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Sample records for microtubule assembly dynamics

  1. Profilin connects actin assembly with microtubule dynamics

    PubMed Central

    Nejedla, Michaela; Sadi, Sara; Sulimenko, Vadym; de Almeida, Francisca Nunes; Blom, Hans; Draber, Pavel; Aspenström, Pontus; Karlsson, Roger

    2016-01-01

    Profilin controls actin nucleation and assembly processes in eukaryotic cells. Actin nucleation and elongation promoting factors (NEPFs) such as Ena/VASP, formins, and WASP-family proteins recruit profilin:actin for filament formation. Some of these are found to be microtubule associated, making actin polymerization from microtubule-associated platforms possible. Microtubules are implicated in focal adhesion turnover, cell polarity establishment, and migration, illustrating the coupling between actin and microtubule systems. Here we demonstrate that profilin is functionally linked to microtubules with formins and point to formins as major mediators of this association. To reach this conclusion, we combined different fluorescence microscopy techniques, including superresolution microscopy, with siRNA modulation of profilin expression and drug treatments to interfere with actin dynamics. Our studies show that profilin dynamically associates with microtubules and this fraction of profilin contributes to balance actin assembly during homeostatic cell growth and affects micro­tubule dynamics. Hence profilin functions as a regulator of microtubule (+)-end turnover in addition to being an actin control element. PMID:27307590

  2. Micropattern-Guided Assembly of Overlapping Pairs of Dynamic Microtubules

    PubMed Central

    Fourniol, Franck J.; Li, Tai-De; Bieling, Peter; Mullins, R. Dyche; Fletcher, Daniel A.; Surrey, Thomas

    2014-01-01

    Interactions between antiparallel microtubules are essential for the organization of spindles in dividing cells. The ability to form immobilized antiparallel microtubule pairs in vitro, combined with the ability to image them via TIRF microscopy, permits detailed biochemical characterization of microtubule cross-linking proteins and their effects on microtubule dynamics. Here, we describe methods for chemical micropatterning of microtubule seeds on glass surfaces in configurations that specifically promote the formation of antiparallel microtubule overlaps in vitro. We demonstrate that this assay is especially well suited for reconstitution of minimal midzone overlaps stabilized by the antiparallel microtubule cross-linking protein PRC1 and its binding partners. The micropatterning method is suitable for use with a broad range of proteins, and the assay is generally applicable to any microtubule cross-linking protein. PMID:24630116

  3. Microtubule dynamics and organization

    NASA Astrophysics Data System (ADS)

    Dogterom, Marileen

    2000-03-01

    Microtubules are rigid biopolymers found in all higher order cells. They are a mayor part of the cytoskeleton, the network of protein polymers that gives the cell its shape and rigidity and allows for various forms of (intra)cellular motility. The intracellular spatial organization of the microtubule network is constantly changing as the microtubules adapt to their different functions. In part, this spatial organization depends on the assembly dynamics (including microtubule nucleation) and forces generated by the microtubules themselves. To understand these mechanisms, we study the physical aspects connected with the assembly, force generation and spatial organization of microtubules in simplified model systems, in the absence of other cellular components. We measure the forces generated by individual microtubules by making them grow against a microfabricated barrier. These experiments show that a single microtubule can generate at least several picoNewton of force, comparable to what is known for motor proteins. Theoretical modeling of force-generation by multi-protofilament polymers is used to predict force-velocity relations that can be compared to experimental data. We study the self-organization of microtubules by confining them to microfabricated chambers that mimic the geometry of living cells. The distribution of microtubule nucleation sites in these chambers is controlled to study its effect on the organization of the microtubule network. We find that so-called microtubule asters position themselves in response to forces generated by dynamic microtubules. Experiments aimed at measuring the forces acting on these asters using optical trapping techniques will be described.

  4. The Dynamics of Microtubule/Motor-Protein Assemblies in Biology and Physics

    NASA Astrophysics Data System (ADS)

    Shelley, Michael J.

    2016-01-01

    Many important processes in the cell are mediated by stiff microtubule polymers and the active motor proteins moving on them. This includes the transport of subcellular structures (nuclei, chromosomes, organelles) and the self-assembly and positioning of the mitotic spindle. Little is understood of these processes, but they present fascinating problems in fluid-structure interactions. Microtubules and motor proteins are also the building blocks of new biosynthetic active suspensions driven by motor-protein activity. These reduced systems can be probed—and modeled—more easily than can the fully biological ones and demonstrate their own aspects of self-assembly and complex dynamics. I review recent work modeling such systems as fluid-structure interaction problems and as multiscale complex fluids.

  5. In vivo microtubule dynamics during experimentally induced conversions between tubulin assembly states in Allogromia laticollaris.

    PubMed

    Welnhofer, E A; Travis, J L

    1996-01-01

    A distinctive property of foraminiferan tubulin is that, in addition to microtubules (MTs), it exists in an alternate assembly state, helical filaments. Here, we have examined in vivo MT dynamics during experimentally induced conversions between these two assembly states in the reticulopods of the marine foraminiferan Allogromia laticollaris. Exposure to high extracellular concentrations of Mg2+ (165 mM) resulted in a complete conversion of MTs into helical filaments. However, Mg2+ treatment also induced a retrograde movement of organelles and cytoplasm, and it was necessary to inhibit this response in order to assess the effects of assembly state changes on individual MTs. This was accomplished by simultaneous treatment with high extracellular Mg2+ and 2,4-dinitrophenol (DNP). The resulting loss in MTs was detected by video enhanced DIC (VEC-DIC) microscopy as either an endwise MT shortening (at an average rate of 474 microns/min) or transformation into one or more irregularly shaped fibrils, which we termed residual fibrils. Correlative immunofluorescence and video microscopy showed residual fibrils to be composed of helical filaments. Removal of extracellular Mg2+/DNP initiated a reversal in assembly state, from helical filaments into MTs, which was completed within 5 min. VEC-DIC microscopy showed that MTs reformed by an endwise lengthening at an average rate of 216 microns/min. These results suggest that conversion between alternate tubulin assembly states provides a more rapid means to build and dismantle MTs than conventional subunit-driven pathways.

  6. Dynamic assembly of polymer nanotube networks via kinesin powered microtubule filaments

    DOE PAGES

    Paxton, Walter F.; Bachand, George D.; Gomez, Andrew; ...

    2015-04-24

    In this study, we describe for the first time how biological nanomotors may be used to actively self-assemble mesoscale networks composed of diblock copolymer nanotubes. The collective force generated by multiple kinesin nanomotors acting on a microtubule filament is large enough to overcome the energy barrier required to extract nanotubes from polymer vesicles comprised of poly(ethylene oxide-b-butadiene) in spite of the higher force requirements relative to extracting nanotubes from lipid vesicles. Nevertheless, large-scale polymer networks were dynamically assembled by the motors. These networks displayed enhanced robustness, persisting more than 24 h post-assembly (compared to 4–5 h for corresponding lipid networks).more » The transport of materials in and on the polymer membranes differs substantially from the transport on analogous lipid networks. Specifically, our data suggest that polymer mobility in nanotubular structures is considerably different from planar or 3D structures, and is stunted by 1D confinement of the polymer subunits. Moreover, quantum dots adsorbed onto polymer nanotubes are completely immobile, which is related to this 1D confinement effect and is in stark contrast to the highly fluid transport observed on lipid tubules.« less

  7. Dynamic assembly of polymer nanotube networks via kinesin powered microtubule filaments

    SciTech Connect

    Paxton, Walter F.; Bachand, George D.; Gomez, Andrew; Henderson, Ian M.; Bouxsein, Nathan F.

    2015-04-24

    In this study, we describe for the first time how biological nanomotors may be used to actively self-assemble mesoscale networks composed of diblock copolymer nanotubes. The collective force generated by multiple kinesin nanomotors acting on a microtubule filament is large enough to overcome the energy barrier required to extract nanotubes from polymer vesicles comprised of poly(ethylene oxide-b-butadiene) in spite of the higher force requirements relative to extracting nanotubes from lipid vesicles. Nevertheless, large-scale polymer networks were dynamically assembled by the motors. These networks displayed enhanced robustness, persisting more than 24 h post-assembly (compared to 4–5 h for corresponding lipid networks). The transport of materials in and on the polymer membranes differs substantially from the transport on analogous lipid networks. Specifically, our data suggest that polymer mobility in nanotubular structures is considerably different from planar or 3D structures, and is stunted by 1D confinement of the polymer subunits. Moreover, quantum dots adsorbed onto polymer nanotubes are completely immobile, which is related to this 1D confinement effect and is in stark contrast to the highly fluid transport observed on lipid tubules.

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

    PubMed

    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.

  9. Assembly and Positioning of Microtubule Asters in Microfabricated Chambers

    NASA Astrophysics Data System (ADS)

    Holy, Timothy E.; Dogterom, Marileen; Yurke, Bernard; Leibler, Stanislas

    1997-06-01

    Intracellular organization depends on a variety of molecular assembly processes; while some of these have been studied in simplified cell-free systems, others depend on the confined geometry of cells and cannot be reconstructed using bulk techniques. To study the latter processes in vitro, we fabricated microscopic chambers that simulate the closed environment of cells. We used these chambers to study the positioning of microtubule asters. Microtubule assembly alone, without the action of molecular motors, is sufficient to position asters. Asters with short microtubules move toward the position expected from symmetry; however, once the microtubules become long enough to buckle, symmetry is broken. Calculations and experiments show that the bending-energy landscape has multiple minima. Microtubule dynamic instability modifies the landscape over time and allows asters to explore otherwise inaccessible configurations.

  10. Microtubule dynamics in fish melanophores

    PubMed Central

    1994-01-01

    We have studied the dynamics of microtubules in black tetra (Gymnocorymbus ternetzi) melanophores to test the possible correlation of microtubule stability and intracellular particle transport. X- rhodamine-or caged fluorescein-conjugated tubulin were microinjected and visualized by fluorescence digital imaging using a cooled charge coupled device and videomicroscopy. Microtubule dynamics were evaluated by determining the time course of tubulin incorporation after pulse injection, by time lapse observation, and by quantitation of fluorescence redistribution after photobleaching and photoactivation. The time course experiments showed that the kinetics of incorporation of labeled tubulin into microtubules were similar for cells with aggregated or dispersed pigment with most microtubules becoming fully labeled within 15-20 min after injection. Quantitation by fluorescence redistribution after photobleaching and photoactivation confirmed that microtubule turnover was rapid in both states, t1/2 = 3.5 +/- 1.5 and 6.1 +/- 3.0 min for cells with aggregated and dispersed pigment, respectively. In addition, immunostaining with antibodies specific to posttranslationally modified alpha-tubulin, which is usually enriched in stable microtubules, showed that microtubules composed exclusively of detyrosinated tubulin were absent and microtubules containing acetylated tubulin were sparse. We conclude that the microtubules of melanophores are very dynamic, that their dynamic properties do not depend critically on the state of pigment distribution, and that their stabilization is not a prerequisite for intracellular transport. PMID:8089178

  11. A coarse-grained model of microtubule self-assembly

    NASA Astrophysics Data System (ADS)

    Regmi, Chola; Cheng, Shengfeng

    Microtubules play critical roles in cell structures and functions. They also serve as a model system to stimulate the next-generation smart, dynamic materials. A deep understanding of their self-assembly process and biomechanical properties will not only help elucidate how microtubules perform biological functions, but also lead to exciting insight on how microtubule dynamics can be altered or even controlled for specific purposes such as suppressing the division of cancer cells. Combining all-atom molecular dynamics (MD) simulations and the essential dynamics coarse-graining method, we construct a coarse-grained (CG) model of the tubulin protein, which is the building block of microtubules. In the CG model a tubulin dimer is represented as an elastic network of CG sites, the locations of which are determined by examining the protein dynamics of the tubulin and identifying the essential dynamic domains. Atomistic MD modeling is employed to directly compute the tubulin bond energies in the surface lattice of a microtubule, which are used to parameterize the interactions between CG building blocks. The CG model is then used to study the self-assembly pathways, kinetics, dynamics, and nanomechanics of microtubules.

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

    NASA Astrophysics Data System (ADS)

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

    2015-12-01

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

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

    PubMed

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

    2015-01-01

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

  14. Dynamic microtubules and the texture of plant cell walls.

    PubMed

    Lloyd, Clive

    2011-01-01

    The relationship between microtubules and cell-wall texture has had a fitful history in which progress in one area has not been matched by progress in the other. For example, the idea that wall texture arises entirely from self-assembly, independently of microtubules, originated with electron microscopic analyses of fixed cells that gave no clue to the ability of microtubules to reorganize. Since then, live-cell studies have established the surprising dynamicity of plant microtubules involving collisions, changes in angle, parallelization, and rotation of microtubule tracks. Combined with proof that cellulose synthases do track along shifting microtubules, this offers more realistic models for the dynamic influence of microtubules on wall texture than could have been imagined in the electron microscopic era-the era from which most ideas on wall texture originate. This review revisits the classical literature on wall organization from the vantage point of current knowledge of microtubule dynamics.

  15. Microtubule dynamics in plant cells.

    PubMed

    Buschmann, Henrik; Sambade, Adrian; Pesquet, Edouard; Calder, Grant; Lloyd, Clive W

    2010-01-01

    This chapter describes some of the choices and unavoidable compromises to be made when studying microtubule dynamics in plant cells. The choice of species still depends very much on the ability to produce transgenic plants and most work has been done in the relatively small cells of Arabidopsis plants or in tobacco BY-2 suspension cells. Fluorescence-tagged microtubule proteins have been used to label entire microtubules, or their plus ends, but there are still few minus-end markers for these acentrosomal cells. Pragmatic decisions have to be made about probes, balancing the efficacy of microtubule labeling against a tendency to overstabilize and bundle the microtubules and even induce helical plant growth. A key limitation in visualizing plant microtubules is the ability to keep plants alive for long periods under the microscope and we describe a biochamber that allows for plant cell growth and development while allowing gas exchange and reducing evaporation. Another major difficulty is the limited fluorescence lifetime and we describe imaging strategies to reduce photobleaching in long-term imaging. We also discuss methods of measuring microtubule dynamics, with emphasis on the behavior of plant-specific microtubule arrays.

  16. Poleward microtubule flux mitotic spindles assembled in vitro

    PubMed Central

    1991-01-01

    In the preceding paper we described pathways of mitotic spindle assembly in cell-free extracts prepared from eggs of Xenopus laevis. Here we demonstrate the poleward flux of microtubules in spindles assembled in vitro, using a photoactivatable fluorescein covalently coupled to tubulin and multi-channel fluorescence videomicroscopy. After local photoactivation of fluorescence by UV microbeam, we observed poleward movement of fluorescein-marked microtubules at a rate of 3 microns/min, similar to rates of chromosome movement and spindle elongation during prometaphase and anaphase. This movement could be blocked by the addition of millimolar AMP-PNP but was not affected by concentrations of vanadate up to 150 microM, suggesting that poleward flux may be driven by a microtubule motor similar to kinesin. In contrast to previous results obtained in vivo (Mitchison, T. J. 1989. J. Cell Biol. 109:637-652), poleward flux in vitro appears to occur independently of kinetochores or kinetochore microtubules, and therefore may be a general property of relatively stable microtubules within the spindle. We find that microtubules moving towards poles are dynamic structures, and we have estimated the average half-life of fluxing microtubules in vitro to be between approximately 75 and 100 s. We discuss these results with regard to the function of poleward flux in spindle movements in anaphase and prometaphase. PMID:1999464

  17. A thermodynamic model of microtubule assembly and disassembly.

    PubMed

    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.

  18. Distinct roles for antiparallel microtubule pairing and overlap during early spindle assembly

    PubMed Central

    Nazarova, Elena; O'Toole, Eileen; Kaitna, Susi; Francois, Paul; Winey, Mark; Vogel, Jackie

    2013-01-01

    During spindle assembly, microtubules may attach to kinetochores or pair to form antiparallel pairs or interpolar microtubules, which span the two spindle poles and contribute to mitotic pole separation and chromosome segregation. Events in the specification of the interpolar microtubules are poorly understood. Using three-dimensional electron tomography and analysis of spindle dynamical behavior in living cells, we investigated the process of spindle assembly. Unexpectedly, we found that the phosphorylation state of an evolutionarily conserved Cdk1 site (S360) in γ-tubulin is correlated with the number and organization of interpolar microtubules. Mimicking S360 phosphorylation (S360D) results in bipolar spindles with a normal number of microtubules but lacking interpolar microtubules. Inhibiting S360 phosphorylation (S360A) results in spindles with interpolar microtubules and high-angle, antiparallel microtubule pairs. The latter are also detected in wild-type spindles <1 μm in length, suggesting that high-angle microtubule pairing represents an intermediate step in interpolar microtubule formation. Correlation of spindle architecture with dynamical behavior suggests that microtubule pairing is sufficient to separate the spindle poles, whereas interpolar microtubules maintain the velocity of pole displacement during early spindle assembly. Our findings suggest that the number of interpolar microtubules formed during spindle assembly is controlled in part through activities at the spindle poles. PMID:23966467

  19. Templated nanocrystal assembly on biodynamic artificial microtubule asters.

    PubMed

    Spoerke, Erik D; Boal, Andrew K; Bachand, George D; Bunker, Bruce C

    2013-03-26

    Microtubules (MTs) and the MT-associated proteins (MAPs) are critical cooperative agents involved in complex nanoassembly processes in biological systems. These biological materials and processes serve as important inspiration in developing new strategies for the assembly of synthetic nanomaterials in emerging techologies. Here, we explore a dynamic biofabrication process, modeled after the form and function of natural aster-like MT assemblies such as centrosomes. Specifically, we exploit the cooperative assembly of MTs and MAPs to form artificial microtubule asters and demonstrate that (1) these three-dimensional biomimetic microtubule asters can be controllably, reversibly assembled and (2) they serve as unique, dynamic biotemplates for the organization of secondary nanomaterials. We describe the MAP-mediated assembly and growth of functionalized MTs onto synthetic particles, the dynamic character of the assembled asters, and the application of these structures as templates for three-dimensional nanocrystal organization across multiple length scales. This biomediated nanomaterials assembly strategy illuminates a promising new pathway toward next-generation nanocomposite development.

  20. Hydrophobic interaction of organic chemicals with microtubule assembly in vitro.

    PubMed

    Stoiber, Thomas; Unger, Eberhard; Dorn, Susanne B; Degen, Gisela H; Bolt, Hermann M

    2008-09-01

    A recent concept connecting the lipophilicity of organic chemicals with their genotoxicity on a chromosomal level implies that the lipophilic character of organic chemicals determines a certain background of chromosomal genotoxicity that can be addressed as "non-specific". This is opposed to compounds with more "specific" modes of action. Such mechanisms influence the processes of karyokinesis and cytokinesis. A critical partial process for the chromosomal segregation is the dynamics of assembly and disassembly of microtubules. To broaden the present database for such interactions, chemicals were selected based on their lipophilicity (log P between -1.5 and +1.0) and on hints from the literature pointing to possibilities of interaction with the tubulin-microtubule system. Thus, acetamide, acrylamide, methylmethane sulfonate, acetonitrile, acrylonitrile and cyclohexanone were assessed as to their potencies to influence the dynamic processes of microtubule assembly and disassembly in a cell-free system in vitro. These compounds covered a range of log P between -1.5 and 1.0, complementary to compounds investigated earlier. The entire body of data supports the general concept that hydrophobic interactions are connected with non-specific processes, which contribute to a background genotoxicity on a chromosomal level. It also points to the dynamics of microtubule assembly and disassembly as a decisive partial process involved.

  1. Video microscopy analysis of the polymerization dynamics of individual microtubules

    NASA Astrophysics Data System (ADS)

    Salmon, E. D.

    1991-05-01

    We have developed methods using video-enhanced differential interference contrast light microscopy (VE-DIC) to measure the association and dissociation rate constants and transition frequencies of microtubule dynamic instability for microtubules assembled from pure tubulin, plus brain microtubule assoicated proteins (MAPs), and for microtubule assembly in living cells and cytosol extracts. Following nucleation, a microtubule end is seen to elongate at constant velocity until it abruptly begins rapid shortening, a transition termed catastrophe. The microtubule either disappears, or converts back to the elongation phase, a transition termed rescue. Catastrophes and rescues occur stochastically and infrequently in comparison to the durations of the elongation and shortening phases. In purified tubulin preparations from both mammalian brain and sea urchin embryos, the elongation and shortening phases exhibit distinctly different association and dissociation rate constants; in particular, the rate of dissocation during rapid shortening can be 100 times or more greater than during elongation particularly at high Mg2+. Brain MAPs (MAP2 and Tau) promote faster elongation, but suppress dynamic instability mainly by decreasing the frequency of catastrophe and increasing the frequency of rescue. In contrast, there are unknown factors in living dividing cells and in extracts from dividing cells which enhance dynamic instability by producing high frequencies of catastrophe (.01-.05 sec-1) at fast elongation velocities (10 μm min-1). Using a microscope perfusion chamber, we have shown for microtubules assembled from pure tubulin that dilution induces rapid shortening within several seconds independent of the elongation velocity or microtubule length. Thus, the stabilizing cap at elongating microtubule ends is small and sensitive to transient changes in the rate of tubulin association, even at high elongation velocities. This means that substantial changes in microtubule

  2. Spatiotemporal control of microtubule nucleation and assembly using magnetic nanoparticles

    NASA Astrophysics Data System (ADS)

    Hoffmann, Céline; Mazari, Elsa; Lallet, Sylvie; Le Borgne, Roland; Marchi, Valérie; Gosse, Charlie; Gueroui, Zoher

    2013-03-01

    Decisions on the fate of cells and their functions are dictated by the spatiotemporal dynamics of molecular signalling networks. However, techniques to examine the dynamics of these intracellular processes remain limited. Here, we show that magnetic nanoparticles conjugated with key regulatory proteins can artificially control, in time and space, the Ran/RCC1 signalling pathway that regulates the cell cytoskeleton. In the presence of a magnetic field, RanGTP proteins conjugated to superparamagnetic nanoparticles can induce microtubule fibres to assemble into asymmetric arrays of polarized fibres in Xenopus laevis egg extracts. The orientation of the fibres is dictated by the direction of the magnetic force. When we locally concentrated nanoparticles conjugated with the upstream guanine nucleotide exchange factor RCC1, the assembly of microtubule fibres could be induced over a greater range of distances than RanGTP particles. The method shows how bioactive nanoparticles can be used to engineer signalling networks and spatial self-organization inside a cell environment.

  3. Microtubule catastrophe from protofilament dynamics.

    PubMed

    Jemseena, V; Gopalakrishnan, Manoj

    2013-09-01

    The disappearance of the guanosine triphosphate- (GTP) tubulin cap is widely believed to be the forerunner event for the growth-shrinkage transition ("catastrophe") in microtubule filaments in eukaryotic cells. We study a discrete version of a stochastic model of the GTP cap dynamics, originally proposed by Flyvbjerg, Holy, and Leibler [Phys. Rev. Lett. 73, 2372 (1994)]. Our model includes both spontaneous and vectorial hydrolysis, as well as dissociation of a nonhydrolyzed dimer from the filament after incorporation. In the first part of the paper, we apply this model to a single protofilament of a microtubule. A catastrophe transition is defined for each protofilament, similarly to the earlier one-dimensional models, the frequency of occurrence of which is then calculated under various conditions but without explicit assumption of steady-state conditions. Using a perturbative approach, we show that the leading asymptotic behavior of the protofilament catastrophe in the limit of large growth velocities is remarkably similar across different models. In the second part of the paper, we extend our analysis to the entire filament by making a conjecture that a minimum number of such transitions are required to occur for the onset of microtubule catastrophe. The frequency of microtubule catastrophe is then determined using numerical simulations and compared with analytical and semianalytical estimates made under steady-state and quasi-steady-state assumptions, respectively, for the protofilament dynamics. A few relevant experimental results are analyzed in detail and compared with predictions from the model. Our results indicate that loss of GTP cap in two to three protofilaments is necessary to trigger catastrophe in a microtubule.

  4. Self-assembly of microtubules and motors

    NASA Astrophysics Data System (ADS)

    Aranson, Igor; Tsimring, Lev

    2005-03-01

    We derive a model describing spatio-temporal assembly of an array of microtubules interacting via molecular motors. Starting from a stochastic model of inelastic polar rods with a generic anisotropic interaction kernel we obtain a set of equations for the local rods concentration and orientation. At large enough mean density of rods and concentration of motors, the model describes orientational instability. We demonstrate that the orientational instability leads to the formation of vortices and (for large density and/or kernel anisotropy) asters seen in recent experiments.

  5. Theoretical Description of Microtubule Dynamics in Fission Yeast During Interphase

    NASA Astrophysics Data System (ADS)

    Oei, Yung-Chin; Jiménez-Dalmaroni, Andrea; Vilfan, Andrej; Duke, Thomas

    2009-03-01

    Fission yeast (S. pombe) is a unicellular organism with a characteristic cylindrical shape. Cell growth during interphase is strongly influenced by microtubule self-organization - a process that has been experimentally well characterised. The microtubules are organized in 3 to 4 bundles, called ``interphase microtubule assemblies'' (IMAs). Each IMA is composed of several microtubules, arranged with their dynamic ``plus'' ends facing the cell tips and their ``minus'' ends overlapping at the cell middle. Although the main protein factors involved in interphase microtubule organization have been identified, an understanding of how their collective interaction with microtubules leads to the organization and structures observed in vivo is lacking. We present a physical model of microtubule dynamics that aims to provide a quantitative description of the self-organization process. First, we solve equations for the microtubule length distribution in steady-state, taking into account the way that a limited tubulin pool affects the nucleation, growth and shrinkage of microtubules. Then we incorporate passive and active crosslinkers (the bundling factor Ase1 and molecular motor Klp2) and investigate the formation of IMA structures. Analytical results are complemented by a 3D stochastic simulation.

  6. Microtubule-depolymerizing kinesins in the regulation of assembly, disassembly, and length of cilia and flagella.

    PubMed

    Hu, Zhangfeng; Liang, Yinwen; Meng, Dan; Wang, Liang; Pan, Junmin

    2015-01-01

    Defects in ciliary assembly, maintenance, and signaling are associated with various human diseases and developmental disorders, termed ciliopathies. Eukaryotic flagella and cilia (interchangeable terms) are microtubule-based organelles. Thus, microtubule dynamics and microtubule-dependent transport are predicted to affect the structural integrity and functionality of cilia profoundly. Kinesin-2 is well known for its role in intraflagellar transport to transport ciliary precursors and signaling molecules. Recently, microtubule-depolymerizing kinesins found in kinesin-8, -13, and -14A families have emerged as regulators of cilia. We first discuss ciliary kinesins identified in the flagellar or ciliary proteome, and then focus on the function and regulation of microtubule-depolymerizing kinesins. Lastly, we review the recent advances of microtubule-depolymerizing kinesins in controlling ciliary assembly, disassembly, and length.

  7. Force Generation by Microtubule Assembly/Disassembly in Mitosis and Related Movements

    PubMed Central

    Inoué, Shinya; Salmon, Edward D.

    1995-01-01

    In this article, we review the dynamic nature of the filaments (microtubules) that make up the labile fibers of the mitotic spindle and asters, we discuss the roles that assembly and disassembly of microtubules play in mitosis, and we consider how such assembling and disassembling polymer filaments can generate forces that are utilized by the living cell in mitosis and related movements. Images PMID:8590794

  8. Microtubule assembly in cytoplasmic extracts of Xenopus oocytes and eggs

    PubMed Central

    1987-01-01

    We have investigated the differences in microtubule assembly in cytoplasm from Xenopus oocytes and eggs in vitro. Extracts of activated eggs could be prepared that assembled extensive microtubule networks in vitro using Tetrahymena axonemes or mammalian centrosomes as nucleation centers. Assembly occurred predominantly from the plus-end of the microtubule with a rate constant of 2 microns.min-1.microM-1 (57 s- 1.microM-1). At the in vivo tubulin concentration, this corresponds to the extraordinarily high rate of 40-50 microns.min-1. Microtubule disassembly rates in these extracts were -4.5 microns.min-1 (128 s-1) at the plus-end and -6.9 microns.min-1 (196 s-1) at the minus-end. The critical concentration for plus-end microtubule assembly was 0.4 microM. These extracts also promoted the plus-end assembly of microtubules from bovine brain tubulin, suggesting the presence of an assembly promoting factor in the egg. In contrast to activated eggs, assembly was never observed in extracts prepared from oocytes, even at tubulin concentrations as high as 20 microM. Addition of oocyte extract to egg extracts or to purified brain tubulin inhibited microtubule assembly. These results suggest that there is a plus-end-specific inhibitor of microtubule assembly in the oocyte and a plus-end-specific promoter of assembly in the eggs. These factors may serve to regulate microtubule assembly during early development in Xenopus. PMID:3680377

  9. Suppression of microtubule assembly kinetics by the mitotic protein TPX2.

    PubMed

    Reid, Taylor A; Schuster, Breanna M; Mann, Barbara J; Balchand, Sai Keshavan; Plooster, Melissa; McClellan, Mark; Coombes, Courtney E; Wadsworth, Pat; Gardner, Melissa K

    2016-04-01

    TPX2 is a widely conserved microtubule-associated protein that is required for mitotic spindle formation and function. Previous studies have demonstrated that TPX2 is required for the nucleation of microtubules around chromosomes; however, the molecular mechanism by which TPX2 promotes microtubule nucleation remains a mystery. In this study, we found that TPX2 acts to suppress tubulin subunit off-rates during microtubule assembly and disassembly, thus allowing for the support of unprecedentedly slow rates of plus-end microtubule growth, and also leading to a dramatically reduced microtubule shortening rate. These changes in microtubule dynamics can be explained in computational simulations by a moderate increase in tubulin-tubulin bond strength upon TPX2 association with the microtubule lattice, which in turn acts to reduce the departure rate of tubulin subunits from the microtubule ends. Thus, the direct suppression of tubulin subunit off-rates by TPX2 during microtubule growth and shortening could provide a molecular mechanism to explain the nucleation of new microtubules in the presence of TPX2.

  10. Filopodia and actin arcs guide the assembly and transport of two populations of microtubules with unique dynamic parameters in neuronal growth cones

    PubMed Central

    Schaefer, Andrew W.; Kabir, Nurul; Forscher, Paul

    2002-01-01

    We have used multimode fluorescent speckle microscopy (FSM) and correlative differential interference contrast imaging to investigate the actin–microtubule (MT) interactions and polymer dynamics known to play a fundamental role in growth cone guidance. We report that MTs explore the peripheral domain (P-domain), exhibiting classical properties of dynamic instability. MT extension occurs preferentially along filopodia, which function as MT polymerization guides. Filopodial bundles undergo retrograde flow and also transport MTs. Thus, distal MT position is determined by the rate of plus-end MT assembly minus the rate of retrograde F-actin flow. Short MT displacements independent of flow are sometimes observed. MTs loop, buckle, and break as they are transported into the T-zone by retrograde flow. MT breakage results in exposure of new plus ends which can regrow, and minus ends which rapidly undergo catastrophes, resulting in efficient MT turnover. We also report a previously undetected presence of F-actin arc structures, which exhibit persistent retrograde movement across the T-zone into the central domain (C-domain) at ∼1/4 the rate of P-domain flow. Actin arcs interact with MTs and transport them into the C-domain. Interestingly, although the MTs associated with arcs are less dynamic than P-domain MTs, they elongate efficiently as a result of markedly lower catastrophe frequencies. PMID:12105186

  11. Tubulin bistability and polymorphic dynamics of microtubules.

    PubMed

    Mohrbach, Hervé; Johner, Albert; Kulić, Igor M

    2010-12-31

    Based on the hypothesis that the GDP-tubulin dimer is a conformationally bistable molecule-rapidly fluctuating between a discrete curved and a straight state-we develop a model for polymorphic dynamics of the microtubule lattice. We show that GDP-tubulin bistability consistently explains unusual dynamic fluctuations, the apparent length-stiffness relation of grafted taxol-stabilized microtubules, and the curved-helical appearance of microtubules in general. When clamped by one end the microtubules undergo an unusual zero energy motion-in its effect reminiscent of a limited rotational hinge. We conclude that microtubules exist in highly cooperative energy-degenerate helical states and discuss possible implications in vivo.

  12. Microtubule bundling and shape transitions: Mechanics, interactions, and self-assemblies

    NASA Astrophysics Data System (ADS)

    Needleman, Daniel Joseph

    Microtubules associate to form bundles in vivo in a wide variety of contexts including the mitotic spindle, neuronal processes, and the cortical array in plant cells. These supramolecular assemblies differ in size and shape, and in their internal structure, but the principles that determine this variation in morphology are not understood. To help elucidate such principals we constructed microtubule bundles in vitro using a variety of bundling agents. We have characterized the structure of these supramolecular assemblies of microtubules from the nanoscale to the mesoscale using synchrotron x-ray scattering and diffraction, video enhanced DIC and fluorescence microscopy, and electron microscopy. In the presence of inert polymers, an osmotic pressure imbalance between the inside and the outside of the microtubules may cause them to buckle to a non-circular cross-section. Depletion effects cause these distorted microtubules to bundle into a lattice with rectangular symmetry. The critical buckling pressure provides a measure of the stiffness of the inter-protofilament bond, and we determined that microtubule associated proteins enhance the strength of this bond, while the chemotherapeutic drug taxol has no effect. Multivalent ions cause microtubules to associate into bundles whose morphology depends on the condensing ion. Tightly packed hexagonal bundles with controllable diameters are observed for large tri-, tetra-, and pentavalent counterions. Unexpectedly, in the presence of small divalent cations, we have discovered a living necklace bundle phase, comprised of dynamical assemblies of MT nematic membranes with linear, branched, and loop topologies. Cations may also cause tubulin to assemble into non-microtubule structures. For example, in the presence of spermine, over time the microtubule bundles transform into a columnar phase of inverted tubules, such that the surface which was facing outside of the microtubules switches to the inside. This rearrangement between

  13. Kinetochore–microtubule attachment is sufficient to satisfy the human spindle assembly checkpoint

    PubMed Central

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

    2015-01-01

    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. PMID:26621779

  14. Tryprostatin A, a specific and novel inhibitor of microtubule assembly.

    PubMed

    Usui, T; Kondoh, M; Cui, C B; Mayumi, T; Osada, H

    1998-08-01

    We have investigated the cell cycle inhibition mechanism and primary target of tryprostatin A (TPS-A) purified from Aspergillus fumigatus. TPS-A inhibited cell cycle progression of asynchronously cultured 3Y1 cells in the M phase in a dose- and time-dependent manner. In contrast, TPS-B (the demethoxy analogue of TPS-A) showed cell-cycle non-specific inhibition on cell growth even though it inhibited cell growth at lower concentrations than TPS-A. TPS-A treatment induced the reversible disruption of the cytoplasmic microtubules of 3Y1 cells as observed by indirect immunofluorescence microscopy in the range of concentrations that specifically inhibited M-phase progression. TPS-A inhibited the assembly in vitro of microtubules purified from bovine brains (40% inhibition at 250 microM); however, there was little or no effect on the self-assembly of purified tubulin when polymerization was induced by glutamate even at 250 microM TPS-A. TPS-A did not inhibit assembly promoted by taxol or by digestion of the C-terminal domain of tubulin. However, TPS-A blocked the tubulin assembly induced by inducers interacting with the C-terminal domain, microtubule-associated protein 2 (MAP2), tau and poly-(l-lysine). These results indicate that TPS-A is a novel inhibitor of MAP-dependent microtubule assembly and, through the disruption of the microtubule spindle, specifically inhibits cell cycle progression at the M phase.

  15. Multiscale polar theory of microtubule and motor-protein assemblies.

    PubMed

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

    2015-01-30

    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.

  16. Multiscale Polar Theory of Microtubule and Motor-Protein Assemblies

    NASA Astrophysics Data System (ADS)

    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.

  17. Multiscale Polar Theory of Microtubule and Motor-Protein Assemblies

    PubMed Central

    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

  18. Multiscale polar theory of microtubule and motor-protein assemblies

    DOE PAGES

    Gao, Tong; Blackwell, Robert; Glaser, Matthew A.; ...

    2015-01-27

    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-specificmore » sources of active destabilizing stress. On larger length scales, our continuum Doi-Onsager theory captures the hydrodynamic flows generated by polarity-dependent active stresses. Finally, the results connect local polar structure to flow structures and defect dynamics.« less

  19. Multiscale polar theory of microtubule and motor-protein assemblies

    SciTech Connect

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

    2015-01-27

    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. Finally, the results connect local polar structure to flow structures and defect dynamics.

  20. Spatial regulation of astral microtubule dynamics by Kif18B in PtK cells.

    PubMed

    Walczak, Claire E; Zong, Hailing; Jain, Sachin; Stout, Jane R

    2016-10-15

    The spatial and temporal control of microtubule dynamics is fundamentally important for proper spindle assembly and chromosome segregation. This is achieved, in part, by the multitude of proteins that bind to and regulate spindle microtubules, including kinesin superfamily members, which act as microtubule-destabilizing enzymes. These fall into two general classes: the kinesin-13 proteins, which directly depolymerize microtubules, and the kinesin-8 proteins, which are plus end-directed motors that either destabilize microtubules or cap the microtubule plus ends. Here we analyze the contribution of a PtK kinesin-8 protein, Kif18B, in the control of mitotic microtubule dynamics. Knockdown of Kif18B causes defects in spindle microtubule organization and a dramatic increase in astral microtubules. Kif18B-knockdown cells had defects in chromosome alignment, but there were no defects in chromosome segregation. The long astral microtubules that occur in the absence of Kif18B are limited in length by the cell cortex. Using EB1 tracking, we show that Kif18B activity is spatially controlled, as loss of Kif18B has the most dramatic effect on the lifetimes of astral microtubules that extend toward the cell cortex. Together our studies provide new insight into how diverse kinesins contribute to spatial microtubule organization in the spindle.

  1. Spatial regulation of astral microtubule dynamics by Kif18B in PtK cells

    PubMed Central

    Walczak, Claire E.; Zong, Hailing; Jain, Sachin; Stout, Jane R.

    2016-01-01

    The spatial and temporal control of microtubule dynamics is fundamentally important for proper spindle assembly and chromosome segregation. This is achieved, in part, by the multitude of proteins that bind to and regulate spindle microtubules, including kinesin superfamily members, which act as microtubule-destabilizing enzymes. These fall into two general classes: the kinesin-13 proteins, which directly depolymerize microtubules, and the kinesin-8 proteins, which are plus end–directed motors that either destabilize microtubules or cap the microtubule plus ends. Here we analyze the contribution of a PtK kinesin-8 protein, Kif18B, in the control of mitotic microtubule dynamics. Knockdown of Kif18B causes defects in spindle microtubule organization and a dramatic increase in astral microtubules. Kif18B-knockdown cells had defects in chromosome alignment, but there were no defects in chromosome segregation. The long astral microtubules that occur in the absence of Kif18B are limited in length by the cell cortex. Using EB1 tracking, we show that Kif18B activity is spatially controlled, as loss of Kif18B has the most dramatic effect on the lifetimes of astral microtubules that extend toward the cell cortex. Together our studies provide new insight into how diverse kinesins contribute to spatial microtubule organization in the spindle. PMID:27559136

  2. MCF7 microtubules: Cancer microtubules with relatively slow and stable dynamic in vitro.

    PubMed

    Feizabadi, Mitra Shojania; Rosario, Brandon

    2017-03-04

    There is known to be significant diversity of β-tubulin isoforms in cells. However, whether the functions of microtubules that are polymerized from different distributions of beta isotypes become distinct from one another are still being explored. Of particular interest, recent studies have identified the role that different beta tubulin isotypes carry in regulating the functions of some of the molecular motors along MCF7, or breast cancer, microtubules. That being said, how the specific distribution of beta tubulin isotypes impacts the MCF7 microtubules' dynamic is not well understood. The current study was initiated to directly quantify the in vitro dynamic and polymerization parameters of single MCF7 microtubules and then compare them with those obtained from neuronal microtubules polymerized from porcine brain tubulin. Surprisingly, unlike porcine brain microtubules, this type of cancer microtubule showed a relatively stable and slow dynamic. The comparison between the subsequently fast and unstable dynamic of porcine brain microtubules with the significantly slow and relatively stable dynamic of MCF7 microtubules suggests that beta tubulin isotypes may not only influence the microtubule based functionalities of some molecular motors, but also may change the microtubule's intrinsic dynamic.

  3. Microtubule-sliding activity of a kinesin-8 promotes spindle assembly and spindle-length control.

    PubMed

    Su, Xiaolei; Arellano-Santoyo, Hugo; Portran, Didier; Gaillard, Jeremie; Vantard, Marylin; Thery, Manuel; Pellman, David

    2013-08-01

    Molecular motors play critical roles in the formation of mitotic spindles, either through controlling the stability of individual microtubules, or by crosslinking and sliding microtubule arrays. Kinesin-8 motors are best known for their regulatory roles in controlling microtubule dynamics. They contain microtubule-destabilizing activities, and restrict spindle length in a wide variety of cell types and organisms. Here, we report an antiparallel microtubule-sliding activity of the budding yeast kinesin-8, Kip3. The in vivo importance of this sliding activity was established through the identification of complementary Kip3 mutants that separate the sliding activity and microtubule-destabilizing activity. In conjunction with Cin8, a kinesin-5 family member, the sliding activity of Kip3 promotes bipolar spindle assembly and the maintenance of genome stability. We propose a slide-disassemble model where the sliding and destabilizing activity of Kip3 balance during pre-anaphase. This facilitates normal spindle assembly. However, the destabilizing activity of Kip3 dominates in late anaphase, inhibiting spindle elongation and ultimately promoting spindle disassembly.

  4. Push or Pull? -- Cryo-Electron Microscopy of Microtubule's Dynamic Instability and Its Roles in the Kinetochore

    NASA Astrophysics Data System (ADS)

    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.

  5. Nup98 regulates bipolar spindle assembly through association with microtubules and opposition of MCAK

    PubMed Central

    Cross, Marie K.; Powers, Maureen A.

    2011-01-01

    During mitosis, the nuclear pore complex is disassembled and, increasingly, nucleoporins are proving to have mitotic functions when released from the pore. We find a contribution of the nucleoporin Nup98 to mitotic spindle assembly through regulation of microtubule dynamics. When added to Xenopus extract spindle assembly assays, the C-terminal domain of Nup98 stimulates uncontrolled growth of microtubules. Conversely, inhibition or depletion of Nup98 leads to formation of stable monopolar spindles. Spindle bipolarity is restored by addition of purified, recombinant Nup98 C-terminus. The minimal required region of Nup98 corresponds to a portion of the C-terminal domain lacking a previously characterized function. We show association between this region of the C-terminus of Nup98 and both Taxol-stabilized microtubules and the microtubule-depolymerizing mitotic centromere–associated kinesin (MCAK). Importantly, we demonstrate that this domain of Nup98 inhibits MCAK depolymerization activity in vitro. These data support a model in which Nup98 interacts with microtubules and antagonizes MCAK activity, thus promoting bipolar spindle assembly. PMID:21209315

  6. Tubulin carbamoylation. Functional amino groups in microtubule assembly.

    PubMed Central

    Mellado, W; Slebe, J C; Maccioni, R B

    1982-01-01

    The characteristics of the carbamoylation of pig brain tubulin were examined by using the modification conditions with cyanate described previously [Mellado, Slebe + Maccioni (1980) Biochem. Int. I, 584--590]. The carbamoylation reaction resulted in an inhibition of microtubule assembly, which was dependent on the concentration of the modifying agent. This tubulin modification appears to inhibit the growth of microtubules. The presence of GTP did not protect tubulin against this inhibition. Electron microscopy showed a marked decrease in the number of tubules after carbamoylation, but no alterations were observed in the microtubule morphology. The incorporation of KN14CO into alpha- and beta-subunits with similar kinetics was also shown, and the carbamoylated residues were identified as epsilon-N-carbamoyl-lysine residues. Images PLATE 1 Fig. 4. PMID:7115308

  7. Physical aspects of the assembly and function of microtubules

    NASA Astrophysics Data System (ADS)

    Holy, Timothy Eric

    Living cells contain polymers called microtubules. Microtubules are used to control cell shape, to generate force for movement, to transport vesicles, and to separate chromosomes during cell division. Microtubule polymerization is governed by a unique, energy-consuming phenomenon called dynamic instability, which leads to large fluctuations in length. This dissertation reports on physical studies (theory and experiment) of microtubules and their roles in living cells. To understand dynamic instability we need to know its mechanism and its function. Experiments on dynamic instability have led to a seeming contradiction as to its mechanism. By introducing a phenomenological model that unites the existing data, we show that this contradiction can be resolved. The model describes the stochastic dynamics of a stabilizing cap which promotes growth, but whose loss leads to disassembly. The theory matches experiments over time scales from seconds to minutes. We address the biological role of dynamic instability, also from a theoretical standpoint. We show that these large length fluctuations are useful: they lead to a rapid search of intracellular space. This search may be an essential step in organizing the cell interior, forging connections between widely-separated components. We show that dynamic instability speeds a search by several orders of magnitude. We also find that the parameters which govern dynamic instability appear to be chosen by the cell so as to minimize the search time. This thesis also reports experiments showing that microtubule polymerization may generate forces for movement and organization. Archetypal movements in living cells (e.g., the movement of the sperm nucleus from the periphery to the center of the egg) were reconstructed in an artificial system. The components of our system are purified proteins and two specially-designed materials: latex beads coated so as to nucleate microtubules, and microscopic chambers fabricated to mimic the confined

  8. Binding of microtubule protein to DNA and chromatin: possibility of simultaneous linkage of microtubule to nucleic and assembly of the microtubule structure.

    PubMed Central

    Villasante, A; Corces, V G; Manso-Martínez, R; Avila, J

    1981-01-01

    Microtubule protein binds to DNA through microtubule associated polypeptides (MAPs). Among MAPs there is one high molecular weight polypeptide (MAP2) which interacts with DNA fundamentally through certain polynucleotide sequences. This interaction is not affected by the presence of histones and other chromosomal proteins. DNA can associate to assembled microtubules and when a determinate DNA/protein ratio is reached the nucleic acid behaves as a microtubule associated molecule. The nucleic acid fragments which preferentially bind to microtubules have been isolated and characterized. These fragments contain DNA regions enriched in repetitive sequences that hybridizes preferentially to the pericentromeric zone of metaphase chromosomes. These results give further support to the model of interaction microtubule-chromosome based upon the mediator function of the microtubule associated proteins. Images PMID:7232207

  9. Prion protein inhibits microtubule assembly by inducing tubulin oligomerization

    SciTech Connect

    Nieznanski, Krzysztof . E-mail: k.nieznanski@nencki.gov.pl; Podlubnaya, Zoya A.; Nieznanska, Hanna

    2006-10-13

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

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

    PubMed

    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.

  11. Dynamic Concentration of Motors in Microtubule Arrays

    NASA Astrophysics Data System (ADS)

    Nédélec, François; Surrey, Thomas; Maggs, A. C.

    2001-04-01

    We present experimental and theoretical studies of the dynamics of molecular motors in microtubule arrays and asters. By solving a convection-diffusion equation we find that the density profile of motors in a two-dimensional aster is characterized by continuously varying exponents. Simulations are used to verify the assumptions of the continuum model. We observe the concentration profiles of kinesin moving in quasi-two-dimensional artificial asters by fluorescent microscopy and compare with our theoretical results.

  12. Microtubule Dynamics Control Tail Retraction in Migrating Vascular Endothelial Cells†

    PubMed Central

    Ganguly, Anutosh; Yang, Hailing; Zhang, Hong; Cabral, Fernando; Patel, Kamala D.

    2014-01-01

    Drugs that target microtubules are potent inhibitors of angiogenesis but their mechanism of action is not well understood. To explore this, we treated human umbilical vein endothelial cells with paclitaxel, vinblastine, and colchicine and measured the effects on microtubule dynamics and cell motility. In general, lower drug concentrations suppressed microtubule dynamics and inhibited cell migration whereas higher concentrations were needed to inhibit cell division; but, surprisingly, large drug-dependent differences were seen in the relative concentrations needed to inhibit these two processes. Suppression of microtubule dynamics did not significantly affect excursions of lamellipodia away from the nucleus or prevent cells from elongating; but, it did inhibit retraction of the trailing edges that are normally enriched in dynamic microtubules, thereby limiting cell locomotion. Complete removal of microtubules with a high vinblastine concentration caused a loss of polarity that resulted in roundish rather than elongated cells, rapid but non-directional membrane activity, and little cell movement. The results are consistent with a model in which more static microtubules stabilize the leading edge of migrating cells while more dynamic microtubules locate to the rear where they can remodel and allow tail retraction. Suppressing microtubule dynamics interferes with tail retraction, but removal of microtubules destroys the asymmetry needed for cell elongation and directional motility. The prediction that suppressing microtubule dynamics might be sufficient to prevent angiogenesis was supported by showing that low concentrations of paclitaxel could prevent the formation of capillary-like structures in an in vitro tube formation assay. PMID:24107446

  13. Mathematical modeling of microtubule dynamics: insights into physiology and disease.

    PubMed

    Buxton, Gavin A; Siedlak, Sandra L; Perry, George; Smith, Mark A

    2010-12-01

    Computer models of microtubule dynamics have provided the basis for many of the theories on the cellular mechanics of the microtubules, their polymerization kinetics, and the diffusion of tubulin and tau. In the three-dimensional model presented here, we include the effects of tau concentration and the hydrolysis of GTP-tubulin to GDP-tubulin and observe the emergence of microtubule dynamic instability. This integrated approach simulates the essential physics of microtubule dynamics in a cellular environment. The model captures the structure of the microtubules as they undergo steady state dynamic instabilities in this simplified geometry, and also yields the average number, length, and cap size of the microtubules. The model achieves realistic geometries and simulates cellular structures found in degenerating neurons in disease states such as Alzheimer disease. Further, this model can be used to simulate microtubule changes following the addition of antimitotic drugs which have recently attracted attention as chemotherapeutic agents.

  14. Neurodegeneration and microtubule dynamics: death by a thousand cuts

    PubMed Central

    Dubey, Jyoti; Ratnakaran, Neena; Koushika, Sandhya P.

    2015-01-01

    Microtubules form important cytoskeletal structures that play a role in establishing and maintaining neuronal polarity, regulating neuronal morphology, transporting cargo, and scaffolding signaling molecules to form signaling hubs. Within a neuronal cell, microtubules are found to have variable lengths and can be both stable and dynamic. Microtubule associated proteins, post-translational modifications of tubulin subunits, microtubule severing enzymes, and signaling molecules are all known to influence both stable and dynamic pools of microtubules. Microtubule dynamics, the process of interconversion between stable and dynamic pools, and the proportions of these two pools have the potential to influence a wide variety of cellular processes. Reduced microtubule stability has been observed in several neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), Amyotrophic Lateral Sclerosis (ALS), and tauopathies like Progressive Supranuclear Palsy. Hyperstable microtubules, as seen in Hereditary Spastic Paraplegia (HSP), also lead to neurodegeneration. Therefore, the ratio of stable and dynamic microtubules is likely to be important for neuronal function and perturbation in microtubule dynamics might contribute to disease progression. PMID:26441521

  15. The effect of human microtubule-associated-protein tau on the assembly structure of microtubules and its ionic strength dependence

    NASA Astrophysics Data System (ADS)

    Choi, M. C.; Raviv, U.; Miller, H. P.; Gaylord, M. R.; Kiris, E.; Ventimiglia, D.; Needleman, D. J.; Chung, P. J.; Deek, J.; Lapointe, N.; Kim, M. W.; Wilson, L.; Feinstein, S. C.; Safinya, C. R.

    2010-03-01

    Microtubules (MTs), 25 nm protein nanotubes, are among the major filamentous elements of the eukaryotic cytoskeleton involved in intracellular trafficking, cell division and the establishment and maintenance of cell shape. Microtubule-associated-protein tau regulates tubulin assembly, MT dynamics and stability. Aberrant tau action has long been correlated with numerous neurodegenerative diseases, including Alzheimer's, and fronto-temporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17) Using synchrotron small angle x-ray scattering (SAXS) and binding assay, we examine the effects of tau on the assembly structure of taxol-stabilized MTs. We find that tau regulates the distribution of protofilament numbers in MTs as reflected in the observed increase in the average radius of MTs with increasing the tau/tubulin molar ratio. Additionally, tau-MT interactions are mediated to a large extent via electrostatic interactions: the binding affinity of tau to MTs is ionic strength dependent. Supported by DOE-BES DE-FG02-06ER46314, NSF DMR-0803103, NIH NS35010, NIH NS13560. (Ref) M.C. Choi, S.C. Feinstein, and C.R. Safinya et al. Biophys. J. 97; 519 (2009).

  16. Vinblastine suppresses dynamics of individual microtubules in living interphase cells.

    PubMed Central

    Dhamodharan, R; Jordan, M A; Thrower, D; Wilson, L; Wadsworth, P

    1995-01-01

    We have characterized the effects of vinblastine on the dynamic instability behavior of individual microtubules in living BS-C-1 cells microinjected with rhodamine-labeled tubulin and have found that at low concentrations (3-64 nM), vinblastine potently suppresses dynamic instability without causing net microtubule depolymerization. Vinblastine suppressed the rates of microtubule growth and shortening, and decreased the frequency of transitions from growth or pause to shortening, also called catastrophe. In vinblastine-treated cells, both the average duration of a pause (a state of attenuated dynamics where neither growth nor shortening could be detected) and the percentage of total time spent in pause were significantly increased. Vinblastine potently decreased dynamicity, a measure of the overall dynamic activity of microtubules, reducing this parameter by 75% at 32 nM. The present work, consistent with earlier in vitro studies, demonstrates that vinblastine kinetically caps the ends of microtubules in living cells and supports the hypothesis that the potent chemotherapeutic action of vinblastine as an antitumor drug is suppression of mitotic spindle microtubule dynamics. Further, the results indicate that molecules that bind to microtubule ends can regulate microtubule dynamic behavior in living cells and suggest that endogenous regulators of microtubule dynamics that work by similar mechanisms may exist in living cells. Images PMID:8534917

  17. Microtubules: dynamically unstable stochastic phase-switching polymers

    NASA Astrophysics Data System (ADS)

    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.

  18. Reassembly of flagellar B (alpha beta) tubulin into singlet microtubules: consequences for cytoplasmic microtubule structure and assembly

    PubMed Central

    1981-01-01

    B(alpha beta) tubulin was obtained from a homogeneous class of microtubules, the incomplete B subfiber of sea urchin sperm flagellar doublet microtubules, by thermal fractionation. The thermally derived soluble B tubulin fraction (100, 000 g-h) repolymerizes in vitro, yielding microtubule-like structures. The microtubule-associated protein (MAP) composition and certain assembly parameters of thermally derived B tubulin are different from those reported for sonication- derived flageller tubulin and purified vertebrate tubulin. The "microtubules" reassembled from thermally prepared B tubulin are composed of 12-15 protofilaments (73% possess 14 protofilaments). A certain number possess a single "adlumenal component" applied to their inside walls, regardless of the number of protofilaments. Following the first cycle of polymerization, 81% of the B tubulin and essentially 100% of the MAPs remain cold insoluble. Evidence suggests that B tubulin assembles faithfully into a B lattice, creating a j seam between two protofilaments that are laterally bonded in a A-lattice configuration. The significance of these seams is discussed in relation to the mechanism of microtubule assembly, the stability of observed ribbons of protofilaments, and the three-dimensional organization of microtubule-associated components. PMID:7251656

  19. Dual detection of chromosomes and microtubules by the chromosomal passenger complex drives spindle assembly.

    PubMed

    Tseng, Boo Shan; Tan, Lei; Kapoor, Tarun M; Funabiki, Hironori

    2010-06-15

    Chromosome-dependent spindle assembly requires the chromosomal recruitment and activation of Aurora B, the kinase subunit of the chromosomal passenger complex (CPC). It remains unclear how the chromosome-activated kinase spatially transmits signals to organize the micron-scale spindle. Here we reveal that the CPC must detect two structures, chromosomes and microtubules, to support spindle assembly in Xenopus egg extracts. While Aurora B is enriched on chromosomes in metaphase, we establish that a fraction of Aurora B is targeted to the metaphase spindle and phosphorylates microtubule-bound substrates. We demonstrate that chromosomally activated Aurora B must be targeted to microtubules to drive spindle assembly. Moreover, although the CPC-microtubule interaction can activate Aurora B, which further promotes microtubule assembly, this positive feedback is not initiated without chromosomes. We propose that the dual detection of chromosomes and microtubules by the CPC is a critical step in assembling spindles around and only around chromosomes.

  20. Signatures of a macroscopic switching transition for a dynamic microtubule

    PubMed Central

    Aparna, J. S.; Padinhateeri, Ranjith; Das, Dibyendu

    2017-01-01

    Characterising complex kinetics of non-equilibrium self-assembly of bio-filaments is of general interest. Dynamic instability in microtubules, consisting of successive catastrophes and rescues, is observed to occur as a result of the non-equilibrium conversion of GTP-tubulin to GDP-tubulin. We study this phenomenon using a model for microtubule kinetics with GTP/GDP state-dependent polymerisation, depolymerisation and hydrolysis of subunits. Our results reveal a sharp switch-like transition in the mean velocity of the filaments, from a growth phase to a shrinkage phase, with an associated co-existence of the two phases. This transition is reminiscent of the discontinuous phase transition across the liquid-gas boundary. We probe the extent of discontinuity in the transition quantitatively using characteristic signatures such as bimodality in velocity distribution, variance and Binder cumulant, and also hysteresis behaviour of the system. We further investigate ageing behaviour in catastrophes of the filament, and find that the multi-step nature of catastrophes is intensified in the vicinity of the switching transition. This assumes importance in the context of Microtubule Associated Proteins which have the potential of altering kinetic parameter values. PMID:28374844

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

    SciTech Connect

    Scaife, R.M. ); Wilson, L. ); Purich, D.L. )

    1992-01-14

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

  2. Regulation of microtubule dynamic instability in vitro by differentially phosphorylated stathmin.

    PubMed

    Manna, Tapas; Thrower, Douglas A; Honnappa, Srinivas; Steinmetz, Michel O; Wilson, Leslie

    2009-06-05

    Stathmin is an important regulator of microtubule polymerization and dynamics. When unphosphorylated it destabilizes microtubules in two ways, by reducing the microtubule polymer mass through sequestration of soluble tubulin into an assembly-incompetent T2S complex (two alpha:beta tubulin dimers per molecule of stathmin), and by increasing the switching frequency (catastrophe frequency) from growth to shortening at plus and minus ends by binding directly to the microtubules. Phosphorylation of stathmin on one or more of its four serine residues (Ser(16), Ser(25), Ser(38), and Ser(63)) reduces its microtubule-destabilizing activity. However, the effects of phosphorylation of the individual serine residues of stathmin on microtubule dynamic instability have not been investigated systematically. Here we analyzed the effects of stathmin singly phosphorylated at Ser(16) or Ser(63), and doubly phosphorylated at Ser(25) and Ser(38), on its ability to modulate microtubule dynamic instability at steady-state in vitro. Phosphorylation at either Ser(16) or Ser(63) strongly reduced or abolished the ability of stathmin to bind to and sequester soluble tubulin and its ability to act as a catastrophe factor by directly binding to the microtubules. In contrast, double phosphorylation of Ser(25) and Ser(38) did not affect the binding of stathmin to tubulin or microtubules or its catastrophe-promoting activity. Our results indicate that the effects of stathmin on dynamic instability are strongly but differently attenuated by phosphorylation at Ser(16) and Ser(63) and support the hypothesis that selective targeting by Ser(16)-specific or Ser(63)-specific kinases provides complimentary mechanisms for regulating microtubule function.

  3. Twelve protofilament taxol-induced microtubules assembled from purified tublin. A synchrotron X-ray scattering study in comparison with glycerol- and map-induced microtubules

    NASA Astrophysics Data System (ADS)

    Andreu, J. M.; Garcia de Ancos, J.; Medrano, F. J.; Gil, R.; Diaz, J. F.; Nogales, E.; Towns-Andrews, E.; Pantos, E.; Bordas, J.

    1991-05-01

    The X-ray solution scattering profiles of taxol microtubules made of purified tubulin and control microtubules, assembled either from purified tubulin in glycerol buffer (a non-specific enhancer of the polymerization of tubulin) or from microtubule protein (a preparation containing tubulin plus microtubule associated proteins), were obtained to 3.3 nm resolution. These profiles show features of the microtubule wall structure which had not been observed in solution before. Comparison of the different profiles indicated that the structure of the microtubule wall is very similar in the three types of microtubules to the resolution of the measurements, however the mean diameter of the taxol microtubules is smaller than that of the control microtubules, by approximately one protofilament less. Actually, only 12 protofilament computer models of microtubules could fit the position of the maxima in the experimental scattering profile of the taxol microtubules. Having only 12 protofilaments implies a discontinuity on the microtubule wall, irrespective of whether the lateral contacts follow the A or B microtubule lattice, and also requires adjustment of the normal lattice to one protofilament axis with respect to the cylinder axis. The fact that the majority of these taxol microtubules assembled from purified tubulin have 12 protofilaments has been visualized by electron micrographs of tannic acid stained microtubule thin sections, and is fully consistent with the microtubule wall projections (fringe patterns) observed in negatively stained and cryo-electron microscopy specimens, which correspond to a 12 protofilament-three start lattice type.

  4. Distinct ECM mechanosensing pathways regulate microtubule dynamics to control endothelial cell branching morphogenesis

    PubMed Central

    Myers, Kenneth A.; Applegate, Kathryn T.

    2011-01-01

    During angiogenesis, cytoskeletal dynamics that mediate endothelial cell branching morphogenesis during vascular guidance are thought to be regulated by physical attributes of the extracellular matrix (ECM) in a process termed mechanosensing. Here, we tested the involvement of microtubules in linking mechanosensing to endothelial cell branching morphogenesis. We used a recently developed microtubule plus end–tracking program to show that specific parameters of microtubule assembly dynamics, growth speed and growth persistence, are globally and regionally modified by, and contribute to, ECM mechanosensing. We demonstrated that engagement of compliant two-dimensional or three-dimensional ECMs induces local differences in microtubule growth speed that require myosin II contractility. Finally, we found that microtubule growth persistence is modulated by myosin II–mediated compliance mechanosensing when cells are cultured on two-dimensional ECMs, whereas three-dimensional ECM engagement makes microtubule growth persistence insensitive to changes in ECM compliance. Thus, compliance and dimensionality ECM mechanosensing pathways independently regulate specific and distinct microtubule dynamics parameters in endothelial cells to guide branching morphogenesis in physically complex ECMs. PMID:21263030

  5. Organization of microtubule assemblies in Dictyostelium syncytia depends on the microtubule crosslinker, Ase1.

    PubMed

    Tikhonenko, Irina; Irizarry, Karen; Khodjakov, Alexey; Koonce, Michael P

    2016-02-01

    It has long been known that the interphase microtubule (MT) array is a key cellular scaffold that provides structural support and directs organelle trafficking in eukaryotic cells. Although in animal cells, a combination of centrosome nucleating properties and polymer dynamics at the distal microtubule ends is generally sufficient to establish a radial, polar array of MTs, little is known about how effector proteins (motors and crosslinkers) are coordinated to produce the diversity of interphase MT array morphologies found in nature. This diversity is particularly important in multinucleated environments where multiple MT arrays must coexist and function. We initiate here a study to address the higher ordered coordination of multiple, independent MT arrays in a common cytoplasm. Deletion of a MT crosslinker of the MAP65/Ase1/PRC1 family disrupts the spatial integrity of multiple arrays in Dictyostelium discoideum, reducing the distance between centrosomes and increasing the intermingling of MTs with opposite polarity. This result, coupled with previous dynein disruptions suggest a robust mechanism by which interphase MT arrays can utilize motors and crosslinkers to sense their position and minimize overlap in a common cytoplasm.

  6. Organization of microtubule assemblies in Dictyostelium syncytia depends on the microtubule crosslinker, Ase1

    PubMed Central

    Tikhonenko, Irina; Irizarry, Karen; Khodjakov, Alexey; Koonce, Michael P.

    2015-01-01

    It has long been known that the interphase microtubule (MT) array is a key cellular scaffold that provides structural support and directs organelle trafficking in eukaryotic cells. Although in animal cells, a combination of centrosome nucleating properties and polymer dynamics at the distal microtubule ends is generally sufficient to establish a radial, polar array of MTs, little is known about how effector proteins (motors and crosslinkers) are coordinated to produce the diversity of interphase MT array morphologies found in nature. This diversity is particularly important in multinucleated environments where multiple MT arrays must coexist and function. We initiate here a study to address the higher ordered coordination of multiple, independent MT arrays in a common cytoplasm. Deletion of a MT crosslinker of the MAP65/Ase1/PRC1 family disrupts the spatial integrity of multiple arrays in Dictyostelium discoideum, reducing the distance between centrosomes and increasing the intermingling of MTs with opposite polarity. This result, coupled with previous dynein disruptions suggest a robust mechanism by which interphase MT arrays can utilize motors and crosslinkers to sense their position and minimize overlap in a common cytoplasm. PMID:26298292

  7. Leading at the Front: How EB Proteins Regulate Microtubule Dynamics

    NASA Astrophysics Data System (ADS)

    Hawkins, Taviare

    2012-02-01

    Microtubules are the most rigid of the cytoskeletal filaments, they provide the cell's scaffolding, form the byways on which motor proteins transport intracellular cargo and reorganize to form the mitotic spindle when the cell needs to divide. These biopolymers are composed of alpha and beta tubulin monomers that create hollow cylindrical nanotubes with an outer diameter of 25 nm and an inner diameter of 17 nm. At steady state concentrations, microtubules undergo a process known as dynamic instability. During dynamic instability the length of individual microtubules is changing as the filament alternates between periods of growth to shrinkage (catastrophe) and shrinkage to growth (rescue). This process can be enhanced or diminished with the addition of microtubule associated proteins (MAPs). MAPs are microtubule binding proteins that stabilize, destabilize, or nucleate microtubules. We will discuss the effects of the stabilizing end-binding proteins (EB1, EB2 and EB3), on microtubule dynamics observed in vitro. The EBs are a unique family of MAPs known to tip track and enhance microtubule growth by stabilizing the ends. This is a different mechanism than those employed by structural MAPs such as tau or MAP4.

  8. Nanomolar concentrations of nocodazole alter microtubule dynamic instability in vivo and in vitro.

    PubMed Central

    Vasquez, R J; Howell, B; Yvon, A M; Wadsworth, P; Cassimeris, L

    1997-01-01

    Previous studies demonstrated that nanomolar concentrations of nocodazole can block cells in mitosis without net microtubule disassembly and resulted in the hypothesis that this block was due to a nocodazole-induced stabilization of microtubules. We tested this hypothesis by examining the effects of nanomolar concentrations of nocodazole on microtubule dynamic instability in interphase cells and in vitro with purified brain tubulin. Newt lung epithelial cell microtubules were visualized by video-enhanced differential interference contrast microscopy and cells were perfused with solutions of nocodazole ranging in concentration from 4 to 400 nM. Microtubules showed a loss of the two-state behavior typical of dynamic instability as evidenced by the addition of a third state where they exhibited little net change in length (a paused state). Nocodazole perfusion also resulted in slower elongation and shortening velocities, increased catastrophe, and an overall decrease in microtubule turnover. Experiments performed on BSC-1 cells that were microinjected with rhodamine-labeled tubulin, incubated in nocodazole for 1 h, and visualized by using low-light-level fluorescence microscopy showed similar results except that nocodazole-treated BSC-1 cells showed a decrease in catastrophe. To gain insight into possible mechanisms responsible for changes in dynamic instability, we examined the effects of 4 nM to 12 microM nocodazole on the assembly of purified tubulin from axoneme seeds. At both microtubule plus and minus ends, perfusion with nocodazole resulted in a dose-dependent decrease in elongation and shortening velocities, increase in pause duration and catastrophe frequency, and decrease in rescue frequency. These effects, which result in an overall decrease in microtubule turnover after nocodazole treatment, suggest that the mitotic block observed is due to a reduction in microtubule dynamic turnover. In addition, the in vitro results are similar to the effects of

  9. Dynamical Length-Regulation of Microtubules

    NASA Astrophysics Data System (ADS)

    Melbinger, Anna; Reese, Louis; Frey, Erwin

    2012-02-01

    Microtubules (MTs) are vital constituents of the cytoskeleton. These stiff filaments are not only needed for mechanical support. They also fulfill highly dynamic tasks. For instance MTs build the mitotic spindle, which pulls the doubled set of chromosomes apart during mitosis. Hence, a well-regulated and adjustable MT length is essential for cell division. Extending a recently introduced model [1], we here study length-regulation of MTs. Thereby we account for both spontaneous polymerization and depolymerization triggered by motor proteins. In contrast to the polymerization rate, the effective depolymerization rate depends on the presence of molecular motors at the tip and thereby on crowding effects which in turn depend on the MT length. We show that these antagonistic effects result in a well-defined MT length. Stochastic simulations and analytic calculations reveal the exact regimes where regulation is feasible. Furthermore, the adjusted MT length and the ensuing strength of fluctuations are analyzed. Taken together, we make quantitative predictions which can be tested experimentally. These results should help to obtain deeper insights in the microscopic mechanisms underlying length-regulation. [4pt] [1] L.Reese, A.Melbinger, E.Frey, Biophys. J., 101, 9, 2190 (2011)

  10. Green's functions and first passage time distributions for dynamic instability of microtubules

    NASA Astrophysics Data System (ADS)

    Bicout, D. J.

    1997-12-01

    It is shown that the dynamic instability process describing the self-assembly and/or disassembly of microtubules is a continuous version of a variant of persistent random walks described by the generalized telegrapher's equation. That is to say, a microtubule is likely to undergo stochastic traveling waves in which catastrophe and rescue events cannot propagate faster than v- and v+, respectively. For this stochastic process, analytic expressions for Green's functions of position and velocity of a microtubule and exact solutions for the first passage time distributions of a microtubule to the nucleating site are obtained. It is shown that, in the ω-->∞ limit, where ω-1 is the persistence time, the dynamic instability process can be described by a diffusion process in the presence of a drift term that, in fact, is the steady-state velocity of the microtubule. As a result, the catastrophe time distribution (i.e., the distribution of microtubule lifetimes to the nucleating site) exhibits a power law with an exponential cutoff as F(t\\|x0)~t-3/2e-t/τc, where τc is the time scale at which the drift term and the diffusive term are comparable.

  11. Assembly of bipolar microtubule structures by passive cross-linkers and molecular motors.

    PubMed

    Johann, D; Goswami, D; Kruse, K

    2016-06-01

    During cell division, sister chromatids are segregated by the mitotic spindle, a bipolar assembly of interdigitating antiparallel polar filaments called microtubules. The spindle contains the midzone, a stable region of overlapping antiparallel microtubules, that is essential for maintaining bipolarity. Although a lot is known about the molecular players involved, the mechanism underlying midzone formation and maintenance is still poorly understood. We study the interaction of polar filaments that are cross-linked by molecular motors moving directionally and by passive cross-linkers diffusing along microtubules. Using a particle-based stochastic model, we find that the interplay of motors and passive cross-linkers can generate a stable finite overlap between a pair of antiparallel polar filaments. We develop a mean-field theory to study this mechanism in detail and investigate the influence of steric interactions between motors and passive cross-linkers on the overlap dynamics. In the presence of interspecies steric interactions, passive cross-linkers mimic the behavior of molecular motors and stable finite overlaps are generated even for non-cross-linking motors. Finally, we develop a mean-field theory for a bundle of aligned polar filaments and show that they can self-organize into a spindlelike pattern. Our work suggests possible ways as to how cells can generate spindle midzones and control their extensions.

  12. Assembly of bipolar microtubule structures by passive cross-linkers and molecular motors

    NASA Astrophysics Data System (ADS)

    Johann, D.; Goswami, D.; Kruse, K.

    2016-06-01

    During cell division, sister chromatids are segregated by the mitotic spindle, a bipolar assembly of interdigitating antiparallel polar filaments called microtubules. The spindle contains the midzone, a stable region of overlapping antiparallel microtubules, that is essential for maintaining bipolarity. Although a lot is known about the molecular players involved, the mechanism underlying midzone formation and maintenance is still poorly understood. We study the interaction of polar filaments that are cross-linked by molecular motors moving directionally and by passive cross-linkers diffusing along microtubules. Using a particle-based stochastic model, we find that the interplay of motors and passive cross-linkers can generate a stable finite overlap between a pair of antiparallel polar filaments. We develop a mean-field theory to study this mechanism in detail and investigate the influence of steric interactions between motors and passive cross-linkers on the overlap dynamics. In the presence of interspecies steric interactions, passive cross-linkers mimic the behavior of molecular motors and stable finite overlaps are generated even for non-cross-linking motors. Finally, we develop a mean-field theory for a bundle of aligned polar filaments and show that they can self-organize into a spindlelike pattern. Our work suggests possible ways as to how cells can generate spindle midzones and control their extensions.

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

    NASA Astrophysics Data System (ADS)

    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.

  14. Regulation of Microtubule Dynamics by TOG-domain proteins XMAP215/Dis1 and CLASP

    PubMed Central

    Al-Bassam, Jawdat; Chang, Fred

    2011-01-01

    The molecular mechanisms by which microtubule-associated proteins (MAPs) regulate the dynamic properties of microtubules (MTs) are still poorly understood. Here, we review recent advances in our understanding of two conserved families of MAPs, the XMAP215/Dis1 and CLASP family of proteins. In vivo and in vitro studies show that XMAP215 proteins act as microtubule polymerases at MT plus ends to accelerate MT assembly, while CLASP proteins promote MT rescue and suppress MT catastrophe events. These are structurally related proteins that use conserved TOG domains to recruit tubulin dimers to MTs. We discuss models for how these proteins might use these individual tubulin dimers to regulate dynamic behaviors of MT plus ends. PMID:21782439

  15. Cooperative lattice dynamics and anomalous fluctuations of microtubules.

    PubMed

    Mohrbach, Hervé; Johner, Albert; Kulić, Igor M

    2012-02-01

    Microtubules have been in the focus of biophysical research for several decades. However, the confusing and mutually contradictory results regarding their elasticity and fluctuations have cast doubt on their present understanding. In this paper, we present the empirical evidence for the existence of discrete guanosine diphosphate (GDP)-tubulin fluctuations between a curved and a straight configuration at room temperature as well as for conformational tubulin cooperativity. Guided by a number of experimental findings, we build the case for a novel microtubule model, with the principal result that microtubules can spontaneously form micron-sized cooperative helical states with unique elastic and dynamic features. The polymorphic dynamics of the microtubule lattice resulting from the tubulin bistability quantitatively explains several experimental puzzles, including anomalous scaling of dynamic fluctuations of grafted microtubules, their apparent length-stiffness relation, and their remarkable curved-helical appearance in general. We point out that the multistability and cooperative switching of tubulin dimers could participate in important cellular processes, and could in particular lead to efficient mechanochemical signaling along single microtubules.

  16. Characterization of rat brain crude extract microtubule assembly: correlation of cold stability with the phosphorylation state of a microtubule-associated 64K protein.

    PubMed

    Margolis, R L; Rauch, C T

    1981-07-21

    We have conducted preliminary investigations into the control of microtubule assembly in rat brain crude extract supernatants. The rationale for these experiments is that microtubules interact with many proteins and are undoubtedly subject to physiological control mechanisms that are lost during tubulin purification. A more complete understanding of the cellular regulation of microtubules must include the physiology of these proteins. Assembly can be monitored in rat brain crude extract high-speed supernatants by measuring the increase in solution turbidity. We find that assembly is maximal in both rate and extent in the absence of added nucleotide. Increasing concentrations of either adenosine 5'-triphosphate (ATP) or guanosine 5'-triphosphate (GTP) inhibit both initiation and elongation of microtubules. GTP appears necessary for assembly and is apparently replenished from an intrinsic energy source during the time course of the assembly reaction. Inhibition of GTP production prevents microtubule assembly, and addition of exogenous GTP will reverse the blockage. Enzymatic removal of GTP at steady state causes a rapid depolymerization to the cold-stable microtubule level. Both GTP production and microtubule assembly display periodic oscillatory maxima. Cold-stable microtubules, which are always present in rat brain crude extract preparations, are rapidly made labile by addition of ATP. Analysis of proteins in cold-stable and cold-labile microtubule fractions shows changes in protein phosphorylation but not in the microtubule-associated protein composition. The tentative conclusion is that the state of phosphorylation of a 64K protein, designated the "switch protein", determines the cold stability or lability, and therefore the dimer association and dissociation rates, of crude extract microtubules.

  17. PHLP2 is essential and plays a role in ciliogenesis and microtubule assembly in Tetrahymena thermophila.

    PubMed

    Bregier, Cezary; Krzemień-Ojak, Lucja; Włoga, Dorota; Jerka-Dziadosz, Maria; Joachimiak, Ewa; Batko, Katarzyna; Filipiuk, Iwona; Smietanka, Urszula; Gaertig, Jacek; Fabczak, Stanisław; Fabczak, Hanna

    2013-11-01

    Recent studies have implicated the phosducin-like protein-2 (PHLP2) in regulation of CCT, a chaperonin whose activity is essential for folding of tubulin and actin. However, the exact molecular function of PHLP2 is unclear. Here we investigate the significance of PHLP2 in a ciliated unicellular model, Tetrahymena thermophila, by deleting its single homolog, Phlp2p. Cells lacking Phlp2p became larger and died within 96 h. Overexpressed Phlp2p-HA localized to cilia, basal bodies, and cytosol without an obvious change in the phenotype. Despite similar localization, overexpressed GFP-Phlp2p caused a dominant-negative effect. Cells overproducing GFP-Phlp2p had decreased rates of proliferation, motility and phagocytosis, as compared to wild type cells or cells overproducing a non-tagged Phlp2p. Growing GFP-Phlp2p-overexpressing cells had fewer cilia and, when deciliated, failed to regenerate cilia, indicating defects in cilia assembly. Paclitaxel-treated GFP-Phlp2p cells failed to elongate cilia, indicating a change in the microtubules dynamics. The pattern of ciliary and cytosolic tubulin isoforms on 2D gels differed between wild type and GFP-Phlp2p-overexpressing cells. Thus, in Tetrahymena, PhLP2 is essential and under specific experimental conditions its activity affects tubulin and microtubule-dependent functions including cilia assembly.

  18. Effects of the fluorescence dye DAPI on microtubule structure in vitro: formation of novel types of tubulin assembly products.

    PubMed

    Vater, W; Böhm, K J; Unger, E

    1993-02-01

    It has been found that the DNA fluorescence dye 4',6-diamidino-2-phenylindole (DAPI) is able to stain also microtubules. However, electron microscopy revealed that DAPI changed microtubule structure and induced the formation of a broad spectrum of polymorphic tubulin assembly products. Upon addition of DAPI to microtubules assembled from 10 to 15 mumol tubulin (molar DAPI/tubulin ratios of 10 to 40) in the presence of microtubule-associated proteins, most of the microtubules were decorated with additional protofilaments usually running parallel to the protofilaments of the microtubule wall (microtubule-protofilament complexes). When DAPI was already present during assembly, curved C- and S-shaped protofilament ribbons and microtubule-ribbon complexes with 6-shaped profiles were the most prominent products, beside microtubules. Additionally, protofilament bundles, some flat sheets, and hoops occurred. Electrophoresis revealed that DAPI lowered the amount of associated proteins, especially of tau-proteins, bound to the assembly products. Nevertheless, DAPI stimulated the assembly, enabled pure tubulin to assemble even at concentrations as low as 10 mumol, and stabilized the assembly products against cold. The microtubule-protofilament complexes, observed for the first time, are interpreted as the result of DAPI-induced protofilament linking as well as of activation of an additional tubulin-tubulin binding site which is possibly identical to that involved in the formation of microtubule doublets.

  19. The sequential activation of the mitotic microtubule assembly pathways favors bipolar spindle formation

    PubMed Central

    Cavazza, Tommaso; Malgaretti, Paolo; Vernos, Isabelle

    2016-01-01

    Centrosome maturation is the process by which the duplicated centrosomes recruit pericentriolar components and increase their microtubule nucleation activity before mitosis. The role of this process in cells entering mitosis has been mostly related to the separation of the duplicated centrosomes and thereby to the assembly of a bipolar spindle. However, spindles can form without centrosomes. In fact, all cells, whether they have centrosomes or not, rely on chromatin-driven microtubule assembly to form a spindle. To test whether the sequential activation of these microtubule assembly pathways, defined by centrosome maturation and nuclear envelope breakdown, plays any role in spindle assembly, we combined experiments in tissue culture cells and Xenopus laevis egg extracts with a mathematical model. We found that interfering with the sequential activation of the microtubule assembly pathways compromises bipolar spindle assembly in tissue culture cells but not in X. laevis egg extracts. Our data suggest a novel function for centrosome maturation that determines the contribution of the chromosomal microtubule assembly pathway and favors bipolar spindle formation in most animal cells in which tubulin is in limiting amounts. PMID:27489339

  20. Mechanisms underlying the active self-assembly of microtubule rings and spools

    DOE PAGES

    VanDelinder, Virginia; Brener, Stephanie; Bachand, George D.

    2016-02-04

    Here, active self-assembly offers a powerful route for the creation of dynamic multiscale structures that are presently inaccessible with standard microfabrication techniques. One such system uses the translation of microtubule filaments by surface-tethered kinesin to actively assemble nanocomposites with bundle, ring, and spool morphologies. Attempts to observe mechanisms involved in this active assembly system have been hampered by experimental difficulties with performing observation during buffer exchange and photodamage from fluorescent excitation. In the present work, we used a custom microfluidic device to remove these limitations and directly study ring/spool formation, including the earliest events (nucleation) that drive subsequent nanocomposite assembly.more » Three distinct formation events were observed: pinning, collisions, and induced curvature. Of these three, collisions accounted for the majority of event leading to ring/spool formation, while the rate of pinning was shown to be dependent on the amount of photodamage in the system. We further showed that formation mechanism directly affects the diameter and rotation direction of the resultant rings and spools. Overall, the fundamental understanding described in this work provides a foundation by which the properties of motor-driven, actively assembled nanocomposites may be tailored toward specific applications.« less

  1. Mechanisms underlying the active self-assembly of microtubule rings and spools

    SciTech Connect

    VanDelinder, Virginia; Brener, Stephanie; Bachand, George D.

    2016-02-04

    Here, active self-assembly offers a powerful route for the creation of dynamic multiscale structures that are presently inaccessible with standard microfabrication techniques. One such system uses the translation of microtubule filaments by surface-tethered kinesin to actively assemble nanocomposites with bundle, ring, and spool morphologies. Attempts to observe mechanisms involved in this active assembly system have been hampered by experimental difficulties with performing observation during buffer exchange and photodamage from fluorescent excitation. In the present work, we used a custom microfluidic device to remove these limitations and directly study ring/spool formation, including the earliest events (nucleation) that drive subsequent nanocomposite assembly. Three distinct formation events were observed: pinning, collisions, and induced curvature. Of these three, collisions accounted for the majority of event leading to ring/spool formation, while the rate of pinning was shown to be dependent on the amount of photodamage in the system. We further showed that formation mechanism directly affects the diameter and rotation direction of the resultant rings and spools. Overall, the fundamental understanding described in this work provides a foundation by which the properties of motor-driven, actively assembled nanocomposites may be tailored toward specific applications.

  2. Tank binding kinase 1 is a centrosome-associated kinase necessary for microtubule dynamics and mitosis.

    PubMed

    Pillai, Smitha; Nguyen, Jonathan; Johnson, Joseph; Haura, Eric; Coppola, Domenico; Chellappan, Srikumar

    2015-12-10

    TANK Binding Kinase 1 (TBK1) is a non-canonical IκB kinase that contributes to KRAS-driven lung cancer. Here we report that TBK1 plays essential roles in mammalian cell division. Specifically, levels of active phospho-TBK1 increase during mitosis and localize to centrosomes, mitotic spindles and midbody, and selective inhibition or silencing of TBK1 triggers defects in spindle assembly and prevents mitotic progression. TBK1 binds to the centrosomal protein CEP170 and to the mitotic apparatus protein NuMA, and both CEP170 and NuMA are TBK1 substrates. Further, TBK1 is necessary for CEP170 centrosomal localization and binding to the microtubule depolymerase Kif2b, and for NuMA binding to dynein. Finally, selective disruption of the TBK1-CEP170 complex augments microtubule stability and triggers defects in mitosis, suggesting that TBK1 functions as a mitotic kinase necessary for microtubule dynamics and mitosis.

  3. Microtubule-based nanomaterials: Exploiting nature's dynamic biopolymers.

    PubMed

    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. Microtubule-based nanomaterials: Exploiting nature's dynamic biopolymers

    SciTech Connect

    Bachand, George D.; Stevens, Mark J.; Spoerke, Erik David

    2015-04-09

    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. In addition, 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.

  5. Microtubule-based nanomaterials: Exploiting nature's dynamic biopolymers

    DOE PAGES

    Bachand, George D.; Stevens, Mark J.; Spoerke, Erik David

    2015-04-09

    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. In addition, 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 onemore » 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.« less

  6. Differential roles of microtubule assembly and sliding in proplatelet formation by megakaryocytes

    PubMed Central

    Patel, Sunita R.; Richardson, Jennifer L.; Schulze, Harald; Kahle, Eden; Galjart, Niels; Drabek, Ksenija; Shivdasani, Ramesh A.; Hartwig, John H.; Italiano, Joseph E.

    2005-01-01

    Megakaryocytes are terminally differentiated cells that, in their final hours, convert their cytoplasm into long, branched proplatelets, which remodel into blood platelets. Proplatelets elongate at an average rate of 0.85 μm/min in a microtubule-dependent process. Addition of rhodamine-tubulin to permeabilized proplatelets, immunofluorescence microscopy of the microtubule plus-end marker end-binding protein 3 (EB3), and fluorescence time-lapse microscopy of EB3–green fluorescent protein (GFP)–expressing megakaryocytes reveal that microtubules, organized as bipolar arrays, continuously polymerize throughout the proplatelet. In immature megakaryocytes lacking proplatelets, microtubule plus-ends initiate and grow by centrosomal nucleation at rates of 8.9 to 12.3 μm/min. In contrast, plus-end growth rates of microtubules within proplatelets are highly variable (1.5-23.5 μm/min) and are both slower and faster than those seen in immature cells. Despite the continuous assembly of microtubules, proplatelets continue to elongate when net microtubule assembly is arrested. One alternative mechanism for force generation is microtubule sliding. Triton X-100–permeabilized proplatelets containing dynein and its regulatory complex, dynactin, but not kinesin, elongate with the addition of adenosine triphosphate (ATP) at a rate of 0.65 μm/min. Retroviral expression in megakaryocytes of dynamitin (p50), which disrupts dynactindynein function, inhibits proplatelet elongation. We conclude that while continuous polymerization of microtubules is necessary to support the enlarging proplatelet mass, the sliding of overlapping microtubules is a vital component of proplatelet elongation. PMID:16118321

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

    PubMed

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

    2016-10-06

    Microtubules are dynamic cytoskeletal elements coordinating and supporting a variety of neuronal processes, including cell division, migration, polarity, intracellular trafficking, and signal transduction. Mutations in genes encoding tubulins and microtubule-associated proteins are known to cause neurodevelopmental and neurodegenerative disorders. Growing evidence suggests that altered microtubule dynamics may also underlie or contribute to neurodevelopmental disorders and neurodegeneration. We report that biallelic mutations in TBCD, encoding one of the five co-chaperones required for assembly and disassembly of the αβ-tubulin heterodimer, the structural unit of microtubules, cause a disease with neurodevelopmental and neurodegenerative features characterized by early-onset cortical atrophy, secondary hypomyelination, microcephaly, thin corpus callosum, developmental delay, intellectual disability, seizures, optic atrophy, and spastic quadriplegia. Molecular dynamics simulations predicted long-range and/or local structural perturbations associated with the disease-causing mutations. Biochemical analyses documented variably reduced levels of TBCD, indicating relative instability of mutant proteins, and defective β-tubulin binding in a subset of the tested mutants. Reduced or defective TBCD function resulted in decreased soluble α/β-tubulin levels and accelerated microtubule polymerization in fibroblasts from affected subjects, demonstrating an overall shift toward a more rapidly growing and stable microtubule population. These cells displayed an aberrant mitotic spindle with disorganized, tangle-shaped microtubules and reduced aster formation, which however did not alter appreciably the rate of cell proliferation. Our findings establish that defective TBCD function underlies a recognizable encephalopathy and drives accelerated microtubule polymerization and enhanced microtubule stability, underscoring an additional cause of altered microtubule dynamics with

  8. The engine of microtubule dynamics comes into focus.

    PubMed

    Mitchison, T J

    2014-05-22

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

  9. Self-Assembled Structures of Tubulin and Microtubules Complexed with Oppositely Charged Molecules

    NASA Astrophysics Data System (ADS)

    Case, Ryan; Pfohl, Thomas; Kim, Joon Heon; Lin, Alison; Safinya, Cyrus R.; Miller, Herb P.; Wilson, Les

    2000-03-01

    Tubulin normally polymerizes into hollow cylindrical microtubules, with outer diameters of about 25 nm, in the presence of Mg^2+ ions and GTP at 37^o C. Microtubules can be stabilized with anticancer agents, such as Taxol. We report on synchotron x-ray studies and confocal imaging data that show that tubulin self-assembles in the presence of cationic lipids at room temperature. These complexes form novel structures with length scales up to three times the diameter of microtubules formed under normal conditions. To improve our understanding of these structures, we use x-ray scattering data of self-assembled structures of Taxol-stabilized microtubule - cationic lipid complexes as a comparison. Supported by NSF DMR-9972246, University of California Biotech Research, and Education Program Training Grant 99-14, DFG Pf 375/1-1.

  10. Probing a self-assembled fd virus membrane with a microtubule

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed

    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.

  12. Contributions of microtubule rotation and dynamic instability to kinetochore capture

    NASA Astrophysics Data System (ADS)

    Sweezy-Schindler, Oliver; Edelmaier, Christopher; Blackwell, Robert; Glaser, Matt; Betterton, Meredith

    2014-03-01

    The capture of lost kinetochores (KCs) by microtubules (MTs) is a crucial part of prometaphase during mitosis. Microtubule dynamic instability has been considered the primary mechanism of KC capture, but recent work discovered that lateral KC attachment to pivoting MTs enabled rapid capture even with significantly reduced MT dynamics. We aim to understand the relative contributions of MT rotational diffusion and dynamic instability to KC capture, as well as KC capture through end-on and/or lateral attachment. Our model consists of rigid MTs and a spherical KC, which are allowed to diffuse inside a spherical nuclear envelope consistent with the geometry of fission yeast. For simplicity, we include a single spindle pole body, which is anchored to the nuclear membrane, and its associated polar MTs. Brownian dynamics treats the diffusion of the MTs and KC and kinetic Monte Carlo models stochastic processes such as dynamic instability. NSF 1546021.

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

    PubMed

    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.

  14. Nonlinear dynamics of dipoles in microtubules: Pseudospin model

    NASA Astrophysics Data System (ADS)

    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.

  15. Effects of organotin compounds on mitosis, spindle structure, toxicity and in vitro microtubule assembly.

    PubMed

    Jensen, K G; Onfelt, A; Wallin, M; Lidums, V; Andersen, O

    1991-09-01

    Di- and tri-methyl, -butyl and phenyl tin, all as chlorides were tested for toxicity and spindle disturbances in V79 Chinese hamster cells and for effects on in vitro assembly of bovine brain tubulin. The V79 cells were treated for 30 min and in general, loss of a stainable spindle could be demonstrated at slightly higher concentrations than c-mitosis. Both these effects were observed at low, non-toxic concentrations. The c-mitotic activity of the compounds was found to increase with increasing lipophilicity and it was best described by a regression on both lipophilicity (partition coefficient octanol/water) and loss of spindle stain. All compounds showed a concentration dependent inhibition of microtubule assembly and all but diphenyltin induced disassembly of preassembled microtubules. An effect on the rate of polymerization was suggested for tributyl- and triphenyltin. The results further indicate that the inhibition of microtubule assembly is through direct interaction with tubulin but does not involve the sulfhydryls of the protein. Thus, the organotins seem to act through two different cooperative mechanisms, inhibition of microtubule assembly and interaction with hydrophobic sites. The latter mechanism might involve Cl-/OH- exchange across cellular membranes. Previous studies have demonstrated chromosomal supercontraction and aneuploidy in human lymphocytes exposed to low concentrations of organotin in vitro and it is suggested that exposure to these compounds may increase the risk of aneuploidy in humans.

  16. Effects of tertiary amine local anesthetics on the assembly and disassembly of brain microtubules in vitro.

    PubMed

    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.

  17. The role of TOG domains in microtubule plus end dynamics.

    PubMed

    Slep, Kevin C

    2009-10-01

    The XMAP215 (Xenopus microtubule-associated protein 215) and CLASP [CLIP-170 (cytoskeletal linker protein 170) associated protein] microtubule plus end tracking families play central roles in the regulation of interphase microtubule dynamics and the proper formation of mitotic spindle architecture and flux. XMAP215 members comprise N-terminally-arrayed hexa-HEAT (huntingtin, elongation factor 3, the PR65/A subunit of protein phosphatase 2A and the lipid kinase Tor) repeats known as TOG (tumour overexpressed gene) domains. Higher eukaryotic XMAP215 members are monomeric and have five TOG domains. Yeast counterparts are dimeric and have two TOG domains. Structure determination of the TOG domain reveals that the six HEAT repeats are aligned to form an oblong scaffold. The TOG domain face composed of intra-HEAT loops forms a contiguous, conserved tubulin-binding surface. Nested within the conserved intra-HEAT loop 1 is an invariant, signature, surface-exposed tryptophan residue that is a prime determinant in the TOG domain-tubulin interaction. The arrayed organization of TOG domains is critical for the processive mechanism of XMAP215, indicative that multiple tubulin/microtubule-binding sites are required for plus end tracking activity. The CLASP family has been annotated as containing a single N-terminal TOG domain. Using XMAP215 TOG domain structure determinants as a metric to analyse CLASP sequence, it is anticipated that CLASP contains two additional cryptic TOGL (TOG-like) domains. The presence of additional TOGL domains implicates CLASP as an ancient XMAP215 relative that uses a similar, multi-TOG-based mechanism to processively track microtubule ends.

  18. Theory of self-assembly of microtubules and motors.

    SciTech Connect

    Aranson, I. S.; Tsimring, L. S.; Materials Science Division; Univ. California at San Diego

    2006-01-01

    We derive a model describing spatiotemporal organization of an array of microtubules interacting via molecular motors. Starting from a stochastic model of inelastic polar rods with a generic anisotropic interaction kernel, we obtain a set of equations for the local rods concentration and orientation. At large enough mean density of rods and concentration of motors, the model describes an orientational instability. We demonstrate that the orientational instability leads to the formation of vortices and (for large density and/or kernel anisotropy) asters seen in recent experiments. We derive the specific form of the interaction kernel from the detailed analysis of microscopic interaction of two filaments mediated by a moving molecular motor and extend our results to include variable motor density and motor attachment to the substrate.

  19. Theory of self-assembly of microtubules and motors

    NASA Astrophysics Data System (ADS)

    Aranson, Igor S.; Tsimring, Lev S.

    2006-09-01

    We derive a model describing spatiotemporal organization of an array of microtubules interacting via molecular motors. Starting from a stochastic model of inelastic polar rods with a generic anisotropic interaction kernel, we obtain a set of equations for the local rods concentration and orientation. At large enough mean density of rods and concentration of motors, the model describes an orientational instability. We demonstrate that the orientational instability leads to the formation of vortices and (for large density and/or kernel anisotropy) asters seen in recent experiments. We derive the specific form of the interaction kernel from the detailed analysis of microscopic interaction of two filaments mediated by a moving molecular motor and extend our results to include variable motor density and motor attachment to the substrate.

  20. Of microtubules and memory: implications for microtubule dynamics in dendrites and spines

    PubMed Central

    Dent, Erik W.

    2017-01-01

    Microtubules (MTs) are cytoskeletal polymers composed of repeating subunits of tubulin that are ubiquitously expressed in eukaryotic cells. They undergo a stochastic process of polymerization and depolymerization from their plus ends termed dynamic instability. MT dynamics is an ongoing process in all cell types and has been the target for the development of several useful anticancer drugs, which compromise rapidly dividing cells. Recent studies also suggest that MT dynamics may be particularly important in neurons, which develop a highly polarized morphology, consisting of a single axon and multiple dendrites that persist throughout adulthood. MTs are especially dynamic in dendrites and have recently been shown to polymerize directly into dendritic spines, the postsynaptic compartment of excitatory neurons in the CNS. These transient polymerization events into dendritic spines have been demonstrated to play important roles in synaptic plasticity in cultured neurons. Recent studies also suggest that MT dynamics in the adult brain function in the essential process of learning and memory and may be compromised in degenerative diseases, such as Alzheimer’s disease. This raises the possibility of targeting MT dynamics in the design of new therapeutic agents. PMID:28035040

  1. Centrosome maturation requires YB-1 to regulate dynamic instability of microtubules for nucleus reassembly

    PubMed Central

    Kawaguchi, Atsushi; Asaka, Masamitsu N.; Matsumoto, Ken; Nagata, Kyosuke

    2015-01-01

    Microtubule formation from the centrosome increases dramatically at the onset of mitosis. This process is termed centrosome maturation. However, regulatory mechanisms of microtubule assembly from the centrosome in response to the centrosome maturation are largely unknown. Here we found that YB-1, a cellular cancer susceptibility protein, is required for the centrosome maturation. Phosphorylated YB-1 accumulated in the centrosome at mitotic phase. By YB-1 knockdown, microtubules were found detached from the centrosome at telophase and an abnormal nuclear shape called nuclear lobulation was found due to defective reassembly of nuclear envelope by mis-localization of non-centrosomal microtubules. In conclusion, we propose that YB-1 is important for the assembly of centrosomal microtubule array for temporal and spatial regulation of microtubules. PMID:25740062

  2. Effect of drugs affecting microtubular assembly on microtubules, phospholipid synthesis and physiological indices (signalling, growth, motility and phagocytosis) in Tetrahymena pyriformis.

    PubMed

    Kovács, P; Csaba, G

    2006-01-01

    Structural changes of microtubules, incorporation of radioactively labelled components into phospholipids, cell motility, growth and phagocytosis were studied under the effect of four drugs affecting microtubular assembly: colchicine, nocodazole, vinblastine and taxol. Although the first three agents influence microtubules in the direction of depolymerization and the fourth stabilizes them, their effects on the structure of microtubules cannot be explained by this. Using confocal microscopy after an acetylated anti-tubulin label, in nocodazole- and colchicine-treated cells, the basal body cages disappear and longitudinal microtubules (LM) became thinner without changing transversal microtubules (TM). After taxol treatment LM also became thinner, however TM disappeared. Under the effect of vinblastine TM became thinner, without influencing LM. These drugs influence the incorporation of components ([(3)H]-serine, [(3)H]-palmitic acid and (32)P) into phospholipids, however their effect is equivocal and cannot be consequently coupled with the effect on the microtubules. Nocodazole, vinblastine and taxol significantly reduced the cell's motility, however colchicine did so to a lesser degree. Vinblastine and nocodazole totally inhibited, and taxol significantly decreased cell growth, while colchicine in a lower concentration increased the multiplication of cells. Phagocytosis was not significantly influenced after 1 min, but after 5 min all the agents studied (except colchicine) significantly inhibited phagocytosis. After 15 and 30 min each molecule caused highly significant inhibition. The experiments demonstrate that drugs affecting microtubular assembly dynamics influence differently the diverse (longitudinal, transversal etc.) microtubular systems of Tetrahymena and also differently influence microtubule-dependent physiological processes. The latter are more dependent on microtubular dynamics than are changes in phospholipid signalling.

  3. Binding of dihydroxynaphthyl aryl ketones to tubulin colchicine site inhibits microtubule assembly.

    PubMed

    Gutierrez, Eunices; Benites, Julio; Valderrama, Jaime A; Calderon, Pedro Buc; Verrax, Julien; Nova, Esteban; Villanelo, Felipe; Maturana, Daniel; Escobar, Cristian; Lagos, Rosalba; Monasterio, Octavio

    2015-10-23

    Dihydroxynaphthyl aryl ketones 1-5 have been evaluated for their abilities to inhibit microtubule assembly and the binding to tubulin. Compounds 3, 4 and 5 displayed competitive inhibition against colchicine binding, and docking analysis showed that they bind to the tubulin colchicine-binding pocket inducing sheets instead of microtubules. Remarkable differences in biological activity observed among the assayed compounds seem to be related to the structure and position of the aryl substituent bonded to the carbonyl group. Compounds 2, 3 and 4, which contain a heterocyclic ring, presented higher affinity for tubulin compared to the carbocyclic analogue 5. Compound 4 showed the best affinity of the series, with an IC50 value of 2.1 μM for microtubule polymerization inhibition and a tubulin dissociation constant of 1.0 ± 0.2 μM, as determined by thermophoresis. Compound 4 was more efficacious in disrupting microtubule assembly in vitro than compound 5 although it contains the trimethoxyphenyl ring present in colchicine. Hydrogen bonds with Asn101 of α-tubulin seem to be responsible for the higher affinity of compound 4 respects to the others.

  4. SCG10, a microtubule destabilizing factor, stimulates the neurite outgrowth by modulating microtubule dynamics in rat hippocampal primary cultured neurons.

    PubMed

    Morii, Hiroshi; Shiraishi-Yamaguchi, Yoko; Mori, Nozomu

    2006-09-01

    Microtubule dynamics, one of the key elements in neurite outgrowth, is regulated by various regulatory factors to determine the behavior of the neuronal growth cone and to form the specialized neuronal shape. SCG10 is a neuron-specific stathmin protein with a potent microtubule destabilizing factor and is enriched in the growth cones of the developing neurons. We investigated the functional role of SCG10 in neurite outgrowth using rat hippocampal primary cultured neurons. Genetic manipulation of SCG10 using a short-interfering RNA duplex markedly decreased the SCG10 expression level and significantly suppressed neurite outgrowth. This result was confirmed by immunodepletion experiments. On the other hand, the protein transduction of SCG10 using a polyarginine tag stimulated neurite outgrowth. Such manipulation of the SCG10 expression level affected microtubule morphology within the growth cones. A decrease in the SCG10 level converted the morphology to a more stable state, while an increase converted the morphology to a more dynamic state. However, an excess of SCG10 induced neurite retraction due to an excess of microtubule disassembly. These results suggest that SCG10 serves as an important regulatory factor of growth cone motility by enhancing microtubule dynamics, possibly through increasing the catastrophe frequency.

  5. Self-repair promotes microtubule rescue

    PubMed Central

    Gaillard, Jérémie; John, Karin; Blanchoin, Laurent; Théry, Manuel

    2016-01-01

    Summary The dynamic instability of microtubules is characterised by slow growth phases stochastically interrupted by rapid depolymerisations called catastrophes. Rescue events can arrest the depolymerisation and restore microtubule elongation. However the origin of these rescue events remain unexplained. Here we show that microtubule lattice self-repair, in structurally damaged sites, is responsible for the rescue of microtubule growth. Tubulin photo-conversion in cells revealed that free tubulin dimers can incorporate along the shafts of microtubules, especially in regions where microtubules cross each other, form bundles or become bent due to mechanical constraints. These incorporation sites appeared to act as effective rescue sites ensuring microtubule rejuvenation. By securing damaged microtubule growth, the self-repair process supports a mechanosensitive growth by specifically promoting microtubule assembly in regions where they are subjected to physical constraints. PMID:27617929

  6. Tau Assembly: The Dominant Role of PHF6 (VQIVYK) in Microtubule Binding Region Repeat R3

    PubMed Central

    Ganguly, Pritam; Do, Thanh D.; Larini, Luca; LaPointe, Nichole E.; Sercel, Alexander J.; Shade, Madeleine F.; Feinstein, Stuart C.; Bowers, Michael T.; Shea, Joan-Emma

    2015-01-01

    Self-aggregation of the microtubule-binding protein Tau reduces its functionality and is tightly associated with Tau-related diseases, termed tauopathies. Tau aggregation is also strongly associated with two nucleating six-residue segments, namely PHF6 (VQIVYK) and PHF6* (VQIINK). In this paper, using experiments and computational modeling, we study the self-assembly of individual and binary mixtures of Tau fragments containing PHF6* (R2/wt; 273GKVQIINKKLDL284) and PHF6 (R3/wt; 306VQIVYKPVDLSK317), and a mutant R2/ΔK280 associated with a neurodegenerative tauopathy. The initial stage of aggregation is probed by ion-mobility mass spectrometry, the kinetics of aggregation monitored with Thioflavin T assays and the morphology of aggregates visualized by transmission electron microscopy. Insights into the structure of early aggregates and the factors stabilizing the aggregates are obtained from replica exchange molecular dynamics simulations. Our data suggest that R3/wt has a much stronger aggregation propensity than either R2/wt or R2/ΔK280. Heterodimers containing R3/wt are less stable than R3/wt homodimers but much more stable than homodimers of R2/wt and R2/ΔK280, suggesting a possible role of PHF6*/PHF6 interactions in initiating the aggregation of full length Tau. Lastly, R2/ΔK280 binds stronger to R3/wt than R2/wt suggesting a possible mechanism for a pathological loss of normal Tau function. PMID:25775228

  7. Bis(1,8-anilinonaphthalenesulfonate). A novel and potent inhibitor of microtubule assembly.

    PubMed

    Horowitz, P; Prasad, V; Luduena, R F

    1984-12-10

    Two related compounds, 1,8-anilinonaphthalenesulfonate (1,8-ANS) and bis(1,8-anilinonaphthalenesulfonate) (Bis-ANS), are useful fluorescent probes for hydrophobic areas on protein molecules. Using fluorescence, we examined the binding of these compounds to bovine brain tubulin and found that Bis-ANS and 1,8-ANS bound to tubulin with Ki values of 2 and 25 microM, respectively. Bis-ANS potently inhibited the polymerization of tubulin into microtubules in vitro. In the presence of microtubule-associated protein 2, half-maximal inhibition of assembly was obtained at 3 microM Bis-ANS. In the presence of tau protein, half-maximal inhibition was obtained at 15 microM Bis-ANS. Surprisingly, 1,8-ANS, even at 200 microM, did not inhibit assembly. Scatchard analysis indicated one binding site for Bis-ANS on tubulin. Previous reports of 1,8-ANS binding to tubulin may have been influenced by the presence of Bis-ANS which until recently was a common contaminant of commercial supplies. Because of its intense fluorescence in addition to its potent inhibitory effects, Bis-ANS appears to be a useful probe to study microtubule assembly and other interactions involving tubulin.

  8. Microtubule dynamic instability: the role of cracks between protofilaments.

    PubMed

    Li, Chunlei; Li, Jun; Goodson, Holly V; Alber, Mark S

    2014-03-28

    Microtubules (MTs) are cytoplasmic protein polymers that are essential for fundamental cellular processes including the maintenance of cell shape, organelle transport and formation of the mitotic spindle. Microtubule dynamic instability is critical for these processes, but it remains poorly understood, in part because the relationship between the structure of the MT tip and the growth/depolymerization transitions is enigmatic. In previous work, we used computational models of dynamic instability to provide evidence that cracks (laterally unbonded regions) between protofilaments play a key role in the regulation of dynamic instability. Here we use computational models to investigate the connection between cracks and dynamic instability in more detail. Our work indicates that while cracks contribute to dynamic instability in a fundamental way, it is not the depth of the cracks per se that governs MT dynamic instability. Instead, what matters more is whether the cracks terminate in GTP-rich or GDP-rich regions of the MT. Based on these observations, we suggest that a functional "GTP cap" (i.e., one capable of promoting MT growth) is one where the cracks terminate in pairs of GTP-bound subunits, and that the likelihood of catastrophe rises significantly with the fraction of crack-terminating subunits that contain GDP. In addition to helping clarify the mechanism of dynamic instability, this idea could also explain how MT stabilizers work: proteins that introduce lateral cross-links between protofilaments would produce islands of GDP-bound tubulin that mimic GTP-rich regions in having strong lateral bonds, thus reducing crack propagation, suppressing catastrophe and promoting rescue.

  9. Tank binding kinase 1 is a centrosome-associated kinase necessary for microtubule dynamics and mitosis

    PubMed Central

    Pillai, Smitha; Nguyen, Jonathan; Johnson, Joseph; Haura, Eric; Coppola, Domenico; Chellappan, Srikumar

    2015-01-01

    TANK Binding Kinase 1 (TBK1) is a non-canonical IκB kinase that contributes to KRAS-driven lung cancer. Here we report that TBK1 plays essential roles in mammalian cell division. Specifically, levels of active phospho-TBK1 increase during mitosis and localize to centrosomes, mitotic spindles and midbody, and selective inhibition or silencing of TBK1 triggers defects in spindle assembly and prevents mitotic progression. TBK1 binds to the centrosomal protein CEP170 and to the mitotic apparatus protein NuMA, and both CEP170 and NuMA are TBK1 substrates. Further, TBK1 is necessary for CEP170 centrosomal localization and binding to the microtubule depolymerase Kif2b, and for NuMA binding to dynein. Finally, selective disruption of the TBK1–CEP170 complex augments microtubule stability and triggers defects in mitosis, suggesting that TBK1 functions as a mitotic kinase necessary for microtubule dynamics and mitosis. PMID:26656453

  10. Identification of a novel microtubule-destabilizing motif in CPAP that binds to tubulin heterodimers and inhibits microtubule assembly.

    PubMed

    Hung, Liang-Yi; Chen, Hua-Ling; Chang, Ching-Wen; Li, Bor-Ran; Tang, Tang K

    2004-06-01

    We have previously identified a new centrosomal protein, centrosomal protein 4.1-associated protein (CPAP), which is associated with the gamma-tubulin complex. Here, we report that CPAP carries a novel microtubule-destabilizing motif that not only inhibits microtubule nucleation from the centrosome but also depolymerizes taxol-stabilized microtubules. Deletion mapping and functional analyses have defined a 112-residue CPAP that is necessary and sufficient for microtubule destabilization. This 112-residue CPAP directly recognizes the plus end of a microtubule and inhibits microtubule nucleation from the centrosome. Biochemical and functional analyses revealed that this 112-residue CPAP also binds to tubulin dimers, resulting in the destabilization of microtubules. Using the tetracycline-controlled system (tet-off), we observed that overexpression of this 112-residue CPAP inhibits cell proliferation and induces apoptosis after G2/M arrest. The possible mechanisms of how this 112-residue motif in CPAP that inhibits microtubule nucleation from the centrosome and disassembles preformed microtubules are discussed.

  11. Inhibition of microtubule dynamics impedes repair of kidney ischemia/reperfusion injury and increases fibrosis

    PubMed Central

    Han, Sang Jun; Kim, Ji-Hyeon; Kim, Jee In; Park, Kwon Moo

    2016-01-01

    The microtubule cytoskeleton is composed of α-tubulin and β-tubulin heterodimers, and it serves to regulate the shape, motility, and division of a cell. Post-translational modifications including acetylation are closely associated with the functional aspects of the microtubule, involving in a number of pathological diseases. However, the role of microtubule acetylation in acute kidney injury (AKI) and progression of AKI to chronic kidney disease have yet to be understood. In this study, ischemia/reperfusion (I/R), a major cause of AKI, resulted in deacetylation of the microtubules with a decrease in α-tubulin acetyltransferase 1 (α-TAT1). Paclitaxel (taxol), an agent that stabilizes microtubules by tubulin acetylation, treatment during the recovery phase following I/R injury inhibited tubular cell proliferation, impaired renal functional recovery, and worsened fibrosis. Taxol induced α-tubulin acetylation and post-I/R cell cycle arrest. Taxol aggregated the microtubule in the cytoplasm, resulting in suppression of microtubule dynamics. Our studies have demonstrated for the first time that I/R induced deacetylation of the microtubules, and that inhibition of microtubule dynamics retarded repair of injured tubular epithelial cells leading to an acceleration of fibrosis. This suggests that microtubule dynamics plays an important role in the processes of repair and fibrosis after AKI. PMID:27270990

  12. TACC3 is a microtubule plus end–tracking protein that promotes axon elongation and also regulates microtubule plus end dynamics in multiple embryonic cell types

    PubMed Central

    Nwagbara, Belinda U.; Faris, Anna E.; Bearce, Elizabeth A.; Erdogan, Burcu; Ebbert, Patrick T.; Evans, Matthew F.; Rutherford, Erin L.; Enzenbacher, Tiffany B.; Lowery, Laura Anne

    2014-01-01

    Microtubule plus end dynamics are regulated by a conserved family of proteins called plus end–tracking proteins (+TIPs). It is unclear how various +TIPs interact with each other and with plus ends to control microtubule behavior. The centrosome-associated protein TACC3, a member of the transforming acidic coiled-coil (TACC) domain family, has been implicated in regulating several aspects of microtubule dynamics. However, TACC3 has not been shown to function as a +TIP in vertebrates. Here we show that TACC3 promotes axon outgrowth and regulates microtubule dynamics by increasing microtubule plus end velocities in vivo. We also demonstrate that TACC3 acts as a +TIP in multiple embryonic cell types and that this requires the conserved C-terminal TACC domain. Using high-resolution live-imaging data on tagged +TIPs, we show that TACC3 localizes to the extreme microtubule plus end, where it lies distal to the microtubule polymerization marker EB1 and directly overlaps with the microtubule polymerase XMAP215. TACC3 also plays a role in regulating XMAP215 stability and localizing XMAP215 to microtubule plus ends. Taken together, our results implicate TACC3 as a +TIP that functions with XMAP215 to regulate microtubule plus end dynamics. PMID:25187649

  13. Maternal Dead-End1 is required for vegetal cortical microtubule assembly during Xenopus axis specification

    PubMed Central

    Mei, Wenyan; Jin, Zhigang; Lai, Fangfang; Schwend, Tyler; Houston, Douglas W.; King, Mary Lou; Yang, Jing

    2013-01-01

    Vertebrate axis specification is an evolutionarily conserved developmental process that relies on asymmetric activation of Wnt signaling and subsequent organizer formation on the future dorsal side of the embryo. Although roles of Wnt signaling during organizer formation have been studied extensively, it is unclear how the Wnt pathway is asymmetrically activated. In Xenopus and zebrafish, the Wnt pathway is triggered by dorsal determinants, which are translocated from the vegetal pole to the future dorsal side of the embryo shortly after fertilization. The transport of dorsal determinants requires a unique microtubule network formed in the vegetal cortex shortly after fertilization. However, molecular mechanisms governing the formation of vegetal cortical microtubule arrays are not fully understood. Here we report that Dead-End 1 (Dnd1), an RNA-binding protein required for primordial germ cell development during later stages of embryogenesis, is essential for Xenopus axis specification. We show that knockdown of maternal Dnd1 specifically interferes with the formation of vegetal cortical microtubules. This, in turn, impairs translocation of dorsal determinants, the initiation of Wnt signaling, organizer formation, and ultimately results in ventralized embryos. Furthermore, we found that Dnd1 binds to a uridine-rich sequence in the 3′-UTR of trim36, a vegetally localized maternal RNA essential for vegetal cortical microtubule assembly. Dnd1 anchors trim36 to the vegetal cortex in the egg, promoting high concentrations of Trim36 protein there. Our work thus demonstrates a novel and surprising function for Dnd1 during early development and provides an important link between Dnd1, mRNA localization, the microtubule cytoskeleton and axis specification. PMID:23615278

  14. Bis-ANS as a specific inhibitor for microtubule-associated protein induced assembly of tubulin.

    PubMed

    Mazumdar, M; Parrack, P K; Mukhopadhyay, K; Bhattacharyya, B

    1992-07-21

    5,5'-Bis[8-(phenylamino)-1-naphthalenesulfonate] (bis-ANS), the fluorescent probe which binds to tubulin, inhibits its assembly into microtubules [Horowitz et al. (1984) J. Biol. Chem. 259, 14647-14650]. The results described in this paper demonstrate that bis-ANS is quite distinct from other well-known microtubule inhibitors in its specificity of action. The inhibitory potentials of bis-ANS and its three structural analogues ANS, Prodan [6-propionyl-2-(dimethylamino)naphthalene], and NSA (naphthalenesulfonic acid) have been compared. It is found that they can be arranged in the following order according to their polymerization inhibitory potentials: bis-ANS approximately equal to Prodan much greater than ANS greater than NSA. Interestingly, the naphthalene nucleus is sufficient to cause inhibition of polymerization. Detailed experiments were carried out to examine the mode of assembly inhibition by aminonaphthalenes at the molecular level, using bis-ANS as a representative. It was found that there was little or no effect of bis-ANS on the assembly of tubulin when polymerization was induced by assembly promoters like taxol, DMSO, or glutamate, or on the assembly of subtilisin-digested protein (tubulin S), for all of which half-maximal inhibition could not be achieved even at 120 microM bis-ANS. On the contrary, bis-ANS acts as an inhibitor in the case of MAP- (MAP2 and tau) and poly(L-lysine)-induced assembly of tubulin, with half-maximal inhibitory concentrations ranging from 1.5 to 7.6 microM. Our results place bis-ANS as a novel inhibitor, which seems to specifically inhibit C-termini-mediated assembly. Of all assembly inhibitors known so far, none exhibits such selection.(ABSTRACT TRUNCATED AT 250 WORDS)

  15. Qualitative Behavior of the Low-Frequency Vibrational Dynamics of Microtubules and the Surrounding Water.

    PubMed

    Moix, Jeremy M; Parker, James E; Echchgadda, Ibtissam

    2017-04-13

    The dynamics of the low-frequency vibrational modes of microtubules play a key role in many theoretical models regarding their biological function. We analyze these dynamics through large scale, classical molecular dynamics simulations of a microtubule composed of 42 tubulin heterodimers to provide insights into the qualitative nature of the vibrational energy absorption and dissipation mechanisms. The computed microtubule absorption spectra and vibrational density of states in the terahertz regime are presented, along with an analysis of the vibrational dephasing rates of the tubulin monomer center of mass dynamics, which are shown to be overdamped. Additionally, the presence of the microtubule modifies the dynamical properties of the solvation shell structure within roughly 10 Å of the protein. These vibrational properties are similar to those seen in other globular proteins and indicate microtubules are unlikely candidates for any large scale collective vibrational processes in the terahertz regime such as Fröhlich condensates.

  16. Contributions of Microtubule Dynamic Instability and Rotational Diffusion to Kinetochore Capture.

    PubMed

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

    2017-02-07

    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 time by 25%. Our results suggest that while microtubule rotational diffusion can speed up kinetochore capture, it is unlikely to be the dominant physical mechanism for typical conditions in fission yeast. In addition, we found that when microtubules undergo dynamic instability, lateral captures predominate even in the absence of rotational diffusion. Counterintuitively, adding rotational diffusion to a dynamic microtubule increases the probability of end-on capture.

  17. Assembly of Caenorhabditis elegans acentrosomal spindles occurs without evident microtubule-organizing centers and requires microtubule sorting by KLP-18/kinesin-12 and MESP-1

    PubMed Central

    Wolff, Ian D.; Tran, Michael V.; Mullen, Timothy J.; Villeneuve, Anne M.; Wignall, Sarah M.

    2016-01-01

    Although centrosomes contribute to spindle formation in most cell types, oocytes of many species are acentrosomal and must organize spindles in their absence. Here we investigate this process in Caenorhabditis elegans, detailing how acentrosomal spindles form and revealing mechanisms required to establish bipolarity. Using high-resolution imaging, we find that in meiosis I, microtubules initially form a “cage-like” structure inside the disassembling nuclear envelope. This structure reorganizes so that minus ends are sorted to the periphery of the array, forming multiple nascent poles that then coalesce until bipolarity is achieved. In meiosis II, microtubules nucleate in the vicinity of chromosomes but then undergo similar sorting and pole formation events. We further show that KLP-18/kinesin-12 and MESP-1, previously shown to be required for spindle bipolarity, likely contribute to bipolarity by sorting microtubules. After their depletion, minus ends are not sorted outward at the early stages of spindle assembly and instead converge. These proteins colocalize on microtubules, are interdependent for localization, and can interact, suggesting that they work together. We propose that KLP-18/kinesin-12 and MESP-1 form a complex that functions to sort microtubules of mixed polarity into a configuration in which minus ends are away from the chromosomes, enabling formation of nascent poles. PMID:27559133

  18. Minimal model for collective kinetochore–microtubule dynamics

    PubMed Central

    Banigan, Edward J.; Chiou, Kevin K.; Ballister, Edward R.; Mayo, Alyssa M.; Lampson, Michael A.; Liu, Andrea J.

    2015-01-01

    Chromosome segregation during cell division depends on interactions of kinetochores with dynamic microtubules (MTs). In many eukaryotes, each kinetochore binds multiple MTs, but the collective behavior of these coupled MTs is not well understood. We present a minimal model for collective kinetochore–MT dynamics, based on in vitro measurements of individual MTs and their dependence on force and kinetochore phosphorylation by Aurora B kinase. For a system of multiple MTs connected to the same kinetochore, the force–velocity relation has a bistable regime with two possible steady-state velocities: rapid shortening or slow growth. Bistability, combined with the difference between the growing and shrinking speeds, leads to center-of-mass and breathing oscillations in bioriented sister kinetochore pairs. Kinetochore phosphorylation shifts the bistable region to higher tensions, so that only the rapidly shortening state is stable at low tension. Thus, phosphorylation leads to error correction for kinetochores that are not under tension. We challenged the model with new experiments, using chemically induced dimerization to enhance Aurora B activity at metaphase kinetochores. The model suggests that the experimentally observed disordering of the metaphase plate occurs because phosphorylation increases kinetochore speeds by biasing MTs to shrink. Our minimal model qualitatively captures certain characteristic features of kinetochore dynamics, illustrates how biochemical signals such as phosphorylation may regulate the dynamics, and provides a theoretical framework for understanding other factors that control the dynamics in vivo. PMID:26417109

  19. The Oligomeric Outer Dynein Arm Assembly Factor CCDC103 Is Tightly Integrated within the Ciliary Axoneme and Exhibits Periodic Binding to Microtubules*

    PubMed Central

    King, Stephen M.; Patel-King, Ramila S.

    2015-01-01

    CCDC103 is an ∼29-kDa protein consisting of a central RPAP3_C domain flanked by N- and C-terminal coiled coils. Defects in CCDC103 lead to primary ciliary dyskinesia caused by the loss of outer dynein arms. This protein is present along the entire length of the ciliary axoneme and does not require other dynein or docking complex components for its integration. Unlike other known dynein assembly factors within the axoneme, CCDC103 is not solubilized by 0.6 m NaCl and requires more chaotropic conditions, such as 0.5 m KI. Alternatively, it can be extracted using 0.3% sarkosyl. CCDC103 forms stable dimers and other oligomers in solution through interactions involving the central domain. The smallest particle observed by dynamic light scattering has a hydrodynamic diameter of ∼25 nm. Furthermore, CCDC103 binds microtubules directly, forming ∼9-nm diameter particles that exhibit a 12-nm spacing on the microtubule lattice, suggesting that there may be two CCDC103 units per outer arm dynein repeat. Although the outer dynein arm docking complex is necessary to form arrays of dyneins along microtubules, it is not sufficient to set up a single array in a precise location on each axonemal doublet. We propose that CCDC103 helps generate a high-affinity site on the doublets for outer arm assembly, either through direct interactions or indirectly, perhaps by modifying the underlying microtubule lattice. PMID:25572396

  20. Steering microtubule shuttle transport with dynamically controlled magnetic fields.

    PubMed

    Mahajan, K D; Ruan, G; Dorcéna, C J; Vieira, G; Nabar, G; Bouxsein, N F; Chalmers, J J; Bachand, G D; Sooryakumar, R; Winter, J O

    2016-04-28

    Nanoscale control of matter is critical to the design of integrated nanosystems. Here, we describe a method to dynamically control directionality of microtubule (MT) motion using programmable magnetic fields. MTs are combined with magnetic quantum dots (i.e., MagDots) that are manipulated by external magnetic fields provided by magnetic nanowires. MT shuttles thus undergo both ATP-driven and externally-directed motion with a fluorescence component that permits simultaneous visualization of shuttle motion. This technology is used to alter the trajectory of MTs in motion and to pin MT motion. Such an approach could be used to evaluate the MT-kinesin transport system and could serve as the basis for improved lab-on-a-chip technologies based on MT transport.

  1. Steering microtubule shuttle transport with dynamically controlled magnetic fields

    SciTech Connect

    Mahajan, K. D.; Ruan, G.; Dorcéna, C. J.; Vieira, G.; Nabar, G.; Bouxsein, N. F.; Chalmers, J. J.; Bachand, G. D.; Sooryakumar, R.; Winter, J. O.

    2016-03-23

    Nanoscale control of matter is critical to the design of integrated nanosystems. Here, we describe a method to dynamically control directionality of microtubule (MT) motion using programmable magnetic fields. MTs are combined with magnetic quantum dots (i.e., MagDots) that are manipulated by external magnetic fields provided by magnetic nanowires. MT shuttles thus undergo both ATP-driven and externally-directed motion with a fluorescence component that permits simultaneous visualization of shuttle motion. This technology is used to alter the trajectory of MTs in motion and to pin MT motion. Ultimately, such an approach could be used to evaluate the MT-kinesin transport system and could serve as the basis for improved lab-on-a-chip technologies based on MT transport.

  2. Steering microtubule shuttle transport with dynamically controlled magnetic fields

    DOE PAGES

    Mahajan, K. D.; Ruan, G.; Dorcéna, C. J.; ...

    2016-03-23

    Nanoscale control of matter is critical to the design of integrated nanosystems. Here, we describe a method to dynamically control directionality of microtubule (MT) motion using programmable magnetic fields. MTs are combined with magnetic quantum dots (i.e., MagDots) that are manipulated by external magnetic fields provided by magnetic nanowires. MT shuttles thus undergo both ATP-driven and externally-directed motion with a fluorescence component that permits simultaneous visualization of shuttle motion. This technology is used to alter the trajectory of MTs in motion and to pin MT motion. Ultimately, such an approach could be used to evaluate the MT-kinesin transport system andmore » could serve as the basis for improved lab-on-a-chip technologies based on MT transport.« less

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

    PubMed

    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.

  4. Steering microtubule shuttle transport with dynamically controlled magnetic fields

    NASA Astrophysics Data System (ADS)

    Mahajan, K. D.; Ruan, G.; Dorcéna, C. J.; Vieira, G.; Nabar, G.; Bouxsein, N. F.; Chalmers, J. J.; Bachand, G. D.; Sooryakumar, R.; Winter, J. O.

    2016-04-01

    Nanoscale control of matter is critical to the design of integrated nanosystems. Here, we describe a method to dynamically control directionality of microtubule (MT) motion using programmable magnetic fields. MTs are combined with magnetic quantum dots (i.e., MagDots) that are manipulated by external magnetic fields provided by magnetic nanowires. MT shuttles thus undergo both ATP-driven and externally-directed motion with a fluorescence component that permits simultaneous visualization of shuttle motion. This technology is used to alter the trajectory of MTs in motion and to pin MT motion. Such an approach could be used to evaluate the MT-kinesin transport system and could serve as the basis for improved lab-on-a-chip technologies based on MT transport.Nanoscale control of matter is critical to the design of integrated nanosystems. Here, we describe a method to dynamically control directionality of microtubule (MT) motion using programmable magnetic fields. MTs are combined with magnetic quantum dots (i.e., MagDots) that are manipulated by external magnetic fields provided by magnetic nanowires. MT shuttles thus undergo both ATP-driven and externally-directed motion with a fluorescence component that permits simultaneous visualization of shuttle motion. This technology is used to alter the trajectory of MTs in motion and to pin MT motion. Such an approach could be used to evaluate the MT-kinesin transport system and could serve as the basis for improved lab-on-a-chip technologies based on MT transport. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr08529b

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

    PubMed

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

    2015-07-15

    ELMO2 belongs to a family of scaffold proteins involved in phagocytosis and cell motility. ELMO2 can simultaneously bind integrin-linked kinase (ILK) and RhoG, forming tripartite ERI complexes. These complexes are involved in promoting β1 integrin-dependent directional migration in undifferentiated epidermal keratinocytes. ELMO2 and ILK have also separately been implicated in microtubule regulation at integrin-containing focal adhesions. During differentiation, epidermal keratinocytes cease to express integrins, but ERI complexes persist. Here we show an integrin-independent role of ERI complexes in modulation of microtubule dynamics in differentiated keratinocytes. Depletion of ERI complexes by inactivating the Ilk gene in these cells reduces microtubule growth and increases the frequency of catastrophe. Reciprocally, exogenous expression of ELMO2 or RhoG stabilizes microtubules, but only if ILK is also present. Mechanistically, activation of Rac1 downstream from ERI complexes mediates their effects on microtubule stability. In this pathway, Rac1 serves as a hub to modulate microtubule dynamics through two different routes: 1) phosphorylation and inactivation of the microtubule-destabilizing protein stathmin and 2) phosphorylation and inactivation of GSK-3β, which leads to the activation of CRMP2, promoting microtubule growth. At the cellular level, the absence of ERI species impairs Ca(2+)-mediated formation of adherens junctions, critical to maintaining mechanical integrity in the epidermis. Our findings support a key role for ERI species in integrin-independent stabilization of the microtubule network in differentiated keratinocytes.

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

    PubMed

    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.

  7. Understanding force-generating microtubule systems through in vitro reconstitution

    PubMed Central

    Kok, Maurits; Dogterom, Marileen

    2016-01-01

    ABSTRACT Microtubules switch between growing and shrinking states, a feature known as dynamic instability. The biochemical parameters underlying dynamic instability are modulated by a wide variety of microtubule-associated proteins that enable the strict control of microtubule dynamics in cells. The forces generated by controlled growth and shrinkage of microtubules drive a large range of processes, including organelle positioning, mitotic spindle assembly, and chromosome segregation. In the past decade, our understanding of microtubule dynamics and microtubule force generation has progressed significantly. Here, we review the microtubule-intrinsic process of dynamic instability, the effect of external factors on this process, and how the resulting forces act on various biological systems. Recently, reconstitution-based approaches have strongly benefited from extensive biochemical and biophysical characterization of individual components that are involved in regulating or transmitting microtubule-driven forces. We will focus on the current state of reconstituting increasingly complex biological systems and provide new directions for future developments. PMID:27715396

  8. Dynamic interplay between nitration and phosphorylation of tubulin cofactor B in the control of microtubule dynamics

    PubMed Central

    Rayala, Suresh K.; Martin, Emil; Sharina, Iraida G.; Molli, Poonam R.; Wang, Xiaoping; Jacobson, Raymond; Murad, Ferid; Kumar, Rakesh

    2007-01-01

    Tubulin cofactor B (TCoB) plays an important role in microtubule dynamics by facilitating the dimerization of α- and β-tubulin. Recent evidence suggests that p21-activated kinase 1 (Pak1), a major signaling nodule in eukaryotic cells, phosphorylates TCoB on Ser-65 and Ser-128 and plays an essential role in microtubule regrowth. However, to date, no upstream signaling molecules have been identified to antagonize the functions of TCoB, which might help in maintaining the equilibrium of microtubules. Here, we discovered that TCoB is efficiently nitrated, mainly on Tyr-64 and Tyr-98, and nitrated-TCoB attenuates the synthesis of new microtubules. In addition, we found that nitration of TCoB antagonizes signaling-dependent phosphorylation of TCoB, whereas optimal nitration of TCoB requires the presence of functional Pak1 phosphorylation sites, thus providing a feedback mechanism to regulate phosphorylation-dependent MT regrowth. Together these findings identified TCoB as the third cytoskeleton protein to be nitrated and suggest a previously undescribed mechanism, whereby growth factor signaling may coordinately integrate nitric oxide signaling in the regulation of microtubule dynamics. PMID:18048340

  9. Important factors determining the nanoscale tracking precision of dynamic microtubule ends

    PubMed Central

    BOHNER, G.; GUSTAFSSON, N.; CADE, N.I.; MAURER, S.P.; GRIFFIN, L.D.

    2016-01-01

    Summary Tracking dynamic microtubule ends in fluorescence microscopy movies provides insight into the statistical properties of microtubule dynamics and is vital for further analysis that requires knowledge of the trajectories of the microtubule ends. Here we analyse the performance of a previously developed automated microtubule end tracking routine; this has been optimized for comparatively low signal‐to‐noise image sequences that are characteristic of microscopy movies of dynamic microtubules growing in vitro. Sequences of simulated microtubule images were generated assuming a variety of different experimental conditions. The simulated movies were then tracked and the tracking errors were characterized. We found that the growth characteristics of the microtubules within realistic ranges had a negligible effect on the tracking precision. The fluorophore labelling density, the pixel size of the images, and the exposure times were found to be important parameters limiting the tracking precision which could be explained using concepts of single molecule localization microscopy. The signal‐to‐noise ratio was found to be a good single predictor of the tracking precision: typical experimental signal‐to‐noise ratios lead to tracking precisions in the range of tens of nanometres, making the tracking program described here a useful tool for dynamic microtubule end tracking with close to molecular precision. PMID:26444439

  10. On and around microtubules: an overview.

    PubMed

    Wade, Richard H

    2009-10-01

    Microtubules are hollow tubes some 25 nm in diameter participating in the eukaryotic cytoskeleton. They are built from alphabeta-tubulin heterodimers that associate to form protofilaments running lengthwise along the microtubule wall with the beta-tubulin subunit facing the microtubule plus end conferring a structural polarity. The alpha- and beta-tubulins are highly conserved. A third member of the tubulin family, gamma-tubulin, plays a role in microtubule nucleation and assembly. Other members of the tubulin family appear to be involved in microtubule nucleation. Microtubule assembly is accompanied by hydrolysis of GTP associated with beta-tubulin so that microtubules consist principally of 'GDP-tubulin' stabilized at the plus end by a short 'cap'. An important property of microtubules is dynamic instability characterized by growth randomly interrupted by pauses and shrinkage. Many proteins interact with microtubules within the cell and are involved in essential functions such as microtubule growth, stabilization, destabilization, and interactions with chromosomes during cell division. The motor proteins kinesin and dynein use microtubules as pathways for transport and are also involved in cell division. Crystallography and electron microscopy are providing a structural basis for understanding the interactions of microtubules with antimitotic drugs, with motor proteins and with plus end tracking proteins.

  11. A divergent canonical WNT-signaling pathway regulates microtubule dynamics

    PubMed Central

    Ciani, Lorenza; Krylova, Olga; Smalley, Matthew J.; Dale, Trevor C.; Salinas, Patricia C.

    2004-01-01

    Dishevelled (DVL) is associated with axonal microtubules and regulates microtubule stability through the inhibition of the serine/threonine kinase, glycogen synthase kinase 3β (GSK-3β). In the canonical WNT pathway, the negative regulator Axin forms a complex with β-catenin and GSK-3β, resulting in β-catenin degradation. Inhibition of GSK-3β by DVL increases β-catenin stability and TCF transcriptional activation. Here, we show that Axin associates with microtubules and unexpectedly stabilizes microtubules through DVL. In turn, DVL stabilizes microtubules by inhibiting GSK-3β through a transcription- and β-catenin–independent pathway. More importantly, axonal microtubules are stabilized after DVL localizes to axons. Increased microtubule stability is correlated with a decrease in GSK-3β–mediated phosphorylation of MAP-1B. We propose a model in which Axin, through DVL, stabilizes microtubules by inhibiting a pool of GSK-3β, resulting in local changes in the phosphorylation of cellular targets. Our data indicate a bifurcation in the so-called canonical WNT-signaling pathway to regulate microtubule stability. PMID:14734535

  12. The role of microtubule dynamics in growth cone motility and axonal growth

    PubMed Central

    1995-01-01

    The growth cone contains dynamic and relatively stable microtubule populations, whose function in motility and axonal growth is uncharacterized. We have used vinblastine at low doses to inhibit microtubule dynamics without appreciable depolymerization to probe the role of these dynamics in growth cone behavior. At doses of vinblastine that interfere only with dynamics, the forward and persistent movement of the growth cone is inhibited and the growth cone wanders without appreciable forward translocation; it quickly resumes forward growth after the vinblastine is washed out. Direct visualization of fluorescently tagged microtubules in these neurons shows that in the absence of dynamic microtubules, the remaining mass of polymer does not invade the peripheral lamella and does not undergo the usual cycle of bundling and splaying and the growth cone stops forward movement. These experiments argue for a role for dynamic microtubules in allowing microtubule rearrangements in the growth cone. These rearrangements seem to be necessary for microtubule bundling, the subsequent coalescence of the cortex around the bundle to form new axon, and forward translocation of the growth cone. PMID:7822411

  13. Lighting up microtubule cytoskeleton dynamics in skeletal muscle

    PubMed Central

    Masedunskas, Andrius; Appaduray, Mark; Gunning, Peter W; Hardeman, Edna C

    2014-01-01

    In the past few decades, live cell microscopy techniques in combination with fluorescent tagging have provided a true explosion in our knowledge of the inner functioning of the cell. Dynamic phenomena can be observed inside living cells and the behavior of individual molecules participating in those events can be documented. However, our preference for simple or easy model systems such as cell culture, has come at a cost of chasing artifacts and missing out on understanding real biology as it happens in complex multicellular organisms. We are now entering a new era where developing meaningful, but also tractable model systems to study biological phenomenon dynamically in vivo in a mammal is not only possible; it will become the gold standard for scientific quality and translational potential.1,2 A study by Oddoux et al. describing the dynamics of the microtubule (MT) cytoskeleton in skeletal muscle is one example that demonstrates the power of developing in vivo/ex vivo models.3 MTs have long attracted attention as targets for cancer therapeutics 4 and more recently as mediators of Duchene muscular dystrophy.5 The muscle fiber MT cytoskeleton forms an intricate rectilinear lattice beneath the sarcolemma and is essential for the structural integrity of the muscle. Cultured cells do not develop such a specialized organization of the MT cytoskeleton and our understanding of it has come from static snapshots of muscle sections.6 In this context, the methodology and the findings reported by Oddoux et al. are a significant step forward. PMID:28243508

  14. Microtubule Dynamics Interacting with Stabilizing Agents in a Cell-like Environment

    NASA Astrophysics Data System (ADS)

    Shojania Feizabadi, Mitra

    2010-03-01

    Microtubules, key components of the cytoskeleton, are involved in several biological functions. They are highly dynamic polymers that stochastically switch between growing and shrinking phases. Due to their critical role in the process of cell division, they are the target of anti-cancer drugs. Antimitotic drugs usually suppress the dynamic instability of microtubules and, therefore, affect the process of cell division. In this work, the dynamic of microtubules interacting with catastrophe suppressing drugs as stabilizing agents, introduced by Mishra et al. (Phys. Rev. E. 72, 51914, 2005), is modified in a confined geometry and associated with the obtained and analyzed steady state solutions.

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

    PubMed

    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.

  16. Effect of Cibacron blue on tubulin assembly in the absence and presence of microtubule-associated proteins.

    PubMed

    Deinum, J; Sörskog, L; Wallin, M

    1982-11-24

    Cibacron blue was found to inhibit assembly and increase the critical concentration of microtubule proteins. In the presence of 4 mol Cibacron blue/mol tubulin, assembly was completely inhibited and pre-formed microtubules disassembled. Addition of 8% (v/v) dimethylsulfoxide to Cibacron blue-inhibited samples induced assembly of normal microtubules in addition to sheets of protofilaments. Disassembly was induced upon addition of 1 mM colchicine or 2 mM Ca2+. No obvious difference was seen in the protein composition of these microtubules compared with controls. GTP exchange was not affected by the presence of Cibacron blue nor was GTP able to counteract its effect. This indicates that the exchangeable GTP site is not involved. The extent of assembly of phosphocellulose purified tubulin in the presence of 8% (v/v) dimethylsulfoxide was only slightly less in the presence of Cibacron blue, although the assembly rate was decreased. These results suggest that Cibacron blue might alter the binding of one or more of the associated proteins stimulating assembly.

  17. Idiotypic mimicry and the assembly of a supramolecular structure: an anti-idiotypic antibody that mimics taxol in its tubulin-microtubule interactions.

    PubMed Central

    Leu, J G; Chen, B X; Diamanduros, A W; Erlanger, B F

    1994-01-01

    Taxol, originally extracted from the bark of the western yew, Taxus brevifolia, is reportedly the first of a new class of anti-cancer agents. It acts by promoting and irreversibly stabilizing microtubule assembly, thus interfering with the dynamic processes required for cell viability and multiplication. With the aim of using immunological techniques to study the mechanism of action of taxol, a monoclonal anti-idiotypic antibody that mimics taxol was prepared, using an auto-anti-idiotypic strategy. It and its Fab fragment inhibited the binding of [3H]taxol to microtubules. Moreover, like taxol, both promoted the assembly of tubulin into microtubules. These findings provide an example of an anti-idiotypic antibody capable of assembling an organized supramolecular structure from soluble cellular components. In addition, it further establishes the ability of anti-idiotypic antibodies to be functional mimics of ligand molecules bearing no structural similarity to immunoglobulins. The variable regions of the antibody have been sequenced. With the exception of the complementarity-determining region 3, the sequence of the heavy chain variable region is strikingly similar to that of an anti-idiotypic antibody raised to anti-insulin. The finding that a polypeptide can mimic taxol raises the possibility that taxol acts as a peptidomimetic compound that interferes with the function of an endogenous polypeptide. Images PMID:7840821

  18. The Dam1 kinetochore complex harnesses microtubule dynamics to produce force and movement.

    PubMed

    Asbury, Charles L; Gestaut, Daniel R; Powers, Andrew F; Franck, Andrew D; Davis, Trisha N

    2006-06-27

    Kinetochores remain attached to microtubule (MT) tips during mitosis even as the tips assemble and disassemble under their grip, allowing filament dynamics to produce force and move chromosomes. The specific proteins that mediate tip attachment are uncertain, and the mechanism of MT-dependent force production is unknown. Recent work suggests that the Dam1 complex, an essential component of kinetochores in yeast, may contribute directly to kinetochore-MT attachment and force production, perhaps by forming a sliding ring encircling the MT. To test these hypotheses, we developed an in vitro motility assay where beads coated with pure recombinant Dam1 complex were bound to the tips of individual dynamic MTs. The Dam1-coated beads remained tip-bound and underwent assembly- and disassembly-driven movement over approximately 3 microm, comparable to chromosome displacements in vivo. Dam1-based attachments to assembling tips were robust, supporting 0.5-3 pN of tension applied with a feedback-controlled optical trap as the MTs lengthened approximately 1 microm. The attachments also harnessed energy from MT disassembly to generate movement against tension. Reversing the direction of force (i.e., switching to compressive force) caused the attachments to disengage the tip and slide over the filament, but sliding was blocked by areas where the MT was anchored to a coverslip, consistent with a coupling structure encircling the filament. Our findings demonstrate how the Dam1 complex may contribute directly to MT-driven chromosome movement.

  19. Phosphoregulation promotes release of kinetochores from dynamic microtubules via multiple mechanisms.

    PubMed

    Sarangapani, Krishna K; Akiyoshi, Bungo; Duggan, Nicole M; Biggins, Sue; Asbury, Charles L

    2013-04-30

    During mitosis, multiprotein complexes called kinetochores orchestrate chromosome segregation by forming load-bearing attachments to dynamic microtubule tips, and by participating in phosphoregulatory error correction. The conserved kinase Aurora B phosphorylates the major microtubule-binding kinetochore subcomplexes, Ndc80 and (in yeast) Dam1, to promote release of erroneous attachments, giving another chance for proper attachments to form. It is unknown whether Aurora B phosphorylation promotes release directly, by increasing the rate of kinetochore detachment, or indirectly, by destabilizing the microtubule tip. Moreover, the relative importance of phosphorylation of Ndc80 vs. Dam1 in the context of whole kinetochores is unclear. To address these uncertainties, we isolated native yeast kinetochore particles carrying phosphomimetic mutations on Ndc80 and Dam1, and applied advanced laser-trapping techniques to measure the strength and stability of their attachments to individual dynamic microtubule tips. Rupture forces were reduced by phosphomimetic mutations on both subcomplexes, in an additive manner, indicating that both subcomplexes make independent contributions to attachment strength. Phosphomimetics on either subcomplex reduced attachment lifetimes under constant force, primarily by accelerating detachment during microtubule growth. Phosphomimetics on Dam1 also increased the likelihood of switches from microtubule growth into shortening, further promoting release in an indirect manner. Taken together, our results suggest that, in vivo, Aurora B releases kinetochores via at least two mechanisms: by weakening the kinetochore-microtubule interface and also by destabilizing the kinetochore-attached microtubule tip.

  20. Microtubule-associated protein-4 controls nanovesicle dynamics and T cell activation.

    PubMed

    Bustos-Morán, Eugenio; Blas-Rus, Noelia; Martin-Cófreces, Noa Beatriz; Sánchez-Madrid, Francisco

    2017-04-01

    The immune synapse (IS) is a specialized structure formed at the contact area between T lymphocytes and antigen-presenting cells (APCs) that is essential for the adaptive immune response. Proper T cell activation requires its polarization towards the APC, which is highly dependent on the tubulin cytoskeleton. Microtubule-associated protein-4 (MAP4) is a microtubule (MT)-stabilizing protein that controls MTs in physiological processes, such as cell division, migration, vesicular transport or primary cilia formation. In this study, we assessed the role of MAP4 in T cell activation. MAP4 decorates the pericentrosomal area and MTs of the T cell, and it is involved in MT detyrosination and stable assembly in response to T cell activation. In addition, MAP4 prompts the timely translocation of the MT-organizing center (MTOC) towards the IS and the dynamics of signaling nanovesicles that sustains T cell activation. However, MAP4 acts as a negative regulator of other T cell activation-related signals, including diacylglycerol (DAG) production and IL2 secretion. Our data indicate that MAP4 acts as a checkpoint molecule that balances positive and negative hallmarks of T cell activation.

  1. Regulation of Microtubule Assembly by Tau and not by Pin1.

    PubMed

    Kutter, Steffen; Eichner, Timo; Deaconescu, Alexandra M; Kern, Dorothee

    2016-05-08

    The molecular mechanism by which the microtubule-associated protein (MAP) tau regulates the formation of microtubules (MTs) is poorly understood. The activity of tau is controlled via phosphorylation at specific Ser/Thr sites. Of those phosphorylation sites, 17 precede a proline, making them potential recognition sites for the peptidyl-prolyl isomerase Pin1. Pin1 binding and catalysis of phosphorylated tau at the AT180 epitope, which was implicated in Alzheimer's disease, has been reported to be crucial for restoring tau's ability to promote MT polymerization in vitro and in vivo [1]. Surprisingly, we discover that Pin1 does not promote phosphorylated tau-induced MT formation in vitro, refuting the commonly accepted model in which Pin1 binding and catalysis on the A180 epitope restores the function of the Alzheimer's associated phosphorylated tau in tubulin assembly [1, 2]. Using turbidity assays, time-resolved small angle X-ray scattering (SAXS), and time-resolved negative stain electron microscopy (EM), we investigate the mechanism of tau-mediated MT assembly and the role of the Thr231 and Ser235 phosphorylation on this process. We discover novel GTP-tubulin ring-shaped species, which are detectable in the earliest stage of tau-induced polymerization and may play a crucial role in the early nucleation phase of MT assembly. Finally, by NMR and SAXS experiments, we show that the tau molecules must be located on the surface of MTs and tubulin rings during the polymerization reaction. The interaction between tau and tubulin is multipartite, with a high affinity interaction of the four tubulin-binding repeats, and a weaker interaction with the proline-rich sequence and the termini of tau.

  2. A minimal model for kinetochore-microtubule dynamics

    NASA Astrophysics Data System (ADS)

    Liu, Andrea

    2014-03-01

    During mitosis, chromosome pairs align at the center of a bipolar microtubule (MT) spindle and oscillate as MTs attaching them to the cell poles polymerize and depolymerize. The cell fixes misaligned pairs by a tension-sensing mechanism. Pairs later separate as shrinking MTs pull each chromosome toward its respective cell pole. We present a minimal model for these processes based on properties of MT kinetics. We apply the measured tension-dependence of single MT kinetics to a stochastic many MT model, which we solve numerically and with master equations. We find that the force-velocity curve for the single chromosome system is bistable and hysteretic. Above some threshold load, tension fluctuations induce MTs to spontaneously switch from a pulling state into a growing, pushing state. To recover pulling from the pushing state, the load must be reduced far below the threshold. This leads to oscillations in the two-chromosome system. Our minimal model quantitatively captures several aspects of kinetochore dynamics observed experimentally. This work was supported by NSF-DMR-1104637.

  3. A Case for Microtubule Vulnerability in Amyotrophic Lateral Sclerosis: Altered Dynamics During Disease

    PubMed Central

    Clark, Jayden A.; Yeaman, Elise J.; Blizzard, Catherine A.; Chuckowree, Jyoti A.; Dickson, Tracey C.

    2016-01-01

    Amyotrophic lateral sclerosis (ALS) is an aggressive multifactorial disease converging on a common pathology: the degeneration of motor neurons (MNs), their axons and neuromuscular synapses. This vulnerability and dysfunction of MNs highlights the dependency of these large cells on their intracellular machinery. Neuronal microtubules (MTs) are intracellular structures that facilitate a myriad of vital neuronal functions, including activity dependent axonal transport. In ALS, it is becoming increasingly apparent that MTs are likely to be a critical component of this disease. Not only are disruptions in this intracellular machinery present in the vast majority of seemingly sporadic cases, recent research has revealed that mutation to a microtubule protein, the tubulin isoform TUBA4A, is sufficient to cause a familial, albeit rare, form of disease. In both sporadic and familial disease, studies have provided evidence that microtubule mediated deficits in axonal transport are the tipping point for MN survivability. Axonal transport deficits would lead to abnormal mitochondrial recycling, decreased vesicle and mRNA transport and limited signaling of key survival factors from the neurons peripheral synapses, causing the characteristic peripheral “die back”. This disruption to microtubule dependant transport in ALS has been shown to result from alterations in the phenomenon of microtubule dynamic instability: the rapid growth and shrinkage of microtubule polymers. This is accomplished primarily due to aberrant alterations to microtubule associated proteins (MAPs) that regulate microtubule stability. Indeed, the current literature would argue that microtubule stability, particularly alterations in their dynamics, may be the initial driving force behind many familial and sporadic insults in ALS. Pharmacological stabilization of the microtubule network offers an attractive therapeutic strategy in ALS; indeed it has shown promise in many neurological disorders, ALS

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

    PubMed

    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.

  5. Uniform polarity microtubule assemblies imaged in native brain tissue by second-harmonic generation microscopy.

    PubMed

    Dombeck, Daniel A; Kasischke, Karl A; Vishwasrao, Harshad D; Ingelsson, Martin; Hyman, Bradley T; Webb, Watt W

    2003-06-10

    Microtubule (MT) ensemble polarity is a diagnostic determinant of the structure and function of neuronal processes. Here, polarized MT structures are selectively imaged with second-harmonic generation (SHG) microscopy in native brain tissue. This SHG is found to colocalize with axons in both brain slices and cultured neurons. Because SHG arises only from noninversion symmetric structures, the uniform polarity of axonal MTs leads to the observed signal, whereas the mixed polarity in dendrites leads to destructive interference. SHG imaging provides a tool to investigate the kinetics and function of MT ensemble polarity in dynamic native brain tissue structures and other subcellular motility structures based on polarized MTs.

  6. Mechanism and Dynamics of Breakage of Fluorescent Microtubules

    PubMed Central

    Guo, Honglian; Xu, Chunhua; Liu, Chunxiang; Qu, E.; Yuan, Ming; Li, Zhaolin; Cheng, Bingying; Zhang, Daozhong

    2006-01-01

    The breakage of fluorescence-labeled microtubules under irradiation of excitation light is found in our experiments. Its mechanism is studied. The results indicate that free radicals are the main reason for the photosensitive breakage. Furthermore, the mechanical properties of the microtubules are probed with a dual-optical tweezers system. It is found that the fluorescence-labeled microtubules are much easier to extend compared with those without fluorescence. Such microtubules can be extended by 30%, and the force for breaking them up is only several piconewtons. In addition, we find that the breakup of the protofilaments is not simultaneous but step-by-step, which further confirms that the interaction between protofilaments is fairly weak. PMID:16387782

  7. Biogenesis of the crystalloid organelle in Plasmodium involves microtubule-dependent vesicle transport and assembly

    PubMed Central

    Saeed, Sadia; Tremp, Annie Z.; Dessens, Johannes T.

    2015-01-01

    Malaria parasites possess unique subcellular structures and organelles. One of these is the crystalloid, a multivesicular organelle that forms during the parasite’s development in vector mosquitoes. The formation and function of these organelles remain poorly understood. A family of six conserved and modular proteins named LCCL-lectin adhesive-like proteins (LAPs), which have essential roles in sporozoite transmission, localise to the crystalloids. In this study we analyse crystalloid formation using transgenic Plasmodium berghei parasites expressing GFP-tagged LAP3. We show that deletion of the LCCL domain from LAP3 causes retarded crystalloid development, while knockout of LAP3 prevents formation of the organelle. Our data reveal that the process of crystalloid formation involves active relocation of endoplasmic reticulum-derived vesicles to common assembly points via microtubule-dependent transport. Inhibition of microtubule-dependent cargo transport disrupts this process and replicates the LCCL domain deletion mutant phenotype in wildtype parasites. These findings provide the first clear insight into crystalloid biogenesis, demonstrating a fundamental role for the LAP family in this process, and identifying the crystalloid and its formation as potential targets for malaria transmission control. PMID:25900212

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

    PubMed

    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."

  9. Assembly of an Intact Golgi Complex Requires Phospholipase A2 (PLA2) Activity, Membrane Tubules, and Dynein-Mediated Microtubule Transport

    PubMed Central

    Judson, Bret L.; Brown, William J.

    2009-01-01

    Previous studies have shown that treatment of mammalian cells with phospholipase A2 (PLA2) antagonists cause the normally interconnected Golgi ribbon to break up into large fragments of stacked Golgi cisternae (“mini-stacks”) that remain located in the juxtanuclear region. Using the reversible PLA2 antagonist, ONO-RS-082 (ONO) and live-cell, time-lapse microscopy to image the Golgi reassembly process, we found that Golgi mini-stacks underwent a burst of membrane tubule formation following washout of ONO: before washout only 4.3 ± 3.8 tubules/cell/10 min were formed, whereas after washout 29.9 ± 11.9 tubules/cell/10 min formed. These membranes tubules formed bridges between physically separate mini-stacks, thus mediating their coalescence into intact Golgi ribbons. Formation of inter-stack tubules and an intact Golgi ribbon was also facilitated by microtubules because treatment with nocodazole significantly inhibited both processes. This microtubule-dependent process was also dependent on dynein because the dynein inhibitor nordihydroguaiaretic acid (NDGA) inhibited reassembly. These studies show that a late stage of Golgi assembly occurs via membrane tubules, whose formation is dependent on PLA2 activity and microtubules. Considering these results together, we concluded that the maintenance and assembly of normal Golgi architecture is dependent on the PLA2-mediated, dynamic formation of inter-Golgi membrane tubules. PMID:19747452

  10. Integrated modeling methodology for microtubule dynamics and Taxol kinetics with experimentally identifiable parameters.

    PubMed

    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.

  11. Differential effects of natural product microtubule stabilizers on microtubule assembly: single agent and combination studies with taxol, epothilone B, and discodermolide.

    PubMed

    Gertsch, Jürg; Meier, Sarah; Müller, Martin; Altmann, Karl-Heinz

    2009-01-05

    A systematic comparison has been performed of the morphology and stability of microtubules (MTs) induced by the potent microtubule-stabilizing agents (MSAs) taxol, epothilone B (Epo B), and discodermolide (DDM) under GTP-free conditions. DDM-induced tubulin polymerization occurred significantly faster than that induced by taxol and Epo B. At the same time, tubulin polymers assembled from soluble tubulin by DDM were morphologically distinct (shorter and less ordered) from those induced by either taxol or Epo B, as demonstrated by electron microscopy. Exposure of MSA-induced tubulin polymers to ultrasound revealed the DDM-based polymers to be less stable to this type of physical stress than those formed with either Epo B or taxol. Interestingly, MT assembly in the presence of both DDM and taxol appeared to produce a distinct new type of MT polymer with a mixed morphology between those of DDM- and taxol-induced structures. The observed differences in MT morphology and stability might be related, at least partly, to differences in intramicrotubular tubulin isotype distribution, as DDM showed a different pattern of beta-tubulin isotype usage in the assembly process.

  12. The molecular dynamics of crawling migration in microtubule-disrupted keratocytes.

    PubMed

    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.

  13. Microtubules, Tubulins and Associated Proteins.

    ERIC Educational Resources Information Center

    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)

  14. Dynamic microtubules at the vegetal cortex predict the embryonic axis in zebrafish.

    PubMed

    Tran, Long Duc; Hino, Hiromu; Quach, Helen; Lim, Shimin; Shindo, Asako; Mimori-Kiyosue, Yuko; Mione, Marina; Ueno, Naoto; Winkler, Christoph; Hibi, Masahiko; Sampath, Karuna

    2012-10-01

    In zebrafish, as in many animals, maternal dorsal determinants are vegetally localized in the egg and are transported after fertilization in a microtubule-dependent manner. However, the organization of early microtubules, their dynamics and their contribution to axis formation are not fully understood. Using live imaging, we identified two populations of microtubules, perpendicular bundles and parallel arrays, which are directionally oriented and detected exclusively at the vegetal cortex before the first cell division. Perpendicular bundles emanate from the vegetal cortex, extend towards the blastoderm, and orient along the animal-vegetal axis. Parallel arrays become asymmetric on the vegetal cortex, and orient towards dorsal. We show that the orientation of microtubules at 20 minutes post-fertilization can predict where the embryonic dorsal structures in zebrafish will form. Furthermore, we find that parallel microtubule arrays colocalize with wnt8a RNA, the candidate maternal dorsal factor. Vegetal cytoplasmic granules are displaced with parallel arrays by ~20°, providing in vivo evidence of a cortical rotation-like process in zebrafish. Cortical displacement requires parallel microtubule arrays, and probably contributes to asymmetric transport of maternal determinants. Formation of parallel arrays depends on Ca(2+) signaling. Thus, microtubule polarity and organization predicts the zebrafish embryonic axis. In addition, our results suggest that cortical rotation-like processes might be more common in early development than previously thought.

  15. Aurora B suppresses microtubule dynamics and limits central spindle size by locally activating KIF4A

    PubMed Central

    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

  16. Microtubule dynamics in the peripheral nervous system: A matter of balance.

    PubMed

    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.

  17. Active sliding between cytoplasmic microtubules.

    PubMed

    Koonce, M P; Tong, J; Euteneuer, U; Schliwa, M

    Microtubules are versatile cellular polymers that play a role in cell shape determination and mediate various motile processes such as ciliary and flagellar bending, chromosome movements and organelle transport. That a sliding microtubule mechanism can generate force has been demonstrated in highly ordered structures such as axonemes, and microtubule-based force generation almost certainly contributes to the function of mitotic and meiotic spindles. Most cytoplasmic microtubule arrays, however, do not exhibit the structural regularity of axonemes and some spindles, and often appear disorganized. Yet many cellular activities (such as shape changes during morphogenesis, axonal extension and spindle assembly) involve highly coordinated microtubule behaviour and possibly require force generated by an intermicrotubule sliding mechanism, or perhaps use sliding to move microtubules rapidly into a protrusion for stabilization. Here we show that active sliding between cytoplasmic microtubules can occur in microtubule bundles of the amoeba Reticulomyxa. A force-producing mechanism of this sort could be used by this organism to facilitate the extension of cell processes and to generate the dynamic movements of the cytoplasmic network.

  18. Kinesin-8 effects on mitotic microtubule dynamics contribute to spindle function in fission yeast

    PubMed Central

    Gergely, Zachary R.; Crapo, Ammon; Hough, Loren E.; McIntosh, J. Richard; Betterton, Meredith D.

    2016-01-01

    Kinesin-8 motor proteins destabilize microtubules. Their absence during cell division is associated with disorganized mitotic chromosome movements and chromosome loss. Despite recent work studying effects of kinesin-8s on microtubule dynamics, it remains unclear whether the kinesin-8 mitotic phenotypes are consequences of their effect on microtubule dynamics, their well-established motor activity, or additional, unknown functions. To better understand the role of kinesin-8 proteins in mitosis, we studied the effects of deletion of the fission yeast kinesin-8 proteins Klp5 and Klp6 on chromosome movements and spindle length dynamics. Aberrant microtubule-driven kinetochore pushing movements and tripolar mitotic spindles occurred in cells lacking Klp5 but not Klp6. Kinesin-8–deletion strains showed large fluctuations in metaphase spindle length, suggesting a disruption of spindle length stabilization. Comparison of our results from light microscopy with a mathematical model suggests that kinesin-8–induced effects on microtubule dynamics, kinetochore attachment stability, and sliding force in the spindle can explain the aberrant chromosome movements and spindle length fluctuations seen. PMID:27146110

  19. Elastic and damping forces generated by confined arrays of dynamic microtubules.

    PubMed

    Howard, J

    2006-02-28

    In addition to serving as structural elements and as tracks for motor proteins, microtubules use chemical energy derived from the hydrolysis of GTP to generate forces when growing and shrinking. These forces are used to push or pull on organelles such as chromosomes and the mitotic spindle. If an array of microtubules grows out from a nucleation site and is confined by the periphery of the cell, pushing and pulling forces can give rise to interesting collective phenomena. In this paper, I show that pushing forces center the array provided that the microtubules are dynamic in the sense that they switch from pushing to shrinking after reaching the periphery. Microtubule dynamics of free ends is neither necessary nor sufficient for centering. Buckling can augment the centering force. For small displacements and velocities, the array can be modeled very simply as a damped spring. The dynamic stiffness of the array is orders of magnitude smaller than its static stiffness, and the relaxation time is on the order of the time that it takes for a microtubule to grow from the center to the periphery. Replacement of a dynamic polymer array with an equivalent mechanical circuit provides a bridge between molecular and cellular mechanics.

  20. Caenorhabditis elegans oocyte meiotic spindle pole assembly requires microtubule severing and the calponin homology domain protein ASPM-1

    PubMed Central

    Connolly, Amy A.; Osterberg, Valerie; Christensen, Sara; Price, Meredith; Lu, Chenggang; Chicas-Cruz, Kathy; Lockery, Shawn; Mains, Paul E.; Bowerman, Bruce

    2014-01-01

    In many animals, including vertebrates, oocyte meiotic spindles are bipolar but assemble in the absence of centrosomes. Although meiotic spindle positioning in oocytes has been investigated extensively, much less is known about their assembly. In Caenorhabditis elegans, three genes previously shown to contribute to oocyte meiotic spindle assembly are the calponin homology domain protein encoded by aspm-1, the katanin family member mei-1, and the kinesin-12 family member klp-18. We isolated temperature-sensitive alleles of all three and investigated their requirements using live-cell imaging to reveal previously undocumented requirements for aspm-1 and mei-1. Our results indicate that bipolar but abnormal oocyte meiotic spindles assemble in aspm-1(-) embryos, whereas klp-18(-) and mei-1(-) mutants assemble monopolar and apolar spindles, respectively. Furthermore, two MEI-1 functions—ASPM-1 recruitment to the spindle and microtubule severing—both contribute to monopolar spindle assembly in klp-18(-) mutants. We conclude that microtubule severing and ASPM-1 both promote meiotic spindle pole assembly in C. elegans oocytes, whereas the kinesin 12 family member KLP-18 promotes spindle bipolarity. PMID:24554763

  1. Calcium-independent disruption of microtubule dynamics by nanosecond pulsed electric fields in U87 human glioblastoma cells

    PubMed Central

    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

  2. Calcium-independent disruption of microtubule dynamics by nanosecond pulsed electric fields in U87 human glioblastoma cells.

    PubMed

    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.

  3. BRCA1 regulates microtubule dynamics and taxane-induced apoptotic cell signaling

    PubMed Central

    Sung, M; Giannakakou, P

    2013-01-01

    The taxanes are effective microtubule-stabilizing chemotherapy drugs used in the treatment of various solid tumors. However, the emergence of drug resistance hampers their clinical efficacy. The molecular basis of clinical taxane resistance remains poorly understood. Breast cancer 1, early onset gene, BRCA1, is a tumor-suppressor gene, whose expression has been correlated with taxane sensitivity in many solid tumors including non-small cell lung cancer. However, the molecular mechanism underlying the relationship between BRCA1 (B1) expression and taxane activity remains unclear. To this end, we created a stable B1 knockdown A549 cell line (B1-KD) to investigate B1’s role in microtubule biology and response to taxane treatment. We show that B1-KD rendered A549 cells resistant to paclitaxel (PTX), phenocopying clinical studies showing that low B1 expression correlated with taxane resistance. As previously reported, we show that loss of B1 enhanced centrosomal γ-tubulin localization and microtubule nucleation. Interestingly, we found that the B1-KD cells exhibited increased microtubule dynamics as compared with parental A549 cells, as assessed by live-cell confocal microscopy using enhanced green fluorescent protein-tagged α-tubulin or EB1 protein. In addition, we showed that loss of B1 impairs the ability of PTX to induce microtubule polymerization using immunofluorescence microscopy and a cell-based tubulin polymerization assay. Furthermore, B1-KD cells exhibited significantly lower intracellular binding of a fluorescently labeled PTX to microtubules. Recent studies have shown that PTX-stabilized microtubules serves as a scaffold for pro-caspase-8 binding and induction of apoptosis downstream of induced-proximity activation of caspase-8. Here we show that loss of B1 reduces the association of pro-caspase-8 with microtubules and subsequently leads to impaired PTX-induced activation of apoptosis. Taken together, our data show that B1 regulates indirectly

  4. Desmosome assembly and dynamics.

    PubMed

    Nekrasova, Oxana; Green, Kathleen J

    2013-11-01

    Desmosomes are intercellular junctions that anchor intermediate filaments (IFs) to the plasma membrane, forming a supracellular scaffold that provides mechanical resilience to tissues. This anchoring function is accomplished by specialized members of the cadherin family and associated cytoskeletal linking proteins, which together form a highly organized membrane core flanked by mirror-image cytoplasmic plaques. Due to the biochemical insolubility of desmosomes, the mechanisms that govern assembly of these components into a functional organelle remained elusive. Recently developed molecular reporters and live cell imaging approaches have provided powerful new tools to monitor this finely tuned process in real time. Here we discuss studies that are beginning to decipher the machinery and regulation governing desmosome assembly and homeostasis in situ and how these mechanisms are affected during disease pathogenesis.

  5. Myosin-Va and dynamic actin oppose microtubules to drive long-range organelle transport.

    PubMed

    Evans, Richard D; Robinson, Christopher; Briggs, Deborah A; Tooth, David J; Ramalho, Jose S; Cantero, Marta; Montoliu, Lluis; Patel, Shyamal; Sviderskaya, Elena V; Hume, Alistair N

    2014-08-04

    In animal cells, microtubule and actin tracks and their associated motors (dynein, kinesin, and myosin) are thought to regulate long- and short-range transport, respectively. Consistent with this, microtubules extend from the perinuclear centrosome to the plasma membrane and allow bidirectional cargo transport over long distances (>1 μm). In contrast, actin often comprises a complex network of short randomly oriented filaments, suggesting that myosin motors move cargo short distances. These observations underpin the "highways and local roads" model for transport along microtubule and actin tracks. The "cooperative capture" model exemplifies this view and suggests that melanosome distribution in melanocyte dendrites is maintained by long-range transport on microtubules followed by actin/myosin-Va-dependent tethering. In this study, we used cell normalization technology to quantitatively examine the contribution of microtubules and actin/myosin-Va to organelle distribution in melanocytes. Surprisingly, our results indicate that microtubules are essential for centripetal, but not centrifugal, transport. Instead, we find that microtubules retard a centrifugal transport process that is dependent on myosin-Va and a population of dynamic F-actin. Functional analysis of mutant proteins indicates that myosin-Va works as a transporter dispersing melanosomes along actin tracks whose +/barbed ends are oriented toward the plasma membrane. Overall, our data highlight the role of myosin-Va and actin in transport, and not tethering, and suggest a new model in which organelle distribution is determined by the balance between microtubule-dependent centripetal and myosin-Va/actin-dependent centrifugal transport. These observations appear to be consistent with evidence coming from other systems showing that actin/myosin networks can drive long-distance organelle transport and positioning.

  6. Myosin-Va and Dynamic Actin Oppose Microtubules to Drive Long-Range Organelle Transport

    PubMed Central

    Evans, Richard D.; Robinson, Christopher; Briggs, Deborah A.; Tooth, David J.; Ramalho, Jose S.; Cantero, Marta; Montoliu, Lluis; Patel, Shyamal; Sviderskaya, Elena V.; Hume, Alistair N.

    2014-01-01

    Summary In animal cells, microtubule and actin tracks and their associated motors (dynein, kinesin, and myosin) are thought to regulate long- and short-range transport, respectively [1–8]. Consistent with this, microtubules extend from the perinuclear centrosome to the plasma membrane and allow bidirectional cargo transport over long distances (>1 μm). In contrast, actin often comprises a complex network of short randomly oriented filaments, suggesting that myosin motors move cargo short distances. These observations underpin the “highways and local roads” model for transport along microtubule and actin tracks [2]. The “cooperative capture” model exemplifies this view and suggests that melanosome distribution in melanocyte dendrites is maintained by long-range transport on microtubules followed by actin/myosin-Va-dependent tethering [5, 9]. In this study, we used cell normalization technology to quantitatively examine the contribution of microtubules and actin/myosin-Va to organelle distribution in melanocytes. Surprisingly, our results indicate that microtubules are essential for centripetal, but not centrifugal, transport. Instead, we find that microtubules retard a centrifugal transport process that is dependent on myosin-Va and a population of dynamic F-actin. Functional analysis of mutant proteins indicates that myosin-Va works as a transporter dispersing melanosomes along actin tracks whose +/barbed ends are oriented toward the plasma membrane. Overall, our data highlight the role of myosin-Va and actin in transport, and not tethering, and suggest a new model in which organelle distribution is determined by the balance between microtubule-dependent centripetal and myosin-Va/actin-dependent centrifugal transport. These observations appear to be consistent with evidence coming from other systems showing that actin/myosin networks can drive long-distance organelle transport and positioning [10, 11]. PMID:25065759

  7. How the transition frequencies of microtubule dynamic instability (nucleation, catastrophe, and rescue) regulate microtubule dynamics in interphase and mitosis: analysis using a Monte Carlo computer simulation.

    PubMed Central

    Gliksman, N R; Skibbens, R V; Salmon, E D

    1993-01-01

    Microtubules (MTs) in newt mitotic spindles grow faster than MTs in the interphase cytoplasmic microtubule complex (CMTC), yet spindle MTs do not have the long lengths or lifetimes of the CMTC microtubules. Because MTs undergo dynamic instability, it is likely that changes in the durations of growth or shortening are responsible for this anomaly. We have used a Monte Carlo computer simulation to examine how changes in the number of MTs and changes in the catastrophe and rescue frequencies of dynamic instability may be responsible for the cell cycle dependent changes in MT characteristics. We used the computer simulations to model interphase-like or mitotic-like MT populations on the basis of the dynamic instability parameters available from newt lung epithelial cells in vivo. We started with parameters that produced MT populations similar to the interphase newt lung cell CMTC. In the simulation, increasing the number of MTs and either increasing the frequency of catastrophe or decreasing the frequency of rescue reproduced the changes in MT dynamics measured in vivo between interphase and mitosis. Images PMID:8298190

  8. Effects of aging in catastrophe on the steady state and dynamics of a microtubule population

    NASA Astrophysics Data System (ADS)

    Jemseena, V.; Gopalakrishnan, Manoj

    2015-05-01

    Several independent observations have suggested that the catastrophe transition in microtubules is not a first-order process, as is usually assumed. Recent in vitro observations by Gardner et al. [M. K. Gardner et al., Cell 147, 1092 (2011), 10.1016/j.cell.2011.10.037] showed that microtubule catastrophe takes place via multiple steps and the frequency increases with the age of the filament. Here we investigate, via numerical simulations and mathematical calculations, some of the consequences of the age dependence of catastrophe on the dynamics of microtubules as a function of the aging rate, for two different models of aging: exponential growth, but saturating asymptotically, and purely linear growth. The boundary demarcating the steady-state and non-steady-state regimes in the dynamics is derived analytically in both cases. Numerical simulations, supported by analytical calculations in the linear model, show that aging leads to nonexponential length distributions in steady state. More importantly, oscillations ensue in microtubule length and velocity. The regularity of oscillations, as characterized by the negative dip in the autocorrelation function, is reduced by increasing the frequency of rescue events. Our study shows that the age dependence of catastrophe could function as an intrinsic mechanism to generate oscillatory dynamics in a microtubule population, distinct from hitherto identified ones.

  9. Mechanism of action of antitumor drugs that interact with microtubules and tubulin.

    PubMed

    Jordan, M A

    2002-01-01

    Microtubules, major structural components in cells, are the target of a large and diverse group of natural product anticancer drugs. Given the success of this class of drugs in cancer treatment, it can be argued that microtubules represent the single best cancer target identified to date. Microtubules are highly dynamic assemblies of the protein tubulin. They readily polymerize and depolymerize in cells, and they undergo two interesting kinds of dynamics called dynamic instability and treadmilling. These dynamic behaviors are crucial to mitosis, the process of chromosomal division to form new cells. Microtubule dynamics are highly regulated during the cell cycle by endogenous cellular regulators. In addition, many antitumor drugs and natural compounds alter the polymerization dynamics of microtubules, blocking mitosis, and consequently, inducing cell death by apoptosis. These drugs include several that inhibit microtubule polymerization at high drug concentrations, namely, the Vinca alkaloids, cryptophycins, halichondrins, estramustine, and colchicine. Another group of these compounds stimulates microtubule polymerization and stabilizes microtubules at high concentrations. These include Taxol, Taxotere, eleutherobins, epothilones, laulimalide, sarcodictyins, and discodermolide. Importantly, considerable evidence indicates that, at lower concentrations, these drugs have a common mechanism of action; they suppress the dynamics of microtubules without appreciably changing the mass of microtubules in the cell. The drugs bind to diverse sites on tubulin and at different positions within the microtubule, and they have diverse effects on microtubule dynamics. However, by their common mechanism of suppression microtubule dynamics, they all block mitosis at the metaphase/anaphase transition, and induce cell death.

  10. Microtubule dynamics of the centrosome-like polar organizers from the basal land plant Marchantia polymorpha.

    PubMed

    Buschmann, Henrik; Holtmannspötter, Michael; Borchers, Agnes; O'Donoghue, Martin-Timothy; Zachgo, Sabine

    2016-02-01

    The liverwort Marchantia employs both modern and ancestral devices during cell division: it forms preprophase bands and in addition it shows centrosome-like polar organizers. We investigated whether polar organizers and preprophase bands cooperate to set up the division plane. To this end, two novel green fluorescent protein-based microtubule markers for dividing cells of Marchantia were developed. Cells of the apical notch formed polar organizers first and subsequently assembled preprophase bands. Polar organizers were formed de novo from multiple mobile microtubule foci localizing to the nuclear envelope. The foci then became concentrated by bipolar aggregation. We determined the comet production rate of polar organizers and show that microtubule plus ends of astral microtubules polymerize faster than those found on cortical microtubules. Importantly, it was observed that conditions increasing polar organizer numbers interfere with preprophase band formation. The data show that polar organizers have much in common with centrosomes, but that they also have specialized features. The results suggest that polar organizers contribute to preprophase band formation and in this way are involved in controlling the division plane. Our analyses of the basal land plant Marchantia shed new light on the evolution of plant cell division.

  11. Microtubule dynamics from mating through the first zygotic division in the budding yeast Saccharomyces cerevisiae.

    PubMed

    Maddox, P; Chin, E; Mallavarapu, A; Yeh, E; Salmon, E D; Bloom, K

    1999-03-08

    We have used time-lapse digital imaging microscopy to examine cytoplasmic astral microtubules (Mts) and spindle dynamics during the mating pathway in budding yeast Saccharomyces cerevisiae. Mating begins when two cells of opposite mating type come into proximity. The cells arrest in the G1 phase of the cell cycle and grow a projection towards one another forming a shmoo projection. Imaging of microtubule dynamics with green fluorescent protein (GFP) fusions to dynein or tubulin revealed that the nucleus and spindle pole body (SPB) became oriented and tethered to the shmoo tip by a Mt-dependent search and capture mechanism. Dynamically unstable astral Mts were captured at the shmoo tip forming a bundle of three or four astral Mts. This bundle changed length as the tethered nucleus and SPB oscillated toward and away from the shmoo tip at growth and shortening velocities typical of free plus end astral Mts (approximately 0.5 micrometer/min). Fluorescent fiduciary marks in Mt bundles showed that Mt growth and shortening occurred primarily at the shmoo tip, not the SPB. This indicates that Mt plus end assembly/disassembly was coupled to pushing and pulling of the nucleus. Upon cell fusion, a fluorescent bar of Mts was formed between the two shmoo tip bundles, which slowly shortened (0.23 +/- 0.07 micrometer/min) as the two nuclei and their SPBs came together and fused (karyogamy). Bud emergence occurred adjacent to the fused SPB approximately 30 min after SPB fusion. During the first mitosis, the SPBs separated as the spindle elongated at a constant velocity (0.75 micrometer/min) into the zygotic bud. There was no indication of a temporal delay at the 2-micrometer stage of spindle morphogenesis or a lag in Mt nucleation by replicated SPBs as occurs in vegetative mitosis implying a lack of normal checkpoints. Thus, the shmoo tip appears to be a new model system for studying Mt plus end dynamic attachments and much like higher eukaryotes, the first mitosis after haploid

  12. The acetylation of alpha-tubulin and its relationship to the assembly and disassembly of microtubules

    PubMed Central

    1986-01-01

    A tight association between Chlamydomonas alpha-tubulin acetyltransferase (TAT) and flagellar axonemes, and the cytoplasmic localization of both tubulin deacetylase (TDA) and an inhibitor of tubulin acetylation have been demonstrated by the use of calf brain tubulin as substrate for these enzymes. A major axonemal TAT of 130 kD has been solubilized by high salt treatment, purified, and characterized. Using the Chlamydomonas TAT with brain tubulin as substrate, we have studied the effects of acetylation on the assembly and disassembly of microtubules in vitro. We also determined the relative rates of acetylation of tubulin dimers and polymers. The acetylation does not significantly affect the temperature-dependent polymerization or depolymerization of tubulin in vitro. Furthermore, polymerization of tubulin is not a prerequisite for the acetylation, although the polymer is a better substrate for TAT than the dimer. The acetylation is sensitive to calcium ions which completely inhibit the acetylation of both dimers and polymers of tubulin. Acetylation of the dimer is not inhibited by colchicine; the effect of colchicine on acetylation of the polymer can be explained by its depolymerizing effect on the polymer. PMID:3733880

  13. The dynamic nature of mollusc egg surface architecture and its relation to the microtubule network.

    PubMed

    Tyler, Sheena E B; Kimber, Susan J

    2006-01-01

    Dynamic changes in the surface architecture pattern of embryos of the slipper limpet (Crepidula fornicata, Mollusca) were found in this study to correlate with the dynamic activity and pattern of the underlying mitotic spindle microtubule network, revealed by fluorescent labelling and confocal imaging techniques. Examination of a series of optical sections indicate that this network appears to be spatially co-ordinated together as a whole throughout the embryo. The microtubule pattern also associated with abnormal multipolar spindles resulting from an applied static magnetic field, indicating that the pattern may be generated by a natural endogenous field source. The patterning characteristics of the surface and microtubule network together provide further morphological evidence for a primary morphogenetic or developmental field system which organises the primary body axis and co-ordinates the pattern of cleavage.

  14. Dynamic interactions of fluorescently labeled microtubule-associated proteins in living cells

    PubMed Central

    1984-01-01

    Microtubule-associated proteins (MAPs) from calf brain were fluorescently labeled with 6-iodoacetamido fluorescein (I-AF). The modified MAPs (especially enriched for MAP2) were fully active in promoting tubulin polymerization in vitro and readily associated with cytoplasmic filaments when microinjected into living cultured cells. Double-labeling experiments indicated that the microinjected AF-MAPs were incorporated predominantly, if not exclusively, into cytoplasmic microtubules in untreated cells or paracrystals induced within vinblastine-treated cells. Similar results were obtained with different cell types (neuronal, epithelial, and fibroblastic) of diverse origin (man, mouse, chicken, and rat kangaroo). Mobility measurements of the microinjected AF-MAPs using the method of fluorescence-photobleaching recovery (FPR) revealed two populations of AF-MAPs with distinct dynamic properties: One fraction represents the soluble pool of MAPs and is mobile with a diffusion coefficient of D = 3 X 10(-9) cm2/s. The other fraction of MAPs is associated with the microtubules and is essentially immobile on the time scale of FPR experiments. However, it showed slow fluorescence recovery with an apparent half time of approximately 5 min. The slow recovery of fluorescence on defined photobleached microtubules occurred most probably by the incorporation of AF-MAPs from the soluble cytoplasmic pool into the bleached area. The bleached spot on defined microtubules remained essentially immobile during the slow recovery phase. These results suggest that MAPs can associate in vivo with microtubules of diverse cell types and that treadmilling of MAP2-containing microtubules in vivo, if it exists, is slower than 4 micron/h. PMID:6547721

  15. Global up-regulation of microtubule dynamics and polarity reversal during regeneration of an axon from a dendrite.

    PubMed

    Stone, Michelle C; Nguyen, Michelle M; Tao, Juan; Allender, Dana L; Rolls, Melissa M

    2010-03-01

    Axon regeneration is crucial for recovery after trauma to the nervous system. For neurons to recover from complete axon removal they must respecify a dendrite as an axon: a complete reversal of polarity. We show that Drosophila neurons in vivo can convert a dendrite to a regenerating axon and that this process involves rebuilding the entire neuronal microtubule cytoskeleton. Two major microtubule rearrangements are specifically induced by axon and not dendrite removal: 1) 10-fold up-regulation of the number of growing microtubules and 2) microtubule polarity reversal. After one dendrite reverses its microtubules, it initiates tip growth and takes on morphological and molecular characteristics of an axon. Only neurons with a single dendrite that reverses polarity are able to initiate tip growth, and normal microtubule plus-end dynamics are required to initiate this growth. In addition, we find that JNK signaling is required for both the up-regulation of microtubule dynamics and microtubule polarity reversal initiated by axon injury. We conclude that regulation of microtubule dynamics and polarity in response to JNK signaling is key to initiating regeneration of an axon from a dendrite.

  16. Global Up-Regulation of Microtubule Dynamics and Polarity Reversal during Regeneration of an Axon from a Dendrite

    PubMed Central

    Stone, Michelle C.; Nguyen, Michelle M.; Tao, Juan; Allender, Dana L.

    2010-01-01

    Axon regeneration is crucial for recovery after trauma to the nervous system. For neurons to recover from complete axon removal they must respecify a dendrite as an axon: a complete reversal of polarity. We show that Drosophila neurons in vivo can convert a dendrite to a regenerating axon and that this process involves rebuilding the entire neuronal microtubule cytoskeleton. Two major microtubule rearrangements are specifically induced by axon and not dendrite removal: 1) 10-fold up-regulation of the number of growing microtubules and 2) microtubule polarity reversal. After one dendrite reverses its microtubules, it initiates tip growth and takes on morphological and molecular characteristics of an axon. Only neurons with a single dendrite that reverses polarity are able to initiate tip growth, and normal microtubule plus-end dynamics are required to initiate this growth. In addition, we find that JNK signaling is required for both the up-regulation of microtubule dynamics and microtubule polarity reversal initiated by axon injury. We conclude that regulation of microtubule dynamics and polarity in response to JNK signaling is key to initiating regeneration of an axon from a dendrite. PMID:20053676

  17. The self-assembly ability of First microtubule-binding repeat from tau and its modulation by phosphorylation

    SciTech Connect

    Zhou Lianxiu; Zeng Zhiyang; Du Jintang; Zhao Yufen; Li Yanmei . E-mail: liym@mail.tsinghua.edu.cn

    2006-09-22

    Aggregation of abnormally phosphorylated tau in the form of tangs of paired helical filaments (PHFs) is one of the hallmarks of Alzheimer's disease (AD) and other tauopathies. It is of fundamental importance to study the mechanism of PHF formation and its modulation by phosphorylation. In this work, we have focused on First microtubule-binding repeat of tau encompassing an abnormal phosphorylation site Ser{sup 262}. The assembly propensities of this repeat and its corresponding phosphorylated form were investigated by turbidity and electron microscopy. Additionally, conformation of the two peptides is also analyzed through circular dichroism (CD) and NMR spectroscopy. Our results reveal that both of them are capable of self-assembly and phosphorylation at Ser{sup 262} could speed up the process of assembly. A possible mechanism of PHF formation is proposed and enhancing effect of phosphorylation on assembly provides an explanation to its toxicity in Alzheimer's disease.

  18. RSK2 signals through stathmin to promote microtubule dynamics and tumor metastasis.

    PubMed

    Alesi, G N; Jin, L; Li, D; Magliocca, K R; Kang, Y; Chen, Z G; Shin, D M; Khuri, F R; Kang, S

    2016-10-13

    Metastasis is responsible for >90% of cancer-related deaths. Complex signaling in cancer cells orchestrates the progression from a primary to a metastatic cancer. However, the mechanisms of these cellular changes remain elusive. We previously demonstrated that p90 ribosomal S6 kinase 2 (RSK2) promotes tumor metastasis. Here we investigated the role of RSK2 in the regulation of microtubule dynamics and its potential implication in cancer cell invasion and tumor metastasis. Stable knockdown of RSK2 disrupted microtubule stability and decreased phosphorylation of stathmin, a microtubule-destabilizing protein, at serine 16 in metastatic human cancer cells. We found that RSK2 directly binds and phosphorylates stathmin at the leading edge of cancer cells. Phosphorylation of stathmin by RSK2 reduced stathmin-mediated microtubule depolymerization. Moreover, overexpression of phospho-mimetic mutant stathmin S16D significantly rescued the decreased invasive and metastatic potential mediated by RSK2 knockdown in vitro and in vivo. Furthermore, stathmin phosphorylation positively correlated with RSK2 expression and metastatic cancer progression in primary patient tumor samples. Our finding demonstrates that RSK2 directly phosphorylates stathmin and regulates microtubule polymerization to provide a pro-invasive and pro-metastatic advantage to cancer cells. Therefore, the RSK2-stathmin pathway represents a promising therapeutic target and a prognostic marker for metastatic human cancers.

  19. Modulation of microtubule dynamics by a TIR domain protein from the intracellular pathogen Brucella melitensis.

    PubMed

    Radhakrishnan, Girish K; Harms, Jerome S; Splitter, Gary A

    2011-10-01

    TIR (Toll/interleukin-1 receptor) domain-containing proteins play a crucial role in innate immunity in eukaryotes. Brucella is a highly infectious intracellular bacterium that encodes a TIR domain protein (TcpB) to subvert host innate immune responses to establish a beneficial niche for pathogenesis. TcpB inhibits NF-κB (nuclear factor κB) activation and pro-inflammatory cytokine secretions mediated by TLR (Toll-like receptor) 2 and TLR4. In the present study, we have demonstrated that TcpB modulates microtubule dynamics by acting as a stabilization factor. TcpB increased the rate of nucleation as well as the polymerization phases of microtubule formation in a similar manner to paclitaxel. TcpB could efficiently inhibit nocodazole- or cold-induced microtubule disassembly. Microtubule stabilization by TcpB is attributed to the BB-loop region of the TIR domain, and a point mutation affected the microtubule stabilization as well as the TLR-suppression properties of TcpB.

  20. Label-Free Imaging of Single Microtubule Dynamics Using Spatial Light Interference Microscopy.

    PubMed

    Kandel, Mikhail E; Teng, Kai Wen; Selvin, Paul R; Popescu, Gabriel

    2017-01-24

    Due to their diameter, of only 24 nm, single microtubules are extremely challenging to image without the use of extrinsic contrast agents. As a result, fluorescence tagging is the common method to visualize their motility. However, such investigation is limited by photobleaching and phototoxicity. We experimentally demonstrate the capability of combining label-free spatial light interference microscopy (SLIM) with numerical processing for imaging single microtubules in a gliding assay. SLIM combines four different intensity images to obtain the optical path length map associated with the sample. Because of the use of broadband fields, the sensitivity to path length is better than 1 nm without (temporal) averaging and better than 0.1 nm upon averaging. Our results indicate that SLIM can image the dynamics of microtubules in a full field of view, of 200 × 200 μm(2), over many hours. Modeling the microtubule transport via the diffusion-advection equation, we found that the dispersion relation yields the standard deviation of the velocity distribution, without the need for tracking individual tubes. Interestingly, during a 2 h window, the microtubules begin to decelerate, at 100 pm/s(2) over a 20 min period. Thus, SLIM is likely to serve as a useful tool for understanding molecular motor activity, especially over large time scales, where fluorescence methods are of limited utility.

  1. An array of nuclear microtubules reorganizes the budding yeast nucleus during quiescence

    PubMed Central

    Laporte, Damien; Courtout, Fabien; Salin, Bénédicte; Ceschin, Johanna

    2013-01-01

    The microtubule cytoskeleton is a highly dynamic network. In dividing cells, its complex architecture not only influences cell shape and movement but is also crucial for chromosome segregation. Curiously, nothing is known about the behavior of this cellular machinery in quiescent cells. Here we show that, upon quiescence entry, the Saccharomyces cerevisiae microtubule cytoskeleton is drastically remodeled. Indeed, while cytoplasmic microtubules vanish, the spindle pole body (SPB) assembles a long and stable monopolar array of nuclear microtubules that spans the entire nucleus. Consequently, the nucleolus is displaced. Kinetochores remain attached to microtubule tips but lose SPB clustering and distribute along the microtubule array, leading to a large reorganization of the nucleus. When cells exit quiescence, the nuclear microtubule array slowly depolymerizes and, by pulling attached centromeres back to the SPB, allows the recovery of a typical Rabl-like configuration. Finally, mutants that do not assemble a nuclear array of microtubules are impaired for both quiescence survival and exit. PMID:24247429

  2. Signaling Scaffold Protein IQGAP1 Interacts with Microtubule Plus-end Tracking Protein SKAP and Links Dynamic Microtubule Plus-end to Steer Cell Migration*

    PubMed Central

    Cao, Dan; Su, Zeqi; Wang, Wenwen; Wu, Huihui; Liu, Xing; Akram, Saima; Qin, Bo; Zhou, Jiajia; Zhuang, Xiaoxuan; Adams, Gregory; Jin, Changjiang; Wang, Xiwei; Liu, Lifang; Hill, Donald L.; Wang, Dongmei; Ding, Xia; Yao, Xuebiao

    2015-01-01

    Cell migration is orchestrated by dynamic interaction of microtubules with the plasma membrane cortex. However, the regulatory mechanisms underlying the cortical actin cytoskeleton and microtubule dynamics are less characterized. Our earlier study showed that small GTPase-activating proteins, IQGAPs, regulate polarized secretion in epithelial cells (1). Here, we show that IQGAP1 links dynamic microtubules to steer cell migration via interacting with the plus-end tracking protein, SKAP. Biochemical characterizations revealed that IQGAP1 and SKAP form a cognate complex and that their binding interfaces map to the WWIQ motif and the C-terminal of SKAP, respectively. The WWIQ peptide disrupts the biochemical interaction between IQGAP1 and SKAP in vitro, and perturbation of the IQGAP1-SKAP interaction in vivo using a membrane-permeable TAT-WWIQ peptide results in inhibition of directional cell migration elicited by EGF. Mechanistically, the N-terminal of SKAP binds to EB1, and its C terminus binds to IQGAP1 in migrating cells. Thus, we reason that a novel IQGAP1 complex orchestrates directional cell migration via coupling dynamic microtubule plus-ends to the cell cortex. PMID:26242911

  3. Titanium dioxide nanoparticles alter cellular morphology via disturbing the microtubule dynamics

    NASA Astrophysics Data System (ADS)

    Mao, Zhilei; Xu, Bo; Ji, Xiaoli; Zhou, Kun; Zhang, Xuemei; Chen, Minjian; Han, Xiumei; Tang, Qiusha; Wang, Xinru; Xia, Yankai

    2015-04-01

    Titanium dioxide (TiO2) nanoparticles (NPs) have been widely used in our daily lives, for example, in the areas of sunscreens, cosmetics, toothpastes, food products, and nanomedical reagents. Recently, increasing concern has been raised about their neurotoxicity, but the mechanisms underlying such toxic effects are still unknown. In this work, we employed a human neuroblastoma cell line (SH-SY5Y) to study the effects of TiO2 NPs on neurological systems. Our results showed that TiO2 NPs did not affect cell viability but induced noticeable morphological changes until 100 μg ml-1. Immunofluorescence detection showed disorder, disruption, retraction, and decreased intensity of the microtubules after TiO2 NPs treatment. Both α and β tubule expressions did not change in the TiO2 NP-treated group, but the percentage of soluble tubules was increased. A microtubule dynamic study in living cells indicated that TiO2 NPs caused a lower growth rate and a higher shortening rate of microtubules as well as shortened lifetimes of de novo microtubules. TiO2 NPs did not cause changes in the expression and phosphorylation state of tau proteins, but a tau-TiO2 NP interaction was observed. TiO2 NPs could interact with tubule heterodimers, microtubules and tau proteins, which led to the instability of microtubules, thus contributing to the neurotoxicity of TiO2 NPs.Titanium dioxide (TiO2) nanoparticles (NPs) have been widely used in our daily lives, for example, in the areas of sunscreens, cosmetics, toothpastes, food products, and nanomedical reagents. Recently, increasing concern has been raised about their neurotoxicity, but the mechanisms underlying such toxic effects are still unknown. In this work, we employed a human neuroblastoma cell line (SH-SY5Y) to study the effects of TiO2 NPs on neurological systems. Our results showed that TiO2 NPs did not affect cell viability but induced noticeable morphological changes until 100 μg ml-1. Immunofluorescence detection showed disorder

  4. Tubulin cofactors and Arl2 are cage-like chaperones that regulate the soluble αβ-tubulin pool for microtubule dynamics

    PubMed Central

    Nithianantham, Stanley; Le, Sinh; Seto, Elbert; Jia, Weitao; Leary, Julie; Corbett, Kevin D; Moore, Jeffrey K; Al-Bassam, Jawdat

    2015-01-01

    Microtubule dynamics and polarity stem from the polymerization of αβ-tubulin heterodimers. Five conserved tubulin cofactors/chaperones and the Arl2 GTPase regulate α- and β-tubulin assembly into heterodimers and maintain the soluble tubulin pool in the cytoplasm, but their physical mechanisms are unknown. Here, we reconstitute a core tubulin chaperone consisting of tubulin cofactors TBCD, TBCE, and Arl2, and reveal a cage-like structure for regulating αβ-tubulin. Biochemical assays and electron microscopy structures of multiple intermediates show the sequential binding of αβ-tubulin dimer followed by tubulin cofactor TBCC onto this chaperone, forming a ternary complex in which Arl2 GTP hydrolysis is activated to alter αβ-tubulin conformation. A GTP-state locked Arl2 mutant inhibits ternary complex dissociation in vitro and causes severe defects in microtubule dynamics in vivo. Our studies suggest a revised paradigm for tubulin cofactors and Arl2 functions as a catalytic chaperone that regulates soluble αβ-tubulin assembly and maintenance to support microtubule dynamics. DOI: http://dx.doi.org/10.7554/eLife.08811.001 PMID:26208336

  5. Brownian dynamics of subunit addition-loss kinetics and thermodynamics in linear polymer self-assembly.

    PubMed

    Castle, Brian T; Odde, David J

    2013-12-03

    The structure and free energy of multistranded linear polymer ends evolves as individual subunits are added and lost. Thus, the energetic state of the polymer end is not constant, as assembly theory has assumed. Here we utilize a Brownian dynamics approach to simulate the addition and loss of individual subunits at the polymer tip. Using the microtubule as a primary example, we examined how the structure of the polymer tip dictates the rate at which units are added to and lost from individual protofilaments. We find that freely diffusing subunits arrive less frequently to lagging protofilaments but bind more efficiently, such that there is no kinetic difference between leading and lagging protofilaments within a tapered tip. However, local structure at the nanoscale has up to an order-of-magnitude effect on the rate of addition. Thus, the kinetic on-rate constant, integrated across the microtubule tip (kon,MT), is an ensemble average of the varying individual protofilament on-rate constants (kon,PF). Our findings have implications for both catastrophe and rescue of the dynamic microtubule end, and provide a subnanoscale framework for understanding the mechanism of action of microtubule-associated proteins and microtubule-directed drugs. Although we utilize the specific example of the microtubule here, the findings are applicable to multistranded polymers generally.

  6. NuSAP modulates the dynamics of kinetochore microtubules by attenuating MCAK depolymerisation activity

    PubMed Central

    Li, Chenyu; Zhang, Yajun; Yang, Qiaoyun; Ye, Fan; Sun, Stella Ying; Chen, Ee Sin; Liou, Yih-Cherng

    2016-01-01

    Nucleolar and spindle-associated protein (NuSAP) is a microtubule-associated protein that functions as a microtubule stabiliser. Depletion of NuSAP leads to severe mitotic defects, however the mechanism by which NuSAP regulates mitosis remains elusive. In this study, we identify the microtubule depolymeriser, mitotic centromere-associated kinesin (MCAK), as a novel binding partner of NuSAP. We show that NuSAP regulates the dynamics and depolymerisation activity of MCAK. Phosphorylation of MCAK by Aurora B kinase, a component of the chromosomal passenger complex, significantly enhances the interaction of NuSAP with MCAK and modulates the effects of NuSAP on the depolymerisation activity of MCAK. Our results reveal an underlying mechanism by which NuSAP controls kinetochore microtubule dynamics spatially and temporally by modulating the depolymerisation function of MCAK in an Aurora B kinase-dependent manner. Hence, this study provides new insights into the function of NuSAP in spindle formation during mitosis. PMID:26733216

  7. Dynamics of assembly production flow

    NASA Astrophysics Data System (ADS)

    Ezaki, Takahiro; Yanagisawa, Daichi; Nishinari, Katsuhiro

    2015-06-01

    Despite recent developments in management theory, maintaining a manufacturing schedule remains difficult because of production delays and fluctuations in demand and supply of materials. The response of manufacturing systems to such disruptions to dynamic behavior has been rarely studied. To capture these responses, we investigate a process that models the assembly of parts into end products. The complete assembly process is represented by a directed tree, where the smallest parts are injected at leaves and the end products are removed at the root. A discrete assembly process, represented by a node on the network, integrates parts, which are then sent to the next downstream node as a single part. The model exhibits some intriguing phenomena, including overstock cascade, phase transition in terms of demand and supply fluctuations, nonmonotonic distribution of stockout in the network, and the formation of a stockout path and stockout chains. Surprisingly, these rich phenomena result from only the nature of distributed assembly processes. From a physical perspective, these phenomena provide insight into delay dynamics and inventory distributions in large-scale manufacturing systems.

  8. Hydrogen peroxide modulates the dynamic microtubule cytoskeleton during the defence responses to Verticillium dahliae toxins in Arabidopsis.

    PubMed

    Yao, Lin-Lin; Zhou, Qun; Pei, Bao-Lei; Li, Ying-Zhang

    2011-09-01

    The molecular mechanisms of signal transduction of plants in response to infection by Verticillium dahliae (VD) are not well understood. We previously showed that NO may act as an upstream signalling molecule to trigger the depolymerization of cortical microtubules in Arabidopsis. In the present study, we used the wild-type, and atrbohD and atrbohF mutants of Arabidopsis to explore the mechanisms of action of H(2)O(2) signals and the dynamic microtubule cytoskeleton in defence responses. We demonstrated that H(2)O(2) may also act as an upstream signalling molecule to regulate cortical microtubule depolymerization. The depolymerization of the cortical microtubules played a functional role in the signalling pathway to mediate the expression of defence genes. The results indicate that H(2)O(2) modulates the dynamic microtubule cytoskeleton to trigger the expression of defence genes against V. dahliae toxins (VD-toxins) in Arabidopsis.

  9. CHK2-BRCA1 tumor-suppressor axis restrains oncogenic Aurora-A kinase to ensure proper mitotic microtubule assembly.

    PubMed

    Ertych, Norman; Stolz, Ailine; Valerius, Oliver; Braus, Gerhard H; Bastians, Holger

    2016-02-16

    BRCA1 (breast cancer type 1 susceptibility protein) is a multifunctional tumor suppressor involved in DNA damage response, DNA repair, chromatin regulation, and mitotic chromosome segregation. Although the nuclear functions of BRCA1 have been investigated in detail, its role during mitosis is little understood. It is clear, however, that loss of BRCA1 in human cancer cells leads to chromosomal instability (CIN), which is defined as a perpetual gain or loss of whole chromosomes during mitosis. Moreover, our recent work has revealed that the mitotic function of BRCA1 depends on its phosphorylation by the tumor-suppressor kinase Chk2 (checkpoint kinase 2) and that this regulation is required to ensure normal microtubule plus end assembly rates within mitotic spindles. Intriguingly, loss of the positive regulation of BRCA1 leads to increased oncogenic Aurora-A activity, which acts as a mediator for abnormal mitotic microtubule assembly resulting in chromosome missegregation and CIN. However, how the CHK2-BRCA1 tumor suppressor axis restrains oncogenic Aurora-A during mitosis to ensure karyotype stability remained an open question. Here we uncover a dual molecular mechanism by which the CHK2-BRCA1 axis restrains oncogenic Aurora-A activity during mitosis and identify BRCA1 itself as a target for Aurora-A relevant for CIN. In fact, Chk2-mediated phosphorylation of BRCA1 is required to recruit the PP6C-SAPS3 phosphatase, which acts as a T-loop phosphatase inhibiting Aurora-A bound to BRCA1. Consequently, loss of CHK2 or PP6C-SAPS3 promotes Aurora-A activity associated with BRCA1 in mitosis. Aurora-A, in turn, then phosphorylates BRCA1 itself, thereby inhibiting the mitotic function of BRCA1 and promoting mitotic microtubule assembly, chromosome missegregation, and CIN.

  10. Erucin, the major isothiocyanate in arugula (Eruca sativa), inhibits proliferation of MCF7 tumor cells by suppressing microtubule dynamics.

    PubMed

    Azarenko, Olga; Jordan, Mary Ann; Wilson, Leslie

    2014-01-01

    Consumption of cruciferous vegetables is associated with reduced risk of various types of cancer. Isothiocyanates including sulforaphane and erucin are believed to be responsible for this activity. Erucin [1-isothiocyanato-4-(methylthio)butane], which is metabolically and structurally related to sulforaphane, is present in large quantities in arugula (Eruca sativa, Mill.), kohlrabi and Chinese cabbage. However, its cancer preventive mechanisms remain poorly understood. We found that erucin inhibits proliferation of MCF7 breast cancer cells (IC50 = 28 µM) in parallel with cell cycle arrest at mitosis (IC50 = 13 µM) and apoptosis, by a mechanism consistent with impairment of microtubule dynamics. Concentrations of 5-15 µM erucin suppressed the dynamic instability of microtubules during interphase in the cells. Most dynamic instability parameters were inhibited, including the rates and extents of growing and shortening, the switching frequencies between growing and shortening, and the overall dynamicity. Much higher erucin concentrations were required to reduce the microtubule polymer mass. In addition, erucin suppressed dynamic instability of microtubules reassembled from purified tubulin in similar fashion. The effects of erucin on microtubule dynamics, like those of sulforaphane, are similar qualitatively to those of much more powerful clinically-used microtubule-targeting anticancer drugs, including taxanes and the vinca alkaloids. The results suggest that suppression of microtubule dynamics by erucin and the resulting impairment of critically important microtubule-dependent cell functions such as mitosis, cell migration and microtubule-based transport may be important in its cancer preventive activities.

  11. Interaction of CK1δ with γTuSC ensures proper microtubule assembly and spindle positioning

    PubMed Central

    Peng, Yutian; Moritz, Michelle; Han, Xuemei; Giddings, Thomas H.; Lyon, Andrew; Kollman, Justin; Winey, Mark; Yates, John; Agard, David A.; Drubin, David G.; Barnes, Georjana

    2015-01-01

    Casein kinase 1δ (CK1δ) family members associate with microtubule-organizing centers (MTOCs) from yeast to humans, but their mitotic roles and targets have yet to be identified. We show here that budding yeast CK1δ, Hrr25, is a γ-tubulin small complex (γTuSC) binding factor. Moreover, Hrr25's association with γTuSC depends on its kinase activity and its noncatalytic central domain. Loss of Hrr25 kinase activity resulted in assembly of unusually long cytoplasmic microtubules and defects in spindle positioning, consistent with roles in regulation of γTuSC-mediated microtubule nucleation and the Kar9 spindle-positioning pathway, respectively. Hrr25 directly phosphorylated γTuSC proteins in vivo and in vitro, and this phosphorylation promoted γTuSC integrity and activity. Because CK1δ and γTuSC are highly conserved and present at MTOCs in diverse eukaryotes, similar regulatory mechanisms are expected to apply generally in eukaryotes. PMID:25971801

  12. Hierarchical Bionanotubes Formed By the Self Assembly of Microtubules With Cationic Membranes Or Polypeptides

    SciTech Connect

    Raviv, U.; Needleman, D.J.; Ewert, K.K.; Safinya, C.R.

    2009-06-05

    At present there is a surge in interest in biophysical research aimed at elucidating collective interactions between cellular proteins and associated biomolecules leading to supramolecular structures, with the ultimate goal of relating structure to function. The nerve cell cytoskeleton provides a rich example of highly ordered bundles and networks of interacting neurofilaments, microtubules and filamentous actin, where the nature of the interactions, structures and structure-function correlations remain poorly understood. We present synchrotron X-ray diffraction and electron microscopy data, in reconstituted protein systems from the bovine central nervous system, which reveal unexpected structures not predicted by current electrostatic theories. By mixing preassembled microtubules with charged membranes or polypeptides we found hierarchical bionanotubes made of microtubules coated by lipid bilayers or polypeptides, which in turn are coated with a third layer of tubulin oligomers forming rings or spirals.

  13. Microtubule-associated protein/microtubule affinity-regulating kinase (p110mark). A novel protein kinase that regulates tau-microtubule interactions and dynamic instability by phosphorylation at the Alzheimer-specific site serine 262.

    PubMed

    Drewes, G; Trinczek, B; Illenberger, S; Biernat, J; Schmitt-Ulms, G; Meyer, H E; Mandelkow, E M; Mandelkow, E

    1995-03-31

    Aberrant phosphorylation of the microtubule-associated protein tau is one of the pathological features of neuronal degeneration in Alzheimer's disease. The phosphorylation of Ser-262 within the microtubule binding region of tau is of particular interest because so far it is observed only in Alzheimer's disease (Hasegawa, M., Morishima-Kawashima, M., Takio, K., Suzuki, M., Titani, K., and Ihara, Y. (1992) J. Biol. Chem. 26, 17047-17054) and because phosphorylation of this site alone dramatically reduces the affinity for microtubules in vitro (Biernat, J., Gustke, N., Drewes, G., Mandelkow, E.-M., and Mandelkow, E. (1993) Neuron 11, 153-163). Here we describe the purification and characterization of a protein-serine kinase from brain tissue with an apparent molecular mass of 110 kDa on SDS gels. This kinase specifically phosphorylates tau on its KIGS or KCGS motifs in the repeat domain, whereas no significant phosphorylation outside this region was detected. Phosphorylation occurs mainly on Ser-262 located in the first repeat. This largely abolishes tau's binding to microtubules and makes them dynamically unstable, in contrast to other protein kinases that phosphorylate tau at or near the repeat domain. The data suggest a role for this novel kinase in cellular events involving rearrangement of the microtuble-associated proteins/microtubule arrays and their pathological degeneration in Alzheimer's disease.

  14. AKAP9, a Regulator of Microtubule Dynamics, Contributes to Blood-Testis Barrier Function

    PubMed Central

    Venkatesh, Deepak; Mruk, Dolores; Herter, Jan M.; Cullere, Xavier; Chojnacka, Katarzyna; Cheng, C. Yan; Mayadas, Tanya N.

    2017-01-01

    The blood-testis barrier (BTB), formed between adjacent Sertoli cells, undergoes extensive remodeling to facilitate the transport of preleptotene spermatocytes across the barrier from the basal to apical compartments of the seminiferous tubules for further development and maturation into spermatozoa. The actin cytoskeleton serves unique structural and supporting roles in this process, but little is known about the role of microtubules and their regulators during BTB restructuring. The large isoform of the cAMP-responsive scaffold protein AKAP9 regulates microtubule dynamics and nucleation at the Golgi. We found that conditional deletion of Akap9 in mice after the initial formation of the BTB at puberty leads to infertility. Akap9 deletion results in marked alterations in the organization of microtubules in Sertoli cells and a loss of barrier integrity despite a relatively intact, albeit more apically localized F-actin and BTB tight junctional proteins. These changes are accompanied by a loss of haploid spermatids due to impeded meiosis. The barrier, however, progressively reseals in older Akap9 null mice, which correlates with a reduction in germ cell apoptosis and a greater incidence of meiosis. However, spermiogenesis remains defective, suggesting additional roles for AKAP9 in this process. Together, our data suggest that AKAP9 and, by inference, the regulation of the microtubule network are critical for BTB function and subsequent germ cell development during spermatogenesis. PMID:26687990

  15. Structural differences between brain beta 1- and beta 2-tubulins: implications for microtubule assembly and colchicine binding.

    PubMed Central

    Little, M; Ludueña, R F

    1985-01-01

    Brain beta 1- and beta 2-tubulins are the major and minor beta-tubulin components of chordate brain tissue, respectively. Two cysteines of beta 1, but not beta 2, can be specifically cross-linked with the bifunctional sulfhydryl reagent N,N'-ethylenebis(iodoacetamide) (EBI). They are in positions 239 and 354. Although separated by 115 amino acid residues along the beta 1-chain, the two sulfur atoms are maximally 9 A apart in the beta 1 tertiary structure. The failure of beta 2 to form a similar cross-bridge is due to the absence of a cysteine in position 239. At least 10 other sequence differences are also present between beta 1 and beta 2. Positions 239 and 354 of beta 1 probably occupy a key part of the tubulin molecule. The microtubule assembly inhibitors colchicine and podophyllotoxin appear to bind on or near this site and EBI is a potent inhibitor of microtubule assembly. Furthermore, the beta 1-cysteine in position 239 appears to be the most reactive in brain tubulin under the given conditions. The marked difference between beta 1 and beta 2 in this critical region suggests that they may have different functions in brain tissue. Images Fig. 1. PMID:4018027

  16. Analysis of microtubule growth dynamics arising from altered actin network structure and contractility in breast tumor cells.

    PubMed

    Ory, Eleanor; Bhandary, Lekhana; Boggs, Amanda; Chakrabarti, Kristi; Parker, Joshua; Losert, Wolfgang; Martin, Stuart S

    2017-01-16

    The periphery of epithelial cells is shaped by opposing cytoskeletal physical forces generated predominately by two dynamic force generating systems - growing microtubule ends push against the boundary from the cell center, and the actin cortex contracts the attached plasma membrane. Here we investigate how changes to the structure and dynamics of the actin cortex alter the dynamics of microtubules. Current drugs target actin polymerization and contraction to reduce cell division and invasiveness; however, the impacts on microtubule dynamics remain incompletely understood. Using human MCF-7 breast tumor cells expressing GFP-tagged microtubule end-binding-protein-1 (EB1) and coexpression of cytoplasmic fluorescent protein mCherry, we map the trajectories of growing microtubule ends and cytoplasmic boundary respectively. Based on EB1 tracks and cytoplasmic boundary outlines, we calculate the speed, distance from cytoplasmic boundary, and straightness of microtubule growth. Actin depolymerization with Latrunculin-A reduces EB1 growth speed as well as allows the trajectories to extend beyond the cytoplasmic boundary. Blebbistatin, a direct myosin-II inhibitor, reduced EB1 speed and yielded less straight EB1 trajectories. Inhibiting signaling upstream of myosin-II contractility via the Rho-kinase inhibitor, Y-27632, altered EB1 dynamics differently from Blebbistatin. These results indicate that reduced actin cortex integrity can induce distinct alterations in microtubule dynamics. Given recent findings that tumor stem cell characteristics are increased by drugs which reduce actin contractility or stabilize microtubules, it remains important to clearly define how cytoskeletal drugs alter the interactions between these two filament systems in tumor cells.

  17. Microtubules are essential for guard-cell function in Vicia and Arabidopsis.

    PubMed

    Eisinger, William; Ehrhardt, David; Briggs, Winslow

    2012-05-01

    Radially arranged cortical microtubules are a prominent feature of guard cells. Guard cells expressing GFP-tubulin 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.

  18. Enhanced dynamic instability of microtubules in a ROS free inert environment.

    PubMed

    Islam, Md Sirajul; Kabir, Arif Md Rashedul; Inoue, Daisuke; Sada, Kazuki; Kakugo, Akira

    2016-04-01

    Reactive oxygen species (ROS), one of the regulators in various biological processes, have recently been suspected to modulate microtubule (MT) dynamics in cells. However due to complicated cellular environment and unavailability of any in vitro investigation, no detail is understood yet. Here, by performing simple in vitro investigations, we have unveiled the effect of ROS on MT dynamics. By studying dynamic instability of MTs in a ROS free environment and comparing with that in the presence of ROS, we disclosed that MTs showed enhanced dynamics in the ROS free environment. All the parameters that define dynamic instability of MTs e.g., growth and shrinkage rates, rescue and catastrophe frequencies were significantly affected by the presence of ROS. This work clearly reveals the role of ROS in modulating MT dynamics in vitro, and would be a great help in understanding the role of ROS in regulation of MT dynamics in cells.

  19. Regulation of Kif15 localization and motility by the C-terminus of TPX2 and microtubule dynamics

    PubMed Central

    Mann, Barbara J.; Balchand, Sai K.; Wadsworth, Patricia

    2017-01-01

    Mitotic motor proteins generate force to establish and maintain spindle bipolarity, but how they are temporally and spatially regulated in vivo is unclear. Prior work demonstrated that a microtubule-associated protein, TPX2, targets kinesin-5 and kinesin-12 motors to spindle microtubules. The C-terminal domain of TPX2 contributes to the localization and motility of the kinesin-5, Eg5, but it is not known whether this domain regulates kinesin-12, Kif15. We found that the C-terminal domain of TPX2 contributes to the localization of Kif15 to spindle microtubules in cells and suppresses motor walking in vitro. Kif15 and Eg5 are partially redundant motors, and overexpressed Kif15 can drive spindle formation in the absence of Eg5 activity. Kif15-dependent bipolar spindle formation in vivo requires the C-terminal domain of TPX2. In the spindle, fluorescent puncta of GFP-Kif15 move toward the equatorial region at a rate equivalent to microtubule growth. Reduction of microtubule growth with paclitaxel suppresses GFP-Kif15 motility, demonstrating that dynamic microtubules contribute to Kif15 behavior. Our results show that the C-terminal region of TPX2 regulates Kif15 in vitro, contributes to motor localization in cells, and is required for Kif15 force generation in vivo and further reveal that dynamic microtubules contribute to Kif15 behavior in vivo. PMID:27852894

  20. Non-equilibrium assembly of microtubules: from molecules to autonomous chemical robots.

    PubMed

    Hess, H; Ross, Jennifer L

    2017-03-22

    Biological systems have evolved to harness non-equilibrium processes from the molecular to the macro scale. It is currently a grand challenge of chemistry, materials science, and engineering to understand and mimic biological systems that have the ability to autonomously sense stimuli, process these inputs, and respond by performing mechanical work. New chemical systems are responding to the challenge and form the basis for future responsive, adaptive, and active materials. In this article, we describe a particular biochemical-biomechanical network based on the microtubule cytoskeletal filament - itself a non-equilibrium chemical system. We trace the non-equilibrium aspects of the system from molecules to networks and describe how the cell uses this system to perform active work in essential processes. Finally, we discuss how microtubule-based engineered systems can serve as testbeds for autonomous chemical robots composed of biological and synthetic components.

  1. The Salmonella effector SseJ disrupts microtubule dynamics when ectopically expressed in normal rat kidney cells

    PubMed Central

    Raines, Sally A.; Hodgkinson, Michael R.; Dowle, Adam A.

    2017-01-01

    Salmonella effector protein SseJ is secreted by Salmonella into the host cell cytoplasm where it can then modify host cell processes. Whilst host cell small GTPase RhoA has previously been shown to activate the acyl-transferase activity of SseJ we show here an un-described effect of SseJ protein production upon microtubule dynamism. SseJ prevents microtubule collapse and this is independent of SseJ’s acyl-transferase activity. We speculate that the effects of SseJ on microtubules would be mediated via its known interactions with the small GTPases of the Rho family. PMID:28235057

  2. FTDP-17 mutations in Tau alter the regulation of microtubule dynamics: an "alternative core" model for normal and pathological Tau action.

    PubMed

    LeBoeuf, Adria C; Levy, Sasha F; Gaylord, Michelle; Bhattacharya, Arnab; Singh, Ambuj K; Jordan, Mary Ann; Wilson, Leslie; Feinstein, Stuart C

    2008-12-26

    Mutations affecting either the structure or regulation of the microtubule-associated protein Tau cause neuronal cell death and dementia. However, the molecular mechanisms mediating these deleterious effects remain unclear. Among the most characterized activities of Tau is the ability to regulate microtubule dynamics, known to be essential for proper cell function and viability. Here we have tested the hypothesis that Tau mutations causing neurodegeneration also alter the ability of Tau to regulate the dynamic instability behaviors of microtubules. Using in vitro microtubule dynamics assays to assess average microtubule growth rates, microtubule growth rate distributions, and catastrophe frequencies, we found that all tested mutants possessing amino acid substitutions or deletions mapping to either the repeat or interrepeat regions of Tau do indeed compromise its ability to regulate microtubule dynamics. Further mutational analyses suggest a novel mechanism of Tau regulatory action based on an "alternative core" of microtubule binding and regulatory activities composed of two repeats and the interrepeat between them. In this model, the interrepeat serves as the primary regulator of microtubule dynamics, whereas the flanking repeats serve as tethers to properly position the interrepeat on the microtubule. Importantly, since there are multiple interrepeats on each Tau molecule, there are also multiple cores on each Tau molecule, each with distinct mechanistic capabilities, thereby providing significant regulatory potential. Taken together, the data are consistent with a microtubule misregulation mechanism for Tau-mediated neuronal cell death and provide a novel mechanistic model for normal and pathological Tau action.

  3. NOCA-1 functions with γ-tubulin and in parallel to Patronin to assemble non-centrosomal microtubule arrays in C. elegans

    PubMed Central

    Wang, Shaohe; Wu, Di; Quintin, Sophie; Green, Rebecca A; Cheerambathur, Dhanya K; Ochoa, Stacy D; Desai, Arshad; Oegema, Karen

    2015-01-01

    Non-centrosomal microtubule arrays assemble in differentiated tissues to perform mechanical and transport-based functions. In this study, we identify Caenorhabditis elegans NOCA-1 as a protein with homology to vertebrate ninein. NOCA-1 contributes to the assembly of non-centrosomal microtubule arrays in multiple tissues. In the larval epidermis, NOCA-1 functions redundantly with the minus end protection factor Patronin/PTRN-1 to assemble a circumferential microtubule array essential for worm growth and morphogenesis. Controlled degradation of a γ-tubulin complex subunit in this tissue revealed that γ-tubulin acts with NOCA-1 in parallel to Patronin/PTRN-1. In the germline, NOCA-1 and γ-tubulin co-localize at the cell surface, and inhibiting either leads to a microtubule assembly defect. γ-tubulin targets independently of NOCA-1, but NOCA-1 targeting requires γ-tubulin when a non-essential putatively palmitoylated cysteine is mutated. These results show that NOCA-1 acts with γ-tubulin to assemble non-centrosomal arrays in multiple tissues and highlight functional overlap between the ninein and Patronin protein families. DOI: http://dx.doi.org/10.7554/eLife.08649.001 PMID:26371552

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

    PubMed

    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.

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

    PubMed

    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.

  6. Cellular inhibition of microtubule assembly by photoactivated sulphonated meso-tetraphenylporphines.

    PubMed

    Berg, K; Moan, J; Bommer, J C; Winkelman, J W

    1990-09-01

    This work relates to studies on modes of phototoxicity by sulphonated mesotetraphenylporphines on cultured cells. Toxicity appears to be related to inhibition of microtubule function. Treatment of human cervix carcinoma cells of the line NHIK 3025 incubated for 18 h with meso-tetraphenylporphine sulphonates (TPPSn where n = 2a, 2o or 4) and exposed to light, inhibits multiplication for the first hours after light exposure, a significant fraction of the cells accumulating in mitosis. The maximal number of cells in mitosis after treatment (approximately 20%) is dependent on the fluence but is similar for all three photosensitizers. For the first hours after treatment the mitotic cells were always mainly in metaphase; mainly seen as c-metaphases and three-group metaphases. During this time anaphase and telophase cells were absent or greatly reduced in number. Indirect immunofluorescence staining of beta-tubulin showed that the spindle apparatus of mitotic cells was perturbed in all cases. Results are presented which indicate that photoactivation of TPPSn located on the plasma membrane destroys microtubules in interphase cells and leads to arrest of the cells in mitosis. The localization of the dye which sensitizes the photoinduced perturbation of microtubules is further discussed.

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

    PubMed Central

    Stubenvoll, Michael D.; Medley, Jeffrey C.; Irwin, Miranda

    2016-01-01

    Centrosomes are critical sites for orchestrating microtubule dynamics, and exhibit dynamic changes in size during the cell cycle. As cells progress to mitosis, centrosomes recruit more microtubules (MT) to form mitotic bipolar spindles that ensure proper chromosome segregation. We report a new role for ATX-2, a C. elegans ortholog of Human Ataxin-2, in regulating centrosome size and MT dynamics. ATX-2, an RNA-binding protein, forms a complex with SZY-20 in an RNA-independent fashion. Depleting ATX-2 results in embryonic lethality and cytokinesis failure, and restores centrosome duplication to zyg-1 mutants. In this pathway, SZY-20 promotes ATX-2 abundance, which inversely correlates with centrosome size. Centrosomes depleted of ATX-2 exhibit elevated levels of centrosome factors (ZYG-1, SPD-5, γ-Tubulin), increasing MT nucleating activity but impeding MT growth. We show that ATX-2 influences MT behavior through γ-Tubulin at the centrosome. Our data suggest that RNA-binding proteins play an active role in controlling MT dynamics and provide insight into the control of proper centrosome size and MT dynamics. PMID:27689799

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

    PubMed

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

    2015-10-21

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

  9. Mechanism of dynamic reorientation of cortical microtubules due to mechanical stress.

    PubMed

    Muratov, Alexander; Baulin, Vladimir A

    2015-12-01

    Directional growth caused by gravitropism and corresponding bending of plant cells has been explored since 19th century, however, many aspects of mechanisms underlying the perception of gravity at the molecular level are still not well known. Perception of gravity in root and shoot gravitropisms is usually attributed to gravisensitive cells, called statocytes, which exploit sedimentation of macroscopic and heavy organelles, amyloplasts, to sense the direction of gravity. Gravity stimulus is then transduced into distal elongation zone, which is several mm far from statocytes, where it causes stretching. It is suggested that gravity stimulus is conveyed by gradients in auxin flux. We propose a theoretical model that may explain how concentration gradients and/or stretching may indirectly affect the global orientation of cortical microtubules, attached to the cell membrane and induce their dynamic reorientation perpendicular to the gradients. In turn, oriented microtubule arrays direct the growth and orientation of cellulose microfibrils, forming part of the cell external skeleton and determine the shape of the cell. Reorientation of microtubules is also observed in reaction to light in phototropism and mechanical bending, thus suggesting universality of the proposed mechanism.

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

    PubMed

    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.

  11. Regulation of Microtubule Dynamics in Axon Regeneration: Insights from C. elegans.

    PubMed

    Tang, Ngang Heok; Chisholm, Andrew D

    2016-01-01

    The capacity of an axon to regenerate is regulated by its external environment and by cell-intrinsic factors. Studies in a variety of organisms suggest that alterations in axonal microtubule (MT) dynamics have potent effects on axon regeneration. We review recent findings on the regulation of MT dynamics during axon regeneration, focusing on the nematode Caenorhabditis elegans. In C. elegans the dual leucine zipper kinase (DLK) promotes axon regeneration, whereas the exchange factor for Arf6 (EFA-6) inhibits axon regeneration. Both DLK and EFA-6 respond to injury and control axon regeneration in part via MT dynamics. How the DLK and EFA-6 pathways are related is a topic of active investigation, as is the mechanism by which EFA-6 responds to axonal injury. We evaluate potential candidates, such as the MT affinity-regulating kinase PAR-1/MARK, in regulation of EFA-6 and axonal MT dynamics in regeneration.

  12. Regulation of Microtubule Dynamics in Axon Regeneration: Insights from C. elegans

    PubMed Central

    Tang, Ngang Heok; Chisholm, Andrew D.

    2016-01-01

    The capacity of an axon to regenerate is regulated by its external environment and by cell-intrinsic factors. Studies in a variety of organisms suggest that alterations in axonal microtubule (MT) dynamics have potent effects on axon regeneration. We review recent findings on the regulation of MT dynamics during axon regeneration, focusing on the nematode Caenorhabditis elegans. In C. elegans the dual leucine zipper kinase (DLK) promotes axon regeneration, whereas the exchange factor for Arf6 (EFA-6) inhibits axon regeneration. Both DLK and EFA-6 respond to injury and control axon regeneration in part via MT dynamics. How the DLK and EFA-6 pathways are related is a topic of active investigation, as is the mechanism by which EFA-6 responds to axonal injury. We evaluate potential candidates, such as the MT affinity-regulating kinase PAR-1/MARK, in regulation of EFA-6 and axonal MT dynamics in regeneration. PMID:27350865

  13. The von Hippel-Lindau tumor suppressor protein influences microtubule dynamics at the cell periphery.

    PubMed

    Lolkema, Martijn P; Mehra, Niven; Jorna, Anita S; van Beest, Moniek; Giles, Rachel H; Voest, Emile E

    2004-12-10

    The von Hippel-Lindau (VHL) protein protects microtubules (MTs) from destabilization by nocodazole treatment. Based on this fixed-cell assay with static end points, VHL has been reported to directly stabilize the MT cytoskeleton. To investigate the dynamic changes in MTs induced by VHL in living cells, we measured the influence of VHL on tubulin turnover using fluorescence recovery after photobleaching (FRAP). To this end, we engineered VHL-deficient renal cell carcinoma cells to constitutively incorporate fluorescently labeled tubulin and to inducibly express VHL. Induction of VHL in these cells resulted in a decrease of tubulin turnover as measured by FRAP at the cell periphery, while minimally influencing MT dynamics around the centrosome. Our data indicates that VHL changes the behavior of MTs dependent on their subcellular localization implying a role for VHL in cellular processes such as migration, polarization, and cell-cell interactions. Here we propose a complementary method to directly measure VHL-induced subcellular changes in microtubule dynamics, which may serve as a tool to study the effect of MT binding proteins such as VHL.

  14. Dynamic pathways for viral capsid assembly

    SciTech Connect

    Hagan, Michael F.; Chandler, David

    2006-02-09

    We develop a class of models with which we simulate the assembly of particles into T1 capsid-like objects using Newtonian dynamics. By simulating assembly for many different values of system parameters, we vary the forces that drive assembly. For some ranges of parameters, assembly is facile, while for others, assembly is dynamically frustrated by kinetic traps corresponding to malformed or incompletely formed capsids. Our simulations sample many independent trajectories at various capsomer concentrations, allowing for statistically meaningful conclusions. Depending on subunit (i.e., capsomer) geometries, successful assembly proceeds by several mechanisms involving binding of intermediates of various sizes. We discuss the relationship between these mechanisms and experimental evaluations of capsid assembly processes.

  15. Dynamic model of the force driving kinesin to move along microtubule-Simulation with a model system

    NASA Astrophysics Data System (ADS)

    Chou, Y. C.; Hsiao, Yi-Feng; To, Kiwing

    2015-09-01

    A dynamic model for the motility of kinesin, including stochastic-force generation and step formation is proposed. The force driving the motion of kinesin motor is generated by the impulse from the collision between the randomly moving long-chain stalk and the ratchet-shaped outer surface of microtubule. Most of the dynamical and statistical features of the motility of kinesin are reproduced in a simulation system, with (a) ratchet structures similar to the outer surface of microtubule, (b) a bead chain connected to two heads, similarly to the stalk of the real kinesin motor, and (c) the interaction between the heads of the simulated kinesin and microtubule. We also propose an experiment to discriminate between the conventional hand-over-hand model and the dynamic model.

  16. γ-Tubulin Ring Complexes and EB1 play antagonistic roles in microtubule dynamics and spindle positioning

    PubMed Central

    Bouissou, Anaїs; Vérollet, Christel; de Forges, Hélène; Haren, Laurence; Bellaїche, Yohanns; Perez, Franck; Merdes, Andreas; Raynaud-Messina, Brigitte

    2014-01-01

    γ-Tubulin is critical for microtubule (MT) assembly and organization. In metazoa, this protein acts in multiprotein complexes called γ-Tubulin Ring Complexes (γ-TuRCs). While the subunits that constitute γ-Tubulin Small Complexes (γ-TuSCs), the core of the MT nucleation machinery, are essential, mutation of γ-TuRC-specific proteins in Drosophila causes sterility and morphological abnormalities via hitherto unidentified mechanisms. Here, we demonstrate a role of γ-TuRCs in controlling spindle orientation independent of MT nucleation activity, both in cultured cells and in vivo, and examine a potential function for γ-TuRCs on astral MTs. γ-TuRCs locate along the length of astral MTs, and depletion of γ-TuRC-specific proteins increases MT dynamics and causes the plus-end tracking protein EB1 to redistribute along MTs. Moreover, suppression of MT dynamics through drug treatment or EB1 down-regulation rescues spindle orientation defects induced by γ-TuRC depletion. Therefore, we propose a role for γ-TuRCs in regulating spindle positioning by controlling the stability of astral MTs. PMID:24421324

  17. The kinesin-2 family member KIF3C regulates microtubule dynamics and is required for axon growth and regeneration.

    PubMed

    Gumy, Laura F; Chew, Daniel J; Tortosa, Elena; Katrukha, Eugene A; Kapitein, Lukas C; Tolkovsky, Aviva M; Hoogenraad, Casper C; Fawcett, James W

    2013-07-10

    Axon regeneration after injury requires the extensive reconstruction, reorganization, and stabilization of the microtubule cytoskeleton in the growth cones. Here, we identify KIF3C as a key regulator of axonal growth and regeneration by controlling microtubule dynamics and organization in the growth cone. KIF3C is developmentally regulated. Rat embryonic sensory axons and growth cones contain undetectable levels of KIF3C protein that is locally translated immediately after injury. In adult neurons, KIF3C is axonally transported from the cell body and is enriched at the growth cone where it preferentially binds to tyrosinated microtubules. Functionally, the interaction of KIF3C with EB3 is necessary for its localization at the microtubule plus-ends in the growth cone. Depletion of KIF3C in adult neurons leads to an increase in stable, overgrown and looped microtubules because of a strong decrease in the microtubule frequency of catastrophes, suggesting that KIF3C functions as a microtubule-destabilizing factor. Adult axons lacking KIF3C, by RNA interference or KIF3C gene knock-out, display an impaired axonal outgrowth in vitro and a delayed regeneration after injury both in vitro and in vivo. Murine KIF3C knock-out embryonic axons grow normally but do not regenerate after injury because they are unable to locally translate KIF3C. These data show that KIF3C is an injury-specific kinesin that contributes to axon growth and regeneration by regulating and organizing the microtubule cytoskeleton in the growth cone.

  18. The human Ino80 binds to microtubule via the E-hook of tubulin: Implications for the role in spindle assembly

    SciTech Connect

    Park, Eun-Jung; Hur, Shin-Kyoung; Lee, Han-Sae; Lee, Shin-Ai; Kwon, Jongbum

    2011-12-16

    Highlights: Black-Right-Pointing-Pointer The N-terminal domain of hIno80 is important for binding to the spindle. Black-Right-Pointing-Pointer The hIno80 N-terminal domain binds to tubulin and microtubule in vitro. Black-Right-Pointing-Pointer The E-hook of tubulin is critical for hIno80 binding to tubulin and microtubule. Black-Right-Pointing-Pointer Tip49a does not bind to microtubule and dispensable for spindle formation. -- Abstract: The human INO80 chromatin remodeling complex, comprising the Ino80 ATPase (hIno80) and the associated proteins such as Tip49a, has been implicated in a variety of nuclear processes other than transcription. We previously have found that hIno80 interacts with tubulin and co-localizes with the mitotic spindle and is required for spindle formation. To better understand the role of hIno80 in spindle formation, we further investigated the interaction between hIno80 and microtubule. Here, we show that the N-terminal domain, dispensable for the nucleosome remodeling activity, is important for hIno80 to interact with tubulin and co-localize with the spindle. The hIno80 N-terminal domain binds to monomeric tubulin and polymerized microtubule in vitro, and the E-hook of tubulin, involved in the polymerization of microtubule, is critical for this binding. Tip49a, which has been reported to associate with the spindle, does not bind to microtubule in vitro and dispensable for spindle formation in vivo. These results suggest that hIno80 can play a direct role in the spindle assembly independent of its chromatin remodeling activity.

  19. Activation of microtubule dynamics increases neuronal growth via the nerve growth factor (NGF)- and Gαs-mediated signaling pathways.

    PubMed

    Sarma, Tulika; Koutsouris, Athanasia; Yu, Jiang Zhu; Krbanjevic, Aleksandar; Hope, Thomas J; Rasenick, Mark M

    2015-04-17

    Signals that activate the G protein Gαs and promote neuronal differentiation evoke Gαs internalization in rat pheochromocytoma (PC12) cells. These agents also significantly increase Gαs association with microtubules, resulting in an increase in microtubule dynamics because of the activation of tubulin GTPase by Gαs. To determine the function of Gαs/microtubule association in neuronal development, we used real-time trafficking of a GFP-Gαs fusion protein. GFP-Gαs concentrates at the distal end of the neurites in differentiated living PC12 cells as well as in cultured hippocampal neurons. Gαs translocates to specialized membrane compartments at tips of growing neurites. A dominant-negative Gα chimera that interferes with Gαs binding to tubulin and activation of tubulin GTPase attenuates neurite elongation and neurite number both in PC12 cells and primary hippocampal neurons. This effect is greatest on differentiation induced by activated Gαs. Together, these data suggest that activated Gαs translocates from the plasma membrane and, through interaction with tubulin/microtubules in the cytosol, is important for neurite formation, development, and outgrowth. Characterization of neuronal G protein dynamics and their contribution to microtubule dynamics is important for understanding the molecular mechanisms by which G protein-coupled receptor signaling orchestrates neuronal growth and differentiation.

  20. The Dynamic Behavior of Individual Microtubules Associated with Chromosomes In Vitro

    PubMed Central

    Hunt, Alan J.; McIntosh, J. Richard

    1998-01-01

    Mitotic movements of chromosomes are usually coupled to the elongation and shortening of the microtubules to which they are bound. The lengths of kinetochore-associated microtubules change by incorporation or loss of tubulin subunits, principally at their chromosome-bound ends. We have reproduced aspects of this phenomenon in vitro, using a real-time assay that displays directly the movements of individual chromosome-associated microtubules as they elongate and shorten. Chromosomes isolated from cultured Chinese hamster ovary cells were adhered to coverslips and then allowed to bind labeled microtubules. In the presence of tubulin and GTP, these microtubules could grow at their chromosome-bound ends, causing the labeled segments to move away from the chromosomes, even in the absence of ATP. Sometimes a microtubule would switch to shortening, causing the direction of movement to change abruptly. The link between a microtubule and a chromosome was mechanically strong; 15 pN of tension was generally insufficient to detach a microtubule, even though it could add subunits at the kinetochore–microtubule junction. The behavior of the microtubules in vitro was regulated by the chromosomes to which they were bound; the frequency of transitions from polymerization to depolymerization was decreased, and the speed of depolymerization-coupled movement toward chromosomes was only one-fifth the rate of shortening for microtubules free in solution. Our results are consistent with a model in which each microtubule interacts with an increasing number of chromosome-associated binding sites as it approaches the kinetochore. PMID:9763448

  1. DNA synthesis and microtubule assembly-related events in fertilized Paracentrotus lividus eggs: reversible inhibition by 10 mM procaine.

    PubMed

    Raymond, M N; Foucault, G; Coffe, G; Pudles, J

    1986-04-01

    This report describes the effects of 10 mM procaine on microtubule assembly and on DNA synthesis, as followed by [3H]colchicine binding assays and [3H]thymidine incorporation respectively, in fertilized Paracentrotus lividus eggs. In the absence of microtubule assembly inhibitors, about 25% of the total egg tubulin is submitted to two cycles of polymerization prior to the first cell division, this polymerization process precedes DNA synthesis. If the zygotes are treated with 10 mM procaine in the course of the cell cycle, tubulin polymerization is inhibited or microtubules are disassembled. DNA synthesis is inhibited when procaine treatment is performed 10 min, before the initiation of the S-period. However, when the drug is applied in the course of this synthetic period, the process is normally accomplished, but the next S-period becomes inhibited. Moreover, procaine treatment increases the cytoplasmic pH of the fertilized eggs by about 0.6 to 0.8 pH units. This pH increase precedes microtubule disassembly and inhibition of DNA synthesis. Washing out the drug induces a decrease of the intracellular pH which returns to about the same value as that of the fertilized egg controls. This pH change is then followed by the reinitiation of microtubule assembly, DNA synthesis and cell division. Our results show that the inhibition of both tubulin polymerization and DNA synthesis in fertilized eggs treated with 10 mM procaine, appears to be related to the drug-induced increase in cytoplasmic pH.

  2. Dual effect of procaine in sea urchin eggs. Inducer and inhibitor of microtubule assembly.

    PubMed

    Coffe, G; Foucault, G; Raymond, M N; Pudles, J

    1985-01-01

    An increase in the amount of cytoplasmic filamentous structures (cytoplasmic matrix and aster) which were recovered after hexylene glycol/Triton X-100 treatment of sea urchin eggs (Paracentrotus lividus) activated by 0.2-2.5 mM procaine was observed. At higher activator concentrations, an opposite effect was observed and formation of these cytoplasmic structures was inhibited in the presence of 10 mM procaine. This inhibitory effect was reversed by diluting the drug in the incubation medium. DNase I inhibition assays on egg homogenates which were performed at different time points of the activation process, show that the same amount of actin was induced to polymerize in eggs activated either by 2.5 or 10 mM procaine. However, colchicine-binding assays on the 100 000 g particulate fractions of these homogenates show that in eggs activated by 10 mM procaine, in contrast to those activated by 2.5 mM, tubulin polymerization was inhibited and microtubules were disassembled. These results show that the dual effect of procaine in the organization of the egg cytoskeleton appears to be related to its effect on the state of tubulin.

  3. Site-specific phosphorylation and microtubule dynamics control Pyrin inflammasome activation

    PubMed Central

    Gao, Wenqing; Yang, Jieling; Liu, Wang; Wang, Yupeng; Shao, Feng

    2016-01-01

    Pyrin, encoded by the MEFV gene, is best known for its gain-of-function mutations causing familial Mediterranean fever (FMF), an autoinflammatory disease. Pyrin forms a caspase-1–activating inflammasome in response to inactivating modifications of Rho GTPases by various bacterial toxins or effectors. Pyrin-mediated innate immunity is unique in that it senses bacterial virulence rather than microbial molecules, but its mechanism of activation is unknown. Here we show that Pyrin was phosphorylated in bone marrow-derived macrophages and dendritic cells. We identified Ser-205 and Ser-241 in mouse Pyrin whose phosphorylation resulted in inhibitory binding by cellular 14-3-3 proteins. The two serines underwent dephosphorylation upon toxin stimulation or bacterial infection, triggering 14-3-3 dissociation, which correlated with Pyrin inflammasome activation. We developed antibodies specific for phosphorylated Ser-205 and Ser-241, which confirmed the stimuli-induced dephosphorylation of endogenous Pyrin. Mutational analyses indicated that both phosphorylation and signal-induced dephosphorylation of Ser-205/241 are important for Pyrin activation. Moreover, microtubule drugs, including colchicine, commonly used to treat FMF, effectively blocked activation of the Pyrin inflammasome. These drugs did not affect Pyrin dephosphorylation and 14-3-3 dissociation but inhibited Pyrin-mediated apoptosis-associated Speck-like protein containing CARD (ASC) aggregation. Our study reveals that site-specific (de)phosphorylation and microtubule dynamics critically control Pyrin inflammasome activation, illustrating a fine and complex mechanism in cytosolic immunity. PMID:27482109

  4. Human TUBB3 mutations perturb microtubule dynamics, kinesin interactions, and axon guidance

    PubMed Central

    Tischfield, Max A.; Baris, Hagit N.; Wu, Chen; Rudolph, Guenther; Van Maldergem, Lionel; He, Wei; Chan, Wai-Man; Andrews, Caroline; Demer, Joseph L.; Robertson, Richard L.; Mackey, David A.; Ruddle, Jonathan B.; Bird, Thomas D.; Gottlob, Irene; Pieh, Christina; Traboulsi, Elias I.; Pomeroy, Scott L.; Hunter, David G.; Soul, Janet S.; Newlin, Anna; Sabol, Louise J.; Doherty, Edward J.; de Uzcátegui, Clara E.; de Uzcátegui, Nicolas; Collins, Mary Louise Z.; Sener, Emin C.; Wabbels, Bettina; Hellebrand, Heide; Meitinger, Thomas; de Berardinis, Teresa; Magli, Adriano; Schiavi, Costantino; Pastore-Trossello, Marco; Koc, Feray; Wong, Agnes M.; Levin, Alex V.; Geraghty, Michael T.; Descartes, Maria; Flaherty, Maree; Jamieson, Robyn V.; Møller, H. U.; Meuthen, Ingo; Callen, David F.; Kerwin, Janet; Lindsay, Susan; Meindl, Alfons; Gupta, Mohan L.; Pellman, David; Engle, Elizabeth C.

    2011-01-01

    We report that eight heterozygous missense mutations in TUBB3, encoding the neuron-specific β-tubulin isotype III, result in a spectrum of human nervous system disorders we now call the TUBB3 syndromes. Each mutation causes the ocular motility disorder CFEOM3, whereas some also result in intellectual and behavioral impairments, facial paralysis, and/or later-onset axonal sensorimotor polyneuropathy. Neuroimaging reveals a spectrum of abnormalities including hypoplasia of oculomotor nerves, and dysgenesis of the corpus callosum, anterior commissure, and corticospinal tracts. A knock-in disease mouse model reveals axon guidance defects without evidence of cortical cell migration abnormalities. We show the disease-associated mutations can impair tubulin heterodimer formation in vitro, although folded mutant heterodimers can still polymerize into microtubules. Modeling each mutation in yeast tubulin demonstrates that all alter dynamic instability whereas a subset disrupts the interaction of microtubules with kinesin motors. These findings demonstrate normal TUBB3 is required for axon guidance and maintenance in mammals. PMID:20074521

  5. Molecular mechanism of action of microtubule-stabilizing anticancer agents.

    PubMed

    Prota, Andrea E; Bargsten, Katja; Zurwerra, Didier; Field, Jessica J; Díaz, José Fernando; Altmann, Karl-Heinz; Steinmetz, Michel O

    2013-02-01

    Microtubule-stabilizing agents (MSAs) are efficacious chemotherapeutic drugs widely used for the treatment of cancer. Despite the importance of MSAs for medical applications and basic research, their molecular mechanisms of action on tubulin and microtubules remain elusive. We determined high-resolution crystal structures of αβ-tubulin in complex with two unrelated MSAs, zampanolide and epothilone A. Both compounds were bound to the taxane pocket of β-tubulin and used their respective side chains to induce structuring of the M-loop into a short helix. Because the M-loop establishes lateral tubulin contacts in microtubules, these findings explain how taxane-site MSAs promote microtubule assembly and stability. Further, our results offer fundamental structural insights into the control mechanisms of microtubule dynamics.

  6. Fluorescent microtubules break up under illumination

    PubMed Central

    1988-01-01

    We have synthesized three new fluorescent analogues of tubulin, using fluorescein or rhodamine groups attached to N-hydroxy-succinimidyl esters, and have partially characterized the properties of these analogues. We have also further characterized the tubulin derivatized with dichlorotriazinyl-aminofluorescein that has previously been used in this and other laboratories. Our results show that all four analogues assemble into microtubules which break up when exposed to light of the wavelengths that excite fluorescence. This sensitivity places severe constraints on the use of these analogues in studies of microtubule dynamics. PMID:3417772

  7. An agent-based model contrasts opposite effects of dynamic and stable microtubules on cleavage furrow positioning

    PubMed Central

    Odell, Garrett M.; Foe, Victoria E.

    2008-01-01

    From experiments by Foe and von Dassow (Foe, V.E., and G. von Dassow. 2008. J. Cell Biol. 183:457–470) and others, we infer a molecular mechanism for positioning the cleavage furrow during cytokinesis. Computer simulations reveal how this mechanism depends on quantitative motor-behavior details and explore how robustly this mechanism succeeds across a range of cell sizes. The mechanism involves the MKLP1 (kinesin-6) component of centralspindlin binding to and walking along microtubules to stimulate cortical contractility where the centralspindlin complex concentrates. The majority of astral microtubules are dynamically unstable. They bind most MKLP1 and suppress cortical Rho/myosin II activation because the tips of unstable microtubules usually depolymerize before MKLP1s reach the cortex. A subset of astral microtubules stabilizes during anaphase, becoming effective rails along which MKLP1 can actually reach the cortex. Because stabilized microtubules aim statistically at the equatorial spindle midplane, that is where centralspindlin accumulates to stimulate furrow formation. PMID:18955556

  8. Downregulation of striatin leads to hyperphosphorylation of MAP2, induces depolymerization of microtubules and inhibits proliferation of HEK293T cells.

    PubMed

    Kaźmierczak-Barańska, Julia; Pęczek, Łukasz; Przygodzka, Patrycja; Cieślak, Marcin J

    2015-01-16

    Microtubules are tubular polymers of α/β-tubulin that are involved in the maintenance of cell shape, motility, and intracellular transport and in the segregation of chromosomes during cell division. Microtubules are dynamic structures, and their assembly is regulated by phosphoproteins called microtubule-associated proteins (MAPs). We propose that striatin, a protein belonging to the striatin family of proteins, is involved in regulation of microtubules. In HEK293T cells, striatin colocalizes with microtubules and stably associates with PP2Ac. Inhibition of striatin expression results in hyperphosphorylation of MAP2 and destabilizes microtubules. Striatin-induced destabilization of microtubules inhibited the proliferation of HEK293T cells and caused the accumulation of cells in the G0/G1 phase of the cell cycle. These results suggest that the PP2A/striatin complex modulates microtubule dynamics by regulating MAP2 phosphorylation.

  9. Supramolecular assembly of biological molecules purified from bovine nerve cells: from microtubule bundles and necklaces to neurofilament networks

    NASA Astrophysics Data System (ADS)

    Needleman, Daniel J.; Jones, Jayna B.; Raviv, Uri; Ojeda-Lopez, Miguel A.; Miller, H. P.; Li, Y.; Wilson, L.; Safinya, C. R.

    2005-11-01

    With the completion of the human genome project, the biosciences community is beginning the daunting task of understanding the structures and functions of a large number of interacting biological macromolecules. Examples include the interacting molecules involved in the process of DNA condensation during the cell cycle, and in the formation of bundles and networks of filamentous actin proteins in cell attachment, motility and cytokinesis. In this proceedings paper we present examples of supramolecular assembly based on proteins derived from the vertebrate nerve cell cytoskeleton. The axonal cytoskeleton in vertebrate neurons provides a rich example of bundles and networks of neurofilaments, microtubules (MTs) and filamentous actin, where the nature of the interactions, structures, and structure-function correlations remains poorly understood. We describe synchrotron x-ray diffraction, electron microscopy, and optical imaging data, in reconstituted protein systems purified from bovine central nervous system, which reveal unexpected structures not predicted by current electrostatic theories of polyelectrolyte bundling, including three-dimensional MT bundles and two-dimensional MT necklaces.

  10. Dynamic microtubule-dependent interactions position homotypic neurones in regular monolayered arrays during retinal development.

    PubMed

    Galli-Resta, Lucia; Novelli, Elena; Viegi, Alessandro

    2002-08-01

    In the vertebrate retina cell layers support serial processing, while monolayered arrays of homotypic neurones tile each layer to allow parallel processing. How neurones form layers and arrays is still largely unknown. We show that monolayered retinal arrays are dynamic structures based on dendritic interactions between the array cells. The analysis of three developing retinal arrays shows that these become regular as a net of dendritic processes links neighbouring array cells. Molecular or pharmacological perturbations of microtubules within dendrites lead to a stereotyped and reversible disruption of array organization: array cells lose their regular spacing and the arrangement in a monolayer. This leads to a micro-mechanical explanation of how monolayers of regularly spaced 'like-cells' are formed.

  11. Activated ADF/cofilin sequesters phosphorylated microtubule-associated-protein during the assembly of Alzheimer-like neuritic cytoskeletal striations

    PubMed Central

    Whiteman, Ineka T.; Gervasio, Othon L.; Cullen, Karen M.; Guillemin, Gilles J.; Jeong, Erica V.; Witting, Paul K.; Antao, Shane T.; Minamide, Laurie S.; Bamburg, James R.; Goldsbury, Claire

    2009-01-01

    In Alzheimer disease (AD), rod-like cofilin aggregates (cofilin-actin rods) and thread-like inclusions containing phosphorylated microtubule-associated protein (pMAP) tau form in the brain (neuropil threads) and the extent of their presence correlates with cognitive decline and disease progression. The assembly mechanism of these respective pathological lesions and the relationship between them is poorly understood, yet vital to understanding the causes of sporadic AD. We demonstrate that during mitochondrial inhibition, activated actin-depolymerizing factor (ADF)/cofilin assemble into rods along processes of cultured primary neurons that recruit pMAP/tau and mimic neuropil threads. Fluorescence Resonance Energy Transfer (FRET) analysis revealed co-localization of cofilin-GFP and pMAP in rods, suggesting their close proximity within a cytoskeletal inclusion complex. The relationship between pMAP and cofilin-actin rods was further investigated using actin-modifying drugs and siRNA knockdown of ADF/cofilin in primary neurons. The results suggest that activation of ADF/cofilin and generation of cofilin-actin rods is required for the subsequent recruitment of pMAP into the inclusions. Additionally we were able to induce the formation of pMAP-positive ADF/cofilin rods by exposing cells to exogenous Aβ peptides. These results reveal a common pathway for pMAP and cofilin accumulation in neuronal processes. The requirement of activated ADF/cofilin for the sequestration of pMAP suggests that neuropil thread structures in the AD brain may be initiated by elevated cofilin activation and F-actin bundling that can be caused by oxidative stress, mitochondrial dysfunction or Aβ peptides, all suspected initiators of synaptic loss and neurodegeneration in AD. PMID:19828813

  12. Gas7b (growth arrest specific protein 7b) regulates neuronal cell morphology by enhancing microtubule and actin filament assembly.

    PubMed

    Gotoh, Aina; Hidaka, Masafumi; Hirose, Keiko; Uchida, Takafumi

    2013-11-29

    Neurons undergo several morphological changes as a part of normal neuron maturation process. Alzheimer disease is associated with increased neuroproliferation and impaired neuronal maturation. In this study, we demonstrated that Gas7b (growth arrest specific protein 7b) expression in a neuronal cell line, Neuro 2A, induces cell maturation by facilitating formation of dendrite-like processes and/or filopodia projections and that Gas7b co-localizes with neurite microtubules. Molecular analysis was performed to evaluate whether Gas7b associates with actin filaments and microtubules, and the data revealed two novel roles of Gas7b in neurite outgrowth: we showed that Gas7b enhances bundling of several microtubule filaments and connects microtubules with actin filaments. These results suggest that Gas7b governs neural cell morphogenesis by enhancing the coordination between actin filaments and microtubules. We conclude that lower neuronal Gas7b levels may impact Alzheimer disease progression.

  13. Microtubules Are Essential for Mitochondrial Dynamics-Fission, Fusion, and Motility-in Dictyostelium discoideum.

    PubMed

    Woods, Laken C; Berbusse, Gregory W; Naylor, Kari

    2016-01-01

    Mitochondrial function is dependent upon mitochondrial structure which is in turn dependent upon mitochondrial dynamics, including fission, fusion, and motility. Here we examined the relationship between mitochondrial dynamics and the cytoskeleton in Dictyostelium discoideum. Using time-lapse analysis, we quantified mitochondrial fission, fusion, and motility in the presence of cytoskeleton disrupting pharmaceuticals and the absence of the potential mitochondria-cytoskeleton linker protein, CluA. Our results indicate that microtubules are essential for mitochondrial movement, as well as fission and fusion; actin plays a less significant role, perhaps selecting the mitochondria for transport. We also suggest that CluA is not a linker protein but plays an unidentified role in mitochondrial fission and fusion. The significance of our work is to gain further insight into the role the cytoskeleton plays in mitochondrial dynamics and function. By better understanding these processes we can better appreciate the underlying mitochondrial contributions to many neurological disorders characterized by altered mitochondrial dynamics, structure, and/or function.

  14. Single-molecule tracking of tau reveals fast kiss-and-hop interaction with microtubules in living neurons.

    PubMed

    Janning, Dennis; Igaev, Maxim; Sündermann, Frederik; Brühmann, Jörg; Beutel, Oliver; Heinisch, Jürgen J; Bakota, Lidia; Piehler, Jacob; Junge, Wolfgang; Brandt, Roland

    2014-11-05

    The microtubule-associated phosphoprotein tau regulates microtubule dynamics and is involved in neurodegenerative diseases collectively called tauopathies. It is generally believed that the vast majority of tau molecules decorate axonal microtubules, thereby stabilizing them. However, it is an open question how tau can regulate microtubule dynamics without impeding microtubule-dependent transport and how tau is also available for interactions other than those with microtubules. Here we address this apparent paradox by fast single-molecule tracking of tau in living neurons and Monte Carlo simulations of tau dynamics. We find that tau dwells on a single microtubule for an unexpectedly short time of ∼40 ms before it hops to the next. This dwell time is 100-fold shorter than previously reported by ensemble measurements. Furthermore, we observed by quantitative imaging using fluorescence decay after photoactivation recordings of photoactivatable GFP-tagged tubulin that, despite this rapid dynamics, tau is capable of regulating the tubulin-microtubule balance. This indicates that tau's dwell time on microtubules is sufficiently long to influence the lifetime of a tubulin subunit in a GTP cap. Our data imply a novel kiss-and-hop mechanism by which tau promotes neuronal microtubule assembly. The rapid kiss-and-hop interaction explains why tau, although binding to microtubules, does not interfere with axonal transport.

  15. Bisphenol A and its analogues disrupt centrosome cycle and microtubule dynamics in prostate cancer.

    PubMed

    Ho, Shuk-Mei; Rao, Rahul; To, Sarah; Schoch, Emma; Tarapore, Pheruza

    2017-02-01

    Humans are increasingly exposed to structural analogues of bisphenol A (BPA), as BPA is being replaced by these compounds in BPA-free consumer products. We have previously shown that chronic and developmental exposure to BPA is associated with increased prostate cancer (PCa) risk in human and animal models. Here, we examine whether exposure of PCa cells (LNCaP, C4-2) to low-dose BPA and its structural analogues (BPS, BPF, BPAF, TBBPA, DMBPA and TMBPA) affects centrosome amplification (CA), a hallmark of cancer initiation and progression. We found that exposure to BPA, BPS, DMBPA and TBBPA, in descending order, increased the number of cells with CA, in a non-monotonic dose-response manner. Furthermore, cells treated with BPA and their analogues initiated centrosome duplication at 8 h after release from serum starvation, significantly earlier in G-1 phase than control cells. This response was attended by earlier release of nucleophosmin from unduplicated centrosomes. BPA-exposed cells exhibited increased expression of cyclin-dependent kinase CDK6 and decreased expression of CDK inhibitors (p21(Waf1/CIP1) and p27(KIP1)). Using specific antagonists for estrogen/androgen receptors, CA in the presence of BPA or its analogues was likely to be mediated via ESR1 signaling. Change in microtubule dynamics was observed on exposure to these analogues, which, for BPA, was accompanied by increased expression of centrosome-associated protein CEP350 Similar to BPA, chronic treatment of cells with DMBPA, but not other analogues, resulted in the enhancement of anchorage-independent growth. We thus conclude that selected BPA analogues, similar to BPA, disrupt centrosome function and microtubule organization, with DMBPA displaying the broadest spectrum of cancer-promoting effects.

  16. Using Photobleaching to Measure Spindle Microtubule Dynamics in Primary Cultures of Dividing Drosophila Meiotic Spermatocytes

    PubMed Central

    2015-01-01

    In dividing animal cells, a microtubule (MT)-based bipolar spindle governs chromosome movement. Current models propose that the spindle facilitates and/or generates translocating forces by regionally depolymerizing the kinetochore fibers (k-fibers) that bind each chromosome. It is unclear how conserved these sites and the resultant chromosome-moving mechanisms are between different dividing cell types because of the technical challenges of quantitatively studying MTs in many specimens. In particular, our knowledge of MT kinetics during the sperm-producing male meiotic divisions remains in its infancy. In this study, I use an easy-to-implement photobleaching-based assay for measuring spindle MT dynamics in primary cultures of meiotic spermatocytes isolated from the fruit fly Drosophila melanogaster. By use of standard scanning confocal microscopy features, fiducial marks were photobleached on fluorescent protein (FP)-tagged MTs. These were followed by time-lapse imaging during different division stages, and their displacement rates were calculated using public domain software. I find that k-fibers continually shorten at their poles during metaphase and anaphase A through the process of MT flux. Anaphase chromosome movement is complemented by Pac-Man, the shortening of the k-fiber at its chromosomal interface. Thus, Drosophila spermatocytes share the sites of spindle dynamism and mechanisms of chromosome movement with mitotic cells. The data reveal the applicability of the photobleaching assay for measuring MT dynamics in primary cultures. This approach can be readily applied to other systems. PMID:25802491

  17. Using Photobleaching to Measure Spindle Microtubule Dynamics in Primary Cultures of Dividing Drosophila Meiotic Spermatocytes.

    PubMed

    Savoian, Matthew S

    2015-07-01

    In dividing animal cells, a microtubule (MT)-based bipolar spindle governs chromosome movement. Current models propose that the spindle facilitates and/or generates translocating forces by regionally depolymerizing the kinetochore fibers (k-fibers) that bind each chromosome. It is unclear how conserved these sites and the resultant chromosome-moving mechanisms are between different dividing cell types because of the technical challenges of quantitatively studying MTs in many specimens. In particular, our knowledge of MT kinetics during the sperm-producing male meiotic divisions remains in its infancy. In this study, I use an easy-to-implement photobleaching-based assay for measuring spindle MT dynamics in primary cultures of meiotic spermatocytes isolated from the fruit fly Drosophila melanogaster. By use of standard scanning confocal microscopy features, fiducial marks were photobleached on fluorescent protein (FP)-tagged MTs. These were followed by time-lapse imaging during different division stages, and their displacement rates were calculated using public domain software. I find that k-fibers continually shorten at their poles during metaphase and anaphase A through the process of MT flux. Anaphase chromosome movement is complemented by Pac-Man, the shortening of the k-fiber at its chromosomal interface. Thus, Drosophila spermatocytes share the sites of spindle dynamism and mechanisms of chromosome movement with mitotic cells. The data reveal the applicability of the photobleaching assay for measuring MT dynamics in primary cultures. This approach can be readily applied to other systems.

  18. Five factors can reconstitute all three phases of microtubule polymerization dynamics.

    PubMed

    Moriwaki, Takashi; Goshima, Gohta

    2016-11-07

    Cytoplasmic microtubules (MTs) undergo growth, shrinkage, and pausing. However, how MT polymerization cycles are produced and spatiotemporally regulated at a molecular level is unclear, as the entire cycle has not been recapitulated in vitro with defined components. In this study, we reconstituted dynamic MT plus end behavior involving all three phases by mixing tubulin with five Drosophila melanogaster proteins (EB1, XMAP215(Msps), Sentin, kinesin-13(Klp10A), and CLASP(Mast/Orbit)). When singly mixed with tubulin, CLASP(Mast/Orbit) strongly inhibited MT catastrophe and reduced the growth rate. However, in the presence of the other four factors, CLASP(Mast/Orbit) acted as an inducer of pausing. The mitotic kinase Plk1(Polo) modulated the activity of CLASP(Mast/Orbit) and kinesin-13(Klp10A) and increased the dynamic instability of MTs, reminiscent of mitotic cells. These results suggest that five conserved proteins constitute the core factors for creating dynamic MTs in cells and that Plk1-dependent phosphorylation is a crucial event for switching from the interphase to mitotic mode.

  19. A designed ankyrin repeat protein selected to bind to tubulin caps the microtubule plus end.

    PubMed

    Pecqueur, Ludovic; Duellberg, Christian; Dreier, Birgit; Jiang, Qiyang; Wang, Chunguang; Plückthun, Andreas; Surrey, Thomas; Gigant, Benoît; Knossow, Marcel

    2012-07-24

    Microtubules are cytoskeleton filaments consisting of αβ-tubulin heterodimers. They switch between phases of growth and shrinkage. The underlying mechanism of this property, called dynamic instability, is not fully understood. Here, we identified a designed ankyrin repeat protein (DARPin) that interferes with microtubule assembly in a unique manner. The X-ray structure of its complex with GTP-tubulin shows that it binds to the β-tubulin surface exposed at microtubule (+) ends. The details of the structure provide insight into the role of GTP in microtubule polymerization and the conformational state of tubulin at the very microtubule end. They show in particular that GTP facilitates the tubulin structural switch that accompanies microtubule assembly but does not trigger it in unpolymerized tubulin. Total internal reflection fluorescence microscopy revealed that the DARPin specifically blocks growth at the microtubule (+) end by a selective end-capping mechanism, ultimately favoring microtubule disassembly from that end. DARPins promise to become designable tools for the dissection of microtubule dynamic properties selective for either of their two different ends.

  20. Glutamylation on alpha-tubulin is not essential but affects the assembly and functions of a subset of microtubules in Tetrahymena thermophila.

    PubMed

    Wloga, Dorota; Rogowski, Krzysztof; Sharma, Neeraj; Van Dijk, Juliette; Janke, Carsten; Eddé, Bernard; Bré, Marie-Hélène; Levilliers, Nicolette; Redeker, Virginie; Duan, Jianming; Gorovsky, Martin A; Jerka-Dziadosz, Maria; Gaertig, Jacek

    2008-08-01

    Tubulin undergoes glutamylation, a conserved posttranslational modification of poorly understood function. We show here that in the ciliate Tetrahymena, most of the microtubule arrays contain glutamylated tubulin. However, the length of the polyglutamyl side chain is spatially regulated, with the longest side chains present on ciliary and basal body microtubules. We focused our efforts on the function of glutamylation on the alpha-tubulin subunit. By site-directed mutagenesis, we show that all six glutamates of the C-terminal tail domain of alpha-tubulin that provide potential sites for glutamylation are not essential but are needed for normal rates of cell multiplication and cilium-based functions (phagocytosis and cell motility). By comparative phylogeny and biochemical assays, we identify two conserved tubulin tyrosine ligase (TTL) domain proteins, Ttll1p and Ttll9p, as alpha-tubulin-preferring glutamyl ligase enzymes. In an in vitro microtubule glutamylation assay, Ttll1p showed a chain-initiating activity while Ttll9p had primarily a chain-elongating activity. GFP-Ttll1p localized mainly to basal bodies, while GFP-Ttll9p localized to cilia. Disruption of the TTLL1 and TTLL9 genes decreased the rates of cell multiplication and phagocytosis. Cells lacking both genes had fewer cortical microtubules and showed defects in the maturation of basal bodies. We conclude that glutamylation on alpha-tubulin is not essential but is required for efficiency of assembly and function of a subset of microtubule-based organelles. Furthermore, the spatial restriction of modifying enzymes appears to be a major mechanism that drives differential glutamylation at the subcellular level.

  1. Dynamic regulation of cortical microtubule organization through prefoldin-DELLA interaction.

    PubMed

    Locascio, Antonella; Blázquez, Miguel A; Alabadí, David

    2013-05-06

    Plant morphogenesis relies on specific patterns of cell division and expansion. It is well established that cortical microtubules influence the direction of cell expansion, but less is known about the molecular mechanisms that regulate microtubule arrangement. Here we show that the phytohormones gibberellins (GAs) regulate microtubule orientation through physical interaction between the nuclear-localized DELLA proteins and the prefoldin complex, a cochaperone required for tubulin folding. In the presence of GA, DELLA proteins are degraded, and the prefoldin complex stays in the cytoplasm and is functional. In the absence of GA, the prefoldin complex is localized to the nucleus, which severely compromises α/β-tubulin heterodimer availability, affecting microtubule organization. The physiological relevance of this molecular mechanism was confirmed by the observation that the daily rhythm of plant growth was accompanied by coordinated oscillation of DELLA accumulation, prefoldin subcellular localization, and cortical microtubule reorientation.

  2. Identification of a novel microtubule binding and assembly domain in the developmentally regulated inter-repeat region of tau

    PubMed Central

    1994-01-01

    Tau is a developmentally regulated microtubule-associated protein that influences microtubule behavior by directly associating with tubulin. The carboxyl terminus of tau contains multiple 18-amino acid repeats that bind microtubules and are separated by 13-14-amino acid inter- repeat (IR) regions previously thought to function as "linkers." Here, we have performed a high resolution deletion analysis of tau and identified the IR region located between repeats 1 and 2 (the R1-R2 IR) as a unique microtubule binding site with more than twice the binding affinity of any individual repeat. Truncation analyses and site- directed mutagenesis reveal that the binding activity of this site is derived primarily from lys265 and lys272, with a lesser contribution from lys271. These results predict strong, discrete electrostatic interactions between the R1-R2 IR and tubulin, in contrast to the distributed array of weak interactions thought to underlie the association between 18-amino acid repeats and microtubules (Butner, K. A., and M. W. Kirschner. J. Cell Biol. 115:717-730). Moreover, competition assays suggest that the R1-R2 IR associates with microtubules at tubulin site(s) distinct from those bound by the repeats. Finally, a synthetic peptide corresponding to just 10 amino acids of the R1-R2 IR is sufficient to promote tubulin polymerization in a sequence-dependent manner. Since the R1-R2 IR is specifically expressed in adult tau, its action may underlie some of the developmental transitions observed in neuronal microtubule organization. We suggest that the R1-R2 IR may establish an adult- specific, high affinity anchor that tethers the otherwise mobile tau molecule to the tubulin lattice, thereby increasing microtubule stability. Moreover, the absence of R1-R2 IR expression during early development may allow for the cytoskeletal plasticity required of immature neurons. PMID:8120098

  3. Identification of a novel microtubule binding and assembly domain in the developmentally regulated inter-repeat region of tau.

    PubMed

    Goode, B L; Feinstein, S C

    1994-03-01

    Tau is a developmentally regulated microtubule-associated protein that influences microtubule behavior by directly associating with tubulin. The carboxyl terminus of tau contains multiple 18-amino acid repeats that bind microtubules and are separated by 13-14-amino acid inter-repeat (IR) regions previously thought to function as "linkers." Here, we have performed a high resolution deletion analysis of tau and identified the IR region located between repeats 1 and 2 (the R1-R2 IR) as a unique microtubule binding site with more than twice the binding affinity of any individual repeat. Truncation analyses and site-directed mutagenesis reveal that the binding activity of this site is derived primarily from lys265 and lys272, with a lesser contribution from lys271. These results predict strong, discrete electrostatic interactions between the R1-R2 IR and tubulin, in contrast to the distributed array of weak interactions thought to underlie the association between 18-amino acid repeats and microtubules (Butner, K. A., and M. W. Kirschner. J. Cell Biol. 115:717-730). Moreover, competition assays suggest that the R1-R2 IR associates with microtubules at tubulin site(s) distinct from those bound by the repeats. Finally, a synthetic peptide corresponding to just 10 amino acids of the R1-R2 IR is sufficient to promote tubulin polymerization in a sequence-dependent manner. Since the R1-R2 IR is specifically expressed in adult tau, its action may underlie some of the developmental transitions observed in neuronal microtubule organization. We suggest that the R1-R2 IR may establish an adult-specific, high affinity anchor that tethers the otherwise mobile tau molecule to the tubulin lattice, thereby increasing microtubule stability. Moreover, the absence of R1-R2 IR expression during early development may allow for the cytoskeletal plasticity required of immature neurons.

  4. Dynamics of centrosome translocation and microtubule organization in neocortical neurons during distinct modes of polarization.

    PubMed

    Sakakibara, Akira; Sato, Toshiyuki; Ando, Ryota; Noguchi, Namiko; Masaoka, Makoto; Miyata, Takaki

    2014-05-01

    Neuronal migration and process formation require cytoskeletal organization and remodeling. Recent studies suggest that centrosome translocation is involved in initial axon outgrowth, while the role of centrosomal positioning is not clear. Here, we examine relations between centrosomal positioning, axonogenesis, and microtubule (MT) polarization in multipolar and bipolar neocortical neurons. We monitored dynamic movements of centrosomes and MT plus ends in migratory neurons in embryonic mouse cerebral slices. In locomoting bipolar neurons, the centrosome oriented toward the pia-directed leading process. Bipolar neurons displayed dense MT plus end dynamics in leading processes, while trailing processes showed clear bidirectional MTs. In migrating multipolar neurons, new processes emerged irrespective of centrosome localization, followed by centrosome reorientations toward the dominant process. Anterograde movements of MT plus ends occurred in growing processes and retrograde movements were observed after retraction of the distal tip. In multipolar neurons, axon formed by tangential extension of a dominant process and the centrosome oriented toward the growing axon, while in locomoting neurons, an axon formed opposite to the direction of migration and the centrosome localized to the base of the leading process. Our data suggest that MT organization may alter centrosomal localization and that centrosomal positioning does not necessarily direct process formation.

  5. Microtubule dynamics analysis using kymographs and variable-rate particle filters.

    PubMed

    Smal, Ihor; Grigoriev, Ilya; Akhmanova, Anna; Niessen, Wiro J; Meijering, Erik

    2010-07-01

    Studying intracellular dynamics is of fundamental importance for understanding healthy life at the molecular level and for developing drugs to target disease processes. One of the key technologies to enable this research is the automated tracking and motion analysis of these objects in microscopy image sequences. To make better use of the spatiotemporal information than common frame-by-frame tracking methods, two alternative approaches have recently been proposed, based upon either Bayesian estimation or space-time segmentation. In this paper, we propose to combine the power of both approaches, and develop a new probabilistic method to segment the traces of the moving objects in kymograph representations of the image data. It is based on variable-rate particle filtering and uses multiscale trend analysis of the extracted traces to estimate the relevant kinematic parameters. Experiments on realistic synthetically generated images as well as on real biological image data demonstrate the improved potential of the new method for the analysis of microtubule dynamics in vitro.

  6. CLASP2 interacts with p120-catenin and governs microtubule dynamics at adherens junctions

    PubMed Central

    Shahbazi, Marta N.; Megias, Diego; Epifano, Carolina; Akhmanova, Anna; Gundersen, Gregg G.; Fuchs, Elaine

    2013-01-01

    Classical cadherins and their connections with microtubules (MTs) are emerging as important determinants of cell adhesion. However, the functional relevance of such interactions and the molecular players that contribute to tissue architecture are still emerging. In this paper, we report that the MT plus end–binding protein CLASP2 localizes to adherens junctions (AJs) via direct interaction with p120-catenin (p120) in primary basal mouse keratinocytes. Reductions in the levels of p120 or CLASP2 decreased the localization of the other protein to cell–cell contacts and altered AJ dynamics and stability. These features were accompanied by decreased MT density and altered MT dynamics at intercellular junction sites. Interestingly, CLASP2 was enriched at the cortex of basal progenitor keratinocytes, in close localization to p120. Our findings suggest the existence of a new mechanism of MT targeting to AJs with potential functional implications in the maintenance of proper cell–cell adhesion in epidermal stem cells. PMID:24368809

  7. Identification of MINUS, a small polypeptide that functions as a microtubule nucleation suppressor.

    PubMed

    Fanara, P; Oback, B; Ashman, K; Podtelejnikov, A; Brandt, R

    1999-02-01

    In eukaryotic cells, tubulin polymerization must be regulated precisely during cell division and differentiation. To identify new mechanisms involved in cellular microtubule formation, we isolated an activity that suppresses microtubule nucleation in vitro. The activity was due to a small acidic polypeptide of 4.7 kDa which we named MINUS (microtubule nucleation suppressor). MINUS inhibited tau- and taxol-mediated microtubule assembly in vitro and was inactivated by dephosphorylation. The protein was purified to homogeneity from cultured neural (PC12) cells and bovine brain. Microinjection of MINUS caused a transient loss of dynamic microtubules in Vero cells. The results suggest that MINUS acts with a novel mechanism on tubulin polymerization, thus regulating microtubule formation in living cells.

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

    PubMed Central

    Melkov, Anna; Simchoni, Yasmin; Alcalay, Yehonatan; Abdu, Uri

    2015-01-01

    ABSTRACT The microtubule (MT) plus-end motor kinesin heavy chain (Khc) is well known for its role in long distance cargo transport. Recent evidence showed that Khc is also required for the organization of the cellular MT network by mediating MT sliding. We found that mutations in Khc and the gene of its adaptor protein, kinesin light chain (Klc) resulted in identical bristle morphology defects, with the upper part of the bristle being thinner and flatter than normal and failing to taper towards the bristle tip. We demonstrate that bristle mitochondria transport requires Khc but not Klc as a competing force to dynein heavy chain (Dhc). Surprisingly, we demonstrate for the first time that Dhc is the primary motor for both anterograde and retrograde fast mitochondria transport. We found that the upper part of Khc and Klc mutant bristles lacked stable MTs. When following dynamic MT polymerization via the use of GFP-tagged end-binding protein 1 (EB1), it was noted that at Khc and Klc mutant bristle tips, dynamic MTs significantly deviated from the bristle parallel growth axis, relative to wild-type bristles. We also observed that GFP-EB1 failed to concentrate as a focus at the tip of Khc and Klc mutant bristles. We propose that the failure of bristle tapering is due to defects in directing dynamic MTs at the growing tip. Thus, we reveal a new function for Khc and Klc in directing dynamic MTs during polarized cell growth. Moreover, we also demonstrate a novel mode of coordination in mitochondrial transport between Khc and Dhc. PMID:26581590

  9. Phosphorylation of α-Tubulin by Protein Kinase C Stimulates Microtubule Dynamics in Human Breast Cells

    PubMed Central

    De, Shatarupa; Tsimounis, Areti; Chen, Xiangyu; Rotenberg, Susan A.

    2014-01-01

    Protein kinase C (PKC) engenders motility through phosphorylation of α-tubulin at Ser-165 in non-transformed MCF-10A cells. Live cell imaging explored the impact of PKC-mediated phosphorylation on microtubule (MT) dynamics. MTs fluorescently labeled with GFP-α-tubulin were treated with diacylglycerol (DAG)-lactone (a membrane-permeable PKC activator), or co-transfected with a pseudo-phosphorylated S165D-α6-tubulin mutant. Each condition increased the dynamicity of MTs by stimulating the rate and duration of the growth phase and decreasing the frequency of catastrophe. In MDA-MB-231 metastatic breast cells where the intrinsic PKC activity is high, these MT growth parameters were also high but could be suppressed by expression of phosphorylation-resistant S165N-α6-tubulin or by treatment with a pan-PKC inhibitor (bis-indoleylmaleimide). Sub-cellular fractionation and immunofluorescence of MCF-10A cells showed that phosphorylation (via DAG-lactone) or pseudo-phosphorylation of α6-tubulin increased its partitioning into MTs as compared to controls, and produced longer, more stable MTs. Following expression of the plus-end binding protein GFP-EB1, DAG-lactone accelerated the formation and increased the number of nascent MTs. Expression of S165D-α6-tubulin promoted Rac1 activation and Rac1-dependent cell motility. These findings call attention to PKC-mediated phosphorylation of α-tubulin as a novel mechanism for controlling the dynamics of MTs that result in cell movement. PMID:24574051

  10. Mechanical Effects of Dynamic Binding between Tau Proteins on Microtubules during Axonal Injury

    PubMed Central

    Ahmadzadeh, Hossein; Smith, Douglas H.; Shenoy, Vivek B.

    2015-01-01

    The viscoelastic nature of axons plays a key role in their selective vulnerability to damage in traumatic brain injury (TBI). Experimental studies have shown that although axons can tolerate 100% strain under slow loading rates, even strain as small as 5% can rupture microtubules (MTs) during the fast loading velocities relevant to TBI. Here, we developed a computational model to examine rate-dependent behavior related to dynamic interactions between MTs and the MT-associated protein tau under varying strains and strain rates. In the model, inverted pairs of tau proteins can dynamically cross-link parallel MTs via the respective MT-binding domain of each tau. The model also incorporates realistic thermodynamic breaking and reformation of the bonds between the connected tau proteins as they respond to mechanical stretch. With simulated stretch of the axon, the model shows that despite the highly dynamic nature of binding and unbinding events, under fast loading rates relevant to TBI, large tensile forces can be transmitted to the MTs that can lead to mechanical rupture of the MT cylinder, in agreement with experimental observations and as inferred in human TBI. In contrast, at slow loading rates, the progressive breaking and reformation of the bonds between the tau proteins facilitate the extension of axons up to ∼100% strain without any microstructural damage. The model also predicts that under fast loading rates, individual MTs detach from MT bundles via sequential breaking of the tau-tau bonds. Finally, the model demonstrates that longer MTs are more susceptible to mechanical rupture, whereas short MTs are more prone to detachment from the MT bundle, leading to disintegration of the axonal MT ultrastructure. Notably, the predictions from the model are in excellent agreement with the findings of the recent in vitro mechanical testing of micropatterned neuronal cultures. PMID:26636944

  11. Kinesin-Binding Protein Controls Microtubule Dynamics and Cargo Trafficking by Regulating Kinesin Motor Activity.

    PubMed

    Kevenaar, Josta T; Bianchi, Sarah; van Spronsen, Myrrhe; Olieric, Natacha; Lipka, Joanna; Frias, Cátia P; Mikhaylova, Marina; Harterink, Martin; Keijzer, Nanda; Wulf, Phebe S; Hilbert, Manuel; Kapitein, Lukas C; de Graaff, Esther; Ahkmanova, Anna; Steinmetz, Michel O; Hoogenraad, Casper C

    2016-04-04

    Kinesin motor proteins play a fundamental role for normal neuronal development by controlling intracellular cargo transport and microtubule (MT) cytoskeleton organization. Regulating kinesin activity is important to ensure their proper functioning, and their misregulation often leads to severe human neurological disorders. Homozygous nonsense mutations in kinesin-binding protein (KBP)/KIAA1279 cause the neurological disorder Goldberg-Shprintzen syndrome (GOSHS), which is characterized by intellectual disability, microcephaly, and axonal neuropathy. Here, we show that KBP regulates kinesin activity by interacting with the motor domains of a specific subset of kinesins to prevent their association with the MT cytoskeleton. The KBP-interacting kinesins include cargo-transporting motors such as kinesin-3/KIF1A and MT-depolymerizing motor kinesin-8/KIF18A. We found that KBP blocks KIF1A/UNC-104-mediated synaptic vesicle transport in cultured hippocampal neurons and in C. elegans PVD sensory neurons. In contrast, depletion of KBP results in the accumulation of KIF1A motors and synaptic vesicles in the axonal growth cone. We also show that KBP regulates neuronal MT dynamics by controlling KIF18A activity. Our data suggest that KBP functions as a kinesin inhibitor that modulates MT-based cargo motility and depolymerizing activity of a subset of kinesin motors. We propose that misregulation of KBP-controlled kinesin motors may represent the underlying molecular mechanism that contributes to the neuropathological defects observed in GOSHS patients.

  12. Altered microtubule dynamics and vesicular transport in mouse and human MeCP2-deficient astrocytes

    PubMed Central

    Delépine, Chloé; Meziane, Hamid; Nectoux, Juliette; Opitz, Matthieu; Smith, Amos B.; Ballatore, Carlo; Saillour, Yoann; Bennaceur-Griscelli, Annelise; Chang, Qiang; Williams, Emily Cunningham; Dahan, Maxime; Duboin, Aurélien; Billuart, Pierre; Herault, Yann; Bienvenu, Thierry

    2016-01-01

    Rett syndrome (RTT) is a rare X-linked neurodevelopmental disorder, characterized by normal post-natal development followed by a sudden deceleration in brain growth with progressive loss of acquired motor and language skills, stereotypic hand movements and severe cognitive impairment. Mutations in the methyl-CpG-binding protein 2 (MECP2) cause more than 95% of classic cases. Recently, it has been shown that the loss of Mecp2 from glia negatively influences neurons in a non-cell-autonomous fashion, and that in Mecp2-null mice, re-expression of Mecp2 preferentially in astrocytes significantly improved locomotion and anxiety levels, restored respiratory abnormalities to a normal pattern and greatly prolonged lifespan compared with globally null mice. We now report that microtubule (MT)-dependent vesicle transport is altered in Mecp2-deficient astrocytes from newborn Mecp2-deficient mice compared with control wild-type littermates. Similar observation has been made in human MECP2 p.Arg294* iPSC-derived astrocytes. Importantly, administration of Epothilone D, a brain-penetrant MT-stabilizing natural product, was found to restore MT dynamics in Mecp2-deficient astrocytes and in MECP2 p.Arg294* iPSC-derived astrocytes in vitro. Finally, we report that relatively low weekly doses of Epothilone D also partially reversed the impaired exploratory behavior in Mecp2308/y male mice. These findings represent a first step toward the validation of an innovative treatment for RTT. PMID:26604147

  13. Physical basis of large microtubule aster growth

    PubMed Central

    Ishihara, Keisuke; Korolev, Kirill S; 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. With increasing nucleation rate, the model predicts an explosive transition from stationary to growing asters with a discontinuous jump of the aster velocity to a nonzero value. Experiments in frog egg extract confirm the main theoretical predictions. Our results suggest that asters observed in large fish and amphibian eggs are a meshwork of short, unstable microtubules maintained by autocatalytic nucleation and provide a paradigm for the assembly of robust and evolvable polymer networks. DOI: http://dx.doi.org/10.7554/eLife.19145.001 PMID:27892852

  14. Electric fields generated by synchronized oscillations of microtubules, centrosomes and chromosomes regulate the dynamics of mitosis and meiosis.

    PubMed

    Zhao, Yue; Zhan, Qimin

    2012-07-02

    Super-macromolecular complexes play many important roles in eukaryotic cells. Classical structural biological studies focus on their complicated molecular structures, physical interactions and biochemical modifications. Recent advances concerning intracellular electric fields generated by cell organelles and super-macromolecular complexes shed new light on the mechanisms that govern the dynamics of mitosis and meiosis. In this review we synthesize this knowledge to provide an integrated theoretical model of these cellular events. We suggest that the electric fields generated by synchronized oscillation of microtubules, centrosomes, and chromatin fibers facilitate several events during mitosis and meiosis, including centrosome trafficking, chromosome congression in mitosis and synapsis between homologous chromosomes in meiosis. These intracellular electric fields are generated under energy excitation through the synchronized electric oscillations of the dipolar structures of microtubules, centrosomes and chromosomes, three of the super-macromolecular complexes within an animal cell.

  15. Physical Modeling of Microtubules Network

    NASA Astrophysics Data System (ADS)

    Allain, Pierre; Kervrann, Charles

    2014-10-01

    Microtubules (MT) are highly dynamic tubulin polymers that are involved in many cellular processes such as mitosis, intracellular cell organization and vesicular transport. Nevertheless, the modeling of cytoskeleton and MT dynamics based on physical properties is difficult to achieve. Using the Euler-Bernoulli beam theory, we propose to model the rigidity of microtubules on a physical basis using forces, mass and acceleration. In addition, we link microtubules growth and shrinkage to the presence of molecules (e.g. GTP-tubulin) in the cytosol. The overall model enables linking cytosol to microtubules dynamics in a constant state space thus allowing usage of data assimilation techniques.

  16. The kinesin-14 Klp2 organizes microtubules into parallel bundles by an ATP-dependent sorting mechanism.

    PubMed

    Braun, Marcus; Drummond, Douglas R; Cross, Robert A; McAinsh, Andrew D

    2009-06-01

    The dynamic organization of microtubules into parallel arrays allows interphase cells to set up multi-lane highways for intracellular transport and M-phase cells to build the mitotic and meiotic spindles. Here we show that a minimally reconstituted system composed of Klp2, a kinesin-14 from the fission yeast Schizosaccharomyces pombe, together with microtubules assembled from purified S. pombe tubulin, autonomously assembles bundles of parallel microtubules. Bundles form by an ATP-dependent sorting mechanism that requires the full-length Klp2 motor. By this mechanism, antiparallel-overlapped microtubules slide over one another until they dissociate from the bundles, whereas parallel-overlapped microtubules are selectively trapped by an energy-dissipating force-balance mechanism. Klp2-driven microtubule sorting provides a robust pathway for the organization of microtubules into parallel arrays. In vivo evidence indicates that Klp2 is required for the proper organization of S. pombe interphase microtubules into bipolar arrays of parallel-overlapped microtubules, suggesting that kinesin-14-dependent microtubule sorting may have wide biological importance.

  17. Notch signalling in adult neurons: a potential target for microtubule stabilization.

    PubMed

    Bonini, Sara Anna; Ferrari-Toninelli, Giulia; Montinaro, Mery; Memo, Maurizio

    2013-11-01

    Cytoskeletal dysfunction has been proposed during the last decade as one of the main mechanisms involved in the aetiology of several neurodegenerative diseases. Microtubules are basic elements of the cytoskeleton and the dysregulation of microtubule stability has been demonstrated to be causative for axonal transport impairment, synaptic contact degeneration, impaired neuronal function leading finally to neuronal loss. Several pathways are implicated in the microtubule assembly/disassembly process. Emerging evidence is focusing on Notch as a microtubule dynamics regulator. We demonstrated that activation of Notch signalling results in increased microtubule stability and changes in axonal morphology and branching. By contrast, Notch inhibition leads to an increase in cytoskeleton plasticity with intense neurite remodelling. Until now, several microtubule-binding compounds have been tested and the results have provided proof of concept that microtubule-binding agents or compounds with the ability to stabilize microtubules may have therapeutic potential for the treatment of Alzheimer's disease and other neurodegenerative diseases. In this review, based on its key role in cytoskeletal dynamics modulation, we propose Notch as a new potential target for microtubule stabilization.

  18. Control of myofibroblast differentiation by microtubule dynamics through a regulated localization of mDia2.

    PubMed

    Sandbo, Nathan; Ngam, Caitlyn; Torr, Elizabeth; Kregel, Steve; Kach, Jacob; Dulin, Nickolai

    2013-05-31

    Myofibroblast differentiation plays a critical role in wound healing and in the pathogenesis of fibrosis. We have previously shown that myofibroblast differentiation is mediated by the activity of serum response factor (SRF), which is tightly controlled by the actin polymerization state. In this study, we investigated the role of the microtubule cytoskeleton in modulating myofibroblast phenotype. Treatment of human lung fibroblasts with the microtubule-destabilizing agent, colchicine, resulted in a formation of numerous stress fibers and expression of myofibroblast differentiation marker proteins. These effects of colchicine were independent of Smad signaling but were mediated by Rho signaling and SRF, as they were attenuated by the Rho kinase inhibitor, Y27632, or by the SRF inhibitor, CCG-1423. TGF-β-induced myofibroblast differentiation was not accompanied by gross changes in the microtubule polymerization state. However, microtubule stabilization by paclitaxel attenuated TGF-β-induced myofibroblast differentiation. Paclitaxel had no effect on TGF-β-induced Smad activation and Smad-dependent gene transcription but inhibited actin polymerization, nuclear accumulation of megakaryoblastic leukemia-1 protein, and SRF activation. The microtubule-associated formin, mDIA2, localized to actin stress fibers upon treatment with TGF-β, and paclitaxel prevented this localization. Treatment with the formin inhibitor, SMI formin homology 2 domain, inhibited stress fiber formation and myofibroblast differentiation induced by TGF-β, without affecting Smad-phosphorylation or microtubule polymerization. Together, these data suggest that (a) TGF-β promotes association of mDia2 with actin stress fibers, which further drives stress fiber formation and myofibroblast differentiation, and (b) microtubule polymerization state controls myofibroblast differentiation through the regulation of mDia2 localization.

  19. Modulation of Microtubule Interprotofilament Interactions by Modified Taxanes

    PubMed Central

    Matesanz, Ruth; Rodríguez-Salarichs, Javier; Pera, Benet; Canales, Ángeles; Andreu, José Manuel; Jiménez-Barbero, Jesús; Bras, Wim; Nogales, Aurora; Fang, Wei-Shuo; Díaz, José Fernando

    2011-01-01

    Microtubules assembled with paclitaxel and docetaxel differ in their numbers of protofilaments, reflecting modification of the lateral association between αβ-tubulin molecules in the microtubule wall. These modifications of microtubule structure, through a not-yet-characterized mechanism, are most likely related to the changes in tubulin-tubulin interactions responsible for microtubule stabilization by these antitumor compounds. We have used a set of modified taxanes to study the structural mechanism of microtubule stabilization by these ligands. Using small-angle x-ray scattering, we have determined how modifications in the shape and size of the taxane substituents result in changes in the interprotofilament angles and in their number. The observed effects have been explained using NMR-aided docking and molecular dynamic simulations of taxane binding at the microtubule pore and luminal sites. Modeling results indicate that modification of the size of substituents at positions C7 and C10 of the taxane core influence the conformation of three key elements in microtubule lateral interactions (the M-loop, the S3 β-strand, and the H3 helix) that modulate the contacts between adjacent protofilaments. In addition, modifications of the substituents at position C2 slightly rearrange the ligand in the binding site, modifying the interaction of the C7 substituent with the M-loop. PMID:22208196

  20. Dynamic Assembly of Magnetic Colloidal Vortices

    SciTech Connect

    Mohorič, Tomaž; Kokot, Gašper; Osterman, Natan; Snezhko, Alexey; Vilfan, Andrej; Babič, Dušan; Dobnikar, Jure

    2016-04-29

    Magnetic colloids in external time-dependent fields are subject to complex induced many-body interactions governing their self-assembly into a variety of equilibrium and out-of-equilibrium structures such as chains, networks, suspended membranes, and colloidal foams. Here, we report experiments, simulations, and theory probing the dynamic assembly of superparamagnetic colloids in precessing external magnetic fields. Within a range of field frequencies, we observe dynamic large-scale structures such as ordered phases composed of precessing chains, ribbons, and rotating fluidic vortices. We show that the structure formation is inherently coupled to the buildup of torque, which originates from internal relaxation of induced dipoles and from transient correlations among the particles as a result of short-lived chain formation. We discuss in detail the physical properties of the vortex phase and demonstrate its potential in particle-coating applications.

  1. The dynamics of nacre self-assembly

    PubMed Central

    Cartwright, Julyan H.E; Checa, Antonio G

    2006-01-01

    We show how nacre and pearl construction in bivalve and gastropod molluscs can be understood in terms of successive processes of controlled self-assembly from the molecular- to the macro-scale. This dynamics involves the physics of the formation of both solid and liquid crystals and of membranes and fluids to produce a nanostructured hierarchically constructed biological composite of polysaccharides, proteins and mineral, whose mechanical properties far surpass those of its component parts. PMID:17251136

  2. Rho GTPases at the crossroad of signaling networks in mammals: impact of Rho-GTPases on microtubule organization and dynamics.

    PubMed

    Wojnacki, José; Quassollo, Gonzalo; Marzolo, María-Paz; Cáceres, Alfredo

    2014-01-01

    Microtubule (MT) organization and dynamics downstream of external cues is crucial for maintaining cellular architecture and the generation of cell asymmetries. In interphase cells RhoA, Rac, and Cdc42, conspicuous members of the family of small Rho GTPases, have major roles in modulating MT stability, and hence polarized cell behaviors. However, MTs are not mere targets of Rho GTPases, but also serve as signaling platforms coupling MT dynamics to Rho GTPase activation in a variety of cellular conditions. In this article, we review some of the key studies describing the reciprocal relationship between small Rho-GTPases and MTs during migration and polarization.

  3. Fission yeast mitochondria are distributed by dynamic microtubules in a motor-independent manner.

    PubMed

    Li, Tianpeng; Zheng, Fan; Cheung, Martin; Wang, Fengsong; Fu, Chuanhai

    2015-06-05

    The cytoskeleton plays a critical role in regulating mitochondria distribution. Similar to axonal mitochondria, the fission yeast mitochondria are distributed by the microtubule cytoskeleton, but this is regulated by a motor-independent mechanism depending on the microtubule associated protein mmb1p as the absence of mmb1p causes mitochondria aggregation. In this study, using a series of chimeric proteins to control the subcellular localization and motility of mitochondria, we show that a chimeric molecule containing a microtubule binding domain and the mitochondria outer membrane protein tom22p can restore the normal interconnected mitochondria network in mmb1-deletion (mmb1∆) cells. In contrast, increasing the motility of mitochondria by using a chimeric molecule containing a kinesin motor domain and tom22p cannot rescue mitochondria aggregation defects in mmb1∆ cells. Intriguingly a chimeric molecule carrying an actin binding domain and tom22p results in mitochondria associated with actin filaments at the actomyosin ring during mitosis, leading to cytokinesis defects. These findings suggest that the passive motor-independent microtubule-based mechanism is the major contributor to mitochondria distribution in wild type fission yeast cells. Hence, we establish that attachment to microtubules, but not kinesin-dependent movement and the actin cytoskeleton, is required and crucial for proper mitochondria distribution in fission yeast.

  4. Asymmetric behavior of severed microtubule ends after ultraviolet-microbeam irradiation of individual microtubules in vitro

    SciTech Connect

    Walker, R.A.; Inoue, S.; Salmon, E.D.

    1989-03-01

    The molecular basis of microtubule dynamic instability is controversial, but is thought to be related to a GTP cap. A key prediction of the GTP cap model is that the proposed labile GDP-tubulin core will rapidly dissociate if the GTP-tubulin cap is lost. We have tested this prediction by using a UV microbeam to cut the ends from elongating microtubules. Phosphocellulose-purified tubulin was assembled onto the plus and minus ends of sea urchin flagellar axoneme fragments at 21-22 degrees C. The assembly dynamics of individual microtubules were recorded in real time using video microscopy. When the tip of an elongating plus end microtubule was cut off, the severed plus end microtubule always rapidly shortened back to the axoneme at the normal plus end rate. However, when the distal tip of an elongating minus end microtubule was cut off, no rapid shortening occurred. Instead, the severed minus end resumed elongation at the normal minus end rate. Our results show that some form of stabilizing cap, possibly a GTP cap, governs the transition (catastrophe) from elongation to rapid shortening at the plus end. At the minus end, a simple GTP cap is not sufficient to explain the observed behavior unless UV induces immediate recapping of minus, but not plus, ends. Another possibility is that a second step, perhaps a structural transformation, is required in addition to GTP cap loss for rapid shortening to occur. This transformation would be favored at plus, but not minus ends, to account for the asymmetric behavior of the ends.

  5. Fission yeast kinesin-8 Klp5 and Klp6 are interdependent for mitotic nuclear retention and required for proper microtubule dynamics.

    PubMed

    Unsworth, Amy; Masuda, Hirohisa; Dhut, Susheela; Toda, Takashi

    2008-12-01

    Fission yeast has two kinesin-8s, Klp5 and Klp6, which associate to form a heterocomplex. Here, we show that Klp5 and Klp6 are mutually dependent on each other for nuclear mitotic localization. During interphase, they are exported to the cytoplasm. In sharp contrast, during mitosis, Klp5 and Klp6 remain in the nucleus, which requires the existence of each counterpart. Canonical nuclear localization signal (NLS) is identified in the nonkinesin C-terminal regions. Intriguingly individual NLS mutants (NLSmut) exhibit loss-of-function phenotypes, suggesting that Klp5 and Klp6 enter the nucleus separately. Indeed, although neither Klp5-NLSmut nor Klp6-NLSmut enters the nucleus, wild-type Klp6 or Klp5, respectively, does so with different kinetics. In the absence of Klp5/6, microtubule catastrophe/rescue frequency and dynamicity are suppressed, whereas growth and shrinkage rates are least affected. Remarkably, chimera strains containing only the N-terminal Klp5 kinesin domains cannot disassemble interphase microtubules during mitosis, leading to the coexistence of cytoplasmic microtubules and nuclear spindles with massive chromosome missegregation. In this strain, a marked reduction of microtubule dynamism, even higher than in klp5/6 deletions, is evident. We propose that Klp5 and Klp6 play a vital role in promoting microtubule dynamics, which is essential for the spatiotemporal control of microtubule morphogenesis.

  6. Silencing of tubulin binding cofactor C modifies microtubule dynamics and cell cycle distribution and enhances sensitivity to gemcitabine in breast cancer cells.

    PubMed

    Hage-Sleiman, Rouba; Herveau, Stéphanie; Matera, Eva-Laure; Laurier, Jean-Fabien; Dumontet, Charles

    2011-02-01

    Tubulin binding cofactor C (TBCC) is essential for the proper folding of α- and β-tubulins into microtubule polymerizable heterodimers. Because microtubules are considered major targets in the treatment of breast cancer, we investigated the influence of TBCC silencing on tubulin pools, microtubule dynamics, and cell cycle distribution of breast cancer cells by developing a variant MCF7 cells with reduced content of TBCC (MC-). MC- cells displayed decreased content in nonpolymerizable tubulins and increased content of polymerizable/microtubule tubulins when compared with control MP6 cells. Microtubules in MC- cells showed stronger dynamics than those of MP6 cells. MC- cells proliferated faster than MP6 cells and showed an altered cell cycle distribution, with a higher percentage in S-phase of the cell cycle. Consequently, MC- cells presented higher sensitivity to the S-phase-targeting agent gemcitabine than MP6 cells in vitro. Although the complete duration of mitosis was shorter in MC- cells and their microtubule dynamics was enhanced, the percentage of cells in G(2)-M phase was not altered nor was there any difference in sensitivity to antimicrotubule-targeting agents when compared with MP6 cells. Xenografts derived from TBCC variants displayed significantly enhanced tumor growth in vivo and increased sensitivity to gemcitabine in comparison to controls. These results are the first to suggest that proteins involved in the proper folding of cytoskeletal components may have an important influence on the cell cycle distribution, proliferation, and chemosensitivity of tumor cells.

  7. Formation of orthopoxvirus cytoplasmic A-type inclusion bodies and embedding of virions are dynamic processes requiring microtubules.

    PubMed

    Howard, Amanda R; Moss, Bernard

    2012-05-01

    In cells infected with some orthopoxviruses, numerous mature virions (MVs) become embedded within large, cytoplasmic A-type inclusions (ATIs) that can protect infectivity after cell lysis. ATIs are composed of an abundant viral protein called ATIp, which is truncated in orthopoxviruses such as vaccinia virus (VACV) that do not form ATIs. To study ATI formation and occlusion of MVs within ATIs, we used recombinant VACVs that express the cowpox full-length ATIp or we transfected plasmids encoding ATIp into cells infected with VACV, enabling ATI formation. ATI enlargement and MV embedment required continued protein synthesis and an intact microtubular network. For live imaging of ATIs and MVs, plasmids expressing mCherry fluorescent protein fused to ATIp were transfected into cells infected with VACV expressing the viral core protein A4 fused to yellow fluorescent protein. ATIs appeared as dynamic, mobile bodies that enlarged by multiple coalescence events, which could be prevented by disrupting microtubules. Coalescence of ATIs was confirmed in cells infected with cowpox virus. MVs were predominantly at the periphery of ATIs early in infection. We determined that coalescence contributed to the distribution of MVs within ATIs and that microtubule-disrupting drugs abrogated coalescence-mediated MV embedment. In addition, MVs were shown to move from viral factories at speeds consistent with microtubular transport to the peripheries of ATIs, whereas disruption of microtubules prevented such trafficking. The data indicate an important role for microtubules in the coalescence of ATIs into larger structures, transport of MVs to ATIs, and embedment of MVs within the ATI matrix.

  8. Self-organization of microtubules and motors

    NASA Astrophysics Data System (ADS)

    Ndlec, F. J.; Surrey, T.; Maggs, A. C.; Leibler, S.

    1997-09-01

    Cellular structures are established and maintained through a dynamic interplay between assembly and regulatory processes. Self-organization of molecular components provides a variety of possible spatial structures: the regulatory machinery chooses the most appropriate to express a given cellular function. Here we study the extent and the characteristics of self-organization using microtubules and molecular motors as a model system. These components are known to participate in the formation of many cellular structures, such as the dynamic asters found in mitotic and meiotic spindles. Purified motors and microtubules have previously been observed to form asters in vitro. We have reproduced this result with a simple system consisting solely of multi-headed constructs of the motor protein kinesin and stabilized microtubules. We show that dynamic asters can also be obtained from a homogeneous solution of tubulin and motors. By varying the relative concentrations of the components, we obtain a variety of self-organized structures. Further, by studying this process in a constrained geometry of micro-fabricated glass chambers, we demonstrate that the same final structure can be reached through different assembly `pathways'.

  9. Interaction of microtubules with the actin cytoskeleton via cross-talk of EB1-containing +TIPs and γ-actin in epithelial cells

    PubMed Central

    Dugina, Vera; Alieva, Irina; Khromova, Natalya; Kireev, Igor; Gunning, Peter W.; Kopnin, Pavel

    2016-01-01

    Actin microfilaments and microtubules are both highly dynamic cytoskeleton components implicated in a wide range of intracellular processes as well as cell-cell and cell-substrate interactions. The interactions of actin filaments with the microtubule system play an important role in the assembly and maintenance of 3D cell structure. Here we demonstrate that cytoplasmic actins are differentially distributed in relation to the microtubule system. LSM, 3D-SIM, proximity ligation assay (PLA) and co-immunoprecipitation methods applied in combination with selective depletion of β- or γ-cytoplasmic actins revealed a selective interaction between microtubules and γ-, but not β-cytoplasmic actin via the microtubule +TIPs protein EB1. EB1-positive comet distribution analysis and quantification have shown more effective microtubule growth in the absence of β-actin. Our data represent the first demonstration that microtubule +TIPs protein EB1 interacts mainly with γ-cytoplasmic actin in epithelial cells. PMID:27683037

  10. Interaction of microtubules with the actin cytoskeleton via cross-talk of EB1-containing +TIPs and γ-actin in epithelial cells.

    PubMed

    Dugina, Vera; Alieva, Irina; Khromova, Natalya; Kireev, Igor; Gunning, Peter W; Kopnin, Pavel

    2016-11-08

    Actin microfilaments and microtubules are both highly dynamic cytoskeleton components implicated in a wide range of intracellular processes as well as cell-cell and cell-substrate interactions. The interactions of actin filaments with the microtubule system play an important role in the assembly and maintenance of 3D cell structure. Here we demonstrate that cytoplasmic actins are differentially distributed in relation to the microtubule system. LSM, 3D-SIM, proximity ligation assay (PLA) and co-immunoprecipitation methods applied in combination with selective depletion of β- or γ-cytoplasmic actins revealed a selective interaction between microtubules and γ-, but not β-cytoplasmic actin via the microtubule +TIPs protein EB1. EB1-positive comet distribution analysis and quantification have shown more effective microtubule growth in the absence of β-actin. Our data represent the first demonstration that microtubule +TIPs protein EB1 interacts mainly with γ-cytoplasmic actin in epithelial cells.

  11. Dynamic changes in microtubule configuration correlate with nuclear migration in the preblastoderm Drosophila embryo

    PubMed Central

    1993-01-01

    Drosophila embryogenesis is initiated by a series of syncytial mitotic divisions. The first nine of these divisions are internal, and are accompanied by two temporally distinct nuclear movements that lead to the formation of a syncytial blastoderm with a uniform monolayer of cortical nuclei. The first of these movements, which we term axial expansion, occurs during division cycles 4-6 and distributes nuclei in a hollow ellipsoid underlying the cortex. This is followed by cortical migration, during cycles 7-10, which places the nuclei in a uniform monolayer at the cortex. Here we report that these two movements differ in their geometry, velocity, cell-cycle dependence, and protein synthesis requirement. We therefore conclude that axial expansion and cortical migration are mechanistically distinct, amplifying a similar conclusion based on pharmacological data (Zalokar and Erk, 1976). We have examined microtubule organization during cortical migration and find that a network of interdigitating microtubules connects the migrating nuclei. These anti-parallel microtubule arrays are observed between migrating nuclei and yolk nuclei located deeper in the embryo. These arrays are present during nuclear movement but break down when the nuclei are not moving. We propose that cortical migration is driven by microtubule-dependent forces that repel adjacent nuclei, leading to an expansion of the nuclear ellipsoid established by axial expansion. PMID:8314839

  12. The control of microtubule stability in vitro and in transfected cells by MAP1B and SCG10.

    PubMed

    Bondallaz, Percy; Barbier, Anne; Soehrman, Sophia; Grenningloh, Gabriele; Riederer, Beat M

    2006-11-01

    In neurons, the regulation of microtubules plays an important role for neurite outgrowth, axonal elongation, and growth cone steering. SCG10 family proteins are the only known neuronal proteins that have a strong destabilizing effect, are highly enriched in growth cones and are thought to play an important role during axonal elongation. MAP1B, a microtubule-stabilizing protein, is found in growth cones as well, therefore it was important to test their effect on microtubules in the presence of both proteins. We used recombinant proteins in microtubule assembly assays and in transfected COS-7 cells to analyze their combined effects in vitro and in living cells, respectively. Individually, both proteins showed their expected activities in microtubule stabilization and destruction respectively. In MAP1B/SCG10 double-transfected cells, MAP1B could not protect microtubules from SCG10-induced disassembly in most cells, in particular not in cells that contained high levels of SCG10. This suggests that SCG10 is more potent to destabilize microtubules than MAP1B to rescue them. In microtubule assembly assays, MAP1B promoted microtubule formation at a ratio of 1 MAP1B per 70 tubulin dimers while a ratio of 1 SCG10 per two tubulin dimers was needed to destroy microtubules. In addition to its known binding to tubulin dimers, SCG10 binds also to purified microtubules in growth cones of dorsal root ganglion neurons in culture. In conclusion, neuronal microtubules are regulated by antagonistic effects of MAP1B and SCG10 and a fine tuning of the balance of these proteins may be critical for the regulation of microtubule dynamics in growth cones.

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

    PubMed

    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.

  14. Stochastic dynamics of macromolecular-assembly networks.

    NASA Astrophysics Data System (ADS)

    Saiz, Leonor; Vilar, Jose

    2006-03-01

    The formation and regulation of macromolecular complexes provides the backbone of most cellular processes, including gene regulation and signal transduction. The inherent complexity of assembling macromolecular structures makes current computational methods strongly limited for understanding how the physical interactions between cellular components give rise to systemic properties of cells. Here we present a stochastic approach to study the dynamics of networks formed by macromolecular complexes in terms of the molecular interactions of their components [1]. Exploiting key thermodynamic concepts, this approach makes it possible to both estimate reaction rates and incorporate the resulting assembly dynamics into the stochastic kinetics of cellular networks. As prototype systems, we consider the lac operon and phage λ induction switches, which rely on the formation of DNA loops by proteins [2] and on the integration of these protein-DNA complexes into intracellular networks. This cross-scale approach offers an effective starting point to move forward from network diagrams, such as those of protein-protein and DNA-protein interaction networks, to the actual dynamics of cellular processes. [1] L. Saiz and J.M.G. Vilar, submitted (2005). [2] J.M.G. Vilar and L. Saiz, Current Opinion in Genetics & Development, 15, 136-144 (2005).

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

    PubMed

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

    2014-12-03

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

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

    NASA Astrophysics Data System (ADS)

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

    2014-12-01

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

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

    PubMed Central

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

    2014-01-01

    As we bring tubulin protein molecules one by one into the vicinity, they self-assemble and entire event we capture live via quantum tunneling. We observe how these molecules form a linear chain and then chains self-assemble into 2D sheet, an essential for microtubule, —fundamental nano-tube in a cellular life form. Even without using GTP, or any chemical reaction, but applying particular ac signal using specially designed antenna around atomic sharp tip we could carry out the self-assembly, however, if there is no electromagnetic pumping, no self-assembly is observed. In order to verify this atomic scale observation, we have built an artificial cell-like environment with nano-scale engineering and repeated spontaneous growth of tubulin protein to its complex with and without electromagnetic signal. We used 64 combinations of plant, animal and fungi tubulins and several doping molecules used as drug, and repeatedly observed that the long reported common frequency region where protein folds mechanically and its structures vibrate electromagnetically. Under pumping, the growth process exhibits a unique organized behavior unprecedented otherwise. Thus, “common frequency point” is proposed as a tool to regulate protein complex related diseases in the future. PMID:25466883

  18. Structural Mutants of the Spindle Pole Body Cause Distinct Alteration of Cytoplasmic Microtubules and Nuclear Dynamics in Multinucleated Hyphae

    PubMed Central

    Lang, Claudia; Grava, Sandrine; Finlayson, Mark; Trimble, Rhonda; Philippsen, Peter

    2010-01-01

    In the multinucleate fungus Ashbya gossypii, cytoplasmic microtubules (cMTs) emerge from the spindle pole body outer plaque (OP) in perpendicular and tangential directions. To elucidate the role of cMTs in forward/backward movements (oscillations) and bypassing of nuclei, we constructed mutants potentially affecting cMT nucleation or stability. Hyphae lacking the OP components AgSpc72, AgNud1, AgCnm67, or the microtubule-stabilizing factor AgStu2 grew like wild- type but showed substantial alterations in the number, length, and/or nucleation sites of cMTs. These mutants differently influenced nuclear oscillation and bypassing. In Agspc72Δ, only long cMTs were observed, which emanate tangentially from reduced OPs; nuclei mainly moved with the cytoplasmic stream but some performed rapid bypassing. Agnud1Δ and Agcnm67Δ lack OPs; short and long cMTs emerged from the spindle pole body bridge/half-bridge structures, explaining nuclear oscillation and bypassing in these mutants. In Agstu2Δ only very short cMTs emanated from structurally intact OPs; all nuclei moved with the cytoplasmic stream. Therefore, long tangential cMTs promote nuclear bypassing and short cMTs are important for nuclear oscillation. Our electron microscopy ultrastructural analysis also indicated that assembly of the OP occurs in a stepwise manner, starting with AgCnm67, followed by AgNud1 and lastly AgSpc72. PMID:20053682

  19. Atomic-resolution structure of the CAP-Gly domain of dynactin on polymeric microtubules determined by magic angle spinning NMR spectroscopy.

    PubMed

    Yan, Si; Guo, Changmiao; Hou, Guangjin; Zhang, Huilan; Lu, Xingyu; Williams, John Charles; Polenova, Tatyana

    2015-11-24

    Microtubules and their associated proteins perform a broad array of essential physiological functions, including mitosis, polarization and differentiation, cell migration, and vesicle and organelle transport. As such, they have been extensively studied at multiple levels of resolution (e.g., from structural biology to cell biology). Despite these efforts, there remain significant gaps in our knowledge concerning how microtubule-binding proteins bind to microtubules, how dynamics connect different conformational states, and how these interactions and dynamics affect cellular processes. Structures of microtubule-associated proteins assembled on polymeric microtubules are not known at atomic resolution. Here, we report a structure of the cytoskeleton-associated protein glycine-rich (CAP-Gly) domain of dynactin motor on polymeric microtubules, solved by magic angle spinning NMR spectroscopy. We present the intermolecular interface of CAP-Gly with microtubules, derived by recording direct dipolar contacts between CAP-Gly and tubulin using double rotational echo double resonance (dREDOR)-filtered experiments. Our results indicate that the structure adopted by CAP-Gly varies, particularly around its loop regions, permitting its interaction with multiple binding partners and with the microtubules. To our knowledge, this study reports the first atomic-resolution structure of a microtubule-associated protein on polymeric microtubules. Our approach lays the foundation for atomic-resolution structural analysis of other microtubule-associated motors.

  20. Tumor suppressor protein DAB2IP participates in chromosomal stability maintenance through activating spindle assembly checkpoint and stabilizing kinetochore-microtubule attachments

    PubMed Central

    Yu, Lan; Shang, Zeng-Fu; Abdisalaam, Salim; Lee, Kyung-Jong; Gupta, Arun; Hsieh, Jer-Tsong; Asaithamby, Aroumougame; Chen, Benjamin P.C.; Saha, Debabrata

    2016-01-01

    Defects in kinetochore-microtubule (KT-MT) attachment and the spindle assembly checkpoint (SAC) during cell division are strongly associated with chromosomal instability (CIN). CIN has been linked to carcinogenesis, metastasis, poor prognosis and resistance to cancer therapy. We previously reported that the DAB2IP is a tumor suppressor, and that loss of DAB2IP is often detected in advanced prostate cancer (PCa) and is indicative of poor prognosis. Here, we report that the loss of DAB2IP results in impaired KT-MT attachment, compromised SAC and aberrant chromosomal segregation. We discovered that DAB2IP directly interacts with Plk1 and its loss inhibits Plk1 kinase activity, thereby impairing Plk1-mediated BubR1 phosphorylation. Loss of DAB2IP decreases the localization of BubR1 at the kinetochore during mitosis progression. In addition, the reconstitution of DAB2IP enhances the sensitivity of PCa cells to microtubule stabilizing drugs (paclitaxel, docetaxel) and Plk1 inhibitor (BI2536). Our findings demonstrate a novel function of DAB2IP in the maintenance of KT-MT structure and SAC regulation during mitosis which is essential for chromosomal stability. PMID:27568005

  1. Cortical microtubule rearrangements and cell wall patterning

    PubMed Central

    Oda, Yoshihisa

    2015-01-01

    Plant cortical microtubules, which form a highly ordered array beneath the plasma membrane, play essential roles in determining cell shape and function by directing the arrangement of cellulosic and non-cellulosic compounds on the cell surface. Interphase transverse arrays of cortical microtubules self-organize through their dynamic instability and inter-microtubule interactions, and by branch-form microtubule nucleation and severing. Recent studies revealed that distinct spatial signals including ROP GTPase, cellular geometry, and mechanical stress regulate the behavior of cortical microtubules at the subcellular and supercellular levels, giving rise to dramatic rearrangements in the cortical microtubule array in response to internal and external cues. Increasing evidence indicates that negative regulators of microtubules also contribute to the rearrangement of the cortical microtubule array. In this review, I summarize recent insights into how the rearrangement of the cortical microtubule array leads to proper, flexible cell wall patterning. PMID:25904930

  2. [Dynamics of cytoskeleton microtubules in higher plant meiosis. II. Perinuclear band formation].

    PubMed

    Shamina, N V; Dorogova, N V; Seriukova, E G

    2003-01-01

    Analyses of correspondent meiotic abnormalities is a good tool for studying cytoskeletal rearrangements during plant cell division. The paper reports on the wheat x wheatgrass F1 hybrids, showing various abnormalities during organization of the prophase perinuclear band of microtubules (PNB) in male meiosis. Based on these data, it may be concluded that the perinuclear system of microtubules (MT) in higher plant meiosis is formed from fibrils of the radial system as a result of their translocation in the cell cytoplasm space. According to our data, at this stage the radial MT arrays pass through the following consequence of events: separating from the nuclear envelope, 2) approaching, 3) tangential orientation to the nuclear surface, 4) bending, 5) co-orientation, lateral interaction. As a result, a flat ring of well organized concentric bent MT bundles encircling the nucleus meridionally is organized.

  3. Toward Discovery of Novel Microtubule Targeting Agents: A SNAP-tag-Based High-Content Screening Assay for the Analysis of Microtubule Dynamics and Cell Cycle Progression.

    PubMed

    Berges, Nina; Arens, Katharina; Kreusch, Verena; Fischer, Rainer; Di Fiore, Stefano

    2017-04-01

    Microtubule targeting agents (MTAs) are used for the treatment of cancer. Novel MTAs could provide additional and beneficial therapeutic options. To improve the sensitivity and throughput of standard immunofluorescence assays for the characterization of MTAs, we used SNAP-tag technology to produce recombinant tubulin monomers. To visualize microtubule filaments, A549 cells transfected with SNAP-tubulin were stained with a membrane-permeable, SNAP-reactive dye. The treatment of SNAP-tubulin cells with stabilizing MTAs such as paclitaxel resulted in the formation of coarsely structured microtubule filaments, whereas depolymerizing MTAs such as nocodazole resulted in diffuse staining patterns in which the tubulin filaments were no longer distinguishable. By combining these components with automated microscopy and image analysis algorithms, we established a robust high-content screening assay for MTAs with a Z' factor of 0.7. Proof of principle was achieved by testing a panel of 10 substances, allowing us to identify MTAs and to distinguish between stabilizing and destabilizing modes of action. By extending the treatment of the cells from 2 to 20 h, our assay also detected abnormalities in cell cycle progression and in the formation of microtubule spindles, providing additional readouts for the discovery of new MTAs and facilitating their early identification during drug-screening campaigns.

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

    PubMed

    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.

  5. Accelerated actin filament polymerization from microtubule plus-ends

    PubMed Central

    Henty-Ridilla, Jessica L.; Rankova, Aneliya; Eskin, Julian A.; Kenny, Katelyn; Goode, Bruce L.

    2016-01-01

    Microtubules govern actin network remodeling in a wide range of biological processes, yet the mechanisms underlying this cytoskeletal crosstalk have remained obscure. Here we used single-molecule fluorescence microscopy to show that the microtubule plus-end associated protein CLIP-170 binds tightly to formins to accelerate actin filament elongation. Furthermore, we observed mDia1 dimers and CLIP-170 dimers co-tracking growing filament ends for minutes. CLIP-170-mDia1 complexes promoted actin polymerization approximately 18 times faster than free barbed end growth, while simultaneously enhancing protection from capping protein. We used a microtubule-actin dynamics co-reconstitution system to observe CLIP-170-mDia1 complexes being recruited to growing microtubule ends by EB1. The complexes triggered rapid growth of actin filaments that remained attached to the microtubule surface. These activities of CLIP-170 were required in primary neurons for normal dendritic morphology. Thus, our results reveal a cellular mechanism whereby growing microtubule plus-ends direct rapid actin assembly. PMID:27199431

  6. Influence of M-phase chromatin on the anisotropy of microtubule asters

    PubMed Central

    1996-01-01

    In many eukaryotic cells going through M-phase, a bipolar spindle is formed by microtubules nucleated from centrosomes. These microtubules, in addition to being "captured" by kinetochores, may be stabilized by chromatin in two different ways: short-range stabilization effects may affect microtubules in close contact with the chromatin, while long- range stabilization effects may "guide" microtubule growth towards the chromatin (e.g., by introducing a diffusive gradient of an enzymatic activity that affects microtubule assembly). Here, we use both meiotic and mitotic extracts from Xenopus laevis eggs to study microtubule aster formation and microtubule dynamics in the presence of chromatin. In "low-speed" meiotic extracts, in the presence of salmon sperm chromatin, we find that short-range stabilization effects lead to a strong anisotropy of the microtubule asters. Analysis of the dynamic parameters of microtubule growth show that this anisotropy arises from a decrease in the catastrophe frequency, an increase in the rescue frequency and a decrease in the growth velocity. In this system we also find evidence for long-range "guidance" effects, which lead to a weak anisotropy of the asters. Statistically relevant results on these long- range effects are obtained in "high-speed" mitotic extracts in the presence of artificially constructed chromatin stripes. We find that aster anisotropy is biased in the direction of the chromatin and that the catastrophe frequency is reduced in its vicinity. In this system we also find a surprising dependence of the catastrophe and the rescue frequencies on the length of microtubules nucleated from centrosomes: the catastrophe frequency increase and the rescue frequency decreases with microtubule length. PMID:8601601

  7. NO serves as a signaling intermediate downstream of H₂O₂ to modulate dynamic microtubule cytoskeleton during responses to VD-toxins in Arabidopsis.

    PubMed

    Yao, Lin-Lin; Pei, Bao-Lei; Zhou, Qun; Li, Ying-Zhang

    2012-02-01

    Although hydrogen peroxide (H₂O₂) and nitric oxide (NO) can act as an upstream signaling molecule to modulate the dynamic microtubule cytoskeleton during the defense responses to Verticillium dahliae (VD) toxins in Arabidopsis, it is not known the relationship between these two signaling molecules. Here, we show that VD-toxin-induced NO accumulation was dependent on prior H₂O₂ production, NO is downstream of H₂O₂ in the signaling process, and that H₂O₂ acted synergistically with NO to modulate the dynamic microtubule cytoskeleton responses to VD-toxins in Arabidopsis.

  8. Revisiting the tubulin cofactors and Arl2 in the regulation of soluble αβ-tubulin pools and their effect on microtubule dynamics.

    PubMed

    Al-Bassam, Jawdat

    2017-02-01

    Soluble αβ-tubulin heterodimers are maintained at high concentration inside eukaryotic cells, forming pools that fundamentally drive microtubule dynamics. Five conserved tubulin cofactors and ADP ribosylation factor-like 2 regulate the biogenesis and degradation of αβ-tubulins to maintain concentrated soluble pools. Here I describe a revised model for the function of three tubulin cofactors and Arl2 as a multisubunit GTP-hydrolyzing catalytic chaperone that cycles to promote αβ-tubulin biogenesis and degradation. This model helps explain old and new data indicating these activities enhance microtubule dynamics in vivo via repair or removal of αβ-tubulins from the soluble pools.

  9. Dynamic self-assembly of 'living' polymeric chains

    NASA Astrophysics Data System (ADS)

    Deng, Binghui; Shi, Yunfeng

    2017-01-01

    We report a dynamic self-assembly system of 'living' polymeric chains sustained by chemistry using reactive molecular dynamics simulations. The linear polymeric chains consist of self-assembled nanoparticles connected by metastable linker molecules. As such, the polymeric chains, once assembled, undergo spontaneous dissociation driven by thermodynamics. However, with a continuous supply of linker molecules and the stored chemical energy therein, the polymeric chains can survive and maintain a steady state averaged chain length. These dynamically self-assembled polymeric chains are analogous to biological systems that both are thermodynamically metastable, yet dynamically stable upon continuous influx of matter and energy.

  10. CYTOPLASMIC MICROTUBULES

    PubMed Central

    Slautterback, David B.

    1963-01-01

    Small cytoplasmic tubules are present in the interstitial cells and cnidoblasts of hydra. They are referred to here as "microtubules." These tubular elements have an outside diameter of 180 A and an inside diameter of 80 A. By difference, the membranous wall is estimated to be 50 A thick. The maximum length of the microtubules cannot be determined from thin sections but is known to exceed 1.5 µ. In the interstitial cells the microtubules are found in the intercellular bridges, free in the cytoplasm and in association with the centrioles. In the cnidoblast they form a framework around the developing nematocyst and in late stages are related to the cnidocil forming a tight skein in the basal part of the cell. Especially in this cell, confluence of microtubules with small spherical vesicles of the Golgi complex has been observed. It is proposed that these tubules function in the transport of water, ions, or small molecules. PMID:14079495

  11. Metaphase Spindle Assembly

    PubMed Central

    Kapoor, Tarun M.

    2017-01-01

    A microtubule-based bipolar spindle is required for error-free chromosome segregation during cell division. In this review I discuss the molecular mechanisms required for the assembly of this dynamic micrometer-scale structure in animal cells. PMID:28165376

  12. Microtubule Severing Stymied by Free Tubulin

    NASA Astrophysics Data System (ADS)

    Ross, Jennifer; Bailey, Megan

    2015-03-01

    Proper organization of the microtubule cytoskeletal network is required to perform many necessary cellular functions including mitosis, cell development, and cell motility. Network organization is achieved through filament remodeling by microtubule-associated proteins (MAPs) that control microtubule dynamics. MAPs that stabilize are relatively well understood, while less is known about destabilizing MAPs, such as severing enzymes. Katanin, the first-discovered microtubule-severing enzyme, is a AAA + enzyme that oligomerizes into hexamers and uses ATP hydrolysis to sever microtubules. Using quantitative fluorescence imaging on reconstituted microtubule severing assays in vitro we investigate how katanin can regulate microtubule dynamics. Interestingly, we find microtubule dynamics inhibits katanin severing activity; dynamic microtubules are not severed. Using systematic experiments introducing free tubulin into the assays we find that free tubulin can compete for microtubule filaments for the katanin proteins. Our work indicates that katanin could function best on stabile microtubules or stabile regions of microtubules in cells in regions where free tubulin is sequesters, low, or depleted.

  13. Dynamics of magnetic nano-particle assembly

    NASA Astrophysics Data System (ADS)

    Kondratyev, V. N.

    2010-11-01

    Ferromagnetically coupled nano-particle assembly is analyzed accounting for inter- and intra- particle electronic structures within the randomly jumping interacting moments model including quantum fluctuations due to the discrete levels and disorder. At the magnetic jump anomalies caused by quantization the magnetic state equation and phase diagram are found to indicate an existence of spinodal regions and critical points. Arrays of magnetized nano-particles with multiple magnetic response anomalies are predicted to display some specific features. In a case of weak coupling such arrays exhibit the well-separated instability regions surrounding the anomaly positions. With increasing coupling we observe further structure modification, plausibly, of bifurcation type. At strong coupling the dynamical instability region become wide while the stable regime arises as a narrow islands at small disorders. It is shown that exploring correlations of magnetic noise amplitudes represents convenient analytical tool for quantitative definition, description and study of supermagnetism, as well as self-organized criticality.

  14. Binding of E-MAP-115 to microtubules is regulated by cell cycle- dependent phosphorylation

    PubMed Central

    1995-01-01

    Expression levels of E-MAP-115, a microtubule-associated protein that stabilizes microtubules, increase with epithelial cell polarization and differentiation (Masson and Kreis, 1993). Although polarizing cells contain significant amounts of this protein, they can still divide and thus all stabilized microtubules must disassemble at the onset of mitosis to allow formation of the dynamic mitotic spindle. We show here that binding of E-MAP-115 to microtubules is regulated by phosphorylation during the cell cycle. Immunolabeling of HeLa cells for E-MAP-115 indicates that the protein is absent from microtubules during early prophase and progressively reassociates with microtubules after late prophase. A fraction of E-MAP-115 from HeLa cells released from a block at the G1/S boundary runs with higher apparent molecular weight on SDS-PAGE, with a peak correlating with the maximal number of cells in early stages of mitosis. E-MAP-115 from nocodazole-arrested mitotic cells, which can be obtained in larger amounts, displays identical modifications and was used for further biochemical characterization. The level of incorporation of 32P into mitotic E-MAP-115 is about 15- fold higher than into the interphase protein. Specific threonine phosphorylation occurs in mitosis, and the amount of phosphate associated with serine also increases. Hyperphosphorylated E-MAP-115 from mitotic cells cannot bind stably to microtubules in vitro. These results suggest that phosphorylation of E-MAP-115 is a prerequisite for increasing the dynamic properties of the interphase microtubules which leads to the assembly of the mitotic spindle at the onset of mitosis. Microtubule-associated proteins are thus most likely key targets for kinases which control changes in microtubule dynamic properties at the G2- to M-phase transition. PMID:7490279

  15. The kinesin KIF21B regulates microtubule dynamics and is essential for neuronal morphology, synapse function and learning and memory

    PubMed Central

    Muhia, Mary; Thies, Edda; Labonté, Dorthe; Ghiretti, Amy E.; Gromova, Kira V.; Xompero, Francesca; Lappe-Siefke, Corinna; Hermans-Borgmeyer, Irm; Kuhl, Dietmar; Schweizer, Michaela; Ohana, Ora; Schwarz, Jürgen R.; Holzbaur, Erika L.F.

    2017-01-01

    SUMMARY The kinesin KIF21B is implicated in several human neurological disorders including delayed cognitive development, yet it remains unclear how KIF21B dysfunction may contribute to pathology. One limitation is that relatively little is known about KIF21B-mediated physiological mechanisms. Here, we generated Kif21b knockout mice and used cellular assays to investigate the relevance of KIF21B in neuronal and in vivo function. We show that KIF21B is a processive motor, and identify an additional role for KIF21B in regulating microtubule dynamics. In neurons lacking KIF21B, microtubules grow more slowly and persistently, leading to tighter packing in dendrites. KIF21B-deficient neurons exhibit decreased dendritic arbor complexity and reduced spine density, which correlate with deficits in synaptic transmission. Consistent with these observations, KIF21B-null mice exhibit behavioral changes involving learning and memory deficits. Collectively, our study provides insight into the cellular function of KIF21B and the basis for cognitive decline resulting from KIF21B dysregulation. PMID:27117409

  16. The acetylenic tricyclic bis(cyano enone), TBE-31, targets microtubule dynamics and cell polarity in migrating cells.

    PubMed

    Chan, Eddie; Saito, Akira; Honda, Tadashi; Di Guglielmo, Gianni M

    2016-04-01

    Cell migration is dependent on the microtubule network for structural support as well as for the proper delivery and positioning of polarity proteins at the leading edge of migrating cells. Identification of drugs that target cytoskeletal-dependent cell migration and protein transport in polarized migrating cells is important in understanding the cell biology of normal and tumor cells and can lead to new therapeutic targets in disease processes. Here, we show that the tricyclic compound TBE-31 directly binds to tubulin and interferes with microtubule dynamics, as assessed by end binding 1 (EB1) live cell imaging. Interestingly, this interference is independent of in vitro tubulin polymerization. Using immunofluorescence microscopy, we also observed that TBE-31 interferes with the polarity of migratory cells. The polarity proteins Rac1, IQGAP and Tiam1 were localized at the leading edge of DMSO-treated migrating cell, but were observed to be in multiple protrusions around the cell periphery of TBE-31-treated cells. Finally, we observed that TBE-31 inhibits the migration of Rat2 fibroblasts with an IC50 of 0.75 μM. Taken together, our results suggest that the inhibition of cell migration by TBE-31 may result from the improper maintenance of cell polarity of migrating cells.

  17. Microtubule-associated proteins from Antarctic fishes.

    PubMed

    Detrich, H W; Neighbors, B W; Sloboda, R D; Williams, R C

    1990-01-01

    Microtubules and presumptive microtubule-associated proteins (MAPs) were isolated from the brain tissues of four Antarctic fishes (Notothenia gibberifrons, N. coriiceps neglecta, Chaenocephalus aceratus, and a Chionodraco sp.) by means of a taxol-dependent, microtubule-affinity procedure (cf. Vallee: Journal of Cell Biology 92:435-442, 1982). MAPs from these fishes were similar to each other in electrophoretic pattern. Prominent in each preparation were proteins in the molecular weight ranges 410,000-430,000, 220,000-280,000, 140,000-155,000, 85,000-95,000, 40,000-45,000, and 32,000-34,000. The surfaces of MAP-rich microtubules were decorated by numerous filamentous projections. Exposure to elevated ionic strength released the MAPs from the microtubules and also removed the filamentous projections. Addition of fish MAPs to subcritical concentrations of fish tubulins at 0-5 degrees C induced the assembly of microtubules. Both the rate and the extent of this assembly increased with increasing concentrations of the MAPs. Sedimentation revealed that approximately six proteins, with apparent molecular weights between 60,000 and 300,000, became incorporated into the microtubule polymer. Bovine MAPs promoted microtubule formation by fish tubulin at 2-5 degrees C, and proteins corresponding to MAPs 1 and 2 co-sedimented with the polymer. MAPs from C. aceratus also enhanced the polymerization of bovine tubulin at 33 degrees C, but the microtubules depolymerized at 0 degrees C. We conclude that MAPs are part of the microtubules of Antarctic fishes, that these proteins promote microtubule assembly in much the same way as mammalian MAPs, and that they do not possess special capacities to promote microtubule assembly at low temperatures or to prevent cold-induced microtubule depolymerization.

  18. Intact Microtubules Preserve Transient Receptor Potential Vanilloid 1 (TRPV1) Functionality through Receptor Binding*

    PubMed Central

    Storti, Barbara; Bizzarri, Ranieri; Cardarelli, Francesco; Beltram, Fabio

    2012-01-01

    The transient receptor potential cation channel subfamily V member 1 (TRPV1) is a protein currently under scrutiny as a pharmacological target for pain management therapies. Recently, the role of TRPV1-microtubule interaction in transducing nociception stimuli to cells by cytoskeletal rearrangement was proposed. In this work, we investigate TRPV1-microtubule interaction in living cells under the resting or activated state of TRPV1, as well as in presence of structurally intact or depolymerized cytoskeletal microtubules. We combined a toolbox of high resolution/high sensitivity fluorescence imaging techniques (such as FRET, correlation spectroscopy, and fluorescence anisotropy) to monitor TRPV1 aggregation status, membrane mobility, and interaction with microtubules. We found that TRPV1 is a dimeric membrane protein characterized by two populations with different diffusion properties in basal condition. After stimulation with resiniferatoxin, TRPV1 dimers tetramerize. The tetramers and the slower population of TRPV1 dimers bind dynamically to intact microtubules but not to tubulin dimers. Upon microtubule disassembly, the interaction with TRPV1 is lost thereby inducing receptor self-aggregation with partial loss of functionality. Intact microtubules play an essential role in maintaining TRPV1 functionality toward activation stimuli. This previously undisclosed property mirrors the recently reported role of TRPV1 in modulating microtubule assembly/disassembly and suggests the participation of these two players in a feedback cycle linking nociception and cytoskeletal remodeling. PMID:22262838

  19. Protein Kinase C Activation Promotes Microtubule Advance in Neuronal Growth Cones by Increasing Average Microtubule Growth Lifetimes

    PubMed Central

    Kabir, Nurul; Schaefer, Andrew W.; Nakhost, Arash; Sossin, Wayne S.; Forscher, Paul

    2001-01-01

    We describe a novel mechanism for protein kinase C regulation of axonal microtubule invasion of growth cones. Activation of PKC by phorbol esters resulted in a rapid, robust advance of distal microtubules (MTs) into the F-actin rich peripheral domain of growth cones, where they are normally excluded. In contrast, inhibition of PKC activity by bisindolylmaleimide and related compounds had no perceptible effect on growth cone motility, but completely blocked phorbol ester effects. Significantly, MT advance occurred despite continued retrograde F-actin flow—a process that normally inhibits MT advance. Polymer assembly was necessary for PKC-mediated MT advance since it was highly sensitive to a range of antagonists at concentrations that specifically interfere with microtubule dynamics. Biochemical evidence is presented that PKC activation promotes formation of a highly dynamic MT pool. Direct assessment of microtubule dynamics and translocation using the fluorescent speckle microscopy microtubule marking technique indicates PKC activation results in a nearly twofold increase in the typical lifetime of a MT growth episode, accompanied by a 1.7-fold increase and twofold decrease in rescue and catastrophe frequencies, respectively. No significant effects on instantaneous microtubule growth, shortening, or sliding rates (in either anterograde or retrograde directions) were observed. MTs also spent a greater percentage of time undergoing retrograde transport after PKC activation, despite overall MT advance. These results suggest that regulation of MT assembly by PKC may be an important factor in determining neurite outgrowth and regrowth rates and may play a role in other cellular processes dependent on directed MT advance. PMID:11238458

  20. High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

    PubMed Central

    Braun, Alexander; Caesar, Nicole M.; Dang, Kyvan; Myers, Kenneth A.

    2016-01-01

    The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration. PMID:27584860

  1. Regulation of Mouse Oocyte Microtubule and Organelle Dynamics by PADI6 and the Cytoplasmic Lattices

    PubMed Central

    Kan, Rui; Yurttas, Piraye; Kim, Boram; Jin, Mei; Wo, Luccie; Lee, Bora; Gosden, Roger; Coonrod, Scott A.

    2010-01-01

    Organelle positioning and movement in oocytes is largely mediated by microtubules (MTs) and their associated motor proteins. While yet to be studied in germ cells, cargo trafficking in somatic cells is also facilitated by specific recognition of acetylated MTs by motor proteins. We have previously shown that oocyte-restricted PADI6 is essential for formation of a novel oocyte-restricted fibrous structure, the cytoplasmic lattices (CPLs). Here, we show that α-tubulin appears to be associated with the PADI6/CPL complex. Next, we demonstrate that organelle positioning and redistribution is defective in PADI6-null oocytes and that alteration of MT polymerization or MT motor activity does not induce organelle redistribution in these oocytes. Finally, we report that levels of acetylated microtubules are dramatically suppressed in the cytoplasm of PADI6-null oocytes, suggesting that the observed organelle redistribution failure is due to defects in stable cytoplasmic MTs. These results demonstrate that the PADI6/CPL superstructure plays a key role in regulating MT-mediated organelle positioning and movement. PMID:21147087

  2. Mechanisms and Dynamics of Collagen Assembly

    NASA Astrophysics Data System (ADS)

    Tao, Jinhui; Friddle, Raymond; Wang, Debin; de Yoreo, Jim

    2013-03-01

    Collagen is the major structural protein of bone, dentine and it template the nucleation of biomineral phases. Both collagen conformation and architecture on substrate are critical for its function. We studied the mechanism of collagen I assembly on mica by in-situ AFM. At acidic condition, assembled architecture evolved from random fibers to co-aligned fibers and finally to bundles as the K+ concentration increased from 100 to 300mM. XPS and NEXAFS showed the concentration of K+ within the collagen layer increased and the intensity of absorption peak due to π*(C =O) resonance decreased with higher K+concentration. The magnitude of collagen-mica (C-M) and collagen-collagen (C-C) interactions were measured by dynamic force spectroscopy. The free energy ΔGb for C-M and C-C at 200mM K+were 13.7kT and 1.4kT, while ΔGb at 300mM K+ were 5.7kT and 12.3kT, respectively. The switch from co-aligned fibers to 3D bundles is driven by the reversal in the magnitude of C-C and C-M interactions. Our results indicate K+ complex with C =O of collagen and its effect on the strength of collagen-collagen bridging is the likely source of architecture control. Authors would like to acknowledge grant no. DK61673 from the National Institutes of Health. Theoretical analysis was supported by Office of Science, Office of Basic Energy Sciences of the U.S. Department of Energy under Contract no. DE-AC02-05CH1123.

  3. A higher-order mathematical modeling for dynamic behavior of protein microtubule shell structures including shear deformation and small-scale effects.

    PubMed

    Daneshmand, Farhang; Farokhi, Hamed; Amabili, Marco

    2014-06-01

    Microtubules in mammalian cells are cylindrical protein polymers which structurally and dynamically organize functional activities in living cells. They are important for maintaining cell structures, providing platforms for intracellular transport, and forming the spindle during mitosis, as well as other cellular processes. Various in vitro studies have shown that microtubules react to applied mechanical loading and physical environment. To investigate the mechanisms underlying such phenomena, a mathematical model based on the orthotropic higher-order shear deformation shell formulation and Hamilton's principle is presented in this paper for dynamic behavior of microtubules. The numerical results obtained by the proposed shell model are verified by the experimental data from the literature, showing great consistency. The nonlocal elasticity theory is also utilized to describe the nano-scale effects of the microtubule structure. The wave propagation and vibration characteristics of the microtubule are examined in the presence and absence of the cytosol employing proposed formulations. The effects of different system parameters such as length, small scale parameter, and cytosol viscosity on vibrational behavior of a microtubule are elucidated. The definitions of critical length and critical viscosity are introduced and the results obtained using the higher order shell model are compared with those obtained employing a first-order shear deformation theory. This comparison shows that the small scale effects become important for higher values of the wave vector and the proposed model gives more accurate results for both small and large values of wave vectors. Moreover, it is shown that for higher circumferential wave number, the torsional wave velocity obtained by the higher-order shell model tend to be higher than the one predicted by the first-order shell model.

  4. A Mutation in γ-Tubulin Alters Microtubule Dynamics and Organization and Is Synthetically Lethal with the Kinesin-like Protein Pkl1pV⃞

    PubMed Central

    Paluh, Janet L.; Nogales, Eva; Oakley, Berl R.; McDonald, Kent; Pidoux, Alison L.; Cande, W. Z.

    2000-01-01

    Mitotic segregation of chromosomes requires spindle pole functions for microtubule nucleation, minus end organization, and regulation of dynamics. γ-Tubulin is essential for nucleation, and we now extend its role to these latter processes. We have characterized a mutation in γ-tubulin that results in cold-sensitive mitotic arrest with an elongated bipolar spindle but impaired anaphase A. At 30°C cytoplasmic microtubule arrays are abnormal and bundle into single larger arrays. Three-dimensional time-lapse video microscopy reveals that microtubule dynamics are altered. Localization of the mutant γ-tubulin is like the wild-type protein. Prediction of γ-tubulin structure indicates that non-α/β-tubulin protein–protein interactions could be affected. The kinesin-like protein (klp) Pkl1p localizes to the spindle poles and spindle and is essential for viability of the γ-tubulin mutant and in multicopy for normal cell morphology at 30°C. Localization and function of Pkl1p in the mutant appear unaltered, consistent with a redundant function for this protein in wild type. Our data indicate a broader role for γ-tubulin at spindle poles in regulating aspects of microtubule dynamics and organization. We propose that Pkl1p rescues an impaired function of γ-tubulin that involves non-tubulin protein–protein interactions, presumably with a second motor, MAP, or MTOC component. PMID:10749926

  5. Microtubule alignment and manipulation using AC electrokinetics.

    PubMed

    Uppalapati, Maruti; Huang, Ying-Ming; Jackson, Thomas N; Hancock, William O

    2008-09-01

    The kinesin-microtubule system plays an important role in intracellular transport and is a model system for integrating biomotor-driven transport into microengineered devices. AC electrokinetics provides a novel tool for manipulating and organizing microtubules in solution, enabling new experimental geometries for investigating and controlling the interactions of microtubules and microtubule motors in vitro. By fabricating microelectrodes on glass substrates and generating AC electric fields across solutions of microtubules in low-ionic-strength buffers, bundles of microtubules are collected and aligned and the electrical properties of microtubules in solution are measured. The AC electric fields result in electro-osmotic flow, electrothermal flow, and dielectrophoresis of microtubules, which can be controlled by varying the solution conductivity, AC frequency, and electrode geometry. By mapping the solution conductivity and AC frequency over which positive dielectrophoresis occurs, the apparent conductivity of taxol-stabilized bovine-brain microtubules in PIPES buffer is measured to be 250 mS m(-1). By maximizing dielectrophoretic forces and minimizing electro-osmotic and electrothermal flow, microtubules are assembled into opposed asters. These experiments demonstrate that AC electrokinetics provides a powerful new tool for kinesin-driven transport applications and for investigating the role of microtubule motors in development and maintenance of the mitotic spindle.

  6. End-binding proteins sensitize microtubules to the action of microtubule-targeting agents.

    PubMed

    Mohan, Renu; Katrukha, Eugene A; Doodhi, Harinath; Smal, Ihor; Meijering, Erik; Kapitein, Lukas C; Steinmetz, Michel O; Akhmanova, Anna

    2013-05-28

    Microtubule-targeting agents (MTAs) are widely used for treatment of cancer and other diseases, and a detailed understanding of the mechanism of their action is important for the development of improved microtubule-directed therapies. Although there is a large body of data on the interactions of different MTAs with purified tubulin and microtubules, much less is known about how the effects of MTAs are modulated by microtubule-associated proteins. Among the regulatory factors with a potential to have a strong impact on MTA activity are the microtubule plus end-tracking proteins, which control multiple aspects of microtubule dynamic instability. Here, we reconstituted microtubule dynamics in vitro to investigate the influence of end-binding proteins (EBs), the core components of the microtubule plus end-tracking protein machinery, on the effects that MTAs exert on microtubule plus-end growth. We found that EBs promote microtubule catastrophe induction in the presence of all MTAs tested. Analysis of microtubule growth times supported the view that catastrophes are microtubule age dependent. This analysis indicated that MTAs affect microtubule aging in multiple ways: destabilizing MTAs, such as colchicine and vinblastine, accelerate aging in an EB-dependent manner, whereas stabilizing MTAs, such as paclitaxel and peloruside A, induce not only catastrophes but also rescues and can reverse the aging process.

  7. The metaphase and anaphase dynamics is dominated by the physical and mechanical properties of both microtubules and chromatin

    NASA Astrophysics Data System (ADS)

    Grisa, Luca; Kilfoil, Maria

    2012-02-01

    One of the most interesting problems in biophysics involves the physical separation of chromosomes and the mechanical properties of both microtubules (MT's) and chromatin. This process involves the polymers MT's and chromatin, each of which has unique physical properties that have been determined extensively in vitro. Of further interest for physicists is the out-of-equilibrium nature of this process involving several force generators from motor proteins and MT depolymerization. We follow the dynamics of spindle pole bodies and centromeres of yeast cells during mitosis in three-dimensions at high spatial resolution. Using this novel approach, we are able to observe spindle oscillations during metaphase, and the three-dimensional dynamics of spindle elongation and chromosome separation during anaphase. With these data, we can separate the dynamics caused by MT depolymerization from those caused by the motors. This allows us to determine the depolymerization rate of the kinetochore MT's in vivo. Furthermore, we determine the temporal profile of the chromatin extension during anaphase we combine with the known force-extension curve of chromatin in vitro, to infer the expected force-velocity curve of the collective motors in vivo, which has never been measured in vivo or in vitro.

  8. A Toll receptor–FoxO pathway represses Pavarotti/MKLP1 to promote microtubule dynamics in motoneurons

    PubMed Central

    Liu, Nan

    2016-01-01

    FoxO proteins are evolutionarily conserved regulators of neuronal structure and function, yet the neuron-specific pathways within which they act are poorly understood. To elucidate neuronal FoxO function in Drosophila melanogaster, we first screened for FoxO’s upstream regulators and downstream effectors. On the upstream side, we present genetic and molecular pathway analyses indicating that the Toll-6 receptor, the Toll/interleukin-1 receptor domain adaptor dSARM, and FoxO function in a linear pathway. On the downstream side, we find that Toll-6–FoxO signaling represses the mitotic kinesin Pavarotti/MKLP1 (Pav-KLP), which itself attenuates microtubule (MT) dynamics. We next probed in vivo functions for this novel pathway and found that it is essential for axon transport and structural plasticity in motoneurons. We demonstrate that elevated expression of Pav-KLP underlies transport and plasticity phenotypes in pathway mutants, indicating that Toll-6–FoxO signaling promotes MT dynamics by limiting Pav-KLP expression. In addition to uncovering a novel molecular pathway, our work reveals an unexpected function for dynamic MTs in enabling rapid activity-dependent structural plasticity. PMID:27502486

  9. Cross-linking of microtubules by microtubule-associated proteins (MAPs) from the brine shrimp, Artemia.

    PubMed

    Campbell, E J; MacKinlay, S A; MacRae, T H

    1989-05-01

    Microtubules induced with taxol to assemble in cell-free extracts of the brine shrimp, Artemia, are cross-linked by microtubule-associated proteins (MAPs). When the MAPs, extracted from taxol-stabilized microtubules with 1 M-NaCl are co-assembled with purified Artemia or mammalian neural tubulin, reconstitution of cross-linking between microtubules occurs. The most prominent non-tubulin protein associated with reconstituted cross-linked microtubules has a molecular weight of 49,000 but we cannot yet exclude the possibility that other proteins may be responsible for the cross-linking. Cross-linkers are separated by varying distances while cross-linked microtubules, prepared under different conditions, are 6.9-7.7 nm apart. Cross-linking of microtubules by MAPs occurs whether MAPs are added to assembling tubulin or to microtubules, and it is not disrupted by ATP. The MAPs are heat-sensitive and do not stabilize microtubules to cold. Immunological characterization of Artemia MAPs on Western blots indicates that Artemia lack MAP 1, MAP 2 and tau. Our results clearly demonstrate that Artemia contain novel MAPs with the ability to cross-link microtubules from phylogenetically disparate organisms in an ATP-independent manner.

  10. Spectrin alpha is important for rear polarization of the microtubule organizing center during migration and spindle pole assembly during division of neointimal smooth muscle cells.

    PubMed

    Silverman-Gavrila, Rosalind V; Silverman-Gavrila, Lorelei B; Bilal, Khawaja Hasan; Bendeck, Michelle P

    2015-04-01

    Directed migration of smooth muscle cells (SMCs) from the media to the intima and their subsequent proliferation are key events in atherosclerosis as these cells contribute to the bulk and stability of atheromatous plaques. We showed previously that two cytoskeleton-associated proteins, RHAMM and ARPC5, play important roles in rear polarization of the microtubule organizing centre (MTOC), directed migration, and in maintaining cell division fidelity. These proteins were analyzed to predict additional potential interacting partners using the bioinformatics programs BLAST, ClustalW, and PPI Spider. We identified spectrin alpha, a protein with a known role in actin polymerization as part of the pathway. We show that in migrating SMCs spectrin alpha localizes at the nodes of the actin net, and it partially colocalizes with RHAMM in the perinuclear region. In dividing SMCs spectrin alpha is present at spindle poles and midbody. Moreover, we show that spectrin alpha and RHAMM interact in a complex. Using siRNA to knockdown spectrin disrupted SMC migration, MTOC polarization, and the assembly of a polygonal actin net dorsolateral of the nucleus. Spectrin alpha knockdown also disrupted the organization of the bipolar spindle, chromosome division, and cytokinesis during cell division. The identification of interacting partners such as spectrin alpha and the decoding of pathways involved in polarity regulation during the migration of smooth muscle cells in atherosclerosis is important for identifying atherosclerosis biomarkers and developing therapeutic agents to block atherosclerotic plaque formation.

  11. Katanin p80, NuMA and cytoplasmic dynein cooperate to control microtubule dynamics

    PubMed Central

    Jin, Mingyue; Pomp, Oz; Shinoda, Tomoyasu; Toba, Shiori; Torisawa, Takayuki; Furuta, Ken’ya; Oiwa, Kazuhiro; Yasunaga, Takuo; Kitagawa, Daiju; Matsumura, Shigeru; Miyata, Takaki; Tan, Thong Teck; Reversade, Bruno; Hirotsune, Shinji

    2017-01-01

    Human mutations in KATNB1 (p80) cause severe congenital cortical malformations, which encompass the clinical features of both microcephaly and lissencephaly. Although p80 plays critical roles during brain development, the underlying mechanisms remain predominately unknown. Here, we demonstrate that p80 regulates microtubule (MT) remodeling in combination with NuMA (nuclear mitotic apparatus protein) and cytoplasmic dynein. We show that p80 shuttles between the nucleus and spindle pole in synchrony with the cell cycle. Interestingly, this striking feature is shared with NuMA. Importantly, p80 is essential for aster formation and maintenance in vitro. siRNA-mediated depletion of p80 and/or NuMA induced abnormal mitotic phenotypes in cultured mouse embryonic fibroblasts and aberrant neurogenesis and neuronal migration in the mouse embryonic brain. Importantly, these results were confirmed in p80-mutant harboring patient-derived induced pluripotent stem cells and brain organoids. Taken together, our findings provide valuable insights into the pathogenesis of severe microlissencephaly, in which p80 and NuMA delineate a common pathway for neurogenesis and neuronal migration via MT organization at the centrosome/spindle pole. PMID:28079116

  12. Force measurements on cargoes in living cells reveal collective dynamics of microtubule motors

    PubMed Central

    Hendricks, Adam G.; Holzbaur, Erika L. F.; Goldman, Yale E.

    2012-01-01

    Many cellular cargoes move bidirectionally along microtubules, driven by teams of plus- and minus-end–directed motor proteins. To probe the forces exerted on cargoes during intracellular transport, we examined latex beads phagocytosed into living mammalian macrophages. These latex bead compartments (LBCs) are encased in membrane and transported along the cytoskeleton by a complement of endogenous kinesin-1, kinesin-2, and dynein motors. The size and refractive index of LBCs makes them well-suited for manipulation with an optical trap. We developed methods that provide in situ calibration of the optical trap in the complex cellular environment, taking into account any variations among cargoes and local viscoelastic properties of the cytoplasm. We found that centrally and peripherally directed forces exerted on LBCs are of similar magnitude, with maximum forces of ∼20 pN. During force events greater than 10 pN, we often observe 8-nm steps in both directions, indicating that the stepping of multiple motors is correlated. These observations suggest bidirectional transport of LBCs is driven by opposing teams of stably bound motors that operate near force balance. PMID:23091040

  13. Katanin p80, NuMA and cytoplasmic dynein cooperate to control microtubule dynamics.

    PubMed

    Jin, Mingyue; Pomp, Oz; Shinoda, Tomoyasu; Toba, Shiori; Torisawa, Takayuki; Furuta, Ken'ya; Oiwa, Kazuhiro; Yasunaga, Takuo; Kitagawa, Daiju; Matsumura, Shigeru; Miyata, Takaki; Tan, Thong Teck; Reversade, Bruno; Hirotsune, Shinji

    2017-01-12

    Human mutations in KATNB1 (p80) cause severe congenital cortical malformations, which encompass the clinical features of both microcephaly and lissencephaly. Although p80 plays critical roles during brain development, the underlying mechanisms remain predominately unknown. Here, we demonstrate that p80 regulates microtubule (MT) remodeling in combination with NuMA (nuclear mitotic apparatus protein) and cytoplasmic dynein. We show that p80 shuttles between the nucleus and spindle pole in synchrony with the cell cycle. Interestingly, this striking feature is shared with NuMA. Importantly, p80 is essential for aster formation and maintenance in vitro. siRNA-mediated depletion of p80 and/or NuMA induced abnormal mitotic phenotypes in cultured mouse embryonic fibroblasts and aberrant neurogenesis and neuronal migration in the mouse embryonic brain. Importantly, these results were confirmed in p80-mutant harboring patient-derived induced pluripotent stem cells and brain organoids. Taken together, our findings provide valuable insights into the pathogenesis of severe microlissencephaly, in which p80 and NuMA delineate a common pathway for neurogenesis and neuronal migration via MT organization at the centrosome/spindle pole.

  14. Force measurements on cargoes in living cells reveal collective dynamics of microtubule motors.

    PubMed

    Hendricks, Adam G; Holzbaur, Erika L F; Goldman, Yale E

    2012-11-06

    Many cellular cargoes move bidirectionally along microtubules, driven by teams of plus- and minus-end-directed motor proteins. To probe the forces exerted on cargoes during intracellular transport, we examined latex beads phagocytosed into living mammalian macrophages. These latex bead compartments (LBCs) are encased in membrane and transported along the cytoskeleton by a complement of endogenous kinesin-1, kinesin-2, and dynein motors. The size and refractive index of LBCs makes them well-suited for manipulation with an optical trap. We developed methods that provide in situ calibration of the optical trap in the complex cellular environment, taking into account any variations among cargoes and local viscoelastic properties of the cytoplasm. We found that centrally and peripherally directed forces exerted on LBCs are of similar magnitude, with maximum forces of ~20 pN. During force events greater than 10 pN, we often observe 8-nm steps in both directions, indicating that the stepping of multiple motors is correlated. These observations suggest bidirectional transport of LBCs is driven by opposing teams of stably bound motors that operate near force balance.

  15. Modulation of RNA polymerase assembly dynamics in transcriptional regulation

    PubMed Central

    Gorski, Stanislaw A.; Snyder, Sara K.; John, Sam; Grummt, Ingrid; Misteli, Tom

    2008-01-01

    The interaction of transcription factors with target genes is highly dynamic. Whether the dynamic nature of these interactions is merely an intrinsic property of transcriptions factors or serves a regulatory role is unknown. Here, we have used single cell fluorescence imaging combined with computational modeling and chromatin immunoprecipitation to analyze transcription complex dynamics in gene regulation during the cell cycle in living cells. We demonstrate a link between the dynamics of RNA polymerase I (RNA pol I) assembly and transcriptional output. We show that transcriptional upregulation is accompanied by prolonged retention of RNA pol I components at the promoter, resulting in longer promoter dwell time, and an increase in the steady state population of assembling polymerase. As a consequence, polymerase assembly efficiency, and ultimately, an rate of entry into processive elongation are elevated. Our results show that regulation of rDNA transcription in vivo occurs via modulation of the efficiency of transcription complex subunit capture and assembly. PMID:18498750

  16. Dynamic Nanoparticle Assemblies for Biomedical Applications.

    PubMed

    Li, Fangyuan; Lu, Jingxiong; Kong, Xueqian; Hyeon, Taeghwan; Ling, Daishun

    2017-02-22

    Designed synthesis and assembly of nanoparticles assisted by their surface ligands can create "smart" materials with programmed responses to external stimuli for biomedical applications. These assemblies can be designed to respond either exogenously (for example, to magnetic field, temperature, ultrasound, light, or electric pulses) or endogenously (to pH, enzymatic activity, or redox gradients) and play an increasingly important role in a diverse range of biomedical applications, such as biosensors, drug delivery, molecular imaging, and novel theranostic systems. In this review, the recent advances and challenges in the development of stimuli-responsive nanoparticle assemblies are summarized; in particular, the application-driven design of surface ligands for stimuli-responsive nanoparticle assemblies that are capable of sensing small changes in the disease microenvironment, which induce the related changes in their physico-chemical properties, is described. Finally, possible future research directions and problems that have to be addressed are briefly discussed.

  17. TTBK2 with EB1/3 regulates microtubule dynamics in migrating cells through KIF2A phosphorylation.

    PubMed

    Watanabe, Takashi; Kakeno, Mai; Matsui, Toshinori; Sugiyama, Ikuko; Arimura, Nariko; Matsuzawa, Kenji; Shirahige, Aya; Ishidate, Fumiyoshi; Nishioka, Tomoki; Taya, Shinichiro; Hoshino, Mikio; Kaibuchi, Kozo

    2015-08-31

    Microtubules (MTs) play critical roles in various cellular events, including cell migration. End-binding proteins (EBs) accumulate at the ends of growing MTs and regulate MT end dynamics by recruiting other plus end-tracking proteins (+TIPs). However, how EBs contribute to MT dynamics through +TIPs remains elusive. We focused on tau-tubulin kinase 2 (TTBK2) as an EB1/3-binding kinase and confirmed that TTBK2 acted as a +TIP. We identified MT-depolymerizing kinesin KIF2A as a novel substrate of TTBK2. TTBK2 phosphorylated KIF2A at S135 in intact cells in an EB1/3-dependent fashion and inactivated its MT-depolymerizing activity in vitro. TTBK2 depletion reduced MT lifetime (facilitated shrinkage and suppressed rescue) and impaired HeLa cell migration, and these phenotypes were partially restored by KIF2A co-depletion. Expression of nonphosphorylatable KIF2A, but not wild-type KIF2A, reduced MT lifetime and slowed down the cell migration. These findings indicate that TTBK2 with EB1/3 phosphorylates KIF2A and antagonizes KIF2A-induced depolymerization at MT plus ends for cell migration.

  18. [Microtubules in the nerve cells: morphological and functional aspects].

    PubMed

    Vorob'ev, V S; Portuganov, V V

    1980-10-01

    The modern literature concerning ultrastructure and cytochemistry of microtubules in the nervous tissue is reviewed. Common features of cytological and biochemical organization of microtubules in different parts of the nervous system of the vertebrates and invertebrates are analysed: the similarity of ultrastructure of microtubules and their molecular organization (tubulin and its alpha- and beta-monomeres), the ability of microtubules to assemble and disassemble, to bind specifically with poisons--colchicine and vinblastine, participation of microtubules in the neuroplastic transport. The authors' data on space arrangement of microtubules within cytoplasm of the neuronal processes (dendrites and unmyelinated axons in the central and peripheral nevous system) are presented. Some literature and personal results concerning ultrastructure of neurofilaments and microtubules in the myelinated nerve fibres are also considered. The functional significance of microtubules in the nervous system is discussed with special reference to facts and hypotheses on a possible role of microtubules in the propagation of nerve impulse.

  19. Dynamics of magnetic assembly of binary colloidal structures

    NASA Astrophysics Data System (ADS)

    Nogueras-Lara, F.; Rodríguez-Arco, L.; López-López, M. T.

    2015-08-01

    Magnetic field (MF)-directed assembly of colloidal particles provides a step towards the bottom-up manufacturing of smart materials whose properties can be precisely modulated by non-contact forces. Here, we study the MF-directed assembly in binary colloids made up of strong ferromagnetic and diamagnetic microparticles dispersed in ferrofluids. We present observations of the aggregation of pairs and small groups of particles to build equilibrium assemblies. We also develop a theoretical model capable of solving the aggregation dynamics and predicting the particle trajectories, a key factor to understand the physics governing the MF-directed assembly.

  20. Specific association of STOP protein with microtubules in vitro and with stable microtubules in mitotic spindles of cultured cells.

    PubMed

    Margolis, R L; Rauch, C T; Pirollet, F; Job, D

    1990-12-01

    STOP (Stable Tubule Only Polypeptide) is a neuronal microtubule associated protein of 145 kd that stabilizes microtubules indefinitely to in vitro disassembly induced by cold temperature, millimolar calcium or by drugs. We have produced monoclonal antibodies against STOP. Using an antibody affinity column, we have produced a homogeneously pure 145 kd protein which has STOP activity as defined by its ability to induce cold stability and resistance to dilution induced disassembly in microtubules in vitro. Western blot analysis, using a specific monoclonal antibody, demonstrates that STOP recycles quantitatively with microtubules through three assembly cycles in vitro. Immunofluorescence analysis demonstrates that STOP is specifically associated with microtubules of mitotic spindles in neuronal cells. Further, and most interestingly, STOP at physiological temperature appears to be preferentially distributed on the distinct microtubule subpopulations that display cold stability; kinetochore-to-pole microtubules and telophase midbody microtubules. The observed distribution suggests that STOP induces the observed cold stability of these microtubule subpopulations in vivo.

  1. The hepatitis E virus open reading frame 3 product interacts with microtubules and interferes with their dynamics.

    PubMed

    Kannan, Harilakshmi; Fan, Sumin; Patel, Deendayal; Bossis, Ioannis; Zhang, Yan-Jin

    2009-07-01

    Hepatitis E virus (HEV) is the causative agent of hepatitis E, a major form of viral hepatitis in developing countries. The open reading frame 3 (ORF3) of HEV encodes a phosphoprotein with a molecular mass of approximately 13 kDa (hereinafter called vp13). vp13 is essential for establishing HEV infections in animals, yet its exact functions are still obscure. Our current study found evidence showing interaction between vp13 and microtubules. Live-cell confocal fluorescence microscopy revealed both filamentous and punctate distribution patterns of vp13 in cells transfected with recombinant ORF3 reporter plasmids. The filamentous pattern of vp13 was altered by a microtubule-destabilizing drug. The vp13 expression led to elevation of acetylated alpha-tubulin, indicating increased microtubule stability. Its association with microtubules was further supported by its presence in microtubule-containing pellets in microtubule isolation assays. Exposure of these pellets to a high-salt buffer caused release of the vp13 to the supernatant, suggesting an electrostatic interaction. Inclusion of ATP and GTP in the lysis buffer during microtubule isolation also disrupted the interaction, indicating its sensitivity to the nucleotides. Further assays showed that motor proteins are needed for the vp13 association with the microtubules because disruption of dynein function abolished the vp13 filamentous pattern. Analysis of ORF3 deletion constructs found that both of the N-terminal hydrophobic domains of vp13 are needed for the interaction. Thus, our findings suggest that the vp13 interaction with microtubules might be needed for establishment of an HEV infection.

  2. Models for microtubule cargo transport coupling the Langevin equation to stochastic stepping motor dynamics: Caring about fluctuations

    NASA Astrophysics Data System (ADS)

    Bouzat, Sebastián

    2016-01-01

    One-dimensional models coupling a Langevin equation for the cargo position to stochastic stepping dynamics for the motors constitute a relevant framework for analyzing multiple-motor microtubule transport. In this work we explore the consistence of these models focusing on the effects of the thermal noise. We study how to define consistent stepping and detachment rates for the motors as functions of the local forces acting on them in such a way that the cargo velocity and run-time match previously specified functions of the external load, which are set on the base of experimental results. We show that due to the influence of the thermal fluctuations this is not a trivial problem, even for the single-motor case. As a solution, we propose a motor stepping dynamics which considers memory on the motor force. This model leads to better results for single-motor transport than the approaches previously considered in the literature. Moreover, it gives a much better prediction for the stall force of the two-motor case, highly compatible with the experimental findings. We also analyze the fast fluctuations of the cargo position and the influence of the viscosity, comparing the proposed model to the standard one, and we show how the differences on the single-motor dynamics propagate to the multiple motor situations. Finally, we find that the one-dimensional character of the models impede an appropriate description of the fast fluctuations of the cargo position at small loads. We show how this problem can be solved by considering two-dimensional models.

  3. On complex, curved trajectories in microtubule gliding

    NASA Astrophysics Data System (ADS)

    Gosselin, Pierre; Mohrbach, Hervé; Kulić, Igor M.; Ziebert, Falko

    2016-04-01

    We study the dynamics of microtubules in gliding assays. These biofilaments are typically considered as purely semiflexible, hence their trajectories under the action of motors covering the substrate have been regarded so far as straight, modulo fluctuations. However, this is not always the case experimentally, where microtubules are known to move on large scale circles or spirals, or even display quite regular wavy trajectories and more complex dynamics. Incorporating recent experimental evidence for a (small) preferred curvature as well as the microtubules' well established lattice twist into a dynamic model for microtubule gliding, we could reproduce both types of trajectories. Interestingly, as a function of the microtubules' length we found length intervals of stable rings alternating with regions where wavy and more complex dynamics prevails. Finally, both types of dynamics (rings and waves) can be rationalized by considering simple limits of the full model.

  4. Ectopic A-lattice seams destabilize microtubules.

    PubMed

    Katsuki, Miho; Drummond, Douglas R; Cross, Robert A

    2014-01-01

    Natural microtubules typically include one A-lattice seam within an otherwise helically symmetric B-lattice tube. It is currently unclear how A-lattice seams influence microtubule dynamic instability. Here we find that including extra A-lattice seams in GMPCPP microtubules, structural analogues of the GTP caps of dynamic microtubules, destabilizes them, enhancing their median shrinkage rate by >20-fold. Dynamic microtubules nucleated by seeds containing extra A-lattice seams have growth rates similar to microtubules nucleated by B-lattice seeds, yet have increased catastrophe frequencies at both ends. Furthermore, binding B-lattice GDP microtubules to a rigor kinesin surface stabilizes them against shrinkage, whereas microtubules with extra A-lattice seams are stabilized only slightly. Our data suggest that introducing extra A-lattice seams into dynamic microtubules destabilizes them by destabilizing their GTP caps. On this basis, we propose that the single A-lattice seam of natural B-lattice MTs may act as a trigger point, and potentially a regulation point, for catastrophe.

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

    PubMed

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

    2014-12-10

    Microtubules, the primary components of the chromosome segregation machinery, are stabilized by longitudinal and lateral noncovalent bonds between the tubulin subunits. However, the thermodynamics of these bonds and the microtubule physicochemical 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 reversible dissociation of lateral bonds followed by irreversible dissociation of the longitudinal bonds. We have determined the free energies of dissociation of the lateral (6.9 ± 0.4 kcal/mol) and longitudinal (14.9 ± 1.5 kcal/mol) tubulin-tubulin bonds. These values in conjunction with the large flexural rigidity of tubulin protofilaments obtained (18,000-26,000 pN·nm(2)) support the idea that the disassembling microtubule is capable of generating a large mechanical force to move chromosomes during cell division. Our computational modeling offers a comprehensive quantitative platform to link molecular tubulin characteristics with the physiological behavior of microtubules. The developed in silico nanoindentation method provides a powerful tool for the exploration of biomechanical properties of other cytoskeletal and multiprotein assemblies.

  6. Depletion of JMJD5 sensitizes tumor cells to microtubule-destabilizing agents by altering microtubule stability.

    PubMed

    Wu, Junyu; He, Zhimin; Wang, Da-Liang; Sun, Fang-Lin

    2016-11-01

    Microtubules play essential roles in mitosis, cell migration, and intracellular trafficking. Drugs that target microtubules have demonstrated great clinical success in cancer treatment due to their capacity to impair microtubule dynamics in both mitotic and interphase stages. In a previous report, we demonstrated that JMJD5 associated with mitotic spindle and was required for proper mitosis. However, it remains elusive whether JMJD5 could regulate the stability of cytoskeletal microtubules and whether it affects the efficacy of microtubule-targeting agents. In this study, we find that JMJD5 localizes not only to the nucleus, a fraction of it also localizes to the cytoplasm. JMJD5 depletion decreases the acetylation and detyrosination of α-tubulin, both of which are markers of microtubule stability. In addition, microtubules in JMJD5-depleted cells are more sensitive to nocodazole-induced depolymerization, whereas JMJD5 overexpression increases α-tubulin detyrosination and enhances the resistance of microtubules to nocodazole. Mechanistic studies revealed that JMJD5 regulates MAP1B protein levels and that MAP1B overexpression rescued the microtubule destabilization induced by JMJD5 depletion. Furthermore, JMJD5 depletion significantly promoted apoptosis in cancer cells treated with the microtubule-targeting anti-cancer drugs vinblastine or colchicine. Together, these findings suggest that JMJD5 is required to regulate the stability of cytoskeletal microtubules and that JMJD5 depletion increases the susceptibility of cancer cells to microtubule-destabilizing agents.

  7. Real-time monitoring of changes in microtubule mechanical properties in response to microtubule-destabilizing drug treatment.

    PubMed

    Han, Sung-Woong; Simona, Patriche; Banu, Mihaela; Adachi, Taiji

    2013-03-01

    Microtubules are cylindrical protein polymers that play important roles in a number of cellular functions. The properties of microtubules are dynamically changed by interacting with many microtubule-related proteins and drugs. In this study, we used atomic force microscopy to evaluate the changes in microtubule mechanical properties induced by treatment with nocodazole, which is a microtubule-destabilizing drug. The average spring constant of the microtubules, which was used as a measure of microtubule lateral stiffness, was drastically decreased by treatment with nocodazole within 30 min from 0.052 +/- 0.014 N/m to 0.029 +/- 0.015 N/m. Our findings will aid in the understanding of microtubule dynamics, protein interactions in response to drug treatment, microtubule-related diseases, and drug development.

  8. Rigidity of microtubules is increased by stabilizing agents

    PubMed Central

    1995-01-01

    Microtubules are rigid polymers that contribute to the static mechanical properties of cells. Because microtubules are dynamic structures whose polymerization is regulated during changes in cell shape, we have asked whether the mechanical properties of microtubules might also be modulated. We measured the flexural rigidity, or bending stiffness, of individual microtubules under a number of different conditions that affect the stability of microtubules against depolymerization. The flexural rigidity of microtubules polymerized with the slowly hydrolyzable nucleotide analogue guanylyl-(alpha, beta)- methylene-diphosphonate was 62 +/- 9 x 10(-24) Nm2 (weighted mean +/- SEM); that of microtubules stabilized with tau protein was 34 +/- 3 x 10(-24) Nm2; and that of microtubules stabilized with the antimitotic drug taxol was 32 +/- 2 x 10(-24) Nm2. For comparison, microtubules that were capped to prevent depolymerization, but were not otherwise stabilized, had a flexural rigidity of 26 +/- 2 x 10(-24) Nm2. Decreasing the temperature from 37 degrees C to approximately 25 degrees C, a condition that makes microtubules less stable, decreased the stiffness of taxol-stabilized microtubules by one-third. We thus find that the more stable a microtubule, the higher its flexural rigidity. This raises the possibility that microtubule rigidity may be regulated in vivo. In addition, the high rigidity of an unstabilized, GDP-containing microtubule suggests that a large amount of energy could be stored as mechanical strain energy in the protein lattice for subsequent force generation during microtubule depolymerization. PMID:7642706

  9. Easy creation of polymeric systems for molecular dynamics with Assemble!

    NASA Astrophysics Data System (ADS)

    Degiacomi, Matteo T.; Erastova, Valentina; Wilson, Mark R.

    2016-05-01

    We present Assemble!, a program greatly simplifying the preparation of molecular dynamics simulations of polymeric systems. The program is controlled either via command line or an intuitive Graphical User Interface, and runs on all major operating systems. Assemble! allows the creation of a desired system of polymer chains from constituent monomers, packs the chains into a box according to the required concentration and returns all the files needed for simulation with Gromacs. We illustrate the capabilities of Assemble! by demonstrating the easy preparation of a 300 monomers-long polyisoprene in hexane, and a heterogeneous mixture of polybutadiene.

  10. Structural investigations into the binding mode of novel neolignans Cmp10 and Cmp19 microtubule stabilizers by in silico molecular docking, molecular dynamics, and binding free energy calculations.

    PubMed

    Tripathi, Shubhandra; Kumar, Akhil; Kumar, B Sathish; Negi, Arvind S; Sharma, Ashok

    2016-06-01

    Microtubule stabilizers provide an important mode of treatment via mitotic cell arrest of cancer cells. Recently, we reported two novel neolignans derivatives Cmp10 and Cmp19 showing anticancer activity and working as microtubule stabilizers at micromolar concentrations. In this study, we have explored the binding site, mode of binding, and stabilization by two novel microtubule stabilizers Cmp10 and Cmp19 using in silico molecular docking, molecular dynamics (MD) simulation, and binding free energy calculations. Molecular docking studies were performed to explore the β-tubulin binding site of Cmp10 and Cmp19. Further, MD simulations were used to probe the β-tubulin stabilization mechanism by Cmp10 and Cmp19. Binding affinity was also compared for Cmp10 and Cmp19 using binding free energy calculations. Our docking results revealed that both the compounds bind at Ptxl binding site in β-tubulin. MD simulation studies showed that Cmp10 and Cmp19 binding stabilizes M-loop (Phe272-Val288) residues of β-tubulin and prevent its dynamics, leading to a better packing between α and β subunits from adjacent tubulin dimers. In addition, His229, Ser280 and Gln281, and Arg278, Thr276, and Ser232 were found to be the key amino acid residues forming H-bonds with Cmp10 and Cmp19, respectively. Consequently, binding free energy calculations indicated that Cmp10 (-113.655 kJ/mol) had better binding compared to Cmp19 (-95.216 kJ/mol). This study provides useful insight for better understanding of the binding mechanism of Cmp10 and Cmp19 and will be helpful in designing novel microtubule stabilizers.

  11. Dynamic self-assembly of coordination polymers in aqueous solution.

    PubMed

    Li, Wen; Kim, Yongju; Li, Jingfang; Lee, Myongsoo

    2014-08-07

    The construction of supramolecular polymers has been intensively pursued because the nanostructures formed through weak non-covalent interactions can be triggered by external stimuli leading to smart materials and sensors. Self-assemblies of coordination polymers consisting of metal ions and organic ligands in aqueous solution also provide particular contributions in this area. The main motivation for developing those coordination polymers originates from the value-added combination between metal ions and ligands. This review highlights the recent progress of the dynamic self-assembly of coordination polymers that result from the sophisticated molecular design, towards fabricating stimuli-responsive systems and bio-related materials. Dynamic structural changes and switchable physical properties triggered by various stimuli are summarized. Finally, the outlook for aqueous nanostructures originated from the dynamic self-assembly of coordination polymers is also presented.

  12. Microtubules self-repair in response to mechanical stress

    NASA Astrophysics Data System (ADS)

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

    2015-11-01

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

  13. Microtubules self-repair in response to mechanical stress

    PubMed Central

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

    2015-01-01

    Microtubules - which define the shape of axons, cilia and flagella, and provide tracks for intracellular transport - can be highly bent by intracellular forces, and microtubule structure and stiffness are thought to be affected by physical constraints. Yet how microtubules tolerate the vast forces exerted on them remains unknown. Here, by using a microfluidic device, we show that microtubule stiffness decreases incrementally with each cycle of bending and release. Similar to other cases of material fatigue, the concentration of mechanical stresses on pre-existing defects in the microtubule lattice is responsible for the generation of larger damages, which further decrease microtubule stiffness. Strikingly, damaged microtubules were able to incorporate new tubulin dimers into their lattice and recover their initial stiffness. Our findings demonstrate that microtubules are ductile materials with self-healing properties, that their dynamics does not exclusively occur at their ends, and that their lattice plasticity enables the microtubules' adaptation to mechanical stresses. PMID:26343914

  14. The microtubule catastrophe promoter Sentin delays stable kinetochore–microtubule attachment in oocytes

    PubMed Central

    Głuszek, A. Agata; Cullen, C. Fiona; Li, Wenjing; Battaglia, Rachel A.; Radford, Sarah J.; Costa, Mariana F.; McKim, Kim S.; Goshima, Gohta

    2015-01-01

    The critical step in meiosis is to attach homologous chromosomes to the opposite poles. In mouse oocytes, stable microtubule end-on attachments to kinetochores are not established until hours after spindle assembly, and phosphorylation of kinetochore proteins by Aurora B/C is responsible for the delay. Here we demonstrated that microtubule ends are actively prevented from stable attachment to kinetochores until well after spindle formation in Drosophila melanogaster oocytes. We identified the microtubule catastrophe-promoting complex Sentin-EB1 as a major factor responsible for this delay. Without this activity, microtubule ends precociously form robust attachments to kinetochores in oocytes, leading to a high proportion of homologous kinetochores stably attached to the same pole. Therefore, regulation of microtubule ends provides an alternative novel mechanism to delay stable kinetochore–microtubule attachment in oocytes. PMID:26668329

  15. End-on microtubule-dynein interactions and pulling-based positioning of microtubule organizing centers

    PubMed Central

    Laan, Liedewij; Roth, Sophie; Dogterom, Marileen

    2012-01-01

    During important cellular processes such as centrosome and spindle positioning, dynein at the cortex interacts with dynamic microtubules in an apparent “end-on” fashion. It is well-established that dynein can generate forces by moving laterally along the microtubule lattice, but much less is known about dynein’s interaction with dynamic microtubule ends. In this paper, we review recent in vitro experiments that show that dynein, attached to an artificial cortex, is able to capture microtubule ends, regulate microtubule dynamics and mediate the generation of pulling forces on shrinking microtubules. We further review existing ideas on the involvement of dynein-mediated cortical pulling forces in the positioning of microtubule organizing centers such as centrosomes. Recent in vitro experiments have demonstrated that cortical pulling forces in combination with pushing forces can lead to reliable centering of microtubule asters in quasi two-dimensional microfabricated chambers. In these experiments, pushing leads to slipping of microtubule ends along the chamber boundaries, resulting in an anisotropic distribution of cortical microtubule contacts that favors centering, once pulling force generators become engaged. This effect is predicted to be strongly geometry-dependent, and we therefore finally discuss ongoing efforts to repeat these experiments in three-dimensional, spherical and deformable geometries. PMID:22895049

  16. Centrophilin: a novel mitotic spindle protein involved in microtubule nucleation

    PubMed Central

    1991-01-01

    A novel protein has been identified which may serve a key function in nucleating spindle microtubule growth in mitosis. This protein, called centrophilin, is sequentially relocated from the centromeres to the centrosomes to the midbody in a manner dependent on the mitotic phase. Centrophilin was initially detected by immunofluorescence with a monoclonal, primate-specific antibody (2D3) raised against kinetochore- enriched chromosome extract from HeLa cells (Valdivia, M. M., and B. R. Brinkley. 1985. J. Cell Biol. 101:1124-1134). Centrophilin forms prominent crescents at the poles of the metaphase spindle, gradually diminishes during anaphase, and bands the equatorial ends of midbody microtubules in telophase. The formation and breakdown of the spindle and midbody correlates in time and space with the aggregation and disaggregation of centrophilin foci. Immunogold EM reveals that centrophilin is a major component of pericentriolar material in metaphase. During recovery from microtubule inhibition, centrophilin foci act as nucleation sites for the assembly of spindle tubules. The 2D3 probe recognizes two high molecular mass polypeptides, 180 and 210 kD, on immunoblots of whole HeLa cell extract. Taken together, these data and the available literature on microtubule dynamics point inevitably to a singular model for control of spindle tubule turnover. PMID:1991791

  17. Microtubule plus-end tracking proteins and their roles in cell division.

    PubMed

    Ferreira, Jorge G; Pereira, Ana L; Maiato, Helder

    2014-01-01

    Microtubules are cellular components that are required for a variety of essential processes such as cell motility, mitosis, and intracellular transport. This is possible because of the inherent dynamic properties of microtubules. Many of these properties are tightly regulated by a number of microtubule plus-end-binding proteins or +TIPs. These proteins recognize the distal end of microtubules and are thus in the right context to control microtubule dynamics. In this review, we address how microtubule dynamics are regulated by different +TIP families, focusing on how functionally diverse +TIPs spatially and temporally regulate microtubule dynamics during animal cell division.

  18. Characterization of the role of calcium in regulating the microtubule-destabilizing activity of MDP25.

    PubMed

    Qin, Tao; Li, Jiejie; Yuan, Ming; Mao, Tonglin

    2012-07-01

    Regulation of cell elongation is important for plant morphogenesis. Many studies have shown that cortical microtubules play crucial roles during cell elongation and that microtubule stability, organization, and dynamics are regulated by microtubule regulatory proteins. Recently, we reported that a novel protein from Arabidopsis, termed microtubule-destabilizing protein 25 (MDP25), functions as a negative regulator of hypocotyl cell elongation. MDP25 destabilizes microtubules and exerts its effect on microtubules as a result of transient elevation of cytosolic calcium levels.

  19. Regulation of microtubule destabilizing activity of Op18/stathmin downstream of Rac1.

    PubMed

    Wittmann, Torsten; Bokoch, Gary M; Waterman-Storer, Clare M

    2004-02-13

    In the leading edge of migrating cells, a subset of microtubules exhibits net growth in a Rac1- and p21-activated kinase-dependent manner. Here, we explore the possibility of whether phosphorylation and inactivation of the microtubule-destabilizing protein Op18/stathmin could be a mechanism regulating microtubule dynamics downstream of Rac1 and p21-activated kinases. We find that, in vitro, Pak1 phosphorylates Op18/stathmin specifically at serine 16 and inactivates its catastrophe promoting activity in biochemical and time lapse microscopy microtubule assembly assays. Furthermore, phosphorylation of either serine 16 or 63 is sufficient to inhibit Op18/stathmin in vitro. In cells, the microtubule-destabilizing effect of an excess of Op18/stathmin can be partially overcome by expression of constitutively active Rac1(Q61L), which is dependent on Pak activity, suggesting that the microtubule cytoskeleton can be regulated through inactivation of Op18/stathmin downstream of Rac1 and Pak in vivo. However, in vivo, Pak1 activity alone is not sufficient to phosphorylate Op18, indicating that additional pathways downstream of Rac1 are required for Op18 regulation.

  20. Stag3 regulates microtubule stability to maintain euploidy during mouse oocyte meiotic maturation

    PubMed Central

    Zhang, Mianqun; Dai, Xiaoxin; Sun, Yalu; Lu, Yajuan; Zhou, Changyin; Miao, Yilong; Wang, Ying; Xiong, Bo

    2017-01-01

    Stag3, a meiosis-specific subunit of cohesin complex, has been demonstrated to function in both male and female reproductive systems in mammals. However, its roles during oocyte meiotic maturation have not been fully defined. In the present study, we report that Stag3 uniquely accumulates on the spindle apparatus and colocalizes with microtubule fibers during mouse oocyte meiotic maturation. Depletion of Stag3 by gene-targeting morpholino disrupts normal spindle assembly and chromosome alignment in oocytes. We also find that depletion of Stag3 reduces the acetylated level of tubulin and microtubule resistance to microtubule depolymerizing drug, suggesting that Stag3 is required for microtubule stability. Consistent with these observations, kinetochore-microtubule attachment, an important mechanism controlling chromosome alignment, is severely impaired in Stag3-depleted oocytes, resultantly causing the significantly increased incidence of aneuploid eggs. Collectively, our data reveal that Stag3 is a novel regulator of microtubule dynamics to ensure euploidy during moue oocyte meiotic maturation. PMID:27906670

  1. Further evidence for microtubule-independent dimerization of TPPP/p25

    PubMed Central

    Oláh, J.; Szénási, T.; Szunyogh, S.; Szabó, A.; Lehotzky, A.; Ovádi, J.

    2017-01-01

    Tubulin Polymerization Promoting Protein (TPPP/p25) is a brain-specific disordered protein that modulates the dynamics and stability of the microtubule network by its assembly promoting, cross-linking and acetylation enhancing activities. In normal brain it is expressed primarily in differentiated oligodendrocytes; however, at pathological conditions it is enriched in inclusions of both neurons and oligodendrocytes characteristic for Parkinson’s disease and multiple system atrophy, respectively. The objective of this paper is to highlight a critical point of a recently published Skoufias’s paper in which the crucial role of the microtubules in TPPP/p25 dimerization leading to microtubule bundling was suggested. However, our previous and present data provide evidence for the microtubule-independent dimerization of TPPP/p25 and its stabilization by disulphide bridges. In addition, our bimolecular fluorescence complementation experiments revealed the dimerization ability of both the full length and the terminal-free (CORE) TPPP/p25 forms, however, while TPPP/p25 aligned along the bundled microtubule network, the associated CORE segments distributed mostly homogeneously within the cytosol. Now, we identified a molecular model from the possible ones suggested in the Skoufias’s paper that could be responsible for stabilization of the microtubule network in the course of the oligodendrocyte differentiation, consequently in the constitution of the myelin sheath. PMID:28074911

  2. Talin-KANK1 interaction controls the recruitment of cortical microtubule stabilizing complexes to focal adhesions

    PubMed Central

    Bouchet, Benjamin P; Gough, Rosemarie E; Ammon, York-Christoph; van de Willige, Dieudonnée; Post, Harm; Jacquemet, Guillaume; Altelaar, AF Maarten; Heck, Albert JR; Goult, Benjamin T; Akhmanova, Anna

    2016-01-01

    The cross-talk between dynamic microtubules and integrin-based adhesions to the extracellular matrix plays a crucial role in cell polarity and migration. Microtubules regulate the turnover of adhesion sites, and, in turn, focal adhesions promote the cortical microtubule capture and stabilization in their vicinity, but the underlying mechanism is unknown. Here, we show that cortical microtubule stabilization sites containing CLASPs, KIF21A, LL5β and liprins are recruited to focal adhesions by the adaptor protein KANK1, which directly interacts with the major adhesion component, talin. Structural studies showed that the conserved KN domain in KANK1 binds to the talin rod domain R7. Perturbation of this interaction, including a single point mutation in talin, which disrupts KANK1 binding but not the talin function in adhesion, abrogates the association of microtubule-stabilizing complexes with focal adhesions. We propose that the talin-KANK1 interaction links the two macromolecular assemblies that control cortical attachment of actin fibers and microtubules. DOI: http://dx.doi.org/10.7554/eLife.18124.001 PMID:27410476

  3. Identification of novel microtubule-binding proteins by taxol-mediated microtubule stabilization and mass spectrometry analysis

    PubMed Central

    He, Xianfei; Liu, Zhu; He, Qianqian; Qin, Juan; Liu, Ningning; Zhang, Linlin; Li, Dengwen; Zhou, Jun; Shui, Wenqing; Liu, Min

    2015-01-01

    Microtubule-binding proteins (MBPs) are structurally and functionally diverse regulators of microtubule-mediated cellular processes. Alteration of MBPs has been implicated in the pathogenesis of human diseases, including cancer. MBPs can stabilize or destabilize microtubules or move along microtubules to transport various cargoes. In addition, MBPs can control microtubule dynamics through direct interaction with microtubules or coordination with other proteins. To better understand microtubule structure and function, it is necessary to identify additional MBPs. In this study, we isolated microtubules and MBPs from mammalian cells by a taxol-based method and then profiled a panel of MBPs by mass spectrometry. We discovered a number of previously uncharacterized MBPs, including several membrane-associated proteins and proteins involved in post-translational modifications, in addition to several structural components. These results support the notion that microtubules have a wide range of functions and may undergo more exquisite regulation than previously recognized. PMID:26445615

  4. Multiscale modeling and simulation of microtubule–motor-protein assemblies

    PubMed Central

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

    2016-01-01

    Microtubules and motor proteins self-organize into biologically important assemblies including the mitotic spindle and the centrosomal microtubule array. Outside of cells, microtubule-motor mixtures can form novel active liquid-crystalline materials driven out of equilibrium by adenosine triphosphate–consuming motor proteins. Microscopic motor activity causes polarity-dependent interactions between motor proteins and microtubules, but how these interactions yield larger-scale dynamical behavior such as complex flows and defect dynamics is not well understood. We develop a multiscale theory for microtubule-motor systems in which Brownian dynamics simulations of polar microtubules driven by motors are used to study microscopic organization and stresses created by motor-mediated microtubule interactions. We identify polarity-sorting and crosslink tether relaxation as two polar-specific sources of active destabilizing stress. We then develop a continuum Doi-Onsager model that captures polarity sorting and the hydrodynamic flows generated by these polar-specific active stresses. In simulations of active nematic flows on immersed surfaces, the active stresses drive turbulent flow dynamics and continuous generation and annihilation of disclination defects. The dynamics follow from two instabilities, and accounting for the immersed nature of the experiment yields unambiguous characteristic length and time scales. When turning off the hydrodynamics in the Doi-Onsager model, we capture formation of polar lanes as observed in the Brownian dynamics simulation. PMID:26764729

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

    PubMed

    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.

  6. Structure, dynamics, assembly, and evolution of protein complexes.

    PubMed

    Marsh, Joseph A; Teichmann, Sarah A

    2015-01-01

    The assembly of individual proteins into functional complexes is fundamental to nearly all biological processes. In recent decades, many thousands of homomeric and heteromeric protein complex structures have been determined, greatly improving our understanding of the fundamental principles that control symmetric and asymmetric quaternary structure organization. Furthermore, our conception of protein complexes has moved beyond static representations to include dynamic aspects of quaternary structure, including conformational changes upon binding, multistep ordered assembly pathways, and structural fluctuations occurring within fully assembled complexes. Finally, major advances have been made in our understanding of protein complex evolution, both in reconstructing evolutionary histories of specific complexes and in elucidating general mechanisms that explain how quaternary structure tends to evolve. The evolution of quaternary structure occurs via changes in self-assembly state or through the gain or loss of protein subunits, and these processes can be driven by both adaptive and nonadaptive influences.

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

    PubMed Central

    Barrack, Keri L.; Fyfe, Paul K.; Hunter, William N.

    2015-01-01

    Tubulin-binding cofactor A (TBCA) participates in microtubule formation, a key process in eukaryotic biology to create the cytoskeleton. There is little information on how TBCA might interact with β-tubulin en route to microtubule biogenesis. To address this, the protozoan Leishmania major was targeted as a model system. The crystal structure of TBCA and comparisons with three orthologous proteins are presented. The presence of conserved features infers that electrostatic interactions that are likely to involve the C-terminal tail of β-tubulin are key to association. This study provides a reagent and template to support further work in this area. PMID:25945706

  8. History-dependent catastrophes regulate axonal microtubule behavior.

    PubMed

    Stepanova, Tatiana; Smal, Ihor; van Haren, Jeffrey; Akinci, Umut; Liu, Zhe; Miedema, Marja; Limpens, Ronald; van Ham, Marco; van der Reijden, Michael; Poot, Raymond; Grosveld, Frank; Mommaas, Mieke; Meijering, Erik; Galjart, Niels

    2010-06-08

    In Chinese hamster ovary cells, microtubules originate at the microtubule organizing center (MTOC) and grow persistently toward the cell edge, where they undergo catastrophe. In axons, microtubule dynamics must be regulated differently because microtubules grow parallel to the plasma membrane and there is no MTOC. GFP-tagged microtubule plus end tracking proteins (+TIPs) mark the ends of growing neuronal microtubules. Their fluorescent "comet-like" pattern reflects turnover of +TIP binding sites. Using GFP-tagged +TIPs and fluorescence-based segmentation and tracking tools, we show that axonal microtubules grow with a constant average velocity and that they undergo catastrophes at random positions, yet in a programmed fashion. Using protein depletion approaches, we find that the +TIPs CLIP-115 and CLIP-170 affect average microtubule growth rate and growth distance in neurons but not the duration of a microtubule growth event. In N1E-115 neuroblastoma cells, we find that EB1, the core +TIP, regulates microtubule growth rate, growth distance, and duration, consistent with in vitro data. Combined, our data suggest that CLIPs influence the axonal microtubule/tubulin ratio, whereas EB1 stimulates microtubule growth and structural transitions at microtubule ends, thereby regulating microtubule catastrophes and the turnover of +TIP binding sites.

  9. Structure and dynamics of layered molecular assemblies

    NASA Astrophysics Data System (ADS)

    Horne, Jennifer Conrad

    This dissertation focuses on the goal of understanding and controlling layered material properties from a molecular perspective. With this understanding, materials can be synthetically tailored to exhibit predetermined bulk properties. This investigation describes the optical response of a family of metal-phosphonate (MP) monolayers and multilayers, materials that are potentially useful because the films are easy to synthesize and are chemically and thermally stable. MP films have shown potential in a variety of chemical sensing and optical applications, and in this dissertation, the suitability of MP films for optical information storage is explored For this application, the extent of photonic energy transport within and between optically active layers is an important factor in determining the stability and specificity of optical modifications made to a material. Intralayer and interlayer energy transport processes can be studied selectively in MP films because the composition, and thus the properties, of each layer are controlled synthetically. It was determined by fluorescence relaxation dynamics in conjunction with atomic force microscopy (AFM) that the substrate and layer morphologies are key factors in determining the layer optical and physical properties. The initial MP layers in a multilayer are structurally heterogeneous, characterized by randomly distributed islands that are ~50 A in diameter. The population dynamics measured for these layers are non-exponential, chromophore concentration-independent, and identical for two different chromophores. The data is explained in the context of an excitation hopping model in a system where the surface is characterized by islands of aggregated chromophores as well as non-aggregated monomers. Within a MP monolayer, the dynamics are dominated by intra-island excitation hopping. Forster (dipolar) energy transfer between the energetically overlapped chromophores does not play a significant role in determining the

  10. Molecular architecture of the Dam1 complex–microtubule interaction

    PubMed Central

    Legal, Thibault; Zou, Juan; Sochaj, Alicja; Rappsilber, Juri

    2016-01-01

    Mitosis is a highly regulated process that allows the equal distribution of the genetic material to the daughter cells. Chromosome segregation requires the formation of a bipolar mitotic spindle and assembly of a multi-protein structure termed the kinetochore to mediate attachments between condensed chromosomes and spindle microtubules. In budding yeast, a single microtubule attaches to each kinetochore, necessitating robustness and processivity of this kinetochore–microtubule attachment. The yeast kinetochore-localized Dam1 complex forms a direct interaction with the spindle microtubule. In vitro, the Dam1 complex assembles as a ring around microtubules and couples microtubule depolymerization with cargo movement. However, the subunit organization within the Dam1 complex, its higher-order oligomerization and how it interacts with microtubules remain under debate. Here, we used chemical cross-linking and mass spectrometry to define the architecture and subunit organization of the Dam1 complex. This work reveals that both the C termini of Duo1 and Dam1 subunits interact with the microtubule and are critical for microtubule binding of the Dam1 complex, placing Duo1 and Dam1 on the inside of the ring structure. Integrating this information with available structural data, we provide a coherent model for how the Dam1 complex self-assembles around microtubules. PMID:26962051

  11. The dynamics of filament assembly define cytoskeletal network morphology

    PubMed Central

    Foffano, Giulia; Levernier, Nicolas; Lenz, Martin

    2016-01-01

    The actin cytoskeleton is a key component in the machinery of eukaryotic cells, and it self-assembles out of equilibrium into a wide variety of biologically crucial structures. Although the molecular mechanisms involved are well characterized, the physical principles governing the spatial arrangement of actin filaments are not understood. Here we propose that the dynamics of actin network assembly from growing filaments results from a competition between diffusion, bundling and steric hindrance, and is responsible for the range of observed morphologies. Our model and simulations thus predict an abrupt dynamical transition between homogeneous and strongly bundled networks as a function of the actin polymerization rate. This suggests that cells may effect dramatic changes to their internal architecture through minute modifications of their nonequilibrium dynamics. Our results are consistent with available experimental data. PMID:28000681

  12. The dynamics of filament assembly define cytoskeletal network morphology

    NASA Astrophysics Data System (ADS)

    Foffano, Giulia; Levernier, Nicolas; Lenz, Martin

    2016-12-01

    The actin cytoskeleton is a key component in the machinery of eukaryotic cells, and it self-assembles out of equilibrium into a wide variety of biologically crucial structures. Although the molecular mechanisms involved are well characterized, the physical principles governing the spatial arrangement of actin filaments are not understood. Here we propose that the dynamics of actin network assembly from growing filaments results from a competition between diffusion, bundling and steric hindrance, and is responsible for the range of observed morphologies. Our model and simulations thus predict an abrupt dynamical transition between homogeneous and strongly bundled networks as a function of the actin polymerization rate. This suggests that cells may effect dramatic changes to their internal architecture through minute modifications of their nonequilibrium dynamics. Our results are consistent with available experimental data.

  13. The KLP-7 Residue S546 Is a Putative Aurora Kinase Site Required for Microtubule Regulation at the Centrosome in C. elegans.

    PubMed

    Han, Xue; Adames, Kelly; Sykes, Ellen M E; Srayko, Martin

    2015-01-01

    Regulation of microtubule dynamics is essential for many cellular processes, including proper assembly and function of the mitotic spindle. The kinesin-13 microtubule-depolymerizing enzymes provide one mechanism to regulate microtubule behaviour temporally and spatially. Vertebrate MCAK locates to chromatin, kinetochores, spindle poles, microtubule tips, and the cytoplasm, implying that the regulation of kinesin-13 activity and subcellular targeting is complex. Phosphorylation of kinesin-13 by Aurora kinase inhibits microtubule depolymerization activity and some Aurora phosphorylation sites on kinesin-13 are required for subcellular localization. Herein, we determine that a C. elegans deletion mutant klp-7(tm2143) causes meiotic and mitotic defects that are consistent with an increase in the amount of microtubules in the cytoplasmic and spindle regions of meiotic embryos, and an increase in microtubules emanating from centrosomes. We show that KLP-7 is phosphorylated by Aurora A and Aurora B kinases in vitro, and that the phosphorylation by Aurora A is stimulated by TPXL-1. Using a structure-function approach, we establish that one putative Aurora kinase site, S546, within the C-terminal part of the core domain is required for the function, but not subcellular localization, of KLP-7 in vivo. Furthermore, FRAP analysis reveals microtubule-dependent differences in the turnover of KLP-7(S546A) and KLP-7(S546E) mutant proteins at the centrosome, suggesting a possible mechanism for the regulation of KLP-7 by Aurora kinase.

  14. The KLP-7 Residue S546 Is a Putative Aurora Kinase Site Required for Microtubule Regulation at the Centrosome in C. elegans

    PubMed Central

    Han, Xue; Adames, Kelly; Sykes, Ellen M. E.; Srayko, Martin

    2015-01-01

    Regulation of microtubule dynamics is essential for many cellular processes, including proper assembly and function of the mitotic spindle. The kinesin-13 microtubule-depolymerizing enzymes provide one mechanism to regulate microtubule behaviour temporally and spatially. Vertebrate MCAK locates to chromatin, kinetochores, spindle poles, microtubule tips, and the cytoplasm, implying that the regulation of kinesin-13 activity and subcellular targeting is complex. Phosphorylation of kinesin-13 by Aurora kinase inhibits microtubule depolymerization activity and some Aurora phosphorylation sites on kinesin-13 are required for subcellular localization. Herein, we determine that a C. elegans deletion mutant klp-7(tm2143) causes meiotic and mitotic defects that are consistent with an increase in the amount of microtubules in the cytoplasmic and spindle regions of meiotic embryos, and an increase in microtubules emanating from centrosomes. We show that KLP-7 is phosphorylated by Aurora A and Aurora B kinases in vitro, and that the phosphorylation by Aurora A is stimulated by TPXL-1. Using a structure-function approach, we establish that one putative Aurora kinase site, S546, within the C-terminal part of the core domain is required for the function, but not subcellular localization, of KLP-7 in vivo. Furthermore, FRAP analysis reveals microtubule-dependent differences in the turnover of KLP-7(S546A) and KLP-7(S546E) mutant proteins at the centrosome, suggesting a possible mechanism for the regulation of KLP-7 by Aurora kinase. PMID:26168236

  15. Dynamic phases, pinning, and pattern formation for driven dislocation assemblies

    DOE PAGES

    Zhou, Caizhi; Reichhardt, Charles; Olson Reichhardt, Cynthia J.; ...

    2015-01-23

    We examine driven dislocation assemblies and show that they can exhibit a set of dynamical phases remarkably similar to those of driven systems with quenched disorder such as vortices in superconductors, magnetic domain walls, and charge density wave materials. These phases include pinned-jammed, fluctuating, and dynamically ordered states, and each produces distinct dislocation patterns as well as specific features in the noise fluctuations and transport properties. Lastly, our work suggests that many of the results established for systems with quenched disorder undergoing plastic depinning transitions can be applied to dislocation systems, providing a new approach for understanding pattern formation andmore » dynamics in these systems.« less

  16. Dynamic phases, pinning, and pattern formation for driven dislocation assemblies

    SciTech Connect

    Zhou, Caizhi; Reichhardt, Charles; Olson Reichhardt, Cynthia J.; Beyerlein, Irene J.

    2015-01-23

    We examine driven dislocation assemblies and show that they can exhibit a set of dynamical phases remarkably similar to those of driven systems with quenched disorder such as vortices in superconductors, magnetic domain walls, and charge density wave materials. These phases include pinned-jammed, fluctuating, and dynamically ordered states, and each produces distinct dislocation patterns as well as specific features in the noise fluctuations and transport properties. Lastly, our work suggests that many of the results established for systems with quenched disorder undergoing plastic depinning transitions can be applied to dislocation systems, providing a new approach for understanding pattern formation and dynamics in these systems.

  17. A novel microtubule inhibitor, MT3-037, causes cancer cell apoptosis by inducing mitotic arrest and interfering with microtubule dynamics

    PubMed Central

    Chang, Ling-Chu; Yu, Yung-Luen; Hsieh, Min-Tsang; Wang, Sheng-Hung; Chou, Ruey-Hwang; Huang, Wei-Chien; Lin, Hui-Yi; Hung, Hsin-Yi; Huang, Li-Jiau; Kuo, Sheng-Chu

    2016-01-01

    We investigated the anticancer potential of a new synthetic compound, 7-(3-fluorophenyl)-4-methylpyrido-[2,3-d]pyrimidin-5(8H)-one (MT3-037). We found that MT3-037 effectively decreased the cancer cell viability by inducing apoptosis. MT3-037 treatments led to cell cycle arrest at M phase, with a marked increase in both expression of cyclin B1 and cyclin-dependent kinase 1 (CDK1) as well as in CDK1 kinase activity. Key proteins that regulate mitotic spindle dynamics, including survivin, Aurora A/B kinases, and polo-like kinase 1 (PLK1), were activated in MT3-037-treated cells. MT3-037-induced apoptosis was accompanied by activation of a pro-apoptotic factor, FADD, and the inactivation of apoptosis inhibitors, Bcl-2 and Bcl-xL, resulting in the cleavage/activation of caspases. The activation of c-Jun N-terminal kinase (JNK) was associated with MT3-037-induced CDK1 and Aurora A/B activation and apoptosis. Immunofluorescence staining of tubulin indicated that MT3-037 altered tubulin networks in cancer cells. Moreover, an in vitro tubulin polymerization assay revealed that MT3-037 inhibited the tubulin polymerization by direct binding to tubulin. Molecular docking studies and binding site completion assays revealed that MT3-037 binds to the colchicine-binding site. Furthermore, MT3-037 significantly inhibited the tumor growth in both MDAMB-468 and Erlotinib-resistant MDA-MB-468 xenograft mouse models. In addition, MT3-037 inhibited the angiogenesis and disrupted the tube formation by human endothelial cells. Our study demonstrates that MT3-037 is a potential tubulin-disrupting agent for antitumor therapy. PMID:27186428

  18. Dynamic self-assembly and control of microfluidic particle crystals

    PubMed Central

    Lee, Wonhee; Amini, Hamed; Stone, Howard A.; Di Carlo, Dino

    2010-01-01

    Engineered two-phase microfluidic systems have recently shown promise for computation, encryption, and biological processing. For many of these systems, complex control of dispersed-phase frequency and switching is enabled by nonlinearities associated with interfacial stresses. Introducing nonlinearity associated with fluid inertia has recently been identified as an easy to implement strategy to control two-phase (solid-liquid) microscale flows. By taking advantage of inertial effects we demonstrate controllable self-assembling particle systems, uncover dynamics suggesting a unique mechanism of dynamic self-assembly, and establish a framework for engineering microfluidic structures with the possibility of spatial frequency filtering. Focusing on the dynamics of the particle–particle interactions reveals a mechanism for the dynamic self-assembly process; inertial lift forces and a parabolic flow field act together to stabilize interparticle spacings that otherwise would diverge to infinity due to viscous disturbance flows. The interplay of the repulsive viscous interaction and inertial lift also allow us to design and implement microfluidic structures that irreversibly change interparticle spacing, similar to a low-pass filter. Although often not considered at the microscale, nonlinearity due to inertia can provide a platform for high-throughput passive control of particle positions in all directions, which will be useful for applications in flow cytometry, tissue engineering, and metamaterial synthesis. PMID:21149674

  19. Transformation and patterning of supermicelles using dynamic holographic assembly

    PubMed Central

    Gould, Oliver E.C.; Qiu, Huibin; Lunn, David J.; Rowden, John; Harniman, Robert L.; Hudson, Zachary M.; Winnik, Mitchell A.; Miles, Mervyn J.; Manners, Ian

    2015-01-01

    Although the solution self-assembly of block copolymers has enabled the fabrication of a broad range of complex, functional nanostructures, their precise manipulation and patterning remain a key challenge. Here we demonstrate that spherical and linear supermicelles, supramolecular structures held together by non-covalent solvophobic and coordination interactions and formed by the hierarchical self-assembly of block copolymer micelle and block comicelle precursors, can be manipulated, transformed and patterned with mediation by dynamic holographic assembly (optical tweezers). This allows the creation of new and stable soft-matter superstructures far from equilibrium. For example, individual spherical supermicelles can be optically held in close proximity and photocrosslinked through controlled coronal chemistry to generate linear oligomeric arrays. The use of optical tweezers also enables the directed deposition and immobilization of supermicelles on surfaces, allowing the precise creation of arrays of soft-matter nano-objects with potentially diverse functionality and a range of applications. PMID:26627644

  20. Arabidopsis AUGMIN Subunit8 Is a Microtubule Plus-End Binding Protein That Promotes Microtubule Reorientation in Hypocotyls[C][W

    PubMed Central

    Cao, Lingyan; Wang, Linhai; Zheng, Min; Cao, Hong; Ding, Lian; Zhang, Xiaolan; Fu, Ying

    2013-01-01

    In plant cells, cortical microtubules provide tracks for cellulose-synthesizing enzymes and regulate cell division, growth, and morphogenesis. The role of microtubules in these essential cellular processes depends on the spatial arrangement of the microtubules. Cortical microtubules are reoriented in response to changes in cell growth status and cell shape. Therefore, an understanding of the mechanism that underlies the change in microtubule orientation will provide insight into plant cell growth and morphogenesis. This study demonstrated that AUGMIN subunit8 (AUG8) in Arabidopsis thaliana is a novel microtubule plus-end binding protein that participates in the reorientation of microtubules in hypocotyls when cell elongation slows down. AUG8 bound to the plus ends of microtubules and promoted tubulin polymerization in vitro. In vivo, AUG8 was recruited to the microtubule branch site immediately before nascent microtubules branched out. It specifically associated with the plus ends of growing cortical microtubules and regulated microtubule dynamics, which facilitated microtubule reorientation when microtubules changed their growth trajectory or encountered obstacle microtubules during microtubule reorientation. This study thus reveals a novel mechanism underlying microtubule reorientation that is critical for modulating cell elongation in Arabidopsis. PMID:23735294

  1. Reeling in chromosomes to spindle poles: The roles of microtubule-destabilizing enzymes in mitotic spindle dynamics

    NASA Astrophysics Data System (ADS)

    Sharp, David

    2004-03-01

    The central purpose of mitosis is achieved during anaphase when sister chromatids disjoin and translocate towards opposite poles of a microtubule-based machine termed mitotic spindle. We have identified two functionally distinct microtubule-destabilizing Kin I kinesin enzymes that are responsible for normal chromatid-to-pole motion during anaphase in Drosophila. One of them, KLP59C, is required to depolymerize MTs specifically at their kinetochore-associated "plus-ends" such that chromosomes 'chew' their way poleward. The second, KLP10A, is required to depolymerize MTs specifically at their pole-associated "minus-ends" thereby 'reeling' chromosomes into spindle poles. These findings provide the first description of the protein machinery that drives anaphase chromatid segregation by actively depolymerizing kinetochore MTs at both ends.

  2. Insights into Antiparallel Microtubule Crosslinking by PRC1, a Conserved Nonmotor Microtubule Binding Protein

    SciTech Connect

    Subramanian, Radhika; Wilson-Kubalek, Elizabeth M.; Arthur, Christopher P.; Bick, Matthew J.; Campbell, Elizabeth A.; Darst, Seth A.; Milligan, Ronald A.; Kapoor, Tarun M.

    2010-09-03

    Formation of microtubule architectures, required for cell shape maintenance in yeast, directional cell expansion in plants and cytokinesis in eukaryotes, depends on antiparallel microtubule crosslinking by the conserved MAP65 protein family. Here, we combine structural and single molecule fluorescence methods to examine how PRC1, the human MAP65, crosslinks antiparallel microtubules. We find that PRC1's microtubule binding is mediated by a structured domain with a spectrin-fold and an unstructured Lys/Arg-rich domain. These two domains, at each end of a homodimer, are connected by a linkage that is flexible on single microtubules, but forms well-defined crossbridges between antiparallel filaments. Further, we show that PRC1 crosslinks are compliant and do not substantially resist filament sliding by motor proteins in vitro. Together, our data show how MAP65s, by combining structural flexibility and rigidity, tune microtubule associations to establish crosslinks that selectively mark antiparallel overlap in dynamic cytoskeletal networks.

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

    SciTech Connect

    Barrack, Keri L.; Fyfe, Paul K.; Hunter, William N.

    2015-04-21

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

  4. Detection and quantification of microtubule detachment from centrosomes and spindle poles.

    PubMed

    Ganguly, Anutosh; Yang, Hailing; Cabral, Fernando

    2013-01-01

    Microtubule detachment from microtubule organizing centers is an important cellular process required for normal cell proliferation. When cells enter mitosis, microtubule turnover increases along with a concurrent increase in microtubule detachment. MCAK, a kinesin-related protein whose abundance is highest during the early stages of mitosis, has been shown to regulate microtubule detachment. Abnormal increases or decreases in the frequency of detachment interfere with spindle function and inhibit cell division. It has been shown that drugs able to promote microtubule assembly (e.g., paclitaxel, epothilones) prevent cell division by suppressing microtubule detachment from centrosomes. Conversely, cytotoxic concentrations of microtubule destabilizing drugs (e.g., vinblastine, nocodazole), tubulin mutations that cause paclitaxel resistance, and specific β-tubulin isotypes increase the frequency of microtubule detachment. In this chapter, we describe a method to calculate the frequency of microtubule detachment by transfecting cells with EGFP-MAP4 and directly observing detachment by live cell imaging.

  5. Human kinetochores are swivel joints that mediate microtubule attachments

    PubMed Central

    Smith, Chris A; McAinsh, Andrew D; Burroughs, Nigel J

    2016-01-01

    Chromosome segregation is a mechanical process that requires assembly of the mitotic spindle – a dynamic microtubule-based force-generating machine. Connections to this spindle are mediated by sister kinetochore pairs, that form dynamic end-on attachments to microtubules emanating from opposite spindle poles. This bi-orientation generates forces that have been reported to stretch the kinetochore itself, which has been suggested to stabilise attachment and silence the spindle checkpoint. We reveal using three dimensional tracking that the outer kinetochore domain can swivel around the inner kinetochore/centromere, which results in large reductions in intra-kinetochore distance (delta) when viewed in lower dimensions. We show that swivel provides a mechanical flexibility that enables kinetochores at the periphery of the spindle to engage microtubules. Swivel reduces as cells approach anaphase, suggesting an organisational change linked to checkpoint satisfaction and/or obligatory changes in kinetochore mechanochemistry may occur before dissolution of sister chromatid cohesion. DOI: http://dx.doi.org/10.7554/eLife.16159.001 PMID:27591356

  6. Evolutionary dynamics in a simple model of self-assembly

    NASA Astrophysics Data System (ADS)

    Johnston, Iain G.; Ahnert, Sebastian E.; Doye, Jonathan P. K.; Louis, Ard A.

    2011-06-01

    We investigate the evolutionary dynamics of an idealized model for the robust self-assembly of two-dimensional structures called polyominoes. The model includes rules that encode interactions between sets of square tiles that drive the self-assembly process. The relationship between the model’s rule set and its resulting self-assembled structure can be viewed as a genotype-phenotype map and incorporated into a genetic algorithm. The rule sets evolve under selection for specified target structures. The corresponding complex fitness landscape generates rich evolutionary dynamics as a function of parameters such as the population size, search space size, mutation rate, and method of recombination. Furthermore, these systems are simple enough that in some cases the associated model genome space can be completely characterized, shedding light on how the evolutionary dynamics depends on the detailed structure of the fitness landscape. Finally, we apply the model to study the emergence of the preference for dihedral over cyclic symmetry observed for homomeric protein tetramers.

  7. Critical dynamics of randomly assembled and diluted threshold networks

    NASA Astrophysics Data System (ADS)

    Kürten, Karl E.; Clark, John W.

    2008-04-01

    The dynamical behavior of a class of randomly assembled networks of binary threshold units subject to random deletion of connections is studied based on the annealed approximation suitable in the thermodynamic limit. The dynamical phase diagram is constructed for several forms of the probability density distribution of nonvanishing connection strengths. The family of power-law distribution functions ρ0(x)=(1-α)/(2|x|α) is found to play a special role in expanding the domain of stable, ordered dynamics at the expense of the disordered, “chaotic” phase. Relationships with other recent studies of the dynamics of complex networks allowing for variable in-degree of the units are explored. The relevance of the pruning of network connections to neural modeling and developmental neurobiology is discussed.

  8. Microtubules regulate disassembly of epithelial apical junctions

    PubMed Central

    Ivanov, Andrei I; McCall, Ingrid C; Babbin, Brian; Samarin, Stanislav N; Nusrat, Asma; Parkos, Charles A

    2006-01-01

    Background Epithelial tight junction (TJ) and adherens junction (AJ) form the apical junctional complex (AJC) which regulates cell-cell adhesion, paracellular permeability and cell polarity. The AJC is anchored on cytoskeletal structures including actin microfilaments and microtubules. Such cytoskeletal interactions are thought to be important for the assembly and remodeling of apical junctions. In the present study, we investigated the role of microtubules in disassembly of the AJC in intestinal epithelial cells using a model of extracellular calcium depletion. Results Calcium depletion resulted in disruption and internalization of epithelial TJs and AJs along with reorganization of perijunctional F-actin into contractile rings. Microtubules reorganized into dense plaques positioned inside such F-actin rings. Depolymerization of microtubules with nocodazole prevented junctional disassembly and F-actin ring formation. Stabilization of microtubules with either docetaxel or pacitaxel blocked contraction of F-actin rings and attenuated internalization of junctional proteins into a subapical cytosolic compartment. Likewise, pharmacological inhibition of microtubule motors, kinesins, prevented contraction of F-actin rings and attenuated disassembly of apical junctions. Kinesin-1 was enriched at the AJC in cultured epithelial cells and it also accumulated at epithelial cell-cell contacts in normal human colonic mucosa. Furthermore, immunoprecipitation experiments demonstrated association of kinesin-1 with the E-cadherin-catenin complex. Conclusion Our data suggest that microtubules play a role in disassembly of the AJC during calcium depletion by regulating formation of contractile F-actin rings and internalization of AJ/TJ proteins. PMID:16509970

  9. Mechanical Properties of Doubly Stabilized Microtubule Filaments

    PubMed Central

    Hawkins, Taviare L.; Sept, David; Mogessie, Binyam; Straube, Anne; Ross, Jennifer L.

    2013-01-01

    Microtubules are cytoskeletal filaments responsible for cell morphology and intracellular organization. Their dynamical and mechanical properties are regulated through the nucleotide state of the tubulin dimers and the binding of drugs and/or microtubule-associated proteins. Interestingly, microtubule-stabilizing factors have differential effects on microtubule mechanics, but whether stabilizers have cumulative effects on mechanics or whether one effect dominates another is not clear. This is especially important for the chemotherapeutic drug Taxol, an important anticancer agent and the only known stabilizer that reduces the rigidity of microtubules. First, we ask whether Taxol will combine additively with another stabilizer or whether one stabilizer will dominate another. We call microtubules in the presence of Taxol and another stabilizer, doubly stabilized. Second, since Taxol is often added to a number of cell types for therapeutic purposes, it is important from a biomedical perspective to understand how Taxol added to these systems affects the mechanical properties in treated cells. To address these questions, we use the method of freely fluctuating filaments with our recently developed analysis technique of bootstrapping to determine the distribution of persistence lengths of a large population of microtubules treated with different stabilizers, including Taxol, guanosine-5′ [(α, β)-methyleno] triphosphate, guanosine-5′-O-(3-thiotriphosphate), tau, and MAP4. We find that combinations of these stabilizers have novel effects on the mechanical properties of microtubules. PMID:23561528

  10. Mechanical properties of doubly stabilized microtubule filaments.

    PubMed

    Hawkins, Taviare L; Sept, David; Mogessie, Binyam; Straube, Anne; Ross, Jennifer L

    2013-04-02

    Microtubules are cytoskeletal filaments responsible for cell morphology and intracellular organization. Their dynamical and mechanical properties are regulated through the nucleotide state of the tubulin dimers and the binding of drugs and/or microtubule-associated proteins. Interestingly, microtubule-stabilizing factors have differential effects on microtubule mechanics, but whether stabilizers have cumulative effects on mechanics or whether one effect dominates another is not clear. This is especially important for the chemotherapeutic drug Taxol, an important anticancer agent and the only known stabilizer that reduces the rigidity of microtubules. First, we ask whether Taxol will combine additively with another stabilizer or whether one stabilizer will dominate another. We call microtubules in the presence of Taxol and another stabilizer, doubly stabilized. Second, since Taxol is often added to a number of cell types for therapeutic purposes, it is important from a biomedical perspective to understand how Taxol added to these systems affects the mechanical properties in treated cells. To address these questions, we use the method of freely fluctuating filaments with our recently developed analysis technique of bootstrapping to determine the distribution of persistence lengths of a large population of microtubules treated with different stabilizers, including Taxol, guanosine-5' [(α, β)-methyleno] triphosphate, guanosine-5'-O-(3-thiotriphosphate), tau, and MAP4. We find that combinations of these stabilizers have novel effects on the mechanical properties of microtubules.

  11. Microinjected fluorescent phalloidin in vivo reveals the F-actin dynamics and assembly in higher plant mitotic cells.

    PubMed Central

    Schmit, A C; Lambert, A M

    1990-01-01

    Endosperm mitotic cells microinjected with fluorescent phalloidin enabled us to follow the in vivo dynamics of the F-actin cytoskeleton. The fluorescent probe immediately bound to plant microfilaments. First, we investigated the active rearrangement of F-actin during chromosome migration, which appeared to be slowed down in the presence of phalloidin. These findings were compared with the actin patterns observed in mitotic cells fixed at different stages. Our second aim was to determine the origin of the actin filaments that appear at the equator during anaphase-telophase transition. It is not clear whether this F-actin is newly assembled at the end of mitosis and could control plant cytokinesis or whether it corresponds to a passive redistribution of broken polymers in response to microtubule dynamics. We microinjected the same cells twice, first in metaphase with rhodamine-phalloidin and then in late anaphase with fluorescein isothiocyanate-phalloidin. This technique enabled us to visualize two F-actin populations that are not co-localized, suggesting that actin is newly assembled during cell plate development. These in vivo data shed new light on the role of actin in plant mitosis and cytokinesis. PMID:2136631

  12. Dual Role for Microtubules in Regulating Cortical Contractility during Cytokinesis

    PubMed Central

    Murthy, Kausalya; Wadsworth, Patricia

    2008-01-01

    Microtubules stimulate contractile ring formation in the equatorial cortex and simultaneously suppress contractility in the polar cortex; how they accomplish these differing activities is incompletely understood. We measured the behavior of GFP-actin in mammalian cells treated with nocodazole under conditions that either completely eliminate microtubules or selectively disassemble astral microtubules. Selective disassembly of astral microtubules resulted functional contractile rings that were wider than controls and had altered dynamic activity, as measured by FRAP. Complete microtubule disassembly or selective loss of astral microtubules resulted in wave-like contractile behavior of actin in the non-equatorial cortex and mislocalization of myosin II and Rho. FRAP experiments showed that both contractility and actin polymerization contributed to the wave-like behavior of actin. Wave-like, contractile behavior in anaphase cells was Rho-dependent. We conclude that dynamic astral microtubules function to suppress Rho activation in the nonequatorial cortex, limiting the contractile activity of the polar cortex. PMID:18559890

  13. Cortical microtubule contacts position the spindle in C. elegans embryos.

    PubMed

    Kozlowski, Cleopatra; Srayko, Martin; Nedelec, Francois

    2007-05-04

    Interactions between microtubules and the cell cortex play a critical role in positioning organelles in a variety of biological contexts. Here we used Caenorhabditis elegans as a model system to study how cortex-microtubule interactions position the mitotic spindle in response to polarity cues. Imaging EBP-2::GFP and YFP::alpha-tubulin revealed that microtubules shrink soon after cortical contact, from which we propose that cortical adaptors mediate microtubule depolymerization energy into pulling forces. We also observe association of dynamic microtubules to form astral fibers that persist, despite the catastrophe events of individual microtubules. Computer simulations show that these effects, which are crucially determined by microtubule dynamics, can explain anaphase spindle oscillations and posterior displacement in 3D.

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

    PubMed

    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.

  15. Lessons from in vitro reconstitution analyses of plant microtubule-associated proteins

    PubMed Central

    Hamada, Takahiro

    2014-01-01

    Plant microtubules, composed of tubulin GTPase, are irreplaceable cellular components that regulate the directions of cell expansion and cell division, chromosome segregation and cell plate formation. To accomplish these functions, plant cells organize microtubule structures by regulating microtubule dynamics. Each microtubule localizes to the proper position with repeated growth and shortening. Although it is possible to reconstitute microtubule dynamics with pure tubulin solution in vitro, many microtubule-associated proteins (MAPs) govern microtubule dynamics in cells. In plants, major MAPs are identified as microtubule stabilizers (CLASP and MAP65 etc.), microtubule destabilizers (kinesin-13, katanin, MAP18 and MDP25), and microtubule dynamics promoters (EB1, MAP215, MOR1, MAP200, SPR2). Mutant analyses with forward and reverse genetics have shown the importance of microtubules and individual MAPs in plants. However, it is difficult to understand how each MAP regulates microtubule dynamics, such as growth and shortening, through mutant analyses. In vitro reconstitution analyses with individual purified MAPs and tubulin are powerful tools to reveal how each MAP regulates microtubule dynamics at the molecular level. In this review, I summarize the results of in vitro reconstitution analyses and introduce current models of how each MAP regulates microtubule dynamic instability. PMID:25202315

  16. Lessons from in vitro reconstitution analyses of plant microtubule-associated proteins.

    PubMed

    Hamada, Takahiro

    2014-01-01

    Plant microtubules, composed of tubulin GTPase, are irreplaceable cellular components that regulate the directions of cell expansion and cell division, chromosome segregation and cell plate formation. To accomplish these functions, plant cells organize microtubule structures by regulating microtubule dynamics. Each microtubule localizes to the proper position with repeated growth and shortening. Although it is possible to reconstitute microtubule dynamics with pure tubulin solution in vitro, many microtubule-associated proteins (MAPs) govern microtubule dynamics in cells. In plants, major MAPs are identified as microtubule stabilizers (CLASP and MAP65 etc.), microtubule destabilizers (kinesin-13, katanin, MAP18 and MDP25), and microtubule dynamics promoters (EB1, MAP215, MOR1, MAP200, SPR2). Mutant analyses with forward and reverse genetics have shown the importance of microtubules and individual MAPs in plants. However, it is difficult to understand how each MAP regulates microtubule dynamics, such as growth and shortening, through mutant analyses. In vitro reconstitution analyses with individual purified MAPs and tubulin are powerful tools to reveal how each MAP regulates microtubule dynamics at the molecular level. In this review, I summarize the results of in vitro reconstitution analyses and introduce current models of how each MAP regulates microtubule dynamic instability.

  17. Evolving tip structures can explain age-dependent microtubule catastrophe.

    PubMed

    Coombes, Courtney E; Yamamoto, Ami; Kenzie, Madeline R; Odde, David J; Gardner, Melissa K

    2013-07-22

    Microtubules are key structural and transport elements in cells. The dynamics at microtubule ends are characterized by periods of slow growth, followed by stochastic switching events termed "catastrophes," in which microtubules suddenly undergo rapid shortening. Growing microtubules are thought to be protected from catastrophe by a GTP-tubulin "cap": GTP-tubulin subunits add to the tips of growing microtubules but are subsequently hydrolyzed to GDP-tubulin subunits once they are incorporated into the microtubule lattice. Loss of the GTP-tubulin cap exposes GDP-tubulin subunits at the microtubule tip, resulting in a catastrophe event. However, the mechanistic basis for sudden loss of the GTP cap, leading to catastrophe, is not known. To investigate microtubule catastrophe events, we performed 3D mechanochemical simulations that account for interactions between neighboring protofilaments. We found that there are two separate factors that contribute to catastrophe events in the 3D simulation: the GTP-tubulin cap size, which settles into a steady-state value that depends on the free tubulin concentration during microtubule growth, and the structure of the microtubule tip. Importantly, 3D simulations predict, and both fluorescence and electron microscopy experiments confirm, that microtubule tips become more tapered as the microtubule grows. This effect destabilizes the tip and ultimately contributes to microtubule catastrophe. Thus, the likelihood of a catastrophe event may be intimately linked to the aging physical structure of the growing microtubule tip. These results have important consequences for catastrophe regulation in cells, as microtubule-associated proteins could promote catastrophe events in part by modifying microtubule tip structures.

  18. Molecular dynamics simulations of evaporation-induced nanoparticle assembly

    NASA Astrophysics Data System (ADS)

    Cheng, Shengfeng; Grest, Gary S.

    2013-02-01

    While evaporating solvent is a widely used technique to assemble nano-sized objects into desired superstructures, there has been limited work on how the assembled structures are affected by the physical aspects of the process. We present large scale molecular dynamics simulations of the evaporation-induced assembly of nanoparticles suspended in a liquid that evaporates in a controlled fashion. The quality of the nanoparticle crystal formed just below the liquid/vapor interface is found to be better at relatively slower evaporation rates, as less defects and grain boundaries appear. This trend is understood as the result of the competition between the accumulation and diffusion times of nanoparticles at the liquid/vapor interface. When the former is smaller, nanoparticles are deposited so fast at the interface that they do not have sufficient time to arrange through diffusion, which leads to the prevalence of defects and grain boundaries. Our results have important implications in understanding assembly of nanoparticles and colloids in non-equilibrium liquid environments.

  19. Uner Tan syndrome caused by a homozygous TUBB2B mutation affecting microtubule stability.

    PubMed

    Breuss, Martin W; Nguyen, Thai; Srivatsan, Anjana; Leca, Ines; Tian, Guoling; Fritz, Tanja; Hansen, Andi H; Musaev, Damir; McEvoy-Venneri, Jennifer; James, Kiely N; Rosti, Rasim O; Scott, Eric; Tan, Uner; Kolodner, Richard D; Cowan, Nicholas J; Keays, David A; Gleeson, Joseph G

    2016-12-23

    The integrity and dynamic properties of the microtubule cytoskeleton are indispensable for the development of the mammalian brain. Consequently, mutations in the genes that encode the structural component (the α/β-tubulin heterodimer) can give rise to severe, sporadic neurodevelopmental disorders. These are commonly referred to as the tubulinopathies. Here we report the addition of recessive quadrupedalism, also known as Uner Tan syndrome (UTS), to the growing list of diseases caused by tubulin variants. Analysis of a consanguineous UTS family identified a biallelic TUBB2B mutation, resulting in a p.R390Q amino acid substitution. In addition to the identifying quadrupedal locomotion, all three patients showed severe cerebellar hypoplasia. None, however, displayed the basal ganglia malformations typically associated with TUBB2B mutations. Functional analysis of the R390Q substitution revealed that it did not affect the ability of β-tubulin to fold or become assembled into the α/β-heterodimer, nor did it influence the incorporation of mutant-containing heterodimers into microtubule polymers. The 390Q mutation in S. cerevisiae TUB2 did not affect growth under basal conditions, but did result in increased sensitivity to microtubule-depolymerizing drugs, indicative of a mild impact of this mutation on microtubule function. The TUBB2B mutation described here represents an unusual recessive mode of inheritance for missense-mediated tubulinopathies and reinforces the sensitivity of the developing cerebellum to microtubule defects.

  20. Potent Antiproliferative Cembrenoids Accumulate in Tobacco upon Infection with Rhodococcus fascians and Trigger Unusual Microtubule Dynamics in Human Glioblastoma Cells

    PubMed Central

    Nacoulma, Aminata P.; Megalizzi, Veronique; Pottier, Laurent R.; De Lorenzi, Manuela; Thoret, Sylviane; Dubois, Joëlle; Vandeputte, Olivier M.; Duez, Pierre; Vereecke, Danny; Jaziri, Mondher El

    2013-01-01

    Aims Though plant metabolic changes are known to occur during interactions with bacteria, these were rarely challenged for pharmacologically active compounds suitable for further drug development. Here, the occurrence of specific chemicals with antiproliferative activity against human cancer cell lines was evidenced in hyperplasia (leafy galls) induced when plants interact with particular phytopathogens, such as the Actinomycete Rhodococcus fascians. Methods We examined leafy galls fraction F3.1.1 on cell proliferation, cell division and cytoskeletal disorganization of human cancer cell lines using time-lapse videomicroscopy imaging, combined with flow cytometry and immunofluorescence analysis. We determined the F3.1.1-fraction composition by gas chromatography coupled to mass spectrometry. Results The leafy galls induced on tobacco by R. fascians yielded fraction F3.1.1 which inhibited proliferation of glioblastoma U373 cells with an IC50 of 4.5 µg/mL, F.3.1.1 was shown to increase cell division duration, cause nuclear morphological deformations and cell enlargement, and, at higher concentrations, karyokinesis defects leading to polyploidization and apoptosis. F3.1.1 consisted of a mixture of isomers belonging to the cembrenoids. The cellular defects induced by F3.1.1 were caused by a peculiar cytoskeletal disorganization, with the occurrence of fragmented tubulin and strongly organized microtubule aggregates within the same cell. Colchicine, paclitaxel, and cembrene also affected U373 cell proliferation and karyokinesis, but the induced microtubule rearrangement was very different from that provoked by F3.1.1. Altogether our data indicate that the cembrenoid isomers in F3.1.1 have a unique mode of action and are able to simultaneously modulate microtubule polymerization and stability. PMID:24167576

  1. Model-adaptive hybrid dynamic control for robotic assembly tasks

    SciTech Connect

    Austin, D.J.; McCarragher, B.J.

    1999-10-01

    A new task-level adaptive controller is presented for the hybrid dynamic control of robotic assembly tasks. Using a hybrid dynamic model of the assembly task, velocity constraints are derived from which satisfactory velocity commands are obtained. Due to modeling errors and parametric uncertainties, the velocity commands may be erroneous and may result in suboptimal performance. Task-level adaptive control schemes, based on the occurrence of discrete events, are used to change the model parameters from which the velocity commands are determined. Two adaptive schemes are presented: the first is based on intuitive reasoning about the vector spaces involved whereas the second uses a search region that is reduced with each iteration. For the first adaptation law, asymptotic convergence to the correct model parameters is proven except for one case. This weakness motivated the development of the second adaptation law, for which asymptotic convergence is proven in all cases. Automated control of a peg-in-hole assembly task is given as an example, and simulations and experiments for this task are presented. These results demonstrate the success of the method and also indicate properties for rapid convergence.

  2. Effect of the microtubule-associated protein tau on dynamics of single-headed motor proteins KIF1A

    NASA Astrophysics Data System (ADS)

    Sparacino, J.; Farías, M. G.; Lamberti, P. W.

    2014-02-01

    Intracellular transport based on molecular motors and its regulation are crucial to the functioning of cells. Filamentary tracks of the cells are abundantly decorated with nonmotile microtubule-associated proteins, such as tau. Motivated by experiments on kinesin-tau interactions [Dixit et al., Science 319, 1086 (2008), 10.1126/science.1152993] we developed a stochastic model of interacting single-headed motor proteins KIF1A that also takes into account the interactions between motor proteins and tau molecules. Our model reproduces experimental observations and predicts significant effects of tau on bound time and run length which suggest an important role of tau in regulation of kinesin-based transport.

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

    PubMed Central

    Tian, Juan; Wang, Xiaohong; Mao, Tonglin; Yuan, Ming; Li, Yunhai

    2016-01-01

    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. PMID:27768706

  4. Dynamic microfluidic control of supramolecular peptide self-assembly

    NASA Astrophysics Data System (ADS)

    Arnon, Zohar A.; Vitalis, Andreas; Levin, Aviad; Michaels, Thomas C. T.; Caflisch, Amedeo; Knowles, Tuomas P. J.; Adler-Abramovich, Lihi; Gazit, Ehud

    2016-10-01

    The dynamic nature of supramolecular polymers has a key role in their organization. Yet, the manipulation of their dimensions and polarity remains a challenge. Here, the minimalistic diphenylalanine building block was applied to demonstrate control of nano-assemblies growth and shrinkage using microfluidics. To fine-tune differential local environments, peptide nanotubes were confined by micron-scale pillars and subjected to monomer flows of various saturation levels to control assembly and disassembly. The small-volume device allows the rapid adjustment of conditions within the system. A simplified kinetic model was applied to calculate parameters of the growth mechanism. Direct real-time microscopy analysis revealed that different peptide derivatives show unidirectional or bidirectional axial dimension variation. Atomistic simulations show that unidirectional growth is dictated by the differences in the axial ends, as observed in the crystalline order of symmetry. This work lays foundations for the rational control of nano-materials dimensions for applications in biomedicine and material science.

  5. Dynamic self-assembly in living systems as computation.

    SciTech Connect

    Bouchard, Ann Marie; Osbourn, Gordon Cecil

    2004-06-01

    Biochemical reactions taking place in living systems that map different inputs to specific outputs are intuitively recognized as performing information processing. Conventional wisdom distinguishes such proteins, whose primary function is to transfer and process information, from proteins that perform the vast majority of the construction, maintenance, and actuation tasks of the cell (assembling and disassembling macromolecular structures, producing movement, and synthesizing and degrading molecules). In this paper, we examine the computing capabilities of biological processes in the context of the formal model of computing known as the random access machine (RAM) [Dewdney AK (1993) The New Turing Omnibus. Computer Science Press, New York], which is equivalent to a Turing machine [Minsky ML (1967) Computation: Finite and Infinite Machines. Prentice-Hall, Englewood Cliffs, NJ]. When viewed from the RAM perspective, we observe that many of these dynamic self-assembly processes - synthesis, degradation, assembly, movement - do carry out computational operations. We also show that the same computing model is applicable at other hierarchical levels of biological systems (e.g., cellular or organism networks as well as molecular networks). We present stochastic simulations of idealized protein networks designed explicitly to carry out a numeric calculation. We explore the reliability of such computations and discuss error-correction strategies (algorithms) employed by living systems. Finally, we discuss some real examples of dynamic self-assembly processes that occur in living systems, and describe the RAM computer programs they implement. Thus, by viewing the processes of living systems from the RAM perspective, a far greater fraction of these processes can be understood as computing than has been previously recognized.

  6. Protein-guided RNA dynamics during early ribosome assembly.

    PubMed

    Kim, Hajin; Abeysirigunawarden, Sanjaya C; Chen, Ke; Mayerle, Megan; Ragunathan, Kaushik; Luthey-Schulten, Zaida; Ha, Taekjip; Woodson, Sarah A

    2014-02-20

    The assembly of 30S ribosomes requires the precise addition of 20 proteins to the 16S ribosomal RNA. How early binding proteins change the ribosomal RNA structure so that later proteins may join the complex is poorly understood. Here we use single-molecule fluorescence resonance energy transfer (FRET) to observe real-time encounters between Escherichia coli ribosomal protein S4 and the 16S 5' domain RNA at an early stage of 30S assembly. Dynamic initial S4-RNA complexes pass through a stable non-native intermediate before converting to the native complex, showing that non-native structures can offer a low free-energy path to protein-RNA recognition. Three-colour FRET and molecular dynamics simulations reveal how S4 changes the frequency and direction of RNA helix motions, guiding a conformational switch that enforces the hierarchy of protein addition. These protein-guided dynamics offer an alternative explanation for induced fit in RNA-protein complexes.

  7. Protein-guided RNA dynamics during early ribosome assembly

    PubMed Central

    Kim, Hajin; Abeysirigunawardena, Sanjaya C.; Chen, Ke; Mayerle, Megan; Ragunathan, Kaushik; Luthey-Schulten, Zaida; Ha, Taekjip; Woodson, Sarah A.

    2014-01-01

    The assembly of 30S ribosomes requires the precise addition of 20 proteins to the 16S ribosomal RNA. How early binding proteins change the rRNA structure so that later proteins may join the complex is poorly understood. Here we use single molecule fluorescence resonance energy transfer (smFRET) to observe real-time encounters between ribosomal protein S4 and the 16S 5′ domain RNA at an early stage of 30S assembly. Dynamic initial S4-RNA complexes pass through a stable non-native intermediate before converting to the native complex, showing that non-native structures can offer a low free energy path to protein-RNA recognition. Three-color FRET and molecular dynamics (MD) simulations reveal how S4 changes the frequency and direction of RNA helix motions, guiding a conformational switch that enforces the hierarchy of protein addition. This protein-guided dynamics offers an alternative explanation for induced fit in RNA-protein complexes. PMID:24522531

  8. Self-assembly of active colloidal molecules with dynamic function

    NASA Astrophysics Data System (ADS)

    Soto, Rodrigo; Golestanian, Ramin

    Catalytically active colloids maintain non-equilibrium conditions in which they produce and deplete chemicals at their surface. While individual colloids that are symmetrically coated do not exhibit dynamical activity, the concentration fields resulting from their chemical activity decay as 1/r and produce gradients that attract or repel other colloids depending on their surface chemistry and ambient variables. This results in a non-equilibrium analogue of ionic systems, but with the remarkable novel feature of action-reaction symmetry breaking. In dilute conditions these active colloids join up to form molecules via generalized ionic bonds. Colloids are found to join up to form self-assembled molecules that could be inert or have spontaneous activity in the form of net translational velocity and spin depending on their symmetry properties and their constituents. As the interactions do not satisfy detailed-balance, it is possible to achieve structures with time dependent functionality. We study a molecule that adopts spontaneous oscillations and another that exhibits a run-and-tumble dynamics similar to bacteria. Our study shows that catalytically active colloids could be used for designing self-assembled structures that posses dynamical functionalities.

  9. Protein-guided RNA dynamics during early ribosome assembly

    NASA Astrophysics Data System (ADS)

    Kim, Hajin; Abeysirigunawarden, Sanjaya C.; Chen, Ke; Mayerle, Megan; Ragunathan, Kaushik; Luthey-Schulten, Zaida; Ha, Taekjip; Woodson, Sarah A.

    2014-02-01

    The assembly of 30S ribosomes requires the precise addition of 20 proteins to the 16S ribosomal RNA. How early binding proteins change the ribosomal RNA structure so that later proteins may join the complex is poorly understood. Here we use single-molecule fluorescence resonance energy transfer (FRET) to observe real-time encounters between Escherichia coli ribosomal protein S4 and the 16S 5' domain RNA at an early stage of 30S assembly. Dynamic initial S4-RNA complexes pass through a stable non-native intermediate before converting to the native complex, showing that non-native structures can offer a low free-energy path to protein-RNA recognition. Three-colour FRET and molecular dynamics simulations reveal how S4 changes the frequency and direction of RNA helix motions, guiding a conformational switch that enforces the hierarchy of protein addition. These protein-guided dynamics offer an alternative explanation for induced fit in RNA-protein complexes.

  10. Model for dynamic self-assembled magnetic surface structures.

    SciTech Connect

    Belkin, M.; Glatz, A.; Snezhko, A.; Aranson, I. S.; Materials Science Division; Northwestern Univ.

    2010-07-07

    We propose a first-principles model for the dynamic self-assembly of magnetic structures at a water-air interface reported in earlier experiments. The model is based on the Navier-Stokes equation for liquids in shallow water approximation coupled to Newton equations for interacting magnetic particles suspended at a water-air interface. The model reproduces most of the observed phenomenology, including spontaneous formation of magnetic snakelike structures, generation of large-scale vortex flows, complex ferromagnetic-antiferromagnetic ordering of the snake, and self-propulsion of bead-snake hybrids.

  11. Expansion and Polarity Sorting in Microtubule-Dynein Bundles

    NASA Astrophysics Data System (ADS)

    Zemel, A.; Mogilner, A.

    Interactions of multiple molecular motors with dynamicpolymers, such as actin and microtubules, form the basis for many processes in the cell cytoskeleton. One example is the active `sorting' of microtubule bundles by dynein molecular motors into aster-like arrays of microtubules; in these bundles dynein motors cross-link and slide neighboring microtubules apart. A number of models have been suggested to quantify the active dynamics of cross-linked bundles of polar filaments. In the case of densely packed bundles, however, a major complication arises from the fact that each microtubule interacts with multiple neighboring filaments. To explicitly take these interactions into account we performed detailed computer simulations in which the equations of motion for all microtubules in the bundle were iteratively solved. Our simulations demonstrate the phenomenon of polarity sorting and reveal the variable-rate of the concurrent bundle expansion and its dependence on the nature of the microtubule-motor interactions.

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

    PubMed

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

    2012-12-21

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

  13. Reovirus Cell Entry Requires Functional Microtubules

    PubMed Central

    Mainou, Bernardo A.; Zamora, Paula F.; Ashbrook, Alison W.; Dorset, Daniel C.; Kim, Kwang S.; Dermody, Terence S.

    2013-01-01

    ABSTRACT Mammalian reovirus binds to cell-surface glycans and junctional adhesion molecule A and enters cells by receptor-mediated endocytosis in a process dependent on β1 integrin. Within the endocytic compartment, reovirus undergoes stepwise disassembly, allowing release of the transcriptionally active viral core into the cytoplasm. To identify cellular mediators of reovirus infectivity, we screened a library of small-molecule inhibitors for the capacity to block virus-induced cytotoxicity. In this screen, reovirus-induced cell killing was dampened by several compounds known to impair microtubule dynamics. Microtubule inhibitors were assessed for blockade of various stages of the reovirus life cycle. While these drugs did not alter reovirus cell attachment or internalization, microtubule inhibitors diminished viral disassembly kinetics with a concomitant decrease in infectivity. Reovirus virions colocalize with microtubules and microtubule motor dynein 1 during cell entry, and depolymerization of microtubules results in intracellular aggregation of viral particles. These data indicate that functional microtubules are required for proper sorting of reovirus virions following internalization and point to a new drug target for pathogens that use the endocytic pathway to invade host cells. PMID:23820395

  14. Assembly dynamics of PML nuclear bodies in living cells.

    PubMed

    Brand, Peter; Lenser, Thorsten; Hemmerich, Peter

    2010-03-05

    The mammalian cell nucleus contains a variety of organelles or nuclear bodies which contribute to key nuclear functions. Promyelocytic leukemia nuclear bodies (PML NBs) are involved in the regulation of apoptosis, antiviral responses, the DNA damage response and chromatin structure, but their precise biochemical function in these nuclear pathways is unknown. One strategy to tackle this problem is to assess the biophysical properties of the component parts of these macromolecular assemblies in living cells. In this study we determined PML NB assembly dynamics by live cell imaging, combined with mathematical modeling. For the first time, dynamics of PML body formation were measured in cells lacking endogenous PML. We show that all six human nuclear PML isoforms are able to form nuclear bodies in PML negative cells. All isoforms exhibit individual exchange rates at NBs in PML positive cells but PML I, II, III and IV are static at nuclear bodies in PML negative cells, suggesting that these isoforms require additional protein partners for efficient exchange. PML V turns over at PML Nbs very slowly supporting the idea of a structural function for this isoform. We also demonstrate that SUMOylation of PML at Lysine positions K160 and/or K490 are required for nuclear body formation in vivo.We propose a model in which the isoform specific residence times of PML provide both, structural stability to function as a scaffold and flexibility to attract specific nuclear proteins for efficient biochemical reactions at the surface of nuclear bodies.MCS code: 92C37.

  15. Microtubules Growth Rate Alteration in Human Endothelial Cells

    PubMed Central

    Alieva, Irina B.; Zemskov, Evgeny A.; Kireev, Igor I.; Gorshkov, Boris A.; Wiseman, Dean A.; Black, Stephen M.; Verin, Alexander D.

    2010-01-01

    To understand how microtubules contribute to the dynamic reorganization of the endothelial cell (EC) cytoskeleton, we established an EC model expressing EB3-GFP, a protein that marks microtubule plus-ends. Using this model, we were able to measure microtubule growth rate at the centrosome region and near the cell periphery of a single human EC and in the EC monolayer. We demonstrate that the majority of microtubules in EC are dynamic, the growth rate of their plus-ends is highest in the internal cytoplasm, in the region of the centrosome. Growth rate of microtubule plus-ends decreases from the cell center toward the periphery. Our data suggest the existing mechanism(s) of local regulation of microtubule plus-ends growth in EC. Microtubule growth rate in the internal cytoplasm of EC in the monolayer is lower than that of single EC suggesting the regulatory effect of cell-cell contacts. Centrosomal microtubule growth rate distribution in single EC indicated the presence of two subpopulations of microtubules with “normal” (similar to those in monolayer EC) and “fast” (three times as much) growth rates. Our results indicate functional interactions between cell-cell contacts and microtubules. PMID:20445745

  16. Microtubules growth rate alteration in human endothelial cells.

    PubMed

    Alieva, Irina B; Zemskov, Evgeny A; Kireev, Igor I; Gorshkov, Boris A; Wiseman, Dean A; Black, Stephen M; Verin, Alexander D

    2010-01-01

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

  17. Dimer model for Tau proteins bound in microtubule bundles

    NASA Astrophysics Data System (ADS)

    Hall, Natalie; Kluber, Alexander; Hayre, N. Robert; Singh, Rajiv; Cox, Daniel

    2013-03-01

    The microtubule associated protein tau is important in nucleating and maintaining microtubule spacing and structure in neuronal axons. Modification of tau is implicated as a later stage process in Alzheimer's disease, but little is known about the structure of tau in microtubule bundles. We present preliminary work on a proposed model for tau dimers in microtubule bundles (dimers are the minimal units since there is one microtubule binding domain per tau). First, a model of tau monomer was created and its characteristics explored using implicit solvent molecular dynamics simulation. Multiple simulations yield a partially collapsed form with separate positively/negatively charged clumps, but which are a factor of two smaller than required by observed microtubule spacing. We argue that this will elongate in dimer form to lower electrostatic energy at a cost of entropic ``spring'' energy. We will present preliminary results on steered molecular dynamics runs on tau dimers to estimate the actual force constant. Supported by US NSF Grant DMR 1207624.

  18. Natural product derivative Bis(4-fluorobenzyl)trisulfide inhibits tumor growth by modification of beta-tubulin at Cys 12 and suppression of microtubule dynamics.

    PubMed

    Xu, Wanhong; Xi, Biao; Wu, Jieying; An, Haoyun; Zhu, Jenny; Abassi, Yama; Feinstein, Stuart C; Gaylord, Michelle; Geng, Baoqin; Yan, Huifang; Fan, Weimin; Sui, Meihua; Wang, Xiaobo; Xu, Xiao

    2009-12-01

    Bis(4-fluorobenzyl)trisulfide (BFBTS) is a synthetic molecule derived from a bioactive natural product, dibenzyltrisulfide, found in a subtropical shrub, Petiveria allieacea. BFBTS has potent anticancer activities to a broad spectrum of tumor cell lines with IC50 values from high nanomolar to low micromolar and showed equal anticancer potency between tumor cell lines overexpressing multidrug-resistant gene, MDR1 (MCF7/adr line and KBv200 line), and their parental MCF7 line and KB lines. BFBTS inhibited microtubule polymerization dynamics in MCF7 cells, at a low nanomolar concentration of 54 nmol/L, while disrupting microtubule filaments in cells at low micromolar concentration of 1 micromol/L. Tumor cells treated with BFBTS were arrested at G2-M phase, conceivably resulting from BFBTS-mediated antimicrotubule activities. Mass spectrometry studies revealed that BFBTS bound and modified beta-tubulin at residue Cys12, forming beta-tubulin-SS-fluorobenzyl. The binding site differs from known antimicrotubule agents, suggesting that BFBTS functions as a novel antimicrotubule agent. BFBTS at a dose of 25 mg/kg inhibited tumor growth with relative tumor growth rates of 19.91%, 18.5%, and 23.42% in A549 lung cancer, Bcap-37 breast cancer, and SKOV3 ovarian cancer xenografts, respectively. Notably, BFBTS was more potent against MDR1-overexpressing MCF7/adr breast cancer xenografts with a relative tumor growth rate of 12.3% than paclitaxel with a rate of 43.0%. BFBTS displays a novel antimicrotubule agent with potentials for cancer therapeutics.

  19. Stable isotope labeling by amino acids in cell culture-based liquid chromatography-mass spectrometry assay to measure microtubule dynamics in neuronal cell cultures.

    PubMed

    Polson, Craig; Cantone, Joseph L; Wei, Cong; Drexler, Dieter M; Meredith, Jere E

    2014-12-01

    Microtubules (MTs) are highly dynamic polymers composed of α- and β-tubulin heterodimers. Dysregulation of MT dynamics in neurons may be a contributing factor in the progression of various neurodegenerative diseases. We developed a stable isotope labeling by amino acids in cell culture (SILAC)-based liquid chromatography-mass spectrometry (LC-MS) method to measure the fraction of [(13)C6]leucine-labeled α-tubulin-derived surrogate peptides. Using this approach, we measured the time course of incorporation of [(13)C6]leucine label into the MT and dimer pools isolated from cycling cells and rat primary hippocampal neurons. We found that the MT pool is in rapid equilibrium with the dimer pool in the cycling cells, consistent with rapid MT polymerization/depolymerization during cell proliferation. Conversely, in neurons, we found that labeling of the MT pool was rapid, whereas the dimer pool was delayed. These results suggest that newly synthesized α-tubulin is first incorporated into MTs or complexes that co-sediment with MTs and that appearance of labeled α-tubulin in the dimer pool may be a consequence of MT depolymerization or breakdown. Our results demonstrate that a SILAC-based approach can be used to measure MT dynamics and may have utility for exploring MT dysregulation in various models of neurodegenerative disease.

  20. CLASP1, astrin and Kif2b form a molecular switch that regulates kinetochore-microtubule dynamics to promote mitotic progression and fidelity

    PubMed Central

    Manning, Amity L; Bakhoum, Samuel F; Maffini, Stefano; Correia-Melo, Clara; Maiato, Helder; Compton, Duane A

    2010-01-01

    Accurate chromosome segregation during mitosis requires precise coordination of various processes, such as chromosome alignment, maturation of proper kinetochore–microtubule (kMT) attachments, correction of erroneous attachments, and silencing of the spindle assembly checkpoint (SAC). How these fundamental aspects of mitosis are coordinately and temporally regulated is poorly understood. In this study, we show that the temporal regulation of kMT attachments by CLASP1, astrin and Kif2b is central to mitotic progression and chromosome segregation fidelity. In early mitosis, a Kif2b–CLASP1 complex is recruited to kinetochores to promote chromosome movement, kMT turnover, correction of attachment errors, and maintenance of SAC signalling. However, during metaphase, this complex is replaced by an astrin–CLASP1 complex, which promotes kMT stability, chromosome alignment, and silencing of the SAC. We show that these two complexes are differentially recruited to kinetochores and are mutually exclusive. We also show that other kinetochore proteins, such as Kif18a, affect kMT attachments and chromosome movement through these proteins. Thus, CLASP1–astrin–Kif2b complex act as a central switch at kinetochores that defines mitotic progression and promotes fidelity by temporally regulating kMT attachments. PMID:20852589

  1. Structure and phosphorylation of microtubule-associated protein 2 (MAP 2).

    PubMed Central

    Vallee, R

    1980-01-01

    Chymotryptic fragments of microtubule-associated protein 2 (MAP 2) containing the portion of the molecule responsible for promoting microtubule assembly were identified. These assembly-promoting fragments displaced intact MAP 2, but not MAP 1, from assembled microtubules. This indicates that the association of MAP 2 with the microtubule surface is reversible. Both the assembly-promoting fragments and fragments representing the portion of the MAP 2 molecule observed as a projection on the microtubule surface were found to contain sites for endogenous cyclic AMP-dependent phosphorylation. The projection fragments were capable of endogenous phosphorylation even after their physical separation from microtubules. This suggests an intimate association of a kinase activity with the projections. Detailed analysis of the properties of the chymotryptic fragments of MAP 2 has led to a map of the molecule showing the major sites of proteolytic attack and the sites of phosphorylation. Images PMID:6251448

  2. Dynamic characterization of satellite assembly for responsive space applications

    NASA Astrophysics Data System (ADS)

    Mascarenas, David; Macknelly, David; Mullins, Josh; Wiest, Heather; Park, Gyuhae

    2013-07-01

    The rapid deployment of satellites for responsive space surveillance applications is hindered by the need to flight-qualify their components and the resulting mechanical assembly. Conventional methods for qualification testing of satellite components are costly and time consuming. Furthermore, full-scale vehicles must be subjected to simulated launch loads during testing, and this harsh testing environment increases the risk of damage to satellite components during qualification. This work focuses on replacing this potentially destructive testing procedure with a non-destructive structural health monitoring (SHM)-based technique while maintaining the same level of confidence in the testing procedure's ability to qualify the satellite for flight. We focus on assessing the performance of SHM techniques to replace the high-cost qualification procedure and to localize faults introduced by improper assembly. The goal of this work is to create a dual-use system that can both assist in the process of qualifying the satellite for launch, as well as provide continuous structural integrity monitoring during manufacture, transport, launch and deployment. SHM techniques were applied on a small-scale structure representative of a responsive satellite. The test structure consisted of an extruded aluminum space-frame covered with aluminum shear plates assembled using bolted joints. Multiple piezoelectric transducers were bonded to the test structure and acted as combined actuators and sensors. Piezoelectric active-sensing based techniques, including measurements of low-frequency global frequency response functions and high-frequency wave propagation techniques, were employed. Using these methods in conjunction with finite element modeling, the dynamic properties of the test structure were established and areas of potential damage could be identified and localized. A procedure for guiding the effective placement of the sensors and actuators is also outlined.

  3. Self-assembly of active colloidal molecules with dynamic function

    NASA Astrophysics Data System (ADS)

    Soto, Rodrigo; Golestanian, Ramin

    2015-05-01

    Catalytically active colloids maintain nonequilibrium conditions in which they produce and deplete chemicals and hence effectively act as sources and sinks of molecules. While individual colloids that are symmetrically coated do not exhibit any form of dynamical activity, the concentration fields resulting from their chemical activity decay as 1 /r and produce gradients that attract or repel other colloids depending on their surface chemistry and ambient variables. This results in a nonequilibrium analog of ionic systems, but with the remarkable novel feature of action-reaction symmetry breaking. We study solutions of such chemically active colloids in dilute conditions when they join up to form molecules via generalized ionic bonds and discuss how we can achieve structures with time-dependent functionality. In particular, we study a molecule that adopts a spontaneous oscillatory pattern of conformations and another that exhibits a run-and-tumble dynamics similar to bacteria. Our study shows that catalytically active colloids could be used for designing self-assembled structures that possess dynamical functionalities that are determined by their prescribed three-dimensional structures, a strategy that follows the design principle of proteins.

  4. Dynamic microfluidic control of supramolecular peptide self-assembly

    PubMed Central

    Arnon, Zohar A.; Vitalis, Andreas; Levin, Aviad; Michaels, Thomas C. T.; Caflisch, Amedeo; Knowles, Tuomas P. J.; Adler-Abramovich, Lihi; Gazit, Ehud

    2016-01-01

    The dynamic nature of supramolecular polymers has a key role in their organization. Yet, the manipulation of their dimensions and polarity remains a challenge. Here, the minimalistic diphenylalanine building block was applied to demonstrate control of nano-assemblies growth and shrinkage using microfluidics. To fine-tune differential local environments, peptide nanotubes were confined by micron-scale pillars and subjected to monomer flows of various saturation levels to control assembly and disassembly. The small-volume device allows the rapid adjustment of conditions within the system. A simplified kinetic model was applied to calculate parameters of the growth mechanism. Direct real-time microscopy analysis revealed that different peptide derivatives show unidirectional or bidirectional axial dimension variation. Atomistic simulations show that unidirectional growth is dictated by the differences in the axial ends, as observed in the crystalline order of symmetry. This work lays foundations for the rational control of nano-materials dimensions for applications in biomedicine and material science. PMID:27779182

  5. Precise and Reversible Protein-Microtubule-Like Structure with Helicity Driven by Dual Supramolecular Interactions.

    PubMed

    Yang, Guang; Zhang, Xiang; Kochovski, Zdravko; Zhang, Yufei; Dai, Bin; Sakai, Fuji; Jiang, Lin; Lu, Yan; Ballauff, Matthias; Li, Xueming; Liu, Cong; Chen, Guosong; Jiang, Ming

    2016-02-17

    Protein microtubule is a significant self-assembled architecture found in nature with crucial biological functions. However, mimicking protein microtubules with precise structure and controllable self-assembly behavior remains highly challenging. In this work, we demonstrate that by using dual supramolecular interactions from a series of well-designed ligands, i.e., protein-sugar interaction and π-π stacking, highly homogeneous protein microtubes were achieved from tetrameric soybean agglutinin without any chemical or biological modification. Using combined cryo-EM single-particle reconstruction and computational modeling, the accurate structure of protein microtube was determined. The helical protein microtube is consisted of three protofilaments, each of which features an array of soybean agglutinin tetramer linked by the designed ligands. Notably, the microtubes resemble the natural microtubules in their structural and dynamic features such as the shape and diameter and the controllable and reversible assembly behavior, among others. Furthermore, the protein microtubes showed an ability to enhance immune response, demonstrating its great potential for biological applications.

  6. Effects of silver ions (Ag+) on contractile ring function and microtubule dynamics during first cleavage in Ilyanassa obsoleta

    NASA Technical Reports Server (NTRS)

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

    1994-01-01

    The terminal phase of cell division involves tight constriction of the cleavage furrow contractile ring, stabilization/elongation of the intercellular bridge, and final separation of the daughter cells. At first cleavage, the fertilized eggs of the mollusk, Ilyanassa obsoleta, form two contractile rings at right angles to each other in the same cytoplasm that constrict to tight necks and partition the egg into a trefoil shape. The cleavage furrow contractile ring (CF) normally constricts around many midbody microtubules (MTs) and results in cleavage; the polar lobe constriction contractile ring (PLC) normally constricts around very few MTs and subsequently relaxes without cleavage. In the presence of Ag+ ions, the PLC 1) begins MT-dependent rapid constriction sooner than controls, 2) encircles more MTs than control egg PLCs, 3) elongates much more than control PLCs, and 4) remains tightly constricted and effectively cleaves the polar lobe from the egg. If Ag(+)-incubated eggs are returned to normal seawater at trefoil, tubulin fluorescence disappears from the PLC neck and the neck relaxes. If nocodazole, a drug that depolymerizes MTs, is added to Ag(+)-incubated eggs during early PLC constriction, the PLC is not stabilized and eventually relaxes. However, if nocodazole is added to Ag(+)-incubated eggs at trefoil, tubulin fluorescence disappears from the PLC neck but the neck remains constricted. These results suggest that Ag+ accelerates and gradually stabilizes the PLC constriction by a mechanism that is initially MT-dependent, but that progressively becomes MT-independent.

  7. Dynamic Characterization of Crystalline Supramolecular Rotors Assembled through Halogen Bonding.

    PubMed

    Catalano, Luca; Pérez-Estrada, Salvador; Terraneo, Giancarlo; Pilati, Tullio; Resnati, Giuseppe; Metrangolo, Pierangelo; Garcia-Garibay, Miguel A

    2015-12-16

    A modular molecular kit for the preparation of crystalline molecular rotors was devised from a set of stators and rotators to gain simple access to a large number of structures with different dynamic performance and physical properties. In this work, we have accomplished this with crystalline molecular rotors self-assembled by halogen bonding of diazabicyclo[2.2.2]octane, acting as a rotator, and a set of five fluorine-substituted iodobenzenes that take the role of the stator. Using variable-temperature (1)H T1 spin-lattice relaxation measurements, we have shown that all structures display ultrafast Brownian rotation with activation energies of 2.4-4.9 kcal/mol and pre-exponential factors of the order of (1-9) × 10(12) s(-1). Line shape analysis of quadrupolar echo (2)H NMR measurements in selected examples indicated rotational trajectories consistent with the 3-fold or 6-fold symmetric potential of the rotator.

  8. Preparation of Segmented Microtubules to Study Motions Driven by the Disassembling Microtubule Ends

    PubMed Central

    Volkov, Vladimir A.; Zaytsev, Anatoly V.; Grishchuk, Ekaterina L.

    2014-01-01

    Microtubule depolymerization can provide force to transport different protein complexes and protein-coated beads in vitro. The underlying mechanisms are thought to play a vital role in the microtubule-dependent chromosome motions during cell division, but the relevant proteins and their exact roles are ill-defined. Thus, there is a growing need to develop assays with which to study such motility in vitro using purified components and defined biochemical milieu. Microtubules, however, are inherently unstable polymers; their switching between growth and shortening is stochastic and difficult to control. The protocols we describe here take advantage of the segmented microtubules that are made with the photoablatable stabilizing caps. Depolymerization of such segmented microtubules can be triggered with high temporal and spatial resolution, thereby assisting studies of motility at the disassembling microtubule ends. This technique can be used to carry out a quantitative analysis of the number of molecules in the fluorescently-labeled protein complexes, which move processively with dynamic microtubule ends. To optimize a signal-to-noise ratio in this and other quantitative fluorescent assays, coverslips should be treated to reduce nonspecific absorption of soluble fluorescently-labeled proteins. Detailed protocols are provided to take into account the unevenness of fluorescent illumination, and determine the intensity of a single fluorophore using equidistant Gaussian fit. Finally, we describe the use of segmented microtubules to study microtubule-dependent motions of the protein-coated microbeads, providing insights into the ability of different motor and nonmotor proteins to couple microtubule depolymerization to processive cargo motion. PMID:24686554

  9. DYNAMIC SHEAR-INFLUENCED COLLAGEN SELF-ASSEMBLY

    PubMed Central

    Saeidi, Nima; Sander, Edward A.

    2011-01-01

    The ability to influence the direction of polymerization of a self-assembling biomolecular system has the potential to generate materials with extremely high anisotropy. In biological systems where highly-oriented cellular populations give rise to aligned and often load-bearing tissue such organized molecular scaffolds could aid in the contact guidance of cells for engineered tissue constructs (e.g cornea and tendon). In this investigation we examine the detailed dynamics of pepsin-extracted type I bovine collagen assembly on a glass surface under the influence of flow between two plates. Differential Interference Contrast (DIC) imaging (60x-1.4NA) with focal plane stabilization was used to resolve and track the growth of collagen aggregates on borosilicate glass for 4 different shear rates (500, 80, 20, and 9 s-1). The detailed morphology of the collagen fibrils/aggregates was examined using Quick Freeze Deep Etch electron microscopy. Nucleation of fibrils on the glass was observed to occur rapidly (~2 min) followed by continued growth of the fibrils. The growth rates were dependent on flow in a complex manner with the highest rate of axial growth (0.1 microns/sec) occurring at a shear rate of 9 s-1. The lowest growth rate occurred at the highest shear. Fibrils were observed to both branch and join during the experiments. The best alignment of fibrils was observed at intermediate shear rates of 20 and 80s-1. However, the investigation revealed that fibril directional growth was not stable. At high shear rates, fibrils would often turn downstream forming what we term “hooks” which are likely the combined result of monomer interaction with the initial collagen layer or “mat” and the high shear rate. Further, QFDE examination of fibril morphology demonstrated that the assembled fibrillar structure did not possess native D-periodicity. Instead, fibrils comprised a collection of generally aligned, monomers which were self-assembled to form a fibril

  10. Microtubules in Plants

    PubMed Central

    Hashimoto, Takashi

    2015-01-01

    Microtubules (MTs) are highly conserved polar polymers that are key elements of the eukaryotic cytoskeleton and are essential for various cell functions. αβ-tubulin, a heterodimer containing one structural GTP and one hydrolysable and exchangeable GTP, is the building block of MTs and is formed by the sequential action of several molecular chaperones. GTP hydrolysis in the MT lattice is mechanistically coupled with MT growth, thus giving MTs a metastable and dynamic nature. MTs adopt several distinct higher-order organizations that function in cell division and cell morphogenesis. Small molecular weight compounds that bind tubulin are used as herbicides and as research tools to investigate MT functions in plant cells. The de novo formation of MTs in cells requires conserved γ-tubulin-containing complexes and targeting/activating regulatory proteins that contribute to the geometry of MT arrays. Various MT regulators and tubulin modifications control the dynamics and organization of MTs throughout the cell cycle and in response to developmental and environmental cues. Signaling pathways that converge on the regulation of versatile MT functions are being characterized. PMID:26019693

  11. Histone H2B monoubiquitination is involved in regulating the dynamics of microtubules during the defense response to Verticillium dahliae toxins in Arabidopsis.

    PubMed

    Hu, Min; Pei, Bao-Lei; Zhang, Li-Fan; Li, Ying-Zhang

    2014-04-01

    Histone H2B monoubiquitination (H2Bub) is being recognized as a regulatory mechanism that controls a range of cellular processes in plants, but the molecular mechanisms of H2Bub that are involved in responses to biotic stress are largely unknown. In this study, we used wild-type and H2Bub loss-of-function mutations of Arabidopsis (Arabidopsis thaliana) to elucidate which of its mechanisms are involved in the regulation of the plant's defense response to Verticillium dahliae (Vd) toxins. We demonstrate that the depolymerization of the cortical microtubules (MTs) was different in the wild type and the mutants in the response to Vd toxins. The loss-of-function alleles of HISTONE MONOUBIQUITINATION1 and HISTONE MONOUBIQUITINATION2 mutations present a weaker depolymerization of the MTs, and protein tyrosine phosphorylation plays a critical role in the regulation of the dynamics of MTs. Moreover, H2Bub is a positive regulator of the gene expression of protein tyrosine phosphatases. These findings provide direct evidence for H2Bub as an important modification with regulatory roles in the defense against Vd toxins and demonstrate that H2Bub is involved in modulating the dynamics of MTs, likely through the protein tyrosine phosphatase-mediated signaling pathway.

  12. Microtubules move the nucleus to quiescence.

    PubMed

    Laporte, Damien; Sagot, Isabelle

    2014-01-01

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

  13. Kinetochore-microtubule interactions during cell division.

    PubMed

    Maiato, Helder; Sunkel, Claudio E

    2004-01-01

    Proper segregation of chromosomes during cell division is essential for the maintenance of genetic stability. During this process chromosomes must establish stable functional interactions with microtubules through the kinetochore, a specialized protein structure located on the surface of the centromeric heterochromatin. Stable attachment of kinetochores to a number of microtubules results in the formation of a kinetochore fibre that mediates chromosome movement. How the kinetochore fibre is formed and how chromosome motion is produced and regulated remain major questions in cell biology. Here we look at some of the history of research devoted to the study of kinetochore-microtubule interaction and attempt to identify significant advances in the knowledge of the basic processes. Ultrastructural work has provided substantial insights into the structure of the kinetochore and associated microtubules during different stages of mitosis. Also, recent in-vivo studies have probed deep into the dynamics of kinetochore-attached microtubules suggesting possible models for the way in which kinetochores harness the capacity of microtubules to do work and turn it into chromosome motion. Much of the research in recent years suggests that indeed multiple mechanisms are involved in both formation of the k-fibre and chromosome motion. Thus, rather than moving to a unified theory, it has become apparent that most cell types have the capacity to build the spindle using multiple and probably redundant mechanisms.

  14. General theory for the mechanics of confined microtubule asters

    NASA Astrophysics Data System (ADS)

    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.

  15. The dual specificity phosphatase Cdc14B bundles and stabilizes microtubules

    SciTech Connect

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

    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.

  16. Kinetochores capture astral microtubules during chromosome attachment to the mitotic spindle: direct visualization in live newt lung cells

    PubMed Central

    1990-01-01

    When viewed by light microscopy the mitotic spindle in newt pneumocytes assembles in an optically clear area of cytoplasm, virtually devoid of mitochondria and other organelles, which can be much larger than the forming spindle. This unique optical property has allowed us to examine the behavior of individual microtubules, at the periphery of asters in highly flattened living prometaphase cells, by video-enhanced differential interference-contrast light microscopy and digital image processing. As in interphase newt pneumocytes (Cassimeris, L., N. K. Pryer, and E. D. Salmon. 1988. J. Cell Biol. 107:2223-2231), centrosomal (i.e., astral) microtubules in prometaphase cells appear to exhibit dynamic instability, elongating at a mean rate of 14.3 +/- 5.1 microns/min (N = 19) and shortening at approximately 16 microns/min. Under favorable conditions the initial interaction between a kinetochore and the forming spindle can be directly observed. During this process the unattached chromosome is repeatedly probed by microtubules projecting from one of the polar regions. When one of these microtubules contacts the primary constriction the chromosome rapidly undergoes poleward translocation. Our observations on living mitotic cells directly demonstrate, for the first time, that chromosome attachment results from an interaction between astral microtubules and the kinetochore. PMID:2391359

  17. Characterization of the role of calcium in regulating the microtubule-destabilizing activity of MDP25

    PubMed Central

    Qin, Tao; Li, Jiejie; Yuan, Ming; Mao, Tonglin

    2012-01-01

    Regulation of cell elongation is important for plant morphogenesis. Many studies have shown that cortical microtubules play crucial roles during cell elongation and that microtubule stability, organization, and dynamics are regulated by microtubule regulatory proteins.1 Recently, we reported that a novel protein from Arabidopsis, termed microtubule-destabilizing protein 25 (MDP25), functions as a negative regulator of hypocotyl cell elongation. MDP25 destabilizes microtubules and exerts its effect on microtubules as a result of transient elevation of cytosolic calcium levels.2 PMID:22751329

  18. Autophagy induction stabilizes microtubules and promotes axon regeneration after spinal cord injury

    PubMed Central

    He, Miao; Ding, Yuetong; Chu, Chen; Tang, Jing; Xiao, Qi; Luo, Zhen-Ge

    2016-01-01

    Remodeling of cytoskeleton structures, such as microtubule assembly, is believed to be crucial for growth cone initiation and regrowth of injured axons. Autophagy plays important roles in maintaining cellular homoeostasis, and its dysfunction causes neuronal degeneration. The role of autophagy in axon regeneration after injury remains speculative. Here we demonstrate a role of autophagy in regulating microtubule dynamics and axon regeneration. We found that autophagy induction promoted neurite outgrowth, attenuated the inhibitory effects of nonpermissive substrate myelin, and decreased the formation of retraction bulbs following axonal injury in cultured cortical neurons. Interestingly, autophagy induction stabilized microtubules by degrading SCG10, a microtubule disassembly protein in neurons. In mice with spinal cord injury, local administration of a specific autophagy-inducing peptide, Tat-beclin1, to lesion sites markedly attenuated axonal retraction of spinal dorsal column axons and cortical spinal tract and promoted regeneration of descending axons following long-term observation. Finally, administration of Tat-beclin1 improved the recovery of motor behaviors of injured mice. These results show a promising effect of an autophagy-inducing reagent on injured axons, providing direct evidence supporting a beneficial role of autophagy in axon regeneration. PMID:27638205

  19. Microtubule-dependent transport of vimentin filament precursors is regulated by actin and by the concerted action of Rho- and p21-activated kinases.

    PubMed

    Robert, Amélie; Herrmann, Harald; Davidson, Michael W; Gelfand, Vladimir I

    2014-07-01

    Intermediate filaments (IFs) form a dense and dynamic network that is functionally associated with microtubules and actin filaments. We used the GFP-tagged vimentin mutant Y117L to study vimentin-cytoskeletal interactions and transport of vimentin filament precursors. This mutant preserves vimentin interaction with other components of the cytoskeleton, but its assembly is blocked at the unit-length filament (ULF) stage. ULFs are easy to track, and they allow a reliable and quantifiable analysis of movement. Our results show that in cultured human vimentin-negative SW13 cells, 2% of vimentin-ULFs move along microtubules bidirectionally, while the majority are stationary and tightly associated with actin filaments. Rapid motor-dependent transport of ULFs along microtubules is enhanced ≥ 5-fold by depolymerization of actin cytoskeleton with latrunculin B. The microtubule-dependent transport of vimentin ULFs is further regulated by Rho-kinase (ROCK) and p21-activated kinase (PAK): ROCK inhibits ULF transport, while PAK stimulates it. Both kinases act on microtubule transport independently of their effects on actin cytoskeleton. Our study demonstrates the importance of the actin cytoskeleton to restrict IF transport and reveals a new role for PAK and ROCK in the regulation of IF precursor transport.-Robert, A., Herrmann, H., Davidson, M. W., and Gelfand, V. I. Microtubule-dependent transport of vimentin filament precursors is regulated by actin and by the concerted action of Rho- and p21-activated kinases.

  20. INSIGHTS INTO ANTI-PARALLEL MICROTUBULE CROSSLINKING BY PRC1, A CONSERVED NON-MOTOR MICROTUBULE BINDING PROTEIN

    PubMed Central

    Subramanian, Radhika; Wilson-Kubalek, Elizabeth M.; Arthur, Christopher P.; Bick, Matthew J.; Campbell, Elizabeth A.; Darst, Seth A.; Milligan, Ronald A.; Kapoor, Tarun M.

    2010-01-01

    SUMMARY Formation of microtubule architectures, required for cell shape maintenance in yeast, directional cell expansion in plants and cytokinesis in eukaryotes, depends on antiparallel microtubule crosslinking by the conserved MAP65 protein family. Here, we combine structural and single molecule fluorescence methods to examine how PRC1, the human MAP65, crosslinks antiparallel microtubules. We find that PRC1's microtubule binding is mediated by a structured domain with a spectrin-fold and an unstructured Lys/Arg-rich domain. These two domains, at each end of a homodimer, are connected by a linkage that is flexible on single microtubules, but forms well-defined crossbridges between antiparallel filaments. Further, we show that PRC1 crosslinks do not substantially resist filament sliding by motor proteins in vitro. Together, our data show how MAP65s, by combining structural flexibility and rigidity, tune microtubule associations to establish compliant crosslinks that selectively `mark' antiparallel overlap in dynamic cytoskeletal networks. PMID:20691902

  1. Sliding of STOP proteins on microtubules: a model system for diffusion-dependent microtubule motility.

    PubMed

    Margolis, R L; Job, D; Pabion, M; Rauch, C T

    1986-01-01

    STOP proteins, of 145 kD, act substoichiometrically to block end-wise disassembly of microtubules. STOPs bind to microtubules either during microtubule assembly or when added at steady state, and when binding to the polymers is apparently irreversible. They are not measurably lost from polymers under competition conditions, and there is no measurable exchange between polymers. Nonetheless, STOP proteins exhibit an extraordinary behavior: they "slide" laterally on the surface of the microtubule. Displacement is assayed by forming hybrid microtubules in which cold stable or cold labile region subunits are labeled. Displacement of STOPs on the polymer with time will cause labeled subunits of cold-stable regions to become increasingly cold labile in a manner reciprocal to cold stabilization of previously cold-labile subunits. Because equilibrium exchange of STOP proteins onto and off the polymers can be ruled out, the displacement of STOPs relative to subunits can only be explained by lateral diffusion or "sliding." Axonal transport and mitotic mechanisms were discussed as implications of such a lateral translocation mechanism for microtubule-dependent motility.

  2. A Refined Reaction-Diffusion Model of Tau-Microtubule Dynamics and Its Application in FDAP Analysis

    PubMed Central

    Igaev, Maxim; Janning, Dennis; Sündermann, Frederik; Niewidok, Benedikt; Brandt, Roland; Junge, Wolfgang

    2014-01-01

    Fluorescence decay after photoactivation (FDAP) and fluorescence recovery after photobleaching (FRAP) are well established approaches for studying the interaction of the microtubule (MT)-associated protein tau with MTs in neuronal cells. Previous interpretations of FDAP/FRAP data have revealed dwell times of tau on MTs in the range of several seconds. However, this is difficult to reconcile with a dwell time recently measured by single-molecule analysis in neuronal processes that was shorter by two orders of magnitude. Questioning the validity of previously used phenomenological interpretations of FDAP/FRAP data, we have generalized the standard two-state reaction-diffusion equations by 1), accounting for the parallel and discrete arrangement of MTs in cell processes (i.e., homogeneous versus heterogeneous distribution of tau-binding sites); and 2), explicitly considering both active (diffusion upon MTs) and passive (piggybacking upon MTs at rates of slow axonal transport) motion of bound tau. For some idealized cases, analytical solutions were derived. By comparing them with the full numerical solution and Monte Carlo simulations, the respective validity domains were mapped. Interpretation of our FDAP data (from processes of neuronally differentiated PC12 cells) in light of the heterogeneous formalism yielded independent estimates for the association (∼2 ms) and dwell (∼100 ms) times of tau to/on a single MT rather than in an MT array. The dwell time was shorter by orders of magnitude than that in a previous report where a homogeneous topology of MTs was assumed. We found that the diffusion of bound tau was negligible in vivo, in contrast to an earlier report that tau diffuses along the MT lattice in vitro. Methodologically, our results demonstrate that the heterogeneity of binding sites cannot be ignored when dealing with reaction-diffusion of cytoskeleton-associated proteins. Physiologically, the results reveal the behavior of tau in cellular processes

  3. An EB1-kinesin complex is sufficient to steer microtubule growth in vitro

    PubMed Central

    Chen, Yalei; Rolls, Melissa M.; Hancock, William O.

    2013-01-01

    Summary Proper microtubule polarity underlies overall neuronal polarity, but mechanisms for maintaining microtubule polarity are not well understood. Previous live imaging in Drosophila dendritic arborization (da) neurons showed that, while microtubules are uniformly plus-end out in axons, dendrites possess uniformly minus-end-out microtubules [1]. Thus, maintaining uniform microtubule polarity in dendrites requires that growing microtubule plus-ends entering branch points must be actively directed towards the cell body. A model was proposed in which EB1 tracks the plus-ends of microtubules growing into a branches and an associated kinesin-2 motor walks along a static microtubule to steer the plus-end toward the cell body. However, the fast plus-end binding dynamics of EB1 [2–5] appear at odds with this proposed mechanical function. To test this model in vitro, we reconstituted the system by artificially dimerizing EB1 to kinesin, growing microtubules from immobilized seeds, and imaging encounters between growing microtubule plus-ends and static microtubules. Consistent with in vivo observations, the EB1-kinesin complex actively steered growing microtubules. Thus EB1 kinetics and mechanics are sufficient to bend microtubules for several seconds. Other kinesins also demonstrated this activity, suggesting this is a general mechanism for organizing and maintaining proper microtubule polarity in cells. PMID:24462004

  4. Pironetin reacts covalently with cysteine-316 of α-tubulin to destabilize microtubule

    NASA Astrophysics Data System (ADS)

    Yang, Jianhong; Wang, Yuxi; Wang, Taijing; Jiang, Jian; Botting, Catherine H.; Liu, Huanting; Chen, Qiang; Yang, Jinliang; Naismith, James H.; Zhu, Xiaofeng; Chen, Lijuan

    2016-06-01

    Molecules that alter the normal dynamics of microtubule assembly and disassembly include many anticancer drugs in clinical use. So far all such therapeutics target β-tubulin, and structural biology has explained the basis of their action and permitted design of new drugs. However, by shifting the profile of β-tubulin isoforms, cancer cells become resistant to treatment. Compounds that bind to α-tubulin are less well characterized and unexploited. The natural product pironetin is known to bind to α-tubulin and is a potent inhibitor of microtubule polymerization. Previous reports had identified that pironetin reacts with lysine-352 residue however analogues designed on this model had much lower potency, which was difficult to explain, hindering further development. We report crystallographic and mass spectrometric data that reveal that pironetin forms a covalent bond to cysteine-316 in α-tubulin via a Michael addition reaction. These data provide a basis for the rational design of α-tubulin targeting chemotherapeutics.

  5. Orientation dynamics of azobenzene-based self-assembled monolayers

    NASA Astrophysics Data System (ADS)

    Fang, Guanjiu

    2007-12-01

    Self-assembled monolayers (SAMs) synthesized on glass and incorporating azobenzene are illuminated with actinic light to study the effect of photo-isomerization on in-plane molecular orientation. Measurements of the monolayer birefringence show that the SAM orientation dynamics are non-first-order kinetics, characterized by an exponential relaxation with a distribution of relaxation times. The orientational birefringence decays as a power law with an exponent that decreases with increasing initial writing intensity, indicative of orientational trapping wells with a distribution of depths. The distribution of trapping well depths, resulting from the distribution of free volumes among the SAM molecules, is modified by exposure to light. This depth modification indicates that photoisomerization is a collective process depending on neighboring molecules. Increasing the light intensity enhances the collective effect so that the orientation dynamics are changed from fractal to normal diffusion. With a further increase in light intensity, the collective effect does not change any more so that the mean width of the trapping well depth distribution, characterized by the power law exponent, becomes constant. The liquid crystal alignment in a photo-buffed SAM/rubbed nylon twisted nematic cell is very stable at room temperature, but it is lost upon cooling if the liquid crystal is heated into the isotropic phase, which is attributed to the thermal orientational relaxation of the SAM molecules. The liquid crystal response to reorienting the SAM with polarized light depends on the initial SAM writing time: the harder the initial writing, the slower subsequent reorientation, which is similar to the behavior of bare SAMs. The reorientation of the liquid crystals shows hysteresis compared with that of bare SAMs, with a larger dose required at higher intensity.

  6. Microtubule defects & Neurodegeneration.

    PubMed

    Baird, Fiona J; Bennett, Craig L

    2013-12-06

    One of the major challenges facing the long term survival of neurons is their requirement to maintain efficient axonal transport over long distances. In humans as large, long-lived vertebrates, the machinery maintaining neuronal transport must remain efficient despite the slow accumulation of cell damage during aging. Mutations in genes encoding proteins which function in the transport system feature prominently in neurologic disorders. Genes known to cause such disorders and showing traditional Mendelian inheritance have been more readily identified. It has been more difficult, however, to isolate factors underlying the complex genetics contributing to the more common idiopathic forms of neurodegenerative disease. At the heart of neuronal transport is the rail network or scaffolding provided by neuron specific microtubules (MTs). The importance of MT dynamics and stability is underscored by the critical role tau protein plays in MT-associated stabilization versus the dysfunction seen in Alzheimer's disease, frontotemporal dementia and other tauopathies. Another example of the requirement for tight regulation of MT dynamics is the need to maintain balanced levels of post-translational modification of key MT building-blocks such as α-tubulin. Tubulins require extensive polyglutamylation at their carboxyl-terminus as part of a novel post-translational modification mechanism to signal MT growth versus destabilization. Dramatically, knock-out of a gene encoding a deglutamylation family member causes an extremely rapid cell death of Purkinje cells in the ataxic mouse model, pcd. This review will examine a range of neurodegenerative conditions where current molecular understanding points to defects in the stability of MTs and axonal transport to emphasize the central role of MTs in neuron survival.

  7. Electrophoretic dynamics of self-assembling branched DNA structures

    NASA Astrophysics Data System (ADS)

    Heuer, Daniel Milton

    This study advances our understanding of the electrophoretic dynamics of branched biopolymers and explores technologies designed to exploit their unique properties. New self-assembly techniques were developed to create branched DNA for visualization via fluorescence microscopy. Experiments in fixed gel networks reveal a distinct trapping behavior, in contrast with linear topologies. The finding that detection can be achieved by introducing a branch point contributes significantly to the field of separation science and can be exploited to develop new applications. Results obtained in polymer solutions point to identical mobilities for branched and linear topologies, despite large differences in their dynamics. This finding led to a new description of electrophoresis based on non-Newtonian viscoelastic effects in the electric double layer surrounding a charged object. This new theoretical framework presents a new outlook important not only to the electrophoretic physics of nucleic acids, but all charged objects including proteins, colloids, and nanoparticles. To study the behavior of smaller biopolymers, such as restriction fragments and recombination intermediates, a library of symmetrically branched DNA was synthesized followed by characterization in gels. The experimental results contribute a large body of information relating molecular architecture and the dynamics of rigid structures in an electric field. The findings allow us to create new separation technologies based on topology. These contributions can also be utilized in a number of different applications including the study of recombination intermediates and the separation of proteins according to structure. To demonstrate the importance of these findings, a sequence and mutation detection technique was envisioned and applied for genetic analysis. Restriction fragments from mutation "hotspots" in the p53 tumor suppressor gene, known to play a role in cancer development, were analyzed with this technique

  8. Dynamic and Kinetic Assembly Studies of an Icosahedral Virus Capsid

    NASA Astrophysics Data System (ADS)

    Lee, Kelly

    2011-03-01

    Hepatitis B virus has an icosahedrally symmetrical core particle (capsid), composed of either 90 or 120 copies of a dimeric protein building block. We are using time-resolved, solution small-angle X-ray scattering and single-molecule fluorescence microscopy to probe the core particle assembly reaction at the ensemble and individual assembly levels. Our experiments to date reveal the assembly process to be highly cooperative with minimal population of stable intermediate species. Solution conditions, particularly salt concentration, appears to influence the partitioning of assembly products into the two sizes of shells. Funding from NIH R00-GM080352 and University of Washington.

  9. Active contraction of microtubule networks

    PubMed Central

    Foster, Peter J; Fürthauer, Sebastian; Shelley, Michael J; Needleman, Daniel J

    2015-01-01

    Many cellular processes are driven by cytoskeletal assemblies. It remains unclear how cytoskeletal filaments and motor proteins organize into cellular scale structures and how molecular properties of cytoskeletal components affect the large-scale behaviors of these systems. Here, we investigate the self-organization of stabilized microtubules in Xenopus oocyte extracts and find that they can form macroscopic networks that spontaneously contract. We propose that these contractions are driven by the clustering of microtubule minus ends by dynein. Based on this idea, we construct an active fluid theory of network contractions, which predicts a dependence of the timescale of contraction on initial network geometry, a development of density inhomogeneities during contraction, a constant final network density, and a strong influence of dynein inhibition on the rate of contraction, all in quantitative agreement with experiments. These results demonstrate that the motor-driven clustering of filament ends is a generic mechanism leading to contraction. DOI: http://dx.doi.org/10.7554/eLife.10837.001 PMID:26701905

  10. The microtubule plus-end tracking protein ARMADILLO-REPEAT KINESIN1 promotes microtubule catastrophe in Arabidopsis.

    PubMed

    Eng, Ryan Christopher; Wasteneys, Geoffrey O

    2014-08-01

    Microtubule dynamics are critically important for plant cell development. Here, we show that Arabidopsis thaliana ARMADILLO-REPEAT KINESIN1 (ARK1) plays a key role in root hair tip growth by promoting microtubule catastrophe events. This destabilizing activity appears to maintain adequate free tubulin concentrations in order to permit rapid microtubule growth, which in turn is correlated with uniform tip growth. Microtubules in ark1-1 root hairs exhibited reduced catastrophe frequency and slower growth velocities, both of which were restored by low concentrations of the microtubule-destabilizing drug oryzalin. An ARK1-GFP (green fluorescent protein) fusion protein expressed under its endogenous promoter localized to growing microtubule plus ends and rescued the ark1-1 root hair phenotype. Transient overexpression of ARK1-RFP (red fluorescent protein) increased microtubule catastrophe frequency. ARK1-fusion protein constructs lacking the N-terminal motor domain still labeled microtubules, suggesting the existence of a second microtubule binding domain at the C terminus of ARK1. ARK1-GFP was broadly expressed in seedlings, but mutant phenotypes were restricted to root hairs, indicating that ARK1's function is redundant in cells other than those forming root hairs.

  11. Dynamics and Statics of DNA-Programmable Nanoparticle Self-Assembly and Crystallization

    NASA Astrophysics Data System (ADS)

    Knorowski, C.; Burleigh, S.; Travesset, A.

    2011-05-01

    DNA linker mediated self-assembly is emerging as a very general strategy for designing new materials. In this Letter, we characterize both the dynamics and thermodynamics of nanoparticle-DNA self-assembly by molecular dynamics simulations from a new coarse-grained model. We establish the general phase diagram and discuss the stability of a previously overlooked crystalline phase (D-bcc). We also characterize universal properties about the dynamics of crystallization. We point out the connection to f-star polymer systems and discuss the implications for ongoing experiments as well as for the general field of DNA mediated self-assembly.

  12. Dynamics and statics of DNA-programmable nanoparticle self-assembly and crystallization.

    PubMed

    Knorowski, C; Burleigh, S; Travesset, A

    2011-05-27

    DNA linker mediated self-assembly is emerging as a very general strategy for designing new materials. In this Letter, we characterize both the dynamics and thermodynamics of nanoparticle-DNA self-assembly by molecular dynamics simulations from a new coarse-grained model. We establish the general phase diagram and discuss the stability of a previously overlooked crystalline phase (D-bcc). We also characterize universal properties about the dynamics of crystallization. We point out the connection to f-star polymer systems and discuss the implications for ongoing experiments as well as for the general field of DNA mediated self-assembly.

  13. Assembly and actuation of nanomaterials using active biomolecules.

    SciTech Connect

    Spoerke, Erik David; Thayer, Gayle Echo; de Boer, Maarten Pieter; Bunker, Bruce Conrad; Liu, Jun; Corwin, Alex David; Gaudioso, Jennifer Marie; Sasaki, Darryl Yoshio; Boal, Andrew Kiskadden; Bachand, George David; Trent, Amanda M.; Bachand, Marlene; Rivera, Susan B.; Koch, Steven John

    2005-11-01

    The formation and functions of living materials and organisms are fundamentally different from those of synthetic materials and devices. Synthetic materials tend to have static structures, and are not capable of adapting to the functional needs of changing environments. In contrast, living systems utilize energy to create, heal, reconfigure, and dismantle materials in a dynamic, non-equilibrium fashion. The overall goal of the project was to organize and reconfigure functional assemblies of nanoparticles using strategies that mimic those found in living systems. Active assembly of nanostructures was studied using active biomolecules to drive the organization and assembly of nanocomposite materials. In this system, kinesin motor proteins and microtubules were used to direct the transport and interactions of nanoparticles at synthetic interfaces. In addition, the kinesin/microtubule transport system was used to actively assemble nanocomposite materials capable of storing significant elastic energy. Novel biophysical measurement tools were also developed for measuring the collective force generated by kinesin motor proteins, which will provide insight on the mechanical constraints of active assembly processes. Responsive reconfiguration of nanostructures was studied in terms of using active biomolecules to mediate the optical properties of quantum dot (QD) arrays through modulation of inter-particle spacing and associated energy transfer interaction. Design rules for kinesin-based transport of a wide range of nanoscale cargo (e.g., nanocrystal quantum dots, micron-sized polymer spheres) were developed. Three-dimensional microtubule organizing centers were assembled in which the polar orientation of the microtubules was controlled by a multi-staged assembly process. Overall, a number of enabling technologies were developed over the course of this project, and will drive the exploitation of energy-driven processes to regulate the assembly, disassembly, and dynamic

  14. Self-organisation and forces in the microtubule cytoskeleton.

    PubMed

    Nédélec, François; Surrey, Thomas; Karsenti, Eric

    2003-02-01

    Modern microscopy techniques allow us to observe specifically tagged proteins in live cells. We can now see directly that many cellular structures, for example mitotic spindles, are in fact dynamic assemblies. Their apparent stability results from out-of-equilibrium stochastic interactions at the molecular level. Recent studies have shown that the spindles can form even after centrosomes are destroyed, and that they can even form around DNA-coated beads devoid of kinetochores. Moreover, conditions have been produced in which microtubule asters interact even in the absence of chromatin. Together, these observations suggest that the spindle can be experimentally deconstructed, and that its defining characteristics can be studied in a simplified context, in the absence of the full division machinery.

  15. Do prokaryotes contain microtubules?

    PubMed Central

    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. Images PMID:7968920

  16. Do prokaryotes contain microtubules?

    NASA Technical Reports Server (NTRS)

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

    PubMed Central

    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

  18. Electric field-induced reversible trapping of microtubules along metallic glass microwire electrodes

    NASA Astrophysics Data System (ADS)

    Kim, Kyongwan; Sikora, Aurélien; Nakayama, Koji S.; Umetsu, Mitsuo; Hwang, Wonmuk; Teizer, Winfried

    2015-04-01

    Microtubules are among bio-polymers providing vital functions in dynamic cellular processes. Artificial organization of these bio-polymers is a requirement for transferring their native functions into device applications. Using electrophoresis, we achieve an accumulation of microtubules along a metallic glass (Pd42.5Cu30Ni7.5P20) microwire in solution. According to an estimate based on migration velocities of microtubules approaching the wire, the electrophoretic mobility of microtubules is around 10-12 m2/Vs. This value is four orders of magnitude smaller than the typical mobility reported previously. Fluorescence microscopy at the individual-microtubule level shows microtubules aligning along the wire axis during the electric field-induced migration. Casein-treated electrodes are effective to reversibly release trapped microtubules upon removal of the external field. An additional result is the condensation of secondary filamentous structures from oriented microtubules.

  19. LIM-kinase 2, a regulator of actin dynamics, is involved in mitotic spindle integrity and sensitivity to microtubule-destabilizing drugs.

    PubMed

    Po'uha, S T; Shum, M S Y; Goebel, A; Bernard, O; Kavallaris, M

    2010-01-28

    LIM-kinase 2 (LIMK2) belongs to the LIMK family of proteins, which comprises LIMK1 and LIMK2. Both proteins regulate actin polymerization through phosphorylation and inactivation of the actin depolymerizing factor cofilin. In this study, we show that the level of LIMK2 protein is increased in neuroblastoma, BE(2)-C cells, selected for resistance to microtubule-destabilizing agents, vincristine and colchicine. However, the level of phosphorylated LIMK1 and LIMK2 was similar in the resistant and parental BE(2)-C cells. In contrast, the level of phospho-cofilin was greatly increased in the drug-resistant cells. Downregulation of LIMK2 expression increases sensitivity of neuroblastoma SH-EP cells to vincristine and vinblastine but not to microtubule-stabilizing agents, while it's overexpression increased its resistance to vincristine. Its vincristine-induced mitotic arrest was moderately inhibited in the LIMK2 knockdown cells, suggesting that the increased drug sensitivity is through an alternative mechanism other then mitotic arrest and apoptosis. Moreover, downregulation of LIMK2 expression induces formation of abnormal mitotic spindles, an effect enhanced in the presence of microtubule-destabilizing agents. LIMK2 is important for normal mitotic spindle formation and altered LIMK2 expression mediates sensitivity to microtubule destabilizing agents. These findings suggest that inhibition of LIMK2 activity may be used for the treatment of tumors resistant to microtubule-destabilizing drugs.

  20. Control of microtubule nucleation and stability in Madin-Darby canine kidney cells: the occurrence of noncentrosomal, stable detyrosinated microtubules

    PubMed Central

    1987-01-01

    The microtubule-nucleating activity of centrosomes was analyzed in fibroblastic (Vero) and in epithelial cells (PtK2, Madin-Darby canine kidney [MDCK]) by double-immunofluorescence labeling with anti- centrosome and antitubulin antibodies. Most of the microtubules emanated from the centrosomes in Vero cells, whereas the microtubule network of MDCK cells appeared to be noncentrosome nucleated and randomly organized. The pattern of microtubule organization in PtK2 cells was intermediate to the patterns observed in the typical fibroblastic and epithelial cells. The two centriole cylinders were tightly associated and located close to the nucleus in Vero and PtK2 cells. In MDCK cells, however, they were clearly separated and electron microscopy revealed that they nucleated only a few microtubules. The stability of centrosomal and noncentrosomal microtubules was examined by treatment of these different cell lines with various concentrations of nocodazole. 1.6 microM nocodazole induced an almost complete depolymerization of microtubules in Vero cells; some centrosome nucleated microtubules remained in PtK2 cells, while many noncentrosomal microtubules resisted that treatment in MDCK cells. Centrosomal and noncentrosomal microtubules regrew in MDCK cells with similar kinetics after release from complete disassembly by high concentrations of nocodazole (33 microM). During regrowth, centrosomal microtubules became resistant to 1.6 microM nocodazole before the noncentrosomal ones, although the latter eventually predominate. We suggest that in MDCK cells, microtubules grow and shrink as proposed by the dynamic instability model but the presence of factors prevents them from complete depolymerization. This creates seeds for reelongation that compete with nucleation off the centrosome. By using specific antibodies, we have shown that the abundant subset of nocodazole- resistant microtubules in MDCK cells contained detyrosinated alpha- tubulin (glu tubulin). On the other hand

  1. Dilution of individual microtubules observed in real time in vitro: evidence that cap size is small and independent of elongation rate

    PubMed Central

    1991-01-01

    Although the mechanism of microtubule dynamic instability is thought to involve the hydrolysis of tubulin-bound GTP, the mechanism of GTP hydrolysis and the basis of microtubule stability are controversial. Video microscopy of individual microtubules and dilution protocols were used to examine the size and lifetime of the stabilizing cap. Purified porcine brain tubulin (7-23 microM) was assembled at 37 degrees C onto both ends of isolated sea urchin axoneme fragments in a miniature flow cell to give a 10-fold variation in elongation rate. The tubulin concentration in the region of microtubule growth could be diluted rapidly (by 84% within 3 s of the onset of dilution). Upon perfusion with buffer containing no tubulin, microtubules experienced a catastrophe (conversion from elongation to rapid shortening) within 4-6 s on average after dilution to 16% of the initial concentration, independent of the predilution rate of elongation and length. Based on extrapolation of catastrophe frequency to zero tubulin concentration, the estimated lifetime of the stable cap after infinite dilution was less than 3-4 s for plus and minus ends, much shorter than the approximately 200 s observed at steady state (Walker, R. A., E. T. O'Brien, N. K. Pryer, M. Soboeiro, W. A. Voter, H. P. Erickson, and E. D. Salmon. 1988. J. Cell Biol. 107:1437-1448.). We conclude that during elongation, both plus and minus ends are stabilized by a short region (approximately 200 dimers or less) and that the size of the stable cap is independent of 10-fold variation in elongation rate. These results eliminate models of dynamic instability which predict extensive "build- up" stabilizing caps and support models which constrain the cap to the elongating tip. We propose that the cell may take advantage of such an assembly mechanism by using "catastrophe factors" that can promote frequent catastrophe even at high elongation rates by transiently binding to microtubule ends and briefly inhibiting GTP

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

    PubMed

    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.

  3. BRIDGES BETWEEN MICROTUBULES

    PubMed Central

    McIntosh, J. R.

    1974-01-01

    Bridges between microtubules have been studied with the electron microscope in the axostyle of Saccinobaculus and in various tubule systems of chicken testis, including the helix of tubules surrounding the elongating spermatid nucleus and the flagellum of the sperm tail. In addition to the previously described periodic bridges, evidence is presented that nonperiodic bridges exist between certain tubules. An analysis of axial spacing between adjacent nonperiodic bridges suggests that these structures are attached to periodic binding sites on the microtubule wall, but that not all the binding sites are filled. The bridges appear nonperiodic as a result of random occupancy of some fraction of the periodic sites. The distribution of these binding sites is related to the substructure of the microtubule wall as seen with negative staining and optical diffraction. PMID:4132065

  4. Predicting out-of-Equilibrium Phase Behavior in the Dynamic Self-Assembly of Colloidal Crystals

    NASA Astrophysics Data System (ADS)

    Swan, James; Sherman, Zachary

    Crystals self-assembled from colloidal particles are useful in an array of well demonstrated applications. During fabrication however, gelation and glassification often leave these materials arrested in defective or disordered metastable states. We show how time-dependent, pulsed interparticle interactions can avoid kinetic barriers and yield well-ordered crystalline domains for a suspension of hard, spherical colloidal particles interacting through short-range attractions. This dynamic self-assembly process is analogous to the flashing Brownian rachet. Although this is an inherently unsteady, out-of-equilibrium process, we can predict its outcome using appropriate time averages of equilibrium equations of state. The predicted phase behavior is tested and validated by examining the fluid/crystal coexistence of such dynamically self-assembling dispersions in Brownian dynamics simulations of sedimentation equilibrium and homogeneous nucleation. We also show that our dynamic self-assembly scheme offers control and tunability over the crystal growth kinetics and can even stabilize nonequilibrium structures.

  5. Dynamic transformation of self-assembled structures using anisotropic magnetized hydrogel microparticles

    NASA Astrophysics Data System (ADS)

    Yoshida, Satoru; Takinoue, Masahiro; Iwase, Eiji; Onoe, Hiroaki

    2016-08-01

    This paper describes a system through which the self-assembly of anisotropic hydrogel microparticles is achieved, which also enables dynamic transformation of the assembled structures. Using a centrifuge-based microfluidic device, anisotropic hydrogel microparticles encapsulating superparamagnetic materials on one side are fabricated, which respond to a magnetic field. We successfully achieve dynamic assembly using these hydrogel microparticles and realize three different self-assembled structures (single and double pearl chain structures, and close-packed structures), which can be transformed to other structures dynamically via tuning of the precessional magnetic field. We believe that the developed system has potential application as an effective platform for a dynamic cell manipulation and cultivation system, in biomimetic autonomous microrobot organization, and that it can facilitate further understanding of the self-organization and complex systems observed in nature.

  6. Brownian dynamics simulation of fission yeast mitotic spindle formation

    NASA Astrophysics Data System (ADS)

    Edelmaier, Christopher

    2014-03-01

    The mitotic spindle segregates chromosomes during mitosis. The dynamics that establish bipolar spindle formation are not well understood. We have developed a computational model of fission-yeast mitotic spindle formation using Brownian dynamics and kinetic Monte Carlo methods. Our model includes rigid, dynamic microtubules, a spherical nuclear envelope, spindle pole bodies anchored in the nuclear envelope, and crosslinkers and crosslinking motor proteins. Crosslinkers and crosslinking motor proteins attach and detach in a grand canonical ensemble, and exert forces and torques on the attached microtubules. We have modeled increased affinity for crosslinking motor attachment to antiparallel microtubule pairs, and stabilization of microtubules in the interpolar bundle. We study parameters controlling the stability of the interpolar bundle and assembly of a bipolar spindle from initially adjacent spindle-pole bodies.

  7. Dynamic Self-Assembly Encodes A Tri-stable Au-TiO2 Photocatalyst.

    PubMed

    Zhang, Qi; Wang, Wen-Zhi; Yu, Jing-Jing; Qu, Da-Hui; Tian, He

    2017-02-01

    A tri-stable switchable catalyst is encoded by pH-controlled dynamic self-assembly of gold and TiO2 nanoparticles (NPs). Through precise adjustment of the integrated dynamic covalent and noncovalent self-assembly process of the two types of nanoparticles, the photocatalytic activity of the hybrid system is modulated by switching pH conditions between tri-stable "highly active", "active", and "inactive" states.

  8. Spindle microtubule dynamics in sea urchin embryos: analysis using a fluorescein-labeled tubulin and measurements of fluorescence redistribution after laser photobleaching

    PubMed Central

    1984-01-01

    The rate of exchange of tubulin that is incorporated into spindle microtubules with dimeric tubulin in the cytoplasm has been measured in sea urchin eggs by studying fluorescence redistribution after photobleaching (FRAP). Dichlorotriazinyl amino fluorescein (DTAF) has been used to label bovine brain tubulin. DTAF-tubulin has been injected into fertilized eggs of Lytechinus variegatus and allowed to equilibrate with the endogenous tubulin pool. Fluorescent spindles formed at the same time that spindles were seen in control eggs, and the injected embryos proceeded through many cycles of division on schedule, suggesting that DTAF-tubulin is a good analogue of tubulin in vivo. A microbeam of argon laser light has been used to bleach parts of the fluorescent spindles, and FRAP has been recorded with a sensitive video camera. Laser bleaching did not affect spindle structure, as seen with polarization optics, nor spindle function, as seen by rate of progress through mitosis, even when one spindle was bleached several times in a single cell cycle. Video image analysis has been used to measure the rate of FRAP and to obtain a low resolution view of the fluorescence redistribution process. The half-time for spindle FRAP is approximately 19 s, even when an entire half-spindle is bleached. Complete exchange of tubulin in nonkinetochore spindle and astral microtubules appeared to occur within 60-80 s at steady state. This rate is too fast to be explained by a simple microtubule end-dependent exchange of tubulin. Efficient microtubule treadmilling would be fast enough, but with current techniques we saw no evidence for movement of the bleached spot during recovery, which we would expect on the basis of Margolis and Wilson's model (Nature (Lond.)., 1981, 293:705)-- fluorescence recovers uniformly. Microtubules may be depolymerizing and repolymerizing rapidly and asynchronously throughout the spindle and asters, but the FRAP data are most compatible with a rapid exchange of

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

    PubMed

    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.

  10. Microtubule stabilising peptides rescue tau phenotypes in-vivo

    PubMed Central

    Quraishe, Shmma; Sealey, Megan; Cranfield, Louise; Mudher, Amritpal

    2016-01-01

    The microtubule cytoskeleton is a highly dynamic, filamentous network underpinning cellular structure and function. In Alzheimer’s disease, the microtubule cytoskeleton is compromised, leading to neuronal dysfunction and eventually cell death. There are currently no disease-modifying therapies to slow down or halt disease progression. However, microtubule stabilisation is a promising therapeutic strategy that is being explored. We previously investigated the disease-modifying potential of a microtubule-stabilising peptide NAP (NAPVSIPQ) in a well-established Drosophila model of tauopathy characterised by microtubule breakdown and axonal transport deficits. NAP prevented as well as reversed these phenotypes even after they had become established. In this study, we investigate the neuroprotective capabilities of an analogous peptide SAL (SALLRSIPA). We found that SAL mimicked NAP’s protective effects, by preventing axonal transport disruption and improving behavioural deficits, suggesting both NAP and SAL may act via a common mechanism. Both peptides contain a putative ‘SIP’ (Ser-Ile-Pro) domain that is important for interactions with microtubule end-binding proteins. Our data suggests this domain may be central to the microtubule stabilising function of both peptides and the mechanism by which they rescue phenotypes in this model of tauopathy. Our observations support microtubule stabilisation as a promising disease-modifying therapeutic strategy for tauopathies like Alzheimer’s disease. PMID:27910888

  11. Microtubule-severing enzymes at the cutting edge.

    PubMed

    Sharp, David J; Ross, Jennifer L

    2012-06-01

    ATP-dependent severing of microtubules was first reported in Xenopus laevis egg extracts in 1991. Two years later this observation led to the purification of the first known microtubule-severing enzyme, katanin. Katanin homologs have now been identified throughout the animal kingdom and in plants. Moreover, members of two closely related enzyme subfamilies, spastin and fidgetin, have been found to sever microtubules and might act alongside katanins in some contexts (Roll-Mecak and McNally, 2010; Yu et al., 2008; Zhang et al., 2007). Over the past few years, it has become clear that microtubule-severing enzymes contribute to a wide range of cellular activities including mitosis and meiosis, morphogenesis, cilia biogenesis and disassembly, and migration. Thus, this group of enzymes is revealing itself to be among the most important of the microtubule regulators. This Commentary focuses on our growing understanding of how microtubule-severing enzymes contribute to the organization and dynamics of diverse microtubule arrays, as well as the structural and biophysical characteristics that afford them the unique capacity to catalyze the removal of tubulin from the interior microtubule lattice. Our goal is to provide a broader perspective, focusing on a limited number of particularly informative, representative and/or timely findings.

  12. Metallic Glass Wire Based Localization of Kinesin/Microtubule Bio-molecular Motility System

    NASA Astrophysics Data System (ADS)

    Kim, K.; Sikora, A.; Yaginuma, S.; Nakayama, K. S.; Nakazawa, H.; Umetsu, M.; Hwang, W.; Teizer, W.

    2014-03-01

    We report electrophoretic accumulation of microtubules along metallic glass (Pd42.5Cu30Ni7.5P20) wires free-standing in solution. Microtubules are dynamic cytoskeletal filaments. Kinesin is a cytoskeletal motor protein. Functions of these bio-molecules are central to various dynamic cellular processes. Functional artificial organization of bio-molecules is a prerequisite for transferring their native functions into device applications. Fluorescence microscopy at the individual-microtubule level reveals microtubules aligning along the wire axis during the electrophoretic migration. Casein-treated electrodes are effective for releasing trapped microtubules upon removal of the external field. Furthermore, we demonstrate gliding motion of microtubules on kinesin-treated metallic glass wires. The reversible manner in the local adsorption of microtubules, the flexibility of wire electrodes, and the compatibility between the wire electrode and the bio-molecules are beneficial for spatio-temporal manipulation of the motility machinery in 3 dimensions.

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

    PubMed

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

    2017-03-24

    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.

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

    PubMed Central

    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

  15. The dynamics and energetics of matrix assembly and mineralization.

    PubMed

    De Yoreo, James J; Chung, Sungwook; Nielsen, Michael H

    2013-10-01

    Formation of biominerals commonly occurs within the context of an organic matrix composed of proteins, polysaccharides, and other macromolecules. Much has been learned about the structure of matrices and the spatial and molecular relationships between matrix and mineral. Only recently has quantitative study of matrix organization and subsequent mineralization been pursued. Here, we review findings from physical studies of matrix assembly in the system of microbial S-layer proteins and of calcium carbonate nucleation on organic templates composed of organothiol self-assembled monolayers on noble metals. Studies on S-layers reveal the importance of multistage assembly pathways and kinetic traps associated with the conformational transformations required to build the basic oligomeric building blocks of the matrix. Experimental investigations of calcium carbonate nucleation on carboxyl-terminated self-assembled monolayers combined with theoretical analyses demonstrate the applicability of classical concepts of nucleation, even when cluster-aggregation pathways are considered, and reveal the underlying energetic and structural source of matrix control over the process. Taken together, these studies highlight the ways in which matrix assembly and mineralization deviate from our classical concepts of crystallization but clearly demonstrate that the concepts of physical chemistry that date back to the days of Gibbs and Ostwald still serve us well in understanding the nucleation and growth of organic matrices and mineral phases.

  16. Switchable static and dynamic self-assembly of magnetic droplets on superhydrophobic surfaces.

    PubMed

    Timonen, Jaakko V I; Latikka, Mika; Leibler, Ludwik; Ras, Robin H A; Ikkala, Olli

    2013-07-19

    Self-assembly is a process in which interacting bodies are autonomously driven into ordered structures. Static structures such as crystals often form through simple energy minimization, whereas dynamic ones require continuous energy input to grow and sustain. Dynamic systems are ubiquitous in nature and biology but have proven challenging to understand and engineer. Here, we bridge the gap from static to dynamic self-assembly by introducing a model system based on ferrofluid droplets on superhydrophobic surfaces. The droplets self-assemble under a static external magnetic field into simple patterns that can be switched to complicated dynamic dissipative structures by applying a time-varying magnetic field. The transition between the static and dynamic patterns involves kinetic trapping and shows complexity that can be directly visualized.

  17. Approach to Dynamic Assembling of Individualized Learning Paths

    ERIC Educational Resources Information Center

    Lubchak, Vladimir; Kupenko, Olena; Kuzikov, Borys

    2012-01-01

    E-learning students are generally heterogeneous and have different capabilities knowledge base and needs. The aim of the Sumy State University (SSU) e-learning system project is to cater to these individual needs by assembling individual learning path. This paper shows current situation with e-learning in Ukraine, state-of-art of development of…

  18. Jupiter, a new Drosophila protein associated with microtubules.

    PubMed

    Karpova, Nina; Bobinnec, Yves; Fouix, Sylvaine; Huitorel, Philippe; Debec, Alain

    2006-05-01

    In this study we describe a novel Drosophila protein Jupiter, which shares properties with several structural microtubule-associated proteins (MAPs) including TAU, MAP2, MAP4. Jupiter is a soluble unfolded molecule with the high net positive charge, rich in Glycine. It possesses two degenerated repeats around the sequence PPGG, separated by a Serine-rich region. Jupiter associates with microtubules in vitro and, fused with the green fluorescent protein (GFP), is an excellent marker to follow microtubule dynamics in vivo. In a jupiter transgenic Drosophila strain generated by the "protein-trap" technique, Jupiter:GFP fusion protein localizes to the microtubule network through the cell cycle at the different stages of development. We found particularly high Jupiter:GFP concentrations in the young embryo, larval nervous system, precursors of eye photoreceptors and adult ovary. Moreover, from jupiter:gfp embryos we have established two permanent cell lines presenting strongly fluorescent microtubules during the whole cell cycle. In these cells, the distribution of the Jupiter:GFP fusion protein reproduces microtubule behavior upon treatment by the drugs colchicine and taxol. The jupiter cell lines and fly strain should be of wide interest for biologists interested in in vivo analysis of microtubule dynamics.

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

    PubMed Central

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

    2013-01-01

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

  20. The Interaction of Microtubules with Stabilizers Characterized at Biochemical and Structural Levels

    NASA Astrophysics Data System (ADS)

    Díaz, J. F.; Andreu, J. M.; Jiménez-Barbero, J.

    Since the discovery of paclitaxel and its peculiar mechanism of cytotoxicity, which has made it and its analogues widely used antitumour drugs, great effort has been made to understand the way they produce their effect in microtubules and to find other products that share this effect without the undesired side effects of low solubility and development of multidrug resistance by tumour cells. This chapter reviews the actual knowledge about the biochemical and structural mechanisms of microtubule stabilization by microtubule stabilizing agents, and illustrates the way paclitaxel and its biomimetics induce microtubule assembly, the thermodynamics of their binding, the way they reach their binding site and the conformation they have when bound.

  1. Reactor Dynamics Experiments with a Sub-Critical Assembly

    SciTech Connect

    Miley, G.H.; Yang, Y.; Wu, L.; Momota, H.

    2004-10-06

    A resurgence in use of nuclear power is now underway worldwide. However due to the shutdown of many university research reactors , student laboratories must rely more heavily on use of sub-critical assemblies. Here a driven sub-critical is described that uses a cylindrical Inertial Electrostatic Confinement (IEC) device to provide a fusion neutron source. The small IEC neutron source would be inserted in a fuel element position, with its power input controlled externally at a control panel. This feature opens the way to use of the critical assembly for a number of transient experiments such as sub-critical pulsing and neutron wave propagation. That in turn adds important new insights and excitement for the student teaching laboratory.

  2. A theory of microtubule catastrophes and their regulation.

    PubMed

    Brun, Ludovic; Rupp, Beat; Ward, Jonathan J; Nédélec, François

    2009-12-15

    Dynamic instability, in which abrupt transitions occur between growing and shrinking states, is an intrinsic property of microtubules that is regulated by both mechanics and specialized proteins. We discuss a model of dynamic instability based on the popular idea that growth is maintained by a cap at the tip of the fiber. The loss of this cap is thought to trigger the transition from growth to shrinkage, called a catastrophe. The model includes longitudinal interactions between the terminal tubulins of each protofilament and "gating rescues" between neighboring protofilaments. These interactions allow individual protofilaments to transiently shorten during a phase of overall microtubule growth. The model reproduces the reported dependency of the catastrophe rate on tubulin concentration, the time between tubulin dilution and catastrophe, and the induction of microtubule catastrophes by walking depolymerases. The model also reproduces the comet tail distribution that is characteristic of proteins that bind to the tips of growing microtubules.

  3. Plant microtubule cytoskeleton complexity: microtubule arrays as fractals.

    PubMed

    Gardiner, John; Overall, Robyn; Marc, Jan

    2012-01-01

    Biological systems are by nature complex and this complexity has been shown to be important in maintaining homeostasis. The plant microtubule cytoskeleton is a highly complex system, with contributing factors through interactions with microtubule-associated proteins (MAPs), expression of multiple tubulin isoforms, and post-translational modification of tubulin and MAPs. Some of this complexity is specific to microtubules, such as a redundancy in factors that regulate microtubule depolymerization. Plant microtubules form partial helical fractals that play a key role in development. It is suggested that, under certain cellular conditions, other categories of microtubule fractals may form including isotropic fractals, triangular fractals, and branched fractals. Helical fractal proteins including coiled-coil and armadillo/beta-catenin repeat proteins and the actin cytoskeleton are important here too. Either alone, or in combination, these fractals may drive much of plant development.

  4. Emergence of reconfigurable wires and spinners via dynamic self-assembly

    DOE PAGES

    Kokot, Gasper; Piet, David; Whitesides, George M.; ...

    2015-03-26

    Dissipative colloidal materials use energy to generate and maintain structural complexity. The energy injection rate, and properties of the environment are important control parameters that influence the outcome of dynamic self-assembly. Here we demonstrate that dispersions of magnetic microparticles confined at the air-liquid interface, and energized by a uniaxial in-plane alternating magnetic field, self-assemble into a variety of structures that range from pulsating clusters and single-particle-thick wires to dynamic arrays of spinners (self-assembled short chains) rotating in either direction. The spinners emerge via spontaneous breaking of the uniaxial symmetry of the energizing magnetic field. Demonstration of the formation and disaggregationmore » of particle assemblies suggests strategies to form new meso-scale structures with the potential to perform functions such as mixing and sensing.« less

  5. Emergence of reconfigurable wires and spinners via dynamic self-assembly

    SciTech Connect

    Kokot, Gasper; Piet, David; Whitesides, George M.; Aranson, Igor S.; Snezhko, Alexey

    2015-03-26

    Dissipative colloidal materials use energy to generate and maintain structural complexity. The energy injection rate, and properties of the environment are important control parameters that influence the outcome of dynamic self-assembly. Here we demonstrate that dispersions of magnetic microparticles confined at the air-liquid interface, and energized by a uniaxial in-plane alternating magnetic field, self-assemble into a variety of structures that range from pulsating clusters and single-particle-thick wires to dynamic arrays of spinners (self-assembled short chains) rotating in either direction. The spinners emerge via spontaneous breaking of the uniaxial symmetry of the energizing magnetic field. Demonstration of the formation and disaggregation of particle assemblies suggests strategies to form new meso-scale structures with the potential to perform functions such as mixing and sensing.

  6. Emergence of reconfigurable wires and spinners via dynamic self-assembly

    PubMed Central

    Kokot, Gasper; Piet, David; Whitesides, George M.; Aranson, Igor S.; Snezhko, Alexey

    2015-01-01

    Dissipative colloidal materials use energy to generate and maintain structural complexity. The energy injection rate, and properties of the environment are important control parameters that influence the outcome of dynamic self-assembly. Here we demonstrate that dispersions of magnetic microparticles confined at the air-liquid interface, and energized by a uniaxial in-plane alternating magnetic field, self-assemble into a variety of structures that range from pulsating clusters and single-particle-thick wires to dynamic arrays of spinners (self-assembled short chains) rotating in either direction. The spinners emerge via spontaneous breaking of the uniaxial symmetry of the energizing magnetic field. Demonstration of the formation and disaggregation of particle assemblies suggests strategies to form new meso-scale structures with the potential to perform functions such as mixing and sensing. PMID:25810144

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

    PubMed

    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. An inner centromere protein that stimulates the microtubule depolymerizing activity of a KinI kinesin.

    PubMed

    Ohi, Ryoma; Coughlin, Margaret L; Lane, William S; Mitchison, Timothy J

    2003-08-01

    Mitosis requires precise control of microtubule dynamics. The KinI kinesin MCAK, a microtubule depolymerase, is critical for this regulation. In a screen to discover previously uncharacterized microtubule-associated proteins, we identified ICIS, a protein that stimulates MCAK activity in vitro. Consistent with this biochemical property, blocking ICIS function in Xenopus extracts with antibodies caused excessive microtubule growth and inhibited spindle formation. Prior to anaphase, ICIS localized in an MCAK-dependent manner to inner centromeres, the chromosomal region located in between sister kinetochores. From Xenopus extracts, ICIS coimmunoprecipitated MCAK and the inner centromere proteins INCENP and Aurora B, which are thought to promote chromosome biorientation. By immunoelectron microscopy, we found that ICIS is present on the surface of inner centromeres, placing it in an ideal location to depolymerize microtubules associated laterally with inner centromeres. At inner centromeres, MCAK-ICIS may destabilize these microtubules and provide a mechanism that prevents kinetochore-microtubule attachment errors.

  9. "Cloud" assemblies: quantum dots form electrostatically bound dynamic nebulae around large gold nanoparticles.

    PubMed

    Lilly, G Daniel; Lee, Jaebeom; Kotov, Nicholas A

    2010-10-14

    Dynamic self-assembled structures of nanoparticles can be produced using predominantly electrostatic interactions. Such assemblies were made from large, positively charged Au metal nanoparticles surrounded by an electrostatically bound cloud of smaller, negatively charged CdSe/ZnS or CdTe quantum dots. At low concentrations they are topologically similar to double electric layers of ions and corona-like assemblies linked by polymer chains. They can also be compared to the topological arrangement of some planetary systems in space. The great advantages of the cloud assemblies are (1) their highly dynamic nature compared to more rigid covalently bound assemblies, (2) simplicity of preparation, and (3) exceptional versatility in components and resulting optical properties. Photoluminescence intensity enhancement originating from quantum resonance between excitons and plasmons was observed for CdSe/ZnS quantum dots, although CdTe dots displayed emission quenching. To evaluate more attentively their dynamic behavior, emission data were collected for the cloud-assemblies with different ratios of the components and ionic strengths of the media. The emission of the system passes through a maximum for 80 QDs ∶ 1 Au NP as determined by the structure of the assemblies and light absorption conditions. Ionic strength dependence of luminescence intensity contradicts the predictions based on the Gouy-Chapman theory and osmotic pressure at high ionic strengths due to formation of larger chaotic colloidally stable assemblies. "Cloud" assemblies made from different nanoscale components can be used both for elucidation of most fundamental aspects of nanoparticle interactions, as well as for practical purposes in sensing and biology.

  10. “Cloud” assemblies: quantum dots form electrostatically bound dynamic nebulae around large gold nanoparticle

    SciTech Connect

    Lilly, G. Daniel; Lee, Jaebeom; Kotov, Nicholas A.

    2010-07-29

    Dynamic self-assembled structures of nanoparticles can be produced using predominantly electrostatic interactions. Such assemblies were made from large, positively charged Au metal nanoparticles surrounded by an electrostatically bound cloud of smaller, negatively charged CdSe/ZnS or CdTe quantum dots. At low concentrations they are topologically similar to double electric layers of ions and corona-like assemblies linked by polymer chains. They can also be compared to the topological arrangement of some planetary systems in space. The great advantages of the cloud assemblies are (1) their highly dynamic nature compared to more rigid covalently bound assemblies, (2) simplicity of preparation, and (3) exceptional versatility in components and resulting optical properties. Photoluminescence intensity enhancement originating from quantum resonance between excitons and plasmons was observed for CdSe/ZnS quantum dots, although CdTe dots displayed emission quenching. To evaluate more attentively their dynamic behavior, emission data were collected for the cloud-assemblies with different ratios of the components and ionic strengths of the media. The emission of the system passes through a maximum for 80 QDs:1 Au NP as determined by the structure of the assemblies and light absorption conditions. Ionic strength dependence of luminescence intensity contradicts the predictions based on the Gouy–Chapman theory and osmotic pressure at high ionic strengths due to formation of larger chaotic colloidally stable assemblies. “Cloud” assemblies made from different nanoscale components can be used both for elucidation of most fundamental aspects of nanoparticle interactions, as well as for practical purposes in sensing and biology.

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

    PubMed

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

    2014-03-01

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

  12. Cell Biology: Microtubule Collisions to the Rescue.

    PubMed

    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.

  13. A Chimeric Cetuximab-Functionalized Corona as a Potent Delivery System for Microtubule-Destabilizing Nanocomplexes to Hepatocellular Carcinoma Cells: A Focus on EGFR and Tubulin Intracellular Dynamics.

    PubMed

    Poojari, Radhika; Kini, Sudarshan; Srivastava, Rohit; Panda, Dulal

    2015-11-02

    In this study, we have developed microtubule destabilizing agents combretastatin A4 (CA4) or 2-methoxyestradiol (2ME) encapsulated poly(d,l-lactide-co-glycolide)-b-poly(ethylene glycol) (PLGA-b-PEG) nanocomplexes for targeted delivery to human hepatocellular carcinoma (HCC) cells. An epidermal growth factor receptor (EGFR) is known to be overexpressed in HCC cells. Therefore, the targeting moiety cetuximab (Cet), an anti-EGFR chimeric monoclonal antibody, is functionalized on the surface of these diblock copolymeric coronas. Cetuximab is associated with the extracellular domain of the EGFR; therefore, the uptake of the cetuximab conjugated nanocomplexes occurred efficiently in EGFR overexpressing HCC cells indicating potent internalization of the complex. The cetuximab targeted-PLGA-b-PEG nanocomplexes encapsulating CA4 or 2ME strongly inhibited phospho-EGFR expression, depolymerized microtubules, produced spindle abnormalities, stalled mitosis, and induced apoptosis in Huh7 cells compared to the free drugs, CA4 or 2ME. Further, the combinatorial strategy of targeted nanocomplexes, Cet-PLGA-b-PEG-CA4 NP and Cet-PLGA-b-PEG-2ME NP, significantly reduced the migration of Huh7 cells, and markedly enhanced the anticancer effects of the microtubule-targeted drugs in Huh7 cells compared to the free drugs, CA4 or 2ME. The results indicated that EGFR receptor-mediated internalization via cetuximab facilitated enhanced uptake of the nanocomplexes leading to potent anticancer efficacy in Huh7 cells. Cetuximab-functionalized PLGA-b-PEG nanocomplexes possess a strong potential for the targeted delivery of CA4 or 2ME in EGFR overexpressed HCC cells, and the strategy may be useful for selectively targeting microtubules in these cells.

  14. Motile Microbots from Dynamically Interacting and Self-Reconfiguring Assemblies of Metallo-Dielectric Janus Microcubes

    NASA Astrophysics Data System (ADS)

    Han, Koohee; Shields, C. Wyatt, IV; Bharti, Bhuvnesh; Lopez, Gabriel P.; Velev, Orlin D.

    A new class of dynamically and reversibly reconfigurable active matter made by magnetic assembly and actuation of metallo-dielectric microcubes will be presented. We describe how magnetically responsive Janus microcubes can be assembled hierarchically into dynamically reconfiguring microclusters. Ferromagnetic cobalt patches of the cubes act as assembly directors. The residual magnetic polarization of the metal-coated facets leads to directional dipole-dipole and field-dipole interactions and reconfiguration of the neighboring cubic particles, which is directed by the conformational restrictions. Dynamic reconfiguration of assembled clusters can be achieved by on-demand switching between the dipole-field interaction and the residual dipole-dipole interaction when the field is turned on and off. We show how pre-assembled Janus microcube clusters can be directionally motile in non-Newtonian fluids by applying asymmetric magnetic fields. The modulation of the viscosity of non-Newtonian fluids upon varying the shear rate allowed demonstrating directional motion, resulting from time-asymmetric stroke patterns (e.g., rapid opening and slow closing). These motile clusters can serve as early prototypes of self-propelling microswimmers capable of in-situ assembly. NSF Grant #DMR-1121107.

  15. Down the Rabbit Hole of Centromere Assembly and Dynamics

    PubMed Central

    Dalal, Yamini; Bui, Minh

    2010-01-01

    The centromere is perhaps the most iconic feature on a eukaryotic chromosome. An amateur enthusiast equipped with a light microscope can easily identify the center of each metacentric chromosome, marking the spot responsible for accurate genome segregation. This review will highlight findings which provide novel insights into how centromeres are assembled and disassembled, the role centromeric proteins play in repair, epigenetic features uniquely found at the centromere, and the three dimensional organization of centromeres caught in the act of mitosis. These advances have unveiled a veritable wonderland of non-canonical features that drive centromere function. PMID:20303726

  16. α-Synuclein is a Novel Microtubule Dynamase.

    PubMed

    Cartelli, Daniele; Aliverti, Alessandro; Barbiroli, Alberto; Santambrogio, Carlo; Ragg, Enzio M; Casagrande, Francesca V M; Cantele, Francesca; Beltramone, Silvia; Marangon, Jacopo; De Gregorio, Carmelita; Pandini, Vittorio; Emanuele, Marco; Chieregatti, Evelina; Pieraccini, Stefano; Holmqvist, Staffan; Bubacco, Luigi; Roybon, Laurent; Pezzoli, Gianni; Grandori, Rita; Arnal, Isabelle; Cappelletti, Graziella

    2016-09-15

    α-Synuclein is a presynaptic protein associated to Parkinson's disease, which is unstructured when free in the cytoplasm and adopts α helical conformation when bound to vesicles. After decades of intense studies, α-Synuclein physiology is still difficult to clear up due to its interaction with multiple partners and its involvement in a pletora of neuronal functions. Here, we looked at the remarkably neglected interplay between α-Synuclein and microtubules, which potentially impacts on synaptic functionality. In order to identify the mechanisms underlying these actions, we investigated the interaction between purified α-Synuclein and tubulin. We demonstrated that α-Synuclein binds to microtubules and tubulin α2β2 tetramer; the latter interaction inducing the formation of helical segment(s) in the α-Synuclein polypeptide. This structural change seems to enable α-Synuclein to promote microtubule nucleation and to enhance microtubule growth rate and catastrophe frequency, both in vitro and in cell. We also showed that Parkinson's disease-linked α-Synuclein variants do not undergo tubulin-induced folding and cause tubulin aggregation rather than polymerization. Our data enable us to propose α-Synuclein as a novel, foldable, microtubule-dynamase, which influences microtubule organisation through its binding to tubulin and its regulating effects on microtubule nucleation and dynamics.

  17. α-Synuclein is a Novel Microtubule Dynamase

    PubMed Central

    Cartelli, Daniele; Aliverti, Alessandro; Barbiroli, Alberto; Santambrogio, Carlo; Ragg, Enzio M.; Casagrande, Francesca V.M.; Cantele, Francesca; Beltramone, Silvia; Marangon, Jacopo; De Gregorio, Carmelita; Pandini, Vittorio; Emanuele, Marco; Chieregatti, Evelina; Pieraccini, Stefano; Holmqvist, Staffan; Bubacco, Luigi; Roybon, Laurent; Pezzoli, Gianni; Grandori, Rita; Arnal, Isabelle; Cappelletti, Graziella

    2016-01-01

    α-Synuclein is a presynaptic protein associated to Parkinson’s disease, which is unstructured when free in the cytoplasm and adopts α helical conformation when bound to vesicles. After decades of intense studies, α-Synuclein physiology is still difficult to clear up due to its interaction with multiple partners and its involvement in a pletora of neuronal functions. Here, we looked at the remarkably neglected interplay between α-Synuclein and microtubules, which potentially impacts on synaptic functionality. In order to identify the mechanisms underlying these actions, we investigated the interaction between purified α-Synuclein and tubulin. We demonstrated that α-Synuclein binds to microtubules and tubulin α2β2 tetramer; the latter interaction inducing the formation of helical segment(s) in the α-Synuclein polypeptide. This structural change seems to enable α-Synuclein to promote microtubule nucleation and to enhance microtubule growth rate and catastrophe frequency, both in vitro and in cell. We also showed that Parkinson’s disease-linked α-Synuclein variants do not undergo tubulin-induced folding and cause tubulin aggregation rather than polymerization. Our data enable us to propose α-Synuclein as a novel, foldable, microtubule-dynamase, which influences microtubule organisation through its binding to tubulin and its regulating effects on microtubule nucleation and dynamics. PMID:27628239

  18. Viscosity Control of the