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Sample records for fast microtubule dynamics

  1. 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.

  2. How to measure microtubule dynamics?

    PubMed

    Straube, Anne

    2011-01-01

    Microtubules are one of the most spectacular features in the cell: long, fairly rigid tubules that provide physical strength while at the same time serving as tracks of the intracellular transport network. In addition, they are the main constituents of the cell division machinery, and guide axonal growth and the direction of cell migration. To be able to fulfil such diverse functions, microtubules have to be arranged into suitable patterns and remodelled according to extra- and intracellular cues. Moreover, the delicate regulation of microtubule dynamics and the dynamic interactions with subcellular structures, such as kinetochores or cell adhesion sites, appear to be of crucial importance to microtubule functions. It is, therefore, important to understand microtubule dynamics and its spatiotemporal regulation at the molecular level. In this chapter, I introduce the concept of microtubule dynamics and discuss the techniques that can be employed to study microtubule dynamics in vitro and in cells, for many of which detailed protocols can be found in this volume. Microtubule dynamics is traditionally assessed by the four parameters of dynamic instability: growth and shrinkage rates, rescue and catastrophe frequencies, sometimes supplemented by pause duration. I discuss emerging issues with and alternatives to this parameter description of microtubule dynamics. PMID:21773917

  3. 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

  4. Microtubule catastrophe from protofilament dynamics

    NASA Astrophysics Data System (ADS)

    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.PRLTAO0031-900710.1103/PhysRevLett.73.2372 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.

  5. Expression of Nucleolin Affects Microtubule Dynamics.

    PubMed

    Gaume, Xavier; Place, Christophe; Delage, Helene; Mongelard, Fabien; Monier, Karine; Bouvet, Philippe

    2016-01-01

    Nucleolin is present in diverse cellular compartments and is involved in a variety of cellular processes from nucleolar structure and function to intracellular trafficking, cell adhesion and migration. Recently, nucleolin has been localized at the mature centriole where it is involved in microtubule nucleation and anchoring. Although this new function of nucleolin linked to microtubule regulation has been identified, the global effects of nucleolin on microtubule dynamics have not been addressed yet. In the present study, we analyzed the roles of nucleolin protein levels on global microtubule dynamics by tracking the EB3 microtubule plus end binding protein in live cells. We have found that during microtubule growth phases, nucleolin affects both the speed and life time of polymerization and by analyzing catastrophe events, we showed that nucleolin reduces catastrophe frequency. This new property of nucleolin was then confirmed in a cold induced microtubule depolymerization experiment in which we have found that cold resistant microtubules were totally destabilized in nucleolin depleted cells. Altogether, our data demonstrate a new function of nucleolin on microtubule stabilization, thus bringing novel insights into understanding the multifunctional properties of nucleolin in healthy and cancer cells. PMID:27309529

  6. Expression of Nucleolin Affects Microtubule Dynamics

    PubMed Central

    Gaume, Xavier; Place, Christophe; Delage, Helene; Mongelard, Fabien; Monier, Karine; Bouvet, Philippe

    2016-01-01

    Nucleolin is present in diverse cellular compartments and is involved in a variety of cellular processes from nucleolar structure and function to intracellular trafficking, cell adhesion and migration. Recently, nucleolin has been localized at the mature centriole where it is involved in microtubule nucleation and anchoring. Although this new function of nucleolin linked to microtubule regulation has been identified, the global effects of nucleolin on microtubule dynamics have not been addressed yet. In the present study, we analyzed the roles of nucleolin protein levels on global microtubule dynamics by tracking the EB3 microtubule plus end binding protein in live cells. We have found that during microtubule growth phases, nucleolin affects both the speed and life time of polymerization and by analyzing catastrophe events, we showed that nucleolin reduces catastrophe frequency. This new property of nucleolin was then confirmed in a cold induced microtubule depolymerization experiment in which we have found that cold resistant microtubules were totally destabilized in nucleolin depleted cells. Altogether, our data demonstrate a new function of nucleolin on microtubule stabilization, thus bringing novel insights into understanding the multifunctional properties of nucleolin in healthy and cancer cells. PMID:27309529

  7. Profilin connects actin assembly with microtubule dynamics.

    PubMed

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

    2016-08-01

    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 micro-tubule dynamics. Hence profilin functions as a regulator of microtubule (+)-end turnover in addition to being an actin control element.

  8. Tubulin Bistability and Polymorphic Dynamics of Microtubules

    NASA Astrophysics Data System (ADS)

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

    2010-12-01

    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.

  9. 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

  10. The role of dynamic instability in microtubule organization

    PubMed Central

    Horio, Tetsuya; Murata, Takashi

    2014-01-01

    Microtubules are one of the three major cytoskeletal components in eukaryotic cells. Heterodimers composed of GTP-bound α- and β-tubulin molecules polymerize to form microtubule protofilaments, which associate laterally to form a hollow microtubule. Tubulin has GTPase activity and the GTP molecules associated with β-tubulin molecules are hydrolyzed shortly after being incorporated into the polymerizing microtubules. GTP hydrolysis alters the conformation of the tubulin molecules and drives the dynamic behavior of microtubules. Periods of rapid microtubule polymerization alternate with periods of shrinkage in a process known as dynamic instability. In plants, dynamic instability plays a key role in determining the organization of microtubules into arrays, and these arrays vary throughout the cell cycle. In this review, we describe the mechanisms that regulate microtubule dynamics and underlie dynamic instability, and discuss how dynamic instability may shape microtubule organization in plant cells. PMID:25339962

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

    PubMed

    Hashimoto, Takashi

    2003-12-01

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

  12. TCTP regulates spindle microtubule dynamics by stabilizing polar microtubules during mouse oocyte meiosis.

    PubMed

    Jeon, Hyuk-Joon; You, Seung Yeop; Park, Yong Seok; Chang, Jong Wook; Kim, Jae-Sung; Oh, Jeong Su

    2016-04-01

    Dynamic changes in spindle structure and function are essential for maintaining genomic integrity during the cell cycle. Spindle dynamics are highly dependent on several microtubule-associated proteins that coordinate the dynamic behavior of microtubules, including microtubule assembly, stability and organization. Here, we show that translationally controlled tumor protein (TCTP) is a novel microtubule-associated protein that regulates spindle dynamics during meiotic maturation. TCTP was expressed and widely distributed in the cytoplasm with strong enrichment at the spindle microtubules during meiosis. TCTP was found to be phosphorylated during meiotic maturation, and was exclusively localized to the spindle poles. Knockdown of TCTP impaired spindle organization without affecting chromosome alignment. These spindle defects were mostly due to the destabilization of the polar microtubules. However, the stability of kinetochore microtubules attached to chromosomes was not affected by TCTP knockdown. Overexpression of a nonphosphorylable mutant of TCTP disturbed meiotic maturation, stabilizing the spindle microtubules. In addition, Plk1 was decreased by TCTP knockdown. Taken together, our results demonstrate that TCTP is a microtubule-associating protein required to regulate spindle microtubule dynamics during meiotic maturation in mouse oocytes.

  13. 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.

  14. 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.

  15. Measuring the Dynamic Parameters of MCF7 Cell Microtubules

    NASA Astrophysics Data System (ADS)

    Winton, Carly; Shojania Feizabadi, Mitra

    2013-03-01

    Microtubules are the key component of the cytoskeleton. They are intrinsically dynamic displaying dynamic instability in which they randomly switch between a phase of growing and shrinking, both in vitro and in vivo. This dynamic is specified by the following parameters: growing rate, shrinking rate, frequency of catastrophe, and frequency of rescue. In this work, we will present our primary results in which we measured the dynamic parameters of a single microtubule polymerized from MCF7 tubulin in vitro. The results are significant since the MCF7 microtubules are non-neural mammalian consisting of different beta tubulin isotypes in their structures as compared to neural mammalian microtubules, such as bovine brain. The unique dynamic parameters of individual MCF7 microtubules in vitro, which are reported for the first time, indicate that non-neural microtubules can be fundamentally different from neural microtubules.

  16. 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

  17. Partial Depletion of Gamma-Actin Suppresses Microtubule Dynamics

    PubMed Central

    Po'uha, Sela T; Honore, Stephane; Braguer, Diane; Kavallaris, Maria

    2013-01-01

    Actin and microtubule interactions are important for many cellular events, however these interactions are poorly described. Alterations in γ-actin are associated with diseases such as hearing loss and cancer. Functional investigations demonstrated that partial depletion of γ-actin affects cell polarity and induces resistance to microtubule-targeted agents. To determine whether γ-actin alterations directly affect microtubule dynamics, microtubule dynamic instability was analyzed in living cells following partial siRNA depletion of γ-actin. Partial depletion of γ-actin suppresses interphase microtubule dynamics by 17.5% due to a decrease in microtubule shortening rates and an increase in microtubule attenuation. γ-Actin partial depletion also increased distance-based microtubule catastrophe and rescue frequencies. In addition, knockdown of γ-actin delayed mitotic progression, partially blocking metaphase–anaphase transition and inhibiting cell proliferation. Interestingly, in the presence of paclitaxel, interphase microtubule dynamics were further suppressed by 24.4% in the γ-actin knockdown cells, which is comparable to 28.8% suppression observed in the control siRNA treated cells. Paclitaxel blocked metaphase–anaphase transition in both the γ-actin knockdown cells and the control siRNA cells. However, the extent of mitotic arrest was much higher in the control cells (28.4%), compared to the γ-actin depleted cells (8.5%). Therefore, suppression of microtubule dynamics by partial depletion of γ-actin is associated with marked delays in metaphase-anaphase transition and not mitotic arrest. This is the first demonstration that γ-actin can modulate microtubule dynamics by reducing the microtubule shortening rate, promoting paused/attenuated microtubules, and increasing transition frequencies suggesting a mechanistic link between γ-actin and microtubules. © 2013 Wiley Periodicals, Inc PMID:23335583

  18. 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

  19. 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

  20. Ahead of the Curve: New Insights into Microtubule Dynamics

    PubMed Central

    Ohi, Ryoma; Zanic, Marija

    2016-01-01

    Microtubule dynamics are fundamental for many aspects of cell physiology, but their mechanistic underpinnings remain unclear despite 40 years of intense research. In recent years, the continued union of reconstitution biochemistry, structural biology, and modeling has yielded important discoveries that deepen our understanding of microtubule dynamics. These studies, which we review here, underscore the importance of GTP hydrolysis-induced changes in tubulin structure as microtubules assemble, and highlight the fact that each aspect of microtubule behavior is the output of complex, multi-step processes. Although this body of work moves us closer to appreciating the key features of microtubule biochemistry that drive dynamic instability, the divide between our understanding of microtubules in isolation versus within the cellular milieu remains vast. Bridging this gap will serve as fertile grounds of cytoskeleton-focused research for many years to come. PMID:26998244

  1. Microtubule-binding agents: a dynamic field of cancer therapeutics

    PubMed Central

    Dumontet, Charles; Jordan, Mary Ann

    2010-01-01

    Preface Microtubules are dynamic filamentous cytoskeletal proteins that are an important therapeutic target in tumor cells. Microtubule binding agents have been part of the pharmacopoeia of cancer for decades, and until the advent of targeted therapy microtubules were the only alternative to DNA as a therapeutic target in cancer. The screening of a variety of botanical species and marine organisms has yielded promising new antitubulin agents with novel properties. Enhanced tumor specificity, reduced neurotoxicity, and insensitivity to chemoresistance mechanisms are the three main objectives in the current search for novel microtubule binding agents. PMID:20885410

  2. 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.

  3. 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.

  4. Reelin promotes microtubule dynamics in processes of developing neurons.

    PubMed

    Meseke, Maurice; Cavus, Ersin; Förster, Eckart

    2013-02-01

    The extracellular matrix protein reelin controls radial migration and layer formation of cortical neurons, in part by modulation of cytoskeletal dynamics. A stabilizing effect of reelin on the actin cytoskeleton has been described recently. However, it is poorly understood how reelin modulates microtubule dynamics. Here, we provide evidence that reelin increases microtubule assembly. This effect is mediated, at least in part, by promoting microtubule plus end dynamics in processes of developing neurons. Thus, we treated primary neuronal cultures with nocodazole to disrupt microtubules. After nocodazole washout, we found microtubule reassembly to be accelerated in the presence of reelin. Moreover, we show that reelin treatment promoted the formation of microtubule plus end binding protein 3 (EB3) comets in developing dendrites, and that EB3 immunostaining in the developing wild-type neocortex is most intense in the reelin-rich marginal zone where leading processes of radially migrating neurons project to. This characteristic EB3 staining pattern was absent in reeler. Also reassembly of nocodazole-dispersed dendritic Golgi apparati, which are closely associated to microtubules, was accelerated by reelin treatment, though with a substantially slower time course when compared to microtubule reassembly. In support of our in vitro results, we found that the subcellular distribution of α-tubulin and acetylated tubulin in reeler cortical sections differed from wild-type and from mice lacking the very low density lipoprotein receptor (VLDLR), known to bind reelin. Taken together, our results suggest that reelin promotes microtubule assembly, at least in part, by increasing microtubule plus end dynamics. PMID:22990595

  5. 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)

  6. 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.

  7. Modulation of host microtubule dynamics by pathogenic bacteria

    PubMed Central

    Radhakrishnan, Girish K.; Splitter, Gary A.

    2013-01-01

    The eukaryotic cytoskeleton is a vulnerable target of many microbial pathogens during the course of infection. Rearrangements of host cytoskeleton benefit microbes in various stages of their infection cycle such as invasion, motility, and persistence. Bacterial pathogens deliver a number of effector proteins into host cells for modulating the dynamics of actin and microtubule cytoskeleton. Alteration of the actin cytoskeleton is generally achieved by bacterial effectors that target the small GTPases of the host. Modulation of microtubule dynamics involves direct interaction of effector proteins with the subunits of microtubules or recruiting cellular proteins that affect microtubule dynamics. This review will discuss effector proteins from animal and human bacterial pathogens that either destabilize or stabilize host micro-tubules to advance the infectious process. A compilation of these research findings will provide an overview of known and unknown strategies used by various bacterial effectors to modulate the host microtubule dynamics. The present review will undoubtedly help direct future research to determine the mechanisms of action of many bacterial effector proteins and contribute to understanding the survival strategies of diverse adherent and invasive bacterial pathogens. PMID:23585820

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

    PubMed

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

    2016-05-10

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

  9. Dynamics of an Idealized Model of Microtubule Growth and Catastrophe

    PubMed Central

    Antal, T.; Krapivsky, P. L.; Redner, S.; Mailman, M.; Chakraborty, B.

    2008-01-01

    We investigate a simple dynamical model of a microtubule that evolves by attachment of guanosine triphosphate (GTP) tubulin to its end, irreversible conversion of GTP to guanosine diphosphate (GDP) tubulin by hydrolysis, and detachment of GDP at the end of a microtubule. As a function of rates of these processes, the microtubule can grow steadily or its length can fluctuate wildly. In the regime where detachment can be neglected, we find exact expressions for the tubule and GTP cap length distributions, as well as power-law length distributions of GTP and GDP islands. In the opposite limit of instantaneous detachment, we find the time between catastrophes, where the microtubule shrinks to zero length, and determine the size distribution of avalanches (sequence of consecutive GDP detachment events). We obtain the phase diagram for general rates and verify our predictions by numerical simulations. PMID:17995026

  10. A dynamical model of kinesin-microtubule motility assays.

    PubMed Central

    Gibbons, F; Chauwin, J F; Despósito, M; José, J V

    2001-01-01

    A two-dimensional stochastic model for the dynamics of microtubules in gliding-assay experiments is presented here, which includes the viscous drag acting on the moving fiber and the interaction with the kinesins. For this purpose, we model kinesin as a spring, and explicitly use parameter values to characterize the model from experimental data. We numerically compute the mean attachment lifetimes of all motors, the total force exerted on the microtubules at all times, the effects of a distribution in the motor speeds, and also the mean velocity of a microtubule in a gliding assay. We find quantitative agreement with the results of J. Howard, A. J. Hudspeth, and R. D. Vale, Nature. 342:154-158. We perform additional numerical analysis of the individual motors, and show how cancellation of the forces exerted by the many motors creates a resultant longitudinal force much smaller than the maximum force that could be exerted by a single motor. We also examine the effects of inhomogeneities in the motor-speeds. Finally, we present a simple theoretical model for microtubules dynamics in gliding assays. We show that the model can be analytically solved in the limit of few motors attached to the microtubule and in the opposite limit of high motor density. We find that the speed of the microtubule goes like the mean speed of the motors in good quantitative agreement with the experimental and numerical results. PMID:11371430

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

    PubMed

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

    2016-08-01

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

  12. Interaction of CDK5RAP2 with EB1 to track growing microtubule tips and to regulate microtubule dynamics.

    PubMed

    Fong, Ka-Wing; Hau, Shiu-Yeung; Kho, Yik-Shing; Jia, Yue; He, Lisheng; Qi, Robert Z

    2009-08-01

    Mutations in cdk5rap2 are linked to autosomal recessive primary microcephaly, and attention has been paid to its function at centrosomes. In this report, we demonstrate that CDK5RAP2 localizes to microtubules and concentrates at the distal tips in addition to centrosomal localization. CDK5RAP2 interacts directly with EB1, a prototypic member of microtubule plus-end tracking proteins, and contains the basic and Ser-rich motif responsible for EB1 binding. The EB1-binding motif is conserved in the CDK5RAP2 sequences of chimpanzee, bovine, and dog but not in those of rat and mouse, suggesting a function gained during the evolution of mammals. The mutation of the Ile/Leu-Pro dipeptide within the motif abolishes EB1 interaction and plus-end attachment. In agreement with the mutational analysis, suppression of EB1 expression inhibits microtubule tip-tracking of CDK5RAP2. We have also found that the CDK5RAP2-EB1 complex regulates microtubule dynamics and stability. CDK5RAP2 depletion by RNA interference impacts the dynamic behaviors of microtubules. The CDK5RAP2-EB1 complex induces microtubule bundling and acetylation when expressed in cell cultures and stimulates microtubule assembly and bundle formation in vitro. Collectively, these results show that CDK5RAP2 targets growing microtubule tips in association with EB1 to regulate microtubule dynamics. PMID:19553473

  13. 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.

  14. 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. PMID:27415303

  15. 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.

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

    PubMed

    Gnanambal K, Mary Elizabeth; Lakshmipathy, Shailaja Vommi

    2016-03-01

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

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

    PubMed

    Gnanambal K, Mary Elizabeth; Lakshmipathy, Shailaja Vommi

    2016-03-01

    Tubulin/microtubule assembly and disassembly is characterized as one of the chief processes during cell growth and division. Hence drugs those perturb these process are considered to be effective in killing fast multiplying cancer cells. There is a collection of natural compounds which disturb microtubule/tubulin dis/assemblage and there have been a lot of efforts concerted in the marine realm too, to surveying such killer molecules. Close to half the natural compounds shooting out from marine invertebrates are generally with no traceable definite mechanisms of action though may be tough anti-cancerous hits at nanogram levels, hence fatefully those discoveries conclude therein without a capacity of translation from laboratory to pharmacy. Astoundingly at least 50% of natural compounds which have definite mechanisms of action causing disorders in tubulin/microtubule kinetics have an isolation history from sponges belonging to the Phylum: Porifera. Poriferans have always been a wonder worker to treat cancers with a choice of, yet precise targets on cancerous tissues. There is a specific order: Dictyoceratida within this Phylum which has contributed to yielding at least 50% of effective compounds possessing this unique mechanism of action mentioned above. However, not much notice is driven to Dictyoceratidans alongside the order: Demospongiae thus dictating the need to know its select microtubule/tubulin irritants since the unearthing of avarol in the year 1974 till date. Hence this review selectively pinpoints all the compounds, noteworthy derivatives and analogs stemming from order: Dictyoceratida focusing on the past, present and future. PMID:26874035

  18. Carbendazim Inhibits Cancer Cell Proliferation by Suppressing Microtubule Dynamics

    PubMed Central

    Yenjerla, Mythili; Cox, Corey; Wilson, Leslie; Jordan, Mary Ann

    2009-01-01

    Carbendazim (methyl 2-benzimidazolecarbamate) is widely used as a systemic fungicide in human food production and appears to act on fungal tubulin. However, it also inhibits proliferation of human cancer cells, including drug- and multidrug-resistant and p53-deficient cell lines. Because of its promising preclinical anti-tumor activity, it has undergone phase I clinical trials and is under further clinical development. Although it weakly inhibits polymerization of brain microtubules and induces G2/M arrest in tumor cells, its mechanism of action in human cells has not been fully elucidated. We examined its mechanism of action in MCF7 human breast cancer cells and found that it inhibits proliferation (IC50, 10 μM) and half-maximally arrests mitosis at a similar concentration (8 μM), in concert with suppression of microtubule dynamic instability without appreciable microtubule depolymerization. It induces mitotic spindle abnormalities and reduces the metaphase intercentromere distance of sister chromatids, indicating reduction of tension on kinetochores, thus leading to metaphase arrest. With microtubules assembled in vitro from pure tubulin, carbendazim also suppresses dynamic instability, reducing the dynamicity by 50% at 10 μM, with only minimal (21%) reduction of polymer mass. Carbendazim binds to mammalian tubulin (Kd, 42.8 ± 4.0 μM). Unlike some benzimidazoles that bind to the colchicine site in tubulin, carbendazim neither competes with colchicine nor competes with vinblastine for binding to brain tubulin. Thus, carbendazim binds to an as yet unidentified site in tubulin and inhibits tumor cell proliferation by suppressing the growing and shortening phases of microtubule dynamic instability, thus inducing mitotic arrest. PMID:19001156

  19. Xenopus TACC1 is a microtubule plus‐end tracking protein that can regulate microtubule dynamics during embryonic development

    PubMed Central

    Lucaj, Christopher M.; Evans, Matthew F.; Nwagbara, Belinda U.; Ebbert, Patrick T.; Baker, Charlie C.; Volk, Joseph G.; Francl, Andrew F.; Ruvolo, Sean P.

    2015-01-01

    Microtubule plus‐end dynamics are regulated by a family of proteins called plus‐end tracking proteins (+TIPs). We recently demonstrated that the transforming acidic coiled‐coil (TACC) domain family member, TACC3, can function as a +TIP to regulate microtubule dynamics in Xenopus laevis embryonic cells. Although it has been previously reported that TACC3 is the only TACC family member that exists in Xenopus, our examination of its genome determined that Xenopus, like all other vertebrates, contains three TACC family members. Here, we investigate the localization and function of Xenopus TACC1, the founding member of the TACC family. We demonstrate that it can act as a +TIP to regulate microtubule dynamics, and that the conserved C‐terminal TACC domain is required for its localization to plus‐ends. We also show that, in Xenopus embryonic mesenchymal cells, TACC1 and TACC3 are each required for maintaining normal microtubule growth speed but exhibit some functional redundancy in the regulation of microtubule growth lifetime. Given the conservation of TACC1 in Xenopus and other vertebrates, we propose that Xenopus laevis is a useful system to investigate unexplored cell biological functions of TACC1 and other TACC family members in the regulation of microtubule dynamics. © 2015 The Authors. Cytoskeleton, Published by Wiley Periodicals, Inc. PMID:26012630

  20. MCAK and Paclitaxel Have Differential Effects on Spindle Microtubule Organization and Dynamics

    PubMed Central

    Rizk, Rania S.; Bohannon, Kevin P.; Wetzel, Laura A.; Powers, James; Shaw, Sidney L.

    2009-01-01

    Within the mitotic spindle, there are multiple populations of microtubules with different turnover dynamics, but how these different dynamics are maintained is not fully understood. MCAK is a member of the kinesin-13 family of microtubule-destabilizing enzymes that is required for proper establishment and maintenance of the spindle. Using quantitative immunofluorescence and fluorescence recovery after photobleaching, we compared the differences in spindle organization caused by global suppression of microtubule dynamics, by treating cells with low levels of paclitaxel, versus specific perturbation of spindle microtubule subsets by MCAK inhibition. Paclitaxel treatment caused a disruption in spindle microtubule organization marked by a significant increase in microtubules near the poles and a reduction in K-fiber fluorescence intensity. This was correlated with a faster t1/2 of both spindle and K-fiber microtubules. In contrast, MCAK inhibition caused a dramatic reorganization of spindle microtubules with a significant increase in astral microtubules and reduction in K-fiber fluorescence intensity, which correlated with a slower t1/2 of K-fibers but no change in the t1/2 of spindle microtubules. Our data support the model that MCAK perturbs spindle organization by acting preferentially on a subset of microtubules, and they support the overall hypothesis that microtubule dynamics is differentially regulated in the spindle. PMID:19158381

  1. Microtubule converging centers-implications for microtubule dynamics in higher plants

    SciTech Connect

    Bajer, A.S.; Mole-Bajer, J.

    1993-12-31

    The reorganization of the microtubular cytoskeleton was studied during telophase-interphase transition and interphase in Haemanthus endosperm cells, and in cell fragments (cytoplasts). This report concerns the role of microtubule (MT) converging centers (MTCCs) in the reorganization of the higher plant cytoskeleton. Microtubules (MTs) were visualized with the immunogold and immunogold-silver enhanced methods. Cells were fixed at room temperature (21{degrees}-24{degrees}C) and after high (35{degrees}-37{degrees}C) and low (4{degrees}-7{degrees}C) temperature shocks. The temperature shocks modify behavior of MTCCs. During early prophase and telophase-interphase transition, the formation of MTCCs is greatly enhanced at elevated temperature. These are stages when a pronounced reorganization of the cytoskeleton takes place. MTCCs are polar structures with remarkably different dynamics and properties at the diverging and converging ends. The indirect evidence shows that the converging tip of MTCC is (-) and the diverging end is (+). Our data imply that the reorganization of the higher plant cytoskeleton is basically a competitive sorting of MTs intrinsic polarity, with MTCCs as principal structural components.

  2. 3-D structure and dynamics of microtubule self-organization

    NASA Astrophysics Data System (ADS)

    Wang, Jing; Ou-Yang, H. Daniel

    2008-03-01

    Laser scanning confocal microscopy was used to study the dynamics of 3D assemblies spontaneously formed in microtubule (MT) solutions. Microtubule solutions prepared by mixing and incubating tubulin in the presence of GTP and Oregon Green conjugated taxol in PM buffer were placed in long, sub-millimeter thin glass cells by the capillary action. Within 24 hours, starting with a uniform distribution, microtubules were found to be gradually separated into a few large ``buckled'' bundles along the long direction, and in the middle plane, of the sample cell. A well-defined wavelength of the buckling sinusoids was around 510 μm. The cross section of these round bundles was approximately 40 μm in diameter and the lengths were several centimeters. Detailed analysis of the 3-D image within the bundles revealed that each bundle seemed to consist of loosely packed MTs. It appeared that MTs were phase separated resulting from attractive interactions between charged MT fibers. The ``buckling'' behavior could be the result of geometrical constraints of the repulsive cell walls and the repulsive interaction between bundles. Detailed 3-D observations of the dynamic evolution of MT assembly could provide insight to the mechanisms of cellular MT organization and phase separation of charged colloidal rods.

  3. The implications of microtubule dynamic instability on chromosome dynamics in metaphase

    NASA Astrophysics Data System (ADS)

    Lubin, David; Chakrabarti, Buddhapriya

    2005-03-01

    We present a model of chromosome oscillations during late metaphase based on the postulates of the stochastic detachment/reattachment of kinetochore microtubules and the dynamic instability model for microtubules dynamics. In this approach the motion of the chromosomes is analyzed by treating them as Brownian particles subject to a fluctuating force arising from the varying number of microtubules attached to the kinetochore at a given time. Furthermore, we predict observable changes in the chromosome dynamics in response to antimitotic drugs (e.g. taxol) that affects the microtubule dynamics. This approach may facilitate the use of the stochastic time series of chromosome position data as a complement to more traditional approaches in the elucidation of the mechanisms of chromosome alignment during metaphase of cell division.

  4. Tau co-organizes dynamic microtubule and actin networks

    PubMed Central

    Elie, Auréliane; Prezel, Elea; Guérin, Christophe; Denarier, Eric; Ramirez-Rios, Sacnicte; Serre, Laurence; Andrieux, Annie; Fourest-Lieuvin, Anne; Blanchoin, Laurent; Arnal, Isabelle

    2015-01-01

    The crosstalk between microtubules and actin is essential for cellular functions. However, mechanisms underlying the microtubule-actin organization by cross-linkers remain largely unexplored. Here, we report that tau, a neuronal microtubule-associated protein, binds to microtubules and actin simultaneously, promoting in vitro co-organization and coupled growth of both networks. By developing an original assay to visualize concomitant microtubule and actin assembly, we show that tau can induce guided polymerization of actin filaments along microtubule tracks and growth of single microtubules along actin filament bundles. Importantly, tau mediates microtubule-actin co-alignment without changing polymer growth properties. Mutagenesis studies further reveal that at least two of the four tau repeated motifs, primarily identified as tubulin-binding sites, are required to connect microtubules and actin. Tau thus represents a molecular linker between microtubule and actin networks, enabling a coordination of the two cytoskeletons that might be essential in various neuronal contexts. PMID:25944224

  5. Microtubule dynamics in vitro are regulated by the tubulin isotype composition.

    PubMed Central

    Panda, D; Miller, H P; Banerjee, A; Ludueña, R F; Wilson, L

    1994-01-01

    The growing and shortening dynamics of individual bovine brain microtubules at their plus ends at steady state in vitro, assembled from isotypically pure alpha beta II, alpha beta III, or alpha beta IV tubulin dimers, were determined by differential interference contrast video microscopy. Microtubules assembled from the purified alpha beta III isotype were considerably more dynamic than microtubules made from the alpha beta II or alpha beta IV isotypes or from unfractionated phosphocellulose-purified tubulin. Furthermore, increasing the proportion of the alpha beta II isotype in a mixture of the alpha beta II and alpha beta III isotypes suppressed microtubule dynamics, demonstrating that microtubule dynamics can be influenced by the tubulin isotype composition. The data support the hypothesis that cells might determine the dynamic properties and functions of its microtubules in part by altering the relative amounts of the different tubulin isotypes. Images PMID:7972064

  6. Tunable dynamics of microtubule-based active isotropic gels

    PubMed Central

    Henkin, Gil; DeCamp, Stephen J.; Chen, Daniel T. N.; Sanchez, Tim; Dogic, Zvonimir

    2014-01-01

    We investigate the dynamics of an active gel of bundled microtubules (MTs) that is driven by clusters of kinesin molecular motors. Upon the addition of ATP, the coordinated action of thousands of molecular motors drives the gel to a highly dynamical turbulent-like state that persists for hours and is only limited by the stability of constituent proteins and the availability of the chemical fuel. We characterize how enhanced transport and emergent macroscopic flows of active gels depend on relevant molecular parameters, including ATP, kinesin motor and depletant concentrations, MT volume fraction, as well as the stoichiometry of the constituent motor clusters. Our results show that the dynamical and structural properties of MT-based active gels are highly tunable. They also indicate existence of an optimal concentration of molecular motors that maximize far-from-equilibrium activity of active isotropic MT gels. PMID:25332391

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

    PubMed Central

    Trogden, Kathryn P.; Rogers, Stephen L.

    2015-01-01

    Microtubules are regulated by a diverse set of proteins that localize to microtubule plus ends (+TIPs) where they regulate dynamic instability and mediate interactions with the cell cortex, actin filaments, and organelles. Although individual +TIPs have been studied in depth and we understand their basic contributions to microtubule dynamics, there is a growing body of evidence that these proteins exhibit cross-talk and likely function to collectively integrate microtubule behavior and upstream signaling pathways. In this study, we have identified a novel protein-protein interaction between the XMAP215 homologue in Drosophila, Mini spindles (Msps), and the CLASP homologue, Orbit. These proteins have been shown to promote and suppress microtubule dynamics, respectively. We show that microtubule dynamics are regionally controlled in cells by Rac acting to suppress GSK3β in the peripheral lamellae/lamellipodium. Phosphorylation of Orbit by GSK3β triggers a relocalization of Msps from the microtubule plus end to the lattice. Mutation of the Msps-Orbit binding site revealed that this interaction is required for regulating microtubule dynamic instability in the cell periphery. Based on our findings, we propose that Msps is a novel Rac effector that acts, in partnership with Orbit, to regionally regulate microtubule dynamics. PMID:26406596

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

    PubMed

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

    1997-03-25

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

  9. Microtubule-dependent transport and dynamics of vimentin intermediate filaments.

    PubMed

    Hookway, Caroline; Ding, Liya; Davidson, Michael W; Rappoport, Joshua Z; Danuser, Gaudenz; Gelfand, Vladimir I

    2015-05-01

    We studied two aspects of vimentin intermediate filament dynamics-transport of filaments and subunit exchange. We observed transport of long filaments in the periphery of cells using live-cell structured illumination microscopy. We studied filament transport elsewhere in cells using a photoconvertible-vimentin probe and total internal reflection microscopy. We found that filaments were rapidly transported along linear tracks in both anterograde and retrograde directions. Filament transport was microtubule dependent but independent of microtubule polymerization and/or an interaction with the plus end-binding protein APC. We also studied subunit exchange in filaments by long-term imaging after photoconversion. We found that converted vimentin remained in small clusters along the length of filaments rather than redistributing uniformly throughout the network, even in cells that divided after photoconversion. These data show that vimentin filaments do not depolymerize into individual subunits; they recompose by severing and reannealing. Together these results show that vimentin filaments are very dynamic and that their transport is required for network maintenance.

  10. 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

    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

    2014-11-01

    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.

  11. Dynamic Change of Cellular Localization of Microtubule-Organizing Center During Conjugation of Ciliate Tetrahymena thermophila.

    PubMed

    Kushida, Yasuharu; Takaine, Masak; Nakano, Kentaro; Sugai, Toshiro; Numata, Osamu

    2015-01-01

    To obtain a comprehensive picture of microtubule dynamics during conjugation, the mode of sexual reproduction in ciliates, we combined indirect immunofluorescence and three-dimensional imaging using confocal laser-scanning microscope to visualize the cellular localization of DNA, microtubules, and γ-tubulin, the main component of the microtubule-organizing center in mating Tetrahymena cells. As the conjugational stages proceeded, the distribution of γ-tubulin changed drastically and microtubules showed dynamic appearance and disappearance during meiosis, nuclear selection, nuclear exchange, and the development of new macronuclei. This study highlights the involvement of cytoskeletal regulation in the modulation of germline nuclear motilities required for ciliate reproduction.

  12. Nonlinear dynamics of microtubules —A longitudinal model

    NASA Astrophysics Data System (ADS)

    Zdravković, S.; Satarić, M. V.; Zeković, S.

    2013-05-01

    In the present letter we describe a model of nonlinear dynamics of microtubules (MTs) assuming a single longitudinal degree of freedom per tubulin dimer. This is a longitudinal displacement of a dimer at a certain position with respect to the neighbouring one. A nonlinear partial differential equation, describing dimer's dynamics within MT, is solved both analytically and numerically. It is shown that such a nonlinear model can lead to the existence of kink solitons moving along the MTs. The internal electrical field strength is calculated using two procedures and a perfect agreement between the results is demonstrated. This enabled the estimation of the total energy, kink velocity and kink width. To simplify the calculation of the total energy we stated and proved a useful auxiliary theorem.

  13. Microtubule dynamics reconstituted in vitro and imaged by single-molecule fluorescence microscopy.

    PubMed

    Gell, Christopher; Bormuth, Volker; Brouhard, Gary J; Cohen, Daniel N; Diez, Stefan; Friel, Claire T; Helenius, Jonne; Nitzsche, Bert; Petzold, Heike; Ribbe, Jan; Schäffer, Erik; Stear, Jeffrey H; Trushko, Anastasiya; Varga, Vladimir; Widlund, Per O; Zanic, Marija; Howard, Jonathon

    2010-01-01

    In vitro assays that reconstitute the dynamic behavior of microtubules provide insight into the roles of microtubule-associated proteins (MAPs) in regulating the growth, shrinkage, and catastrophe of microtubules. The use of total internal reflection fluorescence microscopy with fluorescently labeled tubulin and MAPs has allowed us to study microtubule dynamics at the resolution of single molecules. In this chapter we present a practical overview of how these assays are performed in our laboratory: fluorescent labeling methods, strategies to prolong the time to photo-bleaching, preparation of stabilized microtubules, flow-cells, microtubule immobilization, and finally an overview of the workflow that we follow when performing the experiments. At all stages, we focus on practical tips and highlight potential stumbling blocks.

  14. Microtubule-dependent transport and dynamics of vimentin intermediate filaments

    PubMed Central

    Hookway, Caroline; Ding, Liya; Davidson, Michael W.; Rappoport, Joshua Z.; Danuser, Gaudenz; Gelfand, Vladimir I.

    2015-01-01

    We studied two aspects of vimentin intermediate filament dynamics—transport of filaments and subunit exchange. We observed transport of long filaments in the periphery of cells using live-cell structured illumination microscopy. We studied filament transport elsewhere in cells using a photoconvertible-vimentin probe and total internal reflection microscopy. We found that filaments were rapidly transported along linear tracks in both anterograde and retrograde directions. Filament transport was microtubule dependent but independent of microtubule polymerization and/or an interaction with the plus end–binding protein APC. We also studied subunit exchange in filaments by long-term imaging after photoconversion. We found that converted vimentin remained in small clusters along the length of filaments rather than redistributing uniformly throughout the network, even in cells that divided after photoconversion. These data show that vimentin filaments do not depolymerize into individual subunits; they recompose by severing and reannealing. Together these results show that vimentin filaments are very dynamic and that their transport is required for network maintenance. PMID:25717187

  15. 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

  16. Phase transition analysis of the dynamic instability of microtubules

    NASA Astrophysics Data System (ADS)

    Yarahmadian, Shantia; Yari, Masoud

    2014-09-01

    This paper provides the phase transition analysis of a reaction diffusion equations system modelling the dynamic instability of microtubules (MTs). For this purpose, we have generalized the macroscopic model studied by Mourão et al (2011 Comput. Biol. Chem. 35 269-81). This model investigates the interaction between the MT nucleation, the essential dynamics parameters and extinction, and their impact on the stability of the system. The considered framework encompasses a system of partial differential equations for the elongation and shortening of MTs, where the rates of elongation as well as the lifetimes of the elongating shortening phases are linear functions of GTP-tubulin concentration. In a novel way, this paper investigates the stability analysis and provides a bifurcation analysis for the dynamic instability of MTs in the presence of diffusion and all of the fundamental dynamics parameters. Our stability analysis introduces the phase transition method as a new mathematical tool in the study of MT dynamics. The mathematical tools introduced to handle the problem should be of general use.

  17. Live imaging of microtubule dynamics in organotypic hippocampal slice cultures.

    PubMed

    Schätzle, Philipp; Kapitein, Lukas C; Hoogenraad, Casper C

    2016-01-01

    The microtubule (MT) cytoskeleton plays an active role during different phases of neuronal development and is an essential structure for stable neuronal morphology. MTs determine axon formation, control polarized cargo trafficking, and regulate the dynamics of dendritic spines, the major sites of excitatory synaptic input. Defects in MT function have been linked to various neurological and neurodegenerative diseases and recent studies highlight neuronal MTs as a potential target for therapeutic intervention. Thus, understanding MT dynamics and its regulation is of central importance to study many aspects of neuronal function. The dynamics of MT in neurons can be studied by visualizing fluorescently tagged MT plus-end tracking proteins (+TIPs). Tracking of +TIP trajectories allows analyzing the speeds and directionality of MT growth in axons and dendrites. Numerous labs now use +TIP to track growing MTs in dissociated neuron cultures. This chapter provides detailed methods for live imaging of MT dynamics in organotypic hippocampal slice cultures. We describe protocols for culturing and transducing organotypic slices and imaging MT dynamics by spinning disk confocal microscopy. PMID:26794510

  18. Steering microtubule shuttle transport with dynamically controlled magnetic fields

    DOE PAGESBeta

    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 andmore » could serve as the basis for improved lab-on-a-chip technologies based on MT transport.« less

  19. 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. PMID:27049749

  20. 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

  1. Paired arrangement of kinetochores together with microtubule pivoting and dynamics drive kinetochore capture in meiosis I.

    PubMed

    Cojoc, Gheorghe; Florescu, Ana-Maria; Krull, Alexander; Klemm, Anna H; Pavin, Nenad; Jülicher, Frank; Tolić, Iva M

    2016-05-11

    Kinetochores are protein complexes on the chromosomes, whose function as linkers between spindle microtubules and chromosomes is crucial for proper cell division. The mechanisms that facilitate kinetochore capture by microtubules are still unclear. In the present study, we combine experiments and theory to explore the mechanisms of kinetochore capture at the onset of meiosis I in fission yeast. We show that kinetochores on homologous chromosomes move together, microtubules are dynamic and pivot around the spindle pole, and the average capture time is 3-4 minutes. Our theory describes paired kinetochores on homologous chromosomes as a single object, as well as angular movement of microtubules and their dynamics. For the experimentally measured parameters, the model reproduces the measured capture kinetics and shows that the paired configuration of kinetochores accelerates capture, whereas microtubule pivoting and dynamics have a smaller contribution. Kinetochore pairing may be a general feature that increases capture efficiency in meiotic cells.

  2. Paired arrangement of kinetochores together with microtubule pivoting and dynamics drive kinetochore capture in meiosis I

    PubMed Central

    Cojoc, Gheorghe; Florescu, Ana-Maria; Krull, Alexander; Klemm, Anna H.; Pavin, Nenad; Jülicher, Frank; Tolić, Iva M.

    2016-01-01

    Kinetochores are protein complexes on the chromosomes, whose function as linkers between spindle microtubules and chromosomes is crucial for proper cell division. The mechanisms that facilitate kinetochore capture by microtubules are still unclear. In the present study, we combine experiments and theory to explore the mechanisms of kinetochore capture at the onset of meiosis I in fission yeast. We show that kinetochores on homologous chromosomes move together, microtubules are dynamic and pivot around the spindle pole, and the average capture time is 3–4 minutes. Our theory describes paired kinetochores on homologous chromosomes as a single object, as well as angular movement of microtubules and their dynamics. For the experimentally measured parameters, the model reproduces the measured capture kinetics and shows that the paired configuration of kinetochores accelerates capture, whereas microtubule pivoting and dynamics have a smaller contribution. Kinetochore pairing may be a general feature that increases capture efficiency in meiotic cells. PMID:27166749

  3. Paired arrangement of kinetochores together with microtubule pivoting and dynamics drive kinetochore capture in meiosis I.

    PubMed

    Cojoc, Gheorghe; Florescu, Ana-Maria; Krull, Alexander; Klemm, Anna H; Pavin, Nenad; Jülicher, Frank; Tolić, Iva M

    2016-01-01

    Kinetochores are protein complexes on the chromosomes, whose function as linkers between spindle microtubules and chromosomes is crucial for proper cell division. The mechanisms that facilitate kinetochore capture by microtubules are still unclear. In the present study, we combine experiments and theory to explore the mechanisms of kinetochore capture at the onset of meiosis I in fission yeast. We show that kinetochores on homologous chromosomes move together, microtubules are dynamic and pivot around the spindle pole, and the average capture time is 3-4 minutes. Our theory describes paired kinetochores on homologous chromosomes as a single object, as well as angular movement of microtubules and their dynamics. For the experimentally measured parameters, the model reproduces the measured capture kinetics and shows that the paired configuration of kinetochores accelerates capture, whereas microtubule pivoting and dynamics have a smaller contribution. Kinetochore pairing may be a general feature that increases capture efficiency in meiotic cells. PMID:27166749

  4. Functional Specialization of Stable and Dynamic Microtubules in Protein Traffic in WIF-B Cells

    PubMed Central

    Poüs, C.; Chabin, K.; Drechou, A.; Barbot, L.; Phung-Koskas, T.; Settegrana, C.; Bourguet-Kondracki, M.L.; Maurice, M.; Cassio, D.; Guyot, M.; Durand, G.

    1998-01-01

    We found that the magnesium salt of ilimaquinone, named 201-F, specifically disassembled dynamically unstable microtubules in fibroblasts and various epithelial cell lines. Unlike classical tubulin- interacting drugs such as nocodazole or colchicine which affect all classes of microtubules, 201-F did not depolymerize stable microtubules. In WIF-B–polarized hepatic cells, 201-F disrupted the Golgi complex and inhibited albumin and alpha1-antitrypsin secretion to the same extent as nocodazole. By contrast, 201-F did not impair the transport of membrane proteins to the basolateral surface, which was only affected by the total disassembly of cellular microtubules. Transcytosis of two apical membrane proteins—the alkaline phosphodiesterase B10 and dipeptidyl peptidase IV—was affected to the same extent by 201-F and nocodazole. Taken together, these results indicate that only dynamically unstable microtubules are involved in the transport of secretory proteins to the plasma membrane, and in the transcytosis of membrane proteins to the apical surface. By contrast, stable microtubules, which are not functionally affected by 201-F treatment, are involved in the transport of membrane proteins to the basolateral surface. By specifically disassembling highly dynamic microtubules, 201-F is an invaluable tool with which to study the functional specialization of stable and dynamic microtubules in living cells. PMID:9660870

  5. 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

  6. Microtubule dynamics control HGF-induced lung endothelial barrier enhancement.

    PubMed

    Tian, Xinyong; Tian, Yufeng; Moldobaeva, Nurgul; Sarich, Nicolene; Birukova, Anna A

    2014-01-01

    Microtubules (MT) play a vital role in many cellular functions, but their role in peripheral actin cytoskeletal dynamics which is essential for control of endothelial barrier and monolayer integrity is less understood. We have previously described the enhancement of lung endothelial cell (EC) barrier by hepatocyte growth factor (HGF) which was associated with Rac1-mediated remodeling of actin cytoskeleton. This study investigated involvement of MT-dependent mechanisms in the HGF-induced enhancement of EC barrier. HGF-induced Rac1 activation was accompanied by phosphorylation of stathmin, a regulator of MT dynamics. HGF also stimulated MT peripheral growth monitored by time lapse imaging and tracking analysis of EB-1-decorated MT growing tips, and increased the pool of acetylated tubulin. These effects were abolished by EC pretreatment with HGF receptor inhibitor, downregulation of Rac1 pathway, or by expression of a stathmin-S63A phosphorylation deficient mutant. Expression of stathmin-S63A abolished the HGF protective effects against thrombin-induced activation of RhoA cascade, permeability increase, and EC barrier dysfunction. These results demonstrate a novel MT-dependent mechanism of HGF-induced EC barrier regulation via Rac1/PAK1/stathmin-dependent control of MT dynamics. PMID:25198505

  7. A mutation uncouples the tubulin conformational and GTPase cycles, revealing allosteric control of microtubule dynamics.

    PubMed

    Geyer, Elisabeth A; Burns, Alexander; Lalonde, Beth A; Ye, Xuecheng; Piedra, Felipe-Andres; Huffaker, Tim C; Rice, Luke M

    2015-10-06

    Microtubule dynamic instability depends on the GTPase activity of the polymerizing αβ-tubulin subunits, which cycle through at least three distinct conformations as they move into and out of microtubules. How this conformational cycle contributes to microtubule growing, shrinking, and switching remains unknown. Here, we report that a buried mutation in αβ-tubulin yields microtubules with dramatically reduced shrinking rate and catastrophe frequency. The mutation causes these effects by suppressing a conformational change that normally occurs in response to GTP hydrolysis in the lattice, without detectably changing the conformation of unpolymerized αβ-tubulin. Thus, the mutation weakens the coupling between the conformational and GTPase cycles of αβ-tubulin. By showing that the mutation predominantly affects post-GTPase conformational and dynamic properties of microtubules, our data reveal that the strength of the allosteric response to GDP in the lattice dictates the frequency of catastrophe and the severity of rapid shrinking.

  8. Real-time observations of microtubule dynamic instability in living cells

    PubMed Central

    1988-01-01

    Individual microtubule dynamics were observed in real time in primary cultures of newt lung epithelium using video-enhanced differential interference contrast microscopy and digital image processing. The linear filaments observed in cells corresponded to microtubules based on three criteria: (a) small particles translocated along them; (b) the majority of them disappeared after incubation in nocodazole; (c) and the distribution observed by differential interference contrast correlated with anti-tubulin immunofluorescence staining of the same cell. Microtubules were most clearly observed at the leading edge of cells located at the periphery of the epithelial sheet. Microtubules exhibited dynamic instability behavior: individual microtubules existed in persistent phases of elongation or rapid shortening. Microtubules elongated at a velocity of 7.2 micron/min +/- 0.3 SEM (n = 42) and rapidly shortened at a velocity of 17.3 micron/min +/- 0.7 SEM (n = 35). The transitions between elongation and rapid shortening occurred abruptly and stochastically with a transition frequency of 0.014 s-1 for catastrophe and 0.044 s-1 for rescue. Approximately 70% of the rapidly shortening microtubules were rescued and resumed elongation within the 35 x 35 micron microscopic field. A portion of the microtubule population appeared differentially stable and did not display any measurable elongation or shortening during 10-15-min observations. PMID:3198684

  9. 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.

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

    DOE PAGESBeta

    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

  11. 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.

  12. 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. PMID:25728349

  13. 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.

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

    PubMed

    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 included

  15. 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

  16. 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

  17. 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.

  18. 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

  19. Collapsin response mediator protein 4 regulates growth cone dynamics through the actin and microtubule cytoskeleton.

    PubMed

    Khazaei, Mohamad R; Girouard, Marie-Pier; Alchini, Ricardo; Ong Tone, Stephan; Shimada, Tadayuki; Bechstedt, Susanne; Cowan, Mitra; Guillet, Dominique; Wiseman, Paul W; Brouhard, Gary; Cloutier, Jean Francois; Fournier, Alyson E

    2014-10-24

    Coordinated control of the growth cone cytoskeleton underlies axon extension and guidance. Members of the collapsin response mediator protein (CRMP) family of cytosolic phosphoproteins regulate the microtubule and actin cytoskeleton, but their roles in regulating growth cone dynamics remain largely unexplored. Here, we examine how CRMP4 regulates the growth cone cytoskeleton. Hippocampal neurons from CRMP4-/- mice exhibited a selective decrease in axon extension and reduced growth cone area, whereas overexpression of CRMP4 enhanced the formation and length of growth cone filopodia. Biochemically, CRMP4 can impact both microtubule assembly and F-actin bundling in vitro. Through a structure function analysis of CRMP4, we found that the effects of CRMP4 on axon growth and growth cone morphology were dependent on microtubule assembly, whereas filopodial extension relied on actin bundling. Intriguingly, anterograde movement of EB3 comets, which track microtubule protrusion, slowed significantly in neurons derived from CRMP4-/- mice, and rescue of microtubule dynamics required CRMP4 activity toward both the actin and microtubule cytoskeleton. Together, this study identified a dual role for CRMP4 in regulating the actin and microtubule growth cone cytoskeleton. PMID:25225289

  20. BetaIII-tubulin induces paclitaxel resistance in association with reduced effects on microtubule dynamic instability.

    PubMed

    Kamath, Kathy; Wilson, Leslie; Cabral, Fernando; Jordan, Mary Ann

    2005-04-01

    The development of resistance to paclitaxel in tumors is one of the most significant obstacles to successful therapy. Overexpression of the betaIII-tubulin isotype has been associated with paclitaxel resistance in a number of cancer cell lines and in tumors, but the mechanism of resistance has remained unclear. Paclitaxel inhibits cancer cell proliferation by binding to the beta-subunit of tubulin in microtubules and suppressing microtubule dynamic instability, leading to mitotic arrest and cell death. We hypothesized that betaIII-tubulin overexpression induces resistance to paclitaxel either by constitutively enhancing microtubule dynamic instability in resistant cells or by rendering the microtubules less sensitive to the suppression of dynamics by paclitaxel. Using Chinese hamster ovary cells that inducibly overexpress either betaI- or betaIII-tubulin, we analyzed microtubule dynamic instability during interphase by microinjection of rhodamine-labeled tubulin and time-lapse fluorescence microscopy. In the absence of paclitaxel, there were no differences in any aspect of dynamic instability between the two beta-tubulin-overexpressing cell types. However, in the presence of 150 nm paclitaxel, dynamic instability was suppressed to a significantly lesser extent (suppressed only 12%) in cells overexpressing betaIII-tubulin than in cells overexpressing similar levels of betaI-tubulin (suppressed 47%). The results suggest that overexpression of betaIII-tubulin induces paclitaxel resistance by reducing the ability of paclitaxel to suppress microtubule dynamics. The results also suggest that endogenous regulators of microtubule dynamics may differentially interact with individual tubulin isotypes, supporting the idea that differential expression of tubulin isotypes has functional consequences in cells.

  1. Nucleation and Dynamics of Golgi-derived Microtubules

    PubMed Central

    Sanders, Anna A. W. M.; Kaverina, Irina

    2015-01-01

    Integrity of the Golgi apparatus requires the microtubule (MT) network. A subset of MTs originates at the Golgi itself, which in this case functions as a MT-organizing center (MTOC). Golgi-derived MTs serve important roles in post-Golgi trafficking, maintenance of Golgi integrity, cell polarity and motility, as well as cell type-specific functions, including neurite outgrowth/branching. Here, we discuss possible models describing the formation and dynamics of Golgi-derived MTs. How Golgi-derived MTs are formed is not fully understood. A widely discussed model implicates that the critical step of the process is recruitment of molecular factors, which drive MT nucleation (γ-tubulin ring complex, or γ-TuRC), to the Golgi membrane via specific scaffolding interactions. Based on recent findings, we propose to introduce an additional level of regulation, whereby MT-binding proteins and/or local tubulin dimer concentration at the Golgi helps to overcome kinetic barriers at the initial nucleation step. According to our model, emerging MTs are subsequently stabilized by Golgi-associated MT-stabilizing proteins. We discuss molecular factors potentially involved in all three steps of MT formation. To preserve proper cell functioning, a balance must be maintained between MT subsets at the centrosome and the Golgi. Recent work has shown that certain centrosomal factors are important in maintaining this balance, suggesting a close connection between regulation of centrosomal and Golgi-derived MTs. Finally, we will discuss potential functions of Golgi-derived MTs based on their nucleation site location within a Golgi stack. PMID:26617483

  2. Domains of tau protein, differential phosphorylation, and dynamic instability of microtubules.

    PubMed

    Trinczek, B; Biernat, J; Baumann, K; Mandelkow, E M; Mandelkow, E

    1995-12-01

    The dynamic instability of microtubules is thought to be regulated by MAPs and phosphorylation. Here we describe the effect of the neuronal microtubule-associated protein tau by observing the dynamics of single microtubules by video microscopy. We used recombinant tau isoforms and tau mutants, and we phosphorylated tau by the neuronal kinases MARK (affecting the KXGS motifs within tau's repeat domain) and cdk5 (phosphorylating Ser-Pro motifs in the regions flanking the repeats). The variants of tau can be broadly classified into three categories, depending on their potency to affect microtubule dynamics. "Strong" tau variants have four repeats and both flanking regions. "Medium" variants have one to three repeats and both flanking regions. "Weak" variants lack one or both of the flanking regions, or have no repeats; with such constructs, microtubule dynamics is not significantly different from that of pure tubulin. N- or C-terminal tails of tau have no influence on dynamic instability. The two ends of microtubules (plus and minus) showed different activities but analogous behavior. These results are consistent with the "jaws" model of tau where the flanking regions are considered as targeting domains whereas the addition of repeats makes them catalytically active in terms of microtubule stabilization. The dominant changes in the parameters of dynamic instability induced by tau are those in the dissociation rate and in the catastrophe rate (up to 30-fold). Other rates change only moderately or not at all (association rate increased up to twofold, rates of rescue or rapid shrinkage decreased up to approximately twofold). The order of repeats has little influence on microtubule dynamics (i.e., repeats can be re-arranged or interchanged), arguing in favor of the "distributed weak binding" model proposed by Butner and Kirschner (1991); however, we confirmed the presence of a "hotspot" of binding potential involving Lys274 and Lys281 observed by Goode and Feinstein, 1994

  3. Dynamics of microtubule asters in microfabricated chambers: The role of catastrophes

    PubMed Central

    Faivre-Moskalenko, Cendrine; Dogterom, Marileen

    2002-01-01

    Recent in vivo as well as in vitro experiments have indicated that microtubule pushing alone is sufficient to position a microtubule-organizing center within a cell. Here, we investigate the effect of catastrophes on the dynamics of microtubule asters within microfabricated chambers that mimic the confining geometry of living cells. The use of a glass bead as the microtubule-organizing center allows us to manipulate the aster by using optical tweezers. In the case in which microtubules preexist, we show that because of microtubule buckling, repositioning almost never occurs after relocation with the optical tweezers, although initial microtubule growth always leads the aster to the geometrical center of the chamber. When a catastrophe promoter is added, we find instead that the aster is able to efficiently explore the chamber geometry even after being relocated with the optical tweezers. As predicted by theoretical calculations, the results of our in vitro experiments clearly demonstrate the need for catastrophes for proper positioning in a confining geometry. These findings correlate with recent observations of nuclear positioning in fission yeast cells. PMID:12486218

  4. 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

  5. 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.

  6. Cyclin B interaction with microtubule-associated protein 4 (MAP4) targets p34cdc2 kinase to microtubules and is a potential regulator of M-phase microtubule dynamics

    PubMed Central

    1995-01-01

    We previously demonstrated (Ookata et al., 1992, 1993) that the p34cdc2/cyclin B complex associates with microtubules in the mitotic spindle and premeiotic aster in starfish oocytes, and that microtubule- associated proteins (MAPs) might be responsible for this interaction. In this study, we have investigated the mechanism by which p34cdc2 kinase associates with the microtubule cytoskeleton in primate tissue culture cells whose major MAP is known to be MAP4. Double staining of primate cells with anti-cyclin B and anti-MAP4 antibodies demonstrated these two antigens were colocalized on microtubules and copartitioned following two treatments that altered MAP4 distribution. Detergent extraction before fixation removed cyclin B as well as MAP4 from the microtubules. Depolymerization of some of the cellular microtubules with nocodazole preferentially retained the microtubule localization of both cyclin B and MAP4. The association of p34cdc2/cyclin B kinase with microtubules was also shown biochemically to be mediated by MAP4. Cosedimentation of purified p34cdc2/cyclin B with purified microtubule proteins containing MAP4, but not with MAP-free microtubules, as well as binding of MAP4 to GST-cyclin B fusion proteins, demonstrated an interaction between cyclin B and MAP4. Using recombinant MAP4 fragments, we demonstrated that the Pro-rich C-terminal region of MAP4 is sufficient to mediate the cyclin B-MAP4 interaction. Since p34cdc2/cyclin B physically associated with MAP4, we examined the ability of the kinase complex to phosphorylate MAP4. Incubation of a ternary complex of p34cdc2, cyclin B, and the COOH-terminal domain of MAP4, PA4, with ATP resulted in intracomplex phosphorylation of PA4. Finally, we tested the effects of MAP4 phosphorylation on microtubule dynamics. Phosphorylation of MAP4 by p34cdc2 kinase did not prevent its binding to microtubules, but abolished its microtubule stabilizing activity. Thus, the cyclin B/MAP4 interaction we have described may be

  7. Molecular mechanisms of Tau binding to microtubules and its role in microtubule dynamics in live cells.

    PubMed

    Breuzard, Gilles; Hubert, Pierre; Nouar, Roqiya; De Bessa, Tiphany; Devred, François; Barbier, Pascale; Sturgis, James N; Peyrot, Vincent

    2013-07-01

    Despite extensive studies, the molecular mechanisms of Tau binding to microtubules (MTs) and its consequences on MT stability still remain unclear. It is especially true in cells where the spatiotemporal distribution of Tau-MT interactions is unknown. Using Förster resonance energy transfer (FRET), we showed that the Tau-MT interaction was distributed along MTs in periodic hotspots of high and low FRET intensities. Fluorescence recovery after photobleaching (FRAP) revealed a two-phase exchange of Tau with MTs as a rapid diffusion followed by a slower binding phase. A real-time FRET assay showed that high FRET occurred simultaneously with rescue and pause transitions at MT ends. To further explore the functional interaction of Tau with MTs, the binding of paclitaxel (PTX), tubulin acetylation induced by trichostatin A (TSA), and the expression of non-acetylatable tubulin were used. With PTX and TSA, FRAP curves best fitted a single phase with a long time constant, whereas with non-acetylatable α-tubulin, curves best fitted a two phase recovery. Upon incubation with PTX and TSA, the number of high and low FRET hotspots decreased by up to 50% and no hotspot was observed during rescue and pause transitions. In the presence of non-acetylatable α-tubulin, a 34% increase in low FRET hotspots occurred, and our real-time FRET assay revealed that low FRET hotspots appeared with MTs recovering growth. In conclusion, we have identified, by FRET and FRAP, a discrete Tau-MT interaction, in which Tau could induce conformational changes of MTs, favoring recovery of MT self-assembly. PMID:23659998

  8. Mammalian microtubule P-body dynamics are mediated by nesprin-1.

    PubMed

    Rajgor, Dipen; Mellad, Jason A; Soong, Daniel; Rattner, Jerome B; Fritzler, Marvin J; Shanahan, Catherine M

    2014-05-26

    Nesprins are a multi-isomeric family of spectrin-repeat (SR) proteins, predominantly known as nuclear envelope scaffolds. However, isoforms that function beyond the nuclear envelope remain poorly examined. Here, we characterize p50(Nesp1), a 50-kD isoform that localizes to processing bodies (PBs), where it acts as a microtubule-associated protein capable of linking mRNP complexes to microtubules. Overexpression of dominant-negative p50(Nesp1) caused Rck/p54, but not GW182, displacement from microtubules, resulting in reduced PB movement and cross talk with stress granules (SGs). These cells disassembled canonical SGs induced by sodium arsenite, but not those induced by hydrogen peroxide, leading to cell death and revealing PB-microtubule attachment is required for hydrogen peroxide-induced SG anti-apoptotic functions. Furthermore, p50(Nesp1) was required for miRNA-mediated silencing and interacted with core miRISC silencers Ago2 and Rck/p54 in an RNA-dependent manner and with GW182 in a microtubule-dependent manner. These data identify p50(Nesp1) as a multi-functional PB component and microtubule scaffold necessary for RNA granule dynamics and provides evidence for PB and SG micro-heterogeneity. PMID:24862572

  9. 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

  10. Cooperative dynamics of microtubule ensembles: Polymerization forces and rescue-induced oscillations

    NASA Astrophysics Data System (ADS)

    Zelinski, Björn; Kierfeld, Jan

    2013-01-01

    We investigate the cooperative dynamics of an ensemble of N microtubules growing against an elastic barrier. Microtubules undergo so-called catastrophes, which are abrupt stochastic transitions from a growing to a shrinking state, and rescues, which are transitions back to the growing state. Microtubules can exert pushing or polymerization forces on an obstacle, such as an elastic barrier, if the growing end is in contact with the obstacle. We use dynamical mean-field theory and stochastic simulations to analyze a model where each microtubule undergoes catastrophes and rescues and where microtubules interact by force sharing. For zero rescue rate, cooperative growth terminates in a collective catastrophe. The maximal polymerization force before catastrophes grows linearly with N for small N or a stiff elastic barrier, in agreement with available experimental results, whereas it crosses over to a logarithmic dependence for larger N or a soft elastic barrier. For a nonzero rescue rate and a soft elastic barrier, the dynamics becomes oscillatory with both collective catastrophe and rescue events, which are part of a robust limit cycle. Both the average and maximal polymerization forces then grow linearly with N, and we investigate their dependence on tubulin on-rates and rescue rates, which can be involved in cellular regulation mechanisms. We further investigate the robustness of the collective catastrophe and rescue oscillations with respect to different catastrophe models.

  11. 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.

  12. 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.

  13. A direct interaction between fascin and microtubules contributes to adhesion dynamics and cell migration

    PubMed Central

    Villari, Giulia; Jayo, Asier; Zanet, Jennifer; Fitch, Briana; Serrels, Bryan; Frame, Margaret; Stramer, Brian M.; Goult, Benjamin T.; Parsons, Maddy

    2015-01-01

    ABSTRACT Fascin is an actin-binding and bundling protein that is highly upregulated in most epithelial cancers. Fascin promotes cell migration and adhesion dynamics in vitro and tumour cell metastasis in vivo. However, potential non-actin bundling roles for fascin remain unknown. Here, we show for the first time that fascin can directly interact with the microtubule cytoskeleton and that this does not depend upon fascin-actin bundling. Microtubule binding contributes to fascin-dependent control of focal adhesion dynamics and cell migration speed. We also show that fascin forms a complex with focal adhesion kinase (FAK, also known as PTK2) and Src, and that this signalling pathway lies downstream of fascin–microtubule association in the control of adhesion stability. These findings shed light on new non actin-dependent roles for fascin and might have implications for the design of therapies to target fascin in metastatic disease. PMID:26542021

  14. Dynamic instability 30 years later: complexities in microtubule growth and catastrophe

    PubMed Central

    Brouhard, Gary J.

    2015-01-01

    Microtubules are not like other polymers. Whereas polymers such as F-actin will grow continuously as long as the subunit concentration is high enough, a steadily growing microtubule can suddenly shrink even when there is ample αβ-tubulin around. This remarkable behavior was discovered in 1984 when Tim Mitchison and Marc Kirschner deduced that microtubules switch from growth to shrinkage when they lose their GTP caps. Here, I review the canonical explanation of dynamic instability that was fleshed out in the years after its discovery. Many aspects of this explanation have been recently subverted, particularly those related to how GTP-tubulin forms polymers and why GTP hydrolysis disrupts them. I describe these developments and speculate on how our explanation of dynamic instability can be changed to accommodate them. PMID:25823928

  15. Effects of eribulin, vincristine, paclitaxel and ixabepilone on fast axonal transport and kinesin-1 driven microtubule gliding: Implications for chemotherapy-induced peripheral neuropathy

    PubMed Central

    LaPointe, Nichole E.; Morfini, Gerardo; Brady, Scott T.; Feinstein, Stuart C.; Wilson, Leslie; Jordan, Mary Ann

    2014-01-01

    Chemotherapy-induced peripheral neuropathy (CIPN) is a serious, painful and dose-limiting side effect of cancer drugs that target microtubules. The mechanisms underlying the neuronal damage are unknown, but may include disruption of fast axonal transport, an essential microtubule-based process that moves cellular components over long distances between neuronal cell bodies and nerve terminals. This idea is supported by the “dying back” pattern of degeneration observed in CIPN, and by the selective vulnerability of sensory neurons bearing the longest axonal projections. In this study, we test the hypothesis that microtubule-targeting drugs disrupt fast axonal transport using vesicle motility assays in isolated squid axoplasm and a cell-free microtubule gliding assay with defined components. We compare four clinically-used drugs, eribulin, vincristine, paclitaxel and ixabepilone. Of these, eribulin is associated with a relatively low incidence of severe neuropathy, while vincristine has a relatively high incidence. In vesicle motility assays, we found that all four drugs inhibited anterograde (conventional kinesin-dependent) fast axonal transport, with the potency being vincristine = ixabepilone > paclitaxel = eribulin. Interestingly, eribulin and paclitaxel did not inhibit retrograde (cytoplasmic dynein-dependent) fast axonal transport, in contrast to vincristine and ixabepilone. Similarly, vincristine and ixabepilone both exerted significant inhibitory effects in an in vitro microtubule gliding assay consisting of recombinant kinesin (kinesin-1) and microtubules composed of purified bovine brain tubulin, whereas paclitaxel and eribulin had negligible effects. Our results suggest that (i) inhibition of microtubule-based fast axonal transport may be a significant contributor to neurotoxicity induced by microtubule-targeting drugs, and (ii) that individual microtubule-targeting drugs affect fast axonal transport through different mechanisms. PMID:23711742

  16. 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

  17. Alterations in ovarian cancer cell adhesion drive taxol resistance by increasing microtubule dynamics in a FAK-dependent manner.

    PubMed

    McGrail, Daniel J; Khambhati, Niti N; Qi, Mark X; Patel, Krishan S; Ravikumar, Nithin; Brandenburg, Chandler P; Dawson, Michelle R

    2015-04-17

    Chemorefractory ovarian cancer patients show extremely poor prognosis. Microtubule-stabilizing Taxol (paclitaxel) is a first-line treatment against ovarian cancer. Despite the close interplay between microtubules and cell adhesion, it remains unknown if chemoresistance alters the way cells adhere to their extracellular environment, a process critical for cancer metastasis. To investigate this, we isolated Taxol-resistant populations of OVCAR3 and SKOV3 ovarian cancer cell lines. Though Taxol-resistant cells neither effluxed more drug nor gained resistance to other chemotherapeutics, they did display increased microtubule dynamics. These changes in microtubule dynamics coincided with faster attachment rates and decreased adhesion strength, which correlated with increased surface β1-integrin expression and decreased focal adhesion formation, respectively. Adhesion strength correlated best with Taxol-sensitivity, and was found to be independent of microtubule polymerization but dependent on focal adhesion kinase (FAK), which was up-regulated in Taxol-resistant cells. FAK inhibition also decreased microtubule dynamics to equal levels in both populations, indicating alterations in adhesive signaling are up-stream of microtubule dynamics. Taken together, this work demonstrates that Taxol-resistance dramatically alters how ovarian cancer cells adhere to their extracellular environment causing down-stream increases in microtubule dynamics, providing a therapeutic target that may improve prognosis by not only recovering drug sensitivity, but also decreasing metastasis.

  18. 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.

  19. KATNAL1 Regulation of Sertoli Cell Microtubule Dynamics Is Essential for Spermiogenesis and Male Fertility

    PubMed Central

    Smith, Lee B.; Milne, Laura; Nelson, Nancy; Eddie, Sharon; Brown, Pamela; Atanassova, Nina; O'Bryan, Moira K.; O'Donnell, Liza; Rhodes, Danielle; Wells, Sara; Napper, Diane; Nolan, Patrick; Lalanne, Zuzanna; Cheeseman, Michael; Peters, Josephine

    2012-01-01

    Spermatogenesis is a complex process reliant upon interactions between germ cells (GC) and supporting somatic cells. Testicular Sertoli cells (SC) support GCs during maturation through physical attachment, the provision of nutrients, and protection from immunological attack. This role is facilitated by an active cytoskeleton of parallel microtubule arrays that permit transport of nutrients to GCs, as well as translocation of spermatids through the seminiferous epithelium during maturation. It is well established that chemical perturbation of SC microtubule remodelling leads to premature GC exfoliation demonstrating that microtubule remodelling is an essential component of male fertility, yet the genes responsible for this process remain unknown. Using a random ENU mutagenesis approach, we have identified a novel mouse line displaying male-specific infertility, due to a point mutation in the highly conserved ATPase domain of the novel KATANIN p60-related microtubule severing protein Katanin p60 subunit A-like1 (KATNAL1). We demonstrate that Katnal1 is expressed in testicular Sertoli cells (SC) from 15.5 days post-coitum (dpc) and that, consistent with chemical disruption models, loss of function of KATNAL1 leads to male-specific infertility through disruption of SC microtubule dynamics and premature exfoliation of spermatids from the seminiferous epithelium. The identification of KATNAL1 as an essential regulator of male fertility provides a significant novel entry point into advancing our understanding of how SC microtubule dynamics promotes male fertility. Such information will have resonance both for future treatment of male fertility and the development of non-hormonal male contraceptives. PMID:22654668

  20. Synergistic suppression of microtubule dynamics by discodermolide and paclitaxel in non-small cell lung carcinoma cells.

    PubMed

    Honore, Stéphane; Kamath, Kathy; Braguer, Diane; Horwitz, Susan Band; Wilson, Leslie; Briand, Claudette; Jordan, Mary Ann

    2004-07-15

    Discodermolide is a new microtubule-targeted antimitotic drug in Phase I clinical trials that, like paclitaxel, stabilizes microtubule dynamics and enhances microtubule polymer mass in vitro and in cells. Despite their apparently similar binding sites on microtubules, discodermolide acts synergistically with paclitaxel to inhibit proliferation of A549 human lung cancer cells (L. Martello et al., Clin. Cancer Res., 6: 1978-1987, 2000). To understand their synergy, we examined the effects of the two drugs singly and in combination in A549 cells and found that, surprisingly, their antiproliferative synergy is related to their ability to synergistically inhibit microtubule dynamic instability and mitosis. The combination of discodermolide and paclitaxel at their antiproliferative IC(50)s (7 nm for discodermolide and 2 nm for paclitaxel) altered all of the parameters of dynamic instability synergistically except the time-based rescue frequency. For example, together the drugs inhibited overall microtubule dynamicity by 71%, but each drug individually inhibited dynamicity by only 24%, giving a combination index (CI) of 0.23. Discodermolide and paclitaxel also synergistically blocked cell cycle progression at G(2)-M (41, 9.6, and 16% for both drugs together, for discodermolide alone, and for paclitaxel alone, respectively; CI = 0.59), and they synergistically enhanced apoptosis (CI = 0.85). Microtubules are unique receptors for drugs. The results suggest that ligands that bind to large numbers of binding sites on an individual microtubule can interact in a poorly understood manner to synergistically suppress microtubule dynamic instability and inhibit both mitosis and cell proliferation, with important consequences for combination clinical therapy with microtubule-targeted drugs.

  1. 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

  2. Maytansinoid-antibody conjugates induce mitotic arrest by suppressing microtubule dynamic instability.

    PubMed

    Oroudjev, Emin; Lopus, Manu; Wilson, Leslie; Audette, Charlene; Provenzano, Carmela; Erickson, Hans; Kovtun, Yelena; Chari, Ravi; Jordan, Mary Ann

    2010-10-01

    Maytansine and its analogues (maytansinoids) are potent microtubule-targeted compounds that inhibit proliferation of cells at mitosis. Antibody-maytansinoid conjugates consisting of maytansinoids (DM1 and DM4) attached to tumor-specific antibodies have shown promising clinical results. To determine the mechanism by which the antibody-DM1 conjugates inhibit cell proliferation, we examined the effects of the cleavable anti-EpCAM-SPP-DM1 and uncleavable anti-EpCAM-SMCC-DM1 conjugates on MCF7 human breast tumor cells. We also examined the effects of the free maytansinoids, maytansine and S-methyl DM1 (a version of DM1 that is stable in cell culture medium), for comparison. Both the conjugates and free maytansinoids potently inhibited MCF7 cell proliferation at nanomolar and subnanomolar concentrations, respectively, by arresting the cells in mitotic prometaphase/metaphase. Arrest occurred in concert with the internalization and intracellular processing of both conjugates under conditions that induced abnormal spindle organization and suppressed microtubule dynamic instability. Microtubule depolymerization occurred only at significantly higher drug concentrations. The results indicate that free maytansinoids, antibody-maytansinoid conjugates, and their metabolites exert their potent antimitotic effects through a common mechanism involving suppression of microtubule dynamic instability.

  3. The dynamics of plus end polarization and microtubule assembly during Xenopus cortical rotation.

    PubMed

    Olson, David J; Oh, Denise; Houston, Douglas W

    2015-05-15

    The self-organization of dorsally-directed microtubules during cortical rotation in the Xenopus egg is essential for dorsal axis formation. The mechanisms controlling this process have been problematic to analyze, owing to difficulties in visualizing microtubules in living egg. Also, the order of events occurring at the onset of cortical rotation have not been satisfactorily visualized in vivo and have been inferred from staged fixed samples. To address these issues, we have characterized the dynamics of total microtubule and plus end behavior continuously throughout cortical rotation, as well as in oocytes and unfertilized eggs. Here, we show that the nascent microtubule network forms in the cortex but associates with the deep cytoplasm at the start of rotation. Importantly, plus ends remain cortical and become increasingly more numerous and active prior to rotation, with dorsal polarization occurring rapidly after the onset of rotation. Additionally, we show that vegetally localized Trim36 is required to attenuate dynamic plus end growth, suggesting that vegetal factors are needed to locally coordinate growth in the cortex.

  4. 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

  5. Microtubule Dynamicity Is More Important than Stability in Memory Formation: an In Vivo Study.

    PubMed

    Atarod, Deyhim; Eskandari-Sedighi, Ghazaleh; Pazhoohi, Farid; Karimian, Seyed Morteza; Khajeloo, Mojtaba; Riazi, Gholam Hossein

    2015-06-01

    It has been shown that microtubule (MT) activity and dynamics can have huge impacts on synaptic plasticity and memory formation. This is mainly due to various functions of MTs in neurons; MTs are involved in dendritic spine formation, axonal transportation, neuronal polarity, and receptor trafficking. Recent studies from our group and other labs have suggested the possible role of brain MT dynamicity and activity in memory; however, there is a need for more detailed studies regarding this aspect. In this study, we have tried to evaluate the importance of microtubule dynamicity rather than stability in memory formation in vivo. In order to investigate the role of MT stability in memory formation, we treated mice with paclitaxel-a classic microtubule-stabilizing agent. We then studied the behavior of treated animals using Morris water maze (MWM) test. To measure the effect of injected paclitaxel on MT polymerization kinetics, we conducted polymerization assays on brain extracts of the same paclitaxel-treated animals. Our results show that paclitaxel treatment affects animals' memory in a negative way and treated animals behave poorly in MWM compared to control group. In addition, our kinetics studies show that MT stability is significantly increased in brain extracts from paclitaxel-treated mice, but MT dynamics is reduced. Thus, we suggest that dynamicity is a very important feature of MT protein structures, and regarding memory formation, dynamicity is more important than stability and high activity.

  6. Microtubules Regulate Focal Adhesion Dynamics through MAP4K4

    PubMed Central

    Yue, Jiping; Xie, Min; Gou, Xuewen; Lee, Philbert; Schneider, Michael D; Wu, Xiaoyang

    2014-01-01

    Disassembly of focal adhesions (FAs) allows cell retraction and integrin detachment from the ECM, processes critical for cell movement. Growth of MT (microtubule) can promote FA turnover by serving as tracks to deliver proteins essential for FA disassembly. The molecular nature of this FA “disassembly factor”, however, remains elusive. By quantitative proteomics, we identified MAP4K4 (mitogen-activated protein kinase kinase kinase kinase 4) as a FA regulator that associates with MTs. Conditional knockout (cKO) of MAP4K4 in skin stabilizes FAs and impairs epidermal migration. By exploring underlying mechanisms, we further show that MAP4K4 associates with EB2, a MT binding protein, and IQSEC1, a guanine nucleotide exchange factor (GEF) specific for Arf6, whose activation promotes integrin internalization. Together, our findings provide critical insights into FA disassembly, suggesting that MTs can deliver MAP4K4 toward FAs through EB2, where MAP4K4 can in turn activate Arf6 via IQSEC1 and enhance FA dissolution. PMID:25490267

  7. Microtubules regulate focal adhesion dynamics through MAP4K4.

    PubMed

    Yue, Jiping; Xie, Min; Gou, Xuewen; Lee, Philbert; Schneider, Michael D; Wu, Xiaoyang

    2014-12-01

    Disassembly of focal adhesions (FAs) allows cell retraction and integrin detachment from the extracellular matrix, processes critical for cell movement. Growth of microtubules (MTs) can promote FA turnover by serving as tracks to deliver proteins essential for FA disassembly. The molecular nature of this FA "disassembly factor," however, remains elusive. By quantitative proteomics, we identified mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) as an FA regulator that associates with MTs. Knockout of MAP4K4 stabilizes FAs and impairs cell migration. By exploring underlying mechanisms, we further show that MAP4K4 associates with ending binding 2 (EB2) and IQ motif and SEC7 domain-containing protein 1 (IQSEC1), a guanine nucleotide exchange factor specific for Arf6, whose activation promotes integrin internalization. Together, our findings provide critical insight into FA disassembly, suggesting that MTs can deliver MAP4K4 toward FAs through EB2, where MAP4K4 can, in turn, activate Arf6 via IQSEC1 and enhance FA dissolution. PMID:25490267

  8. Inhibition of microtubule dynamics affects podosome belt formation during osteoclast induction.

    PubMed

    Ti, Yunfan; Zhou, Lingjun; Wang, Rui; Zhao, Jianning

    2015-03-01

    Osteoclast is the only cell that can degrade bone tissue in vivo. Recent studies have shown the important role of cytoskeleton dynamics in osteolysis and the formation of podosome belt in osteoclasts. This process is regulated by the dynamic microtubule (MT) network. We treated osteoclast precursor cells Raw264.7 with low concentration of nocodazole (10 nM) and antineoplastic drug taxol (10 nM) to block MT turnover, and used end binding protein 1 fused to GFP to track the movement of microtubules in induced osteoclasts. We show that low concentrations of nocodazole and taxol interfere with the formation of podosome belt, and reduce TRAP activity of induced osteoclasts. These results suggest that the effect of taxol on MT dynamics may be used clinically to reduce osteoclast activity and potentially prevent development of osteoporosis and other metabolic bone diseases.

  9. Erucin, the Major Isothiocyanate in Arugula (Eruca sativa), Inhibits Proliferation of MCF7 Tumor Cells by Suppressing Microtubule Dynamics

    PubMed Central

    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. PMID:24950293

  10. Human chromokinesins promote chromosome congression and spindle microtubule dynamics during mitosis

    PubMed Central

    Wandke, Cornelia; Barisic, Marin; Sigl, Reinhard; Rauch, Veronika; Wolf, Frank; Amaro, Ana C.; Tan, Chia H.; Pereira, Antonio J.; Kutay, Ulrike; Maiato, Helder; Meraldi, Patrick

    2012-01-01

    Chromokinesins are microtubule plus end–directed motor proteins that bind to chromosome arms. In Xenopus egg cell-free extracts, Xkid and Xklp1 are essential for bipolar spindle formation but the functions of the human homologues, hKID (KIF22) and KIF4A, are poorly understood. By using RNAi-mediated protein knockdown in human cells, we find that only co-depletion delayed progression through mitosis in a Mad2-dependent manner. Depletion of hKID caused abnormal chromosome arm orientation, delayed chromosome congression, and sensitized cells to nocodazole. Knockdown of KIF4A increased the number and length of microtubules, altered kinetochore oscillations, and decreased kinetochore microtubule flux. These changes were associated with failures in establishing a tight metaphase plate and an increase in anaphase lagging chromosomes. Co-depletion of both chromokinesins aggravated chromosome attachment failures, which led to mitotic arrest. Thus, hKID and KIF4A contribute independently to the rapid and correct attachment of chromosomes by controlling the positioning of chromosome arms and the dynamics of microtubules, respectively. PMID:22945934

  11. Human chromokinesins promote chromosome congression and spindle microtubule dynamics during mitosis.

    PubMed

    Wandke, Cornelia; Barisic, Marin; Sigl, Reinhard; Rauch, Veronika; Wolf, Frank; Amaro, Ana C; Tan, Chia H; Pereira, Antonio J; Kutay, Ulrike; Maiato, Helder; Meraldi, Patrick; Geley, Stephan

    2012-09-01

    Chromokinesins are microtubule plus end-directed motor proteins that bind to chromosome arms. In Xenopus egg cell-free extracts, Xkid and Xklp1 are essential for bipolar spindle formation but the functions of the human homologues, hKID (KIF22) and KIF4A, are poorly understood. By using RNAi-mediated protein knockdown in human cells, we find that only co-depletion delayed progression through mitosis in a Mad2-dependent manner. Depletion of hKID caused abnormal chromosome arm orientation, delayed chromosome congression, and sensitized cells to nocodazole. Knockdown of KIF4A increased the number and length of microtubules, altered kinetochore oscillations, and decreased kinetochore microtubule flux. These changes were associated with failures in establishing a tight metaphase plate and an increase in anaphase lagging chromosomes. Co-depletion of both chromokinesins aggravated chromosome attachment failures, which led to mitotic arrest. Thus, hKID and KIF4A contribute independently to the rapid and correct attachment of chromosomes by controlling the positioning of chromosome arms and the dynamics of microtubules, respectively. PMID:22945934

  12. Plasma Membrane Factor XIIIA Transglutaminase Activity Regulates Osteoblast Matrix Secretion and Deposition by Affecting Microtubule Dynamics

    PubMed Central

    Al-Jallad, Hadil F.; Myneni, Vamsee D.; Piercy-Kotb, Sarah A.; Chabot, Nicolas; Mulani, Amina; Keillor, Jeffrey W.; Kaartinen, Mari T.

    2011-01-01

    Transglutaminase activity, arising potentially from transglutaminase 2 (TG2) and Factor XIIIA (FXIIIA), has been linked to osteoblast differentiation where it is required for type I collagen and fibronectin matrix deposition. In this study we have used an irreversible TG-inhibitor to ‘block –and-track’ enzyme(s) targeted during osteoblast differentiation. We show that the irreversible TG-inhibitor is highly potent in inhibiting osteoblast differentiation and mineralization and reduces secretion of both fibronectin and type I collagen and their release from the cell surface. Tracking of the dansyl probe by Western blotting and immunofluorescence microscopy demonstrated that the inhibitor targets plasma membrane-associated FXIIIA. TG2 appears not to contribute to crosslinking activity on the osteoblast surface. Inhibition of FXIIIA with NC9 resulted in defective secretory vesicle delivery to the plasma membrane which was attributable to a disorganized microtubule network and decreased microtubule association with the plasma membrane. NC9 inhibition of FXIIIA resulted in destabilization of microtubules as assessed by cellular Glu-tubulin levels. Furthermore, NC9 blocked modification of Glu-tubulin into 150 kDa high-molecular weight Glu-tubulin form which was specifically localized to the plasma membrane. FXIIIA enzyme and its crosslinking activity were colocalized with plasma membrane-associated tubulin, and thus, it appears that FXIIIA crosslinking activity is directed towards stabilizing the interaction of microtubules with the plasma membrane. Our work provides the first mechanistic cues as to how transglutaminase activity could affect protein secretion and matrix deposition in osteoblasts and suggests a novel function for plasma membrane FXIIIA in microtubule dynamics. PMID:21283799

  13. Structural evidence for cooperative microtubule stabilization by Taxol and the endogenous dynamics regulator MAP4

    PubMed Central

    Xiao, Hui; Wang, Hui; Zhang, Xuechun; Tu, Zongcai; Bulinski, Chloë; Khrapunovich-Baine, Marina; Angeletti, Ruth Hogue; Horwitz, Susan Band

    2012-01-01

    Microtubules (MTs) composed of αβ-tubulin heterodimers are highly dynamic polymers, whose stability can be regulated by numerous endogenous and exogenous factors. Both the anti-mitotic drug, Taxol, and microtubule-associated proteins (MAPs) stabilize this dynamicity by binding to and altering the conformation of MTs. In the current study, amide hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) was used to examine the structural and dynamic properties of the MT complex with the microtubule binding domain of MAP4 (MTB-MAP4) in the presence and absence of Taxol. The changes in the HDX levels indicate that MTB-MAP4 may bind to both the outside and the luminal surfaces of the MTs, and that Taxol reduces both of these interactions. The MTB-MAP4 binding induces conformational rearrangements of α- and β-tubulin that promote an overall stabilization of MTs. Paradoxically, despite Taxol’s negative effects on MAP4 interactions with the MTs, its binding to the MTB-MAP4-MT complex further reduces the overall deuterium incorporation, suggesting that a more stable complex is formed in the presence of the drug. PMID:22270553

  14. Thalidomide (5HPP-33) suppresses microtubule dynamics and depolymerizes the microtubule network by binding at the vinblastine binding site on tubulin.

    PubMed

    Rashid, Aijaz; Kuppa, Annapurna; Kunwar, Ambarish; Panda, Dulal

    2015-03-31

    Thalidomides were initially thought to be broad-range drugs specifically for curing insomnia and relieving morning sickness in pregnant women. However, its use was discontinued because of a major drawback of causing teratogenicity. In this study, we found that a thalidomide derivative, 5-hydroxy-2-(2,6-diisopropylphenyl)-1H-isoindole-1,3-dione (5HPP-33), inhibited the proliferation of MCF-7 with a half-maximal inhibitory concentration of 4.5 ± 0.4 μM. 5HPP-33 depolymerized microtubules and inhibited the reassembly of cold-depolymerized microtubules in MCF-7 cells. Using time-lapse imaging, the effect of 5HPP-33 on the dynamics of individual microtubules in live MCF-7 cells was analyzed. 5HPP-33 (5 μM) decreased the rates of growth and shortening excursions by 34 and 33%, respectively, and increased the time microtubules spent in the pause state by 92% as compared to that of the vehicle-treated MCF-7 cells. 5HPP-33 (5 μM) reduced the dynamicity of microtubules by 62% compared to the control. 5HPP-33 treatment reduced the distance between the two poles of a bipolar spindle, induced multipolarity in some of the treated cells, and blocked cells at mitosis. In vitro, 5HPP-33 bound to tubulin with a weak affinity. Vinblastine inhibited the binding of 5HPP-33 to tubulin, and 5HPP-33 inhibited the binding of BODIPY FL-vinblastine to tubulin. Further, a molecular docking analysis suggested that 5HPP-33 shares its binding site on tubulin with vinblastine. The results provided significant insight into the antimitotic mechanism of action of 5HPP-33 and also suggest a possible mechanism for the teratogenicity of thalidomides.

  15. 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. PMID:26774598

  16. The Kinesin KIF1C and Microtubule Plus Ends Regulate Podosome Dynamics in Macrophages

    PubMed Central

    Kopp, Petra; Lammers, Reiner; Aepfelbacher, Martin; Woehlke, Günther; Rudel, Thomas; Machuy, Nikolaus; Steffen, Walter

    2006-01-01

    Microtubules are important for the turnover of podosomes, dynamic, actin-rich adhesions implicated in migration and invasion of monocytic cells. The molecular basis for this functional dependency, however, remained unclear. Here, we show that contact by microtubule plus ends critically influences the cellular fate of podosomes in primary human macrophages. In particular, we identify the kinesin KIF1C, a member of the Kinesin-3 family, as a plus-end–enriched motor that targets regions of podosome turnover. Expression of mutation constructs or small interfering RNA-/short hairpin RNA-based depletion of KIF1C resulted in decreased podosome dynamics and ultimately in podosome deficiency. Importantly, protein interaction studies showed that KIF1C binds to nonmuscle myosin IIA via its PTPD-binding domain, thus providing an interface between the actin and tubulin cytoskeletons, which may facilitate the subcellular targeting of podosomes by microtubules. This is the first report to implicate a kinesin in podosome regulation and also the first to describe a function for KIF1C in human cells. PMID:16554367

  17. ADP ribosylation factor like 2 (Arl2) protein influences microtubule dynamics in breast cancer cells

    SciTech Connect

    Beghin, Anne . E-mail: anne.beghin@recherche.univ-lyon1.fr; Honore, Stephane; Messana, Celine; Matera, Eva-Laure; Aim, Jennifer; Burlinchon, Sandrine; Braguer, Diane; Dumontet, Charles

    2007-02-01

    ADP ribosylation factor like 2 (Arl2) protein is involved in the folding of tubulin peptides. Variants of the human adenocarcinoma line MCF7 cells with increased or reduced content of Arl2 protein were produced and characterized. Western blot analysis performed after separation of the different fractions of tubulins showed that the content in polymerizable soluble heterodimers was significantly increased in cells with the highest Arl2 expression level (MA+) and reduced in cells with the lowest Arl2 expression level (MA-) in comparison to control cells (MP). Microtubule dynamic instability, measured after microinjection of rhodamine-labelled tubulin in living cells, was significantly enhanced in MA+ cells and reduced in MA- cells. These alterations involved modifications of the microtubule growth and shortening rates, duration of attenuation phases, percentage of time spent in each phase (growth, shortening and attenuation) and catastrophe frequency. We also observed modifications in the expression level of the tumor suppressor protein phosphatase 2Ac, which has been shown to form a complex with Arl2. Finally, cell cycle progression was modified in these cells, particularly in regard to duration of telophase. In summary, alterations in Arl2 protein content were found to be associated with modifications in tubulin pools, microtubule dynamics as well as cell cycle progression.

  18. Mathematical modeling of the intracellular protein dynamics: the importance of active transport along microtubules.

    PubMed

    Szymańska, Zuzanna; Parisot, Martin; Lachowicz, Mirosław

    2014-12-21

    In this paper we propose a mathematical model of protein and mRNA transport inside a cell. The spatio-temporal model takes into account the active transport along microtubules in the cytoplasm as well as diffusion and is able to reproduce the oscillatory changes in protein concentration observed in many experimental data. In the model the protein and the mRNA interact with each other that allows us to classify the model as a simple gene regulatory network. The proposed model is generic and may be adapted to specific signaling pathways. On the basis of numerical simulations, we formulate a new hypothesis that the oscillatory dynamics is allowed by the mRNA active transport along microtubules from the nucleus to distant locations.

  19. 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.

  20. 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

  1. A model for the regulatory network controlling the dynamics of kinetochore microtubule plus-ends and poleward flux in metaphase

    PubMed Central

    Fernandez, Nicolas; Chang, Qiang; Buster, Daniel W.; Sharp, David J.; Ma, Ao

    2009-01-01

    Tight regulation of kinetochore microtubule dynamics is required to generate the appropriate position and movement of chromosomes on the mitotic spindle. A widely studied but mysterious aspect of this regulation occurs during metaphase when polymerization of kinetochore microtubule plus-ends is balanced by depolymerization at their minus-ends. Thus, kinetochore microtubules maintain a constant net length, allowing chromosomes to persist at the spindle equator, but consist of tubulin subunits that continually flux toward spindle poles. Here, we construct a feasible network of regulatory proteins for controlling kinetochore microtubule plus-end dynamics, which was combined with a Monte Carlo algorithm to simulate metaphase tubulin flux. We also test the network model by combining it with a force-balancing model explicitly taking force generators into account. Our data reveal how relatively simple interrelationships among proteins that stimulate microtubule plus-end polymerization, depolymerization, and dynamicity can induce robust flux while accurately predicting apparently contradictory results of knockdown experiments. The model also provides a simple and robust physical mechanism through which the regulatory networks at kinetochore microtubule plus- and minus-ends could communicate. PMID:19416899

  2. Two protein 4.1 domains essential for mitotic spindle and aster microtubule dynamics and organization in vitro.

    PubMed

    Krauss, Sharon Wald; Lee, Gloria; Chasis, Joel Anne; Mohandas, Narla; Heald, Rebecca

    2004-06-25

    Multifunctional structural proteins belonging to the 4.1 family are components of nuclei, spindles, and centrosomes in vertebrate cells. Here we report that 4.1 is critical for spindle assembly and the formation of centrosome-nucleated and motor-dependent self-organized microtubule asters in metaphase-arrested Xenopus egg extracts. Immunodepletion of 4.1 disrupted microtubule arrays and mislocalized the spindle pole protein NuMA. Remarkably, assembly was completely rescued by supplementation with a recombinant 4.1R isoform. We identified two 4.1 domains critical for its function in microtubule polymerization and organization utilizing dominant negative peptides. The 4.1 spectrin-actin binding domain or NuMA binding C-terminal domain peptides caused morphologically disorganized structures. Control peptides with low homology or variant spectrin-actin binding domain peptides that were incapable of binding actin had no deleterious effects. Unexpectedly, the addition of C-terminal domain peptides with reduced NuMA binding caused severe microtubule destabilization in extracts, dramatically inhibiting aster and spindle assembly and also depolymerizing preformed structures. However, the mutant C-terminal peptides did not directly inhibit or destabilize microtubule polymerization from pure tubulin in a microtubule pelleting assay. Our data showing that 4.1 is a crucial factor for assembly and maintenance of mitotic spindles and self-organized and centrosome-nucleated microtubule asters indicates that 4.1 is involved in regulating both microtubule dynamics and organization. These investigations underscore an important functional context for protein 4.1 in microtubule morphogenesis and highlight a previously unappreciated role for 4.1 in cell division.

  3. 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

  4. Suppression of microtubule dynamics by discodermolide by a novel mechanism is associated with mitotic arrest and inhibition of tumor cell proliferation.

    PubMed

    Honore, Stéphane; Kamath, Kathy; Braguer, Diane; Wilson, Leslie; Briand, Claudette; Jordan, Mary Ann

    2003-12-01

    Discodermolide is a new microtubule-targeted drug in Phase I clinical trials that inhibits tumor growth and induces G(2)-M cell cycle arrest. It is effective against paclitaxel-resistant cell lines and acts synergistically in combination with paclitaxel. Suppression of microtubule dynamics by microtubule-targeted drugs has been hypothesized to be responsible for their ability to inhibit mitotic progression and cell proliferation. To determine whether discodermolide blocks mitosis by an effect on microtubule dynamics, we analyzed the effects of discodermolide on microtubule dynamics in living A549 human lung cancer cells during interphase at concentrations that block mitosis and inhibit cell proliferation. We found that discodermolide (7-166 nM) significantly suppressed microtubule dynamic instability. At the IC(50) for proliferation (7 nM discodermolide, 72 h), overall dynamicity was reduced by 23%. The principal parameters of dynamic instability suppressed by discodermolide were the microtubule shortening rate and length shortened. In addition, discodermolide markedly increased the frequency of rescued catastrophes. At the discodermolide concentration that resulted in 50% of maximal mitotic block (83 nM, 20 h), most microtubules were completely non-dynamic, no anaphases occurred, and all spindles were abnormal. The dynamicity of the remaining dynamic microtubules was reduced by 62%. The results indicate that a principal mechanism of inhibition of cell proliferation and mitotic block by discodermolide is suppression of microtubule dynamics. Importantly, the results indicate significant additional stabilizing effects of discodermolide on microtubule dynamics as compared with those of paclitaxel that may in turn reflect differences in their binding sites and their effects on tubulin conformation.

  5. 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.

  6. The linear and rotational motions of the fission yeast nucleus are governed by the stochastic dynamics of spatially distributed microtubules.

    PubMed

    Hui, Tsz Hin; Zheng, Fan; Lin, Yuan; Fu, Chuanhai

    2016-05-01

    Dynamic nuclei are involved in a wide variety of fundamental biological processes including cell migration, cell division and fertilization. Here, we develop a mathematical model, in combination with live-cell imaging at high temporal resolution, to quantitatively elucidate how the linear and rotational motions of the nucleus are governed by the stochastic dynamics of the microtubule cytoskeleton. Our simulation and experimental results demonstrate that microtubule rescue and catastrophe frequencies are the decisive factors in regulating the nuclear movement. Lower rescue and catastrophe frequencies can lead to significantly larger angular and translational oscillations of the nucleus. In addition, our model also suggests that the stochastic dynamics of individual spatially distributed microtubules works collectively as a restoring force to maintain nuclear centering and hence ensures symmetric cell division, in excellent agreement with direct experimental observations.

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

    PubMed Central

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

    2014-01-01

    Hox genes are classically ascribed to function in patterning the anterior-posterior axis of bilaterian animals; however, their role in directing molecular mechanisms underlying morphogenesis at the cellular level remains largely unstudied. We unveil a non-classical role for the zebrafish hoxb1b gene, which shares ancestral functions with mammalian Hoxa1, in controlling progenitor cell shape and oriented cell division during zebrafish anterior hindbrain neural tube morphogenesis. This is likely distinct from its role in cell fate acquisition and segment boundary formation. We show that, without affecting major components of apico-basal or planar cell polarity, Hoxb1b regulates mitotic spindle rotation during the oriented neural keel symmetric mitoses that are required for normal neural tube lumen formation in the zebrafish. This function correlates with a non-cell-autonomous requirement for Hoxb1b in regulating microtubule plus-end dynamics in progenitor cells in interphase. We propose that Hox genes can influence global tissue morphogenesis by control of microtubule dynamics in individual cells in vivo. PMID:24449840

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

    PubMed

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

    2014-02-01

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

  9. The complex dynamic network of microtubule and microfilament cytasters of the leech zygote.

    PubMed

    Cantillana, V; Urrutia, M; Ubilla, A; Fernández, J

    2000-12-01

    The organization of the cytoskeleton in the early first interphase zygote and its involvement in organelle redistribution were studied in the glossiphoniid leech Theromyzon trizonare by confocal and electron microscopy, immunofluorescence, and time-lapse video imaging after microinjection of labeled tubulin and/or actin and loading with a mitotracker. The cytoskeleton consists of an inner or endoplasmic and an outer or ectoplasmic domain. The inner domain consists of a monaster whose fibers retract from the zygote periphery by the end of the early first interphase. The outer domain is built upon a network of microtubules and microfilaments cytasters. Short pulses of microinjected labeled actin or tubulin and Taxol treatment demonstrate that cytasters are centers of microtubule and microfilament nucleation. Immunostaining with anti-centrophilin, anti-BX-63, and anti-AH-6 indicates that the network of cytasters includes centrosomal antigens. Cytasters move in an orderly fashion at speeds of 0.5-2 micrometer/min, in an energy-dependent process retarded and finally blocked by the ATP analogue AMP-PNP and high concentrations of Taxol. Colliding cytasters fuse and form larger cytoskeletal nucleation centers. The leech zygote is a highly compartmentalized cell whose cytasters function as articulated components of a very dynamic cytoskeletal system engaged in bulk transportation of organelles during ooplasmic segregation. PMID:11087633

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

    PubMed

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

    2016-08-16

    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

  11. Kinetic stabilization of microtubule dynamics by indanocine perturbs EB1 localization, induces defects in cell polarity and inhibits migration of MDA-MB-231 cells.

    PubMed

    Kapoor, Sonia; Panda, Dulal

    2012-06-01

    Cell motility is an essential aspect of metastatic spread of cancer. Microtubule-targeted agents exhibit anti-metastatic properties, the underlying mechanism of which remains understudied. In this study, we have investigated the role of microtubule dynamics in migration of cancer cells using indanocine, a synthetic small molecule inhibitor of tubulin. We found that indanocine, at concentrations that did not visibly affect microtubule organization, suppressed dynamic instability of microtubules and reduced the rate of migration of highly metastatic MDA-MB-231 cells. Indanocine-treated cells were defective in lamellipodium formation and could not develop polarized morphology. The kinetic stabilization of microtubules was associated with a marked increase in their acetylation level and a perturbation in the localization of EB1, a microtubule plus end binding protein. Using standard scratch wound healing assay and immunofluorescence analysis; we found that microtubule acetylation occurred in the direction of migration in vehicle-treated cells, whereas indanocine treatment led to a global acetylation of microtubules. The results together suggested that selective stabilization of microtubules was perturbed in the presence of indanocine that possibly resulted in lack of cell polarization and a concurrent reduction in migration of cells. Moreover, microtubule stabilization by indanocine affected adhesion turnover and impaired the polarized pattern of adhesion sites in cells. Together the results indicated that the regulation of microtubule dynamics is required to coordinate cell polarization as well as adhesion asymmetry and support the hypothesis that the perturbation of microtubule dynamics by tubulin-targeted agents can be exploited to restrict the migration of tumor cells.

  12. Dissecting EB1-microtubule interactions from every direction: using single-molecule visualization and static and dynamic binding measurements

    NASA Astrophysics Data System (ADS)

    Lopez, Benjamin

    2015-03-01

    EB1 is an important microtubule associating protein (MAP) that acts as a master coordinator of protein activity at the growing plus-end of the microtubule. We can recapitulate the plus-end binding behavior of EB1 along the entire length of a static microtubule using microtubules polymerized in the presence of the nonhydrolyzable GTP analogs GMPCPP and GTP γS instead of GTP. Through the use of single-molecule TIRF imaging we find that EB1 is highly dynamic (with a sub-second characteristic binding lifetime) and continuously diffusive while bound to the microtubule. We measure the diffusion coefficient, D, through linear fitting to mean-squared displacement of individually labeled proteins, and the binding lifetime, τ, by fitting a single exponential decay to the probability distribution of trajectory lifetimes. In agreement with measurements of other diffusive MAPs, we find that D increases and τ decreases with increasing ionic strength. We also find that D is sensitive to the choice of GTP analog: EB1 proteins bound to GTP γS polymerized microtubules have a D half of that found with GMPCPP polymerized microtubules. To compare these single-molecule measurements to the bulk binding behavior of EB1, we use TIRF imaging to measure the intensity of microtubules coated with EB1-GFP as a function of EB1 concentration. We find that EB1 binding is cooperative and both the quantity of EB1 bound and the dissociation constant are sensitive to GTP analog and ionic concentration. The correlation between binding affinity and D and the cooperative nature of EB1-microtubule binding leads to a decrease in D with increasing EB1 concentration. Interestingly, we also find an increase in τ at high EB1 concentrations, consistent with attractive EB1-microtubule interactions driving the cooperativity. To further understand the nature of the cooperativity we estimate the interaction energy by measuring the association and dissociation rates (kon and koff respectively) at different

  13. Analysis of dynamic instability of steady-state microtubules in vitro by video-enhanced differential interference contrast microscopy with an appendix by Emin Oroudjev.

    PubMed

    Yenjerla, Mythili; Lopus, Manu; Wilson, Leslie

    2010-01-01

    Microtubules are major constituents of the cytoskeleton which display dynamic properties. They exhibit dynamic instability which is defined as the stochastic switching between growing and shortening at microtubule ends. Dynamic instability plays an important role in diverse cellular functions including cell migration and mitosis. Many successful antimitotic drugs and microtubule-associated proteins (MAPs) are known to modulate microtubule dynamics, and it is important to analyze the in vitro dynamic instability of microtubules to study the mechanism of action of microtubule-targeted therapeutics and MAPs. In this chapter, we describe a method to analyze the in vitro dynamic instability of microtubules at steady state using video-enhanced differential contrast (VE-DIC) microscopy in detail. In this method, microtubules are assembled to steady state at 30 degrees C with MAP-free tubulin in a slide chamber in the presence of GTP, using sea urchin axonemes as nucleating seeds. Images of microtubules are enhanced and recorded in real time by a video camera and an image processor connected to a DIC microscope which is maintained at 30 degrees C. We use two software programs to track and analyze the growing and shortening of plus or minus ends of microtubules in the real-time images recorded using VE-DIC. In this chapter, we describe the instructions to use the tracking software Real Time Measurement II (RTM II) program. The instructions to use the analysis software Microtubule Life History Analysis Procedures (MT-LHAP) in Igor Pro software have been described in detail in an appendix (Oroudjev, 2010) following this chapter.

  14. 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

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

    NASA Astrophysics Data System (ADS)

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

    2015-06-01

    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.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

  16. Septin GTPases spatially guide microtubule organization and plus end dynamics in polarizing epithelia

    PubMed Central

    Bowen, Jonathan R.; Hwang, Daniel; Bai, Xiaobo; Roy, Dheeraj

    2011-01-01

    Establishment of epithelial polarity requires the reorganization of the microtubule (MT) cytoskeleton from a radial array into a network positioned along the apicobasal axis of the cell. Little is known about the mechanisms that spatially guide the remodeling of MTs during epithelial polarization. Septins are filamentous guanine triphosphatases (GTPases) that associate with MTs, but the function of septins in MT organization and dynamics is poorly understood. In this paper, we show that in polarizing epithelia, septins guide the directionality of MT plus end movement by suppressing MT catastrophe. By enabling persistent MT growth, two spatially distinct populations of septins, perinuclear and peripheral filaments, steer the growth and capture of MT plus ends. This navigation mechanism is essential for the maintenance of perinuclear MT bundles and for the orientation of peripheral MTs as well as for the apicobasal positioning of MTs. Our results suggest that septins provide the directional guidance cues necessary for polarizing the epithelial MT network. PMID:21788367

  17. 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

  18. Using plusTipTracker software to measure microtubule dynamics in Xenopus laevis growth cones.

    PubMed

    Stout, Alina; D'Amico, Salvatore; Enzenbacher, Tiffany; Ebbert, Patrick; Lowery, Laura Anne

    2014-01-01

    Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics(1,2). One of the most well-known and widely-utilized +TIPs for analyzing MT dynamics is the End-Binding protein, EB1, which binds all growing MT plus-ends, and thus, is a marker for MT polymerization(1). Many studies of EB1 behavior within growth cones have used time-consuming and biased computer-assisted, hand-tracking methods to analyze individual MTs(1-3). Our approach is to quantify global parameters of MT dynamics using the software package, plusTipTracker(4), following the acquisition of high-resolution, live images of tagged EB1 in cultured embryonic growth cones(5). This software is a MATLAB-based, open-source, user-friendly package that combines automated detection, tracking, visualization, and analysis for movies of fluorescently-labeled +TIPs. Here, we present the protocol for using plusTipTracker for the analysis of fluorescently-labeled +TIP comets in cultured Xenopus laevis growth cones. However, this software can also be used to characterize MT dynamics in various cell types(6-8). PMID:25225829

  19. The leading role of microtubules in endothelial barrier dysfunction: disassembly of peripheral microtubules leaves behind the cytoskeletal reorganization.

    PubMed

    Alieva, Irina B; Zemskov, Evgeny A; Smurova, Ksenija M; Kaverina, Irina N; Verin, Alexander D

    2013-10-01

    Disturbance of the endothelial barrier is characterized by dramatic cytoskeleton reorganization, activation of actomyosin contraction and, finally, leads to intercellular gap formation. Here we demonstrate that the edemagenic agent, thrombin, causes a rapid increase in the human pulmonary artery endothelial cell (EC) barrier permeability accompanied by fast decreasing in the peripheral microtubules quantity and reorganization of the microtubule system in the internal cytoplasm of the EC within 5 min of the treatment. The actin stress-fibers formation occurs gradually and the maximal effect is observed relatively later, 30 min of the thrombin treatment. Thus, microtubules reaction develops faster than the reorganization of the actin filaments system responsible for the subsequent changes of the cell shape during barrier dysfunction development. Direct microtubules depolymerization by nocodazole initiates the cascade of barrier dysfunction reactions. Nocodazole-induced barrier disruption is connected directly with the degree of peripheral microtubules depolymerization. Short-term loss of endothelial barrier function occurs at the minimal destruction of peripheral microtubules, when actin filament system is still intact. Specifically, we demonstrate that the EC microtubule dynamics examined by time-lapse imaging of EB3-GFP comets movement has changed under these conditions: microtubule plus ends growth rate significantly decreased near the cell periphery. The microtubules, apparently, are the first target in the circuit of reactions leading to the pulmonary EC barrier compromise. Our results show that dynamic microtubules play an essential role in the barrier function in vitro; peripheral microtubules depolymerization is necessary and sufficient condition for initiation of endothelial barrier dysfunction. PMID:23606375

  20. Formin-Dependent Synaptic Growth; Evidence that Dlar Signals via Diaphanous to Modulate Synaptic Actin and Dynamic Pioneer Microtubules

    PubMed Central

    Pawson, Catherine; Eaton, Benjamin A.; Davis, Graeme W.

    2008-01-01

    The diaphanous gene is the founding member of a family of Diaphanous Related Formin proteins (DRF). We identified diaphanous in a screen for genes that are necessary for the normal growth and stabilization of the Drosophila neuromuscular junction (NMJ). Here we demonstrate that diaphanous mutations perturb synaptic growth at the NMJ. Diaphanous protein is present both pre- and postsynaptically. However, genetic rescue experiments in combination with additional genetic interaction experiments support the conclusion that dia is necessary presynaptically for normal NMJ growth. We then document defects in both the actin and microtubule cytoskeletons in dia mutant nerve terminals. In so doing, we define and characterize a population of dynamic pioneer microtubules within the NMJ that are distinct from the bundled core of microtubules identified by the MAP1b-like protein Futsch. Defects in both synaptic actin and dynamic pioneer MTs are correlated with impaired synaptic growth in dia mutants. Finally, we present genetic evidence that Dia functions downstream of the presynaptic receptor tyrosine phosphatase Dlar and the Rho-type GEF trio to control NMJ growth. Based upon the established function of DRFs as Rho-GTPase dependent regulators of the cell cytoskeleton, we propose a model in which Diaphanous links receptor tyrosine phosphatase signaling at the plasma membrane to growth-dependent modulation of the synaptic actin and microtubule cytoskeletons. PMID:18971454

  1. Dynamic formation of a microchannel array enabling kinesin-driven microtubule transport between separate compartments on a chip.

    PubMed

    Fujimoto, Kazuya; Nagai, Moeto; Shintaku, Hirofumi; Kotera, Hidetoshi; Yokokawa, Ryuji

    2015-05-01

    Microtubules driven by kinesin motors have been utilised as "molecular shuttles" in microfluidic environments with potential applications in autonomous nanoscale manipulations such as capturing, separating, and/or concentrating biomolecules. However, the conventional flow cell-based assay has difficulty in separating bound target molecules from free ones even with buffer flushing because molecular manipulations by molecular shuttles take place on a glass surface and molecular binding occurs stochastically; this makes it difficult to determine whether molecules are carried by molecular shuttles or by diffusion. To address this issue, we developed a microtubule-based transport system between two compartments connected by a single-micrometre-scale channel array that forms dynamically via pneumatic actuation of a polydimethylsiloxane membrane. The device comprises three layers-a control channel layer (top), a microfluidic channel layer (middle), and a channel array layer (bottom)-that enable selective injection of assay solutions into a target compartment and dynamic formation of the microchannel array. The pneumatic channel also serves as a nitrogen supply path to the assay area, which reduces photobleaching of fluorescently labelled microtubules and deactivation of kinesin by oxygen radicals. The channel array suppresses cross-contamination of molecules caused by diffusion or pressure-driven flow between compartments, facilitating unidirectional transport of molecular shuttles from one compartment to another. The method demonstrates, for the first time, efficient and unidirectional microtubule transport by eliminating diffusion of target molecules on a chip and thus may constitute one of the key aspects of motor-driven nanosystems.

  2. Dynamic formation of a microchannel array enabling kinesin-driven microtubule transport between separate compartments on a chip.

    PubMed

    Fujimoto, Kazuya; Nagai, Moeto; Shintaku, Hirofumi; Kotera, Hidetoshi; Yokokawa, Ryuji

    2015-05-01

    Microtubules driven by kinesin motors have been utilised as "molecular shuttles" in microfluidic environments with potential applications in autonomous nanoscale manipulations such as capturing, separating, and/or concentrating biomolecules. However, the conventional flow cell-based assay has difficulty in separating bound target molecules from free ones even with buffer flushing because molecular manipulations by molecular shuttles take place on a glass surface and molecular binding occurs stochastically; this makes it difficult to determine whether molecules are carried by molecular shuttles or by diffusion. To address this issue, we developed a microtubule-based transport system between two compartments connected by a single-micrometre-scale channel array that forms dynamically via pneumatic actuation of a polydimethylsiloxane membrane. The device comprises three layers-a control channel layer (top), a microfluidic channel layer (middle), and a channel array layer (bottom)-that enable selective injection of assay solutions into a target compartment and dynamic formation of the microchannel array. The pneumatic channel also serves as a nitrogen supply path to the assay area, which reduces photobleaching of fluorescently labelled microtubules and deactivation of kinesin by oxygen radicals. The channel array suppresses cross-contamination of molecules caused by diffusion or pressure-driven flow between compartments, facilitating unidirectional transport of molecular shuttles from one compartment to another. The method demonstrates, for the first time, efficient and unidirectional microtubule transport by eliminating diffusion of target molecules on a chip and thus may constitute one of the key aspects of motor-driven nanosystems. PMID:25805147

  3. 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

    1999-03-01

    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

  4. 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

  5. Microtubule Dynamic Instability Controls Podosome Patterning in Osteoclasts through EB1, Cortactin, and Src

    PubMed Central

    Biosse Duplan, Martin; Zalli, Detina; Stephens, Sebastien; Zenger, Serhan; Neff, Lynn; Oelkers, J. Margit; Lai, Frank P. L.; Horne, William; Rottner, Klemens

    2014-01-01

    In osteoclasts (OCs) podosomes are organized in a belt, a feature critical for bone resorption. Although microtubules (MTs) promote the formation and stability of the belt, the MT and/or podosome molecules that mediate the interaction of the two systems are not identified. Because the growing “plus” ends of MTs point toward the podosome belt, plus-end tracking proteins (+TIPs) might regulate podosome patterning. Among the +TIPs, EB1 increased as OCs matured and was enriched in the podosome belt, and EB1-positive MTs targeted podosomes. Suppression of MT dynamic instability, displacement of EB1 from MT ends, or EB1 depletion resulted in the loss of the podosome belt. We identified cortactin as an Src-dependent interacting partner of EB1. Cortactin-deficient OCs presented a defective MT targeting to, and patterning of, podosomes and reduced bone resorption. Suppression of MT dynamic instability or EB1 depletion increased cortactin phosphorylation, decreasing its acetylation and affecting its interaction with EB1. Thus, dynamic MTs and podosomes interact to control bone resorption. PMID:24144981

  6. Effects of eribulin on microtubule binding and dynamic instability are strengthened in the absence of the βIII tubulin isotype.

    PubMed

    Wilson, Leslie; Lopus, Manu; Miller, Herbert P; Azarenko, Olga; Riffle, Stephen; Smith, Jennifer A; Jordan, Mary Ann

    2015-10-27

    Eribulin mesylate (Halaven) is a microtubule-targeted anticancer drug used to treat patients with metastatic breast cancer who have previously received a taxane and an anthracycline. It binds at the plus ends of microtubules and has been shown to suppress plus end growth selectively. Because the class III β tubulin isotype is associated with resistance to microtubule targeting drugs, we sought to determine how βIII tubulin might mechanistically influence the effects of eribulin on microtubules. We found that while [(3)H]eribulin bound to bovine brain soluble tubulin depleted of βIII tubulin in a manner similar to that of unfractionated tubulin, it bound to plus ends of microtubules that were depleted of βIII-depleted tubulin with a maximal stoichiometry (20 ± 3 molecules per microtubule) higher than that of unfractionated microtubules (9 ± 2 molecules per microtubule). In addition, eribulin suppressed the dynamic instability behavior of βIII-depleted microtubules more strongly than and in a manner different from that of microtubules containing βIII tubulin. Specifically, with βIII tubulin present in the microtubules, 100 nM eribulin suppressed the growth rate by 32% and marginally reduced the catastrophe frequency (by 17%) but did not modulate the rescue frequency. However, in the absence of βIII tubulin, eribulin not only reduced the growth rate but also strongly reduced the shortening rate (by 43%) and the catastrophe and the rescue frequencies (by 49 and 32%, respectively). Thus, when present in microtubules, βIII tubulin substantially weakens the effects of eribulin.

  7. Fast internal dynamics in alcohol dehydrogenase.

    PubMed

    Monkenbusch, M; Stadler, A; Biehl, R; Ollivier, J; Zamponi, M; Richter, D

    2015-08-21

    Large-scale domain motions in alcohol dehydrogenase (ADH) have been observed previously by neutron spin-echo spectroscopy (NSE). We have extended the investigation on the dynamics of ADH in solution by using high-resolution neutron time-of-flight (TOF) and neutron backscattering (BS) spectroscopy in the incoherent scattering range. The observed hydrogen dynamics were interpreted in terms of three mobility classes, which allowed a simultaneous description of the measured TOF and BS spectra. In addition to the slow global protein diffusion and domain motions observed by NSE, a fast internal process could be identified. Around one third of the protons in ADH participate in the fast localized diffusive motion. The diffusion coefficient of the fast internal motions is around two third of the value of the surrounding D2O solvent. It is tempting to associate the fast internal process with solvent exposed amino acid residues with dangling side chains. PMID:26298156

  8. Fast internal dynamics in alcohol dehydrogenase

    SciTech Connect

    Monkenbusch, M.; Stadler, A. Biehl, R.; Richter, D.; Ollivier, J.; Zamponi, M.

    2015-08-21

    Large-scale domain motions in alcohol dehydrogenase (ADH) have been observed previously by neutron spin-echo spectroscopy (NSE). We have extended the investigation on the dynamics of ADH in solution by using high-resolution neutron time-of-flight (TOF) and neutron backscattering (BS) spectroscopy in the incoherent scattering range. The observed hydrogen dynamics were interpreted in terms of three mobility classes, which allowed a simultaneous description of the measured TOF and BS spectra. In addition to the slow global protein diffusion and domain motions observed by NSE, a fast internal process could be identified. Around one third of the protons in ADH participate in the fast localized diffusive motion. The diffusion coefficient of the fast internal motions is around two third of the value of the surrounding D{sub 2}O solvent. It is tempting to associate the fast internal process with solvent exposed amino acid residues with dangling side chains.

  9. Quantitative image analysis identifies pVHL as a key regulator of microtubule dynamic instability.

    PubMed

    Thoma, Claudio R; Matov, Alexandre; Gutbrodt, Katrin L; Hoerner, Christian R; Smole, Zlatko; Krek, Wilhelm; Danuser, Gaudenz

    2010-09-20

    Von Hippel-Lindau (VHL) tumor suppressor gene mutations predispose carriers to kidney cancer. The protein pVHL has been shown to interact with microtubules (MTs), which is critical to cilia maintenance and mitotic spindle orientation. However, the function for pVHL in the regulation of MT dynamics is unknown. We tracked MT growth via the plus end marker EB3 (end-binding protein 3)-GFP and inferred additional parameters of MT dynamics indirectly by spatiotemporal grouping of growth tracks from live cell imaging. Our data establish pVHL as a near-optimal MT-stabilizing protein: it attenuates tubulin turnover, both during MT growth and shrinkage, inhibits catastrophe, and enhances rescue frequencies. These functions are mediated, in part, by inhibition of tubulin guanosine triphosphatase activity in vitro and at MT plus ends and along the MT lattice in vivo. Mutants connected to the VHL cancer syndrome are differentially compromised in these activities. Thus, single cell-level analysis of pVHL MT regulatory function allows new predictions for genotype to phenotype associations that deviate from the coarser clinically defined mutant classifications. PMID:20855504

  10. 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

  11. 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.

  12. Mutations in a β-Tubulin Disrupt Spindle Orientation and Microtubule Dynamics in the Early Caenorhabditis elegans EmbryoV⃞

    PubMed Central

    Wright, Amanda J.; Hunter, Craig P.

    2003-01-01

    The early Caenorhabditis elegans embryo contains abundant transcripts for two α- and two β-tubulins, raising the question of whether each isoform performs specialized functions or simply contributes to total tubulin levels. Our identification of two recessive, complementing alleles of a β-tubulin that disrupt nuclear-centrosome centration and rotation in the early embryo originally suggested that this tubulin, tbb-2, has specialized functions. However, embryos from tbb-2 deletion worms do not have defects in nuclear-centrosome centration and rotation suggesting that the complementing alleles are not null mutations. Both complementing alleles have distinct effects on microtubule dynamics and show allele-specific interactions with the two embryonically expressed α-tubulins: One of the alleles causes microtubules to be cold stable and resistant to the microtubule-depolymerizing drug benomyl, whereas the other causes cell cycle-specific defects in microtubule polymerization. Gene-specific RNA interference targeting all four embryonically expressed tubulin genes singly and in all double combinations showed that the tubulin isoforms in the early embryo are largely functionally redundant with the exception of tbb-2. tbb-2 is required for centrosome stabilization during anaphase of the first cell division, suggesting that tbb-2 may be specialized for interactions with the cell cortex. PMID:12937270

  13. EB1-recruited microtubule +TIP complexes coordinate protrusion dynamics during 3D epithelial remodeling

    PubMed Central

    Gierke, Sarah; Wittmann, Torsten

    2012-01-01

    SUMMARY Background Epithelial remodeling, in which apical-basal polarized cells switch to a migratory phenotype, plays a central role in development and disease of multicellular organisms. Although dynamic microtubules (MTs) are required for directed migration on flat surfaces, how MT dynamics are controlled or contribute to epithelial remodeling in a more physiological three-dimensional (3D) environment is not understood. We use confocal live cell imaging to analyze MT function and dynamics during 3D epithelial morphogenesis and remodeling of polarized Madin-Darby canine kidney (MDCK) epithelial cells that undergo partial epithelial-to-mesenchymal transition (EMT) in response to hepatocyte growth factor (HGF). Results We find that HGF treatment increases MT growth rate before morphological changes are evident, and that large numbers of MTs grow into HGF-induced cell extensions independent of centrosome reorientation. Using lentivirus-mediated shRNA, we demonstrate that EB1, an adaptor protein that mediates recruitment of numerous other +TIP proteins to growing MT plus ends, is required for this HGF-induced MT reorganization. We further show that protrusion and adhesion dynamics are disorganized, and that vesicular trafficking to the tip of HGF-induced cell extensions is disrupted in EB1-depleted cells. Conclusions We conclude that EB1-mediated interactions with growing MTs are important to coordinate cell shape changes and directed migration into the surrounding extracellular matrix during epithelial remodeling in a physiological 3D environment. In contrast, EB1 is not required for the establishment or maintenance of apical-basal cell polarity, suggesting different functions of +TIPs and MTs in different types of cell polarity. PMID:22483942

  14. Fast bubble dynamics and sizing

    NASA Astrophysics Data System (ADS)

    Czarnecki, Krzysztof; Fouan, Damien; Achaoui, Younes; Mensah, Serge

    2015-11-01

    Single bubble sizing is usually performed by measuring the resonant bubble response using the Dual Frequency Ultrasound Method. However, in practice, the use of millisecond-duration chirp-like waves yields nonlinear distortions of the bubble oscillations. In comparison with the resonant curve obtained under harmonic excitation, it was observed that the bubble dynamic response shifted by up to 20 percent of the resonant frequency with bubble radii of less than 100 μm. In the case of low pressure waves (P < 5 kPa), an approximate formula for the apparent frequency shift is derived. Simulated and experimental bubble responses are analyzed in the time-frequency domain using an enhanced concentrated (reassigned) spectrogram. The difference in the resonant frequency resulted from the persistence of the resonant mode in the bubble response. Numerical simulations in which these findings are extended to pairs of coupled bubbles and to bubble clouds are also presented.

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

    DOE PAGESBeta

    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).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

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

    PubMed

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

    2015-07-01

    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.

  17. 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.

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

    PubMed

    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 Mecp2(308/y) male mice. These findings represent a first step toward the validation of an innovative treatment for RTT.

  19. 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.

  20. Regulation of microtubule dynamics by DIAPH3 influences amoeboid tumor cell mechanics and sensitivity to taxanes

    PubMed Central

    Morley, Samantha; You, Sungyong; Pollan, Sara; Choi, Jiyoung; Zhou, Bo; Hager, Martin H.; Steadman, Kenneth; Spinelli, Cristiana; Rajendran, Kavitha; Gertych, Arkadiusz; Kim, Jayoung; Adam, Rosalyn M.; Yang, Wei; Krishnan, Ramaswamy; Knudsen, Beatrice S.; Di Vizio, Dolores; Freeman, Michael R.

    2015-01-01

    Taxanes are widely employed chemotherapies for patients with metastatic prostate and breast cancer. Here, we show that loss of Diaphanous-related formin-3 (DIAPH3), frequently associated with metastatic breast and prostate cancers, correlates with increased sensitivity to taxanes. DIAPH3 interacted with microtubules (MT), and its loss altered several parameters of MT dynamics as well as decreased polarized force generation, contractility, and response to substrate stiffness. Silencing of DIAPH3 increased the cytotoxic response to taxanes in prostate and breast cancer cell lines. Analysis of drug activity for tubulin-targeted agents in the NCI-60 cell line panel revealed a uniform positive correlation between reduced DIAPH3 expression and drug sensitivity. Low DIAPH3 expression correlated with improved relapse-free survival in breast cancer patients treated with chemotherapeutic regimens containing taxanes. Our results suggest that inhibition of MT stability arising from DIAPH3 downregulation enhances susceptibility to MT poisons, and that the DIAPH3 network potentially reports taxane sensitivity in human tumors. PMID:26179371

  1. Tension-dependent dynamic microtubule model for metaphase and anaphase phenomena

    NASA Astrophysics Data System (ADS)

    Banigan, Edward; Lampson, Michael; Liu, Andrea

    2013-03-01

    During cell division, 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. Pairs later separate as shrinking MTs pull each chromosome toward its respective cell pole. We present a minimal model for these processes. We use the measured tension-dependence of single MT kinetics and extrapolate for compressed MTs. We apply these to a stochastic many MT model, which we solve numerically and with master equations. We find that tension dependence enhances the speed of chromosome pulling by retracting MTs. 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. Unlike other models, our model also captures breathing oscillations. We also explore how various components control chromosome dynamics through MT rate constants alone.

  2. 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.

  3. Dynein interacts with the neural cell adhesion molecule (NCAM180) to tether dynamic microtubules and maintain synaptic density in cortical neurons.

    PubMed

    Perlson, Eran; Hendricks, Adam G; Lazarus, Jacob E; Ben-Yaakov, Keren; Gradus, Tal; Tokito, Mariko; Holzbaur, Erika L F

    2013-09-27

    Cytoplasmic dynein is well characterized as an organelle motor, but dynein also acts to tether and stabilize dynamic microtubule plus-ends in vitro. Here we identify a novel and direct interaction between dynein and the 180-kDa isoform of the neural cell adhesion molecule (NCAM). Optical trapping experiments indicate that dynein bound to beads via the NCAM180 interaction domain can tether projecting microtubule plus-ends. Live cell assays indicate that the NCAM180-dependent recruitment of dynein to the cortex leads to the selective stabilization of microtubules projecting to NCAM180 patches at the cell periphery. The dynein-NCAM180 interaction also enhances cell-cell adhesion in heterologous cell assays. Dynein and NCAM180 co-precipitate from mouse brain extract and from synaptosomal fractions, consistent with an endogenous interaction in neurons. Thus, we examined microtubule dynamics and synaptic density in primary cortical neurons. We find that depletion of NCAM, inhibition of the dynein-NCAM180 interaction, or dampening of microtubule dynamics with low dose nocodazole all result in significantly decreased in synaptic density. Based on these observations, we propose a working model for the role of dynein at the synapse, in which the anchoring of the motor to the cortex via binding to an adhesion molecule mediates the tethering of dynamic microtubule plus-ends to potentiate synaptic stabilization.

  4. 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. PMID:26467050

  5. N-terminus-modified Hec1 suppresses tumour growth by interfering with kinetochore-microtubule dynamics.

    PubMed

    Orticello, M; Fiore, M; Totta, P; Desideri, M; Barisic, M; Passeri, D; Lenzi, J; Rosa, A; Orlandi, A; Maiato, H; Del Bufalo, D; Degrassi, F

    2015-06-01

    Mitotic proteins are attractive targets to develop molecular cancer therapeutics due to the intimate interdependence between cell proliferation and mitosis. In this work, we have explored the therapeutic potential of the kinetochore (KT) protein Hec1 (Highly Expressed in Cancer protein 1) as a molecular target to produce massive chromosome missegregation and cell death in cancer cells. Hec1 is a constituent of the Ndc80 complex, which mediates KT-microtubule (MT) attachments at mitosis and is upregulated in various cancer types. We expressed Hec1 fused with enhanced green fluorescent protein (EGFP) at its N-terminus MT-interaction domain in HeLa cells and showed that expression of this modified Hec1, which localized at KTs, blocked cell proliferation and promoted apoptosis in tumour cells. EGFP-Hec1 was extremely potent in tumour cell killing and more efficient than siRNA-induced Hec1 depletion. In striking contrast, normal cells showed no apparent cell proliferation defects or cell death following EGFP-Hec1 expression. Live-cell imaging demonstrated that cancer cell death was associated with massive chromosome missegregation within multipolar spindles after a prolonged mitotic arrest. Moreover, EGFP-Hec1 expression was found to increase KT-MT attachment stability, providing a molecular explanation for the abnormal spindle architecture and the cytotoxic activity of this modified protein. Consistent with cell culture data, EGFP-Hec1 expression was found to strongly inhibit tumour growth in a mouse xenograft model by disrupting mitosis and inducing multipolar spindles. Taken together, these findings demonstrate that stimulation of massive chromosome segregation defects can be used as an anti-cancer strategy through the activation of mitotic catastrophe after a multipolar mitosis. Importantly, this study represents a clear proof of concept that targeting KT proteins required for proper KT-MT attachment dynamics constitutes a powerful approach in cancer therapy.

  6. Fast dynamic processes of solar radiation

    SciTech Connect

    Tomson, Teolan

    2010-02-15

    This paper studies dynamic processes of fast-alternating solar radiation which are assessed by alternation of clouds. Most attention is devoted to clouds of type Cumulus Humilis, identified through visual recognition and/or a specially constructed automatic sensor. One second sampling period was used. Recorded data series were analyzed with regard to duration of illuminated 'windows' between shadows, their stochastic intervals, fronts and the magnitude of increments of solar irradiance. (author)

  7. Azaindole derivatives are inhibitors of microtubule dynamics, with anti-cancer and anti-angiogenic activities

    PubMed Central

    Prudent, Renaud; Vassal-Stermann, Émilie; Nguyen, Chi-Hung; Mollaret, Marjorie; Viallet, Jean; Desroches-Castan, Agnès; Martinez, Anne; Barette, Caroline; Pillet, Catherine; Valdameri, Glaucio; Soleilhac, Emmanuelle; Di Pietro, Attilio; Feige, Jean-Jacques; Billaud, Marc; Florent, Jean-Claude; Lafanechère, Laurence

    2013-01-01

    Background and Purpose Drugs targeting microtubules are commonly used for cancer treatment. However, the potency of microtubule inhibitors used clinically is limited by the emergence of resistance. We thus designed a strategy to find new cell-permeable microtubule-targeting agents. Experimental Approach Using a cell-based assay designed to probe for microtubule polymerization status, we screened a chemical library and identified two azaindole derivatives, CM01 and CM02, as cell-permeable microtubule-depolymerizing agents. The mechanism of the anti-tumour effects of these two compounds was further investigated both in vivo and in vitro. Key Results CM01 and CM02 induced G2/M cell cycle arrest and exerted potent cytostatic effects on several cancer cell lines including multidrug-resistant (MDR) cell lines. In vitro experiments revealed that the azaindole derivatives inhibited tubulin polymerization and competed with colchicines for this effect, strongly indicating that tubulin is the cellular target of these azaindole derivatives. In vivo experiments, using a chicken chorioallantoic xenograft tumour assay, established that these compounds exert a potent anti-tumour effect. Furthermore, an assay probing the growth of vessels out of endothelial cell spheroids showed that CM01 and CM02 exert anti-angiogenic activities. Conclusions and Implications CM01 and CM02 are reversible microtubule-depolymerizing agents that exert potent cytostatic effects on human cancer cells of diverse origins, including MDR cells. They were also shown to inhibit angiogenesis and tumour growth in chorioallantoic breast cancer xenografts. Hence, these azaindole derivatives are attractive candidates for further preclinical investigations. PMID:23004938

  8. 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. PMID:18799626

  9. Escape from Mitotic Arrest: An Unexpected Connection Between Microtubule Dynamics and Epigenetic Regulation of Centromeric Chromatin in Schizosaccharomyces pombe.

    PubMed

    George, Anuja A; Walworth, Nancy C

    2015-12-01

    Accurate chromosome segregation is necessary to ensure genomic integrity. Segregation depends on the proper functioning of the centromere, kinetochore, and mitotic spindle microtubules and is monitored by the spindle assembly checkpoint (SAC). In the fission yeast Schizosaccharomyces pombe, defects in Dis1, a microtubule-associated protein that influences microtubule dynamics, lead to mitotic arrest as a result of an active SAC and consequent failure to grow at low temperature. In a mutant dis1 background (dis1-288), loss of function of Msc1, a fission yeast homolog of the KDM5 family of proteins, suppresses the growth defect and promotes normal mitosis. Genetic analysis implicates a histone deacetylase (HDAC)-linked pathway in suppression because HDAC mutants clr6-1, clr3∆, and sir2∆, though not hos2∆, also promote normal mitosis in the dis1-288 mutant. Suppression of the dis phenotype through loss of msc1 function requires the spindle checkpoint protein Mad2 and is limited by the presence of the heterochromatin-associated HP1 protein homolog Swi6. We speculate that alterations in histone acetylation promote a centromeric chromatin environment that compensates for compromised dis1 function by allowing for successful kinetochore-microtubule interactions that can satisfy the SAC. In cells arrested in mitosis by mutation of dis1, loss of function of epigenetic determinants such as Msc1 or specific HDACs can promote cell survival. Because the KDM5 family of proteins has been implicated in human cancers, an appreciation of the potential role of this family of proteins in chromosome segregation is warranted.

  10. Slow dynamics and anomalous nonlinear fast dynamics in diverse solids.

    PubMed

    Johnson, Paul; Sutin, Alexander

    2005-01-01

    Results are reported of the first systematic study of anomalous nonlinear fast dynamics and slow dynamics in a number of solids. Observations are presented from seven diverse materials showing that anomalous nonlinear fast dynamics (ANFD) and slow dynamics (SD) occur together, significantly expanding the nonlinear mesoscopic elasticity class. The materials include samples of gray iron, alumina ceramic, quartzite, cracked Pyrex, marble, sintered metal, and perovskite ceramic. In addition, it is shown that materials which exhibit ANFD have very similar ratios of amplitude-dependent internal-friction to the resonance-frequency shift with strain amplitude. The ratios range between 0.28 and 0.63, except for cracked Pyrex glass, which exhibits a ratio of 1.1, and the ratio appears to be a material characteristic. The ratio of internal friction to resonance frequency shift as a function of time during SD is time independent, ranging from 0.23 to 0.43 for the materials studied. PMID:15704405

  11. Slow dynamics and anomalous nonlinear fast dynamics in diverse solids

    NASA Astrophysics Data System (ADS)

    Johnson, Paul; Sutin, Alexander

    2005-01-01

    Results are reported of the first systematic study of anomalous nonlinear fast dynamics and slow dynamics in a number of solids. Observations are presented from seven diverse materials showing that anomalous nonlinear fast dynamics (ANFD) and slow dynamics (SD) occur together, significantly expanding the nonlinear mesoscopic elasticity class. The materials include samples of gray iron, alumina ceramic, quartzite, cracked Pyrex, marble, sintered metal, and perovskite ceramic. In addition, it is shown that materials which exhibit ANFD have very similar ratios of amplitude-dependent internal-friction to the resonance-frequency shift with strain amplitude. The ratios range between 0.28 and 0.63, except for cracked Pyrex glass, which exhibits a ratio of 1.1, and the ratio appears to be a material characteristic. The ratio of internal friction to resonance frequency shift as a function of time during SD is time independent, ranging from 0.23 to 0.43 for the materials studied. .

  12. Distinctions between dynamic characteristics of the single EG5 motor protein along neural vs. cancerous microtubules.

    PubMed

    Feizabadi, Mitra Shojania; Jun, Yonggun; Reddy, J N Babu

    2016-09-30

    The kinesin 5 motor contributes critically to mitosis, and is often upregulated in cancer. In vitro motility studies of kinesin 5 moving along bovine brain microtubules indicate that the motors have limited processivity. Cancer cells have abnormal mitotic behavior, so one might wonder whether the functional properties of kinesin 5 change in such a background. Because there could be multiple unknown changes in cancerous vs normal cells, we chose to address this question in a controlled in vitro environment. Specifically, through a series of parallel experiments along bovine brain vs. breast cancer microtubules, we quantified the in vitro motility characteristics of single Eg5 molecular motors along these two types of microtubules, combining the utilization of an optical trapping technique with a study of motion in the unloaded regime. The obtained values indicate that Eg5 processivity is 40% less along MCF7 microtubules, compared to that measured on bovine brain MTs. Interestingly, not all single-molecule properties are altered, as the velocity of the single motor doesn't show any significant changes on either track, though the binding time along MCF7 microtubules is almost 25% shorter. The current results, in conjunction with our previously reported outcomes of the evaluation of the Eg5's characteristics under external load, show that in transition from no-load to high-load regime, the Eg5 binding time has less sensitivity on MCF7 as compared to bovine brain MTs. This finding is intriguing, as it suggests that, potentially, groups of Eg5 motors function more effectively in the cancer background of a large ensemble, possibly contributing to faster mitosis in cancer cells.

  13. Distinctions between dynamic characteristics of the single EG5 motor protein along neural vs. cancerous microtubules.

    PubMed

    Feizabadi, Mitra Shojania; Jun, Yonggun; Reddy, J N Babu

    2016-09-30

    The kinesin 5 motor contributes critically to mitosis, and is often upregulated in cancer. In vitro motility studies of kinesin 5 moving along bovine brain microtubules indicate that the motors have limited processivity. Cancer cells have abnormal mitotic behavior, so one might wonder whether the functional properties of kinesin 5 change in such a background. Because there could be multiple unknown changes in cancerous vs normal cells, we chose to address this question in a controlled in vitro environment. Specifically, through a series of parallel experiments along bovine brain vs. breast cancer microtubules, we quantified the in vitro motility characteristics of single Eg5 molecular motors along these two types of microtubules, combining the utilization of an optical trapping technique with a study of motion in the unloaded regime. The obtained values indicate that Eg5 processivity is 40% less along MCF7 microtubules, compared to that measured on bovine brain MTs. Interestingly, not all single-molecule properties are altered, as the velocity of the single motor doesn't show any significant changes on either track, though the binding time along MCF7 microtubules is almost 25% shorter. The current results, in conjunction with our previously reported outcomes of the evaluation of the Eg5's characteristics under external load, show that in transition from no-load to high-load regime, the Eg5 binding time has less sensitivity on MCF7 as compared to bovine brain MTs. This finding is intriguing, as it suggests that, potentially, groups of Eg5 motors function more effectively in the cancer background of a large ensemble, possibly contributing to faster mitosis in cancer cells. PMID:27590585

  14. SPIRAL2 Determines Plant Microtubule Organization by Modulating Microtubule Severing

    PubMed Central

    Wightman, Raymond; Chomicki, Guillaume; Kumar, Manoj; Carr, Paul; Turner, Simon R.

    2013-01-01

    Summary One of the defining characteristics of plant growth and morphology is the pivotal role of cell expansion. While the mechanical properties of the cell wall determine both the extent and direction of cell expansion, the cortical microtubule array plays a critical role in cell wall organization and, consequently, determining directional (anisotropic) cell expansion [1–6]. The microtubule-severing enzyme katanin is essential for plants to form aligned microtubule arrays [7–10]; however, increasing severing activity alone is not sufficient to drive microtubule alignment [11]. Here, we demonstrate that katanin activity depends upon the behavior of the microtubule-associated protein (MAP) SPIRAL2 (SPR2). Petiole cells in the cotyledon epidermis exhibit well-aligned microtubule arrays, whereas adjacent pavement cells exhibit unaligned arrays, even though SPR2 is found at similar levels in both cell types. In pavement cells, however, SPR2 accumulates at microtubule crossover sites, where it stabilizes these crossovers and prevents severing. In contrast, in the adjacent petiole cells, SPR2 is constantly moving along the microtubules, exposing crossover sites that become substrates for severing. Consequently, our study reveals a novel mechanism whereby microtubule organization is determined by dynamics and localization of a MAP that regulates where and when microtubule severing occurs. PMID:24055158

  15. Mal3 masks catastrophe events in Schizosaccharomyces pombe microtubules by inhibiting shrinkage and promoting rescue.

    PubMed

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

    2009-10-23

    Schizosaccharomyces pombe Mal3 is a member of the EB family of proteins, which are proposed to be core elements in a tip-tracking network that regulates microtubule dynamics in cells. How Mal3 itself influences microtubule dynamics is unclear. We tested the effects of full-length recombinant Mal3 on dynamic microtubules assembled in vitro from purified S. pombe tubulin, using dark field video microscopy to avoid fluorescent tagging and data-averaging techniques to improve spatiotemporal resolution. We find that catastrophe occurs stochastically as a fast (<2.2 s) transition from constant speed growth to constant speed shrinkage with a constant probability that is independent of the Mal3 concentration. This implies that Mal3 neither stabilizes nor destabilizes microtubule tips. Mal3 does, however, stabilize the main part of the microtubule lattice, inhibiting shrinkage and increasing the frequency of rescues, consistent with recent models in which Mal3 on the lattice forms stabilizing lateral links between neighboring protofilaments. At high concentrations, Mal3 can entirely block shrinkage and induce very rapid rescue, making catastrophes impossible to detect, which may account for the apparent suppression of catastrophe by Mal3 and other EBs in vivo. Overall, we find that Mal3 stabilizes microtubules not by preventing catastrophe at the microtubule tip but by inhibiting lattice depolymerization and enhancing rescue. We argue that this implies that Mal3 binds microtubules in different modes at the tip and on the lattice. PMID:19740752

  16. 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

  17. The ubiquitin ligase Phr1 regulates axon outgrowth through modulation of microtubule dynamics.

    PubMed

    Lewcock, Joseph W; Genoud, Nicolas; Lettieri, Karen; Pfaff, Samuel L

    2007-11-21

    To discover new genes involved in axon navigation, we conducted a forward genetic screen for recessive alleles affecting motor neuron pathfinding in GFP reporter mice mutagenized with ENU. In Magellan mutant embryos, motor axons were error prone and wandered inefficiently at choice points within embryos, but paradoxically responded to guidance cues with normal sensitivity in vitro. We mapped the Magellan mutation to the Phr1 gene encoding a large multidomain E3 ubiquitin ligase. Phr1 is associated with the microtubule cytoskeleton within neurons and selectively localizes to axons but is excluded from growth cones. Motor and sensory neurons from Magellan mutants display abnormal morphologies due to a breakdown in the polarized distribution of components that segregate between axons and growth cones. The Magellan phenotype can be reversed by stabilizing microtubules with taxol or inhibiting p38MAPK activity. Thus, efficacious pathfinding requires Phr1 activity for coordinating the cytoskeletal organization that distinguishes axons from growth cones.

  18. Intracellular Transport Dynamics of Endosomes Containing DNA Polyplexes along the Microtubule Network

    PubMed Central

    Kulkarni, Rajan P.; Castelino, Kenneth; Majumdar, Arun; Fraser, Scott E.

    2006-01-01

    We have explored the transport of DNA polyplexes enclosed in endosomes within the cellular environment by multiple particle tracking (MPT). The polyplex-loaded endosomes demonstrate enhanced diffusion at short timescales (t < 7 s) with their mean-square displacement (MSD) 〈Δx(t)2〉 scaling as t1.25. For longer time intervals they exhibit subdiffusive transport and have an MSD scaling as t0.7. This crossover from an enhanced diffusion to a subdiffusive regime can be explained by considering the action of motor proteins that actively transport these endosomes along the cellular microtubule network and the thermal bending modes of the microtubule network itself. PMID:16399838

  19. RAB-11 Permissively Regulates Spindle Alignment by Modulating Metaphase Microtubule Dynamics in Caenorhabditis elegans Early Embryos

    PubMed Central

    Zhang, Haining; Squirrell, Jayne M.

    2008-01-01

    Alignment of the mitotic spindle along a preformed axis of polarity is crucial for generating cell diversity in many organisms, yet little is known about the role of the endomembrane system in this process. RAB-11 is a small GTPase enriched in recycling endosomes. When we depleted RAB-11 by RNAi in Caenorhabditis elegans, the spindle of the one-cell embryo failed to align along the axis of polarity in metaphase and underwent violent movements in anaphase. The distance between astral microtubules ends and the anterior cortex was significantly increased in rab-11(RNAi) embryos specifically during metaphase, possibly accounting for the observed spindle alignment defects. Additionally, we found that normal ER morphology requires functional RAB-11, particularly during metaphase. We hypothesize that RAB-11, in conjunction with the ER, acts to regulate cell cycle–specific changes in astral microtubule length to ensure proper spindle alignment in Caenorhabditis elegans early embryos. PMID:18385514

  20. 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.

  1. 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.

  2. 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. PMID:26775215

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

    PubMed

    Lu, Wen; Winding, Michael; Lakonishok, Margot; Wildonger, Jill; Gelfand, Vladimir I

    2016-08-23

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

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

    PubMed

    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

  5. 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

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

    PubMed

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

    2016-04-01

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

  7. Structure and Dynamics of the Kinesin–Microtubule Interaction Revealed by Fluorescence Polarization Microscopy

    PubMed Central

    Sosa, Hernando; Asenjo, Ana B.; Peterman, Erwin J.G.

    2010-01-01

    Fluorescence polarization microscopy (FPM) is the analysis of the polarization of light in a fluorescent microscope in order to determine the angular orientation and rotational mobility of fluorescent molecules. Key advantages of FPM, relative to other structural analysis techniques, are that it allows the detection of conformational changes of fluorescently labeled macromolecules in real time in physiological conditions and at the single-molecule level. In this chapter we describe in detail the FPM experimental set-up and analysis methods we have used to investigate structural intermediates of the motor protein kinesin-1 associated with its walking mechanism along microtubules. We also briefly describe additional FPM methods that have been used to investigate other macromolecular complexes. PMID:20466150

  8. A fast dynamic mode in rare earth based glasses

    NASA Astrophysics Data System (ADS)

    Zhao, L. Z.; Xue, R. J.; Zhu, Z. G.; Ngai, K. L.; Wang, W. H.; Bai, H. Y.

    2016-05-01

    Metallic glasses (MGs) usually exhibit only slow β-relaxation peak, and the signature of the fast dynamic is challenging to be observed experimentally in MGs. We report a general and unusual fast dynamic mode in a series of rare earth based MGs manifested as a distinct fast β'-relaxation peak in addition to slow β-relaxation and α-relaxation peaks. We show that the activation energy of the fast β'-relaxation is about 12RTg and is equivalent to the activation of localized flow event. The coupling of these dynamic processes as well as their relationship with glass transition and structural heterogeneity is discussed.

  9. Wnt5a Evokes Cortical Axon Outgrowth and Repulsive Guidance by Tau Mediated Reorganization of Dynamic Microtubules

    PubMed Central

    Li, Li; Fothergill, Thomas; Hutchins, B Ian; Dent, Erik W; Kali, Katherine

    2014-01-01

    Wnt5a guides cortical axons in vivo by repulsion and in vitro evokes cortical axon outgrowth and repulsion by calcium signaling pathways. Here we examined the role of microtubule (MT) reorganization and dynamics in mediating effects of Wnt5a. Inhibiting MT dynamics with nocodazole and taxol abolished Wnt5a evoked axon outgrowth and repulsion of cultured hamster cortical neurons. EGFP-EB3 labeled dynamic MTs visualized in live cell imaging revealed that growth cone MTs align with the nascent axon. Wnt5a increased axon outgrowth by reorganization of dynamic MTs from a splayed to a bundled array oriented in the direction of axon extension, and Wnt5a gradients induced asymmetric redistribution of dynamic MTs toward the far side of the growth cone. Wnt5a gradients also evoked calcium transients that were highest on the far side of the growth cone. Calcium signaling and the reorganization of dynamic MTs could be linked by tau, a MT associated protein that stabilizes MTs. Tau is phosphorylated at the Ser 262 MT binding site by CaMKII, and is required for Wnt5a induced axon outgrowth and repulsive turning. Phosphorylation of tau at Ser262 is known to detach tau from MTs to increase their dynamics. Using transfection with tau constructs mutated at Ser262, we found that this site is required for the growth and guidance effects of Wnt5a by mediating reorganization of dynamic MTs in cortical growth cones. Moreover, CaMKII inhibition also prevents MT reorganization required for Wnt5a induced axon outgrowth, thus linking Wnt/calcium signaling to tau mediated MT reorganization during growth cone behaviors. © 2013 The Authors. Developmental Neurobiology Published by Wiley Periodicals, Inc.Develop Neurobiol 74: 797–817, 2014 PMID:23818454

  10. Experimental Virus Evolution Reveals a Role of Plant Microtubule Dynamics and TORTIFOLIA1/SPIRAL2 in RNA Trafficking

    PubMed Central

    Buschmann, Henrik; Niehl, Annette; Elena, Santiago F.; Rubio, Luis; Heinlein, Manfred

    2014-01-01

    The cytoskeleton is a dynamic network composed of filamentous polymers and regulatory proteins that provide a flexible structural scaffold to the cell and plays a fundamental role in developmental processes. Mutations that alter the spatial orientation of the cortical microtubule (MT) array of plants are known to cause important changes in the pattern of cell wall synthesis and developmental phenotypes; however, the consequences of such alterations on other MT-network-associated functions in the cytoplasm are not known. In vivo observations suggested a role of cortical MTs in the formation and movement of Tobacco mosaic virus (TMV) RNA complexes along the endoplasmic reticulum (ER). Thus, to probe the significance of dynamic MT behavior in the coordination of MT-network-associated functions related to TMV infection and, thus, in the formation and transport of RNA complexes in the cytoplasm, we performed an evolution experiment with TMV in Arabidopsis thaliana tor1/spr2 and tor2 mutants with specific defects in MT dynamics and asked whether TMV is sensitive to these changes. We show that the altered cytoskeleton induced genetic changes in TMV that were correlated with efficient spread of infection in the mutant hosts. These observations demonstrate a role of dynamic MT rearrangements and of the MT-associated protein TORTIFOLIA1/SPIRAL2 in cellular functions related to virus spread and indicate that MT dynamics and MT-associated proteins represent constraints for virus evolution and adaptation. The results highlight the importance of the dynamic plasticity of the MT network in directing cytoplasmic functions in macromolecular assembly and trafficking and illustrate the value of experimental virus evolution for addressing the cellular functions of dynamic, long-range order systems in multicellular organisms. PMID:25133612

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

    PubMed

    McLaughlin, Colleen N; Nechipurenko, Inna V; Liu, Nan; Broihier, Heather T

    2016-08-15

    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

  12. 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.

  13. Optical imaging of fast, dynamic neurophysiological function.

    SciTech Connect

    Rector, D. M.; Carter, K. M.; Yao, X.; George, J. S.

    2002-01-01

    Fast evoked responses were imaged from rat dorsal medulla and whisker barrel cortex. To investigate the biophysical mechanisms involved, fast optical responses associated with isolated crustacean nerve stimulation were recorded using birefringence and scattered light. Such studies allow optimization of non-invasive imaging techniques being developed for use in humans.

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

    PubMed Central

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

    2015-01-01

    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. PMID:26323690

  15. Control of microtubule dynamics by oncoprotein 18: dissection of the regulatory role of multisite phosphorylation during mitosis.

    PubMed Central

    Larsson, N; Marklund, U; Gradin, H M; Brattsand, G; Gullberg, M

    1997-01-01

    Oncoprotein 18 (Op18; also termed p19, 19K, metablastin, stathmin, and prosolin) is a conserved protein that regulates microtubule (MT) dynamics. Op18 is multisite phosphorylated on four Ser residues during mitosis; two of these Ser residues, Ser-25 and Ser-38, are targets for cyclin-dependent protein kinases (CDKs), and the other two Ser residues, Ser-16 and Ser-63, are targets for an unidentified protein kinase. Mutations of the two CDK sites have recently been shown to result in a mitotic block caused by destabilization of MTs. To understand the role of Op18 in regulation of MT dynamics during mitosis, in this study we dissected the functions of all four phosphorylation sites of Op18 by combining genetic, morphological, and biochemical analyses. The data show that all four phosphorylation sites are involved in switching off Op18 activity during mitosis, an event that appears to be essential for formation of the spindle during metaphase. However, the mechanisms by which specific sites down-regulate Op18 activity differ. Hence, dual phosphorylation on the CDK sites Ser-25 and Ser-38 appears to be required for phosphorylation of Ser-16 and Ser-63; however, by themselves, the CDK sites are of only minor importance in direct regulation of Op18 activity. Subsequent phosphorylation of either Ser-16, Ser-63, or both efficiently switches off Op18 activity. PMID:9271428

  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. Phosphorylation of β-Tubulin by the Down Syndrome Kinase, Minibrain/DYRK1a, Regulates Microtubule Dynamics and Dendrite Morphogenesis.

    PubMed

    Ori-McKenney, Kassandra M; McKenney, Richard J; Huang, Hector H; Li, Tun; Meltzer, Shan; Jan, Lily Yeh; Vale, Ronald D; Wiita, Arun P; Jan, Yuh Nung

    2016-05-01

    Dendritic arborization patterns are consistent anatomical correlates of genetic disorders such as Down syndrome (DS) and autism spectrum disorders (ASDs). In a screen for abnormal dendrite development, we identified Minibrain (MNB)/DYRK1a, a kinase implicated in DS and ASDs, as a regulator of the microtubule cytoskeleton. We show that MNB is necessary to establish the length and cytoskeletal composition of terminal dendrites by controlling microtubule growth. Altering MNB levels disrupts dendrite morphology and perturbs neuronal electrophysiological activity, resulting in larval mechanosensation defects. Using in vivo and in vitro approaches, we uncover a molecular pathway whereby direct phosphorylation of β-tubulin by MNB inhibits tubulin polymerization, a function that is conserved for mammalian DYRK1a. Our results demonstrate that phosphoregulation of microtubule dynamics by MNB/DYRK1a is critical for dendritic patterning and neuronal function, revealing a previously unidentified mode of posttranslational microtubule regulation in neurons and uncovering a conserved pathway for a DS- and ASD-associated kinase. PMID:27112495

  18. Determination of the size and chemical nature of the stabilizing "cap" at microtubule ends using modulators of polymerization dynamics.

    PubMed

    Panda, Dulal; Miller, Herbert P; Wilson, Leslie

    2002-02-01

    The size and chemical nature of the stabilizing cap at microtubule (MT) ends has remained enigmatic, in large part because it has been difficult to detect and measure it directly. By pulsing steady-state suspensions of bovine brain microtubules (MTs) with trace quantities of [gamma(32)P]GTP and sedimenting the MTs through 50% sucrose cushions to reduce background contaminating (32)P to negligible levels, we were able to detect a small number of (32)P molecules that remain stably bound to the MTs (a mean of 25.5 molecules of (32)P per MT). Analysis of the chemical form of the stably bound (32)P by thin-layer chromatography revealed that it was all (32)P-orthophosphate ((32)P(i)). The (32)P(i) was determined to be located at the MT ends because colchicine and vinblastine, drugs that suppress tubulin incorporation into the MT by binding specifically at MT ends, reduced the quantity of the stably bound (32)P(i). Taxol, a drug that stabilizes MT dynamics by binding along the MT surface rather than at the ends, did not affect the stoichiometry of the bound (32)P(i). If the bound (32)P is equally distributed between the two ends, each end would contain 12-13 molecules of (32)P(i). Beryllium fluoride (BeF(3-)) and aluminum fluoride (AlF(4-)), inorganic phosphate analogues, suppressed the dynamic instability behavior of individual MTs and, thus, stabilized them. For example, BeF(3-) (70 microM) reduced the MT shortening rate by 2.5-fold and decreased the transition frequency from the growing or the attenuated state to rapid shortening by 2-fold. The data support the hypothesis that the stabilizing cap at MT ends consists of a single layer of tubulin GDP-P(i) subunits. The data also support the hypothesis that the mechanism giving rise to the destabilized GDP-tubulin core involves release of P(i) rather than hydrolysis of the GTP. PMID:11814355

  19. Role of Epac1, an Exchange Factor for Rap GTPases, in Endothelial Microtubule Dynamics and Barrier Function

    PubMed Central

    Sehrawat, Seema; Cullere, Xavier; Patel, Sunita; Italiano, Joseph

    2008-01-01

    Rap1 GTPase activation by its cAMP responsive nucleotide exchange factor Epac present in endothelial cells increases endothelial cell barrier function with an associated increase in cortical actin. Here, Epac1 was shown to be responsible for these actin changes and to colocalize with microtubules in human umbilical vein endothelial cells. Importantly, Epac activation with a cAMP analogue, 8-pCPT-2′O-Me-cAMP resulted in a net increase in the length of microtubules. This did not require cell–cell interactions or Rap GTPase activation, and it was attributed to microtubule growth as assessed by time-lapse microscopy of human umbilical vein endothelial cell expressing fluorophore-linked microtubule plus-end marker end-binding protein 3. An intact microtubule network was required for Epac-mediated changes in cortical actin and barrier enhancement, but it was not required for Rap activation. Finally, Epac activation reversed microtubule-dependent increases in vascular permeability induced by tumor necrosis factor-α and transforming growth factor-β. Thus, Epac can directly promote microtubule growth in endothelial cells. This, together with Rap activation leads to an increase in cortical actin, which has functional significance for vascular permeability. PMID:18172027

  20. 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. PMID:19369329

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

    PubMed

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

    2015-07-24

    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.

  2. 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

  3. The microtubules dance and the spindle poles swing.

    PubMed

    Munro, Edwin

    2007-05-01

    Using live imaging and computer simulation, Kozlowski et al. (2007) show that an interplay between spindle pole movements, microtubule dynamics, and microtubule bending contribute to asymmetric spindle placement in the C. elegans embryo. PMID:17482539

  4. EXTRACELLULAR MICROTUBULES

    PubMed Central

    Bouck, G. Benjamin

    1969-01-01

    Mastigonemes (Flimmer) from the sperm of Ascophyllum and Fucus were found to consist of a tripartite structure—a ca. 2000-A tapered basal region, a closed microtubular shaft, and a group of terminal filaments. Each of these regions appears to be constructed of globular subunits with a center-to-center distance of about 45 A. The mastigoneme microtubule is of smaller diameter (170–190 A) than cytoplasmic microtubules in these or other plant cells. During the initial stages of flagellar ontogeny, structures similar to mastigonemes (presumptive mastigonemes) are found within membrane-limited sacs in the cytoplasm or within the perinuclear space. Mastigonemes at this time are generally not found on the flagellar surface. Later, when the anterior flagellum acquires mastigonemes, the presumptive mastigonemes are absent from the cytoplasm. The regularity of attachment of mastigonemes to the flagellar surface suggests that specific attachment sites are constructed on the plasma membrane during flagellar ontogeny. No evidence for penetration of the mastigoneme through the plasma membrane was obtained. The origin and structure of mastigonemes are discussed in relation to reports of the origin and structure of other microtubular systems. PMID:5812471

  5. 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.

  6. External electric field effects on the mechanical properties of the αβ-tubulin dimer of microtubules: a molecular dynamics study.

    PubMed

    Saeidi, H R; Lohrasebi, A; Mahnam, K

    2014-08-01

    The mechanical properties of the αβ-tubulin dimer of microtubules was modeled by using the molecular dynamics (MD) simulation method. The effect on the mechanical properties of the dimer of the existence and nonexistence of an applied electric field, either constant or periodic, was studied. Since there are charged or polar groups in the dimer structure, the electric field can interact with the dimer. The elastic constant and Young's modulus of the dimer were decreased when the dimer was exposed to a constant electric field of 0.03 V/nm. Furthermore, applying an oscillating electric field in the 1 GHz range to the dimer increased the elastic constant and Young's modulus of the dimer. These parameters were related to dimer rigidity and, consequently, in this frequency range, the application of electric fields may affect the function of microtubules.

  7. 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

  8. 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.

  9. Persistence Length of Stable Microtubules

    NASA Astrophysics Data System (ADS)

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

    2011-03-01

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

  10. 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.

  11. 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

  12. Microtubules self-repair in response to mechanical stress.

    PubMed

    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. PMID:26343914

  13. Generation of differentially modified microtubules using in vitro enzymatic approaches.

    PubMed

    Vemu, Annapurna; Garnham, Christopher P; Lee, Duck-Yeon; Roll-Mecak, Antonina

    2014-01-01

    Tubulin, the building block of microtubules, is subject to chemically diverse and evolutionarily conserved post-translational modifications that mark microtubules for specific functions in the cell. Here we describe in vitro methods for generating homogenous acetylated, glutamylated, or tyrosinated tubulin and microtubules using recombinantly expressed and purified modification enzymes. The generation of differentially modified microtubules now enables a mechanistic dissection of the effects of tubulin post-translational modifications on the dynamics and mechanical properties of microtubules as well as the behavior of motors and microtubule-associated proteins. PMID:24630106

  14. FAST TRACK COMMUNICATION: Complexified dynamical systems

    NASA Astrophysics Data System (ADS)

    Bender, Carl M.; Holm, Darryl D.; Hook, Daniel W.

    2007-08-01

    Many dynamical systems, such as the Lotka-Volterra predator-prey model and the Euler equations for the free rotation of a rigid body, are {{\\cal P}}{{\\cal T}} symmetric. The standard and well-known real solutions to such dynamical systems constitute an infinitessimal subclass of the full set of complex solutions. This paper examines a subset of the complex solutions that contains the real solutions, namely those having {{\\cal P}}{{\\cal T}} symmetry. The condition of {{\\cal P}}{{\\cal T}} symmetry selects out complex solutions that are periodic.

  15. The Role of Dynein in Microtubule Mechanics

    NASA Astrophysics Data System (ADS)

    Ladd, Tony; Misra, Gaurav; Wu, Jun; Russell, Robert; Lele, Tanmay; Dickinson, Richard

    2012-02-01

    Experiments in Lele's group have shown that microtubules severed by laser ablation do not straighten, as would be expected from the large bending moments along their lengths. Instead, segments near newly created minus ends typically increased in curvature following severing, while segments near new microtubule plus ends depolymerize before any observable change in shape. However, in dynein-inhibited cells, segments near the cut straightened rapidly following severing. These observations suggest that microtubules are subject to significant tangential forces, and that lateral motion of the microtubule is primarily opposed by frictional rather than elastic forces. To interpret the experimental results, we have developed a numerical model for intracellular microtubule mechanics, accounting for dynein-generated forces on the microtubules. We have supplemented the Kirchoff model for an elastic filament with the stochastic growth and collapse of microtubules, and by a model for dynein generated forces. I will present simulations of the dynamics of individual microtubules that show how motor forces result in the localization of short-wavelength buckles near the cell periphery. Our results suggest that microtubule shapes in vivo reflect a dynamic force balance, where bending moments are opposed by dynein-motor forces that include a large effective friction from the stochastic binding and unbinding of the motors. Simulations of the motion of the centrosome are consistent with a mechanism for centrosome centering driven by pulling forces exerted by dynein motors. I will explain how tension on the centrosome can be reconciled with buckled filaments near the cell periphery.

  16. FAST - A multiprocessed environment for visualization of computational fluid dynamics

    NASA Technical Reports Server (NTRS)

    Bancroft, Gordon V.; Merritt, Fergus J.; Plessel, Todd C.; Kelaita, Paul G.; Mccabe, R. Kevin

    1991-01-01

    The paper presents the Flow Analysis Software Toolset (FAST) to be used for fluid-mechanics analysis. The design criteria for FAST including the minimization of the data path in the computational fluid-dynamics (CFD) process, consistent user interface, extensible software architecture, modularization, and the isolation of three-dimensional tasks from the application programmer are outlined. Each separate process communicates through the FAST Hub, while other modules such as FAST Central, NAS file input, CFD calculator, surface extractor and renderer, titler, tracer, and isolev might work together to generate the scene. An interprocess communication package making it possible for FAST to operate as a modular environment where resources could be shared among different machines as well as a single host is discussed.

  17. Microtubule nucleating and severing enzymes for modifying microtubule array organization and cell morphogenesis in response to environmental cues.

    PubMed

    Nakamura, Masayoshi

    2015-02-01

    In higher plants, reorientation of cortical microtubule arrays has been postulated to be of importance for modifying cell growth to adapt to environmental conditions. However, the process of microtubule reorientation is largely unknown. Recent genetic and live cell imaging studies of microtubule dynamics shed light on the regulatory mechanisms of microtubule molecular nucleation and severing apparatuses, which are required for array reorientation in response to blue light signaling. Branching nucleation from γ-tubulin complexes creates a small population of discordant microtubules that are acted on by KATANIN-mediated severing in two ways. KATANIN releases microtubules from nucleation sites and rapidly amplifies discordant microtubules by severing at microtubule crossovers. In this review, I focus on the molecular details of these two enzymes, which enable microtubule array transition.

  18. Dynamic changes in microtubules and early development of reconstructed embryos after somatic cell nuclear transfer in a non-human primate.

    PubMed

    Chen, Naiqing; Liow, Swee-Lian; Yip, Wan-Yue; Tan, Lay-Geok; Tong, Guo-Qing; Ng, Soon-Chye

    2006-01-01

    In order to improve somatic cell nuclear transfer (SCNT) efficiency and to understand cellular changes in SCNT, the dynamic changes in microtubules/DNA and early development of SCNT embryos with single or multiple pronuclei were investigated, along with activation timing on efficiency of SCNT, were studied in the Cynomolgus monkey. The confocal images showed that microtubules assembled around condensed DNA at 1h after cell injection; normal or abnormal reconstructed spindle formed at 2 h after cell injection; and reconstructed spindle separated at 2 h after activation. The results of nuclear formation showed that 61.3% of the reconstructed embryos did not form pronuclei; 19.3% formed a single nucleus, and 11.9% and 7.5% formed two and more than two reconstructed pronuclei, respectively. The cleavage and 8-cell development rates of SCNT embryos with pronuclei were significantly higher than those without pronuclei, but there was no difference in development rates among NT embryos with single, two and more then two pronuclei. Activation at 2 h after cell injection yielded more embryos with pronuclei and yielded 8-cell NT embryos more reliably than did activation at 3-4 h. In conclusion, microtubules assembled around condensed DNA at 1-2 h after cell injection, and formed a spindle at 2 h after SCNT, which separated at 2 h after activation; early development was affected by activation time, but no different between single and multiple pronuclei.

  19. Leiodermatolide, a novel marine natural product, has potent cytotoxic and antimitotic activity against cancer cells, appears to affect microtubule dynamics, and exhibits antitumor activity.

    PubMed

    Guzmán, Esther A; Xu, Qunli; Pitts, Tara P; Mitsuhashi, Kaoru Ogawa; Baker, Cheryl; Linley, Patricia A; Oestreicher, Judy; Tendyke, Karen; Winder, Priscilla L; Suh, Edward M; Wright, Amy E

    2016-11-01

    Pancreatic cancer, the fourth leading cause of cancer death in the United States, has a negative prognosis because metastasis occurs before symptoms manifest. Leiodermatolide, a polyketide macrolide with antimitotic activity isolated from a deep water sponge of the genus Leiodermatium, exhibits potent and selective cytotoxicity toward the pancreatic cancer cell lines AsPC-1, PANC-1, BxPC-3, and MIA PaCa-2, and potent cytotoxicity against skin, breast and colon cancer cell lines. Induction of apoptosis by leiodermatolide was confirmed in the AsPC-1, BxPC-3 and MIA PaCa-2 cells. Leiodermatolide induces cell cycle arrest but has no effects on in vitro polymerization or depolymerization of tubulin alone, while it enhances polymerization of tubulin containing microtubule associated proteins (MAPs). Observations through confocal microscopy show that leiodermatolide, at low concentrations, causes minimal effects on polymerization or depolymerization of the microtubule network in interphase cells, but disruption of spindle formation in mitotic cells. At higher concentrations, depolymerization of the microtubule network is observed. Visualization of the growing microtubule in HeLa cells expressing GFP-tagged plus end binding protein EB-1 showed that leiodermatolide stopped the polymerization of tubulin. These results suggest that leiodermatolide may affect tubulin dynamics without directly interacting with tubulin and hint at a unique mechanism of action. In a mouse model of metastatic pancreatic cancer, leiodermatolide exhibited significant tumor reduction when compared to gemcitabine and controls. The antitumor activities of leiodermatolide, as well as the proven utility of antimitotic compounds against cancer, make leiodermatolide an interesting compound with potential chemotherapeutic effects that may merit further research. PMID:27376928

  20. Leiodermatolide, a novel marine natural product, has potent cytotoxic and antimitotic activity against cancer cells, appears to affect microtubule dynamics, and exhibits antitumor activity.

    PubMed

    Guzmán, Esther A; Xu, Qunli; Pitts, Tara P; Mitsuhashi, Kaoru Ogawa; Baker, Cheryl; Linley, Patricia A; Oestreicher, Judy; Tendyke, Karen; Winder, Priscilla L; Suh, Edward M; Wright, Amy E

    2016-11-01

    Pancreatic cancer, the fourth leading cause of cancer death in the United States, has a negative prognosis because metastasis occurs before symptoms manifest. Leiodermatolide, a polyketide macrolide with antimitotic activity isolated from a deep water sponge of the genus Leiodermatium, exhibits potent and selective cytotoxicity toward the pancreatic cancer cell lines AsPC-1, PANC-1, BxPC-3, and MIA PaCa-2, and potent cytotoxicity against skin, breast and colon cancer cell lines. Induction of apoptosis by leiodermatolide was confirmed in the AsPC-1, BxPC-3 and MIA PaCa-2 cells. Leiodermatolide induces cell cycle arrest but has no effects on in vitro polymerization or depolymerization of tubulin alone, while it enhances polymerization of tubulin containing microtubule associated proteins (MAPs). Observations through confocal microscopy show that leiodermatolide, at low concentrations, causes minimal effects on polymerization or depolymerization of the microtubule network in interphase cells, but disruption of spindle formation in mitotic cells. At higher concentrations, depolymerization of the microtubule network is observed. Visualization of the growing microtubule in HeLa cells expressing GFP-tagged plus end binding protein EB-1 showed that leiodermatolide stopped the polymerization of tubulin. These results suggest that leiodermatolide may affect tubulin dynamics without directly interacting with tubulin and hint at a unique mechanism of action. In a mouse model of metastatic pancreatic cancer, leiodermatolide exhibited significant tumor reduction when compared to gemcitabine and controls. The antitumor activities of leiodermatolide, as well as the proven utility of antimitotic compounds against cancer, make leiodermatolide an interesting compound with potential chemotherapeutic effects that may merit further research.

  1. Fast Responding Voltage Regulator and Dynamic VAR Compensator

    SciTech Connect

    Divan, Deepak; Moghe, Rohit; Tholomier, Damien

    2014-12-31

    The objectives of this project were to develop a dynamic VAR compensator (DVC) for voltage regulation through VAR support to demonstrate the ability to achieve greater levels of voltage control on electricity distribution networks, and faster response compared to existing grid technology. The goal of the project was to develop a prototype Fast Dynamic VAR Compensator (Fast DVC) hardware device, and this was achieved. In addition to developing the dynamic VAR compensator device, Varentec in partnership with researchers at North Carolina State University (NCSU) successfully met the objectives to model the potential positive impact of such DVCs on representative power networks. This modeling activity validated the ability of distributed dynamic VAR compensators to provide fast voltage regulation and reactive power control required to respond to grid disturbances under high penetration of fluctuating and intermittent distributed energy resources (DERs) through extensive simulation studies. Specifically the following tasks were set to be accomplished: 1) Development of dynamic VAR compensator to support dynamic voltage variations on the grid through VAR control 2) Extensive testing of the DVC in the lab environment 3) Present the operational DVC device to the DOE at Varentec’s lab 4) Formulation of a detailed specification sheet, unit assembly document, test setup document, unit bring-up plan, and test plan 5) Extensive simulations of the DVC in a system with high PV penetration. Understanding the operation with many DVC on a single distribution system 6) Creation and submittal of quarterly and final reports conveying the design documents, unit performance data, modeling simulation charts and diagrams, and summary explanations of the satisfaction of program goals. This report details the various efforts that led to the development of the Fast DVC as well as the modeling & simulation results. The report begins with the introduction in Section II which outlines the

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

    PubMed Central

    Drechsler, Hauke; McAinsh, Andrew D.

    2016-01-01

    Human Kinesin-12 (hKif15) plays a crucial role in assembly and maintenance of the mitotic spindle. These functions of hKif15 are partially redundant with Kinesin-5 (Eg5), which can cross-link and drive the extensile sliding of antiparallel microtubules. Although both motors are known to be tetramers, the functional properties of hKif15 are less well understood. Here we reveal how single or multiple Kif15 motors can cross-link, transport, and focus the plus-ends of intersecting microtubules. During transport, Kif15 motors step simultaneously along both microtubules with relative microtubule transport driven by a velocity differential between motor domain pairs. Remarkably, this differential is affected by the underlying intersection geometry: the differential is low on parallel and extreme on antiparallel microtubules where one motor domain pair becomes immobile. As a result, when intersecting microtubules are antiparallel, canonical transport of one microtubule along the other is allowed because one motor is firmly attached to one microtubule while it is stepping on the other. When intersecting microtubules are parallel, however, Kif15 motors can drive (biased) parallel sliding because the motor simultaneously steps on both microtubules that it cross-links. These microtubule rearrangements will focus microtubule plus-ends and finally lead to the formation of parallel bundles. At the same time, Kif15 motors cooperate to suppress catastrophe events at polymerizing microtubule plus-ends, raising the possibility that Kif15 motors may synchronize the dynamics of bundles that they have assembled. Thus, Kif15 is adapted to operate on parallel microtubule substrates, a property that clearly distinguishes it from the other tetrameric spindle motor, Eg5. PMID:26969727

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

    PubMed

    Drechsler, Hauke; McAinsh, Andrew D

    2016-03-22

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

  4. Dissecting microtubule structures by laser ablation.

    PubMed

    Decker, Franziska; Brugués, Jan

    2015-01-01

    Here, we describe a detailed protocol, based on laser ablation and fluorescence optical microscopy, to measure the microtubule organization in spindles, including microtubule length distribution, polarity, and plus and minus end densities. The method uses the asymmetry in microtubule depolymerization after a cut, where the newly created microtubule plus ends depolymerize all the way to the minus ends, whereas the newly created minus ends remain stable. The protocol described in this chapter is optimized for spindles, but can be easily applied to any microtubule-based structure. The chapter is divided into two parts. First, we provide the theoretical basis for the method. Second, we describe in detail all steps necessary to reconstruct the microtubule organization of a spindle assembled in Xenopus laevis egg extract. Compared to electron microscopy, which in theory can resolve individual microtubules in spindles and provide similar structural information, our method is fast and simple enough to allow for a full quantitative reconstruction of the microtubule organization of several X. laevis spindles—which have volumes tens of thousands of times larger than spindles whose structures have been previously solved by electron microscopy—in a single experimental session, as well as to explore how the architecture of these structures changes in response to biochemical perturbations.

  5. A dynamical model of Kara Sea land-fast ice

    NASA Astrophysics Data System (ADS)

    Olason, Einar

    2016-05-01

    This paper introduces modifications to the traditional viscous-plastic sea-ice dynamical model, which are necessary to model land-fast ice in the Kara Sea in a realistic manner. The most important modifications are an increase in the maximum viscosity from the standard value of ζmax=>(2.5×108s>)P to ζmax=>(1013s>)P, and to use a solver for the momentum equation capable of correctly solving for small ice velocities (the limit here is set to 10-4 m/s). Given these modifications, a necessary condition for a realistic fast-ice simulation is that the yield curve give sufficient uniaxial compressive strength. This is consistent with the idea that land-fast ice in the Kara Sea forms primarily via static arching. The modified model is tested and tuned using forcing data and observations from 1997 and 1998. The results show that it is possible to model land-fast ice using this model with the modifications mentioned above. The model performs well in terms of modeled fast-ice extent, but suffers from unrealistic break-ups during the start and end of the fast-ice season. The main results are that fast ice in the Kara Sea is supported by arching of the ice, the arches footers resting on a chain of islands off shore.

  6. Dynamic pricing? Not so fast. a residential consumer perspective

    SciTech Connect

    Alexander, Barbara R.

    2010-07-15

    With the installation of smart metering, will residential customers be moved to ''dynamic'' pricing? Some supporters of changing residential rate design from a fixed and stable rate structure believe customers should be required to take electric service with time-variant price signals. Not so fast, though. There are real implications associated with this strategy. (author)

  7. Bat Dynamics of Female Fast Pitch Softball Batters.

    ERIC Educational Resources Information Center

    Messier, Stephen P.; Owen, Marjorie G.

    1984-01-01

    Female fast pitch softball batters served in an examination of the dynamic characteristics of the bat during the swing through the use of three-dimensional cinematographic analysis techniques. These results were compared with those from previous studies of baseball batting. Findings are listed. (Author/DF)

  8. Quantum dynamics of fast chemical reactions

    SciTech Connect

    Light, J.C.

    1993-12-01

    The aims of this research are to explore, develop, and apply theoretical methods for the evaluation of the dynamics of gas phase collision processes, primarily chemical reactions. The primary theoretical tools developed for this work have been quantum scattering theory, both in time dependent and time independent forms. Over the past several years, the authors have developed and applied methods for the direct quantum evaluation of thermal rate constants, applying these to the evaluation of the hydrogen isotopic exchange reactions, applied wave packet propagation techniques to the dissociation of Rydberg H{sub 3}, incorporated optical potentials into the evaluation of thermal rate constants, evaluated the use of optical potentials for state-to-state reaction probability evaluations, and, most recently, have developed quantum approaches for electronically non-adiabatic reactions which may be applied to simplify calculations of reactive, but electronically adiabatic systems. Evaluation of the thermal rate constants and the dissociation of H{sub 3} were reported last year, and have now been published.

  9. The use of compressive sensing and peak detection in the reconstruction of microtubules length time series in the process of dynamic instability.

    PubMed

    Mahrooghy, Majid; Yarahmadian, Shantia; Menon, Vineetha; Rezania, Vahid; Tuszynski, Jack A

    2015-10-01

    Microtubules (MTs) are intra-cellular cylindrical protein filaments. They exhibit a unique phenomenon of stochastic growth and shrinkage, called dynamic instability. In this paper, we introduce a theoretical framework for applying Compressive Sensing (CS) to the sampled data of the microtubule length in the process of dynamic instability. To reduce data density and reconstruct the original signal with relatively low sampling rates, we have applied CS to experimental MT lament length time series modeled as a Dichotomous Markov Noise (DMN). The results show that using CS along with the wavelet transform significantly reduces the recovery errors comparing in the absence of wavelet transform, especially in the low and the medium sampling rates. In a sampling rate ranging from 0.2 to 0.5, the Root-Mean-Squared Error (RMSE) decreases by approximately 3 times and between 0.5 and 1, RMSE is small. We also apply a peak detection technique to the wavelet coefficients to detect and closely approximate the growth and shrinkage of MTs for computing the essential dynamic instability parameters, i.e., transition frequencies and specially growth and shrinkage rates. The results show that using compressed sensing along with the peak detection technique and wavelet transform in sampling rates reduces the recovery errors for the parameters.

  10. A novel microtubule inhibitor, MT3-037, causes cancer cell apoptosis by inducing mitotic arrest and interfering with microtubule dynamics.

    PubMed

    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

  11. 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

  12. Mechanism of microtubule array expansion in the cytokinetic phragmoplast

    PubMed Central

    Murata, Takashi; Sano, Toshio; Sasabe, Michiko; Nonaka, Shigenori; Higashiyama, Tetsuya; Hasezawa, Seiichiro; Machida, Yasunori; Hasebe, Mitsuyasu

    2013-01-01

    In land plants, the cell plate partitions the daughter cells at cytokinesis. The cell plate initially forms between daughter nuclei and expands centrifugally until reaching the plasma membrane. The centrifugal development of the cell plate is driven by the centrifugal expansion of the phragmoplast microtubule array, but the molecular mechanism underlying this expansion is unknown. Here, we show that the phragmoplast array comprises stable microtubule bundles and dynamic microtubules. We find that the dynamic microtubules are nucleated by γ-tubulin on stable bundles. The dynamic microtubules elongate at the plus ends and form new bundles preferentially at the leading edge of the phragmoplast. At the same time, they are moved away from the cell plate, maintaining a restricted distribution of minus ends. We propose that cycles of attachment of γ-tubulin complexes onto the microtubule bundles, microtubule nucleation and bundling, accompanied by minus-end-directed motility, drive the centrifugal development of the phragmoplast. PMID:23770826

  13. Electron theory of fast and ultrafast dissipative magnetization dynamics.

    PubMed

    Fähnle, M; Illg, C

    2011-12-14

    For metallic magnets we review the experimental and electron-theoretical investigations of fast magnetization dynamics (on a timescale of ns to 100 ps) and of laser-pulse-induced ultrafast dynamics (few hundred fs). It is argued that for both situations the dominant contributions to the dissipative part of the dynamics arise from the excitation of electron-hole pairs and from the subsequent relaxation of these pairs by spin-dependent scattering processes, which transfer angular momentum to the lattice. By effective field theories (generalized breathing and bubbling Fermi-surface models) it is shown that the Gilbert equation of motion, which is often used to describe the fast dissipative magnetization dynamics, must be extended in several aspects. The basic assumptions of the Elliott-Yafet theory, which is often used to describe the ultrafast spin relaxation after laser-pulse irradiation, are discussed very critically. However, it is shown that for Ni this theory probably yields a value for the spin-relaxation time T(1) in good agreement with the experimental value. A relation between the quantity α characterizing the damping of the fast dynamics in simple situations and the time T(1) is derived. PMID:22089491

  14. 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.

  15. The dynamic Allan variance II: a fast computational algorithm.

    PubMed

    Galleani, Lorenzo

    2010-01-01

    The stability of an atomic clock can change with time due to several factors, such as temperature, humidity, radiations, aging, and sudden breakdowns. The dynamic Allan variance, or DAVAR, is a representation of the time-varying stability of an atomic clock, and it can be used to monitor the clock behavior. Unfortunately, the computational time of the DAVAR grows very quickly with the length of the analyzed time series. In this article, we present a fast algorithm for the computation of the DAVAR, and we also extend it to the case of missing data. Numerical simulations show that the fast algorithm dramatically reduces the computational time. The fast algorithm is useful when the analyzed time series is long, or when many clocks must be monitored, or when the computational power is low, as happens onboard satellites and space probes.

  16. A new technique for fast dynamic focusing law computing

    NASA Astrophysics Data System (ADS)

    Fritsch, C.; Cruza, J. F.; Brizuela, J.; Camacho, J.; Moreno, J. M.

    2012-05-01

    Dynamic focusing requires computing the individual delays for every element and every focus in the image. This is an easy and relatively fast task if the inspected medium is homogeneous. Nevertheless, some difficulties arise in presence of interfaces (i.e, wedges, immersion, etc.): refraction effects require computing the Snell's law for every focus and element to find the fastest ray entry point in the interface. The process is easy but takes a long time. This work presents a new technique to compute the focusing delays for an equivalent virtual array that operates in the second medium only, thus avoiding any interface. It is nearly as fast as computing the focal laws in the homogeneous case and an order of magnitude faster than Snell's or Fermat's principle based methods. Furthermore, the technique is completely general and can be applied to any equipment having dynamic focusing capabilities. In fact, the technique is especially well suited for real-time focal law computing hardware.

  17. FAST Simulation Tool Containing Methods for Predicting the Dynamic Response of Wind Turbines

    SciTech Connect

    Jonkman, Jason

    2015-08-12

    FAST is a simulation tool (computer software) for modeling tlie dynamic response of horizontal-axis wind turbines. FAST employs a combined modal and multibody structural-dynamics formulation in the time domain.

  18. Complementary roles of microtubules and microfilaments in the lung fibroblast-mediated contraction of collagen gels: Dynamics and the influence of cell density.

    PubMed

    Redden, Robert A; Doolin, Edward J

    2006-01-01

    Fibroblasts are important cellular components in wound healing, scar formation, and fibrotic disorders; and the fibroblast-populated collagen-gel (FPCG) model allows examination of fibroblast behavior in an in vitro three-dimensional environment similar to that in vivo. Contraction of free-floating FPCGs depends on an active and dynamic cytoskeleton, and the contraction dynamics are highly influenced by cell density. We investigated mechanistic differences between high- and low-cell density FPCG contraction by evaluating contraction dynamics in detail, using specific cytoskeletal disruptors. Collagen gels were seeded with human lung fibroblasts at either high (HD) or low (LD) density, and incubated with or without cytoskeletal disruptors colchicine (microtubules) or cytochalasin D (microfilaments). Gel area was measured daily. FPCG contraction curves were essentially sigmoidal, featuring an initial period of no contraction (lag phase), followed by a period of rapid contraction (log phase). Contraction curves of HD-FPCGs were distinct from those of LD-FPCGs. For example, HD-FPCGs had a negligible lag phase (compared with 3 d for LD-FPCGs) and exhibited a higher rate of log-phase contraction. Both colchicine and cytochalasin dose-dependently inhibited contraction but specifically affected different phases of contraction in HD- and LD-FPCGs; and colchicine inhibited LD-FPCGs much more than HD-FPCGs. The data indicate that LD- and HD-FPCGs contract through different primary mechanisms. Microtubules and microfilaments are both complementarily and dynamically involved in the contraction of FPCGs, and cell density influences primary cytoskeletal mechanisms. These results provide valuable information about fibroblast behavior in healing and fibrosis, and may suggest novel treatment options. PMID:16759151

  19. A note on the theory of fast money flow dynamics

    NASA Astrophysics Data System (ADS)

    Sokolov, A.; Kieu, T.; Melatos, A.

    2010-08-01

    The gauge theory of arbitrage was introduced by Ilinski in [K. Ilinski, preprint arXiv:hep-th/9710148 (1997)] and applied to fast money flows in [A. Ilinskaia, K. Ilinski, preprint arXiv:cond-mat/9902044 (1999); K. Ilinski, Physics of finance: gauge modelling in non-equilibrium pricing (Wiley, 2001)]. The theory of fast money flow dynamics attempts to model the evolution of currency exchange rates and stock prices on short, e.g. intra-day, time scales. It has been used to explain some of the heuristic trading rules, known as technical analysis, that are used by professional traders in the equity and foreign exchange markets. A critique of some of the underlying assumptions of the gauge theory of arbitrage was presented by Sornette in [D. Sornette, Int. J. Mod. Phys. C 9, 505 (1998)]. In this paper, we present a critique of the theory of fast money flow dynamics, which was not examined by Sornette. We demonstrate that the choice of the input parameters used in [K. Ilinski, Physics of finance: gauge modelling in non-equilibrium pricing (Wiley, 2001)] results in sinusoidal oscillations of the exchange rate, in conflict with the results presented in [K. Ilinski, Physics of finance: gauge modelling in non-equilibrium pricing (Wiley, 2001)]. We also find that the dynamics predicted by the theory are generally unstable in most realistic situations, with the exchange rate tending to zero or infinity exponentially.

  20. 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.

  1. Analysis of microtubule curvature.

    PubMed

    Bicek, Andrew D; Tüzel, Erkan; Kroll, Daniel M; Odde, David J

    2007-01-01

    The microtubule cytoskeleton in living cells generate and resist mechanical forces to mediate fundamental cell processes, including cell division and migration. Recent advances in digital fluorescence microscopy have enabled the direct observation of bending of individual microtubules in living cells, which has enabled quantitative estimation of the mechanical state of the microtubule array. Although a variety of mechanisms have been proposed, the precise origins of microtubule deformation in living cells remain largely obscure. To investigate these mechanisms and their relative importance in cellular processes, a method is needed to accurately quantify microtubule bending within living cells. Here we describe a method for quantification of bending, using digital fluorescence microscope images to estimate the distribution of curvature in the microtubule. Digital images of individual microtubules can be used to obtain a set of discrete x-y coordinates along the microtubule contour, which is then used to estimate the curvature distribution. Due to system noise and digitization error, the estimate will be inaccurate to some degree. To quantify the inaccuracy, a computational model is used to simulate both the bending of thermally driven microtubules and their observation by digital fluorescence microscopy. This allows for direct comparison between experimental and simulated images, a method which we call model convolution microscopy. We assess the accuracy of various methods and present a suitable method for estimating the curvature distribution for thermally driven semiflexible polymers. Finally, we discuss extensions of the method to quantify microtubule curvature in living cells. PMID:17613311

  2. EBs Recognize a Nucleotide-Dependent Structural Cap at Growing Microtubule Ends

    PubMed Central

    Maurer, Sebastian P.; Fourniol, Franck J.; Bohner, Gergő; Moores, Carolyn A.; Surrey, Thomas

    2012-01-01

    Summary Growing microtubule ends serve as transient binding platforms for essential proteins that regulate microtubule dynamics and their interactions with cellular substructures. End-binding proteins (EBs) autonomously recognize an extended region at growing microtubule ends with unknown structural characteristics and then recruit other factors to the dynamic end structure. Using cryo-electron microscopy, subnanometer single-particle reconstruction, and fluorescence imaging, we present a pseudoatomic model of how the calponin homology (CH) domain of the fission yeast EB Mal3 binds to the end regions of growing microtubules. The Mal3 CH domain bridges protofilaments except at the microtubule seam. By binding close to the exchangeable GTP-binding site, the CH domain is ideally positioned to sense the microtubule's nucleotide state. The same microtubule-end region is also a stabilizing structural cap protecting the microtubule from depolymerization. This insight supports a common structural link between two important biological phenomena, microtubule dynamic instability and end tracking. PMID:22500803

  3. 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.

  4. Microfilaments and microtubules: the news from yeast.

    PubMed

    Schott, Daniel; Huffaker, Tim; Bretscher, Anthony

    2002-12-01

    New evidence that cortical actin patches and the endocytic machinery share components supports the idea that actin patches are in fact transient membrane coats at the initial stage of endocytosis. Recent studies of actin cables have identified formins as the core of a novel actin-filament-assembling machine. Meanwhile, microtubule-binding proteins have been found in the kinetochore, and factors affecting microtubule dynamic instability have been identified. PMID:12457699

  5. Fast Simulation on Flexible Multibody Dynamics Using Domain Decomposition Technique

    NASA Astrophysics Data System (ADS)

    Imanishi, Etsujiro; Nanjo, Takao; Hirooka, Eiko; Sugano, Naoki

    Recently, HILS (Hardware in the Loop Simulation) has been investigated in the field of the multibody dynamics (MBD). The fast calculation is necessary for the HILS system in order to require the real time simulation. This paper presents a fast simulation technique using the domain decomposition method. The domain decomposition method is widely used in the dynamic simulation for the mechanical system involving the hydraulic control system. This method is, however, not absolutely stable as the numerical integration. Fujikawa proposed a numerical stable solution scheme by introducing the iteration calculation. This paper applies the method to actual simulations of flexible multibody system in which the flexible linkage system and the hydraulic drive system are coupled with each other, and examines the speedup by parallel computing with the common memory in the calculation time. It is shown that using the present method in a multi-degrees-of freedom model can shorten the computing time. The present method is effective for the speedup in the calculation time by applying the dynamic simulation of the actual digging works on the hydraulic excavator.

  6. Optically Resolving Individual Microtubules in Live Axons

    PubMed Central

    Mudrakola, Harsha V.; Zhang, Kai; Cui, Bianxiao

    2010-01-01

    Summary Microtubules are essential cytoskeletal tracks for cargo transportation in axons and also serve as the primary structural scaffold of neurons. Structural assembly, stability, and dynamics of axonal microtubules are of great interest for understanding neuronal functions and pathologies. However, microtubules are so densely packed in axons that their separations are well below the diffraction limit of light, which precludes using optical microscopy for live-cell studies. Here, we present a single-molecule imaging method capable of resolving individual microtubules in live axons. In our method, unlabeled microtubules are revealed by following individual axonal cargos that travel along them. We resolved more than six microtubules in a 1 μm diameter axon by real-time tracking of endosomes containing quantum dots. Our live-cell study also provided direct evidence that endosomes switch between microtubules while traveling along axons, which has been proposed to be the primary means for axonal cargos to effectively navigate through the crowded axoplasmic environment. PMID:19913478

  7. Analysis of microtubules in isolated axoplasm from the squid giant axon.

    PubMed

    Song, Yuyu; Brady, Scott T

    2013-01-01

    Biochemical specialization of cellular microtubules has emerged as a primary mechanism in specifying microtubule dynamics and function. However, study of specific subcellular populations of cytoplasmic microtubules has been limited, particularly in the nervous system. The complexity of nervous tissue makes it difficult to distinguish neuronal microtubules from glial microtubules, and axonal microtubules from dendritic and cell body microtubules. The problem is further compounded by the finding that a large fraction of neuronal tubulin is lost during standard preparations of brain tubulin, and this population of stable microtubules is enriched in axons. Here, we consider a unique biological model that provides a unique opportunity to study axonal microtubules both in situ and in vitro: isolated axoplasm from the squid giant axon. The axoplasm model represents a powerful system for addressing fundamental questions of microtubule structure and function in the axon.

  8. Flexural Rigidity of MCF-7 Microtubules Measured from Thermal Fluctuations in Shape

    NASA Astrophysics Data System (ADS)

    Shojania Feizabadi, Mitra; Mutafopulos, Kiryako; Behr, Adam

    2011-03-01

    Microtubules play a key role in the mechanical and elastic properties of eukaryotic cells. For this reason, measuring the flexural rigidity of bovine brain microtubules have been extensively investigated through different methods of measurement. Beta tubulin isotypes, a noticeable trait to consider as we transfer from mammalian neural microtubules to mammalian non-neural microtubules, are assembled differently in distributions among various types of microtubules. Different studies have shown that microtubules made from different beta-tubulin isotypes express unique polymerization and dynamic behavior. This study focuses on measuring mechanical properties of one of non-neural microtubules, MCF-7. We will discuss the structure differences between brain bovine microtubules and MCF-7, along with the rigidity of single microtubules polymerized from MCF-7 tubulin through monitoring the curvature of microtubule due to thermal fluctuations.

  9. Fast-growing species and sustainability (productivity and site dynamics of three fast-growing species)

    SciTech Connect

    Reddy, A.N.; Sugur, G.V.

    1992-12-31

    Growth of three fast-growing species, raised in a high rainfall zone (2000-2500 mm per annum) has been compared, and the associated site dynamics studies in the Western Ghat area of Karnataka State. Two fast-growing exotics, Acacia auriculiformis and Castuarina equisitifolia, were planted on degraded, open sites at high planting densities (5000 plants ha{sup {minus}1}), and one native fast-growing species. Dendrocalamus strictus, was planted on a good site under seasonal irrigation and wider spacing (500 plants ha{sup {minus}1}). These were studies at the age of 5 years for their comparative productivity, quantity of litter fall and changes in nutrient and microbial status. Among these species, A. auriculiformis recorded the highest total productivity closely followed by D. strictus. However, the MAI after 5 years indicated a higher productivity for D. strictus, when culm production attained harvestable size. C. equisitifolia was a close third. It was also found that D. strictus produced higher biomass at lower planting densities, under better sites and management. The litter fall and changes in nutrient status indicated the highest efficiency in A. auriculiformis, followed by C. equisitifolia. It was concluded that the higher planting density was the major contributing factor; the values were comparatively low for D. strictus mainly owing to a lower stocking density of plants.

  10. 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)

  11. Tau stabilizes microtubules by binding at the interface between tubulin heterodimers

    PubMed Central

    Kadavath, Harindranath; Hofele, Romina V.; Biernat, Jacek; Kumar, Satish; Tepper, Katharina; Urlaub, Henning; Mandelkow, Eckhard; Zweckstetter, Markus

    2015-01-01

    The structure, dynamic behavior, and spatial organization of microtubules are regulated by microtubule-associated proteins. An important microtubule-associated protein is the protein Tau, because its microtubule interaction is impaired in the course of Alzheimer’s disease and several other neurodegenerative diseases. Here, we show that Tau binds to microtubules by using small groups of evolutionary conserved residues. The binding sites are formed by residues that are essential for the pathological aggregation of Tau, suggesting competition between physiological interaction and pathogenic misfolding. Tau residues in between the microtubule-binding sites remain flexible when Tau is bound to microtubules in agreement with a highly dynamic nature of the Tau–microtubule interaction. By binding at the interface between tubulin heterodimers, Tau uses a conserved mechanism of microtubule polymerization and, thus, regulation of axonal stability and cell morphology. PMID:26034266

  12. 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.

  13. EB1 regulates attachment of Ska1 with microtubules by forming extended structures on the microtubule lattice

    PubMed Central

    Thomas, Geethu E.; Bandopadhyay, K.; Sutradhar, Sabyasachi; Renjith, M. R.; Singh, Puja; Gireesh, K. K.; Simon, Steny; Badarudeen, Binshad; Gupta, Hindol; Banerjee, Manidipa; Paul, Raja; Mitra, J.; Manna, Tapas K.

    2016-01-01

    Kinetochore couples chromosome movement to dynamic microtubules, a process that is fundamental to mitosis in all eukaryotes but poorly understood. In vertebrates, spindle-kinetochore-associated (Ska1–3) protein complex plays an important role in this process. However, the proteins that stabilize Ska-mediated kinetochore-microtubule attachment remain unknown. Here we show that microtubule plus-end tracking protein EB1 facilitates Ska localization on microtubules in vertebrate cells. EB1 depletion results in a significant reduction of Ska1 recruitment onto microtubules and defects in mitotic chromosome alignment, which is also reflected in computational modelling. Biochemical experiments reveal that EB1 interacts with Ska1, facilitates Ska1-microtubule attachment and together stabilizes microtubules. Structural studies reveal that EB1 either with Ska1 or Ska complex forms extended structures on microtubule lattice. Results indicate that EB1 promotes Ska association with K-fibres and facilitates kinetochore-microtubule attachment. They also implicate that in vertebrates, chromosome coupling to dynamic microtubules could be mediated through EB1-Ska extended structures. PMID:27225956

  14. Filopodial actin bundles are not necessary for microtubule advance into the peripheral domain of Aplysia neuronal growth cones.

    PubMed

    Burnette, Dylan T; Schaefer, Andrew W; Ji, Lin; Danuser, Gaudenz; Forscher, Paul

    2007-12-01

    Filopodial actin bundles guide microtubule assembly in the growth cone peripheral (P) domain and retrograde actin-network flow simultaneously transports microtubules rearward. Therefore, microtubule-end position is determined by the sum of microtubule assembly and retrograde transport rates. However, how filopodia actually affect microtubule assembly dynamics is unknown. To address this issue we quantitatively assessed microtubule and actin dynamics before and after selective removal of filopodia. Filopodium removal had surprisingly little effect on retrograde actin-flow rates or underlying network structures, but resulted in an approximate doubling of peripheral microtubule density and deeper penetration of microtubules into the P domain. The latter stemmed from less efficient coupling of microtubules to remaining actin networks and not from a change in microtubule polymer dynamics. Loss of filopodia also resulted in increased lateral microtubule movements and a more randomized microtubule distribution in the P domain. In summary, filopodia do not seem to be formally required for microtubule advance; however, their presence ensures radial distribution of microtubules in the P domain and facilitates microtubule transport by retrograde flow. The resulting dynamic steady state has interesting implications for rapid microtubule-positioning responses in the P domain.

  15. 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.

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

    PubMed Central

    Carlier-Grynkorn, Frédérique; Ji, Liang; Fraisier, Vincent; Lombard, Berangère; Dingli, Florent; Loew, Damarys; Paoletti, Anne; Ronot, Xavier; Tran, Phong T.

    2014-01-01

    ABSTRACT The microtubule cytoskeleton plays important roles in cell polarity, motility and division. Microtubules inherently undergo dynamic instability, stochastically switching between phases of growth and shrinkage. In cells, some microtubule-associated proteins (MAPs) and molecular motors can further modulate microtubule dynamics. We present here the fission yeast mtr1+, a new regulator of microtubule dynamics that appears to be not a MAP or a motor. mtr1-deletion (mtr1Δ) primarily results in longer microtubule dwell-time at the cell tip cortex, suggesting that mtr1p acts directly or indirectly as a destabilizer of microtubules. mtr1p is antagonistic to mal3p, the ortholog of mammalian EB1, which stabilizes microtubules. mal3Δ results in short microtubules, but can be partially rescued by mtr1Δ, as the double mutant mal3Δ mtr1Δ exhibits longer microtubules than mal3Δ single mutant. By sequence homology, mtr1p is predicted to be a component of the ribosomal quality control complex. Intriguingly, deletion of a predicted ribosomal gene, rps1801, also resulted in longer microtubule dwell-time similar to mtr1Δ. The double-mutant mal3Δ rps1801Δ also exhibits longer microtubules than mal3Δ single mutant alone. Our study suggests a possible involvement of mtr1p and the ribosome complex in modulating microtubule dynamics. PMID:24928430

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

    PubMed

    Carlier-Grynkorn, Frédérique; Ji, Liang; Fraisier, Vincent; Lombard, Berangère; Dingli, Florent; Loew, Damarys; Paoletti, Anne; Ronot, Xavier; Tran, Phong T

    2014-01-01

    The microtubule cytoskeleton plays important roles in cell polarity, motility and division. Microtubules inherently undergo dynamic instability, stochastically switching between phases of growth and shrinkage. In cells, some microtubule-associated proteins (MAPs) and molecular motors can further modulate microtubule dynamics. We present here the fission yeast mtr1(+), a new regulator of microtubule dynamics that appears to be not a MAP or a motor. mtr1-deletion (mtr1Δ) primarily results in longer microtubule dwell-time at the cell tip cortex, suggesting that mtr1p acts directly or indirectly as a destabilizer of microtubules. mtr1p is antagonistic to mal3p, the ortholog of mammalian EB1, which stabilizes microtubules. mal3Δ results in short microtubules, but can be partially rescued by mtr1Δ, as the double mutant mal3Δ mtr1Δ exhibits longer microtubules than mal3Δ single mutant. By sequence homology, mtr1p is predicted to be a component of the ribosomal quality control complex. Intriguingly, deletion of a predicted ribosomal gene, rps1801, also resulted in longer microtubule dwell-time similar to mtr1Δ. The double-mutant mal3Δ rps1801Δ also exhibits longer microtubules than mal3Δ single mutant alone. Our study suggests a possible involvement of mtr1p and the ribosome complex in modulating microtubule dynamics. PMID:24928430

  18. Fast dynamics in glass-forming polymers revisited

    SciTech Connect

    Colmenero, J.; Arbe, A.; Mijangos, C.; Reinecke, H.

    1997-12-31

    The so called fast-dynamics of glass-forming systems as observed by time of flight (TOF) neutron scattering techniques is revisited. TOF-results corresponding to several glass-forming polymers with different chemical microstructure and glass-transition temperature are presented together with the theoretical framework proposed by the authors to interpret these results. The main conclusion is that the TOF-data can be explained in terms of quasiharmonic vibrations and the particular short time behavior of the segmental dynamics. The segmental dynamics display in the very short time range (t {approx} 2 ps) a crossover from a simple exponential behavior towards a non-exponential regime. The first exponential decay, which is controlled by C-C rotational barriers, can be understood as a trace of the behavior of the system in absence of the effects (correlations, cooperativity, memory effects {hor_ellipsis}) which characterize the dense supercooled liquid like state against the normal liquid state. The non-exponential regime at t > 2 ps corresponds to what is usually understood as {alpha} and {beta} relaxations. Some implications of these results are also discussed.

  19. Cavitation in confined water: ultra-fast bubble dynamics

    NASA Astrophysics Data System (ADS)

    Vincent, Olivier; Marmottant, Philippe

    2012-02-01

    In the hydraulic vessels of trees, water can be found at negative pressure. This metastable state, corresponding to mechanical tension, is achieved by evaporation through a porous medium. It can be relaxed by cavitation, i.e. the sudden nucleation of vapor bubbles. Harmful for the tree due to the subsequent emboli of sap vessels, cavitation is on the contrary used by ferns to eject spores very swiftly. We will focus here on the dynamics of the cavitation bubble, which is of primary importance to explain the previously cited natural phenomena. We use the recently developed method of artificial tress, using transparent hydrogels as the porous medium. Our experiments, on water confined in micrometric hydrogel cavities, show an extremely fast dynamics: bubbles are nucleated at the microsecond timescale. For cavities larger than 100 microns, the bubble ``rings'' with damped oscillations at MHz frequencies, whereas for smaller cavities the oscillations become overdamped. This rich dynamics can be accounted for by a model we developed, leading to a modified Rayleigh-Plesset equation. Interestingly, this model predicts the impossibility to nucleate bubbles above a critical confinement that depends on liquid negative pressure and corresponds to approximately 100 nm for 20 MPa tensions.

  20. 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

  1. Microtubules in plants.

    PubMed

    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.

  2. Dynamic Mode Decomposition of Fast Pressure Sensitive Paint Data.

    PubMed

    Ali, Mohd Y; Pandey, Anshuman; Gregory, James W

    2016-06-11

    Fast-response pressure sensitive paint (PSP) is used in this work to measure and analyze the acoustic pressure field in a rectangular cavity. The high spatial resolution and fast frequency response of PSP effectively captures the spatial and temporal detail of surface pressure resulting in the acoustic pressure field. In this work, a high-speed camera is used to generate a continuous time record of the acoustic pressure fluctuations with PSP. Since the level of the acoustic pressure is near the resolution limit of the sensor system, advanced analysis techniques are used to extract the spatial modes of the pressure field. Both dynamic mode decomposition (DMD) and proper orthogonal decomposition (POD) are compared with phase averaging for data analysis. While all three techniques effectively extract the pressure field and reduce the impact of sensor noise, DMD and POD are more robust techniques that can be applied to aperiodic or multi-frequency signals. Furthermore, DMD is better than POD at suppressing noise in particular regions of the spectrum and at effectively separating spectral energy when multiple acoustic excitation frequencies are present.

  3. Fast-slow climate dynamics and peak global warming

    NASA Astrophysics Data System (ADS)

    Seshadri, Ashwin K.

    2016-06-01

    The dynamics of a linear two-box energy balance climate model is analyzed as a fast-slow system, where the atmosphere, land, and near-surface ocean taken together respond within few years to external forcing whereas the deep-ocean responds much more slowly. Solutions to this system are approximated by estimating the system's time-constants using a first-order expansion of the system's eigenvalue problem in a perturbation parameter, which is the ratio of heat capacities of upper and lower boxes. The solution naturally admits an interpretation in terms of a fast response that depends approximately on radiative forcing and a slow response depending on integrals of radiative forcing with respect to time. The slow response is inversely proportional to the "damping-timescale", the timescale with which deep-ocean warming influences global warming. Applications of approximate solutions are discussed: conditions for a warming peak, effects of an individual pulse emission of carbon dioxide (CO2 ), and metrics for estimating and comparing contributions of different climate forcers to maximum global warming.

  4. Dynamic Mode Decomposition of Fast Pressure Sensitive Paint Data.

    PubMed

    Ali, Mohd Y; Pandey, Anshuman; Gregory, James W

    2016-01-01

    Fast-response pressure sensitive paint (PSP) is used in this work to measure and analyze the acoustic pressure field in a rectangular cavity. The high spatial resolution and fast frequency response of PSP effectively captures the spatial and temporal detail of surface pressure resulting in the acoustic pressure field. In this work, a high-speed camera is used to generate a continuous time record of the acoustic pressure fluctuations with PSP. Since the level of the acoustic pressure is near the resolution limit of the sensor system, advanced analysis techniques are used to extract the spatial modes of the pressure field. Both dynamic mode decomposition (DMD) and proper orthogonal decomposition (POD) are compared with phase averaging for data analysis. While all three techniques effectively extract the pressure field and reduce the impact of sensor noise, DMD and POD are more robust techniques that can be applied to aperiodic or multi-frequency signals. Furthermore, DMD is better than POD at suppressing noise in particular regions of the spectrum and at effectively separating spectral energy when multiple acoustic excitation frequencies are present. PMID:27294939

  5. Dynamic Mode Decomposition of Fast Pressure Sensitive Paint Data

    PubMed Central

    Ali, Mohd Y.; Pandey, Anshuman; Gregory, James W.

    2016-01-01

    Fast-response pressure sensitive paint (PSP) is used in this work to measure and analyze the acoustic pressure field in a rectangular cavity. The high spatial resolution and fast frequency response of PSP effectively captures the spatial and temporal detail of surface pressure resulting in the acoustic pressure field. In this work, a high-speed camera is used to generate a continuous time record of the acoustic pressure fluctuations with PSP. Since the level of the acoustic pressure is near the resolution limit of the sensor system, advanced analysis techniques are used to extract the spatial modes of the pressure field. Both dynamic mode decomposition (DMD) and proper orthogonal decomposition (POD) are compared with phase averaging for data analysis. While all three techniques effectively extract the pressure field and reduce the impact of sensor noise, DMD and POD are more robust techniques that can be applied to aperiodic or multi-frequency signals. Furthermore, DMD is better than POD at suppressing noise in particular regions of the spectrum and at effectively separating spectral energy when multiple acoustic excitation frequencies are present. PMID:27294939

  6. A fast-marching like algorithm for geometrical shock dynamics

    NASA Astrophysics Data System (ADS)

    Noumir, Y.; Le Guilcher, A.; Lardjane, N.; Monneau, R.; Sarrazin, A.

    2015-03-01

    We develop a new algorithm for the computation of the Geometrical Shock Dynamics (GSD) model. The method relies on the fast-marching paradigm and enables the discrete evaluation of the first arrival time of a shock wave and its local velocity on a Cartesian grid. The proposed algorithm is based on a first order upwind finite difference scheme and reduces to a local nonlinear system of two equations solved by an iterative procedure. Reference solutions are built for a smooth radial configuration and for the 2D Riemann problem. The link between the GSD model and p-systems is given. Numerical experiments demonstrate the efficiency of the scheme and its ability to handle singularities.

  7. Wind turbine control systems: Dynamic model development using system identification and the fast structural dynamics code

    SciTech Connect

    Stuart, J.G.; Wright, A.D.; Butterfield, C.P.

    1996-10-01

    Mitigating the effects of damaging wind turbine loads and responses extends the lifetime of the turbine and, consequently, reduces the associated Cost of Energy (COE). Active control of aerodynamic devices is one option for achieving wind turbine load mitigation. Generally speaking, control system design and analysis requires a reasonable dynamic model of {open_quotes}plant,{close_quotes} (i.e., the system being controlled). This paper extends the wind turbine aileron control research, previously conducted at the National Wind Technology Center (NWTC), by presenting a more detailed development of the wind turbine dynamic model. In prior research, active aileron control designs were implemented in an existing wind turbine structural dynamics code, FAST (Fatigue, Aerodynamics, Structures, and Turbulence). In this paper, the FAST code is used, in conjunction with system identification, to generate a wind turbine dynamic model for use in active aileron control system design. The FAST code is described and an overview of the system identification technique is presented. An aileron control case study is used to demonstrate this modeling technique. The results of the case study are then used to propose ideas for generalizing this technique for creating dynamic models for other wind turbine control applications.

  8. Chronic, long-term social stress can cause decreased microtubule protein network activity and dynamics in cerebral cortex of male Wistar rats.

    PubMed

    Eskandari Sedighi, Ghazaleh; Riazi, Gholam Hossein; Vaez Mahdavi, Mohammad Reza; Cheraghi, Tayebe; Atarod, Deyhim; Rafiei, Shahrbanoo

    2015-03-01

    Social stress is viewed as a factor in the etiology of a variety of psychopathologies such as depression and anxiety. Animal models of social stress are well developed and widely used in studying clinical and physiological effects of stress. Stress is known to significantly affect learning and memory, and this effect strongly depends on the type of stress, its intensity, and duration. It has been demonstrated that chronic and acute stress conditions can change neuronal plasticity, characterized by retraction of apical dendrites, reduction in axonogenesis, and decreased neurogenesis. Various behavioral studies have also confirmed a decrease in learning and memory upon exposure of animals to long-term chronic stress. On the other hand, the close relationship between microtubule (MT) protein network and neuroplasticity controlling system suggests the possibility of MT protein alterations in high stressful conditions. In this work, we have studied the kinetics, activity, and dynamicity changes of MT proteins in the cerebral cortex of male Wistar rats that were subjected to social instability for 35 and 100 days. Our results indicate that MT protein network dynamicity and polymerization ability is decreased under long-term (100 days) social stress conditions.

  9. Properties and applications of fast rotors in dynamical optics (abstract)

    SciTech Connect

    Csonka, P. L.

    1989-07-01

    The term ''dynamical optics'' is defined as a set of optical elements at least one of which is nonstationary as seen from the laboratory. Present technology allows the construction of fast rotating mirrors as components in dynamical optical systems. Calculations show that such systems can be used to achieve subpicosecond x-ray pulses and extremely high instantaneous intensities, and, alternatively they can be utilized to saturate the transverse coherence of x-ray beams thereby making possible to perform certain interference experiments. Some properties and additional applications of fast rotating mirrors are discussed: (1) The focusing properties of rotating mirrors can differ significantly from those of stationary reflectors, even though the mirrors are, of course, nonrelativistic. A small (diameter /lt/1 mm) plane mirror when rotating can have a focal length /ital f//sub /ital m// down to a few meters. By changing the speed of rotation, the focal length can be altered continuously from its minimum value, /ital f//sub /ital m//, through infinity (when the mirror is at rest) to /minus//ital f//sub /ital m// (when it's rotation is reversed). Thus, mirrors can be constructed which can be made to both focus and defocus with continuously and accurately variable focal length. (2) Rotating mirrors will induce a frequency change /Delta//omega/ in the reflected beam, allowing one in principle to tune across resonance lines, and also ''active monochromatization'' of x-rays, i.e., increasing the spectral intensity in a selected range, while decreasing it outside that range, all this without any change in the total photon beam intensity. This option is of interest when the total photon intensity cannot be increased beyond a certain limit, high resolution monochromatization is available, and large spectral density is called for in a chosen frequency range.

  10. Colchicine activates actin polymerization by microtubule depolymerization.

    PubMed

    Jung, H I; Shin, I; Park, Y M; Kang, K W; Ha, K S

    1997-06-30

    Swiss 3T3 fibroblasts were treated with the microtubule-disrupting agent colchicine to study any interaction between microtubule dynamics and actin polymerization. Colchicine increased the amount of filamentous actin (F-actin), in a dose- and time-dependent manner with a significant increase at 1 h by about 130% over control level. Confocal microscopic observation showed that colchicine increased F-actin contents by stress fiber formation without inducing membrane ruffling. Colchicine did not activate phospholipase C and phospholipase D, whereas lysophosphatidic acid did, indicating that colchicine may have a different mechanism of actin polymerization regulation from LPA. A variety of microtubule-disrupting agents stimulated actin polymerization in Swiss 3T3 and Rat-2 fibroblasts as did colchicine, but the microtubule-stabilizing agent taxol inhibited actin polymerization induced by the above microtubule-disrupting agents. In addition, colchicine-induced actin polymerization was blocked by two protein phosphatase inhibitors, okadaic acid and calyculin A. These results suggest that microtubule depolymerization activates stress fiber formation by serine/threonine dephosphorylation in fibroblasts. PMID:9264034

  11. Microtubule Elasticity: Connecting All-Atom Simulations with Continuum Mechanics

    NASA Astrophysics Data System (ADS)

    Sept, David; Mackintosh, Fred C.

    2010-01-01

    The mechanical properties of microtubules have been extensively studied using a wide range of biophysical techniques, seeking to understand the mechanics of these cylindrical polymers. Here we develop a method for connecting all-atom molecular dynamics simulations with continuum mechanics and show how this can be applied to understand microtubule mechanics. Our coarse-graining technique applied to the microscopic simulation system yields consistent predictions for the Young’s modulus and persistence length of microtubules, while clearly demonstrating how binding of the drug Taxol decreases the stiffness of microtubules. The techniques we develop should be widely applicable to other macromolecular systems.

  12. 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.

  13. 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.

  14. The family-specific α4-helix of the kinesin-13, MCAK, is critical to microtubule end recognition

    PubMed Central

    Patel, Jennifer T.; Belsham, Hannah R.; Rathbone, Alexandra J.; Wickstead, Bill; Gell, Christopher

    2016-01-01

    Kinesins that influence the dynamics of microtubule growth and shrinkage require the ability to distinguish between the microtubule end and the microtubule lattice. The microtubule depolymerizing kinesin MCAK has been shown to specifically recognize the microtubule end. This ability is key to the action of MCAK in regulating microtubule dynamics. We show that the α4-helix of the motor domain is crucial to microtubule end recognition. Mutation of the residues K524, E525 and R528, which are located in the C-terminal half of the α4-helix, specifically disrupts the ability of MCAK to recognize the microtubule end. Mutation of these residues, which are conserved in the kinesin-13 family and discriminate members of this family from translocating kinesins, impairs the ability of MCAK to discriminate between the microtubule lattice and the microtubule end. PMID:27733589

  15. CLIP-170 facilitates the formation of kinetochore–microtubule attachments

    PubMed Central

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

    2006-01-01

    CLIP-170 is a microtubule ‘plus end tracking' protein involved in several microtubule-dependent processes in interphase. At the onset of mitosis, CLIP-170 localizes to kinetochores, but at metaphase, it is no longer detectable at kinetochores. Although RNA interference (RNAi) experiments have suggested an essential role for CLIP-170 during mitosis, the molecular function of CLIP-170 in mitosis has not yet been revealed. Here, we used a combination of high-resolution microscopy and RNAi-mediated depletion to study the function of CLIP-170 in mitosis. We found that CLIP-170 dynamically localizes to the outer most part of unattached kinetochores and to the ends of growing microtubules. In addition, we provide evidence that a pool of CLIP-170 is transported along kinetochore–microtubules by the dynein/dynactin complex. Interference with CLIP-170 expression results in defective chromosome congression and diminished kinetochore–microtubule attachments, but does not detectibly affect microtubule dynamics or kinetochore–microtubule stability. Taken together, our results indicate that CLIP-170 facilitates the formation of kinetochore–microtubule attachments, possibly through direct capture of microtubules at the kinetochore. PMID:16362039

  16. Fast regional readout CMOS Image Sensor for dynamic MLC tracking

    NASA Astrophysics Data System (ADS)

    Zin, H.; Harris, E.; Osmond, J.; Evans, P.

    2014-03-01

    Advanced radiotherapy techniques such as volumetric modulated arc therapy (VMAT) require verification of the complex beam delivery including tracking of multileaf collimators (MLC) and monitoring the dose rate. This work explores the feasibility of a prototype Complementary metal-oxide semiconductor Image Sensor (CIS) for tracking these complex treatments by utilising fast, region of interest (ROI) read out functionality. An automatic edge tracking algorithm was used to locate the MLC leaves edges moving at various speeds (from a moving triangle field shape) and imaged with various sensor frame rates. The CIS demonstrates successful edge detection of the dynamic MLC motion within accuracy of 1.0 mm. This demonstrates the feasibility of the sensor to verify treatment delivery involving dynamic MLC up to ~400 frames per second (equivalent to the linac pulse rate), which is superior to any current techniques such as using electronic portal imaging devices (EPID). CIS provides the basis to an essential real-time verification tool, useful in accessing accurate delivery of complex high energy radiation to the tumour and ultimately to achieve better cure rates for cancer patients.

  17. Molecular and Mechanical Causes of Microtubule Catastrophe and Aging.

    PubMed

    Zakharov, Pavel; Gudimchuk, Nikita; Voevodin, Vladimir; Tikhonravov, Alexander; Ataullakhanov, Fazoil I; Grishchuk, Ekaterina L

    2015-12-15

    Tubulin polymers, microtubules, can switch abruptly from the assembly to shortening. These infrequent transitions, termed "catastrophes", affect numerous cellular processes but the underlying mechanisms are elusive. We approached this complex stochastic system using advanced coarse-grained molecular dynamics modeling of tubulin-tubulin interactions. Unlike in previous simplified models of dynamic microtubules, the catastrophes in this model arise owing to fluctuations in the composition and conformation of a growing microtubule tip, most notably in the number of protofilament curls. In our model, dynamic evolution of the stochastic microtubule tip configurations over a long timescale, known as the system's "aging", gives rise to the nonexponential distribution of microtubule lifetimes, consistent with experiment. We show that aging takes place in the absence of visible changes in the microtubule wall or tip, as this complex molecular-mechanical system evolves slowly and asymptotically toward the steady-state level of the catastrophe-promoting configurations. This new, to our knowledge, theoretical basis will assist detailed mechanistic investigations of the mechanisms of action of different microtubule-binding proteins and drugs, thereby enabling accurate control over the microtubule dynamics to treat various pathologies. PMID:26682815

  18. 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

  19. Ska3 Ensures Timely Mitotic Progression by Interacting Directly With Microtubules and Ska1 Microtubule Binding Domain

    PubMed Central

    Abad, Maria Alba; Zou, Juan; Medina-Pritchard, Bethan; Nigg, Erich A.; Rappsilber, Juri; Santamaria, Anna; Jeyaprakash, A. Arockia

    2016-01-01

    The establishment of physical attachment between the kinetochore and dynamic spindle microtubules, which undergo cycles of polymerization and depolymerization generating straight and curved microtubule structures, is essential for accurate chromosome segregation. The Ndc80 and Ska complexes are the major microtubule-binding factors of the kinetochore responsible for maintaining chromosome-microtubule coupling during chromosome segregation. We previously showed that the Ska1 subunit of the Ska complex binds dynamic microtubules using multiple contact sites in a mode that allows conformation-independent binding. Here, we show that the Ska3 subunit is required to modulate the microtubule binding capability of the Ska complex (i) by directly interacting with tubulin monomers and (ii) indirectly by interacting with tubulin contacting regions of Ska1 suggesting an allosteric regulation. Perturbing either the Ska3-microtubule interaction or the Ska3-Ska1 interactions negatively influences microtubule binding by the Ska complex in vitro and affects the timely onset of anaphase in cells. Thus, Ska3 employs additional modulatory elements within the Ska complex to ensure robust kinetochore-microtubule attachments and timely progression of mitosis. PMID:27667719

  20. Anti-Microtubule Drugs.

    PubMed

    Florian, Stefan; Mitchison, Timothy J

    2016-01-01

    Small molecule drugs that target microtubules (MTs), many of them natural products, have long been important tools in the MT field. Indeed, tubulin (Tb) was discovered, in part, as the protein binding partner of colchicine. Several anti-MT drug classes also have important medical uses, notably colchicine, which is used to treat gout, familial Mediterranean fever (FMF), and pericarditis, and the vinca alkaloids and taxanes, which are used to treat cancer. Anti-MT drugs have in common that they bind specifically to Tb in the dimer, MT or some other form. However, their effects on polymerization dynamics and on the human body differ markedly. Here we briefly review the most-studied molecules, and comment on their uses in basic research and medicine. Our focus is on practical applications of different anti-MT drugs in the laboratory, and key points that users should be aware of when designing experiments. We also touch on interesting unsolved problems, particularly in the area of medical applications. In our opinion, the mechanism by which any MT drug cures or treats any disease is still unsolved, despite decades of research. Solving this problem for particular drug-disease combinations might open new uses for old drugs, or provide insights into novel routes for treatment. PMID:27193863

  1. Microtubule Self- Assembly

    NASA Astrophysics Data System (ADS)

    Jho, Yongseok; Choi, M. C.; Farago, O.; Kim, Mahnwon; Pincus, P. A.

    2008-03-01

    Microtubules are important structural elements for neurons. Microtubles are cylindrical pipes that are self-assembled from tubulin dimers, These structures are intimately related to the neuron transport system. Abnormal microtubule disintegration contributes to neuro-disease. For several decades, experimentalists investigated the structure of the microtubules using TEM and Cryo-EM. However, the detailed structure at a molecular level remain incompletely understood. . In this presentation, we report numerically studies of the self-assembly process using a toy model for tubulin dimers. We investigate the nature of the interactions which are essential to stabilize such the cylindrical assembly of protofilaments. We use Monte Carlo simulations to suggest the pathways for assembly and disassembly of the microtubules.

  2. 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.

  3. Fast passage dynamic nuclear polarization on rotating solids

    NASA Astrophysics Data System (ADS)

    Mentink-Vigier, Frederic; Akbey, Ümit; Hovav, Yonatan; Vega, Shimon; Oschkinat, Hartmut; Feintuch, Akiva

    2012-11-01

    Magic Angle Spinning (MAS) Dynamic Nuclear Polarization (DNP) has proven to be a very powerful way to improve the signal to noise ratio of NMR experiments on solids. The experiments have in general been interpreted considering the Solid-Effect (SE) and Cross-Effect (CE) DNP mechanisms while ignoring the influence of sample spinning. In this paper, we show experimental data of MAS-DNP enhancements of 1H and 13C in proline and SH3 protein in glass forming water/glycerol solvent containing TOTAPOL. We also introduce a theoretical model that aims at explaining how the nuclear polarization is built in MAS-DNP experiments. By using Liouville space based simulations to include relaxation on two simple spin models, {electron-nucleus} and {electron-electron-nucleus}, we explain how the basic MAS-SE-DNP and MAS-CE-DNP processes work. The importance of fast energy passages and short level anti-crossing is emphasized and the differences between static DNP and MAS-DNP is explained. During a single rotor cycle the enhancement in the {electron-electron-nucleus} system arises from MAS-CE-DNP involving at least three kinds of two-level fast passages: an electron-electron dipolar anti-crossing, a single quantum electron MW encounter and an anti-crossing at the CE condition inducing nuclear polarization in- or decrements. Numerical, powder-averaged, simulations were performed in order to check the influence of the experimental parameters on the enhancement efficiencies. In particular we show that the spinning frequency dependence of the theoretical MAS-CE-DNP enhancement compares favorably with the experimental 1H and 13C MAS-DNP enhancements of proline and SH3.

  4. Fast passage dynamic nuclear polarization on rotating solids.

    PubMed

    Mentink-Vigier, Frederic; Akbey, Umit; Hovav, Yonatan; Vega, Shimon; Oschkinat, Hartmut; Feintuch, Akiva

    2012-11-01

    Magic Angle Spinning (MAS) Dynamic Nuclear Polarization (DNP) has proven to be a very powerful way to improve the signal to noise ratio of NMR experiments on solids. The experiments have in general been interpreted considering the Solid-Effect (SE) and Cross-Effect (CE) DNP mechanisms while ignoring the influence of sample spinning. In this paper, we show experimental data of MAS-DNP enhancements of (1)H and (13)C in proline and SH3 protein in glass forming water/glycerol solvent containing TOTAPOL. We also introduce a theoretical model that aims at explaining how the nuclear polarization is built in MAS-DNP experiments. By using Liouville space based simulations to include relaxation on two simple spin models, {electron-nucleus} and {electron-electron-nucleus}, we explain how the basic MAS-SE-DNP and MAS-CE-DNP processes work. The importance of fast energy passages and short level anti-crossing is emphasized and the differences between static DNP and MAS-DNP is explained. During a single rotor cycle the enhancement in the {electron-electron-nucleus} system arises from MAS-CE-DNP involving at least three kinds of two-level fast passages: an electron-electron dipolar anti-crossing, a single quantum electron MW encounter and an anti-crossing at the CE condition inducing nuclear polarization in- or decrements. Numerical, powder-averaged, simulations were performed in order to check the influence of the experimental parameters on the enhancement efficiencies. In particular we show that the spinning frequency dependence of the theoretical MAS-CE-DNP enhancement compares favorably with the experimental (1)H and (13)C MAS-DNP enhancements of proline and SH3.

  5. Estimation of the diffusion-limited rate of microtubule assembly.

    PubMed Central

    Odde, D J

    1997-01-01

    Microtubule assembly is a complex process with individual microtubules alternating stochastically between extended periods of assembly and disassembly, a phenomenon known as dynamic instability. Since the discovery of dynamic instability, molecular models of assembly have generally assumed that tubulin incorporation into the microtubule lattice is primarily reaction-limited. Recently this assumption has been challenged and the importance of diffusion in microtubule assembly dynamics asserted on the basis of scaling arguments, with tubulin gradients predicted to extend over length scales exceeding a cell diameter, approximately 50 microns. To assess whether individual microtubules in vivo assemble at diffusion-limited rates and to predict the theoretical upper limit on the assembly rate, a steady-state mean-field model for the concentration of tubulin about a growing microtubule tip was developed. Using published parameter values for microtubule assembly in vivo (growth rate = 7 microns/min, diffusivity = 6 x 10(-12) m2/s, tubulin concentration = 10 microM), the model predicted that the tubulin concentration at the microtubule tip was approximately 89% of the concentration far from the tip, indicating that microtubule self-assembly is not diffusion-limited. Furthermore, the gradients extended less than approximately 50 nm (the equivalent of about two microtubule diameters) from the microtubule tip, a distance much less than a cell diameter. In addition, a general relation was developed to predict the diffusion-limited assembly rate from the diffusivity and bulk tubulin concentration. Using this relation, it was estimated that the maximum theoretical assembly rate is approximately 65 microns/min, above which tubulin can no longer diffuse rapidly enough to support faster growth. Images FIGURE 1 PMID:9199774

  6. 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.

  7. Optimization of replica exchange molecular dynamics by fast mimicking.

    PubMed

    Hritz, Jozef; Oostenbrink, Chris

    2007-11-28

    We present an approach to mimic replica exchange molecular dynamics simulations (REMD) on a microsecond time scale within a few minutes rather than the years, which would be required for real REMD. The speed of mimicked REMD makes it a useful tool for "testing" the efficiency of different settings for REMD and then to select those settings, that give the highest efficiency. We present an optimization approach with the example of Hamiltonian REMD using soft-core interactions on two model systems, GTP and 8-Br-GTP. The optimization process using REMD mimicking is very fast. Optimization of Hamiltonian-REMD settings of GTP in explicit water took us less than one week. In our study we focus not only on finding the optimal distances between neighboring replicas, but also on finding the proper placement of the highest level of softness. In addition we suggest different REMD simulation settings at this softness level. We allow several replicas to be simulated at the same Hamiltonian simultaneously and reduce the frequency of switching attempts between them. This approach allows for more efficient conversions from one stable conformation to the other.

  8. Targeting Microtubules for Wound Repair

    PubMed Central

    Charafeddine, Rabab A.; Nosanchuk, Joshua D.; Sharp, David J.

    2016-01-01

    Significance: Fast and seamless healing is essential for both deep and chronic wounds to restore the skin and protect the body from harmful pathogens. Thus, finding new targets that can both expedite and enhance the repair process without altering the upstream signaling milieu and causing serious side effects can improve the way we treat wounds. Since cell migration is key during the different stages of wound healing, it presents an ideal process and intracellular structural machineries to target. Recent Advances and Critical Issues: The microtubule (MT) cytoskeleton is rising as an important structural and functional regulator of wound healing. MTs have been reported to play different roles in the migration of the various cell types involved in wound healing. Specific microtubule regulatory proteins (MRPs) can be targeted to alter a section or subtype of the MT cytoskeleton and boost or hinder cell motility. However, inhibiting intracellular components can be challenging in vivo, especially using unstable molecules, such as small interfering RNA. Nanoparticles can be used to protect these unstable molecules and topically deliver them to the wound. Utilizing this approach, we recently showed that fidgetin-like 2, an uncharacterized MRP, can be targeted to enhance cell migration and wound healing. Future Directions: To harness the full potential of the current MRP therapeutic targets, studies should test them with different delivery platforms, dosages, and skin models. Screening for new MT effectors that boost cell migration in vivo would also help find new targets for skin repair. PMID:27785378

  9. Fast engineering optimization: A novel highly effective control parameterization approach for industrial dynamic processes.

    PubMed

    Liu, Ping; Li, Guodong; Liu, Xinggao

    2015-09-01

    Control vector parameterization (CVP) is an important approach of the engineering optimization for the industrial dynamic processes. However, its major defect, the low optimization efficiency caused by calculating the relevant differential equations in the generated nonlinear programming (NLP) problem repeatedly, limits its wide application in the engineering optimization for the industrial dynamic processes. A novel highly effective control parameterization approach, fast-CVP, is first proposed to improve the optimization efficiency for industrial dynamic processes, where the costate gradient formulae is employed and a fast approximate scheme is presented to solve the differential equations in dynamic process simulation. Three well-known engineering optimization benchmark problems of the industrial dynamic processes are demonstrated as illustration. The research results show that the proposed fast approach achieves a fine performance that at least 90% of the computation time can be saved in contrast to the traditional CVP method, which reveals the effectiveness of the proposed fast engineering optimization approach for the industrial dynamic processes.

  10. 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.

  11. 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

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

    PubMed

    Stepanek, Ludek; Pigino, Gaia

    2016-05-01

    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. PMID:27151870

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

    PubMed Central

    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. PMID:25897332

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

    PubMed

    Stepanek, Ludek; Pigino, Gaia

    2016-05-01

    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.

  15. Kinetics of microtubule catastrophe assessed by probabilistic analysis.

    PubMed

    Odde, D J; Cassimeris, L; Buettner, H M

    1995-09-01

    Microtubules are cytoskeletal filaments whose self-assembly occurs by abrupt switching between states of roughly constant growth and shrinkage, a process known as dynamic instability. Understanding the mechanism of dynamic instability offers potential for controlling microtubule-dependent cellular processes such as nerve growth and mitosis. The growth to shrinkage transitions (catastrophes) and the reverse transitions (rescues) that characterize microtubule dynamic instability have been assumed to be random events with first-order kinetics. By direct observation of individual microtubules in vitro and probabilistic analysis of their distribution of growth times, we found that while the slower growing and biologically inactive (minus) ends obeyed first-order catastrophe kinetics, the faster growing and biologically active (plus) ends did not. The non-first-order kinetics at plus ends imply that growing microtubule plus ends have an effective frequency of catastrophe that depends on how long the microtubules have been growing. This frequency is low initially but then rises asymptotically to a limiting value. Our results also suggest that an additional parameter, beyond the four parameters typically used to describe dynamic instability, is needed to account for the observed behavior and that changing this parameter can significantly affect the distribution of microtubule lengths at steady state. PMID:8519980

  16. Kinetics of microtubule catastrophe assessed by probabilistic analysis.

    PubMed Central

    Odde, D J; Cassimeris, L; Buettner, H M

    1995-01-01

    Microtubules are cytoskeletal filaments whose self-assembly occurs by abrupt switching between states of roughly constant growth and shrinkage, a process known as dynamic instability. Understanding the mechanism of dynamic instability offers potential for controlling microtubule-dependent cellular processes such as nerve growth and mitosis. The growth to shrinkage transitions (catastrophes) and the reverse transitions (rescues) that characterize microtubule dynamic instability have been assumed to be random events with first-order kinetics. By direct observation of individual microtubules in vitro and probabilistic analysis of their distribution of growth times, we found that while the slower growing and biologically inactive (minus) ends obeyed first-order catastrophe kinetics, the faster growing and biologically active (plus) ends did not. The non-first-order kinetics at plus ends imply that growing microtubule plus ends have an effective frequency of catastrophe that depends on how long the microtubules have been growing. This frequency is low initially but then rises asymptotically to a limiting value. Our results also suggest that an additional parameter, beyond the four parameters typically used to describe dynamic instability, is needed to account for the observed behavior and that changing this parameter can significantly affect the distribution of microtubule lengths at steady state. Images FIGURE 1 PMID:8519980

  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. 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.

  19. 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.

  20. Microtubule-severing enzymes at the cutting edge

    PubMed Central

    Sharp, David J.; Ross, Jennifer L.

    2012-01-01

    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. PMID:22595526

  1. 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.

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

    PubMed

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

    2011-11-23

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

  3. Characteristics of the polar assembly and disassembly of microtubules observed in vitro by darkfield light microscopy

    PubMed Central

    1979-01-01

    We describe here the continuous observations of the polymerization of individual microtubules in vitro by darkfield microscopy. In homogeneous preparations we verify that polymerization can occur onto both ends of microtubules. The assembly of microtubules is polar, with one end growing at three times the rate of the other. The differential rate of elongation can be used to determine the polarity of growth off cellular nucleating centers. We show that the microtubules grow off the proximal end of ciliary axonemes at a growth rate equal to that of the slow growing end of free microtubules, while growth off the distal end proceeds at the same rate as the fast growing end. Applying this technique to microtubule growth from metaphase chromosomes isolated from HeLa and CHO cells, we demonstrate that chromosomes initiate polymerization with the fast growing end facing away from the chromosome nucleation site. The opposite ends of free microtubules show different sensitivities to microtubule depolymerizing agents such as low temperature, Ca++ or colchicine as measured directly by darkfield microscopy. The differing rates of assembly and disassembly of each end of a microtubule suggest that a difference in polarity of growth off nucleating sites could serve as one basis for regulating the polymerization of different groups of microtubules in the same cell. PMID:511939

  4. How dynein and microtubules rotate the nucleus.

    PubMed

    Wu, Jun; Lee, Kristen C; Dickinson, Richard B; Lele, Tanmay P

    2011-10-01

    In living cells, a fluctuating torque is exerted on the nuclear surface but the origin of the torque is unclear. In this study, we found that the nuclear rotation angle is directionally persistent on a time scale of tens of minutes, but rotationally diffusive on longer time scales. Rotation required the activity of the microtubule motor dynein. We formulated a model based on microtubules undergoing dynamic instability, with tensional forces between a stationary centrosome and the nuclear surface mediated by dynein. Model simulations suggest that the persistence in rotation angle is due to the transient asymmetric configuration of microtubules exerting a net torque in one direction until the configuration is again randomized by dynamic instability. The model predicts that the rotational magnitude must depend on the distance between the nucleus and the centrosome. To test this prediction, rotation was quantified in patterned cells in which the cell's centrosome was close to the projected nuclear centroid. Consistent with the prediction, the angular displacement was found to decrease in these cells relative to unpatterned cells. This work provides the first mechanistic explanation for how nuclear dynein interactions with discrete microtubules emanating from a stationary centrosome cause rotational torque on the nucleus.

  5. Comment on ``Fast dynamics of glass-forming glycerol''

    NASA Astrophysics Data System (ADS)

    Ngai, K. L.; Roland, C. M.

    1997-02-01

    The coupling model predicts the existence of a prominent fast α process at times <2 ps in fragile glass-forming liquids such as polymers. A further prediction is that this process will be only weakly apparent in intermediate and strong liquids such as glycerol. In light of these predictions, we have reexamined the neutron scattering data of glycerol published by Wuttke, Petry, Coddens, and Fujara (WPCF) [Phys. Rev. E 52, 4026 (1995)], leading to the conclusion that the fast α process is indeed quite weak therein. Thus, the coupling model predictions are fully consistent with the glycerol data, notwithstanding WPCF's statement to the contrary.

  6. 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.

  7. Using dynamic interferometric synthetic aperature radar (InSAR) to image fast-moving surface waves

    DOEpatents

    Vincent, Paul

    2005-06-28

    A new differential technique and system for imaging dynamic (fast moving) surface waves using Dynamic Interferometric Synthetic Aperture Radar (InSAR) is introduced. This differential technique and system can sample the fast-moving surface displacement waves from a plurality of moving platform positions in either a repeat-pass single-antenna or a single-pass mode having a single-antenna dual-phase receiver or having dual physically separate antennas, and reconstruct a plurality of phase differentials from a plurality of platform positions to produce a series of desired interferometric images of the fast moving waves.

  8. Compression dynamics of an indirect drive fast ignition target

    SciTech Connect

    R.B. Stephens; S.P. Hatchett; R.E. Turner; K.A. Tanaka; R. Kodama; R. Kodama; J.M. Soures

    2002-11-12

    In an x-ray driven reentrant cone fast ignition target the x-ray spectrum contains a high energy component that cause preheating of the reentrant cone and mixing of its gold into the collapsing shell. Direct laser drive might avoid this problem.

  9. Suppression of dynamics and frequency synchronization in coupled slow and fast dynamical systems

    NASA Astrophysics Data System (ADS)

    Gupta, Kajari; Ambika, G.

    2016-06-01

    We present our study on the emergent states of two interacting nonlinear systems with differing dynamical time scales. We find that the inability of the interacting systems to fall in step leads to difference in phase as well as change in amplitude. If the mismatch is small, the systems settle to a frequency synchronized state with constant phase difference. But as mismatch in time scale increases, the systems have to compromise to a state of no oscillations. We illustrate this for standard nonlinear systems and identify the regions of quenched dynamics in the parameter plane. The transition curves to this state are studied analytically and confirmed by direct numerical simulations. As an important special case, we revisit the well-known model of coupled ocean-atmosphere system used in climate studies for the interactive dynamics of a fast oscillating atmosphere and slowly changing ocean. Our study in this context indicates occurrence of multi stable periodic states and steady states of convection coexisting in the system, with a complex basin structure.

  10. Mechanism of impaired microtubule-dependent peroxisome trafficking and oxidative stress in SPAST-mutated cells from patients with Hereditary Spastic Paraplegia

    PubMed Central

    Wali, Gautam; Sutharsan, Ratneswary; Fan, Yongjun; Stewart, Romal; Tello Velasquez, Johana; Sue, Carolyn M; Crane, Denis I.; Mackay-Sim, Alan

    2016-01-01

    Hereditary spastic paraplegia (HSP) is an inherited neurological condition that leads to progressive spasticity and gait abnormalities. Adult-onset HSP is most commonly caused by mutations in SPAST, which encodes spastin a microtubule severing protein. In olfactory stem cell lines derived from patients carrying different SPAST mutations, we investigated microtubule-dependent peroxisome movement with time-lapse imaging and automated image analysis. The average speed of peroxisomes in patient-cells was slower, with fewer fast moving peroxisomes than in cells from healthy controls. This was not because of impairment of peroxisome-microtubule interactions because the time-dependent saltatory dynamics of movement of individual peroxisomes was unaffected in patient-cells. Our observations indicate that average peroxisome speeds are less in patient-cells because of the lower probability of individual peroxisome interactions with the reduced numbers of stable microtubules: peroxisome speeds in patient cells are restored by epothilone D, a tubulin-binding drug that increases the number of stable microtubules to control levels. Patient-cells were under increased oxidative stress and were more sensitive than control-cells to hydrogen peroxide, which is primarily metabolised by peroxisomal catalase. Epothilone D also ameliorated patient-cell sensitivity to hydrogen-peroxide. Our findings suggest a mechanism for neurodegeneration whereby SPAST mutations indirectly lead to impaired peroxisome transport and oxidative stress. PMID:27229699

  11. 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.

  12. Electrostatically biased binding of kinesin to microtubules.

    PubMed

    Grant, Barry J; Gheorghe, Dana M; Zheng, Wenjun; Alonso, Maria; Huber, Gary; Dlugosz, Maciej; McCammon, J Andrew; Cross, Robert A

    2011-11-01

    The minimum motor domain of kinesin-1 is a single head. Recent evidence suggests that such minimal motor domains generate force by a biased binding mechanism, in which they preferentially select binding sites on the microtubule that lie ahead in the progress direction of the motor. A specific molecular mechanism for biased binding has, however, so far been lacking. Here we use atomistic Brownian dynamics simulations combined with experimental mutagenesis to show that incoming kinesin heads undergo electrostatically guided diffusion-to-capture by microtubules, and that this produces directionally biased binding. Kinesin-1 heads are initially rotated by the electrostatic field so that their tubulin-binding sites face inwards, and then steered towards a plus-endwards binding site. In tethered kinesin dimers, this bias is amplified. A 3-residue sequence (RAK) in kinesin helix alpha-6 is predicted to be important for electrostatic guidance. Real-world mutagenesis of this sequence powerfully influences kinesin-driven microtubule sliding, with one mutant producing a 5-fold acceleration over wild type. We conclude that electrostatic interactions play an important role in the kinesin stepping mechanism, by biasing the diffusional association of kinesin with microtubules. PMID:22140358

  13. Electrostatically Biased Binding of Kinesin to Microtubules

    PubMed Central

    Zheng, Wenjun; Alonso, Maria; Huber, Gary; Dlugosz, Maciej; McCammon, J. Andrew; Cross, Robert A.

    2011-01-01

    The minimum motor domain of kinesin-1 is a single head. Recent evidence suggests that such minimal motor domains generate force by a biased binding mechanism, in which they preferentially select binding sites on the microtubule that lie ahead in the progress direction of the motor. A specific molecular mechanism for biased binding has, however, so far been lacking. Here we use atomistic Brownian dynamics simulations combined with experimental mutagenesis to show that incoming kinesin heads undergo electrostatically guided diffusion-to-capture by microtubules, and that this produces directionally biased binding. Kinesin-1 heads are initially rotated by the electrostatic field so that their tubulin-binding sites face inwards, and then steered towards a plus-endwards binding site. In tethered kinesin dimers, this bias is amplified. A 3-residue sequence (RAK) in kinesin helix alpha-6 is predicted to be important for electrostatic guidance. Real-world mutagenesis of this sequence powerfully influences kinesin-driven microtubule sliding, with one mutant producing a 5-fold acceleration over wild type. We conclude that electrostatic interactions play an important role in the kinesin stepping mechanism, by biasing the diffusional association of kinesin with microtubules. PMID:22140358

  14. Chromatin-Bound Xenopus Dppa2 Shapes the Nucleus by Locally Inhibiting Microtubule Assembly

    PubMed Central

    Xue, John Z.; Woo, Eileen M.; Postow, Lisa; Chait, Brian T.; Funabiki, Hironori

    2013-01-01

    SUMMARY Nuclear shape and size vary between species, during development and in many tissue pathologies, but the causes and effects of these differences remain poorly understood. During fertilization, sperm nuclei undergo a dramatic conversion from a heavily compacted form into decondensed, spherical pronuclei, accompanied by rapid nucleation of microtubules from centrosomes. Here we report that the assembly of the spherical nucleus depends on a critical balance of microtubule dynamics, which is regulated by the chromatin-binding protein Developmental pluripotency-associated 2 (Dppa2). While microtubules normally promote sperm pronuclear expansion, in Dppa2-depleted Xenopus egg extracts excess microtubules cause pronuclear assembly defects leading to abnormal morphology and disorganized DNA replication. Dppa2 inhibits microtubule polymerization in vitro, and Dppa2 activity is needed at a precise time and location during nascent pronuclear formation. This demonstrates a strict spatiotemporal requirement for local suppression of microtubules during nuclear formation, fulfilled by chromatin-bound microtubule regulators. PMID:24075807

  15. 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.

  16. Tunable PIE and synchronized gating detections by FastFLIM for quantitative microscopy measurements of fast dynamics of single molecules

    NASA Astrophysics Data System (ADS)

    Sun, Yuansheng; Coskun, Ulas; Ferreon, Allan Chris; Barbieri, Beniamino; Liao, Shih-Chu Jeff

    2016-03-01

    The crosstalk between two fluorescent species causes problems in fluorescence microscopy imaging, especially for quantitative measurements such as co-localization, Förster resonance energy transfer (FRET), fluorescence cross correlation spectroscopy (FCCS). In laser scanning confocal microscopy, the lasers can be switched on and off by acousto-optic tunable filters (AOTF) in the microsecond scale for alternative line scanning in order to avoid the crosstalk while minimizing the time delay between two lasers on the same pixel location. In contrast, the pulsed interleaved excitation (PIE) technique synchronizes two pulsed lasers of different wavelengths in the nanosecond scale to enable measuring superfast dynamics of two fluorescent species simultaneously and yet quantitatively without the crosstalk contamination. This feature is critical for many cell biology applications, e.g. accurate determination of stoichiometry in FRET measurements for studying protein-protein interactions or cell signal events, detection of weaker bindings in FCCS by eliminating the false cross correlation due to the crosstalk. The PIE has been used with the time correlated single photon counting (TCSPC) electronics. Here, we describe a novel PIE development using the digital frequency domain (DFD) technique -- FastFLIM, which provides tunable PIE setups and synchronized gating detections, tailored and optimized to specific applications. A few PIE setups by FastFLIM and measurement examples are described. Combined with the sensitivity of Alba and Q2 systems, the PIE allowed us to quantitatively measure the fast dynamics of single molecules.

  17. Compression Dynamics of an Indirect Drive Fast Ignition Target

    NASA Astrophysics Data System (ADS)

    Stephens, R. B.; Hatchett, S. A.; Turner, R. E.; Tanaka, K. A.; Kodama, R.; Soures, J.

    2002-11-01

    We have compared the compression of an indirectly driven cone-in-shell target, a type proposed for the fast ignition concept, with models. The experimental parameters -500 μm diameter plastic shell with 60 μm thick wall were a 1/5 scale realization of a fast ignition target designed for NIF (absorbing 180 kJ for compression and ˜30 kJ for ignition, and yielding ˜30 MJ) [1]. The implosion was backlit with 6.4 keV x-rays, and observed with a framing camera which captured the implosion from ˜2.6 to 3.3 ns after the onset. The collapsing structure was very similar to model predictions except that non-thermal m-band emissions from the hohlraum penetrated the shell and vaporized gold off the reentrant cone. This could be eliminated by changing the hohlraum composition. [1] S. Hatchett, et al., 5th Wkshp on Fast Ignition of Fusion Targets (Satellite Wkshp, 28th EPS Conf. on Contr. Fusion and Plasma Phys.), Madeira, Portugal (2001).

  18. Tau antagonizes end-binding protein tracking at microtubule ends through a phosphorylation-dependent mechanism.

    PubMed

    Ramirez-Rios, Sacnicte; Denarier, Eric; Prezel, Eléa; Vinit, Angélique; Stoppin-Mellet, Virginie; Devred, François; Barbier, Pascale; Peyrot, Vincent; Sayas, Carmen Laura; Avila, Jesus; Peris, Leticia; Andrieux, Annie; Serre, Laurence; Fourest-Lieuvin, Anne; Arnal, Isabelle

    2016-10-01

    Proper regulation of microtubule dynamics is essential for cell functions and involves various microtubule-associated proteins (MAPs). Among them, end-binding proteins (EBs) accumulate at microtubule plus ends, whereas structural MAPs bind along the microtubule lattice. Recent data indicate that the structural MAP tau modulates EB subcellular localization in neurons. However, the molecular determinants of EB/tau interaction remain unknown, as is the effect of this interplay on microtubule dynamics. Here we investigate the mechanisms governing EB/tau interaction in cell-free systems and cellular models. We find that tau inhibits EB tracking at microtubule ends. Tau and EBs form a complex via the C-terminal region of EBs and the microtubule-binding sites of tau. These two domains are required for the inhibitory activity of tau on EB localization to microtubule ends. Moreover, the phosphomimetic mutation S262E within tau microtubule-binding sites impairs EB/tau interaction and prevents the inhibitory effect of tau on EB comets. We further show that microtubule dynamic parameters vary, depending on the combined activities of EBs and tau proteins. Overall our results demonstrate that tau directly antagonizes EB function through a phosphorylation-dependent mechanism. This study highlights a novel role for tau in EB regulation, which might be impaired in neurodegenerative disorders.

  19. 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

    2015-11-01

    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. PMID:26426829

  20. 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

    2015-11-01

    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.

  1. 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.

  2. Wide dynamic range neutron flux monitor having fast time response for the Large Helical Device.

    PubMed

    Isobe, M; Ogawa, K; Miyake, H; Hayashi, H; Kobuchi, T; Nakano, Y; Watanabe, K; Uritani, A; Misawa, T; Nishitani, T; Tomitaka, M; Kumagai, T; Mashiyama, Y; Ito, D; Kono, S; Yamauchi, M; Takeiri, Y

    2014-11-01

    A fast time response, wide dynamic range neutron flux monitor has been developed toward the LHD deuterium operation by using leading-edge signal processing technologies providing maximum counting rate up to ∼5 × 10(9) counts/s. Because a maximum total neutron emission rate over 1 × 10(16) n/s is predicted in neutral beam-heated LHD plasmas, fast response and wide dynamic range capabilities of the system are essential. Preliminary tests have demonstrated successful performance as a wide dynamic range monitor along the design.

  3. Wide dynamic range neutron flux monitor having fast time response for the Large Helical Device

    SciTech Connect

    Isobe, M. Takeiri, Y.; Ogawa, K.; Miyake, H.; Hayashi, H.; Kobuchi, T.; Nakano, Y.; Watanabe, K.; Uritani, A.; Misawa, T.; Nishitani, T.; Tomitaka, M.; Kumagai, T.; Mashiyama, Y.; Ito, D.; Kono, S.; Yamauchi, M.

    2014-11-15

    A fast time response, wide dynamic range neutron flux monitor has been developed toward the LHD deuterium operation by using leading-edge signal processing technologies providing maximum counting rate up to ∼5 × 10{sup 9} counts/s. Because a maximum total neutron emission rate over 1 × 10{sup 16} n/s is predicted in neutral beam-heated LHD plasmas, fast response and wide dynamic range capabilities of the system are essential. Preliminary tests have demonstrated successful performance as a wide dynamic range monitor along the design.

  4. Wide dynamic range neutron flux monitor having fast time response for the Large Helical Devicea)

    NASA Astrophysics Data System (ADS)

    Isobe, M.; Ogawa, K.; Miyake, H.; Hayashi, H.; Kobuchi, T.; Nakano, Y.; Watanabe, K.; Uritani, A.; Misawa, T.; Nishitani, T.; Tomitaka, M.; Kumagai, T.; Mashiyama, Y.; Ito, D.; Kono, S.; Yamauchi, M.; Takeiri, Y.

    2014-11-01

    A fast time response, wide dynamic range neutron flux monitor has been developed toward the LHD deuterium operation by using leading-edge signal processing technologies providing maximum counting rate up to ˜5 × 109 counts/s. Because a maximum total neutron emission rate over 1 × 1016 n/s is predicted in neutral beam-heated LHD plasmas, fast response and wide dynamic range capabilities of the system are essential. Preliminary tests have demonstrated successful performance as a wide dynamic range monitor along the design.

  5. 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

    2016-01-01

    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

  6. 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

  7. 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

  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. Volumetric imaging of fast biological dynamics in deep tissue via wavefront engineering

    NASA Astrophysics Data System (ADS)

    Kong, Lingjie; Tang, Jianyong; Cui, Meng

    2016-03-01

    To reveal fast biological dynamics in deep tissue, we combine two wavefront engineering methods that were developed in our laboratory, namely optical phase-locked ultrasound lens (OPLUL) based volumetric imaging and iterative multiphoton adaptive compensation technique (IMPACT). OPLUL is used to generate oscillating defocusing wavefront for fast axial scanning, and IMPACT is used to compensate the wavefront distortions for deep tissue imaging. We show its promising applications in neuroscience and immunology.

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

    PubMed Central

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

    2015-01-01

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

  11. Ohm's law in the fast lane: general relatiivistic charge dynamics

    NASA Technical Reports Server (NTRS)

    Meier, D.

    2004-01-01

    Fully relativistic and causal equations for the flow of charge in curved spacetime are derived. It is believed that this is the first set of equations to be published that correctly describes the flow of charge, as well as the evolution of the electromagnetic field, in highly dynamical relativistic environments on timescales much shorter than the collapse time (GM/c3).

  12. Diffusion and formation of microtubule asters: physical processes versus biochemical regulation.

    PubMed Central

    Dogterom, M; Maggs, A C; Leibler, S

    1995-01-01

    Microtubule asters forming the mitotic spindle are assembled around two centrosomes through the process of dynamic instability in which microtubules alternate between growing and shrinking states. By modifying the dynamics of this assembly process, cell cycle enzymes, such as cdc2 cyclin kinases, regulate length distributions in the asters. It is believed that the same enzymes control the number of assembled microtubules by changing the "nucleating activity" of the centrosomes. Here we show that assembly of microtubule asters may be strongly altered by effects connected with diffusion of tubulin monomers. Theoretical analysis of a simple model describing assembly of microtubule asters clearly shows the existence of a region surrounding the centrosome depleted in GTP tubulin. The number of assembled microtubules may in some cases be limited by this depletion effect rather than by the number of available nucleation sites on the centrosome. Images Fig. 1 Fig. 2 PMID:7624308

  13. Direct Microtubule-Binding by Myosin-10 Orients Centrosomes toward Retraction Fibers and Subcortical Actin Clouds.

    PubMed

    Kwon, Mijung; Bagonis, Maria; Danuser, Gaudenz; Pellman, David

    2015-08-10

    Positioning of centrosomes is vital for cell division and development. In metazoan cells, spindle positioning is controlled by a dynamic pool of subcortical actin that organizes in response to the position of retraction fibers. These actin "clouds" are proposed to generate pulling forces on centrosomes and mediate spindle orientation. However, the motors that pull astral microtubules toward these actin structures are not known. Here, we report that the unconventional myosin, Myo10, couples actin-dependent forces from retraction fibers and subcortical actin clouds to centrosomes. Myo10-mediated centrosome positioning requires its direct microtubule binding. Computational image analysis of large microtubule populations reveals a direct effect of Myo10 on microtubule dynamics and microtubule-cortex interactions. Myo10's role in centrosome positioning is distinct from, but overlaps with, that of dynein. Thus, Myo10 plays a key role in integrating the actin and microtubule cytoskeletons to position centrosomes and mitotic spindles.

  14. Direct Microtubule-Binding by Myosin-10 Orients Centrosomes toward Retraction Fibers and Subcortical Actin Clouds.

    PubMed

    Kwon, Mijung; Bagonis, Maria; Danuser, Gaudenz; Pellman, David

    2015-08-10

    Positioning of centrosomes is vital for cell division and development. In metazoan cells, spindle positioning is controlled by a dynamic pool of subcortical actin that organizes in response to the position of retraction fibers. These actin "clouds" are proposed to generate pulling forces on centrosomes and mediate spindle orientation. However, the motors that pull astral microtubules toward these actin structures are not known. Here, we report that the unconventional myosin, Myo10, couples actin-dependent forces from retraction fibers and subcortical actin clouds to centrosomes. Myo10-mediated centrosome positioning requires its direct microtubule binding. Computational image analysis of large microtubule populations reveals a direct effect of Myo10 on microtubule dynamics and microtubule-cortex interactions. Myo10's role in centrosome positioning is distinct from, but overlaps with, that of dynein. Thus, Myo10 plays a key role in integrating the actin and microtubule cytoskeletons to position centrosomes and mitotic spindles. PMID:26235048

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

    NASA Astrophysics Data System (ADS)

    Sendek, Austin; Singh, Rajiv; Cox, Daniel

    2013-03-01

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

  16. γ-Tubulin complexes in microtubule nucleation and beyond.

    PubMed

    Oakley, Berl R; Paolillo, Vitoria; Zheng, Yixian

    2015-09-01

    Tremendous progress has been made in understanding the functions of γ-tubulin and, in particular, its role in microtubule nucleation since the publication of its discovery in 1989. The structure of γ-tubulin has been determined, and the components of γ-tubulin complexes have been identified. Significant progress in understanding the structure of the γ-tubulin ring complex and its components has led to a persuasive model for how these complexes nucleate microtubule assembly. At the same time, data have accumulated that γ-tubulin has important but less well understood functions that are not simply a consequence of its function in microtubule nucleation. These include roles in the regulation of plus-end microtubule dynamics, gene regulation, and mitotic and cell cycle regulation. Finally, evidence is emerging that γ-tubulin mutations or alterations of γ-tubulin expression play an important role in certain types of cancer and in other diseases.

  17. γ-Tubulin complexes in microtubule nucleation and beyond

    PubMed Central

    Oakley, Berl R.; Paolillo, Vitoria; Zheng, Yixian

    2015-01-01

    Tremendous progress has been made in understanding the functions of γ-tubulin and, in particular, its role in microtubule nucleation since the publication of its discovery in 1989. The structure of γ-tubulin has been determined, and the components of γ-tubulin complexes have been identified. Significant progress in understanding the structure of the γ-tubulin ring complex and its components has led to a persuasive model for how these complexes nucleate microtubule assembly. At the same time, data have accumulated that γ-tubulin has important but less well understood functions that are not simply a consequence of its function in microtubule nucleation. These include roles in the regulation of plus-end microtubule dynamics, gene regulation, and mitotic and cell cycle regulation. Finally, evidence is emerging that γ-tubulin mutations or alterations of γ-tubulin expression play an important role in certain types of cancer and in other diseases. PMID:26316498

  18. Fluorescent markers of the microtubule cytoskeleton in Zymoseptoria tritici

    PubMed Central

    Schuster, M.; Kilaru, S.; Latz, M.; Steinberg, G.

    2015-01-01

    The microtubule cytoskeleton supports vital processes in fungal cells, including hyphal growth and mitosis. Consequently, it is a target for fungicides, such as benomyl. The use of fluorescent fusion proteins to illuminate microtubules and microtubule-associated proteins has led to a break-through in our understanding of their dynamics and function in fungal cells. Here, we introduce fluorescent markers to visualize microtubules and accessory proteins in the wheat pathogen Zymoseptoria tritici. We fused enhanced green-fluorescent protein to α-tubulin (ZtTub2), to ZtPeb1, a homologue of the mammalian plus-end binding protein EB1, and to ZtGrc1, a component of the minus-end located γ-tubulin ring complex, involved in the nucleation of microtubules. In vivo observation confirms the localization and dynamic behaviour of all three markers. These marker proteins are useful tools for understanding the organization and importance of the microtubule cytoskeleton in Z. tritici. PMID:25857261

  19. Spatial Heat Maps from Fast Information Matching of Fast and Slow Degrees of Freedom: Application to Molecular Dynamics Simulations.

    PubMed

    Kovacs, Julio A; Wriggers, Willy

    2016-08-25

    We introduce a fast information matching (FIM) method for transforming time domain data into spatial images through handshaking between fast and slow degrees of freedom. The analytics takes advantage of the detailed time series available from biomolecular computer simulations, and it yields spatial heat maps that can be visualized on 3D molecular structures or in the form of interaction networks. The speed of our efficient mutual information solver is on the order of a basic Pearson cross-correlation calculation. We demonstrate that the FIM method is superior to linear cross-correlation for the detection of nonlinear dependence in challenging situations where measures for the global dynamics (the "activity") diverge. The analytics is applied to the detection of hinge-bending hot spots and to the prediction of pairwise contacts between residues that are relevant for the global activity exhibited by the molecular dynamics (MD) trajectories. Application examples from various MD laboratories include the millisecond bovine pancreatic trypsin inhibitor (BPTI) trajectory using canonical MD, a Gaussian accelerated MD folding trajectory of chignolin, and the heat-induced unfolding of engrailed homeodomain (EnHD). The FIM implementation will be freely disseminated with our open-source package, TimeScapes.

  20. Proteomic analysis of Brassica stigmatic proteins following the self-incompatibility reaction reveals a role for microtubule dynamics during pollen responses.

    PubMed

    Samuel, Marcus A; Tang, Wenqiang; Jamshed, Muhammad; Northey, Julian; Patel, Darshan; Smith, Daryl; Siu, K W Michael; Muench, Douglas G; Wang, Zhi-Yong; Goring, Daphne R

    2011-12-01

    Mate selection and maintenance of genetic diversity is crucial to successful reproduction and species survival. Plants utilize self-incompatibility system as a genetic barrier to prevent self pollen from developing on the pistil, leading to hybrid vigor and diversity. In Brassica (canola, kale, and broccoli), an allele-specific interaction between the pollen SCR/SP11 (S-locus cysteine rich protein/S locus protein 11) and the pistil S Receptor Kinase, results in the activation of SRK which recruits the Arm Repeat Containing 1 (ARC1) E3 ligase to the proteasome. The targets of Arm Repeat Containing 1 are proposed to be compatibility factors, which when targeted for degradation by Arm Repeat Containing 1 results in pollen rejection. Despite the fact that protein degradation is predicted to be important for successful self-pollen rejection, the identity of the various proteins whose abundance is altered by the SI pathway has remained unknown. To identify potential candidate proteins regulated by the SI response, we have used the two-dimensional difference gel electrophoresis analysis, coupled with matrix-assisted laser desorption ionization/time of flight/MS. We identified 56 differential protein spots with 19 unique candidate proteins whose abundance is down-regulated following self-incompatible pollinations. The identified differentials are predicted to function in various pathways including biosynthetic pathways, signaling, cytoskeletal organization, and exocytosis. From the 19 unique proteins identified, we investigated the role of tubulin and the microtubule network during both self-incompatible and compatible pollen responses. Moderate changes in the microtubule network were observed with self-incompatible pollinations; however, a more distinct localized break-down of the microtubule network was observed during compatible pollinations, that is likely mediated by EXO70A1, leading to successful pollination. PMID:21890472

  1. Anomalous Flexural Behaviors of Microtubules

    PubMed Central

    Liu, Xiaojing; Zhou, Youhe; Gao, Huajian; Wang, Jizeng

    2012-01-01

    Apparent controversies exist on whether the persistence length of microtubules depends on its contour length. This issue is particularly challenging from a theoretical point of view due to the tubular structure and strongly anisotropic material property of microtubules. Here we adopt a higher order continuum orthotropic thin shell model to study the flexural behavior of microtubules. Our model overcomes some key limitations of a recent study based on a simplified anisotropic shell model and results in a closed-form solution for the contour-length-dependent persistence length of microtubules, with predictions in excellent agreement with experimental measurements. By studying the ratio between their contour and persistence lengths, we find that microtubules with length at ∼1.5 μm show the lowest flexural rigidity, whereas those with length at ∼15 μm show the highest flexural rigidity. This finding may provide an important theoretical basis for understanding the mechanical structure of mitotic spindles during cell division. Further analysis on the buckling of microtubules indicates that the critical buckling load becomes insensitive to the tube length for relatively short microtubules, in drastic contrast to the classical Euler buckling. These rich flexural behaviors of microtubules are of profound implication for many biological functions and biomimetic molecular devices. PMID:22768935

  2. Fast molecular tracking maps nanoscale dynamics of plasma membrane lipids

    PubMed Central

    Sahl, Steffen J.; Leutenegger, Marcel; Hilbert, Michael; Hell, Stefan W.; Eggeling, Christian

    2010-01-01

    We describe an optical method capable of tracking a single fluorescent molecule with a flexible choice of high spatial accuracy (∼10–20 nm standard deviation or ∼20–40 nm full-width-at-half-maximum) and temporal resolution (< 1 ms). The fluorescence signal during individual passages of fluorescent molecules through a spot of excitation light allows the sequential localization and thus spatio-temporal tracking of the molecule if its fluorescence is collected on at least three separate point detectors arranged in close proximity. We show two-dimensional trajectories of individual, small organic dye labeled lipids diffusing in the plasma membrane of living cells and directly observe transient events of trapping on < 20 nm spatial scales. The trapping is cholesterol-assisted and much more pronounced for a sphingo- than for a phosphoglycero-lipid, with average trapping times of ∼15 ms and < 4 ms, respectively. The results support previous STED nanoscopy measurements and suggest that, at least for nontreated cells, the transient interaction of a single lipid is confined to macromolecular dimensions. Our experimental approach demonstrates that fast molecular movements can be tracked with minimal invasion, which can reveal new important details of cellular nano-organization. PMID:20351247

  3. Startle auditory stimuli enhance the performance of fast dynamic contractions.

    PubMed

    Fernandez-Del-Olmo, Miguel; Río-Rodríguez, Dan; Iglesias-Soler, Eliseo; Acero, Rafael M

    2014-01-01

    Fast reaction times and the ability to develop a high rate of force development (RFD) are crucial for sports performance. However, little is known regarding the relationship between these parameters. The aim of this study was to investigate the effects of auditory stimuli of different intensities on the performance of a concentric bench-press exercise. Concentric bench-presses were performed by thirteen trained subjects in response to three different conditions: a visual stimulus (VS); a visual stimulus accompanied by a non-startle auditory stimulus (AS); and a visual stimulus accompanied by a startle auditory stimulus (SS). Peak RFD, peak velocity, onset movement, movement duration and electromyography from pectoralis and tricep muscles were recorded. The SS condition induced an increase in the RFD and peak velocity and a reduction in the movement onset and duration, in comparison with the VS and AS condition. The onset activation of the pectoralis and tricep muscles was shorter for the SS than for the VS and AS conditions. These findings point out to specific enhancement effects of loud auditory stimulation on the rate of force development. This is of relevance since startle stimuli could be used to explore neural adaptations to resistance training.

  4. Startle Auditory Stimuli Enhance the Performance of Fast Dynamic Contractions

    PubMed Central

    Fernandez-Del-Olmo, Miguel; Río-Rodríguez, Dan; Iglesias-Soler, Eliseo; Acero, Rafael M.

    2014-01-01

    Fast reaction times and the ability to develop a high rate of force development (RFD) are crucial for sports performance. However, little is known regarding the relationship between these parameters. The aim of this study was to investigate the effects of auditory stimuli of different intensities on the performance of a concentric bench-press exercise. Concentric bench-presses were performed by thirteen trained subjects in response to three different conditions: a visual stimulus (VS); a visual stimulus accompanied by a non-startle auditory stimulus (AS); and a visual stimulus accompanied by a startle auditory stimulus (SS). Peak RFD, peak velocity, onset movement, movement duration and electromyography from pectoralis and tricep muscles were recorded. The SS condition induced an increase in the RFD and peak velocity and a reduction in the movement onset and duration, in comparison with the VS and AS condition. The onset activation of the pectoralis and tricep muscles was shorter for the SS than for the VS and AS conditions. These findings point out to specific enhancement effects of loud auditory stimulation on the rate of force development. This is of relevance since startle stimuli could be used to explore neural adaptations to resistance training. PMID:24489967

  5. Fast and Efficient Dynamic WDM Semiconductor Optical Amplifier Model

    NASA Astrophysics Data System (ADS)

    Mathlouthi, Walid; Lemieux, Pascal; Salsi, Massimiliano; Vannucci, Armando; Bononi, Alberto; Rusch, Leslie A.

    2006-11-01

    A novel state-variable model for semiconductor optical amplifiers (SOAs) that is amenable to block diagram implementation of wavelength division multiplexed (WDM) signals and fast execution times is presented. The novel model is called the reservoir model, in analogy with similar block-oriented models for Raman and erbium-doped fiber amplifiers (EDFAs). A procedure is proposed to extract the needed reservoir model parameters from the parameters of a detailed and accurate space-resolved SOA model due to Connelly, which was extended to cope with the time-resolved gain transient analysis. Several variations of the reservoir model are considered with increasing complexity, which allow the accurate inclusion of scattering losses and gain saturation induced by amplified spontaneous emission. It is shown that at comparable accuracy, the reservoir model can be 20 times faster than the Connelly model in single-channel operation; much more significant time savings are expected for WDM operation. The model neglects intraband SOA phenomena and is thus limited to modulation rates per channel not exceeding 10 Gb/s. The SOA reservoir model provides a unique tool with reasonably short computation times for a reliable analysis of gain transients in WDM optical networks with complex topologies.

  6. Size scaling of microtubule asters in confinement

    NASA Astrophysics Data System (ADS)

    Pelletier, James; Field, Christine; Krutkramelis, Kaspars; Fakhri, Nikta; Oakey, John; Gatlin, Jay; Mitchison, Timothy

    Microtubule asters are radial arrays of microtubules (MTs) nucleated around organizing centers (MTOCs). Across a wide range of cell types and sizes, aster positioning influences cellular organization. To investigate aster size and positioning, we reconstituted dynamic asters in Xenopus cytoplasmic extract, confined in fluorous oil microfluidic emulsions. In large droplets, we observed centering of MTOCs. In small droplets, we observed a breakdown in natural positioning, with MTOCs at the droplet edge and buckled or bundled MTs along the interface. In different systems, asters are positioned by different forces, such as pushing due to MT polymerization, or pulling due to bulk or cortical dynein. To estimate different contributions to aster positioning, we biochemically perturbed dynactin function, or MT or actin polymerization. We used carbon nanotubes to measure molecular motions and forces in asters. These experimental results inform quantitative biophysical models of aster size and positioning in confinement. JFP was supported by a Fannie and John Hertz Graduate Fellowship.

  7. Fast Auroral Snapshot performance using a multi-body dynamic simulation

    NASA Technical Reports Server (NTRS)

    Zimbelman, Darrell; Walker, Mary

    1993-01-01

    This paper examines the complex dynamic interaction between two 2.6 m long stacer booms, four 30 m long flexible wire booms and the attitude control system of the Fast Auroral SnapshoT (FAST) spacecraft. The FAST vehicle will nominally operate as a negative orbit spinner, positioned in a 83 deg inclination, 350 x 4200 km orbit. For this study, a three-axis, non-linear, seven body dynamic simulation is developed using the TREETOPS software package. The significance of this approach is the ability to model each component of the FAST spacecraft as an individual member and connect them together in order to better understand the dynamic coupling between structures and the control system. Both the wire and stacer booms are modeled as separate bodies attached to a rigid central body. The wire booms are oriented perpendicular to the spin axis at right angles relative to each other, whereas the stacer booms are aligned with the spin axis. The analysis consists of a comparison between the simulated in-plane and out-of-plane boom motions with theoretically derived frequencies, and an examination of the dynamic coupling between the control system and boom oscillations. Results show that boom oscillations of up to 0.36 deg are acceptable in order to meet the performance requirements. The dynamic motion is well behaved when the precession coil is operating, however, activation of the spin coil produces an erratic trend in the spin rate which approaches the spin rate requirement.

  8. Fast Distributed Dynamics of Semantic Networks via Social Media

    PubMed Central

    Carrillo, Facundo; Cecchi, Guillermo A.; Sigman, Mariano; Fernández Slezak, Diego

    2015-01-01

    We investigate the dynamics of semantic organization using social media, a collective expression of human thought. We propose a novel, time-dependent semantic similarity measure (TSS), based on the social network Twitter. We show that TSS is consistent with static measures of similarity but provides high temporal resolution for the identification of real-world events and induced changes in the distributed structure of semantic relationships across the entire lexicon. Using TSS, we measured the evolution of a concept and its movement along the semantic neighborhood, driven by specific news/events. Finally, we showed that particular events may trigger a temporary reorganization of elements in the semantic network. PMID:26074953

  9. Fast Distributed Dynamics of Semantic Networks via Social Media.

    PubMed

    Carrillo, Facundo; Cecchi, Guillermo A; Sigman, Mariano; Slezak, Diego Fernández

    2015-01-01

    We investigate the dynamics of semantic organization using social media, a collective expression of human thought. We propose a novel, time-dependent semantic similarity measure (TSS), based on the social network Twitter. We show that TSS is consistent with static measures of similarity but provides high temporal resolution for the identification of real-world events and induced changes in the distributed structure of semantic relationships across the entire lexicon. Using TSS, we measured the evolution of a concept and its movement along the semantic neighborhood, driven by specific news/events. Finally, we showed that particular events may trigger a temporary reorganization of elements in the semantic network.

  10. Fast Distributed Dynamics of Semantic Networks via Social Media.

    PubMed

    Carrillo, Facundo; Cecchi, Guillermo A; Sigman, Mariano; Slezak, Diego Fernández

    2015-01-01

    We investigate the dynamics of semantic organization using social media, a collective expression of human thought. We propose a novel, time-dependent semantic similarity measure (TSS), based on the social network Twitter. We show that TSS is consistent with static measures of similarity but provides high temporal resolution for the identification of real-world events and induced changes in the distributed structure of semantic relationships across the entire lexicon. Using TSS, we measured the evolution of a concept and its movement along the semantic neighborhood, driven by specific news/events. Finally, we showed that particular events may trigger a temporary reorganization of elements in the semantic network. PMID:26074953

  11. Dynamics of pre-ionized fast capillary discharge

    NASA Astrophysics Data System (ADS)

    Hübner, J.; Vrba, P.; Straus, J.; Jancarek, A.; Nevrkla, M.

    2014-05-01

    The goal of this work is to determine the best conditions for pre-ionization of the nitrogen filled capillary plasma column applying an external exponentially damped or high-frequency alternating current. As we supposed, optimal pre-ionization conditions are achieved when the plasma is quiescent, motionless and isothermal, near the local thermodynamical equilibrium. At the time of optimal conditions for the pre-ionization plasma column, the main pulse is applied. This approach enables us to estimate the influence of such prepared plasma on the value of emitted energy during the main current pulse. For modeling of plasma during the pre-pulse and main pulse, the magneto-hydro-dynamics (MHD) NPINCH code [1] and the radiative-MHD Z* code [2] were used. The computer results are used for further improvement of x-ray-ultraviolet-capillary sources designed in IPP ASCR and CTU FNSPE laboratories in Prague.

  12. Fast Search for Dynamic Multi-Relational Graphs

    SciTech Connect

    Choudhury, Sutanay; Holder, Larry; Chin, George; Feo, John T.

    2013-06-23

    Acting on time-critical events by processing ever growing social media or news streams is a major technical challenge. Many of these data sources can be modeled as multi-relational graphs. Continuous queries or techniques to search for rare events that typically arise in monitoring applications have been studied extensively for relational databases. This work is dedicated to answer the question that emerges naturally: how can we efficiently execute a continuous query on a dynamic graph? This paper presents an exact subgraph search algorithm that exploits the temporal characteristics of representative queries for online news or social media monitoring. The algorithm is based on a novel data structure called the that leverages the structural and semantic characteristics of the underlying multi-relational graph. The paper concludes with extensive experimentation on several real-world datasets that demonstrates the validity of this approach.

  13. Fast infrared spectroscopy of protein dynamics: advancing sensitivity and selectivity.

    PubMed

    Koziol, Klemens L; Johnson, Philip J M; Stucki-Buchli, Brigitte; Waldauer, Steven A; Hamm, Peter

    2015-10-01

    2D-IR spectroscopy has matured to a powerful technique to study the structure and dynamics of peptides, but its extension to larger proteins is still in its infancy, the major limitations being sensitivity and selectivity. Site-selective information requires measuring single vibrational probes at sub-millimolar concentrations where most proteins are still stable, which is a severe challenge for conventional (FT)IR spectroscopy. Besides its ultrafast time-resolution, a so far largely underappreciated potential of 2D-IR spectroscopy lies in its sensitivity gain. The present paper sets the goals and outlines strategies how to use that sensitivity gain together with properly designed vibrational labels to make IR spectroscopy a versatile tool to study a wide class of proteins.

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

    Microtubules are dynamic and structural cellular components involved in several cell functions, including cell shape, motility, and intracellular trafficking. In proliferating cells, they are essential components in the division process through the formation of the mitotic spindle. As a result of these functions, tubulin and microtubules are targets for anticancer agents. Microtubule-targeting agents can be divided into two groups: microtubule-stabilizing, and microtubule-destabilizing agents. The former bind to the tubulin polymer and stabilize microtubules, while the latter bind to the tubulin dimers and destabilize microtubules. Alteration of tubulin-microtubule equilibrium determines the disruption of the mitotic spindle, halting the cell cycle at the metaphase-anaphase transition and, eventually, resulting in cell death. Clinical application of earlier microtubule inhibitors, however, unfortunately showed several limits, such as neurological and bone marrow toxicity and the emergence of drug-resistant tumor cells. Here we review several natural and synthetic microtubule-targeting agents, which showed antitumor activity and increased efficacy in comparison to traditional drugs in various preclinical and clinical studies. Cryptophycins, combretastatins, ombrabulin, soblidotin, D-24851, epothilones and discodermolide were used in clinical trials. Some of them showed antiangiogenic and antivascular activity and others showed the ability to overcome multidrug resistance, supporting their possible use in chemotherapy. PMID:26484003

  16. A novel isoform of MAP4 organises the paraxial microtubule array required for muscle cell differentiation

    PubMed Central

    Mogessie, Binyam; Roth, Daniel; Rahil, Zainab; Straube, Anne

    2015-01-01

    The microtubule cytoskeleton is critical for muscle cell differentiation and undergoes reorganisation into an array of paraxial microtubules, which serves as template for contractile sarcomere formation. In this study, we identify a previously uncharacterised isoform of microtubule-associated protein MAP4, oMAP4, as a microtubule organising factor that is crucial for myogenesis. We show that oMAP4 is expressed upon muscle cell differentiation and is the only MAP4 isoform essential for normal progression of the myogenic differentiation programme. Depletion of oMAP4 impairs cell elongation and cell–cell fusion. Most notably, oMAP4 is required for paraxial microtubule organisation in muscle cells and prevents dynein- and kinesin-driven microtubule–microtubule sliding. Purified oMAP4 aligns dynamic microtubules into antiparallel bundles that withstand motor forces in vitro. We propose a model in which the cooperation of dynein-mediated microtubule transport and oMAP4-mediated zippering of microtubules drives formation of a paraxial microtubule array that provides critical support for the polarisation and elongation of myotubes. DOI: http://dx.doi.org/10.7554/eLife.05697.001 PMID:25898002

  17. The effect of temperature on the coupled slow and fast dynamics of an electrochemical oscillator

    PubMed Central

    Zülke, Alana A.; Varela, Hamilton

    2016-01-01

    The coupling among disparate time-scales is ubiquitous in many chemical and biological systems. We have recently investigated the effect of fast and, long-term, slow dynamics in surface processes underlying some electrocatalytic reactions. Herein we report on the effect of temperature on the coupled slow and fast dynamics of a model system, namely the electro-oxidation of formic acid on platinum studied at five temperatures between 5 and 45 °C. The main result was a turning point found at 25 °C, which clearly defines two regions for the temperature dependency on the overall kinetics. In addition, the long-term evolution allowed us to compare reaction steps related to fast and slow evolutions. Results were discussed in terms of the key role of PtO species, which chemically couple slow and fast dynamics. In summary we were able to: (a) identify the competition between two reaction steps as responsible for the occurrence of two temperature domains; (b) compare the relative activation energies of these two steps; and (c) suggest the role of a given reaction step on the period-increasing set of reactions involved in the oscillatory dynamics. The introduced methodology could be applied to other systems to uncover the temperature dependence of complex chemical networks. PMID:27079514

  18. The effect of temperature on the coupled slow and fast dynamics of an electrochemical oscillator

    NASA Astrophysics Data System (ADS)

    Zülke, Alana A.; Varela, Hamilton

    2016-04-01

    The coupling among disparate time-scales is ubiquitous in many chemical and biological systems. We have recently investigated the effect of fast and, long-term, slow dynamics in surface processes underlying some electrocatalytic reactions. Herein we report on the effect of temperature on the coupled slow and fast dynamics of a model system, namely the electro-oxidation of formic acid on platinum studied at five temperatures between 5 and 45 °C. The main result was a turning point found at 25 °C, which clearly defines two regions for the temperature dependency on the overall kinetics. In addition, the long-term evolution allowed us to compare reaction steps related to fast and slow evolutions. Results were discussed in terms of the key role of PtO species, which chemically couple slow and fast dynamics. In summary we were able to: (a) identify the competition between two reaction steps as responsible for the occurrence of two temperature domains; (b) compare the relative activation energies of these two steps; and (c) suggest the role of a given reaction step on the period-increasing set of reactions involved in the oscillatory dynamics. The introduced methodology could be applied to other systems to uncover the temperature dependence of complex chemical networks.

  19. Structural microtubule cap: stability, catastrophe, rescue, and third state.

    PubMed Central

    Jánosi, Imre M; Chrétien, Denis; Flyvbjerg, Henrik

    2002-01-01

    Microtubules polymerize from GTP-liganded tubulin dimers, but are essentially made of GDP-liganded tubulin. We investigate the tug-of-war resulting from the fact that GDP-liganded tubulin favors a curved configuration, but is forced to remain in a straight one when part of a microtubule. We point out that near the end of a microtubule, the proximity of the end shifts the balance in this tug-of-war, with some protofilament bending as result. This somewhat relaxes the microtubule lattice near its end, resulting in a structural cap. This structural cap thus is a simple mechanical consequence of two well-established facts: protofilaments made of GDP-liganded tubulin have intrinsic curvature, and microtubules are elastic, made from material that can yield to forces, in casu its own intrinsic forces. We explore possible properties of this structural cap, and demonstrate 1) how it allows both polymerization from GTP-liganded tubulin and rapid depolymerization in its absence; 2) how rescue can occur; 3) how a third, meta-stable intermediate state is possible and can explain some experimental results; and 4) how the tapered tips observed at polymerizing microtubule ends are stabilized during growth, though unable to accommodate a lateral cap. This scenario thus supports the widely accepted GTP-cap model by suggesting a stabilizing mechanism that explains the many aspects of dynamic instability. PMID:12202357

  20. FAST TRACK COMMUNICATION: Local Hawking temperature for dynamical black holes

    NASA Astrophysics Data System (ADS)

    Hayward, S. A.; Di Criscienzo, R.; Nadalini, M.; Vanzo, L.; Zerbini, S.

    2009-03-01

    A local Hawking temperature is derived for any future outer trapping horizon in spherical symmetry, using a Hamilton Jacobi variant of the Parikh Wilczek tunneling method. It is given by a dynamical surface gravity as defined geometrically. The operational meaning of the temperature is that Kodama observers just outside the horizon measure an invariantly redshifted temperature, diverging at the horizon itself. In static, asymptotically flat cases, the Hawking temperature as usually defined by the Killing vector agrees in standard cases, but generally differs by a relative redshift factor between the horizon and infinity, this being the temperature measured by static observers at infinity. Likewise, the geometrical surface gravity reduces to the Newtonian surface gravity in the Newtonian limit, while the Killing definition instead reflects measurements at infinity. This may resolve a long-standing puzzle concerning the Hawking temperature for the extremal limit of the charged stringy black hole, namely that it is the local temperature which vanishes. In general, this confirms the quasi-stationary picture of black-hole evaporation in early stages. However, the geometrical surface gravity is generally not the surface gravity of a static black hole with the same parameters.

  1. 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.

  2. A ring for all: γ-tubulin-containing nucleation complexes in acentrosomal plant microtubule arrays.

    PubMed

    Hashimoto, Takashi

    2013-12-01

    The construction of dynamic polar microtubules from 13 protofilaments consisting of α-tubulin and β-tubulin heterodimers requires a preformed nucleation seed that specifies subcellular localization and timing of microtubule polymerization in vivo. An evolutionarily conserved γ-tubulin-containing ring complex is recruited to the lateral wall of preexisting microtubules or outer nuclear membranes in plant cells, and is then activated as a template for new microtubules of defined geometry. Specific regulators are thought to target/activate the ring complex to nucleate nascent microtubules in distinct polymerization patterns, as seen in interphase and mitotic arrays. The augmin complex, which was initially identified in metazoan cells, recruits the ring complex to plant mitotic microtubules, where new polymers are abundantly generated at shallow angles.

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

    PubMed Central

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

    2012-01-01

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

  4. Engineering oscillating microtubule bundles.

    PubMed

    Sanchez, Timothy; Dogic, Zvonimir

    2013-01-01

    From motility of simple protists to determining the handedness of complex vertebrates, highly conserved eukaryotic cilia and flagella are essential for the reproduction and survival of many biological organisms. Despite extensive studies, the exact mechanism by which individual components coordinate their activity to produce ciliary beating patterns remains unknown. We describe a novel approach toward studying ciliary beating. Instead of deconstructing a fully functional organelle from the top-down, we describe a process by which synthetic cilia-like structures are assembled from the bottom-up and we present methods for engineering such structures. We demonstrate how simple mixtures of microtubules, kinesin clusters, and a bundling agent assemble into structures that produce spontaneous oscillations, suggesting that self-organized beating may be a generic feature of internally driven bundles. Synthetic cilia-like structures can be assembled at high density, leading to synchronization and metachronal traveling waves, reminiscent of the waves seen in biological ciliary fields.

  5. Tubulin tyrosine nitration regulates microtubule organization in plant cells

    PubMed Central

    Blume, Yaroslav B.; Krasylenko, Yuliya A.; Demchuk, Oleh M.; Yemets, Alla I.

    2013-01-01

    During last years, selective tyrosine nitration of plant proteins gains importance as well-recognized pathway of direct nitric oxide (NO) signal transduction. Plant microtubules are one of the intracellular signaling targets for NO, however, the molecular mechanisms of NO signal transduction with the involvement of cytoskeletal proteins remain to be elucidated. Since biochemical evidence of plant α-tubulin tyrosine nitration has been obtained recently, potential role of this posttranslational modification in regulation of microtubules organization in plant cell is estimated in current paper. It was shown that 3-nitrotyrosine (3-NO2-Tyr) induced partially reversible Arabidopsis primary root growth inhibition, alterations of root hairs morphology and organization of microtubules in root cells. It was also revealed that 3-NO2-Tyr intensively decorates such highly dynamic microtubular arrays as preprophase bands, mitotic spindles and phragmoplasts of Nicotiana tabacum Bright Yellow-2 (BY-2) cells under physiological conditions. Moreover, 3D models of the mitotic kinesin-8 complexes with the tail of detyrosinated, tyrosinated and tyrosine nitrated α-tubulin (on C-terminal Tyr 450 residue) from Arabidopsis were reconstructed in silico to investigate the potential influence of tubulin nitrotyrosination on the molecular dynamics of α-tubulin and kinesin-8 interaction. Generally, presented data suggest that plant α-tubulin tyrosine nitration can be considered as its common posttranslational modification, the direct mechanism of NO signal transduction with the participation of microtubules under physiological conditions and one of the hallmarks of the increased microtubule dynamics. PMID:24421781

  6. Fast-track extreme event attribution: How fast can we disentangle thermodynamic (forced) and dynamic (internal) contributions?

    NASA Astrophysics Data System (ADS)

    Haustein, Karsten; Otto, Friederike; Uhe, Peter; Allen, Myles; Cullen, Heidi

    2016-04-01

    provide sufficient guidance to determine the dynamic contribution to the event on the basis of monthly mean values. No such link can be made (North Atlantic/Western Europe region) for shorter time-scales, unless the observed state of the circulation is taken as reference for the model analysis (e.g. Christidis et al. 2014). We present results from our most recent attribution analysis for the December 2015 UK floods (Storm Desmond and Eva), during which we find a robust teleconnection link between Pacific SSTs and North Atlantic Jetstream anomalies. This is true for both experiments, with forecast and observed SSTs. We propose a fast and simple analysis method based on the comparison of current climatological circulation patterns with actual and natural conditions. Alternative methods are discussed and analysed regarding their potential for fast-track attribution of the role of dynamics. Also, we briefly revisit the issue of internal vs forced dynamic contributions.

  7. Dynamical tunneling versus fast diffusion for a non-convex Hamiltonian

    NASA Astrophysics Data System (ADS)

    Pittman, S. M.; Tannenbaum, E.; Heller, E. J.

    2016-08-01

    This paper attempts to resolve the issue of the nature of the 0.01-0.1 cm-1 peak splittings observed in high-resolution IR spectra of polyatomic molecules. One hypothesis is that these splittings are caused by dynamical tunneling, a quantum-mechanical phenomenon whereby energy flows between two disconnected regions of phase-space across dynamical barriers. However, a competing classical mechanism for energy flow is Arnol'd diffusion, which connects different regions of phase-space by a resonance network known as the Arnol'd web. The speed of diffusion is bounded by the Nekhoroshev theorem, which guarantees stability on exponentially long time scales if the Hamiltonian is steep. Here we consider a non-convex Hamiltonian that contains the characteristics of a molecular Hamiltonian, but does not satisfy the Nekhoroshev theorem. The diffusion along the Arnol'd web is expected to be fast for a non-convex Hamiltonian. While fast diffusion is an unlikely competitor for longtime energy flow in molecules, we show how dynamical tunneling dominates compared to fast diffusion in the nearly integrable regime for a non-convex Hamiltonian, as well as present a new kind of dynamical tunneling.

  8. Quantum computation in brain microtubules: Decoherence and biological feasibility

    NASA Astrophysics Data System (ADS)

    Hagan, S.; Hameroff, S. R.; Tuszyński, J. A.

    2002-06-01

    The Penrose-Hameroff orchestrated objective reduction (orch. OR) model assigns a cognitive role to quantum computations in microtubules within the neurons of the brain. Despite an apparently ``warm, wet, and noisy'' intracellular milieu, the proposal suggests that microtubules avoid environmental decoherence long enough to reach threshold for ``self-collapse'' (objective reduction) by a quantum gravity mechanism put forth by Penrose. The model has been criticized as regards the issue of environmental decoherence, and a recent report by Tegmark finds that microtubules can maintain quantum coherence for only 10-13 s, far too short to be neurophysiologically relevant. Here, we critically examine the decoherence mechanisms likely to dominate in a biological setting and find that (1) Tegmark's commentary is not aimed at an existing model in the literature but rather at a hybrid that replaces the superposed protein conformations of the orch. OR theory with a soliton in superposition along the microtubule; (2) recalculation after correcting for differences between the model on which Tegmark bases his calculations and the orch. OR model (superposition separation, charge vs dipole, dielectric constant) lengthens the decoherence time to 10-5-10-4 s (3) decoherence times on this order invalidate the assumptions of the derivation and determine the approximation regime considered by Tegmark to be inappropriate to the orch. OR superposition; (4) Tegmark's formulation yields decoherence times that increase with temperature contrary to well-established physical intuitions and the observed behavior of quantum coherent states; (5) incoherent metabolic energy supplied to the collective dynamics ordering water in the vicinity of microtubules at a rate exceeding that of decoherence can counter decoherence effects (in the same way that lasers avoid decoherence at room temperature); (6) microtubules are surrounded by a Debye layer of counterions, which can screen thermal fluctuations

  9. Fast-Response Calmodulin-Based Fluorescent Indicators Reveal Rapid Intracellular Calcium Dynamics.

    PubMed

    Helassa, Nordine; Zhang, Xiao-hua; Conte, Ianina; Scaringi, John; Esposito, Elric; Bradley, Jonathan; Carter, Thomas; Ogden, David; Morad, Martin; Török, Katalin

    2015-11-03

    Faithful reporting of temporal patterns of intracellular Ca(2+) dynamics requires the working range of indicators to match the signals. Current genetically encoded calmodulin-based fluorescent indicators are likely to distort fast Ca(2+) signals by apparent saturation and integration due to their limiting fluorescence rise and decay kinetics. A series of probes was engineered with a range of Ca(2+) affinities and accelerated kinetics by weakening the Ca(2+)-calmodulin-peptide interactions. At 37 °C, the GCaMP3-derived probe termed GCaMP3fast is 40-fold faster than GCaMP3 with Ca(2+) decay and rise times, t1/2, of 3.3 ms and 0.9 ms, respectively, making it the fastest to-date. GCaMP3fast revealed discreet transients with significantly faster Ca(2+) dynamics in neonatal cardiac myocytes than GCaMP6f. With 5-fold increased two-photon fluorescence cross-section for Ca(2+) at 940 nm, GCaMP3fast is suitable for deep tissue studies. The green fluorescent protein serves as a reporter providing important novel insights into the kinetic mechanism of target recognition by calmodulin. Our strategy to match the probe to the signal by tuning the affinity and hence the Ca(2+) kinetics of the indicator is applicable to the emerging new generations of calmodulin-based probes.

  10. Fast-Response Calmodulin-Based Fluorescent Indicators Reveal Rapid Intracellular Calcium Dynamics

    PubMed Central

    Helassa, Nordine; Zhang, Xiao-hua; Conte, Ianina; Scaringi, John; Esposito, Elric; Bradley, Jonathan; Carter, Thomas; Ogden, David; Morad, Martin; Török, Katalin

    2015-01-01

    Faithful reporting of temporal patterns of intracellular Ca2+ dynamics requires the working range of indicators to match the signals. Current genetically encoded calmodulin-based fluorescent indicators are likely to distort fast Ca2+ signals by apparent saturation and integration due to their limiting fluorescence rise and decay kinetics. A series of probes was engineered with a range of Ca2+ affinities and accelerated kinetics by weakening the Ca2+-calmodulin-peptide interactions. At 37 °C, the GCaMP3-derived probe termed GCaMP3fast is 40-fold faster than GCaMP3 with Ca2+ decay and rise times, t1/2, of 3.3 ms and 0.9 ms, respectively, making it the fastest to-date. GCaMP3fast revealed discreet transients with significantly faster Ca2+ dynamics in neonatal cardiac myocytes than GCaMP6f. With 5-fold increased two-photon fluorescence cross-section for Ca2+ at 940 nm, GCaMP3fast is suitable for deep tissue studies. The green fluorescent protein serves as a reporter providing important novel insights into the kinetic mechanism of target recognition by calmodulin. Our strategy to match the probe to the signal by tuning the affinity and hence the Ca2+ kinetics of the indicator is applicable to the emerging new generations of calmodulin-based probes. PMID:26527405

  11. Coordinated Action of Fast and Slow Reserves for Optimal Sequential and Dynamic Emergency Reserve Activation

    NASA Astrophysics Data System (ADS)

    Salkuti, Surender Reddy; Bijwe, P. R.; Abhyankar, A. R.

    2016-04-01

    This paper proposes an optimal dynamic reserve activation plan after the occurrence of an emergency situation (generator/transmission line outage, load increase or both). An optimal plan is developed to handle the emergency situation, using coordinated action of fast and slow reserves, for secure operation with minimum overall cost. This paper considers the reserves supplied by generators (spinning reserves) and loads (demand-side reserves). The optimal backing down of costly/fast reserves and bringing up of slow reserves in each sub-interval in an integrated manner is proposed. The simulation studies are performed on IEEE 30, 57 and 300 bus test systems to demonstrate the advantage of proposed integrated/dynamic reserve activation plan over the conventional/sequential approach.

  12. A tethered delivery mechanism explains the catalytic action of a microtubule polymerase

    PubMed Central

    Ayaz, Pelin; Munyoki, Sarah; Geyer, Elisabeth A; Piedra, Felipe-Andrés; Vu, Emily S; Bromberg, Raquel; Otwinowski, Zbyszek; Grishin, Nick V; Brautigam, Chad A; Rice, Luke M

    2014-01-01

    Stu2p/XMAP215 proteins are essential microtubule polymerases that use multiple αβ-tubulin-interacting TOG domains to bind microtubule plus ends and catalyze fast microtubule growth. We report here the structure of the TOG2 domain from Stu2p bound to yeast αβ-tubulin. Like TOG1, TOG2 binds selectively to a fully ‘curved’ conformation of αβ-tubulin, incompatible with a microtubule lattice. We also show that TOG1-TOG2 binds non-cooperatively to two αβ-tubulins. Preferential interactions between TOGs and fully curved αβ-tubulin that cannot exist elsewhere in the microtubule explain how these polymerases localize to the extreme microtubule end. We propose that these polymerases promote elongation because their linked TOG domains concentrate unpolymerized αβ-tubulin near curved subunits already bound at the microtubule end. This tethering model can explain catalyst-like behavior and also predicts that the polymerase action changes the configuration of the microtubule end. DOI: http://dx.doi.org/10.7554/eLife.03069.001 PMID:25097237

  13. Tau interaction with microtubules in vivo.

    PubMed

    Samsonov, Andrey; Yu, Jiang-Zhou; Rasenick, Mark; Popov, Sergey V

    2004-12-01

    Tau is a major microtubule-associated protein which induces bundling and stabilization of axonal microtubules (MTs). To investigate the interaction of tau with MTs in living cells, we expressed GFP-tau fusion protein in cultured Xenopus embryo neurons and performed time-lapse imaging of tau-labeled MTs. Tau uniformly labeled individual MTs regardless of their assembly/disassembly status and location along the axon. Photobleaching experiments indicated that interaction of tau with MTs is very dynamic, with a half-time of fluorescence recovery of the order of 3 seconds. Treatment of cells with taxol, a drug that suppresses MT dynamics, rapidly induced detachment of tau from MTs. Although binding of tau to straight MTs was uniform, there was a heightened concentration of tau at the sites of high MT curvature. Our results suggest that dynamic interaction of tau with MTs may modify local mechanical properties of individual MTs and play a crucial role in the remodeling of the MT cytoskeleton during neuronal plasticity.

  14. The Membrane-Associated Sec1/Munc18 KEULE is Required for Phragmoplast Microtubule Reorganization During Cytokinesis in Arabidopsis.

    PubMed

    Steiner, Alexander; Müller, Lin; Rybak, Katarzyna; Vodermaier, Vera; Facher, Eva; Thellmann, Martha; Ravikumar, Raksha; Wanner, Gerhard; Hauser, Marie-Theres; Assaad, Farhah F

    2016-04-01

    Cytokinesis, the partitioning of the cytoplasm following nuclear division, requires extensive coordination between membrane trafficking and cytoskeletal dynamics. In plants, the onset of cytokinesis is characterized by the assembly of a bipolar microtubule array, the phragmoplast, and of a transient membrane compartment, the cell plate. Little is known about the coordination between membrane deposition at the cell plate and the dynamics of phragmoplast microtubules. In this study, we monitor the localization dynamics of microtubule and membrane markers throughout cytokinesis. Our spatiotemporal resolution is consistent with the general view that microtubule dynamics drive membrane movements. Nonetheless, we provide evidence for active sorting at the cell plate and show that this is, at least in part, mediated by the TRAPPII tethering complex. We also characterize phragmoplast microtubule organization and cell plate formation in a suite of cytokinesis-defective mutants. Of four mutant lines with defects in phragmoplast microtubule organization, only mor1 microtubule-associated mutants exhibited aberrant cell plates. Conversely, the mutants with the strongest impairment in phragmoplast microtubule reorganization are keule alleles, which have a primary defect in membrane fusion. Our findings identify the SEC1/Munc18 protein KEULE as a central regulatory node in the coordination of membrane and microtubule dynamics during plant cytokinesis. PMID:26700031

  15. CLASP1, astrin and Kif2b form a molecular switch that regulates kinetochore-microtubule dynamics to promote mitotic progression and fidelity.

    PubMed

    Manning, Amity L; Bakhoum, Samuel F; Maffini, Stefano; Correia-Melo, Clara; Maiato, Helder; Compton, Duane A

    2010-10-20

    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

  16. The Arabidopsis CLASP Gene Encodes a Microtubule-Associated Protein Involved in Cell Expansion and Division[W

    PubMed Central

    Ambrose, J. Christian; Shoji, Tsubasa; Kotzer, Amanda M.; Pighin, Jamie A.; Wasteneys, Geoffrey O.

    2007-01-01

    Controlling microtubule dynamics and spatial organization is a fundamental requirement of eukaryotic cell function. Members of the ORBIT/MAST/CLASP family of microtubule-associated proteins associate with the plus ends of microtubules, where they promote the addition of tubulin subunits into attached kinetochore fibers during mitosis and stabilize microtubules in the vicinity of the plasma membrane during interphase. To date, nothing is known about their function in plants. Here, we show that the Arabidopsis thaliana CLASP protein is a microtubule-associated protein that is involved in both cell division and cell expansion. Green fluorescent protein–CLASP localizes along the full length of microtubules and shows enrichment at growing plus ends. Our analysis suggests that CLASP promotes microtubule stability. clasp-1 T-DNA insertion mutants are hypersensitive to microtubule-destabilizing drugs and exhibit more sparsely populated, yet well ordered, root cortical microtubule arrays. Overexpression of CLASP promotes microtubule bundles that are resistant to depolymerization with oryzalin. Furthermore, clasp-1 mutants have aberrant microtubule preprophase bands, mitotic spindles, and phragmoplasts, indicating a role for At CLASP in stabilizing mitotic arrays. clasp-1 plants are dwarf, have significantly reduced cell numbers in the root division zone, and have defects in directional cell expansion. We discuss possible mechanisms of CLASP function in higher plants. PMID:17873093

  17. An efficient scheme for sampling fast dynamics at a low average data acquisition rate.

    PubMed

    Philippe, A; Aime, S; Roger, V; Jelinek, R; Prévot, G; Berthier, L; Cipelletti, L

    2016-02-24

    We introduce a temporal scheme for data sampling, based on a variable delay between two successive data acquisitions. The scheme is designed so as to reduce the average data flow rate, while still retaining the information on the data evolution on fast time scales. The practical implementation of the scheme is discussed and demonstrated in light scattering and microscopy experiments that probe the dynamics of colloidal suspensions using CMOS or CCD cameras as detectors.

  18. Cortical microtubule patterning in roots of Arabidopsis thaliana primary cell wall mutants reveals the bidirectional interplay with cell expansion.

    PubMed

    Panteris, Emmanuel; Adamakis, Ioannis-Dimosthenis S; Daras, Gerasimos; Rigas, Stamatis

    2014-04-01

    Cell elongation requires directional deposition of cellulose microfibrils regulated by transverse cortical microtubules. Microtubules respond differentially to suppression of cell elongation along the developmental zones of Arabidopsis thaliana root apex. Cortical microtubule orientation is particularly affected in the fast elongation zone but not in the meristematic or transition zones of thanatos and pom2-4 cellulose-deficient mutants of Arabidopsis thaliana. Here, we report that a uniform phenotype is established among the primary cell wall mutants, as cortical microtubules of root epidermal cells of rsw1 and prc1 mutants exhibit the same pattern described in thanatos and pom2-4. Whether cortical microtubules assume transverse orientation or not is determined by the demand for cellulose synthesis, according to each root zone's expansion rate. It is suggested that cessation of cell expansion may provide a biophysical signal resulting in microtubule reorientation. PMID:24717634

  19. Cortical microtubule patterning in roots of Arabidopsis thaliana primary cell wall mutants reveals the bidirectional interplay with cell expansion

    PubMed Central

    Panteris, Emmanuel; Adamakis, Ioannis-Dimosthenis S; Daras, Gerasimos; Rigas, Stamatis

    2014-01-01

    Cell elongation requires directional deposition of cellulose microfibrils regulated by transverse cortical microtubules. Microtubules respond differentially to suppression of cell elongation along the developmental zones of Arabidopsis thaliana root apex. Cortical microtubule orientation is particularly affected in the fast elongation zone but not in the meristematic or transition zones of thanatos and pom2–4 cellulose-deficient mutants of Arabidopsis thaliana. Here, we report that a uniform phenotype is established among the primary cell wall mutants, as cortical microtubules of root epidermal cells of rsw1 and prc1 mutants exhibit the same pattern described in thanatos and pom2–4. Whether cortical microtubules assume transverse orientation or not is determined by the demand for cellulose synthesis, according to each root zone’s expansion rate. It is suggested that cessation of cell expansion may provide a biophysical signal resulting in microtubule reorientation. PMID:24717634

  20. Cortical microtubule patterning in roots of Arabidopsis thaliana primary cell wall mutants reveals the bidirectional interplay with cell expansion

    PubMed Central

    Panteris, Emmanuel; Adamakis, Ioannis-Dimosthenis S; Daras, Gerasimos; Rigas, Stamatis

    2015-01-01

    Cell elongation requires directional deposition of cellulose microfibrils regulated by transverse cortical microtubules. Microtubules respond differentially to suppression of cell elongation along the developmental zones of Arabidopsis thaliana root apex. Cortical microtubule orientation is particularly affected in the fast elongation zone but not in the meristematic or transition zones of thanatos and pom2–4 cellulose-deficient mutants of Arabidopsis thaliana. Here, we report that a uniform phenotype is established among the primary cell wall mutants, as cortical microtubules of root epidermal cells of rsw1 and prc1 mutants exhibit the same pattern described in thanatos and pom2–4. Whether cortical microtubules assume transverse orientation or not is determined by the demand for cellulose synthesis, according to each root zone's expansion rate. It is suggested that cessation of cell expansion may provide a biophysical signal resulting in microtubule reorientation. PMID:26042727

  1. Cortical microtubule patterning in roots of Arabidopsis thaliana primary cell wall mutants reveals the bidirectional interplay with cell expansion.

    PubMed

    Panteris, Emmanuel; Adamakis, Ioannis-Dimosthenis S; Daras, Gerasimos; Rigas, Stamatis

    2015-01-01

    Cell elongation requires directional deposition of cellulose microfibrils regulated by transverse cortical microtubules. Microtubules respond differentially to suppression of cell elongation along the developmental zones of Arabidopsis thaliana root apex. Cortical microtubule orientation is particularly affected in the fast elongation zone but not in the meristematic or transition zones of thanatos and pom2-4 cellulose-deficient mutants of Arabidopsis thaliana. Here, we report that a uniform phenotype is established among the primary cell wall mutants, as cortical microtubules of root epidermal cells of rsw1 and prc1 mutants exhibit the same pattern described in thanatos and pom2-4. Whether cortical microtubules assume transverse orientation or not is determined by the demand for cellulose synthesis, according to each root zone's expansion rate. It is suggested that cessation of cell expansion may provide a biophysical signal resulting in microtubule reorientation. PMID:26042727

  2. Hsc70 rapidly engages tau after microtubule destabilization.

    PubMed

    Jinwal, Umesh K; O'Leary, John C; Borysov, Sergiy I; Jones, Jeffrey R; Li, Qingyou; Koren, John; Abisambra, Jose F; Vestal, Grant D; Lawson, Lisa Y; Johnson, Amelia G; Blair, Laura J; Jin, Ying; Miyata, Yoshinari; Gestwicki, Jason E; Dickey, Chad A

    2010-05-28

    The microtubule-associated protein Tau plays a crucial role in regulating the dynamic stability of microtubules during neuronal development and synaptic transmission. In a group of neurodegenerative diseases, such as Alzheimer disease and other tauopathies, conformational changes in Tau are associated with the initial stages of disease pathology. Folding of Tau into the MC1 conformation, where the amino acids at residues 7-9 interact with residues 312-342, is one of the earliest pathological alterations of Tau in Alzheimer disease. The mechanism of this conformational change in Tau and the subsequent effect on function and association to microtubules is largely unknown. Recent work by our group and others suggests that members of the Hsp70 family play a significant role in Tau regulation. Our new findings suggest that heat shock cognate (Hsc) 70 facilitates Tau-mediated microtubule polymerization. The association of Hsc70 with Tau was rapidly enhanced following treatment with microtubule-destabilizing agents. The fate of Tau released from the microtubule was found to be dependent on ATPase activity of Hsc70. Microtubule destabilization also rapidly increased the MC1 folded conformation of Tau. An in vitro assay suggests that Hsc70 facilitates formation of MC1 Tau. However, in a hyperphosphorylating environment, the formation of MC1 was abrogated, but Hsc70 binding to Tau was enhanced. Thus, under normal circumstances, MC1 formation may be a protective conformation facilitated by Hsc70. However, in a diseased environment, Hsc70 may preserve Tau in a more unstructured state, perhaps facilitating its pathogenicity.

  3. Katanin maintains meiotic metaphase chromosome alignment and spindle structure in vivo and has multiple effects on microtubules in vitro.

    PubMed

    McNally, Karen; Berg, Evan; Cortes, Daniel B; Hernandez, Veronica; Mains, Paul E; McNally, Francis J

    2014-04-01

    Assembly of Caenorhabditis elegans female meiotic spindles requires both MEI-1 and MEI-2 subunits of the microtubule-severing ATPase katanin. Strong loss-of-function mutants assemble apolar intersecting microtubule arrays, whereas weaker mutants assemble bipolar meiotic spindles that are longer than wild type. To determine whether katanin is also required for spindle maintenance, we monitored metaphase I spindles after a fast-acting mei-1(ts) mutant was shifted to a nonpermissive temperature. Within 4 min of temperature shift, bivalents moved off the metaphase plate, and microtubule bundles within the spindle lengthened and developed a high degree of curvature. Spindles eventually lost bipolar structure. Immunofluorescence of embryos fixed at increasing temperature indicated that MEI-1 was lost from spindle microtubules before loss of ASPM-1, indicating that MEI-1 and ASPM-1 act independently at spindle poles. We quantified the microtubule-severing activity of purified MEI-1/MEI-2 complexes corresponding to six different point mutations and found a linear relationship between microtubule disassembly rate and meiotic spindle length. Previous work showed that katanin is required for severing at points where two microtubules intersect in vivo. We show that purified MEI-1/MEI-2 complexes preferentially sever at intersections between two microtubules and directly bundle microtubules in vitro. These activities could promote parallel/antiparallel microtubule organization in meiotic spindles. PMID:24501424

  4. Imaging Ultra-fast Molecular Dynamics in Free Electron Laser Field

    NASA Astrophysics Data System (ADS)

    Zhang, Y. Z.; Jiang, Y. H.

    The free electron laser (FEL) provides the coherent, brilliant and ultrashort light pulse in short wavelength (extreme ultraviolet and X-ray) regimes, opening up possibilities to study ultra-fast molecular dynamics in photo-induced chemical reactions with new methodologies. In this chapter, we introduce the time-resolved pump-probe experiments on gas-phase targets with FEL facilities to image the nuclear and electronic motions in molecular reactions, which serve as a benchmark for further FEL applications like coherent diffraction imaging and coherent control of functional dynamics in complex molecular reactions.

  5. 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

  6. Modeling of nonlinear physiological systems with fast and slow dynamics. II. Application to cerebral autoregulation.

    PubMed

    Mitsis, G D; Zhang, R; Levine, B D; Marmarelis, V Z

    2002-04-01

    Dynamic autoregulation of cerebral hemodynamics in healthy humans is studied using the novel methodology of the Laguerre-Volterra network for systems with fast and slow dynamics (Mitsis, G. D., and V. Z. Marmarelis, Ann. Biomed. Eng. 30:272-281, 2002). Since cerebral autoregulation is mediated by various physiological mechanisms with significantly different time constants, it is used to demonstrate the efficacy of the new method. Results are presented in the time and frequency domains and reveal that cerebral autoregulation is a nonlinear and dynamic (frequency-dependent) system with considerable nonstationarities. Quantification of the latter reveals greater variability in specific frequency bands for each subject in the low and middle frequency range (below 0.1 Hz). The nonlinear dynamics are prominent also in the low and middle frequency ranges, where the frequency response of the system exhibits reduced gain. PMID:12086006

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

    PubMed

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

    2016-07-11

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

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

    PubMed

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

    2016-07-11

    Microtubules are dynamic polymers built of tubulin dimers that attach in a head-to-tail fashion to form protofilaments, which further associate laterally to form a tube. Asynchronous elongation of individual protofilaments can potentially lead to an altered microtubule-end structure that promotes sudden depolymerization, termed catastrophe [1-4]. However, how the dynamics of individual protofilaments relates to overall growth persistence has remained unclear. Here, we used the microtubule targeting anti-cancer drug Eribulin [5-7] to explore the consequences of stalled protofilament elongation on microtubule growth. Using X-ray crystallography, we first revealed that Eribulin binds to a site on β-tubulin that is required for protofilament plus-end elongation. Based on the structural information, we engineered a fluorescent Eribulin molecule. We demonstrate that single Eribulin molecules specifically interact with microtubule plus ends and are sufficient to either trigger a catastrophe or induce slow and erratic microtubule growth in the presence of EB3. Interestingly, we found that Eribulin increases the frequency of EB3 comet "splitting," transient events where a slow and erratically progressing comet is followed by a faster comet. This observation possibly reflects the "healing" of a microtubule lattice. Because EB3 comet splitting was also observed in control microtubules in the absence of any drugs, we propose that Eribulin amplifies a natural pathway toward catastrophe by promoting the arrest of protofilament elongation. PMID:27321995

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

    PubMed

    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

  10. 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

  11. Diffusible crosslinkers generate directed forces in microtubule networks.

    PubMed

    Lansky, Zdenek; Braun, Marcus; Lüdecke, Annemarie; Schlierf, Michael; ten Wolde, Pieter Rein; Janson, Marcel E; Diez, Stefan

    2015-03-12

    Cytoskeletal remodeling is essential to eukaryotic cell division and morphogenesis. The mechanical forces driving the restructuring are attributed to the action of molecular motors and the dynamics of cytoskeletal filaments, which both consume chemical energy. By contrast, non-enzymatic filament crosslinkers are regarded as mere friction-generating entities. Here, we experimentally demonstrate that diffusible microtubule crosslinkers of the Ase1/PRC1/Map65 family generate directed microtubule sliding when confined between partially overlapping microtubules. The Ase1-generated forces, directly measured by optical tweezers to be in the piconewton-range, were sufficient to antagonize motor-protein driven microtubule sliding. Force generation is quantitatively explained by the entropic expansion of confined Ase1 molecules diffusing within the microtubule overlaps. The thermal motion of crosslinkers is thus harnessed to generate mechanical work analogous to compressed gas propelling a piston in a cylinder. As confinement of diffusible proteins is ubiquitous in cells, the associated entropic forces are likely of importance for cellular mechanics beyond cytoskeletal networks. PMID:25748652

  12. LGN Directs Interphase Endothelial Cell Behavior via the Microtubule Network

    PubMed Central

    Wright, Catherine E.; Kushner, Erich J.; Du, Quansheng; Bautch, Victoria L.

    2015-01-01

    Angiogenic sprouts require coordination of endothelial cell (EC) behaviors as they extend and branch. Microtubules influence behaviors such as cell migration and cell-cell interactions via regulated growth and shrinkage. Here we investigated the role of the mitotic polarity protein LGN in EC behaviors and sprouting angiogenesis. Surprisingly, reduced levels of LGN did not affect oriented division of EC within a sprout, but knockdown perturbed overall sprouting. At the cell level, LGN knockdown compromised cell-cell adhesion and migration. EC with reduced LGN levels also showed enhanced growth and stabilization of microtubules that correlated with perturbed migration. These results fit a model whereby LGN influences interphase microtubule dynamics in endothelial cells to regulate migration, cell adhesion, and sprout extension, and reveal a novel non-mitotic role for LGN in sprouting angiogenesis. PMID:26398908

  13. (Z)-3,5,4'-Trimethoxystilbene Limits Hepatitis C and Cancer Pathophysiology by Blocking Microtubule Dynamics and Cell-Cycle Progression.

    PubMed

    Nguyen, Charles B; Kotturi, Hari; Waris, Gulam; Mohammed, Altaf; Chandrakesan, Parthasarathy; May, Randal; Sureban, Sripathi; Weygant, Nathaniel; Qu, Dongfeng; Rao, Chinthalapally V; Dhanasekaran, Danny N; Bronze, Michael S; Houchen, Courtney W; Ali, Naushad

    2016-08-15

    Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related deaths worldwide. Chronic hepatitis C virus (HCV) infection causes induction of several tumors/cancer stem cell (CSC) markers and is known to be a major risk factor for development of HCC. Therefore, drugs that simultaneously target viral replication and CSC properties are needed for a risk-free treatment of advanced stage liver diseases, including HCC. Here, we demonstrated that (Z)-3,5,4'-trimethoxystilbene (Z-TMS) exhibits potent antitumor and anti-HCV activities without exhibiting cytotoxicity to human hepatocytes in vitro or in mice livers. Diethylnitrosamine (DEN)/carbon tetrachloride (CCl4) extensively induced expression of DCLK1 (a CSC marker) in the livers of C57BL/6 mice following hepatic injury. Z-TMS exhibited hepatoprotective effects against DEN/CCl4-induced injury by reducing DCLK1 expression and improving histologic outcomes. The drug caused bundling of DCLK1 with microtubules and blocked cell-cycle progression at G2-M phase in hepatoma cells via downregulation of CDK1, induction of p21(cip1/waf1) expression, and inhibition of Akt (Ser(473)) phosphorylation. Z-TMS also inhibited proliferation of erlotinib-resistant lung adenocarcinoma cells (H1975) bearing the T790M EGFR mutation, most likely by promoting autophagy and nuclear fragmentation. In conclusion, Z-TMS appears to be a unique therapeutic agent targeting HCV and concurrently eliminating cells with neoplastic potential during chronic liver diseases, including HCC. It may also be a valuable drug for targeting drug-resistant carcinomas and cancers of the lungs, pancreas, colon, and intestine, in which DCLK1 is involved in tumorigenesis. Cancer Res; 76(16); 4887-96. ©2016 AACR. PMID:27287718

  14. Relationship of fast- and slow-timescale neuronal dynamics in human MEG and SEEG.

    PubMed

    Zhigalov, Alexander; Arnulfo, Gabriele; Nobili, Lino; Palva, Satu; Palva, J Matias

    2015-04-01

    A growing body of evidence suggests that the neuronal dynamics are poised at criticality. Neuronal avalanches and long-range temporal correlations (LRTCs) are hallmarks of such critical dynamics in neuronal activity and occur at fast (subsecond) and slow (seconds to hours) timescales, respectively. The critical dynamics at different timescales can be characterized by their power-law scaling exponents. However, insight into the avalanche dynamics and LRTCs in the human brain has been largely obtained with sensor-level MEG and EEG recordings, which yield only limited anatomical insight and results confounded by signal mixing. We investigated here the relationship between the human neuronal dynamics at fast and slow timescales using both source-reconstructed MEG and intracranial stereotactical electroencephalography (SEEG). Both MEG and SEEG revealed avalanche dynamics that were characterized parameter-dependently by power-law or truncated-power-law size distributions. Both methods also revealed robust LRTCs throughout the neocortex with distinct scaling exponents in different functional brain systems and frequency bands. The exponents of power-law regimen neuronal avalanches and LRTCs were strongly correlated across subjects. Qualitatively similar power-law correlations were also observed in surrogate data without spatial correlations but with scaling exponents distinct from those of original data. Furthermore, we found that LRTCs in the autonomous nervous system, as indexed by heart-rate variability, were correlated in a complex manner with cortical neuronal avalanches and LRTCs in MEG but not SEEG. These scalp and intracranial data hence show that power-law scaling behavior is a pervasive but neuroanatomically inhomogeneous property of neuronal dynamics in central and autonomous nervous systems.

  15. Relationship of fast- and slow-timescale neuronal dynamics in human MEG and SEEG.

    PubMed

    Zhigalov, Alexander; Arnulfo, Gabriele; Nobili, Lino; Palva, Satu; Palva, J Matias

    2015-04-01

    A growing body of evidence suggests that the neuronal dynamics are poised at criticality. Neuronal avalanches and long-range temporal correlations (LRTCs) are hallmarks of such critical dynamics in neuronal activity and occur at fast (subsecond) and slow (seconds to hours) timescales, respectively. The critical dynamics at different timescales can be characterized by their power-law scaling exponents. However, insight into the avalanche dynamics and LRTCs in the human brain has been largely obtained with sensor-level MEG and EEG recordings, which yield only limited anatomical insight and results confounded by signal mixing. We investigated here the relationship between the human neuronal dynamics at fast and slow timescales using both source-reconstructed MEG and intracranial stereotactical electroencephalography (SEEG). Both MEG and SEEG revealed avalanche dynamics that were characterized parameter-dependently by power-law or truncated-power-law size distributions. Both methods also revealed robust LRTCs throughout the neocortex with distinct scaling exponents in different functional brain systems and frequency bands. The exponents of power-law regimen neuronal avalanches and LRTCs were strongly correlated across subjects. Qualitatively similar power-law correlations were also observed in surrogate data without spatial correlations but with scaling exponents distinct from those of original data. Furthermore, we found that LRTCs in the autonomous nervous system, as indexed by heart-rate variability, were correlated in a complex manner with cortical neuronal avalanches and LRTCs in MEG but not SEEG. These scalp and intracranial data hence show that power-law scaling behavior is a pervasive but neuroanatomically inhomogeneous property of neuronal dynamics in central and autonomous nervous systems. PMID:25834062

  16. A model for fast axonal transport.

    PubMed

    Blum, J J; Reed, M C

    1985-01-01

    A model for fast axonal transport is developed in which the essential features are that organelles may interact with mechanochemical cross-bridges that in turn interact with microtubules, forming an organelle-engine-microtubule complex which is transported along the microtubules. Computer analysis of the equations derived to describe such a system show that most of the experimental observations on fast axonal transport can be simulated by the model, indicating that the model is useful for the interpretation and design of experiments aimed at clarifying the mechanism of fast axonal transport. PMID:2416456

  17. Fast Imaging Technique to Study Drop Impact Dynamics of Non-Newtonian Fluids

    PubMed Central

    Xu, Qin; Peters, Ivo; Wilken, Sam; Brown, Eric; Jaeger, Heinrich

    2014-01-01

    In the field of fluid mechanics, many dynamical processes not only occur over a very short time interval but also require high spatial resolution for detailed observation, scenarios that make it challenging to observe with conventional imaging systems. One of these is the drop impact of liquids, which usually happens within one tenth of millisecond. To tackle this challenge, a fast imaging technique is introduced that combines a high-speed camera (capable of up to one million frames per second) with a macro lens with long working distance to bring the spatial resolution of the image down to 10 µm/pixel. The imaging technique enables precise measurement of relevant fluid dynamic quantities, such as the flow field, the spreading distance and the splashing speed, from analysis of the recorded video. To demonstrate the capabilities of this visualization system, the impact dynamics when droplets of non-Newtonian fluids impinge on a flat hard surface are characterized. Two situations are considered: for oxidized liquid metal droplets we focus on the spreading behavior, and for densely packed suspensions we determine the onset of splashing. More generally, the combination of high temporal and spatial imaging resolution introduced here offers advantages for studying fast dynamics across a wide range of microscale phenomena. PMID:24637404

  18. Dynamic effect of sodium-water reaction in fast flux test facility power addition sodium pipes

    SciTech Connect

    Huang, S.N.; Anderson, M.J.

    1990-03-01

    The Fast Flux Facility (FFTF) is a demonstration and test facility of the sodium-cooled fast breeder reactor. A power addition'' to the facility is being considered to convert some of the dumped, unused heat into electricity generation. Components and piping systems to be added are sodium-water steam generators, sodium loop extensions from existing dump heat exchangers to sodium-water steam generators, and conventional water/steam loops. The sodium loops can be subjected to the dynamic loadings of pressure pulses that are caused by postulated sodium leaks and subsequent sodium-water reaction in the steam generator. The existing FFTF secondary pipes and the new power addition sodium loops were evaluated for exposure to the dynamic effect of the sodium-water reaction. Elastic and simplified inelastic dynamic analyses were used in this feasibility study. The results indicate that both the maximum strain and strain range are within the allowable limits. Several cycles of the sodium-water reaction can be sustained by the sodium pipes that are supported by ordinary pipe supports and seismic restraints. Expensive axial pipe restraints to withstand the sodium-water reaction loads are not needed, because the pressure-pulse-induced alternating bending stresses act as secondary stresses and the pressure pulse dynamic effect is a deformation-controlled quantity and is self-limiting. 14 refs., 7 figs., 3 tabs.

  19. Fast imaging technique to study drop impact dynamics of non-Newtonian fluids.

    PubMed

    Xu, Qin; Peters, Ivo; Wilken, Sam; Brown, Eric; Jaeger, Heinrich

    2014-01-01

    In the field of fluid mechanics, many dynamical processes not only occur over a very short time interval but also require high spatial resolution for detailed observation, scenarios that make it challenging to observe with conventional imaging systems. One of these is the drop impact of liquids, which usually happens within one tenth of millisecond. To tackle this challenge, a fast imaging technique is introduced that combines a high-speed camera (capable of up to one million frames per second) with a macro lens with long working distance to bring the spatial resolution of the image down to 10 µm/pixel. The imaging technique enables precise measurement of relevant fluid dynamic quantities, such as the flow field, the spreading distance and the splashing speed, from analysis of the recorded video. To demonstrate the capabilities of this visualization system, the impact dynamics when droplets of non-Newtonian fluids impinge on a flat hard surface are characterized. Two situations are considered: for oxidized liquid metal droplets we focus on the spreading behavior, and for densely packed suspensions we determine the onset of splashing. More generally, the combination of high temporal and spatial imaging resolution introduced here offers advantages for studying fast dynamics across a wide range of microscale phenomena.

  20. Calibration of hyperelastic material properties of the human lumbar intervertebral disc under fast dynamic compressive loads.

    PubMed

    Wagnac, Eric; Arnoux, Pierre-Jean; Garo, Anaïs; El-Rich, Marwan; Aubin, Carl-Eric

    2011-10-01

    Under fast dynamic loading conditions (e.g. high-energy impact), the load rate dependency of the intervertebral disc (IVD) material properties may play a crucial role in the biomechanics of spinal trauma. However, most finite element models (FEM) of dynamic spinal trauma uses material properties derived from quasi-static experiments, thus neglecting this load rate dependency. The aim of this study was to identify hyperelastic material properties that ensure a more biofidelic simulation of the IVD under a fast dynamic compressive load. A hyperelastic material law based on a first-order Mooney-Rivlin formulation was implemented in a detailed FEM of a L2-L3 functional spinal unit (FSU) to represent the mechanical behavior of the IVD. Bony structures were modeled using an elasto-plastic Johnson-Cook material law that simulates bone fracture while ligaments were governed by a viscoelastic material law. To mimic experimental studies performed in fast dynamic compression, a compressive loading velocity of 1 m/s was applied to the superior half of L2, while the inferior half of L3 was fixed. An exploratory technique was used to simulate dynamic compression of the FSU using 34 sets of hyperelastic material constants randomly selected using an optimal Latin hypercube algorithm and a set of material constants derived from quasi-static experiments. Selection or rejection of the sets of material constants was based on compressive stiffness and failure parameters criteria measured experimentally. The two simulations performed with calibrated hyperelastic constants resulted in nonlinear load-displacement curves with compressive stiffness (7335 and 7079 N/mm), load (12,488 and 12,473 N), displacement (1.95 and 2.09 mm) and energy at failure (13.5 and 14.7 J) in agreement with experimental results (6551 ± 2017 N/mm, 12,411 ± 829 N, 2.1 ± 0.2 mm and 13.0 ± 1.5 J respectively). The fracture pattern and location also agreed with experimental results. The simulation performed with

  1. Analysis of the Ultra-fast Switching Dynamics in a Hybrid MOSFET/Driver

    SciTech Connect

    Tang, T.; Burkhart, C.; /SLAC

    2011-08-17

    The turn-on dynamics of a power MOSFET during ultra-fast, {approx} ns, switching are discussed in this paper. The testing was performed using a custom hybrid MOSFET/Driver module, which was fabricated by directly assembling die-form components, power MOSFET and drivers, on a printed circuit board. By using die-form components, the hybrid approach substantially reduces parasitic inductance, which facilitates ultra-fast switching. The measured turn on time of the hybrid module with a resistive load is 1.2 ns with an applied voltage of 1000 V and drain current of 33 A. Detailed analysis of the switching waveforms reveals that switching behavior must be interpreted differently in the ultra-fast regime. For example, the gate threshold voltage to turn on the device is observed to increase as the switching time decreases. Further analysis and simulation of MOSFET switching behavior shows that the minimum turn on time scales with the product of the drain-source on resistance and drain-source capacitance, R{sub DS(on)}C{sub OSS}. This information will be useful in power MOSFET selection and gate driver design for ultra-fast switching applications.

  2. Dynamic polarizability of tungsten atoms reconstructed from fast electrical explosion of fine wires in vacuum

    NASA Astrophysics Data System (ADS)

    Sarkisov, G. S.; Rosenthal, S. E.; Struve, K. W.

    2016-10-01

    Nanosecond electrical explosion of fine metal wires in vacuum generates calibrated, radially expanded gas cylinders of metal atoms surrounded by a low-density fast expanding plasma corona. An integrated-phase technique, based on laser interferometry, provides the dynamic dipole polarizability of metal atoms. These data were previously unavailable for tungsten atoms. An extremely high melting temperature and significant premelt electronic emission make these measurements particularly complicated for this refractory metal. Most attempts to vaporize tungsten wire by electrical current pulse result in the disintegration of the sample into macro- and microfragments. However, application of a very fast-rising current, ˜1 kA /ns , can vaporize a thin 10-15 μm-diameter tungsten wire and generate a calibrated gas-plasma cylinder. Adding a dielectric coating to the wire leads to increased energy deposition to the wire core and a reduction of the surrounding plasma corona. Employing the integrated-phase technique on a fast-exploding coated tungsten wire, we find that the dynamic dipole polarizability of tungsten atoms at a wavelength of 532 nm equals 15 ±1.3 Å3 .

  3. Adaptive Movement Compensation for In Vivo Imaging of Fast Cellular Dynamics within a Moving Tissue

    PubMed Central

    Dufour, Hugues; De Koninck, Paul; De Koninck, Yves; Côté, Daniel

    2011-01-01

    In vivo non-linear optical microscopy has been essential to advance our knowledge of how intact biological systems work. It has been particularly enabling to decipher fast spatiotemporal cellular dynamics in neural networks. The power of the technique stems from its optical sectioning capability that in turn also limits its application to essentially immobile tissue. Only tissue not affected by movement or in which movement can be physically constrained can be imaged fast enough to conduct functional studies at high temporal resolution. Here, we show dynamic two-photon Ca2+ imaging in the spinal cord of a living rat at millisecond time scale, free of motion artifacts using an optical stabilization system. We describe a fast, non-contact adaptive movement compensation approach, applicable to rough and weakly reflective surfaces, allowing real-time functional imaging from intrinsically moving tissue in live animals. The strategy involves enslaving the position of the microscope objective to that of the tissue surface in real-time through optical monitoring and a closed feedback loop. The performance of the system allows for efficient image locking even in conditions of random or irregular movements. PMID:21629702

  4. Fast Quantum Molecular Dynamics Simulations of Simple Organic Liquids under Shock Compression

    NASA Astrophysics Data System (ADS)

    Cawkwell, Marc; Niklasson, Anders; Manner, Virginia; McGrane, Shawn; Dattelbaum, Dana

    2013-06-01

    The responses of liquid formic acid, acrylonitrile, and nitromethane to shock compression have been studied using quantum-based molecular dynamics simulations with the self-consistent tight-binding code LATTE. Microcanonical Born-Oppenheimer trajectories with precise energy conservation were computed without relying on an iterative self-consistent field optimization of the electronic degrees of freedom at each time step via the Fast Quantum Mechanical Molecular Dynamics formalism. The input shock pressures required to initiate chemistry in our simulations agree very well with recent laser- and flyer-plate-driven shock compression experiments. On-the-fly analysis of the electronic structure of the liquids over hundreds of picoseconds after dynamic compression revealed that their reactivity is strongly correlated with the temperature and pressure dependence of their HOMO-LUMO gap.

  5. Tau mediates microtubule bundle architectures mimicking fascicles of microtubules found in the axon initial segment

    PubMed Central

    Chung, Peter J.; Song, Chaeyeon; Deek, Joanna; Miller, Herbert P.; Li, Youli; Choi, Myung Chul; Wilson, Leslie; Feinstein, Stuart C.; Safinya, Cyrus R.

    2016-01-01

    Tau, an intrinsically disordered protein confined to neuronal axons, binds to and regulates microtubule dynamics. Although there have been observations of string-like microtubule fascicles in the axon initial segment (AIS) and hexagonal bundles in neurite-like processes in non-neuronal cells overexpressing Tau, cell-free reconstitutions have not replicated either geometry. Here we map out the energy landscape of Tau-mediated, GTP-dependent ‘active' microtubule bundles at 37 °C, as revealed by synchrotron SAXS and TEM. Widely spaced bundles (wall-to-wall distance Dw–w≈25–41 nm) with hexagonal and string-like symmetry are observed, the latter mimicking bundles found in the AIS. A second energy minimum (Dw–w≈16–23 nm) is revealed under osmotic pressure. The wide spacing results from a balance between repulsive forces, due to Tau's projection domain (PD), and a stabilizing sum of transient sub-kBT cationic/anionic charge–charge attractions mediated by weakly penetrating opposing PDs. This landscape would be significantly affected by charge-altering modifications of Tau associated with neurodegeneration. PMID:27452526

  6. Tau mediates microtubule bundle architectures mimicking fascicles of microtubules found in the axon initial segment

    DOE PAGESBeta

    Chung, Peter J.; Song, Chaeyeon; Deek, Joanna; Miller, Herbert P.; Li, Youli; Choi, Myung Chul; Wilson, Leslie; Feinstein, Stuart C.; Safinya, Cyrus R.

    2016-07-25

    Tau, an intrinsically disordered protein confined to neuronal axons, binds to and regulates microtubule dynamics. Although there have been observations of string-like microtubule fascicles in the axon initial segment (AIS) and hexagonal bundles in neurite-like processes in non-neuronal cells overexpressing Tau, cell-free reconstitutions have not replicated either geometry. Here we map out the energy landscape of Tau-mediated, GTP-dependent ‘active’ microtubule bundles at 37°C, as revealed by synchrotron SAXS and TEM. Widely spaced bundles (wall-to-wall distance Dw–w≈25–41nm) with hexagonal and string-like symmetry are observed, the latter mimicking bundles found in the AIS. A second energy minimum (Dw–w≈16–23nm) is revealed under osmoticmore » pressure. The wide spacing results from a balance between repulsive forces, due to Tau’s projection domain (PD), and a stabilizing sum of transient sub-kBT cationic/anionic charge–charge attractions mediated by weakly penetrating opposing PDs. In the end, we find that this landscape would be significantly affected by charge-altering modifications of Tau associated with neurodegeneration.« less

  7. A STRIPAK component Strip regulates neuronal morphogenesis by affecting microtubule stability

    PubMed Central

    Sakuma, Chisako; Okumura, Misako; Umehara, Tomoki; Miura, Masayuki; Chihara, Takahiro

    2015-01-01

    During neural development, regulation of microtubule stability is essential for proper morphogenesis of neurons. Recently, the striatin-interacting phosphatase and kinase (STRIPAK) complex was revealed to be involved in diverse cellular processes. However, there is little evidence that STRIPAK components regulate microtubule dynamics, especially in vivo. Here, we show that one of the core STRIPAK components, Strip, is required for microtubule organization during neuronal morphogenesis. Knockdown of Strip causes a decrease in the level of acetylated α-tubulin in Drosophila S2 cells, suggesting that Strip influences the stability of microtubules. We also found that Strip physically and genetically interacts with tubulin folding cofactor D (TBCD), an essential regulator of α- and β-tubulin heterodimers. Furthermore, we demonstrate the genetic interaction between strip and Down syndrome cell adhesion molecule (Dscam), a cell surface molecule that is known to work with TBCD. Thus, we propose that Strip regulates neuronal morphogenesis by affecting microtubule stability. PMID:26644129

  8. Simulation studies of self-organization of microtubules and molecular motors.

    SciTech Connect

    Jian, Z.; Karpeev, D.; Aranson, I. S.; Bates, P. W.; Michigan State Univ.

    2008-05-01

    We perform Monte Carlo type simulation studies of self-organization of microtubules interacting with molecular motors. We model microtubules as stiff polar rods of equal length exhibiting anisotropic diffusion in the plane. The molecular motors are implicitly introduced by specifying certain probabilistic collision rules resulting in realignment of the rods. This approximation of the complicated microtubule-motor interaction by a simple instant collision allows us to bypass the 'computational bottlenecks' associated with the details of the diffusion and the dynamics of motors and the reorientation of microtubules. Consequently, we are able to perform simulations of large ensembles of microtubules and motors on a very large time scale. This simple model reproduces all important phenomenology observed in in vitro experiments: Formation of vortices for low motor density and raylike asters and bundles for higher motor density.

  9. Simulation studies of self-organization of microtubules and molecular motors

    NASA Astrophysics Data System (ADS)

    Jia, Zhiyuan; Karpeev, Dmitry; Aranson, Igor S.; Bates, Peter W.

    2008-05-01

    We perform Monte Carlo type simulation studies of self-organization of microtubules interacting with molecular motors. We model microtubules as stiff polar rods of equal length exhibiting anisotropic diffusion in the plane. The molecular motors are implicitly introduced by specifying certain probabilistic collision rules resulting in realignment of the rods. This approximation of the complicated microtubule-motor interaction by a simple instant collision allows us to bypass the “computational bottlenecks” associated with the details of the diffusion and the dynamics of motors and the reorientation of microtubules. Consequently, we are able to perform simulations of large ensembles of microtubules and motors on a very large time scale. This simple model reproduces all important phenomenology observed in in vitro experiments: Formation of vortices for low motor density and raylike asters and bundles for higher motor density.

  10. Human Fidgetin is a microtubule severing the enzyme and minus-end depolymerase that regulates mitosis

    PubMed Central

    Mukherjee, Suranjana; Valencia, J. Daniel Diaz; Stewman, Shannon; Metz, Jeremy; Monnier, Sylvain; Rath, Uttama; Asenjo, Ana B.; Charafeddine, Rabab A.; Sosa, Hernando J.; Ross, Jennifer L.; Ma, Ao; Sharp, David J.

    2012-01-01

    Fidgetin is a member of the AAA protein superfamily with important roles in mammalian development. Here we show that human Fidgetin is a potent microtubule severing and depolymerizing the enzyme used to regulate mitotic spindle architecture, dynamics and anaphase A. In vitro, recombinant human Fidgetin severs taxol-stabilized microtubules along their length and promotes depolymerization, primarily from their minus-ends. In cells, human Fidgetin targets to centrosomes, and its depletion with siRNA significantly reduces the velocity of poleward tubulin flux and anaphase A chromatid-to-pole motion. In addition, the loss of Fidgetin induces a microtubule-dependent enlargement of mitotic centrosomes and an increase in the number and length of astral microtubules. Based on these data, we propose that human Fidgetin actively suppresses microtubule growth from and attachment to centrosomes. PMID:22672901

  11. Global solution for a kinetic chemotaxis model with internal dynamics and its fast adaptation limit

    NASA Astrophysics Data System (ADS)

    Liao, Jie

    2015-12-01

    A nonlinear kinetic chemotaxis model with internal dynamics incorporating signal transduction and adaptation is considered. This paper is concerned with: (i) the global solution for this model, and, (ii) its fast adaptation limit to Othmer-Dunbar-Alt type model. This limit gives some insight to the molecular origin of the chemotaxis behaviour. First, by using the Schauder fixed point theorem, the global existence of weak solution is proved based on detailed a priori estimates, under quite general assumptions. However, the Schauder theorem does not provide uniqueness, so additional analysis is required to be developed for uniqueness. Next, the fast adaptation limit of this model is derived by extracting a weak convergence subsequence in measure space. For this limit, the first difficulty is to show the concentration effect on the internal state. Another difficulty is the strong compactness argument on the chemical potential, which is essential for passing the nonlinear kinetic equation to the weak limit.

  12. Towards denoising XMCD movies of fast magnetization dynamics using extended Kalman filter.

    PubMed

    Kopp, M; Harmeling, S; Schütz, G; Schölkopf, B; Fähnle, M

    2015-01-01

    The Kalman filter is a well-established approach to get information on the time-dependent state of a system from noisy observations. It was developed in the context of the Apollo project to see the deviation of the true trajectory of a rocket from the desired trajectory. Afterwards it was applied to many different systems with small numbers of components of the respective state vector (typically about 10). In all cases the equation of motion for the state vector was known exactly. The fast dissipative magnetization dynamics is often investigated by x-ray magnetic circular dichroism movies (XMCD movies), which are often very noisy. In this situation the number of components of the state vector is extremely large (about 10(5)), and the equation of motion for the dissipative magnetization dynamics (especially the values of the material parameters of this equation) is not well known. In the present paper it is shown by theoretical considerations that - nevertheless - there is no principle problem for the use of the Kalman filter to denoise XMCD movies of fast dissipative magnetization dynamics.

  13. Towards denoising XMCD movies of fast magnetization dynamics using extended Kalman filter.

    PubMed

    Kopp, M; Harmeling, S; Schütz, G; Schölkopf, B; Fähnle, M

    2015-01-01

    The Kalman filter is a well-established approach to get information on the time-dependent state of a system from noisy observations. It was developed in the context of the Apollo project to see the deviation of the true trajectory of a rocket from the desired trajectory. Afterwards it was applied to many different systems with small numbers of components of the respective state vector (typically about 10). In all cases the equation of motion for the state vector was known exactly. The fast dissipative magnetization dynamics is often investigated by x-ray magnetic circular dichroism movies (XMCD movies), which are often very noisy. In this situation the number of components of the state vector is extremely large (about 10(5)), and the equation of motion for the dissipative magnetization dynamics (especially the values of the material parameters of this equation) is not well known. In the present paper it is shown by theoretical considerations that - nevertheless - there is no principle problem for the use of the Kalman filter to denoise XMCD movies of fast dissipative magnetization dynamics. PMID:25461588

  14. Numerical continuation of canard orbits in slow-fast dynamical systems

    NASA Astrophysics Data System (ADS)

    Desroches, M.; Krauskopf, B.; Osinga, H. M.

    2010-03-01

    A trajectory of a system with two clearly separated time scales generally consists of fast segments (or jumps) followed by slow segments where the trajectory follows an attracting part of a slow manifold. The switch back to fast dynamics typically occurs when the trajectory passes a fold with respect to a fast direction. A special role is played by trajectories known as canard orbits, which do not jump at a fold but, instead, follow a repelling slow manifold for some time. We concentrate here on the case of a slow-fast system with two slow and one fast variable, where canard orbits arise geometrically as intersection curves of two-dimensional attracting and repelling slow manifolds. Canard orbits are intimately related to the dynamics near special points known as folded singularities, which in turn have been shown to explain small-amplitude oscillations that can be found as part of so-called mixed-mode oscillations. In this paper we present a numerical method to detect and then follow branches of canard orbits in a system parameter. More specifically, we define well-posed two-point boundary value problems (BVPs) that represent orbit segments on the slow manifolds, and we continue their solution families with the package AUTO. In this way, we are able to deal effectively with the numerical challenge of strong attraction to and strong repulsion from the slow manifolds. Canard orbits are detected as the transverse intersection points of the curves along which attracting and repelling slow manifolds intersect a suitable section (near a folded node). These intersection points correspond to a unique pair of orbits segments, one on the attracting and one on the repelling slow manifold. After concatenation of the respective pairs of orbit segments, all detected canard orbits are represented as solutions of a single BVP, which allows us to continue them in system parameters. We demonstrate with two examples—the self-coupled FitzHugh-Nagumo system and a three

  15. Alp7/TACC-Alp14/TOG generates long-lived, fast-growing MTs by an unconventional mechanism

    PubMed Central

    Hussmann, Frauke; Drummond, Douglas R.; Peet, Daniel R.; Martin, Douglas S.; Cross, Robert A.

    2016-01-01

    Alp14 is a TOG-family microtubule polymerase from S. pombe that tracks plus ends and accelerates their growth. To interrogate its mechanism, we reconstituted dynamically unstable single isoform S. pombe microtubules with full length Alp14/TOG and Alp7, the TACC-family binding partner of Alp14. We find that Alp14 can drive microtubule plus end growth at GTP-tubulin concentrations at least 10-fold below the usual critical concentration, at the expense of increased catastrophe. This reveals Alp14 to be a highly unusual enzyme that biases the equilibrium for the reaction that it catalyses. Alp7/TACC enhances the effectiveness of Alp14, by increasing its occupancy. Consistent with this, we show in live cells that Alp7 deletion produces very similar MT dynamics defects to Alp14 deletion. The ability of Alp7/14 to accelerate and bias GTP-tubulin exchange at microtubule plus ends allows it to generate long-lived, fast-growing microtubules at very low cellular free tubulin concentrations. PMID:26864000

  16. Tubulin polymerization promoting protein 1 (Tppp1) phosphorylation by Rho-associated coiled-coil kinase (rock) and cyclin-dependent kinase 1 (Cdk1) inhibits microtubule dynamics to increase cell proliferation.

    PubMed

    Schofield, Alice V; Gamell, Cristina; Suryadinata, Randy; Sarcevic, Boris; Bernard, Ora

    2013-03-15

    Tubulin polymerization promoting protein 1 (Tppp1) regulates microtubule (MT) dynamics via promoting MT polymerization and inhibiting histone deacetylase 6 (Hdac6) activity to increase MT acetylation. Our results reveal that as a consequence, Tppp1 inhibits cell proliferation by delaying the G1/S-phase and the mitosis to G1-phase transitions. We show that phosphorylation of Tppp1 by Rho-associated coiled-coil kinase (Rock) prevents its Hdac6 inhibitory activity to enable cells to enter S-phase. Whereas, our analysis of the role of Tppp1 during mitosis revealed that inhibition of its MT polymerizing and Hdac6 regulatory activities were necessary for cells to re-enter the G1-phase. During this investigation, we also discovered that Tppp1 is a novel Cyclin B/Cdk1 (cyclin-dependent kinase) substrate and that Cdk phosphorylation of Tppp1 inhibits its MT polymerizing activity. Overall, our results show that dual Rock and Cdk phosphorylation of Tppp1 inhibits its regulation of the cell cycle to increase cell proliferation.

  17. [Investigation of molecular machine that integrates microtubules depolymerization and chromosomes movement in mitosis].

    PubMed

    Volkov, V A; Ataullakhanov, F I

    2013-02-01

    The main goal of a dividing cell is to distribute its genetic material equally between two daughter cells. Each of the two sister chromatids comprising each chromosome should go into one of the daughter cells. This is possible thanks to polar protein polymers called microtubules, which form a structure called "spindle" during mitosis. Microtubules are one of the basic elements of the cytoskeleton, but at the same time they are highly dynamic. Minus ends of the microtubules attach to the two poles of the spindle, while their plus ends are constantly growing or shrinking, and are also able to attach to the chromosomes and to move them. Chromosomes attach to the microtubule ends with a special protein super-complex called kinetochore. Each sister chromatid in a pair attaches to the microtubules emanating from a corresponding spindle pole. Kinetochores are attached to the dynamic plus ends of the microtubules, but tubulin subunits are still able to attach and detach to these attached ends. One of the most important questions in mitosis is understanding of the mechanism that allows such attachment to be stable (since unstable attachments lead to chromosome loss and aneuploidy) yet dynamic (arrest of the microtubule dynamics stops mitosis). More than 100 proteins comprising the kinetochore are known today, but there is no clear mechanical view on the molecular machine that forms kinetochore-microtubule attachment. The difficulties in understanding this mechanism are due to the variety of theoretical views on the principle of such machine, as well as limited number of biochemically isolated candidates to test these hypotheses experimentally. Therefore, investigation of the properties of putative couplers of microtubule depolymerization and chromosome movement is an important task.

  18. Tracking the biogenesis and inheritance of subpellicular microtubule in Trypanosoma brucei with inducible YFP-α-tubulin.

    PubMed

    Sheriff, Omar; Lim, Li-Fern; He, Cynthia Y

    2014-01-01

    The microtubule cytoskeleton forms the most prominent structural system in Trypanosoma brucei, undergoing extensive modifications during the cell cycle. Visualization of tyrosinated microtubules leads to a semiconservative mode of inheritance, whereas recent studies employing microtubule plus end tracking proteins have hinted at an asymmetric pattern of cytoskeletal inheritance. To further the knowledge of microtubule synthesis and inheritance during T. brucei cell cycle, the dynamics of the microtubule cytoskeleton was visualized by inducible YFP-α-tubulin expression. During new flagellum/flagellum attachment zone (FAZ) biogenesis and cell growth, YFP-α-tubulin was incorporated mainly between the old and new flagellum/FAZ complexes. Cytoskeletal modifications at the posterior end of the cells were observed with EB1, a microtubule plus end binding protein, particularly during mitosis. Additionally, the newly formed microtubules segregated asymmetrically, with the daughter cell inheriting the new flagellum/FAZ complex retaining most of the new microtubules. Together, our results suggest an intimate connection between new microtubule formation and new FAZ assembly, consequently leading to asymmetric microtubule inheritance and cell division.

  19. Fast Chebyshev-polynomial method for simulating the time evolution of linear dynamical systems.

    PubMed

    Loh, Y L; Taraskin, S N; Elliott, S R

    2001-05-01

    We present a fast method for simulating the time evolution of any linear dynamical system possessing eigenmodes. This method does not require an explicit calculation of the eigenvectors and eigenfrequencies, and is based on a Chebyshev polynomial expansion of the formal operator matrix solution in the eigenfrequency domain. It does not suffer from the limitations of ordinary time-integration methods, and can be made accurate to almost machine precision. Among its possible applications are harmonic classical mechanical systems, quantum diffusion, and stochastic transport theory. An example of its use is given for the problem of vibrational wave-packet propagation in a disordered lattice. PMID:11415044

  20. Ultra-fast dynamic compression technique to study kinetics of phase transformations in Bismuth

    SciTech Connect

    Smith, R F; Kane, J O; Eggert, J H; Saculla, M D; Jankowski, A F; Bastea, M; Hicks, D G; Collins, G W

    2007-12-28

    Pre-heated Bi was ramp compressed within 30 ns to a peak stress of {approx}11 GPa to explore structural phase transformation kinetics under dynamic loading conditions. Under these ultra-fast compression time-scales the equilibrium Bi I-II phase boundary is overpressurized by {Delta}P {approx} 0.8 GPa. {Delta}P is observed to increase logarithmically with strain rate, {var_epsilon}, above 10{sup 6} s{sup -1}. Estimates from a kinetics model predict that the Bi I phase is fully transformed within 3 ns.

  1. Dynamics of dust-free cavities behind fast projectiles in a dusty plasma under microgravity conditions

    SciTech Connect

    Caliebe, D.; Arp, O.; Piel, A.

    2011-07-15

    The penetration of a dusty plasma by fast charged projectiles is studied under microgravity conditions. The mass and charge of the projectiles are larger than those of the target particles. A projectile generates a dust-free cavity in its wake, whose shape strongly depends on the projectile velocity. The faster the projectile the more elongated becomes the cavity while its cross-section decreases. The opening time of the cavity is found independent of the projectile velocity. For supersonic projectiles, the dynamics of the cavity can be decomposed into an initial impulse and a subsequent elastic response that can be modeled by a damped harmonic oscillator.

  2. Anisotropic elastic network modeling of entire microtubules.

    PubMed

    Deriu, Marco A; Soncini, Monica; Orsi, Mario; Patel, Mishal; Essex, Jonathan W; Montevecchi, Franco M; Redaelli, Alberto

    2010-10-01

    Microtubules are supramolecular structures that make up the cytoskeleton and strongly affect the mechanical properties of the cell. Within the cytoskeleton filaments, the microtubule (MT) exhibits by far the highest bending stiffness. Bending stiffness depends on the mechanical properties and intermolecular interactions of the tubulin dimers (the MT building blocks). Computational molecular modeling has the potential for obtaining quantitative insights into this area. However, to our knowledge, standard molecular modeling techniques, such as molecular dynamics (MD) and normal mode analysis (NMA), are not yet able to simulate large molecular structures like the MTs; in fact, their possibilities are normally limited to much smaller protein complexes. In this work, we developed a multiscale approach by merging the modeling contribution from MD and NMA. In particular, MD simulations were used to refine the molecular conformation and arrangement of the tubulin dimers inside the MT lattice. Subsequently, NMA was used to investigate the vibrational properties of MTs modeled as an elastic network. The coarse-grain model here developed can describe systems of hundreds of interacting tubulin monomers (corresponding to up to 1,000,000 atoms). In particular, we were able to simulate coarse-grain models of entire MTs, with lengths up to 350 nm. A quantitative mechanical investigation was performed; from the bending and stretching modes, we estimated MT macroscopic properties such as bending stiffness, Young modulus, and persistence length, thus allowing a direct comparison with experimental data.

  3. Multifunctional Microtubule-Associated Proteins in Plants

    PubMed Central

    Krtková, Jana; Benáková, Martina; Schwarzerová, Kateřina

    2016-01-01

    Microtubules (MTs) are involved in key processes in plant cells, including cell division, growth and development. MT-interacting proteins modulate MT dynamics and organization, mediating functional and structural interaction of MTs with other cell structures. In addition to conventional microtubule-associated proteins (MAPs) in plants, there are many other MT-binding proteins whose primary function is not related to the regulation of MTs. This review focuses on enzymes, chaperones, or proteins primarily involved in other processes that also bind to MTs. The MT-binding activity of these multifunctional MAPs is often performed only under specific environmental or physiological conditions, or they bind to MTs only as components of a larger MT-binding protein complex. The involvement of multifunctional MAPs in these interactions may underlie physiological and morphogenetic events, e.g., under specific environmental or developmental conditions. Uncovering MT-binding activity of these proteins, although challenging, may contribute to understanding of the novel functions of the MT cytoskeleton in plant biological processes. PMID:27148302

  4. Fast dynamics and relaxation of colloidal drops during the drying process using multispeckle diffusing wave spectroscopy.

    PubMed

    Lee, Jeong Yong; Hwang, Ji Won; Jung, Hyun Wook; Kim, Sung Hyun; Lee, Seong Jae; Yoon, Kisun; Weitz, David A

    2013-01-22

    The fast dynamics generated by the Brownian motion of particles in colloidal drops, and the related relaxation during drying, which play key roles in suspension systems, were investigated incorporating multispeckle diffusing wave spectroscopy (MSDWS). MSDWS equipment was implemented to analyze the relaxation properties of suspensions under a nonergodic and nonstationary drying process, which cannot be elucidated by conventional light scattering methods, such as dynamic light scattering and diffusing wave spectroscopy. Rapid particle movement can be identified by the characteristic relaxation time, which is closely related to the Brownian motion due to thermal fluctuations of the particles. In the compacting stage of the drying process, the characteristic relaxation time increased gradually with the drying time because the particles in the colloidal drop were constrained by themselves. Moreover, variations of the initial concentration and particle size considerably affected the complete drying time and characteristic relaxation time, producing a shorter relaxation time for a low concentrated suspension with small particles. PMID:23281633

  5. Kinesin follows the microtubule's protofilament axis

    PubMed Central

    1993-01-01

    We tested the hypothesis that kinesin moves parallel to the microtubule's protofilament axis. We polymerized microtubules with protofilaments that ran either parallel to the microtubule's long axis or that ran along shallow helical paths around the cylindrical surface of the microtubule. When gliding across a kinesin-coated surface, the former microtubules did not rotate. The latter microtubules, those with supertwisted protofilaments, did rotate; the pitch and handedness of the rotation accorded with the supertwist measured by electron cryo- microscopy. The results show that kinesin follows a path parallel to the protofilaments with high fidelity. This implies that the distance between consecutive kinesin-binding sites along the microtubule must be an integral multiple of 4.1 nm, the tubulin monomer spacing along the protofilament, or a multiple of 8.2 nm, the dimer spacing. PMID:8099076

  6. Earthquake signatures from fast slip and dynamic fracture propagation: state of the art

    NASA Astrophysics Data System (ADS)

    Griffith, W. A.; Rowe, C. D.

    2014-12-01

    Earthquakes are dynamic slip events that propagate along fault surfaces, radiating seismic waves. The majority of energy released is expended in heating and breaking of rocks along the fault. The fracture damage is linked to transient stress conditions at the rupture tip which only exist during dynamic slip, while the frictional heating depends on high velocity slip behind the rupture tip to generate heat energy faster than it can be dissipated, causing transient local temperature rise. One might think that extensive damage and alteration would result, creating an unambiguous geological fingerprint, but in fact, there are few unequivocal indicators for past seismic slip. In 1999, the rare fault rock pseudotachylyte (melt formed when frictional heating exceeds the solidus) was the only accepted evidence (Cowan, 1999). Recently, more indicators of fossilized earthquakes have been proposed. We define "evidence of past earthquakes" as evidence of either fast fault slip (at rates that only occur during earthquakes) or evidence of the propagation of dynamic rupture. We first summarize advances made in identifying evidence in the rock record of fast slip. Much of the work during the past 15 years has focused on integrating carefully controlled laboratory friction experiments, and constraints from numerical modeling, with field observations in an attempt to re-create structures observed in fault rocks and then relate these to the specific boundary conditions required to form them in the laboratory. This approach has yielded a number of advances in identifying textures and geochemical signatures diagnostic of fast slip via temperature rises which may not reach the bulk melting temperature of the fault rocks. Next, we focus on damage near the tip of shear ruptures propagating at velocities characteristic of earthquakes, an approach which has received less attention in the geological literature than fast slip but is equally diagnostic. Finally, we try to frame some of the

  7. Centrosome and microtubule functions and dysfunctions in meiosis: implications for age-related infertility and developmental disorders.

    PubMed

    Schatten, Heide; Sun, Qing-Yuan

    2015-07-01

    The effects of oocyte aging on meiotic spindle dynamics have been well recognised, but the mechanisms underlying the effects are not well understood. In this paper we review the role of centrosomes and the microtubule cytoskeleton in meiotic spindle formation and maintenance, and the impact of oocyte aging on spindle integrity resulting in centrosome and microtubule dysfunctions that are associated with aneuploidy. Loss of spindle integrity includes dispersion of proteins from the centrosome core structure and loss of attachment of microtubules to centrosomes and kinetochores, which will result in abnormal chromosome separation. The inability of centrosomal proteins to accurately associate with the centrosome structure may be the result of destabilisation of the core structure itself or of microtubule destabilisation at the centrosome-facing microtubule areas that are acetylated in fresh oocytes but may not be acetylated in aging oocytes. Microtubule destabilisation prevents accurate motor-driven transport of centrosomal proteins along microtubules to form and maintain a functional centrosome. Other factors to form and maintain the MII spindle include signal transductions that affect microtubule dynamics and stability. Understanding the mechanisms underlying centrosome and microtubule dysfunctions during oocyte aging will allow diagnosis and analysis of oocyte quality and abnormalities as important aspects for targeted treatment of aging oocytes to extend or restore viability and developmental capacity. New therapeutic approaches will allow improvements in reproductive success rates in IVF clinics, as well as improvements in reproductive success rates in farm animals. This review is focused on: (1) centrosome and microtubule dynamics in fresh and aging oocytes; (2) regulation of centrosome and/or microtubule dynamics and function; and (3) possible treatments to extend the oocyte's reproductive capacity and viability span. PMID:25903261

  8. Polysome profiling in liver identifies dynamic regulation of endoplasmic reticulum translatome by obesity and fasting.

    PubMed

    Fu, Suneng; Fan, Jason; Blanco, Joshua; Gimenez-Cassina, Alfredo; Danial, Nika N; Watkins, Steve M; Hotamisligil, Gökhan S

    2012-08-01

    Obesity-associated metabolic complications are generally considered to emerge from abnormalities in carbohydrate and lipid metabolism, whereas the status of protein metabolism is not well studied. Here, we performed comparative polysome and associated transcriptional profiling analyses to study the dynamics and functional implications of endoplasmic reticulum (ER)-associated protein synthesis in the mouse liver under conditions of obesity and nutrient deprivation. We discovered that ER from livers of obese mice exhibits a general reduction in protein synthesis, and comprehensive analysis of polysome-bound transcripts revealed extensive down-regulation of protein synthesis machinery, mitochondrial components, and bile acid metabolism in the obese translatome. Nutrient availability also plays an important but distinct role in remodeling the hepatic ER translatome in lean and obese mice. Fasting in obese mice partially reversed the overall translatomic differences between lean and obese nonfasted controls, whereas fasting of the lean mice mimicked many of the translatomic changes induced by the development of obesity. The strongest examples of such regulations were the reduction in Cyp7b1 and Slco1a1, molecules involved in bile acid metabolism. Exogenous expression of either gene significantly lowered plasma glucose levels, improved hepatic steatosis, but also caused cholestasis, indicating the fine balance bile acids play in regulating metabolism and health. Together, our work defines dynamic regulation of the liver translatome by obesity and nutrient availability, and it identifies a novel role for bile acid metabolism in the pathogenesis of metabolic abnormalities associated with obesity.

  9. 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

  10. 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.

  11. 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. PMID:26604305

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

    PubMed Central

    Xue, Chuan; Shtylla, Blerta; Brown, Anthony

    2015-01-01

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

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

    PubMed

    Xue, Chuan; Shtylla, Blerta; Brown, Anthony

    2015-08-01

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

  14. Fast and stable redox reactions of MnO₂/CNT hybrid electrodes for dynamically stretchable pseudocapacitors.

    PubMed

    Gu, Taoli; Wei, Bingqing

    2015-07-21

    Pseudocapacitors, which are energy storage devices that take advantage of redox reactions to store electricity, have a different charge storage mechanism compared to lithium-ion batteries (LIBs) and electric double-layer capacitors (EDLCs), and they could realize further gains if they were used as stretchable power sources. The realization of dynamically stretchable pseudocapacitors and understanding of the underlying fundamentals of their mechanical-electrochemical relationship have become indispensable. We report herein the electrochemical performance of dynamically stretchable pseudocapacitors using buckled MnO2/CNT hybrid electrodes. The extremely small relaxation time constant of less than 0.15 s indicates a fast redox reaction at the MnO2/CNT hybrid electrodes, securing a stable electrochemical performance for the dynamically stretchable pseudocapacitors. This finding and the fundamental understanding gained from the pseudo-capacitive behavior coupled with mechanical deformation under a dynamic stretching mode would provide guidance to further improve their overall performance including a higher power density than LIBs, a higher energy density than EDLCs, and a long-life cycling stability. Most importantly, these results will potentially accelerate the applications of stretchable pseudocapacitors for flexible and biomedical electronics.

  15. Fast dynamics of cortical functional and effective connectivity during word reading.

    PubMed

    Bedo, Nicolas; Ribary, Urs; Ward, Lawrence M

    2014-01-01

    We describe for the first time the fast dynamics of functional and effective (causal) connectivity during word reading. Independent component analysis of high-density EEG recorded during a word reading task recovered multiple sources of electrical brain activity previously identified by fMRI and PET. Results confirmed the ventral occipito-temporal cortex (vOT) as a central hub for word reading, showing a progression of theta-band (3-7 Hz) and gamma-band (30-50 Hz) phase synchronization and directed theta-band and gamma-band information flow with both early visual areas and high-level language-processing areas. These results highlight the interplay between local and long-distance neural dynamics involved at each stage of the reading process. Moreover, these measures of functional and causal connectivity dynamics may be used as a benchmark for comparison with clinical populations (e.g. individuals with developmental dyslexia), such that disturbances in connectivity dynamics may provide insight as to underlying neurological problems with language processing, and their potential remediation.

  16. Role of Ca[sup ++]/calmodulin in the regulation of microtubules in higher plants

    SciTech Connect

    Cyr, R.

    1991-01-01

    This work is aimed at defining the role of calcium/calmodulin in regulating cortical microtubules (MTS) in higher plants. Recent thrust has been to define the effects of calcium upon microtubules in vivo. Using lysed protoplasts, we noted Mts are destabilized by calcium/calmodulin. This effect could be the result of gross depolymerization induced by Ca[sup ++]/calmodulin, or by an increase in the dynamic flux rate. Intact protoplasts exposed to high (10 mM) levels of calcium (which would be expected to increase intercellular calcium levels) contained microtubules that were hypersensitive to Mt inhibitors, compared to control protoplasts exposed to low calcium environments.

  17. Molecular modeling of oscillating GHz electric field influence on the kinesin affinity to microtubule

    NASA Astrophysics Data System (ADS)

    R. Saeidi, H.; S. Setayandeh, S.; Lohrasebi, A.

    2015-08-01

    Kinesin is a microtubule-associated motor protein which can respond to the external electric field due to its polarity. Using a molecular dynamics simulation method, the effect of such a field on the affinity of kinesin to the αβ-tubulin is investigated in this study. To consider kinesin affinity, the system is exposed to an electric field of 0.03 V/nm with frequency values of 1, 2, …, 9, and 10 GHz. It is found that the applied electric field can change kinesin affinity to the microtubule. These changes could perturb the normal operation of kinesin, such as the processive motility of kinesin on the microtubule.

  18. Fast Dynamic Simulation-Based Small Signal Stability Assessment and Control

    SciTech Connect

    Acharya, Naresh; Baone, Chaitanya; Veda, Santosh; Dai, Jing; Chaudhuri, Nilanjan; Leonardi, Bruno; Sanches-Gasca, Juan; Diao, Ruisheng; Wu, Di; Huang, Zhenyu; Zhang, Yu; Jin, Shuangshuang; Zheng, Bin; Chen, Yousu

    2014-12-31

    Power grid planning and operation decisions are made based on simulation of the dynamic behavior of the system. Enabling substantial energy savings while increasing the reliability of the aging North American power grid through improved utilization of existing transmission assets hinges on the adoption of wide-area measurement systems (WAMS) for power system stabilization. However, adoption of WAMS alone will not suffice if the power system is to reach its full entitlement in stability and reliability. It is necessary to enhance predictability with "faster than real-time" dynamic simulations that will enable the dynamic stability margins, proactive real-time control, and improve grid resiliency to fast time-scale phenomena such as cascading network failures. Present-day dynamic simulations are performed only during offline planning studies, considering only worst case conditions such as summer peak, winter peak days, etc. With widespread deployment of renewable generation, controllable loads, energy storage devices and plug-in hybrid electric vehicles expected in the near future and greater integration of cyber infrastructure (communications, computation and control), monitoring and controlling the dynamic performance of the grid in real-time would become increasingly important. The state-of-the-art dynamic simulation tools have limited computational speed and are not suitable for real-time applications, given the large set of contingency conditions to be evaluated. These tools are optimized for best performance of single-processor computers, but the simulation is still several times slower than real-time due to its computational complexity. With recent significant advances in numerical methods and computational hardware, the expectations have been rising towards more efficient and faster techniques to be implemented in power system simulators. This is a natural expectation, given that the core solution algorithms of most commercial simulators were developed

  19. A dynamic prescribed vortex wake model for the FAST/AeroDyn wind energy conversion simulation code

    NASA Astrophysics Data System (ADS)

    Currin, Hugh D.

    2007-12-01

    A Dynamic Prescribed Vortex Wake model for analysis of horizontal axis wind turbines has been developed. This model extends the HAWTDAWG steady state prescribed wake code to dynamic flows. This extension assumes wake vortices follow prescription functions valid at the time each vortex is generated. This allows modeling of dynamic wake effects known to exist. This assumption is supported through analysis and comparison to UAE Phase VI test data. The new Dynamic Prescribed Vortex Wake model is built into AeroDyn as a third aerodynamic model and uses the FAST structural dynamic model. It implements the wind models and dynamic stall model in AeroDyn. FAST structural degrees of freedom are implemented. Comparisons are made to UAE Phase VI wind tunnel data, and to the other two AeroDyn models, Blade Element Momentum and Generalized Dynamic Wake. Both steady, to verify the base model, and dynamic, to validate the extension to dynamic flow, conditions are considered. Both axial and yawed flow are analyzed. Dynamic UAE test data analyzed include rapid pitch, Sequence Q, and yaw release, Sequence E. The Dynamic Prescribed Wake model compares favorably to test data and to other AeroDyn models. Small rapid dynamic response is noted in each model and the test data. The new Dynamic Prescribed Vortex Wake model shows promise. Release of the code for experimental use and further validation is recommended.

  20. Tau Induces Cooperative Taxol Binding to Microtubules

    NASA Astrophysics Data System (ADS)

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

    2004-03-01

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

  1. Active Contraction of Microtubule Networks

    NASA Astrophysics Data System (ADS)

    Foster, Peter; Fürthauer, Sebastian; Shelley, Michael; Needleman, Daniel

    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.

  2. Detailed Per-residue Energetic Analysis Explains the Driving Force for Microtubule Disassembly

    PubMed Central

    Ayoub, Ahmed T.; Klobukowski, Mariusz; Tuszynski, Jack A.

    2015-01-01

    Microtubules are long filamentous hollow cylinders whose surfaces form lattice structures of αβ-tubulin heterodimers. They perform multiple physiological roles in eukaryotic cells and are targets for therapeutic interventions. In our study, we carried out all-atom molecular dynamics simulations for arbitrarily long microtubules that have either GDP or GTP molecules in the E-site of β-tubulin. A detailed energy balance of the MM/GBSA inter-dimer interaction energy per residue contributing to the overall lateral and longitudinal structural stability was performed. The obtained results identified the key residues and tubulin domains according to their energetic contributions. They also identified the molecular forces that drive microtubule disassembly. At the tip of the plus end of the microtubule, the uneven distribution of longitudinal interaction energies within a protofilament generates a torque that bends tubulin outwardly with respect to the cylinder's axis causing disassembly. In the presence of GTP, this torque is opposed by lateral interactions that prevent outward curling, thus stabilizing the whole microtubule. Once GTP hydrolysis reaches the tip of the microtubule (lateral cap), lateral interactions become much weaker, allowing tubulin dimers to bend outwards, causing disassembly. The role of magnesium in the process of outward curling has also been demonstrated. This study also showed that the microtubule seam is the most energetically labile inter-dimer interface and could serve as a trigger point for disassembly. Based on a detailed balance of the energetic contributions per amino acid residue in the microtubule, numerous other analyses could be performed to give additional insights into the properties of microtubule dynamic instability. PMID:26030285

  3. A surface ice module for wind turbine dynamic response simulation using FAST

    DOE PAGESBeta

    Yu, Bingbin; Karr, Dale G.; Song, Huimin; Sirnivas, Senu

    2016-06-03

    It is a fact that developing offshore wind energy has become more and more serious worldwide in recent years. Many of the promising offshore wind farm locations are in cold regions that may have ice cover during wintertime. The challenge of possible ice loads on offshore wind turbines raises the demand of modeling capacity of dynamic wind turbine response under the joint action of ice, wind, wave, and current. The simulation software FAST is an open source computer-aided engineering (CAE) package maintained by the National Renewable Energy Laboratory. In this paper, a new module of FAST for assessing the dynamicmore » response of offshore wind turbines subjected to ice forcing is presented. In the ice module, several models are presented which involve both prescribed forcing and coupled response. For conditions in which the ice forcing is essentially decoupled from the structural response, ice forces are established from existing models for brittle and ductile ice failure. For conditions in which the ice failure and the structural response are coupled, such as lock-in conditions, a rate-dependent ice model is described, which is developed in conjunction with a new modularization framework for FAST. In this paper, analytical ice mechanics models are presented that incorporate ice floe forcing, deformation, and failure. For lower speeds, forces slowly build until the ice strength is reached and ice fails resulting in a quasi-static condition. For intermediate speeds, the ice failure can be coupled with the structural response and resulting in coinciding periods of the ice failure and the structural response. A third regime occurs at high speeds of encounter in which brittle fracturing of the ice feature occurs in a random pattern, which results in a random vibration excitation of the structure. An example wind turbine response is simulated under ice loading of each of the presented models. This module adds to FAST the capabilities for analyzing the response of wind

  4. Salt stress-induced disassembly of Arabidopsis cortical microtubule arrays involves 26S proteasome-dependent degradation of SPIRAL1.

    PubMed

    Wang, Songhu; Kurepa, Jasmina; Hashimoto, Takashi; Smalle, Jan A

    2011-09-01

    The dynamic instability of cortical microtubules (MTs) (i.e., their ability to rapidly alternate between phases of growth and shrinkage) plays an essential role in plant growth and development. In addition, recent studies have revealed a pivotal role for dynamic instability in the response to salt stress conditions. The salt stress response includes a rapid depolymerization of MTs followed by the formation of a new MT network that is believed to be better suited for surviving high salinity. Although this initial depolymerization response is essential for the adaptation to salt stress, the underlying molecular mechanism has remained largely unknown. Here, we show that the MT-associated protein SPIRAL1 (SPR1) plays a key role in salt stress-induced MT disassembly. SPR1, a microtubule stabilizing protein, is degraded by the 26S proteasome, and its degradation rate is accelerated in response to high salinity. We show that accelerated SPR1 degradation is required for a fast MT disassembly response to salt stress and for salt stress tolerance.

  5. Microtubule-Depolymerizing Kinesin KLP10A Restricts the Length of the Acentrosomal Meiotic Spindle in Drosophila Females

    PubMed Central

    Radford, Sarah J.; Harrison, Andrew M.; McKim, Kim S.

    2012-01-01

    During cell division, a bipolar array of microtubules forms the spindle through which the forces required for chromosome segregation are transmitted. Interestingly, the spindle as a whole is stable enough to support these forces even though it is composed of dynamic microtubules, which are constantly undergoing periods of growth and shrinkage. Indeed, the regulation of microtubule dynamics is essential to the integrity and function of the spindle. We show here that a member of an important class of microtubule-depolymerizing kinesins, KLP10A, is required for the proper organization of the acentrosomal meiotic spindle in Drosophila melanogaster oocytes. In the absence of KLP10A, microtubule length is not controlled, resulting in extraordinarily long and disorganized spindles. In addition, the interactions between chromosomes and spindle microtubules are disturbed and can result in the loss of contact. These results indicate that the regulation of microtubule dynamics through KLP10A plays a critical role in restricting the length and maintaining bipolarity of the acentrosomal meiotic spindle and in promoting the contacts that the chromosomes make with microtubules required for meiosis I segregation. PMID:22865737

  6. Microtubule disruption targets HIF-1α mRNA to cytoplasmic P-bodies for translational repression

    PubMed Central

    Carbonaro, Marisa; O'Brate, Aurora

    2011-01-01

    The hypoxia inducible factor 1α (HIF-1α) is overexpressed in solid tumors, driving tumor angiogenesis and survival. However, the mechanisms regulating HIF-1α expression in solid tumors are not fully understood. In this study, we find that microtubule integrity and dynamics are intricately involved in orchestrating HIF-1α translation. HIF-1α messenger RNA (mRNA) traffics on dynamic microtubules when it is actively translated. Microtubule perturbation by taxol (TX) and other microtubule-targeting drugs stalls HIF-1α mRNA transport and releases it from polysomes, suppressing its translation. Immunoprecipitation of the P-body component Argonaute 2 (Ago2) after microtubule disruption shows significant enrichment of HIF-1α mRNAs and HIF-targeting microRNAs (miRNAs). Inhibition of HIF-repressing miRNAs or Ago2 knockdown abrogates TX’s ability to suppress HIF-1α translation. Interestingly, microtubule repolymerization after nocodazole washout allows HIF-1α mRNA to reenter active translation, suggesting that microtubule dynamics exert tight yet reversible control over HIF-1α translation. Collectively, we provide evidence for a new mechanism of microtubule-dependent HIF-1α translation with important implications for cell biology. PMID:21220510

  7. Microtubule Bundling and Shape Transitions

    NASA Astrophysics Data System (ADS)

    Needleman, Daniel

    2005-03-01

    Microtubules (MTs) are hollow cylindrical polymers composed of heterodimers of the protein tubulin that align end-to-end in the MT wall, forming linear protofilaments that interact laterally. Placing MTs under osmotic pressure causes them to reversibly buckle to a noncircular shape and pack into rectangular bundles at a critical osmotic pressure; further increases in pressure continue to distort MTs elastically. At higher osmotic pressures stressing polymers may be forced into the MT lumen causing the MTs to revert to a circle cross-section and pack into hexagonal bundles. This SAXRD-osmotic stress study provides a probe of the inter-protofilament bond strength and gives insight into the mechanisms by which microtubule associated proteins and the cancer chemotherapeutic drug Taxol stabilize MTs. We present further measurements of the mechanical properties of MT walls, MT-MT interactions, and the entry of polymers into the microtubule lumen. Supported by NSF DMR- 0203755, NIH GM-59288 and NS-13560, and CTS-0103516. SSRL is supported by the U.S. DOE.

  8. Microtubule sliding in reactivated flagella.

    PubMed

    Takahashi, K; Shingyoji, C; Kamimura, S

    1982-01-01

    Recent experimental studies of microtubule sliding in demembranated sea urchin sperm flagella are described. A local iontophoretic application of ATP to a Triton-extracted flagellum elicits a local bending response whose form is in exact conformity with the predictions of the sliding microtubule model. Cinematographic analysis of the microtubule sliding initiated by treating fragments of demembranated flagella with trypsin in the presence of ATP reveals that the speed of sliding is almost constant. This implies that the speed does not depend on the number of dynein arms participating in the generation of sliding force. The distribution of apparent sliding velocities indicates that there is no difference in sliding velocity among the doublets. The sliding velocity depends on MgATP concentration in a manner consistent with Michaelis-Menten kinetics. The sliding velocity of doublets in trypsin-treated axonemes is close to the maximum velocity of relative sliding taking place between adjacent doublets in beating flagella reactivated at the same MgATP concentration.

  9. 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

  10. Coupled slow and fast surface dynamics in an electrocatalytic oscillator: Model and simulations

    SciTech Connect

    Nascimento, Melke A.; Nagao, Raphael; Eiswirth, Markus; Varela, Hamilton

    2014-12-21

    The co-existence of disparate time scales is pervasive in many systems. In particular for surface reactions, it has been shown that the long-term evolution of the core oscillator is decisively influenced by slow surface changes, such as progressing deactivation. Here we present an in-depth numerical investigation of the coupled slow and fast surface dynamics in an electrocatalytic oscillator. The model consists of four nonlinear coupled ordinary differential equations, investigated over a wide parameter range. Besides the conventional bifurcation analysis, the system was studied by means of high-resolution period and Lyapunov diagrams. It was observed that the bifurcation diagram changes considerably as the irreversible surface poisoning evolves, and the oscillatory region shrinks. The qualitative dynamics changes accordingly and the chaotic oscillations are dramatically suppressed. Nevertheless, periodic cascades are preserved in a confined region of the resistance vs. voltage diagram. Numerical results are compared to experiments published earlier and the latter reinterpreted. Finally, the comprehensive description of the time-evolution in the period and Lyapunov diagrams suggests further experimental studies correlating the evolution of the system's dynamics with changes of the catalyst structure.

  11. Mitosis. Microtubule detyrosination guides chromosomes during mitosis.

    PubMed

    Barisic, Marin; Silva e Sousa, Ricardo; Tripathy, Suvranta K; Magiera, Maria M; Zaytsev, Anatoly V; Pereira, Ana L; Janke, Carsten; Grishchuk, Ekaterina L; Maiato, Helder

    2015-05-15

    Before chromosomes segregate into daughter cells, they align at the mitotic spindle equator, a process known as chromosome congression. Centromere-associated protein E (CENP-E)/Kinesin-7 is a microtubule plus-end-directed kinetochore motor required for congression of pole-proximal chromosomes. Because the plus-ends of many astral microtubules in the spindle point to the cell cortex, it remains unknown how CENP-E guides pole-proximal chromosomes specifically toward the equator. We found that congression of pole-proximal chromosomes depended on specific posttranslational detyrosination of spindle microtubules that point to the equator. In vitro reconstitution experiments demonstrated that CENP-E-dependent transport was strongly enhanced on detyrosinated microtubules. Blocking tubulin tyrosination in cells caused ubiquitous detyrosination of spindle microtubules, and CENP-E transported chromosomes away from spindle poles in random directions. Thus, CENP-E-driven chromosome congression is guided by microtubule detyrosination.

  12. Microtubule detyrosination guides chromosomes during mitosis

    PubMed Central

    Barisic, Marin; Silva e Sousa, Ricardo; Tripathy, Suvranta K.; Magiera, Maria M.; Zaytsev, Anatoly V.; Pereira, Ana L.; Janke, Carsten; Grishchuk, Ekaterina L.; Maiato, Helder

    2015-01-01

    Before chromosomes segregate into daughter cells they align at the mitotic spindle equator, a process known as chromosome congression. CENP-E/Kinesin-7 is a microtubule plus-end-directed kinetochore motor required for congression of pole-proximal chromosomes. Because the plus-ends of many astral microtubules in the spindle point to the cell cortex, it remains unknown how CENP-E guides pole-proximal chromosomes specifically towards the equator. Here we found that congression of pole-proximal chromosomes depended on specific post-translational detyrosination of spindle microtubules that point to the equator. In vitro reconstitution experiments demonstrated that CENP-E-dependent transport was strongly enhanced on detyrosinated microtubules. Blocking tubulin tyrosination in cells caused ubiquitous detyrosination of spindle microtubules and CENP-E transported chromosomes away from spindle poles in random directions. Thus, CENP-E-driven chromosome congression is guided by microtubule detyrosination. PMID:25908662

  13. Mitosis. Microtubule detyrosination guides chromosomes during mitosis.

    PubMed

    Barisic, Marin; Silva e Sousa, Ricardo; Tripathy, Suvranta K; Magiera, Maria M; Zaytsev, Anatoly V; Pereira, Ana L; Janke, Carsten; Grishchuk, Ekaterina L; Maiato, Helder

    2015-05-15

    Before chromosomes segregate into daughter cells, they align at the mitotic spindle equator, a process known as chromosome congression. Centromere-associated protein E (CENP-E)/Kinesin-7 is a microtubule plus-end-directed kinetochore motor required for congression of pole-proximal chromosomes. Because the plus-ends of many astral microtubules in the spindle point to the cell cortex, it remains unknown how CENP-E guides pole-proximal chromosomes specifically toward the equator. We found that congression of pole-proximal chromosomes depended on specific posttranslational detyrosination of spindle microtubules that point to the equator. In vitro reconstitution experiments demonstrated that CENP-E-dependent transport was strongly enhanced on detyrosinated microtubules. Blocking tubulin tyrosination in cells caused ubiquitous detyrosination of spindle microtubules, and CENP-E transported chromosomes away from spindle poles in random directions. Thus, CENP-E-driven chromosome congression is guided by microtubule detyrosination. PMID:25908662

  14. Actin filaments and microtubules play different roles during bristle elongation in Drosophila.

    PubMed

    Tilney, L G; Connelly, P S; Vranich, K A; Shaw, M K; Guild, G M

    2000-04-01

    Developing bristles in Drosophila pupae contain 7-11 bundles of crosslinked actin filaments and a large population of microtubules. During bristle growth the rate of cell elongation increases with bristle length. Thin section EM shows that bundle size is correlated with the amount of cytoplasm at all points along the bristle. Thus, as the bristle elongates and tapers, fewer actin filaments are used. To ensure penetration of inhibitors we isolated thoraces and cultured them in vitro; bristles elongate at rates identical to bristles growing in situ. Interestingly, inhibitors of actin filament assembly (cytochalasin D and latrunculin A) dramatically curtailed bristle elongation while a filament stabilizer (jasplakinolide) accelerated elongation. In contrast, inhibitors of microtubule dynamics (nocodazole, vinblastine, colchicine and taxol) did not affect bristle elongation. Surprisingly, the bristle microtubules are stable and do not turn over. Furthermore, the density of microtubules decreases as the bristle elongates. These two facts coupled with calculations and kinetics of elongation and the fact that the microtubules are short indicate that the microtubules are assembled early in development and then transported distally as the bristle grows. We conclude that actin assembly is crucial for bristle cell elongation and that microtubules must furnish other functions such as to provide bulk to the bristle cytoplasm as well as playing a role in vesicle transport.

  15. Microtubules mediate germ-nuclear behavior after meiosis in conjugation of Paramecium caudatum.

    PubMed

    Nakajima, Yuka; Ishida, Masaki; Mikami, Kazuyuki

    2002-01-01

    Microtubule dynamics in Paramecium caudatum were investigated with an anti-alpha-tubulin antibody and a microinjection technique to determine the function of microtubules on micronuclear behavior during conjugation. After meiosis, all four haploid micronuclei were connected by microtubular filaments to the paroral region and moved close to this region. This nuclear movement was micronucleus-specific, because some small macronuclear fragments transplanted from exconjugants never moved to the region. Only one of the four germ nuclei moved into the paroral cone and was covered by microtubule assembly (the so-called first assembly of microtubules, AM-I). This nucleus survived there, while the other three not in this region degenerated. The movement of germ nucleus was inhibited by the injection of the anti-alpha-tubulin antibody. The surviving germ nucleus divided once and produced a migratory pronucleus and a stationary pronucleus. Prior to the reciprocal exchange of the migratory nuclei, microtubules assembled around the migratory pronuclei again (the so-called second assembly of microtubules, AM-II). Then, the migratory pronucleus moved into the partner cell and fused with the stationary pronucleus. Thus, microtubules appear to be indispensable for nuclear behavior: they enable migration of postmeiotic nuclei to the paroral region and they permit the survival of the nucleus at the paroral cone. PMID:11908901

  16. Actin- and microtubule-dependent regulation of Golgi morphology by FHDC1

    PubMed Central

    Copeland, Sarah J.; Thurston, Susan F.; Copeland, John W.

    2016-01-01

    The Golgi apparatus is the central hub of intracellular trafficking and consists of tethered stacks of cis, medial, and trans cisternae. In mammalian cells, these cisternae are stitched together as a perinuclear Golgi ribbon, which is required for the establishment of cell polarity and normal subcellular organization. We previously identified FHDC1 (also known as INF1) as a unique microtubule-binding member of the formin family of cytoskeletal-remodeling proteins. We show here that endogenous FHDC1 regulates Golgi ribbon formation and has an apparent preferential association with the Golgi-derived microtubule network. Knockdown of FHDC1 expression results in defective Golgi assembly and suggests a role for FHDC1 in maintenance of the Golgi-derived microtubule network. Similarly, overexpression of FHDC1 induces dispersion of the Golgi ribbon into functional ministacks. This effect is independent of centrosome-derived microtubules and instead likely requires the interaction between the FHDC1 microtubule-binding domain and the Golgi-derived microtubule network. These effects also depend on the interaction between the FHDC1 FH2 domain and the actin cytoskeleton. Thus our results suggest that the coordination of actin and microtubule dynamics by FHDC1 is required for normal Golgi ribbon formation. PMID:26564798

  17. Pulsed holographic microscopy as a measurement method of dynamic fracture toughness for fast propagating cracks

    NASA Astrophysics Data System (ADS)

    Suzuki, Shinichi; Homma, Hiroomi; Kusaka, Riichiro

    A METHOD OF pulsed holographic microscopy is applied to take instantaneous microscopic photographs of the neighborhoods of crack tips propagating through PMMA or through AISI 4340 steel specimens at a speed of several hundred meters per second. The cracks are in the opening mode. A fast propagating crack is recorded as a hologram at an instant during its propagation. A microscopic photograph of the crack is taken with a conventional microscope to magnify the reconstructed image from the hologram. From the microscopic photograph, crack opening displacement (COD) is measured along the crack in the vicinity of the crack tip. The COD is of the order often to one hundred microns, and in proportion to the square root of the distance from the crack tip. The dynamic fracture toughness KID is obtained using the formula for COD in the singular stress field of a fast propagating crack. Simultaneous KID measurement both through pulsed holographic microscopy and through the caustic method is furthermore carried out with PMMA specimens. The values of KID obtained through pulsed holographic microscopy are in agreement with those through the caustic method. Microcracks accompanied by a main crack are also photographed with the method of pulsed holographic microscopy.

  18. Dynamic measurements at THz frequencies with a fast rotary delay line

    NASA Astrophysics Data System (ADS)

    Guerboukha, Hichem; Markov, Andrey; Qu, Hang; Skorobogatiy, Maksim

    2016-02-01

    Fabrication, characterization, and applications of a fast rotary linear optical delay line (FRLODL) for THz time-domain spectroscopy are presented. The FRLODL features two reflective surfaces with spatially separated incoming and outgoing beams. It has been manufactured using CNC machining. A linear dependence of the optical delay on the rotation angle allows a straightforward extraction of the conversion factor between the acquisition time (in ms) and the terahertz pulse time (in ps). The FRLODL has been tested using rotation speeds of up to 48 Hz, corresponding to an acquisition rate of up to 192 Hz with four blades incorporated on the same disk. At high speeds we observe a decrease of the bandwidth due to the limitations of the electronics, in particular, the transimpedance amplifier. An error analysis is performed by experimentally evaluating the signal-to-noise ratio and the dynamic range. With regard to the applications of the FRLODL, we first present observation of the evaporation of liquids, namely water, acetone and methanol. We then demonstrate monitoring of the spray painting process. Finally, detection of fast moving objects at 1 m/s and their thickness characterization are presented.

  19. Metal hydrides reactors with improved dynamic characteristics for a fast cycling hydrogen compressor

    NASA Astrophysics Data System (ADS)

    Popeneciu, G.; Coldea, I.; Lupu, D.; Misan, I.; Ardelean, O.

    2009-08-01

    This paper presents an investigation of coupled heat and mass transfer process in metal hydrides hydrogen storage reactors. Hydrogen storage and compression performance of our designed and developed reactors are studied by varying the operating parameters and analyzing the effects of metal hydride bed parameters. The metal alloy selected to characterize the cycling behaviour of reactors is LaNi5, material synthesized and characterized by us in the range 20-80°C. Four types of metal hydride reactors were tested with the aim to provide a fast hydrogen absorption-desorption cycle, able to be thermally cycled at rapid rates. Some new technical solutions have been studied to make a step forward in reducing the duration of the reactors cycle, which combines the effective increase of the thermal conductivity and good permeability to hydrogen gas. Dynamic characteristic of developed fast metal hydride reactors is improved using our novel mixture metal hydride-CA conductive additive due to the increased effective thermal conductivity of the alloy bed. The advanced hydride bed design with high heat transfer capabilities can be thermally cycled at a rapid rate, under 120 seconds, in order to process high hydrogen flow rates.

  20. FAST: A multi-processed environment for visualization of computational fluid dynamics

    NASA Technical Reports Server (NTRS)

    Bancroft, Gordon V.; Merritt, Fergus J.; Plessel, Todd C.; Kelaita, Paul G.; Mccabe, R. Kevin

    1991-01-01

    Three-dimensional, unsteady, multi-zoned fluid dynamics simulations over full scale aircraft are typical of the problems being investigated at NASA Ames' Numerical Aerodynamic Simulation (NAS) facility on CRAY2 and CRAY-YMP supercomputers. With multiple processor workstations available in the 10-30 Mflop range, we feel that these new developments in scientific computing warrant a new approach to the design and implementation of analysis tools. These larger, more complex problems create a need for new visualization techniques not possible with the existing software or systems available as of this writing. The visualization techniques will change as the supercomputing environment, and hence the scientific methods employed, evolves even further. The Flow Analysis Software Toolkit (FAST), an implementation of a software system for fluid mechanics analysis, is discussed.

  1. Fast and accurate quantum molecular dynamics of dense plasmas across temperature regimes

    DOE PAGESBeta

    Sjostrom, Travis; Daligault, Jerome

    2014-10-10

    Here, we develop and implement a new quantum molecular dynamics approximation that allows fast and accurate simulations of dense plasmas from cold to hot conditions. The method is based on a carefully designed orbital-free implementation of density functional theory. The results for hydrogen and aluminum are in very good agreement with Kohn-Sham (orbital-based) density functional theory and path integral Monte Carlo calculations for microscopic features such as the electron density as well as the equation of state. The present approach does not scale with temperature and hence extends to higher temperatures than is accessible in the Kohn-Sham method and lowermore » temperatures than is accessible by path integral Monte Carlo calculations, while being significantly less computationally expensive than either of those two methods.« less

  2. Fast and accurate quantum molecular dynamics of dense plasmas across temperature regimes

    SciTech Connect

    Sjostrom, Travis; Daligault, Jerome

    2014-10-10

    Here, we develop and implement a new quantum molecular dynamics approximation that allows fast and accurate simulations of dense plasmas from cold to hot conditions. The method is based on a carefully designed orbital-free implementation of density functional theory. The results for hydrogen and aluminum are in very good agreement with Kohn-Sham (orbital-based) density functional theory and path integral Monte Carlo calculations for microscopic features such as the electron density as well as the equation of state. The present approach does not scale with temperature and hence extends to higher temperatures than is accessible in the Kohn-Sham method and lower temperatures than is accessible by path integral Monte Carlo calculations, while being significantly less computationally expensive than either of those two methods.

  3. Fast and reliable decisions for a dynamic song parameter in field crickets.

    PubMed

    Trobe, Daniela; Schuster, Richard; Römer, Heiner

    2011-01-01

    We investigated the choice of female crickets for a dynamic song parameter (chirp rate) on a walking compensator, and the underlying neuronal basis for the choice in the form of discharge differences in the pair of AN1-neurons driving the phonotactic steering behaviour. Our analysis reveals that decisions about chirp rate in a choice situation are made fast and reliably by female crickets. They steered towards the higher chirp rate after a delay of only 2.2-6 s, depending on the rate difference between the song alternatives. In this time period, the female experienced only one to two additional chirps in the song model with the higher rate. There was a strong correlation between the accumulated AN1 discharge difference and the amount of steering towards the side with the stronger response. PMID:20878165

  4. Fast and reliable decisions for a dynamic song parameter in field crickets

    PubMed Central

    Trobe, Daniela; Schuster, Richard; Römer, Heiner

    2014-01-01

    We investigated the choice of female crickets for a dynamic song parameter (chirp rate) on a walking compensator, and the underlying neuronal basis for the choice in the form of discharge differences in the pair of AN1-neurons driving the phonotactic steering behaviour. Our analysis reveals that decisions about chirp rate in a choice situation are made fast and reliably by female crickets. They steered towards the higher chirp rate after a delay of only 2.2–6 s, depending on the rate difference between the song alternatives. In this time period, the female experienced only one to two additional chirps in the song model with the higher rate. There was a strong correlation between the accumulated AN1 discharge difference and the amount of steering towards the side with the stronger response. PMID:20878165

  5. Fast and reliable decisions for a dynamic song parameter in field crickets.

    PubMed

    Trobe, Daniela; Schuster, Richard; Römer, Heiner

    2011-01-01

    We investigated the choice of female crickets for a dynamic song parameter (chirp rate) on a walking compensator, and the underlying neuronal basis for the choice in the form of discharge differences in the pair of AN1-neurons driving the phonotactic steering behaviour. Our analysis reveals that decisions about chirp rate in a choice situation are made fast and reliably by female crickets. They steered towards the higher chirp rate after a delay of only 2.2-6 s, depending on the rate difference between the song alternatives. In this time period, the female experienced only one to two additional chirps in the song model with the higher rate. There was a strong correlation between the accumulated AN1 discharge difference and the amount of steering towards the side with the stronger response.

  6. SPH modeling of adhesion in fast dynamics: Application to the Cold Spray process

    NASA Astrophysics Data System (ADS)

    Profizi, Paul; Combescure, Alain; Ogawa, Kahuziro

    2016-04-01

    The objective of this paper is to show, in a specific case, the importance of modeling adhesive forces when simulating the bouncing of very small particles impacting a substrate at high speed. The implementation of this model into a fast-dynamics SPH code is described. Taking the example of an impacted elastic cylinder, we show that the adhesive forces, which are surface forces, play a significant role only if the particles are sufficiently small. The effect of the choice of the type of interaction law in the cohesive zone is studied and some conclusions on the relevance of the modeling of the adhesive forces for fast-dynamics impacts are drawn. Then, the adhesion model is used to simulate the Cold Spray process. An aluminum particle is projected against a substrate made of the same material at a velocity ranging from 200 to 1000 m ṡs-1. We study the effects of the various modeling assumptions on the final result: bouncing or sticking. Increasingly complex models are considered. At a 200 m ṡs-1 impact velocity, elastic behavior is assumed, the substrate being simply supported at its base and supplied with absorbing boundaries. The same absorbing boundaries are also used for all the other simulations. Then, plasticity is introduced and the impact velocity is increased up to 1000 m ṡs-1. At the highest velocities, the resulting strains are very significant. The calculations show that if the adhesion model is appropriately chosen, it is possible to reproduce the experimental observations: the particles stick to the substrate in a range of impact velocities surrounded by two velocity ranges in which the particles bounce.

  7. Microtubule nucleation and organization in dendrites.

    PubMed

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

    2016-07-01

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

  8. Impact of Fast Sodium Channel Inactivation on Spike Threshold Dynamics and Synaptic Integration

    PubMed Central

    Platkiewicz, Jonathan; Brette, Romain

    2011-01-01

    Neurons spike when their membrane potential exceeds a threshold value. In central neurons, the spike threshold is not constant but depends on the stimulation. Thus, input-output properties of neurons depend both on the effect of presynaptic spikes on the membrane potential and on the dynamics of the spike threshold. Among the possible mechanisms that may modulate the threshold, one strong candidate is Na channel inactivation, because it specifically impacts spike initiation without affecting the membrane potential. We collected voltage-clamp data from the literature and we found, based on a theoretical criterion, that the properties of Na inactivation could indeed cause substantial threshold variability by itself. By analyzing simple neuron models with fast Na inactivation (one channel subtype), we found that the spike threshold is correlated with the mean membrane potential and negatively correlated with the preceding depolarization slope, consistent with experiments. We then analyzed the impact of threshold dynamics on synaptic integration. The difference between the postsynaptic potential (PSP) and the dynamic threshold in response to a presynaptic spike defines an effective PSP. When the neuron is sufficiently depolarized, this effective PSP is briefer than the PSP. This mechanism regulates the temporal window of synaptic integration in an adaptive way. Finally, we discuss the role of other potential mechanisms. Distal spike initiation, channel noise and Na activation dynamics cannot account for the observed negative slope-threshold relationship, while adaptive conductances (e.g. K+) and Na inactivation can. We conclude that Na inactivation is a metabolically efficient mechanism to control the temporal resolution of synaptic integration. PMID:21573200

  9. Anomalous motor mediated cargo transport in microtubule networks

    NASA Astrophysics Data System (ADS)

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

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

  10. Tubulin Bond Energies and Microtubule Biomechanics Determined from Nanoindentation in Silico

    PubMed Central

    2015-01-01

    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·nm2) 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. PMID:25389565

  11. 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.

  12. Dissecting the Nanoscale Distributions and Functions of Microtubule-End-Binding Proteins EB1 and ch-TOG in Interphase HeLa Cells

    PubMed Central

    Nakamura, Satoko; Grigoriev, Ilya; Nogi, Taisaku; Hamaji, Tomoko; Cassimeris, Lynne; Mimori-Kiyosue, Yuko

    2012-01-01

    Recently, the EB1 and XMAP215/TOG families of microtubule binding proteins have been demonstrated to bind autonomously to the growing plus ends of microtubules and regulate their behaviour in in vitro systems. However, their functional redundancy or difference in cells remains obscure. Here, we compared the nanoscale distributions of EB1 and ch-TOG along microtubules using high-resolution microscopy techniques, and also their roles in microtubule organisation in interphase HeLa cells. The ch-TOG accumulation sites protruded ∼100 nm from the EB1 comets. Overexpression experiments showed that ch-TOG and EB1 did not interfere with each other’s localisation, confirming that they recognise distinct regions at the ends of microtubules. While both EB1 and ch-TOG showed similar effects on microtubule plus end dynamics and additively increased microtubule dynamicity, only EB1 exhibited microtubule-cell cortex attachment activity. These observations indicate that EB1 and ch-TOG regulate microtubule organisation differently via distinct regions in the plus ends of microtubules. PMID:23251535

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

    PubMed

    Nakamura, Satoko; Grigoriev, Ilya; Nogi, Taisaku; Hamaji, Tomoko; Cassimeris, Lynne; Mimori-Kiyosue, Yuko

    2012-01-01

    Recently, the EB1 and XMAP215/TOG families of microtubule binding proteins have been demonstrated to bind autonomously to the growing plus ends of microtubules and regulate their behaviour in in vitro systems. However, their functional redundancy or difference in cells remains obscure. Here, we compared the nanoscale distributions of EB1 and ch-TOG along microtubules using high-resolution microscopy techniques, and also their roles in microtubule organisation in interphase HeLa cells. The ch-TOG accumulation sites protruded ∼100 nm from the EB1 comets. Overexpression experiments showed that ch-TOG and EB1 did not interfere with each other's localisation, confirming that they recognise distinct regions at the ends of microtubules. While both EB1 and ch-TOG showed similar effects on microtubule plus end dynamics and additively increased microtubule dynamicity, only EB1 exhibited microtubule-cell cortex attachment activity. These observations indicate that EB1 and ch-TOG regulate microtubule organisation differently via distinct regions in the plus ends of microtubules. PMID:23251535

  14. Fast Quantum Molecular Dynamics Simulations of Shock-induced Chemistry in Organic Liquids

    NASA Astrophysics Data System (ADS)

    Cawkwell, Marc

    2014-03-01

    The responses of liquid formic acid and phenylacetylene to shock compression have been investigated via quantum-based molecular dynamics simulations with the self-consistent tight-binding code LATTE. Microcanonical Born-Oppenheimer trajectories with precise conservation of the total energy were computed without relying on an iterative self-consistent field optimization of the electronic degrees of freedom at each time step via the Fast Quantum Mechanical Molecular Dynamics formalism [A. M. N. Niklasson and M. J. Cawkwell, Phys. Rev. B, 86, 174308 (2012)]. The conservation of the total energy in our trajectories was pivotal for the capture of adiabatic shock heating as well as temperature changes arising from endo- or exothermic chemistry. Our self-consistent tight-binding parameterizations yielded very good predictions for the gas-phase geometries of formic acid and phenylacetylene molecules and the principal Hugoniots of the liquids. In accord with recent flyer-plate impact experiments, our simulations revealed i) that formic acid reacts at relatively low impact pressures but with no change in volume between products and reactants, and ii) a two-step polymerization process for phenylacetylene. Furthermore, the evolution of the HOMO-LUMO gap tracked on-the-fly during our simulations could be correlated with changes transient absorption measured during laser-driven shock compression experiments on these liquids.

  15. Portable low-coherence interferometry for quantitatively imaging fast dynamics with extended field of view

    NASA Astrophysics Data System (ADS)

    Shaked, Natan T.; Girshovitz, Pinhas; Frenklach, Irena

    2014-06-01

    We present our recent advances in the development of compact, highly portable and inexpensive wide-field interferometric modules. By a smart design of the interferometric system, including the usage of low-coherence illumination sources and common-path off-axis geometry of the interferometers, spatial and temporal noise levels of the resulting quantitative thickness profile can be sub-nanometric, while processing the phase profile in real time. In addition, due to novel experimentally-implemented multiplexing methods, we can capture low-coherence off-axis interferograms with significantly extended field of view and in faster acquisition rates. Using these techniques, we quantitatively imaged rapid dynamics of live biological cells including sperm cells and unicellular microorganisms. Then, we demonstrated dynamic profiling during lithography processes of microscopic elements, with thicknesses that may vary from several nanometers to hundreds of microns. Finally, we present new algorithms for fast reconstruction (including digital phase unwrapping) of off-axis interferograms, which allow real-time processing in more than video rate on regular single-core computers.

  16. Dynamic fast terminal sliding mode control of a shape memory alloy actuated system

    NASA Astrophysics Data System (ADS)

    Marathe, Meeshawn S.; Srinivasan, S. M.

    2016-04-01

    In this paper we address the chattering phenomenon which is a common drawback associated with the normal Sliding Mode Control (SMC) law for a basic shape memory alloy (SMA) actuated system. A new method has been proposed to counter this effect by combining the concepts of Fast Terminal SMC and Dynamic controller. A phenomenological model is developed for the SMA which incorporates a piecewise linear hysteresis behavior. This model is used for both open loop as well as closed loop simulations for a linear motion control system. Based on this model, a dynamic terminal sliding mode control law is derived and applied to the system. A normal SMC law with saturation function which is known to reduce chattering is compared with the proposed control law for its effectiveness to curb the issue of chattering versus its ability to faithfully track a desired trajectory. Numerical Simulations indicate that the proposed law is able to reduce the chattering effect sufficiently and at par with the control technique involving saturation function.

  17. Forces from the rear: deformed microtubules in neuronal growth cones influence retrograde flow and advancement

    NASA Astrophysics Data System (ADS)

    Rauch, Philipp; Heine, Paul; Goettgens, Barbara; Käs, Josef A.

    2013-01-01

    The directed motility of growth cones at the tip of neuronal processes is a key function in neuronal path-finding and relies on a complex system of interacting cytoskeletal components. Despite intensive research in this field, many aspects of the mechanical roles of actin structures and, in particular, of microtubules throughout this process remain unclear. Mostly, force generation is ascribed to actin-myosin-based structures such as filopodia bundles and the dynamic polymer gel within the lamellipodium. Our analysis of microtubule buckling and deformation in motile growth cones reveals that extending microtubule filaments contribute significantly to the overall protrusion force. In this study, we establish a relationship of the local variations in stored bending energy and deformation characteristics to growth cone morphology and retrograde actin flow. This implies the relevance of microtubule pushing and deformation for general neurite advancement as well as steering processes.

  18. Chromosome position at the spindle equator is regulated by chromokinesin and a bipolar microtubule array.

    PubMed

    Takagi, Jun; Itabashi, Takeshi; Suzuki, Kazuya; Ishiwata, Shin'ichi

    2013-09-30

    The chromosome alignment is mediated by polar ejection and poleward forces acting on the chromosome arm and kinetochores, respectively. Although components of the motile machinery such as chromokinesin have been characterized, their dynamics within the spindle is poorly understood. Here we show that a quantum dot (Qdot) binding up to four Xenopus chromokinesin (Xkid) molecules behaved like a nanosize chromosome arm in the meiotic spindle, which is self-organized in cytoplasmic egg extracts. Xkid-Qdots travelled long distances along microtubules by changing several tracks, resulting in their accumulation toward and distribution around the metaphase plate. The analysis indicated that the direction of motion and velocity depend on the distribution of microtubule polarity within the spindle. Thus, this mechanism is governed by chromokinesin motors, which is dependent on symmetrical microtubule orientation that may allow chromosomes to maintain their position around the spindle equator until correct microtubule-kinetochore attachment is established.

  19. Chromosome position at the spindle equator is regulated by chromokinesin and a bipolar microtubule array.

    PubMed

    Takagi, Jun; Itabashi, Takeshi; Suzuki, Kazuya; Ishiwata, Shin'ichi

    2013-01-01

    The chromosome alignment is mediated by polar ejection and poleward forces acting on the chromosome arm and kinetochores, respectively. Although components of the motile machinery such as chromokinesin have been characterized, their dynamics within the spindle is poorly understood. Here we show that a quantum dot (Qdot) binding up to four Xenopus chromokinesin (Xkid) molecules behaved like a nanosize chromosome arm in the meiotic spindle, which is self-organized in cytoplasmic egg extracts. Xkid-Qdots travelled long distances along microtubules by changing several tracks, resulting in their accumulation toward and distribution around the metaphase plate. The analysis indicated that the direction of motion and velocity depend on the distribution of microtubule polarity within the spindle. Thus, this mechanism is governed by chromokinesin motors, which is dependent on symmetrical microtubule orientation that may allow chromosomes to maintain their position around the spindle equator until correct microtubule-kinetochore attachment is established. PMID:24077015

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

    SciTech Connect

    Fanarraga, M.L.

    2009-02-01

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

  1. Polymerization of microtubules in the presence of diffusing tubulins: a boundary layer analysis

    NASA Astrophysics Data System (ADS)

    Yadav, Vandana; Mukherji, Sutapa

    2015-02-01

    Microtubules are biopolymers with an interesting growth dynamics that alternates between persistent phases of polymerization and depolymerization. The growth dynamics is expected to be influenced by the dynamics of the tubulin molecules around the microtubule. In this paper, we present a boundary layer based method that allows us to study systematically a nonlinear growth model coupling the growth dynamics of microtubule and the diffusive dynamics of free tubulins in the neighboring environment. Following previous experimental studies, we consider the growth and the rescue rates to be dependent on the tubulin density in the neighborhood of the microtubule tip. Considering the effect of various regulatory proteins that facilitate the motion of tubulin molecules along the direction of growth, we incorporate an effective biased diffusion of tubulin molecules. Based on the values of the bias velocity and other parameters, the boundary layer analysis shows two regions with distinct nature of microtubule length distributions. In order to show the wider applicability of the method, we briefly discuss the case of tubulin density dependent catastrophe rate. Some of our results are supported by numerical simulations.

  2. Energy efficient low power shared-memory Fast Fourier Transform (FFT) processor with dynamic voltage scaling

    NASA Astrophysics Data System (ADS)

    Fitrio, D.; Singh, J.; Stojcevski, A.

    2005-12-01

    Reduction of power dissipations in CMOS circuits needs to be addressed for portable battery devices. Selection of appropriate transistor library to minimise leakage current, implementation of low power design architectures, power management implementation, and the choice of chip packaging, all have impact on power dissipation and are important considerations in design and implementation of integrated circuits for low power applications. Energy-efficient architecture is highly desirable for battery operated systems, which operates in a wide variation of operating scenarios. Energy-efficient design aims to reconfigure its own architectures to scale down energy consumption depending upon the throughput and quality requirement. An energy efficient system should be able to decide its minimum power requirements by dynamically scaling its own operating frequency, supply voltage or the threshold voltage according to a variety of operating scenarios. The increasing product demand for application specific integrated circuit or processor for independent portable devices has influenced designers to implement dedicated processors with ultra low power requirements. One of these dedicated processors is a Fast Fourier Transform (FFT) processor, which is widely used in signal processing for numerous applications such as, wireless telecommunication and biomedical applications where the demand for extended battery life is extremely high. This paper presents the design and performance analysis of a low power shared memory FFT processor incorporating dynamic voltage scaling. Dynamic voltage scaling enables power supply scaling into various supply voltage levels. The concept behind the proposed solution is that if the speed of the main logic core can be adjusted according to input load or amount of processor's computation "just enough" to meet the r