Science.gov

Sample records for microtubule assembly dynamics

  1. 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. PMID:27307590

  2. Harnessing microtubule dynamic instability for nanostructure assembly.

    SciTech Connect

    Bouchard, Ann Marie; Osbourn, Gordon Cecil

    2004-06-01

    Intracellular molecular machines synthesize molecules, tear apart others, transport materials, transform energy into different forms, and carry out a host of other coordinated processes. Many molecular processes have been shown to work outside of cells, and the idea of harnessing these molecular machines to build nanostructures is attractive. Two examples are microtubules and motor proteins, which aid cell movement, help determine cell shape and internal structure, and transport vesicles and organelles within the cell. These molecular machines work in a stochastic, noisy fashion: microtubules switch randomly between growing and shrinking in a process known as dynamic instability; motor protein movement along microtubules is randomly interrupted by the motor proteins falling off. A common strategy in attempting to gain control over these highly dynamic, stochastic processes is to eliminate some processes (e.g., work with stabilized microtubules) in order to focus on others (interaction of microtubules with motor proteins). In this paper, we illustrate a different strategy for building nanostructures, which, rather than attempting to control or eliminate some dynamic processes, uses them to advantage in building nanostructures. Specifically, using stochastic agent-based simulations, we show how the natural dynamic instability of microtubules can be harnessed in building nanostructures, and discuss strategies for ensuring that 'unreliable' stochastic processes yield a robust outcome.

  3. 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

  4. 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.

  5. 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.

  6. 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

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

    SciTech Connect

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

    2015-04-24

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

  8. 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

  9. 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. PMID:25753733

  10. 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

  11. Role of Microtubules in Stress Granule Assembly

    PubMed Central

    Chernov, Konstantin G.; Barbet, Aurélie; Hamon, Loic; Ovchinnikov, Lev P.; Curmi, Patrick A.; Pastré, David

    2009-01-01

    Following exposure to various stresses (arsenite, UV, hyperthermia, and hypoxia), mRNAs are assembled into large cytoplasmic bodies known as “stress granules,” in which mRNAs and associated proteins may be processed by specific enzymes for different purposes like transient storing, sorting, silencing, or other still unknown processes. To limit mRNA damage during stress, the assembly of micrometric granules has to be rapid, and, indeed, it takes only ∼10–20 min in living cells. However, such a rapid assembly breaks the rules of hindered diffusion in the cytoplasm, which states that large cytoplasmic bodies are almost immobile. In the present work, using HeLa cells and YB-1 protein as a stress granule marker, we studied three hypotheses to understand how cells overcome the limitation of hindered diffusion: shuttling of small messenger ribonucleoprotein particles from small to large stress granules, sliding of messenger ribonucleoprotein particles along microtubules, microtubule-mediated stirring of large stress granules. Our data favor the two last hypotheses and underline that microtubule dynamic instability favors the formation of micrometric stress granules. PMID:19843517

  12. 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

  13. 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.

  14. 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.

  15. 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.

  16. 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. PMID:26802898

  17. A Thermodynamic Model of Microtubule Assembly and Disassembly

    PubMed Central

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

    2009-01-01

    Microtubules are self-assembling polymers whose dynamics are essential for the normal function of cellular processes including chromosome separation and cytokinesis. Therefore understanding what factors effect microtubule growth is fundamental to our understanding of the control of microtubule based processes. An important factor that determines the status of a microtubule, whether it is growing or shrinking, is the length of the GTP tubulin microtubule cap. Here, we derive a Monte Carlo model of the assembly and disassembly of microtubules. We use thermodynamic laws to reduce the number of parameters of our model and, in particular, we take into account the contribution of water to the entropy of the system. We fit all parameters of the model from published experimental data using the GTP tubulin dimer attachment rate and the lateral and longitudinal binding energies of GTP and GDP tubulin dimers at both ends. Also we calculate and incorporate the GTP hydrolysis rate. We have applied our model and can mimic published experimental data, which formerly suggested a single layer GTP tubulin dimer microtubule cap, to show that these data demonstrate that the GTP cap can fluctuate and can be several microns long. PMID:19668378

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

    PubMed Central

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

    2013-01-01

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

  19. Templated nanocrystal assembly on biodynamic artificial microtubule asters.

    PubMed

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

    2013-03-26

    Microtubules (MTs) and the MT-associated proteins (MAPs) are critical cooperative agents involved in complex nanoassembly processes in biological systems. These biological materials and processes serve as important inspiration in developing new strategies for the assembly of synthetic nanomaterials in emerging techologies. Here, we explore a dynamic biofabrication process, modeled after the form and function of natural aster-like MT assemblies such as centrosomes. Specifically, we exploit the cooperative assembly of MTs and MAPs to form artificial microtubule asters and demonstrate that (1) these three-dimensional biomimetic microtubule asters can be controllably, reversibly assembled and (2) they serve as unique, dynamic biotemplates for the organization of secondary nanomaterials. We describe the MAP-mediated assembly and growth of functionalized MTs onto synthetic particles, the dynamic character of the assembled asters, and the application of these structures as templates for three-dimensional nanocrystal organization across multiple length scales. This biomediated nanomaterials assembly strategy illuminates a promising new pathway toward next-generation nanocomposite development. PMID:23363365

  20. Drugs That Target Dynamic Microtubules: A New Molecular Perspective

    PubMed Central

    Stanton, Richard A.; Gernert, Kim M.; Nettles, James H.; Aneja, Ritu

    2011-01-01

    Microtubules have long been considered an ideal target for anticancer drugs because of the essential role they play in mitosis, forming the dynamic spindle apparatus. As such, there is a wide variety of compounds currently in clinical use and in development that act as antimitotic agents by altering microtubule dynamics. Although these diverse molecules are known to affect microtubule dynamics upon binding to one of the three established drug domains (taxane, vinca alkaloid, or colchicine site), the exact mechanism by which each drug works is still an area of intense speculation and research. In this study, we review the effects of microtubule-binding chemotherapeutic agents from a new perspective, considering how their mode of binding induces conformational changes and alters biological function relative to the molecular vectors of microtubule assembly or disassembly. These “biological vectors” can thus be used as a spatiotemporal context to describe molecular mechanisms by which microtubule-targeting drugs work. PMID:21381049

  1. 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

  2. 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

  3. Self-assembly and sorting of acentrosomal microtubules by TACC3 facilitate kinetochore capture during the mitotic spindle assembly.

    PubMed

    Fu, Wenxiang; Chen, Hao; Wang, Gang; Luo, Jia; Deng, Zhaoxuan; Xin, Guangwei; Xu, Nan; Guo, Xiao; Lei, Jun; Jiang, Qing; Zhang, Chuanmao

    2013-09-17

    Kinetochore capture by dynamic kinetochore microtubule fibers (K fibers) is essential for proper chromosome alignment and accurate distribution of the replicated genome during cell division. Although this capture process has been extensively studied, the mechanisms underlying the initiation of this process and the proper formation of the K fibers remain largely unknown. Here we show that transforming acidic coiled-coil-containing protein 3 (TACC3) is essential for kinetochore capture and proper K-fiber formation in HeLa cells. To observe the assembly of acentrosomal microtubules more clearly, the cells were released from higher concentrations of nocodazole into zero or lower concentrations. We find that small acentrosomal TACC3-microtubule aster formation near the kinetochores and binding of the asters with the kinetochores are the initial steps of the kinetochore capture by the acentrosomal microtubules, and that the sorting of kinetochore-captured acentrosomal microtubules with centrosomal microtubules leads to the capture of kinetochore by centrosomal microtubules from both spindle poles. We demonstrate that the sorting of the TACC3-associated microtubules with the centrosomal microtubules is a crucial process for spindle assembly and chromosome movement. These findings, which are also supported in the unperturbed mitosis without nocodazole, reveal a critical TACC3-dependent acentrosomal microtubule nucleation and sorting process to regulate kinetochore-microtubule connections and provide deep insight into the mechanisms of mitotic spindle assembly and chromosome alignment. PMID:24003142

  4. Self-assembly of microtubules and motors

    NASA Astrophysics Data System (ADS)

    Aranson, Igor; Tsimring, Lev

    2005-03-01

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

  5. Dynamics of Antarctic fish microtubules at low temperatures

    SciTech Connect

    Himes, R.H.; Detrich, H.W. III )

    1989-06-13

    The tubulins of Antarctic fishes, purified from brain tissue and depleted of microtubule-associated proteins (MAPs), polymerized efficiently in vitro to yield microtubules at near-physiological and supraphysiological temperatures (5, 10, and 20{degree}C). The dynamics of the microtubules at these temperatures were examined through the use of labeled guanosine 5{prime}-triphosphate (GTP) as a marker for the incorporation, retention, and loss of tubulin dimers. Following attainment of a steady state in microtubule mass at 20{degree}C, the rate of incorporation of ({sup 3}H)GTP (i.e., tubulin dimers) during pulses of constant duration decreased asymptotically toward a constant, nonzero value as the interval prior to label addition to the microtubule solution increased. Concomitant with the decreasing rate of label incorporation, the average length of the microtubules increased, and the number concentration of microtubules decreased. Thus, redistribution of microtubule lengths appears to be responsible for the time-dependent decrease in the rate of tubulin uptake. At each temperature, most of the incorporated label was retained by the microtubules during a subsequent chase with excess unlabeled GTP. In contrast, when microtubules were assembled do novo in the presence of ({alpha}-{sup 32}P)GTP at 5{degree}C and then exposed to a pulse of ({sup 3}H)GTP, the {sup 32}P label was lost over time during a subsequent chase with unlabeled GTP, whereas the {sup 3}H label was retained. Together, these results indicate that the microtubules of Antarctic fishes exhibit, at low temperatures, behaviors consistent both with subunit treadmilling and with dynamic instability and/or microtubule annealing.

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

    PubMed Central

    Chernov, Konstantin G; Mechulam, Alain; Popova, Nadezhda V; Pastre, David; Nadezhdina, Elena S; Skabkina, Olga V; Shanina, Nina A; Vasiliev, Victor D; Tarrade, Anne; Melki, Judith; Joshi, Vandana; Baconnais, Sonia; Toma, Flavio; Ovchinnikov, Lev P; Curmi, Patrick A

    2008-01-01

    Background YB-1 is a major regulator of gene expression in eukaryotic cells. In addition to its role in transcription, YB-1 plays a key role in translation and stabilization of mRNAs. Results We show here that YB-1 interacts with tubulin and microtubules and stimulates microtubule assembly in vitro. High resolution imaging via electron and atomic force microscopy revealed that microtubules assembled in the presence of YB-1 exhibited a normal single wall ultrastructure and indicated that YB-1 most probably coats the outer microtubule wall. Furthermore, we found that YB-1 also promotes the assembly of MAPs-tubulin and subtilisin-treated tubulin. Finally, we demonstrated that tubulin interferes with RNA:YB-1 complexes. Conclusion These results suggest that YB-1 may regulate microtubule assembly in vivo and that its interaction with tubulin may contribute to the control of mRNA translation. PMID:18793384

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

    PubMed Central

    Bartoli, Kristen M.; Bishop, Darryl L.; Saunders, William S.

    2011-01-01

    Stress granules (SGs) are cytoplasmic foci that appear in cells exposed to stress-induced translational inhibition. SGs function as a triage center, where mRNAs are sorted for storage, degradation, and translation reinitiation. The underlying mechanisms of SGs dynamics are still being characterized, although many key players have been identified. The main components of SGs are stalled 48S preinitiation complexes. To date, many other proteins have also been found to localize in SGs and are hypothesized to function in SG dynamics. Most recently, the microtubule cytoskeleton and associated motor proteins have been demonstrated to function in SG dynamics. In this paper, we will discuss current literature examining the function of microtubules and the molecular microtubule motors in SG assembly, coalescence, movement, composition, organization, and disassembly. PMID:21760798

  8. Microtubule dynamics in neuronal morphogenesis.

    PubMed

    Sakakibara, Akira; Ando, Ryota; Sapir, Tamar; Tanaka, Teruyuki

    2013-07-01

    Microtubules (MTs) are essential for neuronal morphogenesis in the developing brain. The MT cytoskeleton provides physical support to shape the fine structure of neuronal processes. MT-based motors play important roles in nucleokinesis, process formation and retraction. Regulation of MT stability downstream of extracellular cues is proposed to be critical for axonogenesis. Axons and dendrites exhibit different patterns of MT organization, underlying the divergent functions of these processes. Centrosomal positioning has drawn the attention of researchers because it is a major clue to understanding neuronal MT organization. In this review, we focus on how recent advances in live imaging have revealed the dynamics of MT organization and centrosome positioning during neural development. PMID:23864552

  9. Microtubule dynamics in neuronal morphogenesis

    PubMed Central

    Sakakibara, Akira; Ando, Ryota; Sapir, Tamar; Tanaka, Teruyuki

    2013-01-01

    Microtubules (MTs) are essential for neuronal morphogenesis in the developing brain. The MT cytoskeleton provides physical support to shape the fine structure of neuronal processes. MT-based motors play important roles in nucleokinesis, process formation and retraction. Regulation of MT stability downstream of extracellular cues is proposed to be critical for axonogenesis. Axons and dendrites exhibit different patterns of MT organization, underlying the divergent functions of these processes. Centrosomal positioning has drawn the attention of researchers because it is a major clue to understanding neuronal MT organization. In this review, we focus on how recent advances in live imaging have revealed the dynamics of MT organization and centrosome positioning during neural development. PMID:23864552

  10. 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

  11. 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

  12. Association of Microtubule Dynamics with Chronic Epilepsy.

    PubMed

    Xu, Xin; Hu, Yida; Xiong, Yan; Li, Zhonggui; Wang, Wei; Du, Chao; Yang, Yong; Zhang, Yanke; Xiao, Fei; Wang, Xuefeng

    2016-09-01

    Approximately 30 % of epilepsy cases are refractory to current pharmacological treatments through unknown mechanisms. Much work has been done on the role of synaptic components in the pathogenesis of epilepsy, but relatively little attention has been given to the potential role of the microtubules. We investigated the level of microtubule dynamic in 30 human epileptic tissues and two different chronic epilepsy rat models. The administration of microtubule-modulating agent attenuated the progression of chronic epilepsy. By contrast, microtubule-depolymerizing agent aggravated the progression of chronic epilepsy. The electrophysiological index by whole-cell clamp was used to investigate the neuronal excitation and inhibitory synaptic transmission in brain slices after administration of microtubule-modulating agent and microtubule-depolymerizing agent. Interestingly, we found that microtubule-modulating agent significantly increased the frequency of action potential firing in interneurons, and significantly promoted the amplitudes and frequencies of miniature inhibitory postsynaptic currents. Microtubule-depolymerizing agent had an opposite effect. These findings suggest that modulating hyperdynamic microtubules may take an anti-epileptic effect via postsynaptic mechanisms in interneurons. It could represent a potential pharmacologic target in epilepsy treatment. PMID:26377107

  13. 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

  14. Dynamics of Actively Driven Crosslinked Microtubule Networks

    NASA Astrophysics Data System (ADS)

    Yadav, Vikrant; Stanhope, Kasimira; Evans, Arthur A.; Ross, Jennifer L.

    We have designed a model experiment to explore dynamics of crosslinked active microtubule clusters crosslinked with MAP65. Microtubule clusters are allowed to settle on a slide coated with kinesin-1 molecular motors, which move microtubules. We systematically tune either concentration of cross linkers bound to microtubule (ρc) or the global concentration of microtubules (ρMT) . We quantified the shape of the cluster by measuring the standard deviation (σ) of the cluster outline. At low ρMTor ρc the network is in an expanding state. At higher ρMTor ρc expansion slows down, reaches zero at a critical density, and become negative indicating contraction. Further increase of ρMTor ρc halts any kind of dynamics. The ρMT-ρc phase space shows distinct regions of extensile, contractile and static regimes. We model these results using active hydrodynamic theory. Microtubules are modeled as active rods whereas effect of crosslinkers is modeled using a collision term that prefers anti-parallel alignment of microtubules. A linearized analysis of hydrodynamic equation predicts existence of density driven expanding, contracting, and static phases for microtubule clusters.

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

    PubMed

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

    2013-03-01

    Decisions on the fate of cells and their functions are dictated by the spatiotemporal dynamics of molecular signalling networks. However, techniques to examine the dynamics of these intracellular processes remain limited. Here, we show that magnetic nanoparticles conjugated with key regulatory proteins can artificially control, in time and space, the Ran/RCC1 signalling pathway that regulates the cell cytoskeleton. In the presence of a magnetic field, RanGTP proteins conjugated to superparamagnetic nanoparticles can induce microtubule fibres to assemble into asymmetric arrays of polarized fibres in Xenopus laevis egg extracts. The orientation of the fibres is dictated by the direction of the magnetic force. When we locally concentrated nanoparticles conjugated with the upstream guanine nucleotide exchange factor RCC1, the assembly of microtubule fibres could be induced over a greater range of distances than RanGTP particles. The method shows how bioactive nanoparticles can be used to engineer signalling networks and spatial self-organization inside a cell environment. PMID:23334169

  16. Kinetic model for colchicine inhibition of microtubule assembly

    SciTech Connect

    Sternlicht, H.; Ringel, I.; Szasz, J.

    1980-10-01

    Colchicine is a potent drug used to probe microtubule dependent processes. We have recently shown that substoichiometric concentrations of colchicine-tubulin complex (CD), a 1:1 tight binding complex of drug with tubulin, copolymerizes with tubulin to form microtubule copolymers. The affinity of the microtubule ends for tublin decreased as the CD mole fraction in the microtubule increased. Mole fraction ratios as small as 1 CD to approx. 50 to 100 tubulins in the copolymers were accompanied by a significant change in binding affinities and polymerization rates. We have further extended our investigation of the CD-tubulin copolymerization reaction. A kinetic model was derived which relates the composition of the microtubule copolymer to the composition of the reaction mixture. This model allowed a predictive correlation to be made between copolymer composition and the extent of assembly inhibition.

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

    PubMed Central

    Hinow, Peter; Rezania, Vahid; Lopus, Manu; Jordan, Mary Ann; Tuszyński, Jack A.

    2011-01-01

    We propose a stochastic model that accounts for the growth, catastrophe and rescue processes of steady state microtubules assembled from MAP-free tubulin in the possible presence of a microtubule associated drug. As an example for the latter, we both experimentally and theoretically study the perturbation of microtubule dynamic instability by S-methyl-D-DM1, a synthetic derivative of the microtubule-targeted agent maytansine and a potential anticancer agent. Our model predicts that among drugs that act locally at the microtubule tip, primary inhibition of the loss of GDP tubulin results in stronger damping of microtubule dynamics than inhibition of GTP tubulin addition. On the other hand, drugs whose action occurs in the interior of the microtubule need to be present in much higher concentrations to have visible effects. PMID:21836336

  18. Effect of Aluminum, Iron, and Zinc Ions on the Assembly of Microtubules from Brain Microtubule Proteins.

    PubMed

    Shevtsov, P N; Shevtsova, E F; Burbaeva, G Sh

    2016-08-01

    Al(3+), Fe(3+), and Zn(2+) ions can disturb microtubule assembly from tubulin and microtubuleassociated proteins in rat brain. The main structural forms of these microtubules are rings and tangled bundles. These structures are formed only in the presence of Al(3+) and Fe(3+) ions. Therefore, Zn(2+) ions can be excluded from possible causes of structural abnormalities in microtubules during Alzheimer's disease. Al(3+) ions are the most probable etiological cause of Alzheimer's disease. The concentration of Al(3+) ions affecting the structure of microtubules is one order of magnitude lower than that of Fe(3+) ions (10 and 100 μM, respectively), which corresponds to their brain concentration reported in Alzheimer's disease. PMID:27591874

  19. 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

  20. 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

  1. 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.

  2. Microtubule segment stabilization by RASSF1A is required for proper microtubule dynamics and Golgi integrity

    PubMed Central

    Arnette, Christopher; Efimova, Nadia; Zhu, Xiaodong; Clark, Geoffrey J.; Kaverina, Irina

    2014-01-01

    The tumor suppressor and microtubule-associated protein Ras association domain family 1A (RASSF1A) has a major effect on many cellular processes, such as cell cycle progression and apoptosis. RASSF1A expression is frequently silenced in cancer and is associated with increased metastasis. Therefore we tested the hypothesis that RASSF1A regulates microtubule organization and dynamics in interphase cells, as well as its effect on Golgi integrity and cell polarity. Our results show that RASSF1A uses a unique microtubule-binding pattern to promote site-specific microtubule rescues, and loss of RASSF1A leads to decreased microtubule stability. Furthermore, RASSF1A-associated stable microtubule segments are necessary to prevent Golgi fragmentation and dispersal in cancer cells and maintain a polarized cell front. These results indicate that RASSF1A is a key regulator in the fine tuning of microtubule dynamics in interphase cells and proper Golgi organization and cell polarity. PMID:24478455

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

    PubMed Central

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

    1998-01-01

    We have investigated the cell cycle inhibition mechanism and primary target of tryprostatin A (TPS-A) purified from Aspergillus fumigatus. TPS-A inhibited cell cycle progression of asynchronously cultured 3Y1 cells in the M phase in a dose- and time-dependent manner. In contrast, TPS-B (the demethoxy analogue of TPS-A) showed cell-cycle non-specific inhibition on cell growth even though it inhibited cell growth at lower concentrations than TPS-A. TPS-A treatment induced the reversible disruption of the cytoplasmic microtubules of 3Y1 cells as observed by indirect immunofluorescence microscopy in the range of concentrations that specifically inhibited M-phase progression. TPS-A inhibited the assembly in vitro of microtubules purified from bovine brains (40% inhibition at 250 microM); however, there was little or no effect on the self-assembly of purified tubulin when polymerization was induced by glutamate even at 250 microM TPS-A. TPS-A did not inhibit assembly promoted by taxol or by digestion of the C-terminal domain of tubulin. However, TPS-A blocked the tubulin assembly induced by inducers interacting with the C-terminal domain, microtubule-associated protein 2 (MAP2), tau and poly-(l-lysine). These results indicate that TPS-A is a novel inhibitor of MAP-dependent microtubule assembly and, through the disruption of the microtubule spindle, specifically inhibits cell cycle progression at the M phase. PMID:9677311

  4. 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

  5. 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.

  6. 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.

  7. 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.

  8. Prion protein inhibits microtubule assembly by inducing tubulin oligomerization

    SciTech Connect

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

    2006-10-13

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

  9. Contribution of Noncentrosomal Microtubules to Spindle Assembly in Drosophila Spermatocytes

    PubMed Central

    2004-01-01

    Previous data suggested that anastral spindles, morphologically similar to those found in oocytes, can assemble in a centrosome-independent manner in cells that contain centrosomes. It is assumed that the microtubules that build these acentrosomal spindles originate over the chromatin. However, the actual processes of centrosome-independent microtubule nucleation, polymerisation, and sorting have not been documented in centrosome-containing cells. We have identified two experimental conditions in which centrosomes are kept close to the plasma membrane, away from the nuclear region, throughout meiosis I in Drosophila spermatocytes. Time-lapse confocal microscopy of these cells labelled with fluorescent chimeras reveals centrosome-independent microtubule nucleation, growth, and sorting into a bipolar spindle array over the nuclear region, away from the asters. The onset of noncentrosomal microtubule nucleation is significantly delayed with respect to nuclear envelope breakdown and coincides with the end of chromosome condensation. It takes place in foci that are close to the membranes that ensheath the nuclear region, not over the condensed chromosomes. Metaphase plates are formed in these spindles, and, in a fraction of them, some degree of polewards chromosome segregation takes place. In these cells that contain both membrane-bound asters and an anastral spindle, the orientation of the cytokinesis furrow correlates with the position of the asters and is independent of the orientation of the spindle. We conclude that the fenestrated nuclear envelope may significantly contribute to the normal process of spindle assembly in Drosophila spermatocytes. We also conclude that the anastral spindles that we have observed are not likely to provide a robust back-up able to ensure successful cell division. We propose that these anastral microtubule arrays could be a constitutive component of wild-type spindles, normally masked by the abundance of centrosome-derived microtubules

  10. Dynamic microtubules: Experimental observation and computer simulation of polar microtubule behaviour with lateral cap model mechanisms

    NASA Astrophysics Data System (ADS)

    Bayley, P. M.; Martin, S. R.; Sharma, K. K.

    1991-05-01

    Microtubule dynamic instability involves the existence, within a population of microtubules, of sub-populations of growing and shrinking microtubules which interconvert apparently at random. We consider the scope and limitation of experimental observations of individual microtubules by video enhanced dark-field microscopy. This unique experimental phenomenon has been rationalized by the presence of a ``cap'' of tubulin-GTP which can stabilize the growing state. We have modelled this process quantitatively by numerical simulation and illustrate the basic principles by computer graphics. The inherent α-β asymmetry of the microtubule lattice determines that the relationship between the addition reaction of tubulin-GTP and the related hydrolysis of a polymer tubulin-GTP is different at the two ends of the microtubule. In the single layer, Lateral Cap model for microtubule dynamic instability, a plausible mechanism has been proposed for the dynamic properties at the ``active'' (presumed β-out) end in which the tubulin-GTP which is hydrolyzed is related longitudinally to the binding site by the 13-start protofilament helix. [1,2]. We now show a similar but distinct mechanism could hold for the ``inactive'' (presumed α-out) end of the microtubule. Lateral hydrolysis rules (related to 5- or 8- start helical contacts) predict that the α-end could in fact be less dynamic and cooperative in terms of reduced amplitudes of growth and shrinking. This would make a distinctive contribution to the J(c) plot of microtubule growth versus [tubulin-GTP]. These predictions are thus amenable to experimental verification. This approach illustrates how the helical lattice symmetry of the microtubule polymer can confer unique dynamic characteristics, which derive from the heterodimeric structure and guanine nucleotide binding properties of the component protein tubulin. It also provides a basis for the interpretation of the interactions of microtubules with anti-mitotic drugs used in

  11. 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

  12. An assay to image neuronal microtubule dynamics in mice

    PubMed Central

    Kleele, Tatjana; Marinković, Petar; Williams, Philip R.; Stern, Sina; Weigand, Emily E.; Engerer, Peter; Naumann, Ronald; Hartmann, Jana; Karl, Rosa M.; Bradke, Frank; Bishop, Derron; Herms, Jochen; Konnerth, Arthur; Kerschensteiner, Martin; Godinho, Leanne; Misgeld, Thomas

    2014-01-01

    Microtubule dynamics in neurons play critical roles in physiology, injury and disease and determine microtubule orientation, the cell biological correlate of neurite polarization. Several microtubule binding proteins, including end-binding protein 3 (EB3), specifically bind to the growing plus tip of microtubules. In the past, fluorescently tagged end-binding proteins have revealed microtubule dynamics in vitro and in non-mammalian model organisms. Here, we devise an imaging assay based on transgenic mice expressing yellow fluorescent protein-tagged EB3 to study microtubules in intact mammalian neurites. Our approach allows measurement of microtubule dynamics in vivo and ex vivo in peripheral nervous system and central nervous system neurites under physiological conditions and after exposure to microtubule-modifying drugs. We find an increase in dynamic microtubules after injury and in neurodegenerative disease states, before axons show morphological indications of degeneration or regrowth. Thus increased microtubule dynamics might serve as a general indicator of neurite remodelling in health and disease. PMID:25219969

  13. GIT1 enhances neurite outgrowth by stimulating microtubule assembly

    PubMed Central

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

    2016-01-01

    GIT1, a G-protein-coupled receptor kinase interacting protein, has been reported to be involved in neurite outgrowth. However, the neurobiological functions of the protein remain unclear. In this study, we found that GIT1 was highly expressed in the nervous system, and its expression was maintained throughout all stages of neuritogenesis in the brain. In primary cultured mouse hippocampal neurons from GIT1 knockout mice, there was a significant reduction in total neurite length per neuron, as well as in the average length of axon-like structures, which could not be prevented by nerve growth factor treatment. Overexpression of GIT1 significantly promoted axon growth and fully rescued the axon outgrowth defect in the primary hippocampal neuron cultures from GIT1 knockout mice. The GIT1 N terminal region, including the ADP ribosylation factor-GTPase activating protein domain, the ankyrin domains and the Spa2 homology domain, were sufficient to enhance axonal extension. Importantly, GIT1 bound to many tubulin proteins and microtubule-associated proteins, and it accelerated microtubule assembly in vitro. Collectively, our findings suggest that GIT1 promotes neurite outgrowth, at least partially by stimulating microtubule assembly. This study provides new insight into the cellular and molecular pathogenesis of GIT1-associated neurological diseases. PMID:27127481

  14. 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

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

    NASA Astrophysics Data System (ADS)

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

    2010-03-01

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

  16. Analysis of centrosome function and microtubule dynamics by time-lapse microscopy in Xenopus egg extracts.

    PubMed

    Wiese, Christiane; Mayers, Jonathan R; Albee, Alison J

    2009-01-01

    Centrosomes are essential organelles that organize the microtubule cytoskeleton during interphase and mitosis. Centrosomes are assembled from tens to hundreds of proteins, but how these proteins are organized into functional microtubule nucleating and organizing centers is not yet clear. An important step in understanding the role of individual proteins in centrosome function is to understand whether they are involved in forming, stabilizing, or anchoring microtubules. It is becoming increasingly clear that the analysis of fixed samples is inadequate for a true understanding of the dynamics that drive cell biological processes. In this chapter we focus on methods to analyze microtubule nucleation, organization, and dynamics using assays based on mitotic Xenopus egg extracts and in vitro reactions. These methods can easily be adapted to the study of interphase processes, or to the study of other cytoskeletal proteins and their dynamics. PMID:19768426

  17. The STARD9/Kif16a Kinesin Associates With Mitotic Microtubules and Regulates Spindle Pole Assembly

    PubMed Central

    Torres, Jorge Z.; Summers, Matthew K.; Peterson, David; Brauer, Matthew J.; Lee, James; Senese, Silvia; Gholkar, Ankur A.; Lo, Yu-Chen; Lei, Xingye; Jung, Kenneth; Anderson, David C.; Davis, David P.; Belmont, Lisa; Jackson, Peter K.

    2011-01-01

    SUMMARY During cell division cells form the microtubule-based mitotic spindle, a highly specialized and dynamic structure that mediates proper chromosome transmission to daughter cells. Cancer cells can show perturbed mitotic spindles and an approach in cancer treatment has been to trigger cell killing by targeting microtubule dynamics or spindle assembly. To identify and characterize proteins necessary for spindle assembly, and potential antimitotic targets, we performed a proteomic and genetic analysis of 592 mitotic microtubule co-purifying proteins (MMCPs). Screening for regulators that affect both mitosis and apoptosis, we report the identification and characterization of STARD9, a kinesin-3 family member, which localizes to centrosomes and stabilizes the pericentriolar material (PCM). STARD9-depleted cells have fragmented PCM, form multipolar spindles, activate the spindle assembly checkpoint (SAC), arrest in mitosis, and undergo apoptosis. Interestingly, STARD9-depletion synergizes with the chemotherapeutic agent taxol to increase mitotic death, demonstrating that STARD9 is a mitotic kinesin and a potential anti-mitotic target. PMID:22153075

  18. Force-generation and dynamic instability of microtubule bundles

    PubMed Central

    Laan, Liedewij; Husson, Julien; Munteanu, E. Laura; Kerssemakers, Jacob W. J.; Dogterom, Marileen

    2008-01-01

    Individual dynamic microtubules can generate pushing or pulling forces when their growing or shrinking ends are in contact with cellular objects such as the cortex or chromosomes. These microtubules can operate in parallel bundles, for example when interacting with mitotic chromosomes. Here, we investigate the force-generating capabilities of a bundle of growing microtubules and study the effect that force has on the cooperative dynamics of such a bundle. We used an optical tweezers setup to study microtubule bundles growing against a microfabricated rigid barrier in vitro. We show that multiple microtubules can generate a pushing force that increases linearly with the number of microtubules present. In addition, the bundle can cooperatively switch to a shrinking state, due to a force-induced coupling of the dynamic instability of single microtubules. In the presence of GMPCPP, bundle catastrophes no longer occur, and high bundle forces are reached more effectively. We reproduce the observed behavior with a simple simulation of microtubule bundle dynamics that takes into account previously measured force effects on single microtubules. Using this simulation, we also show that a constant compressive force on a growing bundle leads to oscillations in bundle length that are of potential relevance for chromosome oscillations observed in living cells. PMID:18577596

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

    SciTech Connect

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

    1992-01-14

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

  20. Thermodynamic and structural analysis of microtubule assembly: the role of GTP hydrolysis.

    PubMed Central

    Vulevic, B; Correia, J J

    1997-01-01

    Different models have been proposed that link the tubulin heterodimer nucleotide content and the role of GTP hydrolysis with microtubule assembly and dynamics. Here we compare the thermodynamics of microtubule assembly as a function of nucleotide content by van't Hoff analysis. The thermodynamic parameters of tubulin assembly in 30-100 mM piperazine-N,N'-bis(2-ethanesulfonic acid), 1 mM MgSO4, 2 mM EGTA, pH 6.9, in the presence of a weakly hydrolyzable analog, GMPCPP, the dinucleotide analog GMPCP plus 2 M glycerol, and GTP plus 2 M glycerol were obtained together with data for taxol-GTP/GDP tubulin assembly (GMPCPP and GMPCP are the GTP and GDP nucleotide analogs where the alpha beta oxygen has been replaced by a methylene, -CH2-). All of the processes studied are characterized by a positive enthalpy, a positive entropy, and a large, negative heat capacity change. GMPCP-induced assembly has the largest negative heat capacity change and GMPCPP has the second largest, whereas GTP/2 M glycerol- and taxol-induced assembly have more positive values, respectively. A large, negative heat capacity is most consistent with the burial of water-accessible hydrophobic surface area, which gives rise to the release of bound water. The heat capacity changes observed with GTP/2 M glycerol-induced and with taxol-induced assembly are very similar, -790 +/- 190 cal/mol/k, and correspond to the burial of 3330 +/- 820 A2 of nonpolar surface area. This value is shown to be very similar to an estimate of the buried nonpolar surface in a reconstructed microtubule lattice. Polymerization data from GMPCP- and GMPCPP-induced assembly are consistent with buried nonpolar surface areas that are 3 and 6 times larger. A linear enthalpy-entropy and enthalpy-free energy plot for tubulin polymerization reactions verifies that enthalpy-entropy compensation for this system is based upon true biochemical correlation, most likely corresponding to a dominant hydrophobic effect. Entropy analysis suggests

  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. Effects of anti-Alzheimer drugs on phosphorylation and assembly of microtubules from brain microtubular proteins.

    PubMed

    Shevtsov, P N; Shevtsova, E F; Burbaeva, G Sh; Bachurin, S O

    2014-04-01

    We studied the effects of anti-Alzheimer drugs (tacrine, amiridine, and memantine) on phosphorylation of tubulin and microtubule-associated proteins isolated from rat brain, evaluated the capacity of these proteins to polymerize into microtubules after addition of study pharmacological agents, and analyzed the structure of generated microtubules. It was shown that test substances impair assembly of microtubules to a different extent. Dose-dependent effects of these agents on phosphorylation of tubulin and microtubule-associated proteins were observed. Triazolam (not approved for clinical use as anti-Alzheimer drug) in the same concentrations was used as the reference substance in the same tests. It was observed that this substance even in minimal concentration induced the most pronounced changes in microtubule structure. A direct correlation between the capacity of the test substances to modulate tubulin phosphorylation and to impair microtubule structure was found: the more the substance inhibited tubulin phosphorylation, the more it disordered microtubule structure. PMID:24824692

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

    PubMed

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

    2016-02-01

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

  5. Theoretical analysis of microtubule dynamics at all times.

    PubMed

    Li, Xin; Kolomeisky, Anatoly B

    2014-12-01

    Microtubules are biopolymers consisting of tubulin dimer subunits. As a major component of cytoskeleton they are essential for supporting most important cellular processes such as cell division, signaling, intracellular transport and cell locomotion. The hydrolysis of guanosine triphosphate (GTP) molecules attached to each tubulin subunit supports the nonequilibrium nature of microtubule dynamics. One of the most spectacular properties of microtubules is their dynamic instability when their growth from continuous attachment of tubulin dimers stochastically alternates with periods of shrinking. Despite the critical importance of this process to all cellular activities, its mechanism remains not fully understood. We investigated theoretically microtubule dynamics at all times by analyzing explicitly temporal evolution of various length clusters of unhydrolyzed subunits. It is found that the dynamic behavior of microtubules depends strongly on initial conditions. Our theoretical findings provide a microscopic explanation for recent experiments which found that the frequency of catastrophes increases with the lifetime of microtubules. It is argued that most growing microtubule configurations cannot transit in one step into a shrinking state, leading to a complex overall temporal behavior. Theoretical calculations combined with Monte Carlo computer simulations are also directly compared with experimental observations, and good agreement is found. PMID:25390471

  6. Curcumin suppresses the dynamic instability of microtubules, activates the mitotic checkpoint and induces apoptosis in MCF-7 cells.

    PubMed

    Banerjee, Mithu; Singh, Parminder; Panda, Dulal

    2010-08-01

    In this study, curcumin, a potential anticancer agent, was found to dampen the dynamic instability of individual microtubules in living MCF-7 cells. It strongly reduced the rate and extent of shortening states, and modestly reduced the rate and extent of growing states. In addition, curcumin decreased the fraction of time microtubules spent in the growing state and strongly increased the time microtubules spent in the pause state. Brief treatment with curcumin depolymerized mitotic microtubules, perturbed microtubule-kinetochore attachment and disturbed the mitotic spindle structure. Curcumin also perturbed the localization of the kinesin protein Eg5 and induced monopolar spindle formation. Further, curcumin increased the accumulation of Mad2 and BubR1 at the kinetochores, indicating that it activated the mitotic checkpoint. In addition, curcumin treatment increased the metaphase/anaphase ratio, indicating that it can delay mitotic progression from the metaphase to anaphase. We provide evidence suggesting that the affected cells underwent apoptosis via the p53-dependent apoptotic pathway. The results support the idea that kinetic stabilization of microtubule dynamics assists in the nuclear translocation of p53. Curcumin exerted additive effects when combined with vinblastine, a microtubule depolymerizing drug, whereas the combination of curcumin with paclitaxel, a microtubule-stabilizing drug, produced an antagonistic effect on the inhibition of MCF-7 cell proliferation. The results together suggested that curcumin inhibited MCF-7 cell proliferation by inhibiting the assembly dynamics of microtubules. PMID:20646066

  7. 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)

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

    PubMed Central

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

    1997-01-01

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

  9. 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.

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

    PubMed

    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. PMID:27475079

  11. 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

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

    NASA Astrophysics Data System (ADS)

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

    2016-06-01

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

  13. Mechanical breaking of microtubules in axons during dynamic stretch injury underlies delayed elasticity, microtubule disassembly, and axon degeneration

    PubMed Central

    Tang-Schomer, Min D.; Patel, Ankur R.; Baas, Peter W.; Smith, Douglas H.

    2010-01-01

    Little is known about which components of the axonal cytoskeleton might break during rapid mechanical deformation, such as occurs in traumatic brain injury. Here, we micropatterned neuronal cell cultures on silicone membranes to induce dynamic stretch exclusively of axon fascicles. After stretch, undulating distortions formed along the axons that gradually relaxed back to a straight orientation, demonstrating a delayed elastic response. Subsequently, swellings developed, leading to degeneration of almost all axons by 24 h. Stabilizing the microtubules with taxol maintained the undulating geometry after injury but greatly reduced axon degeneration. Conversely, destabilizing microtubules with nocodazole prevented undulations but greatly increased the rate of axon loss. Ultrastructural analyses of axons postinjury revealed immediate breakage and buckling of microtubules in axon undulations and progressive loss of microtubules. Collectively, these data suggest that dynamic stretch of axons induces direct mechanical failure at specific points along microtubules. This microtubule disorganization impedes normal relaxation of the axons, resulting in undulations. However, this physical damage also triggers progressive disassembly of the microtubules around the breakage points. While the disintegration of microtubules allows delayed recovery of the “normal” straight axon morphology, it comes at a great cost by interrupting axonal transport, leading to axonal swelling and degeneration.—Tang-Schomer, M. D., Patel, A. R,, Baas, P. W., Smith, D. H. Mechanical breaking of microtubules in axons during dynamic stretch injury underlies delayed elasticity, microtubule disassembly, and axon degeneration. PMID:20019243

  14. Microtubule plus tips: A dynamic route to chromosomal instability

    PubMed Central

    Stolz, Ailine; Ertych, Norman; Bastians, Holger

    2015-01-01

    Although chromosomal instability (CIN) is a recognized hallmark of cancer the underlying mechanisms and consequences are largely unknown. However, it is accepted that lagging chromosomes represent a major prerequisite for chromosome missegregation in cancer cells. Here, we discuss how lagging chromosomes are generated and our recent findings establishing increased microtubule assembly rates as a source of CIN.

  15. Dynamics and Organization of Cortical Microtubules as Revealed by Superresolution Structured Illumination Microscopy1[W

    PubMed Central

    Komis, George; Mistrik, Martin; Šamajová, Olga; Doskočilová, Anna; Ovečka, Miroslav; Illés, Peter; Bartek, Jiri; Šamaj, Jozef

    2014-01-01

    Plants employ acentrosomal mechanisms to organize cortical microtubule arrays essential for cell growth and differentiation. Using structured illumination microscopy (SIM) adopted for the optimal documentation of Arabidopsis (Arabidopsis thaliana) hypocotyl epidermal cells, dynamic cortical microtubules labeled with green fluorescent protein fused to the microtubule-binding domain of the mammalian microtubule-associated protein MAP4 and with green fluorescent protein-fused to the alpha tubulin6 were comparatively recorded in wild-type Arabidopsis plants and in the mitogen-activated protein kinase mutant mpk4 possessing the former microtubule marker. The mpk4 mutant exhibits extensive microtubule bundling, due to increased abundance and reduced phosphorylation of the microtubule-associated protein MAP65-1, thus providing a very useful genetic tool to record intrabundle microtubule dynamics at the subdiffraction level. SIM imaging revealed nano-sized defects in microtubule bundling, spatially resolved microtubule branching and release, and finally allowed the quantification of individual microtubules within cortical bundles. Time-lapse SIM imaging allowed the visualization of subdiffraction, short-lived excursions of the microtubule plus end, and dynamic instability behavior of both ends during free, intrabundle, or microtubule-templated microtubule growth and shrinkage. Finally, short, rigid, and nondynamic microtubule bundles in the mpk4 mutant were observed to glide along the parent microtubule in a tip-wise manner. In conclusion, this study demonstrates the potential of SIM for superresolution time-lapse imaging of plant cells, showing unprecedented details accompanying microtubule dynamic organization. PMID:24686112

  16. Functional role of ɛ-tubulin in the assembly of the centriolar microtubule scaffold

    PubMed Central

    Dupuis-Williams, Pascale; Fleury-Aubusson, Anne; de Loubresse, Nicole Garreau; Geoffroy, Hélène; Vayssié, Laurence; Galvani, Angélique; Espigat, Aude; Rossier, Jean

    2002-01-01

    Centrioles and basal bodies fascinate by their spectacular architecture, featuring an arrangement of nine microtubule triplets into an axial symmetry, whose biogenesis relies on yet elusive mechanisms. However, the recent discovery of new tubulins, such as δ-, ɛ-, or η-tubulin, could constitute a breakthrough for deciphering the assembly steps of this unconventional microtubule scaffold. Here, we report the functional analysis in vivo of ɛ-tubulin, based on gene silencing in Paramecium, which demonstrates that this protein, which localizes at the basal bodies, is essential for the assembly and anchorage of the centriolar microtubules. PMID:12356863

  17. α-Synuclein Fibrils Exhibit Gain of Toxic Function, Promoting Tau Aggregation and Inhibiting Microtubule Assembly.

    PubMed

    Oikawa, Takayuki; Nonaka, Takashi; Terada, Makoto; Tamaoka, Akira; Hisanaga, Shin-Ichi; Hasegawa, Masato

    2016-07-15

    α-Synuclein is the major component of Lewy bodies and Lewy neurites in Parkinson disease and dementia with Lewy bodies and of glial cytoplasmic inclusions in multiple system atrophy. It has been suggested that α-synuclein fibrils or intermediate protofibrils in the process of fibril formation may have a toxic effect on neuronal cells. In this study, we investigated the ability of soluble monomeric α-synuclein to promote microtubule assembly and the effects of conformational changes of α-synuclein on Tau-promoted microtubule assembly. In marked contrast to previous findings, monomeric α-synuclein had no effect on microtubule polymerization. However, both α-synuclein fibrils and protofibrils inhibited Tau-promoted microtubule assembly. The inhibitory effect of α-synuclein fibrils was greater than that of the protofibrils. Dot blot overlay assay and spin-down techniques revealed that α-synuclein fibrils bind to Tau and inhibit microtubule assembly by depleting the Tau available for microtubule polymerization. Using various deletion mutants of α-synuclein and Tau, the acidic C-terminal region of α-synuclein and the basic central region of Tau were identified as regions involved in the binding. Furthermore, introduction of α-synuclein fibrils into cultured cells overexpressing Tau protein induced Tau aggregation. These results raise the possibility that α-synuclein fibrils interact with Tau, inhibit its function to stabilize microtubules, and also promote Tau aggregation, leading to dysfunction of neuronal cells. PMID:27226637

  18. Regulation of actin assembly by microtubules in fission yeast cell polarity.

    PubMed

    Chang, Fred; Feierbach, Becket; Martin, Sophie

    2005-01-01

    It has been speculated that microtubule plus ends function to regulate the actin cytoskeleton in processes such as cytokinesis, cell polarization and cell migration. In the fission yeast Schizosaccharomyces pombe, interphase microtubules regulate cell polarity through proteins such as tea1p, a kelch repeat protein, and for3p, a formin that nucleates actin cable assembly at cell tips. Here, we review recent progress on understanding tea1p regulation and function. Microtubules may govern the localization of tea1p by transporting it on the plus ends of microtubules and depositing it directly onto the cell tip when the microtubule catastrophes. The interaction of tea1p with the CLIP170 protein tip1p is responsible for its localization at growing microtubule plus ends. Tea1p may regulate cell polarity by associating with large 'polarisome' complexes that include for3p. For3p is present at both cell tips, but is not on the microtubules. Tea1p is needed to localize the formin to establish polarized cell growth at cell tips that have not grown previously. These studies begin to elucidate a molecular pathway for how microtubules contribute to the proper spatial regulation of actin assembly and polarized cell growth. PMID:16355535

  19. 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. PMID:27317686

  20. 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

  1. 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.

  2. The engine of microtubule dynamics comes into focus.

    PubMed

    Mitchison, T J

    2014-05-22

    In this issue, Alushin et al. report high-resolution structures of three states of the microtubule lattice: GTP-bound, which is stable to depolymerization; unstable GDP-bound; and stable Taxol and GDP-bound. By comparing these structures at near-atomic resolution, they are able to propose a detailed model for how GTP hydrolysis destabilizes the microtubule and thus powers dynamic instability and chromosome movement. Destabilization of cytoskeleton filaments by nucleotide hydrolysis is an important general principle in cell dynamics, and this work represents a major step forward on a problem with a long history. PMID:24855939

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

    DOE PAGESBeta

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

    2016-02-04

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

  4. Mechanisms Underlying the Active Self-Assembly of Microtubule Rings and Spools.

    PubMed

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

    2016-03-14

    Active self-assembly offers a powerful route for the creation of dynamic multiscale structures that are presently inaccessible with standard microfabrication techniques. One such system uses the translation of microtubule filaments by surface-tethered kinesin to actively assemble nanocomposites with bundle, ring, and spool morphologies. Attempts to observe mechanisms involved in this active assembly system have been hampered by experimental difficulties with performing observation during buffer exchange and photodamage from fluorescent excitation. In the present work, we used a custom microfluidic device to remove these limitations and directly study ring/spool formation, including the earliest events (nucleation) that drive subsequent nanocomposite assembly. Three distinct formation events were observed: pinning, collisions, and induced curvature. Of these three, collisions accounted for the majority of event leading to ring/spool formation, while the rate of pinning was shown to be dependent on the amount of photodamage in the system. We further showed that formation mechanism directly affects the diameter and rotation direction of the resultant rings and spools. Overall, the fundamental understanding described in this work provides a foundation by which the properties of motor-driven, actively assembled nanocomposites may be tailored toward specific applications. PMID:26842978

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

    NASA Astrophysics Data System (ADS)

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

    2000-03-01

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

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

    NASA Astrophysics Data System (ADS)

    Xie, Sheng; Pelcovits, Robert A.; Hagan, Michael F.

    2016-06-01

    The self-assembly of highly anisotropic colloidal particles leads to a rich variety of morphologies whose properties are just beginning to be understood. This article uses computer simulations to probe a particle-scale perturbation of a commonly studied colloidal assembly, a monolayer membrane composed of rodlike fd viruses in the presence of a polymer depletant. Motivated by experiments currently in progress, we simulate the interaction between a microtubule and a monolayer membrane as the microtubule "pokes" and penetrates the membrane face-on. Both the viruses and the microtubule are modeled as hard spherocylinders of the same diameter, while the depletant is modeled using ghost spheres. We find that the force exerted on the microtubule by the membrane is zero either when the microtubule is completely outside the membrane or when it has fully penetrated the membrane. The microtubule is initially repelled by the membrane as it begins to penetrate but experiences an attractive force as it penetrates further. We assess the roles played by translational and rotational fluctuations of the viruses and the osmotic pressure of the polymer depletant. We find that rotational fluctuations play a more important role than the translational ones. The dependence on the osmotic pressure of the depletant of the width and height of the repulsive barrier and the depth of the attractive potential well is consistent with the assumed depletion-induced attractive interaction between the microtubule and viruses. We discuss the relevance of these studies to the experimental investigations.

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

    PubMed

    Fridén, B; Wallin, M

    1991-07-10

    Microtubule-associated proteins (MAPs) were separated from tubulin with several different methods. The ability of the isolated MAPs to reinduce assembly of phosphocellulose purified tubulin differed markedly between the different methods. MAPs isolated by addition of 0.35 M NaCl to taxol-stabilized microtubules stimulated tubulin assembly most effectively, while addition of 0.6 M NaCl produced MAPs with a substantially lower ability to stimulate tubulin assembly. The second best preparation was achieved with phosphocellulose chromatographic separation of MAPs with 0.6 M NaCl elution. The addition of estramustine phosphate to microtubules reconstituted of MAPs prepared by 0.35 M NaCl or phosphocellulose chromatography, induced less disassembly than for microtubules assembled from unseparated proteins, and was almost without effect on microtubules reconstituted from MAPs prepared by taxol and 0.6 M NaCl. Estramustine phosphate binds to the tubulin binding part of the MAPs, and the results do therefore indicate that the MAPs are altered by the separation methods. Since the MAPs are regarded as highly stable molecules, one probable alteration could be aggregation of the MAPs, as also indicated by the results. The purified tubulin itself seemed not to be affected by the phosphocellulose purification, since the microtubule proteins were unchanged by the low buffer strenght used during the cromatography. However, the assembly competence after a prolonged incubation of the microtubule proteins at 4 degrees C was dependent on intact bindings between the tubulin and MAPs. PMID:1681420

  8. A genetic analysis of microtubule assembly and function in yeast

    SciTech Connect

    Solomon, F.; Guenette, S.; Kirkpatrick, D.; Praitis, V.; Weinstein, B.; Archer, J.

    1993-12-31

    The major goal of our laboratory`s research is to understand how cells organize their cytoskeletons to produce motility: specific patterns of shape change, intracellular motility and locomotion. We focus primarily on microtubules. We appreciate that results from several laboratories including our own, suggest that microtubule function is expressed in part through interactions with other elements of the cytoskeleton and other cellular compartments, such as the plasma membrane. However, focusing on microtubules represents a justifiable reduction, since a wide variety of drug interference and localization experiments support the notion that intact microtubules are essential for each of these motile phenomena. The primary problem facing this field is understanding how microtubule structure and function is regulated in vivo. Although there are a variety of excellent experimental systems which permit detailed analyses of behavior in vitro, the extrapolation of these results to the situation in the cytoplasm is problematic. These efforts have been boosted significantly in the last several years by two advances: first, traditionally excellent genetic organisms, such as the yeasts, have been enlisted in the study of motility; second, molecular biology has enabled {open_quotes}pseudo-genetic{close_quotes} approaches in animal cells which display the most interesting of motile phenomena. Our laboratory is involved in both of these efforts. In the present report, we will summarize our present approaches using yeast.

  9. 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.

  10. 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

  11. 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.

  12. Increased microtubule assembly rates influence chromosomal instability in colorectal cancer cells.

    PubMed

    Ertych, Norman; Stolz, Ailine; Stenzinger, Albrecht; Weichert, Wilko; Kaulfuß, Silke; Burfeind, Peter; Aigner, Achim; Wordeman, Linda; Bastians, Holger

    2014-08-01

    Chromosomal instability (CIN) is defined as the perpetual missegregation of whole chromosomes during mitosis and represents a hallmark of human cancer. However, the mechanisms influencing CIN and its consequences on tumour growth are largely unknown. We identified an increase in microtubule plus-end assembly rates as a mechanism influencing CIN in colorectal cancer cells. This phenotype is induced by overexpression of the oncogene AURKA or by loss of the tumour suppressor gene CHK2, a genetic constitution found in 73% of human colorectal cancers. Increased microtubule assembly rates are associated with transient abnormalities in mitotic spindle geometry promoting the generation of lagging chromosomes and influencing CIN. Reconstitution of proper microtubule assembly rates by chemical or genetic means suppresses CIN and thereby, unexpectedly, accelerates tumour growth in vitro and in vivo. Thus, we identify a fundamental mechanism influencing CIN in cancer cells and reveal its adverse consequence on tumour growth. PMID:24976383

  13. Kinetics of self-assembling microtubules: an "inverse problem" in biochemistry.

    PubMed

    Flyvbjerg, H; Jobs, E; Leibler, S

    1996-06-11

    Experimental time series for a nonequilibrium reaction may in some cases contain sufficient data to determine a unique kinetic model for the reaction by a systematic mathematical analysis. As an example, a kinetic model for the self-assembly of microtubules is derived here from turbidity time series for solutions in which microtubules assemble. The model may be seen as a generalization of Oosawa's classical nucleation-polymerization model. It reproduces the experimental data with a four-stage nucleation process and a critical nucleus of 15 monomers. PMID:8650204

  14. Dissecting the Function and Assembly of Acentriolar Microtubule Organizing Centers in Drosophila Cells In Vivo

    PubMed Central

    Baumbach, Janina; Novak, Zsofia Anna; Raff, Jordan W.; Wainman, Alan

    2015-01-01

    Acentriolar microtubule organizing centers (aMTOCs) are formed during meiosis and mitosis in several cell types, but their function and assembly mechanism is unclear. Importantly, aMTOCs can be overactive in cancer cells, enhancing multipolar spindle formation, merotelic kinetochore attachment and aneuploidy. Here we show that aMTOCs can form in acentriolar Drosophila somatic cells in vivo via an assembly pathway that depends on Asl, Cnn and, to a lesser extent, Spd-2—the same proteins that appear to drive mitotic centrosome assembly in flies. This finding enabled us to ablate aMTOC formation in acentriolar cells, and so perform a detailed genetic analysis of the contribution of aMTOCs to acentriolar mitotic spindle formation. Here we show that although aMTOCs can nucleate microtubules, they do not detectably increase the efficiency of acentriolar spindle assembly in somatic fly cells. We find that they are required, however, for robust microtubule array assembly in cells without centrioles that also lack microtubule nucleation from around the chromatin. Importantly, aMTOCs are also essential for dynein-dependent acentriolar spindle pole focusing and for robust cell proliferation in the absence of centrioles and HSET/Ncd (a kinesin essential for acentriolar spindle pole focusing in many systems). We propose an updated model for acentriolar spindle pole coalescence by the molecular motors Ncd/HSET and dynein in conjunction with aMTOCs. PMID:26020779

  15. Dissecting the function and assembly of acentriolar microtubule organizing centers in Drosophila cells in vivo.

    PubMed

    Baumbach, Janina; Novak, Zsofia Anna; Raff, Jordan W; Wainman, Alan

    2015-05-01

    Acentriolar microtubule organizing centers (aMTOCs) are formed during meiosis and mitosis in several cell types, but their function and assembly mechanism is unclear. Importantly, aMTOCs can be overactive in cancer cells, enhancing multipolar spindle formation, merotelic kinetochore attachment and aneuploidy. Here we show that aMTOCs can form in acentriolar Drosophila somatic cells in vivo via an assembly pathway that depends on Asl, Cnn and, to a lesser extent, Spd-2--the same proteins that appear to drive mitotic centrosome assembly in flies. This finding enabled us to ablate aMTOC formation in acentriolar cells, and so perform a detailed genetic analysis of the contribution of aMTOCs to acentriolar mitotic spindle formation. Here we show that although aMTOCs can nucleate microtubules, they do not detectably increase the efficiency of acentriolar spindle assembly in somatic fly cells. We find that they are required, however, for robust microtubule array assembly in cells without centrioles that also lack microtubule nucleation from around the chromatin. Importantly, aMTOCs are also essential for dynein-dependent acentriolar spindle pole focusing and for robust cell proliferation in the absence of centrioles and HSET/Ncd (a kinesin essential for acentriolar spindle pole focusing in many systems). We propose an updated model for acentriolar spindle pole coalescence by the molecular motors Ncd/HSET and dynein in conjunction with aMTOCs. PMID:26020779

  16. Divergent microtubule assembly rates after short- versus long-term loss of end-modulating kinesins

    PubMed Central

    Wordeman, Linda; Decarreau, Justin; Vicente, Juan Jesus; Wagenbach, Michael

    2016-01-01

    Depletion of microtubule (MT) regulators can initiate stable alterations in MT assembly rates that affect chromosome instability and mitotic spindle function, but the manner by which cellular MT assembly rates can stably increase or decrease is not understood. To investigate this phenomenon, we measured the response of microtubule assembly to both rapid and long-term loss of MT regulators MCAK/Kif2C and Kif18A. Depletion of MCAK/Kif2C by siRNA stably decreases MT assembly rates in mitotic spindles, whereas depletion of Kif18A stably increases rates of assembly. Surprisingly, this is not phenocopied by rapid rapamycin-dependent relocalization of MCAK/Kif2C and Kif18A to the plasma membrane. Instead, this treatment yields opposite affects on MT assembly. Rapidly increased MT assembly rates are balanced by a decrease in nucleated microtubules, whereas nucleation appears to be maximal and limiting for decreased MT assembly rates and also for long-term treatments. We measured amplified tubulin synthesis during long-term depletion of MT regulators and hypothesize that this is the basis for different phenotypes arising from long-term versus rapid depletion of MT regulators. PMID:26912793

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

    PubMed

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

    2015-05-01

    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. PMID:26012630

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

    PubMed

    Genna, J M; Coffe, G; Pudles, J

    1980-09-01

    From kinetic and electron microscopy studies on the effects of procaine, tetracaine and dibucaine on the polymerization and depolymerization of the microtubules isolated from pig and rat brains the following results were obtained. 1. Procaine or tetracaine, at the concentration range of 0.5--20 mM and of 0.5--5 mM respectively, increases the rate of tubulin polymerization (24 degrees C or 37 degrees C) and of microtubule depolymerization (4 degrees C) as a linear function of the concentration of the anesthetics, while identical amounts of microtubules are formed. In the absence of microtubule-associated proteins the polymerization of tubulin is not induced by 10 mM procaine, furthermore, the critical concentration of microtubule proteins necessary for assembly into microtubules is not affected at this concentration level of the anesthetic. This suggests that procaine affects not the nucleation, but rather the elongation process. 2. Dibucaine, from 0.5 mM to 3 mM increases the lag time of the polymerization reaction, while from 0.5 mM to 2 mM it linearly decreases both tubulin polymerization (24 degrees C) and microtubule depolymerization (4 degrees C) rates. Dibucaine, up to mM concentration, does not affect the extent of tubulin polymerization; however, above this concentration it induces the formation of amorphous aggregates. 3. Procaine or tetracaine enhances the depolymerizing effect of calcium on microtubules. The half-maximal values for the depolymerizing effect of calcium were 0.96, 0.71 and 0.51 mM for the control, in the presence of 10 mM procaine and 5 mM tetracaine respectively. PMID:7439170

  19. Theory of self-assembly of microtubules and motors

    NASA Astrophysics Data System (ADS)

    Aranson, Igor S.; Tsimring, Lev S.

    2006-09-01

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

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

    SciTech Connect

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

    2006-01-01

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

  1. Steady-state theory of the interference of GTP hydrolysis in the mechanism of microtubule assembly.

    PubMed Central

    Hill, T L; Carlier, M F

    1983-01-01

    A model is presented for the interference of GTP hydrolysis in the mechanism of microtubule assembly. This model is suggested by previous results showing that both GTP and GDP are present at microtubule ends because of GTP hydrolysis and that tubulin does not bind to a GDP-bound end. The analytical theory developed here is aimed at calculation of the steady-state subunit flux at one end of the polymer. The GTP/GDP features just mentioned result in a nonlinear plot of the flux versus tubulin concentration. Microtubules are predicted to exhibit a different kinetic behavior below and above the critical concentration, which can be considered as a transition between two regimes. PMID:6580643

  2. Phosphorylation of tau by glycogen synthase kinase 3beta affects the ability of tau to promote microtubule self-assembly.

    PubMed Central

    Utton, M A; Vandecandelaere, A; Wagner, U; Reynolds, C H; Gibb, G M; Miller, C C; Bayley, P M; Anderton, B H

    1997-01-01

    To study the effects of phosphorylation by glycogen synthase kinase-3beta (GSK-3beta) on the ability of the microtubule-associated protein tau to promote microtubule self-assembly, tau isoform 1 (foetal tau) and three mutant forms of this tau isoform were investigated. The three mutant forms of tau had the following serine residues, known to be phosphorylated by GSK-3, replaced with alanine residues so as to preclude their phosphorylation: (1) Ser-199 and Ser-202 (Ser-199/202-->Ala), (2) Ser-235 (Ser-235-->Ala) and (3) Ser-396 and Ser-404 (Ser-396/404-->Ala). Wild-type tau and the mutant forms of tau were phosphorylated with GSK-3beta, and their ability to promote microtubule self-assembly was compared with the corresponding non-phosphorylated tau species. In the non-phosphorylated form, wild-type tau and all of the mutants affected the mean microtubule length and number concentrations of assembled microtubules in a manner consistant with enhanced microtubule nucleation. Phosphorylation of these tau species with GSK-3beta consistently reduced the ability of a given tau species to promote microtubule self-assembly, although the affinity of the tau for the microtubules was not greatly affected by phosphorylation since the tau species remained largely associated with the microtubules. This suggests that the regulation of microtubule assembly can be controlled by phosphorylation of tau at sites accessible to GSK-3beta by a mechanism that does not necessarily involve the dissociation of tau from the microtubules. PMID:9169608

  3. Stable kinetochore–microtubule attachment is sufficient to silence the spindle assembly checkpoint in human cells

    PubMed Central

    Tauchman, Eric C.; Boehm, Frederick J.; DeLuca, Jennifer G.

    2015-01-01

    During mitosis, duplicated sister chromatids attach to microtubules emanating from opposing sides of the bipolar spindle through large protein complexes called kinetochores. In the absence of stable kinetochore–microtubule attachments, a cell surveillance mechanism known as the spindle assembly checkpoint (SAC) produces an inhibitory signal that prevents anaphase onset. Precisely how the inhibitory SAC signal is extinguished in response to microtubule attachment remains unresolved. To address this, we induced formation of hyper-stable kinetochore–microtubule attachments in human cells using a non-phosphorylatable version of the protein Hec1, a core component of the attachment machinery. We find that stable attachments are sufficient to silence the SAC in the absence of sister kinetochore bi-orientation and strikingly in the absence of detectable microtubule pulling forces or tension. Furthermore, we find that SAC satisfaction occurs despite the absence of large changes in intra-kinetochore distance, suggesting that substantial kinetochore stretching is not required for quenching the SAC signal. PMID:26620470

  4. Kinesin-13 regulates flagellar, interphase, and mitotic microtubule dynamics in Giardia intestinalis.

    PubMed

    Dawson, Scott C; Sagolla, Meredith S; Mancuso, Joel J; Woessner, David J; House, Susan A; Fritz-Laylin, Lillian; Cande, W Zacheus

    2007-12-01

    Microtubule depolymerization dynamics in the spindle are regulated by kinesin-13, a nonprocessive kinesin motor protein that depolymerizes microtubules at the plus and minus ends. Here we show that a single kinesin-13 homolog regulates flagellar length dynamics, as well as other interphase and mitotic dynamics in Giardia intestinalis, a widespread parasitic diplomonad protist. Both green fluorescent protein-tagged kinesin-13 and EB1 (a plus-end tracking protein) localize to the plus ends of mitotic and interphase microtubules, including a novel localization to the eight flagellar tips, cytoplasmic anterior axonemes, and the median body. The ectopic expression of a kinesin-13 (S280N) rigor mutant construct caused significant elongation of the eight flagella with significant decreases in the median body volume and resulted in mitotic defects. Notably, drugs that disrupt normal interphase and mitotic microtubule dynamics also affected flagellar length in Giardia. Our study extends recent work on interphase and mitotic kinesin-13 functioning in metazoans to include a role in regulating flagellar length dynamics. We suggest that kinesin-13 universally regulates both mitotic and interphase microtubule dynamics in diverse microbial eukaryotes and propose that axonemal microtubules are subject to the same regulation of microtubule dynamics as other dynamic microtubule arrays. Finally, the present study represents the first use of a dominant-negative strategy to disrupt normal protein function in Giardia and provides important insights into giardial microtubule dynamics with relevance to the development of antigiardial compounds that target critical functions of kinesins in the giardial life cycle. PMID:17766466

  5. DDA3 associates with microtubule plus ends and orchestrates microtubule dynamics and directional cell migration

    PubMed Central

    Zhang, Liangyu; Shao, Hengyi; Zhu, Tongge; Xia, Peng; Wang, Zhikai; Liu, Lifang; Yan, Maomao; Hill, Donald L.; Fang, Guowei; Chen, Zhengjun; Wang, Dongmei; Yao, Xuebiao

    2013-01-01

    Cell motility and adhesion involve orchestrated interaction of microtubules (MTs) with their plus-end tracking proteins (+TIPs). However, the mechanisms underlying regulations of MT dynamics and directional cell migration are still elusive. Here, we show that DDA3-EB1 interaction orchestrates MT plus-end dynamics and facilitates directional cell migration. Biochemical characterizations reveal that DDA3 interacts with EB1 via its SxIP motif within the C-terminal Pro/Ser-rich region. Time-lapse and total internal reflection fluorescence (TIRF) microscopic assays demonstrate that DDA3 exhibits EB1-dependent, MT plus-end loading and tracking. The EB1-based loading of DDA3 is responsible for MT plus-ends stabilization at the cell cortex, which in turn orchestrates directional cell migration. Interestingly, the DDA3-EB1 interaction is potentially regulated by EB1 acetylation, which may account for physiological regulation underlying EGF-elicited cell migration. Thus, the EB1-based function of DDA3 links MT dynamics to directional cell migration. PMID:23652583

  6. Dynamic Nanoparticles Assemblies

    PubMed Central

    WANG, LIBING; XU, LIGUANG; KUANG, HUA; XU, CHUANLAI; KOTOV, NICHOLAS A.

    2012-01-01

    CONSPECTUS Importance Although nanoparticle (NP) assemblies are at the beginning of their development, their unique geometrical shapes and media-responsive optical, electronic and magnetic properties have attracted significant interest. Nanoscale assembly bridges multiple sizes of materials: individual nanoparticles, discrete molecule-like or virus-like nanoscale agglomerates, microscale devices, and macroscale materials. The capacity to self-assemble can greatly facilitate the integration of nanotechnology with other technologies and, in particular, with microscale fabrication. In this Account, we describe developments in the emerging field of dynamic NP assemblies, which are spontaneously formed superstructures containing more than two inorganic nanoscale particles that display ability to change their geometrical, physical, chemical, and other attributes. In many ways, dynamic assemblies can represent a bottleneck in the ‘bottom-up’ fabrication of NP-based devices because they can produce a much greater variety of assemblies, but they also provide a convenient tool for variation of geometries and dimensions of nanoparticle assemblies. Classification Superstructures of NPs (and those held together by similar intrinsic forces) are classified into two groups: Class 1 where media and external fields can alter shape, conformation, and order of stable superstructures with a nearly constant number same. The future development of successful dynamic assemblies requires understanding the equilibrium in dynamic NP systems. The dynamic nature of Class 1 assemblies is associated with the equilibrium between different conformations of a superstructure and is comparable to the isomerization in classical chemistry. Class 2 assemblies involve the formation and/or breakage of linkages between the NPs, which is analogous to the classical chemical equilibrium for the formation of a molecule from atoms. Finer classification of NP assemblies in accord with established conventions

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

    PubMed

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

    2014-03-28

    Microtubules (MTs) are cytoplasmic protein polymers that are essential for fundamental cellular processes including the maintenance of cell shape, organelle transport and formation of the mitotic spindle. Microtubule dynamic instability is critical for these processes, but it remains poorly understood, in part because the relationship between the structure of the MT tip and the growth/depolymerization transitions is enigmatic. In previous work, we used computational models of dynamic instability to provide evidence that cracks (laterally unbonded regions) between protofilaments play a key role in the regulation of dynamic instability. Here we use computational models to investigate the connection between cracks and dynamic instability in more detail. Our work indicates that while cracks contribute to dynamic instability in a fundamental way, it is not the depth of the cracks per se that governs MT dynamic instability. Instead, what matters more is whether the cracks terminate in GTP-rich or GDP-rich regions of the MT. Based on these observations, we suggest that a functional "GTP cap" (i.e., one capable of promoting MT growth) is one where the cracks terminate in pairs of GTP-bound subunits, and that the likelihood of catastrophe rises significantly with the fraction of crack-terminating subunits that contain GDP. In addition to helping clarify the mechanism of dynamic instability, this idea could also explain how MT stabilizers work: proteins that introduce lateral cross-links between protofilaments would produce islands of GDP-bound tubulin that mimic GTP-rich regions in having strong lateral bonds, thus reducing crack propagation, suppressing catastrophe and promoting rescue. PMID:24652487

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

    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

  11. 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

  12. 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

  13. Self-Organization of Anastral Spindles by Synergy of Dynamic Instability, Autocatalytic Microtubule Production, and a Spatial Signaling Gradient

    PubMed Central

    Clausen, Thomas; Ribbeck, Katharina

    2007-01-01

    Assembly of the mitotic spindle is a classic example of macromolecular self-organization. During spindle assembly, microtubules (MTs) accumulate around chromatin. In centrosomal spindles, centrosomes at the spindle poles are the dominating source of MT production. However, many systems assemble anastral spindles, i.e., spindles without centrosomes at the poles. How anastral spindles produce and maintain a high concentration of MTs in the absence of centrosome-catalyzed MT production is unknown. With a combined biochemistry-computer simulation approach, we show that the concerted activity of three components can efficiently concentrate microtubules (MTs) at chromatin: (1) an external stimulus in form of a RanGTP gradient centered on chromatin, (2) a feed-back loop where MTs induce production of new MTs, and (3) continuous re-organization of MT structures by dynamic instability. The mechanism proposed here can generate and maintain a dissipative MT super-structure within a RanGTP gradient. PMID:17330139

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  16. TMAO promotes fibrillization and microtubule assembly activity in the C-terminal repeat region of tau.

    PubMed

    Scaramozzino, Francesca; Peterson, Dylan W; Farmer, Patrick; Gerig, J T; Graves, Donald J; Lew, John

    2006-03-21

    Alzheimer's disease most closely correlates with the appearance of the neurofibrillary tangles (NFTs), intracellular fibrous aggregates of the microtubule-associated protein, tau. Under native conditions, tau is an unstructured protein, and its physical characterization has revealed no clues about the three-dimensional structural determinants essential for aggregation or microtubule binding. We have found that the natural osmolyte trimethylamine N-oxide (TMAO) induces secondary structure in a C-terminal fragment of tau (tau(187)) and greatly promotes both self-aggregation and microtubule (MT) assembly activity. These processes could be distinguished, however, by a single-amino acid substitution (Tyr(310) --> Ala), which severely inhibited aggregation but had no effect on MT assembly activity. The inability of this mutant to aggregate could be completely reversed by TMAO. We propose a model in which TMAO induces partial order in tau(187), resulting in conformers that may correspond to on-pathway intermediates of either aggregation or tau-dependent MT assembly or both. These studies set the stage for future high-resolution structural characterization of these intermediates and the basis by which Tyr(310) may direct pathologic versus normal tau function. PMID:16533051

  17. 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

  18. The contribution of αβ-tubulin curvature to microtubule dynamics

    PubMed Central

    2014-01-01

    Microtubules are dynamic polymers of αβ-tubulin that form diverse cellular structures, such as the mitotic spindle for cell division, the backbone of neurons, and axonemes. To control the architecture of microtubule networks, microtubule-associated proteins (MAPs) and motor proteins regulate microtubule growth, shrinkage, and the transitions between these states. Recent evidence shows that many MAPs exert their effects by selectively binding to distinct conformations of polymerized or unpolymerized αβ-tubulin. The ability of αβ-tubulin to adopt distinct conformations contributes to the intrinsic polymerization dynamics of microtubules. αβ-Tubulin conformation is a fundamental property that MAPs monitor and control to build proper microtubule networks. PMID:25385183

  19. 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

  20. Suppression of microtubule dynamic instability and turnover in MCF7 breast cancer cells by sulforaphane

    PubMed Central

    Azarenko, Olga; Okouneva, Tatiana; Singletary, Keith W.; Jordan, Mary Ann; Wilson, Leslie

    2008-01-01

    Sulforaphane (SFN), a prominent isothiocyanate present in cruciferous vegetables, is believed to be responsible along with other isothiocyanates for the cancer preventive activity of such vegetables. SFN arrests mitosis, possibly by affecting spindle microtubule function. A critical property of microtubules is their rapid and time-sensitive growth and shortening dynamics (dynamic instability), and suppression of dynamics by antimitotic anticancer drugs (e.g. taxanes and the vinca alkaloids) is central to the anticancer mechanisms of such drugs. We found that at concentrations that inhibited proliferation and mitosis of MCF7-green fluorescent protein-α-tubulin breast tumor cells by ∼50% (∼15 μM), SFN significantly modified microtubule organization in arrested spindles without modulating the spindle microtubule mass, in a manner similar to that of much more powerful antimitotic drugs. By using quantitative fluorescence video microscopy, we determined that at its mitotic concentration required to inhibit mitosis by 50%, SFN suppressed the dynamic instability of the interphase microtubules in these cells, strongly reducing the rate and extent of growth and shortening and decreasing microtubule turnover, without affecting the polymer mass. SFN suppressed the dynamics of purified microtubules in a similar fashion at concentrations well below those required to depolymerize microtubules, indicating that the suppression of dynamic instability by SFN in cells is due to a direct effect on the microtubules. The results indicate that SFN arrests proliferation and mitosis by stabilizing microtubules in a manner weaker than but similar to more powerful clinically used antimitotic anticancer drugs and strongly support the hypothesis that inhibition of mitosis by microtubule stabilization is important for SFN's chemopreventive activity. PMID:18952594

  1. 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.

  2. 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

  3. Minimal model for collective kinetochore-microtubule dynamics.

    PubMed

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

    2015-10-13

    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

  4. 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

  5. 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

  6. 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

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

    PubMed Central

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

    2015-01-01

    ELMO2 belongs to a family of scaffold proteins involved in phagocytosis and cell motility. ELMO2 can simultaneously bind integrin-linked kinase (ILK) and RhoG, forming tripartite ERI complexes. These complexes are involved in promoting β1 integrin–dependent directional migration in undifferentiated epidermal keratinocytes. ELMO2 and ILK have also separately been implicated in microtubule regulation at integrin-containing focal adhesions. During differentiation, epidermal keratinocytes cease to express integrins, but ERI complexes persist. Here we show an integrin-independent role of ERI complexes in modulation of microtubule dynamics in differentiated keratinocytes. Depletion of ERI complexes by inactivating the Ilk gene in these cells reduces microtubule growth and increases the frequency of catastrophe. Reciprocally, exogenous expression of ELMO2 or RhoG stabilizes microtubules, but only if ILK is also present. Mechanistically, activation of Rac1 downstream from ERI complexes mediates their effects on microtubule stability. In this pathway, Rac1 serves as a hub to modulate microtubule dynamics through two different routes: 1) phosphorylation and inactivation of the microtubule-destabilizing protein stathmin and 2) phosphorylation and inactivation of GSK-3β, which leads to the activation of CRMP2, promoting microtubule growth. At the cellular level, the absence of ERI species impairs Ca2+-mediated formation of adherens junctions, critical to maintaining mechanical integrity in the epidermis. Our findings support a key role for ERI species in integrin-independent stabilization of the microtubule network in differentiated keratinocytes. PMID:25995380

  8. 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

  9. 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

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

    PubMed

    Bohner, G; Gustafsson, N; Cade, N I; Maurer, S P; Griffin, L D; Surrey, T

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2007-01-01

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

  13. 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

  14. Tau assembly: the dominant role of PHF6 (VQIVYK) in microtubule binding region repeat R3.

    PubMed

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

    2015-04-01

    Self-aggregation of the microtubule-binding protein Tau reduces its functionality and is tightly associated with Tau-related diseases, termed tauopathies. Tau aggregation is also strongly associated with two nucleating six-residue segments, namely PHF6 (VQIVYK) and PHF6* (VQIINK). In this paper, using experiments and computational modeling, we study the self-assembly of individual and binary mixtures of Tau fragments containing PHF6* (R2/wt; (273)GKVQIINKKLDL(284)) and PHF6 (R3/wt; (306)VQIVYKPVDLSK(317)) and a mutant R2/ΔK280 associated with a neurodegenerative tauopathy. The initial stage of aggregation is probed by ion-mobility mass spectrometry, the kinetics of aggregation monitored with Thioflavin T assays, and the morphology of aggregates visualized by transmission electron microscopy. Insights into the structure of early aggregates and the factors stabilizing the aggregates are obtained from replica exchange molecular dynamics simulations. Our data suggest that R3/wt has a much stronger aggregation propensity than either R2/wt or R2/ΔK280. Heterodimers containing R3/wt are less stable than R3/wt homodimers but much more stable than homodimers of R2/wt and R2/ΔK280, suggesting a possible role of PHF6*-PHF6 interactions in initiating the aggregation of full-length Tau. Lastly, R2/ΔK280 binds more strongly to R3/wt than R2/wt, suggesting a possible mechanism for a pathological loss of normal Tau function. PMID:25775228

  15. The oligomeric outer dynein arm assembly factor CCDC103 is tightly integrated within the ciliary axoneme and exhibits periodic binding to microtubules.

    PubMed

    King, Stephen M; Patel-King, Ramila S

    2015-03-20

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

  16. The Spontaneous Alignment of Microtubules in Vitro.

    NASA Astrophysics Data System (ADS)

    Hitt, Anne Louise

    Microtubules assembled at 37^circ C in vitro from tubulin, with or without microtubule associated proteins (MAPs), spontaneously form macroscopic domains of intense birefringence. Because the intrinsic birefringence of microtubules is small, the observed effect must be due to form birefringence, caused by a mutually parallel disposition of microtubules. The observed birefringence cannot be accounted for by multiple light scattering. Birefringence and microtubule polymerization are observed to be temporally coupled. The development of multiple macroscopic birefringent domains is mirrored by the formation of large domains which scatter light strongly. Because these solutions are not homogeneous, Beer's law may not apply. These turbid domains may account for the turbidity overshoot observed by several laboratories. Electron micrographs of sections of gluteraldehyde -fixed microtubule solutions which exhibited birefringence before and after fixation displayed a directionality. This confirms that microtubules in solution are aligned with respect to each other. Centrifugation of birefringent microtubule solutions yields an isotropic supernatant and an intensely birefringent pellet, suggesting that the birefringent domains are dense and sediment intact. If MAPs are present, the birefringent domains can be observed in dilute solution after more than 20 hours at 37^circ C. Polymerization conditions which result in oscillations in microtubule assembly due to dynamic instability also result in oscillations in microtubule alignment. These observations, taken together, indicate that microtubule solutions become nematic liquid crystals exhibiting a polydomain schlieren texture upon polymerization in vitro. These domains appear to be stable, dense constructs of microtubules, which are liquid-crystalline in character. Assembly of microtubules initially results in the formation of many small microtubules; with time, however, fewer but longer microtubules are observed. Recently, two

  17. Spindle assembly checkpoint proteins are positioned close to core microtubule attachment sites at kinetochores

    PubMed Central

    Wan, Xiaohu; Cheerambathur, Dhanya; Gassmann, Reto; Suzuki, Aussie; Lawrimore, Josh; Desai, Arshad; Salmon, E.D.

    2013-01-01

    Spindle assembly checkpoint proteins have been thought to reside in the peripheral corona region of the kinetochore, distal to microtubule attachment sites at the outer plate. However, recent biochemical evidence indicates that checkpoint proteins are closely linked to the core kinetochore microtubule attachment site comprised of the Knl1–Mis12–Ndc80 (KMN) complexes/KMN network. In this paper, we show that the Knl1–Zwint1 complex is required to recruit the Rod–Zwilch–Zw10 (RZZ) and Mad1–Mad2 complexes to the outer kinetochore. Consistent with this, nanometer-scale mapping indicates that RZZ, Mad1–Mad2, and the C terminus of the dynein recruitment factor Spindly are closely juxtaposed with the KMN network in metaphase cells when their dissociation is blocked and the checkpoint is active. In contrast, the N terminus of Spindly is ∼75 nm outside the calponin homology domain of the Ndc80 complex. These results reveal how checkpoint proteins are integrated within the substructure of the kinetochore and will aid in understanding the coordination of microtubule attachment and checkpoint signaling during chromosome segregation. PMID:23979716

  18. MACF1 regulates the migration of pyramidal neurons via microtubule dynamics and GSK-3 signaling.

    PubMed

    Ka, Minhan; Jung, Eui-Man; Mueller, Ulrich; Kim, Woo-Yang

    2014-11-01

    Neuronal migration and subsequent differentiation play critical roles for establishing functional neural circuitry in the developing brain. However, the molecular mechanisms that regulate these processes are poorly understood. Here, we show that microtubule actin crosslinking factor 1 (MACF1) determines neuronal positioning by regulating microtubule dynamics and mediating GSK-3 signaling during brain development. First, using MACF1 floxed allele mice and in utero gene manipulation, we find that MACF1 deletion suppresses migration of cortical pyramidal neurons and results in aberrant neuronal positioning in the developing brain. The cell autonomous deficit in migration is associated with abnormal dynamics of leading processes and centrosomes. Furthermore, microtubule stability is severely damaged in neurons lacking MACF1, resulting in abnormal microtubule dynamics. Finally, MACF1 interacts with and mediates GSK-3 signaling in developing neurons. Our findings establish a cellular mechanism underlying neuronal migration and provide insights into the regulation of cytoskeleton dynamics in developing neurons. PMID:25224226

  19. Centriolar satellite– and hMsd1/SSX2IP-dependent microtubule anchoring is critical for centriole assembly

    PubMed Central

    Hori, Akiko; Peddie, Christopher J.; Collinson, Lucy M.; Toda, Takashi

    2015-01-01

    Centriolar satellites are numerous electron-dense granules dispersed around the centrosome. Mutations in their components are linked to various human diseases, but their molecular roles remain elusive. In particular, the significance of spatial communication between centriolar satellites and the centrosome is unknown. hMsd1/SSX2IP localizes to both the centrosome and centriolar satellites and is required for tethering microtubules to the centrosome. Here we show that hMsd1/SSX2IP-mediated microtubule anchoring is essential for proper centriole assembly and duplication. On hMsd1/SSX2IP knockdown, the centriolar satellites become stuck at the microtubule minus end near the centrosome. Intriguingly, these satellites contain many proteins that normally localize to the centrosome. Of importance, microtubule structures, albeit not being anchored properly, are still required for the emergence of abnormal satellites, as complete microtubule depolymerization results in the disappearance of these aggregates from the vicinity of the centrosome. We highlighted, using superresolution and electron microscopy, that under these conditions, centriole structures are faulty. Remarkably, these cells are insensitive to Plk4 overproduction–induced ectopic centriole formation, yet they accelerate centrosome reduplication upon hydroxyurea arrest. Finally, the appearance of satellite aggregates is cancer cell specific. Together our findings provide novel insights into the mechanism of centriole assembly and microtubule anchoring. PMID:25833712

  20. Centriolar satellite- and hMsd1/SSX2IP-dependent microtubule anchoring is critical for centriole assembly.

    PubMed

    Hori, Akiko; Peddie, Christopher J; Collinson, Lucy M; Toda, Takashi

    2015-06-01

    Centriolar satellites are numerous electron-dense granules dispersed around the centrosome. Mutations in their components are linked to various human diseases, but their molecular roles remain elusive. In particular, the significance of spatial communication between centriolar satellites and the centrosome is unknown. hMsd1/SSX2IP localizes to both the centrosome and centriolar satellites and is required for tethering microtubules to the centrosome. Here we show that hMsd1/SSX2IP-mediated microtubule anchoring is essential for proper centriole assembly and duplication. On hMsd1/SSX2IP knockdown, the centriolar satellites become stuck at the microtubule minus end near the centrosome. Intriguingly, these satellites contain many proteins that normally localize to the centrosome. Of importance, microtubule structures, albeit not being anchored properly, are still required for the emergence of abnormal satellites, as complete microtubule depolymerization results in the disappearance of these aggregates from the vicinity of the centrosome. We highlighted, using superresolution and electron microscopy, that under these conditions, centriole structures are faulty. Remarkably, these cells are insensitive to Plk4 overproduction-induced ectopic centriole formation, yet they accelerate centrosome reduplication upon hydroxyurea arrest. Finally, the appearance of satellite aggregates is cancer cell specific. Together our findings provide novel insights into the mechanism of centriole assembly and microtubule anchoring. PMID:25833712

  1. Kinesin superfamily proteins and the regulation of microtubule dynamics in morphogenesis.

    PubMed

    Niwa, Shinsuke

    2015-01-01

    Kinesin superfamily proteins (KIFs) are microtubule-dependent molecular motors that serve as sources of force for intracellular transport and cell division. Recent studies have revealed new roles of KIFs as microtubule stabilizers and depolymerizers, and these activities are fundamental to cellular morphogenesis and mammalian development. KIF2A and KIF19A have microtubule-depolymerizing activities and regulate the neuronal morphology and cilia length, respectively. KIF21A and KIF26A work as microtubule stabilizers that regulate axonal morphology. Morphological defects that are similar to human diseases are observed in mice in which these KIF genes have been deleted. Actually, KIF2A and KIF21A have been identified as causes of human neuronal diseases. In this review, the functions of these atypical KIFs that regulate microtubule dynamics are discussed. Moreover, some interesting unanswered questions and hypothetical answers to them are discussed. PMID:25347970

  2. 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. PMID:26235140

  3. 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.

  4. Rapid movements of vimentin on microtubule tracks: kinesin-dependent assembly of intermediate filament networks.

    PubMed

    Prahlad, V; Yoon, M; Moir, R D; Vale, R D; Goldman, R D

    1998-10-01

    The assembly and maintenance of an extended intermediate filament (IF) network in fibroblasts requires microtubule (MT) integrity. Using a green fluorescent protein-vimentin construct, and spreading BHK-21 cells as a model system to study IF-MT interactions, we have discovered a novel mechanism involved in the assembly of the vimentin IF cytoskeleton. This entails the rapid, discontinuous, and MT-dependent movement of IF precursors towards the peripheral regions of the cytoplasm where they appear to assemble into short fibrils. These precursors, or vimentin dots, move at speeds averaging 0.55 +/- 0.24 micrometer/s. The vimentin dots colocalize with MT and their motility is inhibited after treatment with nocodazole. Our studies further implicate a conventional kinesin in the movement of the vimentin dots. The dots colocalize with conventional kinesin as shown by indirect immunofluorescence, and IF preparations from spreading cells are enriched in kinesin. Furthermore, microinjection of kinesin antibodies into spreading cells prevents the assembly of an extended IF network. These studies provide insights into the interactions between the IF and MT systems. They also suggest a role for conventional kinesin in the distribution of non-membranous protein cargo, and the local regulation of IF assembly. PMID:9763428

  5. Rapid Movements of Vimentin on Microtubule Tracks: Kinesin-dependent Assembly of Intermediate Filament Networks

    PubMed Central

    Prahlad, Veena; Yoon, Miri; Moir, Robert D.; Vale, Ronald D.; Goldman, Robert D.

    1998-01-01

    The assembly and maintenance of an extended intermediate filament (IF) network in fibroblasts requires microtubule (MT) integrity. Using a green fluorescent protein–vimentin construct, and spreading BHK-21 cells as a model system to study IF–MT interactions, we have discovered a novel mechanism involved in the assembly of the vimentin IF cytoskeleton. This entails the rapid, discontinuous, and MT-dependent movement of IF precursors towards the peripheral regions of the cytoplasm where they appear to assemble into short fibrils. These precursors, or vimentin dots, move at speeds averaging 0.55 ± 0.24 μm/s. The vimentin dots colocalize with MT and their motility is inhibited after treatment with nocodazole. Our studies further implicate a conventional kinesin in the movement of the vimentin dots. The dots colocalize with conventional kinesin as shown by indirect immunofluorescence, and IF preparations from spreading cells are enriched in kinesin. Furthermore, microinjection of kinesin antibodies into spreading cells prevents the assembly of an extended IF network. These studies provide insights into the interactions between the IF and MT systems. They also suggest a role for conventional kinesin in the distribution of non-membranous protein cargo, and the local regulation of IF assembly. PMID:9763428

  6. Mechanism and Dynamics of Breakage of Fluorescent Microtubules

    PubMed Central

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

    2006-01-01

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

  7. Xorbit/CLASP links dynamic microtubules to chromosomes in the Xenopus meiotic spindle

    PubMed Central

    Hannak, Eva; Heald, Rebecca

    2006-01-01

    A family of microtubule (MT)-binding proteins, Orbit/multiple asters/cytoplasmic linker protein–associated protein, has emerged as an important player during mitosis, but their functional mechanisms are poorly understood. In this study, we used meiotic egg extracts to gain insight into the role of the Xenopus laevis homologue Xorbit in spindle assembly and function. Xorbit immunodepletion or its inhibition by a dominant-negative fragment resulted in chromosome alignment defects and aberrant MT structures, including monopolar and small spindles. Xorbit-depleted extracts failed to nucleate MTs around chromatin-coated beads, indicating its essential requirement for spindle assembly in the absence of centrosomes and kinetochores. Xorbit's MT stabilizing effect was most apparent during anaphase, when spindle MTs depolymerized rapidly upon Xorbit inhibition. Biochemical interaction between a COOH-terminal Xorbit fragment and the kinetochore-associated kinesin centromeric protein E may contribute to Xorbit's role in chromosome congression. We propose that Xorbit tethers dynamic MT plus ends to kinetochores and chromatin, providing a stabilizing activity that is crucial for spindle assembly and chromosome segregation. PMID:16390996

  8. The effect of multivalent cations and Tau on paclitaxel-stabilized microtubule assembly, disassembly, and structure.

    PubMed

    Safinya, Cyrus R; Chung, Peter J; Song, Chaeyeon; Li, Youli; Ewert, Kai K; Choi, Myung Chul

    2016-06-01

    In this review we describe recent studies directed at understanding the formation of novel nanoscale assemblies in biological materials systems. In particular, we focus on the effects of multivalent cations, and separately, of microtubule-associated protein (MAP) Tau, on microtubule (MT) ordering (bundling), MT disassembly, and MT structure. Counter-ion directed bundling of paclitaxel-stabilized MTs is a model electrostatic system, which parallels efforts to understand MT bundling by intrinsically disordered proteins (typically biological polyampholytes) expressed in neurons. We describe studies, which reveal an unexpected transition from tightly spaced MT bundles to loose bundles consisting of strings of MTs as the valence of the cationic counter-ion decreases from Z=3 to Z=2. This transition is not predicted by any current theories of polyelectrolytes. Notably, studies of a larger series of divalent counter-ions reveal strong ion specific effects. Divalent counter-ions may either bundle or depolymerize paclitaxel-stabilized MTs. The ion concentration required for depolymerization decreases with increasing atomic number. In a more biologically related system we review synchrotron small angle x-ray scattering (SAXS) studies on the effect of the Tau on the structure of paclitaxel-stabilized MTs. The electrostatic binding of MAP Tau isoforms leads to an increase in the average radius of microtubules with increasing Tau coverage (i.e. a re-distribution of protofilament numbers in MTs). Finally, inspired by MTs as model nanotubes, we briefly describe other more robust lipid-based cylindrical nanostructures, which may have technological applications, for example, in drug encapsulation and delivery. PMID:26684364

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

    PubMed

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

    2016-05-01

    The molecular mechanism by which the microtubule-associated protein (MAP) tau regulates the formation of microtubules (MTs) is poorly understood. The activity of tau is controlled via phosphorylation at specific Ser/Thr sites. Of those phosphorylation sites, 17 precede a proline, making them potential recognition sites for the peptidyl-prolyl isomerase Pin1. Pin1 binding and catalysis of phosphorylated tau at the AT180 epitope, which was implicated in Alzheimer's disease, has been reported to be crucial for restoring tau's ability to promote MT polymerization in vitro and in vivo [1]. Surprisingly, we discover that Pin1 does not promote phosphorylated tau-induced MT formation in vitro, refuting the commonly accepted model in which Pin1 binding and catalysis on the A180 epitope restores the function of the Alzheimer's associated phosphorylated tau in tubulin assembly [1, 2]. Using turbidity assays, time-resolved small angle X-ray scattering (SAXS), and time-resolved negative stain electron microscopy (EM), we investigate the mechanism of tau-mediated MT assembly and the role of the Thr231 and Ser235 phosphorylation on this process. We discover novel GTP-tubulin ring-shaped species, which are detectable in the earliest stage of tau-induced polymerization and may play a crucial role in the early nucleation phase of MT assembly. Finally, by NMR and SAXS experiments, we show that the tau molecules must be located on the surface of MTs and tubulin rings during the polymerization reaction. The interaction between tau and tubulin is multipartite, with a high affinity interaction of the four tubulin-binding repeats, and a weaker interaction with the proline-rich sequence and the termini of tau. PMID:26996940

  10. Assembly and bundling of marginal band microtubule protein: role of tau.

    PubMed

    Sanchez, I; Cohen, W D

    1994-01-01

    Microtubule protein extracted from dogfish erythrocyte cytoskeletons by disassembly of marginal bands at low temperature formed linear microtubule (MT) bundles upon reassembly at 22 degrees C. The bundles, which were readily visible by video-enhanced phase contrast or DIC microscopy, increased in length and thickness with time. At steady state after 1 hour, most bundles were 6-11 microns in length and 2-5 MTs in thickness. No inter-MT cross-bridges were visible by negative staining. The bundles exhibited mechanical stability in flow as well as flexibility, in this respect resembling native marginal bands. As analyzed by SDS-PAGE and immunoblotting, our standard extraction conditions yielded MT protein preparations and bundles containing tau protein but not high molecular weight MAPs such as MAP-2 or syncolin. In addition, late fractions of MT protein obtained by gel filtration were devoid of high molecular weight proteins but still produced MT bundles. The marginal band tau was salt-extractable and heat-stable, bound antibodies to mammalian brain tau, and formed aggregates upon desalting. Antibodies to tau blocked MT assembly, but both assembly and bundling occurred in the presence of antibodies to actin or syncolin. The MTs were "unbundled" by subtilisin or by high salt (0.5-1 M KCl or NaCl), consistent with tau involvement in bundling. High salt extracts retained bundling activity, and salt-induced unbundling was reversible with desalting. However, reversibility was observed only after salt-induced MT disassembly had occurred. Reconstitution experiments showed that addition of marginal band tau to preassembled MTs did not produce bundles, whereas tau presence during MT reassembly did yield bundles. Thus, in this system, tau appears to play a role in both MT assembly and bundling, serving in the latter function as a coassembly factor. PMID:7820858

  11. Rac1 Modulates Stimulus-evoked Ca2+ Release in Neuronal Growth Cones via Parallel Effects on Microtubule/Endoplasmic Reticulum Dynamics and Reactive Oxygen Species Production

    PubMed Central

    Zhang, Xiao-Feng

    2009-01-01

    The small G protein Rac regulates cytoskeletal protein dynamics in neuronal growth cones and has been implicated in axon growth, guidance, and branching. Intracellular Ca2+ is another well known regulator of growth cone function; however, effects of Rac activity on intracellular Ca2+ metabolism have not been well characterized. Here, we investigate how Rac1 activity affects release of Ca2+ from intracellular endoplasmic reticulum (ER) stores stimulated by application of serotonin (5-hydroxytriptamine). We also address how Rac1 effects on microtubule assembly dynamics affect distribution of Ca2+ release sites. Multimode fluorescent microscopy was used to correlate microtubule and ER behavior, and ratiometric imaging was used to assess intracellular Ca2+ dynamics. We report that Rac1 activity both promotes Ca2+ release and affects its spatial distribution in neuronal growth cones. The underlying mechanism involves synergistic Rac1 effects on microtubule assembly and reactive oxygen species (ROS) production. Rac1 activity modulates Ca2+ by 1) enhancing microtubule assembly which in turn promotes spread of the ER-based Ca2+ release machinery into the growth cone periphery, and 2) by increasing ROS production which facilitated inositol 1,4,5-trisphosphate-dependent Ca2+ release. These results cast Rac1 as a key modulator of intracellular Ca2+ function in the neuronal growth cone. PMID:19570918

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

    Nakashima, Hitomi; Okimura, Chika; Iwadate, Yoshiaki

    2015-01-01

    Cell-crawling migration plays an essential role in complex biological phenomena. It is now generally believed that many processes essential to such migration are regulated by microtubules in many cells, including fibroblasts and neurons. However, keratocytes treated with nocodazole, which is an inhibitor of microtubule polymerization – and even keratocyte fragments that contain no microtubules – migrate at the same velocity and with the same directionality as normal keratocytes. In this study, we discovered that not only these migration properties, but also the molecular dynamics that regulate such properties, such as the retrograde flow rate of actin filaments, distributions of vinculin and myosin II, and traction forces, are also the same in nocodazole-treated keratocytes as those in untreated keratocytes. These results suggest that microtubules are not in fact required for crawling migration of keratocytes, either in terms of migrating properties or of intracellular molecular dynamics. PMID:27493851

  14. 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)

  15. 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

  16. 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

  17. A Web Interface for the Quantification of Microtubule Dynamics

    PubMed Central

    Kong, Koon Yin; Marcus, Adam I.; Giaanakakou, Paraskevi; Wang, May D.

    2016-01-01

    We propose a web interface that allows researchers to quantify and analyze microtubule confocal images online. Most analyses of microtubule confocal images are performed manually using very simple software or tools. Analysis results are stored locally within each collaborator with different styles and formats. This has limited the sharing of data and results when collaborating among different research parties. A web interface provides a simple way for users to process data online. It also allows easy sharing of both data and results among different participating groups. Analysis workflow of the interface is made similar to existing manual protocols. We demonstrate the integration of image processing algorithm in the current workflow to aid the analysis. Our design also allows integration of novel automated analysis algorithms and modules to re-evaluate existing data. This interface can provide a validation platform for new automated algorithm and allow collaboration on microtubule image analysis from different locations.

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

    PubMed Central

    Nunes Bastos, Ricardo; Gandhi, Sapan R.; Baron, Ryan D.; Gruneberg, Ulrike; Nigg, Erich A.

    2013-01-01

    Anaphase central spindle formation is controlled by the microtubule-stabilizing factor PRC1 and the kinesin KIF4A. We show that an MKlp2-dependent pool of Aurora B at the central spindle, rather than global Aurora B activity, regulates KIF4A accumulation at the central spindle. KIF4A phosphorylation by Aurora B stimulates the maximal microtubule-dependent ATPase activity of KIF4A and promotes its interaction with PRC1. In the presence of phosphorylated KIF4A, microtubules grew more slowly and showed long pauses in growth, resulting in the generation of shorter PRC1-stabilized microtubule overlaps in vitro. Cells expressing only mutant forms of KIF4A lacking the Aurora B phosphorylation site overextended the anaphase central spindle, demonstrating that this regulation is crucial for microtubule length control in vivo. Aurora B therefore ensures that suppression of microtubule dynamic instability by KIF4A is restricted to a specific subset of microtubules and thereby contributes to central spindle size control in anaphase. PMID:23940115

  19. Macroscopic stiffening of embryonic tissues via microtubules, RhoGEF and the assembly of contractile bundles of actomyosin

    PubMed Central

    Zhou, Jian; Kim, Hye Young; Wang, James H.-C.; Davidson, Lance A.

    2010-01-01

    During morphogenesis, forces generated by cells are coordinated and channeled by the viscoelastic properties of the embryo. Microtubules and F-actin are considered to be two of the most important structural elements within living cells accounting for both force production and mechanical stiffness. In this paper, we investigate the contribution of microtubules to the stiffness of converging and extending dorsal tissues in Xenopus laevis embryos using cell biological, biophysical and embryological techniques. Surprisingly, we discovered that depolymerizing microtubules stiffens embryonic tissues by three- to fourfold. We attribute tissue stiffening to Xlfc, a previously identified RhoGEF, which binds microtubules and regulates the actomyosin cytoskeleton. Combining drug treatments and Xlfc activation and knockdown lead us to the conclusion that mechanical properties of tissues such as viscoelasticity can be regulated through RhoGTPase pathways and rule out a direct contribution of microtubules to tissue stiffness in the frog embryo. We can rescue nocodazole-induced stiffening with drugs that reduce actomyosin contractility and can partially rescue morphogenetic defects that affect stiffened embryos. We support these conclusions with a multi-scale analysis of cytoskeletal dynamics, tissue-scale traction and measurements of tissue stiffness to separate the role of microtubules from RhoGEF activation. These findings suggest a re-evaluation of the effects of nocodazole and increased focus on the role of Rho family GTPases as regulators of the mechanical properties of cells and their mechanical interactions with surrounding tissues. PMID:20630946

  20. Photoswitchable Inhibitors of Microtubule Dynamics Optically Control Mitosis and Cell Death.

    PubMed

    Borowiak, Malgorzata; Nahaboo, Wallis; Reynders, Martin; Nekolla, Katharina; Jalinot, Pierre; Hasserodt, Jens; Rehberg, Markus; Delattre, Marie; Zahler, Stefan; Vollmar, Angelika; Trauner, Dirk; Thorn-Seshold, Oliver

    2015-07-16

    Small molecules that interfere with microtubule dynamics, such as Taxol and the Vinca alkaloids, are widely used in cell biology research and as clinical anticancer drugs. However, their activity cannot be restricted to specific target cells, which also causes severe side effects in chemotherapy. Here, we introduce the photostatins, inhibitors that can be switched on and off in vivo by visible light, to optically control microtubule dynamics. Photostatins modulate microtubule dynamics with a subsecond response time and control mitosis in living organisms with single-cell spatial precision. In longer-term applications in cell culture, photostatins are up to 250 times more cytotoxic when switched on with blue light than when kept in the dark. Therefore, photostatins are both valuable tools for cell biology, and are promising as a new class of precision chemotherapeutics whose toxicity may be spatiotemporally constrained using light. PMID:26165941

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

    PubMed

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

    2014-08-01

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

  2. 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

  3. 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.

  4. 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.

  5. 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

  6. 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. PMID:26969727

  7. 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

  8. APC is an RNA-Binding Protein and its Interactome Provides a Link to Neural Development and Microtubule Assembly

    PubMed Central

    Preitner, Nicolas; Quan, Jie; Nowakowski, Dan W.; Hancock, Melissa L.; Shi, Jianhua; Tcherkezian, Joseph; Young-Pearse, Tracy L.; Flanagan, John G.

    2014-01-01

    SUMMARY Adenomatous polyposis coli (APC) is a microtubule plus-end scaffolding protein important in biology and disease. APC is implicated in RNA localization, although the mechanisms and functional significance remain unclear. We show that APC is an RNA-binding protein, and identify an RNA interactome by HITS-CLIP. Targets were highly enriched for APC-related functions, including microtubule organization, cell motility, cancer and neurologic disease. Among the targets is β2B-tubulin, known to be required in human neuron and axon migration. We show β2B-tubulin is synthesized in axons and localizes preferentially to dynamic microtubules in the growth cone periphery. APC binds the β2B-tubulin 3'UTR; treatments interfering with this interaction reduced β2B-tubulin mRNA axonal localization and expression, depleted dynamic microtubules and the growth cone periphery, and impaired neuron migration. These results identify APC as a platform binding functionally-related protein and RNA networks, and suggest a self-organizing model for the microtubule to localize synthesis of its own subunits. PMID:25036633

  9. 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

  10. 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

  11. 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. PMID:26510788

  12. 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. PMID:27046833

  13. Cryo-EM Studies of Microtubule Structural Intermediates and Kinetochore–Microtubule Interactions

    PubMed Central

    Nogales, Eva; Ramey, Vincent H.; Wang, Hong-Wei

    2014-01-01

    The existence of structural intermediates in the processes of microtubule assembly and disassembly, and their relationship with the nucleotide state of tubulin, have been the subject of significant study and recent controversy. The first part of this chapter describes experiments and methods designed to characterize, using cryo-electron microscopy (cryo-EM) and image analysis, the structure of stabilized tubulin assemblies that we propose mimic the growth and shortening states at microtubule ends. We further put forward the idea that these intermediates have important biological functions, especially during cellular processes where the dynamic character of microtubules is essential. One such process is the attachment of spindle microtubules to kinetochores in eukaryotic cell division. The second part of this chapter is consequently dedicated to studies of the yeast Dam1 kinetochore complex and its interaction with microtubules. This complex is essential for accurate chromosome segregation and is an important target of the Aurora B spindle check-point kinase. The Dam1 complex self-assembles in a microtubule-dependent manner into rings and spirals. The rings are able to track microtubule-depolymerizing ends against a load and in a highly processive manner, an essential property for their function in vivo. We describe the experimental in vitro protocols to produce biologically relevant self-assembled structures of Dam1 around microtubules and their structural characterization by cryo-EM. PMID:20466133

  14. Microtubule-dependent membrane dynamics in Ustilago maydis

    PubMed Central

    Göhre, Vera; Vollmeister, Evelyn; Bölker, Michael; Feldbrügge, Michael

    2012-01-01

    Long-distance trafficking of membranous structures along the cytoskeleton is crucial for secretion and endocytosis in eukaryotes. Molecular motors are transporting both secretory and endocytic vesicles along polarized microtubules. Here, we review the transport mechanism and biological function of a distinct subset of large vesicles marked by the G-protein Rab5a in the model microorganism Ustilago maydis. These Rab5a-positive endosomes shuttle bi-directionally along microtubules mediated by the Unc104/KIF1A-related motor Kin3 and dynein Dyn1/2. Rab5a-positive endosomes exhibit diverse functions during the life cycle of U. maydis. In haploid budding cells they are involved in cytokinesis and pheromone signaling. During filamentous growth endosomes are used for long-distance transport of mRNA, a prerequisite to maintain polarity most likely via local translation of specific proteins at both the apical and distal ends of filaments. Endosomal co-transport of mRNA constitutes a novel function of these membrane compartments supporting the view that endosomes function as multipurpose platforms. PMID:23181166

  15. Desmosome assembly and dynamics

    PubMed Central

    Nekrasova, Oxana; Green, Kathleen J.

    2013-01-01

    Desmosomes are intercellular junctions that anchor intermediate filaments to the plasma membrane, forming a supracellular scaffold that provides mechanical resilience to tissues. This anchoring function is accomplished by specialized members of the cadherin family and associated cytoskeletal linking proteins, which together form a highly organized membrane core flanked by mirror image cytoplasmic plaques. Due to the biochemical insolubility of desmosomes, the mechanisms that govern assembly of these components into a functional organelle remained elusive. Recently developed molecular reporters and live cell imaging approaches have provided powerful new tools to monitor this finely-tuned process in real time. Here we discuss studies that are beginning to decipher the machinery and regulation governing desmosome assembly and homeostasis in situ, and how these mechanisms are affected during disease pathogenesis. PMID:23891292

  16. 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

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

    SciTech Connect

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

    2006-09-22

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

  18. XMAP215 activity sets spindle length by controlling the total mass of spindle microtubules.

    PubMed

    Reber, Simone B; Baumgart, Johannes; Widlund, Per O; Pozniakovsky, Andrei; Howard, Jonathon; Hyman, Anthony A; Jülicher, Frank

    2013-09-01

    Metaphase spindles are microtubule-based structures that use a multitude of proteins to modulate their morphology and function. Today, we understand many details of microtubule assembly, the role of microtubule-associated proteins, and the action of molecular motors. Ultimately, the challenge remains to understand how the collective behaviour of these nanometre-scale processes gives rise to a properly sized spindle on the micrometre scale. By systematically engineering the enzymatic activity of XMAP215, a processive microtubule polymerase, we show that Xenopus laevis spindle length increases linearly with microtubule growth velocity, whereas other parameters of spindle organization, such as microtubule density, lifetime and spindle shape, remain constant. We further show that mass balance can be used to link the global property of spindle size to individual microtubule dynamic parameters. We propose that spindle length is set by a balance of non-uniform nucleation and global microtubule disassembly in a liquid-crystal-like arrangement of microtubules. PMID:23974040

  19. 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

  20. 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

  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

    2015-01-01

    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. PMID:26208336

  2. 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

  3. 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

  4. Spatio-temporal Dynamics and Mechanisms of Stress Granule Assembly

    PubMed Central

    Ohshima, Daisuke; Arimoto-Matsuzaki, Kyoko; Tomida, Taichiro; Takekawa, Mutsuhiro; Ichikawa, Kazuhisa

    2015-01-01

    Stress granules (SGs) are non-membranous cytoplasmic aggregates of mRNAs and related proteins, assembled in response to environmental stresses such as heat shock, hypoxia, endoplasmic reticulum (ER) stress, chemicals (e.g. arsenite), and viral infections. SGs are hypothesized as a loci of mRNA triage and/or maintenance of proper translation capacity ratio to the pool of mRNAs. In brain ischemia, hippocampal CA3 neurons, which are resilient to ischemia, assemble SGs. In contrast, CA1 neurons, which are vulnerable to ischemia, do not assemble SGs. These results suggest a critical role SG plays in regards to cell fate decisions. Thus SG assembly along with its dynamics should determine the cell fate. However, the process that exactly determines the SG assembly dynamics is largely unknown. In this paper, analyses of experimental data and computer simulations were used to approach this problem. SGs were assembled as a result of applying arsenite to HeLa cells. The number of SGs increased after a short latent period, reached a maximum, then decreased during the application of arsenite. At the same time, the size of SGs grew larger and became localized at the perinuclear region. A minimal mathematical model was constructed, and stochastic simulations were run to test the modeling. Since SGs are discrete entities as there are only several tens of them in a cell, commonly used deterministic simulations could not be employed. The stochastic simulations replicated observed dynamics of SG assembly. In addition, these stochastic simulations predicted a gamma distribution relative to the size of SGs. This same distribution was also found in our experimental data suggesting the existence of multiple fusion steps in the SG assembly. Furthermore, we found that the initial steps in the SG assembly process and microtubules were critical to the dynamics. Thus our experiments and stochastic simulations presented a possible mechanism regulating SG assembly. PMID:26115353

  5. Pharmacological and genetic evidence for a role of rootlet and phycoplast microtubules in the positioning and assembly of cleavage furrows in Chlamydomonas reinhardtii.

    PubMed

    Ehler, L L; Dutcher, S K

    1998-01-01

    In Chlamydomonas reinhardtii, specialized cytoskeletal structures known as rootlet microtubules are present throughout interphase and mitosis. During cytokinesis, an array of microtubules termed the phycoplast is nucleated from rootlet microtubules and forms coincidentally with the cleavage furrow [Johnson and Porter, 1968: J. Cell Biol. 38:403-425; Holmes and Dutcher, 1989: J. Cell Sci. 94:273-285; Gaffel and el-Gammel, 1990: Protoplasma 156:139-148; Schibler and Huang, 1991: J. Cell Biol. 113:605-614]. We have obtained two independent lines of evidence that support the hypothesis that the rootlet and phycoplast microtubules play a direct role in cleavage furrow placement and assembly. First, the destabilization of spindle and phycoplast microtubules by pharmacological agents was accompanied by the aberrant distribution of actin and a failure of cytokinesis. Second, we characterized mutant strains that failed to complete cytokinesis properly. Actin and myosin were mislocalized to additional rootlet microtubules in the cyt2-1 strain, and this mislocalization was correlated with the presence of additional cleavage furrows. This evidence suggests that microtubules are necessary for the correct positioning and assembly of functional cleavage furrows in C. reinhardtii. PMID:9634216

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

    PubMed

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

    2016-02-01

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

  7. 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

  8. 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

  9. Regulation of tubulin levels and microtubule assembly in Saccharomyces cerevisiae: consequences of altered tubulin gene copy number.

    PubMed Central

    Katz, W; Weinstein, B; Solomon, F

    1990-01-01

    Microtubule organization in the cytoplasm is in part a function of the number and length of the assembled polymers. The intracellular concentration of tubulin could specify those parameters. Saccharomyces cerevisiae strains constructed with moderately decreased or increased copy numbers of tubulin genes provide an opportunity to study the cellular response to a steady-state change in tubulin concentration. We found no evidence of a mechanism for adjusting tubulin concentrations upward from a deficit, nor did we find a need for such a mechanism: cells with no more than 50% of the wild-type tubulin level were normal with respect to a series of microtubule-dependent properties. Strains with increased copies of both alpha- and beta-tubulin genes, or of alpha-tubulin genes alone, apparently did down regulate their tubulin levels. As a result, they contained greater than normal concentrations of tubulin but much less than predicted from the increase in gene number. Some of this down regulation occurred at the level of protein. These strains were also phenotypically normal. Cells could contain excess alpha-tubulin protein without detectable consequences, but perturbations resulting in excess beta-tubulin genes may have affected microtubule-dependent functions. All of the observed regulation of levels of tubulin can be explained as a response to toxicity associated with excess tubulin proteins, especially if beta-tubulin is much more toxic than alpha-tubulin. Images PMID:2204811

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

    PubMed Central

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

    2015-01-01

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

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

    SciTech Connect

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

    2009-06-05

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

  12. Microtubule plus-end and minus-end capture at adherens junctions is involved in the assembly of apico-basal arrays in polarised epithelial cells.

    PubMed

    Bellett, Gemma; Carter, Jane M; Keynton, Jennifer; Goldspink, Deborah; James, Colin; Moss, David K; Mogensen, Mette M

    2009-10-01

    Apico-basal polarisation of epithelial cells involves a dramatic reorganisation of the microtubule cytoskeleton. The classic radial array of microtubules focused on a centrally located centrosome typical of many animal cells is lost or greatly reduced and a non-centrosomal apico-basal array develops. The molecules and mechanisms responsible for the assembly and positioning of these non-centrosomal microtubules have not been fully elucidated. Using a Nocodazole induced regrowth assay in invitro culture (MDCK) and in situ epithelial (cochlear Kolliker's) cell models we establish that the apico-basal array originates from the centrosome and that the non-centrosomal microtubule minus-end anchoring sites do not contribute significantly to their nucleation. Confocal and electron microscopy revealed that an extended radial array assembles with microtubule plus-ends targeting cadheren sites at adherens junctions and EB1 and CLIP-170 co-localising with beta-catenin and dynein clusters at the junction sites. The extended radial array is likely to be a vital intermediate step in the assembly process with cortical anchored dynein providing the mechanical force required for microtubule release, translocation and capture. Ultrastructural analyses of the apico-basal arrays in fully polarised MDCK and Kolliker's cells revealed microtubule minus-end association with the most apical adherens junction (Zonula adherens). We propose that a release and capture model involving both microtubule plus- and minus-end capture at adherens junctions is responsible for the generation of non-centrosomal apico-basal arrays in most centrosome containing polarised epithelial cells. PMID:19479825

  13. Kinesin-5 is a microtubule polymerase

    PubMed Central

    Chen, Yalei; Hancock, William O

    2015-01-01

    Kinesin-5 slides antiparallel microtubules during spindle assembly, and regulates the branching of growing axons. Besides the mechanical activities enabled by its tetrameric configuration, the specific motor properties of kinesin-5 that underlie its cellular function remain unclear. Here by engineering a stable kinesin-5 dimer and reconstituting microtubule dynamics in vitro, we demonstrate that kinesin-5 promotes microtubule polymerization by increasing the growth rate and decreasing the catastrophe frequency. Strikingly, microtubules growing in the presence of kinesin-5 have curved plus ends, suggesting that the motor stabilizes growing protofilaments. Single-molecule fluorescence experiments reveal that kinesin-5 remains bound to the plus ends of static microtubules for 7 s, and tracks growing microtubule plus ends in a manner dependent on its processivity. We propose that kinesin-5 pauses at microtubule plus ends and enhances polymerization by stabilizing longitudinal tubulin–tubulin interactions, and that these activities underlie the ability kinesin-5 to slide and stabilize microtubule bundles in cells. PMID:26437877

  14. 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

  15. Mechanistic Origin of Microtubule Dynamic Instability and Its Modulation by EB Proteins.

    PubMed

    Zhang, Rui; Alushin, Gregory M; Brown, Alan; Nogales, Eva

    2015-08-13

    Microtubule (MT) dynamic instability is driven by GTP hydrolysis and regulated by microtubule-associated proteins, including the plus-end tracking end-binding protein (EB) family. We report six cryo-electron microscopy (cryo-EM) structures of MTs, at 3.5 Å or better resolution, bound to GMPCPP, GTPγS, or GDP, either decorated with kinesin motor domain after polymerization or copolymerized with EB3. Subtle changes around the E-site nucleotide during hydrolysis trigger conformational changes in α-tubulin around an "anchor point," leading to global lattice rearrangements and strain generation. Unlike the extended lattice of the GMPCPP-MT, the EB3-bound GTPγS-MT has a compacted lattice that differs in lattice twist from that of the also compacted GDP-MT. These results and the observation that EB3 promotes rapid hydrolysis of GMPCPP suggest that EB proteins modulate structural transitions at growing MT ends by recognizing and promoting an intermediate state generated during GTP hydrolysis. Our findings explain both EBs end-tracking behavior and their effect on microtubule dynamics. PMID:26234155

  16. 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.

  17. 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

  18. Dynamics of assembly production flow

    NASA Astrophysics Data System (ADS)

    Ezaki, Takahiro; Yanagisawa, Daichi; Nishinari, Katsuhiro

    2015-06-01

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

  19. 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

  20. 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

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

    PubMed

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

    2015-01-01

    Noscapine is an orally administrable drug used worldwide for cough suppression and has recently been demonstrated to disrupt microtubule dynamics and possess anticancer activity. However, the molecular mechanisms regulating noscapine activity remain poorly defined. Here we demonstrate that cylindromatosis (CYLD), a microtubule-associated tumor suppressor protein, modulates the activity of noscapine both in cell lines and in primary cells of acute lymphoblastic leukemia (ALL). Flow cytometry and immunofluorescence microscopy reveal that CYLD increases the ability of noscapine to induce mitotic arrest and apoptosis. Examination of cellular microtubules as well as in vitro assembled microtubules shows that CYLD enhances the effect of noscapine on microtubule polymerization. Microtubule cosedimentation and fluorescence titration assays further reveal that CYLD interacts with microtubule outer surface and promotes noscapine binding to microtubules. These findings thus demonstrate CYLD as a critical regulator of noscapine activity and have important implications for ALL treatment. PMID:25897332

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

    PubMed

    Muratov, Alexander; Baulin, Vladimir A

    2015-12-01

    Directional growth caused by gravitropism and corresponding bending of plant cells has been explored since 19th century, however, many aspects of mechanisms underlying the perception of gravity at the molecular level are still not well known. Perception of gravity in root and shoot gravitropisms is usually attributed to gravisensitive cells, called statocytes, which exploit sedimentation of macroscopic and heavy organelles, amyloplasts, to sense the direction of gravity. Gravity stimulus is then transduced into distal elongation zone, which is several mm far from statocytes, where it causes stretching. It is suggested that gravity stimulus is conveyed by gradients in auxin flux. We propose a theoretical model that may explain how concentration gradients and/or stretching may indirectly affect the global orientation of cortical microtubules, attached to the cell membrane and induce their dynamic reorientation perpendicular to the gradients. In turn, oriented microtubule arrays direct the growth and orientation of cellulose microfibrils, forming part of the cell external skeleton and determine the shape of the cell. Reorientation of microtubules is also observed in reaction to light in phototropism and mechanical bending, thus suggesting universality of the proposed mechanism. PMID:26422460

  3. 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

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

    PubMed Central

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

    2015-01-01

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

  5. Distinct roles of doublecortin modulating the microtubule cytoskeleton

    PubMed Central

    Moores, Carolyn A; Perderiset, Mylène; Kappeler, Caroline; Kain, Susan; Drummond, Douglas; Perkins, Stephen J; Chelly, Jamel; Cross, Rob; Houdusse, Anne; Francis, Fiona

    2006-01-01

    Doublecortin is a neuronal microtubule-stabilising protein, mutations of which cause mental retardation and epilepsy in humans. How doublecortin influences microtubule dynamics, and thereby brain development, is unclear. We show here by video microscopy that purified doublecortin has no effect on the growth rate of microtubules. However, it is a potent anti-catastrophe factor that stabilises microtubules by linking adjacent protofilaments and counteracting their outward bending in depolymerising microtubules. We show that doublecortin-stabilised microtubules are substrates for kinesin translocase motors and for depolymerase kinesins. In addition, doublecortin does not itself oligomerise and does not bind to tubulin heterodimers but does nucleate microtubules. In cells, doublecortin is enriched at the distal ends of neuronal processes and our data raise the possibility that the function of doublecortin in neurons is to drive assembly and stabilisation of non-centrosomal microtubules in these doublecortin-enriched distal zones. These distinct properties combine to give doublecortin a unique function in microtubule regulation, a role that cannot be compensated for by other microtubule-stabilising proteins and nucleating factors. PMID:16957770

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

    PubMed Central

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

    2015-01-01

    The key step for faithful chromosome segregation during meiosis is kinetochore assembly. Defects in this process result in aneuploidy, leading to miscarriages, infertility and various birth defects. However, the roles of kinetochores in homologous chromosome segregation during meiosis are ill-defined. Here we found that Zwint-1 is required for homologous chromosome segregation during meiosis. Knockdown of Zwint-1 accelerated the first meiosis by abrogating the kinetochore recruitment of Mad2, leading to chromosome misalignment and a high incidence of aneuploidy. Although Zwint-1 knockdown did not affect Aurora C kinase activity, the meiotic defects following Zwint-1 knockdown were similar to those observed with ZM447439 treatment. Importantly, the chromosome misalignment following Aurora C kinase inhibition was not restored after removing the inhibitor in Zwint-1-knockdown oocytes, whereas the defect was rescued after the inhibitor washout in the control oocytes. These results suggest that Aurora C kinase-mediated correction of erroneous kinetochore-microtubule attachment is primarily regulated by Zwint-1. Our results provide the first evidence that Zwint-1 is required to correct erroneous kinetochore-microtubule attachment and regulate spindle checkpoint function during meiosis. PMID:26486467

  7. 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

  8. Experimental virus evolution reveals a role of plant microtubule dynamics and TORTIFOLIA1/SPIRAL2 in RNA trafficking.

    PubMed

    Peña, Eduardo José; Ferriol, Inmaculada; Sambade, Adrián; 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

  9. 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

  10. 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. PMID:26921917

  11. 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.

  12. 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

  13. Microtubule teardrop patterns

    NASA Astrophysics Data System (ADS)

    Okeyoshi, Kosuke; Kawamura, Ryuzo; Yoshida, Ryo; Osada, Yoshihito

    2015-03-01

    Several strategies for controlling microtubule patterns are developed because of the rigidity determined from the molecular structure and the geometrical structure. In contrast to the patterns in co-operation with motor proteins or associated proteins, microtubules have a huge potential for patterns via their intrinsic flexural rigidity. We discover that a microtubule teardrop pattern emerges via self-assembly under hydrodynamic flow from the parallel bundles without motor proteins. In the growth process, the bundles ultimately bend according to the critical bending curvature. Such protein pattern formation utilizing the intrinsic flexural rigidity will provide broad understandings of self-assembly of rigid rods, not only in biomolecules, but also in supramolecules.

  14. Bundling, sliding, and pulling microtubules in cells and in silico

    PubMed Central

    Howard, Jonathon; Tolić-Nørrelykke, Iva M.

    2007-01-01

    Microtubules and other proteins self-organize into complex dynamic structures such as the mitotic spindle, which separates the chromosomes during cell division. Much is known about the individual molecular players involved in assembly and positioning of the mitotic spindle, but how they act together to generate the often unexpected behavior of the whole microtubule system is not understood. Two recent papers use a combination of experimental (imaging) and theoretical (computer simulation) methods to explore the formation of bipolar linear microtubule arrays in fission yeast and the oscillatory movement of the mitotic spindle in the nematode worm. In the simulation approach, the rules for the interactions of the components (microtubules and microtubule-associated proteins) are specified and the evolution of the system is followed, with the aim of identifying the minimal set of components that can mimic the real system. The work on fission yeast concludes that bipolar microtubule structures can arise from self-organization of microtubules through nucleators, bundlers, and sliders, without a requirement for a special microtubule-organizing center. The work on the worm embryo suggests that both the positive feedback that drives oscillations and the centering force that limits their amplitude may arise from microtubule pulling forces. The systems approach exemplified by these papers should stimulate new experiments aimed at discovering the principles of cellular organization. PMID:19404456

  15. 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

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

    PubMed Central

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

    2011-01-01

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

  17. 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

  18. 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

  19. Patronin/Shot Cortical Foci Assemble the Noncentrosomal Microtubule Array that Specifies the Drosophila Anterior-Posterior Axis.

    PubMed

    Nashchekin, Dmitry; Fernandes, Artur Ribeiro; St Johnston, Daniel

    2016-07-11

    Noncentrosomal microtubules play an important role in polarizing differentiated cells, but little is known about how these microtubules are organized. Here we identify the spectraplakin, Short stop (Shot), as the cortical anchor for noncentrosomal microtubule organizing centers (ncMTOCs) in the Drosophila oocyte. Shot interacts with the cortex through its actin-binding domain and recruits the microtubule minus-end-binding protein, Patronin, to form cortical ncMTOCs. Shot/Patronin foci do not co-localize with γ-tubulin, suggesting that they do not nucleate new microtubules. Instead, they capture and stabilize existing microtubule minus ends, which then template new microtubule growth. Shot/Patronin foci are excluded from the oocyte posterior by the Par-1 polarity kinase to generate the polarized microtubule network that localizes axis determinants. Both proteins also accumulate apically in epithelial cells, where they are required for the formation of apical-basal microtubule arrays. Thus, Shot/Patronin ncMTOCs may provide a general mechanism for organizing noncentrosomal microtubules in differentiated cells. PMID:27404359

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

    PubMed

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

    1986-04-01

    This report describes the effects of 10 mM procaine on microtubule assembly and on DNA synthesis, as followed by [3H]colchicine binding assays and [3H]thymidine incorporation respectively, in fertilized Paracentrotus lividus eggs. In the absence of microtubule assembly inhibitors, about 25% of the total egg tubulin is submitted to two cycles of polymerization prior to the first cell division, this polymerization process precedes DNA synthesis. If the zygotes are treated with 10 mM procaine in the course of the cell cycle, tubulin polymerization is inhibited or microtubules are disassembled. DNA synthesis is inhibited when procaine treatment is performed 10 min, before the initiation of the S-period. However, when the drug is applied in the course of this synthetic period, the process is normally accomplished, but the next S-period becomes inhibited. Moreover, procaine treatment increases the cytoplasmic pH of the fertilized eggs by about 0.6 to 0.8 pH units. This pH increase precedes microtubule disassembly and inhibition of DNA synthesis. Washing out the drug induces a decrease of the intracellular pH which returns to about the same value as that of the fertilized egg controls. This pH change is then followed by the reinitiation of microtubule assembly, DNA synthesis and cell division. Our results show that the inhibition of both tubulin polymerization and DNA synthesis in fertilized eggs treated with 10 mM procaine, appears to be related to the drug-induced increase in cytoplasmic pH. PMID:3709552

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

    PubMed

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

    1985-01-01

    An increase in the amount of cytoplasmic filamentous structures (cytoplasmic matrix and aster) which were recovered after hexylene glycol/Triton X-100 treatment of sea urchin eggs (Paracentrotus lividus) activated by 0.2-2.5 mM procaine was observed. At higher activator concentrations, an opposite effect was observed and formation of these cytoplasmic structures was inhibited in the presence of 10 mM procaine. This inhibitory effect was reversed by diluting the drug in the incubation medium. DNase I inhibition assays on egg homogenates which were performed at different time points of the activation process, show that the same amount of actin was induced to polymerize in eggs activated either by 2.5 or 10 mM procaine. However, colchicine-binding assays on the 100 000 g particulate fractions of these homogenates show that in eggs activated by 10 mM procaine, in contrast to those activated by 2.5 mM, tubulin polymerization was inhibited and microtubules were disassembled. These results show that the dual effect of procaine in the organization of the egg cytoskeleton appears to be related to its effect on the state of tubulin. PMID:4038386

  2. 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

  3. Dynamic Pathways for Viral Capsid Assembly

    PubMed Central

    Hagan, Michael F.; Chandler, David

    2006-01-01

    We develop a class of models with which we simulate the assembly of particles into T1 capsidlike objects using Newtonian dynamics. By simulating assembly for many different values of system parameters, we vary the forces that drive assembly. For some ranges of parameters, assembly is facile; for others, assembly is dynamically frustrated by kinetic traps corresponding to malformed or incompletely formed capsids. Our simulations sample many independent trajectories at various capsomer concentrations, allowing for statistically meaningful conclusions. Depending on subunit (i.e., capsomer) geometries, successful assembly proceeds by several mechanisms involving binding of intermediates of various sizes. We discuss the relationship between these mechanisms and experimental evaluations of capsid assembly processes. PMID:16565055

  4. Dynamic pathways for viral capsid assembly

    SciTech Connect

    Hagan, Michael F.; Chandler, David

    2006-02-09

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

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

    PubMed Central

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

    2016-01-01

    Spindle assembly and function require precise control of microtubule nucleation and dynamics. The chromatin-driven spindle assembly pathway exerts such control locally in the vicinity of chromosomes. One of the key targets of this pathway is TPX2. The molecular mechanism of how TPX2 stimulates microtubule nucleation is not understood. Using microscopy-based dynamic in vitro reconstitution assays with purified proteins, we find that human TPX2 directly stabilises growing microtubule ends and stimulates microtubule nucleation by stabilising early microtubule nucleation intermediates. Human microtubule polymerase chTOG (XMAP215/Msps/Stu2p/Dis1/Alp14 homolog) only weakly promotes nucleation, but acts synergistically with TPX2. Hence, a combination of distinct and complementary activities is sufficient for efficient microtubule formation in vitro. Importins control the efficiency of the microtubule nucleation by selectively blocking TPX2’s interaction with microtubule nucleation intermediates. This in vitro reconstitution reveals the molecular mechanism of regulated microtubule formation by a minimal nucleation module essential for chromatin-dependent microtubule nucleation in cells. PMID:26414402

  6. 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

  7. 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

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

    PubMed Central

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

    2014-01-01

    The microtubule-associated phosphoprotein tau regulates microtubule dynamics and is involved in neurodegenerative diseases collectively called tauopathies. It is generally believed that the vast majority of tau molecules decorate axonal microtubules, thereby stabilizing them. However, it is an open question how tau can regulate microtubule dynamics without impeding microtubule-dependent transport and how tau is also available for interactions other than those with microtubules. Here we address this apparent paradox by fast single-molecule tracking of tau in living neurons and Monte Carlo simulations of tau dynamics. We find that tau dwells on a single microtubule for an unexpectedly short time of ∼40 ms before it hops to the next. This dwell time is 100-fold shorter than previously reported by ensemble measurements. Furthermore, we observed by quantitative imaging using fluorescence decay after photoactivation recordings of photoactivatable GFP–tagged tubulin that, despite this rapid dynamics, tau is capable of regulating the tubulin–microtubule balance. This indicates that tau's dwell time on microtubules is sufficiently long to influence the lifetime of a tubulin subunit in a GTP cap. Our data imply a novel kiss-and-hop mechanism by which tau promotes neuronal microtubule assembly. The rapid kiss-and-hop interaction explains why tau, although binding to microtubules, does not interfere with axonal transport. PMID:25165145

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

    PubMed Central

    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 dynamics1,2. 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 polymerization1. Many studies of EB1 behavior within growth cones have used time-consuming and biased computer-assisted, hand-tracking methods to analyze individual MTs1-3. Our approach is to quantify global parameters of MT dynamics using the software package, plusTipTracker4, following the acquisition of high-resolution, live images of tagged EB1 in cultured embryonic growth cones5. 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 types6-8. PMID:25225829

  10. 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

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

    NASA Astrophysics Data System (ADS)

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

    2005-11-01

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

  12. Micropatterning microtubules.

    PubMed

    Portran, Didier

    2014-01-01

    The following protocol describes a method to control the orientation and polarity of polymerizing microtubules (MTs). Reconstitution of specific geometries of dynamic MT networks is achieved using a ultraviolet (UV) micropatterning technique in combination with stabilized MT microseeds. The process is described in three main parts. First, the surface is passivated to avoid the non-specific absorption of proteins, using different polyethylene glycol (PEG)-based surface treatment. Second, specific adhesive surfaces (the micropatterns) are imprinted through a photomask using deep UVs. Lastly, MT microseeds are adhered to the micropatterns followed by MT polymerization. PMID:24484656

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

    PubMed Central

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

    2009-01-01

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

  14. 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

  15. 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

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

    PubMed Central

    2015-01-01

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

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

    PubMed

    Savoian, Matthew S

    2015-07-01

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

  18. Atomic water channel controlling remarkable properties of a single brain microtubule: correlating single protein to its supramolecular assembly.

    PubMed

    Sahu, Satyajit; Ghosh, Subrata; Ghosh, Batu; Aswani, Krishna; Hirata, Kazuto; Fujita, Daisuke; Bandyopadhyay, Anirban

    2013-09-15

    Microtubule nanotubes are found in every living eukaryotic cells; these are formed by reversible polymerization of the tubulin protein, and their hollow fibers are filled with uniquely arranged water molecules. Here we measure single tubulin molecule and single brain-neuron extracted microtubule nanowire with and without water channel inside to unravel their unique electronic and optical properties for the first time. We demonstrate that the energy levels of a single tubulin protein and single microtubule made of 40,000 tubulin dimers are identical unlike conventional materials. Moreover, the transmitted ac power and the transient fluorescence decay (single photon count) are independent of the microtubule length. Even more remarkable is the fact that the microtubule nanowire is more conducting than a single protein molecule that constitutes the nanowire. Microtubule's vibrational peaks condense to a single mode that controls the emergence of size independent electronic/optical properties, and automated noise alleviation, which disappear when the atomic water core is released from the inner cylinder. We have carried out several tricky state-of-the-art experiments and identified the electromagnetic resonance peaks of single microtubule reliably. The resonant vibrations established that the condensation of energy levels and periodic oscillation of unique energy fringes on the microtubule surface, emerge as the atomic water core resonantly integrates all proteins around it such that the nanotube irrespective of its size functions like a single protein molecule. Thus, a monomolecular water channel residing inside the protein-cylinder displays an unprecedented control in governing the tantalizing electronic and optical properties of microtubule. PMID:23567633

  19. 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

  20. Mean-field study of the role of lateral cracks in microtubule dynamics

    NASA Astrophysics Data System (ADS)

    Margolin, Gennady; Goodson, Holly V.; Alber, Mark S.

    2011-04-01

    A link between dimer-scale processes and microtubule (MT) dynamics at macroscale is studied by comparing simulations obtained using computational dimer-scale model with its mean-field approximation. The novelty of the mean-field model (MFM) is in its explicit representation of inter-protofilament cracks, as well as in the direct incorporation of the dimer-level kinetics. Due to inclusion of both longitudinal and lateral dimer interactions, the MFM is two dimensional, in contrast to previous theoretical models of MTs. It is the first analytical model that predicts and quantifies crucial features of MT dynamics such as (i) existence of a minimal soluble tubulin concentration needed for the polymerization (with concentration represented as a function of model parameters), (ii) existence of steady-state growth and shortening phases (given with their respective velocities), and (iii) existence of an unstable pause state near zero velocity. In addition, the size of the GTP cap of a growing MT is estimated. Theoretical predictions are shown to be in good agreement with the numerical simulations.

  1. p120-catenin regulates microtubule dynamics and cell migration in a cadherin-independent manner.

    PubMed

    Ichii, Tetsuo; Takeichi, Masatoshi

    2007-07-01

    p120-catenin (p120) has been shown to be essential for cadherin stability. Here, we show that p120 is capable of regulating microtubule (MT) dynamics in a cadherin-independent manner. When p120 was depleted in cadherin-deficient Neuro-2a (N2a) cells, MT stability was reduced, as assessed by the nocodazole sensitivity of MTs. On the contrary, over-expression of p120 caused MTs to become resistant to nocodazole. Time-lapse recording of GFP-tagged EB1, a protein which binds the growing plus-ends of MTs, introduced into these cells demonstrated that the plus ends underwent more frequent catastrophe in p120-depleted cells. In addition, p120 knockdown up-regulated the motility of isolated cells, whereas it down-regulated the directional migration of cells from wound edges; and these migratory behaviors of cells were mimicked by nocodazole-induced MT depolymerization. These results suggest that p120 has the ability to regulate MT dynamics and that this activity, in turn, affects cell motility independently of the cadherin adhesion system. PMID:17584295

  2. 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

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

    PubMed

    Shahbazi, Marta N; Megias, Diego; Epifano, Carolina; Akhmanova, Anna; Gundersen, Gregg G; Fuchs, Elaine; Perez-Moreno, Mirna

    2013-12-23

    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

  4. Fission yeast MOZART1/Mzt1 is an essential γ-tubulin complex component required for complex recruitment to the microtubule organizing center, but not its assembly

    PubMed Central

    Masuda, Hirohisa; Mori, Risa; Yukawa, Masashi; Toda, Takashi

    2013-01-01

    γ-Tubulin plays a universal role in microtubule nucleation from microtubule organizing centers (MTOCs) such as the animal centrosome and fungal spindle pole body (SPB). γ-Tubulin functions as a multiprotein complex called the γ-tubulin complex (γ-TuC), consisting of GCP1–6 (GCP1 is γ-tubulin). In fungi and flies, it has been shown that GCP1–3 are core components, as they are indispensable for γ-TuC complex assembly and cell division, whereas the other three GCPs are not. Recently a novel conserved component, MOZART1, was identified in humans and plants, but its precise functions remain to be determined. In this paper, we characterize the fission yeast homologue Mzt1, showing that it is essential for cell viability. Mzt1 is present in approximately equal stoichiometry with Alp4/GCP2 and localizes to all the MTOCs, including the SPB and interphase and equatorial MTOCs. Temperature-sensitive mzt1 mutants display varying degrees of compromised microtubule organization, exhibiting multiple defects during both interphase and mitosis. Mzt1 is required for γ-TuC recruitment, but not sufficient to localize to the SPB, which depends on γ-TuC integrity. Intriguingly, the core γ-TuC assembles in the absence of Mzt1. Mzt1 therefore plays a unique role within the γ-TuC components in attachment of this complex to the major MTOC site. PMID:23885124

  5. 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

  6. Glutamylation on α-Tubulin Is Not Essential but Affects the Assembly and Functions of a Subset of Microtubules in Tetrahymena thermophila▿ †

    PubMed Central

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

    2008-01-01

    Tubulin undergoes glutamylation, a conserved posttranslational modification of poorly understood function. We show here that in the ciliate Tetrahymena, most of the microtubule arrays contain glutamylated tubulin. However, the length of the polyglutamyl side chain is spatially regulated, with the longest side chains present on ciliary and basal body microtubules. We focused our efforts on the function of glutamylation on the α-tubulin subunit. By site-directed mutagenesis, we show that all six glutamates of the C-terminal tail domain of α-tubulin that provide potential sites for glutamylation are not essential but are needed for normal rates of cell multiplication and cilium-based functions (phagocytosis and cell motility). By comparative phylogeny and biochemical assays, we identify two conserved tubulin tyrosine ligase (TTL) domain proteins, Ttll1p and Ttll9p, as α-tubulin-preferring glutamyl ligase enzymes. In an in vitro microtubule glutamylation assay, Ttll1p showed a chain-initiating activity while Ttll9p had primarily a chain-elongating activity. GFP-Ttll1p localized mainly to basal bodies, while GFP-Ttll9p localized to cilia. Disruption of the TTLL1 and TTLL9 genes decreased the rates of cell multiplication and phagocytosis. Cells lacking both genes had fewer cortical microtubules and showed defects in the maturation of basal bodies. We conclude that glutamylation on α-tubulin is not essential but is required for efficiency of assembly and function of a subset of microtubule-based organelles. Furthermore, the spatial restriction of modifying enzymes appears to be a major mechanism that drives differential glutamylation at the subcellular level. PMID:18586949

  7. Morlin, an inhibitor of cortical microtubule dynamics and cellulose synthase movement

    PubMed Central

    DeBolt, Seth; Gutierrez, Ryan; Ehrhardt, David W.; Melo, Carlos V.; Ross, Loretta; Cutler, Sean R.; Somerville, Christopher; Bonetta, Dario

    2007-01-01

    Morlin (7-ethoxy-4-methyl chromen-2-one) was discovered in a screen of 20,000 compounds for small molecules that cause altered cell morphology resulting in swollen root phenotype in Arabidopsis. Live-cell imaging of fluorescently labeled cellulose synthase (CESA) and microtubules showed that morlin acts on the cortical microtubules and alters the movement of CESA. Morlin caused a novel syndrome of cytoskeletal defects, characterized by cortical array reorientation and compromised rates of both microtubule elongation and shrinking. Formation of shorter and more bundled microtubules and detachment from the cell membrane resulted when GFP::MAP4-MBP was used to visualize microtubules during morlin treatment. Cytoskeletal effects were accompanied by a reduction in the velocity and redistribution of CESA complexes labeled with YFP::CESA6 at the cell cortex. Morlin caused no inhibition of mouse myoblast, bacterial or fungal cell proliferation at concentrations that inhibit plant cell growth. By contrast, morlin stimulated microtubule disassembly in cultured hippocampal neurons but had no significant effect on cell viability. Thus, morlin appears to be a useful new probe of the mechanisms that regulate microtubule cortical array organization and its functional interaction with CESA. PMID:17389408

  8. Fatostatin Inhibits Cancer Cell Proliferation by Affecting Mitotic Microtubule Spindle Assembly and Cell Division.

    PubMed

    Gholkar, Ankur A; Cheung, Keith; Williams, Kevin J; Lo, Yu-Chen; Hamideh, Shadia A; Nnebe, Chelsea; Khuu, Cindy; Bensinger, Steven J; Torres, Jorge Z

    2016-08-12

    The sterol regulatory element-binding protein (SREBP) transcription factors have become attractive targets for pharmacological inhibition in the treatment of metabolic diseases and cancer. SREBPs are critical for the production and metabolism of lipids and cholesterol, which are essential for cellular homeostasis and cell proliferation. Fatostatin was recently discovered as a specific inhibitor of SREBP cleavage-activating protein (SCAP), which is required for SREBP activation. Fatostatin possesses antitumor properties including the inhibition of cancer cell proliferation, invasion, and migration, and it arrests cancer cells in G2/M phase. Although Fatostatin has been viewed as an antitumor agent due to its inhibition of SREBP and its effect on lipid metabolism, we show that Fatostatin's anticancer properties can also be attributed to its inhibition of cell division. We analyzed the effect of SREBP activity inhibitors including Fatostatin, PF-429242, and Betulin on the cell cycle and determined that only Fatostatin possessed antimitotic properties. Fatostatin inhibited tubulin polymerization, arrested cells in mitosis, activated the spindle assembly checkpoint, and triggered mitotic catastrophe and reduced cell viability. Thus Fatostatin's ability to inhibit SREBP activity and cell division could prove beneficial in treating aggressive types of cancers such as glioblastomas that have elevated lipid metabolism and fast proliferation rates and often develop resistance to current anticancer therapies. PMID:27378817

  9. 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

  10. Dynamic Models for Templated Viral Capsid Assembly

    NASA Astrophysics Data System (ADS)

    Hagan, Michael

    2008-03-01

    The replication of many viruses with single-stranded genomes requires the simultaneous assembly of an ordered protein shell, or capsid, and encapsidation of the genome. In this talk, I will present coarse-grained computational and theoretical models that describe the assembly of viral capsid proteins around interior cores, such as polymers and rigid spheres. These models are motivated by two recently developed experimental model systems in which viral proteins dynamically encapsidate inorganic nanoparticles and polyelectrolytes. Model predictions suggest that some forms of cooperative interactions between subunits and cores can dramatically enhance rates and robustness of assembly, as compared to the spontaneous assembly of subunits into empty capsids. For large core-subunit interactions, subunits adsorb onto a core en masse in a disordered manner, and then undergo a cooperative rearrangement into an ordered capsid structure. These assembly pathways are unlike any seen for empty capsids formation. While model predictions suggest that cooperative interactions between disparate assembling components can overcome some limitations of spontaneous assembly, the complexity of multicomponent assembly introduces new forms of kinetic traps that can frustrate assembly, and hence introduces new limitations. These findings have implications for a mechanism in which viruses use interactions between proteins and genomic molecules to promote and control assembly, and thereby control the replication process.

  11. Spindle microtubule dysfunction and cancer predisposition

    PubMed Central

    Stumpff, Jason; Ghule, Prachi N.; Shimamura, Akiko; Stein, Janet L.; Greenblatt, Marc

    2014-01-01

    Chromosome segregation and spindle microtubule dynamics are strictly coordinated during cell division in order to preserve genomic integrity. Alterations in the genome that affect microtubule stability and spindle assembly during mitosis may contribute to genomic instability and cancer predisposition, but directly testing this potential link poses a significant challenge. Germ-line mutations in tumor suppressor genes that predispose patients to cancer and alter spindle microtubule dynamics offer unique opportunities to investigate the relationship between spindle dysfunction and carcinogenesis. Mutations in two such tumor suppressors, adenomatous polyposis coli (APC) and Shwachman-Bodian-Diamond syndrome (SBDS), affect multifunctional proteins that have been well characterized for their roles in Wnt signaling and interphase ribosome assembly, respectively. Less understood, however, is how their shared involvement in stabilizing the microtubules that comprise the mitotic spindle contributes to cancer predisposition. Here, we briefly discuss the potential for mutations in APC and SBDS as informative tools for studying the impact of mitotic spindle dysfunction on cellular transformation. PMID:24905602

  12. CLASP2 Has Two Distinct TOG Domains That Contribute Differently to Microtubule Dynamics.

    PubMed

    Maki, Takahisa; Grimaldi, Ashley D; Fuchigami, Sotaro; Kaverina, Irina; Hayashi, Ikuko

    2015-07-17

    CLIP-associated proteins CLASPs are mammalian microtubule (MT) plus-end tracking proteins (+TIPs) that promote MT rescue in vivo. Their plus-end localization is dependent on other +TIPs, EB1 and CLIP-170, but in the leading edge of the cell, CLASPs display lattice-binding activity. MT association of CLASPs is suggested to be regulated by multiple TOG (tumor overexpressed gene) domains and by the serine-arginine (SR)-rich region, which contains binding sites for EB1. Here, we report the crystal structures of the two TOG domains of CLASP2. Both domains consist of six HEAT repeats, which are similar to the canonical paddle-like tubulin-binding TOG domains, but have arched conformations. The degrees and directions of curvature are different between the two TOG domains, implying that they have distinct roles in MT binding. Using biochemical, molecular modeling and cell biological analyses, we have investigated the interactions between the TOG domains and αβ-tubulin and found that each domain associates differently with αβ-tubulin. Our findings suggest that, by varying the degrees of domain curvature, the TOG domains may distinguish the structural conformation of the tubulin dimer, discriminate between different states of MT dynamic instability and thereby function differentially as stabilizers of MTs. PMID:26003921

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

    PubMed

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

    2016-04-01

    Kinesin motor proteins play a fundamental role for normal neuronal development by controlling intracellular cargo transport and microtubule (MT) cytoskeleton organization. Regulating kinesin activity is important to ensure their proper functioning, and their misregulation often leads to severe human neurological disorders. Homozygous nonsense mutations in kinesin-binding protein (KBP)/KIAA1279 cause the neurological disorder Goldberg-Shprintzen syndrome (GOSHS), which is characterized by intellectual disability, microcephaly, and axonal neuropathy. Here, we show that KBP regulates kinesin activity by interacting with the motor domains of a specific subset of kinesins to prevent their association with the MT cytoskeleton. The KBP-interacting kinesins include cargo-transporting motors such as kinesin-3/KIF1A and MT-depolymerizing motor kinesin-8/KIF18A. We found that KBP blocks KIF1A/UNC-104-mediated synaptic vesicle transport in cultured hippocampal neurons and in C. elegans PVD sensory neurons. In contrast, depletion of KBP results in the accumulation of KIF1A motors and synaptic vesicles in the axonal growth cone. We also show that KBP regulates neuronal MT dynamics by controlling KIF18A activity. Our data suggest that KBP functions as a kinesin inhibitor that modulates MT-based cargo motility and depolymerizing activity of a subset of kinesin motors. We propose that misregulation of KBP-controlled kinesin motors may represent the underlying molecular mechanism that contributes to the neuropathological defects observed in GOSHS patients. PMID:26948876

  14. 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. PMID:26604147

  15. 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

  16. 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.

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

    PubMed Central

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

    2016-01-01

    BRCA1 (breast cancer type 1 susceptibility protein) is a multifunctional tumor suppressor involved in DNA damage response, DNA repair, chromatin regulation, and mitotic chromosome segregation. Although the nuclear functions of BRCA1 have been investigated in detail, its role during mitosis is little understood. It is clear, however, that loss of BRCA1 in human cancer cells leads to chromosomal instability (CIN), which is defined as a perpetual gain or loss of whole chromosomes during mitosis. Moreover, our recent work has revealed that the mitotic function of BRCA1 depends on its phosphorylation by the tumor-suppressor kinase Chk2 (checkpoint kinase 2) and that this regulation is required to ensure normal microtubule plus end assembly rates within mitotic spindles. Intriguingly, loss of the positive regulation of BRCA1 leads to increased oncogenic Aurora-A activity, which acts as a mediator for abnormal mitotic microtubule assembly resulting in chromosome missegregation and CIN. However, how the CHK2–BRCA1 tumor suppressor axis restrains oncogenic Aurora-A during mitosis to ensure karyotype stability remained an open question. Here we uncover a dual molecular mechanism by which the CHK2–BRCA1 axis restrains oncogenic Aurora-A activity during mitosis and identify BRCA1 itself as a target for Aurora-A relevant for CIN. In fact, Chk2-mediated phosphorylation of BRCA1 is required to recruit the PP6C–SAPS3 phosphatase, which acts as a T-loop phosphatase inhibiting Aurora-A bound to BRCA1. Consequently, loss of CHK2 or PP6C-SAPS3 promotes Aurora-A activity associated with BRCA1 in mitosis. Aurora-A, in turn, then phosphorylates BRCA1 itself, thereby inhibiting the mitotic function of BRCA1 and promoting mitotic microtubule assembly, chromosome missegregation, and CIN. PMID:26831064

  18. 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

  19. Thirty years of search and capture: The complex simplicity of mitotic spindle assembly

    PubMed Central

    2015-01-01

    Cell division is enacted by a microtubule-based, self-assembling macromolecular machine known as the mitotic spindle. In 1986, Kirschner and Mitchison proposed that by undergoing dynamic cycles of growth and disassembly, microtubules search for chromosomes. Capture of microtubules by the kinetochores progressively connects chromosomes to the bipolar spindle. 30 years later, “search and capture” remains the cornerstone of spindle assembly. However, a variety of facilitating mechanisms such as regulation of microtubule dynamics by diffusible gradients, spatially selective motor activities, and adaptive changes in chromosome architecture have been discovered. We discuss how these mechanisms ensure that the spindle assembles rapidly and with a minimal number of errors. PMID:26668328

  20. Assembly and dynamics of synthetic cilia

    NASA Astrophysics Data System (ADS)

    Sanchez, Tim

    2012-02-01

    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 to produce ciliary beating patterns remains unknown. We describe a novel approach towards 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. We find that simple mixtures of microtubules, kinesin clusters, and a bundling agent produce spontaneous oscillations in MT bundles, suggesting that self-organized beating may be a generic feature of internally driven bundles. Furthermore, bundles in close proximity spontaneously coordinate their beating to generate metachronal traveling waves, reminiscent of the waves seen in ciliary fields. These findings and future refinements of the system can potentially provide insights into general design principles required for engineering synthetic cilia as well as understanding the biological analogues.

  1. 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

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

    PubMed Central

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

    2015-01-01

    The cytoskeleton plays a critical role in regulating mitochondria distribution. Similar to axonal mitochondria, the fission yeast mitochondria are distributed by the microtubule cytoskeleton, but this is regulated by a motor-independent mechanism depending on the microtubule associated protein mmb1p as the absence of mmb1p causes mitochondria aggregation. In this study, using a series of chimeric proteins to control the subcellular localization and motility of mitochondria, we show that a chimeric molecule containing a microtubule binding domain and the mitochondria outer membrane protein tom22p can restore the normal interconnected mitochondria network in mmb1-deletion (mmb1∆) cells. In contrast, increasing the motility of mitochondria by using a chimeric molecule containing a kinesin motor domain and tom22p cannot rescue mitochondria aggregation defects in mmb1∆ cells. Intriguingly a chimeric molecule carrying an actin binding domain and tom22p results in mitochondria associated with actin filaments at the actomyosin ring during mitosis, leading to cytokinesis defects. These findings suggest that the passive motor-independent microtubule-based mechanism is the major contributor to mitochondria distribution in wild type fission yeast cells. Hence, we establish that attachment to microtubules, but not kinesin-dependent movement and the actin cytoskeleton, is required and crucial for proper mitochondria distribution in fission yeast. PMID:26046468

  3. 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.

  4. Regulation of Microtubule Dynamics through Phosphorylation on Stathmin by Epstein-Barr Virus Kinase BGLF4*

    PubMed Central

    Chen, Po-Wen; Lin, Sue-Jane; Tsai, Shu-Chun; Lin, Jiun-Han; Chen, Mei-Ru; Wang, Jiin-Tarng; Lee, Chung-Pei; Tsai, Ching-Hwa

    2010-01-01

    Stathmin is an important microtubule (MT)-destabilizing protein, and its activity is differently attenuated by phosphorylation at one or more of its four phosphorylatable serine residues (Ser-16, Ser-25, Ser-38, and Ser-63). This phosphorylation of stathmin plays important roles in mitotic spindle formation. We observed increasing levels of phosphorylated stathmin in Epstein-Barr virus (EBV)-harboring lymphoblastoid cell lines (LCLs) and nasopharyngeal carcinoma (NPC) cell lines during the EBV lytic cycle. These suggest that EBV lytic products may be involved in the regulation of stathmin phosphorylation. BGLF4 is an EBV-encoded kinase and has similar kinase activity to cdc2, an important kinase that phosphorylates serine residues 25 and 38 of stathmin during mitosis. Using an siRNA approach, we demonstrated that BGLF4 contributes to the phosphorylation of stathmin in EBV-harboring NPC. Moreover, we confirmed that BGLF4 interacts with and phosphorylates stathmin using an in vitro kinase assay and an in vivo two-dimensional electrophoresis assay. Interestingly, unlike cdc2, BGLF4 was shown to phosphorylate non-proline directed serine residues of stathmin (Ser-16) and it mediated phosphorylation of stathmin predominantly at serines 16, 25, and 38, indicating that BGLF4 can down-regulate the activity of stathmin. Finally, we demonstrated that the pattern of MT organization was changed in BGLF4-expressing cells, possibly through phosphorylation of stathmin. In conclusion, we have shown that a viral Ser/Thr kinase can directly modulate the activity of stathmin and this contributes to alteration of cellular MT dynamics and then may modulate the associated cellular processes. PMID:20110360

  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. PMID

  6. Multispecies population dynamics of prebiotic compositional assemblies.

    PubMed

    Markovitch, Omer; Lancet, Doron

    2014-09-21

    Present life portrays a two-tier phenomenology: molecules compose supramolecular structures, such as cells or organisms, which in turn portray population behaviors, including selection, evolution and ecological dynamics. Prebiotic models have often focused on evolution in populations of self-replicating molecules, without explicitly invoking the intermediate molecular-to-supramolecular transition. Here, we explore a prebiotic model that allows one to relate parameters of chemical interaction networks within molecular assemblies to emergent population dynamics. We use the graded autocatalysis replication domain (GARD) model, which simulates the network dynamics within amphiphile-containing molecular assemblies, and exhibits quasi-stationary compositional states termed compotype species. These grow by catalyzed accretion, divide and propagate their compositional information to progeny in a replication-like manner. The model allows us to ask how molecular network parameters influence assembly evolution and population dynamics parameters. In 1000 computer simulations, each embodying different parameter set of the global chemical interaction network parameters, we observed a wide range of behaviors. These were analyzed by a multi species logistic model often used for analyzing population ecology (r-K or Lotka-Volterra competition model). We found that compotypes with a larger intrinsic molecular repertoire show a higher intrinsic growth (r) and lower carrying capacity (K), as well as lower replication fidelity. This supports a prebiotic scenario initiated by fast-replicating assemblies with a high molecular diversity, evolving into more faithful replicators with narrower molecular repertoires. PMID:24831416

  7. 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

  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. 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

  10. Functional coupling of microtubules to membranes - implications for membrane structure and dynamics.

    PubMed

    Stephens, David J

    2012-06-15

    The microtubule network dictates much of the spatial patterning of the cytoplasm, and the coupling of microtubules to membranes controls the structure and positioning of organelles and directs membrane trafficking between them. The connection between membranes and the microtubule cytoskeleton, and the way in which organelles are shaped and moved by interactions with the cytoskeleton, have been studied intensively in recent years. In particular, recent work has expanded our thinking of this topic to include the mechanisms by which membranes are shaped and how cargo is selected for trafficking as a result of coupling to the cytoskeleton. In this Commentary, I will discuss the molecular basis for membrane-motor coupling and the physiological outcomes of this coupling, including the way in which microtubule-based motors affect membrane structure, cargo sorting and vectorial trafficking between organelles. Whereas many core concepts of these processes are now well understood, key questions remain about how the coupling of motors to membranes is established and controlled, about the regulation of cargo and/or motor loading and about the control of directionality. PMID:22736043

  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. Dynamic Assembly of Magnetic Colloidal Vortices.

    PubMed

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

    2016-05-24

    Magnetic colloids in external time-dependent fields are subject to complex induced many-body interactions governing their self-assembly into a variety of equilibrium and out-of-equilibrium structures such as chains, networks, suspended membranes, and colloidal foams. Here, we report experiments, simulations, and theory probing the dynamic assembly of superparamagnetic colloids in precessing external magnetic fields. Within a range of field frequencies, we observe dynamic large-scale structures such as ordered phases composed of precessing chains, ribbons, and rotating fluidic vortices. We show that the structure formation is inherently coupled to the buildup of torque, which originates from internal relaxation of induced dipoles and from transient correlations among the particles as a result of short-lived chain formation. We discuss in detail the physical properties of the vortex phase and demonstrate its potential in particle-coating applications. PMID:27128501

  13. The dynamics of nacre self-assembly

    PubMed Central

    Cartwright, Julyan H.E; Checa, Antonio G

    2006-01-01

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

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

    PubMed

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

    2016-05-23

    Centrioles are fundamental and evolutionarily conserved microtubule-based organelles whose assembly is characterized by microtubule growth rates that are orders of magnitude slower than those of cytoplasmic microtubules. Several centriolar proteins can interact with tubulin or microtubules, but how they ensure the exceptionally slow growth of centriolar microtubules has remained mysterious. Here, we bring together crystallographic, biophysical, and reconstitution assays to demonstrate that the human centriolar protein CPAP (SAS-4 in worms and flies) binds and "caps" microtubule plus ends by associating with a site of β-tubulin engaged in longitudinal tubulin-tubulin interactions. Strikingly, we uncover that CPAP activity dampens microtubule growth and stabilizes microtubules by inhibiting catastrophes and promoting rescues. We further establish that the capping function of CPAP is important to limit growth of centriolar microtubules in cells. Our results suggest that CPAP acts as a molecular lid that ensures slow assembly of centriolar microtubules and, thereby, contributes to organelle length control. PMID:27219064

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

    NASA Astrophysics Data System (ADS)

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

    2014-12-01

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

  16. Adenomatous polyposis coli mutants dominantly activate Hsf1-dependent cell stress pathways through inhibition of microtubule dynamics

    PubMed Central

    Davies, Alexander E.; Kortright, Kaitlyn; Kaplan, Kenneth B.

    2015-01-01

    Cancer cells up-regulate cell stress pathways, including the protein chaperone Hsp90. Increases in Hsp90 are believed “buffer” mutant protein activities necessary for cancer phenotypes. Activation of the cell stress pathway also alters the transcriptional landscape of cells in ways that are critical for cancer progression. However, it is unclear when and how the cell stress pathway is de-regulated during cancer progression. Here we report that mutations in adenomatous polyposis coli (APC) found in colorectal cancer activate cell stress pathways in mouse intestinal crypt cells, prior to loss of heterozygosity at APC or to the appearance of canonical intestinal cancer markers. Hsp90 levels are elevated in normal APC heterozygote crypt cells and further elevated in non-cancer cells adjacent to dysplasias, suggesting that the Hsp90 stress pathway marks the “cancer-field” effect. Expression of mutant APC in normal human epithelial cells is sufficient to activate a cell stress pathway via perturbations in microtubule dynamics. Inhibition of microtubule dynamics is sufficient to activate an Hsf1-dependent increase in gene transcription and protein levels. We suggest that the early activation of this Hsf1 dependent cell stress pathway by mono-allelic mutations in APC can affect cell programming in a way that contributes to cancer onset. PMID:26320184

  17. Actin–microtubule coordination at growing microtubule ends

    PubMed Central

    López, Magdalena Preciado; Huber, Florian; Grigoriev, Ilya; Steinmetz, Michel O.; Akhmanova, Anna; Koenderink, Gijsje H.; Dogterom, Marileen

    2014-01-01

    To power dynamic processes in cells, the actin and microtubule cytoskeletons organize into complex structures. Although it is known that cytoskeletal coordination is vital for cell function, the mechanisms by which cross-linking proteins coordinate actin and microtubule activities remain poorly understood. In particular, it is unknown how the distinct mechanical properties of different actin architectures modulate the outcome of actin–microtubule interactions. To address this question, we engineered the protein TipAct, which links growing microtubule ends via end-binding proteins to actin filaments. We show that growing microtubules can be captured and guided by stiff actin bundles, leading to global actin–microtubule alignment. Conversely, growing microtubule ends can transport, stretch and bundle individual actin filaments, thereby globally defining actin filament organization. Our results provide a physical basis to understand actin–microtubule cross-talk, and reveal that a simple cross-linker can enable a mechanical feedback between actin and microtubule organization that is relevant to diverse biological contexts. PMID:25159196

  18. 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.

  19. Isolation and characterization of polybrominated diphenyl ethers as inhibitors of microtubule assembly from the marine sponge Phyllospongia dendyi collected at Palau.

    PubMed

    Liu, Hongwei; Namikoshi, Michio; Meguro, Shiori; Nagai, Hiroshi; Kobayashi, Hisayoshi; Yao, Xinsheng

    2004-03-01

    Two new polybrominated diphenyl ethers (1 and 2) were isolated by bioassay-guided separations together with nine known compounds (3-11) from the marine sponge Phyllospongia dendyi collected from Palau. The structures were assigned on the basis of their spectral data. Compounds 3, 7, and 10 showed inhibitory activities to the assembly of microtubule proteins (IC(50): 29.6, 33.5, and 20.9 microM, respectively) and to the meiotic maturation of starfish oocytes (IC(50): 3.6, 4.2, and 4.2 microM, respectively), while 1, 2, 4-6, 8, 9, and 11 were not active at 100 microM. Two phenolic hydroxyls are required for their bioactivities. PMID:15043436

  20. Bundling of bovine and brine shrimp (Artemia) microtubules in vitro.

    PubMed

    MacRae, T H

    1984-06-01

    Cell-free extracts from embryos of the brine shrimp (Artemia) induced bundling of bovine microtubules assembled in the presence of glycerol and Mg++. Sedimentation of microtubules through sucrose cushions and subsequent electrophoresis revealed that bundling occurred independently of accessory proteins tightly bound to the microtubules. Bovine microtubules containing microtubule-associated proteins (MAPS) or assembled with taxol did not bundle. The unusual polymerization properties of homogeneous Artemia tubulin, bundling in the absence of added factors and the small number of microtubules assembled in crude embryo extracts upon addition of taxol precluded a complete comparative study of Artemia and bovine microtubule bundling. Interesting properties of the in vitro assembly of Artemia microtubules were, however, elaborated and putative Artemia MAPs were observed as a consequence of the work with brine shrimp embryos. PMID:6744423

  1. The Kinesin KIF21B Regulates Microtubule Dynamics and Is Essential for Neuronal Morphology, Synapse Function, and Learning and Memory.

    PubMed

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

    2016-05-01

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

  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. Stochastic dynamics of macromolecular-assembly networks.

    NASA Astrophysics Data System (ADS)

    Saiz, Leonor; Vilar, Jose

    2006-03-01

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

  4. Microtubules continuously dictate distribution of actin filaments and positioning of cell cleavage in grasshopper spermatocytes.

    PubMed

    Alsop, G Bradley; Zhang, Dahong

    2004-03-15

    We systematically examined the impact of microtubules on distribution of actin filaments and positioning of cell cleavage using micromanipulation to progressively alter the symmetric distribution of spindle microtubules in grasshopper spermatocytes. The initial microtubule asymmetry was induced by placing a single chromosome at one spindle pole using a microneedle, which facilitates regional assembly of spindle microtubules. We augmented chromosome-induced microtubule asymmetry by further removing the aster from the achromosomal pole, producing unichromosome-bearing monopolar spindles. We created the highest spindle asymmetry by cutting early anaphase cells in two, each containing a full set of segregating chromosomes in a half-spindle. We demonstrate that the location of the spindle midzone, distribution of actin filaments, and position of cell cleavage depend on the amount of microtubule asymmetry generated, shifting up to 48.6+/-3.8% away from the spindle equator in cut cells. The positional shift is dynamic, changing incessantly as spindle microtubules reorganize during cytokinesis. These results suggest that microtubules continuously dictate the distribution of actin filaments and positioning of cell cleavage in grasshopper spermatocytes. PMID:15020685

  5. 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

  6. Nonlinear ionic pulses along microtubules.

    PubMed

    Sekulić, D L; Satarić, B M; Tuszynski, J A; Satarić, M V

    2011-05-01

    Microtubules are cylindrically shaped cytoskeletal biopolymers that are essential for cell motility, cell division and intracellular trafficking. Here, we investigate their polyelectrolyte character that plays a very important role in ionic transport throughout the intra-cellular environment. The model we propose demonstrates an essentially nonlinear behavior of ionic currents which are guided by microtubules. These features are primarily due to the dynamics of tubulin C-terminal tails which are extended out of the surface of the microtubule cylinder. We also demonstrate that the origin of nonlinearity stems from the nonlinear capacitance of each tubulin dimer. This brings about conditions required for the creation and propagation of solitonic ionic waves along the microtubule axis. We conclude that a microtubule plays the role of a biological nonlinear transmission line for ionic currents. These currents might be of particular significance in cell division and possibly also in cognitive processes taking place in nerve cells. PMID:21604102

  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. 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

  9. Growth and Shortening of Microtubules

    PubMed Central

    Zhang, Yunxin

    2011-01-01

    In this study, a two-state mechanochemical model is presented to describe the dynamic instability of microtubules (MTs) in cells. The MT switches between two states, the assembly and disassembly states. In assembly state, the growth of MTs includes two processes: free GTP-tubulin binding to the tip of protofilament (PF) and conformation change of PF, during which the first tubulin unit that curls outwards is rearranged onto the MT surface, using the energy released from the hydrolysis of GTP in the penultimate tubulin unit. In the disassembly state, the shortening of MTs also includes two processes, the release of GDP-tubulin from the tip of PF and the curling of one new tubulin unit out of the MT surface. Switches between these two states, which are usually called rescue and catastrophe, happen stochastically with external force-dependent rates. Using this two-state model with parameters obtained by fitting the recent experimental data, detailed properties of MT growth are obtained. I find that MT is mainly in the assembly state, its mean growth velocity increases with both the external force and the GTP-tubulin concentration, and an MT will shorten on average without an external force. To know more about the external force and GTP-tubulin concentration-dependent properties of MT growth, and for future experimental verification of this two-state model, 11 critical forces are defined and discussed numerically. PMID:21903577

  10. Controls of interaction dynamics of orbital assembly

    NASA Technical Reports Server (NTRS)

    Su, Renjeng

    1991-01-01

    Building structures and spacecraft in orbit will require technologies for positioning, docking/berthing, and joining orbital structures. A fundamental problem underlying the operation of docking and berthing is that of controlling the contact dynamics of mechanical structures actuated by active mechanisms such as robotic devices. Control systems must be designed to control these active mechanisms so that both the free space motions and contact motions are stable and satisfy specifications on position accuracy and bounds on contact forces. For the large orbital structures of the future, the problem of interactive dynamics and control is fundamentally different in several ways than it was for spacecraft docking in the past. First, future space structures must be treated as flexible structures - the operations of docking, berthing, and assembly will need to respect the vibrations of the structures. Second, the assembly of these structures will require multiple-point contact, rather than the essentially single-point positioning of conventional spacecraft docking. Third, some assembly operations require the subassemblies to be brought and held in contact so that successful joining can be accomplished. A preliminary study of contact stability and compliance control design has resulted in the development of an analytical method and a design method to analyze stability. The analytical method analyzes the problem of stability when an actively-controlled structure contacts a passive structure. This method makes it possible to accurately estimate the stiffness of the passive structures with which the contact motion will become unstable. The analytic results suggest that passivity is neither achievable in practice, nor necessary as a design concept. A contact control system need only be passive up to a certain frequency; beyond that frequency the system can be stabilized with sufficiently small gains. With this concept the Center developed a design methodology for achieving

  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. Crowding of Molecular Motors Determines Microtubule Depolymerization

    PubMed Central

    Reese, Louis; Melbinger, Anna; Frey, Erwin

    2011-01-01

    The assembly and disassembly dynamics of microtubules (MTs) is tightly controlled by MT-associated proteins. Here, we investigate how plus-end-directed depolymerases of the kinesin-8 family regulate MT depolymerization dynamics. Using an individual-based model, we reproduce experimental findings. Moreover, crowding is identified as the key regulatory mechanism of depolymerization dynamics. Our analysis reveals two qualitatively distinct regimes. For motor densities above a particular threshold, a macroscopic traffic jam emerges at the plus-end and the MT dynamics become independent of the motor concentration. Below this threshold, microscopic traffic jams at the tip arise that cancel out the effect of the depolymerization kinetics such that the depolymerization speed is solely determined by the motor density. Because this density changes over the MT length, length-dependent regulation is possible. Remarkably, motor cooperativity affects only the end-residence time of depolymerases and not the depolymerization speed. PMID:22067158

  14. 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

  15. 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

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

    PubMed

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

    2015-08-31

    Microtubules (MTs) play critical roles in various cellular events, including cell migration. End-binding proteins (EBs) accumulate at the ends of growing MTs and regulate MT end dynamics by recruiting other plus end-tracking proteins (+TIPs). However, how EBs contribute to MT dynamics through +TIPs remains elusive. We focused on tau-tubulin kinase 2 (TTBK2) as an EB1/3-binding kinase and confirmed that TTBK2 acted as a +TIP. We identified MT-depolymerizing kinesin KIF2A as a novel substrate of TTBK2. TTBK2 phosphorylated KIF2A at S135 in intact cells in an EB1/3-dependent fashion and inactivated its MT-depolymerizing activity in vitro. TTBK2 depletion reduced MT lifetime (facilitated shrinkage and suppressed rescue) and impaired HeLa cell migration, and these phenotypes were partially restored by KIF2A co-depletion. Expression of nonphosphorylatable KIF2A, but not wild-type KIF2A, reduced MT lifetime and slowed down the cell migration. These findings indicate that TTBK2 with EB1/3 phosphorylates KIF2A and antagonizes KIF2A-induced depolymerization at MT plus ends for cell migration. PMID:26323690

  17. 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

  18. 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

  19. 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

  20. 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

  1. Shape-Conserved Dynamic Condensation in the Process of Aster Formation from a System of Microtubules and Cross-Linked Kinesin Motors

    NASA Astrophysics Data System (ADS)

    Kim, K.; Sikora, A.; Nakazawa, H.; Umetsu, M.; Hwang, W.; Teizer, W.

    2015-03-01

    We report fluorescence microscopy studies of a cellular element-based active system that is composed of rhodamine-labeled microtubules and functionalized kinesin motor proteins, cross-linked via streptavidin-coated quantum dots. The motor proteins organize microtubules into aster-like structures containing core aggregations of the quantum dot-motor protein complexes. The cores result from the dynamic condensation of sub-clusters that are connected to each other randomly. The inter-cluster distance decays exponentially with time during the condensation. Intriguingly, the shape defined by lines connecting the clusters is well conserved while the dynamic process reduces the size. This shape conservation is governed by a scaling behavior during the condensation, following a power law with respect to the distance between sub-clusters. We explain this isomorphic contraction during the aster formation process using a simple mechanistic model.

  2. Assembly, maintenance and dynamics of peroxisomes.

    PubMed

    Erdmann, Ralf

    2016-05-01

    Peroxisomes are ubiquitous organelles of eukaryotic cells, and it is becoming increasingly clear that the biogenesis of these multi-purpose organelles is more complex than initially anticipated. Along this line, peroxisomes exhibit features, which clearly distinguish them from other cellular organelles, like their ability to import folded proteins or their capability to form de novo. However, further insight into the cellular life of peroxisomes also revealed features that they share with other organelles, such as organelle fission or regulated degradation by autophagy, that are similar for peroxisomes, mitochondria and chloroplasts. This special issue highlights recent progress in the understanding of the biogenesis of peroxisomes with emphasis on the assembly, maintenance and dynamics of the organelles. In particular, it focuses on the following areas: (i) topogenesis of peroxisomal matrix proteins as well as the structure and function of peroxisomal protein import machineries. (ii) Peroxisomal targeting of membrane proteins and de novo formation of peroxisomes. (iii) Maintenance of peroxisomes in health and disease. (iv) Proliferation and regulated degradation of peroxisomes. (v) Motility and inheritance of peroxisomes. (vi) Role of peroxisomes in the cellular context. PMID:26851075

  3. AMP Kinase Activation Alters Oxidant-Induced Stress Granule Assembly by Modulating Cell Signaling and Microtubule Organization.

    PubMed

    Mahboubi, Hicham; Koromilas, Antonis E; Stochaj, Ursula

    2016-10-01

    Eukaryotic cells assemble stress granules (SGs) when translation initiation is inhibited. Different cell signaling pathways regulate SG production. Particularly relevant to this process is 5'-AMP-activated protein kinase (AMPK), which functions as a stress sensor and is transiently activated by adverse physiologic conditions. Here, we dissected the role of AMPK for oxidant-induced SG formation. Our studies identified multiple steps of de novo SG assembly that are controlled by the kinase. Single-cell analyses demonstrated that pharmacological AMPK activation prior to stress exposure changed SG properties, because the granules became more abundant and smaller in size. These altered SG characteristics correlated with specific changes in cell survival, cell signaling, cytoskeletal organization, and the abundance of translation initiation factors. Specifically, AMPK activation increased stress-induced eukaryotic initiation factor (eIF) 2α phosphorylation and reduced the concentration of eIF4F complex subunits eIF4G and eIF4E. At the same time, the abundance of histone deacetylase 6 (HDAC6) was diminished. This loss of HDAC6 was accompanied by increased acetylation of α-tubulin on Lys40. Pharmacological studies further confirmed this novel AMPK-HDAC6 interplay and its importance for SG biology. Taken together, we provide mechanistic insights into the regulation of SG formation. We propose that AMPK activation stimulates oxidant-induced SG formation but limits their fusion into larger granules. PMID:27430620

  4. Ectopic A-lattice seams destabilize microtubules

    PubMed Central

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

    2014-01-01

    Natural microtubules typically include one A-lattice seam within an otherwise helically symmetric B-lattice tube. It is currently unclear how A-lattice seams influence microtubule dynamic instability. Here we find that including extra A-lattice seams in GMPCPP microtubules, structural analogues of the GTP caps of dynamic microtubules, destabilizes them, enhancing their median shrinkage rate by >20-fold. Dynamic microtubules nucleated by seeds containing extra A-lattice seams have growth rates similar to microtubules nucleated by B-lattice seeds, yet have increased catastrophe frequencies at both ends. Furthermore, binding B-lattice GDP microtubules to a rigor kinesin surface stabilizes them against shrinkage, whereas microtubules with extra A-lattice seams are stabilized only slightly. Our data suggest that introducing extra A-lattice seams into dynamic microtubules destabilizes them by destabilizing their GTP caps. On this basis, we propose that the single A-lattice seam of natural B-lattice MTs may act as a trigger point, and potentially a regulation point, for catastrophe. PMID:24463734

  5. On complex, curved trajectories in microtubule gliding

    NASA Astrophysics Data System (ADS)

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

    2016-04-01

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

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

    NASA Astrophysics Data System (ADS)

    Bouzat, Sebastián

    2016-01-01

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

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

    PubMed

    Bouzat, Sebastián

    2016-01-01

    One-dimensional models coupling a Langevin equation for the cargo position to stochastic stepping dynamics for the motors constitute a relevant framework for analyzing multiple-motor microtubule transport. In this work we explore the consistence of these models focusing on the effects of the thermal noise. We study how to define consistent stepping and detachment rates for the motors as functions of the local forces acting on them in such a way that the cargo velocity and run-time match previously specified functions of the external load, which are set on the base of experimental results. We show that due to the influence of the thermal fluctuations this is not a trivial problem, even for the single-motor case. As a solution, we propose a motor stepping dynamics which considers memory on the motor force. This model leads to better results for single-motor transport than the approaches previously considered in the literature. Moreover, it gives a much better prediction for the stall force of the two-motor case, highly compatible with the experimental findings. We also analyze the fast fluctuations of the cargo position and the influence of the viscosity, comparing the proposed model to the standard one, and we show how the differences on the single-motor dynamics propagate to the multiple motor situations. Finally, we find that the one-dimensional character of the models impede an appropriate description of the fast fluctuations of the cargo position at small loads. We show how this problem can be solved by considering two-dimensional models. PMID:26871095

  8. A TOG Protein Confers Tension Sensitivity to Kinetochore-Microtubule Attachments.

    PubMed

    Miller, Matthew P; Asbury, Charles L; Biggins, Sue

    2016-06-01

    The development and survival of all organisms depends on equal partitioning of their genomes during cell division. Accurate chromosome segregation requires selective stabilization of kinetochore-microtubule attachments that come under tension due to opposing pulling forces exerted on sister kinetochores by dynamic microtubule tips. Here, we show that the XMAP215 family member, Stu2, makes a major contribution to kinetochore-microtubule coupling. Stu2 and its human ortholog, ch-TOG, exhibit a conserved interaction with the Ndc80 kinetochore complex that strengthens its attachment to microtubule tips. Strikingly, Stu2 can either stabilize or destabilize kinetochore attachments, depending on the level of kinetochore tension and whether the microtubule tip is assembling or disassembling. These dichotomous effects of Stu2 are independent of its previously studied regulation of microtubule dynamics. Altogether, our results demonstrate how a kinetochore-associated factor can confer opposing, tension-dependent effects to selectively stabilize tension-bearing attachments, providing mechanistic insight into the basis for accuracy during chromosome segregation. PMID:27156448

  9. 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

  10. Microparticle assembly and contact line dynamics

    NASA Astrophysics Data System (ADS)

    Ghosh, Moniraj

    This thesis addresses three topics. First, microparticle assembly on solid surfaces from an evaporative suspension is studied. It is well known that microparticles collect near three phase contact lines owing to evaporative fluxes. In a dip coating configuration, if the evaporative flux and plate withdrawal velocity U are matched, large colloidal crystals form. Here, I investigate the consequences of varying the plate withdrawal rate, and find that periodic striped patterns emerge which depend strongly on U. The stripes form when three phase contact lines "jump", or recede rapidly, upon detaching from well-wet particle aggregates on less wet substrates. Stripe width, spacing and height change abruptly at a transition velocity which can be related to a Landau-Levich transition in the flow. The second part of my thesis is a numerical simulation of drop spreading and retraction as a function of drop scale. The drop moves over a thin liquid film, and drop motion is initiated by an impulsive change in surface wettability. Owing to the presence of the film, these simulations require no closure condition at the 'apparent' contact line. Rather, relationships emerge between the contact line velocity and the dynamic contact angle. For nanoscopic drops, molecular effects dominate the drop motion. For drops an order of magnitude larger than the thin film, regimes emerge in which drops move according to Tanner's law, a relationship derived for macroscopic drops. Drop retraction is considerably more rapid than spreading owing to rapid dewetting events near the contact line. This thesis concludes with a discussion of a technique for creating multifunctional surfaces presenting discrete patches of several proteins. The technique relies on microcontact printing (microCP) to define active regions, and the use of a microfluidics device to deliver proteins to those regions. The surfaces are used to capture cells from a suspension, to sort cells from a mixed suspension, and to study

  11. Root-secreted allelochemical in the noxious weed Phragmites australis deploys a reactive oxygen species response and microtubule assembly disruption to execute rhizotoxicity.

    PubMed

    Rudrappa, Thimmaraju; Bonsall, Justin; Gallagher, John L; Seliskar, Denise M; Bais, Harsh P

    2007-10-01

    Phragmites australis is considered the most invasive plant in marsh and wetland communities in the eastern United States. Although allelopathy has been considered as a possible displacing mechanism in P. australis, there has been minimal success in characterizing the responsible allelochemical. We tested the occurrence of root-derived allelopathy in the invasiveness of P. australis. To this end, root exudates of two P. australis genotypes, BB (native) and P38 (an exotic) were tested for phytotoxicity on different plant species. The treatment of the susceptible plants with P. australis root exudates resulted in acute rhizotoxicity. It is interesting to note that the root exudates of P38 were more effective in causing root death in susceptible plants compared to the native BB exudates. The active ingredient in the P. australis exudates was identified as 3,4,5-trihydroxybenzoic acid (gallic acid). We tested the phytotoxic efficacy of gallic acid on various plant systems, including the model plant Arabidopsis thaliana. Most tested plants succumbed to the gallic acid treatment with the exception of P. australis itself. Mechanistically, gallic acid treatment generated elevated levels of reactive oxygen species (ROS) in the treated plant roots. Furthermore, the triggered ROS mediated the disruption of the root architecture of the susceptible plants by damaging the microtubule assembly. The study also highlights the persistence of the exuded gallic acid in P. australis's rhizosphere and its inhibitory effects against A. thaliana in the soil. In addition, gallic acid demonstrated an inhibitory effect on Spartina alterniflora, one of the salt marsh species it successfully invades. PMID:17899282

  12. 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. PMID:26212016

  13. 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.

  14. 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

  15. 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

  16. 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.

  17. 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

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

    PubMed Central

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

    2015-01-01

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

  19. The microtubule catastrophe promoter Sentin delays stable kinetochore-microtubule attachment in oocytes.

    PubMed

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

    2015-12-21

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

  20. Transient assembly of active materials fueled by a chemical reaction

    NASA Astrophysics Data System (ADS)

    Boekhoven, Job; Hendriksen, Wouter E.; Koper, Ger J. M.; Eelkema, Rienk; van Esch, Jan H.

    2015-09-01

    Fuel-driven self-assembly of actin filaments and microtubules is a key component of cellular organization. Continuous energy supply maintains these transient biomolecular assemblies far from thermodynamic equilibrium, unlike typical synthetic systems that spontaneously assemble at thermodynamic equilibrium. Here, we report the transient self-assembly of synthetic molecules into active materials, driven by the consumption of a chemical fuel. In these materials, reaction rates and fuel levels, instead of equilibrium composition, determine properties such as lifetime, stiffness, and self-regeneration capability. Fibers exhibit strongly nonlinear behavior including stochastic collapse and simultaneous growth and shrinkage, reminiscent of microtubule dynamics.

  1. Dynamics of magnetic assembly of binary colloidal structures

    NASA Astrophysics Data System (ADS)

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

    2015-08-01

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

  2. Talin-KANK1 interaction controls the recruitment of cortical microtubule stabilizing complexes to focal adhesions

    PubMed Central

    Bouchet, Benjamin P; Gough, Rosemarie E; Ammon, York-Christoph; van de Willige, Dieudonnée; Post, Harm; Jacquemet, Guillaume; Altelaar, AF Maarten; Heck, Albert JR; Goult, Benjamin T; Akhmanova, Anna

    2016-01-01

    The cross-talk between dynamic microtubules and integrin-based adhesions to the extracellular matrix plays a crucial role in cell polarity and migration. Microtubules regulate the turnover of adhesion sites, and, in turn, focal adhesions promote the cortical microtubule capture and stabilization in their vicinity, but the underlying mechanism is unknown. Here, we show that cortical microtubule stabilization sites containing CLASPs, KIF21A, LL5β and liprins are recruited to focal adhesions by the adaptor protein KANK1, which directly interacts with the major adhesion component, talin. Structural studies showed that the conserved KN domain in KANK1 binds to the talin rod domain R7. Perturbation of this interaction, including a single point mutation in talin, which disrupts KANK1 binding but not the talin function in adhesion, abrogates the association of microtubule-stabilizing complexes with focal adhesions. We propose that the talin-KANK1 interaction links the two macromolecular assemblies that control cortical attachment of actin fibers and microtubules. DOI: http://dx.doi.org/10.7554/eLife.18124.001 PMID:27410476

  3. Evidence for two distinct binding sites for tau on microtubules

    PubMed Central

    Makrides, Victoria; Massie, Michelle R.; Feinstein, Stuart C.; Lew, John

    2004-01-01

    The microtubule-associated protein tau regulates diverse and essential microtubule functions, from the nucleation and promotion of microtubule polymerization to the regulation of microtubule polarity and dynamics, as well as the spacing and bundling of axonal microtubules. Thermodynamic studies show that tau interacts with microtubules in the low- to mid-nanomolar range, implying moderate binding affinity. At the same time, it is well established that microtubule-bound tau does not undergo exchange with the bulk medium readily, suggesting that the tau-microtubule interaction is essentially irreversible. Given this dilemma, we investigated the mechanism of interaction between tau and microtubules in kinetic detail. Stopped-flow kinetic analysis reveals moderate binding affinity between tau and preassembled microtubules and rapid dissociation/association kinetics. In contrast, when microtubules are generated by copolymerization of tubulin and tau, a distinct population of microtubule-bound tau is observed, the binding of which seems irreversible. We propose that reversible binding occurs between tau and the surface of preassembled microtubules, whereas irreversible binding results when tau is coassembled with tubulin into a tau-microtubule copolymer. Because the latter is expected to be physiologically relevant, its characterization is of central importance. PMID:15096589

  4. 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

  5. Computing Nonequilibrium Conformational Dynamics of Structured Nucleic Acid Assemblies.

    PubMed

    Sedeh, Reza Sharifi; Pan, Keyao; Adendorff, Matthew Ralph; Hallatschek, Oskar; Bathe, Klaus-Jürgen; Bathe, Mark

    2016-01-12

    Synthetic nucleic acids can be programmed to form precise three-dimensional structures on the nanometer-scale. These thermodynamically stable complexes can serve as structural scaffolds to spatially organize functional molecules including multiple enzymes, chromophores, and force-sensing elements with internal dynamics that include substrate reaction-diffusion, excitonic energy transfer, and force-displacement response that often depend critically on both the local and global conformational dynamics of the nucleic acid assembly. However, high molecular weight assemblies exhibit long time-scale and large length-scale motions that cannot easily be sampled using all-atom computational procedures such as molecular dynamics. As an alternative, here we present a computational framework to compute the overdamped conformational dynamics of structured nucleic acid assemblies and apply it to a DNA-based tweezer, a nine-layer DNA origami ring, and a pointer-shaped DNA origami object, which consist of 204, 3,600, and over 7,000 basepairs, respectively. The framework employs a mechanical finite element model for the DNA nanostructure combined with an implicit solvent model to either simulate the Brownian dynamics of the assembly or alternatively compute its Brownian modes. Computational results are compared with an all-atom molecular dynamics simulation of the DNA-based tweezer. Several hundred microseconds of Brownian dynamics are simulated for the nine-layer ring origami object to reveal its long time-scale conformational dynamics, and the first ten Brownian modes of the pointer-shaped structure are predicted. PMID:26636351

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

    PubMed Central

    King, Matthew; Petry, Sabine

    2016-01-01

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

  7. Molecular architecture of the Dam1 complex–microtubule interaction

    PubMed Central

    Legal, Thibault; Zou, Juan; Sochaj, Alicja; Rappsilber, Juri

    2016-01-01

    Mitosis is a highly regulated process that allows the equal distribution of the genetic material to the daughter cells. Chromosome segregation requires the formation of a bipolar mitotic spindle and assembly of a multi-protein structure termed the kinetochore to mediate attachments between condensed chromosomes and spindle microtubules. In budding yeast, a single microtubule attaches to each kinetochore, necessitating robustness and processivity of this kinetochore–microtubule attachment. The yeast kinetochore-localized Dam1 complex forms a direct interaction with the spindle microtubule. In vitro, the Dam1 complex assembles as a ring around microtubules and couples microtubule depolymerization with cargo movement. However, the subunit organization within the Dam1 complex, its higher-order oligomerization and how it interacts with microtubules remain under debate. Here, we used chemical cross-linking and mass spectrometry to define the architecture and subunit organization of the Dam1 complex. This work reveals that both the C termini of Duo1 and Dam1 subunits interact with the microtubule and are critical for microtubule binding of the Dam1 complex, placing Duo1 and Dam1 on the inside of the ring structure. Integrating this information with available structural data, we provide a coherent model for how the Dam1 complex self-assembles around microtubules. PMID:26962051

  8. Microtubule-Associated Protein 2, a Marker of Neuronal Differentiation, Induces Mitotic Defects, Inhibits Growth of Melanoma Cells, and Predicts Metastatic Potential of Cutaneous Melanoma

    PubMed Central

    Soltani, Mohammad H.; Pichardo, Rita; Song, Ziqui; Sangha, Namrata; Camacho, Fabian; Satyamoorthy, Kapaettu; Sangueza, Omar P.; Setaluri, Vijayasaradhi

    2005-01-01

    Dynamic instability of microtubules is critical for mitotic spindle assembly and disassembly during cell division, especially in rapidly dividing tumor cells. Microtubule-associated proteins (MAPs) are a family of proteins that influence this property. We showed previously that MAP2, a neuron-specific protein that stabilizes microtubules in the dendrites of postmitotic neurons, is induced in primary cutaneous melanoma but is absent in metastatic melanomas. We proposed that induction of a microtubule-stabilizing protein in primary melanoma could disrupt the dynamic instability of microtubules, inhibit cell division and prevent or delay tumor progression. Here we show, by Kaplan-Meier survival and multivariate Cox regression analysis, that patients diagnosed with MAP2+ primary melanomas have significantly better metastatic disease-free survival than those with MAP2− disease. Investigation of the mechanisms that underlie the effect of MAP2 on melanoma progression showed that MAP2 expression in metastatic melanoma cell lines leads to microtubule stabilization, cell cycle arrest in G2-M phase and growth inhibition. Disruption of microtubule dynamics by MAP2 resulted in multipolar mitotic spindles, defects in cytokinesis and accumulation of cells with large nuclei, similar to those seen in vivo in MAP2+ primary melanomas cells. These data suggest that ectopic activation of a neuronal differentiation gene in melanoma during early tumor progression inhibits cell division and correlates with inhibition or delay of metastasis. PMID:15920168

  9. The KLP-7 Residue S546 Is a Putative Aurora Kinase Site Required for Microtubule Regulation at the Centrosome in C. elegans

    PubMed Central

    Han, Xue; Adames, Kelly; Sykes, Ellen M. E.; Srayko, Martin

    2015-01-01

    Regulation of microtubule dynamics is essential for many cellular processes, including proper assembly and function of the mitotic spindle. The kinesin-13 microtubule-depolymerizing enzymes provide one mechanism to regulate microtubule behaviour temporally and spatially. Vertebrate MCAK locates to chromatin, kinetochores, spindle poles, microtubule tips, and the cytoplasm, implying that the regulation of kinesin-13 activity and subcellular targeting is complex. Phosphorylation of kinesin-13 by Aurora kinase inhibits microtubule depolymerization activity and some Aurora phosphorylation sites on kinesin-13 are required for subcellular localization. Herein, we determine that a C. elegans deletion mutant klp-7(tm2143) causes meiotic and mitotic defects that are consistent with an increase in the amount of microtubules in the cytoplasmic and spindle regions of meiotic embryos, and an increase in microtubules emanating from centrosomes. We show that KLP-7 is phosphorylated by Aurora A and Aurora B kinases in vitro, and that the phosphorylation by Aurora A is stimulated by TPXL-1. Using a structure-function approach, we establish that one putative Aurora kinase site, S546, within the C-terminal part of the core domain is required for the function, but not subcellular localization, of KLP-7 in vivo. Furthermore, FRAP analysis reveals microtubule-dependent differences in the turnover of KLP-7(S546A) and KLP-7(S546E) mutant proteins at the centrosome, suggesting a possible mechanism for the regulation of KLP-7 by Aurora kinase. PMID:26168236

  10. Dynamic covalent assembly and disassembly of nanoparticle aggregates.

    PubMed

    Borsley, Stefan; Kay, Euan R

    2016-07-12

    The quantitative assembly and disassembly of a new type of dynamic covalent nanoparticle (NP) building block is reported. In situ spectroscopic characterization reveals constitutionally adaptive NP-bound monolayers of boronate esters. Ditopic linker molecules are used to produce covalently connected AuNP assemblies, displaying open dendritic morphologies, and which, despite being linked by covalent bonds, can be fully disassembled on application of an appropriate chemical stimulus. PMID:27001937

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

    NASA Astrophysics Data System (ADS)

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

    2016-05-01

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

  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. Arabidopsis AUGMIN Subunit8 Is a Microtubule Plus-End Binding Protein That Promotes Microtubule Reorientation in Hypocotyls[C][W

    PubMed Central

    Cao, Lingyan; Wang, Linhai; Zheng, Min; Cao, Hong; Ding, Lian; Zhang, Xiaolan; Fu, Ying

    2013-01-01

    In plant cells, cortical microtubules provide tracks for cellulose-synthesizing enzymes and regulate cell division, growth, and morphogenesis. The role of microtubules in these essential cellular processes depends on the spatial arrangement of the microtubules. Cortical microtubules are reoriented in response to changes in cell growth status and cell shape. Therefore, an understanding of the mechanism that underlies the change in microtubule orientation will provide insight into plant cell growth and morphogenesis. This study demonstrated that AUGMIN subunit8 (AUG8) in Arabidopsis thaliana is a novel microtubule plus-end binding protein that participates in the reorientation of microtubules in hypocotyls when cell elongation slows down. AUG8 bound to the plus ends of microtubules and promoted tubulin polymerization in vitro. In vivo, AUG8 was recruited to the microtubule branch site immediately before nascent microtubules branched out. It specifically associated with the plus ends of growing cortical microtubules and regulated microtubule dynamics, which facilitated microtubule reorientation when microtubules changed their growth trajectory or encountered obstacle microtubules during microtubule reorientation. This study thus reveals a novel mechanism underlying microtubule reorientation that is critical for modulating cell elongation in Arabidopsis. PMID:23735294

  14. Models, Regulations, and Functions of Microtubule Severing by Katanin

    PubMed Central

    Ghosh, Debasish Kumar; Dasgupta, Debdeep; Guha, Abhishek

    2012-01-01

    Regulation of microtubule dynamics depends on stochastic balance between polymerization and severing process which lead to differential spatiotemporal abundance and distribution of microtubules during cell development, differentiation, and morphogenesis. Microtubule severing by a conserved AAA family protein Katanin has emerged as an important microtubule architecture modulating process in cellular functions like division, migration, shaping and so on. Regulated by several factors, Katanin manifests connective crosstalks in network motifs in regulation of anisotropic severing pattern of microtubule protofilaments in cell type and stage dependent way. Mechanisms of structural disintegration of microtubules by Katanin involve heterogeneous mechanochemical processes and sensitivity of microtubules to Katanin plays significant roles in mitosis/meiosis, neurogenesis, cilia/flagella formation, cell wall development and so on. Deregulated and uncoordinated expression of Katanin has been shown to have implications in pathophysiological conditions. In this paper, we highlight mechanistic models and regulations of microtubule severing by Katanin in context of structure and various functions of Katanin in different organisms.

  15. A general modeling and visualization tool for comparing different members of a group: application to studying tau-mediated regulation of microtubule dynamics

    PubMed Central

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

    2008-01-01

    Background Innumerable biological investigations require comparing collections of molecules, cells or organisms to one another with respect to one or more of their properties. Almost all of these comparisons are performed manually, which can be susceptible to inadvertent bias as well as miss subtle effects. The development and application of computer-assisted analytical and interpretive tools could help address these issues and thereby dramatically improve these investigations. Results We have developed novel computer-assisted analytical and interpretive tools and applied them to recent studies examining the ability of 3-repeat and 4-repeat tau to regulate the dynamic behavior of microtubules in vitro. More specifically, we have developed an automated and objective method to define growth, shortening and attenuation events from real time videos of dynamic microtubules, and demonstrated its validity by comparing it to manually assessed data. Additionally, we have used the same data to develop a general strategy of building different models of interest, computing appropriate dissimilarity functions to compare them, and embedding them on a two-dimensional plot for visualization and easy comparison. Application of these methods to assess microtubule growth rates and growth rate distributions established the validity of the embedding procedure and revealed non-linearity in the relationship between the tau:tubulin molar ratio and growth rate distribution. Conclusion This work addresses the need of the biological community for rigorously quantitative and generally applicable computational tools for comparative studies. The two-dimensional embedding method retains the inherent structure of the data, and yet markedly simplifies comparison between models and parameters of different samples. Most notably, even in cases where numerous parameters exist by which to compare the different samples, our embedding procedure provides a generally applicable computational strategy to

  16. 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

  17. 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

  18. Microtubule networks for plant cell division.

    PubMed

    de Keijzer, Jeroen; Mulder, Bela M; Janson, Marcel E

    2014-09-01

    During cytokinesis the cytoplasm of a cell is divided to form two daughter cells. In animal cells, the existing plasma membrane is first constricted and then abscised to generate two individual plasma membranes. Plant cells on the other hand divide by forming an interior dividing wall, the so-called cell plate, which is constructed by localized deposition of membrane and cell wall material. Construction starts in the centre of the cell at the locus of the mitotic spindle and continues radially towards the existing plasma membrane. Finally the membrane of the cell plate and plasma membrane fuse to form two individual plasma membranes. Two microtubule-based cytoskeletal networks, the phragmoplast and the pre-prophase band (PPB), jointly control cytokinesis in plants. The bipolar microtubule array of the phragmoplast regulates cell plate deposition towards a cortical position that is templated by the ring-shaped microtubule array of the PPB. In contrast to most animal cells, plants do not use centrosomes as foci of microtubule growth initiation. Instead, plant microtubule networks are striking examples of self-organizing systems that emerge from physically constrained interactions of dispersed microtubules. Here we will discuss how microtubule-based activities including growth, shrinkage, severing, sliding, nucleation and bundling interrelate to jointly generate the required ordered structures. Evidence mounts that adapter proteins sense the local geometry of microtubules to locally modulate the activity of proteins involved in microtubule growth regulation and severing. Many of the proteins and mechanisms involved have roles in other microtubule assemblies as well, bestowing broader relevance to insights gained from plants. PMID:25136380

  19. 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.

  20. Self-assembly of dynamic orthoester cryptates

    PubMed Central

    Brachvogel, René-Chris; Hampel, Frank; von Delius, Max

    2015-01-01

    The discovery of coronands and cryptands, organic compounds that can accommodate metal ions in a preorganized two- or three-dimensional environment, was a milestone in supramolecular chemistry, leading to countless applications from organic synthesis to metallurgy and medicine. These compounds are typically prepared via multistep organic synthesis and one of their characteristic features is the high stability of their covalent framework. Here we report the use of a dynamic covalent exchange reaction for the one-pot template synthesis of a new class of coronates and cryptates, in which acid-labile O,O,O-orthoesters serve as bridgeheads. In contrast to their classic analogues, the compounds described herein are constitutionally dynamic in the presence of acid and can be induced to release their guest via irreversible deconstruction of the cage. These properties open up a wide range of application opportunities, from systems chemistry to molecular sensing and drug delivery. PMID:25997913

  1. Dynamic, Directed Self-Assembly of Nanoparticles via Toggled Interactions.

    PubMed

    Sherman, Zachary M; Swan, James W

    2016-05-24

    Crystals self-assembled from nanoparticles have useful properties such as optical activity and sensing capability. During fabrication, however, gelation and glassification often leave these materials arrested in defective or disordered metastable states. This is a key difficulty preventing adoption of self-assembled nanoparticle materials at scale. Processes which suppress kinetic arrest and defect formation while accelerating growth of ordered materials are essential for bottom-up approaches to creating nanomaterials. Dynamic, directed self-assembly processes in which the interactions between self-assembling components are actuated temporally offer one promising methodology for accelerating and controlling bottom-up growth of nanostructures. In this article, we show through simulation and theory how time-dependent, periodically toggled interparticle attractions can avoid kinetic barriers and yield well-ordered crystalline domains for a dispersion of nanoparticles interacting via a short-ranged, isotropic potential. The growth mechanism and terminal structure of the dispersion are controlled by parameters of the toggling protocol. This control allows for selection of processes that yield rapid self-assembled, low defect crystals. Although self-assembly via periodically toggled attractions is inherently unsteady and out-of-equilibrium, its outcome is predicted by a first-principles theory of nonequilibrium thermodynamics. The theory necessitates equality of the time average of pressure and chemical potential in coexisting phases of the dispersion. These quantities are evaluated using well known equations of state. The phase behavior predicted by this theory agrees well with measurements made in Brownian dynamics simulations of sedimentation equilibrium and homogeneous nucleation. The theory can easily be extended to model dynamic self-assembly directed by other toggled conservative force fields. PMID:27096705

  2. Artificial Photosynthesis at Dynamic Self-Assembled Interfaces in Water.

    PubMed

    Hansen, Malte; Troppmann, Stefan; König, Burkhard

    2016-01-01

    Artificial photosynthesis is one of the big scientific challenges of today. Self-assembled dynamic interfaces, such as vesicles or micelles, have been used as microreactors to mimic biological photosynthesis. These aggregates can help to overcome typical problems of homogeneous photocatalytic water splitting. Microheterogeneous environments organize catalyst-photosensitizer assemblies at the interface in close proximity and thus enhance intermolecular interactions. Thereby vesicles and micelles may promote photoinitiated charge separation and suppress back electron transfer. The dynamic self-assembled interfaces solubilize non-polar compounds and protect sensitive catalytic units and intermediates against degradation. In addition, vesicles provide compartmentation that was used to separate different redox environments needed for an overall water splitting system. This Minireview provides an overview of the applications of micellar and vesicular microheterogeneous systems for solar energy conversion by photosensitized water oxidation and hydrogen generation. PMID:26552728

  3. Environmental and Endogenous Control of Cortical Microtubule Orientation.

    PubMed

    Chen, Xu; Wu, Shuang; Liu, Zengyu; Friml, Jiří

    2016-06-01

    Plant growth requires a tight coordination of cell shape and anisotropic expansion. Owing to their immobility, plant cells determine body architecture through the orientation of cell division and cell expansion. Microtubule cytoskeleton represents a versatile cellular structure essential for coordinating flexible cell morphogenesis. Previous studies have identified a large number of microtubule-associated regulators that control microtubule dynamics; however, the mechanisms by which microtubule reorientation responds to exogenous and environmental stimuli are largely unknown. In this review, we describe the molecular details of microtubule dynamics that are required for cortical microtubule array pattern formation, and recapitulate current knowledge on the mechanisms by which various environmental and endogenous stimuli control cortical microtubule reorientation. PMID:26951762

  4. Reconstituting the kinetochore-microtubule interface: what, why, and how

    PubMed Central

    Akiyoshi, Bungo; Biggins, Sue

    2012-01-01

    The kinetochore is the proteinaceous complex that governs the movement of duplicated chromosomes by interacting with spindle microtubules during mitosis and meiosis. Faithful chromosome segregation requires that kinetochores form robust load-bearing attachments to the tips of dynamic spindle microtubules, correct microtubule attachment errors, and delay the onset of anaphase until all chromosomes have made proper attachments. To understand how this macromolecular machine operates to segregate duplicated chromosomes with exquisite accuracy, it is critical to reconstitute and study kinetochore-microtubule interactions in vitro using defined components. Here, we review the current status of reconstitution as well as recent progress in understanding the microtubule binding functions of kinetochores in vivo. PMID:22289864

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

    SciTech Connect

    Barrack, Keri L.; Fyfe, Paul K.; Hunter, William N.

    2015-04-21

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

  6. Lessons from in vitro reconstitution analyses of plant microtubule-associated proteins

    PubMed Central

    Hamada, Takahiro

    2014-01-01

    Plant microtubules, composed of tubulin GTPase, are irreplaceable cellular components that regulate the directions of cell expansion and cell division, chromosome segregation and cell plate formation. To accomplish these functions, plant cells organize microtubule structures by regulating microtubule dynamics. Each microtubule localizes to the proper position with repeated growth and shortening. Although it is possible to reconstitute microtubule dynamics with pure tubulin solution in vitro, many microtubule-associated proteins (MAPs) govern microtubule dynamics in cells. In plants, major MAPs are identified as microtubule stabilizers (CLASP and MAP65 etc.), microtubule destabilizers (kinesin-13, katanin, MAP18 and MDP25), and microtubule dynamics promoters (EB1, MAP215, MOR1, MAP200, SPR2). Mutant analyses with forward and reverse genetics have shown the importance of microtubules and individual MAPs in plants. However, it is difficult to understand how each MAP regulates microtubule dynamics, such as growth and shortening, through mutant analyses. In vitro reconstitution analyses with individual purified MAPs and tubulin are powerful tools to reveal how each MAP regulates microtubule dynamics at the molecular level. In this review, I summarize the results of in vitro reconstitution analyses and introduce current models of how each MAP regulates microtubule dynamic instability. PMID:25202315

  7. 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

  8. Solid-State and Solution NMR Studies of the CAP-Gly Domain of Mammalian Dynactin and Its Interaction with Microtubules

    SciTech Connect

    Sun, Shangjin; Siglin, Amanda; Williams, John C.; Polenova, Tatyana E.

    2009-07-29

    Microtubules (MTs) and microtubule binding proteins (MTBPs) play fundamental physiological roles including vesicle and organelle transport, cell motility, and cell division. Despite the importance of the MT/MTBP assemblies, there remains virtually no structural or dynamic information about their interaction at the atomic level due to the inherent insolubility and lack of long-range order of MTs. In this study, we present a combined magic angle spinning solid-state and solution NMR study of the MTBP CAP-Gly domain of mammalian dynactin and its interaction with paclitaxel-stabilized microtubules. We report resonance assignments and secondary structure analysis of the free CAP-Gly in solution and in the solid state by a combination of two- and three-dimensional homo- and heteronuclear correlation spectra. In solution, binding of CAP-Gly to microtubules is accompanied by the broadening of the majority of the peaks in HSQC spectra except for the residues at the termini, precluding further structural analysis of the CAP-Gly/microtubule complexes. In the solid state, DARR spectra of free CAP-Gly and its complex with microtubules display well-resolved lines, permitting residue-specific resonance assignments. Interestingly, a number of chemical shifts in the solid-state DARR spectra of the CAP-Gly/microtubule complex are perturbed compared to those of the free CAP-Gly, suggesting that conformational changes occur in the protein upon binding to the microtubules. These results indicate that CAP-Gly/microtubule assemblies are amenable to detailed structural characterization by magic angle spinning NMR spectroscopy and that solid-state NMR is a viable technique to study MT/protein interactions in general.

  9. Novel Piperazine-based Compounds Inhibit Microtubule Dynamics and Sensitize Colon Cancer Cells to Tumor Necrosis Factor-induced Apoptosis*

    PubMed Central

    Chopra, Avijeet; Anderson, Amy; Giardina, Charles

    2014-01-01

    We recently identified a series of mitotically acting piperazine-based compounds that potently increase the sensitivity of colon cancer cells to apoptotic ligands. Here we describe a structure-activity relationship study on this compound class and identify a highly active derivative ((4-(3-chlorophenyl)piperazin-1-yl)(2-ethoxyphenyl)methanone), referred to as AK301, the activity of which is governed by the positioning of functional groups on the phenyl and benzoyl rings. AK301 induced mitotic arrest in HT29 human colon cancer cells with an ED50 of ≈115 nm. Although AK301 inhibited growth of normal lung fibroblast cells, mitotic arrest was more pronounced in the colon cancer cells (50% versus 10%). Cells arrested by AK301 showed the formation of multiple microtubule organizing centers with Aurora kinase A and γ-tubulin. Employing in vitro and in vivo assays, tubulin polymerization was found to be slowed (but not abolished) by AK301. In silico molecular docking suggests that AK301 binds to the colchicine-binding domain on β-tubulin, but in a novel orientation. Cells arrested by AK301 expressed elevated levels of TNFR1 on their surface and more readily activated caspases-8, -9, and -3 in the presence of TNF. Relative to other microtubule destabilizers, AK301 was the most active TNF-sensitizing agent and also stimulated Fas- and TRAIL-induced apoptosis. In summary, we report a new class of mitosis-targeting agents that effectively sensitizes cancer cells to apoptotic ligands. These compounds should help illuminate the role of microtubules in regulating apoptotic ligand sensitivity and may ultimately be useful for developing agents that augment the anti-cancer activities of the immune response. PMID:24338023

  10. 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.

  11. Active contraction of microtubule networks.

    PubMed

    Foster, Peter J; Fürthauer, Sebastian; Shelley, Michael J; Needleman, Daniel J

    2015-01-01

    Many cellular processes are driven by cytoskeletal assemblies. It remains unclear how cytoskeletal filaments and motor proteins organize into cellular scale structures and how molecular properties of cytoskeletal components affect the large-scale behaviors of these systems. Here, we investigate the self-organization of stabilized microtubules in Xenopus oocyte extracts and find that they can form macroscopic networks that spontaneously contract. We propose that these contractions are driven by the clustering of microtubule minus ends by dynein. Based on this idea, we construct an active fluid theory of network contractions, which predicts a dependence of the timescale of contraction on initial network geometry, a development of density inhomogeneities during contraction, a constant final network density, and a strong influence of dynein inhibition on the rate of contraction, all in quantitative agreement with experiments. These results demonstrate that the motor-driven clustering of filament ends is a generic mechanism leading to contraction. PMID:26701905

  12. Dynamic phases, pinning, and pattern formation for driven dislocation assemblies

    SciTech Connect

    Zhou, Caizhi; Reichhardt, Charles; Olson Reichhardt, Cynthia J.; Beyerlein, Irene J.

    2015-01-23

    We examine driven dislocation assemblies and show that they can exhibit a set of dynamical phases remarkably similar to those of driven systems with quenched disorder such as vortices in superconductors, magnetic domain walls, and charge density wave materials. These phases include pinned-jammed, fluctuating, and dynamically ordered states, and each produces distinct dislocation patterns as well as specific features in the noise fluctuations and transport properties. Lastly, our work suggests that many of the results established for systems with quenched disorder undergoing plastic depinning transitions can be applied to dislocation systems, providing a new approach for understanding pattern formation and dynamics in these systems.

  13. Dynamic phases, pinning, and pattern formation for driven dislocation assemblies

    DOE PAGESBeta

    Zhou, Caizhi; Reichhardt, Charles; Olson Reichhardt, Cynthia J.; Beyerlein, Irene J.

    2015-01-23

    We examine driven dislocation assemblies and show that they can exhibit a set of dynamical phases remarkably similar to those of driven systems with quenched disorder such as vortices in superconductors, magnetic domain walls, and charge density wave materials. These phases include pinned-jammed, fluctuating, and dynamically ordered states, and each produces distinct dislocation patterns as well as specific features in the noise fluctuations and transport properties. Lastly, our work suggests that many of the results established for systems with quenched disorder undergoing plastic depinning transitions can be applied to dislocation systems, providing a new approach for understanding pattern formation andmore » dynamics in these systems.« less

  14. Dynamic Phases, Pinning, and Pattern Formation for Driven Dislocation Assemblies

    PubMed Central

    Zhou, Caizhi; Reichhardt, Charles; Olson Reichhardt, Cynthia J.; Beyerlein, Irene J.

    2015-01-01

    We examine driven dislocation assemblies and show that they can exhibit a set of dynamical phases remarkably similar to those of driven systems with quenched disorder such as vortices in superconductors, magnetic domain walls, and charge density wave materials. These phases include pinned-jammed, fluctuating, and dynamically ordered states, and each produces distinct dislocation patterns as well as specific features in the noise fluctuations and transport properties. Our work suggests that many of the results established for systems with quenched disorder undergoing plastic depinning transitions can be applied to dislocation systems, providing a new approach for understanding pattern formation and dynamics in these systems. PMID:25613839

  15. Dynamic Phases, Pinning, and Pattern Formation for Driven Dislocation Assemblies

    NASA Astrophysics Data System (ADS)

    Zhou, Caizhi; Reichhardt, Charles; Olson Reichhardt, Cynthia J.; Beyerlein, Irene J.

    2015-01-01

    We examine driven dislocation assemblies and show that they can exhibit a set of dynamical phases remarkably similar to those of driven systems with quenched disorder such as vortices in superconductors, magnetic domain walls, and charge density wave materials. These phases include pinned-jammed, fluctuating, and dynamically ordered states, and each produces distinct dislocation patterns as well as specific features in the noise fluctuations and transport properties. Our work suggests that many of the results established for systems with quenched disorder undergoing plastic depinning transitions can be applied to dislocation systems, providing a new approach for understanding pattern formation and dynamics in these systems.

  16. 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.

  17. Dynamic self-assembly and control of microfluidic particle crystals

    PubMed Central

    Lee, Wonhee; Amini, Hamed; Stone, Howard A.; Di Carlo, Dino

    2010-01-01

    Engineered two-phase microfluidic systems have recently shown promise for computation, encryption, and biological processing. For many of these systems, complex control of dispersed-phase frequency and switching is enabled by nonlinearities associated with interfacial stresses. Introducing nonlinearity associated with fluid inertia has recently been identified as an easy to implement strategy to control two-phase (solid-liquid) microscale flows. By taking advantage of inertial effects we demonstrate controllable self-assembling particle systems, uncover dynamics suggesting a unique mechanism of dynamic self-assembly, and establish a framework for engineering microfluidic structures with the possibility of spatial frequency filtering. Focusing on the dynamics of the particle–particle interactions reveals a mechanism for the dynamic self-assembly process; inertial lift forces and a parabolic flow field act together to stabilize interparticle spacings that otherwise would diverge to infinity due to viscous disturbance flows. The interplay of the repulsive viscous interaction and inertial lift also allow us to design and implement microfluidic structures that irreversibly change interparticle spacing, similar to a low-pass filter. Although often not considered at the microscale, nonlinearity due to inertia can provide a platform for high-throughput passive control of particle positions in all directions, which will be useful for applications in flow cytometry, tissue engineering, and metamaterial synthesis. PMID:21149674

  18. Transformation and patterning of supermicelles using dynamic holographic assembly

    NASA Astrophysics Data System (ADS)

    Gould, Oliver E. C.; Qiu, Huibin; Lunn, David J.; Rowden, John; Harniman, Robert L.; Hudson, Zachary M.; Winnik, Mitchell A.; Miles, Mervyn J.; Manners, Ian

    2015-12-01

    Although the solution self-assembly of block copolymers has enabled the fabrication of a broad range of complex, functional nanostructures, their precise manipulation and patterning remain a key challenge. Here we demonstrate that spherical and linear supermicelles, supramolecular structures held together by non-covalent solvophobic and coordination interactions and formed by the hierarchical self-assembly of block copolymer micelle and block comicelle precursors, can be manipulated, transformed and patterned with mediation by dynamic holographic assembly (optical tweezers). This allows the creation of new and stable soft-matter superstructures far from equilibrium. For example, individual spherical supermicelles can be optically held in close proximity and photocrosslinked through controlled coronal chemistry to generate linear oligomeric arrays. The use of optical tweezers also enables the directed deposition and immobilization of supermicelles on surfaces, allowing the precise creation of arrays of soft-matter nano-objects with potentially diverse functionality and a range of applications.

  19. Transformation and patterning of supermicelles using dynamic holographic assembly

    PubMed Central

    Gould, Oliver E.C.; Qiu, Huibin; Lunn, David J.; Rowden, John; Harniman, Robert L.; Hudson, Zachary M.; Winnik, Mitchell A.; Miles, Mervyn J.; Manners, Ian

    2015-01-01

    Although the solution self-assembly of block copolymers has enabled the fabrication of a broad range of complex, functional nanostructures, their precise manipulation and patterning remain a key challenge. Here we demonstrate that spherical and linear supermicelles, supramolecular structures held together by non-covalent solvophobic and coordination interactions and formed by the hierarchical self-assembly of block copolymer micelle and block comicelle precursors, can be manipulated, transformed and patterned with mediation by dynamic holographic assembly (optical tweezers). This allows the creation of new and stable soft-matter superstructures far from equilibrium. For example, individual spherical supermicelles can be optically held in close proximity and photocrosslinked through controlled coronal chemistry to generate linear oligomeric arrays. The use of optical tweezers also enables the directed deposition and immobilization of supermicelles on surfaces, allowing the precise creation of arrays of soft-matter nano-objects with potentially diverse functionality and a range of applications. PMID:26627644

  20. Microtubule-like Properties of the Bacterial Actin Homolog ParM-R1*

    PubMed Central

    Popp, David; Narita, Akihiro; Lee, Lin Jie; Larsson, Mårten; Robinson, Robert C.

    2012-01-01

    In preparation for mammalian cell division, microtubules repeatedly probe the cytoplasm to capture chromosomes and assemble the mitotic spindle. Critical features of this microtubule system are the formation of radial arrays centered at the centrosomes and dynamic instability, leading to persistent cycles of polymerization and depolymerization. Here, we show that actin homolog, ParM-R1 that drives segregation of the R1 multidrug resistance plasmid from Escherichia coli, can also self-organize in vitro into asters, which resemble astral microtubules. ParM-R1 asters grow from centrosome-like structures consisting of interconnected nodes related by a pseudo 8-fold symmetry. In addition, we show that ParM-R1 is able to perform persistent microtubule-like oscillations of assembly and disassembly. In vitro, a whole population of ParM-R1 filaments is synchronized between phases of growth and shrinkage, leading to prolonged synchronous oscillations even at physiological ParM-R1 concentrations. These results imply that the selection pressure to reliably segregate DNA during cell division has led to common mechanisms within diverse segregation machineries. PMID:22908230

  1. Regulation of kinesin-transport by microtubule age and polymerization conditions

    NASA Astrophysics Data System (ADS)

    Xu, Jing; Liang, Winnie; King, Stephen; Faysal, K.

    2015-03-01

    Microtubules are fundamental biopolymers in cells, formed via self-assembly (``polymerization'') of tubulin dimers. Microtubule polymerization conditions have been shown to alter the presence of defects in microtubule lattices, including point defects (missing tubulin dimers) and line defects (protofilament disruption). Potential impact of these lattice defects on molecular motor-based transport is not yet understood. Here we investigate the impact of microtubule polymerization conditions on multiple-kinesin transport, using single-molecule-type optical trapping experiments. We find that kinesin-based cargoes pause preferentially at specific locations along individual microtubules, and that the pause frequency and duration is strongly dependent on microtubule age and polymerization condition. Within each polymerization condition and for fresh microtubules, we also observe significant variations in multiple-kinesin travel distances, depending on which microtubules the motors travel along. Taken together, our study suggests an important role of microtubule lattice defect in regulating intracellular transport.

  2. Targeting, Capture, and Stabilization of Microtubules at Early Focal Adhesions

    PubMed Central

    Kaverina, Irina; Rottner, Klemens; Small, J. Victor

    1998-01-01

    By co-injecting fluorescent tubulin and vinculin into fish fibroblasts we have revealed a “cross talk” between microtubules and early sites of substrate contact. This mutuality was first indicated by the targeting of vinculin-rich foci by microtubules during their growth towards the cell periphery. In addition to passing directly over contact sites, the ends of single microtubules could be observed to target several contacts in succession or the same contact repetitively, with intermittent withdrawals. Targeting sometimes involved side-stepping, or the major re-routing of a microtubule, indicative of a guided, rather than a random process. The paths that microtubules followed into contacts were unrelated to the orientation of stress fiber assemblies and targeting occurred also in mouse fibroblasts that lacked a system of intermediate filaments. Further experiments with microtubule inhibitors showed that adhesion foci can: (a) capture microtubules and stabilize them against disassembly by nocodazole; and (b), act as preferred sites of microtubule polymerization, during either early recovery from nocodazole, or brief treatment with taxol. From these and other findings we speculate that microtubules are guided into substrate contact sites and through the motor-dependent delivery of signaling molecules serve to modulate their development. It is further proposed this modulation provides the route whereby microtubules exert their influence on cell shape and polarity. PMID:9660872

  3. Negative regulation of EB1 turnover at microtubule plus ends by interaction with microtubule-associated protein ATIP3

    PubMed Central

    Rodrigues-Ferreira, Sylvie; Nehlig, Anne; Bouchet, Benjamin Pierre; Morel, Marina; Leconte, Ludovic; Serre, Laurence; Arnal, Isabelle; Braguer, Diane; Savina, Ariel; Honore, Stéphane; Nahmias, Clara

    2015-01-01

    The regulation of microtubule dynamics is critical to ensure essential cell functions. End binding protein 1 (EB1) is a master regulator of microtubule dynamics that autonomously binds an extended GTP/GDP-Pi structure at growing microtubule ends and recruits regulatory proteins at this location. However, negative regulation of EB1 association with growing microtubule ends remains poorly understood. We show here that microtubule-associated tumor suppressor ATIP3 interacts with EB1 through direct binding of a non-canonical proline-rich motif. Results indicate that ATIP3 does not localize at growing microtubule ends and that in situ ATIP3-EB1 molecular complexes are mostly detected in the cytosol. We present evidence that a minimal EB1-interacting sequence of ATIP3 is both necessary and sufficient to prevent EB1 accumulation at growing microtubule ends in living cells and that EB1-interaction is involved in reducing cell polarity. By fluorescence recovery of EB1-GFP after photobleaching, we show that ATIP3 silencing accelerates EB1 turnover at microtubule ends with no modification of EB1 diffusion in the cytosol. We propose a novel mechanism by which ATIP3-EB1 interaction indirectly reduces the kinetics of EB1 exchange on its recognition site, thereby accounting for negative regulation of microtubule dynamic instability. Our findings provide a unique example of decreased EB1 turnover at growing microtubule ends by cytosolic interaction with a tumor suppressor. PMID:26498358

  4. Negative regulation of EB1 turnover at microtubule plus ends by interaction with microtubule-associated protein ATIP3.

    PubMed

    Velot, Lauriane; Molina, Angie; Rodrigues-Ferreira, Sylvie; Nehlig, Anne; Bouchet, Benjamin Pierre; Morel, Marina; Leconte, Ludovic; Serre, Laurence; Arnal, Isabelle; Braguer, Diane; Savina, Ariel; Honore, Stéphane; Nahmias, Clara

    2015-12-22

    The regulation of microtubule dynamics is critical to ensure essential cell functions. End binding protein 1 (EB1) is a master regulator of microtubule dynamics that autonomously binds an extended GTP/GDP-Pi structure at growing microtubule ends and recruits regulatory proteins at this location. However, negative regulation of EB1 association with growing microtubule ends remains poorly understood. We show here that microtubule-associated tumor suppressor ATIP3 interacts with EB1 through direct binding of a non-canonical proline-rich motif. Results indicate that ATIP3 does not localize at growing microtubule ends and that in situ ATIP3-EB1 molecular complexes are mostly detected in the cytosol. We present evidence that a minimal EB1-interacting sequence of ATIP3 is both necessary and sufficient to prevent EB1 accumulation at growing microtubule ends in living cells and that EB1-interaction is involved in reducing cell polarity. By fluorescence recovery of EB1-GFP after photobleaching, we show that ATIP3 silencing accelerates EB1 turnover at microtubule ends with no modification of EB1 diffusion in the cytosol. We propose a novel mechanism by which ATIP3-EB1 interaction indirectly reduces the kinetics of EB1 exchange on its recognition site, thereby accounting for negative regulation of microtubule dynamic instability. Our findings provide a unique example of decreased EB1 turnover at growing microtubule ends by cytosolic interaction with a tumor suppressor. PMID:26498358

  5. Evolutionary dynamics in a simple model of self-assembly

    NASA Astrophysics Data System (ADS)

    Johnston, Iain G.; Ahnert, Sebastian E.; Doye, Jonathan P. K.; Louis, Ard A.

    2011-06-01

    We investigate the evolutionary dynamics of an idealized model for the robust self-assembly of two-dimensional structures called polyominoes. The model includes rules that encode interactions between sets of square tiles that drive the self-assembly process. The relationship between the model’s rule set and its resulting self-assembled structure can be viewed as a genotype-phenotype map and incorporated into a genetic algorithm. The rule sets evolve under selection for specified target structures. The corresponding complex fitness landscape generates rich evolutionary dynamics as a function of parameters such as the population size, search space size, mutation rate, and method of recombination. Furthermore, these systems are simple enough that in some cases the associated model genome space can be completely characterized, shedding light on how the evolutionary dynamics depends on the detailed structure of the fitness landscape. Finally, we apply the model to study the emergence of the preference for dihedral over cyclic symmetry observed for homomeric protein tetramers.

  6. Critical dynamics of randomly assembled and diluted threshold networks

    NASA Astrophysics Data System (ADS)

    Kürten, Karl E.; Clark, John W.

    2008-04-01

    The dynamical behavior of a class of randomly assembled networks of binary threshold units subject to random deletion of connections is studied based on the annealed approximation suitable in the thermodynamic limit. The dynamical phase diagram is constructed for several forms of the probability density distribution of nonvanishing connection strengths. The family of power-law distribution functions ρ0(x)=(1-α)/(2|x|α) is found to play a special role in expanding the domain of stable, ordered dynamics at the expense of the disordered, “chaotic” phase. Relationships with other recent studies of the dynamics of complex networks allowing for variable in-degree of the units are explored. The relevance of the pruning of network connections to neural modeling and developmental neurobiology is discussed.

  7. Microtubule-associated protein-like binding of the kinesin-1 tail to microtubules.

    PubMed

    Seeger, Mark A; Rice, Sarah E

    2010-03-12

    The kinesin-1 molecular motor contains an ATP-dependent microtubule-binding site in its N-terminal head domain and an ATP-independent microtubule-binding site in its C-terminal tail domain. Here we demonstrate that a kinesin-1 tail fragment associates with microtubules with submicromolar affinity. Binding is largely electrostatic in nature, and is facilitated by a region of basic amino acids in the tail and the acidic E-hook at the C terminus of tubulin. The tail binds to a site on tubulin that is independent of the head domain-binding site but overlaps with the binding site of the microtubule-associated protein Tau. Surprisingly, the kinesin tail domain stimulates microtubule assembly and stability in a manner similar to Tau. The biological function of this strong kinesin tail-microtubule interaction remains to be seen, but it is likely to play an important role in kinesin regulation due to the close proximity of the microtubule-binding region to the conserved regulatory and cargo-binding domains of the tail. PMID:20071331

  8. Microtubule-associated Protein-like Binding of the Kinesin-1 Tail to Microtubules*

    PubMed Central

    Seeger, Mark A.; Rice, Sarah E.

    2010-01-01

    The kinesin-1 molecular motor contains an ATP-dependent microtubule-binding site in its N-terminal head domain and an ATP-independent microtubule-binding site in its C-terminal tail domain. Here we demonstrate that a kinesin-1 tail fragment associates with microtubules with submicromolar affinity. Binding is largely electrostatic in nature, and is facilitated by a region of basic amino acids in the tail and the acidic E-hook at the C terminus of tubulin. The tail binds to a site on tubulin that is independent of the head domain-binding site but overlaps with the binding site of the microtubule-associated protein Tau. Surprisingly, the kinesin tail domain stimulates microtubule assembly and stability in a manner similar to Tau. The biological function of this strong kinesin tail-microtubule interaction remains to be seen, but it is likely to play an important role in kinesin regulation due to the close proximity of the microtubule-binding region to the conserved regulatory and cargo-binding domains of the tail. PMID:20071331

  9. 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.

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

    PubMed

    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

  11. 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

  12. Model-adaptive hybrid dynamic control for robotic assembly tasks

    SciTech Connect

    Austin, D.J.; McCarragher, B.J.

    1999-10-01

    A new task-level adaptive controller is presented for the hybrid dynamic control of robotic assembly tasks. Using a hybrid dynamic model of the assembly task, velocity constraints are derived from which satisfactory velocity commands are obtained. Due to modeling errors and parametric uncertainties, the velocity commands may be erroneous and may result in suboptimal performance. Task-level adaptive control schemes, based on the occurrence of discrete events, are used to change the model parameters from which the velocity commands are determined. Two adaptive schemes are presented: the first is based on intuitive reasoning about the vector spaces involved whereas the second uses a search region that is reduced with each iteration. For the first adaptation law, asymptotic convergence to the correct model parameters is proven except for one case. This weakness motivated the development of the second adaptation law, for which asymptotic convergence is proven in all cases. Automated control of a peg-in-hole assembly task is given as an example, and simulations and experiments for this task are presented. These results demonstrate the success of the method and also indicate properties for rapid convergence.

  13. Continuous monitoring of structural dynamics in polymer assemblies

    NASA Astrophysics Data System (ADS)

    Guzman Sepulveda, Jose Rafael; Deng, Jinan; Fang, Jiyu; Dogariu, Aristide

    Due to their flexibility, optical methods are preferred approaches for monitoring the dynamics and mechanical properties of scattering systems such as polymer solutions, colloidal suspensions, and complex media in general. In particular, their potential noninvasiveness is critical for the passive assessment of dynamic processes. Practical implementations however suffer sometimes from limitations due to effects such as multiple scattering or strong attenuation. Here we introduce an optical technique that overcomes some of these limitations and permits accessing the dynamics of complex colloidal systems under realistic conditions and inherent external influences. This interferometric technique operates based on the coherence-gated isolation of single scattering and allows for the spatially-resolved evaluation of the system's dynamics in optically isolated picoliter-sized volumes. This effective isolation permits a fully passive characterization of nonstationary dynamic processes in complex systems including aggregation and self-assembling, sedimentation, structural evolution and phase transitions, interface dynamics, and dynamics in inhomogeneous or stratified solvents. CREOL, The College of Optics and Photonics.

  14. Space Station/Orbiter berthing dynamics during an assembly flight

    NASA Technical Reports Server (NTRS)

    Cooper, Paul A.; Stockwell, Alan E.; Wu, Shih-Chin

    1993-01-01

    A large-angle, multi-body, dynamic modeling capability was developed to help validate numerical simulations of the dynamic motion and control forces which occur while berthing Space Station Freedom to the Shuttle Orbiter during early assembly flights. The paper describes the dynamics and control of the station, the attached Shuttle Remote Manipulator System, and the Orbiter during a maneuver from a gravity-gradient attitude to a torque equilibrium attitude using the station reaction control jets. The influence of the elastic behavior of the station and of the remote manipulator system on the attitude control of the station/Orbiter system during the maneuver is investigated. The flexibility of the station and the arm had only a minor influence on the attitude control of the system during the maneuver.

  15. Linker-Mediated Self-Assembly Dynamics of Charged Nanoparticles.

    PubMed

    Lin, Guanhua; Chee, See Wee; Raj, Sanoj; Král, Petr; Mirsaidov, Utkur

    2016-08-23

    Using in situ liquid cell transmission electron microscopy (TEM), we visualized a stepwise self-assembly of surfactant-coated and hydrated gold nanoparticles (NPs) into linear chains or branched networks. The NP binding is facilitated by linker molecules, ethylenediammonium, which form hydrogen bonds with surfactant molecules of neighboring NPs. The observed spacing between bound neighboring NPs, ∼15 Å, matches the combined length of two surfactants and one linker molecule. Molecular dynamics simulations reveal that for lower concentrations of linkers, NPs with charged surfactants cannot be fully neutralized by strongly binding divalent linkers, so that NPs carry higher effective charges and tend to form chains, due to poor screening. The highly polar NP surfaces polarize and partly immobilize nearby water molecules, which promotes NPs binding. The presented experimental and theoretical approach allows for detail observation and explanation of self-assembly processes in colloidal nanosystems. PMID:27494560

  16. Preparation of Segmented Microtubules to Study Motions Driven by the Disassembling Microtubule Ends

    PubMed Central

    Volkov, Vladimir A.; Zaytsev, Anatoly V.; Grishchuk, Ekaterina L.

    2014-01-01

    Microtubule depolymerization can provide force to transport different protein complexes and protein-coated beads in vitro. The underlying mechanisms are thought to play a vital role in the microtubule-dependent chromosome motions during cell division, but the relevant proteins and their exact roles are ill-defined. Thus, there is a growing need to develop assays with which to study such motility in vitro using purified components and defined biochemical milieu. Microtubules, however, are inherently unstable polymers; their switching between growth and shortening is stochastic and difficult to control. The protocols we describe here take advantage of the segmented microtubules that are made with the photoablatable stabilizing caps. Depolymerization of such segmented microtubules can be triggered with high temporal and spatial resolution, thereby assisting studies of motility at the disassembling microtubule ends. This technique can be used to carry out a quantitative analysis of the number of molecules in the fluorescently-labeled protein complexes, which move processively with dynamic microtubule ends. To optimize a signal-to-noise ratio in this and other quantitative fluorescent assays, coverslips should be treated to reduce nonspecific absorption of soluble fluorescently-labeled proteins. Detailed protocols are provided to take into account the unevenness of fluorescent illumination, and determine the intensity of a single fluorophore using equidistant Gaussian fit. Finally, we describe the use of segmented microtubules to study microtubule-dependent motions of the protein-coated microbeads, providing insights into the ability of different motor and nonmotor proteins to couple microtubule depolymerization to processive cargo motion. PMID:24686554

  17. 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.

  18. Dynamic colloidal assembly pathways via low dimensional models.

    PubMed

    Yang, Yuguang; Thyagarajan, Raghuram; Ford, David M; Bevan, Michael A

    2016-05-28

    Here we construct a low-dimensional Smoluchowski model for electric field mediated colloidal crystallization using Brownian dynamic simulations, which were previously matched to experiments. Diffusion mapping is used to infer dimensionality and confirm the use of two order parameters, one for degree of condensation and one for global crystallinity. Free energy and diffusivity landscapes are obtained as the coefficients of a low-dimensional Smoluchowski equation to capture the thermodynamics and kinetics of microstructure evolution. The resulting low-dimensional model quantitatively captures the dynamics of different assembly pathways between fluid, polycrystal, and single crystals states, in agreement with the full N-dimensional data as characterized by first passage time distributions. Numerical solution of the low-dimensional Smoluchowski equation reveals statistical properties of the dynamic evolution of states vs. applied field amplitude and system size. The low-dimensional Smoluchowski equation and associated landscapes calculated here can serve as models for predictive control of electric field mediated assembly of colloidal ensembles into two-dimensional crystalline objects. PMID:27250328

  19. Self-assembly of active colloidal molecules with dynamic function

    NASA Astrophysics Data System (ADS)

    Soto, Rodrigo; Golestanian, Ramin

    Catalytically active colloids maintain non-equilibrium conditions in which they produce and deplete chemicals at their surface. While individual colloids that are symmetrically coated do not exhibit dynamical activity, the concentration fields resulting from their chemical activity decay as 1/r and produce gradients that attract or repel other colloids depending on their surface chemistry and ambient variables. This results in a non-equilibrium analogue of ionic systems, but with the remarkable novel feature of action-reaction symmetry breaking. In dilute conditions these active colloids join up to form molecules via generalized ionic bonds. Colloids are found to join up to form self-assembled molecules that could be inert or have spontaneous activity in the form of net translational velocity and spin depending on their symmetry properties and their constituents. As the interactions do not satisfy detailed-balance, it is possible to achieve structures with time dependent functionality. We study a molecule that adopts spontaneous oscillations and another that exhibits a run-and-tumble dynamics similar to bacteria. Our study shows that catalytically active colloids could be used for designing self-assembled structures that posses dynamical functionalities.

  20. Dynamic multiprotein assemblies shape the spatial structure of cell signaling.

    PubMed

    Nussinov, Ruth; Jang, Hyunbum

    2014-01-01

    Cell signaling underlies critical cellular decisions. Coordination, efficiency as well as fail-safe mechanisms are key elements. How the cell ensures that these hallmarks are at play are important questions. Cell signaling is often viewed as taking place through discrete and cross-talking pathways; oftentimes these are modularized to emphasize distinct functions. While simple, convenient and clear, such models largely neglect the spatial structure of cell signaling; they also convey inter-modular (or inter-protein) spatial separation that may not exist. Here our thesis is that cell signaling is shaped by a network of multiprotein assemblies. While pre-organized, the assemblies and network are loose and dynamic. They contain transiently-associated multiprotein complexes which are often mediated by scaffolding proteins. They are also typically anchored in the membrane, and their continuum may span the cell. IQGAP1 scaffolding protein which binds proteins including Raf, calmodulin, Mek, Erk, actin, and tens more, with actin shaping B-cell (and likely other) membrane-anchored nanoclusters and allosterically polymerizing in dynamic cytoskeleton formation, and Raf anchoring in the membrane along with Ras, provides a striking example. The multivalent network of dynamic proteins and lipids, with specific interactions forming and breaking, can be viewed as endowing gel-like properties. Collectively, this reasons that efficient, productive and reliable cell signaling takes place primarily through transient, preorganized and cooperative protein-protein interactions spanning the cell rather than stochastic, diffusion-controlled processes. PMID:25046855

  1. Protein-guided RNA dynamics during early ribosome assembly

    NASA Astrophysics Data System (ADS)

    Kim, Hajin; Abeysirigunawarden, Sanjaya C.; Chen, Ke; Mayerle, Megan; Ragunathan, Kaushik; Luthey-Schulten, Zaida; Ha, Taekjip; Woodson, Sarah A.

    2014-02-01

    The assembly of 30S ribosomes requires the precise addition of 20 proteins to the 16S ribosomal RNA. How early binding proteins change the ribosomal RNA structure so that later proteins may join the complex is poorly understood. Here we use single-molecule fluorescence resonance energy transfer (FRET) to observe real-time encounters between Escherichia coli ribosomal protein S4 and the 16S 5' domain RNA at an early stage of 30S assembly. Dynamic initial S4-RNA complexes pass through a stable non-native intermediate before converting to the native complex, showing that non-native structures can offer a low free-energy path to protein-RNA recognition. Three-colour FRET and molecular dynamics simulations reveal how S4 changes the frequency and direction of RNA helix motions, guiding a conformational switch that enforces the hierarchy of protein addition. These protein-guided dynamics offer an alternative explanation for induced fit in RNA-protein complexes.

  2. Dynamic colloidal assembly pathways via low dimensional models

    NASA Astrophysics Data System (ADS)

    Yang, Yuguang; Thyagarajan, Raghuram; Ford, David M.; Bevan, Michael A.

    2016-05-01

    Here we construct a low-dimensional Smoluchowski model for electric field mediated colloidal crystallization using Brownian dynamic simulations, which were previously matched to experiments. Diffusion mapping is used to infer dimensionality and confirm the use of two order parameters, one for degree of condensation and one for global crystallinity. Free energy and diffusivity landscapes are obtained as the coefficients of a low-dimensional Smoluchowski equation to capture the thermodynamics and kinetics of microstructure evolution. The resulting low-dimensional model quantitatively captures the dynamics of different assembly pathways between fluid, polycrystal, and single crystals states, in agreement with the full N-dimensional data as characterized by first passage time distributions. Numerical solution of the low-dimensional Smoluchowski equation reveals statistical properties of the dynamic evolution of states vs. applied field amplitude and system size. The low-dimensional Smoluchowski equation and associated landscapes calculated here can serve as models for predictive control of electric field mediated assembly of colloidal ensembles into two-dimensional crystalline objects.

  3. Large fluctuations in the disassembly rate of microtubules revealed by atomic force microscopy.

    PubMed

    Thomson, Neil H; Kasas, Sandor; Riederer, Beat M; Catsicas, Stefan; Dietler, Giovanni; Kulik, Andrzej J; Forró, László

    2003-01-01

    Atomic force microscopy (AFM) in situ has been used to observe the cold disassembly dynamics of microtubules at a previously unrealised spatial resolution. Microtubules either electrostatically or covalently bound to aminosilane surfaces disassembled at room temperature under buffer solutions with no free tubulin present. This process was followed by taking sequential tapping-mode AFM images and measuring the change in the microtubule end position as a function of time, with an spatial accuracy down to +/-20nm and a temporal accuracy of +/-1s. As well as giving average disassembly rates on the order of 1-10 tubulin monomers per second, large fluctuations in the disassembly rate were revealed, indicating that the process is far from smooth and linear under these experimental conditions. The surface bound rates measured here are comparable to the rates for GMPCPP-tubulin microtubules free in solution, suggesting that inhibition of tubulin curvature through steric hindrance controls the average, relatively low disassembly rate. The large fluctuations in this rate are thought to be due to multiple pathways in the kinetics of disassembly with differing rate constants and/or stalling due to defects in the microtubule lattice. Microtubules that were covalently bound to the surface left behind the protofilaments covalently cross-linked to the aminosilane via glutaraldehyde during the disassembly process. Further work is needed to quantitatively assess the effects of surface binding on protofibril disassembly rates, reveal any differences in disassembly rates between the plus and minus ends and to enable assembly as well as disassembly to be imaged in the microscope fluid cell in real-time. PMID:12801676

  4. 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

  5. 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

  6. 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

  7. Molecular mechanisms of kinetochore capture by spindle microtubules.

    PubMed

    Tanaka, Kozo; Mukae, Naomi; Dewar, Hilary; van Breugel, Mark; James, Euan K; Prescott, Alan R; Antony, Claude; Tanaka, Tomoyuki U

    2005-04-21

    For high-fidelity chromosome segregation, kinetochores must be properly captured by spindle microtubules, but the mechanisms underlying initial kinetochore capture have remained elusive. Here we visualized individual kinetochore-microtubule interactions in Saccharomyces cerevisiae by regulating the activity of a centromere. Kinetochores are captured by the side of microtubules extending from spindle poles, and are subsequently transported poleward along them. The microtubule extension from spindle poles requires microtubule plus-end-tracking proteins and the Ran GDP/GTP exchange factor. Distinct kinetochore components are used for kinetochore capture by microtubules and for ensuring subsequent sister kinetochore bi-orientation on the spindle. Kar3, a kinesin-14 family member, is one of the regulators that promote transport of captured kinetochores along microtubules. During such transport, kinetochores ensure that they do not slide off their associated microtubules by facilitating the conversion of microtubule dynamics from shrinkage to growth at the plus ends. This conversion is promoted by the transport of Stu2 from the captured kinetochores to the plus ends of microtubules. PMID:15846338

  8. Interface dynamics explain assembly dependency of influenza neuraminidase catalytic activity

    PubMed Central

    von Grafenstein, Susanne; Wallnoefer, Hannes G.; Kirchmair, Johannes; Fuchs, Julian E.; Huber, Roland G.; Schmidtke, Michaela; Sauerbrei, Andreas; Rollinger, Judith M.; Liedl, Klaus R.

    2015-01-01

    Influenza virus neuraminidase (iNA) is a homotetrameric surface protein of the influenza virus and an established target for antiviral drugs. In contrast to neuraminidases (NAs) of other biological systems (non-iNAs), enzymatic activity of iNA is only observed in a quaternary assembly and iNA needs the tetramerization to mediate enzymatic activity. Obviously, differences on a molecular level between iNA and non-iNAs are responsible for this intriguing observation. Comparison between protein structures and multiple sequence alignment allow the identification of differences in amino acid composition in crucial regions of the enzyme, such as next to the conserved D151 and the 150-loop. These differences in amino acid sequence and protein tetramerization are likely to alter the dynamics of the system. Therefore, we performed molecular dynamics simulations to investigate differences in the molecular flexibility of monomers, dimers, and tetramers of iNAs of subtype N1 (avian 2004, pandemic 1918 and pandemic 2009 iNA) and as comparison the non-iNA monomer from Clostridium perfringens. We show that conformational transitions of iNA are crucially influenced by its assembly state. The protein–protein interface induces a complex hydrogen-bonding network between the 110-helix and the 150-loop, which consequently stabilizes the structural arrangement of the binding site. Therefore, we claim that these altered dynamics are responsible for the dependence of iNA’s catalytic activity on the tetrameric assembly. Only the tetramerization-induced balance between stabilization and altered local flexibility in the binding site provides the appropriate arrangement of key residues for iNA’s catalytic activity. PMID:24279589

  9. Model for dynamic self-assembled magnetic surface structures.

    SciTech Connect

    Belkin, M.; Glatz, A.; Snezhko, A.; Aranson, I. S.; Materials Science Division; Northwestern Univ.

    2010-07-07

    We propose a first-principles model for the dynamic self-assembly of magnetic structures at a water-air interface reported in earlier experiments. The model is based on the Navier-Stokes equation for liquids in shallow water approximation coupled to Newton equations for interacting magnetic particles suspended at a water-air interface. The model reproduces most of the observed phenomenology, including spontaneous formation of magnetic snakelike structures, generation of large-scale vortex flows, complex ferromagnetic-antiferromagnetic ordering of the snake, and self-propulsion of bead-snake hybrids.

  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. 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.

  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. Phylogenetic diversity of the expression of the microtubule-associated protein tau: implications for neurodegenerative disorders.

    PubMed

    Janke, C; Beck, M; Stahl, T; Holzer, M; Brauer, K; Bigl, V; Arendt, T

    1999-05-01

    The microtubule-associated protein tau regulates the dynamic stability of the neuronal cytoskeleton by interacting with microtubules. It is encoded by a single gene, but expressed in a variety of isoforms due to differential RNA splicing. Six isoforms can be found in the human central nervous system. These isoforms differ in their ability to promote the assembly of microtubules as well as in their capacity to stabilize existing microtubule structures. Furthermore, some of the isoforms of tau are specifically involved in the pathogenesis of neurodegenerative disorders. Thus, splicing of tau might critically influence the physiological functions of tau protein as well as the pathogenesis of neurodegenerative diseases with tauopathy. The present study addresses the differential expression of the six isoforms of tau in the central nervous system of 12 mammalian species including Homo sapiens. The occurrence of each of the six tau isoforms was highly variable. However, species that were phylogenetically related expressed a similar pattern of tau isoforms. These results suggest a phylogenetic descent of splicing paradigms, which can be matched with known phylogenetic concepts based on morphological and molecular genetical studies. Especially, the unique expression pattern of tau isoforms in the human central nervous system implicates a possible link to the particular vulnerability of humans to neurodegenerative disorders with tauopathy, namely Alzheimer's disease, frontotemporal dementia and Pick's disease. PMID:10320789

  14. Measuring Microtubule Polarity in Spindles with Second-Harmonic Generation

    PubMed Central

    Yu, Che-Hang; Langowitz, Noah; Wu, Hai-Yin; Farhadifar, Reza; Brugues, Jan; Yoo, Tae Yeon; Needleman, Daniel

    2014-01-01

    The spatial organization of microtubule polarity, and the interplay between microtubule polarity and protein localization, is thought to be crucial for spindle assembly, anaphase, and cytokinesis, but these phenomena remain poorly understood, in part due to the difficulty of measuring microtubule polarity in spindles. We develop and implement a method to nonperturbatively and quantitatively measure microtubule polarity throughout spindles using a combination of second-harmonic generation and two-photon fluorescence. We validate this method using computer simulations and by comparison to structural data on spindles obtained from electron tomography and laser ablation. This method should provide a powerful tool for studying spindle organization and function, and may be applicable for investigating microtubule polarity in other systems. PMID:24739157

  15. Microtubule-dependent transport of vimentin filament precursors is regulated by actin and by the concerted action of Rho- and p21-activated kinases

    PubMed Central

    Robert, Amélie; Herrmann, Harald; Davidson, Michael W.; Gelfand, Vladimir I.

    2014-01-01

    Intermediate filaments (IFs) form a dense and dynamic network that is functionally associated with microtubules and actin filaments. We used the GFP-tagged vimentin mutant Y117L to study vimentin-cytoskeletal interactions and transport of vimentin filament precursors. This mutant preserves vimentin interaction with other components of the cytoskeleton, but its assembly is blocked at the unit-length filament (ULF) stage. ULFs are easy to track, and they allow a reliable and quantifiable analysis of movement. Our results show that in cultured human vimentin-negative SW13 cells, 2% of vimentin-ULFs move along microtubules bidirectionally, while the majority are stationary and tightly associated with actin filaments. Rapid motor-dependent transport of ULFs along microtubules is enhanced ≥5-fold by depolymerization of actin cytoskeleton with latrunculin B. The microtubule-dependent transport of vimentin ULFs is further regulated by Rho-kinase (ROCK) and p21-activated kinase (PAK): ROCK inhibits ULF transport, while PAK stimulates it. Both kinases act on microtubule transport independently of their effects on actin cytoskeleton. Our study demonstrates the importance of the actin cytoskeleton to restrict IF transport and reveals a new role for PAK and ROCK in the regulation of IF precursor transport.—Robert, A., Herrmann, H., Davidson, M. W., and Gelfand, V. I. Microtubule-dependent transport of vimentin filament precursors is regulated by actin and by the concerted action of Rho- and p21-activated kinases. PMID:24652946

  16. 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

  17. 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

  18. Analysis and inverse substructuring computation on dynamic quality of mechanical assembly

    NASA Astrophysics Data System (ADS)

    Lü, Guangqing; Yi, Chuijie; Fang, Ke

    2016-05-01

    Mechanical assembly has its own dynamic quality directly affecting the dynamic quality of whole product and should be considered in quality inspection and estimation of mechanical assembly. Based on functional relations between dynamic characteristics involved in mechanical assembly, the effects of assembling process on dynamic characteristics of substructural components of an assembly system are investigated by substructuring analysis. Assembly-coupling dynamic stiffness is clarified as the dominant factor of the effects and can be used as a quantitative measure of assembly dynamic quality. Two computational schemes using frequency response functions(FRFs) to determine the stiffness are provided and discussed by inverse substructuring analysis, including their applicable conditions and implementation procedure in application. Eigenvalue analysis on matrix-ratios of FRFs before and after assembling is employed and well validates the analytical outcomes and the schemes via both a lumped-parameter model and its analogic experimental counterpart. Applying the two schemes to inspect the dynamic quality provides the message of dynamic performance of the assembly system, and therefore improves conventional quality inspection and estimation of mechanical assembly in completeness.

  19. Analysis and inverse substructuring computation on dynamic quality of mechanical assembly

    NASA Astrophysics Data System (ADS)

    Lü, Guangqing; Yi, Chuijie; Fang, Ke

    2016-04-01

    Mechanical assembly has its own dynamic quality directly affecting the dynamic quality of whole product and should be considered in quality inspection and estimation of mechanical assembly. Based on functional relations between dynamic characteristics involved in mechanical assembly, the effects of assembling process on dynamic characteristics of substructural components of an assembly system are investigated by substructuring analysis. Assembly-coupling dynamic stiffness is clarified as the dominant factor of the effects and can be used as a quantitative measure of assembly dynamic quality. Two computational schemes using frequency response functions(FRFs) to determine the stiffness are provided and discussed by inverse substructuring analysis, including their applicable conditions and implementation procedure in application. Eigenvalue analysis on matrix-ratios of FRFs before and after assembling is employed and well validates the analytical outcomes and the schemes via both a lumped-parameter model and its analogic experimental counterpart. Applying the two schemes to inspect the dynamic quality provides the message of dynamic performance of the assembly system, and therefore improves conventional quality inspection and estimation of mechanical assembly in completeness.

  20. Dynamic characterization of satellite assembly for responsive space applications

    NASA Astrophysics Data System (ADS)

    Mascarenas, David; Macknelly, David; Mullins, Josh; Wiest, Heather; Park, Gyuhae

    2013-07-01

    The rapid deployment of satellites for responsive space surveillance applications is hindered by the need to flight-qualify their components and the resulting mechanical assembly. Conventional methods for qualification testing of satellite components are costly and time consuming. Furthermore, full-scale vehicles must be subjected to simulated launch loads during testing, and this harsh testing environment increases the risk of damage to satellite components during qualification. This work focuses on replacing this potentially destructive testing procedure with a non-destructive structural health monitoring (SHM)-based technique while maintaining the same level of confidence in the testing procedure's ability to qualify the satellite for flight. We focus on assessing the performance of SHM techniques to replace the high-cost qualification procedure and to localize faults introduced by improper assembly. The goal of this work is to create a dual-use system that can both assist in the process of qualifying the satellite for launch, as well as provide continuous structural integrity monitoring during manufacture, transport, launch and deployment. SHM techniques were applied on a small-scale structure representative of a responsive satellite. The test structure consisted of an extruded aluminum space-frame covered with aluminum shear plates assembled using bolted joints. Multiple piezoelectric transducers were bonded to the test structure and acted as combined actuators and sensors. Piezoelectric active-sensing based techniques, including measurements of low-frequency global frequency response functions and high-frequency wave propagation techniques, were employed. Using these methods in conjunction with finite element modeling, the dynamic properties of the test structure were established and areas of potential damage could be identified and localized. A procedure for guiding the effective placement of the sensors and actuators is also outlined.

  1. Dynamic self-assembly of motile bacteria in liquid crystals

    PubMed Central

    Mushenheim, Peter C.; Trivedi, Rishi R.; Tuson, Hannah H.

    2014-01-01

    This paper reports an investigation of dynamical behaviors of motile rod-shaped bacteria within anisotropic viscoelastic environments defined by lyotropic liquid crystals (LCs). In contrast to passive microparticles (including non-motile bacteria) that associate irreversibly in LCs via elasticity-mediated forces, we report that motile Proteus mirabilis bacteria form dynamic and reversible multi-cellular assemblies when dispersed in a lyotropic LC. By measuring the velocity of the bacteria through the LC (8.8 +/− 0.2 μm/s) and by characterizing the ordering of the LC about the rod-shaped bacteria (tangential anchoring), we conclude that the reversibility of the inter-bacterial interaction emerges from the interplay of forces generated by the flagella of the bacteria and the elasticity of the LC, both of which are comparable in magnitude (tens of pN) for motile Proteus mirabilis cells. We also measured the dissociation process, which occurs in a direction determined by the LC, to bias the size distribution of multi-cellular bacterial complexes in a population of motile Proteus mirabilis relative to a population of non-motile cells. Overall, these observations and others reported in this paper provide insight into the fundamental dynamical behaviors of bacteria in complex anisotropic environments and suggest that motile bacteria in LCs are an exciting model system for exploration of principles for the design of active materials. PMID:24652584

  2. Self-assembly of active colloidal molecules with dynamic function

    NASA Astrophysics Data System (ADS)

    Soto, Rodrigo; Golestanian, Ramin

    2015-05-01

    Catalytically active colloids maintain nonequilibrium conditions in which they produce and deplete chemicals and hence effectively act as sources and sinks of molecules. While individual colloids that are symmetrically coated do not exhibit any form of dynamical activity, the concentration fields resulting from their chemical activity decay as 1 /r and produce gradients that attract or repel other colloids depending on their surface chemistry and ambient variables. This results in a nonequilibrium analog of ionic systems, but with the remarkable novel feature of action-reaction symmetry breaking. We study solutions of such chemically active colloids in dilute conditions when they join up to form molecules via generalized ionic bonds and discuss how we can achieve structures with time-dependent functionality. In particular, we study a molecule that adopts a spontaneous oscillatory pattern of conformations and another that exhibits a run-and-tumble dynamics similar to bacteria. Our study shows that catalytically active colloids could be used for designing self-assembled structures that possess dynamical functionalities that are determined by their prescribed three-dimensional structures, a strategy that follows the design principle of proteins.

  3. Pironetin reacts covalently with cysteine-316 of α-tubulin to destabilize microtubule

    NASA Astrophysics Data System (ADS)

    Yang, Jianhong; Wang, Yuxi; Wang, Taijing; Jiang, Jian; Botting, Catherine H.; Liu, Huanting; Chen, Qiang; Yang, Jinliang; Naismith, James H.; Zhu, Xiaofeng; Chen, Lijuan

    2016-06-01

    Molecules that alter the normal dynamics of microtubule assembly and disassembly include many anticancer drugs in clinical use. So far all such therapeutics target β-tubulin, and structural biology has explained the basis of their action and permitted design of new drugs. However, by shifting the profile of β-tubulin isoforms, cancer cells become resistant to treatment. Compounds that bind to α-tubulin are less well characterized and unexploited. The natural product pironetin is known to bind to α-tubulin and is a potent inhibitor of microtubule polymerization. Previous reports had identified that pironetin reacts with lysine-352 residue however analogues designed on this model had much lower potency, which was difficult to explain, hindering further development. We report crystallographic and mass spectrometric data that reveal that pironetin forms a covalent bond to cysteine-316 in α-tubulin via a Michael addition reaction. These data provide a basis for the rational design of α-tubulin targeting chemotherapeutics.

  4. Active contraction of microtubule networks

    PubMed Central

    Foster, Peter J; Fürthauer, Sebastian; Shelley, Michael J; Needleman, Daniel J

    2015-01-01

    Many cellular processes are driven by cytoskeletal assemblies. It remains unclear how cytoskeletal filaments and motor proteins organize into cellular scale structures and how molecular properties of cytoskeletal components affect the large-scale behaviors of these systems. Here, we investigate the self-organization of stabilized microtubules in Xenopus oocyte extracts and find that they can form macroscopic networks that spontaneously contract. We propose that these contractions are driven by the clustering of microtubule minus ends by dynein. Based on this idea, we construct an active fluid theory of network contractions, which predicts a dependence of the timescale of contraction on initial network geometry, a development of density inhomogeneities during contraction, a constant final network density, and a strong influence of dynein inhibition on the rate of contraction, all in quantitative agreement with experiments. These results demonstrate that the motor-driven clustering of filament ends is a generic mechanism leading to contraction. DOI: http://dx.doi.org/10.7554/eLife.10837.001 PMID:26701905

  5. The Microtubule Plus-End Tracking Protein ARMADILLO-REPEAT KINESIN1 Promotes Microtubule Catastrophe in Arabidopsis[W][OPEN

    PubMed Central

    Eng, Ryan Christopher; Wasteneys, Geoffrey O.

    2014-01-01

    Microtubule dynamics are critically important for plant cell development. Here, we show that Arabidopsis thaliana ARMADILLO-REPEAT KINESIN1 (ARK1) plays a key role in root hair tip growth by promoting microtubule catastrophe events. This destabilizing activity appears to maintain adequate free tubulin concentrations in order to permit rapid microtubule growth, which in turn is correlated with uniform tip growth. Microtubules in ark1-1 root hairs exhibited reduced catastrophe frequency and slower growth velocities, both of which were restored by low concentrations of the microtubule-destabilizing drug oryzalin. An ARK1-GFP (green fluorescent protein) fusion protein expressed under its endogenous promoter localized to growing microtubule plus ends and rescued the ark1-1 root hair phenotype. Transient overexpression of ARK1-RFP (red fluorescent protein) increased microtubule catastrophe frequency. ARK1-fusion protein constructs lacking the N-terminal motor domain still labeled microtubules, suggesting the existence of a second microtubule binding domain at the C terminus of ARK1. ARK1-GFP was broadly expressed in seedlings, but mutant phenotypes were restricted to root hairs, indicating that ARK1’s function is redundant in cells other than those forming root hairs. PMID:25159991

  6. Dynamic shear-influenced collagen self-assembly.

    PubMed

    Saeidi, Nima; Sander, Edward A; Ruberti, Jeffrey W

    2009-12-01

    The ability to influence the direction of polymerization of a self-assembling biomolecular system has the potential to generate materials with extremely high anisotropy. In biological systems where highly-oriented cellular populations give rise to aligned and often load-bearing tissue such organized molecular scaffolds could aid in the contact guidance of cells for engineered tissue constructs (e.g. cornea and tendon). In this investigation we examine the detailed dynamics of pepsin-extracted type I bovine collagen assembly on a glass surface under the influence of flow between two plates. Differential Interference Contrast (DIC) imaging (60x-1.4NA) with focal plane stabilization was used to resolve and track the growth of collagen aggregates on borosilicate glass for 4 different shear rates (500, 80, 20, and 9s(-1)). The detailed morphology of the collagen fibrils/aggregates was examined using Quick Freeze Deep Etch (QFDE) electron microscopy. Nucleation of fibrils on the glass was observed to occur rapidly (approximately 2 min) followed by continued growth of the fibrils. The growth rates were dependent on flow in a complex manner with the highest rate of axial growth (0.1 micro/s) occurring at a shear rate of 9s(-1). The lowest growth rate occurred at the highest shear. Fibrils were observed to both branch and join during the experiments. The best alignment of fibrils was observed at intermediate shear rates of 20 and 80s(-1). However, the investigation revealed that fibril directional growth was not stable. At high shear rates, fibrils would often turn downstream forming what we term "hooks" which are likely the combined result of monomer interaction with the initial collagen layer or "mat" and the high shear rate. Further, QFDE examination of fibril morphology demonstrated that the assembled fibrillar structure did not possess native D-periodicity. Instead, fibrils comprised a collection of generally aligned, monomers which were self-assembled to form a fibril

  7. Concentration dependence of variability in growth rates of microtubules.

    PubMed Central

    Pedigo, Susan; Williams, Robley C

    2002-01-01

    Growth and shortening of microtubules in the course of their polymerization and depolymerization have previously been observed to occur at variable rates. To gain insight into the meaning of this prominent variability, we studied the way in which its magnitude depends on the growth rate of experimentally observed and computer-simulated microtubules. The dynamic properties of plus-ended microtubules nucleated by pieces of Chlamydomonas flagellar axonemes were observed in real time by video-enhanced differential interference contrast light microscopy at differing tubulin concentrations. By means of a Monte Carlo algorithm, populations of microtubules were simulated that had similar growth and dynamic properties to the experimentally observed microtubules. By comparison of the experimentally observed and computer-simulated populations of microtubules, we found that 1) individual microtubules displayed an intrinsic variability that did not change as the rate of growth for a population increased, and 2) the variability was approximately fivefold greater than predicted by a simple model of subunit addition and loss. The model used to simulate microtubule growth has no provision for incorporation of lattice defects of any type, nor sophisticated geometry of the growing end. Thus, these as well as uncontrolled experimental variables were eliminated as causes for the prominent variability. PMID:12324403

  8. 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.

  9. 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

  10. DNA/Fusogenic Lipid Nanocarrier Assembly: Millisecond Structural Dynamics.

    PubMed

    Angelov, Borislav; Angelova, Angelina; Filippov, Sergey K; Narayanan, Theyencheri; Drechsler, Markus; Štěpánek, Petr; Couvreur, Patrick; Lesieur, Sylviane

    2013-06-01

    Structural changes occurring on a millisecond time scale during uptake of DNA by cationic lipid nanocarriers are monitored by time-resolved small-angle X-ray scattering (SAXS) coupled to a rapid-mixing stopped-flow technique. Nanoparticles (NPs) of nanochannel organization are formed by PEGylation, hydration, and dispersion of a lipid film of the fusogenic lipid monoolein in a mixture with positively charged (DOMA) and PEGylated (DOPE-PEG2000) amphiphiles and are characterized by the inner cubic structure of very large nanochannels favorable for DNA upload. Ultrafast structural dynamics of complexation and assembly of these cubosome particles with neurotrophic plasmid DNA (pDNA) is revealed thanks to the high brightness of the employed synchrotron X-ray beam. The rate constant of the pDNA/lipid NP complexation is estimated from dynamic roentgenograms recorded at 4 ms time resolution. pDNA upload into the vastly hydrated channels of the cubosome carriers leads to a fast nanoparticle-nanoparticle structural transition and lipoplex formation involving tightly packed pDNA. PMID:26283134

  11. Electric field-induced reversible trapping of microtubules along metallic glass microwire electrodes

    NASA Astrophysics Data System (ADS)

    Kim, Kyongwan; Sikora, Aurélien; Nakayama, Koji S.; Umetsu, Mitsuo; Hwang, Wonmuk; Teizer, Winfried

    2015-04-01

    Microtubules are among bio-polymers providing vital functions in dynamic cellular processes. Artificial organization of these bio-polymers is a requirement for transferring their native functions into device applications. Using electrophoresis, we achieve an accumulation of microtubules along a metallic glass (Pd42.5Cu30Ni7.5P20) microwire in solution. According to an estimate based on migration velocities of microtubules approaching the wire, the electrophoretic mobility of microtubules is around 10-12 m2/Vs. This value is four orders of magnitude smaller than the typical mobility reported previously. Fluorescence microscopy at the individual-microtubule level shows microtubules aligning along the wire axis during the electric field-induced migration. Casein-treated electrodes are effective to reversibly release trapped microtubules upon removal of the external field. An additional result is the condensation of secondary filamentous structures from oriented microtubules.

  12. Dynamic self-assembly of non-Brownian spheres studied by molecular dynamics simulations

    NASA Astrophysics Data System (ADS)

    Carvente, O.; Salazar-Cruz, M.; Peñuñuri, F.; Ruiz-Suárez, J. C.

    2016-02-01

    Granular self-assembly of confined non-Brownian spheres under gravity is studied by molecular dynamics simulations. Starting from a disordered phase, dry or cohesive spheres organize, by vibrational annealing, into body-centered-tetragonal or face-centered-cubic structures, respectively. During the self-assembling process, isothermal and isodense points are observed. The existence of such points indicates that both granular temperature and packing fraction undergo an inversion process that may be in the core of crystal nucleation. Around the isothermal point, a sudden growth of granular clusters having the maximum coordination number takes place, indicating the outcome of a first-order phase transition. We propose a heuristic equation that successfully describes the dynamic evolution of the local packing fraction in terms of the local granular temperature, along the entire crystallization process.

  13. Early Spindle Assembly in Drosophila Embryos: Role of a Force Balance Involving Cytoskeletal Dynamics and Nuclear MechanicsD⃞V⃞

    PubMed Central

    Cytrynbaum, E. N.; Sommi, P.; Brust-Mascher, I.; Scholey, J. M.; Mogilner, A.

    2005-01-01

    Mitotic spindle morphogenesis depends upon the action of microtubules (MTs), motors and the cell cortex. Previously, we proposed that cortical- and MT-based motors acting alone can coordinate early spindle assembly in Drosophila embryos. Here, we tested this model using microscopy of living embryos to analyze spindle pole separation, cortical reorganization, and nuclear dynamics in interphase-prophase of cycles 11-13. We observe that actin caps remain flat as they expand and that furrows do not ingress. As centrosomes separate, they follow a linear trajectory, maintaining a constant pole-to-furrow distance while the nucleus progressively deforms along the elongating pole-pole axis. These observations are incorporated into a model in which outward forces generated by zones of active cortical dynein are balanced by inward forces produced by nuclear elasticity and during cycle 13, by Ncd, which localizes to interpolar MTs. Thus, the force-balance driving early spindle morphogenesis depends upon MT-based motors acting in concert with the cortex and nucleus. PMID:16079179

  14. Signaling function of alpha-catenin in microtubule regulation.

    PubMed

    Shtutman, Michael; Chausovsky, Alexander; Prager-Khoutorsky, Masha; Schiefermeier, Natalia; Boguslavsky, Shlomit; Kam, Zvi; Fuchs, Elaine; Geiger, Benjamin; Borisy, Gary G; Bershadsky, Alexander D

    2008-08-01

    Centrosomes control microtubule dynamics in many cell types, and their removal from the cytoplasm leads to a shift from dynamic instability to treadmilling behavior and to a dramatic decrease of microtubule mass (Rodionov et al., 1999; PNAS 96:115). In cadherin-expressing cells, these effects can be reversed:non-centrosomal cytoplasts that form cadherin-mediated adherens junctions display dense arrays of microtubules (Chausovsky et al., 2000; Nature Cell Biol 2:797). In adherens junctions, cadherin's cytoplasmic domain binds p120 catenin and beta-catenin, which in turn binds alpha-catenin. To elucidate the roles of the cadherin-associated proteins in regulating microtubule dynamics, we prepared GFP-tagged, plasma membrane targeted or untargeted p120 catenin, alpha-catenin and beta-catenin and tested their ability to rescue the loss of microtubule mass caused by centrosomal removal in the poorly adhesive cell line CHO-K1. Only membrane targeting of alpha-catenin led to a significant increase in microtubule length and density in centrosome-free cytoplasts. Expression of non-membrane-targeted alpha-catenin produced only a slight effect, while both membrane-targeted and non-targeted p120 and beta-catenin were ineffective in this assay. Together, these findings suggest that alpha-catenin is able to regulate microtubule dynamics in a centrosome-independent manner. PMID:18677116

  15. Signaling function of α-catenin in microtubule regulation

    PubMed Central

    Shtutman, Michael; Chausovsky, Alexander; Prager-Khoutorsky, Masha; Schiefermeier, Natalia; Boguslavsky, Shlomit; Kam, Zvi; Fuchs, Elaine; Geiger, Benjamin; Borisy, Gary G.; Bershadsky, Alexander D.

    2009-01-01

    Centrosomes control microtubule dynamics in many cell types, and their removal from the cytoplasm leads to a shift from dynamic instability to treadmilling behavior and to a dramatic decrease of microtubule mass (Rodionov et al., 1999; PNAS 96:115). In cadherin-expressing cells, these effects can be reversed: non-centrosomal cytoplasts that form cadherin-mediated adherens junctions display dense arrays of microtubules (Chausovsky et al., 2000; Nature Cell Biol 2:797). In adherens junctions, cadherin’s cytoplasmic domain binds p120 catenin and β-catenin, which in turn binds α-catenin. To elucidate the roles of the cadherin-associated proteins in regulating microtubule dynamics, we prepared GFP-tagged, plasma membrane targeted or untargeted p120 catenin, α-catenin and β-catenin and tested their ability to rescue the loss of microtubule mass caused by centrosomal removal in the poorly adhesive cell line CHO-K1. Only membrane targeting of α-catenin led to a significant increase in microtubule length and density in centrosome-free cytoplasts. Expression of non-membrane-targeted α-catenin produced only a slight effect, while both membrane-targeted and non-targeted p120 and β-catenin were ineffective in this assay. Together, these findings suggest that α-catenin is able to regulate microtubule dynamics in a centrosome-independent manner. PMID:18677116

  16. Microtubules and control of macronuclear 'amitosis' in Paramecium.

    PubMed

    Tucker, J B; Beisson, J; Roche, D L; Cohen, J

    1980-08-01

    The 'amitotic' division of the macronucleus during binary fission in P. tetraurelia includes a detailed sequence of shape changes that are temporally coordinated with the adoption of a series of well-defined positions and orientations inside the cell. The deployment of nucleoplasmic microtubules that is spatially correlated with the shaping ritual is more complex and precise than has been reported previously. Macronuclear division is not amitotic. It is not a simple constriction into two halves. As a dividing macronucleus starts to elongate it becomes dorsoventrally flattened against the dorsal cortex of the organism and assumes an elliptical shape. Concurrently, an elliptical marginal band of intranuclear microtubules assembles that has the same spatial relationship to nuclear shape as the marginal microtubules assembles that has the same spatial relationship to nuclear shape as the marginal microtubule bands of certain elliptical vertebrate blood cells have to cell shape. The band breaks down as further elongation occurs and the nucleus adopts the shape of a straight and slender sausage. Most of the intranuclear microtubules assemble as elongation starts and break down shortly after elongation is completed; the majority are oriented parallel to the longitudinal axis of the nucleus throughout elongation. Some of them are attached to nucleoli and are coated with granules which are almost certainly derived from the cortices of nucleoli. The peripheral concentration, interconnexion, orientation, and overlapping arrangement of microtubules, and the reduction in microtubule number per nuclear cross-section as elongation proceeds at a rate of about 40 micrometers min-1, are all compatible with the provision of a microtubule sliding mechanism as the main skeletal basis for elongation. There are indications that this mechanism is augmented by anchorage and/or active propulsion of nucleoli that may perhaps facilitate fairly equitable segregation of chromosomal material to

  17. Kinks and bell-type solitons in microtubules

    NASA Astrophysics Data System (ADS)

    Zdravković, Slobodan; Gligorić, Goran

    2016-06-01

    In the present paper, we study the nonlinear dynamics of microtubules relying on the known u-model. As a mathematical procedure, we use the simplest equation method. We recover some solutions obtained earlier using less general methods. These are kink solitons. In addition, we show that the solution of the crucial differential equation, describing nonlinear dynamics of microtubules, can be a bell-type soliton. The discovery of this new solution is supported by numerical analysis.

  18. Kinks and bell-type solitons in microtubules.

    PubMed

    Zdravković, Slobodan; Gligorić, Goran

    2016-06-01

    In the present paper, we study the nonlinear dynamics of microtubules relying on the known u-model. As a mathematical procedure, we use the simplest equation method. We recover some solutions obtained earlier using less general methods. These are kink solitons. In addition, we show that the solution of the crucial differential equation, describing nonlinear dynamics of microtubules, can be a bell-type soliton. The discovery of this new solution is supported by numerical analysis. PMID:27368766

  19. Differential radiolabeling of opposite microtubule ends: methodology, equilibrium exchange-flux analysis, and drug poisoning

    SciTech Connect

    Jordan, M.A.; Farrell, K.W.

    1983-04-01

    We describe a method which allows opposite microtubule ends to be distinguished by differentially labeling the microtubules with (/sup 3/H)- and (/sup 14/C)guanine nucleotides. Assembly-disassembly reaction at opposite microtubule ends can therefore be measured simultaneously and without modification of the tubulin dimers or microtubules. The method is predicated on experimental observations wich demonstated that net dimer addition to steady-state microtubules mst be predominantly unidirectional. This does not preclude, however, some bidirectional dimer addition to steady-state microtubules by an equilibrium-exchange mechanism. We therefore calculated the relative contribution to dimer incorporation of bidirectional equilibrium exchange in a unidirectional microtubule system (s=0.06). Under our conditions bidirectional dimer incorporation is negligible; net dimer additional to steady-state microtubules is overwhelmingly unidirectional. We used this method to study the effects of colchicine and podophyllotoxin on assembly-disassembly at opposite microtubule ends. Both drugs inhibit substoichiometrically net dimer addition to one microtubule end and, to a lesser extent, net dimer loss from the opposite end.

  20. 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.

  1. Dynamics and Self-Assembly of Nanoparticles on Biomembranes

    NASA Astrophysics Data System (ADS)

    Bhattacharya, Rupak; Maingi, Vishal; Kanchi, Subbarao; Rahul Suresh, Bagul; Jayaraman, N.; Maity, Prabal; Ayappa, K. G.; Basu, Jaydeep

    2013-03-01

    We have recently been investigating the diffusion mediated self-assembly of various types of Dendrimers on supported DMPC lipid bilayer. Atomic Force Microscopy is used to study the pattern formation for PETIM dendrimers of different core composition as well as of generations. Extensive studies have been carried out using different concentration and different packing of lipid molecules constituting the lipid bilayer. Interestingly Oxygen Core dendrimer forms regular circular patterns on membranes whereas the Nitrogen Core dendrimer do not. A fully atomistic Molecular Dynamics simulation with implicit water clearly shows the evidence of domain formation for O-core dendrimers on bilayer, which is absent in the other one. Different generation for Oxygen core dendrimers forms patterns with a pore inside. The reduction of the diameter of these patterns with decreasing packing of lipid molecules indicates the possible role of lipid molecules in aggregation process. Further study using Confocal Fluorescence Correlation Spectroscopy is underway to correlate this type of membrane mediated pattern formation with underlying lipid diffusion. CSIR and DST for financial Support

  2. Dynamic Characterization of Crystalline Supramolecular Rotors Assembled through Halogen Bonding.

    PubMed

    Catalano, Luca; Pérez-Estrada, Salvador; Terraneo, Giancarlo; Pilati, Tullio; Resnati, Giuseppe; Metrangolo, Pierangelo; Garcia-Garibay, Miguel A

    2015-12-16

    A modular molecular kit for the preparation of crystalline molecular rotors was devised from a set of stators and rotators to gain simple access to a large number of structures with different dynamic performance and physical properties. In this work, we have accomplished this with crystalline molecular rotors self-assembled by halogen bonding of diazabicyclo[2.2.2]octane, acting as a rotator, and a set of five fluorine-substituted iodobenzenes that take the role of the stator. Using variable-temperature (1)H T1 spin-lattice relaxation measurements, we have shown that all structures display ultrafast Brownian rotation with activation energies of 2.4-4.9 kcal/mol and pre-exponential factors of the order of (1-9) × 10(12) s(-1). Line shape analysis of quadrupolar echo (2)H NMR measurements in selected examples indicated rotational trajectories consistent with the 3-fold or 6-fold symmetric potential of the rotator. PMID:26583701

  3. Properties of microtubule bundles induced by Glyceraldehyde-3-phosphate dehydrogenase

    NASA Astrophysics Data System (ADS)

    Somers, Marijke; Engelborghs, Yves

    1991-05-01

    The binding of Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; E.C. 1.2.1.12) to microtubules causes the microtubules to assemble into large bundles. This bundling can be considered as a further step in the assembly of supramolecular structures. The rate of bundle formation, after addition of GAPDH to preformed microtubules, is not dependent on the GAPDH concentration and reflects bundling kinetics. Bundle disassembly can be studied by the addition of 1 mM adenosine 5'-(β, -imidotri-phosphate) (AMPPNP) to bundled microtubules, and is extremely fast. Bundling reduces the rate of association of tubulin dimers to microtubules, as well as the dissocation from the microtubles. Both rates are reduced to the same extent. This is in agreement with the fact that the critical concentration of tubulin is practically not influenced by the binding of the enzyme. Adding microtubule associated proteins (at I=0.1 M) does not appreciably influence the affinity for GAPDH, but reduces bundle formation possibly for sterical reasons.

  4. Space shuttling in the cell: nucleocytoplasmic transport and microtubule organization during the cell cycle.

    PubMed

    Sato, Masamitsu; Toda, Takashi

    2010-01-01

    Microtubules form a multifunctional filamentous structure essential for the cell. In interphase, microtubules form networks in the cytoplasm and play pivotal roles in cell polarity and intracellular transport of various biomolecules. In mitosis, microtubules dramatically change their morphology to assemble the mitotic spindle, thereby pulling the chromosomes toward the spindle poles. One long-standing question is how microtubules are reorganized upon mitotic entry. Yeast cells undergo closed mitosis, in which the nuclear envelope persists, whereas higher eukaryotes undergo open mitosis, in which the nuclear envelope breaks down. Microtubule reorganization must be controlled by selective localization of microtubule-assembly factors. Recent findings in fission yeast indicate that several microtubule-associated proteins (MAPs) shuttle between the cytoplasm and the nucleus through regulation by Ran GTPase, the universal organizer of nucleocytoplasmic transport. Furthermore, the synergistic interplay of Ran and cyclin-dependent kinase (CDK) induces the critical spatiotemporal shift of modes in microtubule assembly from cytoplasmic arrays to nuclear spindles. A MAP complex Alp7/TACC-Alp14/TOG undergoes nucleocytoplasmic shuttling in interphase, whereas it is retained in the mitotic nucleus through a decrease of its nuclear export by CDK. Our understanding of how microtubules are reorganized during the cell cycle is beginning to emerge. PMID:21327068

  5. Assembly and actuation of nanomaterials using active biomolecules.

    SciTech Connect

    Spoerke, Erik David; Thayer, Gayle Echo; de Boer, Maarten Pieter; Bunker, Bruce Conrad; Liu, Jun; Corwin, Alex David; Gaudioso, Jennifer Marie; Sasaki, Darryl Yoshio; Boal, Andrew Kiskadden; Bachand, George David; Trent, Amanda M.; Bachand, Marlene; Rivera, Susan B.; Koch, Steven John

    2005-11-01

    The formation and functions of living materials and organisms are fundamentally different from those of synthetic materials and devices. Synthetic materials tend to have static structures, and are not capable of adapting to the functional needs of changing environments. In contrast, living systems utilize energy to create, heal, reconfigure, and dismantle materials in a dynamic, non-equilibrium fashion. The overall goal of the project was to organize and reconfigure functional assemblies of nanoparticles using strategies that mimic those found in living systems. Active assembly of nanostructures was studied using active biomolecules to drive the organization and assembly of nanocomposite materials. In this system, kinesin motor proteins and microtubules were used to direct the transport and interactions of nanoparticles at synthetic interfaces. In addition, the kinesin/microtubule transport system was used to actively assemble nanocomposite materials capable of storing significant elastic energy. Novel biophysical measurement tools were also developed for measuring the collective force generated by kinesin motor proteins, which will provide insight on the mechanical constraints of active assembly processes. Responsive reconfiguration of nanostructures was studied in terms of using active biomolecules to mediate the optical properties of quantum dot (QD) arrays through modulation of inter-particle spacing and associated energy transfer interaction. Design rules for kinesin-based transport of a wide range of nanoscale cargo (e.g., nanocrystal quantum dots, micron-sized polymer spheres) were developed. Three-dimensional microtubule organizing centers were assembled in which the polar orientation of the microtubules was controlled by a multi-staged assembly process. Overall, a number of enabling technologies were developed over the course of this project, and will drive the exploitation of energy-driven processes to regulate the assembly, disassembly, and dynamic

  6. Brownian dynamics simulation of fission yeast mitotic spindle formation

    NASA Astrophysics Data System (ADS)

    Edelmaier, Christopher

    2014-03-01

    The mitotic spindle segregates chromosomes during mitosis. The dynamics that establish bipolar spindle formation are not well understood. We have developed a computational model of fission-yeast mitotic spindle formation using Brownian dynamics and kinetic Monte Carlo methods. Our model includes rigid, dynamic microtubules, a spherical nuclear envelope, spindle pole bodies anchored in the nuclear envelope, and crosslinkers and crosslinking motor proteins. Crosslinkers and crosslinking motor proteins attach and detach in a grand canonical ensemble, and exert forces and torques on the attached microtubules. We have modeled increased affinity for crosslinking motor attachment to antiparallel microtubule pairs, and stabilization of microtubules in the interpolar bundle. We study parameters controlling the stability of the interpolar bundle and assembly of a bipolar spindle from initially adjacent spindle-pole bodies.

  7. Microtubule-associated protein 1B interaction with tubulin tyrosine ligase contributes to the control of microtubule tyrosination.

    PubMed

    Utreras, Elías; Jiménez-Mateos, Eva Maria; Contreras-Vallejos, Erick; Tortosa, Elena; Pérez, Mar; Rojas, Sebastián; Saragoni, Lorena; Maccioni, Ricardo B; Avila, Jesús; González-Billault, Christian

    2008-01-01

    Microtubule-associated protein 1B (MAP1B) is the first microtubule-associated protein to be expressed during nervous system development. MAP1B belongs to a large family of proteins that contribute to the stabilization and/or enhancement of microtubule polymerization. These functions are related to the control of the dynamic properties of microtubules. The C-terminal domain of the neuronal alpha-tubulin isotype is characterized by the presence of an acidic polypeptide, with the last amino acid being tyrosine. This tyrosine residue may be enzymatically removed from the protein by an unknown carboxypeptidase activity. Subsequently, the tyrosine residue is again incorporated into this tubulin by another enzyme, tubulin tyrosine ligase, to yield tyrosinated tubulin. Because neurons lacking MAP1B have a reduced proportion of tyrosinated microtubules, we analyzed the possible interaction between MAP1B and tubulin tyrosine ligase. Our results show that these proteins indeed interact and that the interaction is not affected by MAP1B phosphorylation. Additionally, neurons lacking MAP1B, when exposed to drugs that reversibly depolymerize microtubules, do not fully recover tyrosinated microtubules upon drug removal. These results suggest that MAP1B regulates tyrosination of alpha-tubulin in neuronal microtubules. This regulation may be important for general processes involved in nervous system development such as axonal guidance and neuronal migration. PMID:18075266

  8. Large-scale dissipative particle dynamics simulations of self-assembly amphiphilic systems†

    PubMed Central

    Li, Xuejin; Tang, Yu-Hang

    2014-01-01

    We present large-scale simulation results on the self-assembly of amphiphilic systems in bulk solution and under soft confinement. Self-assembled unilamellar and multilamellar vesicles are formed from amphiphilic molecules in bulk solution. The system is simulated by placing amphiphilic molecules inside large unilamellar vesicles (LUVs) and the dynamic soft confinement-induced self-assembled vesicles are investigated. Moreover, the self-assembly of sickle hemoglobin (HbS) is simulated in a crowded and fluctuating intracellular space and our results demonstrate that the HbS self-assemble into polymer fibers causing the LUV shape to be distorted. PMID:24938634

  9. Electrophoretic dynamics of self-assembling branched DNA structures

    NASA Astrophysics Data System (ADS)

    Heuer, Daniel Milton

    This study advances our understanding of the electrophoretic dynamics of branched biopolymers and explores technologies designed to exploit their unique properties. New self-assembly techniques were developed to create branched DNA for visualization via fluorescence microscopy. Experiments in fixed gel networks reveal a distinct trapping behavior, in contrast with linear topologies. The finding that detection can be achieved by introducing a branch point contributes significantly to the field of separation science and can be exploited to develop new applications. Results obtained in polymer solutions point to identical mobilities for branched and linear topologies, despite large differences in their dynamics. This finding led to a new description of electrophoresis based on non-Newtonian viscoelastic effects in the electric double layer surrounding a charged object. This new theoretical framework presents a new outlook important not only to the electrophoretic physics of nucleic acids, but all charged objects including proteins, colloids, and nanoparticles. To study the behavior of smaller biopolymers, such as restriction fragments and recombination intermediates, a library of symmetrically branched DNA was synthesized followed by characterization in gels. The experimental results contribute a large body of information relating molecular architecture and the dynamics of rigid structures in an electric field. The findings allow us to create new separation technologies based on topology. These contributions can also be utilized in a number of different applications including the study of recombination intermediates and the separation of proteins according to structure. To demonstrate the importance of these findings, a sequence and mutation detection technique was envisioned and applied for genetic analysis. Restriction fragments from mutation "hotspots" in the p53 tumor suppressor gene, known to play a role in cancer development, were analyzed with this technique

  10. Dynamic and Kinetic Assembly Studies of an Icosahedral Virus Capsid

    NASA Astrophysics Data System (ADS)

    Lee, Kelly

    2011-03-01

    Hepatitis B virus has an icosahedrally symmetrical core particle (capsid), composed of either 90 or 120 copies of a dimeric protein building block. We are using time-resolved, solution small-angle X-ray scattering and single-molecule fluorescence microscopy to probe the core particle assembly reaction at the ensemble and individual assembly levels. Our experiments to date reveal the assembly process to be highly cooperative with minimal population of stable intermediate species. Solution conditions, particularly salt concentration, appears to influence the partitioning of assembly products into the two sizes of shells. Funding from NIH R00-GM080352 and University of Washington.

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

    PubMed Central

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

    2013-01-01

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

  12. A theory of microtubule catastrophes and their regulation

    PubMed Central

    Brun, Ludovic; Rupp, Beat; Ward, Jonathan J.; Nédélec, François

    2009-01-01

    Dynamic instability, in which abrupt transitions occur between growing and shrinking states, is an intrinsic property of microtubules that is regulated by both mechanics and specialized proteins. We discuss a model of dynamic instability based on the popular idea that growth is maintained by a cap at the tip of the fiber. The loss of this cap is thought to trigger the transition from growth to shrinkage, called a catastrophe. The model includes longitudinal interactions between the terminal tubulins of each protofilament and “gating rescues” between neighboring protofilaments. These interactions allow individual protofilaments to transiently shorten during a phase of overall microtubule growth. The model reproduces the reported dependency of the catastrophe rate on tubulin concentration, the time between tubulin dilution and catastrophe, and the induction of microtubule catastrophes by walking depolymerases. The model also reproduces the comet tail distribution that is characteristic of proteins that bind to the tips of growing microtubules. PMID:19948965

  13. Invited review: Microtubule severing enzymes couple atpase activity with tubulin GTPase spring loading.

    PubMed

    Bailey, Megan E; Jiang, Nan; Dima, Ruxandra I; Ross, Jennifer L

    2016-08-01

    Microtubules are amazing filaments made of GTPase enzymes that store energy used for their own self-destruction to cause a stochastically driven dynamics called dynamic instability. Dynamic instability can be reproduced in vitro with purified tubulin, but the dynamics do not mimic that observed in cells. This is because stabilizers and destabilizers act to alter microtubule dynamics. One interesting and understudied class of destabilizers consists of the microtubule-severing enzymes from the ATPases Associated with various cellular Activities (AAA+) family of ATP-enzymes. Here we review current knowledge about GTP-driven microtubule dynamics and how that couples to ATP-driven destabilization by severing enzymes. We present a list of challenges regarding the mechanism of severing, which require development of experimental and modeling approaches to shed light as to how severing enzymes can act to regulate microtubule dynamics in cells. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 547-556, 2016. PMID:27037673

  14. CYLD - a deubiquitylase that acts to fine-tune microtubule properties and functions.

    PubMed

    Yang, Yunfan; Zhou, Jun

    2016-06-15

    Microtubules are dynamic structures that are crucially involved in a variety of cellular activities. The dynamic properties and functions of microtubules are regulated by various factors, such as tubulin isotype composition and microtubule-binding proteins. Initially identified as a deubiquitylase with tumor-suppressing functions, the protein cylindromatosis (CYLD) has recently been revealed to interact with microtubules, modulate microtubule dynamics, and participate in the regulation of cell migration, cell cycle progression, chemotherapeutic drug sensitivity and ciliogenesis. These findings have greatly enriched our understanding of the roles of CYLD in physiological and pathological conditions. Here, we focus on recent literature that shows how CYLD impacts on microtubule properties and functions in various biological processes, and discuss the challenges we face when interpreting results obtained from different experimental systems. PMID:27173491

  15. The Structure and Dynamics of Higher-Order Assemblies: Amyloids, Signalosomes, and Granules.

    PubMed

    Wu, Hao; Fuxreiter, Monika

    2016-05-19

    We here attempt to achieve an integrated understanding of the structure and dynamics of a number of higher-order assemblies, including amyloids, various kinds of signalosomes, and cellular granules. We propose that the synergy between folded domains, linear motifs, and intrinsically disordered regions regulates the formation and intrinsic fuzziness of all higher-order assemblies, creating a structural and dynamic continuum. We describe how such regulatory mechanisms could be influenced under pathological conditions. PMID:27203110

  16. 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

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

    SciTech Connect

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

    2012-02-01

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

  18. Spatial organization of the Ran pathway by microtubules in mitosis

    PubMed Central

    Oh, Doogie; Yu, Che-Hang; Needleman, Daniel J.

    2016-01-01

    Concentration gradients of soluble proteins are believed to be responsible for control of morphogenesis of subcellular systems, but the mechanisms that generate the spatial organization of these subcellular gradients remain poorly understood. Here, we use a newly developed multipoint fluorescence fluctuation spectroscopy technique to study the ras-related nuclear protein (Ran) pathway, which forms soluble gradients around chromosomes in mitosis and is thought to spatially regulate microtubule behaviors during spindle assembly. We found that the distribution of components of the Ran pathway that influence microtubule behaviors is determined by their interactions with microtubules, resulting in microtubule nucleators being localized by the microtubules whose formation they stimulate. Modeling and perturbation experiments show that this feedback makes the length of the spindle insensitive to the length scale of the Ran gradient, allows the spindle to assemble outside the peak of the Ran gradient, and explains the scaling of the spindle with cell size. Such feedback between soluble signaling pathways and the mechanics of the cytoskeleton may be a general feature of subcellular organization. PMID:27439876

  19. Spatial organization of the Ran pathway by microtubules in mitosis.

    PubMed

    Oh, Doogie; Yu, Che-Hang; Needleman, Daniel J

    2016-08-01

    Concentration gradients of soluble proteins are believed to be responsible for control of morphogenesis of subcellular systems, but the mechanisms that generate the spatial organization of these subcellular gradients remain poorly understood. Here, we use a newly developed multipoint fluorescence fluctuation spectroscopy technique to study the ras-related nuclear protein (Ran) pathway, which forms soluble gradients around chromosomes in mitosis and is thought to spatially regulate microtubule behaviors during spindle assembly. We found that the distribution of components of the Ran pathway that influence microtubule behaviors is determined by their interactions with microtubules, resulting in microtubule nucleators being localized by the microtubules whose formation they stimulate. Modeling and perturbation experiments show that this feedback makes the length of the spindle insensitive to the length scale of the Ran gradient, allows the spindle to assemble outside the peak of the Ran gradient, and explains the scaling of the spindle with cell size. Such feedback between soluble signaling pathways and the mechanics of the cytoskeleton may be a general feature of subcellular organization. PMID:27439876

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

    PubMed Central

    Hutchins, B. Ian; Wray, Susan

    2014-01-01

    Microtubules are a critical part of neuronal polarity and leading process extension, thus microtubule movement plays an important role in neuronal migration. However, the dynamics of microtubules during the forward movement of the nucleus into the leading process (nucleokinesis) is unclear and may be dependent on the cell type and mode of migration used. In particular, little is known about cytoskeletal changes during axophilic migration, commonly used in anteroposterior neuronal migration. We recently showed that leading process actin flow in migrating GnRH neurons is controlled by a signaling cascade involving IP3 receptors, CaMKK, AMPK, and RhoA. In the present study, microtubule dynamics were examined in GnRH neurons. Failure of the migration of these cells leads to the neuroendocrine disorder Kallmann Syndrome. Microtubules translocated forward along the leading process shaft during migration, but reversed direction and moved toward the nucleus when migration stalled. Blocking calcium release through IP3 receptors halted migration and induced the same reversal of microtubule translocation, while blocking cortical actin flow prevented microtubules from translocating toward the distal leading process. Super-resolution imaging revealed that microtubule plus-end tips are captured at the actin cortex through calcium-dependent mechanisms. This work shows that cortical actin flow draws the microtubule network forward through calcium-dependent capture in order to promote nucleokinesis, revealing a novel mechanism engaged by migrating neurons to facilitate movement. PMID:25505874

  1. 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

  2. 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

  3. 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

  4. The γ-tubulin-specific inhibitor gatastatin reveals temporal requirements of microtubule nucleation during the cell cycle

    PubMed Central

    Chinen, Takumi; Liu, Peng; Shioda, Shuya; Pagel, Judith; Cerikan, Berati; Lin, Tien-chen; Gruss, Oliver; Hayashi, Yoshiki; Takeno, Haruka; Shima, Tomohiro; Okada, Yasushi; Hayakawa, Ichiro; Hayashi, Yoshio; Kigoshi, Hideo; Usui, Takeo; Schiebel, Elmar

    2015-01-01

    Inhibitors of microtubule (MT) assembly or dynamics that target α/β-tubulin are widely exploited in cancer therapy and biological research. However, specific inhibitors of the MT nucleator γ-tubulin that would allow testing temporal functions of γ-tubulin during the cell cycle are yet to be identified. By evolving β-tubulin-binding drugs we now find that the glaziovianin A derivative gatastatin is a γ-tubulin-specific inhibitor. Gatastatin decreased interphase MT dynamics of human cells without affecting MT number. Gatastatin inhibited assembly of the mitotic spindle in prometaphase. Addition of gatastatin to preformed metaphase spindles altered MT dynamics, reduced the number of growing MTs and shortened spindle length. Furthermore, gatastatin prolonged anaphase duration by affecting anaphase spindle structure, indicating the continuous requirement of MT nucleation during mitosis. Thus, gatastatin facilitates the dissection of the role of γ-tubulin during the cell cycle and reveals the sustained role of γ-tubulin. PMID:26503935

  5. Approach to Dynamic Assembling of Individualized Learning Paths

    ERIC Educational Resources Information Center

    Lubchak, Vladimir; Kupenko, Olena; Kuzikov, Borys

    2012-01-01

    E-learning students are generally heterogeneous and have different capabilities knowledge base and needs. The aim of the Sumy State University (SSU) e-learning system project is to cater to these individual needs by assembling individual learning path. This paper shows current situation with e-learning in Ukraine, state-of-art of development of…

  6. Structural basis for the association of MAP6 protein with microtubules and its regulation by calmodulin.

    PubMed

    Lefèvre, Julien; Savarin, Philippe; Gans, Pierre; Hamon, Loïc; Clément, Marie-Jeanne; David, Marie-Odile; Bosc, Christophe; Andrieux, Annie; Curmi, Patrick A

    2013-08-23

    Microtubules are highly dynamic αβ-tubulin polymers. In vitro and in living cells, microtubules are most often cold- and nocodazole-sensitive. When present, the MAP6/STOP family of proteins protects microtubules from cold- and nocodazole-induced depolymerization but the molecular and structure determinants by which these proteins stabilize microtubules remain under debate. We show here that a short protein fragment from MAP6-N, which encompasses its Mn1 and Mn2 modules (MAP6(90-177)), recapitulates the function of the full-length MAP6-N protein toward microtubules, i.e. its ability to stabilize microtubules in vitro and in cultured cells in ice-cold conditions or in the presence of nocodazole. We further show for the first time, using biochemical assays and NMR spectroscopy, that these effects result from the binding of MAP6(90-177) to microtubules with a 1:1 MAP6(90-177):tubulin heterodimer stoichiometry. NMR data demonstrate that the binding of MAP6(90-177) to microtubules involve its two Mn modules but that a single one is also able to interact with microtubules in a closely similar manner. This suggests that the Mn modules represent each a full microtubule binding domain and that MAP6 proteins may stabilize microtubules by bridging tubulin heterodimers from adjacent protofilaments or within a protofilament. Finally, we demonstrate that Ca(2+)-calmodulin competes with microtubules for MAP6(90-177) binding and that the binding mode of MAP6(90-177) to microtubules and Ca(2+)-calmodulin involves a common stretch of amino acid residues on the MAP6(90-177) side. This result accounts for the regulation of microtubule stability in cold condition by Ca(2+)-calmodulin. PMID:23831686

  7. Quantifying cadherin mechanotransduction machinery assembly/disassembly dynamics using fluorescence covariance analysis

    PubMed Central

    Vedula, Pavan; Cruz, Lissette A.; Gutierrez, Natasha; Davis, Justin; Ayee, Brian; Abramczyk, Rachel; Rodriguez, Alexis J.

    2016-01-01

    Quantifying multi-molecular complex assembly in specific cytoplasmic compartments is crucial to understand how cells use assembly/disassembly of these complexes to control function. Currently, biophysical methods like Fluorescence Resonance Energy Transfer and Fluorescence Correlation Spectroscopy provide quantitative measurements of direct protein-protein interactions, while traditional biochemical approaches such as sub-cellular fractionation and immunoprecipitation remain the main approaches used to study multi-protein complex assembly/disassembly dynamics. In this article, we validate and quantify multi-protein adherens junction complex assembly in situ using light microscopy and Fluorescence Covariance Analysis. Utilizing specific fluorescently-labeled protein pairs, we quantified various stages of adherens junction complex assembly, the multiprotein complex regulating epithelial tissue structure and function following de novo cell-cell contact. We demonstrate: minimal cadherin-catenin complex assembly in the perinuclear cytoplasm and subsequent localization to the cell-cell contact zone, assembly of adherens junction complexes, acto-myosin tension-mediated anchoring, and adherens junction maturation following de novo cell-cell contact. Finally applying Fluorescence Covariance Analysis in live cells expressing fluorescently tagged adherens junction complex proteins, we also quantified adherens junction complex assembly dynamics during epithelial monolayer formation. PMID:27357130

  8. Getting a Grip on Microtubules.

    PubMed

    Schaletzky, Julia; Rape, Michael

    2016-02-25

    Posttranslational modifications control microtubule behavior, yet assigning roles to particular signals was hampered by lack of defined in vitro systems. In this issue of Cell, Valenstein and Roll-Mecak establish a biochemical platform to interrogate consequences of microtubule polyglutamylation, thereby providing important insights into the specificity and quantitative nature of cellular information transfer. PMID:26919420

  9. 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

  10. Dynamic spectrin/ankyrin-G microdomains promote lateral membrane assembly by opposing endocytosis

    PubMed Central

    Jenkins, Paul M.; He, Meng; Bennett, Vann

    2015-01-01

    Current physical models for plasma membranes emphasize dynamic 10- to 300-nm compartments at thermodynamic equilibrium but subject to thermal fluctuations. However, epithelial lateral membranes contain micrometer-sized domains defined by an underlying membrane skeleton composed of spectrin and its partner ankyrin-G. We demonstrate that these spectrin/ankyrin-G domains exhibit local microtubule-dependent movement on a time scale of minutes and encounter most of the lateral membranes within an hour. Spectrin/ankyrin-G domains exclude clathrin and clathrin-dependent cargo, and inhibit both receptor-mediated and bulk endocytosis. Moreover, inhibition of endocytosis fully restores lateral membrane height in spectrin- or ankyrin-G–depleted cells. These findings support a non-equilibrium cellular-scale model for epithelial lateral membranes, where spectrin/ankyrin-G domains actively patrol the plasma membrane, analogous to “window washers,” and promote columnar morphology by blocking membrane uptake. PMID:26523289

  11. 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

  12. 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.

  13. Microtubule self-organization is gravity-dependent

    PubMed Central

    Papaseit, Cyril; Pochon, Nathalie; Tabony, James

    2000-01-01

    Although weightlessness is known to affect living cells, the manner by which this occurs is unknown. Some reaction-diffusion processes have been theoretically predicted as being gravity-dependent. Microtubules, a major constituent of the cellular cytoskeleton, self-organize in vitro by way of reaction-diffusion processes. To investigate how self-organization depends on gravity, microtubules were assembled under low gravity conditions produced during space flight. Contrary to the samples formed on an in-flight 1 × g centrifuge, the samples prepared in microgravity showed almost no self-organization and were locally disordered. PMID:10880562

  14. Large-scale dynamic assembly of metal nanostructures in plasmofluidic field.

    PubMed

    Patra, Partha Pratim; Chikkaraddy, Rohit; Thampi, Sreeja; Tripathi, Ravi P N; Kumar, G V Pavan

    2016-04-12

    We discuss two aspects of the plasmofluidic assembly of plasmonic nanostructures at the metal-fluid interface. First, we experimentally show how three and four spot evanescent-wave excitation can lead to unconventional assembly of plasmonic nanoparticles at the metal-fluid interface. We observed that the pattern of assembly was mainly governed by the plasmon interference pattern at the metal-fluid interface, and further led to interesting dynamic effects within the assembly. The interference patterns were corroborated by 3D finite-difference time-domain simulations. Secondly, we show how anisotropic geometry, such as Ag nanowires, can be assembled and aligned in unstructured and structured plasmofluidic fields. We found that by structuring the metal-film, Ag nanowires can be aligned at the metal-fluid interface with a single evanescent-wave excitation, thus highlighting the prospect of assembling plasmonic circuits in a fluid. An interesting aspect of our method is that we obtain the assembly at locations away from the excitation points, thus leading to remote assembly of nanostructures. The results discussed herein may have implications in realizing a platform for reconfigurable plasmonic metamaterials, and a test-bed to understand the effect of plasmon interference on assembly of nanostructures in fluids. PMID:26765282

  15. Dynamic self-assembly of microscale rotors and swimmers

    NASA Astrophysics Data System (ADS)

    Davies Wykes, Megan S.; Palacci, Jérémie; Adachi, Takuji; Ristroph, Leif; Zhong, Xiao; Ward, Michael D.; Zhang, Jun; Shelley, Michael J.

    Biological systems often involve the self-assembly of basic components into complex and function- ing structures. Artificial systems that mimic such processes can provide a well-controlled setting to explore the principles involved and also synthesize useful micromachines. Our experiments show that immotile, but active, components self-assemble into two types of structure that exhibit the fundamental forms of motility: translation and rotation. Specifically, micron-scale metallic rods are designed to induce extensile surface flows in the presence of a chemical fuel; these rods interact with each other and pair up to form either a swimmer or a rotor. Such pairs can transition reversibly be- tween these two configurations, leading to kinetics reminiscent of bacterial run-and-tumble motion.

  16. Dynamic self-assembly of microscale rotors and swimmers.

    PubMed

    Davies Wykes, Megan S; Palacci, Jérémie; Adachi, Takuji; Ristroph, Leif; Zhong, Xiao; Ward, Michael D; Zhang, Jun; Shelley, Michael J

    2016-05-18

    Biological systems often involve the self-assembly of basic components into complex and functioning structures. Artificial systems that mimic such processes can provide a well-controlled setting to explore the principles involved and also synthesize useful micromachines. Our experiments show that immotile, but active, components self-assemble into two types of structure that exhibit the fundamental forms of motility: translation and rotation. Specifically, micron-scale metallic rods are designed to induce extensile surface flows in the presence of a chemical fuel; these rods interact with each other and pair up to form either a swimmer or a rotor. Such pairs can transition reversibly between these two configurations, leading to kinetics reminiscent of bacterial run-and-tumble motion. PMID:27121100

  17. Hydrolysis of GTP associated with the formation of tubulin oligomers is involved in microtubule nucleation.

    PubMed Central

    Carlier, M F; Didry, D; Pantaloni, D

    1997-01-01

    Hydrolysis of GTP is known to accompany microtubule assembly. Here we show that hydrolysis of GTP is also associated with the formation of linear oligomers of tubulin, which are precursors (prenuclei) in microtubule assembly. The hydrolysis of GTP on these linear oligomers inhibits the lateral association of GTP-tubulin that leads to the formation of a bidimensional lattice. Therefore GTP hydrolysis interferes with the nucleation of microtubules. Linear oligomers are also formed in mixtures of GTP-tubulin and GDP-tubulin. The hydrolysis of GTP associated with heterologous interactions between GTP-tubulin and GDP-tubulin in the cooligomer takes place at a threefold faster rate than upon homologous interactions between GTP-tubulins. The implication of these results in a model of vectorial GTP hydrolysis in microtubule assembly is discussed. Images FIGURE 7 PMID:9199805

  18. Emergence of reconfigurable wires and spinners via dynamic self-assembly

    PubMed Central

    Kokot, Gasper; Piet, David; Whitesides, George M.; Aranson, Igor S.; Snezhko, Alexey

    2015-01-01

    Dissipative colloidal materials use energy to generate and maintain structural complexity. The energy injection rate, and properties of the environment are important control parameters that influence the outcome of dynamic self-assembly. Here we demonstrate that dispersions of magnetic microparticles confined at the air-liquid interface, and energized by a uniaxial in-plane alternating magnetic field, self-assemble into a variety of structures that range from pulsating clusters and single-particle-thick wires to dynamic arrays of spinners (self-assembled short chains) rotating in either direction. The spinners emerge via spontaneous breaking of the uniaxial symmetry of the energizing magnetic field. Demonstration of the formation and disaggregation of particle assemblies suggests strategies to form new meso-scale structures with the potential to perform functions such as mixing and sensing. PMID:25810144

  19. Emergence of reconfigurable wires and spinners via dynamic self-assembly

    DOE PAGESBeta

    Kokot, Gasper; Piet, David; Whitesides, George M.; Aranson, Igor S.; Snezhko, Alexey

    2015-03-26

    Dissipative colloidal materials use energy to generate and maintain structural complexity. The energy injection rate, and properties of the environment are important control parameters that influence the outcome of dynamic self-assembly. Here we demonstrate that dispersions of magnetic microparticles confined at the air-liquid interface, and energized by a uniaxial in-plane alternating magnetic field, self-assemble into a variety of structures that range from pulsating clusters and single-particle-thick wires to dynamic arrays of spinners (self-assembled short chains) rotating in either direction. The spinners emerge via spontaneous breaking of the uniaxial symmetry of the energizing magnetic field. Demonstration of the formation and disaggregationmore » of particle assemblies suggests strategies to form new meso-scale structures with the potential to perform functions such as mixing and sensing.« less

  20. Emergence of reconfigurable wires and spinners via dynamic self-assembly

    SciTech Connect

    Kokot, Gasper; Piet, David; Whitesides, George M.; Aranson, Igor S.; Snezhko, Alexey

    2015-03-26

    Dissipative colloidal materials use energy to generate and maintain structural complexity. The energy injection rate, and properties of the environment are important control parameters that influence the outcome of dynamic self-assembly. Here we demonstrate that dispersions of magnetic microparticles confined at the air-liquid interface, and energized by a uniaxial in-plane alternating magnetic field, self-assemble into a variety of structures that range from pulsating clusters and single-particle-thick wires to dynamic arrays of spinners (self-assembled short chains) rotating in either direction. The spinners emerge via spontaneous breaking of the uniaxial symmetry of the energizing magnetic field. Demonstration of the formation and disaggregation of particle assemblies suggests strategies to form new meso-scale structures with the potential to perform functions such as mixing and sensing.

  1. "Cloud" assemblies: quantum dots form electrostatically bound dynamic nebulae around large gold nanoparticles.

    PubMed

    Lilly, G Daniel; Lee, Jaebeom; Kotov, Nicholas A

    2010-10-14

    Dynamic self-assembled structures of nanoparticles can be produced using predominantly electrostatic interactions. Such assemblies were made from large, positively charged Au metal nanoparticles surrounded by an electrostatically bound cloud of smaller, negatively charged CdSe/ZnS or CdTe quantum dots. At low concentrations they are topologically similar to double electric layers of ions and corona-like assemblies linked by polymer chains. They can also be compared to the topological arrangement of some planetary systems in space. The great advantages of the cloud assemblies are (1) their highly dynamic nature compared to more rigid covalently bound assemblies, (2) simplicity of preparation, and (3) exceptional versatility in components and resulting optical properties. Photoluminescence intensity enhancement originating from quantum resonance between excitons and plasmons was observed for CdSe/ZnS quantum dots, although CdTe dots displayed emission quenching. To evaluate more attentively their dynamic behavior, emission data were collected for the cloud-assemblies with different ratios of the components and ionic strengths of the media. The emission of the system passes through a maximum for 80 QDs ∶ 1 Au NP as determined by the structure of the assemblies and light absorption conditions. Ionic strength dependence of luminescence intensity contradicts the predictions based on the Gouy-Chapman theory and osmotic pressure at high ionic strengths due to formation of larger chaotic colloidally stable assemblies. "Cloud" assemblies made from different nanoscale components can be used both for elucidation of most fundamental aspects of nanoparticle interactions, as well as for practical purposes in sensing and biology. PMID:20672150

  2. “Cloud” assemblies: quantum dots form electrostatically bound dynamic nebulae around large gold nanoparticle

    SciTech Connect

    Lilly, G. Daniel; Lee, Jaebeom; Kotov, Nicholas A.

    2010-07-29

    Dynamic self-assembled structures of nanoparticles can be produced using predominantly electrostatic interactions. Such assemblies were made from large, positively charged Au metal nanoparticles surrounded by an electrostatically bound cloud of smaller, negatively charged CdSe/ZnS or CdTe quantum dots. At low concentrations they are topologically similar to double electric layers of ions and corona-like assemblies linked by polymer chains. They can also be compared to the topological arrangement of some planetary systems in space. The great advantages of the cloud assemblies are (1) their highly dynamic nature compared to more rigid covalently bound assemblies, (2) simplicity of preparation, and (3) exceptional versatility in components and resulting optical properties. Photoluminescence intensity enhancement originating from quantum resonance between excitons and plasmons was observed for CdSe/ZnS quantum dots, although CdTe dots displayed emission quenching. To evaluate more attentively their dynamic behavior, emission data were collected for the cloud-assemblies with different ratios of the components and ionic strengths of the media. The emission of the system passes through a maximum for 80 QDs:1 Au NP as determined by the structure of the assemblies and light absorption conditions. Ionic strength dependence of luminescence intensity contradicts the predictions based on the Gouy–Chapman theory and osmotic pressure at high ionic strengths due to formation of larger chaotic colloidally stable assemblies. “Cloud” assemblies made from different nanoscale components can be used both for elucidation of most fundamental aspects of nanoparticle interactions, as well as for practical purposes in sensing and biology.

  3. The peroxisomal multifunctional protein interacts with cortical microtubules in plant cells

    PubMed Central

    2005-01-01

    Background The plant peroxisomal multifunctional protein (MFP) possesses up to four enzymatic activities that are involved in catalyzing different reactions of fatty acid β-oxidation in the peroxisome matrix. In addition to these peroxisomal activities, in vitro assays revealed that rice MFP possesses microtubule- and RNA-binding activities suggesting that this protein also has important functions in the cytosol. Results We demonstrate that MFP is an authentic microtubule-binding protein, as it localized to the cortical microtubule array in vivo, in addition to its expected targeting to the peroxisome matrix. MFP does not, however, interact with the three mitotic microtubule arrays. Microtubule co-sedimentation assays of truncated versions of MFP revealed that multiple microtubule-binding domains are present on the MFP polypeptide. This indicates that these regions function together to achieve high-affinity binding of the full-length protein. Real-time imaging of a transiently expressed green fluorescent protein-MFP chimera in living plant cells illustrated that a dynamic, spatial interaction exits between peroxisomes and cortical microtubules as peroxisomes move along actin filaments or oscillate at fixed locations. Conclusion Plant MFP is associated with the cortical microtubule array, in addition to its expected localization in the peroxisome. This observation, coupled with apparent interactions that frequently occur between microtubules and peroxisomes in the cell cortex, supports the hypothesis that MFP is concentrated on microtubules in order to facilitate the regulated import of MFP into peroxisomes. PMID:16313672

  4. 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

  5. Microtubule organization by the antagonistic mitotic motors kinesin-5 and kinesin-14

    PubMed Central

    Hentrich, Christian

    2010-01-01

    During cell division, different molecular motors act synergistically to rearrange microtubules. Minus end–directed motors are thought to have a dual role: focusing microtubule ends to poles and establishing together with plus end–directed motors a balance of force between antiparallel microtubules in the spindle. We study here the competing action of Xenopus laevis kinesin-14 and -5 in vitro in situations in which these motors with opposite directionality cross-link and slide microtubules. We find that full-length kinesin-14 can form microtubule asters without additional factors, whereas kinesin-5 does not, likely reflecting an adaptation to mitotic function. A stable balance of force is not established between two antiparallel microtubules with these motors. Instead, directional instability is generated, promoting efficient motor and microtubule sorting. A nonmotor microtubule cross-linker can suppress directional instability but also impedes microtubule sorting, illustrating a conflict between stability and dynamicity of organization. These results establish the basic organizational properties of these antagonistic mitotic motors and a microtubule bundler. PMID:20439998

  6. 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

  7. Numerical simulation of gas dynamics and heat exchange tasks in fuel assemblies of the nuclear reactors

    SciTech Connect

    Zhuchenko, S. V.

    2014-11-12

    This report presents a PC-based program for solution gas dynamics and heat exchange mathematical tasks in fuel assemblies of the fast-neutron nuclear reactors. A fuel assembly consisting of bulk heat-generating elements, which are integrated together by the system of supply and pressure manifolds, is examined. Spherical heat-generating microelements, which contain nuclear fuel, are pulled into the heat-generating elements. Gaseous coolant proceed from supply manifolds to heat-generating elements, where it withdraws the nuclear reaction heat and assembles in pressure manifolds.

  8. Network-selectivity and stimulus-discrimination in the primary visual cortex: cell-assembly dynamics.

    PubMed

    Bharmauria, Vishal; Bachatene, Lyes; Cattan, Sarah; Brodeur, Simon; Chanauria, Nayan; Rouat, Jean; Molotchnikoff, Stéphane

    2016-01-01

    Visual neurons coordinate their responses in relation to the stimulus; however, the complex interplay between a stimulus and the functional dynamics of an assembly still eludes neuroscientists. To this aim, we recorded cell assemblies from multi-electrodes in the primary visual cortex of anaesthetized cats in response to randomly presented sine-wave drifting gratings whose orientation tilted in 22.5° steps. Cross-correlograms revealed the functional connections at all the tested orientations. We show that a cell-assembly discriminates between orientations by recruiting a 'salient' functional network at every presented orientation, wherein the connections and their strengths (peak-probabilities in the cross-correlogram) change from one orientation to another. Within these assemblies, closely tuned neurons exhibited increased connectivity and connection-strengths compared with differently tuned neurons. Minimal connectivity between untuned neurons suggests the significance of neuronal selectivity in assemblies. This study reflects upon the dynamics of functional connectivity, and brings to the fore the importance of a 'signature' functional network in an assembly that is strictly related to a specific stimulus. It appears that an assembly is the major 'functional unit' of information processing in cortical circuits, rather than the individual neurons. PMID:26469525

  9. Motile Microbots from Dynamically Interacting and Self-Reconfiguring Assemblies of Metallo-Dielectric Janus Microcubes

    NASA Astrophysics Data System (ADS)

    Han, Koohee; Shields, C. Wyatt, IV; Bharti, Bhuvnesh; Lopez, Gabriel P.; Velev, Orlin D.

    A new class of dynamically and reversibly reconfigurable active matter made by magnetic assembly and actuation of metallo-dielectric microcubes will be presented. We describe how magnetically responsive Janus microcubes can be assembled hierarchically into dynamically reconfiguring microclusters. Ferromagnetic cobalt patches of the cubes act as assembly directors. The residual magnetic polarization of the metal-coated facets leads to directional dipole-dipole and field-dipole interactions and reconfiguration of the neighboring cubic particles, which is directed by the conformational restrictions. Dynamic reconfiguration of assembled clusters can be achieved by on-demand switching between the dipole-field interaction and the residual dipole-dipole interaction when the field is turned on and off. We show how pre-assembled Janus microcube clusters can be directionally motile in non-Newtonian fluids by applying asymmetric magnetic fields. The modulation of the viscosity of non-Newtonian fluids upon varying the shear rate allowed demonstrating directional motion, resulting from time-asymmetric stroke patterns (e.g., rapid opening and slow closing). These motile clusters can serve as early prototypes of self-propelling microswimmers capable of in-situ assembly. NSF Grant #DMR-1121107.

  10. Performance evaluation of dynamic assembly period algorithm in TCP over OBS networks

    NASA Astrophysics Data System (ADS)

    Peng, Shuping; Li, Zhengbin; He, Yongqi; Xu, Anshi

    2007-11-01

    Dynamic Assembly Period (DAP) is a novel assembly algorithm, which is based on the dynamic TCP window. The assembly algorithm can track the variation of the current TCP window aroused by the burst loss events, and update the assembly period dynamically for the next assembly. The analytical model provides the theoretical foundation for the proposed assembly algorithm. Nowadays, there are several kinds of TCP flavors proposed to enhance the performance of TCP, such as Default, Tahoe, Reno, New Reno, SACK, etc., which are adopted in the current internet. In this paper, we evaluated the performance of DAP under the different TCP flavors. The simulation results show that the performance of DAP under Default TCP flavor is the best. The difference in the performance of DAP under such flavors is correlated with the inside mechanism of the flavors. We also compared the performance of DAP and FAP under the same TCP flavor. It indicates that the performance of DAP is better than that of FAP in a wide range of burst loss rate.

  11. 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

  12. Down the Rabbit Hole of Centromere Assembly and Dynamics

    PubMed Central

    Dalal, Yamini; Bui, Minh

    2010-01-01

    The centromere is perhaps the most iconic feature on a eukaryotic chromosome. An amateur enthusiast equipped with a light microscope can easily identify the center of each metacentric chromosome, marking the spot responsible for accurate genome segregation. This review will highlight findings which provide novel insights into how centromeres are assembled and disassembled, the role centromeric proteins play in repair, epigenetic features uniquely found at the centromere, and the three dimensional organization of centromeres caught in the act of mitosis. These advances have unveiled a veritable wonderland of non-canonical features that drive centromere function. PMID:20303726

  13. Dynamic characteristics of an assembly of prop-fan blades

    NASA Technical Reports Server (NTRS)

    Srinivasan, A. V.; Kielb, R. E.; Lawrence, C.

    1986-01-01

    In contrast to conventional propellers, propfan blades are thin and highly swept-back, thereby giving rise to large bending and twisting deformations and complex vibratory characteristics. Aerodynamic performance depends on the extent of steady state deformation, and the aeroelastic response depends on the vibratory frequency and mode shape. Attention is presently given to the principal results of structural analyses for a five-bladed propfan assembly; these results are compared with test data. The results encompass both steady deformations and vibratory frequencies and mode shapes in a vacuum centrifugal environment.

  14. Viscosity Control of the Dynamic Self-Assembly in Ferromagnetic Suspensions

    NASA Astrophysics Data System (ADS)

    Piet, D. L.; Straube, A. V.; Snezhko, A.; Aranson, I. S.

    2013-05-01

    Recent studies of dynamic self-assembly in ferromagnetic colloids suspended in liquid-air or liquid-liquid interfaces revealed a rich variety of dynamic structures ranging from linear snakes to axisymmetric asters, which exhibit novel morphology of the magnetic ordering accompanied by large-scale hydrodynamic flows. Based on controlled experiments and first principles theory, we argue that the transition from snakes to asters is governed by the viscosity of the suspending liquid where less viscous liquids favor snakes and more viscous, asters. By obtaining analytic solutions of the time-averaged Navier-Stokes equations, we gain insight into the role of mean hydrodynamic flows and an overall balance of forces governing the self-assembly. Our results illustrate that the viscosity can be used to control the outcome of the dynamic self-assembly in magnetic colloidal suspensions.

  15. Viscosity control of the dynamic self-assembly in ferromagnetic suspensions.

    PubMed

    Piet, D L; Straube, A V; Snezhko, A; Aranson, I S

    2013-05-10

    Recent studies of dynamic self-assembly in ferromagnetic colloids suspended in liquid-air or liquid-liquid interfaces revealed a rich variety of dynamic structures ranging from linear snakes to axisymmetric asters, which exhibit novel morphology of the magnetic ordering accompanied by large-scale hydrodynamic flows. Based on controlled experiments and fir