah; Alexandre, Paula; Greene, Nicholas D E; Copp, Andrew J

    2016-07-01

    Microtubules (MTs) are key cellular components, long known to participate in morphogenetic events that shape the developing embryo. However, the links between the cellular functions of MTs, their effects on cell shape and polarity, and their role in large-scale morphogenesis remain poorly understood. Here, these relationships were examined with respect to two strategies for generating the vertebrate neural tube: bending and closure of the mammalian neural plate; and cavitation of the teleost neural rod. The latter process has been compared with 'secondary' neurulation that generates the caudal spinal cord in mammals. MTs align along the apico-basal axis of the mammalian neuroepithelium early in neural tube closure, participating functionally in interkinetic nuclear migration, which indirectly impacts on cell shape. Whether MTs play other functional roles in mammalian neurulation remains unclear. In the zebrafish, MTs are important for defining the neural rod midline prior to its cavitation, both by localizing apical proteins at the tissue midline and by orienting cell division through a mirror-symmetric MT apparatus that helps to further define the medial localization of apical polarity proteins. Par proteins have been implicated in centrosome positioning in neuroepithelia as well as in the control of polarized morphogenetic movements in the neural rod. Understanding of MT functions during early nervous system development has so far been limited, partly by techniques that fail to distinguish 'cause' from 'effect'. Future developments will likely rely on novel ways to selectively impair MT function in order to investigate the roles they play.

  13. 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; Gelfand, Vladimir I

    2016-08-23

    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.

  14. Dirichlet boundary conditions for arbitrary-shaped boundaries in stellarator-like magnetic fields for the Flux-Coordinate Independent method

    Science.gov (United States)

    Hill, Peter; Shanahan, Brendan; Dudson, Ben

    2017-04-01

    We present a technique for handling Dirichlet boundary conditions with the Flux Coordinate Independent (FCI) parallel derivative operator with arbitrary-shaped material geometry in general 3D magnetic fields. The FCI method constructs a finite difference scheme for ∇∥ by following field lines between poloidal planes and interpolating within planes. Doing so removes the need for field-aligned coordinate systems that suffer from singularities in the metric tensor at null points in the magnetic field (or equivalently, when q → ∞). One cost of this method is that as the field lines are not on the mesh, they may leave the domain at any point between neighbouring planes, complicating the application of boundary conditions. The Leg Value Fill (LVF) boundary condition scheme presented here involves an extrapolation/interpolation of the boundary value onto the field line end point. The usual finite difference scheme can then be used unmodified. We implement the LVF scheme in BOUT++ and use the Method of Manufactured Solutions to verify the implementation in a rectangular domain, and show that it does not modify the error scaling of the finite difference scheme. The use of LVF for arbitrary wall geometry is outlined. We also demonstrate the feasibility of using the FCI approach in no n-axisymmetric configurations for a simple diffusion model in a "straight stellarator" magnetic field. A Gaussian blob diffuses along the field lines, tracing out flux surfaces. Dirichlet boundary conditions impose a last closed flux surface (LCFS) that confines the density. Including a poloidal limiter moves the LCFS to a smaller radius. The expected scaling of the numerical perpendicular diffusion, which is a consequence of the FCI method, in stellarator-like geometry is recovered. A novel technique for increasing the parallel resolution during post-processing, in order to reduce artefacts in visualisations, is described.

  15. The reciprocal coordination and mechanics of molecular motors in living cells.

    Science.gov (United States)

    Laib, Jeneva A; Marin, John A; Bloodgood, Robert A; Guilford, William H

    2009-03-03

    Molecular motors in living cells are involved in whole-cell locomotion, contractility, developmental shape changes, and organelle movement and positioning. Whether motors of different directionality are functionally coordinated in cells or operate in a semirandom "tug of war" is unclear. We show here that anterograde and retrograde microtubule-based motors in the flagella of Chlamydomonas are regulated such that only motors of a common directionality are engaged at any single time. A laser trap was used to position microspheres on the plasma membrane of immobilized paralyzed Chlamydomonas flagella. The anterograde and retrograde movements of the microsphere were measured with nanometer resolution as microtubule-based motors engaged the transmembrane protein FMG-1. An average of 10 motors acted to move the microsphere in either direction. Reversal of direction during a transport event was uncommon, and quiescent periods separated every transport event, suggesting the coordinated and exclusive action of only a single motor type. After a jump to 32 degrees C, temperature-sensitive mutants of kinesin-2 (fla10) showed exclusively retrograde transport events, driven by 7 motors on average. These data suggest that molecular motors in living cells can be reciprocally coordinated to engage simultaneously in large numbers and for exclusive transport in a single direction, even when a mixed population of motors is present. This offers a unique model for studying the mechanics, regulation, and directional coordination of molecular motors in a living intracellular environment.

  16. Rational assembly of Pb(II)/Cd(II)/Mn(II) coordination polymers based on flexible V-shaped dicarboxylate ligand: Syntheses, helical structures and properties

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Gao-Shan [School of Environment and Chemical Engineering, Nanchang Hangkong University, Nanchang 330063 (China); Liu, Chong-Bo, E-mail: cbliu@nchu.edu.cn [School of Environment and Chemical Engineering, Nanchang Hangkong University, Nanchang 330063 (China); School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332 (United States); Liu, Hong [School of Environment and Chemical Engineering, Nanchang Hangkong University, Nanchang 330063 (China); Robbins, Julianne; Zhang, Z. John [School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332 (United States); Yin, Hong-Shan [School of Environment and Chemical Engineering, Nanchang Hangkong University, Nanchang 330063 (China); Wen, Hui-Liang [State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047 (China); Wang, Yu-Hua [School of Environment and Chemical Engineering, Nanchang Hangkong University, Nanchang 330063 (China)

    2015-05-15

    Six new coordination polymers, namely, [Pb(L)(H{sub 2}O)] (1), [Pb(L)(phen)] (2), [Pb{sub 2}(L){sub 2}(4,4′-bipy){sub 0.5}] (3), [Cd(L)(phen)] (4), [Cd(L)(4,4′-bipy)]·H{sub 2}O (5) and [Mn(L)(4,4′-bipy)]·H{sub 2}O (6) have been synthesized by the hydrothermal reaction of 2,2′-[hexafluoroisopropylidenebis(p-phenyleneoxy)]diacetic acid (H{sub 2}L) with Pb(II)/Cd(II)/Mn(II) in the presence of ancillary ligands 4,4′-bipyridine (4,4′-bipy) or 1,10-phenanthroline (phen). Complexes 1 and 4–6 exhibit 2-D structures, and complexes 2–3 display 3-D frameworks, of which L{sup 2−} ligands join metal ions to single-stranded helical chains of 1, 3–6 and double-stranded helical chains of 2. Complexes 2 and 3 also contain double-stranded Metal–O helices. Topology analysis reveals that complexes 1 and 4 both represent 4-connected sql net, 2 represents 6-connected pcu net, 3 exhibits a novel (3,12)-connected net, while 5 and 6 display (3,5)-connected gek1 net. The six complexes exhibit two kinds of inorganic–organic connectivities: I{sup 0}O{sup 2} for 1, 4–6, and I{sup 1}O{sup 2} for 2–3. The photoluminescent properties of 4–5 and the magnetic properties of 6 have been investigated. - Graphical abstract: Six new Pb(II)/Cd(II)/Mn(II) coordination polymers with helical structures based on flexible V-shaped dicarboxylate ligand have been synthesized and structurally characterized. Photoluminescent and magnetic properties have been investigated. - Highlights: • Six novel M(II) coordination polymers with 2,2′-[hexafluoroisopropylidenebis(p-phenyleneoxy)]diacetic acid and N-donor ligands. • Complexes 1–6 show diverse intriguing helical characters. • The luminescent properties of complexes 1–5 were investigated. • Complex 6 shows antiferromagnetic coupling.

  17. Microtubule as nanobioelectronic nonlinear circuit

    Directory of Open Access Journals (Sweden)

    Sekulić Dalibor L.

    2012-01-01

    Full Text Available In recent years, the use of biological molecules has offered exciting alternatives to conventional synthetic methods. Specific methods use various biological templates to direct the deposition and patterning of inorganic materials. Here we have established a new electrical model of microtubules as a biological nanoscale circuit based on polyelectrolyte features of cylindrical biopolymers. Our working hypothesis is that microtubules play an active role in sub-cellular computation and signaling via electronic and protonic conductivity and can thus be made useful in hybrid materials that offer novel electronic characteristics. We verify these hypotheses both computationally and analytically through a quantitative model based on the atomic resolution structures of the key functional proteins.

  18. Microtubules in Dendritic Spine Development

    OpenAIRE

    2008-01-01

    It is generally believed that only the actin cytoskeleton resides in dendritic spines and controls spine morphology and plasticity. Here we report that microtubules (MTs) are present in spines and that shRNA knockdown of the MT-plus end binding protein EB3 significantly reduces spine formation. Furthermore, stabilization and inhibition of MTs by low doses of taxol and nocodazole enhance and impair spine formation elicited by BDNF, respectively. Therefore, MTs play an important role in the con...

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

    DEFF Research Database (Denmark)

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

    1996-01-01

    individual tubulin dimers, an ignored. In this cap model, GTP hydrolysis is assumed to be stochastic and uncoupled to microtubule growth. Different rates of hydrolysis are assumed for GTP in the cap's interior and for GTP at its boundary with hydrolyzed parts of the microtubule. Expectation values...... and probability distributions relating to available experimental data are derived. Caps are found to be short and the total rate of hydrolysis at a microtubule end is found to be dynamically coupled to growth. The so-called catastrophe rate is a simple function of the microtubule growth rare and fits experimental...... of microtubule growth before dilution. The GTP content of microtubules is found and its rare of hydrolysis is determined under the circumstances created in an experiment designed to measure this GTP content. It is concluded that this experiment's failure to register any GTP content is consistent with the model...

  20. Proteomic Analysis of Microtubule Interacting Proteins over the Course of Xylem Tracheary Element Formation in Arabidopsis.

    Science.gov (United States)

    Derbyshire, Paul; Ménard, Delphine; Green, Porntip; Saalbach, Gerhard; Buschmann, Henrik; Lloyd, Clive W; Pesquet, Edouard

    2015-10-01

    Plant vascular cells, or tracheary elements (TEs), rely on circumferential secondary cell wall thickenings to maintain sap flow. The patterns in which TE thickenings are organized vary according to the underlying microtubule bundles that guide wall deposition. To identify microtubule interacting proteins present at defined stages of TE differentiation, we exploited the synchronous differentiation of TEs in Arabidopsis thaliana suspension cultures. Quantitative proteomic analysis of microtubule pull-downs, using ratiometric (14)N/(15)N labeling, revealed 605 proteins exhibiting differential accumulation during TE differentiation. Microtubule interacting proteins associated with membrane trafficking, protein synthesis, DNA/RNA binding, and signal transduction peaked during secondary cell wall formation, while proteins associated with stress peaked when approaching TE cell death. In particular, CELLULOSE SYNTHASE-INTERACTING PROTEIN1, already associated with primary wall synthesis, was enriched during secondary cell wall formation. RNAi knockdown of genes encoding several of the identified proteins showed that secondary wall formation depends on the coordinated presence of microtubule interacting proteins with nonoverlapping functions: cell wall thickness, cell wall homogeneity, and the pattern and cortical location of the wall are dependent on different proteins. Altogether, proteins linking microtubules to a range of metabolic compartments vary specifically during TE differentiation and regulate different aspects of wall patterning.

  1. Symmetry Breaking in an Edgeless Epithelium by Fat2-Regulated Microtubule Polarity

    Directory of Open Access Journals (Sweden)

    Dong-Yuan Chen

    2016-05-01

    Full Text Available Planar cell polarity (PCP information is a critical determinant of organ morphogenesis. While PCP in bounded epithelial sheets is increasingly well understood, how PCP is organized in tubular and acinar tissues is not. Drosophila egg chambers (follicles are an acinus-like “edgeless epithelium” and exhibit a continuous, circumferential PCP that does not depend on pathways active in bounded epithelia; this follicle PCP directs formation of an ellipsoid rather than a spherical egg. Here, we apply an imaging algorithm to “unroll” the entire 3D tissue surface and comprehensively analyze PCP onset. This approach traces chiral symmetry breaking to plus-end polarity of microtubules in the germarium, well before follicles form and rotate. PCP germarial microtubules provide chiral information that predicts the direction of whole-tissue rotation as soon as independent follicles form. Concordant microtubule polarity, but not microtubule alignment, requires the atypical cadherin Fat2, which acts at an early stage to translate plus-end bias into coordinated actin-mediated collective cell migration. Because microtubules are not required for PCP or migration after follicle rotation initiates, while dynamic actin and extracellular matrix are, polarized microtubules lie at the beginning of a handoff mechanism that passes early chiral PCP of the cytoskeleton to a supracellular planar polarized extracellular matrix and elongates the organ.

  2. Expression of Nucleolin Affects Microtubule Dynamics.

    Directory of Open Access Journals (Sweden)

    Xavier Gaume

    Full Text Available Nucleolin is present in diverse cellular compartments and is involved in a variety of cellular processes from nucleolar structure and function to intracellular trafficking, cell adhesion and migration. Recently, nucleolin has been localized at the mature centriole where it is involved in microtubule nucleation and anchoring. Although this new function of nucleolin linked to microtubule regulation has been identified, the global effects of nucleolin on microtubule dynamics have not been addressed yet. In the present study, we analyzed the roles of nucleolin protein levels on global microtubule dynamics by tracking the EB3 microtubule plus end binding protein in live cells. We have found that during microtubule growth phases, nucleolin affects both the speed and life time of polymerization and by analyzing catastrophe events, we showed that nucleolin reduces catastrophe frequency. This new property of nucleolin was then confirmed in a cold induced microtubule depolymerization experiment in which we have found that cold resistant microtubules were totally destabilized in nucleolin depleted cells. Altogether, our data demonstrate a new function of nucleolin on microtubule stabilization, thus bringing novel insights into understanding the multifunctional properties of nucleolin in healthy and cancer cells.

  3. Mechanical stress induced mechanism of microtubule catastrophes.

    Science.gov (United States)

    Hunyadi, Viktória; Chrétien, Denis; Jánosi, Imre M

    2005-05-13

    Microtubules assembled in vitro from pure tubulin can switch occasionally from growing to shrinking states or resume assembly, an unusual behavior termed "dynamic instability of microtubule growth". Its origin remains unclear and several models have been proposed, including occasional switching of the microtubules into energetically unfavorable configurations during assembly. In this study, we have asked whether the excess energy accumulated in these configurations would be of sufficient magnitude to destabilize the capping region that must exist at the end of growing microtubules. For this purpose, we have analyzed the frequency distribution of microtubules assembled in vitro from pure tubulin, and modeled the different mechanical constraints accumulated in their wall. We find that the maximal excess energy that the microtubule lattice can store is in the order of 11 kBT per dimer. Configurations that require distortions up to approximately 20 kBT are allowed at the expense of a slight conformational change, and larger distortions are not observed. Modeling of the different elastic deformations suggests that the excess energy is essentially induced by protofilament skewing, microtubule radial curvature change and inter-subunit shearing, distortions that must destabilize further the tubulin subunits interactions. These results are consistent with the hypothesis that unfavorable closure events may trigger the catastrophes observed at low tubulin concentration in vitro. In addition, we propose a novel type of representation that describes the stability of microtubule assembly systems, and which might be of considerable interest to study the effects of stabilizing and destabilizing factors on microtubule structure and dynamics.

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

    Directory of Open Access Journals (Sweden)

    Maria Kwiatkowska

    2011-07-01

    Full Text Available Lipotubuloids, structures containing lipid bodies and microtubules, are described in ovary epidermalcells of Ornithogalum umbellatum. Microtubules of lipotubuloids can be fixed in electron microscope fixativecontaining only buffered OsO4 or in glutaraldehyde with OsO4 post-fixation, or in a mixture of OsO4 and glutaraldehyde[1]. None of these substances fixes cortical microtubules of ovary epidermis of this plant which ischaracterized by dynamic longitudinal growth. However, cortical microtubules can be fixed with cold methanolaccording immunocytological methods with the use of b-tubulin antibodies and fluorescein. The existence ofcortical microtubules has also been evidenced by EM observations solely after the use of taxol, microtubulestabilizer, and fixation in a glutaraldehyde/OsO4 mixture. These microtubules mostly lie transversely, sometimesobliquely, and rarely parallel to the cell axis. Staining, using Ruthenium Red and silver hexamine, has revealedthat lipotubuloid microtubules surface is covered with polysaccharides. The presumption has been made thatthe presence of a polysaccharide layer enhances the stability of lipotubuloid microtubules.

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

    Science.gov (United States)

    Nishida, Tomoki; Yoshimura, Ryoichi; Endo, Yasuhisa

    2017-06-23

    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.

  6. Structural diversity and photocatalytic properties of Cd(II) coordination polymers constructed by a flexible V-shaped bipyridyl benzene ligand and dicarboxylate derivatives.

    Science.gov (United States)

    Liu, Lei-Lei; Yu, Cai-Xia; Ma, Feng-Ji; Li, Ya-Ru; Han, Jing-Jing; Lin, Lu; Ma, Lu-Fang

    2015-01-28

    Hydrothermal reactions of Cd(OAc)2·2H2O with a flexible V-shaped bipyridyl benzene ligand and five benzenedicarboxylic acid derivatives gave rise to five new coordination polymers i.e., [Cd(1,4-BDC)(bpmb)(H2O)]n (1), {[Cd(1,3-BDC)(bpmb)]·0.125H2O}n (2), [Cd2(5-Me-1,3-BDC)2(bpmb)2]n (3), [Cd(5-NO2-1,3-BDC)(bpmb)(H2O)]n (4) and [Cd(5-OH-1,3-BDC)(bpmb)(H2O)]n (5) (bpmb = 1,3-bis(pyridine-3-ylmethoxy)benzene, 1,4-H2BDC = 1,4-benzenedicarboxylic acid, 1,3-H2BDC = 1,3-benzenedicarboxylic acid, 5-Me-1,3-H2BDC = 5-methyl-1,3-benzenedicarboxylic acid, 5-NO2-1,3-H2BDC = 5-nitro-1,3-benzenedicarboxylic acid, 5-OH-1,3-H2BDC = 5-hydroxy-1,3-benzenedicarboxylic acid). Their structures have been determined by single-crystal X-ray diffraction analyses, elemental analyses, IR spectra, powder X-ray diffraction (PXRD) and thermogravimetric analyses (TGA). Compound 1 is a two-fold interpenetrating network showing the coexistence of polyrotaxane and polycatenane characters. Compounds 2 and 3 exhibit similar 2D (3,5)-connected (4(2)·6(7)·8)(4(2)·6) nets in which the bpmb ligands work as lockers in interlocking 1D [Cd(1,3-BDC/5-Me-1,3-BDC)]n chains. Compound 4 shows a 2D 4-connected (6(6)) sandwich-like structure with differently oriented [Cd(5-NO2-1,3-BDC)]n chains. Compound 5 is a 3D supramolecular pcu net based on a 1D ladder-shaped chain. These results suggest that the substituted positions of carboxylate groups and changes in substituted R groups in the 5-position of BDC ligands have significant effect on the final structures. These compounds exhibited relatively good photocatalytic activity towards the degradation of methylene blue (MB) in aqueous solution under UV irradiation. Moreover, solid-state photoluminescence properties of 1-5 were also investigated.

  7. Stochastic Model of Microtubule Dynamics

    Science.gov (United States)

    Hryniv, Ostap; Martínez Esteban, Antonio

    2017-10-01

    We introduce a continuous time stochastic process on strings made of two types of particle, whose dynamics mimics that of microtubules in a living cell. The long term behaviour of the system is described in terms of the velocity v of the string end. We show that v is an analytic function of its parameters and study its monotonicity properties. We give a complete characterisation of the phase diagram of the model and derive several criteria of the growth (v>0) and the shrinking (v<0) regimes of the dynamics.

  8. Emerging roles for microtubules in angiosperm pollen tube growth highlight new research cues

    Directory of Open Access Journals (Sweden)

    Alessandra eMoscatelli

    2015-02-01

    Full Text Available In plants, actin filaments have an important role in organelle movement and cytoplasmic streaming. Otherwise microtubules have a role in restricting organelles to specific areas of the cell and in maintaining organelle morphology. In somatic plant cells, microtubules also participate in cell division and morphogenesis, allowing cells to take their definitive shape in order to perform specific functions. In the latter case, microtubules influence assembly of the cell wall, controlling the delivery of enzymes involved in cellulose synthesis and of wall modulation material to the proper sites.In angiosperm pollen tubes, organelle movement is generally attributed to the acto-myosin system, the main role of which is in distributing organelles in the cytoplasm and in carrying secretory vesicles to the apex for polarized growth. Recent data on membrane trafficking suggests a role of microtubules in fine delivery and repositioning of vesicles to sustain pollen tube growth. This review examines the role of microtubules in secretion and endocytosis, highlighting new research cues regarding cell wall construction and pollen tube-pistil crosstalk, that help unravel the role of microtubules in polarized growth.

  9. Movement of chromosomes with severed kinetochore microtubules.

    Science.gov (United States)

    Forer, Arthur; Johansen, Kristen M; Johansen, Jørgen

    2015-05-01

    Experiments dating from 1966 and thereafter showed that anaphase chromosomes continued to move poleward after their kinetochore microtubules were severed by ultraviolet microbeam irradiation. These observations were initially met with scepticism as they contradicted the prevailing view that kinetochore fibre microtubules pulled chromosomes to the pole. However, recent experiments using visible light laser microbeam irradiations have corroborated these earlier experiments as anaphase chromosomes again were shown to move poleward after their kinetochore microtubules were severed. Thus, multiple independent studies using different techniques have shown that chromosomes can indeed move poleward without direct microtubule connections to the pole, with only a kinetochore 'stub' of microtubules. An issue not yet settled is: what propels the disconnected chromosome? There are two not necessarily mutually exclusive proposals in the literature: (1) chromosome movement is propelled by the kinetochore stub interacting with non-kinetochore microtubules and (2) chromosome movement is propelled by a spindle matrix acting on the stub. In this review, we summarise the data indicating that chromosomes can move with severed kinetochore microtubules and we discuss proposed mechanisms for chromosome movement with severed kinetochore microtubules.

  10. Kinesin-12 motors cooperate to suppress microtubule catastrophes and drive the formation of parallel microtubule bundles.

    Science.gov (United States)

    Drechsler, Hauke; McAinsh, Andrew D

    2016-03-22

    Human Kinesin-12 (hKif15) plays a crucial role in assembly and maintenance of the mitotic spindle. These functions of hKif15 are partially redundant with Kinesin-5 (Eg5), which can cross-link and drive the extensile sliding of antiparallel microtubules. Although both motors are known to be tetramers, the functional properties of hKif15 are less well understood. Here we reveal how single or multiple Kif15 motors can cross-link, transport, and focus the plus-ends of intersecting microtubules. During transport, Kif15 motors step simultaneously along both microtubules with relative microtubule transport driven by a velocity differential between motor domain pairs. Remarkably, this differential is affected by the underlying intersection geometry: the differential is low on parallel and extreme on antiparallel microtubules where one motor domain pair becomes immobile. As a result, when intersecting microtubules are antiparallel, canonical transport of one microtubule along the other is allowed because one motor is firmly attached to one microtubule while it is stepping on the other. When intersecting microtubules are parallel, however, Kif15 motors can drive (biased) parallel sliding because the motor simultaneously steps on both microtubules that it cross-links. These microtubule rearrangements will focus microtubule plus-ends and finally lead to the formation of parallel bundles. At the same time, Kif15 motors cooperate to suppress catastrophe events at polymerizing microtubule plus-ends, raising the possibility that Kif15 motors may synchronize the dynamics of bundles that they have assembled. Thus, Kif15 is adapted to operate on parallel microtubule substrates, a property that clearly distinguishes it from the other tetrameric spindle motor, Eg5.

  11. Mobility of Taxol in Microtubule Bundles

    Science.gov (United States)

    Ross, J.

    2003-06-01

    Mobility of taxol inside microtubules was investigated using fluorescence recovery after photobleaching (FRAP) on flow-aligned bundles. Bundles were made of microtubules with either GMPCPP or GTP at the exchangeable site on the tubulin dimer. Recovery times were sensitive to bundle thickness and packing, indicating that taxol molecules are able to move laterally through the bundle. The density of open binding sites along a microtubule was varied by controlling the concentration of taxol in solution for GMPCPP samples. With > 63% sites occupied, recovery times were independent of taxol concentration and, therefore, inversely proportional to the microscopic dissociation rate, k_{off}. It was found that 10*k_{off} (GMPCPP) ~ k_{off} (GTP), consistent with, but not fully accounting for, the difference in equilibrium constants for taxol on GMPCPP and GTP microtubules. With taxol along the microtubule interior is hindered by rebinding events when open sites are within ~7 nm of each other.

  12. A polarised population of dynamic microtubules mediates homeostatic length control in animal cells.

    Directory of Open Access Journals (Sweden)

    Remigio Picone

    Full Text Available Because physical form and function are intimately linked, mechanisms that maintain cell shape and size within strict limits are likely to be important for a wide variety of biological processes. However, while intrinsic controls have been found to contribute to the relatively well-defined shape of bacteria and yeast cells, the extent to which individual cells from a multicellular animal control their plastic form remains unclear. Here, using micropatterned lines to limit cell extension to one dimension, we show that cells spread to a characteristic steady-state length that is independent of cell size, pattern width, and cortical actin. Instead, homeostatic length control on lines depends on a population of dynamic microtubules that lead during cell extension, and that are aligned along the long cell axis as the result of interactions of microtubule plus ends with the lateral cell cortex. Similarly, during the development of the zebrafish neural tube, elongated neuroepithelial cells maintain a relatively well-defined length that is independent of cell size but dependent upon oriented microtubules. A simple, quantitative model of cellular extension driven by microtubules recapitulates cell elongation on lines, the steady-state distribution of microtubules, and cell length homeostasis, and predicts the effects of microtubule inhibitors on cell length. Together this experimental and theoretical analysis suggests that microtubule dynamics impose unexpected limits on cell geometry that enable cells to regulate their length. Since cells are the building blocks and architects of tissue morphogenesis, such intrinsically defined limits may be important for development and homeostasis in multicellular organisms.

  13. An epigenetic regulator emerges as microtubule minus-end binding and stabilizing factor in mitosis.

    Science.gov (United States)

    Meunier, Sylvain; Shvedunova, Maria; Van Nguyen, Nhuong; Avila, Leonor; Vernos, Isabelle; Akhtar, Asifa

    2015-08-05

    The evolutionary conserved NSL complex is a prominent epigenetic regulator controlling expression of thousands of genes. Here we uncover a novel function of the NSL complex members in mitosis. As the cell enters mitosis, KANSL1 and KANSL3 undergo a marked relocalisation from the chromatin to the mitotic spindle. By stabilizing microtubule minus ends in a RanGTP-dependent manner, they are essential for spindle assembly and chromosome segregation. Moreover, we identify KANSL3 as a microtubule minus-end-binding protein, revealing a new class of mitosis-specific microtubule minus-end regulators. By adopting distinct functions in interphase and mitosis, KANSL proteins provide a link to coordinate the tasks of faithful expression and inheritance of the genome during different phases of the cell cycle.

  14. Intermediate filament-associated cytolinker plectin 1c destabilizes microtubules in keratinocytes

    Science.gov (United States)

    Valencia, Rocio G.; Walko, Gernot; Janda, Lubomir; Novacek, Jirka; Mihailovska, Eva; Reipert, Siegfried; Andrä-Marobela, Kerstin; Wiche, Gerhard

    2013-01-01

    The transition of microtubules (MTs) from an assembled to a disassembled state plays an essential role in several cellular functions. While MT dynamics are often linked to those of actin filaments, little is known about whether intermediate filaments (IFs) have an influence on MT dynamics. We show here that plectin 1c (P1c), one of the multiple isoforms of the IF-associated cytolinker protein plectin, acts as an MT destabilizer. We found that MTs in P1c-deficient (P1c−/−) keratinocytes are more resistant toward nocodazole-induced disassembly and display increased acetylation. In addition, live imaging of MTs in P1c−/−, as well as in plectin-null, cells revealed decreased MT dynamics. Increased MT stability due to P1c deficiency led to changes in cell shape, increased velocity but loss of directionality of migration, smaller-sized focal adhesions, higher glucose uptake, and mitotic spindle aberrations combined with reduced growth rates of cells. On the basis of ex vivo and in vitro experimental approaches, we suggest a mechanism for MT destabilization in which isoform-specific binding of P1c to MTs antagonizes the MT-stabilizing and assembly-promoting function of MT-associated proteins through an inhibitory function exerted by plectin's SH3 domain. Our results open new perspectives on cytolinker-coordinated IF-MT interaction and its physiological significance. PMID:23363598

  15. Decoherence Time of a Microtubule

    CERN Document Server

    Hiramatsu, T; Sakakibara, K; Hiramatsu, Takashi; Matsui, Tetsuo; Sakakibara, Kazuhiko

    2006-01-01

    We formulate and study a quantum field theory of a microtubule, a basic element of living cells. Following the quantum theory of consciousness by Hameroff and Penrose, we let the system to make self-reductions, and measure the decoherence time $\\tau_N$ (the mean interval between two successive reductions) of a cluster consisting of more than $N$ neighboring cells (tubulins). $\\tau_N$ is interpreted as an instance of the stream of consciousness. For a sufficiently small electron hopping amplitude, $\\tau_N$ obeys an exponential law, $\\tau_N \\sim \\exp(c' N)$, and may take realistic values $\\tau_N $ \\raisebox{-0.5ex} {$\\stackrel{>}{\\sim}$} $ 10^{-2}$ sec for $N \\raisebox{-0.5ex} {$\\stackrel{>}{\\sim}$} 1100$.

  16. A mechanism for reorientation of cortical microtubule arrays driven by microtubule severing.

    Science.gov (United States)

    Lindeboom, Jelmer J; Nakamura, Masayoshi; Hibbel, Anneke; Shundyak, Kostya; Gutierrez, Ryan; Ketelaar, Tijs; Emons, Anne Mie C; Mulder, Bela M; Kirik, Viktor; Ehrhardt, David W

    2013-12-06

    Environmental and hormonal signals cause reorganization of microtubule arrays in higher plants, but the mechanisms driving these transitions have remained elusive. The organization of these arrays is required to direct morphogenesis. We discovered that microtubule severing by the protein katanin plays a crucial and unexpected role in the reorientation of cortical arrays, as triggered by blue light. Imaging and genetic experiments revealed that phototropin photoreceptors stimulate katanin-mediated severing specifically at microtubule intersections, leading to the generation of new microtubules at these locations. We show how this activity serves as the basis for a mechanism that amplifies microtubules orthogonal to the initial array, thereby driving array reorientation. Our observations show how severing is used constructively to build a new microtubule array.

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

    Science.gov (United States)

    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

    2016-05-23

    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 they ensure the exceptionally slow growth of centriolar microtubules has remained mysterious. Here, we bring together crystallographic, biophysical, and reconstitution assays to demonstrate that the human centriolar protein CPAP (SAS-4 in worms and flies) binds and "caps" microtubule plus ends by associating with a site of β-tubulin engaged in longitudinal tubulin-tubulin interactions. Strikingly, we uncover that CPAP activity dampens microtubule growth and stabilizes microtubules by inhibiting catastrophes and promoting rescues. We further establish that the capping function of CPAP is important to limit growth of centriolar microtubules in cells. Our results suggest that CPAP acts as a molecular lid that ensures slow assembly of centriolar microtubules and, thereby, contributes to organelle length control.

  18. Transient increase in the levels of γ-tubulin complex and katanin are responsible for reorientation by ethylene and hypergravity of cortical microtubules.

    Science.gov (United States)

    Soga, Kouichi; Yamaguchi, Aya; Kotake, Toshihisa; Wakabayashi, Kazuyuki; Hoson, Takayuki

    2010-11-01

    The body shape of a plant is primarily regulated by orientation of cortical microtubules. γ-Tubulin complex and katanin are required for the nucleation and the severing of microtubules, respectively. Here we discuss the role of γ-tubulin complex and katanin during reorientation of cortical microtubules. 1-Aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene, modifies growth anisotropy of azuki bean epicotyls; it inhibits elongation growth and promotes lateral growth. The ACC-induced reorientation of cortical microtubules from transverse to longitudinal directions preceded the modification of growth anisotropy. The transcript level of γ-tubulin complex (VaTUG and VaGCP3) and katanin (VaKTN1) was increased transiently by ACC treatment. During reorientation of cortical microtubules by hypergravity, which also modifies growth anisotropy of shoots, the expression levels of both γ-tubulin complex and katanin genes were increased transiently. The increase in the number of the nucleated microtubule branch as well as the microtubule-severing activity via upregulation of γ-tubulin complex genes and katanin genes may be involved in the reorientation of cortical microtubules, and contribute to the regulation of the shape of plant body.

  19. Shaping the meristem by mechanical forces

    Science.gov (United States)

    Peaucelle, Alexis; Höfte, Herman

    2009-01-01

    A recent report shows that cells in the Arabidopsis apical meristem orientate their cortical microtubules along mechanical stress patterns generated during tissue morphogenesis. This in turn is expected to influence the mechanical properties of the cell via the modification of the cortical microtubule network and the cell wall. This feedback loop controlling the shape of the meristem may act in parallel with auxin signalling, which determines the site of organ primordium formation. PMID:20948640

  20. Shaping the meristem by mechanical forces

    OpenAIRE

    Laufs, Patrick; Peaucelle, Alexis; Höfte, Herman

    2009-01-01

    A recent report shows that cells in the Arabidopsis apical meristem orientate their cortical microtubules along mechanical stress patterns generated during tissue morphogenesis. This in turn is expected to influence the mechanical properties of the cell via the modification of the cortical microtubule network and the cell wall. This feedback loop controlling the shape of the meristem may act in parallel with auxin signalling, which determines the site of organ primordium formation.

  1. The smallest active fragment of microtubule-associated protein 4 and its interaction with microtubules in phosphate buffer.

    Science.gov (United States)

    Hashi, Yurika; Nagase, Lisa; Matsushima, Kazuyuki; Kotani, Susumu

    2012-01-01

    To analyze the interaction between microtubule-associated protein (MAP) 4 and microtubules physicochemically, a MAP4 active site fragment was designed for nuclear magnetic resonance (NMR) use. The fragment was bacterially expressed and purified to homogeneity. The buffer conditions for NMR were optimized to support microtubule assembly. The fragment was found to bind to microtubules under the optimized buffer conditions.

  2. A Novel Copper(Ⅱ) Coordination Polymer with Figure-of-eight-shaped Channels Occupied by Flexible Monodentate and Bidentate N,N'-bipyrdyl Ligands%A Novel Copper(Ⅱ) Coordination Polymer with Figure-of-eight-shaped Channels Occupied by Flexible Monodentate and Bidentate N,N'-bipyrdyl Ligands

    Institute of Scientific and Technical Information of China (English)

    PAN Peng-Bin; ZHANG Xin; YANG Jin-Xia; YIN Pei-Xiu; YAO Yuan-Gen

    2011-01-01

    A copper(Ⅱ) coordination polymer, [Cu3(HOA)2(bpp)4]n (1, HnOA = 3,3',4,4'- oxidiphthalic acid, bpp = 1,3-bis(4-pyridyl) propane), has been hydrothermally synthesized. The crystal structure is of monoclinic, space group P21/c with a = 11.544(3), b = 16.129(4), c = 21.181 (6) A, β = 98.848(5), V= 3897.0(2) A3, C84018N8H70Cu3, Mr = 1670.13, Z = 2, De = 1.423 g/cm^3, F(000) = 1722, μ = 0.887 mm^-1, R = 0.0878 and wR = 0.2583 for 8496 observed reflections (I 〉 2σ(I)). Single-crystal X-ray structural analysis reveals that 1 is a three-dimensional (3D) framework based on a two-dimensional (2D) mesomeric layer of alternating left- and fight-handed helical chains. The monodentate terminal and bidentate bridging bpp ligands in the same conformation are all encapsulated in the figure-of-eight-shaped channels, thus further stabilizing the whole three-dimensional framework.

  3. One-parameter nonrelativistic supersymmetry for microtubules

    CERN Document Server

    Rosu, H C

    2003-01-01

    The simple supersymmetric model of Caticha [PRA 51, 4264 (1995)], as used by Rosu [PRE 55, 2038 (1997)] for microtubules, is generalized to the case of Mielnik's one-parameter nonrelativistic susy [JMP 25, 3387 (1984)

  4. Motor function in interpolar microtubules during metaphase

    OpenAIRE

    Deutsch, J. M.; Lewis, Ian P.

    2013-01-01

    We analyze experimental observations of microtubules undergoing small fluctuations about a "balance point" when mixed in solution of two different kinesin motor proteins, KLP61F and Ncd. It has been proposed that the microtubule movement is due to stochastic variations in the densities of the two species of motor proteins. We test this hypothesis here by showing how it maps onto a one-dimensional random walk in a random environment. Our estimate of the amplitude of the fluctuations agrees wit...

  5. Wave->Diffusion Transition in Microtubules

    OpenAIRE

    2005-01-01

    In this paper the heat transport in microtubules (MT) is investigated. When the dimension of the structure is of the order of the de Broglie wave length the transport phenomena must be analyzed within quantum mechanics. In this paper we developed the Dirac type thermal equation for MT .The solution of the equation-the temperature fields for electrons can be wave type or diffusion type depending on the dynamics of the scattering. Key words: Microtubules ultrashort laser pulses, Dirac thermal e...

  6. A novel p21-activated kinase binds the actin and microtubule networks and induces microtubule stabilization

    Science.gov (United States)

    Cau, Julien; Faure, Sandrine; Comps, Michel; Delsert, Claude; Morin, Nathalie

    2001-01-01

    Coordination of the different cytoskeleton networks in the cell is of central importance for morphogenesis, organelle transport, and motility. The Rho family proteins are well characterized for their effects on the actin cytoskeleton, but increasing evidence indicates that they may also control microtubule (MT) dynamics. Here, we demonstrate that a novel Cdc42/Rac effector, X-p21-activated kinase (PAK)5, colocalizes and binds to both the actin and MT networks and that its subcellular localization is regulated during cell cycle progression. In transfected cells, X-PAK5 promotes the formation of stabilized MTs that are associated in bundles and interferes with MTs dynamics, slowing both the elongation and shrinkage rates and inducing long paused periods. X-PAK5 subcellular localization is regulated tightly, since coexpression with active Rac or Cdc42 induces its shuttling to actin-rich structures. Thus, X-PAK5 is a novel MT-associated protein that may communicate between the actin and MT networks during cellular responses to environmental conditions. PMID:11733543

  7. Spatiotemporal Regulation of Nuclear Transport Machinery and Microtubule Organization

    Directory of Open Access Journals (Sweden)

    Naoyuki Okada

    2015-08-01

    Full Text Available 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.

  8. Dynamics and regulation of plant interphase microtubules: a comparative view.

    Science.gov (United States)

    Hashimoto, Takashi

    2003-12-01

    Microtubule and actin cytoskeletons are fundamental to a variety of cellular activities within eukaryotic organisms. Extensive information on the dynamics and functions of microtubules, as well as on their regulatory proteins, have been revealed in fungi and animals, and corresponding pictures are now slowly emerging in plants. During interphase, plant cells contain highly dynamic cortical microtubules that organize into ordered arrays, which are apparently regulated by distinct groups of microtubule regulators. Comparison with fungal and animal microtubules highlights both conserved and unique mechanisms for the regulation of the microtubule cytoskeleton in plants.

  9. The actin-microtubule cross-linking activity of Drosophila Short stop is regulated by intramolecular inhibition.

    Science.gov (United States)

    Applewhite, Derek A; Grode, Kyle D; Duncan, Mara C; Rogers, Stephen L

    2013-09-01

    Actin and microtubule dynamics must be precisely coordinated during cell migration, mitosis, and morphogenesis--much of this coordination is mediated by proteins that physically bridge the two cytoskeletal networks. We have investigated the regulation of the Drosophila actin-microtubule cross-linker Short stop (Shot), a member of the spectraplakin family. Our data suggest that Shot's cytoskeletal cross-linking activity is regulated by an intramolecular inhibitory mechanism. In its inactive conformation, Shot adopts a "closed" conformation through interactions between its NH(2)-terminal actin-binding domain and COOH-terminal EF-hand-GAS2 domain. This inactive conformation is targeted to the growing microtubule plus end by EB1. On activation, Shot binds along the microtubule through its COOH-terminal GAS2 domain and binds to actin with its NH(2)-terminal tandem CH domains. We propose that this mechanism allows Shot to rapidly cross-link dynamic microtubules in response to localized activating signals at the cell cortex.

  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. A study of microtubule dipole lattices

    Science.gov (United States)

    Nandi, Shubhendu

    Microtubules are cytoskeletal protein polymers orchestrating a host of important cellular functions including, but not limited to, cell support, cell division, cell motility and cell transport. In this thesis, we construct a toy-model of the microtubule lattice composed of vector Ising spins representing tubulin molecules, the building block of microtubules. Nearest-neighbor and next-to-nearest neighbor interactions are considered within an anisotropic dielectric medium. As a consequence of the helical topology, we observe that certain spin orientations render the lattice frustrated with nearest neighbor ferroelectric and next-to-nearest neighbor antiferroelectric bonds. Under these conditions, the lattice displays the remarkable property of stabilizing certain spin patterns that are robust to thermal fluctuations. We model this behavior in the framework of a generalized Ising model known as the J1 - J2 model and theoretically determine the set of stable patterns. Employing Monte-Carlo methods, we demonstrate the stability of such patterns in the microtubule lattice at human physiological temperatures. This suggests a novel biological mechanism for storing information in living organisms, whereby the tubulin spin (dipole moment) states become information bits and information gets stored in microtubules in a way that is robust to thermal fluctuations.

  12. Chromator is required for proper microtubule spindle formation and mitosis in Drosophila.

    Science.gov (United States)

    Ding, Yun; Yao, Changfu; Lince-Faria, Mariana; Rath, Uttama; Cai, Weili; Maiato, Helder; Girton, Jack; Johansen, Kristen M; Johansen, Jørgen

    2009-10-01

    The chromodomain protein, Chromator, has been shown to have multiple functions that include regulation of chromatin structure as well as coordination of muscle remodeling during metamorphosis depending on the developmental context. In this study we show that mitotic neuroblasts from brain squash preparations from larvae heteroallelic for the two Chromator loss-of-function alleles Chro(71) and Chro(612) have severe microtubule spindle and chromosome segregation defects that were associated with a reduction in brain size. The microtubule spindles formed were incomplete, unfocused, and/or without clear spindle poles and at anaphase chromosomes were lagging and scattered. Time-lapse analysis of mitosis in S2 cells depleted of Chromator by RNAi treatment suggested that the lagging and scattered chromosome phenotypes were caused by incomplete alignment of chromosomes at the metaphase plate, possibly due to a defective spindle-assembly checkpoint, as well as of frayed and unstable microtubule spindles during anaphase. Expression of full-length Chromator transgenes under endogenous promoter control restored both microtubule spindle morphology as well as brain size strongly indicating that the observed mutant defects were directly attributable to lack of Chromator function.

  13. Measurement of Binding Force between Microtubule-Associated Protein and Microtubule

    Institute of Scientific and Technical Information of China (English)

    XU Chun-Hua; GUO Hong-Lian; QU E; LI Zhao-Lin; YUAN Ming; CHENG Bing-Ying; ZHANG Dao-Zhong

    2007-01-01

    Microtubule-associated proteins (MAPs) are important proteins in cells. They can regulate the organization,dynamics and function of microtubules. We measure the binding force between microtubule and a new plant MAP, i.e. AtMAP65-1, by dual-optical tweezers. The force is obtained to be 14.6±3.5 pN from the data statistics and analysis. This force measurement is helpful to understand the function and mechanism of MAPs from the mechanical point of view and lays the groundwork for future measurements of the mechanical properties of other biological macro-molecules.

  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. 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."

  16. GDP-Tubulin Incorporation into Growing Microtubules Modulates Polymer Stability*

    Science.gov (United States)

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

    2010-01-01

    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.” PMID:20371874

  17. Mmb1p binds mitochondria to dynamic microtubules

    Science.gov (United States)

    Fu, Chuanhai; Jain, Deeptee; Costa, Judite; Velve-Casquillas, Guilhem; Tran, Phong T.

    2015-01-01

    Summary Background Mitochondria form a dynamics tubular network within the cell. Proper mitochondria movement and distribution are critical for their localized function in cell metabolism, growth, and survival. In mammalian cells, mechanisms of mitochondria positioning appear dependent on the microtubule cytoskeleton, with kinesin or dynein motors carrying mitochondria as cargos and distributing them throughout the microtubule network. Interestingly, the timescale of microtubule dynamics occurs in seconds, and the timescale of mitochondria distribution occurs in minutes. How does the cell couple these two time constants? Results Fission yeast also relies on microtubules for mitochondria distribution. We report here a new microtubule-dependent but motor-independent mechanism for proper mitochondria positioning in fission yeast. We identify the protein mmb1p, which binds to mitochondria and microtubules. Mmb1p attaches the tubular mitochondria to the microtubule lattice at multiple discrete interaction sites. Mmb1 deletion causes mitochondria to aggregate, with the long-term consequence of defective mitochondria distribution and cell death. Mmb1p decreases microtubule dynamicity. Conclusion Mmb1p is a new microtubule-mitochondria binding protein. We propose that mmb1p act to couple long-term mitochondria distribution to short-term microtubule dynamics by attenuating microtubule dynamics, thus enhancing the mitochondria-microtubule interaction time. PMID:21856157

  18. Studies on the role of microtubules in myofibrillogenesis

    Institute of Scientific and Technical Information of China (English)

    LINZHONGXIANG; HOWARDHOLTZER

    1990-01-01

    Co-localization of microtubule (MT) and muscle myosin (MHC) myofibril immunofluoresoonoe in developing myotubes of chicken skeletal muscle cultures was observed by using double staining of tubulin and MHC indirect immunofluorescence.120-tetradecanoyl-phorbol-12-acetate (TPA) selectively and reversibly blocks myofibrillogenesis and alters the morphology of myotubes in to myosacs where MTs are present in radiating pattern.When the arrested myogenic cells recover and start myofibrillogenesis after released from TPA,prior to the emergence of myofibrils,the pre-ecisting MTs become bipolarly aligned coincidently with the tubular restoration of cell shape.Single nascent myofibrils overlapping with MTs extend into the base of growth tips where MTs go farther to the end of the tips.That MT might act as scaffold in guiding the bipolar elongation of the growing myofibrils was suggested.Taxol and colcemid disturbed MT polymerization and disposition,and interfered with the normal spatial assembly of myofibrils in developing myotubes.

  19. Stathmin regulates microtubule dynamics and microtubule organizing center polarization in activated T cells.

    Science.gov (United States)

    Filbert, Erin L; Le Borgne, Marie; Lin, Joseph; Heuser, John E; Shaw, Andrey S

    2012-06-01

    Polarization of T cells involves reorientation of the microtubule organizing center (MTOC). Because activated ERK is localized at the immunological synapse, we investigated its role by showing that ERK activation is important for MTOC polarization. Suspecting that ERK phosphorylates a regulator of microtubules, we next focused on stathmin, a known ERK substrate. Our work indicates that during T cell activation, ERK is recruited to the synapse, allowing it to phosphorylate stathmin molecules near the immunological synapse. Supporting an important role of stathmin phosphorylation in T cell activation, we showed that T cell activation results in increased microtubule growth rate dependent on the presence of stathmin. The significance of this finding was demonstrated by results showing that CTLs from stathmin(-/-) mice displayed defective MTOC polarization and defective target cell cytolysis. These data implicate stathmin as a regulator of the microtubule network during T cell activation.

  20. Immunohistological labeling of microtubules in sensory neuron dendrites, tracheae, and muscles in the Drosophila larva body wall.

    Science.gov (United States)

    Yalgin, Cagri; Karim, M Rezaul; Moore, Adrian W

    2011-11-10

    To understand how differences in complex cell shapes are achieved, it is important to accurately follow microtubule organization. The Drosophila larval body wall contains several cell types that are models to study cell and tissue morphogenesis. For example tracheae are used to examine tube morphogenesis(1), and the dendritic arborization (DA) sensory neurons of the Drosophila larva have become a primary system for the elucidation of general and neuron-class-specific mechanisms of dendritic differentiation(2-5) and degeneration(6). The shape of dendrite branches can vary significantly between neuron classes, and even among different branches of a single neuron(7,8). Genetic studies in DA neurons suggest that differential cytoskeletal organization can underlie morphological differences in dendritic branch shape(4,9-11). We provide a robust immunological labeling method to assay in vivo microtubule organization in DA sensory neuron dendrite arbor (Figures 1, 2, Movie 1). This protocol illustrates the dissection and immunostaining of first instar larva, a stage when active sensory neuron dendrite outgrowth and branching organization is occurring (12,13). In addition to staining sensory neurons, this method achieves robust labeling of microtubule organization in muscles (Movies 2, 3), trachea (Figure 3, Movie 3), and other body wall tissues. It is valuable for investigators wishing to analyze microtubule organization in situ in the body wall when investigating mechanisms that control tissue and cell shape.

  1. A family of entangled coordination polymers constructed from a flexible V-shaped long bicarboxylic acid and auxiliary N-donor ligands: Luminescent sensing

    Science.gov (United States)

    Wang, Jun; Bai, Chao; Hu, Huai-Ming; Yuan, Fei; Xue, Gang-Lin

    2017-05-01

    Eight Zn(II)-based coordination polymers, namely, [Zn2L2(2,2'-bipy)]n·nH2O (1), [Zn2L2(phen)]n·nH2O (2), [ZnL(phen)(H2O)]n (3), [Zn3L3(4,4'-bipy)]n (4), [Zn2L2(4,4'-bipy)2]n [Zn2L2(H2O)2]n·2nH2O (5), [Zn4L4(bpp)2]n (6), [ZnL(bbi)0.5]n (7), [ZnL(bpz)]n (8) (H2L=4,4'-{[1,2-phenylenebis-(methylene)]bis(oxy)}dibenzoic acid, 2,2'-bipy =2,2'-bipyridine, phen =1,10-phenanthroline, 4,4'-bpy=4,4'-bipyridine, bpp =1,3-bis(4-pyridyl)propane, bbi=1,4-bis(imidazol-1-yl)butane, bpz=3,3‧,5,5‧-tetramethyl-4,4‧-bipyrazole), have been hydrothermally synthesized and structurally characterized. 1-8 display various coordination motifs with different entangled forms and conformations due to the effect of the assistant N-donor ligands. The photoluminescent properties of compounds 1-8 in solid state were studied. Interestingly, 3 exhibits highly efficient luminescent sensing for Cu2+ cations and CrO42- anions, as well as detection ability for the different organic solvents and nitro explosives. These results indicated that it could be utilized as a multi-responsive luminescent sensor. Furthermore, compound 3 also shows good chemical resistance to both acidity and alkalinity solutions with pH ranging from 2 to 13. Thus, multi-photofunctionality and fluorescent response to pH have been combined in the 3, which is the first example in the Zn-based hybrid materials.

  2. The formin DIAPH1 (mDia1) regulates megakaryocyte proplatelet formation by remodeling the actin and microtubule cytoskeletons.

    Science.gov (United States)

    Pan, Jiajia; Lordier, Larissa; Meyran, Deborah; Rameau, Philippe; Lecluse, Yann; Kitchen-Goosen, Susan; Badirou, Idinath; Mokrani, Hayat; Narumiya, Shuh; Alberts, Arthur S; Vainchenker, William; Chang, Yunhua

    2014-12-18

    Megakaryocytes are highly specialized precursor cells that produce platelets via cytoplasmic extensions called proplatelets. Proplatelet formation (PPF) requires profound changes in microtubule and actin organization. In this work, we demonstrated that DIAPH1 (mDia1), a mammalian homolog of Drosophila diaphanous that works as an effector of the small GTPase Rho, negatively regulates PPF by controlling the dynamics of the actin and microtubule cytoskeletons. Moreover, we showed that inhibition of both DIAPH1 and the Rho-associated protein kinase (Rock)/myosin pathway increased PPF via coordination of both cytoskeletons. We provide evidence that 2 major effectors of the Rho GTPase pathway (DIAPH1 and Rock/myosin II) are involved not only in Rho-mediated stress fibers assembly, but also in the regulation of microtubule stability and dynamics during PPF.

  3. Microtubules guide root hair tip growth

    NARCIS (Netherlands)

    Sieberer, B.; Ketelaar, M.J.; Esseling, J.J.; Emons, A.M.C.

    2005-01-01

    The ability to establish cell polarity is crucial to form and function of an individual cell. Polarity underlies critical processes during cell development, such as cell growth, cell division, cell differentiation and cell signalling. Interphase cytoplasmic microtubules in tip-growing fission yeast

  4. Microtubules guide root hair tip growth

    NARCIS (Netherlands)

    Sieberer, B.; Ketelaar, M.J.; Esseling, J.J.; Emons, A.M.C.

    2005-01-01

    The ability to establish cell polarity is crucial to form and function of an individual cell. Polarity underlies critical processes during cell development, such as cell growth, cell division, cell differentiation and cell signalling. Interphase cytoplasmic microtubules in tip-growing fission yeast

  5. Electrostatically biased binding of kinesin to microtubules.

    Directory of Open Access Journals (Sweden)

    Barry J Grant

    2011-11-01

    Full Text Available The minimum motor domain of kinesin-1 is a single head. Recent evidence suggests that such minimal motor domains generate force by a biased binding mechanism, in which they preferentially select binding sites on the microtubule that lie ahead in the progress direction of the motor. A specific molecular mechanism for biased binding has, however, so far been lacking. Here we use atomistic Brownian dynamics simulations combined with experimental mutagenesis to show that incoming kinesin heads undergo electrostatically guided diffusion-to-capture by microtubules, and that this produces directionally biased binding. Kinesin-1 heads are initially rotated by the electrostatic field so that their tubulin-binding sites face inwards, and then steered towards a plus-endwards binding site. In tethered kinesin dimers, this bias is amplified. A 3-residue sequence (RAK in kinesin helix alpha-6 is predicted to be important for electrostatic guidance. Real-world mutagenesis of this sequence powerfully influences kinesin-driven microtubule sliding, with one mutant producing a 5-fold acceleration over wild type. We conclude that electrostatic interactions play an important role in the kinesin stepping mechanism, by biasing the diffusional association of kinesin with microtubules.

  6. Discodermolide interferes with the binding of tau protein to microtubules.

    Science.gov (United States)

    Kar, Santwana; Florence, Gordon J; Paterson, Ian; Amos, Linda A

    2003-03-27

    We investigated whether discodermolide, a novel antimitotic agent, affects the binding to microtubules of tau protein repeat motifs. Like taxol, the new drug reduces the proportion of tau that pellets with microtubules. Despite their differing structures, discodermolide, taxol and tau repeats all bind to a site on beta-tubulin that lies within the microtubule lumen and is crucial in controlling microtubule assembly. Low concentrations of tau still bind strongly to the outer surfaces of preformed microtubules when the acidic C-terminal regions of at least six tubulin dimers are available for interaction with each tau molecule; otherwise binding is very weak.

  7. Cell cycle regulation of tubulin RNA level, tubulin protein synthesis, and assembly of microtubules in Physarum.

    Science.gov (United States)

    Schedl, T; Burland, T G; Gull, K; Dove, W F

    1984-07-01

    The temporal relationship between tubulin expression and the assembly of the mitotic spindle microtubules has been investigated during the naturally synchronous cell cycle of the Physarum plasmodium. The cell cycle behavior of the tubulin isoforms was examined by two-dimensional gel electrophoresis of proteins labeled in vivo and by translation of RNA in vitro. alpha 1-, alpha 2-, beta 1-, and beta 2-tubulin synthesis increases coordinately until metaphase, and then falls, with beta 2 falling more rapidly than beta 1. Nucleic acid hybridization demonstrated that alpha- and beta-tubulin RNAs accumulate coordinately during G2, peaking at metaphase. Quantitative analysis demonstrated that alpha-tubulin RNA increases with apparent exponential kinetics, peaking with an increase over the basal level of greater than 40-fold. After metaphase, tubulin RNA levels fall exponentially, with a short half-life (19 min). Electron microscopic analysis of the plasmodium showed that the accumulation of tubulin RNA begins long before the polymerization of mitotic spindle microtubules. By contrast, the decay of tubulin RNA after metaphase coincides with the depolymerization of the spindle microtubules.

  8. Integrin-linked kinase regulates interphase and mitotic microtubule dynamics.

    Directory of Open Access Journals (Sweden)

    Simin Lim

    Full Text Available Integrin-linked kinase (ILK localizes to both focal adhesions and centrosomes in distinct multiprotein complexes. Its dual function as a kinase and scaffolding protein has been well characterized at focal adhesions, where it regulates integrin-mediated cell adhesion, spreading, migration and signaling. At the centrosomes, ILK regulates mitotic spindle organization and centrosome clustering. Our previous study showed various spindle defects after ILK knockdown or inhibition that suggested alteration in microtubule dynamics. Since ILK expression is frequently elevated in many cancer types, we investigated the effects of ILK overexpression on microtubule dynamics. We show here that overexpressing ILK in HeLa cells was associated with a shorter duration of mitosis and decreased sensitivity to paclitaxel, a chemotherapeutic agent that suppresses microtubule dynamics. Measurement of interphase microtubule dynamics revealed that ILK overexpression favored microtubule depolymerization, suggesting that microtubule destabilization could be the mechanism behind the decreased sensitivity to paclitaxel, which is known to stabilize microtubules. Conversely, the use of a small molecule inhibitor selective against ILK, QLT-0267, resulted in suppressed microtubule dynamics, demonstrating a new mechanism of action for this compound. We further show that treatment of HeLa cells with QLT-0267 resulted in higher inter-centromere tension in aligned chromosomes during mitosis, slower microtubule regrowth after cold depolymerization and the presence of a more stable population of spindle microtubules. These results demonstrate that ILK regulates microtubule dynamics in both interphase and mitotic cells.

  9. 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.

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

    Directory of Open Access Journals (Sweden)

    Jason E Duncan

    Full Text Available 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.

  11. A seven-coordinated manganese(II) complex with V-shaped ligand bis(N-benzylbenzimidazol-2-ylmethyl)benzylamine: synthesis, structure, DNA-binding properties and antioxidant activities.

    Science.gov (United States)

    Wu, Huilu; Yuan, Jingkun; Bai, Ying; Wang, Hua; Pan, Guolong; Kong, Jin

    2012-11-05

    A manganese(II) complex of the type, [MnL(pic)(2)]·H(2)O, was obtained by the reaction of the V-shaped ligand bis(N-benzylbenzimidazol-2-ylmethyl)benzylamine (L) with Mn(pic)(2) (pic=picrate). The ligand L and Mn(II) complex were confirmed on the basis of elemental analysis, molarconductivities, (1)H NMR, IR, UV-vis spectra and X-ray crystallography. Single-crystal X-ray revealed that central Mn(II) atom is seven-coordinate with a MnN(3)O(4) environment, in which ligand L acts as a tridentate N-donor. The remaining coordination sites were occupied by four O atoms afforded by two picrate anion. Interaction of the free ligand L and Mn(II) complex with DNA were investigated by spectrophotometric methods and viscosity measurements. The results suggested that both ligand L and Mn(II) complex bind to DNA in an intercalative binding mode, and DNA-binding affinity of the Mn(II) complex is stronger than that of ligand L. Moreover, antioxidant assay in vitro shows the Mn(II) complex possesses significant antioxidant activities.

  12. Resolving bundled microtubules using anti-tubulin nanobodies.

    Science.gov (United States)

    Mikhaylova, Marina; Cloin, Bas M C; Finan, Kieran; van den Berg, Robert; Teeuw, Jalmar; Kijanka, Marta M; Sokolowski, Mikolaj; Katrukha, Eugene A; Maidorn, Manuel; Opazo, Felipe; Moutel, Sandrine; Vantard, Marylin; Perez, Frank; van Bergen en Henegouwen, Paul M P; Hoogenraad, Casper C; Ewers, Helge; Kapitein, Lukas C

    2015-08-11

    Microtubules are hollow biopolymers of 25-nm diameter and are key constituents of the cytoskeleton. In neurons, microtubules are organized differently between axons and dendrites, but their precise organization in different compartments is not completely understood. Super-resolution microscopy techniques can detect specific structures at an increased resolution, but the narrow spacing between neuronal microtubules poses challenges because most existing labelling strategies increase the effective microtubule diameter by 20-40 nm and will thereby blend neighbouring microtubules into one structure. Here we develop single-chain antibody fragments (nanobodies) against tubulin to achieve super-resolution imaging of microtubules with a decreased apparent diameter. To test the resolving power of these novel probes, we generate microtubule bundles with a known spacing of 50-70 nm and successfully resolve individual microtubules. Individual bundled microtubules can also be resolved in different mammalian cells, including hippocampal neurons, allowing novel insights into fundamental mechanisms of microtubule organization in cell- and neurobiology.

  13. How do Plants Organize Microtubules Without a Centrosome?

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    A microtubule nucleates from a γ-tubulin complex, which consists of γ-tubulin, proteins from the SPC97/SPC98 family, and the WD40 motif protein GCP-WD. We analyzed the phylogenetic relationships of the genes encoding these proteins and found that the components of this complex are widely conserved among land plants and other eukaryotes. By contrast,the interphase and mitotic arrays of microtubules in land plants differ from those in other eukaryotes. In the interphase cortical array, the majority of microtubules nucleate on existing microtubules in the absence of conspicuous microtubule organizing centers (MTOCs), such as a centrosome. During mitosis, the spindle also forms in the absence of conspicuous MTOCs. Both poles of the spindle are broad, and branched structures of microtubules called microtubule converging centers form at the poles. In this review, we hypothesize that the microtubule converging centers form via microtubuledependent microtubule nucleation, as in the case of the interphase arrays. The evolutionary insights arising from the molecular basis of the diversity in microtubule organization are discussed.

  14. Motor protein accumulation on antiparallel microtubule overlaps

    CERN Document Server

    Kuan, Hui-Shun

    2015-01-01

    Biopolymers serve as one-dimensional tracks on which motor proteins move to perform their biological roles. Motor protein phenomena have inspired theoretical models of one-dimensional transport, crowding, and jamming. Experiments studying the motion of Xklp1 motors on reconstituted antiparallel microtubule overlaps demonstrated that motors recruited to the overlap walk toward the plus end of individual microtubules and frequently switch between filaments. We study a model of this system that couples the totally asymmetric simple exclusion process (TASEP) for motor motion with switches between antiparallel filaments and binding kinetics. We determine steady-state motor density profiles for fixed-length overlaps using exact and approximate solutions of the continuum differential equations and compare to kinetic Monte Carlo simulations. The center region, far from the overlap ends, has a constant motor density as one would na\\"ively expect. However, rather than following a simple binding equilibrium, the center ...

  15. CSPP and CSPP-L associate with centrosomes and microtubules and differently affect microtubule organization.

    Science.gov (United States)

    Patzke, Sebastian; Stokke, Trond; Aasheim, Hans-Christian

    2006-10-01

    We recently described the identification of a centrosome/spindle pole associated protein, CSPP, involved in cell cycle progression. Here we report a CSPP isoform denoted CSPP-L, with a 294 amino acids longer N-terminus and a 51 amino acids insertion located in the coiled-coil mid-domain. Expression analysis indicates an inverse cell cycle dependent regulation. CSPP mRNA expression is highest in G1 whereas CSPP-L expression is highest in G2/M. Ectopic expression of CSPP-L impairs cell cycle progression weaker in G1 than CSPP. Furthermore, normal mitotic phenotypes were observed in CSPP-L but not in CSPP transfectants. CSPP-L relocates from spindle microtubules and poles in metaphase to the mid-spindle in anaphase and concentrates at the mid-body in telophase/cytokinesis. CSPP-L high-expressing mitotic cells were predominantly characterized by lagging chromosomes or monopolar spindles, in contrast to the predominant multipolar spindles observed with CSPP expression. The different effects of CSPP and CSPP-L on microtubule organization in mitosis depend on the coiled-coil mid-domain insertion. The common C-terminal domain is required to repress that activity until mitosis. Notably, this C-terminal domain alone can associate with centrosomes in a microtubule independent manner. Taken together, CSPP and CSPP-L interact with centrosomes and microtubules and can differently affect microtubule organization. Copyright 2006 Wiley-Liss, Inc.

  16. Microtubule-Targeting Therapy for Prostate Cancer

    Science.gov (United States)

    2007-02-01

    Cancer1828 Mol Cancer Ther 2005;4(12). December 2005 22. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning : a laboratory manual. 2nd ed. Cold Spring...Harbor (NY): Cold Spring Harbor Laboratory; 1989. 23. Zhu XX, Kozarsky K, Strahler JR, et al. Molecular cloning of a novel human leukemia-associated...of Cancer Research, Abstract #4940, 2005. 3. Mistry, SJ, Atweh, GF. Microtubule targeting therapy: Anti-stathmin based molecular cancer

  17. Effect of weightlessness on colloidal particle transport and segregation in self-organising microtubule preparations.

    Science.gov (United States)

    Tabony, James; Rigotti, Nathalie; Glade, Nicolas; Cortès, Sandra

    2007-05-01

    Weightlessness is known to effect cellular functions by as yet undetermined processes. Many experiments indicate a role of the cytoskeleton and microtubules. Under appropriate conditions in vitro microtubule preparations behave as a complex system that self-organises by a combination of reaction and diffusion. This process also results in the collective transport and organisation of any colloidal particles present. In large centimetre-sized samples, self-organisation does not occur when samples are exposed to a brief early period of weightlessness. Here, we report both space-flight and ground-based (clinorotation) experiments on the effect of weightlessness on the transport and segregation of colloidal particles and chromosomes. In centimetre-sized containers, both methods show that a brief initial period of weightlessness strongly inhibits particle transport. In miniature cell-sized containers under normal gravity conditions, the particle transport that self-organisation causes results in their accumulation into segregated regions of high and low particle density. The gravity dependence of this behaviour is strongly shape dependent. In square wells, neither self-organisation nor particle transport and segregation occur under conditions of weightlessness. On the contrary, in rectangular canals, both phenomena are largely unaffected by weightlessness. These observations suggest, depending on factors such as cell and embryo shape, that major biological functions associated with microtubule driven particle transport and organisation might be strongly perturbed by weightlessness.

  18. Microtubule self-organisation depends upon gravity

    Science.gov (United States)

    Tabony, J.; Pochon, N.; Papaseit, C.

    2001-01-01

    The molecular processes by which gravity is transduced into biological systems are poorly, if at all, understood. Under equilibrium conditions, chemical and biochemical structures do not depend upon gravity. It has been proposed that biological systems might show a gravity dependence by way of the bifurcation properties of certain types of non-linear chemical reactions that are far-from-equilibrium. We have found that in-vitro preparations of microtubules, an important element of the cellular cytoskeleton, show this type of behaviour. On earth, the solutions show macroscopic self-ordering, and the morphology of the structures that form depend upon the orientation of the sample with respect to gravity at a critical moment at an early stage in the development of the self-organised state. An experiment carried out in a sounding rocket, showed that as predicted by theories of this type, no self-organisation occurs when the microtubules are assembled under low gravity conditions. This is an experimental demonstration of how a very simple biochemical system, containing only two molecules, can be gravity sensitive. At a molecular level this behaviour results from an interaction of gravity with macroscopic concentration and density fluctuations that arise from the processes of microtubule contraction and elongation.

  19. MICROTUBULE ORGANIZATION 1 regulates structure and function of microtubule arrays during mitosis and cytokinesis in the Arabidopsis root.

    Science.gov (United States)

    Kawamura, Eiko; Himmelspach, Regina; Rashbrooke, Madeleine C; Whittington, Angela T; Gale, Kevin R; Collings, David A; Wasteneys, Geoffrey O

    2006-01-01

    MICROTUBULE ORGANIZATION 1 (MOR1) is a plant member of the highly conserved MAP215/Dis1 family of microtubule-associated proteins. Prior studies with the temperature-sensitive mor1 mutants of Arabidopsis (Arabidopsis thaliana), which harbor single amino acid substitutions in an N-terminal HEAT repeat, proved that MOR1 regulates cortical microtubule organization and function. Here we demonstrate by use of live cell imaging and immunolabeling that the mor1-1 mutation generates specific defects in the microtubule arrays of dividing vegetative cells. Unlike the universal cortical microtubule disorganization in elongating mor1-1 cells, disruption of mitotic and cytokinetic microtubule arrays was not detected in all dividing cells. Nevertheless, quantitative analysis identified distinct defects in preprophase bands (PPBs), spindles, and phragmoplasts. In nearly one-half of dividing cells at the restrictive temperature of 30 degrees C, PPBs were not detected prior to spindle formation, and those that did form were often disrupted. mor1-1 spindles and phragmoplasts were short and abnormally organized and persisted for longer times than in wild-type cells. The reduced length of these arrays predicts that the component microtubule lengths are also reduced, suggesting that microtubule length is a critical determinant of spindle and phragmoplast structure, orientation, and function. Microtubule organizational defects led to aberrant chromosomal arrangements, misaligned or incomplete cell plates, and multinucleate cells. Antiserum raised against an N-terminal MOR1 sequence labeled the full length of microtubules in interphase arrays, PPBs, spindles, and phragmoplasts. Continued immunolabeling of the disorganized and short microtubules of mor1-1 at the restrictive temperature demonstrated that the mutant mor1-1(L174F) protein loses function without dissociating from microtubules, providing important insight into the mechanism by which MOR1 may regulate microtubule length.

  20. Interconnections between cell wall polymers, wall mechanics, and cortical microtubules: Teasing out causes and consequences.

    Science.gov (United States)

    Xiao, Chaowen; Anderson, Charles T

    2016-09-01

    In plants, cell wall components including cellulose, hemicelluloses, and pectins interact with each other to form complex extracellular network structures that control cell growth and maintain cell shape. However, it is still not clear exactly how different wall polymers interact, how the conformations and interactions of cell wall polymers relate to wall mechanics, and how these factors impinge on intracellular structures such as the cortical microtubule cytoskeleton. Here, based on studies of Arabidopsis thaliana xxt1 xxt2 mutants, which lack detectable xyloglucan in their walls and display aberrant wall mechanics, altered cellulose patterning and biosynthesis, and reduced cortical microtubule stability, we discuss the potential relationships between cell wall biosynthesis, wall mechanics, and cytoskeletal dynamics in an effort to better understand their roles in controlling plant growth and morphogenesis.

  1. 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.

  2. Cellular Samurai: katanin and the severing of microtubules.

    Science.gov (United States)

    Quarmby, L

    2000-08-01

    Recent biochemical studies of the AAA ATPase, katanin, provide a foundation for understanding how microtubules might be severed along their length. These in vitro studies are complemented by a series of recent reports of direct in vivo observation of microtubule breakage, which indicate that the in vitro phenomenon of catalysed microtubule severing is likely to be physiological. There is also new evidence that microtubule severing by katanin is important for the production of non-centrosomal microtubules in cells such as neurons and epithelial cells. Although it has been difficult to establish the role of katanin in mitosis, new genetic evidence indicates that a katanin-like protein, MEI-1, plays an essential role in meiosis in C. elegans. Finally, new proteins involved in the severing of axonemal microtubules have been discovered in the deflagellation system of Chlamydomonas.

  3. Microtubule Associated Proteins in Plants and the Processes They Manage

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Microtubule associated proteins (MAPs) are proteins that physically bind to microtubules in eukaryotes. MAPs play important roles in regulating the polymerization and organization of microtubules and in using the ensuing microtubule arrays to carry out a variety of cellular functions. In plants, MAPs manage the construction, repositioning, and dismantling of four distinct microtubule arrays throughout the cell cycle. Three of these arrays, the cortical array, the preprophase band,and the phragmoplast, are prominent to plants and are responsible for facilitating cell wall deposition and modification,transducing signals, demarcating the plane of cell division, and forming the new cell plate during cytokinesis, This review highlights important aspects of how MAPs in plants establish and maintain microtubule arrays as well as regulate cell growth, cell division, and cellular responses to the environment.

  4. Cortical microtubule arrays are initiated from a nonrandom prepattern driven by atypical microtubule initiation.

    Science.gov (United States)

    Lindeboom, Jelmer J; Lioutas, Antonios; Deinum, Eva E; Tindemans, Simon H; Ehrhardt, David W; Emons, Anne Mie C; Vos, Jan W; Mulder, Bela M

    2013-03-01

    The ordered arrangement of cortical microtubules in growing plant cells is essential for anisotropic cell expansion and, hence, for plant morphogenesis. These arrays are dismantled when the microtubule cytoskeleton is rearranged during mitosis and reassembled following completion of cytokinesis. The reassembly of the cortical array has often been considered as initiating from a state of randomness, from which order arises at least partly through self-organizing mechanisms. However, some studies have shown evidence for ordering at early stages of array assembly. To investigate how cortical arrays are initiated in higher plant cells, we performed live-cell imaging studies of cortical array assembly in tobacco (Nicotiana tabacum) Bright Yellow-2 cells after cytokinesis and drug-induced disassembly. We found that cortical arrays in both cases did not initiate randomly but with a significant overrepresentation of microtubules at diagonal angles with respect to the cell axis, which coincides with the predominant orientation of the microtubules before their disappearance from the cell cortex in preprophase. In Arabidopsis (Arabidopsis thaliana) root cells, recovery from drug-induced disassembly was also nonrandom and correlated with the organization of the previous array, although no diagonal bias was observed in these cells. Surprisingly, during initiation, only about one-half of the new microtubules were nucleated from locations marked by green fluorescent protein-γ-tubulin complex protein2-tagged γ-nucleation complexes (γ-tubulin ring complex), therefore indicating that a large proportion of early polymers was initiated by a noncanonical mechanism not involving γ-tubulin ring complex. Simulation studies indicate that the high rate of noncanonical initiation of new microtubules has the potential to accelerate the rate of array repopulation.

  5. Effects of spermine binding on Taxol-stabilized microtubules

    Science.gov (United States)

    Cheng, Shengfeng; Regmi, Chola

    Previous studies have shown that polyamines such as spermine present in cells at physiological concentrations can facilitate the polymerization of tubulins into microtubules (MTs). A recent experiment demonstrates that in the presence of high-concentration spermine, Taxol-stabilized MTs undergo a shape transformation into inverted tubulin tubules (ITTs), the outside surface of which corresponds to the inside surface of a regular MT. However, the molecular mechanism underlying the shape transformation of MTs into ITTs is unclear. We perform all atom molecular dynamics simulations on Taxol-stabilized MT sheets containing two protofilaments surrounded by spermine ions. The spermine concentration is varied from 0 to 25mM to match the range probed experimentally. We identify important spermine binding regions on the MT surface and the influence of the spermine binding on the structure and dynamics of MTs. In contrast to Taxol, our results show that spermine binding seems to decrease the flexibility of tubulin proteins, resulting in weaker tubulin-tubulin contacts and promoting the bending of protofilaments into curved protofilaments, inverted rings, and eventually inverted tubules.

  6. CLIP-170 facilitates the formation of kinetochore-microtubule attachments.

    Science.gov (United States)

    Tanenbaum, Marvin E; Galjart, Niels; van Vugt, Marcel A T M; Medema, René H

    2006-01-11

    CLIP-170 is a microtubule 'plus end tracking' protein involved in several microtubule-dependent processes in interphase. At the onset of mitosis, CLIP-170 localizes to kinetochores, but at metaphase, it is no longer detectable at kinetochores. Although RNA interference (RNAi) experiments have suggested an essential role for CLIP-170 during mitosis, the molecular function of CLIP-170 in mitosis has not yet been revealed. Here, we used a combination of high-resolution microscopy and RNAi-mediated depletion to study the function of CLIP-170 in mitosis. We found that CLIP-170 dynamically localizes to the outer most part of unattached kinetochores and to the ends of growing microtubules. In addition, we provide evidence that a pool of CLIP-170 is transported along kinetochore-microtubules by the dynein/dynactin complex. Interference with CLIP-170 expression results in defective chromosome congression and diminished kinetochore-microtubule attachments, but does not detectibly affect microtubule dynamics or kinetochore-microtubule stability. Taken together, our results indicate that CLIP-170 facilitates the formation of kinetochore-microtubule attachments, possibly through direct capture of microtubules at the kinetochore.

  7. Deceivingly dynamic: Learning-dependent changes in stathmin and microtubules.

    Science.gov (United States)

    Uchida, Shusaku; Shumyatsky, Gleb P

    2015-10-01

    Microtubules, one of the major cytoskeletal structures, were previously considered stable and only indirectly involved in synaptic structure and function in mature neurons. However, recent evidence demonstrates that microtubules are dynamic and have an important role in synaptic structure, synaptic plasticity, and memory. In particular, learning induces changes in microtubule turnover and stability, and pharmacological manipulation of microtubule dynamics alters synaptic plasticity and long-term memory. These learning-induced changes in microtubules are controlled by the phosphoprotein stathmin, whose only known cellular activity is to negatively regulate microtubule formation. During the first eight hours following learning, changes in the phosphorylation of stathmin go through two phases causing biphasic shifts in microtubules stability/instability. These shifts, in turn, regulate memory formation by controlling in the second phase synaptic transport of the GluA2 subunit of AMPA receptors. Improper regulation of stathmin and microtubule dynamics has been observed in aged animals and in patients with Alzheimer's disease and depression. Thus, recent work on stathmin and microtubules has identified new molecular players in the early stages of memory encoding.

  8. Calculation of the Electromagnetic Field Around a Microtubule

    Directory of Open Access Journals (Sweden)

    D. Havelka

    2009-01-01

    Full Text Available Microtubules are important structures in the cytoskeleton which organizes the cell. A single microtubule is composed of electrically polar structures, tubulin heterodimers, which have a strong electric dipole moment. Vibrations are expected to be generated in microtubules, thus tubulin heterodimers oscillate as electric dipoles. This gives rise to an electromagnetic field which is detected around the cells. We calculate here the electromagnetic field of microtubules if they are excited at 1 GHz. This paper includes work done for the bachelor thesis of the first author. 

  9. Microtubules Modulate F-actin Dynamics during Neuronal Polarization.

    Science.gov (United States)

    Zhao, Bing; Meka, Durga Praveen; Scharrenberg, Robin; König, Theresa; Schwanke, Birgit; Kobler, Oliver; Windhorst, Sabine; Kreutz, Michael R; Mikhaylova, Marina; Calderon de Anda, Froylan

    2017-08-29

    Neuronal polarization is reflected by different dynamics of microtubule and filamentous actin (F-actin). Axonal microtubules are more stable than those in the remaining neurites, while dynamics of F-actin in axonal growth cones clearly exceed those in their dendritic counterparts. However, whether a functional interplay exists between the microtubule network and F-actin dynamics in growing axons and whether this interplay is instrumental for breaking cellular symmetry is currently unknown. Here, we show that an increment on microtubule stability or number of microtubules is associated with increased F-actin dynamics. Moreover, we show that Drebrin E, an F-actin and microtubule plus-end binding protein, mediates this cross talk. Drebrin E segregates preferentially to growth cones with a higher F-actin treadmilling rate, where more microtubule plus-ends are found. Interruption of the interaction of Drebrin E with microtubules decreases F-actin dynamics and arrests neuronal polarization. Collectively the data show that microtubules modulate F-actin dynamics for initial axon extension during neuronal development.

  10. 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.

  11. Queueing induced by bidirectional motor motion near the end of a microtubule

    Science.gov (United States)

    Ashwin, Peter; Lin, Congping; Steinberg, Gero

    2010-11-01

    Recent live observations of motors in long-range microtubule (MT) dependent transport in the fungus Ustilago maydis have reported bidirectional motion of dynein and an accumulation of the motors at the polymerization-active (the plus-end) of the microtubule. Quantitative data derived from in vivo observation of dynein has enabled us to develop an accurate, quantitatively-valid asymmetric simple exclusion process (ASEP) model that describes the coordinated motion of anterograde and retrograde motors sharing a single oriented microtubule. We give approximate expressions for the size and distribution of the accumulation, and discuss queueing properties for motors entering this accumulation. We show for this ASEP model, that the mean accumulation can be modeled as an M/M/∞ queue that is Poisson distributed with mean Farr/pd , where Farr is the flux of motors that arrives at the tip and pd is the rate at which individual motors change direction from anterograde to retrograde motion. Deviations from this can in principle be used to gain information about other processes at work in the accumulation. Furthermore, our work is a significant step toward a mathematical description of the complex interactions of motors in cellular long-range transport of organelles.

  12. Inter-domain Cooperation in INCENP Promotes Aurora B Relocation from Centromeres to Microtubules

    Directory of Open Access Journals (Sweden)

    Armando van der Horst

    2015-07-01

    Full Text Available The chromosomal passenger complex is essential for error-free chromosome segregation and proper execution of cytokinesis. To coordinate nuclear division with cytoplasmic division, its enzymatic subunit, Aurora B, relocalizes from centromeres in metaphase to the spindle midzone in anaphase. In budding yeast, this requires dephosphorylation of the microtubule-binding (MTB domain of the INCENP analog Sli15. The mechanistic basis for this relocalization in metazoans is incompletely understood. We demonstrate that the putative coiled-coil domain within INCENP drives midzone localization of Aurora B via a direct, electrostatic interaction with microtubules. Furthermore, we provide evidence that the CPC multimerizes via INCENP’s centromere-targeting domain (CEN box, which increases the MTB affinity of INCENP. In (prometaphase, the MTB affinity of INCENP is outcompeted by the affinity of its CEN box for centromeres, while at anaphase onset—when the histone mark H2AT120 is dephosphorylated—INCENP and Aurora B switch from centromere to microtubule localization.

  13. Human SAS-6 C-Terminus Nucleates and Promotes Microtubule Assembly in Vitro by Binding to Microtubules.

    Science.gov (United States)

    Gupta, Hindol; Badarudeen, Binshad; George, Athira; Thomas, Geethu Emily; Gireesh, K K; Manna, Tapas K

    2015-10-20

    Centrioles are essential components of the animal centrosome and play crucial roles in the formation of cilia and flagella. They are cylindrical structures composed of nine triplet microtubules organized around a central cartwheel. Recent studies have identified spindle assembly abnormal protein SAS-6 as a critical component necessary for formation of the cartwheel. However, the molecular details of how the cartwheel participates in centriolar microtubule assembly have not been clearly understood. In this report, we show that the C-terminal tail (residues 470-657) of human SAS-6, HsSAS-6 C, the region that has been shown to extend toward the centriolar wall where the microtubule triplets are organized, nucleated and induced microtubule polymerization in vitro. The N-terminus (residues 1-166) of HsSAS-6, the domain known to be involved in formation of the central hub of the cartwheel, did not, however, exert any effect on microtubule polymerization. HsSAS-6 C bound to the microtubules and localized along the lengths of the microtubules in vitro. Microtubule pull-down and coimmunoprecipitation (Co-IP) experiments with S-phase synchronized HeLa cell lysates showed that the endogenous HsSAS-6 coprecipitated with the microtubules, and it mediated interaction with tubulin. Isothermal calorimetry titration and size exclusion chromatography showed that HsSAS-6 C bound to the αβ-tubulin dimer in vitro. The results demonstrate that HsSAS-6 possesses an intrinsic microtubule assembly promoting activity and further implicate that its outer exposed C-terminal tail may play critical roles in microtubule assembly and stabilizing microtubule attachment with the centriolar cartwheel.

  14. Interplay between kinesin-1 and cortical dynein during axonal outgrowth and microtubule organization in Drosophila neurons.

    Science.gov (United States)

    del Castillo, Urko; Winding, Michael; Lu, Wen; Gelfand, Vladimir I

    2015-12-28

    In this study, we investigated how microtubule motors organize microtubules in Drosophila neurons. We showed that, during the initial stages of axon outgrowth, microtubules display mixed polarity and minus-end-out microtubules push the tip of the axon, consistent with kinesin-1 driving outgrowth by sliding antiparallel microtubules. At later stages, the microtubule orientation in the axon switches from mixed to uniform polarity with plus-end-out. Dynein knockdown prevents this rearrangement and results in microtubules of mixed orientation in axons and accumulation of microtubule minus-ends at axon tips. Microtubule reorganization requires recruitment of dynein to the actin cortex, as actin depolymerization phenocopies dynein depletion, and direct recruitment of dynein to the membrane bypasses the actin requirement. Our results show that cortical dynein slides 'minus-end-out' microtubules from the axon, generating uniform microtubule arrays. We speculate that differences in microtubule orientation between axons and dendrites could be dictated by differential activity of cortical dynein.

  15. Arabidopsis phospholipase D alpha 1-derived phosphatidic acid regulates microtubule organization and cell development under microtubule-interacting drugs treatment.

    Science.gov (United States)

    Zhang, Qun; Qu, Yana; Wang, Qing; Song, Ping; Wang, Peipei; Jia, Qianru; Guo, Jinhe

    2017-01-01

    Phospholipase D (PLD) and its product phosphatidic acid (PA) are emerging as essential regulators of cytoskeleton organization in plants. However, the underlying molecular mechanisms of PA-mediated microtubule reorganization in plants remain largely unknown. In this study, we used pharmacological and genetic approaches to analyze the function of Arabidopsis thaliana PLDα1 in the regulation of microtubule organization and cell development in response to microtubule-affecting drugs. Treatment with the microtubule-stabilizing drug paclitaxel resulted in less growth inhibition and decreased rightward slant of roots, longitudinal alignment of microtubules, and enhanced length of hypocotyl epidermal cells in the pldα1 mutant, the phenotype of which was rescued by exogenous application of PA. Moreover, the pldα1 mutant was sensitive to the microtubule-disrupting drugs oryzalin and propyzamide in terms of seedling survival ratio, left-skewing angle of roots and microtubule organization. In addition, both disruption and stabilization of microtubules induced by drugs activated PLDα1 activity. Our findings demonstrate that in A. thaliana, PLDα1/PA might regulate cell development by modulating microtubule organization in an activity-dependent manner.

  16. Highly Transient Molecular Interactions Underlie the Stability of Kinetochore–Microtubule Attachment During Cell Division

    OpenAIRE

    Zaytsev, Anatoly V.; Ataullakhanov, Fazly I; Grishchuk, Ekaterina L.

    2013-01-01

    Chromosome segregation during mitosis is mediated by spindle microtubules that attach to chromosomal kinetochores with strong yet labile links. The exact molecular composition of the kinetochore–microtubule interface is not known but microtubules are thought to bind to kinetochores via the specialized microtubule-binding sites, which contain multiple microtubule-binding proteins. During prometaphase the lifetime of microtubule attachments is short but in metaphase it increases 3-fold, presuma...

  17. Studying neuronal microtubule organization and microtubule-associated proteins using single molecule localization microscopy

    NARCIS (Netherlands)

    Chazeau, Anaël; Katrukha, Eugene A; Hoogenraad, Casper C; Kapitein, Lukas C

    2016-01-01

    The formation and maintenance of highly polarized neurons critically depends on the proper organization of the microtubule (MT) cytoskeleton. In axons, MTs are uniformly oriented with their plus-end pointing outward whereas in mature dendrites MTs have mixed orientations. MT organization and dynamic

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

    DEFF Research Database (Denmark)

    Zhang, Gang; Kelstrup, Christian D; Hu, Xiao-Wen;

    2012-01-01

    the chromosomes and stable kinetochore fibers are absent. Through quantitative mass spectrometry and immunofluorescence we find that the binding of the 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......The Ndc80 complex establishes end-on attachment of kinetochores to microtubules essential for chromosome segregation. The Ndc80 subunit is characterized by an N-terminal region, that binds directly to microtubules, and a long coiled-coil region that interacts with Nuf2. A loop region in Ndc80...

  19. Microtubules: dynamically unstable stochastic phase-switching polymers

    Science.gov (United States)

    Zakharov, P. N.; Arzhanik, V. K.; Ulyanov, E. V.; Gudimchuk, N. B.; Ataullakhanov, F. I.

    2016-08-01

    One of the simplest molecular motors, a biological microtubule, is reviewed as an example of a highly nonequilibrium molecular machine capable of stochastic transitions between slow growth and rapid disassembly phases. Basic properties of microtubules are described, and various approaches to simulating their dynamics, from statistical chemical kinetics models to molecular dynamics models using the Metropolis Monte Carlo and Brownian dynamics methods, are outlined.

  20. Resolving bundled microtubules using anti-tubulin nanobodies

    NARCIS (Netherlands)

    Mikhaylova, Marina; Cloin, Bas M C; Finan, Kieran; van den Berg, Robert; Teeuw, Jalmar; Kijanka, Marta M; Sokolowski, Mikolaj; Katrukha, Eugene A; Maidorn, Manuel; Opazo, Felipe; Moutel, Sandrine; Vantard, Marylin; Perez, Frank; van Bergen en Henegouwen, Paul M P; Hoogenraad, Casper C; Ewers, Helge; Kapitein, Lukas C

    2015-01-01

    Microtubules are hollow biopolymers of 25-nm diameter and are key constituents of the cytoskeleton. In neurons, microtubules are organized differently between axons and dendrites, but their precise organization in different compartments is not completely understood. Super-resolution microscopy techn

  1. EB1 targets to kinetochores with attached, polymerizing microtubules.

    Science.gov (United States)

    Tirnauer, Jennifer S; Canman, Julie C; Salmon, E D; Mitchison, Timothy J

    2002-12-01

    Microtubule polymerization dynamics at kinetochores is coupled to chromosome movements, but its regulation there is poorly understood. The plus end tracking protein EB1 is required both for regulating microtubule dynamics and for maintaining a euploid genome. To address the role of EB1 in aneuploidy, we visualized its targeting in mitotic PtK1 cells. Fluorescent EB1, which localized to polymerizing ends of astral and spindle microtubules, was used to track their polymerization. EB1 also associated with a subset of attached kinetochores in late prometaphase and metaphase, and rarely in anaphase. Localization occurred in a narrow crescent, concave toward the centromere, consistent with targeting to the microtubule plus end-kinetochore interface. EB1 did not localize to kinetochores lacking attached kinetochore microtubules in prophase or early prometaphase, or upon nocodazole treatment. By time lapse, EB1 specifically targeted to kinetochores moving antipoleward, coupled to microtubule plus end polymerization, and not during plus end depolymerization. It localized independently of spindle bipolarity, the spindle checkpoint, and dynein/dynactin function. EB1 is the first protein whose targeting reflects kinetochore directionality, unlike other plus end tracking proteins that show enhanced kinetochore binding in the absence of microtubules. Our results suggest EB1 may modulate kinetochore microtubule polymerization and/or attachment.

  2. Dynamic microtubules regulate dendritic spine morphology and synaptic plasticity

    NARCIS (Netherlands)

    Jaworski, J.; Kapitein, L.C.; Montenegro Gouveia, S.; Dortland, B.R.; Wulf, P.S.; Grigoriev, I.; Camera, P.; Spangler, S.A.; Di Stefano, P.; Demmers, J.; Krugers, H.; Defilippi, P.; Akhmanova, A.; Hoogenraad, C.C.

    2009-01-01

    Dendritic spines are the major sites of excitatory synaptic input, and their morphological changes have been linked to learning and memory processes. Here, we report that growing microtubule plus ends decorated by the microtubule tip-tracking protein EB3 enter spines and can modulate spine morpholog

  3. Molecular Pathway of Microtubule Organization at the Golgi Apparatus

    NARCIS (Netherlands)

    Wu, Jingchao; de Heus, Cecilia; Liu, Qingyang; Bouchet, Benjamin P; Noordstra, Ivar; Jiang, Kai; Hua, Shasha; Martin, Maud; Yang, Chao; Grigoriev, Ilya; Katrukha, Eugene A; Altelaar, A F Maarten; Hoogenraad, Casper C; Qi, Robert Z; Klumperman, Judith; Akhmanova, Anna

    2016-01-01

    The Golgi apparatus controls the formation of non-centrosomal microtubule arrays important for Golgi organization, polarized transport, cell motility, and cell differentiation. Here, we show that CAMSAP2 stabilizes and attaches microtubule minus ends to the Golgi through a complex of AKAP450 and myo

  4. CLIP-170 facilitates the formation of kinetochore-microtubule attachments

    NARCIS (Netherlands)

    Tanenbaum, ME; Galjart, N; van Vugt, MATM; Medema, RH

    2006-01-01

    CLIP-170 is a microtubule 'plus end tracking' protein involved in several microtubule-dependent processes in interphase. At the onset of mitosis, CLIP-170 localizes to kinetochores, but at metaphase, it is no longer detectable at kinetochores. Although RNA interference (RNAi) experiments have

  5. Cloning, expression, purification and refolding of microtubule affinity-regulating kinase 4 expressed in Escherichia coli.

    Science.gov (United States)

    Naz, Farha; Asad, Mohd; Malhotra, Pawan; Islam, Asimul; Ahmad, Faizan; Hassan, Md Imtaiyaz

    2014-03-01

    Microtubule-associated protein/microtubule affinity-regulating kinase 4 (MARK4) is a member of the family Ser/Thr kinase and involved in numerous biological functions including microtubule bundle formation, nervous system development, positive regulation of programmed cell death, cell cycle control, cell polarity determination, cell shape alterations, cell division etc. For various biophysical and structural studies, we need this protein in adequate quantity. In this paper, we report a novel cloning strategy for MARK4. We have cloned MARK4 catalytic domain including 59 N-terminal extra residues with unknown function and catalytic domain alone in PQE30 vector. The recombinant MARK4 was expressed in the inclusion bodies in M15 cells. The inclusion bodies were solubilized effectively with 1.5% N-lauroylsarcosine in alkaline buffer and subsequently purified using Ni-NTA affinity chromatography in a single step with high purity and good concentration. Purity of protein was checked on sodium dodecyl sulphate-polyacrylamide gel electrophoresis and identified by using mass spectrometry immunoblotting. Refolding of the recombinant protein was validated by ATPase assay. Our purification procedure is quick, simple and produces adequate quantity of proteins with high purity in a limited step.

  6. ICIS and Aurora B coregulate the microtubule depolymerase Kif2a.

    Science.gov (United States)

    Knowlton, Anne L; Vorozhko, Valeriya V; Lan, Weijie; Gorbsky, Gary J; Stukenberg, P Todd

    2009-05-12

    Kinesins in the mitotic spindle play major roles in determining spindle shape, size, and bipolarity, although specific regulation of these kinesins at distinct locations on the spindle is poorly understood. So that the forces that are required for spindle bipolarity are balanced, microtubule-depolymerizing kinesins are tightly regulated. Aurora B kinase phosphorylates the neck regions of the kinesin-13 family microtubule depolymerases Kif2a and mitotic centromere-associated kinesin (MCAK) and inhibits their depolymerase activities. How they are reactivated and how this is controlled independently on different kinetochore fibers is unknown. We show that inner centromere Kin-I stimulator (ICIS), which stimulates the related depolymerase MCAK, can reactivate Kif2a after Aurora B inhibition. When antibodies that block the ability of ICIS to activate Kif2a are injected into cells, monopolar spindles are generated. This phenotype is rescued by coinjection of anti-Nuf2 antibodies. We have performed a structure-function analysis of the ICIS protein and find that the N terminus of ICIS binds Aurora B and its regulators INCENP and TD60, whereas a central region binds MCAK, Kif2a, and microtubules, suggesting a scaffold function for ICIS. These data argue that ICIS and the chromosomal passenger complex (CPC) regulate Kif2a depolymerase activity.

  7. Multimodal microtubule binding by the Ndc80 kinetochore complex.

    Science.gov (United States)

    Alushin, Gregory M; Musinipally, Vivek; Matson, Daniel; Tooley, John; Stukenberg, P Todd; Nogales, Eva

    2012-11-01

    The Ndc80 complex is a key site of kinetochore-microtubule attachment during cell division. The human complex engages microtubules with a globular 'head' formed by tandem calponin-homology domains and an 80-amino-acid unstructured 'tail' that contains sites of phosphoregulation by the Aurora B kinase. Using biochemical, cell biological and electron microscopy analyses, we dissected the roles of the tail in binding of microtubules and mediation of cooperative interactions between Ndc80 complexes. Two segments of the tail that contain Aurora B phosphorylation sites become ordered at interfaces; one with tubulin and the second with an adjacent Ndc80 head on the microtubule surface, forming interactions that are disrupted by phosphorylation. We propose a model in which Ndc80's interaction with either growing or shrinking microtubule ends can be tuned by the phosphorylation state of its tail.

  8. Quantum optical coherence in cytoskeletal microtubules: implications for brain function.

    Science.gov (United States)

    Jibu, M; Hagan, S; Hameroff, S R; Pribram, K H; Yasue, K

    1994-01-01

    'Laser-like,' long-range coherent quantum phenomena may occur biologically within cytoskeletal microtubules. This paper presents a theoretical prediction of the occurrence in biological media of the phenomena which we term 'superradiance' and 'self-induced transparency'. Interactions between the electric dipole field of water molecules confined within the hollow core of microtubules and the quantized electromagnetic radiation field are considered, and microtubules are theorized to play the roles of non-linear coherent optical devices. Superradiance is a specific quantum mechanical ordering phenomenon with characteristic times much shorter than those of thermal interaction. Consequently, optical signalling (and computation) in microtubules would be free from both thermal noise and loss. Superradiant optical computing in networks of microtubules and other cytoskeletal structures may provide a basis for biomolecular cognition and a substrate for consciousness.

  9. Tensile stress stimulates microtubule outgrowth in living cells

    Science.gov (United States)

    Kaverina, Irina; Krylyshkina, Olga; Beningo, Karen; Anderson, Kurt; Wang, Yu-Li; Small, J. Victor

    2002-01-01

    Cell motility is driven by the sum of asymmetric traction forces exerted on the substrate through adhesion foci that interface with the actin cytoskeleton. Establishment of this asymmetry involves microtubules, which exert a destabilising effect on adhesion foci via targeting events. Here, we demonstrate the existence of a mechano-sensing mechanism that signals microtubule polymerisation and guidance of the microtubules towards adhesion sites under increased stress. Stress was applied either by manipulating the body of cells moving on glass with a microneedle or by stretching a flexible substrate that cells were migrating on. We propose a model for this mechano-sensing phenomenon whereby microtubule polymerisation is stimulated and guided through the interaction of a microtubule tip complex with actin filaments under tension.

  10. Novel Coordination Chemistry of Aluminum Borohydride

    Science.gov (United States)

    2014-08-01

    four different crystal shapes were identified under a microscope.  Super thin plates are not a preferred crystal shape for X-ray analysis. Anion ...coordination of various ligands. Previously we investigated an IL based upon the [Al(BH4)4]- anion . This material showed a much improved air and...moisture stability compared to ABH. Here we present research on the coordination of the cyanoborohydride anion (NCBH3-) with ABH. The coordination

  11. Stochastic models for plant microtubule self-organization and structure.

    Science.gov (United States)

    Eren, Ezgi C; Dixit, Ram; Gautam, Natarajan

    2015-12-01

    One of the key enablers of shape and growth in plant cells is the cortical microtubule (CMT) system, which is a polymer array that forms an appropriately-structured scaffolding in each cell. Plant biologists have shown that stochastic dynamics and simple rules of interactions between CMTs can lead to a coaligned CMT array structure. However, the mechanisms and conditions that cause CMT arrays to become organized are not well understood. It is prohibitively time-consuming to use actual plants to study the effect of various genetic mutations and environmental conditions on CMT self-organization. In fact, even computer simulations with multiple replications are not fast enough due to the spatio-temporal complexity of the system. To redress this shortcoming, we develop analytical models and methods for expeditiously computing CMT system metrics that are related to self-organization and array structure. In particular, we formulate a mean-field model to derive sufficient conditions for the organization to occur. We show that growth-prone dynamics itself is sufficient to lead to organization in presence of interactions in the system. In addition, for such systems, we develop predictive methods for estimation of system metrics such as expected average length and number of CMTs over time, using a stochastic fluid-flow model, transient analysis, and approximation algorithms tailored to our problem. We illustrate the effectiveness of our approach through numerical test instances and discuss biological insights.

  12. The exchangeability of shape

    Directory of Open Access Journals (Sweden)

    Kaba Dramane

    2010-10-01

    Full Text Available Abstract Background Landmark based geometric morphometrics (GM allows the quantitative comparison of organismal shapes. When applied to systematics, it is able to score shape changes which often are undetectable by traditional morphological studies and even by classical morphometric approaches. It has thus become a fast and low cost candidate to identify cryptic species. Due to inherent mathematical properties, shape variables derived from one set of coordinates cannot be compared with shape variables derived from another set. Raw coordinates which produce these shape variables could be used for data exchange, however they contain measurement error. The latter may represent a significant obstacle when the objective is to distinguish very similar species. Results We show here that a single user derived dataset produces much less classification error than a multiple one. The question then becomes how to circumvent the lack of exchangeability of shape variables while preserving a single user dataset. A solution to this question could lead to the creation of a relatively fast and inexpensive systematic tool adapted for the recognition of cryptic species. Conclusions To preserve both exchangeability of shape and a single user derived dataset, our suggestion is to create a free access bank of reference images from which one can produce raw coordinates and use them for comparison with external specimens. Thus, we propose an alternative geometric descriptive system that separates 2-D data gathering and analyzes.

  13. The Gas2 family protein Pigs is a microtubule +TIP that affects cytoskeleton organisation.

    Science.gov (United States)

    Girdler, Gemma C; Applewhite, Derek A; Perry, Wick M G; Rogers, Stephen L; Röper, Katja

    2016-01-01

    Coordination between different cytoskeletal systems is crucial for many cell biological functions, including cell migration and mitosis, and also plays an important role during tissue morphogenesis. Proteins of the class of cytoskeletal crosslinkers, or cytolinkers, have the ability to interact with more than one cytoskeletal system at a time and are prime candidates to mediate any coordination. One such class comprises the Gas2-like proteins, combining a conserved calponin-homology-type actin-binding domain and a Gas2 domain predicted to bind microtubules (MTs). This domain combination is also found in spectraplakins, huge cytolinkers that play important roles in many tissues in both invertebrates and vertebrates. Here, we dissect the ability of the single Drosophila Gas2-like protein Pigs to interact with both actin and MT cytoskeletons, both in vitro and in vivo, and illustrate complex regulatory interactions that determine the localisation of Pigs to and its effects on the cytoskeleton.

  14. A thermodynamic model of microtubule assembly and disassembly.

    Directory of Open Access Journals (Sweden)

    Bernard M A G Piette

    Full Text Available Microtubules are self-assembling polymers whose dynamics are essential for the normal function of cellular processes including chromosome separation and cytokinesis. Therefore understanding what factors effect microtubule growth is fundamental to our understanding of the control of microtubule based processes. An important factor that determines the status of a microtubule, whether it is growing or shrinking, is the length of the GTP tubulin microtubule cap. Here, we derive a Monte Carlo model of the assembly and disassembly of microtubules. We use thermodynamic laws to reduce the number of parameters of our model and, in particular, we take into account the contribution of water to the entropy of the system. We fit all parameters of the model from published experimental data using the GTP tubulin dimer attachment rate and the lateral and longitudinal binding energies of GTP and GDP tubulin dimers at both ends. Also we calculate and incorporate the GTP hydrolysis rate. We have applied our model and can mimic published experimental data, which formerly suggested a single layer GTP tubulin dimer microtubule cap, to show that these data demonstrate that the GTP cap can fluctuate and can be several microns long.

  15. A thermodynamic model of microtubule assembly and disassembly.

    Science.gov (United States)

    Piette, Bernard M A G; Liu, Junli; Peeters, Kasper; Smertenko, Andrei; Hawkins, Timothy; Deeks, Michael; Quinlan, Roy; Zakrzewski, Wojciech J; Hussey, Patrick J

    2009-08-11

    Microtubules are self-assembling polymers whose dynamics are essential for the normal function of cellular processes including chromosome separation and cytokinesis. Therefore understanding what factors effect microtubule growth is fundamental to our understanding of the control of microtubule based processes. An important factor that determines the status of a microtubule, whether it is growing or shrinking, is the length of the GTP tubulin microtubule cap. Here, we derive a Monte Carlo model of the assembly and disassembly of microtubules. We use thermodynamic laws to reduce the number of parameters of our model and, in particular, we take into account the contribution of water to the entropy of the system. We fit all parameters of the model from published experimental data using the GTP tubulin dimer attachment rate and the lateral and longitudinal binding energies of GTP and GDP tubulin dimers at both ends. Also we calculate and incorporate the GTP hydrolysis rate. We have applied our model and can mimic published experimental data, which formerly suggested a single layer GTP tubulin dimer microtubule cap, to show that these data demonstrate that the GTP cap can fluctuate and can be several microns long.

  16. An improved quantitative analysis method for plant cortical microtubules.

    Science.gov (United States)

    Lu, Yi; Huang, Chenyang; Wang, Jia; Shang, Peng

    2014-01-01

    The arrangement of plant cortical microtubules can reflect the physiological state of cells. However, little attention has been paid to the image quantitative analysis of plant cortical microtubules so far. In this paper, Bidimensional Empirical Mode Decomposition (BEMD) algorithm was applied in the image preprocessing of the original microtubule image. And then Intrinsic Mode Function 1 (IMF1) image obtained by decomposition was selected to do the texture analysis based on Grey-Level Cooccurrence Matrix (GLCM) algorithm. Meanwhile, in order to further verify its reliability, the proposed texture analysis method was utilized to distinguish different images of Arabidopsis microtubules. The results showed that the effect of BEMD algorithm on edge preserving accompanied with noise reduction was positive, and the geometrical characteristic of the texture was obvious. Four texture parameters extracted by GLCM perfectly reflected the different arrangements between the two images of cortical microtubules. In summary, the results indicate that this method is feasible and effective for the image quantitative analysis of plant cortical microtubules. It not only provides a new quantitative approach for the comprehensive study of the role played by microtubules in cell life activities but also supplies references for other similar studies.

  17. An Improved Quantitative Analysis Method for Plant Cortical Microtubules

    Directory of Open Access Journals (Sweden)

    Yi Lu

    2014-01-01

    Full Text Available The arrangement of plant cortical microtubules can reflect the physiological state of cells. However, little attention has been paid to the image quantitative analysis of plant cortical microtubules so far. In this paper, Bidimensional Empirical Mode Decomposition (BEMD algorithm was applied in the image preprocessing of the original microtubule image. And then Intrinsic Mode Function 1 (IMF1 image obtained by decomposition was selected to do the texture analysis based on Grey-Level Cooccurrence Matrix (GLCM algorithm. Meanwhile, in order to further verify its reliability, the proposed texture analysis method was utilized to distinguish different images of Arabidopsis microtubules. The results showed that the effect of BEMD algorithm on edge preserving accompanied with noise reduction was positive, and the geometrical characteristic of the texture was obvious. Four texture parameters extracted by GLCM perfectly reflected the different arrangements between the two images of cortical microtubules. In summary, the results indicate that this method is feasible and effective for the image quantitative analysis of plant cortical microtubules. It not only provides a new quantitative approach for the comprehensive study of the role played by microtubules in cell life activities but also supplies references for other similar studies.

  18. Microtubules in the Cerebral Cortex: Role in Memory and Consciousness

    Science.gov (United States)

    Woolf, Nancy J.

    This chapter raises the question whether synaptic connections in the cerebral cortex are adequate in accounting for higher cognition, especially cognition involving multimodal processing. A recent and novel approach to brain mechanics is outlined, one that involves microtubules and microtubule-associated protein-2 (MAP2). In addition to effects on the neuronal membrane, neurotransmitters exert actions on microtubules. These neurotransmitter effects alter the MAP2 phosphorylation state and rates of microtubule polymerization and transport. It is argued that these processes are important to the physical basis of memory and consciousness. In support of this argument, MAP2 is degraded with learning in discrete cortical modules. How this relates to synaptic change related to learning is unknown. The specific proposal is advanced that learning alters microtubules in the subsynaptic zone lying beneath the synapse, and that this forms the physical basis of long-term memory storage because microtubule networks determine the synapse strength by directing contacts with actin filaments and transport of synaptic proteins. It is argued that this is more probable than memory-related physical storage in the synapse itself. Comparisons to consciousness are made and it is concluded that there is a link between microtubules, memory and consciousness.

  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. 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.

  1. Tau Induces Cooperative Taxol Binding to Microtubules

    Science.gov (United States)

    Ross, Jennifer; Santangelo, Christian; Victoria, Makrides; Fygenson, Deborah

    2004-03-01

    Taxol and tau are two ligands which stabilize the microtubule (MT) lattice. Taxol is an anti-mitotic drug that binds β tubulin in the MT interior. Tau is a MT-associated protein that binds both α and β tubulin on the MT exterior. Both taxol and tau reduce MT dynamics and promote tubulin polymerization. Tau alone also acts as a buttress to bundle, stiffen, and space MTs. A structural study recently suggested that taxol and tau may interact by binding to the same site. Using fluorescence recovery after photobleaching, we find that tau induces taxol to bind MTs cooperatively depending on the tau concentration. We develop a model that correctly fits the data in the absence of tau and yields a measure of taxol cooperativity when tau is present.

  2. Crowding of molecular motors determines microtubule depolymerization

    CERN Document Server

    Reese, Louis; Frey, Erwin

    2011-01-01

    Assembly and disassembly dynamics of microtubules (MTs) is tightly controlled by MT associated proteins. Here, we investigate how plus-end-directed depolymerases of the kinesin-8 family regulate MT depolymerization dynamics. Employing an individual-based model, we reproduce experimental findings. Moreover, crowding is identified as the key regulatory mechanism of depolymerization dynamics. Our analysis gives two qualitatively distinct regimes. For motor densities above a particular threshold, a macroscopic traffic jam emerges at the plus-end and the MT dynamics become independent of the motor concentration. Below this threshold, microscopic traffic jams at the tip arise which cancel out the effect of the depolymerization kinetics such that the depolymerization speed is solely determined by the motor density. Because this density changes over the MT length, length-dependent regulation is possible. Remarkably, motor cooperativity does not affect the depolymerization speed but only the end-residence time of depo...

  3. Regulation of localization and activity of the microtubule depolymerase MCAK.

    Science.gov (United States)

    Tanenbaum, Marvin E; Medema, René H; Akhmanova, Anna

    2011-03-01

    Mitotic Centromere Associated Kinesin (MCAK) is a potent microtubule depolymerizing and catastrophe-inducing factor, which uses the energy of ATP hydrolysis to destabilize microtubule ends. MCAK is localized to inner centromeres, kinetochores and spindle poles of mitotic cells, and is also present in the cytoplasm. Both in interphase and in mitosis, MCAK can specifically accumulate at the growing microtubule ends. Here we discuss the mechanisms, which modulate subcellular localization and activity of MCAK through the interaction with the End Binding (EB) proteins and phosphorylation.

  4. Fluorescent taxoids as probes of the microtubule cytoskeleton.

    Science.gov (United States)

    Evangelio, J A; Abal, M; Barasoain, I; Souto, A A; Lillo, M P; Acuña, A U; Amat-Guerri, F; Andreu, J M

    1998-01-01

    Microtubules are specifically and efficiently visualized with the new fluorescent taxoids 7-O-[N-(4'-fluoresceincarbonyl)-L-alanyl]taxol (FLUTAX) and 7-O-[N-(4'-tetramethylrhodaminecarbonyl)-L-alanyl]taxol (ROTAX). Similarly to taxol, FLUTAX and ROTAX are able to drive inactive GDP-liganded tubulin into microtubule assembly. One molecule of FLUTAX binds per alphabeta-tubulin dimer assembled, competing with taxol for the same microtubule binding site with an eightfold smaller relative affinity. FLUTAX-induced microtubule elongation is markedly Mg2+-dependent, encompassing the binding of one Mg2+ ion more per tubulin dimer polymerized than in the case of taxol. A small perturbation of the absorption spectrum of bound FLUTAX is consistent with a cationic microenvironment relative to the solution. The fluorescence anisotropy of FLUTAX increases by an order of magnitude upon binding to microtubules and time-resolved measurements indicate that the fluorescein moiety remains considerably mobile on a protein surface. The rate of labeling suggests that this is the outer microtubule wall. Alternatively, the microtubule lumen would be functional. FLUTAX- and ROTAX-induced microtubules, radial structures, and organized microtubule bundles are readily observed under the fluorescence microscope. Rapid and accurate visualization of native (or very mildly fixed) cytoplasmic and spindle microtubules of a variety of permeabilized cells is simply obtained with micromolar FLUTAX, with an advantage over immunofluorescence. In addition, FLUTAX labels the centrosomes of PtK2 cells more intensely than antibodies to alpha- or beta-tubulin, and co-localizing with antibodies to gamma-tubulin. Two brightly fluorescent spots, probably separating or duplicating centrioles, can be resolved in the centrosomes of interphase cells. This finding indicates that centrosomes may well be additional targets of action of taxoids. FLUTAX strongly labels microtubules near the spindle poles, as well as

  5. Acentrosomal Microtubule Assembly in Mitosis: The Where, When, and How.

    Science.gov (United States)

    Meunier, Sylvain; Vernos, Isabelle

    2016-02-01

    In mitosis the cell assembles the bipolar spindle, a microtubule (MT)-based apparatus that segregates the duplicated chromosomes into two daughter cells. Most animal cells enter mitosis with duplicated centrosomes that provide an active source of dynamic MTs. However, it is now established that spindle assembly relies on the nucleation of acentrosomal MTs occurring around the chromosomes after nuclear envelope breakdown, and on pre-existing microtubules. Where chromosome-dependent MT nucleation occurs, when MT amplification takes place and how the two pathways function are still key questions that generate some controversies. We reconcile the data and present an integrated model accounting for acentrosomal microtubule assembly in the dividing cell.

  6. Microtubule Dynamics and Oscillating State for Mitotic Spindle

    CERN Document Server

    Rashid-Shomali, Safura

    2010-01-01

    We present a physical mechanism that can cause the mitotic spindle to oscillate. The driving force for this mechanism emerges from the polymerization of astral microtubules interacting with the cell cortex. We show that Brownian ratchet model for growing microtubules reaching the cell cortex, mediate an effective mass to the spindle body and therefore force it to oscillate. We compare the predictions of this mechanism with the previous mechanisms which were based on the effects of motor proteins. Finally we combine the effects of microtubules polymerization and motor proteins, and present the detailed phase diagram for possible oscillating states.

  7. Structural microtubule cap: Stability, catastrophe, rescue, and third state

    DEFF Research Database (Denmark)

    Flyvbjerg, H.; Chretien, D.; Janosi, I.M.

    2002-01-01

    Microtubules polymerize from GTP-liganded tubulin dinners, but are essentially made of GDP-liganded tubulin. We investigate the tug-of-war resulting from the fact that GDP-liganded tubulin favors a curved configuration, but is forced to remain in a straight one when part of a microtubule. We point...... of two well-established facts: protofilaments made of GDP-liganded tubulin have intrinsic curvature, and microtubules are elastic, made from material that can yield to forces, in casu its own intrinsic forces. We explore possible properties of this structural cap, and demonstrate 1) how it allows both...

  8. Mitochondrial inheritance is mediated by microtubules in mammalian cell division.

    Science.gov (United States)

    Lawrence, Elizabeth; Mandato, Craig

    2013-11-01

    The mitochondrial network fragments and becomes uniformly dispersed within the cytoplasm when mammalian cells enter mitosis. Such morphology and distribution of mitochondria was previously thought to facilitate the stochastic inheritance of mitochondria by daughter cells. In contrast, we recently reported that mitochondria in dividing mammalian cells are inherited by an ordered mechanism of inheritance mediated by microtubules. We showed that mitochondria are progressively enriched at the cell equator and depleted at the poles throughout division. Furthermore, the mitochondrial distribution during division is dependent on microtubules, indicating an ordered inheritance strategy. The microtubule-mediated positioning of mitochondria in dividing mammalian cells may have functional consequences for cell division and/or mitochondrial inheritance.

  9. Visualizing and Analyzing Branching Microtubule Nucleation Using Meiotic Xenopus Egg Extracts and TIRF Microscopy

    Science.gov (United States)

    King, Matthew; Petry, Sabine

    2016-01-01

    Mitotic and meiotic spindles consist primarily of microtubules, which originate from centrosomes and within the vicinity of chromatin. Indirect evidence suggested that microtubules also originate throughout the spindle, but the high microtubule density within the spindle precludes the direct observation of this phenomenon. By using meiotic Xenopus laevis egg extract and employing total internal reflection (TIRF) microscopy, microtubule nucleation from preexisting microtubules could be demonstrated and analyzed. Branching microtubule nucleation is an ideal mechanism to assemble and maintain a mitotic spindle, because microtubule numbers are amplified while preserving their polarity. Here, we describe the assays that made these findings possible and the experiments that helped identify the key molecular players involved. PMID:27193844

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

    Science.gov (United States)

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

    2013-04-01

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

  11. Modeling the Effects of Drug Binding on the Dynamic Instability of Microtubules

    CERN Document Server

    Hinow, Peter; Lopus, Manu; Jordan, Mary Ann; Tuszynski, Jack A

    2010-01-01

    We propose a stochastic model that accounts for the growth, catastrophe and rescue processes of steady state microtubules assembled from MAP-free tubulin. Both experimentally and theoretically we study the perturbation of microtubule dynamic instability by S-methyl-D-DM1, a synthetic derivative of the microtubule-targeted agent maytansine and a potential anticancer agent. We find that to be an effective suppressor of microtubule dynamics a drug must primarily suppress the loss of GDP tubulin from the microtubule tip.

  12. Multiscale Polar Theory of Microtubule and 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 are building blocks of self-organized subcellular biological structures such as the mitotic spindle and the centrosomal microtubule array. These same ingredients can form new “bioactive” liquid-crystalline fluids that are intrinsically out of equilibrium and which display complex flows and defect dynamics. It is not yet well understood how microscopic activity, which involves polarity-dependent interactions between motor proteins and microtubules, yields such larger-scale dynamical structures. In our multiscale theory, Brownian dynamics simulations of polar microtubule ensembles driven by cross-linking motors allow us to study microscopic organization and stresses. Polarity sorting and cross-link relaxation emerge as two polar-specific sources of active destabilizing stress. On larger length scales, our continuum Doi-Onsager theory captures the hydrodynamic flows generated by polarity-dependent active stresses. The results connect local polar structure to flow structures and defect dynamics. PMID:25679909

  13. The Role of Microtubule Movement in Bidirectional Organelle Transport

    National Research Council Canada - National Science Library

    Igor M. Kulić; André E. X. Brown; Hwajin Kim; Comert Kural; Benjamin Blehm; Paul R. Selvin; Philip C. Nelson; Vladimir I. Gelfand

    2008-01-01

    We study the role of microtubule movement in bidirectional organelle transport in Drosophila S2 cells and show that EGFP-tagged peroxisomes in cells serve as sensitive probes of motor induced, noisy cytoskeletal motions...

  14. Insights from an erroneous kinetochore-microtubule attachment state.

    Science.gov (United States)

    Cane, Stuart; McGilvray, Philip T; Maresca, Thomas J

    2013-01-01

    Faithful distribution of the genome requires that sister kinetochores, which assemble on each chromatid during cell division, interact with dynamic microtubules from opposite spindle poles in a configuration called chromosome biorientation. Biorientation produces tension that increases the affinity of kinetochores for microtubules via ill-defined mechanisms. Non-bioriented kinetochore-microtubule (kt-MT) interactions are prevalent but short-lived due to an error correction pathway that reduces the affinity of kinetochores for microtubules. Interestingly, incorrect kt-MT interactions can be stabilized by experimentally applying force to misoriented chromosomes. Here, a live-cell force assay is utilized to characterize the molecular composition of a common type of improper kt-MT attachment. Our force-related studies are also discussed in the context of current models for tension-dependent stabilization of kt-MT interactions.

  15. Mechanical Models of Microtubule Bundle Collapse in Alzheimer's Disease

    Science.gov (United States)

    Sendek, Austin; Singh, Rajiv; Cox, Daniel

    2013-03-01

    Amyloid-beta aggregates initiate Alzheimer's disease, and downstream trigger degradation of tau proteins that act as microtubule bundle stabilizers and mechanical spacers. Currently it is unclear which of tau cutting by proteases, tau phosphorylation, or tau aggregation are responsible for cytoskeleton degradation., We construct a percolation simulation of the microtubule bundle using a molecular spring model for the taus and including depletion force attraction between microtubules and membrane/actin cytoskeletal surface tension. The simulation uses a fictive molecular dynamics to model the motion of the individual microtubules within the bundle as a result of random tau removal, and calculates the elastic modulus of the bundle as the tau concentration falls. We link the tau removal steps to kinetic tau steps in various models of tau degradation. Supported by US NSF Grant DMR 1207624

  16. The cell morphogenesis gene ANGUSTIFOLIA encodes a CtBP/BARS-like protein and is involved in the control of the microtubule cytoskeleton.

    Science.gov (United States)

    Folkers, U; Kirik, V; Schöbinger, U; Falk, S; Krishnakumar, S; Pollock, M A; Oppenheimer, D G; Day, I; Reddy, A S M; Jürgens, G; Hülskamp, M; Reddy, A R

    2002-03-15

    The ANGUSTIFOLIA (AN) gene is required for leaf hair (trichome) branching and is also involved in polarized expansion underlying organ shape. Here we show that the AN gene encodes a C-terminal binding proteins/brefeldin A ADP-ribosylated substrates (CtBP/BARS) related protein. AN is expressed at low levels in all organs and the AN protein is localized in the cytoplasm. In an mutant trichomes, the organization of the actin cytoskeleton is normal but the distribution of microtubules is aberrant. A role of AN in the control of the microtubule cytoskeleton is further supported by the finding that AN genetically and physically interacts with ZWICHEL, a kinesin motor molecule involved in trichome branching. Our data suggest that CtBP/BARS-like protein function in plants is directly associated with the microtubule cytoskeleton.

  17. The Dynamic Pollen Tube Cytoskeleton: Live Cell Studies Using Actin-Binding and Microtubule-Binding Reporter Proteins

    Institute of Scientific and Technical Information of China (English)

    Alice Y. Cheung; Qiao-hong Duan; Silvia Santos Costa; Barend H.J.de Graaf; Veronica S.Di Stilio; Jose Feijo; Hen-Ming Wu

    2008-01-01

    Pollen tubes elongate within the pistil to transport sperm cells to the embryo sac for fertilization.Growth occurs exclusively at the tube apex,rendering pollen tube elongation a most dramatic polar cell growth process.A hall-mark pollen tube feature is its cytoskeleton,which comprises elaborately organized and dynamic actin microfilaments and microtubules.Pollen tube growth is dependent on the actin cytoskeleton;its organization and regulation have been exalined extensively by various approaches.including fluorescent protein labeled actin-binding proteins in live cell studies.Using the previously described GFP-NtADF1 and GFP-LIADF1, and a new actin reporter protein NtPLIM2b-GFP,we re-affirm that the predominant actin structures in elongating tobacco and lily pollen tubes are long,streaming actin cables along the pollen tube shank,and a subapical structure comprising shorter actin cables.The subapical collection of actin microfilaments undergoes dynamic changes,giving rise to the appearance of structures that range from basket-or funnel-shaped,mesh-like to a subtle ring.NtPLIM2b-GFP is used in combination with a guanine nucleotide exchange factor for the Rho GTPases,AtROP-GEF1,to illustrate the use of these actin reporter proteins to explore the linkage between the polar cell growth process and its actin cytoskeleton.Contrary to the actin cytoskeleton,microtubules appear not to play a direct role in supporting the polar cell growth process in angiosperm pollen tubes.Using a microtubule reporter protein based on the microtubule end-binding protein from Arabidopsis AtEB1,GFP-AtEB1,we show that the extensive microtubule network in elongating pollen tubes displays varying degrees of dynamics.These reporter proteins provide versatile tools to explore the functional connection between major structural and signaling components of the polar pollen tube growth process.

  18. 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.

  19. Dynein prevents erroneous kinetochore-microtubule attachments in mitosis.

    Science.gov (United States)

    Barisic, Marin; Maiato, Helder

    2015-01-01

    Equal distribution of the genetic material during cell division relies on efficient congression of chromosomes to the metaphase plate. Prior to their alignment, the Dynein motor recruited to kinetochores transports a fraction of laterally-attached chromosomes along microtubules toward the spindle poles. By doing that, Dynein not only contributes to chromosome movements, but also prevents premature stabilization of end-on kinetochore-microtubule attachments. This is achieved by 2 parallel mechanisms: 1) Dynein-mediated poleward movement of chromosomes counteracts opposite polar-ejection forces (PEFs) on chromosome arms by the microtubule plus-end-directed motors chromokinesins. Otherwise, they could stabilize erroneous syntelic kinetochore-microtubule attachments and lead to the random ejection of chromosomes away from the spindle poles; and 2) By transporting chromosomes to the spindle poles, Dynein brings the former to the zone of highest Aurora A kinase activity, further destabilizing kinetochore-microtubule attachments. Thus, Dynein plays an important role in keeping chromosome segregation error-free by preventing premature stabilization of kinetochore-microtubule attachments near the spindle poles.

  20. Cortical microtubules are responsible for gravity resistance in plants.

    Science.gov (United States)

    Hoson, Takayuki; Matsumoto, Shouhei; Soga, Kouichi; Wakabayashi, Kazuyuki

    2010-06-01

    Mechanical resistance to the gravitational force is a principal gravity response in plants distinct from gravitropism. In the final step of gravity resistance, plants increase the rigidity of their cell walls. Here we discuss the role of cortical microtubules, which sustain the function of the cell wall, in gravity resistance. Hypocotyls of Arabidopsis tubulin mutants were shorter and thicker than the wild-type, and showed either left-handed or right-handed helical growth at 1 g. The degree of twisting phenotype was intensified under hypergravity conditions. Hypergravity also induces reorientation of cortical microtubules from transverse to longitudinal directions in epidermal cells. In tubulin mutants, the percentage of cells with longitudinal microtubules was high even at 1 g, and it was further increased by hypergravity. The left-handed helical growth mutants had right-handed microtubule arrays, whereas the right-handed mutant had left-handed arrays. Moreover, blockers of mechanoreceptors suppressed both the twisting phenotype and reorientation of microtubules in tubulin mutants. These results support the hypothesis that cortical microtubules play an essential role in maintenance of normal growth phenotype against the gravitational force, and suggest that mechanoreceptors are involved in signal perception in gravity resistance. Space experiments will confirm whether this view is applicable to plant resistance to 1 g gravity, as to the resistance to hypergravity.

  1. TECHNICAL COORDINATION

    CERN Multimedia

    A. Ball

    Overview From a technical perspective, CMS has been in “beam operation” state since 6th November. The detector is fully closed with all components operational and the magnetic field is normally at the nominal 3.8T. The UXC cavern is normally closed with the radiation veto set. Access to UXC is now only possible during downtimes of LHC. Such accesses must be carefully planned, documented and carried out in agreement with CMS Technical Coordination, Experimental Area Management, LHC programme coordination and the CCC. Material flow in and out of UXC is now strictly controlled. Access to USC remains possible at any time, although, for safety reasons, it is necessary to register with the shift crew in the control room before going down.It is obligatory for all material leaving UXC to pass through the underground buffer zone for RP scanning, database entry and appropriate labeling for traceability. Technical coordination (notably Stephane Bally and Christoph Schaefer), the shift crew and run ...

  2. Microtubules and associated molecular motors in Neurospora crassa.

    Science.gov (United States)

    Mouriño-Pérez, Rosa Reyna; Riquelme, Meritxell; Callejas-Negrete, Olga Alicia; Galván-Mendoza, José Iván

    2016-01-01

    The cytoskeleton provides structure, shape and movement to various cells. Microtubules (MTs) are tubular structures made of α and β-tubulin heterodimers organized in 13 protofilaments, forming a hollow cylinder. A vast group of MT-associated proteins determines the function, behavior and interaction of the MTs with other cellular components. Among these proteins, molecular motors such as the dynein-dynactin complex and kinesin superfamily play roles in MT organization and organelle transport. This article focuses on the MT cytoskeleton and associated molecular motors in the filamentous fungus Neurospora crassa In addition to reviewing current available information for this fungus and contrasting it with knowledge of other fungal species, we present new experimental results that support the role of dynein, dynactin and conventional kinesin in MT organization, dynamics and transport of subcellular structures (nuclei and secretory vesicles). In wild type hyphae of N. crassa, cytoplasmic MTs are arranged longitudinally along hyphae and display a helical curvature. They interlace with one another to form a network throughout the cytoplasm. N. crassa dynein and dynactin mutants have a scant and disorganized MT cytoskeleton, an erratic and reduced Spitzenkörper (Spk) and distorted hyphal morphology. In contrast, hyphae of mutants with defective conventional kinesin exhibit only minor disruptions in MT and Spk organization. Although nuclear positioning is affected in all mutants, the MT-associated motor proteins are not major contributors to nuclear movement during hyphal growth. Cytoplasmic bulk flow is the vehicle for nuclear displacement in growing hyphal regions of N. crassa Motors are involved in nuclei saltatory movements in both retrograde or anterograde direction. In the dynein and kinesin mutants, micro and macrovesicles can reach the Spk, although growth is slightly impaired and the Spk displays an erratic path. Hyphal growth requires MTs, and their associated

  3. Balance of microtubule stiffness and cortical tension determines the size of blood cells with marginal band across species

    CERN Document Server

    Dmitrieff, Serge; Mathur, Aastha; Nédélec, François

    2016-01-01

    The fast blood stream of animals is associated with large shear stresses. Consequently, blood cells have evolved a special morphology and a specific internal architecture allowing them to maintain their integrity over several weeks. For instance, non-mammalian red blood cells, mammalian erythroblasts and platelets have a peripheral ring of microtubules, called the marginal band, that flattens the overall cell morphology by pushing on the cell cortex. In this article, we model how the shape of these cells stems from the balance between marginal band elasticity and cortical tension. We predict that the diameter of the cell scales with the total microtubule polymer, and verify the predicted law across a wide range of species. Our analysis also shows that the combination of the marginal band rigidity and cortical tension increases the ability of the cell to withstand forces without deformation. Finally, we model the marginal band coiling that occurs during the disc-to-sphere transition observed for instance at th...

  4. Tau neurofibrillary pathology and microtubule stability.

    Science.gov (United States)

    Michaelis, Mary L; Dobrowsky, Rick T; Li, Guibin

    2002-12-01

    We previously reported that nonomolar concentrations of Taxol and several structurally diverse microtubule (MT)-stabilizing agents significantly enhanced the survival of neurons in the presence of fibrils of amyloid beta peptide (Abeta). Pretreatment of neurons with MT-stabilizing drugs also blocked Abeta-induced activation of tau hyperphosphorylation. Although tau is a substrate for several kinases, we initially focused on cdk5, as this tau kinase has been shown to be activated in Abeta-treated neurons and Alzheimer's disease (AD) brain. In an in vitro kinase assay, Taxol inhibited activation of cdk5 by Abeta. In addition, the proposed cellular cascade in which calpain activation leads to cleavage of the cdk5 regulator, p35, to the strong kinase activator p25 was also prevented. Taxol did not directly inhibit the activity of either cdk5 or calpain, indicating that other cellular components are required for the effect of the drug on Abeta activation of tau phosphorylation. Our results suggest that drugs that interact with MTs can alter signaling events in neurons, possibly because some MTs play a role in organizing protein complexes involved in responses to Abeta. Thus the cytoskeletal network may serve as a biosensor of cellular well-being.

  5. The Katanin Microtubule Severing Protein in Plants

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Katanin is a heterodimeric microtubule (MT) severing protein that uses energy from ATP hydrolysis to generate internal breaks along MTs. Katanin p60, one of the two subunits, possesses ATPase and MT-bindinglsevering activities, and the p80 subunit is responsible for targeting of katanin to certain subcellular locations. In animals, katanin plays an important role in the release of MTs from their nucleation sites in the centrosome. It is also involved in severing MTs into smaller fragments which can serve as templates for further polymerization to increase MT number during meiotic and mitotic spindle assembly. Katanin homologs are present in a wide variety of plant species. The Arabidopsis katanin homolog has been shown to possess ATP-dependent MT severing activity in vitro and exhibit a punctate localization pattern at the cell cortex and the perinuclear region. Disruption of katanin functions by genetic mutations causes a delay in the disappearance of the perinuclear MT array and results in an aberrant organization of cortical MTs in elongating cells. Consequently, katanin mutations lead to defects in cell elongation, cellulose microfibril deposition, and hormonal responses. Studies of katanin in plants provide new insights into our understanding of its roles in cellular functions.

  6. In vitro assembly of plant tubulin in the absence of microtubule-stabilizing reagents

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The assembly of microtubules is essential for physiological functions of microtubules. Addition of microtubule-stabilizing reagents or microtubule "seeds" is usually necessary for plant tubulin assembly in vitro, which hinders the investigation of plant microtubule dynamics. In the present note, highly purified plant tubulins have been obtained from lily pollen, a non-microtubule-stabilizing reagent or microtubule "seed" system for plant tubulin assembly has been established and the analysis of plant tubulin assembly performed. Experiment results showed that purified tubulin polymerized in vitro, and a typical microtubule structure was observed with electron microscopy. The kinetics curve of tubulin assembly exhibited typical "parabola". The presence of taxol significantly altered the character of plant tubulin assembly, including that abnormal microtubules were assembled and the critical concentration for plant tubulin assembly was decreased exceedingly from 3 mg/mL in the absence of taxol to 0.043 mg/mL in the presence of taxol.

  7. The dual specificity phosphatase Cdc14B bundles and stabilizes microtubules

    Energy Technology Data Exchange (ETDEWEB)

    Plumley, Hyekyung [ORNL; Liu, Yie [ORNL; Gomez, Marla V [ORNL; Wang, Yisong [ORNL

    2005-01-01

    The Cdc14 dual-specificity phosphatases regulate key events in the eukaryotic cell cycle. However, little is known about the function of mammalian CDC14B family members. Here, we demonstrate that subcellular localization of CDC14B protein is cell cycle regulated. CDC14B can bind, bundle, and stabilize microtubules in vitro independently of its catalytic activity. Basic amino acid residues within the nucleolar targeting domain are important for both retaining CDC14B in the nucleolus and preventing microtubule bundling. Overexpression of CDC14B resulted in the formation of cytoplasmic CDC14B and microtubule bundles in interphase cells. These microtubule bundles were resistant to microtubule depolymerization reagents and enriched in acetylated -tubulin. Expression of cytoplasmic forms of CDC14B impaired microtubule nucleation from the microtubule organization center. CDC14B is thus a novel microtubule-bundling and -stabilizing protein, whose regulated subcellular localization may help modulate spindle and microtubule dynamics in mitosis.

  8. On the significance of microtubule flexural behavior in cytoskeletal mechanics.

    Directory of Open Access Journals (Sweden)

    Mehrdad Mehrbod

    Full Text Available Quantitative description of cell mechanics has challenged biological scientists for the past two decades. Various structural models have been attempted to analyze the structure of the cytoskeleton. One important aspect that has been largely ignored in all these modeling approaches is related to the flexural and buckling behavior of microtubular filaments. The objective of this paper is to explore the influence of this flexural and buckling behavior in cytoskeletal mechanics.In vitro the microtubules are observed to buckle in the first mode, reminiscent of a free, simply-supported beam. In vivo images of microtubules, however, indicate that the buckling mostly occurs in higher modes. This buckling mode switch takes place mostly because of the lateral support of microtubules via their connections to actin and intermediate filaments. These lateral loads are exerted throughout the microtubule length and yield a considerable bending behavior that, unless properly accounted for, would produce erroneous results in the modeling and analysis of the cytoskeletal mechanics.One of the promising attempts towards mechanical modeling of the cytoskeleton is the tensegrity model, which simplifies the complex network of cytoskeletal filaments into a combination merely of tension-bearing actin filaments and compression-bearing microtubules. Interestingly, this discrete model can qualitatively explain many experimental observations in cell mechanics. However, evidence suggests that the simplicity of this model may undermine the accuracy of its predictions, given the model's underlying assumption that "every single member bears solely either tensile or compressive behavior," i.e. neglecting the flexural behavior of the microtubule filaments. We invoke an anisotropic continuum model for microtubules and compare the bending energy stored in a single microtubule with its axial strain energy at the verge of buckling. Our results suggest that the bending energy can

  9. Association between microtubules and Golgi vesicles isolated from rat parotid glands.

    Science.gov (United States)

    Coffe, G; Raymond, M N

    1990-01-01

    We report an isolation procedure of trans-Golgi vesicles (GVs) from rat parotid glands. Various organelle markers were used, particularly galactosyl transferase as a trans-Golgi marker, to test the purity of the GV fraction. A quantitative in vitro binding assay between microtubules and GVs is described. The vesicles were incubated with taxol-induced microtubules, layered between 50% and 43% sucrose cushions and subjected to centrifugation. Unlike free microtubules which were sedimented, the GV-bound microtubules co-migrated upward with GVs. Quantification of these bound microtubules was carried out by densitometric scanning of Coomassie blue-stained gels. The association between microtubules and GVs followed a saturation curve, with a plateau value of 20 micrograms of microtubule protein bound to 500 micrograms of GV fraction. The half-saturation of the GV sites was obtained with a microtubule concentration of 20 micrograms/ml. Electron microscopy of negatively stained re-floated material showed numerous microtubule-vesicle complexes. Coating of microtubules with an excess of brain microtubule-associated proteins (MAPs) abolished binding. In the absence of exogenous microtubules, we showed that the GV fraction was already interacting with a class of endogenous rat parotid microtubules. This class of colcemid and cold-stable microtubules represents 10-20% of the total tubulin content of the parotid cell.

  10. Microtubules Are Essential for Guard-Cell Function in Vicia and Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    William Eisinger; David Ehrhardt; Winslow Briggs

    2012-01-01

    Radially arranged cortical microtubules are a prominent feature of guard cells.Guard cells expressing GFPtubulin showed consistent changes in the appearance of microtubules when stomata opened or closed.Guard cells showed fewer microtubule structures as stomata closed,whether induced by transfer to darkness,ABA,hydrogen peroxide,or sodium hydrogen carbonate.Guard cells kept in the dark (closed stomata) showed increases in microtubule structures and stomatal aperture on light treatment.GFP-EB1,marking microtubule growing plus ends,showed no change in number of plus ends or velocity of assembly on stomatal closure.Since the number of growing plus ends and the rate of plus-end growth did not change when microtubule structure numbers declined,microtubule instability and/or rearrangement must be responsible for the apparent loss of microtubules.Guard cells with closed stomata showed more cytosolic GFP-fluorescence than those with open stomata as cortical microtubules became disassembled,although with a large net loss in total fluorescence.Microtubule-targeted drugs blocked guard-cell function in Vicia and Arabidopsis.Oryzalin disrupted guard-cell microtubules and prevented stomatal opening and taxol stabilized guard-cell microtubules and delayed stomatal closure.Gas exchange measurements indicated that the transgenes for fluorescent-labeled proteins did not disrupt normal stomatal function.These dynamic changes in guard-cell microtubules combined with our inhibitor studies provide evidence for an active role of microtubules in guard-cell function.

  11. LKB1 destabilizes microtubules in myoblasts and contributes to myoblast differentiation.

    Directory of Open Access Journals (Sweden)

    Isma Mian

    Full Text Available BACKGROUND: Skeletal muscle myoblast differentiation and fusion into multinucleate myotubes is associated with dramatic cytoskeletal changes. We find that microtubules in differentiated myotubes are highly stabilized, but premature microtubule stabilization blocks differentiation. Factors responsible for microtubule destabilization in myoblasts have not been identified. FINDINGS: We find that a transient decrease in microtubule stabilization early during myoblast differentiation precedes the ultimate microtubule stabilization seen in differentiated myotubes. We report a role for the serine-threonine kinase LKB1 in both microtubule destabilization and myoblast differentiation. LKB1 overexpression reduced microtubule elongation in a Nocodazole washout assay, and LKB1 RNAi increased it, showing LKB1 destabilizes microtubule assembly in myoblasts. LKB1 levels and activity increased during myoblast differentiation, along with activation of the known LKB1 substrates AMP-activated protein kinase (AMPK and microtubule affinity regulating kinases (MARKs. LKB1 overexpression accelerated differentiation, whereas RNAi impaired it. CONCLUSIONS: Reduced microtubule stability precedes myoblast differentiation and the associated ultimate microtubule stabilization seen in myotubes. LKB1 plays a positive role in microtubule destabilization in myoblasts and in myoblast differentiation. This work suggests a model by which LKB1-induced microtubule destabilization facilitates the cytoskeletal changes required for differentiation. Transient destabilization of microtubules might be a useful strategy for enhancing and/or synchronizing myoblast differentiation.

  12. A hopping mechanism for cargo transport by molecular motors in crowded microtubules

    CERN Document Server

    Goldman, Carla

    2010-01-01

    Most models designed to study the bidirectional movement of cargos as they are driven by molecular motors rely on the idea that motors of different polarities can be coordinated by external agents if arranged into a motor-cargo complex to perform the necessary work [gross04]. Although these models have provided us with important insights into these phenomena, there are still many unanswered questions regarding the mechanisms through which the movement of the complex takes place on crowded microtubules. For example (i) how does cargo-binding affect motor motility? and in connection with that - (ii) how does the presence of other motors (and also other cargos) on the microtubule affect the motility of the motor-cargo complex? We discuss these questions from a different perspective. The movement of a cargo is conceived here as a hopping process resulting from the transference of cargo between neighboring motors. In the light of this, we examine the conditions under which cargo might display bidirectional movemen...

  13. PDK1-Akt pathway regulates radial neuronal migration and microtubules in the developing mouse neocortex.

    Science.gov (United States)

    Itoh, Yasuhiro; Higuchi, Maiko; Oishi, Koji; Kishi, Yusuke; Okazaki, Tomohiko; Sakai, Hiroshi; Miyata, Takaki; Nakajima, Kazunori; Gotoh, Yukiko

    2016-05-24

    Neurons migrate a long radial distance by a process known as locomotion in the developing mammalian neocortex. During locomotion, immature neurons undergo saltatory movement along radial glia fibers. The molecular mechanisms that regulate the speed of locomotion are largely unknown. We now show that the serine/threonine kinase Akt and its activator phosphoinositide-dependent protein kinase 1 (PDK1) regulate the speed of locomotion of mouse neocortical neurons through the cortical plate. Inactivation of the PDK1-Akt pathway impaired the coordinated movement of the nucleus and centrosome, a microtubule-dependent process, during neuronal migration. Moreover, the PDK1-Akt pathway was found to control microtubules, likely by regulating the binding of accessory proteins including the dynactin subunit p150(glued) Consistent with this notion, we found that PDK1 regulates the expression of cytoplasmic dynein intermediate chain and light intermediate chain at a posttranscriptional level in the developing neocortex. Our results thus reveal an essential role for the PDK1-Akt pathway in the regulation of a key step of neuronal migration.

  14. TECHNICAL COORDINATION

    CERN Multimedia

    A. Ball

    2010-01-01

    Operational Experience At the end of the first full-year running period of LHC, CMS is established as a reliable, robust and mature experiment. In particular common systems and infrastructure faults accounted for <0.6 % CMS downtime during LHC pp physics. Technical operation throughout the entire year was rather smooth, the main faults requiring UXC access being sub-detector power systems and rack-cooling turbines. All such problems were corrected during scheduled technical stops, in the shadow of tunnel access needed by the LHC, or in negotiated accesses or access extensions. Nevertheless, the number of necessary accesses to the UXC averaged more than one per week and the technical stops were inevitably packed with work packages, typically 30 being executed within a few days, placing a high load on the coordination and area management teams. It is an appropriate moment for CMS Technical Coordination to thank all those in many CERN departments and in the Collaboration, who were involved in CMS techni...

  15. A Quantitative Method for Microtubule Analysis in Fluorescence Images.

    Science.gov (United States)

    Lan, Xiaodong; Li, Lingfei; Hu, Jiongyu; Zhang, Qiong; Dang, Yongming; Huang, Yuesheng

    2015-12-01

    Microtubule analysis is of significant value for a better understanding of normal and pathological cellular processes. Although immunofluorescence microscopic techniques have proven useful in the study of microtubules, comparative results commonly rely on a descriptive and subjective visual analysis. We developed an objective and quantitative method based on image processing and analysis of fluorescently labeled microtubular patterns in cultured cells. We used a multi-parameter approach by analyzing four quantifiable characteristics to compose our quantitative feature set. Then we interpreted specific changes in the parameters and revealed the contribution of each feature set using principal component analysis. In addition, we verified that different treatment groups could be clearly discriminated using principal components of the multi-parameter model. High predictive accuracy of four commonly used multi-classification methods confirmed our method. These results demonstrated the effectiveness and efficiency of our method in the analysis of microtubules in fluorescence images. Application of the analytical methods presented here provides information concerning the organization and modification of microtubules, and could aid in the further understanding of structural and functional aspects of microtubules under normal and pathological conditions.

  16. Intracellular spatial localization regulated by the microtubule network.

    Directory of Open Access Journals (Sweden)

    Jing Chen

    Full Text Available The commonly recognized mechanisms for spatial regulation inside the cell are membrane-bounded compartmentalization and biochemical association with subcellular organelles. We use computational modeling to investigate another spatial regulation mechanism mediated by the microtubule network in the cell. Our results demonstrate that the mitotic spindle can impose strong sequestration and concentration effects on molecules with binding affinity for microtubules, especially dynein-directed cargoes. The model can recapitulate the essence of three experimental observations on distinct microtubule network morphologies: the sequestration of germ plasm components by the mitotic spindles in the Drosophila syncytial embryo, the asymmetric cell division initiated by the time delay in centrosome maturation in the Drosophila neuroblast, and the diffusional block between neighboring energids in the Drosophila syncytial embryo. Our model thus suggests that the cell cycle-dependent changes in the microtubule network are critical for achieving different spatial regulation effects. The microtubule network provides a spatially extensive docking platform for molecules and gives rise to a "structured cytoplasm", in contrast to a free and fluid environment.

  17. The feasibility of coherent energy transfer in microtubules.

    Science.gov (United States)

    Craddock, Travis John Adrian; Friesen, Douglas; Mane, Jonathan; Hameroff, Stuart; Tuszynski, Jack A

    2014-11-06

    It was once purported that biological systems were far too 'warm and wet' to support quantum phenomena mainly owing to thermal effects disrupting quantum coherence. However, recent experimental results and theoretical analyses have shown that thermal energy may assist, rather than disrupt, quantum coherent transport, especially in the 'dry' hydrophobic interiors of biomolecules. Specifically, evidence has been accumulating for the necessary involvement of quantum coherent energy transfer between uniquely arranged chromophores in light harvesting photosynthetic complexes. The 'tubulin' subunit proteins, which comprise microtubules, also possess a distinct architecture of chromophores, namely aromatic amino acids, including tryptophan. The geometry and dipolar properties of these aromatics are similar to those found in photosynthetic units indicating that tubulin may support coherent energy transfer. Tubulin aggregated into microtubule geometric lattices may support such energy transfer, which could be important for biological signalling and communication essential to living processes. Here, we perform a computational investigation of energy transfer between chromophoric amino acids in tubulin via dipole excitations coupled to the surrounding thermal environment. We present the spatial structure and energetic properties of the tryptophan residues in the microtubule constituent protein tubulin. Plausibility arguments for the conditions favouring a quantum mechanism of signal propagation along a microtubule are provided. Overall, we find that coherent energy transfer in tubulin and microtubules is biologically feasible.

  18. Fission yeast mtr1p regulates interphase microtubule cortical dwell-time

    Directory of Open Access Journals (Sweden)

    Frédérique Carlier-Grynkorn

    2014-06-01

    Full Text Available The microtubule cytoskeleton plays important roles in cell polarity, motility and division. Microtubules inherently undergo dynamic instability, stochastically switching between phases of growth and shrinkage. In cells, some microtubule-associated proteins (MAPs and molecular motors can further modulate microtubule dynamics. We present here the fission yeast mtr1+, a new regulator of microtubule dynamics that appears to be not a MAP or a motor. mtr1-deletion (mtr1Δ primarily results in longer microtubule dwell-time at the cell tip cortex, suggesting that mtr1p acts directly or indirectly as a destabilizer of microtubules. mtr1p is antagonistic to mal3p, the ortholog of mammalian EB1, which stabilizes microtubules. mal3Δ results in short microtubules, but can be partially rescued by mtr1Δ, as the double mutant mal3Δ mtr1Δ exhibits longer microtubules than mal3Δ single mutant. By sequence homology, mtr1p is predicted to be a component of the ribosomal quality control complex. Intriguingly, deletion of a predicted ribosomal gene, rps1801, also resulted in longer microtubule dwell-time similar to mtr1Δ. The double-mutant mal3Δ rps1801Δ also exhibits longer microtubules than mal3Δ single mutant alone. Our study suggests a possible involvement of mtr1p and the ribosome complex in modulating microtubule dynamics.

  19. Kinetochore-Dependent Microtubule Rescue Ensures Their Efficient and Sustained Interactions in Early Mitosis

    Science.gov (United States)

    Gandhi, Sapan R.; Gierliński, Marek; Mino, Akihisa; Tanaka, Kozo; Kitamura, Etsushi; Clayton, Lesley; Tanaka, Tomoyuki U.

    2011-01-01

    Summary How kinetochores regulate microtubule dynamics to ensure proper kinetochore-microtubule interactions is unknown. Here, we studied this during early mitosis in Saccharomyces cerevisiae. When a microtubule shrinks and its plus end reaches a kinetochore bound to its lateral surface, the microtubule end attempts to tether the kinetochore. This process often fails and, responding to this failure, microtubule rescue (conversion from shrinkage to growth) occurs, preventing kinetochore detachment from the microtubule end. This rescue is promoted by Stu2 transfer (ortholog of vertebrate XMAP215/ch-TOG) from the kinetochore to the microtubule end. Meanwhile, microtubule rescue distal to the kinetochore is also promoted by Stu2, which is transported by a kinesin-8 motor Kip3 along the microtubule from the kinetochore. Microtubule extension following rescue facilitates interaction with other widely scattered kinetochores, diminishing long delays in collecting the complete set of kinetochores by microtubules. Thus, kinetochore-dependent microtubule rescue ensures efficient and sustained kinetochore-microtubule interactions in early mitosis. PMID:22075150

  20. 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

  1. Pseudomonas aeruginosa exotoxin Y-mediated tau hyperphosphorylation impairs microtubule assembly in pulmonary microvascular endothelial cells.

    Directory of Open Access Journals (Sweden)

    Ron Balczon

    Full Text Available Pseudomonas aeruginosa uses a type III secretion system to introduce the adenylyl and guanylyl cyclase exotoxin Y (ExoY into the cytoplasm of endothelial cells. ExoY induces Tau hyperphosphorylation and insolubility, microtubule breakdown, barrier disruption and edema, although the mechanism(s responsible for microtubule breakdown remain poorly understood. Here we investigated both microtubule behavior and centrosome activity to test the hypothesis that ExoY disrupts microtubule dynamics. Fluorescence microscopy determined that infected pulmonary microvascular endothelial cells contained fewer microtubules than control cells, and further studies demonstrated that the microtubule-associated protein Tau was hyperphosphorylated following infection and dissociated from microtubules. Disassembly/reassembly studies determined that microtubule assembly was disrupted in infected cells, with no detectable effects on either microtubule disassembly or microtubule nucleation by centrosomes. This effect of ExoY on microtubules was abolished when the cAMP-dependent kinase phosphorylation site (Ser-214 on Tau was mutated to a non-phosphorylatable form. These studies identify Tau in microvascular endothelial cells as the target of ExoY in control of microtubule architecture following pulmonary infection by Pseudomonas aeruginosa and demonstrate that phosphorylation of tau following infection decreases microtubule assembly.

  2. Coordination Capacity

    CERN Document Server

    Cuff, Paul; Cover, Thomas

    2009-01-01

    We develop elements of a theory of cooperation and coordination in networks. Rather than considering a communication network as a means of distributing information, or of reconstructing random processes at remote nodes, we ask what dependence can be established among the nodes given the communication constraints. Specifically, in a network with communication rates between the nodes, we ask what is the set of all achievable joint distributions p(x1, ..., xm) of actions at the nodes on the network. Several networks are solved, including arbitrarily large cascade networks. Distributed cooperation can be the solution to many problems such as distributed games, distributed control, and establishing mutual information bounds on the influence of one part of a physical system on another.

  3. Statistical mechanics provides novel insights into microtubule stability and mechanism of shrinkage.

    Science.gov (United States)

    Jain, Ishutesh; Inamdar, Mandar M; Padinhateeri, Ranjith

    2015-02-01

    Microtubules are nano-machines that grow and shrink stochastically, making use of the coupling between chemical kinetics and mechanics of its constituent protofilaments (PFs). We investigate the stability and shrinkage of microtubules taking into account inter-protofilament interactions and bending interactions of intrinsically curved PFs. Computing the free energy as a function of PF tip position, we show that the competition between curvature energy, inter-PF interaction energy and entropy leads to a rich landscape with a series of minima that repeat over a length-scale determined by the intrinsic curvature. Computing Langevin dynamics of the tip through the landscape and accounting for depolymerization, we calculate the average unzippering and shrinkage velocities of GDP protofilaments and compare them with the experimentally known results. Our analysis predicts that the strength of the inter-PF interaction (E(s)(m)) has to be comparable to the strength of the curvature energy (E(b)(m)) such that E(s)(m) - E(b)(m) ≈ 1kBT, and questions the prevalent notion that unzippering results from the domination of bending energy of curved GDP PFs. Our work demonstrates how the shape of the free energy landscape is crucial in explaining the mechanism of MT shrinkage where the unzippered PFs will fluctuate in a set of partially peeled off states and subunit dissociation will reduce the length.

  4. Statistical mechanics provides novel insights into microtubule stability and mechanism of shrinkage.

    Directory of Open Access Journals (Sweden)

    Ishutesh Jain

    2015-02-01

    Full Text Available Microtubules are nano-machines that grow and shrink stochastically, making use of the coupling between chemical kinetics and mechanics of its constituent protofilaments (PFs. We investigate the stability and shrinkage of microtubules taking into account inter-protofilament interactions and bending interactions of intrinsically curved PFs. Computing the free energy as a function of PF tip position, we show that the competition between curvature energy, inter-PF interaction energy and entropy leads to a rich landscape with a series of minima that repeat over a length-scale determined by the intrinsic curvature. Computing Langevin dynamics of the tip through the landscape and accounting for depolymerization, we calculate the average unzippering and shrinkage velocities of GDP protofilaments and compare them with the experimentally known results. Our analysis predicts that the strength of the inter-PF interaction (E(s(m has to be comparable to the strength of the curvature energy (E(b(m such that E(s(m - E(b(m ≈ 1kBT, and questions the prevalent notion that unzippering results from the domination of bending energy of curved GDP PFs. Our work demonstrates how the shape of the free energy landscape is crucial in explaining the mechanism of MT shrinkage where the unzippered PFs will fluctuate in a set of partially peeled off states and subunit dissociation will reduce the length.

  5. Waves of actin and microtubule polymerization drive microtubule-based transport and neurite growth before single axon formation

    Science.gov (United States)

    Winans, Amy M; Collins, Sean R; Meyer, Tobias

    2016-01-01

    Many developing neurons transition through a multi-polar state with many competing neurites before assuming a unipolar state with one axon and multiple dendrites. Hallmarks of the multi-polar state are large fluctuations in microtubule-based transport into and outgrowth of different neurites, although what drives these fluctuations remains elusive. We show that actin waves, which stochastically migrate from the cell body towards neurite tips, direct microtubule-based transport during the multi-polar state. Our data argue for a mechanical control system whereby actin waves transiently widen the neurite shaft to allow increased microtubule polymerization to direct Kinesin-based transport and create bursts of neurite extension. Actin waves also require microtubule polymerization, arguing that positive feedback links these two components. We propose that actin waves create large stochastic fluctuations in microtubule-based transport and neurite outgrowth, promoting competition between neurites as they explore the environment until sufficient external cues can direct one to become the axon. DOI: http://dx.doi.org/10.7554/eLife.12387.001 PMID:26836307

  6. Taxol(®): The First Microtubule Stabilizing Agent.

    Science.gov (United States)

    Yang, Chia-Ping Huang; Horwitz, Susan Band

    2017-08-09

    Taxol(®), an antitumor drug with significant activity, is the first microtubule stabilizing agent described in the literature. This short review of the mechanism of action of Taxol(®) emphasizes the research done in the Horwitz' laboratory. It discusses the contribution of photoaffinity labeled analogues of Taxol(®) toward our understanding of the binding site of the drug on the microtubule. The importance of hydrogen/deuterium exchange experiments to further our insights into the stabilization of microtubules by Taxol(®) is addressed. The development of drug resistance, a major problem that arises in the clinic, is discussed. Studies describing differential drug binding to distinct β-tubulin isotypes are presented. Looking forward, it is suggested that the β-tubulin isotype content of a tumor may influence its responses to Taxol(®).

  7. Quantum Computation in Brain Microtubules? Decoherence and Biological Feasibility

    CERN Document Server

    Hagan, S; Tuszynski, J A

    2000-01-01

    The Penrose-Hameroff (`Orch OR') model of quantum computation in brain microtubules has been criticized as regards the issue of environmental decoherence. A recent report by Tegmark finds that microtubules can maintain quantum coherence for only $10^{-13}$ s, far too short to be neurophysiologically relevant. Here, we critically examine the assumptions behind Tegmark's calculation and find that: 1) Tegmark's commentary is not aimed at an existing model in the literature but rather at a hybrid that replaces the superposed protein conformations of the `Orch OR' theory with a soliton in superposition along the microtubule, 2) Tegmark predicts decreasing decoherence times at lower temperature, in direct contradiction of the observed behavior of quantum states, 3) recalculation after correcting Tegmark's equation for differences between his model and the `Orch OR' model (superposition separation, charge vs. dipole, dielectric constant) lengthens the decoherence time to $10^{-5} - 10^{-4}$ s and invalidates a criti...

  8. Disruption of microtubule integrity initiates mitosis during CNS repair.

    Science.gov (United States)

    Bossing, Torsten; Barros, Claudia S; Fischer, Bettina; Russell, Steven; Shepherd, David

    2012-08-14

    Mechanisms of CNS repair have vital medical implications. We show that traumatic injury to the ventral midline of the embryonic Drosophila CNS activates cell divisions to replace lost cells. A pilot screen analyzing transcriptomes of single cells during repair pointed to downregulation of the microtubule-stabilizing GTPase mitochondrial Rho (Miro) and upregulation of the Jun transcription factor Jun-related antigen (Jra). Ectopic Miro expression can prevent midline divisions after damage, whereas Miro depletion destabilizes cortical β-tubulin and increases divisions. Disruption of cortical microtubules, either by chemical depolymerization or by overexpression of monomeric tubulin, triggers ectopic mitosis in the midline and induces Jra expression. Conversely, loss of Jra renders midline cells unable to replace damaged siblings. Our data indicate that upon injury, the integrity of the microtubule cytoskeleton controls cell division in the CNS midline, triggering extra mitosis to replace lost cells. The conservation of the identified molecules suggests that similar mechanisms may operate in vertebrates.

  9. Microtubule stabilization reduces scarring and causes axon regeneration after spinal cord injury

    NARCIS (Netherlands)

    F. Hellal (Farida); A. Hurtado (Andres); J. Ruschel (Jörg); K.C. Flynn (Kevin); C.J. Laskowski (Claudia); M. Umlauf (Martina); L.C. Kapitein (Lukas); D. Strikis (Dinara); V. Lemmon (Vance); J. Bixby (John); C.C. Hoogenraad (Casper); F. Bradke (Frank)

    2011-01-01

    textabstractHypertrophic scarring and poor intrinsic axon growth capacity constitute major obstacles for spinal cord repair. These processes are tightly regulated by microtubule dynamics. Here, moderate microtubule stabilization decreased scar formation after spinal cord injury in rodents through va

  10. Active self-organization of microtubules in an inert chamber system

    National Research Council Canada - National Science Library

    Arif Md Rashedul Kabir; Daisuke Inoue; Akira Kakugo; Kazuki Sada; Jian Ping Gong

    2012-01-01

    Microtubule-kinesin system is considered as a building block for the construction of artificial biomachines, and active self-organization of microtubules has been used to integrate their structural...

  11. CYLD Regulates Noscapine Activity in Acute Lymphoblastic Leukemia via a Microtubule-Dependent Mechanism.

    Science.gov (United States)

    Yang, Yunfan; Ran, Jie; Sun, Lei; Sun, Xiaodong; Luo, Youguang; Yan, Bing; Tala; Liu, Min; Li, Dengwen; Zhang, Lei; Bao, Gang; Zhou, Jun

    2015-01-01

    Noscapine is an orally administrable drug used worldwide for cough suppression and has recently been demonstrated to disrupt microtubule dynamics and possess anticancer activity. However, the molecular mechanisms regulating noscapine activity remain poorly defined. Here we demonstrate that cylindromatosis (CYLD), a microtubule-associated tumor suppressor protein, modulates the activity of noscapine both in cell lines and in primary cells of acute lymphoblastic leukemia (ALL). Flow cytometry and immunofluorescence microscopy reveal that CYLD increases the ability of noscapine to induce mitotic arrest and apoptosis. Examination of cellular microtubules as well as in vitro assembled microtubules shows that CYLD enhances the effect of noscapine on microtubule polymerization. Microtubule cosedimentation and fluorescence titration assays further reveal that CYLD interacts with microtubule outer surface and promotes noscapine binding to microtubules. These findings thus demonstrate CYLD as a critical regulator of noscapine activity and have important implications for ALL treatment.

  12. Microtubule stability and MAPI B upregulation control neuritogenesis in CAD cells

    Institute of Scientific and Technical Information of China (English)

    Wen LI; Jin-tang XIA; Yue FENG

    2006-01-01

    Aim: To study the role of microtubule dynamics and microtubule associated protein 1B (MAP1B) in regulation of the neurite extension in CAD catecholaminergic neuronal cell line. Methods: The neuritogenesis of the CAD cells was abolished by inhibiting microtubule polymerization with nocodazole and by blocking microtubule depolymerization with taxol. MAP1B and tubulin protein expression was detected by Western blot. Immunofluorescent staining of tubulins was observed by fluorescent and confocal microscopy. Results: Microtubule dynamics was essential for CAD neurite extension. Dosage analysis revealed that neurite extension was much more sensitive to nocodazole than to taxol, suggesting a functional requirement for highly active microtubule assembly. A remarkable upregulation of MAP1B protein was detected during neurite extension accompanied with increased microtubule stability. Conclusion: Upregulation of MAP1B leads to the stabilization of newly formed microtubules in the developing neurites, which in turn promotes neurite extension.

  13. Septins localize to microtubules during nutritional limitation in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Vázquez de Aldana Carlos R

    2008-10-01

    Full Text Available Abstract Background In Saccharomyces cerevisiae, nutrient limitation stimulates diploid cells to undergo DNA replication and meiosis, followed by the formation of four haploid spores. Septins are a family of proteins that assemble a ring structure at the mother-daughter neck during vegetative growth, where they control cytokinesis. In sporulating cells, the septin ring disassembles and septins relocalize to the prospore membrane. Results Here, we demonstrate that nutrient limitation triggers a change in the localization of at least two vegetative septins (Cdc10 and Cdc11 from the bud neck to the microtubules. The association of Cdc10 and Cdc11 with microtubules persists into meiosis, and they are found associated with the meiotic spindle until the end of meiosis II. In addition, the meiosis-specific septin Spr28 displays similar behavior, suggesting that this is a common feature of septins. Septin association to microtubules is a consequence of the nutrient limitation signal, since it is also observed when haploid cells are incubated in sporulation medium and when haploid or diploid cells are grown in medium containing non-fermentable carbon sources. Moreover, during meiosis II, when the nascent prospore membrane is formed, septins moved from the microtubules to this membrane. Proper organization of the septins on the membrane requires the sporulation-specific septins Spr3 and Spr28. Conclusion Nutrient limitation in S. cerevisiae triggers the sporulation process, but it also induces the disassembly of the septin bud neck ring and relocalization of the septin subunits to the nucleus. Septins remain associated with microtubules during the meiotic divisions and later, during spore morphogenesis, they are detected associated to the nascent prospore membranes surrounding each nuclear lobe. Septin association to microtubules also occurs during growth in non-fermentable carbon sources.

  14. Balance of microtubule stiffness and cortical tension determines the size of blood cells with marginal band across species.

    Science.gov (United States)

    Dmitrieff, Serge; Alsina, Adolfo; Mathur, Aastha; Nédélec, François J

    2017-04-25

    The fast bloodstream of animals is associated with large shear stresses. To withstand these conditions, blood cells have evolved a special morphology and a specific internal architecture to maintain their integrity over several weeks. For instance, nonmammalian red blood cells, mammalian erythroblasts, and platelets have a peripheral ring of microtubules, called the marginal band, that flattens the overall cell morphology by pushing on the cell cortex. In this work, we model how the shape of these cells stems from the balance between marginal band rigidity and cortical tension. We predict that the diameter of the cell scales with the total microtubule polymer and verify the predicted law across a wide range of species. Our analysis also shows that the combination of the marginal band rigidity and cortical tension increases the ability of the cell to withstand forces without deformation. Finally, we model the marginal band coiling that occurs during the disk-to-sphere transition observed, for instance, at the onset of blood platelet activation. We show that when cortical tension increases faster than cross-linkers can unbind, the marginal band will coil, whereas if the tension increases more slowly, the marginal band may shorten as microtubules slide relative to each other.

  15. Three-dimensional reconstruction of tubulin sheets and re-investigation of microtubule surface lattice.

    Science.gov (United States)

    Schultheiss, R; Mandelkow, E

    1983-10-25

    Sheets are incomplete microtubule walls observed as polymorphic variants of microtubule assembly. Their substructure is similar to that of microtubules, as shown by two-dimensional optical and computer reconstruction. We have extended earlier studies by computing a three-dimensional reconstruction. From a re-investigation of the surface lattice it appears that the three-start helix of microtubules is right-handed rather than left-handed.

  16. 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.

  17. Kinesin-3 in the basidiomycete Ustilago maydis transports organelles along the entire microtubule array.

    Science.gov (United States)

    Steinberg, Gero

    2015-01-01

    The molecular motor kinesin-3 transports early endosomes along microtubules in filamentous fungi. It was reported that kinesin-3 from the ascomycete fungi Aspergillus nidulans and Neurospora crassa use a subset of post-translationally modified and more stable microtubules. Here, I show that kinesin-3 from the basidiomycete Ustilago maydis moves along all hyphal microtubules. This difference is likely due to variation in cell cycle control and associated organization of the microtubule array.

  18. In vivo and in vitro effects of the mitochondrial uncoupler FCCP on microtubules.

    OpenAIRE

    Maro, B.; Marty, M C; Bornens, M

    1982-01-01

    FCCP (carbonylcyanide-p-trifluoromethoxyphenylhydrazone), a potent uncoupler of oxidative phosphorylation, induces the complete disruption of cellular microtubules. A further analysis of this effect on BHK21 cells has shown that a decrease in the number of microtubules can be observed 15 min after adding FCCP and there is complete disruption after 60 min. Regrowth of microtubules was initiated 30 min after removal of FCCP, in marked contrast with the rapid reversion observed when microtubules...

  19. RUN COORDINATION

    CERN Multimedia

    C. Delaere

    2013-01-01

    Since the LHC ceased operations in February, a lot has been going on at Point 5, and Run Coordination continues to monitor closely the advance of maintenance and upgrade activities. In the last months, the Pixel detector was extracted and is now stored in the pixel lab in SX5; the beam pipe has been removed and ME1/1 removal has started. We regained access to the vactank and some work on the RBX of HB has started. Since mid-June, electricity and cooling are back in S1 and S2, allowing us to turn equipment back on, at least during the day. 24/7 shifts are not foreseen in the next weeks, and safety tours are mandatory to keep equipment on overnight, but re-commissioning activities are slowly being resumed. Given the (slight) delays accumulated in LS1, it was decided to merge the two global runs initially foreseen into a single exercise during the week of 4 November 2013. The aim of the global run is to check that we can run (parts of) CMS after several months switched off, with the new VME PCs installed, th...

  20. RUN COORDINATION

    CERN Multimedia

    Christophe Delaere

    2013-01-01

    The focus of Run Coordination during LS1 is to monitor closely the advance of maintenance and upgrade activities, to smooth interactions between subsystems and to ensure that all are ready in time to resume operations in 2015 with a fully calibrated and understood detector. After electricity and cooling were restored to all equipment, at about the time of the last CMS week, recommissioning activities were resumed for all subsystems. On 7 October, DCS shifts began 24/7 to allow subsystems to remain on to facilitate operations. That culminated with the Global Run in November (GriN), which   took place as scheduled during the week of 4 November. The GriN has been the first centrally managed operation since the beginning of LS1, and involved all subdetectors but the Pixel Tracker presently in a lab upstairs. All nights were therefore dedicated to long stable runs with as many subdetectors as possible. Among the many achievements in that week, three items may be highlighted. First, the Strip...

  1. Sliding of microtubules by a team of dynein motors: Understanding the effect of spatial distribution of motor tails and mutual exclusion of motor heads on microtubules

    Science.gov (United States)

    Singh, Hanumant Pratap; Takshak, Anjneya; Mall, Utkarsh; Kunwar, Ambarish

    2016-06-01

    Molecular motors are natural nanomachines that use the free energy released from ATP hydrolysis to generate mechanical forces. Cytoplasmic dynein motors often work collectively as a team to drive important processes such as axonal growth, proplatelet formation and mitosis, as forces generated by single motors are insufficient. A large team of dynein motors is used to slide cytoskeletal microtubules with respect to one another during the process of proplatelet formation and axonal growth. These motors attach to a cargo microtubule via their tail domains, undergo the process of detachment and reattachment of their head domains on another track microtubule, while sliding the cargo microtubule along the track. Traditional continuum/mean-field approaches used in the past are not ideal for studying the sliding mechanism of microtubules, as they ignore spatial and temporal fluctuations due to different possible distributions of motor tails on cargo filament, as well as binding/unbinding of motors from their track. Therefore, these models cannot be used to address important questions such as how the distribution of motor tails on microtubules, or how the mutual exclusion of motor heads on microtubule tracks affects the sliding velocity of cargo microtubule. To answer these, here we use a computational stochastic model where we model each dynein motor explicitly. In our model, we use both random as well as uniform distributions of dynein motors on cargo microtubule, as well as mutual exclusion of motors on microtubule tracks. We find that sliding velocities are least affected by the distribution of motor tails on microtubules, whereas they are greatly affected by mutual exclusion of motor heads on microtubule tracks. We also find that sliding velocity depends on the length of cargo microtubule if mutual exclusion among motor heads is considered.

  2. Phospholipase D activation correlates with microtubule reorganization in living plant cells

    NARCIS (Netherlands)

    P.B. Dhonukshe; A.M. Laxalt; J. Goedhart; Th.W.J. Gadella; T. Munnik

    2003-01-01

    A phospholipase D (PLD) was shown recently to decorate microtubules in plant cells. Therefore, we used tobacco BY-2 cells expressing the microtubule reporter GFP-MAP4 to test whether PLD activation affects the organization of plant microtubules. Within 30 min of adding n-butanol, a potent activator

  3. 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...

  4. 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...

  5. The von Hippel-Lindau tumour suppressor interacts with microtubules through kinesin-2

    NARCIS (Netherlands)

    Lolkema, M.P.J.K.; Mans, D.A.; Snijckers, C.M.J.T.; Noort, Mascha van; Beest, M. van; Voest, E.E.; Giles, R.H.

    2007-01-01

    Synthesis and maintenance of primary cilia are regulated by the von Hippel-Lindau (VHL) tumour suppressor protein. Recent studies indicate that this regulation is linked to microtubule-dependent functions of pVHL such as orienting microtubule growth and increasing plus-end microtubule stability, how

  6. Cortical Microtubule Arrays Are Initiated from a Nonrandom Prepattern Driven by Atypical Microtubule Initiation1[W][OA

    Science.gov (United States)

    Lindeboom, Jelmer J.; Lioutas, Antonios; Deinum, Eva E.; Tindemans, Simon H.; Ehrhardt, David W.; Emons, Anne Mie C.; Vos, Jan W.; Mulder, Bela M.

    2013-01-01

    The ordered arrangement of cortical microtubules in growing plant cells is essential for anisotropic cell expansion and, hence, for plant morphogenesis. These arrays are dismantled when the microtubule cytoskeleton is rearranged during mitosis and reassembled following completion of cytokinesis. The reassembly of the cortical array has often been considered as initiating from a state of randomness, from which order arises at least partly through self-organizing mechanisms. However, some studies have shown evidence for ordering at early stages of array assembly. To investigate how cortical arrays are initiated in higher plant cells, we performed live-cell imaging studies of cortical array assembly in tobacco (Nicotiana tabacum) Bright Yellow-2 cells after cytokinesis and drug-induced disassembly. We found that cortical arrays in both cases did not initiate randomly but with a significant overrepresentation of microtubules at diagonal angles with respect to the cell axis, which coincides with the predominant orientation of the microtubules before their disappearance from the cell cortex in preprophase. In Arabidopsis (Arabidopsis thaliana) root cells, recovery from drug-induced disassembly was also nonrandom and correlated with the organization of the previous array, although no diagonal bias was observed in these cells. Surprisingly, during initiation, only about one-half of the new microtubules were nucleated from locations marked by green fluorescent protein-γ-tubulin complex protein2-tagged γ-nucleation complexes (γ-tubulin ring complex), therefore indicating that a large proportion of early polymers was initiated by a noncanonical mechanism not involving γ-tubulin ring complex. Simulation studies indicate that the high rate of noncanonical initiation of new microtubules has the potential to accelerate the rate of array repopulation. PMID:23300168

  7. Optimal shapes for self-propelled swimmers

    Science.gov (United States)

    Koumoutsakos, Petros; van Rees, Wim; Gazzola, Mattia

    2011-11-01

    We optimize swimming shapes of three-dimensional self-propelled swimmers by combining the CMA- Evolution Strategy with a remeshed vortex method. We analyze the robustness of optimal shapes and discuss the near wake vortex dynamics for optimal speed and efficiency at Re=550. We also report preliminary results of optimal shapes and arrangements for multiple coordinated swimmers.

  8. Stabilization of coordinated motion for underwater vehicles

    Institute of Scientific and Technical Information of China (English)

    Fan Wu; Zhi-Yong Geng

    2011-01-01

    This paper presents a coordinating and stabilizing control law for a group of underwater vehicles with unstable dynamics. The coordinating law is derived from a potential that only depends on the relative configuration of the underwater vehicles. Being coordinated, the group behaves like one mechanical system with symmetry, and we focus on stabilizing a family of coordinated motions, called relative equilibria. The stabilizing law is derived using energy shaping to stabilize the relative equilibria which involve each vehicle translating along its longest (unstable) axis without spinning,while maintaining a relative configuration within the group.The proposed control law is physically motivated and avoids the linearization or cancellation of nonlinearities.

  9. Microtubules accelerate the kinase activity of Aurora-B by a reduction in dimensionality.

    Science.gov (United States)

    Noujaim, Michael; Bechstedt, Susanne; Wieczorek, Michal; Brouhard, Gary J

    2014-01-01

    Aurora-B is the kinase subunit of the Chromosome Passenger Complex (CPC), a key regulator of mitotic progression that corrects improper kinetochore attachments and establishes the spindle midzone. Recent work has demonstrated that the CPC is a microtubule-associated protein complex and that microtubules are able to activate the CPC by contributing to Aurora-B auto-phosphorylation in trans. Aurora-B activation is thought to occur when the local concentration of Aurora-B is high, as occurs when Aurora-B is enriched at centromeres. It is not clear, however, whether distributed binding to large structures such as microtubules would increase the local concentration of Aurora-B. Here we show that microtubules accelerate the kinase activity of Aurora-B by a "reduction in dimensionality." We find that microtubules increase the kinase activity of Aurora-B toward microtubule-associated substrates while reducing the phosphorylation levels of substrates not associated to microtubules. Using the single molecule assay for microtubule-associated proteins, we show that a minimal CPC construct binds to microtubules and diffuses in a one-dimensional (1D) random walk. The binding of Aurora-B to microtubules is salt-dependent and requires the C-terminal tails of tubulin, indicating that the interaction is electrostatic. We show that the rate of Aurora-B auto-activation is faster with increasing concentrations of microtubules. Finally, we demonstrate that microtubules lose their ability to stimulate Aurora-B when their C-terminal tails are removed by proteolysis. We propose a model in which microtubules act as scaffolds for the enzymatic activity of Aurora-B. The scaffolding activity of microtubules enables rapid Aurora-B activation and efficient phosphorylation of microtubule-associated substrates.

  10. 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

  11. Plk phosphorylation regulates the microtubule-stabilizing protein TCTP.

    Science.gov (United States)

    Yarm, Frederic R

    2002-09-01

    The mitotic polo-like kinases have been implicated in the formation and function of bipolar spindles on the basis of their respective localizations and mutant phenotypes. To date, this putative regulation has been limited to a kinesin-like motor protein, a centrosomal structural protein, and two microtubule-associated proteins (MAPs). In this study, another spindle-regulating protein, the mammalian non-MAP microtubule-binding and -stabilizing protein, the translationally controlled tumor protein (TCTP), was identified as a putative Plk-interacting clone by a two-hybrid screen. Plk phosphorylates TCTP on two serine residues in vitro and cofractionates with the majority of kinase activity toward TCTP in mitotic cell lysates. In addition, these sites were demonstrated to be phosphorylated in vivo. Overexpression of a Plk phosphorylation site-deficient mutant of TCTP induced a dramatic increase in the number of multinucleate cells, rounded cells with condensed ball-like nuclei, and cells undergoing cell death, similar to both the reported anti-Plk antibody microinjection and the low-concentration taxol treatment phenotypes. These results suggest that phosphorylation decreases the microtubule-stabilizing activity of TCTP and promotes the increase in microtubule dynamics that occurs after metaphase.

  12. Regulation of microtubule stability and mitotic progression by survivin.

    Science.gov (United States)

    Giodini, Alessandra; Kallio, Marko J; Wall, Nathan R; Gorbsky, Gary J; Tognin, Simona; Marchisio, Pier Carlo; Symons, Marc; Altieri, Dario C

    2002-05-01

    Survivin is a member of the inhibitor of apoptosis (IAP) gene family, which has been implicated in both preservation of cell viability and regulation of mitosis in cancer cells. Here, we show that HeLa cells microinjected with a polyclonal antibody to survivin exhibited delayed progression in prometaphase (31.5 +/- 6.9 min) and metaphase (126.8 +/- 73.8 min), as compared with control injected cells (prometaphase, 21.5 +/- 3.3 min; metaphase, 18.9 +/- 4.5 min; P mitotic spindles severely depleted of microtubules and occasionally underwent apoptosis without exiting the mitotic block or thereafter. Forced expression of survivin in HeLa cells profoundly influenced microtubule dynamics with reduction of pole-to-pole distance at metaphase (8.57 +/- 0.21 microm versus 10.58 +/- 0.19 microm; P microtubules against nocodazole-induced depolymerization in vivo. These data demonstrate that survivin functions at cell division to control microtubule stability and assembly of a normal mitotic spindle. This pathway may facilitate checkpoint evasion and promote resistance to chemotherapy in cancer.

  13. BMP signaling and microtubule organization regulate synaptic strength.

    Science.gov (United States)

    Ball, R W; Peled, E S; Guerrero, G; Isacoff, E Y

    2015-04-16

    The strength of synaptic transmission between a neuron and multiple postsynaptic partners can vary considerably. We have studied synaptic heterogeneity using the glutamatergic Drosophila neuromuscular junction (NMJ), which contains multiple synaptic connections of varying strengths between a motor axon and muscle fiber. In larval NMJs, there is a gradient of synaptic transmission from weak proximal to strong distal boutons. We imaged synaptic transmission with the postsynaptically targeted fluorescent calcium sensor SynapCam, to investigate the molecular pathways that determine synaptic strength and set up this gradient. We discovered that mutations in the Bone Morphogenetic Protein (BMP) signaling pathway disrupt production of strong distal boutons. We find that strong connections contain unbundled microtubules in the boutons, suggesting a role for microtubule organization in transmission strength. The spastin mutation, which disorganizes microtubules, disrupted the transmission gradient, supporting this interpretation. We propose that the BMP pathway, shown previously to function in the homeostatic regulation of synaptic growth, also boosts synaptic transmission in a spatially selective manner that depends on the microtubule system.

  14. Total synthesis of the potent microtubule-stabilizing agent (+)-discodermolide.

    Science.gov (United States)

    Harried, Scott S; Lee, Christopher P; Yang, Ge; Lee, Tony I H; Myles, David C

    2003-08-22

    The total synthesis of the potent microtubule-stabilizing, antimitotic agent (+)-discodermolide is described. The convergent synthetic strategy takes advantage of the diastereoselective alkylation of a ketone enolate to establish the key C15-C16 bond. The synthesis is amenable to preparation of gram-scale quantities of (+)-discodermolide and analogues.

  15. Direct Modulation of Microtubule Stability Contributes to Anthracene General Anesthesia

    Science.gov (United States)

    Emerson, Daniel J.; Weiser, Brian P.; Psonis, John; Liao, Zhengzheng; Taratula, Olena; Fiamengo, Ashley; Wang, Xiaozhao; Sugasawa, Keizo; Smith, Amos B.; Eckenhoff, Roderic G; Dmochowski, Ivan J.

    2013-01-01

    Recently, we identified 1-aminoanthracene as a fluorescent general anesthetic. To investigate the mechanism of action, a photoactive analogue, 1-azidoanthracene, was synthesized. Administration of 1-azidoanthracene to albino stage 40–47 tadpoles was found to immobilize animals upon near-UV irradiation of the forebrain region. The immobilization was often reversible, but it was characterized by a longer duration consistent with covalent attachment of the ligand to functionally important targets. IEF/SDS-PAGE examination of irradiated tadpole brain homogenate revealed labeled protein, identified by mass spectrometry as β-tubulin. In vitro assays with aminoanthracene-cross-linked tubulin indicated inhibition of microtubule polymerization, similar to colchicine. Tandem mass spectrometry confirmed anthracene binding near the colchicine site. Stage 40–47 tadpoles were also incubated 1 h with microtubule stabilizing agents, epothilone D or discodermolide, followed by dosing with 1-aminoanthracene. The effective concentration of 1-aminoanthracene required to immobilize the tadpoles was significantly increased in the presence of either microtubule stabilizing agent. Epothilone D similarly mitigated the effects of a clinical neurosteroid general anesthetic, allopregnanolone, believed to occupy the colchicine site in tubulin. We conclude that neuronal microtubules are “on-pathway” targets for anthracene general anesthetics and may also represent functional targets for some neurosteroid general anesthetics. PMID:23484901

  16. Fission yeast Scp3 potentially maintains microtubule orientation through bundling.

    Directory of Open Access Journals (Sweden)

    Kanako Ozaki

    Full Text Available Microtubules play important roles in organelle transport, the maintenance of cell polarity and chromosome segregation and generally form bundles during these processes. The fission yeast gene scp3+ was identified as a multicopy suppressor of the cps3-81 mutant, which is hypersensitive to isopropyl N-3-chlorophenylcarbamate (CIPC, a poison that induces abnormal multipolar spindle formation in higher eukaryotes. In this study, we investigated the function of Scp3 along with the effect of CIPC in the fission yeast Schizosaccharomyces pombe. Microscopic observation revealed that treatment with CIPC, cps3-81 mutation and scp3+ gene deletion disturbed the orientation of microtubules in interphase cells. Overexpression of scp3+ suppressed the abnormal orientation of microtubules by promoting bundling. Functional analysis suggested that Scp3 functions independently from Ase1, a protein largely required for the bundling of the mitotic spindle. A strain lacking the ase1+ gene was more sensitive to CIPC, with the drug affecting the integrity of the mitotic spindle, indicating that CIPC has a mitotic target that has a role redundant with Ase1. These results suggested that multiple systems are independently involved to ensure microtubule orientation by bundling in fission yeast.

  17. 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

  18. EWSR1 regulates mitosis by dynamically influencing microtubule acetylation.

    Science.gov (United States)

    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.

  19. Structure of the microtubule-binding domain of flagellar dynein.

    Science.gov (United States)

    Kato, Yusuke S; Yagi, Toshiki; Harris, Sarah A; Ohki, Shin-ya; Yura, Kei; Shimizu, Youské; Honda, Shinya; Kamiya, Ritsu; Burgess, Stan A; Tanokura, Masaru

    2014-11-04

    Flagellar dyneins are essential microtubule motors in eukaryotes, as they drive the beating motions of cilia and flagella. Unlike myosin and kinesin motors, the track binding mechanism of dyneins and the regulation between the strong and weak binding states remain obscure. Here we report the solution structure of the microtubule-binding domain of flagellar dynein-c/DHC9 (dynein-c MTBD). The structure reveals a similar overall helix-rich fold to that of the MTBD of cytoplasmic dynein (cytoplasmic MTBD), but dynein-c MTBD has an additional flap, consisting of an antiparallel b sheet. The flap is positively charged and highly flexible. Despite the structural similarity to cytoplasmic MTBD, dynein-c MTBD shows only a small change in the microtubule- binding affinity depending on the registry change of coiled coil-sliding, whereby lacks the apparent strong binding state. The surface charge distribution of dynein-c MTBD also differs from that of cytoplasmic MTBD, which suggests a difference in the microtubule-binding mechanism.

  20. Microtubule-dependent modulation of adhesion complex composition.

    Science.gov (United States)

    Ng, Daniel H J; Humphries, Jonathan D; Byron, Adam; Millon-Frémillon, Angélique; Humphries, Martin J

    2014-01-01

    The microtubule network regulates the turnover of integrin-containing adhesion complexes to stimulate cell migration. Disruption of the microtubule network results in an enlargement of adhesion complex size due to increased RhoA-stimulated actomyosin contractility, and inhibition of adhesion complex turnover; however, the microtubule-dependent changes in adhesion complex composition have not been studied in a global, unbiased manner. Here we used label-free quantitative mass spectrometry-based proteomics to determine adhesion complex changes that occur upon microtubule disruption with nocodazole. Nocodazole-treated cells displayed an increased abundance of the majority of known adhesion complex components, but no change in the levels of the fibronectin-binding α5β1 integrin. Immunofluorescence analyses confirmed these findings, but revealed a change in localisation of adhesion complex components. Specifically, in untreated cells, α5-integrin co-localised with vinculin at peripherally located focal adhesions and with tensin at centrally located fibrillar adhesions. In nocodazole-treated cells, however, α5-integrin was found in both peripherally located and centrally located adhesion complexes that contained both vinculin and tensin, suggesting a switch in the maturation state of adhesion complexes to favour focal adhesions. Moreover, the switch to focal adhesions was confirmed to be force-dependent as inhibition of cell contractility with the Rho-associated protein kinase inhibitor, Y-27632, prevented the nocodazole-induced conversion. These results highlight a complex interplay between the microtubule cytoskeleton, adhesion complex maturation state and intracellular contractile force, and provide a resource for future adhesion signaling studies. The proteomics data have been deposited in the ProteomeXchange with identifier PXD001183.

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

    Science.gov (United States)

    Hutchins, B Ian; Wray, Susan

    2014-01-01

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

  2. Motor coordination: a local hub for coordination.

    Science.gov (United States)

    Calabrese, Ronald L

    2014-03-31

    A local interneuron of a crayfish central pattern generator serves as a hub that integrates ascending and descending coordinating information and passes it on to a local oscillatory microcircuit to coordinate a series of segmental appendages known as swimmerets.

  3. Sites of glucose transporter-4 vesicle fusion with the plasma membrane correlate spatially with microtubules.

    Directory of Open Access Journals (Sweden)

    Jennine M Dawicki-McKenna

    Full Text Available In adipocytes, vesicles containing glucose transporter-4 (GLUT4 redistribute from intracellular stores to the cell periphery in response to insulin stimulation. Vesicles then fuse with the plasma membrane, facilitating glucose transport into the cell. To gain insight into the details of microtubule involvement, we examined the spatial organization and dynamics of microtubules in relation to GLUT4 vesicle trafficking in living 3T3-L1 adipocytes using total internal reflection fluorescence (TIRF microscopy. Insulin stimulated an increase in microtubule density and curvature within the TIRF-illuminated region of the cell. The high degree of curvature and abrupt displacements of microtubules indicate that substantial forces act on microtubules. The time course of the microtubule density increase precedes that of the increase in intensity of fluorescently-tagged GLUT4 in this same region of the cell. In addition, portions of the microtubules are highly curved and are pulled closer to the cell cortex, as confirmed by Parallax microscopy. Microtubule disruption delayed and modestly reduced GLUT4 accumulation at the plasma membrane. Quantitative analysis revealed that fusions of GLUT4-containing vesicles with the plasma membrane, detected using insulin-regulated aminopeptidase with a pH-sensitive GFP tag (pHluorin, preferentially occur near microtubules. Interestingly, long-distance vesicle movement along microtubules visible at the cell surface prior to fusion does not appear to account for this proximity. We conclude that microtubules may be important in providing spatial information for GLUT4 vesicle fusion.

  4. A novel role for aquaporin-5 in enhancing microtubule organization and stability.

    Directory of Open Access Journals (Sweden)

    Venkataramana K Sidhaye

    Full Text Available Aquaporin-5 (AQP5 is a water-specific channel located on the apical surface of airway epithelial cells. In addition to regulating transcellular water permeability, AQP5 can regulate paracellular permeability, though the mechanisms by which this occurs have not been determined. Microtubules also regulate paracellular permeability. Here, we report that AQP5 promotes microtubule assembly and helps maintain the assembled microtubule steady state levels with slower turnover dynamics in cells. Specifically, reduced levels of AQP5 correlated with lower levels of assembled microtubules and decreased paracellular permeability. In contrast, overexpression of AQP5 increased assembly of microtubules, with evidence of increased MT stability, and promoted the formation of long straight microtubules in the apical domain of the epithelial cells. These findings indicate that AQP5-mediated regulation of microtubule dynamics modulates airway epithelial barrier properties and epithelial function.

  5. Tubulin bond energies and microtubule biomechanics determined from nanoindentation in silico

    CERN Document Server

    Kononova, Olga; Theisen, Kelly E; Marx, Kenneth A; Dima, Ruxandra I; Ataullakhanov, Fazly I; Grishchuk, Ekaterina L; Barsegov, Valeri

    2015-01-01

    Microtubules, the primary components of the chromosome segregation machinery, are stabilized by longitudinal and lateral non-covalent bonds between the tubulin subunits. However, the thermodynamics of these bonds and the microtubule physico-chemical properties are poorly understood. Here, we explore the biomechanics of microtubule polymers using multiscale computational modeling and nanoindentations in silico of a contiguous microtubule fragment. A close match between the simulated and experimental force-deformation spectra enabled us to correlate the microtubule biomechanics with dynamic structural transitions at the nanoscale. Our mechanical testing revealed that the compressed MT behaves as a system of rigid elements interconnected through a network of lateral and longitudinal elastic bonds. The initial regime of continuous elastic deformation of the microtubule is followed by the transition regime, during which the microtubule lattice undergoes discrete structural changes, which include first the reversib...

  6. Sulfo-SMCC Prevents Annealing of Taxol-Stabilized Microtubules In Vitro

    CERN Document Server

    Prabhune, Meenakshi; Schmidt, Christoph F

    2015-01-01

    Microtubule structure and functions have been widely studied in vitro and in cells. Research has shown that cysteines on tubulin play a crucial role in the polymerization of microtubules. Here, we show that blocking sulfhydryl groups of cysteines in taxol-stabilized polymerized microtubules with a commonly used chemical crosslinker prevents temporal end-to-end annealing of microtubules in vitro. This can dramatically affect the length distribution of the microtubules. The crosslinker sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, sulfo-SMCC, consists of a maleimide and a N-hydroxysuccinimide ester group to bind to sulfhydryl groups and primary amines, respectively. Interestingly, addition of a maleimide dye alone does not show the same prevention of annealing in stabilized microtubules. This study shows that the sulfhydryl groups of cysteines of tubulin that are vital for the polymerization are also important for the subsequent annealing of microtubules.

  7. Globally visualizing the microtubule-dependent transport behaviors of influenza virus in live cells.

    Science.gov (United States)

    Liu, Shu-Lin; Zhang, Li-Juan; Wang, Zhi-Gang; Zhang, Zhi-Ling; Wu, Qiu-Mei; Sun, En-Ze; Shi, Yun-Bo; Pang, Dai-Wen

    2014-04-15

    Understanding the microtubule-dependent behaviors of viruses in live cells is very meaningful for revealing the mechanisms of virus infection and endocytosis. Herein, we used a quantum dots-based single-particle tracking technique to dynamically and globally visualize the microtubule-dependent transport behaviors of influenza virus in live cells. We found that the intersection configuration of microtubules can interfere with the transport behaviors of the virus in live cells, which lead to the changing and long-time pausing of the transport behavior of viruses. Our results revealed that most of the viruses moved along straight microtubules rapidly and unidirectionally from the cell periphery to the microtubule organizing center (MTOC) near the bottom of the cell, and the viruses were confined in the grid of microtubules near the top of the cell and at the MTOC near the bottom of the cell. These results provided deep insights into the influence of entire microtubule geometry on the virus infection.

  8. Cell edges accumulate gamma tubulin complex components and nucleate microtubules following cytokinesis in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Chris Ambrose

    Full Text Available Microtubules emanate from distinct organizing centers in fungal and animal cells. In plant cells, by contrast, microtubules initiate from dispersed sites in the cell cortex, where they then self-organize into parallel arrays. Previous ultrastructural evidence suggested that cell edges participate in microtubule nucleation but so far there has been no direct evidence for this. Here we use live imaging to show that components of the gamma tubulin nucleation complex (GCP2 and GCP3 localize at distinct sites along the outer periclinal edge of newly formed crosswalls, and that microtubules grow predominantly away from these edges. These data confirm a role for cell edges in microtubule nucleation, and suggest that an asymmetric distribution of microtubule nucleation factors contributes to cortical microtubule organization in plants, in a manner more similar to other kingdoms than previously thought.

  9. PACSIN1, a Tau-interacting protein, regulates axonal elongation and branching by facilitating microtubule instability.

    Science.gov (United States)

    Liu, Yingying; Lv, Kaosheng; Li, Zenglong; Yu, Albert C H; Chen, Jianguo; Teng, Junlin

    2012-11-16

    Tau is a major member of the neuronal microtubule-associated proteins. It promotes tubulin assembly and stabilizes axonal microtubules. Previous studies have demonstrated that Tau forms cross-bridges between microtubules, with some particles located on cross-bridges, suggesting that some proteins interact with Tau and might be involved in regulating Tau-related microtubule dynamics. This study reports that PACSIN1 interacts with Tau in axon. PACSIN1 blockade results in impaired axonal elongation and a higher number of primary axonal branches in mouse dorsal root ganglia neurons, which is induced by increasing the binding ability of Tau to microtubules. In PACSIN1-blocked dorsal root ganglia neurons, a greater amount of Tau is inclined to accumulate in the central domain of growth cones, and it promotes the stability of the microtubule network. Taken together, these results suggest that PACSIN1 is an important Tau binding partner in regulating microtubule dynamics and forming axonal plasticity.

  10. Alignment between PIN1 polarity and microtubule orientation in the shoot apical meristem reveals a tight coupling between morphogenesis and auxin transport.

    Directory of Open Access Journals (Sweden)

    Marcus G Heisler

    Full Text Available Morphogenesis during multicellular development is regulated by intercellular signaling molecules as well as by the mechanical properties of individual cells. In particular, normal patterns of organogenesis in plants require coordination between growth direction and growth magnitude. How this is achieved remains unclear. Here we show that in Arabidopsis thaliana, auxin patterning and cellular growth are linked through a correlated pattern of auxin efflux carrier localization and cortical microtubule orientation. Our experiments reveal that both PIN1 localization and microtubule array orientation are likely to respond to a shared upstream regulator that appears to be biomechanical in nature. Lastly, through mathematical modeling we show that such a biophysical coupling could mediate the feedback loop between auxin and its transport that underlies plant phyllotaxis.

  11. 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.

  12. Three luminescent d{sup 10} metal coordination polymers assembled from a semirigid V-shaped ligand with high selective detecting of Cu{sup 2+} ion and nitrobenzene

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Wei-Ping [Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of the Ministry of Education, Shaanxi Key Laboratory of Physico-Inorg. Chem., College of Chemistry & Materials Science, Northwest University, Xi’an 710069 (China); Institute of Functional Materials, College of Chemistry and Pharmaceutical Engineering, Sichuan University of Science & Engineering, Zigong 643000 (China); Liu, Ping; Liang, Yu-Tong; Cui, Lin; Xi, Zheng-Ping [Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of the Ministry of Education, Shaanxi Key Laboratory of Physico-Inorg. Chem., College of Chemistry & Materials Science, Northwest University, Xi’an 710069 (China); Wang, Yao-Yu, E-mail: wyaoyu@nwu.edu.cn [Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of the Ministry of Education, Shaanxi Key Laboratory of Physico-Inorg. Chem., College of Chemistry & Materials Science, Northwest University, Xi’an 710069 (China)

    2015-08-15

    Three 2D luminescent coordination polymers with helical frameworks, [ZnL{sub 2}]{sub n} (1) and ([ML{sub 2}]·(H{sub 2}O)){sub n} (M=Zn (2), Cd (3)) (HL=4-((2-methyl-1 H-imidazol-1-yl)methyl)benzoic acid), have been assembled under hydro(solvo)thermal conditions. Complex 1 is in chiral space group and displays a rare 2D→2D 2-fold parallel interpenetrated layer network with two types of chiral double helixes. Interestingly, the single crystal structure analyses indicate the coexistence of enantiomers la and 1b in one pot, while the bulk crystallization of 1 are racemic mixtures based on the CD measurement. 2 and 3 are isostructural, in the structure, there are two kinds of 2D chiral helical-layers which stack in an -ABAB- sequence leading to the overall structure are mesomer and achiral. All compounds display intense luminescence in solid state at room temperature with high chemical and thermal stability. More importantly, 1 has been successfully applied in the detection of Cu{sup 2+} ions in aqueous media and nitrobenzene and the probable detecting mechanism was also discussed. - Graphical abstract: Three luminescent d{sup 10} metal coordination polymers with helical-layer based on 4-((2-methyl-1H-imidazol-1-yl)methyl)benzoic acid have been obtained. Compound 1 shows high selective detecting for Cu{sup 2+} ion in aqueous and nitrobenzene. - Highlights: • Three coordination polymers with chiral helical-layer have been obtained. • 1 Can luminescent detect Cu{sup 2+} ion in aqueous media and nitrobenzene. • Racemic mixture or mesomer compounds can be obtained by controlling the reaction conditions.

  13. 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

  14. Arabidopsis GCP3-interacting protein 1/MOZART 1 is an integral component of the γ-tubulin-containing microtubule nucleating complex.

    Science.gov (United States)

    Nakamura, Masayoshi; Yagi, Noriyoshi; Kato, Takehide; Fujita, Satoshi; Kawashima, Noriyuki; Ehrhardt, David W; Hashimoto, Takashi

    2012-07-01

    Microtubules in eukaryotic cells are nucleated from ring-shaped complexes that contain γ-tubulin and a family of homologous γ-tubulin complex proteins (GCPs), but the subunit composition of the complexes can vary among fungi, animals and plants. Arabidopsis GCP3-interacting protein 1 (GIP1), a small protein with no homology to the GCP family, interacts with GCP3 in vitro, and is a plant homolog of vertebrate mitotic-spindle organizing protein associated with a ring of γ-tubulin 1 (MOZART1), a recently identified component of the γ-tubulin complex in human cell lines. In this study, we characterized two closely related Arabidopsis GIP1s: GIP1a and GIP1b. Single mutants of gip1a and gip1b were indistinguishable from wild-type plants, but their double mutant was embryonic lethal, and showed impaired development of male gametophytes. Functional fusions of GIP1a with green fluorescent protein (GFP) were used to purify GIP1a-containing complexes from Arabidopsis plants, which contained all the subunits (except NEDD1) previously identified in the Arabidopsis γ-tubulin complexes. GIP1a and GIP1b interacted specifically with Arabidopsis GCP3 in yeast. GFP-GIP1a labeled mitotic microtubule arrays in a pattern largely consistent with, but partly distinct from, the localization of the γ-tubulin complex containing GCP2 or GCP3 in planta. In interphase cortical arrays, the labeled complexes were preferentially recruited to existing microtubules, from which new microtubules were efficiently nucleated. However, in contrast to complexes labeled with tagged GCP2 or GCP3, their recruitment to cortical areas with no microtubules was rarely observed. These results indicate that GIP1/MOZART1 is an integral component of a subset of the Arabidopsis γ-tubulin complexes. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.

  15. Schapiro Shapes

    Science.gov (United States)

    O'Connell, Emily

    2009-01-01

    This article describes a lesson on Schapiro Shapes. Schapiro Shapes is based on the art of Miriam Schapiro, who created a number of works of figures in action. Using the basic concepts of this project, students learn to create their own figures and styles. (Contains 1 online resource.)

  16. Cyclin G-associated kinase promotes microtubule outgrowth from chromosomes during spindle assembly.

    Science.gov (United States)

    Tanenbaum, Marvin E; Vallenius, Tea; Geers, Erica F; Greene, Lois; Mäkelä, Tomi P; Medema, Rene H

    2010-08-01

    During mitosis, all chromosomes must attach to microtubules of the mitotic spindle to ensure correct chromosome segregation. Microtubule attachment occurs at specialized structures at the centromeric region of chromosomes, called kinetochores. These kinetochores can generate microtubule attachments through capture of centrosome-derived microtubules, but in addition, they can generate microtubules themselves, which are subsequently integrated with centrosome-derived microtubules to form the mitotic spindle. Here, we have performed a large scale RNAi screen and identify cyclin G-associated kinase (GAK) as a novel regulator of microtubule generation at kinetochores/chromatin. This function of GAK requires its C-terminal J-domain, which is essential for clathrin recycling from endocytic vesicles. Consistently, cells lacking GAK show strongly reduced levels of clathrin on the mitotic spindle, and reduction of clathrin levels also inhibits microtubule generation at kinetochores/chromosomes. Finally, we present evidence that association of clathrin with the spindle is promoted by a signal coming from the chromosomes. These results identify a role for GAK and clathrin in microtubule outgrowth from kinetochores/chromosomes and suggest that GAK acts through clathrin to control microtubule outgrowth around chromosomes.

  17. Taxol differentially modulates the dynamics of microtubules assembled from unfractionated and purified beta-tubulin isotypes.

    Science.gov (United States)

    Derry, W B; Wilson, L; Khan, I A; Luduena, R F; Jordan, M A

    1997-03-25

    Substoichiometric binding of taxol to tubulin in microtubules potently suppresses microtubule dynamics, which appears to be the most sensitive antiproliferative mechanism of taxol. To determine whether the beta-tubulin isotype composition of a microtubule can modulate sensitivity to taxol, we measured the effects of substoichiometric ratios of taxol bound to tubulin in microtubules on the dynamics of microtubules composed of purified alphabeta(II)-, alphabeta(III)-, or alphabeta(IV)-tubulin isotypes and compared the results with the effects of taxol on microtubules assembled from unfractionated tubulin. Substoichiometric ratios of bound taxol in microtubules assembled from purified beta-tubulin isotypes or unfractionated tubulin potently suppressed the shortening rates and the lengths shortened per shortening event. Correlation of the suppression of the shortening rate with the stoichiometry of bound taxol revealed that microtubules composed of purified alphabeta(II)-, alphabeta(III)-, and alphabeta(IV)-tubulin were, respectively, 1.6-, 7.4-, and 7.2-fold less sensitive to the effects of bound taxol than microtubules assembled from unfractionated tubulin. These results indicate that taxol differentially modulates microtubule dynamics depending upon the beta-tubulin isotype composition. The results are consistent with recent studies correlating taxol resistance in tumor cells with increased levels of beta(III0- and beta(IV)-tubulin expression and suggest that altered cellular expression of beta-tubulin isotypes can be an important mechanism by which tumor cells develop resistance to taxol.

  18. The Ndc80 complex uses a tripartite attachment point to couple microtubule depolymerization to chromosome movement.

    Science.gov (United States)

    Tooley, John G; Miller, Stephanie A; Stukenberg, P Todd

    2011-04-15

    In kinetochores, the Ndc80 complex couples the energy in a depolymerizing microtubule to perform the work of moving chromosomes. The complex directly binds microtubules using an unstructured, positively charged N-terminal tail located on Hec1/Ndc80. Hec1/Ndc80 also contains a calponin homology domain (CHD) that increases its affinity for microtubules in vitro, yet whether it is required in cells and how the tail and CHD work together are critical unanswered questions. Human kinetochores containing Hec1/Ndc80 with point mutations in the CHD fail to align chromosomes or form productive microtubule attachments. Kinetochore architecture and spindle checkpoint protein recruitment are unaffected in these mutants, and the loss of CHD function cannot be rescued by removing Aurora B sites from the tail. The interaction between the Hec1/Ndc80 CHD and a microtubule is facilitated by positively charged amino acids on two separate regions of the CHD, and both are required for kinetochores to make stable attachments to microtubules. Chromosome congression in cells also requires positive charge on the Hec1 tail to facilitate microtubule contact. In vitro binding data suggest that charge on the tail regulates attachment by directly increasing microtubule affinity as well as driving cooperative binding of the CHD. These data argue that in vertebrates there is a tripartite attachment point facilitating the interaction between Hec1/Ndc80 and microtubules. We discuss how such a complex microtubule-binding interface may facilitate the coupling of depolymerization to chromosome movement.

  19. Push-me-pull-you: how microtubules organize the cell interior.

    Science.gov (United States)

    Tolić-Nørrelykke, Iva M

    2008-09-01

    Dynamic organization of the cell interior, which is crucial for cell function, largely depends on the microtubule cytoskeleton. Microtubules move and position organelles by pushing, pulling, or sliding. Pushing forces can be generated by microtubule polymerization, whereas pulling typically involves microtubule depolymerization or molecular motors, or both. Sliding between a microtubule and another microtubule, an organelle, or the cell cortex is also powered by molecular motors. Although numerous examples of microtubule-based pushing and pulling in living cells have been observed, it is not clear why different cell types and processes employ different mechanisms. This review introduces a classification of microtubule-based positioning strategies and discusses the efficacy of pushing and pulling. The positioning mechanisms based on microtubule pushing are efficient for movements over small distances, and for centering of organelles in symmetric geometries. Mechanisms based on pulling, on the other hand, are typically more elaborate, but are necessary when the distances to be covered by the organelles are large, and when the geometry is asymmetric and complex. Thus, taking into account cell geometry and the length scale of the movements helps to identify general principles of the intracellular layout based on microtubule forces.

  20. The non-catalytic domains of Drosophila katanin regulate its abundance and microtubule-disassembly activity.

    Directory of Open Access Journals (Sweden)

    Kyle D Grode

    Full Text Available Microtubule severing is a biochemical reaction that generates an internal break in a microtubule and regulation of microtubule severing is critical for cellular processes such as ciliogenesis, morphogenesis, and meiosis and mitosis. Katanin is a conserved heterodimeric ATPase that severs and disassembles microtubules, but the molecular determinants for regulation of microtubule severing by katanin remain poorly defined. Here we show that the non-catalytic domains of Drosophila katanin regulate its abundance and activity in living cells. Our data indicate that the microtubule-interacting and trafficking (MIT domain and adjacent linker region of the Drosophila katanin catalytic subunit Kat60 cooperate to regulate microtubule severing in two distinct ways. First, the MIT domain and linker region of Kat60 decrease its abundance by enhancing its proteasome-dependent degradation. The Drosophila katanin regulatory subunit Kat80, which is required to stabilize Kat60 in cells, conversely reduces the proteasome-dependent degradation of Kat60. Second, the MIT domain and linker region of Kat60 augment its microtubule-disassembly activity by enhancing its association with microtubules. On the basis of our data, we propose that the non-catalytic domains of Drosophila katanin serve as the principal sites of integration of regulatory inputs, thereby controlling its ability to sever and disassemble microtubules.

  1. Changes in microtubule stability and density in myelin-deficient shiverer mouse CNS axons

    Science.gov (United States)

    Kirkpatrick, L. L.; Witt, A. S.; Payne, H. R.; Shine, H. D.; Brady, S. T.

    2001-01-01

    Altered axon-Schwann cell interactions in PNS myelin-deficient Trembler mice result in changed axonal transport rates, neurofilament and microtubule-associated protein phosphorylation, neurofilament density, and microtubule stability. To determine whether PNS and CNS myelination have equivalent effects on axons, neurofilaments, and microtubules in CNS, myelin-deficient shiverer axons were examined. The genetic defect in shiverer is a deletion in the myelin basic protein (MBP) gene, an essential component of CNS myelin. As a result, shiverer mice have little or no compact CNS myelin. Slow axonal transport rates in shiverer CNS axons were significantly increased, in contrast to the slowing in demyelinated PNS nerves. Even more striking were substantial changes in the composition and properties of microtubules in shiverer CNS axons. The density of axonal microtubules is increased, reflecting increased expression of tubulin in shiverer, and the stability of microtubules is drastically reduced in shiverer axons. Shiverer transgenic mice with two copies of a wild-type myelin basic protein transgene have an intermediate level of compact myelin, making it possible to determine whether the actual level of compact myelin is an important regulator of axonal microtubules. Both increased microtubule density and reduced microtubule stability were still observed in transgenic mouse nerves, indicating that signals beyond synaptogenesis and the mere presence of compact myelin are required for normal regulation of the axonal microtubule cytoskeleton.

  2. Push-me-pull-you: how microtubules organize the cell interior

    Science.gov (United States)

    2008-01-01

    Dynamic organization of the cell interior, which is crucial for cell function, largely depends on the microtubule cytoskeleton. Microtubules move and position organelles by pushing, pulling, or sliding. Pushing forces can be generated by microtubule polymerization, whereas pulling typically involves microtubule depolymerization or molecular motors, or both. Sliding between a microtubule and another microtubule, an organelle, or the cell cortex is also powered by molecular motors. Although numerous examples of microtubule-based pushing and pulling in living cells have been observed, it is not clear why different cell types and processes employ different mechanisms. This review introduces a classification of microtubule-based positioning strategies and discusses the efficacy of pushing and pulling. The positioning mechanisms based on microtubule pushing are efficient for movements over small distances, and for centering of organelles in symmetric geometries. Mechanisms based on pulling, on the other hand, are typically more elaborate, but are necessary when the distances to be covered by the organelles are large, and when the geometry is asymmetric and complex. Thus, taking into account cell geometry and the length scale of the movements helps to identify general principles of the intracellular layout based on microtubule forces. PMID:18404264

  3. Doublecortin Is Excluded from Growing Microtubule Ends and Recognizes the GDP-Microtubule Lattice.

    Science.gov (United States)

    Ettinger, Andreas; van Haren, Jeffrey; Ribeiro, Susana A; Wittmann, Torsten

    2016-06-20

    Many microtubule (MT) functions are mediated by a diverse class of proteins (+TIPs) at growing MT plus ends that control intracellular MT interactions and dynamics and depend on end-binding proteins (EBs) [1]. Cryoelectron microscopy has recently identified the EB binding site as the interface of four tubulin dimers that undergoes a conformational change in response to β-tubulin GTP hydrolysis [2, 3]. Doublecortin (DCX), a MT-associated protein (MAP) required for neuronal migration during cortical development [4, 5], binds to the same site as EBs [6], and recent in vitro studies proposed DCX localization to growing MT ends independent of EBs [7]. Because this conflicts with observations in neurons [8, 9] and the molecular function of DCX is not well understood, we revisited intracellular DCX dynamics at low expression levels. Here, we report that DCX is not a +TIP in cells but, on the contrary, is excluded from the EB1 domain. In addition, we find that DCX-MT interactions are highly sensitive to MT geometry. In cells, DCX binding was greatly reduced at MT segments with high local curvature. Remarkably, this geometry-dependent binding to MTs was completely reversed in the presence of taxanes, which reconciles incompatible observations in cells [9] and in vitro [10]. We propose a model explaining DCX specificity for different MT geometries based on structural changes induced by GTP hydrolysis that decreases the spacing between adjacent tubulin dimers [11]. Our data are consistent with a unique mode of MT interaction in which DCX specifically recognizes this compacted GDP-like MT lattice.

  4. In vivo and in vitro effects of the mitochondrial uncoupler FCCP on microtubules.

    Science.gov (United States)

    Maro, B; Marty, M C; Bornens, M

    1982-01-01

    FCCP (carbonylcyanide-p-trifluoromethoxyphenylhydrazone), a potent uncoupler of oxidative phosphorylation, induces the complete disruption of cellular microtubules. A further analysis of this effect on BHK21 cells has shown that a decrease in the number of microtubules can be observed 15 min after adding FCCP and there is complete disruption after 60 min. Regrowth of microtubules was initiated 30 min after removal of FCCP, in marked contrast with the rapid reversion observed when microtubules are disrupted by nocodazole. A similar delay was required for the recovery of mitochondrial function as assessed by rhodamine 123 labelling. The effect of FCCP on microtubules was partially inhibited by preincubation of the cells with NaN3, suggesting that FCCP acts on microtubules through mitochondria. FCCP did not depolymerize microtubules of cells permeabilized with Triton X-100. In vitro polymerisation of microtubule protein was only slightly diminished by concentrations of FCCP which provoke complete disassembly in vivo. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the microtubules polymerized in vitro in the presence of FCCP showed a reduced amount of high mol. wt. proteins, mainly MAP 2, associated with them. In an attempt to reproduce the mitochondrial effects of FCCP in vitro, we checked the effects of alkaline pH and calcium on microtubule protein polymerization in the presence of FCCP. FCCP did not influence the calcium inhibitory effect but did significantly increase the inhibitory effect of alkaline pH. We conclude that FCCP could depolymerise microtubules in vivo through a dual operation: increasing the intracellular pH by the disruption of the mitochondrial H+ gradient and decreasing the stability of microtubules by impairing the binding of microtubule-associated proteins.

  5. Effects of 3-repeat tau on taxol mobility through microtubules

    Science.gov (United States)

    Park, Hyunjoo; Fygenson, Deborah; Kim, Mahn Won

    2005-03-01

    Both the anti-cancer drug taxol and the microtubule-associated protein tau suppress dynamics of microtubules (MT). We have observed taxol mobility with full-length 3-repeat tau, one of six tau isoforms, using fluorescence recovery after photobleaching (FRAP) on MTs and compare with earlier results on recombinant full-length adult 4-repeat tau. Taxol mobility becomes highly sensitive to taxol concentration in the presence of 3-repeat tau (up to 1:1 molar ratio) as it does in the presence of 4-repeat tau, but is 2 to 3 times faster at low taxol concentrations. Fitting to a mean-field binding reaction model [J.L. Ross et.al, PNAS 101:12910-5 (2004)] suggests that the presence of 3-repeat tau enhances taxol movement through pores in the MT walls.

  6. Nonlinear Dynamics of Dipoles in Microtubules: Pseudo-Spin Model

    CERN Document Server

    Nesterov, Alexander I; Berman, Gennady P; Mavromatos, Nick E

    2016-01-01

    We perform a theoretical study of the dynamics of the electric field excitations in a microtubule by taking into consideration the realistic cylindrical geometry, dipole-dipole interactions of the tubulin-based protein heterodimers, the radial electric field produced by the solvent, and a possible degeneracy of energy states of individual heterodimers. The consideration is done in the frames of the classical pseudo-spin model. We derive the system of nonlinear dynamical ordinary differential equations of motion for interacting dipoles, and the continuum version of these equations. We obtain the solutions of these equations in the form of snoidal waves, solitons, kinks, and localized spikes. Our results will help to a better understanding of the functional properties of microtubules including the motor protein dynamics and the information transfer processes. Our considerations are based on classical dynamics. Some speculations on the role of possible quantum effects are also made.

  7. Nonlinear dynamics of dipoles in microtubules: Pseudospin model.

    Science.gov (United States)

    Nesterov, Alexander I; Ramírez, Mónica F; Berman, Gennady P; Mavromatos, Nick E

    2016-06-01

    We perform a theoretical study of the dynamics of the electric field excitations in a microtubule by taking into consideration the realistic cylindrical geometry, dipole-dipole interactions of the tubulin-based protein heterodimers, the radial electric field produced by the solvent, and a possible degeneracy of energy states of individual heterodimers. The consideration is done in the frame of the classical pseudospin model. We derive the system of nonlinear dynamical partial differential equations of motion for interacting dipoles and the continuum version of these equations. We obtain the solutions of these equations in the form of snoidal waves, solitons, kinks, and localized spikes. Our results will help to achieve a better understanding of the functional properties of microtubules including the motor protein dynamics and the information transfer processes. Our considerations are based on classical dynamics. Some speculations on the role of possible quantum effects are also made.

  8. The Feasibility of Coherent Energy Transfer in Microtubules

    CERN Document Server

    Craddock, Travis John Adrian; Mane, Jonathan; Hameroff, Stuart; Tuszynski, Jack A

    2014-01-01

    It was once purported that biological systems were far too warm and wet to support quantum phenomena mainly due to thermal effects disrupting quantum coherence. However recent experimental results and theoretical analyses have shown that thermal energy may assist, rather than disrupt, quantum coherence, especially in the dry hydrophobic interiors of biomolecules. Specifically, evidence has been accumulating for the necessary involvement of quantum coherence and entanglement between uniquely arranged chromophores in light harvesting photosynthetic complexes. Amazingly, the tubulin subunit proteins, which comprise microtubules, also possess a distinct architecture of chromophores, namely aromatic amino acids including tryptophan. The geometry and dipolar properties of these aromatics are similar to those found in photosynthetic units indicating that tubulin may support coherent energy transfer. Tubulin aggregated into microtubule geometric lattices may support such energy transfer, which could be of import for ...

  9. The octarepeat region of hamster PrP (PrP51-91) enhances the formation of microtubule and antagonize Cu~(2+)-induced microtubule-disrupting activity

    Institute of Scientific and Technical Information of China (English)

    Xiaoli Li; Chenfang Dong; Song Shi; Guirong Wang; Yuan Li; Xin Wang; Qi Shi; Chan Tian; Ruimin Zhou; Chen Gao; Xiaoping Dong

    2009-01-01

    Prion protein (PrP) is considered to associate with microtubule and its major component, tubulin. In the present study, octarepeat region of PrP (PrP51-91) was expressed in prokaryotic-expressing system. Using GST pull-down assay and co-immunoprecipitation, the mol-ecular interaction between PrP51-91 and tubulin was observed. Our data also demonstrated that PrP51-91 could efficiently stimulate microtubule assembly in vitro, indicating a potential effect of PrP on microtu-bule dynamics. Moreover, PrP51-91 was confirmed to be able to antagonize Cu~(2+)-induced microtubule-disrupt-ing activity in vivo, partially protecting against Cu~(2+) intoxication to culture cells and stabilize cellular micro-tubule structure. The association of the octarepeat region of PrP with tubulin may further provide insight into the biological function of PrP in the neurons.

  10. Oxidative stress decreases microtubule growth and stability in ventricular myocytes

    OpenAIRE

    Drum, BML; Yuan, C.; Li, L; Liu, Q.; Wordeman, L; Santana, LF

    2016-01-01

    © 2016 Elsevier Ltd.Microtubules (MTs) have many roles in ventricular myocytes, including structural stability, morphological integrity, and protein trafficking. However, despite their functional importance, dynamic MTs had never been visualized in living adult myocytes. Using adeno-associated viral vectors expressing the MT-associated protein plus end binding protein 3 (EB3) tagged with EGFP, we were able to perform live imaging and thus capture and quantify MT dynamics in ventricular myocyt...

  11. Kinetochore microtubule establishment is defective in oocytes from aged mice

    OpenAIRE

    Shomper, Maria; Lappa, Christina; FitzHarris, Greg

    2014-01-01

    Errors in chromosome segregation in mammalian oocytes increase in number with advancing maternal age, and are a major cause of pregnancy loss. Why chromosome segregation errors are more common in oocytes from older females remains poorly understood. In mitosis, accurate chromosome segregation is enabled by attachment of kinetochores to microtubules from appropriate spindle poles, and erroneous attachments increase the likelihood of mis-segregation. Whether attachment errors are responsible fo...

  12. The role of microtubules in contractile ring function

    Science.gov (United States)

    Conrad, A. H.; Paulsen, A. Q.; Conrad, G. W.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    During cytokinesis, a cortical contractile ring forms around a cell, constricts to a stable tight neck and terminates in separation of the daughter cells. At first cleavage, Ilyanassa obsoleta embryos form two contractile rings simultaneously. The cleavage furrow (CF), in the animal hemisphere between the spindle poles, constricts to a stable tight neck and separates the daughter cells. The third polar lobe constriction (PLC-3), in the vegetal hemisphere below the spindle, constricts to a transient tight neck, but then relaxes, allowing the polar lobe cytoplasm to merge with one daughter cell. Eggs exposed to taxol, a drug that stabilizes microtubules, before the CF or the PLC-3 develop, fail to form CFs, but form stabilized tight PLCs. Eggs exposed to taxol at the time of PLC-3 formation develop varied numbers of constriction rings in their animal hemispheres and one PLC in their vegetal hemisphere, none of which relax. Eggs exposed to taxol after PLC-3 initiation form stabilized tight CFs and PLCs. At maximum constriction, control embryos display immunolocalization of nonextractable alpha-tubulin in their CFs, but not in their PLCs, and reveal, via electron microscopy, many microtubules extending through their CFs, but not through their PLCs. Embryos which form stabilized tightly constricted CFs and PLCs in the presence of taxol display immunolocalization of nonextractable alpha-tubulin in both constrictions and show many polymerized microtubules extending through both CFs and PLCs. These results suggest that the extension of microtubules through a tight contractile ring may be important for stabilizing that constriction and facilitating subsequent cytokinesis.

  13. Limitations of Radar Coordinates

    OpenAIRE

    Bini, Donato; Lusanna, Luca; Mashhoon, Bahram

    2004-01-01

    The construction of a radar coordinate system about the world line of an observer is discussed. Radar coordinates for a hyperbolic observer as well as a uniformly rotating observer are described in detail. The utility of the notion of radar distance and the admissibility of radar coordinates are investigated. Our results provide a critical assessment of the physical significance of radar coordinates.

  14. Ethanol exposure disrupts extraembryonic microtubule cytoskeleton and embryonic blastomere cell adhesion, producing epiboly and gastrulation defects

    Directory of Open Access Journals (Sweden)

    Swapnalee Sarmah

    2013-08-01

    Fetal alcohol spectrum disorder (FASD occurs when pregnant mothers consume alcohol, causing embryonic ethanol exposure and characteristic birth defects that include craniofacial, neural and cardiac defects. Gastrulation is a particularly sensitive developmental stage for teratogen exposure, and zebrafish is an outstanding model to study gastrulation and FASD. Epiboly (spreading blastomere cells over the yolk cell, prechordal plate migration and convergence/extension cell movements are sensitive to early ethanol exposure. Here, experiments are presented that characterize mechanisms of ethanol toxicity on epiboly and gastrulation. Epiboly mechanisms include blastomere radial intercalation cell movements and yolk cell microtubule cytoskeleton pulling the embryo to the vegetal pole. Both of these processes were disrupted by ethanol exposure. Ethanol effects on cell migration also indicated that cell adhesion was affected, which was confirmed by cell aggregation assays. E-cadherin cell adhesion molecule expression was not affected by ethanol exposure, but E-cadherin distribution, which controls epiboly and gastrulation, was changed. E-cadherin was redistributed into cytoplasmic aggregates in blastomeres and dramatically redistributed in the extraembryonic yolk cell. Gene expression microarray analysis was used to identify potential causative factors for early development defects, and expression of the cell adhesion molecule protocadherin-18a (pcdh18a, which controls epiboly, was significantly reduced in ethanol exposed embryos. Injecting pcdh18a synthetic mRNA in ethanol treated embryos partially rescued epiboly cell movements, including enveloping layer cell shape changes. Together, data show that epiboly and gastrulation defects induced by ethanol are multifactorial, and include yolk cell (extraembryonic tissue microtubule cytoskeleton disruption and blastomere adhesion defects, in part caused by reduced pcdh18a expression.

  15. Autoinhibition of TBCB regulates EB1-mediated microtubule dynamics.

    Science.gov (United States)

    Carranza, Gerardo; Castaño, Raquel; Fanarraga, Mónica L; Villegas, Juan Carlos; Gonçalves, João; Soares, Helena; Avila, Jesus; Marenchino, Marco; Campos-Olivas, Ramón; Montoya, Guillermo; Zabala, Juan Carlos

    2013-01-01

    Tubulin cofactors (TBCs) participate in the folding, dimerization, and dissociation pathways of the tubulin dimer. Among them, TBCB and TBCE are two CAP-Gly domain-containing proteins that together efficiently interact with and dissociate the tubulin dimer. In the study reported here we showed that TBCB localizes at spindle and midzone microtubules during mitosis. Furthermore, the motif DEI/M-COO(-) present in TBCB, which is similar to the EEY/F-COO(-) element characteristic of EB proteins, CLIP-170, and α-tubulin, is required for TBCE-TBCB heterodimer formation and thus for tubulin dimer dissociation. This motif is responsible for TBCB autoinhibition, and our analysis suggests that TBCB is a monomer in solution. Mutants of TBCB lacking this motif are derepressed and induce microtubule depolymerization through an interaction with EB1 associated with microtubule tips. TBCB is also able to bind to the chaperonin complex CCT containing α-tubulin, suggesting that it could escort tubulin to facilitate its folding and dimerization, recycling or degradation.

  16. Anomalous motor mediated cargo transport in microtubule networks

    Science.gov (United States)

    Vandal, Steven; Macveigh-Fierro, Daniel; Shen, Zhiyuan; Lemoi, Kyle; Vidali, Luis; Ross, Jennifer; Tuzel, Erkan

    Cargo transport is an important biological mechanism by which cells locomote, self-organize, and actively transport organelles. This transport is mediated by the cytoskeletal network and molecular motors; however, it is not known how network self-organization and dynamics affect these transport processes. In order to develop a mechanistic understanding of cargo transport, we use a coarse-grained Brownian dynamics model that incorporates the dynamics of these networks, as well as experimentally determined motor properties. We will test these models with two experimental systems: (1) in vitro microtubule networks with kinesin-1 motors, and quantum dot cargos on recreated microtubule networks, and (2) an excellent model organism, the moss Physcomitrella patens, in which chloroplasts are transported via the microtubule network by means of kinesin-like proteins. Phenomenological network characterizations are made, both in vivo and in vitro, and cargo motility is characterized using Mean Squared Displacement (MSD) measurements. Our simulations shed light on the role of network density and motor properties on the observed transport behavior, and improve our understanding of cargo transport in cells.

  17. GIT1 enhances neurite outgrowth by stimulating microtubule assembly

    Institute of Scientific and Technical Information of China (English)

    Yi-sheng Li; Li-xia Qin; Jie Liu; Wei-liang Xia; Jian-ping Li; Hai-lian Shen; Wei-Qiang Gao

    2016-01-01

    GIT1, a G-protein-coupled receptor kinase interacting protein, has been reported to be involved in neurite outgrowth. However, the neu-robiological 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 signiifcant reduction in total neurite length per neuron, as well as in the average length of axon-like struc-tures, which could not be prevented by nerve growth factor treatment. Overexpression of GIT1 signiifcantly 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 sufifcient to enhance axonal extension. Importantly, GIT1 bound to many tubulin proteins and microtubule-associated proteins, and it accelerated microtubule assemblyin vitro. Collectively, our ifndings 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 neuro-logical diseases.

  18. Two-state mechanochemical model for microtubule growth

    CERN Document Server

    Zhang, Yunxin

    2011-01-01

    In this study, a two-state mechanochemical model is presented to describe the dynamic properties of microtubule (MT) growth in cells. The MT switches between two states, assembly state and disassembly state. In assembly state, the growth of microtubule includes two processes: GTP-tubulin binding to the tip of protofilament (PF) and conformational change of PF, during which the penultimate GTP is hydrolyzed and the first tubulin unit that curls out the MT surface is rearranged into MT surface using the energy released from GTP hydrolysis. In disassembly state, the shortening of microtubule is also described by two processes, the release of GDP-tibulin from the tip of PF and one new tubulin unit curls out from the MT surface. Switches between these two states, which are usually called rescue and catastrophe, happen stochastically with external force dependent rates. Using this two-state model with parameters obtained by fitting the recent experimental data, detailed properties of MT growth are obtained, we find...

  19. 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.

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

    Directory of Open Access Journals (Sweden)

    Gang Zhang

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

  1. Polo-like kinase 1 regulates Nlp, a centrosome protein involved in microtubule nucleation.

    Science.gov (United States)

    Casenghi, Martina; Meraldi, Patrick; Weinhart, Ulrike; Duncan, Peter I; Körner, Roman; Nigg, Erich A

    2003-07-01

    In animal cells, most microtubules are nucleated at centrosomes. At the onset of mitosis, centrosomes undergo a structural reorganization, termed maturation, which leads to increased microtubule nucleation activity. Centrosome maturation is regulated by several kinases, including Polo-like kinase 1 (Plk1). Here, we identify a centrosomal Plk1 substrate, termed Nlp (ninein-like protein), whose properties suggest an important role in microtubule organization. Nlp interacts with two components of the gamma-tubulin ring complex and stimulates microtubule nucleation. Plk1 phosphorylates Nlp and disrupts both its centrosome association and its gamma-tubulin interaction. Overexpression of an Nlp mutant lacking Plk1 phosphorylation sites severely disturbs mitotic spindle formation. We propose that Nlp plays an important role in microtubule organization during interphase, and that the activation of Plk1 at the onset of mitosis triggers the displacement of Nlp from the centrosome, allowing the establishment of a mitotic scaffold with enhanced microtubule nucleation activity.

  2. Relative velocity of sliding of microtubules by the action of Kinesin-5

    CERN Document Server

    Roy, Sthitadhi

    2011-01-01

    Kinesin-5, also known as Eg5 in vertebrates is a processive motor with 4 heads, which moves on filamentous tracks called microtubules. The basic function of Kinesin-5 is to slide apart two anti-parallel microtubules by simultaneously walking on both the microtubules. We develop an analytical expression for the steady-state relative velocity of this sliding in terms of the rates of attachments and detachments of motor heads with the ATPase sites on the microtubules. We first analyse the motion of one pair of motor heads on one microtubule and then couple it to the motion of the other pair of motor heads of the same motor on the second microtubule to get the relative velocity of sliding.

  3. Mitosis and microtubule organizational changes in rice root-tip cells

    Institute of Scientific and Technical Information of China (English)

    XUSHIXIONG(SYZEE); CHUNGUILI; CHENGZHU

    1993-01-01

    The pattern of change of the microtubule cytoskeleton of the root-tip cells of rice during mitosis was studied using immunofluorescence technic and confocal laser scanning microscopy. All the major stages of ceil division including preprophase, prophase, metaphase, anaphase and telophase were observed. The most significant finding was that in the preprophase cells microtubules radiating from the nuclear surface to the cortex were frequently seen. During development these microtubules became closely associated with the preprophase band and prophase spindie indicating that the microtubules radiating from the nuclear surface, the preprophase band and the prophazc spindle were structurally and functionally closely related to each other. Granule-like anchorage sites for the radiating microtubules at the muclear surface were often seen and the possibility that these gramle-like anchorage sites might represent the microtubule organizing centres was discussed.

  4. High rectifying efficiencies of microtubule motility on kinesin-coated gold nanostructures.

    Science.gov (United States)

    van den Heuvel, Martin G L; Butcher, Christopher T; Smeets, Ralph M M; Diez, Stefan; Dekker, Cees

    2005-06-01

    We demonstrate highly efficient rectification of microtubule motility on gold nanofabricated structures. First, we present a novel nanofabrication process for the creation of gold tracks for microtubule motility recessed in silicon oxide. This approach is particularly useful because it enables the use of the well-understood PEG-silane chemistry on SiO2 for the blocking of kinesin, whereas the gold tracks allow possible electrical control. We demonstrate excellent confinement of microtubule motility to the gold nanostructures and that microtubules move on the gold with speeds comparable to that on glass. Second, we present designs of three advanced rectifier geometries. We analyze the microtubule pathways through the geometries, and we demonstrate highly efficient rectification with up to 92% efficiency. As a result, we find that up to 97% of the microtubules move unidirectionally.

  5. Kinesin-1 Translocation along Human Breast Cancer Cell Microtubules in Vitro

    Science.gov (United States)

    Shojania Feizabadi, Mitra; Jun, Yonggun

    2015-03-01

    A principle approach to better understand intra-cellular microtubule based transport is to study such it in vitro. Such in vitro examinations have predominantly used microtubules polymerized from bovine brain tubulin, but motor function can also in principle be affected by the specific tubulin isotypes present in different cells. The human breast cancer cells carry different beta tubulin isotype distribution. However, it is entirely unknown whether transport along the microtubules is different in these cells. In this work we have characterized, for the first time, the translocation specifications of kinesin-1 along human breast cancer cell microtubules polymerized in vitro. We found that as compared with the translocation along bovine brain microtubules, kinesin-1 shows a fifty percent shorter processive run length and slightly slower velocity under similar experimental conditions. These first time results support the regulatory role of tubulin isotypes in regards to motor protein translocations, and quantify the translocation specifications of kinesin-1 along microtubules of human breast cancer cells.

  6. Selective extraction of isolated mitotic apparatus. Evidence that typical microtubule protein is extracted by organic mercurial.

    Science.gov (United States)

    Bibring, T; Baxandall, J

    1971-02-01

    Mitotic apparatus isolated from sea urchin eggs has been treated with meralluride sodium under conditions otherwise resembling those of its isolation. The treatment causes a selective morphological disappearance of microtubules while extracting a major protein fraction, probably consisting of two closely related proteins, which constitutes about 10% of mitotic apparatus protein. Extraction of other cell particulates under similar conditions yields much less of this protein. The extracted protein closely resembles outer doublet microtubule protein from sea urchin sperm tail in properties considered typical of microtubule proteins: precipitation by calcium ion and vinblastine, electrophoretic mobility in both acid and basic polyacrylamide gels, sedimentation coefficient, molecular weight, and, according to a preliminary determination, amino acid composition. An antiserum against a preparation of sperm tail outer doublet microtubules cross-reacts with the extract from mitotic apparatus. On the basis of these findings it appears that microtubule protein is selectively extracted from isolated mitotic apparatus by treatment with meralluride, and is a typical microtubule protein.

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

    Directory of Open Access Journals (Sweden)

    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.

  8. Xenopus oocyte wound healing as a model system for analysis of microtubule-actin interactions.

    Science.gov (United States)

    Zhang, Tong; Mandato, Craig A

    2007-01-01

    Microtubule-actin interactions are fundamental to many cellular processes such as cytokinesis and cellular locomotion. Investigating the mechanism of microtubule-actin interactions is the key to understand the cellular morphogenesis and related pathological processes. The abundance and highly dynamic nature of microtubules and F-actin raise a serious challenge when trying to distinguish between the real and fortuitous interactions within a cell. Xenopus oocyte wound model represents an ideal system to study microtubule-actin interactions as well as microtubule-dependent control of the actin polymerization. Here, we describe a series of cytoskeleton specific treatments in Xenopus oocyte wound healing experiments and use confocal fluorescence microscopy to analyze fixed oocytes to examine microtubule-actin interactions.

  9. Regulatory volume decrease in Leishmania mexicana: effect of anti-microtubule drugs

    Directory of Open Access Journals (Sweden)

    Francehuli Dagger

    2013-02-01

    Full Text Available The trypanosomatid cytoskeleton is responsible for the parasite's shape and it is modulated throughout the different stages of the parasite's life cycle. When parasites are exposed to media with reduced osmolarity, they initially swell, but subsequently undergo compensatory shrinking referred to as regulatory volume decrease (RVD. We studied the effects of anti-microtubule (Mt drugs on the proliferation of Leishmania mexicana promastigotes and their capacity to undergo RVD. All of the drugs tested exerted antiproliferative effects of varying magnitudes [ansamitocin P3 (AP3> trifluoperazine > taxol > rhizoxin > chlorpromazine]. No direct relationship was found between antiproliferative drug treatment and RVD. Similarly, Mt stability was not affected by drug treatment. Ansamitocin P3, which is effective at nanomolar concentrations, blocked amastigote-promastigote differentiation and was the only drug that impeded RVD, as measured by light dispersion. AP3 induced 2 kinetoplasts (Kt 1 nucleus cells that had numerous flagella-associated Kts throughout the cell. These results suggest that the dramatic morphological changes induced by AP3 alter the spatial organisation and directionality of the Mts that are necessary for the parasite's hypotonic stress-induced shape change, as well as its recovery.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-02-01

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

  11. 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.

  12. Tension-dependent regulation of microtubule dynamics at kinetochores can explain metaphase congression in yeast

    National Research Council Canada - National Science Library

    Gardner, Melissa K; Pearson, Chad G; Sprague, Brian L; Zarzar, Ted R; Bloom, Kerry; Salmon, E D; Odde, David J

    2005-01-01

    During metaphase in budding yeast mitosis, sister kinetochores are tethered to opposite poles and separated, stretching their intervening chromatin, by singly attached kinetochore microtubules (kMTs...

  13. TOG Proteins Are Spatially Regulated by Rac-GSK3β to Control Interphase Microtubule Dynamics.

    Directory of Open Access Journals (Sweden)

    Kathryn P Trogden

    Full Text Available Microtubules are regulated by a diverse set of proteins that localize to microtubule plus ends (+TIPs where they regulate dynamic instability and mediate interactions with the cell cortex, actin filaments, and organelles. Although individual +TIPs have been studied in depth and we understand their basic contributions to microtubule dynamics, there is a growing body of evidence that these proteins exhibit cross-talk and likely function to collectively integrate microtubule behavior and upstream signaling pathways. In this study, we have identified a novel protein-protein interaction between the XMAP215 homologue in Drosophila, Mini spindles (Msps, and the CLASP homologue, Orbit. These proteins have been shown to promote and suppress microtubule dynamics, respectively. We show that microtubule dynamics are regionally controlled in cells by Rac acting to suppress GSK3β in the peripheral lamellae/lamellipodium. Phosphorylation of Orbit by GSK3β triggers a relocalization of Msps from the microtubule plus end to the lattice. Mutation of the Msps-Orbit binding site revealed that this interaction is required for regulating microtubule dynamic instability in the cell periphery. Based on our findings, we propose that Msps is a novel Rac effector that acts, in partnership with Orbit, to regionally regulate microtubule dynamics.

  14. Inhibition of kinesin-5 improves regeneration of injured axons by a novel microtubule-based mechanism

    Institute of Scientific and Technical Information of China (English)

    Peter W. Baas; Andrew J. Matamoros

    2015-01-01

    Microtubules have been identiifed as a powerful target for augmenting regeneration of injured adult axons in the central nervous system. Drugs that stabilize microtubules have shown some promise, but there are concerns that abnormally stabilizing microtubules may have only limited beneifts for regeneration, while at the same time may be detrimental to the normal work that microtubules perform for the axon. Kinesin-5 (also called kif11 or Eg5), a molecular motor protein best known for its crucial role in mitosis, acts as a brake on microtubule movements by other motor proteins in the axon. Drugs that inhibit kinesin-5, originally developed to treat cancer, result in greater mobility of microtubules in the axon and an overall shift in the forces on the microtubule array. As a result, the axon grows faster, retracts less, and more readily enters environments that are inhibitory to axonal regeneration. Thus, drugs that inhibit kinesin-5 offer a novel microtubule-based means to boost axonal regeneration without the concerns that ac-company abnormal stabilization of the microtubule array. Even so, inhibiting kinesin-5 is not without its own caveats, such as potential problems with navigation of the regenerating axon to its target, as well as morphological effects on dendrites that could affect learning and memory if the drugs reach the brain.

  15. A plus-end raft to control microtubule dynamics and function.

    Science.gov (United States)

    Galjart, Niels; Perez, Franck

    2003-02-01

    Cells require a properly oriented and organised microtubule array to transmit positional information. Recent data have revealed a heterogeneous population of microtubule-binding proteins that accumulates mainly at distal ends of polymerising microtubules. Two mechanisms may account for this concentration: transient immobilisation, which involves association of proteins with growing ends, followed by release more proximally; and deposition at ends via a molecular motor. As with lipid rafts, protein concentration at distal ends may allow a cascade of interactions in the restricted area of a microtubule plus end. This may, in turn, control the dynamic behaviour of this cytoskeletal network and its anchoring to other structures.

  16. 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.

  17. Myomegalin is necessary for the formation of centrosomal and Golgi-derived microtubules

    Directory of Open Access Journals (Sweden)

    Régine Roubin

    2012-12-01

    The generation of cellular microtubules is initiated at specific sites such as the centrosome and the Golgi apparatus that contain nucleation complexes rich in γ-tubulin. The microtubule growing plus-ends are stabilized by plus-end tracking proteins (+TIPs, mainly EB1 and associated proteins. Myomegalin was identified as a centrosome/Golgi protein associated with cyclic nucleotide phosphodiesterase. We show here that Myomegalin exists as several isoforms. We characterize two of them. One isoform, CM-MMG, harbors a conserved domain (CM1, recently described as a nucleation activator, and is related to a family of γ-tubulin binding proteins, which includes Drosophila centrosomin. It localizes at the centrosome and at the cis-Golgi in an AKAP450-dependent manner. It recruits γ-tubulin nucleating complexes and promotes microtubule nucleation. The second isoform, EB-MMG, is devoid of CM1 domain and has a unique N-terminus with potential EB1-binding sites. It localizes at the cis-Golgi and can localize to microtubule plus-ends. EB-MMG binds EB1 and affects its loading on microtubules and microtubule growth. Depletion of Myomegalin by small interfering RNA delays microtubule growth from the centrosome and Golgi apparatus, and decreases directional migration of RPE1 cells. In conclusion, the Myomegalin gene encodes different isoforms that regulate microtubules. At least two of these have different roles, demonstrating a previously unknown mechanism to control microtubules in vertebrate cells.

  18. Microtubule polarity and the direction of pigment transport reverse simultaneously in surgically severed melanophore arms.

    Science.gov (United States)

    McNiven, M A; Wang, M; Porter, K R

    1984-07-01

    The transport of pigment through the long cytoplasmic extensions (arms) of teleost melanophores is a microtubule-dependent event. We have severed the arms from melanophores to test whether microtubules isolated from the centrosome maintain their original polarity and disposition. In addition, we have tested whether arms containing microtubules of mixed polarities alter the direction of pigment transport. We find that microtubules within severed arms eventually change their polarity and reorganize from the arm center as if to form a new minicell. Concomitant with this change is a reversal in the direction of pigment transport.

  19. Measurement of Breaking Force of Fluorescence Labelled Microtubules with Optical Tweezers

    Institute of Scientific and Technical Information of China (English)

    LIU Chun-Xiang; GUO Hong-Lian; XU Chun-Hua; YUAN Ming; LI Znao-Lin; CHENG Bing-Ying; ZHANG Dao-Zhong

    2005-01-01

    @@ Under illumination of excitation light, the force that can make fluorescent dye-labelled microtubules break up is measured by using dual-beam optical tweezers. It is found that this force is about several piconewtons, which is two orders of magnitude smaller than that without fluorescence label. Microtubules can be elongated about 20% and the increase of the tensile force is nonlinear with the microtubule elongation. Some qualitative explanations are given for the mechanisms about the breakup and elongation of microtubules exposed to excitation light.

  20. Inhibition of kinesin-5 improves regeneration of injured axons by a novel microtubule-based mechanism

    Directory of Open Access Journals (Sweden)

    Peter W Baas

    2015-01-01

    Full Text Available Microtubules have been identified as a powerful target for augmenting regeneration of injured adult axons in the central nervous system. Drugs that stabilize microtubules have shown some promise, but there are concerns that abnormally stabilizing microtubules may have only limited benefits for regeneration, while at the same time may be detrimental to the normal work that microtubules perform for the axon. Kinesin-5 (also called kif11 or Eg5, a molecular motor protein best known for its crucial role in mitosis, acts as a brake on microtubule movements by other motor proteins in the axon. Drugs that inhibit kinesin-5, originally developed to treat cancer, result in greater mobility of microtubules in the axon and an overall shift in the forces on the microtubule array. As a result, the axon grows faster, retracts less, and more readily enters environments that are inhibitory to axonal regeneration. Thus, drugs that inhibit kinesin-5 offer a novel microtubule-based means to boost axonal regeneration without the concerns that accompany abnormal stabilization of the microtubule array. Even so, inhibiting kinesin-5 is not without its own caveats, such as potential problems with navigation of the regenerating axon to its target, as well as morphological effects on dendrites that could affect learning and memory if the drugs reach the brain.

  1. Conjugation in S. pombe: identification of a microtubule-organising centre, a requirement for microtubules and a role for Mad2.

    Science.gov (United States)

    Petersen, J; Heitz, M J; Hagan, I M

    1998-08-27

    During the G1 phase of the cell cycle, cells of the fission yeast Schizosaccharomyces pombe can be induced to mate by nitrogen starvation and the presence of mating pheromones. Polarised growth towards cells of the opposite mating type (P or M) leads to the formation of a projection tip and, upon contact, localised cell wall degradation results in conjugation and cell fusion [1]. Here, we have investigated the role of microtubules in this process. We describe a previously unidentified microtubule-organising centre (MTOC) that forms at projection tips upon cell-to-cell contact, before cells fuse. Treatment of mating cells with the microtubule-destabilising drug thiabendazole (TBZ) showed that microtubule integrity was required for mating at two distinct stages: during projection tip formation and cell fusion. Projection tip formation requires filamentous (F) actin function [2] and microtubules are required for the localisation of F actin to the projection tip. We also identify a role during mating for Mad2--a mitotic checkpoint protein that is required in all eukaryotes to maintain the mitotic state in response to microtubule depolymerisation [3]. S. pombe mad2 mutant cells were compromised in their ability to mate upon removal of TBZ, indicating that in fission yeast, in the absence of microtubules, Mad2 is also required to maintain mating competence.

  2. Dependency of microtubule-associated proteins (MAPs) for tubulin stability and assembly; use of estramustine phosphate in the study of microtubules.

    Science.gov (United States)

    Fridén, B; Wallin, M

    1991-07-10

    Microtubule-associated proteins (MAPs) were separated from tubulin with several different methods. The ability of the isolated MAPs to reinduce assembly of phosphocellulose purified tubulin differed markedly between the different methods. MAPs isolated by addition of 0.35 M NaCl to taxol-stabilized microtubules stimulated tubulin assembly most effectively, while addition of 0.6 M NaCl produced MAPs with a substantially lower ability to stimulate tubulin assembly. The second best preparation was achieved with phosphocellulose chromatographic separation of MAPs with 0.6 M NaCl elution. The addition of estramustine phosphate to microtubules reconstituted of MAPs prepared by 0.35 M NaCl or phosphocellulose chromatography, induced less disassembly than for microtubules assembled from unseparated proteins, and was almost without effect on microtubules reconstituted from MAPs prepared by taxol and 0.6 M NaCl. Estramustine phosphate binds to the tubulin binding part of the MAPs, and the results do therefore indicate that the MAPs are altered by the separation methods. Since the MAPs are regarded as highly stable molecules, one probable alteration could be aggregation of the MAPs, as also indicated by the results. The purified tubulin itself seemed not to be affected by the phosphocellulose purification, since the microtubule proteins were unchanged by the low buffer strenght used during the cromatography. However, the assembly competence after a prolonged incubation of the microtubule proteins at 4 degrees C was dependent on intact bindings between the tubulin and MAPs.

  3. Movement and Coordination

    Science.gov (United States)

    ... Español Text Size Email Print Share Movement and Coordination Page Content Article Body At this age, your ... level will strengthen his body and develop his coordination. In the months ahead, your child’s running will ...

  4. Developmental coordination disorder

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/article/001533.htm Developmental coordination disorder To use the sharing features on this page, please enable JavaScript. Developmental coordination disorder is a childhood disorder. It leads to ...

  5. Influence of Anion and Mole Ratio on the Coordination Behavior of an NO2S3-Macrocycle: The Formation of a Dumbbell-Shaped Macrocyclic Cadmium(II) Iodide Complex.

    Science.gov (United States)

    Lee, Hyeong-Hwan; Lee, Eunji; Ju, Huiyeong; Kim, Seulgi; Park, In-Hyeok; Lee, Shim Sung

    2016-03-01

    Anion and mole ratio dependent formations of cadmium(II) complexes with an NO2S3-macrocycle (L) incorporating a pyridine subunit are reported. When the cadmium(II) salts (1-10 equiv) with different halide ions (Br(-) or I(-)) were reacted with L, CdBr2 afforded a monomer complex, [Cd(L)Br]2[Cd2Br6]·CH2Cl2 (1), with three separated parts in the whole mole ratio range: two 1:1 stoichiometric complex cation parts and one Cd2Br6 cluster anion part. After separation of 1 by filtration, [Cd(L)Br]2[CdBr4]·CH2Cl2 (2) with similar composition was afforded, except the cluster was isolated from the filtrate. Unlike the CdBr2 complexation, CdI2 afforded the mole ratio dependent products (3-5). Below 2.5 equiv of CdI2, [Cd(L)I]2[CdI4]·CH2Cl2 (3) and [Cd(L)I]2[Cd2I6]·0.5CH2Cl2 (4) with different cadmium(II) iodide clusters were isolated as kinetic (3) and a thermodynamic (4) products. Notably, the use of 3 equiv or above amount of CdI2 gave a dumbbell-shaped complex, {[Cd(L)]2(μ-Cd4I12)} (5), in which two mononuclear macrocyclic complex units are linked by a (μ-Cd4I12)(4-) cluster. To monitor the mole ratio dependency as well as their reactivities, the systematic powder X-ray diffraction (PXRD) analysis has also been applied.

  6. A microtubule interactome: complexes with roles in cell cycle and mitosis.

    Directory of Open Access Journals (Sweden)

    Julian R Hughes

    2008-04-01

    Full Text Available The microtubule (MT cytoskeleton is required for many aspects of cell function, including the transport of intracellular materials, the maintenance of cell polarity, and the regulation of mitosis. These functions are coordinated by MT-associated proteins (MAPs, which work in concert with each other, binding MTs and altering their properties. We have used a MT cosedimentation assay, combined with 1D and 2D PAGE and mass spectrometry, to identify over 250 MAPs from early Drosophila embryos. We have taken two complementary approaches to analyse the cellular function of novel MAPs isolated using this approach. First, we have carried out an RNA interference (RNAi screen, identifying 21 previously uncharacterised genes involved in MT organisation. Second, we have undertaken a bioinformatics analysis based on binary protein interaction data to produce putative interaction networks of MAPs. By combining both approaches, we have identified and validated MAP complexes with potentially important roles in cell cycle regulation and mitosis. This study therefore demonstrates that biologically relevant data can be harvested using such a multidisciplinary approach, and identifies new MAPs, many of which appear to be important in cell division.

  7. Coordination and Cooperation

    OpenAIRE

    Janssen, Maarten

    2003-01-01

    textabstractThis comment makes four related points. First, explaining coordination is different from explaining cooperation. Second, solving the coordination problem is more important for the theory of games than solving the cooperation problem. Third, a version of the Principle of Coordination can be rationalized on individualistic grounds. Finally, psychological game theory should consider how players perceive their gaming situation. ---------------------------------------------------------...

  8. Processing Coordination Ambiguity

    Science.gov (United States)

    Engelhardt, Paul E.; Ferreira, Fernanda

    2010-01-01

    We examined temporarily ambiguous coordination structures such as "put the butter in the bowl and the pan on the towel." Minimal Attachment predicts that the ambiguous noun phrase "the pan" will be interpreted as a noun-phrase coordination structure because it is syntactically simpler than clausal coordination. Constraint-based theories assume…

  9. Processing Coordination Ambiguity

    Science.gov (United States)

    Engelhardt, Paul E.; Ferreira, Fernanda

    2010-01-01

    We examined temporarily ambiguous coordination structures such as "put the butter in the bowl and the pan on the towel." Minimal Attachment predicts that the ambiguous noun phrase "the pan" will be interpreted as a noun-phrase coordination structure because it is syntactically simpler than clausal coordination. Constraint-based…

  10. Coordination and Cooperation

    NARCIS (Netherlands)

    M.C.W. Janssen (Maarten)

    2003-01-01

    textabstractThis comment makes four related points. First, explaining coordination is different from explaining cooperation. Second, solving the coordination problem is more important for the theory of games than solving the cooperation problem. Third, a version of the Principle of Coordination can

  11. Coordination and Cooperation

    NARCIS (Netherlands)

    M.C.W. Janssen (Maarten)

    2003-01-01

    textabstractThis comment makes four related points. First, explaining coordination is different from explaining cooperation. Second, solving the coordination problem is more important for the theory of games than solving the cooperation problem. Third, a version of the Principle of Coordination can

  12. 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.

  13. Connections between microtubules and endoplasmic reticulum in mitotic spindle

    Directory of Open Access Journals (Sweden)

    J. A. Tarkowska

    2015-01-01

    Full Text Available Dividing endosperm cells of Haemanthus katherinae Bak. were treated with an 0.025 per cent aqueous solution of an oleander glycosides mixture which produces severe disturtaances in the mitotic spindle and high hypertrophy of the endoplasmic reticulum (ER in the whole cells. There appear between the kinetochore microtubules (MTs numerous elongated and narrow ER cisterns, particularly well visible when the number of kinetochore MTs is reduced. Both these structures (MTs and ER are frequently connected by cross-bridges. The presumable role of these connections is discused.

  14. Buckling of microtubules: An insight by molecular and continuum mechanics

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Jin; Meguid, S. A., E-mail: meguid@mie.utoronto.ca [Mechanics and Aerospace Design Laboratory, Department of Mechanical and Industrial Engineering, University of Toronto, Toronto, Ontario M5S 3G8 (Canada)

    2014-10-27

    The molecular structural mechanics method has been extended to investigate the buckling of microtubules (MTs) with various configurations. The results indicate that for relative short MTs the shear deformation effect, rather than the nonlocal effect, is mainly responsible for the limitation of their widely used Euler beam description and the observed length-dependence of their bending stiffness. In addition, the configuration effect of MTs is also studied and considered as an explanation for the large scattering of the critical buckling force and bending stiffness observed in existing experiments. This configuration effect is also found to mainly originate from the geometry of the MTs and is mainly determined by the protofilament number.

  15. Pseudo-Spin Model for the Cytoskeletal Microtubule Surface

    Institute of Scientific and Technical Information of China (English)

    CHEN Ying; QIU Xi-Jun; LI Ru-Xin

    2004-01-01

    @@ Due to the inherent symmetry structures and the electric properties in the microtubule (MT), we treat the MT as a one-dimensional ferroelectric system and describe the nonlinear dynamics of dimer electric dipoles in one protofilament of the MT by virtue of the double-well potential. Consequently, the physical problem has been mapped onto the pseudo-spin system, and the mean-field approximation has been taken to obtain some physical results, including the expression for the phase transition temperature Tc and the estimated value of Tc (≈ 312 K).

  16. Biochemical characterization of tektins from sperm flagellar doublet microtubules

    OpenAIRE

    1987-01-01

    Tektins, protein components of stable protofilaments from sea urchin sperm flagellar outer doublet microtubules (Linck, R. W., and G. L. Langevin, 1982, J. Cell Sci., 58:1-22), are separable by preparative SDS PAGE into 47-, 51-, and 55-kD equimolar components. High resolution two-dimensional tryptic peptide mapping reveals 63-67% coincidence among peptides of the 51-kD tektin chain and its 47- and 55-kD counterparts, greater than 70% coincidence between the 47- and 55-kD tektins, but little ...

  17. Adsorption Kinetics in Nanoscale Porous Coordination Polymers

    Energy Technology Data Exchange (ETDEWEB)

    Nune, Satish K.; Thallapally, Praveen K.; McGrail, Benard Peter; Annapureddy, Harsha V. R.; Dang, Liem X.; Mei, Donghai; Karri, Naveen; Alvine, Kyle J.; Olszta, Matthew J.; Arey, Bruce W.; Dohnalkova, Alice

    2015-10-07

    Nanoscale porous coordination polymers were synthesized using simple wet chemical method. The effect of various polymer surfactants on colloidal stability and shape selectivity was investigated. Our results suggest that the nanoparticles exhibited significantly improved adsorption kinetics compared to bulk crystals due to decreased diffusion path lengths and preferred crystal plane interaction.

  18. Teenagers’ Shape

    Institute of Scientific and Technical Information of China (English)

    亚玲

    2007-01-01

    <正>Teenagers have been of a new shape these days. They are about 20 pounds heavier than teenagers were 60 years ago. They are about four inches taller, too. These facts come from J. M. Tanner, a professor in England.

  19. Motor-mediated bidirectional transport along an antipolar microtubule bundle: a mathematical model.

    Science.gov (United States)

    Lin, Congping; Ashwin, Peter; Steinberg, Gero

    2013-05-01

    Long-distance bidirectional transport of organelles depends on the coordinated motion of various motor proteins on the cytoskeleton. Recent quantitative live cell imaging in the elongated hyphal cells of Ustilago maydis has demonstrated that long-range motility of motors and their endosomal cargo occurs on unipolar microtubules (MTs) near the extremities of the cell. These MTs are bundled into antipolar bundles within the central part of the cell. Dynein and kinesin-3 motors coordinate their activity to move early endosomes (EEs) in a bidirectional fashion where dynein drives motility towards MT minus ends and kinesin towards MT plus ends. Although this means that one can easily assign the drivers of bidirectional motion in the unipolar section, the bipolar orientations in the bundle mean that it is possible for either motor to drive motion in either direction. In this paper we use a multilane asymmetric simple exclusion process modeling approach to simulate and investigate phases of bidirectional motility in a minimal model of an antipolar MT bundle. In our model, EE cargos (particles) change direction on each MT with a turning rate Ω and there is switching between MTs in the bundle at the minus ends. At these ends, particles can hop between MTs with rate q(1) on passing from a unipolar to a bipolar section (the obstacle-induced switching rate) or q(2) on passing in the other direction (the end-induced switching rate). By a combination of numerical simulations and mean-field approximations, we investigate the distribution of particles along the MTs for different values of these parameters and of Θ, the overall density of particles within this closed system. We find that even if Θ is low, the system can exhibit a variety of phases with shocks in the density profiles near plus and minus ends caused by queuing of particles. We discuss how the parameters influence the type of particle that dominates active transport in the bundle.

  20. Motor-mediated bidirectional transport along an antipolar microtubule bundle: A mathematical model

    Science.gov (United States)

    Lin, Congping; Ashwin, Peter; Steinberg, Gero

    2013-05-01

    Long-distance bidirectional transport of organelles depends on the coordinated motion of various motor proteins on the cytoskeleton. Recent quantitative live cell imaging in the elongated hyphal cells of Ustilago maydis has demonstrated that long-range motility of motors and their endosomal cargo occurs on unipolar microtubules (MTs) near the extremities of the cell. These MTs are bundled into antipolar bundles within the central part of the cell. Dynein and kinesin-3 motors coordinate their activity to move early endosomes (EEs) in a bidirectional fashion where dynein drives motility towards MT minus ends and kinesin towards MT plus ends. Although this means that one can easily assign the drivers of bidirectional motion in the unipolar section, the bipolar orientations in the bundle mean that it is possible for either motor to drive motion in either direction. In this paper we use a multilane asymmetric simple exclusion process modeling approach to simulate and investigate phases of bidirectional motility in a minimal model of an antipolar MT bundle. In our model, EE cargos (particles) change direction on each MT with a turning rate Ω and there is switching between MTs in the bundle at the minus ends. At these ends, particles can hop between MTs with rate q1 on passing from a unipolar to a bipolar section (the obstacle-induced switching rate) or q2 on passing in the other direction (the end-induced switching rate). By a combination of numerical simulations and mean-field approximations, we investigate the distribution of particles along the MTs for different values of these parameters and of Θ, the overall density of particles within this closed system. We find that even if Θ is low, the system can exhibit a variety of phases with shocks in the density profiles near plus and minus ends caused by queuing of particles. We discuss how the parameters influence the type of particle that dominates active transport in the bundle.

  1. Analysis of Soybean Microtubule Persistence Length; New Evidence on the Correlation between Structural Composition and Mechanical Properties

    Science.gov (United States)

    Shojania Feizabadi, Mitra; Winton, Carly; Barrientos, Jimmy

    2012-02-01

    Recent studies on microtubules composed of different β tubulin isotypes indicate their different functionality in terms of their dynamical behavior or the mechanism of their interaction with chemotherapeutic drugs. Along these lines, the result of our recent study measuring the rigidity of neural and non-neural samples of microtubules with different β tubulin isotype compositions suggests that the distinguished mechanical properties of microtubules, such as rigidity, may also be associated with the different distribution of their β tubulin isotypes. In our current study, we have reported the persistence length of a single soybean microtubule. This plant microtubule has a structural composition different from that of mammalian microtubules. Under the same experimental methods of measurement, the soybean microtubules showed a different persistence length as compared to the value of the persistence length that we estimated in the study of both single Bovine Brain and MCF7 microtubules.

  2. 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

    chTOG is a conserved microtubule polymerase that catalyses the addition of tubulin dimers to promote microtubule growth. chTOG interacts with TACC3, a member of the transforming acidic coiled-coil (TACC) family. Here we analyse their association using the Xenopus homologues, XTACC3 (TACC3) and XM...

  3. Small scale effects on the mechanical behaviors of protein microtubules based on the nonlocal elasticity theory

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Yuanwen, E-mail: ywgao@lzu.edu.cn [Key Laboratory of Mechanics on Western Disaster and Environment, Ministry of Education, Department of Mechanics and Engineering Science, College of Civil Engineering and Mechanics, Lanzhou University, Lanzhou 730000 (China); Lei, Fang-Ming [Key Laboratory of Mechanics on Western Disaster and Environment, Ministry of Education, Department of Mechanics and Engineering Science, College of Civil Engineering and Mechanics, Lanzhou University, Lanzhou 730000 (China)

    2009-09-25

    Based on the nonlocal elastic theory, small scale effects are considered in the investigation of the mechanical properties of protein microtubules. A new prediction formula for the persistence lengths of microtubules with the consideration of the small scale effect is presented. Subsequently, the buckling of microtubules is studied based on a nonlocal elastic beam model. The predicted results of our model indicate that the length-dependence of persistence length is related not only to the shear terms, but also to the small scale effect. The Eular beam model, which is always considered unable to explain the length-dependence of microtubules, can capture the length-dependence of the persistence length of microtubules with the consideration of the small scale effect. The elastic buckling behaviors of microtubules in viscoelastic surrounding cytoplasm are also considered using the nonlocal Timoshenko beam model in this paper, and the results indicate that the small scale effect of microtubules also plays an important role in the buckling of microtubules.

  4. Termination of Protofilament Elongation by Eribulin Induces Lattice Defects that Promote Microtubule Catastrophes

    NARCIS (Netherlands)

    Doodhi, Harinath; Prota, Andrea E; Rodríguez-García, Ruddi; Xiao, Hui; Custar, Daniel W; Bargsten, Katja; Katrukha, Eugene A; Hilbert, Manuel; Hua, Shasha; Jiang, Kai; Grigoriev, Ilya; Yang, Chia-Ping H; Cox, David; Horwitz, Susan Band; Kapitein, Lukas C; Akhmanova, Anna; Steinmetz, Michel O

    2016-01-01

    Microtubules are dynamic polymers built of tubulin dimers that attach in a head-to-tail fashion to form protofilaments, which further associate laterally to form a tube. Asynchronous elongation of individual protofilaments can potentially lead to an altered microtubule-end structure that promotes su

  5. Termination of Protofilament Elongation by Eribulin Induces Lattice Defects that Promote Microtubule Catastrophes.

    Science.gov (United States)

    Doodhi, Harinath; Prota, Andrea E; Rodríguez-García, Ruddi; Xiao, Hui; Custar, Daniel W; Bargsten, Katja; Katrukha, Eugene A; Hilbert, Manuel; Hua, Shasha; Jiang, Kai; Grigoriev, Ilya; Yang, Chia-Ping H; Cox, David; Horwitz, Susan Band; Kapitein, Lukas C; Akhmanova, Anna; Steinmetz, Michel O

    2016-07-11

    Microtubules are dynamic polymers built of tubulin dimers that attach in a head-to-tail fashion to form protofilaments, which further associate laterally to form a tube. Asynchronous elongation of individual protofilaments can potentially lead to an altered microtubule-end structure that promotes sudden depolymerization, termed catastrophe [1-4]. However, how the dynamics of individual protofilaments relates to overall growth persistence has remained unclear. Here, we used the microtubule targeting anti-cancer drug Eribulin [5-7] to explore the consequences of stalled protofilament elongation on microtubule growth. Using X-ray crystallography, we first revealed that Eribulin binds to a site on β-tubulin that is required for protofilament plus-end elongation. Based on the structural information, we engineered a fluorescent Eribulin molecule. We demonstrate that single Eribulin molecules specifically interact with microtubule plus ends and are sufficient to either trigger a catastrophe or induce slow and erratic microtubule growth in the presence of EB3. Interestingly, we found that Eribulin increases the frequency of EB3 comet "splitting," transient events where a slow and erratically progressing comet is followed by a faster comet. This observation possibly reflects the "healing" of a microtubule lattice. Because EB3 comet splitting was also observed in control microtubules in the absence of any drugs, we propose that Eribulin amplifies a natural pathway toward catastrophe by promoting the arrest of protofilament elongation.

  6. Kinetochore-microtubule attachment is sufficient to satisfy the human spindle assembly checkpoint.

    Science.gov (United States)

    Etemad, Banafsheh; Kuijt, Timo E F; Kops, Geert J P L

    2015-12-01

    The spindle assembly checkpoint (SAC) is a genome surveillance mechanism that protects against aneuploidization. Despite profound progress on understanding mechanisms of its activation, it remains unknown what aspect of chromosome-spindle interactions is monitored by the SAC: kinetochore-microtubule attachment or the force generated by dynamic microtubules that signals stable biorientation of chromosomes? To answer this, we uncoupled these two processes by expressing a non-phosphorylatable version of the main microtubule-binding protein at kinetochores (HEC1-9A), causing stabilization of incorrect kinetochore-microtubule attachments despite persistent activity of the error-correction machinery. The SAC is fully functional in HEC1-9A-expressing cells, yet cells in which chromosomes cannot biorient but are stably attached to microtubules satisfy the SAC and exit mitosis. SAC satisfaction requires neither intra-kinetochore stretching nor dynamic microtubules. Our findings support the hypothesis that in human cells the end-on interactions of microtubules with kinetochores are sufficient to satisfy the SAC without the need for microtubule-based pulling forces.

  7. Nonlocal shear deformable shell model for bending buckling of microtubules embedded in an elastic medium

    Energy Technology Data Exchange (ETDEWEB)

    Shen Huishen, E-mail: hsshen@mail.sjtu.edu.c [Department of Engineering Mechanics, Shanghai Jiao Tong University, Shanghai 200030 (China); State Key Laboratory of Ocean Engineering, Shanghai Jiao Tong University, Shanghai 200030 (China)

    2010-08-30

    A nonlocal shear deformable shell model is developed for buckling of microtubules embedded in an elastic matrix of cytoplasm under bending in thermal environments. The results reveal that the lateral constraint has a significant effect on the buckling moments of a microtubule when the foundation stiffness is sufficiently large.

  8. Microtubule drugs: action, selectivity, and resistance across the kingdoms of life.

    Science.gov (United States)

    Dostál, V; Libusová, L

    2014-09-01

    Microtubule drugs such as paclitaxel, colchicine, vinblastine, trifluralin, or oryzalin form a chemically diverse group that has been reinforced by a large number of novel compounds over time. They all share the ability to change microtubule properties. The profound effects of disrupted microtubule systems on cell physiology can be used in research as well as anticancer treatment and agricultural weed control. The activity of microtubule drugs generally depends on their binding to α- and β-tubulin subunits. The microtubule drugs are often effective only in certain taxonomic groups, while other organisms remain resistant. Available information on the molecular basis of this selectivity is summarized. In addition to reviewing published data, we performed sequence data mining, searching for kingdom-specific signatures in plant, animal, fungal, and protozoan tubulin sequences. Our findings clearly correlate with known microtubule drug resistance determinants and add more amino acid positions with a putative effect on drug-tubulin interaction. The issue of microtubule network properties in plant cells producing microtubule drugs is also addressed.

  9. 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.

  10. The XMAP215-family protein DdCP224 is required for cortical interactions of microtubules

    Directory of Open Access Journals (Sweden)

    Hestermann Andrea

    2004-06-01

    Full Text Available Abstract Background Interactions of peripheral microtubule tips with the cell cortex are of crucial importance for nuclear migration, spindle orientation, centrosome positioning and directional cell movement. Microtubule plus end binding proteins are thought to mediate interactions of microtubule tips with cortical actin and membrane proteins in a dynein-dependent manner. XMAP215-family proteins are main regulators of microtubule plus end dynamics but so far they have not been implicated in the interactions of microtubule tips with the cell cortex. Results Here we show that overexpression of an N-terminal fragment of DdCP224, the Dictyostelium XMAP215 homologue, caused a collapse of the radial microtubule cytoskeleton, whereby microtubules lost contact with the cell cortex and were dragged behind like a comet tail of an unusually motile centrosome. This phenotype was indistinguishable from mutants overexpressing fragments of the dynein heavy chain or intermediate chain. Moreover, it was accompanied by dispersal of the Golgi apparatus and reduced cortical localization of the dynein heavy chain indicating a disrupted dynein/dynactin interaction. The interference of DdCP224 with cortical dynein function is strongly supported by the observations that DdCP224 and its N-terminal fragment colocalize with dynein and coimmunoprecipitate with dynein and dynactin. Conclusions Our data show that XMAP215-like proteins are required for the interaction of microtubule plus ends with the cell cortex in interphase cells and strongly suggest that this function is mediated by dynein.

  11. Encoding the microtubule structure: Allosteric interactions between the microtubule +TIP complex master regulators and TOG-domain proteins

    Science.gov (United States)

    Grimaldi, Ashley D; Zanic, Marija; Kaverina, Irina

    2015-01-01

    Since their initial discovery, the intriguing proteins of the +TIP network have been the focus of intense investigation. Although many of the individual +TIP functions have been revealed, the capacity for +TIP proteins to regulate each other has not been widely addressed. Importantly, recent studies involving EBs, the master regulators of the +TIP complex, and several TOG-domain proteins have uncovered a novel mechanism of mutual +TIP regulation: allosteric interactions through changes in microtubule structure. These findings have added another level of complexity to the existing evidence on +TIP regulation and highlight the cooperative nature of the +TIP protein network. PMID:25895033

  12. Monte Carlo Simulation on Coordinated Movement of Kinesin and Dynein Motors

    Institute of Scientific and Technical Information of China (English)

    WANG Hong; DOU Shuo-Xing; WANG Peng-Ye

    2008-01-01

    Kinesin and dynein are two important classes of molecular motors which are responsible for active organelle trafficking and cell division.They call work together to carry a cargo,moving along the microtubule in a coordinated way.We use Monte Carlo method to simulate the dynamics of this coordinated movement.Based on four essential assumptions,our simulations reproduce some features of the recent in vivo experiments.The fast moving speed of the cargo js simulated and the speed distribution is presented.

  13. Interpolation of microtubules into cortical arrays during cell elongation and differentiation in roots of Azolla pinnata.

    Science.gov (United States)

    Hardham, A R; Gunning, B E

    1979-06-01

    Longitudinal sections of roots of Azolla pinnata R. Br. were prepared for electron microscopy so that cortical microtubules could be counted along the longitudinal walls in cell files in the endodermis, pericycle, and inner and outer cortex, and in sieve and xylem elements. With the exception of the xylem, where there are no transverse cell divisions, each file of cells commences with its initial cell and then possesses a zone of concomitant cell expansion and transverse cell division, followed, after completion of the divisions, by a zone of terminal cell differentiation. The cells augment their population of cortical microtubules as they elongate and divide, showing a net increase of up to 0.6 micron of polymerized microtubule length per min. Two main sub-processes were found: (i) When a longitudinal wall is first formed it is supplied with a higher number of microtubules per unit length of wall than it will have later, when it is being expanded. This initial quota becomes diluted as the second sub-process commences. (ii) The cells interpolate new microtubules at a rate which is characteristic of the cell, and, in the endodermis, of the face of the cell, while the cell elongates. Most cell types thus maintain a set density of cortical microtubules while they elongate and divide. Comparisons of endodermal cells in untreated controls, and roots that had been treated with colchicine, low temperature, or high pressure indicate that the initial quota of microtubules, and the later interpolations, and differentially sensitive to microtuble perturbations. Three types of behaviour, all related to changes in the cell walls, were noted as cortex, xylem and sieve element cells entered their respective phases of cell differentiation. The cortical cells expanded in all dimensions, and the interpolation of microtubules diminished or ceased. The sieve elements continued to elongate, and interpolated at a high rate, reaching unusually high densities of microtubules when the cell

  14. Capu and Spire assemble a cytoplasmic actin mesh that maintains microtubule organization in the Drosophila oocyte.

    Science.gov (United States)

    Dahlgaard, Katja; Raposo, Alexandre A S F; Niccoli, Teresa; St Johnston, Daniel

    2007-10-01

    Mutants in the actin nucleators Cappuccino and Spire disrupt the polarized microtubule network in the Drosophila oocyte that defines the anterior-posterior axis, suggesting that microtubule organization depends on actin. Here, we show that Cappuccino and Spire organize an isotropic mesh of actin filaments in the oocyte cytoplasm. capu and spire mutants lack this mesh, whereas overexpressed truncated Cappuccino stabilizes the mesh in the presence of Latrunculin A and partially rescues spire mutants. Spire overexpression cannot rescue capu mutants, but prevents actin mesh disassembly at stage 10B and blocks late cytoplasmic streaming. We also show that the actin mesh regulates microtubules indirectly, by inhibiting kinesin-dependent cytoplasmic flows. Thus, the Capu pathway controls alternative states of the oocyte cytoplasm: when active, it assembles an actin mesh that suppresses kinesin motility to maintain a polarized microtubule cytoskeleton. When inactive, unrestrained kinesin movement generates flows that wash microtubules to the cortex.

  15. Steady-state EB cap size fluctuations are determined by stochastic microtubule growth and maturation.

    Science.gov (United States)

    Rickman, Jamie; Duellberg, Christian; Cade, Nicholas I; Griffin, Lewis D; Surrey, Thomas

    2017-03-28

    Growing microtubules are protected from depolymerization by the presence of a GTP or GDP/Pi cap. End-binding proteins of the EB1 family bind to the stabilizing cap, allowing monitoring of its size in real time. The cap size has been shown to correlate with instantaneous microtubule stability. Here we have quantitatively characterized the properties of cap size fluctuations during steady-state growth and have developed a theory predicting their timescale and amplitude from the kinetics of microtubule growth and cap maturation. In contrast to growth speed fluctuations, cap size fluctuations show a characteristic timescale, which is defined by the lifetime of the cap sites. Growth fluctuations affect the amplitude of cap size fluctuations; however, cap size does not affect growth speed, indicating that microtubules are far from instability during most of their time of growth. Our theory provides the basis for a quantitative understanding of microtubule stability fluctuations during steady-state growth.

  16. Antiproliferative Activity of Crocin Involves Targeting of Microtubules in Breast Cancer Cells

    Science.gov (United States)

    Hire, Rupali R.; Srivastava, Shalini; Davis, Melissa B.; Kumar Konreddy, Ananda; Panda, Dulal

    2017-01-01

    Crocin, a component of saffron spice, is known to have an anticancer activity. However, the targets of crocin are not known. In this study, crocin was found to inhibit the proliferation of HCC70, HCC1806, HeLa and CCD1059sk cells by targeting microtubules. Crocin depolymerized both the interphase and mitotic microtubules of different cancer cells, inhibited mitosis and induced multipolar spindle formation in these cells. In vitro, crocin inhibited the assembly of pure tubulin as well as the assembly of microtubule-associated protein rich tubulin. Electron microscopic analysis showed that crocin inhibited microtubule assembly while it induced aggregation of tubulin at higher concentrations. Crocin co-eluted with tubulin suggesting that it binds to tubulin. Vinblastine inhibited the binding of crocin to tubulin while podophyllotoxin did not inhibit the crocin binding indicating that crocin binds at the vinblastine site on tubulin. The results suggested that crocin inhibited cell proliferation mainly by disrupting the microtubule network. PMID:28337976

  17. Multi-modal microtubule binding by the Ndc80 kinetochore complex

    Science.gov (United States)

    Alushin, Gregory M.; Musinipally, Vivek; Matson, Daniel; Tooley, John; Stukenberg, P. Todd; Nogales, Eva

    2012-01-01

    Summary The Ndc80 complex is a key site of kinetochore-microtubule attachment during cell division. The human complex engages microtubules with a globular “head” formed by tandem calponin-homology domains and an 80 amino-acid unstructured “tail” that contains sites of phospho-regulation by the Aurora B kinase. Using biochemical, cell biological, and electron microscopy analyses, we have dissected the tail’s roles in microtubule binding and mediating cooperative interactions between Ndc80 complexes. Two segments of the tail that contain Aurora B sites become ordered at interfaces; one with tubulin and the second with an adjacent Ndc80 head on the microtubule surface, forming interactions which are disrupted by phosphorylation. We propose a model in which Ndc80’s interaction with either growing or shrinking microtubule ends can be tuned by the phosphorylation state of its tail. PMID:23085714

  18. Specific polar subpopulations of astral microtubules control spindle orientation and symmetric neural stem cell division.

    Science.gov (United States)

    Mora-Bermúdez, Felipe; Matsuzaki, Fumio; Huttner, Wieland B

    2014-07-04

    Mitotic spindle orientation is crucial for symmetric vs asymmetric cell division and depends on astral microtubules. Here, we show that distinct subpopulations of astral microtubules exist, which have differential functions in regulating spindle orientation and division symmetry. Specifically, in polarized stem cells of developing mouse neocortex, astral microtubules reaching the apical and basal cell cortex, but not those reaching the central cell cortex, are more abundant in symmetrically than asymmetrically dividing cells and reduce spindle orientation variability. This promotes symmetric divisions by maintaining an apico-basal cleavage plane. The greater abundance of apical/basal astrals depends on a higher concentration, at the basal cell cortex, of LGN, a known spindle-cell cortex linker. Furthermore, newly developed specific microtubule perturbations that selectively decrease apical/basal astrals recapitulate the symmetric-to-asymmetric division switch and suffice to increase neurogenesis in vivo. Thus, our study identifies a novel link between cell polarity, astral microtubules, and spindle orientation in morphogenesis.

  19. Contributions of microtubule dynamic instability and rotational diffusion to kinetochore capture

    CERN Document Server

    Blackwell, Robert; Edelmaier, Christopher; Gergely, Zachary R; Flynn, Patrick J; Montes, Salvador; Crapo, Ammon; Doostan, Alireza; McIntosh, J Richard; Glaser, Matthew A; Betterton, Meredith D

    2016-01-01

    Microtubule dynamic instability allows search and capture of kinetochores during spindle formation, an important process for accurate chromosome segregation during cell division. Recent work has found that microtubule rotational diffusion about minus-end attachment points contributes to kinetochore capture in fission yeast, but the relative contributions of dynamic instability and rotational diffusion are not well understood. We have developed a biophysical model of kinetochore capture in small fission-yeast nuclei using hybrid Brownian dynamics/kinetic Monte Carlo simulation techniques. With this model, we have studied the importance of dynamic instability and microtubule rotational diffusion for kinetochore capture, both to the lateral surface of a microtubule and at or near its end. Over a range of biologically relevant parameters, microtubule rotational diffusion decreased capture time, but made a relatively small contribution compared to dynamic instability. At most, rotational diffusion reduced capture ...

  20. Light-microscopic observations of individual microtubules reconstituted from brain tubulin.

    Science.gov (United States)

    Kuriyama, R; Miki-Noumura, T

    1975-12-01

    The course of polymerization of individual brain microtubules could be observed with a light microscope employing dark-field illumination. Statistical analysis of the increase in microtubule length during the polymerization was in accordance with the time course of viscosity change of the tubulin solution. After a plateau level in viscosity was attained, there was no significant change in histograms showing length distribution. These observations were confirmed with fixed and stained microtubules, using a phase-contrast microscope. Observations with dark-field illumination revealed that reconstituted microtubules depolymerized and disappeared immediately upon exposure to buffer containing CaCl2 or sulphydryl reagents such as p-chloromercuriphenyl sulphonic acid (PCMPS) and p-chloromercuribenzoic acid (PCMB). They were also cold-labile. The growth of heterogeneous microtubules which were assembled by mixing purified tubulin dimers with ciliary outer fibres could also be followed with these optical systems.

  1. Thymoquinone disrupts the microtubule dynamics in fission yeast Schizosaccharomyces pombe

    Directory of Open Access Journals (Sweden)

    Nusrat Masood

    2016-11-01

    Full Text Available Mad2 deletion strain of Schizosaccharomyces pombe was found to be sensitive to thymoquinone, a signature molecule present in Nigella sativa in a dose-dependent manner. Mad2 protein is an indispensable part of mitotic spindle checkpoint complex and is required for the cell cycle arrest in response to the spindle defects. Although the expression of α tubulin was not affected in thymoquinone treated cells, but the expression of β-tubulin was reduced. Further, the absence of microtubule in thymoquinone treated cells suggests its involvement in tubulin polymerization. Molecular docking studies revealed that thymoquinone specifically binds to β-tubulin near the Taxotere binding site of Tub1 (Tubulin α-β dimer. These studies additionally showed that thymoquinone interacts with the residues present in chain B, which is an inherent part of Mad2 protein of mitotic checkpoint complex (MCC. We concluded that the thymoquinone disrupts the microtubule polymerization that leads to the requirement of spindle checkpoint protein for the cell survival.

  2. Oxidative stress decreases microtubule growth and stability in ventricular myocytes.

    Science.gov (United States)

    Drum, Benjamin M L; Yuan, Can; Li, Lei; Liu, Qinghang; Wordeman, Linda; Santana, L Fernando

    2016-04-01

    Microtubules (MTs) have many roles in ventricular myocytes, including structural stability, morphological integrity, and protein trafficking. However, despite their functional importance, dynamic MTs had never been visualized in living adult myocytes. Using adeno-associated viral vectors expressing the MT-associated protein plus end binding protein 3 (EB3) tagged with EGFP, we were able to perform live imaging and thus capture and quantify MT dynamics in ventricular myocytes in real time under physiological conditions. Super-resolution nanoscopy revealed that EB1 associated in puncta along the length of MTs in ventricular myocytes. The vast (~80%) majority of MTs grew perpendicular to T-tubules at a rate of 0.06μm∗s(-1) and growth was preferentially (82%) confined to a single sarcomere. Microtubule catastrophe rate was lower near the Z-line than M-line. Hydrogen peroxide increased the rate of catastrophe of MTs ~7-fold, suggesting that oxidative stress destabilizes these structures in ventricular myocytes. We also quantified MT dynamics after myocardial infarction (MI), a pathological condition associated with increased production of reactive oxygen species (ROS). Our data indicate that the catastrophe rate of MTs increases following MI. This contributed to decreased transient outward K(+) currents by decreasing the surface expression of Kv4.2 and Kv4.3 channels after MI. On the basis of these data, we conclude that, under physiological conditions, MT growth is directionally biased and that increased ROS production during MI disrupts MT dynamics, decreasing K(+) channel trafficking.

  3. 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.

  4. Kindlin1 regulates microtubule function to ensure normal mitosis.

    Science.gov (United States)

    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.

  5. Microrheology of single microtubule filaments and synthesized cytoskeletal networks

    Science.gov (United States)

    Koch, Matthias; Rohrbach, Alexander

    2015-03-01

    The ability to sense and respond to external mechanical forces is crucial for cells in many processes such as cell growth and division. Common models on mechanotransduction rely on the conversion of mechanical stimuli to chemical signals in the cell periphery and their translocation by diffusion (passive) or molecular motors (active). These processes are rather slow (~ seconds) and it has been argued that the cytoskeleton itself might be able to transport a mechanical signal within microseconds via stress waves. Microtubules are the stiffest component of the cytoskeleton and thus ideal candidates for this purpose. We study the frequency dependent response of single microtubule filaments and small networks thereof in a bottom-up approach using several (N =2-10) time-multiplexed optical tweezers together with back focal plane interferometry. Small synthesized networks with a defined geometry are constructed using trapped Neutravidin beads as anchor points for biotinylated filaments. The network is then probed by a defined oscillation of one anchor (actor). The frequency dependent response of the remaining beads (sensors) is analyzed experimentally and modeled theoretically over a wide frequency range.

  6. Joint shape segmentation with linear programming

    KAUST Repository

    Huang, Qixing

    2011-01-01

    We present an approach to segmenting shapes in a heterogenous shape database. Our approach segments the shapes jointly, utilizing features from multiple shapes to improve the segmentation of each. The approach is entirely unsupervised and is based on an integer quadratic programming formulation of the joint segmentation problem. The program optimizes over possible segmentations of individual shapes as well as over possible correspondences between segments from multiple shapes. The integer quadratic program is solved via a linear programming relaxation, using a block coordinate descent procedure that makes the optimization feasible for large databases. We evaluate the presented approach on the Princeton segmentation benchmark and show that joint shape segmentation significantly outperforms single-shape segmentation techniques. © 2011 ACM.

  7. The microtubule plus-end-tracking protein CLIP-170 associates with the spermatid manchette and is essential for spermatogenesis.

    NARCIS (Netherlands)

    A.S. Akhmanova (Anna); A.L. Mausset-Bonnefont (Anne-Laure); W.A. van Cappellen (Gert); N. Keijzer (Nanda); C.C. Hoogenraad (Casper); T. Stepanova (Tatiana); K. Drabek (Ksenija); J. van der Wees (Jacqueline); M. Mommaas (Mieke); J. Onderwater (Jos); H. van der Meulen (Hans); M.E. Tanenbaum (Marvin); R.H. Medema (Rene); J.W. Hoogerbrugge (Jos); J.T.M. Vreeburg (Jan); E.J. Uringa; J.A. Grootegoed (Anton); F.G. Grosveld (Frank); N.J. Galjart (Niels)

    2005-01-01

    textabstractCLIP-170 is a microtubule "plus-end-tracking protein" implicated in the control of microtubule dynamics, dynactin localization, and the linking of endosomes to microtubules. To investigate the function of mouse CLIP-170, we generated CLIP-170 knockout and GFP-CLIP-170 knock-in alleles. R

  8. The microtubule plus-end-tracking protein CLIP-170 associates with the spermatid manchette and is essential for spermatogenesis

    NARCIS (Netherlands)

    Akhmanova, A.S.; Mausset-Bonnefont, A.-L.; Cappellen, W. van; Keijzer, N.; Hoogenraad, C.C.; Stepanova, T.; Drabek, K.; Wees, J. van der; Mommaas, M.; Onderwater, J.; Meulen, H. van der; Tanenbaum, M.E.; Medema, R.H.; Hoogerbrugge, J.; Vreeburg, J.; Uringa, E.-J.; Grootegoed, J.A.; Grosveld, F.; Galjart, N.

    2005-01-01

    CLIP-170 is a microtubule "plus-end-tracking protein" implicated in the control of microtubule dynamics, dynactin localization, and the linking of endosomes to microtubules. To investigate the function of mouse CLIP-170, we generated CLIP-170 knockout and GFP-CLIP-170 knock-in alleles. Residual CLIP

  9. A Stochastic Multiscale Model That Explains the Segregation of Axonal Microtubules and Neurofilaments in Neurological Diseases.

    Directory of Open Access Journals (Sweden)

    Chuan Xue

    2015-08-01

    Full Text Available The organization of the axonal cytoskeleton is a key determinant of the normal function of an axon, which is a long thin projection of a neuron. Under normal conditions two axonal cytoskeletal polymers, microtubules and neurofilaments, align longitudinally in axons and are interspersed in axonal cross-sections. However, in many neurotoxic and neurodegenerative disorders, microtubules and neurofilaments segregate apart from each other, with microtubules and membranous organelles clustered centrally and neurofilaments displaced to the periphery. This striking segregation precedes the abnormal and excessive neurofilament accumulation in these diseases, which in turn leads to focal axonal swellings. While neurofilament accumulation suggests an impairment of neurofilament transport along axons, the underlying mechanism of their segregation from microtubules remains poorly understood for over 30 years. To address this question, we developed a stochastic multiscale model for the cross-sectional distribution of microtubules and neurofilaments in axons. The model describes microtubules, neurofilaments and organelles as interacting particles in a 2D cross-section, and is built upon molecular processes that occur on a time scale of seconds or shorter. It incorporates the longitudinal transport of neurofilaments and organelles through this domain by allowing stochastic arrival and departure of these cargoes, and integrates the dynamic interactions of these cargoes with microtubules mediated by molecular motors. Simulations of the model demonstrate that organelles can pull nearby microtubules together, and in the absence of neurofilament transport, this mechanism gradually segregates microtubules from neurofilaments on a time scale of hours, similar to that observed in toxic neuropathies. This suggests that the microtubule-neurofilament segregation can be a consequence of the selective impairment of neurofilament transport. The model generates the

  10. Interaction of an Overexpressed gamma-Tubulin with Microtubules In Vivo and In Vitro.

    Science.gov (United States)

    Kofron, M; Nadezdina, E; Vassilev, A; Matuliene, J; Essner, R; Kato, J; Kuriyama, R

    1998-08-01

    gamma-Tubulin is an ubiquitous MTOC (microtubule-organizing center) component essential for the regulation of microtubule functions. A 1.8 kb cDNA coding for gamma-tubulin was isolated from CHO cells. Analysis of nucleotide sequence predicts a protein of 451 amino acids, which is over 97% identical to human and Xenopus gamma-tubulin. When CHO cells were transiently transfected with the gamma-tubulin clone, epitope-tagged full-length, as well as truncated polypeptides (amino acids 1-398 and 1-340), resulted in the formation of cytoplasmic foci of various sizes. Although one of the foci was identified as the centrosome, the rest of the dots were not associated with any other centrosomal components tested so far. The pattern of microtubule organization was not affected by induction of such gamma-tubulin-containing dots in transfected cells. In addition, the cytoplasmic foci were unable to serve as the site for microtubule regrowth in nocodazole-treated cells upon removal of the drug, suggesting that gamma-tubulin-containing foci were not involved in the activity for microtubule formation and organization. Using the monomeric form of Chlamydomonas gamma-tubulin purified from insect Sf9 cells (), interaction between gamma-tubulin and microtubules was further investigated by immunoelectron microscopy. Microtubules incubated with gamma-tubulin monomers in vitro were associated with more gold particles conjugated with gamma-tubulin than in controls where no exogenous gamma-tubulin was added. However, binding of gamma-tubulin to microtubules was not extensive and was easily lost during sample preparation. Although gamma-tubulin was detected at the minus end of microtubules several times more frequently than the plus end, the majority of gold particles were seen along the microtubule length. These results contradict the previous reports (; ), which might be ascribed to the difference in the level of protein expression in transfected cells.

  11. Phospholipase d activation correlates with microtubule reorganization in living plant cells.

    Science.gov (United States)

    Dhonukshe, Pankaj; Laxalt, Ana M; Goedhart, Joachim; Gadella, Theodorus W J; Munnik, Teun

    2003-11-01

    A phospholipase D (PLD) was shown recently to decorate microtubules in plant cells. Therefore, we used tobacco BY-2 cells expressing the microtubule reporter GFP-MAP4 to test whether PLD activation affects the organization of plant microtubules. Within 30 min of adding n-butanol, a potent activator of PLD, cortical microtubules were released from the plasma membrane and partially depolymerized, as visualized with four-dimensional confocal imaging. The isomers sec- and tert-butanol, which did not activate PLD, did not affect microtubule organization. The effect of treatment on PLD activation was monitored by the in vivo formation of phosphatidylbutanol, a specific reporter of PLD activity. Tobacco cells also were treated with mastoparan, xylanase, NaCl, and hypoosmotic stress as reported activators of PLD. We confirmed the reports and found that all treatments induced microtubule reorganization and PLD activation within the same time frame. PLD still was activated in microtubule-stabilized (taxol) and microtubule-depolymerized (oryzalin) situations, suggesting that PLD activation triggers microtubular reorganization and not vice versa. Exogenously applied water-soluble synthetic phosphatidic acid did not affect the microtubular cytoskeleton. Cell cycle studies revealed that n-butanol influenced not just interphase cortical microtubules but also those in the preprophase band and phragmoplast, but not those in the spindle structure. Cell growth and division were inhibited in the presence of n-butanol, whereas sec- and tert-butanol had no such effects. Using these novel insights, we propose a model for the mechanism by which PLD activation triggers microtubule reorganization in plant cells.

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

    Directory of Open Access Journals (Sweden)

    Joy G Ghosh

    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

  13. Do Environmental Education School Coordinators Have a Mission?

    Science.gov (United States)

    Šimonová, Petra; Cincera, Jan

    2016-01-01

    Teachers who are specialized in environmental education (environmental education school coordinators) can play an important role in empowering students to shape a sustainable future. In this study, the authors examined a group of Czech environmental education school coordinators. The authors aimed to clarify how they interpret their role at their…

  14. Do Environmental Education School Coordinators Have a Mission?

    Science.gov (United States)

    Šimonová, Petra; Cincera, Jan

    2016-01-01

    Teachers who are specialized in environmental education (environmental education school coordinators) can play an important role in empowering students to shape a sustainable future. In this study, the authors examined a group of Czech environmental education school coordinators. The authors aimed to clarify how they interpret their role at their…

  15. Isomorphic coalescence of aster cores formed in vitro from microtubules and kinesin motors

    Science.gov (United States)

    Kim, K.; Sikora, A.; Nakazawa, H.; Umetsu, M.; Hwang, W.; Teizer, W.

    2016-10-01

    We report fluorescence microscopy studies of the formation of aster-like structures emerging from a cellular element-based active system and a novel analysis of the aster condensation. The system consists of rhodamine labeled microtubules which are dynamically coupled by functionalized kinesin motor proteins cross-linked via streptavidin-coated quantum dots (QDs). The aster-shaped objects contain core structures. The cores are aggregates of the QD-motor protein complexes, and result from the dynamic condensation of sub-clusters that are connected to each other randomly. The structural specificity of the aster core reflects a configuration of the initial connectivity between sub-clusters. Detailed image analysis allows us to extract a novel correlation between the condensation speed and the sub-cluster separation. The size of the core is scaled down during the condensation process, following a power law dependence on the distance between sub-clusters. The exponent of the power law is close to two, as expected from a geometric model. This single exponent common to all the contractile lines implies that there exists a time regime during which an isomorphic contraction of the aster core continues during the condensation process. We analyze the observed contraction by using a model system with potential applicability in a wide range of emergent phenomena in randomly coupled active networks, which are prevalent in the cellular environment.

  16. Mitosis-coupled, microtubule-dependent clustering of endosomal vesicles around centrosomes.

    Science.gov (United States)

    Takatsu, Hiroyuki; Katoh, Yohei; Ueda, Tomoko; Waguri, Satoshi; Murayama, Takashi; Takahashi, Senye; Shin, Hye-Won; Nakayama, Kazuhisa

    2013-01-01

    Upon cell division, not only cells themselves but also their organelles undergo drastic shape changes, although the behaviors of organelles other than the Golgi apparatus remain poorly understood. We followed the spatiotemporal changes in the localization of transferrin receptor (TfnR) and other proteins. In early mitotic phases, a population of proteins cycling through the endocytic recycling compartment (ERC) exhibits a distinct spatiotemporal change from that of Golgi proteins. In prophase/prometaphase, when the cell surface-to-volume ratio is reaching its minimum, the ERC proteins are transiently assembled around the centrated centrosome in a microtubule- and dynein-dependent manner, and soon separated polewards into two clusters concomitant with separation of duplicated centrosomes. Electron microscopic analysis revealed that endosomal vesicles containing endocytosed transferrin cluster tightly around centrosomes without fusing with one another. As cytokinesis proceeds, the clusters gradually collapse, and the ERC proteins reassemble around the furrowing equatorial region. FRAP (fluorescence recovery after photobleaching) analyses of EGFP-TfnR-expressing cells revealed minimal membrane exchange between the endosomal clusters and other cellular compartments until anaphase/telophase, when membrane traffic resumes. Our observations indicate that ERC clustering around centrosomes plays a fundamental role in restricting membrane delivery to the plasma membrane during early mitotic phases, when the cell surface-to-volume ratio reaches its minimum.

  17. 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.

  18. FoxO regulates microtubule dynamics and polarity to promote dendrite branching in Drosophila sensory neurons.

    Science.gov (United States)

    Sears, James C; Broihier, Heather T

    2016-10-01

    The size and shape of dendrite arbors are defining features of neurons and critical determinants of neuronal function. The molecular mechanisms establishing arborization patterns during development are not well understood, though properly regulated microtubule (MT) dynamics and polarity are essential. We previously found that FoxO regulates axonal MTs, raising the question of whether it also regulates dendritic MTs and morphology. Here we demonstrate that FoxO promotes dendrite branching in all classes of Drosophila dendritic arborization (da) neurons. FoxO is required both for initiating growth of new branches and for maintaining existing branches. To elucidate FoxO function, we characterized MT organization in both foxO null and overexpressing neurons. We find that FoxO directs MT organization and dynamics in dendrites. Moreover, it is both necessary and sufficient for anterograde MT polymerization, which is known to promote dendrite branching. Lastly, FoxO promotes proper larval nociception, indicating a functional consequence of impaired da neuron morphology in foxO mutants. Together, our results indicate that FoxO regulates dendrite structure and function and suggest that FoxO-mediated pathways control MT dynamics and polarity.

  19. Mechanics of kinetochore microtubules and their interactions with chromosomes during cell division

    Science.gov (United States)

    Nazockdast, Ehssan; Fürthauer, Sebastian; Redemann, Stephanie; Baumgart, Johannes; Lindow, Norbert; Kratz, Andrea; Prohaska, Steffen; Müller-Reichert, Thomas; Shelley, Michael

    2016-11-01

    The accurate segregation of chromosomes, and subsequent cell division, in Eukaryotic cells is achieved by the interactions of an assembly of microtubules (MTs) and motor-proteins, known as the mitotic spindle. We use a combination of our computational platform for simulating cytoskeletal assemblies and our structural data from high-resolution electron tomography of the mitotic spindle, to study the kinetics and mechanics of MTs in the spindle, and their interactions with chromosomes during chromosome segregation in the first cell division in C.elegans embryo. We focus on kinetochore MTs, or KMTs, which have one end attached to a chromosome. KMTs are thought to be a key mechanical component in chromosome segregation. Using exploratory simulations of MT growth, bending, hydrodynamic interactions, and attachment to chromosomes, we propose a mechanical model for KMT-chromosome interactions that reproduces observed KMT length and shape distributions from electron tomography. We find that including detailed hydrodynamic interactions between KMTs is essential for agreement with the experimental observations.

  20. Mammalian SEPT9 isoforms direct microtubule-dependent arrangements of septin core heteromers.

    Science.gov (United States)

    Sellin, Mikael E; Stenmark, Sonja; Gullberg, Martin

    2012-11-01

    Septin-family proteins assemble into rod-shaped heteromeric complexes that form higher-order arrangements at the cell cortex, where they serve apparently conserved functions as diffusion barriers and molecular scaffolds. There are 13 confirmed septin paralogues in mammals, which may be ubiquitous or tissue specific. Septin hetero-oligomerization appears homology subgroup directed, which in turn determines the subunit arrangement of six- to eight-subunit core heteromers. Here we address functional properties of human SEPT9, which, due to variable mRNA splicing, exists as multiple isoforms that differ between tissues. Myeloid K562 cells express three SEPT9 isoforms, all of which have an equal propensity to hetero-oligomerize with SEPT7-containing hexamers to generate octameric heteromers. However, due to limiting amounts of SEPT9, K562 cells contain both hexameric and octameric heteromers. To generate cell lines with controllable hexamer-to-octamer ratios and that express single SEPT9 isoforms, we developed a gene product replacement strategy. By this means we identified SEPT9 isoform-specific properties that either facilitate septin heteromer polymerization along microtubules or modulate the size range of submembranous septin disks-a prevalent septin structure in nonadhered cells. Our findings show that the SEPT9 expression level directs the hexamer-to-octamer ratio, and that the isoform composition and expression level together determine higher-order arrangements of septins.

  1. Positioning of microtubule organizing centers by cortical pushing and pulling forces

    Science.gov (United States)

    Pavin, Nenad; Laan, Liedewij; Ma, Rui; Dogterom, Marileen; Jülicher, Frank

    2012-10-01

    Positioning of microtubule (MT) organizing centers with respect to the confining geometry of cells depends on pushing and/or pulling forces generated by MTs that interact with the cell cortex (Dogterom et al 2005 Curr. Opin. Cell Biol. 17 67-74). How, in living cells, these forces lead to proper positioning is still largely an open question. Recently, it was shown by in vitro experiments using artificial microchambers that in a square geometry, MT asters center more reliably by a combination of pulling and pushing forces than by pushing forces alone (Laan et al 2012a Cell 148 502-14). These findings were explained by a physical description of aster mechanics that includes slipping of pushing MT ends along chamber boundaries. In this paper, we extend that theoretical work by studying the influence of the shape of the confining geometry on the positioning process. We find that pushing and pulling forces can have centering or off-centering behavior in different geometries. Pushing forces center in a one-dimensional and a square geometry, but lead to off-centering in a circle if slipping is sufficiently pronounced. Pulling forces, however, do not center in a one-dimensional geometry, but improve centering in a circle and a square. In an elongated stadium geometry, positioning along the short axis depends mainly on pulling forces, while positioning along the long axis depends mainly on pushing forces. Our theoretical results suggest that different positioning strategies could be used by different cell types.

  2. The cell morphogenesis gene ANGUSTIFOLIA encodes a CtBP/BARS-like protein and is involved in the control of the microtubule cytoskeleton

    OpenAIRE

    Folkers, U; Kirik, V.; Schöbinger, U.; Falk, S; Krishnakumar, S; Pollock, M A; Oppenheimer, D.G.; Day, I.; Reddy, A.R.; Jürgens, G; Hülskamp, M

    2002-01-01

    The ANGUSTIFOLIA (AN) gene is required for leaf hair (trichome) branching and is also involved in polarized expansion underlying organ shape. Here we show that the AN gene encodes a C-terminal binding proteins/brefeldin A ADP-ribosylated substrates (CtBP/BARS) related protein. AN is expressed at low levels in all organs and the AN protein is localized in the cytoplasm. In an mutant trichomes, the organization of the actin cytoskeleton is normal but the distribution of microtubules is aberrant...

  3. Shape shifting

    DEFF Research Database (Denmark)

    Gottlieb, Stefan; Hundebøl, Jesper; Jensen, Jens Stissing;

    2009-01-01

    In this paper we are concerned with the specific effects of a 3D building model produced and reproduced in the planning and construction phases on a particular construction project. We contrast these effects with the policy intentions expressed within a state funded, public initiative that aims...... to promote so-called ‘digital construction' in Denmark. One of the main objectives of this initiative is to ensure better coordination between the different phases of the building project through the application of 3D Building Information Models (BIM). The intentions are to improve the construction phase...... by providing pervasive on-site planning and logistics, where the 3D building model combined with process-data developed and maintained by the individual contractor should facilitate the production of detailed step-by-step production planning in the form of ‘production cards'. The empirical findings from...

  4. Shape Shifting

    DEFF Research Database (Denmark)

    Gottlieb, Stefan; Hundebøl, Jesper; Jensen, Jens Stissing

    2009-01-01

    the case-study reveal a much less pervasive and more coin cidental utilisation of the 3D model than envisioned programmatically. We show how the BIM is introduced into the construction phase in the form of paper based 3D prints - as a supplement only to the existing practices. While this modest utilization......In this paper we are concerned with the specific effects of a 3D building model produced and reproduced in the planning and construction phases on a particular construction project. We contrast these effects with the policy intentions expressed within a state funded, public initiative that aims...... to promote so-called ‘digital construction' in Denmark. One of the main objectives of this initiative is to ensure better coordination between the different phases of the building project through the application of 3D Building Information Models (BIM). The intentions are to improve the construction phase...

  5. Shape Shifting

    DEFF Research Database (Denmark)

    Gottlieb, Stefan; Hundebøl, Jesper; Larsen, Casper Schultz

    the case-study reveal a much less pervasive and more coin cidental utilisation of the 3D model than envisioned programmatically. We show how the BIM is introduced into the construction phase in the form of paper based 3D prints - as a supplement only to the existing practices. While this modest utilization......In this paper we are concerned with the specific effects of a 3D building model produced and reproduced in the planning and construction phases on a particular construction project. We contrast these effects with the policy intentions expressed within a state funded, public initiative that aims...... to promote so-called ‘digital construction' in Denmark. One of the main objectives of this initiative is to ensure better coordination between the different phases of the building project through the application of 3D Building Information Models (BIM). The intentions are to improve the construction phase...

  6. Enhanced time overcurrent coordination

    Energy Technology Data Exchange (ETDEWEB)

    Enriquez, Arturo Conde; Martinez, Ernesto Vazquez [Universidad Autonoma de Nuevo Leon, Facultad de Ingenieria Mecanica y Electrica, Apdo. Postal 114-F, Ciudad Universitaria, CP 66450 San Nicolas de los Garza, Nuevo Leon (Mexico)

    2006-04-15

    In this paper, we recommend a new coordination system for time overcurrent relays. The purpose of the coordination process is to find a time element function that allows it to operate using a constant back-up time delay, for any fault current. In this article, we describe the implementation and coordination results of time overcurrent relays, fuses and reclosers. Experiments were carried out in a laboratory test situation using signals of a power electrical system physics simulator. (author)

  7. Probing a self-assembled fd virus membrane with a microtubule

    Science.gov (United States)

    Xie, Sheng; Pelcovits, Robert A.; Hagan, Michael F.

    2016-06-01

    The self-assembly of highly anisotropic colloidal particles leads to a rich variety of morphologies whose properties are just beginning to be understood. This article uses computer simulations to probe a particle-scale perturbation of a commonly studied colloidal assembly, a monolayer membrane composed of rodlike fd viruses in the presence of a polymer depletant. Motivated by experiments currently in progress, we simulate the interaction between a microtubule and a monolayer membrane as the microtubule "pokes" and penetrates the membrane face-on. Both the viruses and the microtubule are modeled as hard spherocylinders of the same diameter, while the depletant is modeled using ghost spheres. We find that the force exerted on the microtubule by the membrane is zero either when the microtubule is completely outside the membrane or when it has fully penetrated the membrane. The microtubule is initially repelled by the membrane as it begins to penetrate but experiences an attractive force as it penetrates further. We assess the roles played by translational and rotational fluctuations of the viruses and the osmotic pressure of the polymer depletant. We find that rotational fluctuations play a more important role than the translational ones. The dependence on the osmotic pressure of the depletant of the width and height of the repulsive barrier and the depth of the attractive potential well is consistent with the assumed depletion-induced attractive interaction between the microtubule and viruses. We discuss the relevance of these studies to the experimental investigations.

  8. Aurora B suppresses microtubule dynamics and limits central spindle size by locally activating KIF4A

    Science.gov (United States)

    Nunes Bastos, Ricardo; Gandhi, Sapan R.; Baron, Ryan D.; Gruneberg, Ulrike; Nigg, Erich A.

    2013-01-01

    Anaphase central spindle formation is controlled by the microtubule-stabilizing factor PRC1 and the kinesin KIF4A. We show that an MKlp2-dependent pool of Aurora B at the central spindle, rather than global Aurora B activity, regulates KIF4A accumulation at the central spindle. KIF4A phosphorylation by Aurora B stimulates the maximal microtubule-dependent ATPase activity of KIF4A and promotes its interaction with PRC1. In the presence of phosphorylated KIF4A, microtubules grew more slowly and showed long pauses in growth, resulting in the generation of shorter PRC1-stabilized microtubule overlaps in vitro. Cells expressing only mutant forms of KIF4A lacking the Aurora B phosphorylation site overextended the anaphase central spindle, demonstrating that this regulation is crucial for microtubule length control in vivo. Aurora B therefore ensures that suppression of microtubule dynamic instability by KIF4A is restricted to a specific subset of microtubules and thereby contributes to central spindle size control in anaphase. PMID:23940115

  9. Long astral microtubules and RACK-1 stabilize polarity domains during maintenance phase in Caenorhabditis elegans embryos.

    Directory of Open Access Journals (Sweden)

    Erkang Ai

    Full Text Available Cell polarity is a very well conserved process important for cell differentiation, cell migration, and embryonic development. After the establishment of distinct cortical domains, polarity cues have to be stabilized and maintained within a fluid and dynamic membrane to achieve proper cell asymmetry. Microtubules have long been thought to deliver the signals required to polarize a cell. While previous studies suggest that microtubules play a key role in the establishment of polarity, the requirement of microtubules during maintenance phase remains unclear. In this study, we show that depletion of Caenorhabditis elegans RACK-1, which leads to short astral microtubules during prometaphase, specifically affects maintenance of cortical PAR domains and Dynamin localization. We then investigated the consequence of knocking down other factors that also abolish astral microtubule elongation during polarity maintenance phase. We found a correlation between short astral microtubules and the instability of PAR-6 and PAR-2 domains during maintenance phase. Our data support a necessary role for astral microtubules in the maintenance phase of cell polarity.

  10. Single Molecule Investigation of Kinesin-1 Motility Using Engineered Microtubule Defects

    Science.gov (United States)

    Gramlich, Michael W.; Conway, Leslie; Liang, Winnie H.; Labastide, Joelle A.; King, Stephen J.; Xu, Jing; Ross, Jennifer L.

    2017-01-01

    The structure of the microtubule is tightly regulated in cells via a number of microtubule associated proteins and enzymes. Microtubules accumulate structural defects during polymerization, and defect size can further increase under mechanical stresses. Intriguingly, microtubule defects have been shown to be targeted for removal via severing enzymes or self-repair. The cell’s control in defect removal suggests that defects can impact microtubule-based processes, including molecular motor-based intracellular transport. We previously demonstrated that microtubule defects influence cargo transport by multiple kinesin motors. However, mechanistic investigations of the observed effects remained challenging, since defects occur randomly during polymerization and are not directly observable in current motility assays. To overcome this challenge, we used end-to-end annealing to generate defects that are directly observable using standard epi-fluorescence microscopy. We demonstrate that the annealed sites recapitulate the effects of polymerization-derived defects on multiple-motor transport, and thus represent a simple and appropriate model for naturally-occurring defects. We found that single kinesins undergo premature dissociation, but not preferential pausing, at the annealed sites. Our findings provide the first mechanistic insight to how defects impact kinesin-based transport. Preferential dissociation on the single-molecule level has the potential to impair cargo delivery at locations of microtubule defect sites in vivo. PMID:28287156

  11. α-Synuclein Fibrils Exhibit Gain of Toxic Function, Promoting Tau Aggregation and Inhibiting Microtubule Assembly*

    Science.gov (United States)

    Oikawa, Takayuki; Nonaka, Takashi; Terada, Makoto; Tamaoka, Akira; Hisanaga, Shin-ichi; Hasegawa, Masato

    2016-01-01

    α-Synuclein is the major component of Lewy bodies and Lewy neurites in Parkinson disease and dementia with Lewy bodies and of glial cytoplasmic inclusions in multiple system atrophy. It has been suggested that α-synuclein fibrils or intermediate protofibrils in the process of fibril formation may have a toxic effect on neuronal cells. In this study, we investigated the ability of soluble monomeric α-synuclein to promote microtubule assembly and the effects of conformational changes of α-synuclein on Tau-promoted microtubule assembly. In marked contrast to previous findings, monomeric α-synuclein had no effect on microtubule polymerization. However, both α-synuclein fibrils and protofibrils inhibited Tau-promoted microtubule assembly. The inhibitory effect of α-synuclein fibrils was greater than that of the protofibrils. Dot blot overlay assay and spin-down techniques revealed that α-synuclein fibrils bind to Tau and inhibit microtubule assembly by depleting the Tau available for microtubule polymerization. Using various deletion mutants of α-synuclein and Tau, the acidic C-terminal region of α-synuclein and the basic central region of Tau were identified as regions involved in the binding. Furthermore, introduction of α-synuclein fibrils into cultured cells overexpressing Tau protein induced Tau aggregation. These results raise the possibility that α-synuclein fibrils interact with Tau, inhibit its function to stabilize microtubules, and also promote Tau aggregation, leading to dysfunction of neuronal cells. PMID:27226637

  12. Dynamics of microtubule asters in microfabricated chambers: The role of catastrophes

    Science.gov (United States)

    Faivre-Moskalenko, Cendrine; Dogterom, Marileen

    2002-01-01

    Recent in vivo as well as in vitro experiments have indicated that microtubule pushing alone is sufficient to position a microtubule-organizing center within a cell. Here, we investigate the effect of catastrophes on the dynamics of microtubule asters within microfabricated chambers that mimic the confining geometry of living cells. The use of a glass bead as the microtubule-organizing center allows us to manipulate the aster by using optical tweezers. In the case in which microtubules preexist, we show that because of microtubule buckling, repositioning almost never occurs after relocation with the optical tweezers, although initial microtubule growth always leads the aster to the geometrical center of the chamber. When a catastrophe promoter is added, we find instead that the aster is able to efficiently explore the chamber geometry even after being relocated with the optical tweezers. As predicted by theoretical calculations, the results of our in vitro experiments clearly demonstrate the need for catastrophes for proper positioning in a confining geometry. These findings correlate with recent observations of nuclear positioning in fission yeast cells. PMID:12486218

  13. Deposition features of Ni on self-assembled microtubule template from biolipid by electroless method

    Institute of Scientific and Technical Information of China (English)

    FU; Yubin; ZHANG; Lide; ZHENG; Jiyong; FU; Shangang; ZHU; M

    2004-01-01

    Diacetylenic glycero-phosphatidylcholine is a chiral molecule with amphiphilic property, and it can self-assembly into a lipid microtubular structure. The lipid microtubule is a stable structure formed by tightly wound helical ribbons, and the ribbon-wrapping patterns have a significant effect on their chemical deposition on the microtubules. The deposition of colloidal Pd catalyst occurs mainly on the helical edge of the wound helical ribbons to form helical deposition lines of colloidal Pd particles in the interior and exterior of the lipid microtubules, resulting in an uneven chemical deposition of Ni on the microtubules. Catalyzed by as-deposited colloidal Pd, metallized Ni microtubules are characterized by a helical form, which may be in relation to inner stress due to the thickness difference or the different deposition processes. The observation of microtom shows that metallized tubules have a hollow structure. Some metallized tubules have a kind of coaxial double layer structure observed in the direct experiment evidence, indicating that metallization can occur in the inner and outer surface of the lipid tubules. Both lipid microtubules and metallized microtubules can be used as vehicles for encapsulating biological active molecules to control their release and to develop micro-components in biological and mechanical systems.

  14. Hypergravity induces reorientation of cortical microtubules and modifies growth anisotropy in azuki bean epicotyls.

    Science.gov (United States)

    Soga, Kouichi; Wakabayashi, Kazuyuki; Kamisaka, Seiichiro; Hoson, Takayuki

    2006-11-01

    We examined the changes in the orientation of cortical microtubules during the hypergravity-induced modification of growth anisotropy (inhibition of elongation growth and promotion of lateral growth) in azuki bean (Vigna angularis Ohwi et Ohashi) epicotyls. The percentage of cells with transverse microtubules was decreased, while that with longitudinal microtubules was increased, in proportion to the logarithm of the magnitude of gravity. The percentage of cells with longitudinal microtubules showed an increase within 0.5 h of transfer of the 1g-grown seedlings to a 300g-hypergravity condition. Lanthanum and gadolinium, blockers of calcium channels, nullified the modification of growth anisotropy and reorientation of microtubules by hypergravity. Horizontal and acropetal hypergravity modified growth anisotropy and reorientation of microtubules, as did basipetal hypergravity, and these changes were not seen in the presence of lanthanum or gadolinium. These results suggest that hypergravity changes activities of lanthanum- and gadolinium-sensitive calcium channels independently of its direction, which may lead to reorientation of cortical microtubules and modification of growth anisotropy in azuki bean epicotyls.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2017-06-26

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

  16. Variational Principles for Buckling of Microtubules Modeled as Nonlocal Orthotropic Shells

    Directory of Open Access Journals (Sweden)

    Sarp Adali

    2014-01-01

    Full Text Available A variational principle for microtubules subject to a buckling load is derived by semi-inverse method. The microtubule is modeled as an orthotropic shell with the constitutive equations based on nonlocal elastic theory and the effect of filament network taken into account as an elastic surrounding. Microtubules can carry large compressive forces by virtue of the mechanical coupling between the microtubules and the surrounding elastic filament network. The equations governing the buckling of the microtubule are given by a system of three partial differential equations. The problem studied in the present work involves the derivation of the variational formulation for microtubule buckling. The Rayleigh quotient for the buckling load as well as the natural and geometric boundary conditions of the problem is obtained from this variational formulation. It is observed that the boundary conditions are coupled as a result of nonlocal formulation. It is noted that the analytic solution of the buckling problem for microtubules is usually a difficult task. The variational formulation of the problem provides the basis for a number of approximate and numerical methods of solutions and furthermore variational principles can provide physical insight into the problem.

  17. Laulimalide induces dose-dependent modulation of microtubule behaviour in the C. elegans embryo.

    Directory of Open Access Journals (Sweden)

    Megha Bajaj

    Full Text Available Laulimalide is a microtubule-binding drug that was originally isolated from marine sponges. High concentrations of laulimalide stabilize microtubules and inhibit cell division similarly to paclitaxel; however, there are important differences with respect to the nature of the specific cellular defects between these two drugs and their binding sites on the microtubule. In this study, we used Caenorhabditis elegans embryos to investigate the acute effects of laulimalide on microtubules in vivo, with a direct comparison to paclitaxel. We observed surprising dose-dependent effects for laulimalide, whereby microtubules were stabilized at concentrations above 100 nM, but destabilized at concentrations between 50 and 100 nM. Despite this behaviour at low concentrations, laulimalide acted synergistically with paclitaxel to stabilize microtubules when both drugs were used at sub-effective concentrations, consistent with observations of synergistic interactions between these two drugs in other systems. Our results indicate that laulimalide induces a concentration-dependent, biphasic change in microtubule polymer dynamics in the C. elegans embryo.

  18. Arl2- and Msps-dependent microtubule growth governs asymmetric division.

    Science.gov (United States)

    Chen, Keng; Koe, Chwee Tat; Xing, Zhanyuan Benny; Tian, Xiaolin; Rossi, Fabrizio; Wang, Cheng; Tang, Quan; Zong, Wenhui; Hong, Wan Jin; Taneja, Reshma; Yu, Fengwei; Gonzalez, Cayetano; Wu, Chunlai; Endow, Sharyn; Wang, Hongyan

    2016-03-14

    Asymmetric division of neural stem cells is a fundamental strategy to balance their self-renewal and differentiation. It is long thought that microtubules are not essential for cell polarity in asymmetrically dividing Drosophila melanogaster neuroblasts (NBs; neural stem cells). Here, we show that Drosophila ADP ribosylation factor like-2 (Arl2) and Msps, a known microtubule-binding protein, control cell polarity and spindle orientation of NBs. Upon arl2 RNA intereference, Arl2-GDP expression, or arl2 deletions, microtubule abnormalities and asymmetric division defects were observed. Conversely, overactivation of Arl2 leads to microtubule overgrowth and depletion of NBs. Arl2 regulates microtubule growth and asymmetric division through localizing Msps to the centrosomes in NBs. Moreover, Arl2 regulates dynein function and in turn centrosomal localization of D-TACC and Msps. Arl2 physically associates with tubulin cofactors C, D, and E. Arl2 functions together with tubulin-binding cofactor D to control microtubule growth, Msps localization, and NB self-renewal. Therefore, Arl2- and Msps-dependent microtubule growth is a new paradigm regulating asymmetric division of neural stem cells.

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

    Directory of Open Access Journals (Sweden)

    Carsten Schwan

    2009-10-01

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

  20. Adding Curvature to Minimum Description Length Shape Models

    DEFF Research Database (Denmark)

    Thodberg, Hans Henrik; Ólafsdóttir, Hildur

    2003-01-01

    The Minimum Description Length (MDL) approach to shape modelling seeks a compact description of a set of shapes in terms of the coordinates of marks on the shapes. It has been shown that the mark positions resulting from this optimisation to a large extent solve the so-called point correspondence...

  1. 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.

  2. Conformational mechanism for the stability of microtubule-kinetochore attachments

    CERN Document Server

    Bertalan, Zsolt; Maiato, Helder; Zapperi, Stefano

    2014-01-01

    Regulating the stability of microtubule(MT)-kinetochore attachments is fundamental to avoiding mitotic errors and ensure proper chromosome segregation during cell division. While biochemical factors involved in this process have been identified, its mechanics still needs to be better understood. Here we introduce and simulate a mechanical model of MT-kinetochore interactions in which the stability of the attachment is ruled by the geometrical conformations of curling MT-protofilaments entangled in kinetochore fibrils. The model allows us to reproduce with good accuracy in vitro experimental measurements of the detachment times of yeast kinetochores from MTs under external pulling forces. Numerical simulations suggest that geometrical features of MT-protofilaments may play an important role in the switch between stable and unstable attachments.

  3. Mechanism for the catastrophe-promoting activity of the microtubule destabilizer Op18/stathmin.

    Science.gov (United States)

    Gupta, Kamlesh K; Li, Chunlei; Duan, Aranda; Alberico, Emily O; Kim, Oleg V; Alber, Mark S; Goodson, Holly V

    2013-12-17

    Regulation of microtubule dynamic instability is crucial for cellular processes, ranging from mitosis to membrane transport. Stathmin (also known as oncoprotein 18/Op18) is a prominent microtubule destabilizer that acts preferentially on microtubule minus ends. Stathmin has been studied intensively because of its association with multiple types of cancer, but its mechanism of action remains controversial. Two models have been proposed. One model is that stathmin promotes microtubule catastrophe indirectly, and does so by sequestering tubulin; the other holds that stathmin alters microtubule dynamics by directly destabilizing growing microtubules. Stathmin's sequestration activity is well established, but the mechanism of any direct action is mysterious because stathmin binds to microtubules very weakly. To address these issues, we have studied interactions between stathmin and varied tubulin polymers. We show that stathmin binds tightly to Dolastatin-10 tubulin rings, which mimic curved tubulin protofilaments, and that stathmin depolymerizes stabilized protofilament-rich polymers. These observations lead us to propose that stathmin promotes catastrophe by binding to and acting upon protofilaments exposed at the tips of growing microtubules. Moreover, we suggest that stathmin's minus-end preference results from interactions between stathmin's N terminus and the surface of α-tubulin that is exposed only at the minus end. Using computational modeling of microtubule dynamics, we show that these mechanisms could account for stathmin's observed activities in vitro, but that both the direct and sequestering activities are likely to be relevant in a cellular context. Taken together, our results suggest that stathmin can promote catastrophe by direct action on protofilament structure and interactions.

  4. Detailed Per-residue Energetic Analysis Explains the Driving Force for Microtubule Disassembly.

    Directory of Open Access Journals (Sweden)

    Ahmed T Ayoub

    2015-06-01

    Full Text Available Microtubules are long filamentous hollow cylinders whose surfaces form lattice structures of αβ-tubulin heterodimers. They perform multiple physiological roles in eukaryotic cells and are targets for therapeutic interventions. In our study, we carried out all-atom molecular dynamics simulations for arbitrarily long microtubules that have either GDP or GTP molecules in the E-site of β-tubulin. A detailed energy balance of the MM/GBSA inter-dimer interaction energy per residue contributing to the overall lateral and longitudinal structural stability was performed. The obtained results identified the key residues and tubulin domains according to their energetic contributions. They also identified the molecular forces that drive microtubule disassembly. At the tip of the plus end of the microtubule, the uneven distribution of longitudinal interaction energies within a protofilament generates a torque that bends tubulin outwardly with respect to the cylinder's axis causing disassembly. In the presence of GTP, this torque is opposed by lateral interactions that prevent outward curling, thus stabilizing the whole microtubule. Once GTP hydrolysis reaches the tip of the microtubule (lateral cap, lateral interactions become much weaker, allowing tubulin dimers to bend outwards, causing disassembly. The role of magnesium in the process of outward curling has also been demonstrated. This study also showed that the microtubule seam is the most energetically labile inter-dimer interface and could serve as a trigger point for disassembly. Based on a detailed balance of the energetic contributions per amino acid residue in the microtubule, numerous other analyses could be performed to give additional insights into the properties of microtubule dynamic instability.

  5. A novel role for doublecortin and doublecortin-like kinase in regulating growth cone microtubules.

    Science.gov (United States)

    Jean, Daphney C; Baas, Peter W; Black, Mark M

    2012-12-15

    Doublecortin (DCX) and doublecortin-like kinase (DCLK), closely related family members, are microtubule-associated proteins with overlapping functions in both neuronal migration and axonal outgrowth. In growing axons, these proteins appear to have their primary functions in the growth cone. Here, we used siRNA to deplete these proteins from cultured rat sympathetic neurons. Normally, microtubules in the growth cone exhibit a gently curved contour as they extend from the base of the cone toward its periphery. However, following depletion of DCX and DCLK, microtubules throughout the growth cone become much more curvy, with many microtubules exhibiting multiple prominent bends over relatively short distances, creating a configuration that we termed wave-like folds. Microtubules with these folds appeared as if they were buckling in response to powerful forces. Indeed, inhibition of myosin-II, which generates forces on the actin cytoskeleton to push microtubules in the growth cone back toward the axonal shaft, significantly decreases the frequency of these wave-like folds. In addition, in the absence of DCX and DCLK, the depth of microtubule invasion into filopodia is reduced compared with controls, and at a functional level, growth cone responses to substrate guidance cues are altered. Conversely, overexpression of DCX results in microtubules that are straighter than usual, suggesting that higher levels of these proteins can enable an even greater resistance to folding. These findings support a role for DCX and DCLK in enabling microtubules to overcome retrograde actin-based forces, thereby facilitating the ability of the growth cone to carry out its crucial path-finding functions.

  6. 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

  7. Coordinate transformations and matter waves cloaking

    Science.gov (United States)

    Mohammadi, G. R.; Moghaddam, A. G.; Mohammadkhani, R.

    2016-03-01

    Transformation method provides an efficient tool to control wave propagation inside the materials. Using the coordinate transformation approach, we study invisibility cloaks with sphere, cylinder and ellipsoid structures for electronic waves propagation. The underlying physics behind this investigation is the fact that Schrödinger equation with position dependent mass tensor and potentials has a covariant form which follows the coordinate transformation. Using this technique we obtain the exact spatial form of the mass tensor and potentials for a variety of cloaks with different shapes.

  8. Calcium-independent disruption of microtubule dynamics by nanosecond pulsed electric fields in U87 human glioblastoma cells

    Science.gov (United States)

    Carr, Lynn; Bardet, Sylvia M.; Burke, Ryan C.; Arnaud-Cormos, Delia; Leveque, Philippe; O’Connor, Rodney P.

    2017-01-01

    High powered, nanosecond duration, pulsed electric fields (nsPEF) cause cell death by a mechanism that is not fully understood and have been proposed as a targeted cancer therapy. Numerous chemotherapeutics work by disrupting microtubules. As microtubules are affected by electrical fields, this study looks at the possibility of disrupting them electrically with nsPEF. Human glioblastoma cells (U87-MG) treated with 100, 10 ns, 44 kV/cm pulses at a frequency of 10 Hz showed a breakdown of their interphase microtubule network that was accompanied by a reduction in the number of growing microtubules. This effect is temporally linked to loss of mitochondrial membrane potential and independent of cellular swelling and calcium influx, two factors that disrupt microtubule growth dynamics. Super-resolution microscopy revealed microtubule buckling and breaking as a result of nsPEF application, suggesting that nsPEF may act directly on microtubules. PMID:28117459

  9. Calcium-independent disruption of microtubule dynamics by nanosecond pulsed electric fields in U87 human glioblastoma cells.

    Science.gov (United States)

    Carr, Lynn; Bardet, Sylvia M; Burke, Ryan C; Arnaud-Cormos, Delia; Leveque, Philippe; O'Connor, Rodney P

    2017-01-24

    High powered, nanosecond duration, pulsed electric fields (nsPEF) cause cell death by a mechanism that is not fully understood and have been proposed as a targeted cancer therapy. Numerous chemotherapeutics work by disrupting microtubules. As microtubules are affected by electrical fields, this study looks at the possibility of disrupting them electrically with nsPEF. Human glioblastoma cells (U87-MG) treated with 100, 10 ns, 44 kV/cm pulses at a frequency of 10 Hz showed a breakdown of their interphase microtubule network that was accompanied by a reduction in the number of growing microtubules. This effect is temporally linked to loss of mitochondrial membrane potential and independent of cellular swelling and calcium influx, two factors that disrupt microtubule growth dynamics. Super-resolution microscopy revealed microtubule buckling and breaking as a result of nsPEF application, suggesting that nsPEF may act directly on microtubules.

  10. Synthesis of arylpyrazole linked benzimidazole conjugates as potential microtubule disruptors.

    Science.gov (United States)

    Kamal, Ahmed; Shaik, Anver Basha; Polepalli, Sowjanya; Kumar, G Bharath; Reddy, Vangala Santhosh; Mahesh, Rasala; Garimella, Srujana; Jain, Nishant

    2015-03-01

    In an attempt to develop potent and selective anticancer agents, a series of twenty arylpyrazole linked benzimidazole conjugates (10a-t) were designed and synthesized as microtubule destabilizing agents. The joining of arylpyrazole to the benzimidazole moiety resulted in a four ring (A, B, C and D) molecular scaffold that comprises of polar heterocyclic rings in the middle associated with rotatable single bonds and substituted aryl rings placed in the opposite directions. These conjugates were evaluated for their ability to inhibit the growth of sixty cancer cell line panel of the NCI. Among these some conjugates like 10a, 10b, 10d, 10e, 10p and 10r exhibited significant growth inhibitory activity against most of the cell lines ranging from 0.3 to 13μM. Interestingly, the conjugate 10b with methoxy group on D-ring expressed appreciable cytotoxic potential. A549 cells treated with some of the potent conjugates like 10a, 10b and 10d arrested cells at G2/M phase apart from activating cyclin-B1 protein levels and disrupting microtubule network. Moreover, these conjugates effectively inhibited tubulin polymerization with IC50 values of 1.3-3.8μM. Whereas, the caspase assay revealed that they activate the casepase-3 leading to apoptosis. Particularly 10b having methoxy substituent induced activity almost 3 folds higher than CA-4. Furthermore, a competitive colchicine binding assay and molecular modeling analysis suggests that these conjugates bind to the tubulin successfully at the colchicine binding site. These investigations reveal that such conjugates having pyrazole and benzimidazole moieties have the potential in the development of newer chemotherapeutic agents.

  11. The 3Ms of central spindle assembly: microtubules, motors and MAPs.

    Science.gov (United States)

    Glotzer, Michael

    2009-01-01

    During metaphase, sister chromatids are positioned at the midpoint of the microtubule-based mitotic spindle in preparation for their segregation. The onset of anaphase triggers inactivation of the key mitotic kinase cyclin-dependent kinase 1 (CDK1) and the polewards movement of sister chromatids. During anaphase, the mitotic spindle reorganizes in preparation for cytokinesis. Kinesin motor proteins and microtubule-associated proteins bundle the plus ends of interpolar microtubules and generate the central spindle, which regulates cleavage furrow initiation and the completion of cytokinesis. Complementary approaches, including cell biology, genetics and computational modelling, have provided new insights into the mechanism and regulation of central spindle assembly.

  12. [The role of cortical microtubules in moss protonemal cells during dehydration/rehydration cycle].

    Science.gov (United States)

    Chen, Zhi-Ling; Ouyang, Hao-Miao; Liu, Xiang-Lin; Xia, Gui-Xian

    2003-05-01

    Plant cells response to water deficit through a variety of physiological processes. In this work, we studied the function of microtubule cytoskeleton during dehydration/rehydration cycle in moss (Atrichum undulatum) protonemal cells as a model system. The morphological and cytological change of protonemal cells during dehydration and rehydration cycle were first investigated. Under normal conditions, protonemal cells showed bright green colour and appeared wet and fresh. Numerous chloroplasts distributed regularly throughout the cytoplasm in each cell. After dehydration treatment, protonemal cells lost most of their chlorophylls and turned to look yellow and dry. In addition, dehydration caused plasmolysis in these cells. Upon rehydration, the cells could recover completely from the dehydrated state. These results indicated that moss had a remarkable intrinsic ability to survive from the extreme drought stress. Microtubule, an important component of cytoskeleton, is considered to play crucial roles in the responses to some environmental stresses such as cold and light. To see if it is also involved in the drought tolerance, dynamic organization of microtubules in protonemal cells of Atrichum undulatum subjected to drought and rehydration were examined by indirect immunofluorescence combined with confocal lasersharp scanning microscopy. The cortical microtubules were arranged into a fine structure with a predominant orientation parallel to the long axis of the cells in the control cells. After dehydration, the microtubule organization was remarkablly altered and the fine microtubule structure disappeared whereas some thicker cables formed. When the cells were grown under rehydration conditions, the fine microtubule arrays reappeared. These results provided a piece of evidence that microtubules play a role in the cellular responses to drought stress in moss. Furthermore, we analyzed the effects of the microtubule-disrupting agent colchicine on the morphology recovery

  13. Neutralizing antibody blocks adenovirus infection by arresting microtubule-dependent cytoplasmic transport.

    Science.gov (United States)

    Smith, Jason G; Cassany, Aurelia; Gerace, Larry; Ralston, Robert; Nemerow, Glen R

    2008-07-01

    Neutralizing antibodies are commonly elicited by viral infection. Most antibodies that have been characterized block early stages of virus entry that occur before membrane penetration, whereas inhibition of late stages in entry that occurs after membrane penetration has been poorly characterized. Here we provide evidence that the neutralizing antihexon monoclonal antibody 9C12 inhibits adenovirus infection by blocking microtubule-dependent translocation of the virus to the microtubule-organizing center following endosome penetration. These studies identify a previously undescribed mechanism by which neutralizing antibodies block virus infection, a situation that may be relevant for other nonenveloped viruses that use microtubule-dependent transport during cell entry.

  14. Neutralizing Antibody Blocks Adenovirus Infection by Arresting Microtubule-Dependent Cytoplasmic Transport▿

    Science.gov (United States)

    Smith, Jason G.; Cassany, Aurelia; Gerace, Larry; Ralston, Robert; Nemerow, Glen R.

    2008-01-01

    Neutralizing antibodies are commonly elicited by viral infection. Most antibodies that have been characterized block early stages of virus entry that occur before membrane penetration, whereas inhibition of late stages in entry that occurs after membrane penetration has been poorly characterized. Here we provide evidence that the neutralizing antihexon monoclonal antibody 9C12 inhibits adenovirus infection by blocking microtubule-dependent translocation of the virus to the microtubule-organizing center following endosome penetration. These studies identify a previously undescribed mechanism by which neutralizing antibodies block virus infection, a situation that may be relevant for other nonenveloped viruses that use microtubule-dependent transport during cell entry. PMID:18448546

  15. Characterizing traits of coordination

    NARCIS (Netherlands)

    Poss, R.

    2013-01-01

    How can one recognize coordination languages and technologies? As this report shows, the common approach that contrasts coordination with computation is intellectually unsound: depending on the selected understanding of the word "computation", it either captures too many or too few programming

  16. Coordination models and languages

    NARCIS (Netherlands)

    Papadopoulos, G.A.; Arbab, F.

    1998-01-01

    A new class of models, formalisms and mechanisms has recently evolved for describing concurrent and distributed computations based on the concept of ``coordination''. The purpose of a coordination model and associated language is to provide a means of integrating a number of possibly heterogeneous c

  17. Team coordination dynamics.

    Science.gov (United States)

    Gorman, Jamie C; Amazeen, Polemnia G; Cooke, Nancy J

    2010-07-01

    Team coordination consists of both the dynamics of team member interaction and the environmental dynamics to which a team is subjected. Focusing on dynamics, an approach is developed that contrasts with traditional aggregate-static concepts of team coordination as characterized by the shared mental model approach. A team coordination order parameter was developed to capture momentary fluctuations in coordination. Team coordination was observed in three-person uninhabited air vehicle teams across two experimental sessions. The dynamics of the order parameter were observed under changes of a team familiarity control parameter. Team members returned for the second session to either the same (Intact) or different (Mixed) team. 'Roadblock' perturbations, or novel changes in the task environment, were introduced in order to probe the stability of team coordination. Nonlinear dynamic methods revealed differences that a traditional approach did not: Intact and Mixed team coordination dynamics looked very different; Mixed teams were more stable than Intact teams and explored the space of solutions without the need for correction. Stability was positively correlated with the number of roadblock perturbations that were overcome successfully. The novel and non-intuitive contribution of a dynamical analysis was that Mixed teams, who did not have a long history working together, were more adaptive. Team coordination dynamics carries new implications for traditional problems such as training adaptive teams.

  18. Coordinate measuring machines

    DEFF Research Database (Denmark)

    De Chiffre, Leonardo

    This document is used in connection with three exercises of 2 hours duration as a part of the course GEOMETRICAL METROLOGY AND MACHINE TESTING. The exercises concern three aspects of coordinate measuring: 1) Measuring and verification of tolerances on coordinate measuring machines, 2) Traceability...

  19. Social Postural Coordination

    Science.gov (United States)

    Varlet, Manuel; Marin, Ludovic; Lagarde, Julien; Bardy, Benoit G.

    2011-01-01

    The goal of the current study was to investigate whether a visual coupling between two people can produce spontaneous interpersonal postural coordination and change their intrapersonal postural coordination involved in the control of stance. We examined the front-to-back head displacements of participants and the angular motion of their hip and…

  20. Social Postural Coordination

    Science.gov (United States)

    Varlet, Manuel; Marin, Ludovic; Lagarde, Julien; Bardy, Benoit G.

    2011-01-01

    The goal of the current study was to investigate whether a visual coupling between two people can produce spontaneous interpersonal postural coordination and change their intrapersonal postural coordination involved in the control of stance. We examined the front-to-back head displacements of participants and the angular motion of their hip and…

  1. Supramolecular Chirality in Dynamic Coordination Chemistry

    Directory of Open Access Journals (Sweden)

    Hiroyuki Miyake

    2014-10-01

    Full Text Available Labile metal complexes have a useful coordination bond; which is weaker than a covalent C–C bond and is reversibly and dynamically formed and dissociated. Such labile metal complexes also can be used to construct chiral shapes and offer dynamic conversion of chiral molecular shapes in response to external stimuli. This review provides recent examples of chirality induction and describes the dynamic conversion systems produced by chiral metal complexes including labile metal centers, most of which respond to external stimuli by exhibiting sophisticated conversion phenomena.

  2. The molecular dynamics of crawling migration in microtubule-disrupted keratocytes.

    Science.gov (United States)

    Nakashima, Hitomi; Okimura, Chika; Iwadate, Yoshiaki

    2015-01-01

    Cell-crawling migration plays an essential role in complex biological phenomena. It is now generally believed that many processes essential to such migration are regulated by microtubules in many cells, including fibroblasts and neurons. However, keratocytes treated with nocodazole, which is an inhibitor of microtubule polymerization - and even keratocyte fragments that contain no microtubules - migrate at the same velocity and with the same directionality as normal keratocytes. In this study, we discovered that not only these migration properties, but also the molecular dynamics that regulate such properties, such as the retrograde flow rate of actin filaments, distributions of vinculin and myosin II, and traction forces, are also the same in nocodazole-treated keratocytes as those in untreated keratocytes. These results suggest that microtubules are not in fact required for crawling migration of keratocytes, either in terms of migrating properties or of intracellular molecular dynamics.

  3. Microtubule organization within mitotic spindles revealed by serial block face scanning electron microscopy and image analysis.

    Science.gov (United States)

    Nixon, Faye M; Honnor, Thomas R; Clarke, Nicholas I; Starling, Georgina P; Beckett, Alison J; Johansen, Adam M; Brettschneider, Julia A; Prior, Ian A; Royle, Stephen J

    2017-05-15

    Serial block face scanning electron microscopy (SBF-SEM) is a powerful method to analyze cells in 3D. Here, working at the resolution limit of the method, we describe a correlative light-SBF-SEM workflow to resolve microtubules of the mitotic spindle in human cells. We present four examples of uses for this workflow that are not practical by light microscopy and/or transmission electron microscopy. First, distinguishing closely associated microtubules within K-fibers; second, resolving bridging fibers in the mitotic spindle; third, visualizing membranes in mitotic cells, relative to the spindle apparatus; and fourth, volumetric analysis of kinetochores. Our workflow also includes new computational tools for exploring the spatial arrangement of microtubules within the mitotic spindle. We use these tools to show that microtubule order in mitotic spindles is sensitive to the level of TACC3 on the spindle. © 2017. Published by The Company of Biologists Ltd.

  4. Clostridium difficile toxin CDT hijacks microtubule organization and reroutes vesicle traffic to increase pathogen adherence.

    Science.gov (United States)

    Schwan, Carsten; Kruppke, Anna S; Nölke, Thilo; Schumacher, Lucas; Koch-Nolte, Friedrich; Kudryashev, Mikhail; Stahlberg, Henning; Aktories, Klaus

    2014-02-11

    Clostridium difficile causes antibiotic-associated diarrhea and pseudomembranous colitis by the actions of Rho-glucosylating toxins A and B. Recently identified hypervirulent strains, which are associated with increased morbidity and mortality, additionally produce the actin-ADP-ribosylating toxin C. difficile transferase (CDT). CDT depolymerizes actin, causes formation of microtubule-based protrusions, and increases pathogen adherence. Here we show that CDT-induced protrusions allow vesicle traffic and contain endoplasmic reticulum tubules, connected to microtubules via the calcium sensor Stim1. The toxin reroutes Rab11-positive vesicles containing fibronectin, which is involved in bacterial adherence, from basolateral to the apical membrane sides in a microtubule- and Stim1-dependent manner. The data yield a model of C. difficile adherence regulated by actin depolymerization, microtubule restructuring, subsequent Stim1-dependent Ca(2+) signaling, vesicle rerouting, and secretion of ECM proteins to increase bacterial adherence.

  5. Joined at the hip: kinetochores, microtubules, and spindle assembly checkpoint signaling.

    Science.gov (United States)

    Sacristan, Carlos; Kops, Geert J P L

    2015-01-01

    Error-free chromosome segregation relies on stable connections between kinetochores and spindle microtubules. The spindle assembly checkpoint (SAC) monitors such connections and relays their absence to the cell cycle machinery to delay cell division. The molecular network at kinetochores that is responsible for microtubule binding is integrated with the core components of the SAC signaling system. Molecular-mechanistic understanding of how the SAC is coupled to the kinetochore-microtubule interface has advanced significantly in recent years. The latest insights not only provide a striking view of the dynamics and regulation of SAC signaling events at the outer kinetochore but also create a framework for understanding how that signaling may be terminated when kinetochores and microtubules connect.

  6. Microtubule dynamics in the peripheral nervous system: A matter of balance.

    Science.gov (United States)

    Almeida-Souza, Leonardo; Timmerman, Vincent; Janssens, Sophie

    2011-11-01

    The special architecture of neurons in the peripheral nervous system, with axons extending for long distances, represents a major challenge for the intracellular transport system. Two recent studies show that mutations in the small heat shock protein HSPB1, which cause an axonal type of Charcot-Marie-Tooth (CMT) neuropathy, affect microtubule dynamics and impede axonal transport. Intriguingly, while at presymptomatic age the neurons in the mutant HSPB1 mouse show a hyperstable microtubule network, at postsymptomatic age, the microtubule network completely lost its stability as reflected by a marked decrease in tubulin acetylation levels. We here propose a model explaining the role of microtubule stabilization and tubulin acetylation in the pathogenesis of HSPB1 mutations.

  7. Protein kinase Darkener of apricot and its substrate EF1γ regulate organelle transport along microtubules.

    Science.gov (United States)

    Serpinskaya, Anna S; Tuphile, Karine; Rabinow, Leonard; Gelfand, Vladimir I

    2014-01-01

    Regulation of organelle transport along microtubules is important for proper distribution of membrane organelles and protein complexes in the cytoplasm. RNAi-mediated knockdown in cultured Drosophila S2 cells demonstrates that two microtubule-binding proteins, a unique isoform of Darkener of apricot (DOA) protein kinase, and its substrate, translational elongation factor EF1γ, negatively regulate transport of several classes of membrane organelles along microtubules. Inhibition of transport by EF1γ requires its phosphorylation by DOA on serine 294. Together, our results indicate a new role for two proteins that have not previously been implicated in regulation of the cytoskeleton. These results further suggest that the biological role of some of the proteins binding to the microtubule track is to regulate cargo transport along these tracks.

  8. Uranyl ion coordination

    Science.gov (United States)

    Evans, H.T.

    1963-01-01

    A review of the known crystal structures containing the uranyl ion shows that plane-pentagon coordination is equally as prevalent as plane-square or plane-hexagon. It is suggested that puckered-hexagon configurations of OH - or H2O about the uranyl group will tend to revert to plane-pentagon coordination. The concept of pentagonal coordination is invoked for possible explanations of the complex crystallography of the natural uranyl hydroxides and the unusual behavior of polynuclear ions in hydrolyzed uranyl solutions.

  9. An epigenetic regulator emerges as microtubule minus-end binding and stabilizing factor in mitosis

    OpenAIRE

    Meunier, Sylvain; Shvedunova, Maria; Van Nguyen, Nhuong; Ávila, Leonor; Vernos, Isabelle; Akhtar, Asifa

    2015-01-01

    The evolutionary conserved NSL complex is a prominent epigenetic regulator controlling expression of thousands of genes. Here we uncover a novel function of the NSL complex members in mitosis. As the cell enters mitosis, KANSL1 and KANSL3 undergo a marked relocalisation from the chromatin to the mitotic spindle. By stabilizing microtubule minus ends in a RanGTP-dependent manner, they are essential for spindle assembly and chromosome segregation. Moreover, we identify KANSL3 as a microtubule m...

  10. Effects of tertiary amine local anesthetics on the assembly and disassembly of brain microtubules in vitro.

    Science.gov (United States)

    Genna, J M; Coffe, G; Pudles, J

    1980-09-01

    From kinetic and electron microscopy studies on the effects of procaine, tetracaine and dibucaine on the polymerization and depolymerization of the microtubules isolated from pig and rat brains the following results were obtained. 1. Procaine or tetracaine, at the concentration range of 0.5--20 mM and of 0.5--5 mM respectively, increases the rate of tubulin polymerization (24 degrees C or 37 degrees C) and of microtubule depolymerization (4 degrees C) as a linear function of the concentration of the anesthetics, while identical amounts of microtubules are formed. In the absence of microtubule-associated proteins the polymerization of tubulin is not induced by 10 mM procaine, furthermore, the critical concentration of microtubule proteins necessary for assembly into microtubules is not affected at this concentration level of the anesthetic. This suggests that procaine affects not the nucleation, but rather the elongation process. 2. Dibucaine, from 0.5 mM to 3 mM increases the lag time of the polymerization reaction, while from 0.5 mM to 2 mM it linearly decreases both tubulin polymerization (24 degrees C) and microtubule depolymerization (4 degrees C) rates. Dibucaine, up to mM concentration, does not affect the extent of tubulin polymerization; however, above this concentration it induces the formation of amorphous aggregates. 3. Procaine or tetracaine enhances the depolymerizing effect of calcium on microtubules. The half-maximal values for the depolymerizing effect of calcium were 0.96, 0.71 and 0.51 mM for the control, in the presence of 10 mM procaine and 5 mM tetracaine respectively.

  11. Kinetochore–microtubule error correction is driven by differentially regulated interaction modes

    OpenAIRE

    Kalantzaki, Maria; Kitamura, Etsushi; Zhang, Tongli; Mino, Akihisa; Novák, Béla; Tanaka, Tomoyuki U

    2015-01-01

    For proper chromosome segregation, sister kinetochores must interact with microtubules from opposite spindle poles (bi-orientation). To establish bi-orientation, aberrant kinetochore–microtubule attachments are disrupted (error correction) by Aurora B kinase (Ipl1 in budding yeast). Paradoxically, during this disruption, new attachments are still formed efficiently to allow fresh attempts at bi-orientation. How this is possible remains an enigma. Here we show that kinetochore attachment to th...

  12. Kar3Vik1 uses a minus-end directed powerstroke for movement along microtubules.

    Directory of Open Access Journals (Sweden)

    Julia Cope

    Full Text Available We have used cryo-electron microscopy (cryo-EM and helical averaging to examine the 3-D structure of the heterodimeric kinesin-14 Kar3Vik1 complexed to microtubules at a resolution of 2.5 nm. 3-D maps were obtained at key points in Kar3Vik1's nucleotide hydrolysis cycle to gain insight into the mechanism that this motor uses for retrograde motility. In all states where Kar3Vik1 maintained a strong interaction with the microtubule, we found, as observed by cryo-EM, that the motor bound with one head domain while the second head extended outwards. 3-D reconstructions of Kar3Vik1-microtubule complexes revealed that in the nucleotide-free state, the motor's coiled-coil stalk points toward the plus-end of the microtubule. In the ATP-state, the outer head is shown to undergo a large rotation that reorients the stalk ∼75° to point toward the microtubule minus-end. To determine which of the two heads binds to tubulin in each nucleotide state, we employed specific Nanogold®-labeling of Vik1. The resulting maps confirmed that in the nucleotide-free, ATP and ADP+Pi states, Kar3 maintains contact with the microtubule surface, while Vik1 extends away from the microtubule and tracks with the coiled-coil as it rotates towards the microtubule minus-end. While many previous investigations have focused on the mechanisms of homodimeric kinesins, this work presents the first comprehensive study of the powerstroke of a heterodimeric kinesin. The stalk rotation shown here for Kar3Vik1 is highly reminiscent of that reported for the homodimeric kinesin-14 Ncd, emphasizing the conservation of a mechanism for minus-end directed motility.

  13. Identification of a TPX2-like microtubule-associated protein in Drosophila.

    Directory of Open Access Journals (Sweden)

    Gohta Goshima

    Full Text Available Chromosome segregation during mitosis and meiosis relies on the spindle and the functions of numerous microtubule-associated proteins (MAPs. One of the best-studied spindle MAPs is the highly conserved TPX2, which has been reported to have characteristic intracellular dynamics and molecular activities, such as nuclear localisation in interphase, poleward movement in the metaphase spindle, microtubule nucleation, microtubule stabilisation, microtubule bundling, Aurora A kinase activation, kinesin-5 binding, and kinesin-12 recruitment. This protein has been shown to be essential for spindle formation in every cell type analysed so far. However, as yet, TPX2 homologues have not been found in the Drosophila genome. In this study, I found that the Drosophila protein Ssp1/Mei-38 has significant homology to TPX2. Sequence conservation was limited to the putative spindle microtubule-associated region of TPX2, and intriguingly, D-TPX2 (Ssp1/Mei-38 lacks Aurora A- and kinesin-5-binding domains, which are highly conserved in other animal and plant species, including many insects such as ants and bees. D-TPX2 uniformly localised to kinetochore microtubule-enriched regions of the metaphase spindle in the S2 cell line, and it had microtubule binding and bundling activities in vitro. In comparison with other systems, the contribution of D-TPX2 to cell division seems to be minor; live cell imaging of microtubules and chromosomes after RNAi knockdown identified significant delay in chromosome congression in only 18% of the cells. Thus, while this conserved spindle protein is present in Drosophila, other mechanisms may largely compensate for its spindle assembly and chromosome segregation functions.

  14. CYLD Regulates Noscapine Activity in Acute Lymphoblastic Leukemia via a Microtubule-Dependent Mechanism

    OpenAIRE

    Yang, Yunfan; Ran, Jie; Sun, Lei; Sun, Xiaodong; Luo, Youguang; Yan, Bing; Tala,; Liu, Min; Li, Dengwen; Zhang, Lei; Bao, Gang; Zhou, Jun

    2015-01-01

    Noscapine is an orally administrable drug used worldwide for cough suppression and has recently been demonstrated to disrupt microtubule dynamics and possess anticancer activity. However, the molecular mechanisms regulating noscapine activity remain poorly defined. Here we demonstrate that cylindromatosis (CYLD), a microtubule-associated tumor suppressor protein, modulates the activity of noscapine both in cell lines and in primary cells of acute lymphoblastic leukemia (ALL). Flow cytometry a...

  15. Early stages of spindle formation and independence of chromosome and microtubule cycles in Haemanthus endosperm.

    Science.gov (United States)

    Smirnova, E A; Bajer, A S

    1998-01-01

    We analyzed transformation of the interphase microtubular cytoskeleton into the prophase spindle and followed the pattern of spindle axis determination. Microtubules in endosperm of the higher plant Haemanthus (Scadoxus) were stained by the immunogold and immunogold silver-enhanced methods. Basic structural units involved in spindle morphogenesis were "microtubule converging centers." We emphasized the importance of relative independence of chromosomal and microtubular cycles, and the influence of these cycles on the progress of mitosis. Cells with moderately desynchronized cycles were functional, but extreme desynchronization led to aberrant mitosis. There were three distinct phases of spindle development. The first one comprised interphase and early to mid-prophase. During this phase, the interphase microtubule meshwork radiating from the nuclear surface into the cytoplasm rearranged and formed a dense microtubule cage around the nucleus. The second phase comprised mid to late prophase, and resulted in the formation of normal (bipolar) or transitory aberrant (apolar or multipolar) prophase spindles. The third phase comprised late prophase with prometaphase. The onset of prometaphase was accompanied by a rapid association of microtubule converging centers with kinetochores. In this stage aberrant spindles transformed invariably into bipolar ones. Lateral association of a few bipolar kinetochore fibers at early prometaphase established the core of the bipolar spindle and its alignment. We concluded that (1) spindle formation is a largely independent microtubular process modified by the chromosomal/kinetochore cycle; and (2) the initial polarity of the spindle is established by microtubule converging centers, which are a functional substitute of the centrosome/MTOC. We believe that the dynamics of microtubule converging centers is an expression of microtubule self-organization driven by motor proteins as proposed by Mitchison [1992: Philos. Trans. R. Soc. Lond. B

  16. ICP0 dismantles microtubule networks in herpes simplex virus-infected cells.

    Directory of Open Access Journals (Sweden)

    Mingyu Liu

    Full Text Available Infected-cell protein 0 (ICP0 is a RING finger E3 ligase that regulates herpes simplex virus (HSV mRNA synthesis, and strongly influences the balance between latency and replication of HSV. For 25 years, the nuclear functions of ICP0 have been the subject of intense scrutiny. To obtain new clues about ICP0's mechanism of action, we constructed HSV-1 viruses that expressed GFP-tagged ICP0. To our surprise, both GFP-tagged and wild-type ICP0 were predominantly observed in the cytoplasm of HSV-infected cells. Although ICP0 is exclusively nuclear during the immediate-early phase of HSV infection, further analysis revealed that ICP0 translocated to the cytoplasm during the early phase where it triggered a previously unrecognized process; ICP0 dismantled the microtubule network of the host cell. A RING finger mutant of ICP0 efficiently bundled microtubules, but failed to disperse microtubule bundles. Synthesis of ICP0 proved to be necessary and sufficient to disrupt microtubule networks in HSV-infected and transfected cells. Plant and animal viruses encode many proteins that reorganize microtubules. However, this is the first report of a viral E3 ligase that regulates microtubule stability. Intriguingly, several cellular E3 ligases orchestrate microtubule disassembly and reassembly during mitosis. Our results suggest that ICP0 serves a dual role in the HSV life cycle, acting first as a nuclear regulator of viral mRNA synthesis and acting later, in the cytoplasm, to dismantle the host cell's microtubule network in preparation for virion synthesis and/or egress.

  17. Detailed Per-residue Energetic Analysis Explains the Driving Force for Microtubule Disassembly

    OpenAIRE

    Ayoub, Ahmed T.; Mariusz Klobukowski; Tuszynski, Jack A

    2015-01-01

    Microtubules are long filamentous hollow cylinders whose surfaces form lattice structures of αβ-tubulin heterodimers. They perform multiple physiological roles in eukaryotic cells and are targets for therapeutic interventions. In our study, we carried out all-atom molecular dynamics simulations for arbitrarily long microtubules that have either GDP or GTP molecules in the E-site of β-tubulin. A detailed energy balance of the MM/GBSA inter-dimer interaction energy per residue contributing to t...

  18. 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).

  19. Interactions of the HSV-1 UL25 Capsid Protein with Cellular Microtubule-associated Protein

    Institute of Scientific and Technical Information of China (English)

    Lei GUO; Ying ZHANG; Yan-chun CHE; Wen-juan WU; Wei-zhong LI; Li-chun WANG; Yun LIAO; Long-ding LIU; Qi-han LI

    2008-01-01

    An interaction between the HSV-1 UL25 capsid protein and cellular microtubule-associated protein was found using a yeast two-hybrid screen and β-D-galactosidase activity assays. Immunofluorescence microscopy of the UL25 protein demonstrated its co-localization with cellular microtubule-associated protein in the plasma membrane. Further investigations with deletion mutants suggest that UL25 is likely to have a function in the nucleus.

  20. A stochastic model of kinetochore–microtubule attachment accurately describes fission yeast chromosome segregation

    OpenAIRE

    Gay, Guillaume; Courtheoux, Thibault; Reyes, Céline; Tournier, Sylvie; Gachet, Yannick

    2012-01-01

    In fission yeast, erroneous attachments of spindle microtubules to kinetochores are frequent in early mitosis. Most are corrected before anaphase onset by a mechanism involving the protein kinase Aurora B, which destabilizes kinetochore microtubules (ktMTs) in the absence of tension between sister chromatids. In this paper, we describe a minimal mathematical model of fission yeast chromosome segregation based on the stochastic attachment and detachment of ktMTs. The model accurately reproduce...

  1. Push or Pull? -- Cryo-Electron Microscopy of Microtubule's Dynamic Instability and Its Roles in the Kinetochore

    Science.gov (United States)

    Wang, Hong-Wei

    2009-03-01

    Microtubule is a biopolymer made up of alpha-beta-tubulin heterodimers. The tubulin dimers assemble head-to-tail as protofilaments and about 13 protofilaments interact laterally to form a hollow cylindrical structure which is the microtubule. As the major cytoskeleton in all eukaryotic cells, microtubules have the intrinsic property to switch stochastically between growth and shrinkage phases, a phenomenon termed as their dynamic instability. Microtubule's dynamic instability is closely related to the types of nucleotide (GTP or GDP) that binds to the beta-tubulin. We have biochemically trapped two types of assembly states of tubulin with GTP or GDP bound representing the polymerizing and depolymerizing ends of microtubules respectively. Using cryo-electron microscopy, we have elucidated the structures of these intermediate assemblies, showing that tubulin protofilaments demonstrate various curvatures and form different types of lateral interactions depending on the nucleotide states of tubulin and the temperature. Our work indicates that during the microtubule's dynamic cycle, tubulin undergoes various assembly states. These states, different from the straight microtubule, lend the highly dynamic and complicated behavior of microtubules. Our study of microtubule's interaction with certain kinetochore complexes suggests that the intermediate assemblies are responsible for specific mechanical forces that are required during the mitosis or meiosis. Our discoveries strongly suggest that a microtubule is a molecular machine rather than a simple cellular scaffold.

  2. Microtubule assembly affects bone mass by regulating both osteoblast and osteoclast functions: stathmin deficiency produces an osteopenic phenotype in mice.

    Science.gov (United States)

    Liu, Hongbin; Zhang, Rongrong; Ko, Seon-Yle; Oyajobi, Babatunde O; Papasian, Christopher J; Deng, Hong-Wen; Zhang, Shujun; Zhao, Ming

    2011-09-01

    Cytoskeleton microtubules regulate various cell signaling pathways that are involved in bone cell function. We recently reported that inhibition of microtubule assembly by microtubule-targeting drugs stimulates osteoblast differentiation and bone formation. To further elucidate the role of microtubules in bone homeostasis, we characterized the skeletal phenotype of mice null for stathmin, an endogenous protein that inhibits microtubule assembly. In vivo micro-computed tomography (µCT) and histology revealed that stathmin deficiency results in a significant reduction of bone mass in adult mice concurrent with decreased osteoblast and increased osteoclast numbers in bone tissues. Phenotypic analyses of primary calvarial cells and bone marrow cells showed that stathmin deficiency inhibited osteoblast differentiation and induced osteoclast formation. In vitro overexpression studies showed that increased stathmin levels enhanced osteogenic differentiation of preosteoblast MC3T3-E1 cells and mouse bone marrow-derived cells and attenuated osteoclast formation from osteoclast precursor Raw264.7 cells and bone marrow cells. Results of immunofluorescent studies indicated that overexpression of stathmin disrupted radial microtubule filaments, whereas deficiency of stathmin stabilized the microtubule network structure in these bone cells. In addition, microtubule-targeting drugs that inhibit microtubule assembly and induce osteoblast differentiation lost these effects in the absence of stathmin. Collectively, these results suggest that stathmin, which alters microtubule dynamics, plays an essential role in maintenance of postnatal bone mass by regulating both osteoblast and osteoclast functions in bone. \\

  3. The microtubule aster formation and its role in nuclear envelope assembly around the sperm chromatin in Xenopus egg extracts

    Institute of Scientific and Technical Information of China (English)

    YANG Ning; CHEN Zhongcai; LU Ping; ZHANG Chuanmao; ZHAI Zhonghe; TANG Xiaowei

    2003-01-01

    Nuclear envelope is a dynamic structure in the cell cycle. At the beginning of mitosis, nuclear envelope breaks down and its components disperse into the cytoplasm. At the end of mitosis, nuclear envelope reassembles using the dispersed components. Searching for the mechanisms of the nuclear disassembly and reassembly has for a long time been one of the key projects for cell biologists. In this report we show that microtubules take a role in the nuclear envelope assembly around the sperm chromatin in Xenopus egg extracts. Microtubule cytoskeleton has been demonstrated to take roles in the transport of intracellular membranes such as Golgi and ER vesicles. We found that the nuclear envelope assembly needs functional microtubules. At the beginning of the nuclear assembly, microtubules nucleated to form a microtubule aster around the centrosome at the base of the sperm head. Using the microtubule drug colchicine to disrupt the microtubule nucleation, nuclear envelope reassembly was seriously inhibited. If the microtubules were stabilized by taxol, another microtubule drug, the nuclear envelope reassembly was also interfered, although a significantly large aster formed around the chromatin. Based on these observations, we propose that microtubules play an important role in the nuclear envelope reassembly maybe by transporting the nuclear envelope precursors to the chromatin surfaces.

  4. Magnetic Coordinate Systems

    CERN Document Server

    Laundal, K M

    2016-01-01

    Geospace phenomena such as the aurora, plasma motion, ionospheric currents and associated magnetic field disturbances are highly organized by Earth's main magnetic field. This is due to the fact that the charged particles that comprise space plasma can move almost freely along magnetic field lines, but not across them. For this reason it is sensible to present such phenomena relative to Earth's magnetic field. A large variety of magnetic coordinate systems exist, designed for different purposes and regions, ranging from the magnetopause to the ionosphere. In this paper we review the most common magnetic coordinate systems and describe how they are defined, where they are used, and how to convert between them. The definitions are presented based on the spherical harmonic expansion coefficients of the International Geomagnetic Reference Field (IGRF) and, in some of the coordinate systems, the position of the Sun which we show how to calculate from the time and date. The most detailed coordinate systems take the...

  5. Supercritical Airfoil Coordinates

    Data.gov (United States)

    National Aeronautics and Space Administration — Rectangular Supercritical Wing (Ricketts) - design and measured locations are provided in an Excel file RSW_airfoil_coordinates_ricketts.xls . One sheet is with Non...

  6. Understanding social motor coordination.

    Science.gov (United States)

    Schmidt, R C; Fitzpatrick, Paula; Caron, Robert; Mergeche, Joanna

    2011-10-01

    Recently there has been much interest in social coordination of motor movements, or as it is referred to by some researchers, joint action. This paper reviews the cognitive perspective's common coding/mirror neuron theory of joint action, describes some of its limitations and then presents the behavioral dynamics perspective as an alternative way of understanding social motor coordination. In particular, behavioral dynamics' ability to explain the temporal coordination of interacting individuals is detailed. Two experiments are then described that demonstrate how dynamical processes of synchronization are apparent in the coordination underlying everyday joint actions such as martial art exercises, hand-clapping games, and conversations. The import of this evidence is that emergent dynamic patterns such as synchronization are the behavioral order that any neural substrate supporting joint action (e.g., mirror systems) would have to sustain.

  7. Environmental Compliance Issue Coordination

    Science.gov (United States)

    An order to establish the Department of Energy (DOE) requirements for coordination of significant environmental compliance issues to ensure timely development and consistent application of Departmental environmental policy and guidance

  8. Integrated modeling methodology for microtubule dynamics and Taxol kinetics with experimentally identifiable parameters.

    Science.gov (United States)

    Zhao, He; Sokhansanj, Bahrad A

    2007-10-01

    Microtubule dynamics play a critical role in cell function and stress response, modulating mitosis, morphology, signaling, and transport. Drugs such as paclitaxel (Taxol) can impact tubulin polymerization and affect microtubule dynamics. While theoretical methods have been previously proposed to simulate microtubule dynamics, we develop a methodology here that can be used to compare model predictions with experimental data. Our model is a hybrid of (1) a simple two-state stochastic formulation of tubulin polymerization kinetics and (2) an equilibrium approximation for the chemical kinetics of Taxol drug binding to microtubule ends. Model parameters are biologically realistic, with values taken directly from experimental measurements. Model validation is conducted against published experimental data comparing optical measurements of microtubule dynamics in cultured cells under normal and Taxol-treated conditions. To compare model predictions with experimental data requires applying a "windowing" strategy on the spatiotemporal resolution of the simulation. From a biological perspective, this is consistent with interpreting the microtubule "pause" phenomenon as at least partially an artifact of spatiotemporal resolution limits on experimental measurement.

  9. Molecular mechanisms of antitumor activity of taxanes. I. Interaction of docetaxel with microtubules

    Directory of Open Access Journals (Sweden)

    Sabina Tabaczar

    2010-11-01

    Full Text Available Docetaxel (Taxotere, a new semisynthetic taxoid, is a mitotic inhibitor, widely used in monotherapy or in combination with other anticancer drugs against many types of cancer. The structure and dynamics of microtubules as the main target for docetaxel activity inside the cell and the taxane-binding site on β-tubulin are discussed. Microtubules are highly dynamic assemblies of α- and β-tubulin. They readily polymerize and depolymerize in cells and these dynamic behaviours are crucial to cell mitosis. Microtubule instability is attributed to their capability to hydrolyze GTP to GDP, which causes their depolymerization. Addition of new α-, β-tubulin heterodimer bound to GTP leads to tubulin polymerization, which increases the length of the microtubule. Docetaxel alters the polymerization dynamics of microtubules, which causes blockage of cell mitosis, and consequently induces apoptotic and non-apoptotic cell death. Docetaxel specifically acts on the S, M and G2 phases of the cell cycle. This paper reviews the current state of knowledge related to the molecular mechanisms of docetaxel action on the cell cycle and microtubule dynamics. In addition, a brief survey of the present state of research on the new generation (2nd and 3rd of taxanes is presented.

  10. Laminin/β1 integrin signal triggers axon formation by promoting microtubule assembly and stabilization

    Institute of Scientific and Technical Information of China (English)

    Wen-Liang Lei; Shi-Ge Xing; Cai-Yun Deng; Xiang-Chun Ju; Xing-Yu Jiang; Zhen-Ge Luo

    2012-01-01

    Axon specification during neuronal polarization is closely associated with increased microtubule stabilization in one of the neurites of unpolarized neuron,but how this increased microtubule stability is achieved is unclear.Here,we show that extracellular matrix (ECM) component laminin promotes neuronal polarization via regulating directional microtubule assembly through β1 integrin (Itgb1).Contact with laminin coated on culture substrate or polystyrene beads was sufficient for axon specification of undifferentiated neurites in cultured hippocampal neurons and cortical slices.Active Itgb1 was found to be concentrated in laminin-contacting neurites.Axon formation was promoted and abolished by enhancing and attenuating Itgbl signaling,respectively.Interestingly,laminin contact promoted plus-end microtubule assembly in a manner that required Itgbl.Moreover,stabilizing microtubules partially prevented polarization defects caused by ltgbl downregulation.Finally,genetic ablation of ltgbl in dorsal telencephalic progenitors caused deficits in axon development of cortical pyramidal neurons.Thus,laminin/Itgb1 signaling plays an instructive role in axon initiation and growth,both in vitro and in vivo,through the regulation of microtubule assembly.This study has established a linkage between an extrinsic factor and intrinsic cytoskeletai dynamics during neuronal polarization.

  11. 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.

  12. Quantitative Changes in Microtubule Distribution Correlate with Guard Cell Function in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    William R. Eisinger; Viktor Kirik; Charlotte Lewis; David W. Ehrhardt; Winslow R. Briggs

    2012-01-01

    Radially arranged cortical microtubules are a prominent feature of guard cells.We observed guard cells expressing GFP-tubulin (GFP-TUA6) with confocal microscopy and found recognizable changes in the appearance of microtubules when stomata open or close (Eisinger et al.,2012).In the present study,analysis of fluorescence distribution showed a dramatic increase in peak intensities of microtubule bundles within guard cells as stomata open.This increase was correlated with an increase in the total fluorescence that could be attributed to polymerized tubulin.Adjacent pavement cells did not show similar changes in peak intensities or integrated fluorescence when stomatal apertures changed.Imaging of RFP-tagged end binding protein 1 (EB1) and YFP-tagged α-tubulin expressed in the same cell revealed that the number of microtubules with growing ends remained constant,although the total amount of polymerized tubulin was higher in open than in closed guard cells.Taken together,these results indicate that the changes in microtubule array organization that are correlated with and required for normal guard cell function are characterized by changes in microtubule clustering or bundling.

  13. Familial Mediterranean fever mutations lift the obligatory requirement for microtubules in Pyrin inflammasome activation.

    Science.gov (United States)

    Van Gorp, Hanne; Saavedra, Pedro H V; de Vasconcelos, Nathalia M; Van Opdenbosch, Nina; Vande Walle, Lieselotte; Matusiak, Magdalena; Prencipe, Giusi; Insalaco, Antonella; Van Hauwermeiren, Filip; Demon, Dieter; Bogaert, Delfien J; Dullaers, Melissa; De Baere, Elfride; Hochepied, Tino; Dehoorne, Joke; Vermaelen, Karim Y; Haerynck, Filomeen; De Benedetti, Fabrizio; Lamkanfi, Mohamed

    2016-12-13

    Familial Mediterranean fever (FMF) is the most common monogenic autoinflammatory disease worldwide. It is caused by mutations in the inflammasome adaptor Pyrin, but how FMF mutations alter signaling in FMF patients is unknown. Herein, we establish Clostridium difficile and its enterotoxin A (TcdA) as Pyrin-activating agents and show that wild-type and FMF Pyrin are differentially controlled by microtubules. Diverse microtubule assembly inhibitors prevented Pyrin-mediated caspase-1 activation and secretion of IL-1β and IL-18 from mouse macrophages and human peripheral blood mononuclear cells (PBMCs). Remarkably, Pyrin inflammasome activation persisted upon microtubule disassembly in PBMCs of FMF patients but not in cells of patients afflicted with other autoinflammatory diseases. We further demonstrate that microtubules control Pyrin activation downstream of Pyrin dephosphorylation and that FMF mutations enable microtubule-independent assembly of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) micrometer-sized perinuclear structures (specks). The discovery that Pyrin mutations remove the obligatory requirement for microtubules in inflammasome activation provides a conceptual framework for understanding FMF and enables immunological screening of FMF mutations.

  14. SPIN90 Depletion and Microtubule Acetylation Mediate Stromal Fibroblast Activation in Breast Cancer Progression.

    Science.gov (United States)

    You, Eunae; Huh, Yun Hyun; Kwon, Ahreum; Kim, So Hee; Chae, In Hee; Lee, Ok-Jun; Ryu, Je-Hwang; Park, Min Ho; Kim, Ga-Eon; Lee, Ji Shin; Lee, Kun Ho; Lee, Yong-Seok; Kim, Jung-Woong; Rhee, Sangmyung; Song, Woo Keun

    2017-09-01

    Biomechanical remodeling of stroma by cancer-associated fibroblasts (CAF) in early stages of cancer is critical for cancer progression, and mechanical cues such as extracellular matrix stiffness control cell differentiation and malignant progression. However, the mechanism by which CAF activation occurs in low stiffness stroma in early stages of cancer is unclear. Here, we investigated the molecular mechanism underlying CAF regulation by SPIN90 and microtubule acetylation under conditions of mechanically soft matrices corresponding to normal stromal rigidity. SPIN90 was downregulated in breast cancer stroma but not tumor, and this low stromal expression correlated with decreased survival in breast cancer patients. Spin90 deficiency facilitated recruitment of mDia2 and APC complex to microtubules, resulting in increased microtubule acetylation. This increased acetylation promoted nuclear localization of YAP, which upregulated expression of myofibroblast marker genes on soft matrices. Spin90 depletion enhanced tumor progression, and blockade of microtubule acetylation in CAF significantly inhibited tumor growth in mice. Together, our data demonstrate that loss of SPIN90-mediated microtubule acetylation is a key step in CAF activation in low stiffness stroma. Moreover, correlation among these factors in human breast cancer tissue supports the clinical relevance of SPIN90 and microtubule acetylation in tumor development. Cancer Res; 77(17); 4710-22. ©2017 AACR. ©2017 American Association for Cancer Research.

  15. Drosophila spastin regulates synaptic microtubule networks and is required for normal motor function.

    Directory of Open Access Journals (Sweden)

    Nina Tang Sherwood

    2004-12-01

    Full Text Available The most common form of human autosomal dominant hereditary spastic paraplegia (AD-HSP is caused by mutations in the SPG4 (spastin gene, which encodes an AAA ATPase closely related in sequence to the microtubule-severing protein Katanin. Patients with AD-HSP exhibit degeneration of the distal regions of the longest axons in the spinal cord. Loss-of-function mutations in the Drosophila spastin gene produce larval neuromuscular junction (NMJ phenotypes. NMJ synaptic boutons in spastin mutants are more numerous and more clustered than in wild-type, and transmitter release is impaired. spastin-null adult flies have severe movement defects. They do not fly or jump, they climb poorly, and they have short lifespans. spastin hypomorphs have weaker behavioral phenotypes. Overexpression of Spastin erases the muscle microtubule network. This gain-of-function phenotype is consistent with the hypothesis that Spastin has microtubule-severing activity, and implies that spastin loss-of-function mutants should have an increased number of microtubules. Surprisingly, however, we observed the opposite phenotype: in spastin-null mutants, there are fewer microtubule bundles within the NMJ, especially in its distal boutons. The Drosophila NMJ is a glutamatergic synapse that resembles excitatory synapses in the mammalian spinal cord, so the reduction of organized presynaptic microtubules that we observe in spastin mutants may be relevant to an understanding of human Spastin's role in maintenance of axon terminals in the spinal cord.

  16. Structure of growing microtubule ends: two-dimensional sheets close into tubes at variable rates.

    Science.gov (United States)

    Chrétien, D; Fuller, S D; Karsenti, E

    1995-06-01

    Observation of microtubule growth at different rates by cryo-electron microscopy reveals that the ends range from blunt to long, gently curved sheets. The mean sheet length increases with the growth rate while the width of the distributions increases with the extent of assembly. The combination of a concentration dependent growth rate of the tubulin sheet with a variable closure rate of the microtubule cylinder, results in a model in which stochastic fluctuations in sheet length and tubulin conformation confine GTP-tubulins to microtubule ends. We propose that the variability of microtubule growth rate observed by video microscopy (Gildersleeve, R. F., A. R. Cross, K. E. Cullen, A. P. Fagen, and R. C. Williams. 1992. J. Biol. Chem. 267: 7995-8006, and this study) is due to the variation in the rate of cylinder closure. The curvature of the sheets at the end of growing microtubules and the small oligomeric structures observed at the end of disassembling microtubules, indicate that tubulin molecules undergo conformational changes both during assembly and disassembly.

  17. [Coordination and donation].

    Science.gov (United States)

    Elizalde, J; Lorente, M

    2006-01-01

    The progressive incorporation of organ transplants as a therapeutic resource resulted in organisational adaptation and overall transplant management, leading to the emergence of the figure of the transplant coordinator in the mid-1980s. In Spain, the National Organisation of Transplants (Organización Nacional de Transplantes - ONT) was created, establishing a system - called the "Spanish model" - based on a network of coordinators at three levels: national, the autonomous community and the hospital. This organisational structure is a point of reference at the world level. The prevalence of the Intensive Medicine specialisation amongst hospital transplant coordinators is remarkable. The majority of organs proceed from brain-dead patients with beating hearts and this requires the infrastructure offered by intensive care units. The functions of the coordinator can be summarised in guaranteeing a synchrony of all the elements and teams that come together in an organisational chain that has come to be called the "process of donation". Schematically, the crucial points that the hospital coordinator develops are the following: - Detection of the potential donor. - Maintenance of the donor. - Diagnosis of brain death. - Family consent. - Preparation of the hospital logistics. - Helping the relatives. - Direct involvement in the Program of Guarantee of Quality. - Person of reference in any activity related to the transplant. It would be desirable to achieve the creation of transplant coordination teams, with univocal messages, professionalism and a permanent input of the so-called "human factor", which is so necessary and also so close to the transplant world.

  18. Continuous parallel coordinates.

    Science.gov (United States)

    Heinrich, Julian; Weiskopf, Daniel

    2009-01-01

    Typical scientific data is represented on a grid with appropriate interpolation or approximation schemes,defined on a continuous domain. The visualization of such data in parallel coordinates may reveal patterns latently contained in the data and thus can improve the understanding of multidimensional relations. In this paper, we adopt the concept of continuous scatterplots for the visualization of spatially continuous input data to derive a density model for parallel coordinates. Based on the point-line duality between scatterplots and parallel coordinates, we propose a mathematical model that maps density from a continuous scatterplot to parallel coordinates and present different algorithms for both numerical and analytical computation of the resulting density field. In addition, we show how the 2-D model can be used to successively construct continuous parallel coordinates with an arbitrary number of dimensions. Since continuous parallel coordinates interpolate data values within grid cells, a scalable and dense visualization is achieved, which will be demonstrated for typical multi-variate scientific data.

  19. Magnetic Coordinate Systems

    Science.gov (United States)

    Laundal, K. M.; Richmond, A. D.

    2017-03-01

    Geospace phenomena such as the aurora, plasma motion, ionospheric currents and associated magnetic field disturbances are highly organized by Earth's main magnetic field. This is due to the fact that the charged particles that comprise space plasma can move almost freely along magnetic field lines, but not across them. For this reason it is sensible to present such phenomena relative to Earth's magnetic field. A large variety of magnetic coordinate systems exist, designed for different purposes and regions, ranging from the magnetopause to the ionosphere. In this paper we review the most common magnetic coordinate systems and describe how they are defined, where they are used, and how to convert between them. The definitions are presented based on the spherical harmonic expansion coefficients of the International Geomagnetic Reference Field (IGRF) and, in some of the coordinate systems, the position of the Sun which we show how to calculate from the time and date. The most detailed coordinate systems take the full IGRF into account and define magnetic latitude and longitude such that they are constant along field lines. These coordinate systems, which are useful at ionospheric altitudes, are non-orthogonal. We show how to handle vectors and vector calculus in such coordinates, and discuss how systematic errors may appear if this is not done correctly.

  20. Linear shaped charge

    Energy Technology Data Exchange (ETDEWEB)

    Peterson, David; Stofleth, Jerome H.; Saul, Venner W.

    2017-07-11

    Linear shaped charges are described herein. In a general embodiment, the linear shaped charge has an explosive with an elongated arrowhead-shaped profile. The linear shaped charge also has and an elongated v-shaped liner that is inset into a recess of the explosive. Another linear shaped charge includes an explosive that is shaped as a star-shaped prism. Liners are inset into crevices of the explosive, where the explosive acts as a tamper.

  1. Cardiac-specific deletion of the microtubule-binding protein CENP-F causes dilated cardiomyopathy

    Directory of Open Access Journals (Sweden)

    Ellen Dees

    2012-07-01

    CENP-F is a large multifunctional protein with demonstrated regulatory roles in cell proliferation, vesicular transport and cell shape through its association with the microtubule (MT network. Until now, analysis of CENP-F has been limited to in vitro analysis. Here, using a Cre-loxP system, we report the in vivo disruption of CENP-F gene function in murine cardiomyocytes, a cell type displaying high levels of CENP-F expression. Loss of CENP-F function in developing myocytes leads to decreased cell division, blunting of trabeculation and an initially smaller, thin-walled heart. Still, embryos are born at predicted mendelian ratios on an outbred background. After birth, hearts lacking CENP-F display disruption of their intercalated discs and loss of MT integrity particularly at the costamere; these two structures are essential for cell coupling/electrical conduction and force transduction in the heart. Inhibition of myocyte proliferation and cell coupling as well as loss of MT maintenance is consistent with previous reports of generalized CENP-F function in isolated cells. One hundred percent of these animals develop progressive dilated cardiomyopathy with heart block and scarring, and there is a 20% mortality rate. Importantly, although it has long been postulated that the MT cytoskeleton plays a role in the development of heart disease, this study is the first to reveal a direct genetic link between disruption of this network and cardiomyopathy. Finally, this study has broad implications for development and disease because CENP-F loss of function affects a diverse array of cell-type-specific activities in other organs.

  2. 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.

  3. Microtubule-based nanomaterials: Exploiting nature's dynamic biopolymers.

    Science.gov (United States)

    Bachand, George D; Spoerke, Erik D; Stevens, Mark J

    2015-06-01

    For more than a decade now, biomolecular systems have served as an inspiration for the development of synthetic nanomaterials and systems that are capable of reproducing many of unique and emergent behaviors of living systems. One intriguing element of such systems may be found in a specialized class of proteins known as biomolecular motors that are capable of performing useful work across multiple length scales through the efficient conversion of chemical energy. Microtubule (MT) filaments may be considered within this context as their dynamic assembly and disassembly dissipate energy, and perform work within the cell. MTs are one of three cytoskeletal filaments in eukaryotic cells, and play critical roles in a range of cellular processes including mitosis and vesicular trafficking. Based on their function, physical attributes, and unique dynamics, MTs also serve as a powerful archetype of a supramolecular filament that underlies and drives multiscale emergent behaviors. In this review, we briefly summarize recent efforts to generate hybrid and composite nanomaterials using MTs as biomolecular scaffolds, as well as computational and synthetic approaches to develop synthetic one-dimensional nanostructures that display the enviable attributes of the natural filaments.

  4. Feedback Mechanism for Microtubule Length Regulation by Stathmin Gradients

    Science.gov (United States)

    Zeitz, Maria; Kierfeld, Jan

    2014-12-01

    We formulate and analyze a theoretical model for the regulation of microtubule (MT) polymerization dynamics by the signaling proteins Rac1 and stathmin. In cells, the MT growth rate is inhibited by cytosolic stathmin, which, in turn, is inactivated by Rac1. Growing MTs activate Rac1 at the cell edge, which closes a positive feedback loop. We investigate both tubulin sequestering and catastrophe promotion as mechanisms for MT growth inhibition by stathmin. For a homogeneous stathmin concentration in the absence of Rac1, we find a switch-like regulation of the MT mean length by stathmin. For constitutively active Rac1 at the cell edge, stathmin is deactivated locally, which establishes a spatial gradient of active stathmin. In this gradient, we find a stationary bimodal MT length distributions for both mechanisms of MT growth inhibition by stathmin. One subpopulation of the bimodal length distribution can be identified with fast growing and long pioneering MTs in the region near the cell edge, which have been observed experimentally. The feedback loop is closed through Rac1 activation by MTs. For tubulin sequestering by stathmin, this establishes a bistable switch with two stable states: one stable state corresponds to upregulated MT mean length and bimodal MT length distributions, i.e., pioneering MTs; the other stable state corresponds to an interrupted feedback with short MTs. Stochastic effects as well as external perturbations can trigger switching events. For catastrophe promoting stathmin we do not find bistability.

  5. Microtubule dynamics control HGF-induced lung endothelial barrier enhancement.

    Directory of Open Access Journals (Sweden)

    Xinyong Tian

    Full Text Available Microtubules (MT play a vital role in many cellular functions, but their role in peripheral actin cytoskeletal dynamics which is essential for control of endothelial barrier and monolayer integrity is less understood. We have previously described the enhancement of lung endothelial cell (EC barrier by hepatocyte growth factor (HGF which was associated with Rac1-mediated remodeling of actin cytoskeleton. This study investigated involvement of MT-dependent mechanisms in the HGF-induced enhancement of EC barrier. HGF-induced Rac1 activation was accompanied by phosphorylation of stathmin, a regulator of MT dynamics. HGF also stimulated MT peripheral growth monitored by time lapse imaging and tracking analysis of EB-1-decorated MT growing tips, and increased the pool of acetylated tubulin. These effects were abolished by EC pretreatment with HGF receptor inhibitor, downregulation of Rac1 pathway, or by expression of a stathmin-S63A phosphorylation deficient mutant. Expression of stathmin-S63A abolished the HGF protective effects against thrombin-induced activation of RhoA cascade, permeability increase, and EC barrier dysfunction. These results demonstrate a novel MT-dependent mechanism of HGF-induced EC barrier regulation via Rac1/PAK1/stathmin-dependent control of MT dynamics.

  6. 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.

  7. Microtubule length dependence of motor traffic in cells.

    Science.gov (United States)

    Zhang, Yunxin

    2012-10-01

    Motor proteins in living cells, such as kinesin and dynein, can move processively along the microtubule (MT), and can also detach from or attach to MT stochastically. Experiments found that the traffic of motors along MT may be jammed; thus various theoretical models were designed to understand this process. However, previous studies mainly focused on motor attachment/detachment rate dependent properties. Leduc et al. recently found that the traffic jam of motor protein Kip3 depends on MT length (Proc. Natl. Acad. Sci. U.S.A. 109, 6100 (2012)). Therefore, this study discusses the MT length-dependent properties of motor traffic. The results showed that MT length has one critical value N(c); a traffic jam occurs only when MT length N > N(c). The jammed MT length increases with total MT length N , whereas the non-jammed MT length may not change monotonically with N . The critical value N(c) increases with motor detachment rate from MT, but decreases with motor attachment rate to MT. Therefore, the traffic of motors will be more likely to be jammed when the MT is long, motor detachment rate is high, and motor detachment rate is low.

  8. Microtubule length dependence of motor traffic in cells

    CERN Document Server

    Zhang, Yunxin

    2012-01-01

    In living cells, motor proteins, such as kinesin and dynein can move processively along microtubule (MT), and also detach from or attach to MT stochastically. Experiments have found that, the traffic of motor might be jammed, and various theoretical models have been designed to understand this traffic jam phenomenon. But previous studies mainly focus on motor attachment/detachment rate dependent properties. Recent experiment of Leduc {\\it et al.} found that the traffic jam formation of motor protein kinesin depends also on the length of MT [Proc. Natl. Acad. Sci. U.S.A. {\\bf 109}, 6100-6105 (2012)]. In this study, the MT length dependent properties of motor traffic will be analyzed. We found that MT length has one {\\it critical value} $N_c$, traffic jam occurs only when MT length $N>N_c$. The jammed length of MT increases with total MT length, while the non-jammed MT length might not change monotonically with the total MT length. The critical value $N_c$ increases with motor detachment rate from MT, but decre...

  9. Toxicity and interaction of titanium dioxide nanoparticles with microtubule protein

    Institute of Scientific and Technical Information of China (English)

    Zahra Naghdi Gheshlaghi; Gholam Hossein Riazi; Shahin Ahmadian; Mahmoud Ghafari; Roya Mahinpour

    2008-01-01

    Titanium dioxide (TiO2) nanoparticles (NPs) are widely used in several manufactured products. The small size of NPs facilitates their uptake into cells as well as transcytosis across epithelial cells into blood and lymph circulation to reach different sites, such as the central nervous system. Different studies have shown the risks that TiO2 NPs in the neuronal system and other organs present. As membranebound layer aggregates or single particles, TiO2 NPs can enter not only cells, but also mitochondria and nuclei.Therefore these particles can interact with cytoplasmic proteins such as microtubules (MTs). MTs are cytoskeletal proteins that are essential in eukaryotic cells for a variety of functions, such as cellular transport, cell motility and mitosis. MTs in neurons are used to transport substances such as neurotransmitters. Single TiO2 NPs in cytoplasm can interact with these proteins and affect their crucial functions in different tissues. In this study, we showed the effects of TiO2 NPs on MT polymerization and structure using ultraviolet spectrophotometer and fluorometry. The fluorescent spectroscopy showed a significant tubulin conformational change in the presence of TiO2 NPs and the ultraviolet spectroscopy results showed that TiO2 NPs affect tubulin polymerization and decrease it. The aim of this study was to find the potential risks that TiO2 NPs pose to human organs and cells.

  10. Microtubule-stabilizing agents as potential therapeutics for neurodegenerative disease.

    Science.gov (United States)

    Brunden, Kurt R; Trojanowski, John Q; Smith, Amos B; Lee, Virginia M-Y; Ballatore, Carlo

    2014-09-15

    Microtubules (MTs), cytoskeletal elements found in all mammalian cells, play a significant role in cell structure and in cell division. They are especially critical in the proper functioning of post-mitotic central nervous system neurons, where MTs serve as the structures on which key cellular constituents are trafficked in axonal projections. MTs are stabilized in axons by the MT-associated protein tau, and in several neurodegenerative diseases, including Alzheimer's disease, frontotemporal lobar degeneration, and Parkinson's disease, tau function appears to be compromised due to the protein dissociating from MTs and depositing into insoluble inclusions referred to as neurofibrillary tangles. This loss of tau function is believed to result in alterations of MT structure and function, resulting in aberrant axonal transport that likely contributes to the neurodegenerative process. There is also evidence of axonal transport deficiencies in other neurodegenerative diseases, including amyotrophic lateral sclerosis and Huntington's disease, which may result, at least in part, from MT alterations. Accordingly, a possible therapeutic strategy for such neurodegenerative conditions is to treat with MT-stabilizing agents, such as those that have been used in the treatment of cancer. Here, we review evidence of axonal transport and MT deficiencies in a number of neurodegenerative diseases, and summarize the various classes of known MT-stabilizing agents. Finally, we highlight the growing evidence that small molecule MT-stabilizing agents provide benefit in animal models of neurodegenerative disease and discuss the desired features of such molecules for the treatment of these central nervous system disorders.

  11. Enantioselective effects of (+)- and (-)-citronellal on animal and plant microtubules.

    Science.gov (United States)

    Altshuler, Osnat; Abu-Abied, Mohamad; Chaimovitsh, David; Shechter, Alona; Frucht, Hilla; Dudai, Nativ; Sadot, Einat

    2013-09-27

    Citronellal is a major component of Corymbia citriodora and Cymbopogon nardus essential oils. Herein it is shown that whereas (+)-citronellal (1) is an effective microtubule (MT)-disrupting compound, (-)-citronellal (2) is not. Quantitative image analysis of fibroblast cells treated with 1 showed total fluorescence associated with fibers resembling that in cells treated with the MT-disrupting agents colchicine and vinblastine; in the presence of 2, the fluorescence more closely resembled that in control cells. The distribution of tubulin in soluble and insoluble fractions in the presence of 1 also resembled that in the presence of colchicine, whereas similar tubulin distribution was obtained in the presence of 2 and in control cells. In vitro polymerization of MTs was inhibited by 1 but not 2. Measurements of MT dynamics in plant cells showed similar MT elongation and shortening rates in control and 2-treated cells, whereas in the presence of 1, much fewer and shorter MTs were observed and no elongation or shrinkage was detected. Taken together, the MT system is suggested to be able to discriminate between different enantiomers of the same compound. In addition, the activity of essential oils rich in citronellal is affected by the relative content of the two enantiomers of this monoterpenoid.

  12. Microtubule modification influences cellular response to amyloid-β exposure

    Directory of Open Access Journals (Sweden)

    Nicole Shamitko-Klingensmith

    2016-05-01

    Full Text Available During the normal aging process, cytoskeletal changes such as a reduction in density or disruption of cytoskeletal components occur that can affect neuronal function. As aging is the biggest risk factor for Alzheimer's disease (AD, this study sought to determine how microtubule (MT modification influences cellular response upon exposure to β-amyloid1-42 (Aβ1-42, which is implicated in AD. The MT networks of hypothalamic GT1-7 neurons were modified by common disrupting or stabilizing drugs, and then the physical and mechanical properties of the modified neurons were determined. The MT modified neurons were then exposed to Aβ1-42 and the ability of the neurons to cope with this exposure was determined by a variety of biochemical assays. Flow cytometry studies indicated that MT disruption reduced the binding of Aβ1-42 to the plasma membrane by 45% per cell compared to neurons with stabilized or unaltered MTs. Although the cells with disrupted MTs experienced less peptide-membrane binding, they experienced similar or increased levels of cytotoxicity caused by the Aβ1-42 exposure. In contrast, MT stabilization delayed toxicity caused by Aβ1-42. These results demonstrate that MT modification significantly influences the ability of neurons to cope with toxicity induced by Aβ1-42.

  13. 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.

  14. 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.

  15. Dynein and mast/orbit/CLASP have antagonistic roles in regulating kinetochore-microtubule plus-end dynamics.

    Science.gov (United States)

    Reis, Rita; Feijão, Tália; Gouveia, Susana; Pereira, António J; Matos, Irina; Sampaio, Paula; Maiato, Helder; Sunkel, Claudio E

    2009-07-15

    Establishment and maintenance of the mitotic spindle requires the balanced activity of microtubule-associated proteins and motors. In this study we have addressed how the microtubule plus-end tracking protein mast/orbit/CLASP and cytoplasmic dynein regulate this process in Drosophila melanogaster embryos and S2 cells. We show that mast accumulates at kinetochores early in mitosis, which is followed by a poleward streaming upon microtubule attachment. This leads to a reduction of mast levels at kinetochores during metaphase and anaphase that depends largely on the microtubule minus end-directed motor cytoplasmic dynein. Surprisingly, we also found that co-depletion of dynein rescues spindle bipolarity in mast-depleted cells, while restoring normal microtubule poleward flux. Our results suggest that mast and dynein have antagonistic roles in the local regulation of microtubule plus-end dynamics at kinetochores, which are important for the maintenance of spindle bipolarity and normal spindle length.

  16. 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.

  17. The N-DRC forms a conserved biochemical complex that maintains outer doublet alignment and limits microtubule sliding in motile axonemes.

    Science.gov (United States)

    Bower, Raqual; Tritschler, Douglas; Vanderwaal, Kristyn; Perrone, Catherine A; Mueller, Joshua; Fox, Laura; Sale, Winfield S; Porter, M E

    2013-04-01

    The nexin-dynein regulatory complex (N-DRC) is proposed to coordinate dynein arm activity and interconnect doublet microtubules. Here we identify a conserved region in DRC4 critical for assembly of the N-DRC into the axoneme. At least 10 subunits associate with DRC4 to form a discrete complex distinct from other axonemal substructures. Transformation of drc4 mutants with epitope-tagged DRC4 rescues the motility defects and restores assembly of missing DRC subunits and associated inner-arm dyneins. Four new DRC subunits contain calcium-signaling motifs and/or AAA domains and are nearly ubiquitous in species with motile cilia. However, drc mutants are motile and maintain the 9 + 2 organization of the axoneme. To evaluate the function of the N-DRC, we analyzed ATP-induced reactivation of isolated axonemes. Rather than the reactivated bending observed with wild-type axonemes, ATP addition to drc-mutant axonemes resulted in splaying of doublets in the distal region, followed by oscillatory bending between pairs of doublets. Thus the N-DRC provides some but not all of the resistance to microtubule sliding and helps to maintain optimal alignment of doublets for productive flagellar motility. These findings provide new insights into the mechanisms that regulate motility and further highlight the importance of the proximal region of the axoneme in generating flagellar bending.

  18. Disruption of microtubules in rat skeletal muscle does not inhibit insulin- or contraction-stimulated glucose transport

    DEFF Research Database (Denmark)

    Ai, Hua; Ralston, Evelyn; Lauritzen, Hans P M M

    2003-01-01

    or epitrochlearis muscles. In contrast, nocodazole, another microtubule-disrupting drug, rapidly and dose dependently blocked insulin- and contraction-stimulated glucose transport. A similar discrepancy between colchicine and nocodazole was also found in their ability to block glucose transport in muscle giant...... "ghost" vesicles. This suggests that the ability of insulin and contractions to stimulate glucose transport in muscle does not require an intact microtubule network and that nocodazole inhibits glucose transport independently of its microtubule-disrupting effect....

  19. Analysis of the hominoid os coxae by Cartesian coordinates.

    Science.gov (United States)

    McHenry, H M; Corruccini, R S

    1978-02-01

    This study is based upon 48 3-dimensional coordinates taken on 4 fossil hominid and 127 extant hominoid coxal bones. The follis include Sts 14, SK 3155, MLD 7, and MLD 25. The comparative sample consists of 42 Homo sapiens, 27 Pan troglodytes, 29 Gorilla gorilla and 29 Pongo pygmaeus. The coordinates improve the metrical representation of the bone beyond what can be done with linear measurements because the shape complexity of the os coxae is so great. The coordinates are rotated and translated so that all bones are in a standard position. The coordinates are then standardized for each specimen by dividing all coordinates by the pooled standard deviation of X, Y, and Z coordinates. These data are treated to standard statistical analyses including analysis of variance, Penrose size and shape statistics, principal coordinates and components, and canonical variates analysis. The data are then further altered by using some specimen as a standard and rotating each specimen until the total squared distance between its coordinates and those of the standard are minimized. The same statistics are applied to these "best fit" data. The results show a high degree of agreement between the methods. The hominid os coxae are dundamentally different from the other hominoids and the fossil hominids share the basic hominid configuration but with some unique differences.

  20. Distinct pose of discodermolide in taxol binding pocket drives a complementary mode of microtubule stabilization.

    Science.gov (United States)

    Khrapunovich-Baine, Marina; Menon, Vilas; Verdier-Pinard, Pascal; Smith, Amos B; Angeletti, Ruth Hogue; Fiser, Andras; Horwitz, Susan Band; Xiao, Hui

    2009-12-15

    The microtubule cytoskeleton has proven to be an effective target for cancer therapeutics. One class of drugs, known as microtubule stabilizing agents (MSAs), binds to microtubule polymers and stabilizes them against depolymerization. The prototype of this group of drugs, Taxol, is an effective chemotherapeutic agent used extensively in the treatment of human ovarian, breast, and lung carcinomas. Although electron crystallography and photoaffinity labeling experiments determined that the binding site for Taxol is in a hydrophobic pocket in beta-tubulin, little was known about the effects of this drug on the conformation of the entire microtubule. A recent study from our laboratory utilizing hydrogen-deuterium exchange (HDX) in concert with various mass spectrometry (MS) techniques has provided new information on the structure of microtubules upon Taxol binding. In the current study we apply this technique to determine the binding mode and the conformational effects on chicken erythrocyte tubulin (CET) of another MSA, discodermolide, whose synthetic analogues may have potential use in the clinic. We confirmed that, like Taxol, discodermolide binds to the taxane binding pocket in beta-tubulin. However, as opposed to Taxol, which has major interactions with the M-loop, discodermolide orients itself away from this loop and toward the N-terminal H1-S2 loop. Additionally, discodermolide stabilizes microtubules mainly via its effects on interdimer contacts, specifically on the alpha-tubulin side, and to a lesser extent on interprotofilament contacts between adjacent beta-tubulin subunits. Also, our results indicate complementary stabilizing effects of Taxol and discodermolide on the microtubules, which may explain the synergy observed between the two drugs in vivo.