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Sample records for kinesin-dependent microtubule motility

  1. Chlorpyrifos, chlorpyrifos-oxon, and diisopropylfluorophosphate inhibit kinesin-dependent microtubule motility

    SciTech Connect

    Gearhart, Debra A. . E-mail: dgearhar@mcg.edu; Sickles, Dale W.; Buccafusco, Jerry J.; Prendergast, Mark A.; Terry, Alvin V.

    2007-01-01

    Diisopropylfluorophosphate, originally developed as a chemical warfare agent, is structurally similar to nerve agents, and chlorpyrifos has extensive worldwide use as an agricultural pesticide. While inhibition of cholinesterases underlies the acute toxicity of these organophosphates, we previously reported impaired axonal transport in the sciatic nerves from rats treated chronically with subthreshold doses of chlorpyrifos. Those data indicate that chlorpyrifos (and/or its active metabolite, chlorpyrifos-oxon) might directly affect the function of kinesin and/or microtubules-the principal proteins that mediate anterograde axonal transport. The current report describes in vitro assays to assess the concentration-dependent effects of chlorpyrifos (0-10 {mu}M), chlorpyrifos-oxon (0-10 {mu}M), and diisopropylfluorophosphate (0-0.59 nM) on kinesin-dependent microtubule motility. Preincubating bovine brain microtubules with the organophosphates did not alter kinesin-mediated microtubule motility. In contrast, preincubation of bovine brain kinesin with diisopropylfluorophosphate, chlorpyrifos, or chlorpyrifos-oxon produced a concentration-dependent increase in the number of locomoting microtubules that detached from the kinesin-coated glass cover slip. Our data suggest that the organophosphates-chlorpyrifos-oxon, chlorpyrifos, and diisopropylfluorophosphate-directly affect kinesin, thereby disrupting kinesin-dependent transport on microtubules. Kinesin-dependent movement of vesicles, organelles, and other cellular components along microtubules is fundamental to the organization of all eukaryotic cells, especially in neurons where organelles and proteins synthesized in the cell body must move down long axons to pre-synaptic sites in nerve terminals. We postulate that disruption of kinesin-dependent intracellular transport could account for some of the long-term effects of organophosphates on the peripheral and central nervous system.

  2. Tubulin protofilaments and kinesin-dependent motility

    PubMed Central

    1992-01-01

    . This indicates that even if these microtubules differ in surface lattice, this does not affect the motility. PMID:1500429

  3. Highly sensitive kinesin-microtubule motility assays using SLIM

    NASA Astrophysics Data System (ADS)

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

    2016-03-01

    We provide an experimental demonstration of Spatial Light Interference Microscopy (SLIM) as a tool for measuring the motion of 25 nm tubulin structures without the use of florescence labels. Compared to intensity imaging methods such as phase contrast or DIC, our imaging technique relies on the ratios of images associated with optically introduced phase shifts, thus implicitly removing background illumination. To demonstrate our new found capabilities, we characterize kinesin-based motility continuously over periods of time where fluorescence would typically photobleach. We exploit this new method to compare the motility of microtubules at low ATP concentrations, with and without the tagging proteins formerly required to perform these studies. Our preliminary results show that the tags have a non-negligible effect on the microtubule motility, slowing the process down by more than 10%.

  4. Sliding of STOP proteins on microtubules: a model system for diffusion-dependent microtubule motility.

    PubMed

    Margolis, R L; Job, D; Pabion, M; Rauch, C T

    1986-01-01

    STOP proteins, of 145 kD, act substoichiometrically to block end-wise disassembly of microtubules. STOPs bind to microtubules either during microtubule assembly or when added at steady state, and when binding to the polymers is apparently irreversible. They are not measurably lost from polymers under competition conditions, and there is no measurable exchange between polymers. Nonetheless, STOP proteins exhibit an extraordinary behavior: they "slide" laterally on the surface of the microtubule. Displacement is assayed by forming hybrid microtubules in which cold stable or cold labile region subunits are labeled. Displacement of STOPs on the polymer with time will cause labeled subunits of cold-stable regions to become increasingly cold labile in a manner reciprocal to cold stabilization of previously cold-labile subunits. Because equilibrium exchange of STOP proteins onto and off the polymers can be ruled out, the displacement of STOPs relative to subunits can only be explained by lateral diffusion or "sliding." Axonal transport and mitotic mechanisms were discussed as implications of such a lateral translocation mechanism for microtubule-dependent motility.

  5. Purified Kinesin Promotes Vesicle Motility and Induces Active Sliding Between Microtubules In vitro

    NASA Astrophysics Data System (ADS)

    Urrutia, Raul; McNiven, Mark A.; Albanesi, Joseph P.; Murphy, Douglas B.; Kachar, Bechara

    1991-08-01

    We examined the ability of kinesin to support the movement of adrenal medullary chromaffin granules on microtubules in a defined in vitro system. We found that kinesin and ATP are all that is required to support efficient (33% vesicle motility) and rapid (0.4-0.6 μ m/s) translocation of secretory granule membranes on microtubules in the presence of a low-salt motility buffer. Kinesin also induced the formation of microtubule asters in this buffer, with the plus ends of microtubules located at the center of each aster. This observation indicates that kinesin is capable of promoting active sliding between microtubules toward their respective plus ends, a movement analogous to that of anaphase b in the mitotic spindle. The fact that vesicle translocation, microtubule sliding, and microtubule-dependent kinesin ATPase activities are all enhanced in low-salt buffer establishes a functional parallel between this translocator and other motility ATPases, myosin, and dynein.

  6. 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.

  7. Microtubule stability affects the unique motility of F-actin in Marchantia polymorpha.

    PubMed

    Era, Atsuko; Kutsuna, Natsumaro; Higaki, Takumi; Hasezawa, Seiichiro; Nakano, Akihiko; Ueda, Takashi

    2013-01-01

    Actin microfilaments play crucial roles in diverse plant functions. Some specific cellular processes require interaction between F-actin and microtubules, and it is believed that there are direct or indirect connections between F-actin and microtubules. We previously reported that actin microfilaments exhibit unique dynamic motility in cells of the liverwort, Marchantia polymorpha; the relevance of this activity to microtubules has not been explored. To examine whether the dynamics of F-actin in M. polymorpha were somehow regulated by microtubules, we investigated the effects of stabilization or destabilization of microtubules on dynamics of actin bundles, which were visualized by Lifeact-Venus. To our surprise, both stabilization and destabilization of microtubules exerted similar effects on F-actin motility; apparent sliding movement of F-actin in M. polymorpha cells was accelerated by both oryzalin and paclitaxel, with the effect of paclitaxel more evident than that of oryzalin. Immunofluorescence staining revealed that some F-actin bundles were arrayed along with microtubules in M. polymorpha thallus cells. These results suggest that microtubules play regulatory roles in the unique F-actin dynamics in M. polymorpha.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

    1995-01-01

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

  10. Single Molecule Investigation of Kinesin-1 Motility Using Engineered Microtubule Defects

    PubMed Central

    Gramlich, Michael W.; Conway, Leslie; Liang, Winnie H.; Labastide, Joelle A.; King, Stephen J.; Xu, Jing; Ross, Jennifer L.

    2017-01-01

    The structure of the microtubule is tightly regulated in cells via a number of microtubule associated proteins and enzymes. Microtubules accumulate structural defects during polymerization, and defect size can further increase under mechanical stresses. Intriguingly, microtubule defects have been shown to be targeted for removal via severing enzymes or self-repair. The cell’s control in defect removal suggests that defects can impact microtubule-based processes, including molecular motor-based intracellular transport. We previously demonstrated that microtubule defects influence cargo transport by multiple kinesin motors. However, mechanistic investigations of the observed effects remained challenging, since defects occur randomly during polymerization and are not directly observable in current motility assays. To overcome this challenge, we used end-to-end annealing to generate defects that are directly observable using standard epi-fluorescence microscopy. We demonstrate that the annealed sites recapitulate the effects of polymerization-derived defects on multiple-motor transport, and thus represent a simple and appropriate model for naturally-occurring defects. We found that single kinesins undergo premature dissociation, but not preferential pausing, at the annealed sites. Our findings provide the first mechanistic insight to how defects impact kinesin-based transport. Preferential dissociation on the single-molecule level has the potential to impair cargo delivery at locations of microtubule defect sites in vivo. PMID:28287156

  11. Single Molecule Investigation of Kinesin-1 Motility Using Engineered Microtubule Defects

    NASA Astrophysics Data System (ADS)

    Gramlich, Michael W.; Conway, Leslie; Liang, Winnie H.; Labastide, Joelle A.; King, Stephen J.; Xu, Jing; Ross, Jennifer L.

    2017-03-01

    The structure of the microtubule is tightly regulated in cells via a number of microtubule associated proteins and enzymes. Microtubules accumulate structural defects during polymerization, and defect size can further increase under mechanical stresses. Intriguingly, microtubule defects have been shown to be targeted for removal via severing enzymes or self-repair. The cell’s control in defect removal suggests that defects can impact microtubule-based processes, including molecular motor-based intracellular transport. We previously demonstrated that microtubule defects influence cargo transport by multiple kinesin motors. However, mechanistic investigations of the observed effects remained challenging, since defects occur randomly during polymerization and are not directly observable in current motility assays. To overcome this challenge, we used end-to-end annealing to generate defects that are directly observable using standard epi-fluorescence microscopy. We demonstrate that the annealed sites recapitulate the effects of polymerization-derived defects on multiple-motor transport, and thus represent a simple and appropriate model for naturally-occurring defects. We found that single kinesins undergo premature dissociation, but not preferential pausing, at the annealed sites. Our findings provide the first mechanistic insight to how defects impact kinesin-based transport. Preferential dissociation on the single-molecule level has the potential to impair cargo delivery at locations of microtubule defect sites in vivo.

  12. A genome-wide RNAi screen for microtubule bundle formation and lysosome motility regulation in Drosophila S2 cells

    PubMed Central

    Jolly, Amber L.; Luan, Chi-Hao; Dusel, Brendon E.; Dunne, Sara Fernandez; Winding, Michael; Dixit, Vishrut J.; Robins, Chloe; Saluk, Jennifer L.; Logan, David J.; Carpenter, Anne E.; Sharma, Manu; Dean, Deborah; Cohen, Andrew R.; Gelfand, Vladimir I.

    2016-01-01

    Summary Long-distance intracellular transport of organelles, mRNA, and proteins (“cargo”) occurs along the microtubule cytoskeleton by the action of kinesin and dynein motor proteins; the vast network of factors involved in regulating intracellular cargo transport are still unknown. We capitalize on the Drosophila melanogaster S2 model cell system to monitor lysosome transport along microtubule bundles, which require enzymatically active kinesin-1 motor protein for their formation. We use an automated tracking program and a naïve Bayesian classifier for the multivariate motility data to analyze 15,683 gene phenotypes, and find 98 proteins involved in regulating lysosome motility along microtubules and 48 involved in the formation of microtubule filled processes in S2 cells. We identify innate immunity genes, ion channels and signaling proteins having a role in lysosome motility regulation, and find an unexpected relationship between the dynein motor, Rab7a and lysosome motility regulation. PMID:26774481

  13. A Genome-wide RNAi Screen for Microtubule Bundle Formation and Lysosome Motility Regulation in Drosophila S2 Cells.

    PubMed

    Jolly, Amber L; Luan, Chi-Hao; Dusel, Brendon E; Dunne, Sara F; Winding, Michael; Dixit, Vishrut J; Robins, Chloe; Saluk, Jennifer L; Logan, David J; Carpenter, Anne E; Sharma, Manu; Dean, Deborah; Cohen, Andrew R; Gelfand, Vladimir I

    2016-01-26

    Long-distance intracellular transport of organelles, mRNA, and proteins ("cargo") occurs along the microtubule cytoskeleton by the action of kinesin and dynein motor proteins, but the vast network of factors involved in regulating intracellular cargo transport are still unknown. We capitalize on the Drosophila melanogaster S2 model cell system to monitor lysosome transport along microtubule bundles, which require enzymatically active kinesin-1 motor protein for their formation. We use an automated tracking program and a naive Bayesian classifier for the multivariate motility data to analyze 15,683 gene phenotypes and find 98 proteins involved in regulating lysosome motility along microtubules and 48 involved in the formation of microtubule filled processes in S2 cells. We identify innate immunity genes, ion channels, and signaling proteins having a role in lysosome motility regulation and find an unexpected relationship between the dynein motor, Rab7a, and lysosome motility regulation.

  14. Merkel Cell Polyomavirus Small T Antigen Mediates Microtubule Destabilization To Promote Cell Motility and Migration

    PubMed Central

    Knight, Laura M.; Stakaityte, Gabriele; Wood, Jennifer, J.; Abdul-Sada, Hussein; Griffiths, David A.; Howell, Gareth J.; Wheat, Rachel; Blair, G. Eric; Steven, Neil M.; Macdonald, Andrew; Blackbourn, David J.

    2014-01-01

    ABSTRACT Merkel cell carcinoma (MCC) is an aggressive skin cancer of neuroendocrine origin with a high propensity for recurrence and metastasis. Merkel cell polyomavirus (MCPyV) causes the majority of MCC cases due to the expression of the MCPyV small and large tumor antigens (ST and LT, respectively). Although a number of molecular mechanisms have been attributed to MCPyV tumor antigen-mediated cellular transformation or replication, to date, no studies have investigated any potential link between MCPyV T antigen expression and the highly metastatic nature of MCC. Here we use a quantitative proteomic approach to show that MCPyV ST promotes differential expression of cellular proteins implicated in microtubule-associated cytoskeletal organization and dynamics. Intriguingly, we demonstrate that MCPyV ST expression promotes microtubule destabilization, leading to a motile and migratory phenotype. We further highlight the essential role of the microtubule-associated protein stathmin in MCPyV ST-mediated microtubule destabilization and cell motility and implicate the cellular phosphatase catalytic subunit protein phosphatase 4C (PP4C) in the regulation of this process. These findings suggest a possible molecular mechanism for the highly metastatic phenotype associated with MCC. IMPORTANCE Merkel cell polyomavirus (MCPyV) causes the majority of cases of Merkel cell carcinoma (MCC), an aggressive skin cancer with a high metastatic potential. However, the molecular mechanisms leading to virally induced cancer development have yet to be fully elucidated. In particular, no studies have investigated any potential link between the virus and the highly metastatic nature of MCC. We demonstrate that the MCPyV small tumor antigen (ST) promotes the destabilization of the host cell microtubule network, which leads to a more motile and migratory cell phenotype. We further show that MCPyV ST induces this process by regulating the phosphorylation status of the cellular microtubule

  15. Inhibition of kinesin-driven microtubule motility by monoclonal antibodies to kinesin heavy chains

    PubMed Central

    1988-01-01

    We have prepared and characterized seven mouse monoclonal antibodies (SUK 1-7) to the 130-kD heavy chain of sea urchin egg kinesin. On immunoblots, SUK 3 and SUK 4 cross-reacted with Drosophila embryo 116- kD heavy chains, and SUK 4, SUK 5, SUK 6, and SUK 7 bound to the 120-kD heavy chains of bovine brain kinesin. Three out of seven monoclonal antikinesins (SUK 4, SUK 6, and SUK 7) caused a dose-dependent inhibition of sea urchin egg kinesin-induced microtubule translocation, whereas the other four monoclonal antibodies had no detectable effect on this motility. The inhibitory monoclonal antibodies (SUK 4, SUK 6, and SUK 7) appear to bind to spatially related sites on an ATP- sensitive microtubule binding 45-kD chymotryptic fragment of the 130-kD heavy chain, whereas SUK 2 binds to a spatially distinct site. None of the monoclonal antikinesins inhibited the microtubule activated MgATPase activity of kinesin, suggesting that SUK 4, SUK 6, and SUK 7 uncouple this MgATPase activity from motility. PMID:2974459

  16. Control and gating of kinesin-microtubule motility on electrically heated thermo-chips.

    PubMed

    Ramsey, Laurence; Schroeder, Viktor; van Zalinge, Harm; Berndt, Michael; Korten, Till; Diez, Stefan; Nicolau, Dan V

    2014-06-01

    First lab-on-chip devices based on active transport by biomolecular motors have been demonstrated for basic detection and sorting applications. However, to fully employ the advantages of such hybrid nanotechnology, versatile spatial and temporal control mechanisms are required. Using a thermo-responsive polymer, we demonstrated a temperature controlled gate that either allows or disallows the passing of microtubules through a topographically defined channel. The gate is addressed by a narrow gold wire, which acts as a local heating element. It is shown that the electrical current flowing through a narrow gold channel can control the local temperature and as a result the conformation of the polymer. This is the first demonstration of a spatially addressable gate for microtubule motility which is a key element of nanodevices based on biomolecular motors.

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    2016-01-01

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

  19. Role of the Kinesin Neck Region in Processive Microtubule-based Motility

    PubMed Central

    Romberg, Laura; Pierce, Daniel W.; Vale, Ronald D.

    1998-01-01

    Kinesin is a dimeric motor protein that can move along a microtubule for several microns without releasing (termed processive movement). The two motor domains of the dimer are thought to move in a coordinated, hand-over-hand manner. A region adjacent to kinesin's motor catalytic domain (the neck) contains a coiled coil that is sufficient for motor dimerization and has been proposed to play an essential role in processive movement. Recent models have suggested that the neck enables head-to-head communication by creating a stiff connection between the two motor domains, but also may unwind during the mechanochemical cycle to allow movement to new tubulin binding sites. To test these ideas, we mutated the neck coiled coil in a 560-amino acid (aa) dimeric kinesin construct fused to green fluorescent protein (GFP), and then assayed processivity using a fluorescence microscope that can visualize single kinesin–GFP molecules moving along a microtubule. Our results show that replacing the kinesin neck coiled coil with a 28-aa residue peptide sequence that forms a highly stable coiled coil does not greatly reduce the processivity of the motor. This result argues against models in which extensive unwinding of the coiled coil is essential for movement. Furthermore, we show that deleting the neck coiled coil decreases processivity 10-fold, but surprisingly does not abolish it. We also demonstrate that processivity is increased by threefold when the neck helix is elongated by seven residues. These results indicate that structural features of the neck coiled coil, although not essential for processivity, can tune the efficiency of single molecule motility. PMID:9508773

  20. Glucose regulated proteins 78 and 75 bind to the receptor for hyaluronan mediated motility in interphase microtubules

    SciTech Connect

    Kuwabara, Hiroko . E-mail: pa2020@art.osaka-med.ac.jp; Yoneda, Masahiko; Hayasaki, Hana; Nakamura, Toshiya; Mori, Hiroshi

    2006-01-20

    The receptor for hyaluronan mediated motility (RHAMM), which is a hyaluronan-binding protein, is a centrosomal and microtubal protein. Here, we have identified two RHAMM-binding proteins, glucose regulated protein (GRP) 78 and GRP75, using co-immunoprecipitation analysis. These two proteins directly bound to glutathione-S-transferase-RHAMM fusion proteins. By double immunostaining, GRP78 and GRP75 colocalized with RHAMM in interphase microtubules, but were separated in mitotic spindles. Prevention of microtubule polymerization by TN-16 and vincristine sulfate induced RHAMM overexpression without a significant change in GRP78/75. Taken together, GRP78/75 and RHAMM complexes may stabilize microtubules in the interphase, associated with a downregulation of RHAMM. These results reveal a new biochemical activity of RHAMM.

  1. Simultaneous Visualization of Peroxisomes and Cytoskeletal Elements Reveals Actin and Not Microtubule-Based Peroxisome Motility in Plants1[w

    PubMed Central

    Mathur, Jaideep; Mathur, Neeta; Hülskamp, Martin

    2002-01-01

    Peroxisomes were visualized in living plant cells using a yellow fluorescent protein tagged with a peroxisomal targeting signal consisting of the SKL motif. Simultaneous visualization of peroxisomes and microfilaments/microtubules was accomplished in onion (Allium cepa) epidermal cells transiently expressing the yellow fluorescent protein-peroxi construct, a green fluorescent protein-mTalin construct that labels filamentous-actin filaments, and a green fluorescent protein-microtubule-binding domain construct that labels microtubules. The covisualization of peroxisomes and cytoskeletal elements revealed that, contrary to the reports from animal cells, peroxisomes in plants appear to associate with actin filaments and not microtubules. That peroxisome movement is actin based was shown by pharmacological studies. For this analysis we used onion epidermal cells and various cell types of Arabidopsis including trichomes, root hairs, and root cortex cells exhibiting different modes of growth. In transient onion epidermis assay and in transgenic Arabidopsis plants, an interference with the actin cytoskeleton resulted in progressive loss of saltatory movement followed by the aggregation and a complete cessation of peroxisome motility within 30 min of drug application. Microtubule depolymerization or stabilization had no effect. PMID:11891258

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

    PubMed

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

    2015-06-05

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

  3. 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

  4. Comparison of the motile and enzymatic properties of two microtubule minus-end-directed motors, ncd and cytoplasmic dynein.

    PubMed

    Shimizu, T; Toyoshima, Y Y; Edamatsu, M; Vale, R D

    1995-02-07

    Cytoplasmic dynein and ncd, a kinesin-related protein from Drosophila, are motor proteins that move toward the minus ends of microtubules, while kinesin moves to the microtubule plus end. In previous work, we examined the nucleotide dependence of motility and enzymatic activity by kinesin [Shimizu, T., Furusawa, K., Ohashi, S., Toyoshima, Y. Y., Okuno, M., Malik, F., & Vale, R. D., (1991) J. Cell Biol. 112, 1189-1197]. In this study, we examined these activities of the cytoplasmic dynein from bovine brain and ncd in order to explore what enzymatic features might be shared by these two minus-end-directed motors. Both ncd and cytoplasmic dynein demonstrated an activation of ATPase activity upon the addition of microtubules (30-fold and 6-fold, respectively). A significant difference between ncd and cytoplasmic dynein was their relative sensitivity to vanadate and to aluminum fluoride. In contrast to cytoplasmic dynein, ncd polypeptide was not cleaved by UV-vanadate treatment, and its ATPase and motility were unaffected by vanadate (up to 0.1 mM). When the nucleotide requirement for movement as examined using a battery of 20 nucleotides and nucleotide analogues, cytoplasmic dynein was found to exhibit a specificity very similar to that of axonemal dyneins from Tetrahymena. Surprisingly, however, the nucleotide specificities of in vitro motility produced by ncd or its construct, GST/MC1 (a fusion protein of glutathione S-transferase and 210-700 of the predicted ncd amino acid sequence), were quite distinct from that of kinesin. Thus, the nucleotide specificity profiles of members of the kinesin motor superfamily do not appear to be identical.

  5. High-resolution tracking of microtubule motility driven by a single kinesin motor.

    PubMed Central

    Malik, F; Brillinger, D; Vale, R D

    1994-01-01

    Kinesin is a microtubule-based motor protein that contains two identical force-generating subunits. The kinesin binding sites along the microtubule lie 8 nm apart (the dimension of the tubulin dimer), which implies that kinesin must translocate a minimum distance of 8 nm per hydrolysis cycle. Measurements of kinesin's microtubule-stimulated ATPase activity (approximately 20 ATP per sec) and velocity of transport (approximately 0.6 micron/sec), however, suggest that the net distance moved per ATP (approximately 30 nm) may be greater than one tubulin dimer under zero load conditions. To explore how kinesin translocates during its ATPase cycle, we constructed a microscope capable of tracking movement with 1-nm resolution at a bandwidth of 200 Hz and used this device to examine microtubule movement driven by a single kinesin motor. Regular stepwise movements were not observed in displacement traces of moving microtubules, although Brownian forces acting on elastic elements within the kinesin motor precluded detection of steps that were < 12 nm. Though individual steps of approximately 16 nm were occasionally observed, their infrequent occurrence suggests that kinesin rarely moves abruptly by distances of two or more tubulin subunits during its ATP hydrolysis cycle. Instead it is more likely that kinesin moves forward by the distance of only a single tubulin subunit under zero load conditions. Images PMID:8183952

  6. Regulation of early endocytic vesicle motility and fission in a reconstituted system.

    PubMed

    Bananis, Eustratios; Murray, John W; Stockert, Richard J; Satir, Peter; Wolkoff, Allan W

    2003-07-01

    We previously established conditions to reconstitute kinesin-dependent early endocytic vesicle motility and fission on microtubules in vitro. The present study examined the question whether motility and fission are regulated in this system. Screening for proteins by immunofluorescence microscopy revealed that the small G protein, Rab4, was associated with 80% of hepatocyte-derived early endocytic vesicles that contain the ligand asialoorosomucoid (ASOR). By contrast, other markers for early endocytic vesicles including clathrin, Rab5 and EEA1 were present in the preparation but did not colocalize with the ASOR vesicles. Guanine nucleotides exchanged into the Rab4 present on the vesicles as shown by solubilization of Rab4 by Rab-GDI; solubilization was inhibited by incubation with GTP-gamma-S and promoted by GDP. Pre-incubation of vesicles with GDP increased the number of vesicles moving on microtubules and markedly increased vesicle fission. This increase in motility from GDP was shown to be towards the minus end of microtubules, possibly through activation of the minus-end-directed kinesin, KIFC2. Pre-incubation of vesicles with GTP-gamma-S, by contrast, repressed motility. Addition of exogenous GST-Rab4- GTP-gamma-S led to a further repression of motility and fission. Repression was not seen with addition of GST-Rab4-GDP. Treatment of vesicles with Rab4 antibody also repressed motility, and repression was not seen when vesicles were pre-incubated with GDP. Based on these results we hypothesize that endogenous Rab4-GTP suppresses motility of ASOR-containing vesicles in hepatocytes and that conversion of Rab4-GTP to Rab4-GDP serves as a molecular switch that activates minus-end kinesin-based motility, facilitating early endosome fission and consequent receptor-ligand segregation.

  7. Microtubule-dependent Plus- and Minus End–directed Motilities Are Competing Processes for Nuclear Targeting of Adenovirus

    PubMed Central

    Suomalainen, Maarit; Nakano, Michel Y.; Keller, Stephan; Boucke, Karin; Stidwill, Robert P.; Greber, Urs F.

    1999-01-01

    Adenovirus (Ad) enters target cells by receptor-mediated endocytosis, escapes to the cytosol, and then delivers its DNA genome into the nucleus. Here we analyzed the trafficking of fluorophore-tagged viruses in HeLa and TC7 cells by time-lapse microscopy. Our results show that native or taxol-stabilized microtubules (MTs) support alternating minus- and plus end–directed movements of cytosolic virus with elementary speeds up to 2.6 μm/s. No directed movement was observed in nocodazole-treated cells. Switching between plus- and minus end–directed elementary speeds at frequencies up to 1 Hz was observed in the periphery and near the MT organizing center (MTOC) after recovery from nocodazole treatment. MT-dependent motilities allowed virus accumulation near the MTOC at population speeds of 1–10 μm/min, depending on the cell type. Overexpression of p50/dynamitin, which is known to affect dynein-dependent minus end–directed vesicular transport, significantly reduced the extent and the frequency of minus end–directed migration of cytosolic virus, and increased the frequency, but not the extent of plus end–directed motility. The data imply that a single cytosolic Ad particle engages with two types of MT-dependent motor activities, the minus end– directed cytoplasmic dynein and an unknown plus end– directed activity. PMID:10037788

  8. Motility of microtubules on the inner surface of water-in-oil emulsion droplets.

    PubMed

    Tsuji, Mikako; Kabir, Arif Md Rashedul; Ito, Masaki; Inoue, Daisuke; Kokado, Kenta; Sada, Kazuki; Kakugo, Akira

    2017-10-03

    The water-in-oil emulsion systems have recently been attracting much attention in various fields. However, functionalization of the water-in-oil emulsion systems, which is required for expanding their applications in industry and research, has been challenging. We now demonstrate functionalization of a water-in-oil emulsion system by anchoring a target protein molecule. A microtubule associated motor protein kinesin-1 was successfully anchored to the inner surface of water-in-oil emulsion droplets by employing the specific interaction of the nickel-nitrilotriacetic acid (NTA)-histidine tag. The microtubules exhibited a gliding motion on the kinesin functionalized inner surface of the emulsion droplets, which confirmed success of the functionalization of the water-in-oil emulsion system. This result would be beneficial in exploring the roles of biomolecular motor systems in the cellular events that take place at the cell membrane and might also contribute to expanding the nanotechnological applications of biomolecular motors and water-in-oil emulsion systems in the future.

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

    PubMed

    Kovács, P; Csaba, G

    2006-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  11. The N-DRC forms a conserved biochemical complex that maintains outer doublet alignment and limits microtubule sliding in motile axonemes

    PubMed Central

    Bower, Raqual; Tritschler, Douglas; VanderWaal, Kristyn; Perrone, Catherine A.; Mueller, Joshua; Fox, Laura; Sale, Winfield S.; Porter, M. E.

    2013-01-01

    The nexin–dynein regulatory complex (N-DRC) is proposed to coordinate dynein arm activity and interconnect doublet microtubules. Here we identify a conserved region in DRC4 critical for assembly of the N-DRC into the axoneme. At least 10 subunits associate with DRC4 to form a discrete complex distinct from other axonemal substructures. Transformation of drc4 mutants with epitope-tagged DRC4 rescues the motility defects and restores assembly of missing DRC subunits and associated inner-arm dyneins. Four new DRC subunits contain calcium-signaling motifs and/or AAA domains and are nearly ubiquitous in species with motile cilia. However, drc mutants are motile and maintain the 9 + 2 organization of the axoneme. To evaluate the function of the N-DRC, we analyzed ATP-induced reactivation of isolated axonemes. Rather than the reactivated bending observed with wild-type axonemes, ATP addition to drc-mutant axonemes resulted in splaying of doublets in the distal region, followed by oscillatory bending between pairs of doublets. Thus the N-DRC provides some but not all of the resistance to microtubule sliding and helps to maintain optimal alignment of doublets for productive flagellar motility. These findings provide new insights into the mechanisms that regulate motility and further highlight the importance of the proximal region of the axoneme in generating flagellar bending. PMID:23427265

  12. Microtubule catastrophe and rescue.

    PubMed

    Gardner, Melissa K; Zanic, Marija; Howard, Jonathon

    2013-02-01

    Microtubules are long cylindrical polymers composed of tubulin subunits. In cells, microtubules play an essential role in architecture and motility. For example, microtubules give shape to cells, serve as intracellular transport tracks, and act as key elements in important cellular structures such as axonemes and mitotic spindles. To accomplish these varied functions, networks of microtubules in cells are very dynamic, continuously remodeling through stochastic length fluctuations at the ends of individual microtubules. The dynamic behavior at the end of an individual microtubule is termed 'dynamic instability'. This behavior manifests itself by periods of persistent microtubule growth interrupted by occasional switching to rapid shrinkage (called microtubule 'catastrophe'), and then by switching back from shrinkage to growth (called microtubule 'rescue'). In this review, we summarize recent findings which provide new insights into the mechanisms of microtubule catastrophe and rescue, and discuss the impact of these findings in regards to the role of microtubule dynamics inside of cells. Copyright © 2012 Elsevier Ltd. All rights reserved.

  13. Microtubule Catastrophe and Rescue

    PubMed Central

    Gardner, Melissa K.; Zanic, Marija; Howard, Jonathon

    2012-01-01

    Microtubules are long cylindrical polymers composed of tubulin subunits. In cells, microtubules play an essential role in architecture and motility. For example, microtubules give shape to cells, serve as intracellular transport tracks, and act as key elements in important cellular structures such as axonemes and mitotic spindles. To accomplish these varied functions, networks of microtubules in cells are very dynamic, continuously remodeling through stochastic length fluctuations at the ends of individual microtubules. The dynamic behavior at the end of an individual microtubule is termed “dynamic instability”. This behavior manifests itself by periods of persistent microtubule growth interrupted by occasional switching to rapid shrinkage (called microtubule `catastrophe'), and then by switching back from shrinkage to growth (called microtubule `rescue'). In this review, we summarize recent findings which provide new insights into the mechanisms of microtubule catastrophe and rescue, and discuss the impact of these findings in regards to the role of microtubule dynamics inside of cells. PMID:23092753

  14. Video-enhanced contrast, differential interference contrast (AVEC-DIC) microscopy: a new method capable of analyzing microtubule-related motility in the reticulopodial network of Allogromia laticollaris.

    PubMed

    Allen, R D; Allen, N S; Travis, J L

    1981-01-01

    A new method called Allen Video-enhanced Contrast, Differential Interference Contrast (AVEC-DIC) microscopy is shown to be sufficiently sensitive to detect several new features of microtubule-related motility in the reticulopodial network of the foraminifer, Allogromia. The method takes advantage of the variable gain and offset features of a binary video camera to operate the DIC microscope under conditions highly favorable for video imaging, but in which the optical image is virtually invisible to the eye yet retains its full information when viewed by a suitable video camera. The improvements are made possible by setting a dé Senarmont compensator to lambda/9-lambda/4 at maximal working aperture of internally corrected planapochromatic objectives. Under these conditions, the offset feature of the video camera can reject so much stray light from the instrument and specimen that contrast compares favorably with that observed in high-extinction images, and polarizing rectifiers offer scarcely any advantage. Freed from the constraints of the light-limited conditions of DIC microscopy, video images can be recorded 60 times per second, or over 1,000 times the rate of photomicrographs at comparable magnifications under high-extinction conditions. Application of this method to the reticulopodial network of Allogromia has shown that cytoplasmic organelles are translocated only in contact with single microtubules or bundles of microtubules, and that these organelles fail to move when separated from microtubules. Microtubules themselves undergo both axial translatory ("sliding") and lateral "zipping and unzipping" movements that have been suggested to occur during mitosis and other biological processes.

  15. Purification and assay of a 145-kDa protein (STOP145) with microtubule-stabilizing and motility behavior.

    PubMed

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

    1986-02-01

    The capacity of microtubules to disassemble in vitro is profoundly affected by a protein factor designated STOP (stable tubule only polypeptide). Here we report the isolation of STOP protein and confirm that its activity is, as predicted, highly substoichiometric to the tubulin in microtubules. The isolation of the 145-kDa STOP (STOP145) protein has been effected from isolated cold-stable microtubules by two column steps: DEAE ion-exchange and a calmodulin affinity column. To confirm the protein's activity we have produced an antibody against STOP145 and have used the antibody to specifically remove the protein and the activity using an antibody-linked affinity column. We conclude that the STOP145 protein accounts for the observed in vitro stabilization of microtubules.

  16. The Cauliflower Mosaic Virus Protein P6 Forms Motile Inclusions That Traffic along Actin Microfilaments and Stabilize Microtubules1[W][OA

    PubMed Central

    Harries, Phillip A.; Palanichelvam, Karuppaiah; Yu, Weichang; Schoelz, James E.; Nelson, Richard S.

    2009-01-01

    The gene VI product (P6) of Cauliflower mosaic virus (CaMV) is a multifunctional protein known to be a major component of cytoplasmic inclusion bodies formed during CaMV infection. Although these inclusions are known to contain virions and are thought to be sites of translation from the CaMV 35S polycistronic RNA intermediate, the precise role of these bodies in the CaMV infection cycle remains unclear. Here, we examine the functionality and intracellular location of a fusion between P6 and GFP (P6-GFP). We initially show that the ability of P6-GFP to transactivate translation is comparable to unmodified P6. Consequently, our work has direct application for the large body of literature in which P6 has been expressed ectopically and its functions characterized. We subsequently found that P6-GFP forms highly motile cytoplasmic inclusion bodies and revealed through fluorescence colocalization studies that these P6-GFP bodies associate with the actin/endoplasmic reticulum network as well as microtubules. We demonstrate that while P6-GFP inclusions traffic along microfilaments, those associated with microtubules appear stationary. Additionally, inhibitor studies reveal that the intracellular movement of P6-GFP inclusions is sensitive to the actin inhibitor, latrunculin B, which also inhibits the formation of local lesions by CaMV in Nicotiana edwardsonii leaves. The motility of P6 along microfilaments represents an entirely new property for this protein, and these results imply a role for P6 in intracellular and cell-to-cell movement of CaMV. PMID:19028879

  17. 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.

  18. Down-Regulation of Desmosomes in Cultured Cells: The Roles of PKC, Microtubules and Lysosomal/Proteasomal Degradation

    PubMed Central

    McHarg, Selina; Hopkins, Gemma; Lim, Lusiana; Garrod, David

    2014-01-01

    Desmosomes are intercellular adhesive junctions of major importance for tissue integrity. To allow cell motility and migration they are down-regulated in epidermal wound healing. Electron microscopy indicates that whole desmosomes are internalised by cells in tissues, but the mechanism of down-regulation is unclear. In this paper we provide an overview of the internalisation of half-desmosomes by cultured cells induced by calcium chelation. Our results show that: (i) half desmosome internalisation is dependent on conventional PKC isoforms; (ii) microtubules transport internalised half desmosomes to the region of the centrosome by a kinesin-dependent mechanism; (iii) desmosomal proteins remain colocalised after internalisation and are not recycled to the cell surface; (iv) internalised desmosomes are degraded by the combined action of lysosomes and proteasomes. We also confirm that half desmosome internalisation is dependent upon the actin cytoskeleton. These results suggest that half desmosomes are not disassembled and recycled during or after internalisation but instead are transported to the centrosomal region where they are degraded. These findings may have significance for the down-regulation of desmosomes in wounds. PMID:25291180

  19. 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.

  20. 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.

  1. 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

  2. Pathogenic Forms of Tau Inhibit Kinesin-Dependent Axonal Transport through a Mechanism Involving Activation of Axonal Phosphotransferases

    PubMed Central

    Kanaan, Nicholas M.; Morfini, Gerardo A.; LaPointe, Nichole E.; Pigino, Gustavo F.; Patterson, Kristina R.; Song, Yuyu; Andreadis, Athena; Fu, Yifan; Brady, Scott T.; Binder, Lester I.

    2012-01-01

    Aggregated filamentous forms of hyperphosphorylated tau (a microtubule-associated protein) represent pathological hallmarks of Alzheimer’s disease (AD) and other tauopathies. While axonal transport dysfunction is thought to represent a primary pathogenic factor in AD and other neurodegenerative diseases, the direct molecular link between pathogenic forms of tau and deficits in axonal transport remain unclear. Recently, we demonstrated that filamentous, but not soluble, forms of wild-type tau inhibit anterograde, kinesin-based fast axonal transport (FAT) by activating axonal protein phosphatase 1 (PP1) and glycogen synthase kinase 3 (GSK3), independent of microtubule binding. Here, we demonstrate that amino acids 2–18 of tau, comprising a phosphatase-activating domain (PAD), are necessary and sufficient for activation of this pathway in axoplasms isolated from squid giant axons. Various pathogenic forms of tau displaying increased exposure of PAD inhibited anterograde FAT in squid axoplasm. Importantly, immunohistochemical studies using a novel PAD-specific monoclonal antibody in human postmortem tissue indicated that increased PAD exposure represents an early pathogenic event in AD that closely associates in time with AT8 immunoreactivity, an early marker of pathological tau. We propose a model of pathogenesis in which disease-associated changes in tau conformation lead to increased exposure of PAD, activation of PP1-GSK3, and inhibition of FAT. Results from these studies reveal a novel role for tau in modulating axonal phosphotransferases and provide a molecular basis for a toxic gain-of-function associated with pathogenic forms of tau. PMID:21734277

  3. Pathogenic forms of tau inhibit kinesin-dependent axonal transport through a mechanism involving activation of axonal phosphotransferases.

    PubMed

    Kanaan, Nicholas M; Morfini, Gerardo A; LaPointe, Nichole E; Pigino, Gustavo F; Patterson, Kristina R; Song, Yuyu; Andreadis, Athena; Fu, Yifan; Brady, Scott T; Binder, Lester I

    2011-07-06

    Aggregated filamentous forms of hyperphosphorylated tau (a microtubule-associated protein) represent pathological hallmarks of Alzheimer's disease (AD) and other tauopathies. While axonal transport dysfunction is thought to represent a primary pathogenic factor in AD and other neurodegenerative diseases, the direct molecular link between pathogenic forms of tau and deficits in axonal transport remain unclear. Recently, we demonstrated that filamentous, but not soluble, forms of wild-type tau inhibit anterograde, kinesin-based fast axonal transport (FAT) by activating axonal protein phosphatase 1 (PP1) and glycogen synthase kinase 3 (GSK3), independent of microtubule binding. Here, we demonstrate that amino acids 2-18 of tau, comprising a phosphatase-activating domain (PAD), are necessary and sufficient for activation of this pathway in axoplasms isolated from squid giant axons. Various pathogenic forms of tau displaying increased exposure of PAD inhibited anterograde FAT in squid axoplasm. Importantly, immunohistochemical studies using a novel PAD-specific monoclonal antibody in human postmortem tissue indicated that increased PAD exposure represents an early pathogenic event in AD that closely associates in time with AT8 immunoreactivity, an early marker of pathological tau. We propose a model of pathogenesis in which disease-associated changes in tau conformation lead to increased exposure of PAD, activation of PP1-GSK3, and inhibition of FAT. Results from these studies reveal a novel role for tau in modulating axonal phosphotransferases and provide a molecular basis for a toxic gain-of-function associated with pathogenic forms of tau.

  4. Active sliding between cytoplasmic microtubules.

    PubMed

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

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

  5. 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.

  6. Optical approaches to the study of foraminiferan motility.

    PubMed

    Travis, J L; Bowser, S S

    1988-01-01

    Microtubules are the major cytoskeletal component of foraminiferan reticulopodia. Video-enhanced differential interference contrast light microscopy has demonstrated that the microtubules serve as the intracellular tracks along which rapid bidirectional organelle transport and cell surface motility occurs. Microtubules appear to move, both axially and laterally within the pseudopodial cytoplasm, and these microtubule translocations appear to drive the various reticulopodial movements. F-actin is localized to discrete filament plaques form at sites of pseudopod-substrate adhesion. Correlative immunofluorescence and electron microscopy reveals a structural interaction between microtubules and the actin-containing filament plaques. Our recent data on reticulopodial motility are discussed in an historical context, and a model for foram motility, based on motile microtubules, is presented.

  7. How dynein moves along microtubules

    PubMed Central

    Bhabha, Gira; Johnson, Graham T.; Schroeder, Courtney M.; Vale, Ronald D.

    2015-01-01

    Cytoplasmic dynein, a member of the AAA family of ATPases, drives the processive movement of numerous intracellular cargos towards the minus end of microtubules. Here, we summarize the structural and motile properties of dynein and highlight features that distinguish this motor from kinesin-1 and myosin V, two well-studied transport motors. Integrating information from recent crystal and cryo-EM structures as well as high-resolution single molecule studies, we also discuss models for how dynein biases its movement in one direction along a microtubule track, and present a movie that illustrates these principles. PMID:26678005

  8. CYTOPLASMIC MICROTUBULES

    PubMed Central

    Slautterback, David B.

    1963-01-01

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

  9. Cellulose-Microtubule Uncoupling Proteins Prevent Lateral Displacement of Microtubules during Cellulose Synthesis in Arabidopsis.

    PubMed

    Liu, Zengyu; Schneider, Rene; Kesten, Christopher; Zhang, Yi; Somssich, Marc; Zhang, Youjun; Fernie, Alisdair R; Persson, Staffan

    2016-08-08

    Cellulose is the most abundant biopolymer on Earth and is the major contributor to plant morphogenesis. Cellulose is synthesized by plasma membrane-localized cellulose synthase complexes (CSCs). Nascent cellulose microfibrils become entangled in the cell wall, and further catalysis therefore drives the CSC forward through the membrane: a process guided by cortical microtubules via the protein CSI1/POM2. Still, it is unclear how the microtubules can withstand the forces generated by the motile CSCs to effectively direct CSC movement. Here, we identified a family of microtubule-associated proteins, the cellulose synthase-microtubule uncouplings (CMUs), that located as static puncta along cortical microtubules. Functional disruption of the CMUs caused lateral microtubule displacement and compromised microtubule-based guidance of CSC movement. CSCs that traversed the microtubules interacted with the microtubules via CSI1/POM2, which prompted the lateral microtubule displacement. Hence, we have revealed how microtubules can withstand the propulsion of the CSCs during cellulose biosynthesis and thus sustain anisotropic plant cell growth. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Visualizing individual microtubules by bright field microscopy

    NASA Astrophysics Data System (ADS)

    Gutiérrez-Medina, Braulio; Block, Steven M.

    2010-11-01

    Microtubules are slender (˜25 nm diameter), filamentous polymers involved in cellular structure and organization. Individual microtubules have been visualized via fluorescence imaging of dye-labeled tubulin subunits and by video-enhanced, differential interference-contrast microscopy of unlabeled polymers using sensitive CCD cameras. We demonstrate the imaging of unstained microtubules using a microscope with conventional bright field optics in conjunction with a webcam-type camera and a light-emitting diode illuminator. The light scattered by microtubules is image-processed to remove the background, reduce noise, and enhance contrast. The setup is based on a commercial microscope with a minimal set of inexpensive components, suitable for implementation in a student laboratory. We show how this approach can be used in a demonstration motility assay, tracking the gliding motions of microtubules driven by the motor protein kinesin.

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

    PubMed

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

    2014-01-01

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

  12. Organelles are transported on sliding microtubules in Reticulomyxa.

    PubMed

    Orokos, D D; Cole, R W; Travis, J L

    2000-12-01

    Organelles and plasma membrane domains appear to be transported along Reticulomyxa's microtubule cytoskeleton. Previously we demonstrated that organelle and cell surface transport share the same enzymatic properties and suggested that both are powered by the same cytoplasmic dynein. Motility analysis in Reticulomyxa is complicated by the fact that the microtubules also are motile and appear to "slide" bidirectionally throughout the network. We have utilized laser ablation to address this frame-of-reference problem as to how each transport component (microtubule sliding vs. organelle translocations) contributes to reactivated bidirectional translocation of organelles along the microtubule cytoskeleton. Laser ablation was used to cut microtubule bundles from lysed networks into 4-15-microm segments. After examining these reactivated cut fragments, it appears that the majority of organelles did not move relative to microtubule fragments, but remained attached to microtubules and moved as the microtubules slid. Microtubule sliding stops after 1-2 min and cannot be reactivated even when perfused with fresh ATP. Furthermore, once sliding stops, organelle transport also stops. Our findings indicate that the majority of Reticulomyxa pseudopodial organelles do not move along the surface of the microtubules, rather it is the sliding of the microtubules to which they are attached that moves them.

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

    PubMed Central

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

    2014-01-01

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

  14. Organization of microtubules in cochlear hair cells.

    PubMed

    Furness, D N; Hackney, C M; Steyger, P S

    1990-07-01

    The organization of microtubules in hair cells of the guinea-pig cochlea has been investigated using transmission electron microscopy and correlated with the location of tubulin-associated immunofluorescence in surface preparations of the organ of Corti. Results from both techniques reveal consistent distributions of microtubules in inner and outer hair cells. In the inner hair cells, microtubules are most concentrated in the apex. Reconstruction from serial sections shows three main groups: firstly, in channels through the cuticular plate and in a discontinuous belt around its upper perimeter; secondly, forming a ring inside a rim extending down from the lower perimeter of the plate; and thirdly, in a meshwork underlying the main body of the plate. In the cell body, microtubules line the inner face of the subsurface cistern and extend longitudinally through a tubulo-vesicular track between the apex and base. In outer hair cells, the pattern of microtubules associated with the cuticular plate is similar, although there are fewer present than in inner hair cells. In outer hair cells from the apex of the cochlea, microtubules occur around an infracuticular protrusion of cuticular plate material. In the cell body, many more microtubules occur in the region below the nucleus compared with inner hair cells. The possible functions of microtubules in hair cells are discussed by comparison with those found in other systems. These include morphogenesis and maintenance of cell shape; intracellular transport, e.g., of neurotransmitter vesicles; providing a possible substrate for motility; mechanical support of structures associated with sensory transduction.

  15. Axoneme Structure from Motile Cilia.

    PubMed

    Ishikawa, Takashi

    2017-01-03

    The axoneme is the main extracellular part of cilia and flagella in eukaryotes. It consists of a microtubule cytoskeleton, which normally comprises nine doublets. In motile cilia, dynein ATPase motor proteins generate sliding motions between adjacent microtubules, which are integrated into a well-orchestrated beating or rotational motion. In primary cilia, there are a number of sensory proteins functioning on membranes surrounding the axoneme. In both cases, as the study of proteomics has elucidated, hundreds of proteins exist in this compartmentalized biomolecular system. In this article, we review the recent progress of structural studies of the axoneme and its components using electron microscopy and X-ray crystallography, mainly focusing on motile cilia. Structural biology presents snapshots (but not live imaging) of dynamic structural change and gives insights into the force generation mechanism of dynein, ciliary bending mechanism, ciliogenesis, and evolution of the axoneme.

  16. Statistical physical models of cellular motility

    NASA Astrophysics Data System (ADS)

    Banigan, Edward J.

    Cellular motility is required for a wide range of biological behaviors and functions, and the topic poses a number of interesting physical questions. In this work, we construct and analyze models of various aspects of cellular motility using tools and ideas from statistical physics. We begin with a Brownian dynamics model for actin-polymerization-driven motility, which is responsible for cell crawling and "rocketing" motility of pathogens. Within this model, we explore the robustness of self-diffusiophoresis, which is a general mechanism of motility. Using this mechanism, an object such as a cell catalyzes a reaction that generates a steady-state concentration gradient that propels the object in a particular direction. We then apply these ideas to a model for depolymerization-driven motility during bacterial chromosome segregation. We find that depolymerization and protein-protein binding interactions alone are sufficient to robustly pull a chromosome, even against large loads. Next, we investigate how forces and kinetics interact during eukaryotic mitosis with a many-microtubule model. Microtubules exert forces on chromosomes, but since individual microtubules grow and shrink in a force-dependent way, these forces lead to bistable collective microtubule dynamics, which provides a mechanism for chromosome oscillations and microtubule-based tension sensing. Finally, we explore kinematic aspects of cell motility in the context of the immune system. We develop quantitative methods for analyzing cell migration statistics collected during imaging experiments. We find that during chronic infection in the brain, T cells run and pause stochastically, following the statistics of a generalized Levy walk. These statistics may contribute to immune function by mimicking an evolutionarily conserved efficient search strategy. Additionally, we find that naive T cells migrating in lymph nodes also obey non-Gaussian statistics. Altogether, our work demonstrates how physical

  17. Microtubules Remodel Actomyosin Networks in Xenopus Egg Extracts via Two Mechanisms of F-Actin Transport

    PubMed Central

    Waterman-Storer, Clare; Duey, Devin Y.; Weber, Kari L.; Keech, John; Cheney, Richard E.; Salmon, E.D.; Bement, William M.

    2000-01-01

    Interactions between microtubules and filamentous actin (F-actin) are crucial for many cellular processes, including cell locomotion and cytokinesis, but are poorly understood. To define the basic principles governing microtubule/F-actin interactions, we used dual-wavelength digital fluorescence and fluorescent speckle microscopy to analyze microtubules and F-actin labeled with spectrally distinct fluorophores in interphase Xenopus egg extracts. In the absence of microtubules, networks of F-actin bundles zippered together or exhibited serpentine gliding along the coverslip. When microtubules were nucleated from Xenopus sperm centrosomes, they were released and translocated away from the aster center. In the presence of microtubules, F-actin exhibited two distinct, microtubule-dependent motilities: rapid (∼250–300 nm/s) jerking and slow (∼50 nm/s), straight gliding. Microtubules remodeled the F-actin network, as F-actin jerking caused centrifugal clearing of F-actin from around aster centers. F-actin jerking occurred when F-actin bound to motile microtubules powered by cytoplasmic dynein. F-actin straight gliding occurred when F-actin bundles translocated along the microtubule lattice. These interactions required Xenopus cytosolic factors. Localization of myosin-II to F-actin suggested it may power F-actin zippering, while localization of myosin-V on microtubules suggested it could mediate interactions between microtubules and F-actin. We examine current models for cytokinesis and cell motility in light of these findings. PMID:10908578

  18. EXTRACELLULAR MICROTUBULES

    PubMed Central

    Bouck, G. Benjamin

    1969-01-01

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

  19. Microtubule-dependent control of cell shape and pseudopodial activity is inhibited by the antibody to kinesin motor domain

    PubMed Central

    1993-01-01

    One of the major functions of cytoplasmic microtubules is their involvement in maintenance of asymmetric cell shape. Microtubules were considered to perform this function working as rigid structural elements. At the same time, microtubules play a critical role in intracellular organelle transport, and this fact raises the possibility that the involvement of microtubules in maintenance of cell shape may be mediated by directed transport of certain cellular components to a limited area of the cell surface (e.g., to the leading edge) rather than by their functioning as a mechanical support. To test this hypothesis we microinjected cultured human fibroblasts with the antibody (called HD antibody) raised against kinesin motor domain highly conserved among the different members of kinesin superfamily. As was shown before this antibody inhibits kinesin-dependent microtubule gliding in vitro and interferes with a number of microtubule-dependent transport processes in living cells. Preimmune IgG fraction was used for control experiments. Injections of fibroblasts with HD antibody but not with preimmune IgG significantly reduced their asymmetry, resulting in loss of long processes and elongated cell shape. In addition, antibody injection suppressed pseudopodial activity at the leading edge of fibroblasts moving into an experimentally made wound. Analysis of membrane organelle distribution showed that kinesin antibody induced clustering of mitochondria in perinuclear region and their withdrawal from peripheral parts of the cytoplasm. HD antibody does not affect either density or distribution of cytoplasmic microtubules. The results of our experiments show that many changes of phenotype induced in cells by microtubule-depolymerizing agents can be mimicked by the inhibition of motor proteins, and therefore microtubule functions in maintaining of the cell shape and polarity are mediated by motor proteins rather than by being provided by rigidity of tubulin polymer itself. PMID

  20. Bidirectional motility of the fission yeast kinesin-5, Cut7

    SciTech Connect

    Edamatsu, Masaki

    2014-03-28

    Highlights: • Motile properties of Cut7 (fission yeast kinesin-5) were studied for the first time. • Half-length Cut7 moved toward plus-end direction of microtubule. • Full-length Cut7 moved toward minus-end direction of microtubule. • N- and C-terminal microtubule binding sites did not switch the motile direction. - Abstract: Kinesin-5 is a homotetrameric motor with its motor domain at the N-terminus. Kinesin-5 crosslinks microtubules and functions in separating spindle poles during mitosis. In this study, the motile properties of Cut7, fission yeast kinesin-5, were examined for the first time. In in vitro motility assays, full-length Cut7 moved toward minus-end of microtubules, but the N-terminal half of Cut7 moved toward the opposite direction. Furthermore, additional truncated constructs lacking the N-terminal or C-terminal regions, but still contained the motor domain, did not switch the motile direction. These indicated that Cut7 was a bidirectional motor, and microtubule binding regions at the N-terminus and C-terminus were not involved in its directionality.

  1. Role of the microtubule cytoskeleton in gravisensing Chara rhizoids.

    PubMed

    Braun, M; Sievers, A

    1994-04-01

    The arrangement of the microtubule cytoskeleton in tip-growing and gravisensing Chara rhizoids has been documented by immunofluorescence microscopy. Predominantly axially oriented undulating bundles of cortical microtubules were found in the basal zone of the rhizoids and colocalized with the microfilament bundles underlying the cytoplasmic streaming. Microtubules penetrate the subapical zone, forming a three-dimensional network that envelops the nucleus and organelles. Microtubules are present up to 5 to 10 microns basal from the apical cytoplasmic region containing the statoliths. No microtubules were found in the apical zone of the rhizoid which is the site of tip growth and gravitropism. Depolymerization of microtubules by application of oryzalin does not affect cytoplasmic streaming and gravitropic growth until the relatively stationary and polarly organized apical and subapical cytoplasm is converted into streaming cytoplasm. When the statoliths and the apical cytoplasm are included in the cytoplasmic streaming, tip growth and gravitropism are stopped. Oryzalin-induced disruption of the microtubule cytoskeleton also results in a rearrangement of the dense network of apical and subapical microfilaments into thicker bundles, whereas disruption of the microfilament cytoskeleton by cytochalasin D had no effect on the organization of the microtubule cytoskeleton. It is, therefore, concluded that the arrangement of microtubules is essential for the polar cytoplasmic zonation and the functionally polar organization of the actin cytoskeleton which is responsible for the motile processes in rhizoids. Microtubules are not involved in the primary events of gravitropism in Chara rhizoids.

  2. Modeling of chromosome motility during mitosis.

    PubMed

    Gardner, Melissa K; Odde, David J

    2006-12-01

    Chromosome motility is a highly regulated and complex process that ultimately achieves proper segregation of the replicated genome. Recent modeling studies provide a computational framework for investigating how microtubule assembly dynamics, motor protein activity and mitotic spindle mechanical properties are integrated to drive chromosome motility. Among other things, these studies show that metaphase chromosome oscillations can be explained by a range of assumptions, and that non-oscillatory states can be achieved with modest changes to the model parameters. In addition, recent microscopy studies provide new insight into the nature of the coupling between force on the kinetochore and kinetochore-microtubule assembly/disassembly. Together, these studies facilitate advancement toward a unified model that quantitatively predicts chromosome motility.

  3. Heterotrimeric Kinesin II Is the Microtubule Motor Protein Responsible for Pigment Dispersion in Xenopus Melanophores

    PubMed Central

    Tuma, M. Carolina; Zill, Andrew; Le Bot, Nathalie; Vernos, Isabelle; Gelfand, Vladimir

    1998-01-01

    Melanophores move pigment organelles (melanosomes) from the cell center to the periphery and vice-versa. These bidirectional movements require cytoplasmic microtubules and microfilaments and depend on the function of microtubule motors and a myosin. Earlier we found that melanosomes purified from Xenopus melanophores contain the plus end microtubule motor kinesin II, indicating that it may be involved in dispersion (Rogers, S.L., I.S. Tint, P.C. Fanapour, and V.I. Gelfand. 1997. Proc. Natl. Acad. Sci. USA. 94: 3720–3725). Here, we generated a dominant-negative construct encoding green fluorescent protein fused to the stalk-tail region of Xenopus kinesin-like protein 3 (Xklp3), the 95-kD motor subunit of Xenopus kinesin II, and introduced it into melanophores. Overexpression of the fusion protein inhibited pigment dispersion but had no effect on aggregation. To control for the specificity of this effect, we studied the kinesin-dependent movement of lysosomes. Neither dispersion of lysosomes in acidic conditions nor their clustering under alkaline conditions was affected by the mutant Xklp3. Furthermore, microinjection of melanophores with SUK4, a function-blocking kinesin antibody, inhibited dispersion of lysosomes but had no effect on melanosome transport. We conclude that melanosome dispersion is powered by kinesin II and not by conventional kinesin. This paper demonstrates that kinesin II moves membrane-bound organelles. PMID:9852150

  4. Drosophila Katanin is a microtubule depolymerase that regulates cortical-microtubule plus-end interactions and cell migration

    PubMed Central

    Zhang, Dong; Grode, Kyle D.; Stewman, Shannon; Diaz, Daniel; Liebling, Emily; Rath, Uttama; Riera, Tania; Currie, Joshua; Buster, Daniel W.; Asenjo, Ana B.; Sosa, Hernando J.; Ross, Jennifer; Ma, Ao; Rogers, Stephen L.; Sharp, David J.

    2011-01-01

    Regulation of microtubule dynamics at the cell cortex is important for cell motility, morphogenesis and division. Here we show that the Drosophila Katanin, Dm-Kat60, functions to generate a dynamic cortical-microtubule interface in interphase cells. In S2 cells, Dm-Kat60 concentrates at the interphase cell cortex where it suppresses the polymerization of microtubule plus-ends thereby preventing the formation of aberrantly dense cortical arrays. Dm-Kat60 also localizes to the leading edge migratory D17 cells and negatively regulates multiple parameters of their motility. Finally, in vitro, Dm-Kat60 severs and depolymerizes MTs from their ends. Based on these data, we propose that Dm-Kat60 removes tubulin from microtubule ends or lattice that contact specific cortical sites to preventing stable and/or lateral attachments. The asymmetric distribution of such an activity could help generate regional variations in MT behaviors involved in cell migration. PMID:21378981

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

    PubMed

    Stepanek, Ludek; Pigino, Gaia

    2016-05-06

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

  6. Molecular Pathway of Microtubule Organization at the Golgi Apparatus.

    PubMed

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

    2016-10-10

    The Golgi apparatus controls the formation of non-centrosomal microtubule arrays important for Golgi organization, polarized transport, cell motility, and cell differentiation. Here, we show that CAMSAP2 stabilizes and attaches microtubule minus ends to the Golgi through a complex of AKAP450 and myomegalin. CLASPs stabilize CAMSAP2-decorated microtubules but are not required for their Golgi tethering. AKAP450 is also essential for Golgi microtubule nucleation, and myomegalin and CDK5RAP2 but not CAMSAP2 contribute to this function. In the absence of centrosomes, AKAP450- and CAMSAP2-dependent pathways of microtubule minus-end organization become dominant, and the presence of at least one of them is needed to maintain microtubule density. Strikingly, a compact Golgi can be assembled in the absence of both centrosomal and Golgi microtubules. However, CAMSAP2- and AKAP450-dependent Golgi microtubules facilitate Golgi reorientation and cell invasion in a 3D matrix. We propose that Golgi-anchored microtubules are important for polarized cell movement but not for coalescence of Golgi membranes. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Tensile stress stimulates microtubule outgrowth in living cells

    NASA Technical Reports Server (NTRS)

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

    2002-01-01

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

  8. Tensile stress stimulates microtubule outgrowth in living cells

    NASA Technical Reports Server (NTRS)

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

    2002-01-01

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

  9. Taxol Crystals Can Masquerade as Stabilized Microtubules

    PubMed Central

    Alsop, G. Bradley; Zhang, Dahong

    2008-01-01

    Taxol is a potent anti-mitotic drug used in chemotherapy, angioplastic stents, and cell biology research. By binding and stabilizing microtubules, Taxol inhibits their dynamics, crucial for cell division, motility, and survival. The drug has also been reported to induce formation of asters and bundles composed of stabilized microtubules. Surprisingly, at commonly used concentrations, Taxol forms crystals that rapidly bind fluorescent tubulin subunits, generating structures with an uncanny resemblance to microtubule asters and bundles. Kinetic and topological considerations suggest that tubulin subunits, rather than microtubules, bind the crystals. This sequestration of tubulin from the subunit pool would be expected to shift the equilibrium of free to polymerized tubulin to disfavor assembly. Our results imply that some previously reported Taxol-induced asters or bundles could include or be composed of tubulin-decorated Taxol crystals. Thus, reevaluation of certain morphological, chemical, and physical properties of Taxol-treated microtubules may be necessary. Moreover, our findings suggest a novel mechanism for chemotherapy-induced cytotoxicity in non-dividing cells, with far-reaching medical implications. PMID:18213384

  10. A study of microtubule dipole lattices

    NASA Astrophysics Data System (ADS)

    Nandi, Shubhendu

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

  11. Learn About GI Motility

    MedlinePlus

    ... Disorders of the Large Intestine Disorders of the Pelvic Floor Motility Testing Personal Stories Contact About GI Motility ... Disorders of the Large Intestine Disorders of the Pelvic Floor Motility Testing Personal Stories Contact About GI Motility ...

  12. About GI Motility

    MedlinePlus

    ... Disorders of the Large Intestine Disorders of the Pelvic Floor Motility Testing Personal Stories Contact About GI Motility ... Disorders of the Large Intestine Disorders of the Pelvic Floor Motility Testing Personal Stories Contact About GI Motility ...

  13. Ice Recovery Assay for Detection of Golgi-Derived Microtubules

    PubMed Central

    Grimaldi, Ashley D.; Fomicheva, Maria; Kaverina, Irina

    2014-01-01

    Proper organization of the microtubule cytoskeleton is essential for many cellular processes including maintenance of Golgi organization and cell polarity. Traditionally, the centrosome is considered to be the major microtubule organizing center (MTOC) of the cell; however, microtubule nucleation can also occur through centrosome-independent mechanisms. Recently, the Golgi has been described as an additional, centrosome-independent, MTOC with distinct cellular functions. Golgi-derived microtubules contribute to the formation of an asymmetric microtubule network, control Golgi organization, and support polarized trafficking and directed migration in motile cells. In this chapter, we present an assay using recovery from ice treatment to evaluate the potential of the Golgi, or other MTOCs, to nucleate microtubules. This technique allows for clear separation of distinct MTOCs and observation of newly nucleated microtubules at these locations, which are normally obscured by the dense microtubule network present at steady-state conditions. This type of analysis is important for discovery and characterization of noncentrosomal MTOCs and, ultimately, understanding of their unique cellular functions. PMID:24295320

  14. Ice recovery assay for detection of Golgi-derived microtubules.

    PubMed

    Grimaldi, Ashley D; Fomicheva, Maria; Kaverina, Irina

    2013-01-01

    Proper organization of the microtubule cytoskeleton is essential for many cellular processes including maintenance of Golgi organization and cell polarity. Traditionally, the centrosome is considered to be the major microtubule organizing center (MTOC) of the cell; however, microtubule nucleation can also occur through centrosome-independent mechanisms. Recently, the Golgi has been described as an additional, centrosome-independent, MTOC with distinct cellular functions. Golgi-derived microtubules contribute to the formation of an asymmetric microtubule network, control Golgi organization, and support polarized trafficking and directed migration in motile cells. In this chapter, we present an assay using recovery from ice treatment to evaluate the potential of the Golgi, or other MTOCs, to nucleate microtubules. This technique allows for clear separation of distinct MTOCs and observation of newly nucleated microtubules at these locations, which are normally obscured by the dense microtubule network present at steady-state conditions. This type of analysis is important for discovery and characterization of noncentrosomal MTOCs and, ultimately, understanding of their unique cellular functions. Copyright © 2013 Elsevier Inc. All rights reserved.

  15. Targeting Microtubules for Wound Repair

    PubMed Central

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

    2016-01-01

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

  16. 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)

  17. 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)

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

    PubMed

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

    2015-01-01

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

  19. Selective adhesion of functional microtubules to patterned silane surfaces.

    PubMed Central

    Turner, D C; Chang, C; Fang, K; Brandow, S L; Murphy, D B

    1995-01-01

    We show that microtubule polymers can be immobilized selectively on lithographically patterned silane surfaces while retaining their native properties. Silane films were chemisorbed on polished silicon wafers or glass coverslips and patterned using a deep UV lithographic process developed at the Naval Research Laboratory. Hydrocarbon and fluorocarbon alkyl silanes, as well as amino and thiol terminal alkyl silanes, were investigated as substrates for microtubule adhesion with retention of biological activity. Microtubules were found to adhere strongly to amine terminal silanes while retaining the ability to act as substrates for the molecular motor protein kinesin. Aminosilane patterns with linewidths varying from 1 to 50 microns were produced lithographically and used to produce patterns of selectively adhered microtubules. Microtubules were partially aligned on the patterned lines by performing the immobilization in a fluid flow field. Patterns were imaged with atomic force microscopy and differential interference contrast microscopy. Motility assays were carried out using kinesin-coated beads and observed with differential interference contrast microscopy. Kinesin bead movement on the patterned microtubules was comparable to movement on microtubule control surfaces. Images FIGURE 1 FIGURE 2 FIGURE 3 FIGURE 4 PMID:8599684

  20. Learn About GI Motility

    MedlinePlus

    ... eNewsletter Sidebar × MOBILE MENU About Us Learn About GI Motility Digestive Tract Disorders of the Esophagus Disorders ... Pelvic Floor Motility Testing Personal Stories Contact About GI Motility Twitter Facebook YouTube Search Search ... About Us ...

  1. About GI Motility

    MedlinePlus

    ... eNewsletter Sidebar × MOBILE MENU About Us Learn About GI Motility Digestive Tract Disorders of the Esophagus Disorders ... Pelvic Floor Motility Testing Personal Stories Contact About GI Motility Twitter Facebook YouTube Search Search ... About Us ...

  2. Effects of colchicine, vinblastine and nocodazole on polarity, motility, chemotaxis and cAMP levels of human polymorphonuclear leukocytes.

    PubMed

    Keller, H U; Naef, A; Zimmermann, A

    1984-07-01

    We present evidence for intrinsic polymorphonuclear leukocyte (PMN) polarity manifested in presence of microtubule-disrupting drugs. Polarization in response to colchicine correlated with the known dose-dependent effects of this drug on microtubule disassembly. The response to 10(-5) M colchicine, 10(-5) M vinblastine and 10(-6) M nocodazole was associated with stimulated motility and random locomotion. Responses elicited by microtubule-disrupting drugs differed from f-Met-Leu-Phe (fMLP)-induced polarization by functional and morphological criteria. Polarization, motility and orthokinesis responses were much weaker. Furthermore, ruffling was almost absent in PMNs polarized in response to colchicine, vinblastine or nocodazole. The response was inhibited by cytochalasin B, indicating that it is microfilament-dependent. We suggest that microtubule-disrupting drugs induce motility via structural changes in the cytoskeleton which act as signals for the motor apparatus. The intrinsic polarity manifested in the presence of microtubule-disrupting drugs could be reversed by an extracellular chemotactic gradient. Stimulated locomotion and motility in response to microtubule-disrupting drugs was only observed with initially spherical PMNs but not with initially motile cells. The findings provide an explanation for the numerous conflicting statements on the chemokinetic activities of these drugs. The role of cAMP in stimulated polarization and motility has been studied. Colchicine, vinblastine and nocodazole elicited a transient elevation of cAMP levels within 1 min of stimulation. cAMP elevation and stimulated motility were not quantitatively correlated.

  3. The Elephant in the Room: The Role of Microtubules in Cancer.

    PubMed

    Cirillo, Luca; Gotta, Monica; Meraldi, Patrick

    2017-01-01

    Microtubules are the backbone of all eukaryotic cells cytoskeleton. Their dynamic behaviour constitutes the basis for many biological processes such as cellular motility, cytoplasmic transport and cell division. Some the most effective chemotherapeutics, such as the taxanes, are microtubule interfering drugs. Moreover, many studies suggest that microtubule dynamics are altered in cancer cell divisions and linked to chromosomal instability, aneuploidy and development of drug resistances. The elephant in the room, however, is that despite all these evidences, the exact role of microtubules in malignancies remains elusive, partially due to the lack of clear genetic alterations linking microtubules to cancer. This review will discuss the molecular mechanisms that might alter microtubule dynamics in cancer cells, the pro and cons of the different theories linking these alterations to cancer progression, and the possible directions to address future key questions.

  4. Microtubule motors mediate endosomal sorting by maintaining functional domain organization.

    PubMed

    Hunt, Sylvie D; Townley, Anna K; Danson, Chris M; Cullen, Peter J; Stephens, David J

    2013-06-01

    Many microtubule motors have been shown to couple to endosomal membranes. These motors include dynein in addition to many different kinesin family members. Sorting nexins (SNXs) are central to the organization and function of endosomes. These proteins can actively shape endosomal membranes and couple directly or indirectly to the minus-end microtubule motor dynein. Motor proteins acting on endosomes drive their motility, dictate their morphology and affect cargo segregation. We have used well-characterized members of the SNX family to elucidate motor coupling using high-resolution light microscopy coupled with depletion of specific microtubule motors. Endosomal domains labelled with SNX1, SNX4 and SNX8 couple to discrete combinations of dynein and kinesin motors. These specific combinations govern the structure and motility of each SNX-coated membrane in addition to the segregation of distinct functional endosomal subdomains. Taken together, our data show that these key features of endosome dynamics are governed by the same set of opposing microtubule motors. Thus, microtubule motors help to define the mosaic layout of endosomes that underpins cargo sorting.

  5. Swimming with protists: perception, motility and flagellum assembly.

    PubMed

    Ginger, Michael L; Portman, Neil; McKean, Paul G

    2008-11-01

    In unicellular and multicellular eukaryotes, fast cell motility and rapid movement of material over cell surfaces are often mediated by ciliary or flagellar beating. The conserved defining structure in most motile cilia and flagella is the '9+2' microtubule axoneme. Our general understanding of flagellum assembly and the regulation of flagellar motility has been led by results from seminal studies of flagellate protozoa and algae. Here we review recent work relating to various aspects of protist physiology and cell biology. In particular, we discuss energy metabolism in eukaryotic flagella, modifications to the canonical assembly pathway and flagellum function in parasite virulence.

  6. A mathematical model of stress generation in microtubule pair interactions

    NASA Astrophysics Data System (ADS)

    Fang, Fang; Betterton, Meredith; Shelley, Michael

    2014-11-01

    Microtubules and motor proteins are basic ingredients in many cellular structures and of new biosynthetic ``active'' suspensions. The interaction of microtubules with their surrounding fluid medium depends fundamentally upon the force generation afforded them through cross-linking motile motor proteins. Here we develop a simple mathematical model, based on the statistical mechanics, motor proteins binding and unbinding, to study the generation of active fluid stresses. We study the role and contributions of ``polarity sorting'' and ``tether'' relaxation on the generation of intrinsic, destabilizing stresses.

  7. Reconstitution of microtubule-dependent organelle transport.

    PubMed

    Barak, Pradeep; Rai, Ashim; Dubey, Alok Kumar; Rai, Priyanka; Mallik, Roop

    2014-01-01

    Microtubule (MT)-based motor proteins transport many cellular factors to their functionally relevant locations within cells, and defects in transport are linked to human disease. Understanding the mechanism and regulation of this transport process in living cells is difficult because of the complex in vivo environment and limited means to manipulate the system. On the other hand, in vitro motility assays using purified motors attached to beads does not recapitulate the full complexity of cargo transport in vivo. Assaying motility of organelles in cell extracts is therefore attractive, as natural cargoes are being examined, but in an environment that is more amenable to manipulation. Here, we describe the purification and in vitro MT-based motility of phagosomes from Dictyostelium and lipid droplets from rat liver. These assays have the potential to address diverse questions related to endosome/phagosome maturation, fatty acid regulation, and could also serve as a starting point for reconstituting the motility of other types of organelles. © 2014 Elsevier Inc. All rights reserved.

  8. 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

  9. 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

  10. Microtubule acetylation promotes kinesin-1 binding and transport.

    PubMed

    Reed, Nathan A; Cai, Dawen; Blasius, T Lynne; Jih, Gloria T; Meyhofer, Edgar; Gaertig, Jacek; Verhey, Kristen J

    2006-11-07

    Long-distance intracellular delivery is driven by kinesin and dynein motor proteins that ferry cargoes along microtubule tracks . Current models postulate that directional trafficking is governed by known biophysical properties of these motors-kinesins generally move to the plus ends of microtubules in the cell periphery, whereas cytoplasmic dynein moves to the minus ends in the cell center. However, these models are insufficient to explain how polarized protein trafficking to subcellular domains is accomplished. We show that the kinesin-1 cargo protein JNK-interacting protein 1 (JIP1) is localized to only a subset of neurites in cultured neuronal cells. The mechanism of polarized trafficking appears to involve the preferential recognition of microtubules containing specific posttranslational modifications (PTMs) by the kinesin-1 motor domain. Using a genetic approach to eliminate specific PTMs, we show that the loss of a single modification, alpha-tubulin acetylation at Lys-40, influences the binding and motility of kinesin-1 in vitro. In addition, pharmacological treatments that increase microtubule acetylation cause a redirection of kinesin-1 transport of JIP1 to nearly all neurite tips in vivo. These results suggest that microtubule PTMs are important markers of distinct microtubule populations and that they act to control motor-protein trafficking.

  11. BORC Functions Upstream of Kinesins 1 and 3 to Coordinate Regional Movement of Lysosomes Along Different Microtubule Tracks

    PubMed Central

    Guardia, Carlos M.; Farías, Ginny G.; Jia, Rui; Pu, Jing; Bonifacino, Juan S.

    2016-01-01

    Summary The multiple functions of lysosomes are critically dependent on their ability to undergo bidirectional movement along microtubules between the center and the periphery of the cell. Centrifugal and centripetal movement of lysosomes is mediated by kinesin and dynein motors, respectively. We recently described a multisubunit complex named BORC that recruits the small GTPase Arl8 to lysosomes to promote their kinesin-dependent movement toward the cell periphery. Here we show that BORC and Arl8 function upstream of two structurally distinct kinesin types: kinesin-1 (KIF5B) and kinesin-3 (KIF1Bβ and KIF1A). Remarkably, KIF5B preferentially moves lysosomes on perinuclear tracks enriched in acetylated α-tubulin, whereas KIF1Bβ and KIF1A drive lysosome movement on more rectilinear, peripheral tracks enriched in tyrosinated α-tubulin. These findings establish BORC as a master regulator of lysosome positioning through coupling to different kinesins and microtubule tracks. Common regulation by BORC enables coordinate control of lysosome movement in different regions of the cell. PMID:27851960

  12. Forces from the rear: deformed microtubules in neuronal growth cones influence retrograde flow and advancement

    NASA Astrophysics Data System (ADS)

    Rauch, Philipp; Heine, Paul; Goettgens, Barbara; Käs, Josef A.

    2013-01-01

    The directed motility of growth cones at the tip of neuronal processes is a key function in neuronal path-finding and relies on a complex system of interacting cytoskeletal components. Despite intensive research in this field, many aspects of the mechanical roles of actin structures and, in particular, of microtubules throughout this process remain unclear. Mostly, force generation is ascribed to actin-myosin-based structures such as filopodia bundles and the dynamic polymer gel within the lamellipodium. Our analysis of microtubule buckling and deformation in motile growth cones reveals that extending microtubule filaments contribute significantly to the overall protrusion force. In this study, we establish a relationship of the local variations in stored bending energy and deformation characteristics to growth cone morphology and retrograde actin flow. This implies the relevance of microtubule pushing and deformation for general neurite advancement as well as steering processes.

  13. Cytoplasmic streaming in Drosophila oocytes varies with kinesin activity and correlates with the microtubule cytoskeleton architecture

    PubMed Central

    Ganguly, Sujoy; Williams, Lucy S.; Palacios, Isabel M.; Goldstein, Raymond E.

    2012-01-01

    Cells can localize molecules asymmetrically through the combined action of cytoplasmic streaming, which circulates their fluid contents, and specific anchoring mechanisms. Streaming also contributes to the distribution of nutrients and organelles such as chloroplasts in plants, the asymmetric position of the meiotic spindle in mammalian embryos, and the developmental potential of the zygote, yet little is known quantitatively about the relationship between streaming and the motor activity which drives it. Here we use Particle Image Velocimetry to quantify the statistical properties of Kinesin-dependent streaming during mid-oogenesis in Drosophila. We find that streaming can be used to detect subtle changes in Kinesin activity and that the flows reflect the architecture of the microtubule cytoskeleton. Furthermore, based on characterization of the rheology of the cytoplasm in vivo, we establish estimates of the number of Kinesins required to drive the observed streaming. Using this in vivo data as the basis of a model for transport, we suggest that the disordered character of transport at mid-oogenesis, as revealed by streaming, is an important component of the localization dynamics of the body plan determinant oskar mRNA. PMID:22949706

  14. Cytoplasmic streaming in Drosophila oocytes varies with kinesin activity and correlates with the microtubule cytoskeleton architecture.

    PubMed

    Ganguly, Sujoy; Williams, Lucy S; Palacios, Isabel M; Goldstein, Raymond E

    2012-09-18

    Cells can localize molecules asymmetrically through the combined action of cytoplasmic streaming, which circulates their fluid contents, and specific anchoring mechanisms. Streaming also contributes to the distribution of nutrients and organelles such as chloroplasts in plants, the asymmetric position of the meiotic spindle in mammalian embryos, and the developmental potential of the zygote, yet little is known quantitatively about the relationship between streaming and the motor activity which drives it. Here we use Particle Image Velocimetry to quantify the statistical properties of Kinesin-dependent streaming during mid-oogenesis in Drosophila. We find that streaming can be used to detect subtle changes in Kinesin activity and that the flows reflect the architecture of the microtubule cytoskeleton. Furthermore, based on characterization of the rheology of the cytoplasm in vivo, we establish estimates of the number of Kinesins required to drive the observed streaming. Using this in vivo data as the basis of a model for transport, we suggest that the disordered character of transport at mid-oogenesis, as revealed by streaming, is an important component of the localization dynamics of the body plan determinant oskar mRNA.

  15. FUS inclusions disrupt RNA localization by sequestering kinesin-1 and inhibiting microtubule detyrosination.

    PubMed

    Yasuda, Kyota; Clatterbuck-Soper, Sarah F; Jackrel, Meredith E; Shorter, James; Mili, Stavroula

    2017-04-03

    Cytoplasmic inclusions of the RNA-binding protein fused in sarcoma (FUS) represent one type of membraneless ribonucleoprotein compartment. Formation of FUS inclusions is promoted by amyotrophic lateral sclerosis (ALS)-linked mutations, but the cellular functions affected upon inclusion formation are poorly defined. In this study, we find that FUS inclusions lead to the mislocalization of specific RNAs from fibroblast cell protrusions and neuronal axons. This is mediated by recruitment of kinesin-1 mRNA and protein within FUS inclusions, leading to a loss of detyrosinated glutamate (Glu)-microtubules (MTs; Glu-MTs) and an inability to support the localization of RNAs at protrusions. Importantly, dissolution of FUS inclusions using engineered Hsp104 disaggregases, or overexpression of kinesin-1, reverses these effects. We further provide evidence that kinesin-1 affects MT detyrosination not through changes in MT stability, but rather through targeting the tubulin carboxypeptidase enzyme onto specific MTs. Interestingly, other pathological inclusions lead to similar outcomes, but through apparently distinct mechanisms. These results reveal a novel kinesin-dependent mechanism controlling the MT cytoskeleton and identify loss of Glu-MTs and RNA mislocalization as common outcomes of ALS pathogenic mutations.

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

    PubMed

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

    2015-01-16

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

  17. Zinc Deficiency and Microtubule Function in Prostate Cells

    DTIC Science & Technology

    2006-04-01

    in rat models of zinc deficiency with no reported change between control and deficient conditions [Oteiza et al., 1990]. However, microtubule...polymerization rates were decreased in zinc deficient rats in that study. The authors suggested that zinc deficiency might cause a change in the expression of...spermatozoan motility I. Distribution of iron, zinc and copper in sea urchin spermatozoa . 1972 Exp Cell Res. 70(2):311-316, 1972. Oteiza PI, Hurley LS

  18. Motor domain-based motility system and motile properties of alpha heavy chain in Tetrahymena outer arm dynein.

    PubMed

    Edamatsu, Masaki

    2014-10-24

    Axonemal dynein plays an essential role in ciliary motility, and impaired ciliary motility causes human diseases such as primary ciliary dyskinesia (PCD). The motor domain of axonemal dynein powers ciliary motility and its function is regulated by several accessary proteins bound to the tail region. Therefore, to understand the essential properties of dynein motility, examining the motile properties of the motor domain without the tail is necessary. In this study, the functional motor domain of the alpha heavy chain in Tetrahymena outer arm dynein was purified, and its motile properties were examined using an in vitro motility system. The purified protein caused microtubules to glide at a velocity of 5.0μm/s with their minus-end trailing, and motility was inhibited in an ATP concentration-dependent manner, which is in contrast with kinesin1. This method could be applicable to other axonemal dyneins and will enable further molecular studies on diverse axonemal dyneins and ciliary motility. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. BRIDGES BETWEEN MICROTUBULES

    PubMed Central

    McIntosh, J. R.

    1974-01-01

    Bridges between microtubules have been studied with the electron microscope in the axostyle of Saccinobaculus and in various tubule systems of chicken testis, including the helix of tubules surrounding the elongating spermatid nucleus and the flagellum of the sperm tail. In addition to the previously described periodic bridges, evidence is presented that nonperiodic bridges exist between certain tubules. An analysis of axial spacing between adjacent nonperiodic bridges suggests that these structures are attached to periodic binding sites on the microtubule wall, but that not all the binding sites are filled. The bridges appear nonperiodic as a result of random occupancy of some fraction of the periodic sites. The distribution of these binding sites is related to the substructure of the microtubule wall as seen with negative staining and optical diffraction. PMID:4132065

  20. Microtubule-based force generation.

    PubMed

    Kent, Ian A; Lele, Tanmay P

    2017-05-01

    Microtubules are vital to many important cell processes, such as cell division, transport of cellular cargo, organelle positioning, and cell migration. Owing to their diverse functions, understanding microtubule function is an important part of cell biological research that can help in combating various diseases. For example, microtubules are an important target of chemotherapeutic drugs such as paclitaxel because of their pivotal role in cell division. Many functions of microtubules relate to the generation of mechanical forces. These forces are generally either a direct result of microtubule polymerization/depolymerization or generated by motor proteins that move processively along microtubules. In this review, we summarize recent efforts to quantify and model force generation by microtubules in the context of microtubule function. WIREs Nanomed Nanobiotechnol 2017, 9:e1428. doi: 10.1002/wnan.1428 For further resources related to this article, please visit the WIREs website. © 2016 Wiley Periodicals, Inc.

  1. 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

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

    PubMed

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

    2015-07-15

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

  3. The functional expression and motile properties of recombinant outer arm dynein from Tetrahymena.

    PubMed

    Edamatsu, Masaki

    2014-05-16

    Cilia and flagella are motile organelles that play various roles in eukaryotic cells. Ciliary movement is driven by axonemal dyneins (outer arm and inner arm dyneins) that bind to peripheral microtubule doublets. Elucidating the molecular mechanism of ciliary movement requires the genetic engineering of axonemal dyneins; however, no expression system for axonemal dyneins has been previously established. This study is the first to purify recombinant axonemal dynein with motile activity. In the ciliated protozoan Tetrahymena, recombinant outer arm dynein purified from ciliary extract was able to slide microtubules in a gliding assay. Furthermore, the recombinant dynein moved processively along microtubules in a single-molecule motility assay. This expression system will be useful for investigating the unique properties of diverse axonemal dyneins and will enable future molecular studies on ciliary movement. Copyright © 2014 Elsevier Inc. All rights reserved.

  4. Gliding motility powers invasion and egress in Apicomplexa.

    PubMed

    Frénal, Karine; Dubremetz, Jean-François; Lebrun, Maryse; Soldati-Favre, Dominique

    2017-09-04

    Protozoan parasites have developed elaborate motility systems that facilitate infection and dissemination. For example, amoebae use actin-rich membrane extensions called pseudopodia, whereas Kinetoplastida are propelled by microtubule-containing flagella. By contrast, the motile and invasive stages of the Apicomplexa - a phylum that contains the important human pathogens Plasmodium falciparum (which causes malaria) and Toxoplasma gondii (which causes toxoplasmosis) - have a unique machinery called the glideosome, which is composed of an actomyosin system that underlies the plasma membrane. The glideosome promotes substrate-dependent gliding motility, which powers migration across biological barriers, as well as active host cell entry and egress from infected cells. In this Review, we discuss the discovery of the principles that govern gliding motility, the characterization of the molecular machinery involved, and its impact on parasite invasion and egress from infected cells.

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

    PubMed

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

    2006-09-01

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

  6. Microtubule dynamics in fish melanophores

    PubMed Central

    1994-01-01

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

  7. Plant microtubule cytoskeleton complexity: microtubule arrays as fractals.

    PubMed

    Gardiner, John; Overall, Robyn; Marc, Jan

    2012-01-01

    Biological systems are by nature complex and this complexity has been shown to be important in maintaining homeostasis. The plant microtubule cytoskeleton is a highly complex system, with contributing factors through interactions with microtubule-associated proteins (MAPs), expression of multiple tubulin isoforms, and post-translational modification of tubulin and MAPs. Some of this complexity is specific to microtubules, such as a redundancy in factors that regulate microtubule depolymerization. Plant microtubules form partial helical fractals that play a key role in development. It is suggested that, under certain cellular conditions, other categories of microtubule fractals may form including isotropic fractals, triangular fractals, and branched fractals. Helical fractal proteins including coiled-coil and armadillo/beta-catenin repeat proteins and the actin cytoskeleton are important here too. Either alone, or in combination, these fractals may drive much of plant development.

  8. A Structural Basis for How Motile Cilia Beat

    PubMed Central

    Satir, Peter; Heuser, Thomas; Sale, Winfield S.

    2014-01-01

    The motile cilium is a mechanical wonder, a cellular nanomachine that produces a high-speed beat based on a cycle of bends that move along an axoneme made of 9+2 microtubules. The molecular motors, dyneins, power the ciliary beat. The dyneins are compacted into inner and outer dynein arms, whose activity is highly regulated to produce microtubule sliding and axonemal bending. The switch point hypothesis was developed long ago to account for how sliding in the presence of axonemal radial spoke–central pair interactions causes the ciliary beat. Since then, a new genetic, biochemical, and structural complexity has been discovered, in part, with Chlamydomonas mutants, with high-speed, high-resolution analysis of movement and with cryoelectron tomography. We stand poised on the brink of new discoveries relating to the molecular control of motility that extend and refine our understanding of the basic events underlying the switching of arm activity and of bend formation and propagation. PMID:26955066

  9. A Structural Basis for How Motile Cilia Beat.

    PubMed

    Satir, Peter; Heuser, Thomas; Sale, Winfield S

    2014-12-01

    The motile cilium is a mechanical wonder, a cellular nanomachine that produces a high-speed beat based on a cycle of bends that move along an axoneme made of 9+2 microtubules. The molecular motors, dyneins, power the ciliary beat. The dyneins are compacted into inner and outer dynein arms, whose activity is highly regulated to produce microtubule sliding and axonemal bending. The switch point hypothesis was developed long ago to account for how sliding in the presence of axonemal radial spoke-central pair interactions causes the ciliary beat. Since then, a new genetic, biochemical, and structural complexity has been discovered, in part, with Chlamydomonas mutants, with high-speed, high-resolution analysis of movement and with cryoelectron tomography. We stand poised on the brink of new discoveries relating to the molecular control of motility that extend and refine our understanding of the basic events underlying the switching of arm activity and of bend formation and propagation.

  10. Cell Biology: Microtubule Collisions to the Rescue.

    PubMed

    Gardner, Melissa K

    2016-12-19

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

  11. The motility of axonemal dynein is regulated by the tubulin code.

    PubMed

    Alper, Joshua D; Decker, Franziska; Agana, Bernice; Howard, Jonathon

    2014-12-16

    Microtubule diversity, arising from the utilization of different tubulin genes and from posttranslational modifications, regulates many cellular processes including cell division, neuronal differentiation and growth, and centriole assembly. In the case of cilia and flagella, multiple cell biological studies show that microtubule diversity is important for axonemal assembly and motility. However, it is not known whether microtubule diversity directly influences the activity of the axonemal dyneins, the motors that drive the beating of the axoneme, nor whether the effects on motility are indirect, perhaps through regulatory pathways upstream of the motors, such as the central pair, radial spokes, or dynein regulatory complex. To test whether microtubule diversity can directly regulate the activity of axonemal dyneins, we asked whether in vitro acetylation or deacetylation of lysine 40 (K40), a major posttranslational modification of α-tubulin, or whether proteolytic cleavage of the C-terminal tail (CTT) of α- and β-tubulin, the location of detyrosination, polyglutamylation, and polyglycylation modifications as well as most of the genetic diversity, can influence the activity of outer arm axonemal dynein in motility assays using purified proteins. By quantifying the motility with displacement-weighted velocity analysis and mathematically modeling the results, we found that K40 acetylation increases and CTTs decrease axonemal dynein motility. These results show that axonemal dynein directly deciphers the tubulin code, which has important implications for eukaryotic ciliary beat regulation.

  12. The Motility of Axonemal Dynein Is Regulated by the Tubulin Code

    PubMed Central

    Alper, Joshua D.; Decker, Franziska; Agana, Bernice; Howard, Jonathon

    2014-01-01

    Microtubule diversity, arising from the utilization of different tubulin genes and from posttranslational modifications, regulates many cellular processes including cell division, neuronal differentiation and growth, and centriole assembly. In the case of cilia and flagella, multiple cell biological studies show that microtubule diversity is important for axonemal assembly and motility. However, it is not known whether microtubule diversity directly influences the activity of the axonemal dyneins, the motors that drive the beating of the axoneme, nor whether the effects on motility are indirect, perhaps through regulatory pathways upstream of the motors, such as the central pair, radial spokes, or dynein regulatory complex. To test whether microtubule diversity can directly regulate the activity of axonemal dyneins, we asked whether in vitro acetylation or deacetylation of lysine 40 (K40), a major posttranslational modification of α-tubulin, or whether proteolytic cleavage of the C-terminal tail (CTT) of α- and β-tubulin, the location of detyrosination, polyglutamylation, and polyglycylation modifications as well as most of the genetic diversity, can influence the activity of outer arm axonemal dynein in motility assays using purified proteins. By quantifying the motility with displacement-weighted velocity analysis and mathematically modeling the results, we found that K40 acetylation increases and CTTs decrease axonemal dynein motility. These results show that axonemal dynein directly deciphers the tubulin code, which has important implications for eukaryotic ciliary beat regulation. PMID:25658008

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

    PubMed

    Benseddik, Khedidja; Sen Nkwe, Nadine; Daou, Pascale; Verdier-Pinard, Pascal; Badache, Ali

    2013-01-01

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

  14. Active diffusion and microtubule-based transport oppose myosin forces to position organelles in cells

    NASA Astrophysics Data System (ADS)

    Lin, Congping; Schuster, Martin; Guimaraes, Sofia Cunha; Ashwin, Peter; Schrader, Michael; Metz, Jeremy; Hacker, Christian; Gurr, Sarah Jane; Steinberg, Gero

    2016-06-01

    Even distribution of peroxisomes (POs) and lipid droplets (LDs) is critical to their role in lipid and reactive oxygen species homeostasis. How even distribution is achieved remains elusive, but diffusive motion and directed motility may play a role. Here we show that in the fungus Ustilago maydis ~95% of POs and LDs undergo diffusive motions. These movements require ATP and involve bidirectional early endosome motility, indicating that microtubule-associated membrane trafficking enhances diffusion of organelles. When early endosome transport is abolished, POs and LDs drift slowly towards the growing cell end. This pole-ward drift is facilitated by anterograde delivery of secretory cargo to the cell tip by myosin-5. Modelling reveals that microtubule-based directed transport and active diffusion support distribution, mobility and mixing of POs. In mammalian COS-7 cells, microtubules and F-actin also counteract each other to distribute POs. This highlights the importance of opposing cytoskeletal forces in organelle positioning in eukaryotes.

  15. Active diffusion and microtubule-based transport oppose myosin forces to position organelles in cells

    PubMed Central

    Lin, Congping; Schuster, Martin; Guimaraes, Sofia Cunha; Ashwin, Peter; Schrader, Michael; Metz, Jeremy; Hacker, Christian; Gurr, Sarah Jane; Steinberg, Gero

    2016-01-01

    Even distribution of peroxisomes (POs) and lipid droplets (LDs) is critical to their role in lipid and reactive oxygen species homeostasis. How even distribution is achieved remains elusive, but diffusive motion and directed motility may play a role. Here we show that in the fungus Ustilago maydis ∼95% of POs and LDs undergo diffusive motions. These movements require ATP and involve bidirectional early endosome motility, indicating that microtubule-associated membrane trafficking enhances diffusion of organelles. When early endosome transport is abolished, POs and LDs drift slowly towards the growing cell end. This pole-ward drift is facilitated by anterograde delivery of secretory cargo to the cell tip by myosin-5. Modelling reveals that microtubule-based directed transport and active diffusion support distribution, mobility and mixing of POs. In mammalian COS-7 cells, microtubules and F-actin also counteract each other to distribute POs. This highlights the importance of opposing cytoskeletal forces in organelle positioning in eukaryotes. PMID:27251117

  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. Regulation of Eukaryotic Flagellar Motility

    NASA Astrophysics Data System (ADS)

    Mitchell, David R.

    2005-03-01

    The central apparatus is essential for normal eukaryotic flagellar bend propagation as evidenced by the paralysis associated with mutations that prevent central pair (CP) assembly. Interactions between doublet-associated radial spokes and CP projections are thought to modulate spoke-regulated protein kinases and phosphatases on outer doublets, and these enzymes in turn modulate dynein activity. To better understand CP control mechanisms, we determined the three-dimensional structure of the Chlamydomonas reinhardtii CP complex and analyzed CP orientation during formation and propagation of flagellar bending waves. We show that a single CP microtubule, C1, is near the outermost doublet in curved regions of the flagellum, and this orientation is maintained by twists between successive principal and reverse bends. The Chlamydomonas CP is inherently twisted; twists are not induced by bend formation, and do not depend on forces or signals transmitted through spoke-central pair interactions. We hypothesize that CP orientation passively responds to bend formation, and that bend propagation drives rotation of the CP and maintains a constant CP orientation in bends, which in turn permits signal transduction between specific CP projections and specific doublet-associated dyneins through radial spokes. The central pair kinesin, Klp1, although essential for normal motility, is therefore not the motor that drives CP rotation. The CP also acts as a scaffold for enzymes that maintain normal intraflagellar ATP concentration.

  18. Chromosome position at the spindle equator is regulated by chromokinesin and a bipolar microtubule array.

    PubMed

    Takagi, Jun; Itabashi, Takeshi; Suzuki, Kazuya; Ishiwata, Shin'ichi

    2013-09-30

    The chromosome alignment is mediated by polar ejection and poleward forces acting on the chromosome arm and kinetochores, respectively. Although components of the motile machinery such as chromokinesin have been characterized, their dynamics within the spindle is poorly understood. Here we show that a quantum dot (Qdot) binding up to four Xenopus chromokinesin (Xkid) molecules behaved like a nanosize chromosome arm in the meiotic spindle, which is self-organized in cytoplasmic egg extracts. Xkid-Qdots travelled long distances along microtubules by changing several tracks, resulting in their accumulation toward and distribution around the metaphase plate. The analysis indicated that the direction of motion and velocity depend on the distribution of microtubule polarity within the spindle. Thus, this mechanism is governed by chromokinesin motors, which is dependent on symmetrical microtubule orientation that may allow chromosomes to maintain their position around the spindle equator until correct microtubule-kinetochore attachment is established.

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

    PubMed Central

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

    2001-01-01

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

  20. Physics of protein motility and motor proteins

    NASA Astrophysics Data System (ADS)

    Kolomeisky, Anatoly B.

    2013-09-01

    Motor proteins are enzymatic molecules that transform chemical energy into mechanical motion and work. They are critically important for supporting various cellular activities and functions. In the last 15 years significant progress in understanding the functioning of motor proteins has been achieved due to revolutionary breakthroughs in single-molecule experimental techniques and strong advances in theoretical modelling. However, microscopic mechanisms of protein motility are still not well explained, and the collective efforts of many scientists are needed in order to solve these complex problems. In this special section the reader will find the latest advances on the difficult road to mapping motor proteins dynamics in various systems. Recent experimental developments have allowed researchers to monitor and to influence the activity of single motor proteins with a high spatial and temporal resolution. It has stimulated significant theoretical efforts to understand the non-equilibrium nature of protein motility phenomena. The latest results from all these advances are presented and discussed in this special section. We would like to thank the scientists from all over the world who have reported their latest research results for this special section. We are also grateful to the staff and editors of Journal of Physics: Condensed Matter for their invaluable help in handling all the administrative and refereeing activities. The field of motor proteins and protein motility is fast moving, and we hope that this collection of articles will be a useful source of information in this highly interdisciplinary area. Physics of protein motility and motor proteins contents Physics of protein motility and motor proteinsAnatoly B Kolomeisky Identification of unique interactions between the flexible linker and the RecA-like domains of DEAD-box helicase Mss116 Yuan Zhang, Mirkó Palla, Andrew Sun and Jung-Chi Liao The load dependence of the physical properties of a molecular motor

  1. Folding of the Tau Protein on Microtubules.

    PubMed

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

    2015-08-24

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

  2. 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

  3. Microtubule dynamics in plant cells.

    PubMed

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

    2010-01-01

    This chapter describes some of the choices and unavoidable compromises to be made when studying microtubule dynamics in plant cells. The choice of species still depends very much on the ability to produce transgenic plants and most work has been done in the relatively small cells of Arabidopsis plants or in tobacco BY-2 suspension cells. Fluorescence-tagged microtubule proteins have been used to label entire microtubules, or their plus ends, but there are still few minus-end markers for these acentrosomal cells. Pragmatic decisions have to be made about probes, balancing the efficacy of microtubule labeling against a tendency to overstabilize and bundle the microtubules and even induce helical plant growth. A key limitation in visualizing plant microtubules is the ability to keep plants alive for long periods under the microscope and we describe a biochamber that allows for plant cell growth and development while allowing gas exchange and reducing evaporation. Another major difficulty is the limited fluorescence lifetime and we describe imaging strategies to reduce photobleaching in long-term imaging. We also discuss methods of measuring microtubule dynamics, with emphasis on the behavior of plant-specific microtubule arrays. 2010 Elsevier Inc. All rights reserved.

  4. Kar3Vik1 uses a minus-end directed powerstroke for movement along microtubules.

    PubMed

    Cope, Julia; Rank, Katherine C; Gilbert, Susan P; Rayment, Ivan; Hoenger, Andreas

    2013-01-01

    We have used cryo-electron microscopy (cryo-EM) and helical averaging to examine the 3-D structure of the heterodimeric kinesin-14 Kar3Vik1 complexed to microtubules at a resolution of 2.5 nm. 3-D maps were obtained at key points in Kar3Vik1's nucleotide hydrolysis cycle to gain insight into the mechanism that this motor uses for retrograde motility. In all states where Kar3Vik1 maintained a strong interaction with the microtubule, we found, as observed by cryo-EM, that the motor bound with one head domain while the second head extended outwards. 3-D reconstructions of Kar3Vik1-microtubule complexes revealed that in the nucleotide-free state, the motor's coiled-coil stalk points toward the plus-end of the microtubule. In the ATP-state, the outer head is shown to undergo a large rotation that reorients the stalk ∼75° to point toward the microtubule minus-end. To determine which of the two heads binds to tubulin in each nucleotide state, we employed specific Nanogold®-labeling of Vik1. The resulting maps confirmed that in the nucleotide-free, ATP and ADP+Pi states, Kar3 maintains contact with the microtubule surface, while Vik1 extends away from the microtubule and tracks with the coiled-coil as it rotates towards the microtubule minus-end. While many previous investigations have focused on the mechanisms of homodimeric kinesins, this work presents the first comprehensive study of the powerstroke of a heterodimeric kinesin. The stalk rotation shown here for Kar3Vik1 is highly reminiscent of that reported for the homodimeric kinesin-14 Ncd, emphasizing the conservation of a mechanism for minus-end directed motility.

  5. A "MICROTUBULE" IN A BACTERIUM

    PubMed Central

    van Iterson, Woutera; Hoeniger, Judith F. M.; van Zanten, Eva Nijman

    1967-01-01

    A study of the anchorage of the flagella in swarmers of Proteus mirabilis led to the incidental observation of microtubules. These microtubules were found in thin sections and in whole mount preparations of cells from which most of the content had been released by osmotic shock before staining negatively with potassium phosphotungstate (PTA). The microtubules are in negatively stained preparations about 200 A wide, i.e. somewhat thicker than the flagella (approximately 130 A). They are thus somewhat thinner than most microtubules recorded for other cells. They are referred to as microtubules because of their smooth cylindrical wall, or cortex, surrounding a hollow core which is readily filled with PTA when stained negatively. Since this is probably the first time that such a structure is described inside a bacterium, we do not know for certain whether it represents a normal cell constituent or an abnormality, for instance of the type of "polysheaths" (16). PMID:10976198

  6. Identification and Characterization of a Novel Microtubule-Based Motor Associated with Membranous Organelles in Tobacco Pollen Tubes

    PubMed Central

    Cai, Giampiero; Romagnoli, Silvia; Moscatelli, Alessandra; Ovidi, Elisa; Gambellini, Gabriella; Tiezzi, Antonio; Cresti, Mauro

    2000-01-01

    Pollen tube growth depends on the differential distribution of organelles and vesicles along the tube. The role of microtubules in organelle movement is uncertain, mainly because information at the molecular level is limited. In an effort to understand the molecular basis of microtubule-based movement, we isolated from tobacco pollen tubes polypeptides that cosediment with microtubules in an ATP-dependent manner. Major polypeptides released from microtubules by ATP (ATP-MAPs) had molecular masses of 90, 80, and 41 kD. Several findings indicate that the 90-kD ATP-MAP is a kinesin-related motor: binding of the polypeptide to microtubules was enhanced by the nonhydrolyzable ATP analog AMP-PNP; the 90-kD polypeptide reacted specifically with a peptide antibody directed against a highly conserved region in the motor domain of the kinesin superfamily; purified 90-kD ATP-MAP induced microtubules to glide in motility assays in vitro; and the 90-kD ATP-MAP cofractionated with microtubule-activated ATPase activity. Immunolocalization studies indicated that the 90-kD ATP-MAP binds to organelles associated with microtubules in the cortical region of the pollen tube. These findings suggest that the 90-kD ATP-MAP is a kinesin-related microtubule motor that moves organelles in the cortex of growing pollen tubes. PMID:11006343

  7. Equimolar heterodimers in microtubules

    PubMed Central

    1982-01-01

    Two equimolar beta chains can be resolved from sea urchin sperm flagellar and scallop gill ciliary tubulins, and from certain brain tubulins as well, using the Triton X-100-acid-urea polyacrylamide gel system commonly used for histone analysis. The beta chains are identified as such from their mobility on urea-free SDS PAGE, from amino acid composition, and from tryptic peptide distribution. Scallop beta chains have almost identical amino acid profiles but they differ by one tryptic peptide. Optimal conditions for beta chain resolution are very species-dependent, with some closely related species showing either maximal or no beta chain separation. In addition, beef brain tubulin on Triton X-100-acid-urea electrophoresis and scallop gill ciliary tubulin upon isoelectric focusing in the presence of SDS show two approximately equimolar alpha chains. These data, indicating equimolar amounts of two potentially different tubulin heterodimers from a variety of microtubule types, support a model for microtubule structure wherein protofilaments consist of alternating heterodimers of two kinds, generating a 16-nm (2-dimer) axial repeat. PMID:7202008

  8. Targeting Toxoplasma Tubules: Tubulin, Microtubules, and Associated Proteins in a Human Pathogen

    PubMed Central

    2014-01-01

    Toxoplasma gondii is an obligate intracellular parasite that causes serious opportunistic infections, birth defects, and blindness in humans. Microtubules are critically important components of diverse structures that are used throughout the Toxoplasma life cycle. As in other eukaryotes, spindle microtubules are required for chromosome segregation during replication. Additionally, a set of membrane-associated microtubules is essential for the elongated shape of invasive “zoites,” and motility follows a spiral trajectory that reflects the path of these microtubules. Toxoplasma zoites also construct an intricate, tubulin-based apical structure, termed the conoid, which is important for host cell invasion and associates with proteins typically found in the flagellar apparatus. Last, microgametes specifically construct a microtubule-containing flagellar axoneme in order to fertilize macrogametes, permitting genetic recombination. The specialized roles of these microtubule populations are mediated by distinct sets of associated proteins. This review summarizes our current understanding of the role of tubulin, microtubule populations, and associated proteins in Toxoplasma; these components are used for both novel and broadly conserved processes that are essential for parasite survival. PMID:25380753

  9. Targeting Toxoplasma tubules: tubulin, microtubules, and associated proteins in a human pathogen.

    PubMed

    Morrissette, Naomi

    2015-01-01

    Toxoplasma gondii is an obligate intracellular parasite that causes serious opportunistic infections, birth defects, and blindness in humans. Microtubules are critically important components of diverse structures that are used throughout the Toxoplasma life cycle. As in other eukaryotes, spindle microtubules are required for chromosome segregation during replication. Additionally, a set of membrane-associated microtubules is essential for the elongated shape of invasive "zoites," and motility follows a spiral trajectory that reflects the path of these microtubules. Toxoplasma zoites also construct an intricate, tubulin-based apical structure, termed the conoid, which is important for host cell invasion and associates with proteins typically found in the flagellar apparatus. Last, microgametes specifically construct a microtubule-containing flagellar axoneme in order to fertilize macrogametes, permitting genetic recombination. The specialized roles of these microtubule populations are mediated by distinct sets of associated proteins. This review summarizes our current understanding of the role of tubulin, microtubule populations, and associated proteins in Toxoplasma; these components are used for both novel and broadly conserved processes that are essential for parasite survival.

  10. Rapid Microtubule-dependent Induction of Neurite-like Extensions in NIH 3T3 Fibroblasts by Inhibition of ROCK and Cbl

    PubMed Central

    Scaife, Robin M.; Job, Didier; Langdon, Wallace Y.

    2003-01-01

    A number of key cellular functions, such as morphological differentiation and cell motility, are closely associated with changes in cytoskeletal dynamics. Many of the principal signaling components involved in actin cytoskeletal dynamics have been identified, and these have been shown to be critically involved in cell motility. In contrast, signaling to microtubules remains relatively uncharacterized, and the importance of signaling pathways in modulation of microtubule dynamics has so far not been established clearly. We report here that the Rho-effector ROCK and the multiadaptor proto-oncoprotein Cbl can profoundly affect the microtubule cytoskeleton. Simultaneous inhibition of these two signaling molecules induces a dramatic rearrangement of the microtubule cytoskeleton into microtubule bundles. The formation of these microtubule bundles, which does not involve signaling by Rac, Cdc42, Crk, phosphatidylinositol 3-kinase, and Abl, is sufficient to induce distinct neurite-like extensions in NIH 3T3 fibroblasts, even in the absence of microfilaments. This novel microtubule-dependent function that promotes neurite-like extensions is not dependent on net changes in microtubule polymerization or stabilization, but rather involves selective elongation and reorganization of microtubules into long bundles. PMID:12960437

  11. Mutation of Growth Arrest Specific 8 Reveals a Role in Motile Cilia Function and Human Disease

    PubMed Central

    Lewis, Wesley R.; Malarkey, Erik B.; Tritschler, Douglas; Bower, Raqual; Pasek, Raymond C.; Porath, Jonathan D.; Birket, Susan E.; Saunier, Sophie; Antignac, Corinne; Leigh, Margaret W.; Zariwala, Maimoona A.; Drummond, Iain A.; Parant, John M.; Hildebrandt, Friedhelm; Yoder, Bradley K.

    2016-01-01

    Ciliopathies are genetic disorders arising from dysfunction of microtubule-based cellular appendages called cilia. Different cilia types possess distinct stereotypic microtubule doublet arrangements with non-motile or ‘primary’ cilia having a 9+0 and motile cilia have a 9+2 array of microtubule doublets. Primary cilia are critical sensory and signaling centers needed for normal mammalian development. Defects in their structure/function result in a spectrum of clinical and developmental pathologies including abnormal neural tube and limb patterning. Altered patterning phenotypes in the limb and neural tube are due to perturbations in the hedgehog (Hh) signaling pathway. Motile cilia are important in fluid movement and defects in motility result in chronic respiratory infections, altered left-right asymmetry, and infertility. These features are the hallmarks of Primary Ciliary Dyskinesia (PCD, OMIM 244400). While mutations in several genes are associated with PCD in patients and animal models, the genetic lesion in many cases is unknown. We assessed the in vivo functions of Growth Arrest Specific 8 (GAS8). GAS8 shares strong sequence similarity with the Chlamydomonas Nexin-Dynein Regulatory Complex (NDRC) protein 4 (DRC4) where it is needed for proper flagella motility. In mammalian cells, the GAS8 protein localizes not only to the microtubule axoneme of motile cilia, but also to the base of non-motile cilia. Gas8 was recently implicated in the Hh signaling pathway as a regulator of Smoothened trafficking into the cilium. Here, we generate the first mouse with a Gas8 mutation and show that it causes severe PCD phenotypes; however, there were no overt Hh pathway phenotypes. In addition, we identified two human patients with missense variants in Gas8. Rescue experiments in Chlamydomonas revealed a subtle defect in swim velocity compared to controls. Further experiments using CRISPR/Cas9 homology driven repair (HDR) to generate one of these human missense variants

  12. Length dependence of displacement fluctuations and velocity in microtubule sliding movement driven by sea urchin sperm outer arm beta dynein in vitro.

    PubMed

    Imafuku, Y; Toyoshima, Y Y; Tawada, K

    1997-09-01

    We have studied the dependence on microtubule length of sliding velocity and positional fluctuation from recorded trajectories of microtubules sliding over sea urchin sperm outer arm beta dynein in a motility assay in vitro. The positional fluctuation was quantified by calculating the mean-square displacement deviation from the average, the calculation of which yields an effective diffusion coefficient. We have found that (1) the sliding velocity depends hyperbolically on the microtubule length, and (2) the effective diffusion coefficients do not depend on the length for sufficiently long microtubules. The length dependence of the sliding velocity indicates that the duty ratio, defined as the force producing period over the total cycle time of beta dynein interaction with microtubule, is very small. The length independence of the effective diffusion coefficient indicates that there is a correlation in the sliding movement fluctuation of microtubules.

  13. Teamwork in microtubule motors.

    PubMed

    Mallik, Roop; Rai, Arpan K; Barak, Pradeep; Rai, Ashim; Kunwar, Ambarish

    2013-11-01

    Diverse cellular processes are driven by the collective force from multiple motor proteins. Disease-causing mutations cause aberrant function of motors, but the impact is observed at a cellular level and beyond, therefore necessitating an understanding of cell mechanics at the level of motor molecules. One way to do this is by measuring the force generated by ensembles of motors in vivo at single-motor resolution. This has been possible for microtubule motor teams that transport intracellular organelles, revealing unexpected differences between collective and single-molecule function. Here we review how the biophysical properties of single motors, and differences therein, may translate into collective motor function during organelle transport and perhaps in other processes outside transport. Copyright © 2013 Elsevier Ltd. All rights reserved.

  14. The kinesin-8 Kip3 scales anaphase spindle length by suppression of midzone microtubule polymerization

    PubMed Central

    Rizk, Rania S.; DiScipio, Katherine A.; Proudfoot, Kathleen G.

    2014-01-01

    Mitotic spindle function is critical for cell division and genomic stability. During anaphase, the elongating spindle physically segregates the sister chromatids. However, the molecular mechanisms that determine the extent of anaphase spindle elongation remain largely unclear. In a screen of yeast mutants with altered spindle length, we identified the kinesin-8 Kip3 as essential to scale spindle length with cell size. Kip3 is a multifunctional motor protein with microtubule depolymerase, plus-end motility, and antiparallel sliding activities. Here we demonstrate that the depolymerase activity is indispensable to control spindle length, whereas the motility and sliding activities are not sufficient. Furthermore, the microtubule-destabilizing activity is required to counteract Stu2/XMAP215-mediated microtubule polymerization so that spindle elongation terminates once spindles reach the appropriate final length. Our data support a model where Kip3 directly suppresses spindle microtubule polymerization, limiting midzone length. As a result, sliding forces within the midzone cannot buckle spindle microtubules, which allows the cell boundary to define the extent of spindle elongation. PMID:24616221

  15. The kinesin-8 Kip3 scales anaphase spindle length by suppression of midzone microtubule polymerization.

    PubMed

    Rizk, Rania S; Discipio, Katherine A; Proudfoot, Kathleen G; Gupta, Mohan L

    2014-03-17

    Mitotic spindle function is critical for cell division and genomic stability. During anaphase, the elongating spindle physically segregates the sister chromatids. However, the molecular mechanisms that determine the extent of anaphase spindle elongation remain largely unclear. In a screen of yeast mutants with altered spindle length, we identified the kinesin-8 Kip3 as essential to scale spindle length with cell size. Kip3 is a multifunctional motor protein with microtubule depolymerase, plus-end motility, and antiparallel sliding activities. Here we demonstrate that the depolymerase activity is indispensable to control spindle length, whereas the motility and sliding activities are not sufficient. Furthermore, the microtubule-destabilizing activity is required to counteract Stu2/XMAP215-mediated microtubule polymerization so that spindle elongation terminates once spindles reach the appropriate final length. Our data support a model where Kip3 directly suppresses spindle microtubule polymerization, limiting midzone length. As a result, sliding forces within the midzone cannot buckle spindle microtubules, which allows the cell boundary to define the extent of spindle elongation.

  16. Laser irradiation of centrosomes in newt eosinophils: evidence of centriole role in motility

    SciTech Connect

    Koonce, M.P.; Cloney, R.A.; Berns, M.W.

    1984-06-01

    Newt eosinophils are motile granulated leukocytes that uniquely display a highly visible centrosomal area. Electron microscope and tubulin antibody fluorescence confirms the presence of centrioles, pericentriolar material, and radiating microtubules within this visible area. Actin antibodies intensely stain the advancing cell edges and tail but only weakly stain pseudopods being withdrawn into the cell. Randomly activated eosinophils follow a roughly consistent direction with an average rate of 22.5 ..mu..m/min. The position of the centrosome is always located between the trailing cell nucleus and advancing cell edge. If the cell extends more than one pseudopod, the one closest to or containing the centrosome is always the one in which motility continues. Laser irradiation of the visible centrosomal area resulted in rapid cell rounding. After several minutes following irradiation, most cells flattened and movement continued. However, postirradiation motility was uncoordinated and directionless, and the rate decreased to an average of 14.5 ..mu..m/min. Electron microscopy and tubulin immunofluorescence indicated that an initial disorganization of microtubules resulted from the laser microirradiations. After several minutes, organized microtubules reappeared, but the centrioles appeared increasingly damaged. The irregularities in motility due to irradiation are probably related to the damaged centrioles. The results presented in this paper suggest that the centrosome is an important structure in controlling the rate and direction of newt eosinophil motility.

  17. Biological Information Processing in Single Microtubules

    DTIC Science & Technology

    2011-08-20

    electronic properties of a single Microtubule Google Mountain view campus, workshop on quantum biology 22 October 2010 http://www.youtube.com/watch?v...Chemists, Tsukuba, Japan March 1-3, (2011) 3. Quantum aspects of microtubule: Direct experimental evidence for the existence of quantum states in...microtubule, Towards a science of consciousness May 2-8 (2011), Sweden 4. Electromagnetic energy of cells and microtubule: how microtubule research will

  18. Artificial microtubule cytoskeleton construction, manipulation, and modeling via holographic trapping of network nodes

    NASA Astrophysics Data System (ADS)

    Bergman, J.; Doval, F.; Vershinin, M.

    2016-09-01

    Cytoskeletal networks are 3D arrangements of filaments whose complex spatial structure contributes significantly to their intracellular functions, e.g. biomechanics and cargo motility. Microtubule networks in cells are a particular challenge for in vitro modeling because they are sparse and possess overall structure and so cannot be approximated experimentally as a random hydrogel. We have used holographic optical trapping to precisely position and hold multiple microtubule filaments in an in vitro assay, where chemical and environmental variables can be carefully controlled. Below we describe the relevant practical details of the approach and demonstrate how our approach can scale to accommodate modeling of molecular motor transport and biomechanics experiments.

  19. In vitro motility from recombinant dynein heavy chain.

    PubMed Central

    Mazumdar, M; Mikami, A; Gee, M A; Vallee, R B

    1996-01-01

    The dyneins are a class of motor protein involved in ciliary and flagellar motility, organelle transport, and chromosome segregation. Because of their large size and subunit complexity, relatively little is known about their mechanisms of force production and regulation. We report here on the expression and analysis of the entire rat cytoplasmic dynein heavy chain (Mr 532,000). Full-length cDNAs were constructed from a series of partial clones and tagged at the C terminus with either a FLAG-epitope tag or a His6-tag. The recombinant polypeptides were expressed either in insect cells by baculovirus infection or in COS-7 cells by transient transfection. The recombinant protein was mostly soluble and showed good microtubule binding. It exhibited a broad sedimentation profile, indicative of the formation of dimers as well as higher order multimers. Good microtubule gliding motility activity was observed in assays of heavy chain expressed in either insect or COS-7 cells. Average microtubule gliding velocities of 1.2-1.8 microm/sec were observed, comparable with the rates determined for calf brain cytoplasmic dynein. These results represent the first indication that recombinant heavy chain alone is capable of force production, and should lead to rapid progress in defining the dynein motor domain. Images Fig. 1 Fig. 2 Fig. 3 PMID:8692854

  20. Actin-based motility drives baculovirus transit to the nucleus and cell surface.

    PubMed

    Ohkawa, Taro; Volkman, Loy E; Welch, Matthew D

    2010-07-26

    Most viruses move intracellularly to and from their sites of replication using microtubule-based mechanisms. In this study, we show that nucleocapsids of the baculovirus Autographa californica multiple nucleopolyhedrovirus undergo intracellular motility driven by actin polymerization. Motility requires the viral P78/83 capsid protein and the host Arp2/3 complex. Surprisingly, the virus directs two sequential and coordinated phases of actin-based motility. Immediately after cell entry, motility enables exploration of the cytoplasm and collision with the nuclear periphery, speeding nuclear entry and the initiation of viral gene expression. Nuclear entry itself requires transit through nuclear pore complexes. Later, after the onset of early gene expression, motility is required for accumulation of a subpopulation of nucleocapsids in the tips of actin-rich surface spikes. Temporal coordination of actin-based nuclear and surface translocation likely enables rapid transmission to neighboring cells during infection in insects and represents a distinctive evolutionary strategy for overcoming host defenses.

  1. Microtubule and Motor-Dependent Endocytic Vesicle Sorting in Vitro

    PubMed Central

    Bananis, Eustratios; Murray, John W.; Stockert, Richard J.; Satir, Peter; Wolkoff, Allan W.

    2000-01-01

    Endocytic vesicles undergo fission to sort ligand from receptor. Using quantitative immunofluorescence and video imaging, we provide the first in vitro reconstitution of receptor–ligand sorting in early endocytic vesicles derived from rat liver. We show that to undergo fission, presegregation vesicles must bind to microtubules (MTs) and move upon addition of ATP. Over 13% of motile vesicles elongate and are capable of fission. After fission, one vesicle continues to move, whereas the other remains stationary, resulting in their separation. On average, almost 90% receptor is found in one daughter vesicle, whereas ligand is enriched by ∼300% with respect to receptor in the other daughter vesicle. Although studies performed on polarity marked MTs showed approximately equal plus and minus end–directed motility, immunofluorescence microscopy revealed that kinesins, but not dynein, were associated with these vesicles. Motility and fission were prevented by addition of 1 mM 5′-adenylylimido-diphosphate (AMP-PNP, an inhibitor of kinesins) or incubation with kinesin antibodies, but were unaffected by addition of 5 μM vanadate (a dynein inhibitor) or dynein antibodies. These studies indicate an essential role of kinesin-based MT motility in endocytic vesicle sorting, providing a system in which factors required for endocytic vesicle processing can be identified and characterized. PMID:11018063

  2. Microtubules for Nonlinear Optical Limiting

    DTIC Science & Technology

    1993-12-03

    microtubule alignment and centering was accomplished by attaching a l-mL glass syringe and 26 gauge needle to one or both ends of a glass capillary prefilled ...with liquid crystal or a microtubule/K24 mixture. The capillary ends were epoxy sealed to the syringe needles and then pressure or vacuum was applied...to the capillary by pushing or pulling the syringe plungers. All photographs were taken using a Wild MPS 11 35 mm camera attached to Nikon Optiphot

  3. Microtubule organization during human parthenogenesis.

    PubMed

    Terada, Yukihiro; Hasegawa, Hisataka; Ugajin, Tomohisa; Murakami, Takashi; Yaegashi, Nobuo; Okamura, Kunihiro

    2009-04-01

    In human fertilization, the sperm centrosome plays a crucial role as a microtubule organizing center (MTOC). We studied microtubule organization during human parthenogenesis, which occurs when a human egg undergoes cleavage without a sperm centrosome. Multiple cytoplasmic asters were organized in the human oocyte after parthenogenetic activation, indicating that multiple MTOC are present in the human oocyte cytoplasm and function like a human sperm centrosome during parthenogenesis.

  4. 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.

  5. Unconventional functions of microtubule motors.

    PubMed

    Muresan, Virgil; Muresan, Zoia

    2012-04-01

    With the functional characterization of proteins advancing at fast pace, the notion that one protein performs different functions - often with no relation to each other - emerges as a novel principle of how cells work. Molecular motors are no exception to this new development. Here, we provide an account on recent findings revealing that microtubule motors are multifunctional proteins that regulate many cellular processes, in addition to their main function in transport. Some of these functions rely on their motor activity, but others are independent of it. Of the first category, we focus on the role of microtubule motors in organelle biogenesis, and in the remodeling of the cytoskeleton, especially through the regulation of microtubule dynamics. Of the second category, we discuss the function of microtubule motors as static anchors of the cargo at the destination, and their participation in regulating signaling cascades by modulating interactions between signaling proteins, including transcription factors. We also review atypical forms of transport, such as the cytoplasmic streaming in the oocyte, and the movement of cargo by microtubule fluctuations. Our goal is to provide an overview of these unexpected functions of microtubule motors, and to incite future research in this expanding field.

  6. Kar3Vik1, a member of the kinesin-14 superfamily, shows a novel kinesin microtubule binding pattern.

    PubMed

    Rank, Katherine C; Chen, Chun Ju; Cope, Julia; Porche, Ken; Hoenger, Andreas; Gilbert, Susan P; Rayment, Ivan

    2012-06-25

    Kinesin-14 motors generate microtubule minus-end-directed force used in mitosis and meiosis. These motors are dimeric and operate with a nonprocessive powerstroke mechanism, but the role of the second head in motility has been unclear. In Saccharomyces cerevisiae, the Kinesin-14 Kar3 forms a heterodimer with either Vik1 or Cik1. Vik1 contains a motor homology domain that retains microtubule binding properties but lacks a nucleotide binding site. In this case, both heads are implicated in motility. Here, we show through structural determination of a C-terminal heterodimeric Kar3Vik1, electron microscopy, equilibrium binding, and motility that at the start of the cycle, Kar3Vik1 binds to or occludes two αβ-tubulin subunits on adjacent protofilaments. The cycle begins as Vik1 collides with the microtubule followed by Kar3 microtubule association and ADP release, thereby destabilizing the Vik1-microtubule interaction and positioning the motor for the start of the powerstroke. The results indicate that head-head communication is mediated through the adjoining coiled coil.

  7. Memo mediates ErbB2-driven cell motility.

    PubMed

    Marone, Romina; Hess, Daniel; Dankort, David; Muller, William J; Hynes, Nancy E; Badache, Ali

    2004-06-01

    Clinical studies have revealed that cancer patients whose tumours have increased ErbB2 expression tend to have more aggressive, metastatic disease, which is associated with parameters predicting a poor outcome. The molecular basis underlying ErbB2-dependent cell motility and metastases formation, however, still remains poorly understood. In this study, we show that activation of a set of signalling molecules, including MAPK, phosphatidylinositol-3-OH kinase (PI(3)K) and Src, is required for Neu/ErbB2-dependent lamellipodia formation and for motility of breast carcinoma cells. Stimulation of these molecules, however, failed to induce efficient cell migration in the absence of Neu/ErbB2 phosphorylation at Tyr 1201 or Tyr 1227. We describe a novel molecule, Memo (mediator of ErbB2-driven cell motility), that interacts with a phospho-Tyr 1227-containing peptide, most probably through the Shc adaptor protein. After Neu/ErbB2 activation, Memo-defective cells form actin fibres and grow lamellipodia, but fail to extend microtubules towards the cell cortex. Our data suggest that Memo controls cell migration by relaying extracellular chemotactic signals to the microtubule cytoskeleton.

  8. Improved Plasmids for Fluorescent Protein Tagging of Microtubules in Saccharomyces cerevisiae

    PubMed Central

    Baranowski, Kaitlyn; Lee, Wei-Lih

    2016-01-01

    The ability to fluorescently label microtubules in live cells has enabled numerous studies of motile and mitotic processes. Such studies are particularly useful in budding yeast due to the ease with which they can be genetically manipulated and imaged by live cell fluorescence microscopy. Due to problems associated with fusing genes encoding fluorescent proteins (FPs) to the native a-tubulin (TUB1) gene, the FP-Tub1 fusion is generally integrated into the genome such that the endogenous TUB1 locus is left intact. Although such modifications have no apparent consequences on cell viability, it is unknown if these genome integrated FP-tubulin fusions negatively affect microtubule functions. Thus, a simple, economical, and highly sensitive assay of microtubule function is required. Furthermore, the current plasmids available for generation of FP-Tub1 fusions have not kept pace with the development of improved FPs. Here, we have developed a simple and sensitive assay of microtubule function that is sufficient to identify microtubule defects that were not apparent by fluorescence microscopy or cell growth assays. Using results obtained from this assay, we have engineered a new family of thirty FP-Tub1 plasmids that employ various improved FPs and numerous selectable markers that upon genome integration have no apparent defect on microtubule function. PMID:25711127

  9. Microtubules in Bacteria: Ancient Tubulins Build a Five-Protofilament Homolog of the Eukaryotic Cytoskeleton

    PubMed Central

    Pilhofer, Martin; Ladinsky, Mark S.; McDowall, Alasdair W.; Petroni, Giulio; Jensen, Grant J.

    2011-01-01

    Microtubules play crucial roles in cytokinesis, transport, and motility, and are therefore superb targets for anti-cancer drugs. All tubulins evolved from a common ancestor they share with the distantly related bacterial cell division protein FtsZ, but while eukaryotic tubulins evolved into highly conserved microtubule-forming heterodimers, bacterial FtsZ presumably continued to function as single homopolymeric protofilaments as it does today. Microtubules have not previously been found in bacteria, and we lack insight into their evolution from the tubulin/FtsZ ancestor. Using electron cryomicroscopy, here we show that the tubulin homologs BtubA and BtubB form microtubules in bacteria and suggest these be referred to as “bacterial microtubules” (bMTs). bMTs share important features with their eukaryotic counterparts, such as straight protofilaments and similar protofilament interactions. bMTs are composed of only five protofilaments, however, instead of the 13 typical in eukaryotes. These and other results suggest that rather than being derived from modern eukaryotic tubulin, BtubA and BtubB arose from early tubulin intermediates that formed small microtubules. Since we show that bacterial microtubules can be produced in abundance in vitro without chaperones, they should be useful tools for tubulin research and drug screening. PMID:22162949

  10. Microtubules in plants.

    PubMed

    Hashimoto, Takashi

    2015-01-01

    Microtubules (MTs) are highly conserved polar polymers that are key elements of the eukaryotic cytoskeleton and are essential for various cell functions. αβ-tubulin, a heterodimer containing one structural GTP and one hydrolysable and exchangeable GTP, is the building block of MTs and is formed by the sequential action of several molecular chaperones. GTP hydrolysis in the MT lattice is mechanistically coupled with MT growth, thus giving MTs a metastable and dynamic nature. MTs adopt several distinct higher-order organizations that function in cell division and cell morphogenesis. Small molecular weight compounds that bind tubulin are used as herbicides and as research tools to investigate MT functions in plant cells. The de novo formation of MTs in cells requires conserved γ-tubulin-containing complexes and targeting/activating regulatory proteins that contribute to the geometry of MT arrays. Various MT regulators and tubulin modifications control the dynamics and organization of MTs throughout the cell cycle and in response to developmental and environmental cues. Signaling pathways that converge on the regulation of versatile MT functions are being characterized.

  11. 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

  12. 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.

  13. PACRG, a protein linked to ciliary motility, mediates cellular signaling.

    PubMed

    Loucks, Catrina M; Bialas, Nathan J; Dekkers, Martijn P J; Walker, Denise S; Grundy, Laura J; Li, Chunmei; Inglis, P Nick; Kida, Katarzyna; Schafer, William R; Blacque, Oliver E; Jansen, Gert; Leroux, Michel R

    2016-07-01

    Cilia are microtubule-based organelles that project from nearly all mammalian cell types. Motile cilia generate fluid flow, whereas nonmotile (primary) cilia are required for sensory physiology and modulate various signal transduction pathways. Here we investigate the nonmotile ciliary signaling roles of parkin coregulated gene (PACRG), a protein linked to ciliary motility. PACRG is associated with the protofilament ribbon, a structure believed to dictate the regular arrangement of motility-associated ciliary components. Roles for protofilament ribbon-associated proteins in nonmotile cilia and cellular signaling have not been investigated. We show that PACRG localizes to a small subset of nonmotile cilia in Caenorhabditis elegans, suggesting an evolutionary adaptation for mediating specific sensory/signaling functions. We find that it influences a learning behavior known as gustatory plasticity, in which it is functionally coupled to heterotrimeric G-protein signaling. We also demonstrate that PACRG promotes longevity in C. elegans by acting upstream of the lifespan-promoting FOXO transcription factor DAF-16 and likely upstream of insulin/IGF signaling. Our findings establish previously unrecognized sensory/signaling functions for PACRG and point to a role for this protein in promoting longevity. Furthermore, our work suggests additional ciliary motility-signaling connections, since EFHC1 (EF-hand containing 1), a potential PACRG interaction partner similarly associated with the protofilament ribbon and ciliary motility, also positively regulates lifespan.

  14. PACRG, a protein linked to ciliary motility, mediates cellular signaling

    PubMed Central

    Loucks, Catrina M.; Bialas, Nathan J.; Dekkers, Martijn P. J.; Walker, Denise S.; Grundy, Laura J.; Li, Chunmei; Inglis, P. Nick; Kida, Katarzyna; Schafer, William R.; Blacque, Oliver E.; Jansen, Gert; Leroux, Michel R.

    2016-01-01

    Cilia are microtubule-based organelles that project from nearly all mammalian cell types. Motile cilia generate fluid flow, whereas nonmotile (primary) cilia are required for sensory physiology and modulate various signal transduction pathways. Here we investigate the nonmotile ciliary signaling roles of parkin coregulated gene (PACRG), a protein linked to ciliary motility. PACRG is associated with the protofilament ribbon, a structure believed to dictate the regular arrangement of motility-associated ciliary components. Roles for protofilament ribbon–associated proteins in nonmotile cilia and cellular signaling have not been investigated. We show that PACRG localizes to a small subset of nonmotile cilia in Caenorhabditis elegans, suggesting an evolutionary adaptation for mediating specific sensory/signaling functions. We find that it influences a learning behavior known as gustatory plasticity, in which it is functionally coupled to heterotrimeric G-protein signaling. We also demonstrate that PACRG promotes longevity in C. elegans by acting upstream of the lifespan-promoting FOXO transcription factor DAF-16 and likely upstream of insulin/IGF signaling. Our findings establish previously unrecognized sensory/signaling functions for PACRG and point to a role for this protein in promoting longevity. Furthermore, our work suggests additional ciliary motility-signaling connections, since EFHC1 (EF-hand containing 1), a potential PACRG interaction partner similarly associated with the protofilament ribbon and ciliary motility, also positively regulates lifespan. PMID:27193298

  15. Cortical microtubule rearrangements and cell wall patterning

    PubMed Central

    Oda, Yoshihisa

    2015-01-01

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

  16. Self-repair promotes microtubule rescue

    PubMed Central

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

    2016-01-01

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

  17. 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.

  18. Displacement-Weighted Velocity Analysis of Gliding Assays Reveals that Chlamydomonas Axonemal Dynein Preferentially Moves Conspecific Microtubules

    PubMed Central

    Alper, Joshua D.; Tovar, Miguel; Howard, Jonathon

    2013-01-01

    In vitro gliding assays, in which microtubules are observed to glide over surfaces coated with motor proteins, are important tools for studying the biophysics of motility. Gliding assays with axonemal dyneins have the unusual feature that the microtubules exhibit large variations in gliding speed despite measures taken to eliminate unsteadiness. Because axonemal dynein gliding assays are usually done using heterologous proteins, i.e., dynein and tubulin from different organisms, we asked whether the source of tubulin could underlie the unsteadiness. By comparing gliding assays with microtubules polymerized from Chlamydomonas axonemal tubulin with those from porcine brain tubulin, we found that the unsteadiness is present despite matching the source of tubulin to the source of dynein. We developed a novel, to our knowledge, displacement-weighted velocity analysis to quantify both the velocity and the unsteadiness of gliding assays systematically and without introducing bias toward low motility. We found that the quantified unsteadiness is independent of tubulin source. In addition, we found that the short Chlamydomonas microtubules translocate significantly faster than their porcine counterparts. By modeling the effect of length on velocity, we propose that the observed effect may be due to a higher rate of binding of Chlamydomonas axonemal dynein to Chlamydomonas microtubules than to porcine microtubules. PMID:23663842

  19. Regulation of microtubule motors by tubulin isotypes and posttranslational modifications

    PubMed Central

    Sirajuddin, Minhajuddin; Rice, Luke M.; Vale, Ronald D.

    2014-01-01

    The ‘tubulin-code’ hypothesis proposes that different tubulin genes or posttranslational modifications (PTMs), which mainly confer variation in the carboxy-terminal tail (CTT), result in unique interactions with microtubule-associated proteins for specific cellular functions. However, the inability to isolate distinct and homogenous tubulin species has hindered biochemical testing of this hypothesis. Here, we have engineered 25 α/β tubulin heterodimers with distinct CTTs and PTMs and tested their interactions with four different molecular motors using single molecule assays. Our results show that tubulin isotypes and PTMs can govern motor velocity, processivity and microtubule depolymerization rates, with substantial changes conferred by even single amino acid variation. Revealing the importance and specificity of PTMs, we show that kinesin-1 motility on neuronal β-tubulin (TUBB3) is increased by polyglutamylation and that robust kinesin-2 motility requires detyrosination of α-tubulin. Our results also show that different molecular motors recognize distinctive tubulin “signatures”, which supports the premise of tubulin-code hypothesis. PMID:24633327

  20. Single molecule analysis of cytoplasmic dynein motility

    NASA Astrophysics Data System (ADS)

    Yildiz, Ahmet

    2014-03-01

    Cytoplasmic dynein is a homodimeric AAA + motor that transports a multitude of cargos towards the microtubule (MT) minus end. The mechanism of dynein motility remains unclear, due to its large size (2.6 MDa) and the complexity of its structure. By tracking the stepping motion of both heads at nanometer resolution, we observed that dynein heads move independently along the MT, in contrast to hand over hand movement of kinesins and myosin. Stepping behavior of the heads varies as a function of interhead separation and establishing the basis of high variability in dynein step size. By engineering the mechanical and catalytic properties of the dynein motor domain, we show that a rigid linkage between monomers and dimerization between N-terminal tail domains are not essential for processive movement. Instead, dynein processivity minimally requires the linker domain of one active monomer to be attached to an inert MT tether retaining only the MT-binding domain. The release of a dynein monomer from the MT can be mediated either by nucleotide binding or external load. Nucleotide dependent release is inhibited by the tension on the linker domain at high interhead separations. Tension dependent release is highly asymmetric, with faster release towards the minus-end. Reversing the asymmetry of the MT binding interface results in plus end directed motility, even though the force was generated by the dynein motor activity. On the basis of these measurements, we propose a model that describes the basis of dynein processivity, directionality and force generation.

  1. Single Fungal Kinesin Motor Molecules Move Processively along Microtubules

    PubMed Central

    Lakämper, Stefan; Kallipolitou, Athina; Woehlke, Günther; Schliwa, Manfred; Meyhöfer, Edgar

    2003-01-01

    Conventional kinesins are two-headed molecular motors that move as single molecules micrometer-long distances on microtubules by using energy derived from ATP hydrolysis. The presence of two heads is a prerequisite for this processive motility, but other interacting domains, like the neck and K-loop, influence the processivity and are implicated in allowing some single-headed kinesins to move processively. Neurospora kinesin (NKin) is a phylogenetically distant, dimeric kinesin from Neurospora crassa with high gliding speed and an unusual neck domain. We quantified the processivity of NKin and compared it to human kinesin, HKin, using gliding and fluorescence-based processivity assays. Our data show that NKin is a processive motor. Single NKin molecules translocated microtubules in gliding assays on average 2.14 μm (N = 46). When we tracked single, fluorescently labeled NKin motors, they moved on average 1.75 μm (N = 182) before detaching from the microtubule, whereas HKin motors moved shorter distances (0.83 μm, N = 229) under identical conditions. NKin is therefore at least twice as processive as HKin. These studies, together with biochemical work, provide a basis for experiments to dissect the molecular mechanisms of processive movement. PMID:12609885

  2. Anomalous motor mediated cargo transport in microtubule networks

    NASA Astrophysics Data System (ADS)

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

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

  3. Structural Heterogeneity of Mitochondria Induced by the Microtubule Cytoskeleton

    PubMed Central

    Sukhorukov, Valerii M.; Meyer-Hermann, Michael

    2015-01-01

    By events of fusion and fission mitochondria generate a partially interconnected, irregular network of poorly specified architecture. Here, its organization is examined theoretically by taking into account the physical association of mitochondria with microtubules. Parameters of the cytoskeleton mesh are derived from the mechanics of single fibers. The model of the mitochondrial reticulum is formulated in terms of a dynamic spatial graph. The graph dynamics is modulated by the density of microtubules and their crossings. The model reproduces the full spectrum of experimentally found mitochondrial configurations. In centrosome-organized cells, the chondriome is predicted to develop strong structural inhomogeneity between the cell center and the periphery. An integrated analysis of the cytoskeletal and the mitochondrial components reveals that the structure of the reticulum depends on the balance between anterograde and retrograde motility of mitochondria on microtubules, in addition to fission and fusion. We propose that it is the combination of the two processes that defines synergistically the mitochondrial structure, providing the cell with ample capabilities for its regulative adaptation. PMID:26355039

  4. Microtubule defects & Neurodegeneration.

    PubMed

    Baird, Fiona J; Bennett, Craig L

    2013-12-06

    One of the major challenges facing the long term survival of neurons is their requirement to maintain efficient axonal transport over long distances. In humans as large, long-lived vertebrates, the machinery maintaining neuronal transport must remain efficient despite the slow accumulation of cell damage during aging. Mutations in genes encoding proteins which function in the transport system feature prominently in neurologic disorders. Genes known to cause such disorders and showing traditional Mendelian inheritance have been more readily identified. It has been more difficult, however, to isolate factors underlying the complex genetics contributing to the more common idiopathic forms of neurodegenerative disease. At the heart of neuronal transport is the rail network or scaffolding provided by neuron specific microtubules (MTs). The importance of MT dynamics and stability is underscored by the critical role tau protein plays in MT-associated stabilization versus the dysfunction seen in Alzheimer's disease, frontotemporal dementia and other tauopathies. Another example of the requirement for tight regulation of MT dynamics is the need to maintain balanced levels of post-translational modification of key MT building-blocks such as α-tubulin. Tubulins require extensive polyglutamylation at their carboxyl-terminus as part of a novel post-translational modification mechanism to signal MT growth versus destabilization. Dramatically, knock-out of a gene encoding a deglutamylation family member causes an extremely rapid cell death of Purkinje cells in the ataxic mouse model, pcd. This review will examine a range of neurodegenerative conditions where current molecular understanding points to defects in the stability of MTs and axonal transport to emphasize the central role of MTs in neuron survival.

  5. Sperm Motility in Flow

    NASA Astrophysics Data System (ADS)

    Guasto, Jeffrey; Juarez, Gabriel; Stocker, Roman

    2012-11-01

    A wide variety of plants and animals reproduce sexually by releasing motile sperm that seek out a conspecific egg, for example in the reproductive tract for mammals or in the water column for externally fertilizing organisms. Sperm are aided in their quest by chemical cues, but must also contend with hydrodynamic forces, resulting from laminar flows in reproductive tracts or turbulence in aquatic habitats. To understand how velocity gradients affect motility, we subjected swimming sperm to a range of highly-controlled straining flows using a cross-flow microfluidic device. The motion of the cell body and flagellum were captured through high-speed video microscopy. The effects of flow on swimming are twofold. For moderate velocity gradients, flow simply advects and reorients cells, quenching their ability to cross streamlines. For high velocity gradients, fluid stresses hinder the internal bending of the flagellum, directly inhibiting motility. The transition between the two regimes is governed by the Sperm number, which compares the external viscous stresses with the internal elastic stresses. Ultimately, unraveling the role of flow in sperm motility will lead to a better understanding of population dynamics among aquatic organisms and infertility problems in humans.

  6. Chromosome position at the spindle equator is regulated by chromokinesin and a bipolar microtubule array

    PubMed Central

    Takagi, Jun; Itabashi, Takeshi; Suzuki, Kazuya; Ishiwata, Shin'ichi

    2013-01-01

    The chromosome alignment is mediated by polar ejection and poleward forces acting on the chromosome arm and kinetochores, respectively. Although components of the motile machinery such as chromokinesin have been characterized, their dynamics within the spindle is poorly understood. Here we show that a quantum dot (Qdot) binding up to four Xenopus chromokinesin (Xkid) molecules behaved like a nanosize chromosome arm in the meiotic spindle, which is self-organized in cytoplasmic egg extracts. Xkid-Qdots travelled long distances along microtubules by changing several tracks, resulting in their accumulation toward and distribution around the metaphase plate. The analysis indicated that the direction of motion and velocity depend on the distribution of microtubule polarity within the spindle. Thus, this mechanism is governed by chromokinesin motors, which is dependent on symmetrical microtubule orientation that may allow chromosomes to maintain their position around the spindle equator until correct microtubule–kinetochore attachment is established. PMID:24077015

  7. Defining motility in the Staphylococci.

    PubMed

    Pollitt, Eric J G; Diggle, Stephen P

    2017-08-01

    The ability of bacteria to move is critical for their survival in diverse environments and multiple ways have evolved to achieve this. Two forms of motility have recently been described for Staphylococcus aureus, an organism previously considered to be non-motile. One form is called spreading, which is a type of sliding motility and the second form involves comet formation, which has many observable characteristics associated with gliding motility. Darting motility has also been observed in Staphylococcus epidermidis. This review describes how motility is defined and how we distinguish between passive and active motility. We discuss the characteristics of the various forms of Staphylococci motility, the molecular mechanisms involved and the potential future research directions.

  8. Role of the RNA-binding protein IMP-2 in muscle cell motility.

    PubMed

    Boudoukha, Selim; Cuvellier, Sylvain; Polesskaya, Anna

    2010-12-01

    Insulin-like growth factor 2 (IGF-2) mRNA-binding proteins (IMPs) are a family of posttranscriptional regulatory factors with well-understood roles in embryonic development and cancer but with poorly characterized functions in normal adult cells and tissues. We now show that IMP-2, the most ubiquitously expressed member of the family, is abundant in human and mouse adult skeletal myoblasts, where it is indispensable for cell motility and for stabilization of microtubules. To explore the functions of IMP-2, we analyzed the transcripts that were differentially regulated in IMP-2-depleted myoblasts and bound to IMP-2 in normal myoblasts. Among them were the mRNAs of PINCH-2, an important mediator of cell adhesion and motility, and MURF-3, a microtubule-stabilizing protein. By gain- and loss-of-function assays and gel shift experiments, we show that IMP-2 regulates the expression of PINCH-2 and MURF-3 proteins via direct binding to their mRNAs. Upregulation of PINCH-2 in IMP-2-depleted myoblasts is the key event responsible for their decreased motility. Our data reveal how the posttranscriptional regulation of gene expression by IMP-2 contributes to the control of adhesion structures and stable microtubules and demonstrate an important function for IMP-2 in cellular motility.

  9. Optimization of isopolar microtubule arrays.

    PubMed

    Agayan, Rodney R; Tucker, Robert; Nitta, Takahiro; Ruhnow, Felix; Walter, Wilhelm J; Diez, Stefan; Hess, Henry

    2013-02-19

    Isopolar arrays of aligned cytoskeletal filaments are components in a number of designs of hybrid nanodevices incorporating biomolecular motors. For example, a combination of filament arrays and motor arrays can form an actuator or a molecular engine resembling an artificial muscle. Here, isopolar arrays of microtubules are fabricated by flow alignment, and their quality is characterized by their degree of alignment. We find, in agreement with our analytical models, that the degree of alignment is ultimately limited by thermal forces, while the kinetics of the alignment process are influenced by the flow strength, the microtubule stiffness, the gliding velocity, and the tip length. Strong flows remove microtubules from the surface and reduce the filament density, suggesting that there is an optimal flow strength for the fabrication of ordered arrays.

  10. Rapid Microtubule Self-assembly Kinetics

    PubMed Central

    Gardner, Melissa K.; Charlebois, Blake D.; Jánosi, Imre M.; Howard, Jonathon; Hunt, Alan J.; Odde, David J.

    2011-01-01

    Microtubule assembly is vital for many fundamental cellular processes. Current models for microtubule assembly kinetics assume that the subunit disassociation rate from a microtubule tip is independent of free subunit concentration. Using Total-Internal-Reflection-Fluorescence (TIRF) microscopy and a laser tweezers assay to measure in vitro microtubule assembly with nanometer resolution, we find that the subunit dissociation rate from a microtubule tip increases as the free subunit concentration increases. These data are consistent with a two-dimensional model for microtubule assembly, and are explained by a shift in microtubule tip structure from a relatively blunt shape at low free concentrations to relatively tapered at high free concentrations. Because both the association and the dissociation rates increase at higher free subunit concentrations, we find that the kinetics of microtubule assembly are an order-of-magnitude higher than currently estimated in the literature. PMID:21854983

  11. Rapid microtubule self-assembly kinetics.

    PubMed

    Gardner, Melissa K; Charlebois, Blake D; Jánosi, Imre M; Howard, Jonathon; Hunt, Alan J; Odde, David J

    2011-08-19

    Microtubule assembly is vital for many fundamental cellular processes. Current models for microtubule assembly kinetics assume that the subunit dissociation rate from a microtubule tip is independent of free subunit concentration. Total-Internal-Reflection-Fluorescence (TIRF) microscopy experiments and data from a laser tweezers assay that measures in vitro microtubule assembly with nanometer resolution, provides evidence that the subunit dissociation rate from a microtubule tip increases as the free subunit concentration increases. These data are consistent with a two-dimensional model for microtubule assembly, and are explained by a shift in microtubule tip structure from a relatively blunt shape at low free concentrations to relatively tapered at high free concentrations. We find that because both the association and the dissociation rates increase at higher free subunit concentrations, the kinetics of microtubule assembly are an order-of-magnitude higher than currently estimated in the literature. Copyright © 2011 Elsevier Inc. All rights reserved.

  12. Cytoskeleton-dependent endomembrane organization in plant cells: an emerging role for microtubules.

    PubMed

    Brandizzi, Federica; Wasteneys, Geoffrey O

    2013-07-01

    Movement of secretory organelles is a fascinating yet largely mysterious feature of eukaryotic cells. Microtubule-based endomembrane and organelle motility utilizing the motor proteins dynein and kinesin is commonplace in animal cells. In contrast, it has been long accepted that intracellular motility in plant cells is predominantly driven by myosin motors dragging organelles and endomembrane-bounded cargo along actin filament bundles. Consistent with this, defects in the acto-myosin cytoskeleton compromise plant growth and development. Recent findings, however, challenge the actin-centric view of the motility of critical secretory organelles and distribution of associated protein machinery. In this review, we provide an overview of the current knowledge on actin-mediated organelle movement within the secretory pathway of plant cells, and report on recent and exciting findings that support a critical role of microtubules in plant cell development, in fine-tuning the positioning of Golgi stacks, as well as their involvement in cellulose synthesis and auxin polar transport. These emerging aspects of the biology of microtubules highlight adaptations of an ancestral machinery that plants have specifically evolved to support the functioning of the acto-myosin cytoskeleton, and mark new trends in our global appreciation of the complexity of organelle movement within the plant secretory pathway.

  13. Biological Information Processing in Single Microtubules

    DTIC Science & Technology

    2014-03-05

    single Microtubule Google Mountain view campus, workshop on quantum biology 22 October 2010 3. Paul Davies Beyond Center at Arizona State University...Phoenix) Phoenix, workshop on quantum biology and cancer research, Experimental studies on single microtubule, 25-27 October 2010, Tempe, Arizona...State University, USA 4. Quantum aspects of microtubule: Direct experimental evidence for the existence of quantum states in microtubule, Towards a

  14. The dynein cortical anchor Num1 activates dynein motility by relieving Pac1/LIS1-mediated inhibition

    PubMed Central

    Lammers, Lindsay G.

    2015-01-01

    Cortically anchored dynein orients the spindle through interactions with astral microtubules. In budding yeast, dynein is offloaded to Num1 receptors from microtubule plus ends. Rather than walking toward minus ends, dynein remains associated with plus ends due in part to its association with Pac1/LIS1, an inhibitor of dynein motility. The mechanism by which dynein is switched from “off” at the plus ends to “on” at the cell cortex remains unknown. Here, we show that overexpression of the coiled-coil domain of Num1 specifically depletes dynein–dynactin–Pac1/LIS1 complexes from microtubule plus ends and reduces dynein-Pac1/LIS1 colocalization. Depletion of dynein from plus ends requires its microtubule-binding domain, suggesting that motility is required. An enhanced Pac1/LIS1 affinity mutant of dynein or overexpression of Pac1/LIS1 rescues dynein plus end depletion. Live-cell imaging reveals minus end–directed dynein–dynactin motility along microtubules upon overexpression of the coiled-coil domain of Num1, an event that is not observed in wild-type cells. Our findings indicate that dynein activity is directly switched “on” by Num1, which induces Pac1/LIS1 removal. PMID:26483554

  15. A viscoelastic model for axonal microtubule rupture.

    PubMed

    Shamloo, Amir; Manuchehrfar, Farid; Rafii-Tabar, Hashem

    2015-05-01

    Axon is an important part of the neuronal cells and axonal microtubules are bundles in axons. In axons, microtubules are coated with microtubule-associated protein tau, a natively unfolded filamentous protein in the central nervous system. These proteins are responsible for cross-linking axonal microtubule bundles. Through complimentary dimerization with other tau proteins, bridges are formed between nearby microtubules creating bundles. Formation of bundles of microtubules causes their transverse reinforcement and has been shown to enhance their ability to bear compressive loads. Though microtubules are conventionally regarded as bearing compressive loads, in certain circumstances during traumatic brain injuries, they are placed in tension. In our model, microtubule bundles were formed from a large number of discrete masses. We employed Standard Linear Solid model (SLS), a viscoelastic model, to computationally simulate microtubules. In this study, we investigated the dynamic responses of two dimensional axonal microtubules under suddenly applied end forces by implementing discrete masses connected to their neighboring masses with a Standard Linear Solid unit. We also investigated the effect of the applied force rate and magnitude on the deformation of bundles. Under tension, a microtubule fiber may rupture as a result of a sudden force. Using the developed model, we could predict the critical regions of the axonal microtubule bundles in the presence of varying end forces. We finally analyzed the nature of microtubular failure under varying mechanical stresses. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Microtubule Control of Metabolism in Prostate Cancer

    DTIC Science & Technology

    2013-06-01

    that increase cell death when combined with docetaxel.Here we tested whether two metabolic inhibitors, metformin or 2- deoxy-glucose, function...microtubule cytoskeleton (docetaxel, paclitaxel, or nocodazole) singly, or in combination with metabolic inhibitors ( metformin or 2-deoxy-glucose...Microtubule-targeted drugs, which either stabilize or destabilize microtubules, acted synergistically with either metformin or 2-deoxy-glucose to

  17. On and around microtubules: an overview.

    PubMed

    Wade, Richard H

    2009-10-01

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

  18. Kinetochore–microtubule attachment throughout mitosis potentiated by the elongated stalk of the kinetochore kinesin CENP-E

    PubMed Central

    Vitre, Benjamin; Gudimchuk, Nikita; Borda, Ranier; Kim, Yumi; Heuser, John E.; Cleveland, Don W.; Grishchuk, Ekaterina L.

    2014-01-01

    Centromere protein E (CENP-E) is a highly elongated kinesin that transports pole-proximal chromosomes during congression in prometaphase. During metaphase, it facilitates kinetochore–microtubule end-on attachment required to achieve and maintain chromosome alignment. In vitro CENP-E can walk processively along microtubule tracks and follow both growing and shrinking microtubule plus ends. Neither the CENP-E–dependent transport along microtubules nor its tip-tracking activity requires the unusually long coiled-coil stalk of CENP-E. The biological role for the CENP-E stalk has now been identified through creation of “Bonsai” CENP-E with significantly shortened stalk but wild-type motor and tail domains. We demonstrate that Bonsai CENP-E fails to bind microtubules in vitro unless a cargo is contemporaneously bound via its C-terminal tail. In contrast, both full-length and truncated CENP-E that has no stalk and tail exhibit robust motility with and without cargo binding, highlighting the importance of CENP-E stalk for its activity. Correspondingly, kinetochore attachment to microtubule ends is shown to be disrupted in cells whose CENP-E has a shortened stalk, thereby producing chromosome misalignment in metaphase and lagging chromosomes during anaphase. Together these findings establish an unexpected role of CENP-E elongated stalk in ensuring stability of kinetochore–microtubule attachments during chromosome congression and segregation. PMID:24920822

  19. Polarity orientation of microtubules and its applications with motor proteins

    NASA Astrophysics Data System (ADS)

    Yokokawa, Ryuji

    2010-12-01

    We have studied integrations of micro/nano machining (MEMS/NEMS) technologies and biomaterials. One of our research directions is to utilize biomaterials in MEMS/NEMS to reveal new functions that could not be achieved by MEMS/NEMS alone. Here a motor protein system will be introduced as a nano actuator. The motility of kinesin and dynein motor proteins has been integrated with MEMS/NEMS or a microfluidic system. Since these motor proteins move on cytoskeletal filaments—microtubules (MTs)—depending on MT polarity, a key technology is to develop methods to orient MT polarities and then immobilize them. We have developed three methods to define MT polarities by (i) shared flow in a microfluidic channel, (ii) nanostructures and (iii) MEMS tweezers. Once MT polarities were oriented and fixed on a chip, they were ready to serve as rails for nano transport by kinesin and dynein motility. The motility was visualized by attaching cargos to motors, where the cargos were microbeads, silicon structures and quantum dots (Q-dots). This nano transport system can achieve a transport distance of up to ~100 μm, which enables us to focus on the transport of molecules not on bulk molecular flow by conventional microfluidics. Such a bio-hybrid system will be a key factor in realizing nano-scale system integration at the molecular scale.

  20. Modeling collective cell motility

    NASA Astrophysics Data System (ADS)

    Rappel, Wouter-Jan

    Eukaryotic cells often move in groups, a critical aspect of many biological and medical processes including wound healing, morphogenesis and cancer metastasis. Modeling can provide useful insights into the fundamental mechanisms of collective cell motility. Constructing models that incorporate the physical properties of the cells, however, is challenging. Here, I discuss our efforts to build a comprehensive cell motility model that includes cell membrane properties, cell-substrate interactions, cell polarity, and cell-cell interaction. The model will be applied to a variety of systems, including motion on micropatterned substrates and the migration of border cells in Drosophila. This work was supported by NIH Grant No. P01 GM078586 and NSF Grant No. 1068869.

  1. Motility of Mollicutes

    NASA Astrophysics Data System (ADS)

    Wolgemuth, Charles; Igoshin, Oleg; Oster, George

    2003-03-01

    Recent experiments show that the conformation of filament proteins play a role in the motility and morphology of many different types of bacteria. Conformational changes in the protein subunits may produce forces to drive propulsion and cell division. Here we present a molecular mechanism by which these forces can drive cell motion. Coupling of a biochemical cycle, such as ATP hydrolysis, to the dynamics of elastic filaments enable elastic filaments to propagate deformations that generate propulsive forces. We demonstrate this possibility for two classes of wall-less bacteria called mollicutes: the swimming of helical shaped Spiroplasma, and the gliding motility of Mycoplasma. Similar mechanisms may explain the locomotion of other prokaryotes, including the swimming of Synechococcus and the gliding of some myxobacteria.

  2. NCAM regulates cell motility.

    PubMed

    Prag, Søren; Lepekhin, Eugene A; Kolkova, Kateryna; Hartmann-Petersen, Rasmus; Kawa, Anna; Walmod, Peter S; Belman, Vadym; Gallagher, Helen C; Berezin, Vladimir; Bock, Elisabeth; Pedersen, Nina

    2002-01-15

    Cell migration is required during development of the nervous system. The regulatory mechanisms for this process, however, are poorly elucidated. We show here that expression of or exposure to the neural cell adhesion molecule (NCAM) strongly affected the motile behaviour of glioma cells independently of homophilic NCAM interactions. Expression of the transmembrane 140 kDa isoform of NCAM (NCAM-140) caused a significant reduction in cellular motility, probably through interference with factors regulating cellular attachment, as NCAM-140-expressing cells exhibited a decreased attachment to a fibronectin substratum compared with NCAM-negative cells. Ectopic expression of the cytoplasmic part of NCAM-140 also inhibited cell motility, presumably via the non-receptor tyrosine kinase p59(fyn) with which NCAM-140 interacts. Furthermore, we showed that the extracellular part of NCAM acted as a paracrine inhibitor of NCAM-negative cell locomotion through a heterophilic interaction with a cell-surface receptor. As we showed that the two N-terminal immunoglobulin modules of NCAM, which are known to bind to heparin, were responsible for this inhibition, we presume that this receptor is a heparan sulfate proteoglycan. A model for the inhibitory effect of NCAM is proposed, which involves competition between NCAM and extracellular components for the binding to membrane-associated heparan sulfate proteoglycan.

  3. Motility of Mycoplasma pneumoniae.

    PubMed Central

    Radestock, U; Bredt, W

    1977-01-01

    Cell of Mycoplasma pneumoniae FH gliding on a glass surface in liquid medium were examined by microscopic observation and quantitatively by microcinematography (30 frames per min). Comparisons were made only within the individual experiments. The cells moved in an irregular pattern with numerous narrow bends and circles. They never changed their leading end. The average speed (without pauses) was relatively constant between o.2 and 0.5 mum/s. The maximum speed was about 1.5 to 2.0 mum/s. The movements were interrupted by resting periods of different lengths and frequency. Temperature, viscosity, pH, and the presence of yeast extract in the medium influenced the motility significantly; changes in glucose, calcium ions, and serum content were less effective. The movements were affected by iodoacetate, p-mercuribenzoate, and mitomycin C at inhibitory or subinhibitory concentrations. Sodium fluoride, sodium cyanide, dinitrophenol, chloramphenicol, puromycin, cholchicin, and cytochalasin B at minimal inhibitory concentrations did not affect motility. The movements were effectively inhibited by anti-M. pneumoniae antiserum. Studies with absorbed antiserum suggested that the surface components involved in motility are heat labile. The gliding of M. pneumoniae cells required an intact energy metabolism and the proteins involved seemed to have a low turnover. Images PMID:14925

  4. In Vitro Assays Demonstrate That Pollen Tube Organelles Use Kinesin-Related Motor Proteins to Move along MicrotubulesW⃞

    PubMed Central

    Romagnoli, Silvia; Cai, Giampiero; Cresti, Mauro

    2003-01-01

    The movement of pollen tube organelles relies on cytoskeletal elements. Although the movement of organelles along actin filaments in the pollen tube has been studied widely and is becoming progressively clear, it remains unclear what role microtubules play. Many uncertainties about the role of microtubules in the active transport of pollen tube organelles and/or in the control of this process remain to be resolved. In an effort to determine if organelles are capable of moving along microtubules in the absence of actin, we extracted organelles from tobacco pollen tubes and analyzed their ability to move along in vitro–polymerized microtubules under different experimental conditions. Regardless of their size, the organelles moved at different rates along microtubules in the presence of ATP. Cytochalasin D did not inhibit organelle movement, indicating that actin filaments are not required for organelle transport in our assay. The movement of organelles was cytosol independent, which suggests that soluble factors are not necessary for the organelle movement to occur and that microtubule-based motor proteins are present on the organelle surface. By washing organelles with KI, it was possible to release proteins capable of gliding carboxylated beads along microtubules. Several membrane fractions, which were separated by Suc density gradient centrifugation, showed microtubule-based movement. Proteins were extracted by KI treatment from the most active organelle fraction and then analyzed with an ATP-sensitive microtubule binding assay. Proteins isolated by the selective binding to microtubules were tested for the ability to glide microtubules in the in vitro motility assay, for the presence of microtubule-stimulated ATPase activity, and for cross-reactivity with anti-kinesin antibodies. We identified and characterized a 105-kD organelle-associated motor protein that is functionally, biochemically, and immunologically related to kinesin. This work provides clear

  5. Dear microtubule, I see you

    DOE PAGES

    Nogales, Eva

    2016-11-01

    This essay summarizes my personal journey toward the atomic visualization of microtubules and a mechanistic understanding of how these amazing polymers work. During this journey, I have been witness and partaker in the blooming of a technique I love—cryo-electron microscopy.

  6. Dear microtubule, I see you

    PubMed Central

    Nogales, Eva

    2016-01-01

    This essay summarizes my personal journey toward the atomic visualization of microtubules and a mechanistic understanding of how these amazing polymers work. During this journey, I have been witness and partaker in the blooming of a technique I love—cryo-electron microscopy. PMID:27799495

  7. Microtubule catastrophe from protofilament dynamics.

    PubMed

    Jemseena, V; Gopalakrishnan, Manoj

    2013-09-01

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

  8. Xyloglucan Deficiency Disrupts Microtubule Stability and Cellulose Biosynthesis in Arabidopsis, Altering Cell Growth and Morphogenesis

    SciTech Connect

    Xiao, Chaowen; Zhang, Tian; Zheng, Yunzhen; Cosgrove, Daniel J.; Anderson, Charles T.

    2015-11-02

    Xyloglucan constitutes most of the hemicellulose in eudicot primary cell walls and functions in cell wall structure and mechanics. Although Arabidopsis (Arabidopsis thaliana) xxt1 xxt2 mutants lacking detectable xyloglucan are viable, they display growth defects that are suggestive of alterations in wall integrity. To probe the mechanisms underlying these defects, we analyzed cellulose arrangement, microtubule patterning and dynamics, microtubule- and wall-integrity-related gene expression, and cellulose biosynthesis in xxt1 xxt2 plants. We found that cellulose is highly aligned in xxt1 xxt2 cell walls, that its three-dimensional distribution is altered, and that microtubule patterning and stability are aberrant in etiolated xxt1 xxt2 hypocotyls. We also found that the expression levels of microtubule-associated genes, such as MAP70-5 and CLASP, and receptor genes, such as HERK1 and WAK1, were changed in xxt1 xxt2 plants and that cellulose synthase motility is reduced in xxt1 xxt2 cells, corresponding with a reduction in cellulose content. Our results indicate that loss of xyloglucan affects both the stability of the microtubule cytoskeleton and the production and patterning of cellulose in primary cell walls. These findings establish, to our knowledge, new links between wall integrity, cytoskeletal dynamics, and wall synthesis in the regulation of plant morphogenesis.

  9. Fluctuation in the microtubule sliding movement driven by kinesin in vitro.

    PubMed

    Imafuku, Y; Toyoshima, Y Y; Tawada, K

    1996-02-01

    We studied the fluctuation in the translational sliding movement of microtubules driven by kinesin in a motility assay in vitro. By calculating the mean-square displacement deviation from the average as a function of time, we obtained motional diffusion coefficients for microtubules and analyzed the dependence of the coefficients on microtubule length. Our analyses suggest that 1) the motional diffusion coefficient consists of the sum of two terms, one that is proportional to the inverse of the microtubule length (as the longitudinal diffusion coefficient of a filament in Brownian movement is) and another that is independent of the length, and 2) the length-dependent term decreases with increasing kinesin concentration. This latter term almost vanishes within the length range we studied at high kinesin concentrations. From the length-dependence relationship, we evaluated the friction coefficient for sliding microtubules. This value is much larger than the solvent friction and thus consistent with protein friction. The length independence of the motional diffusion coefficient observed at sufficiently high kinesin concentrations indicates the presence of correlation in the sliding movement fluctuation. This places significant constraint on the possible mechanisms of the sliding movement generation by kinesin motors in vitro.

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

    PubMed

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

    2017-01-24

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

  11. Microtubule alignment and manipulation using AC electrokinetics.

    PubMed

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

    2008-09-01

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

  12. Characterization of microtubule buckling in living cells.

    PubMed

    Pallavicini, Carla; Monastra, Alejandro; Bardeci, Nicolás González; Wetzler, Diana; Levi, Valeria; Bruno, Luciana

    2017-09-01

    Microtubules are filamentous biopolymers involved in essential biological processes. They form key structures in eukaryotic cells, and thus it is very important to determine the mechanisms involved in the formation and maintenance of the microtubule network. Microtubule bucklings are transient and localized events commonly observed in living cells and characterized by a fast bending and its posterior relaxation. Active forces provided by molecular motors have been indicated as responsible for most of these rapid deformations. However, the factors that control the shape amplitude and the time scales of the rising and release stages remain unexplored. In this work, we study microtubule buckling in living cells using Xenopus laevis melanophores as a model system. We tracked single fluorescent microtubules from high temporal resolution (0.3-2 s) confocal movies. We recovered the center coordinates of the filaments with 10-nm precision and analyzed the amplitude of the deformation as a function of time. Using numerical simulations, we explored different force mechanisms resulting in microtubule bending. The simulated events reproduce many features observed for microtubules, suggesting that a mechanistic model captures the essential processes underlying microtubule buckling. Also, we studied the interplay between actively transported vesicles and the microtubule network using a two-color technique. Our results suggest that microtubules may affect transport indirectly besides serving as tracks of motor-driven organelles. For example, they could obstruct organelles at microtubule intersections or push them during filament mechanical relaxation.

  13. Optomechanical proposal for monitoring microtubule mechanical vibrations

    NASA Astrophysics Data System (ADS)

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

    2017-07-01

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

  14. Arabidopsis MICROTUBULE-ASSOCIATED PROTEIN18 functions in directional cell growth by destabilizing cortical microtubules.

    PubMed

    Wang, Xia; Zhu, Lei; Liu, Baoquan; Wang, Che; Jin, Lifeng; Zhao, Qian; Yuan, Ming

    2007-03-01

    Microtubule-associated proteins (MAPs) play important roles in the regulation of microtubule function in cells. We describe Arabidopsis thaliana MAP18, which binds to microtubules and inhibits tubulin polymerization in vitro and colocalizes along cortical microtubules as patches of dot-like structures. MAP18 is expressed mostly in the expanding cells. Cells overexpressing MAP18 in Arabidopsis exhibit various growth phenotypes with loss of polarity. Cortical microtubule arrays were significantly altered in cells either overexpressing MAP18 or where it had been downregulated by RNA interference (RNAi). The cortical microtubules were more sensitive to treatment with microtubule-disrupting drugs when MAP18 was overexpressed, but more resistant when MAP18 was eliminated in cells expressing MAP18 RNAi. Our study demonstrated that MAP18 may play a role in regulating directional cell growth and cortical microtubule organization by destabilizing microtubules.

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

    PubMed

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

    2016-08-23

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

  16. MOTILE MARINE BACTERIA I.

    PubMed Central

    Leifson, Einar; Cosenza, B. J.; Murchelano, R.; Cleverdon, R. C.

    1964-01-01

    Leifson, Einar (Loyola University, Chicago, Ill.), B. J. Cosenza, R. Murchelano, and R. C. Cleverdon. Motile marine bacteria. I. Techniques, ecology, and general characteristics. J. Bacteriol. 87:652–666. 1964.—Aerobic, heterotrophic bacteria were isolated from the waters of the Long Island Sound, Narragansett Bay, Atlantic Ocean, and from the intestine of a variety of marine animals found along the shore of the Long Island Sound. A total of about 600 cultures of motile bacteria were studied morphologically and physiologically, with special emphasis on flagellar characteristics. The great majority of the bacteria isolated from the water were polar flagellate, nonfermentative, nonpigmented, and gramnegative. Most of these were straight, capsulated rods, but a considerable number were curved like vibrios. Yellow-pigmented isolates were often nonmotile, and the motile forms were most frequently subpolar flagellate. Several rosette-forming bacteria, including Caulobacter species, were isolated. Two typical spirilla and one flagellated coccus were found. Peritrichous flagellate bacteria, both gram-positive and gram-negative, were rare except in bottom mud. The normal intestinal flora of marine animals, such as fish and shellfish, consisted of polar flagellate, fermentative, non-pigmented, gram-negative, straight rods. Curved forms, like vibrios, were less common. Polar multitrichous flagellate forms were not uncommon and included all the luminescent types isolated. A considerable proportion of the polar monotrichous flagellate rods swarmed over the surface of agar media. When grown on solid media, all of these showed mixed polar and lateral flagellation; in liquid media, mainly polar flagellation was found. The ecology and general taxonomy of marine bacteria are discussed. Images PMID:14129669

  17. Symmetry-Breaking Motility

    NASA Astrophysics Data System (ADS)

    Lee, Allen; Lee, Ha Youn; Kardar, Mehran

    2005-09-01

    Locomotion of bacteria by actin polymerization and in vitro motion of spherical beads coated with a protein catalyzing polymerization are examples of active motility. Starting from a simple model of forces locally normal to the surface of a bead, we construct a phenomenological equation for its motion. The singularities at a continuous transition between moving and stationary beads are shown to be related to the symmetries of its shape. Universal features of the phase behavior are calculated analytically and confirmed by simulations. Fluctuations in velocity are shown to be generically non-Maxwellian and correlated to the shape of the bead.

  18. Cellular mechanics and motility

    NASA Astrophysics Data System (ADS)

    Hénon, Sylvie; Sykes, Cécile

    2015-10-01

    The term motility defines the movement of a living organism. One widely known example is the motility of sperm cells, or the one of flagellar bacteria. The propulsive element of such organisms is a cilium(or flagellum) that beats. Although cells in our tissues do not have a flagellum in general, they are still able to move, as we will discover in this chapter. In fact, in both cases of movement, with or without a flagellum, cell motility is due to a dynamic re-arrangement of polymers inside the cell. Let us first have a closer look at the propulsion mechanism in the case of a flagellum or a cilium, which is the best known, but also the simplest, and which will help us to define the hydrodynamic general conditions of cell movement. A flagellum is sustained by cellular polymers arranged in semi-flexible bundles and flagellar beating generates cell displacement. These polymers or filaments are part of the cellular skeleton, or "cytoskeleton", which is, in this case, external to the cellular main body of the organism. In fact, bacteria move in a hydrodynamic regime in which viscosity dominates over inertia. The system is thus in a hydrodynamic regime of low Reynolds number (Box 5.1), which is nearly exclusively the case in all cell movements. Bacteria and their propulsion mode by flagella beating are our unicellular ancestors 3.5 billion years ago. Since then, we have evolved to form pluricellular organisms. However, to keep the ability of displacement, to heal our wounds for example, our cells lost their flagellum, since it was not optimal in a dense cell environment: cells are too close to each other to leave enough space for the flagella to accomplish propulsion. The cytoskeleton thus developed inside the cell body to ensure cell shape changes and movement, and also mechanical strength within a tissue. The cytoskeleton of our cells, like the polymers or filaments that sustain the flagellum, is also composed of semi-flexible filaments arranged in bundles, and also in

  19. Kinematic analysis of Toxoplasma gondii motility.

    PubMed

    Frixione, E; Mondragón, R; Meza, I

    1996-01-01

    Toxoplasma gondii tachyzoites execute a complex and little understood combination of rapid movements to reach and penetrate human or other animals cells. In the present study, computer-assisted simulation was used to quantitatively analyze the motility of these parasites in three-dimensional space with spatial and temporal resolutions in the micrometer and subsecond ranges. A digital model based on electron-micrographs of a serially sectioned tachyzoite was animated according to a videomicrographed sequence of a characteristic repetitive movement. Keyframe animation defined over 150 frames by a total of 36 kinematic parameters for specific motions, of both the whole model and particular domains, resulted in a real-time life-like simulation of the videorecorded tachyzoite movement. The kinematic values indicate that a full revolution of the model is composed of three half-turns accomplished in nearly 5 s with two phases: a relatively slow 180 degrees tilting with regard to the substratum plane, followed by fast (over 200 degrees/s) spinning almost simultaneous with pivoting around the posterior end, each clockwise and for about 180 degrees. Maximal flexing of the body, as well as bowing and retraction of its anterior end, occur at midway during the tilting phase. An estimated 70 degrees. clockwise torsion of the body seems to precede the spinning-pivoting phase. The results suggest the operation of two basic forces in the motility of T. gondii tachyzoites: (1) a clockwise torque that causes torsion, spinning, and pivoting; and (2) a longitudinal pull that contracts, bends and tilts the parasite. We discuss the possibility that both of these forces might result from the action of an actin-myosin system enveloping the twisted framework of microtubules characteristic of these organisms.

  20. Microtubule-associated proteins from Antarctic fishes.

    PubMed

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

    1990-01-01

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

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

    PubMed Central

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

    2001-01-01

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

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

    PubMed

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

    2013-05-28

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

  3. Spirochete motility and morpholgy

    NASA Astrophysics Data System (ADS)

    Charon, Nyles

    2004-03-01

    Spirochetes have a unique structure, and as a result their motility is different from that of other bacteria. These organisms can swim in a highly viscous, gel-like medium, such as that found in connective tissue, that inhibits the motility of most other bacteria. In spirochetes, the organelles for motility, the periplasmic flagella, reside inside the cell within the periplasmic space. A given periplasmic flagellum is attached only at one end of the cell, and depending on the species, may or may not overlap in the center of the cell. The number of periplasmic flagella varies from species to species. These structures have been shown to be directly involved in motility and function by rotating within the periplasmic space (1). The present talk focuses on the spirochete that causes Lyme disease, Borrelia burgdorferi. In many bacterial species, cell shape is usually dictated by the peptidoyglycan layer of the cell wall. In the first part of the talk, results will be presented that the morphology of B. burgdorferi is the result of a complex interaction between the cell cylinder and the internal periplasmic flagella resulting in a cell with a flat-wave morphology. Backward moving, propagating waves enable these bacteria to swim and translate in a given direction. Using targeted mutagenesis, we inactivated the gene encoding the major periplasmic flagellar filament protein FlaB. The resulting flaB mutants not only were non-motile, but were rod-shaped (2). Western blot analysis indicated that flaB was no longer synthesized, and electron microscopy revealed that the mutants were completely deficient in periplasmic flagella. Our results indicate that the periplasmic flagella of B. burgdorferi have a skeletal function. These organelles dynamically interact with the rod-shaped cell cylinder to enable the cell to swim, and to confer in part its flat-wave morphology The latter part of the talk concerns the basis for asymmetrical rotation of the periplasmic flagella of B

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

    PubMed Central

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

    2016-01-01

    Spatiotemporal cytoskeleton remodeling is pivotal for cell adhesion and migration. Here we investigated the function of Gas2-related protein on chromosome 22 (GAR22β), a poorly characterized protein that interacts with actin and microtubules. Primary and immortalized GAR22β−/− Sertoli cells moved faster than wild-type cells. In addition, GAR22β−/− cells showed a more prominent focal adhesion turnover. GAR22β overexpression or its reexpression in GAR22β−/− cells reduced cell motility and focal adhesion turnover. GAR22β–actin interaction was stronger than GAR22β–microtubule interaction, resulting in GAR22β localization and dynamics that mirrored those of the actin cytoskeleton. Mechanistically, GAR22β interacted with the regulator of microtubule dynamics end-binding protein 1 (EB1) via a novel noncanonical amino acid sequence, and this GAR22β–EB1 interaction was required for the ability of GAR22β to modulate cell motility. We found that GAR22β is highly expressed in mouse testes, and its absence resulted in reduced spermatozoa generation, lower actin levels in testes, and impaired motility and ultrastructural disorganization of spermatozoa. Collectively our findings identify GAR22β as a novel regulator of cell adhesion and migration and provide a foundation for understanding the molecular basis of diverse cytoskeleton-dependent processes. PMID:26564797

  5. Microtubule Bundling and Shape Transitions

    NASA Astrophysics Data System (ADS)

    Needleman, Daniel

    2005-03-01

    Microtubules (MTs) are hollow cylindrical polymers composed of heterodimers of the protein tubulin that align end-to-end in the MT wall, forming linear protofilaments that interact laterally. Placing MTs under osmotic pressure causes them to reversibly buckle to a noncircular shape and pack into rectangular bundles at a critical osmotic pressure; further increases in pressure continue to distort MTs elastically. At higher osmotic pressures stressing polymers may be forced into the MT lumen causing the MTs to revert to a circle cross-section and pack into hexagonal bundles. This SAXRD-osmotic stress study provides a probe of the inter-protofilament bond strength and gives insight into the mechanisms by which microtubule associated proteins and the cancer chemotherapeutic drug Taxol stabilize MTs. We present further measurements of the mechanical properties of MT walls, MT-MT interactions, and the entry of polymers into the microtubule lumen. Supported by NSF DMR- 0203755, NIH GM-59288 and NS-13560, and CTS-0103516. SSRL is supported by the U.S. DOE.

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

    PubMed

    Kwiatkowska, Maria; Stępiński, Dariusz; Polit, Justyna T; Popłońska, Katarzyna; Wojtczak, Agnieszka

    2011-01-01

    Lipotubuloids, structures containing lipid bodies and microtubules, are described in ovary epidermal cells of Ornithogalum umbellatum. Microtubules of lipotubuloids can be fixed in electron microscope fixative containing only buffered OsO(4) or in glutaraldehyde with OsO(4) post-fixation, or in a mixture of OsO(4) and glutaraldehyde. None of these substances fixes cortical microtubules of ovary epidermis of this plant which is characterized by dynamic longitudinal growth. However, cortical microtubules can be fixed with cold methanol according immunocytological methods with the use of β-tubulin antibodies and fluorescein. The existence of cortical microtubules has also been evidenced by EM observations solely after the use of taxol, microtubule stabilizer, and fixation in a glutaraldehyde/OsO(4) mixture. These microtubules mostly lie transversely, sometimes obliquely, and rarely parallel to the cell axis. Staining, using Ruthenium Red and silver hexamine, has revealed that lipotubuloid microtubules surface is covered with polysaccharides. The presumption has been made that the presence of a polysaccharide layer enhances the stability of lipotubuloid microtubules.

  7. Mechanics of motility initiation and motility arrest in crawling cells

    NASA Astrophysics Data System (ADS)

    Recho, Pierre; Putelat, Thibaut; Truskinovsky, Lev

    2015-11-01

    Motility initiation in crawling cells requires transformation of a symmetric state into a polarized state. In contrast, motility arrest is associated with re-symmetrization of the internal configuration of a cell. Experiments on keratocytes suggest that polarization is triggered by the increased contractility of motor proteins but the conditions of re-symmetrization remain unknown. In this paper we show that if adhesion with the extra-cellular substrate is sufficiently low, the progressive intensification of motor-induced contraction may be responsible for both transitions: from static (symmetric) to motile (polarized) at a lower contractility threshold and from motile (polarized) back to static (symmetric) at a higher contractility threshold. Our model of lamellipodial cell motility is based on a 1D projection of the complex intra-cellular dynamics on the direction of locomotion. In the interest of analytical transparency we also neglect active protrusion and view adhesion as passive. Despite the unavoidable oversimplifications associated with these assumptions, the model reproduces quantitatively the motility initiation pattern in fish keratocytes and reveals a crucial role played in cell motility by the nonlocal feedback between the mechanics and the transport of active agents. A prediction of the model that a crawling cell can stop and re-symmetrize when contractility increases sufficiently far beyond the motility initiation threshold still awaits experimental verification.

  8. Monoclonal antibodies to kinesin heavy and light chains stain vesicle- like structures, but not microtubules, in cultured cells

    PubMed Central

    1989-01-01

    Kinesin, a microtubule-activated ATPase and putative motor protein for the transport of membrane-bounded organelles along microtubules, was purified from bovine brain and used as an immunogen for the production of murine monoclonal antibodies. Hybridoma lines that secreted five distinct antikinesin IgGs were cloned. Three of the antibodies reacted on immunoblots with the 124-kD heavy chain of kinesin, while the other two antibodies recognized the 64-kD light chain. When used for immunofluorescence microscopy, the antibodies stained punctate, cytoplasmic structures in a variety of cultured mammalian cell types. Consistent with the identification of these structures as membrane- bounded organelles was the observation that cells which had been extracted with Triton X-100 before fixation contained little or no immunoreactive material. Staining of microtubules in the interphase cytoplasm or mitotic spindle was never observed, nor were associated structures, such as centrosomes and primary cilia, labeled by any of the antibodies. Nevertheless, in double-labeling experiments using antibodies to kinesin and tubulin, kinesin-containing particles were most abundant in regions where microtubules were most highly concentrated and the particles often appeared to be aligned on microtubules. These results constitute the first direct evidence for the association of kinesin with membrane-bounded organelles, and suggest a molecular mechanism for organelle motility based on transient interactions of organelle-bound kinesin with the microtubule surface. PMID:2522455

  9. Microtubule detyrosination guides chromosomes during mitosis

    PubMed Central

    Barisic, Marin; Silva e Sousa, Ricardo; Tripathy, Suvranta K.; Magiera, Maria M.; Zaytsev, Anatoly V.; Pereira, Ana L.; Janke, Carsten; Grishchuk, Ekaterina L.; Maiato, Helder

    2015-01-01

    Before chromosomes segregate into daughter cells they align at the mitotic spindle equator, a process known as chromosome congression. CENP-E/Kinesin-7 is a microtubule plus-end-directed kinetochore motor required for congression of pole-proximal chromosomes. Because the plus-ends of many astral microtubules in the spindle point to the cell cortex, it remains unknown how CENP-E guides pole-proximal chromosomes specifically towards the equator. Here we found that congression of pole-proximal chromosomes depended on specific post-translational detyrosination of spindle microtubules that point to the equator. In vitro reconstitution experiments demonstrated that CENP-E-dependent transport was strongly enhanced on detyrosinated microtubules. Blocking tubulin tyrosination in cells caused ubiquitous detyrosination of spindle microtubules and CENP-E transported chromosomes away from spindle poles in random directions. Thus, CENP-E-driven chromosome congression is guided by microtubule detyrosination. PMID:25908662

  10. Spirochete periplasmic flagella and motility.

    PubMed

    Li, C; Motaleb, A; Sal, M; Goldstein, S F; Charon, N W

    2000-10-01

    Spirochetes have a unique structure, and as a result their motility is different from that of other bacteria. They also have a special attribute: spirochetes can swim in a highly viscous, gel-like medium, such as that found in connective tissue, that inhibits the motility of most other bacteria. In spirochetes, the organelles for motility, the periplasmic flagella, reside inside the cell within the periplasmic space. A given periplasmic flagellum is attached only at one end of the cell, and depending on the species, may or may not overlap in the center of the cell with those attached at the other end. The number of periplasmic flagella varies from species to species. These structures have been shown to be directly involved in spirochete motility, and they function by rotating within the periplasmic space. The mechanics of motility also vary among the spirochetes. In Leptospira, a motility model developed several years ago has been extensively tested, and the evidence supporting this model is convincing. Borrelia burgdorferi swims differently, and a model of its motility has been recently put forward. This model is based on analyzing the motion of swimming cells, high voltage electron microscopy of fixed cells, and mutant analysis. To better understand spirochete motility on a more molecular level, the proteins and genes involved in motility are being analyzed. Spirochete periplasmic flagellar filaments are among the most complex of bacterial flagella. They are composed of the FlaA sheath proteins, and in many species, multiple FlaB core proteins. Allelic exchange mutagenesis of the genes which encode these proteins is beginning to yield important information with respect to periplasmic flagellar structure and function. Although we are at an early stage with respect to analyzing the function, organization, and regulation of many of the genes involved in spirochete motility, unique aspects have already become evident. Future studies on spirochete motility should be

  11. Ectopic A-lattice seams destabilize microtubules.

    PubMed

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

    2014-01-01

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

  12. 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

  13. Mechanical stress induced mechanism of microtubule catastrophes.

    PubMed

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

    2005-05-13

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

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

    PubMed Central

    Patel-Hett, Sunita; Richardson, Jennifer L.; Schulze, Harald; Drabek, Ksenija; Isaac, Natasha A.; Hoffmeister, Karin; Shivdasani, Ramesh A.; Bulinski, J. Chloë; Galjart, Niels; Hartwig, John H.

    2008-01-01

    The marginal band of microtubules maintains the discoid shape of resting blood platelets. Although studies of platelet microtubule coil structure conclude that it is composed of a single microtubule, no investigations of its dynamics exist. In contrast to previous studies, permeabilized platelets incubated with GTP-rhodamine-tubulin revealed tubulin incorporation at 7.9 (± 1.9) points throughout the coil, and anti-EB1 antibodies stained 8.7 (± 2.0) sites, indicative of multiple free microtubules. To pursue this result, we expressed the microtubule plus-end marker EB3-GFP in megakaryocytes and examined its behavior in living platelets released from these cells. Time-lapse microscopy of EB3-GFP in resting platelets revealed multiple assembly sites within the coil and a bidirectional pattern of assembly. Consistent with these findings, tyrosinated tubulin, a marker of newly assembled microtubules, localized to resting platelet microtubule coils. These results suggest that the resting platelet marginal band contains multiple highly dynamic microtubules of mixed polarity. Analysis of microtubule coil diameters in newly formed resting platelets indicates that microtubule coil shrinkage occurs with aging. In addition, activated EB3-GFP–expressing platelets exhibited a dramatic increase in polymerizing microtubules, which travel outward and into filopodia. Thus, the dynamic microtubules associated with the marginal band likely function during both resting and activated platelet states. PMID:18230754

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

    PubMed

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

    2016-11-01

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

  16. Stochastic Model of Microtubule Dynamics

    NASA Astrophysics Data System (ADS)

    Hryniv, Ostap; Martínez Esteban, Antonio

    2017-10-01

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

  17. Insights into the Structure and Function of Ciliary and Flagellar Doublet Microtubules

    PubMed Central

    Linck, Richard; Fu, Xiaofeng; Lin, Jianfeng; Ouch, Christna; Schefter, Alexandra; Steffen, Walter; Warren, Peter; Nicastro, Daniela

    2014-01-01

    Cilia and flagella are conserved, motile, and sensory cell organelles involved in signal transduction and human disease. Their scaffold consists of a 9-fold array of remarkably stable doublet microtubules (DMTs), along which motor proteins transmit force for ciliary motility and intraflagellar transport. DMTs possess Ribbons of three to four hyper-stable protofilaments whose location, organization, and specialized functions have been elusive. We performed a comprehensive analysis of the distribution and structural arrangements of Ribbon proteins from sea urchin sperm flagella, using quantitative immunobiochemistry, proteomics, immuno-cryo-electron microscopy, and tomography. Isolated Ribbons contain acetylated α-tubulin, β-tubulin, conserved protein Rib45, >95% of the axonemal tektins, and >95% of the calcium-binding proteins, Rib74 and Rib85.5, whose human homologues are related to the cause of juvenile myoclonic epilepsy. DMTs contain only one type of Ribbon, corresponding to protofilaments A11-12-13-1 of the A-tubule. Rib74 and Rib85.5 are associated with the Ribbon in the lumen of the A-tubule. Ribbons contain a single ∼5-nm wide filament, composed of equimolar tektins A, B, and C, which interact with the nexin-dynein regulatory complex. A summary of findings is presented, and the functions of Ribbon proteins are discussed in terms of the assembly and stability of DMTs, ciliary motility, and other microtubule systems. PMID:24794867

  18. Novel mitochondrial extensions provide evidence for a link between microtubule-directed movement and mitochondrial fission

    SciTech Connect

    Bowes, Timothy; Gupta, Radhey S.

    2008-11-07

    Mitochondrial dynamics play an important role in a large number of cellular processes. Previously, we reported that treatment of mammalian cells with the cysteine-alkylators, N-ethylmaleimide and ethacrynic acid, induced rapid mitochondrial fusion forming a large reticulum approximately 30 min after treatment. Here, we further investigated this phenomenon using a number of techniques including live-cell confocal microscopy. In live cells, drug-induced fusion coincided with a cessation of fast mitochondrial movement which was dependent on microtubules. During this loss of movement, thin mitochondrial tubules extending from mitochondria were also observed, which we refer to as 'mitochondrial extensions'. The formation of these mitochondrial extensions, which were not observed in untreated cells, depended on microtubules and was abolished by pretreatment with nocodazole. In this study, we provide evidence that these extensions result from of a block in mitochondrial fission combined with continued application of motile force by microtubule-dependent motor complexes. Our observations strongly suggest the existence of a link between microtubule-based mitochondrial trafficking and mitochondrial fission.

  19. Tuning microtubule-based transport via filamentous MAPs: the problem of dynein

    PubMed Central

    Vershinin, Michael; Xu, Jing; Razafsky, David S.; King, Stephen J.; Gross, Steven P.

    2010-01-01

    We recently proposed that regulating the single-to-multiple motor transition was a likely strategy for regulating kinesin-based transport in vivo. Here, we use an in vitro bead assay coupled with an optical trap to investigate how this proposed regulatory mechanism affects dynein-based transport. We show that tau’s regulation of kinesin function can proceed without interfering with dynein-based transport. Surprisingly, at extremely high tau levels—where kinesin cannot bind microtubules—dynein can still contact microtubules. The difference between tau’s effects on kinesin- and dynein-based motility suggests that tau can be used to tune relative amounts of plus-end and minus-end directed transport. As in the case of kinesin, we find that the 3RS isoform of tau is a more potent inhibitor of dynein binding to microtubules. We show that this isoform-specific effect is not due to steric interference of tau’s projection domains, but rather due to tau’s interactions with the motor at the microtubule surface. Nonetheless, we do observe a modest steric interference effect of tau away from the microtubule and discuss the potential implications of this for molecular motor structure. PMID:18373727

  20. Wolbachia Utilizes Host Microtubules and Dynein for Anterior Localization in the Drosophila Oocyte

    PubMed Central

    Li, Jennifer M; Cao, Jian; Wieschaus, Eric; Sullivan, William

    2005-01-01

    To investigate the role of the host cytoskeleton in the maternal transmission of the endoparasitic bacteria Wolbachia, we have characterized their distribution in the female germ line of Drosophila melanogaster. In the germarium, Wolbachia are distributed to all germ cells of the cyst, establishing an early infection in the cell destined to become the oocyte. During mid-oogenesis, Wolbachia exhibit a distinct concentration between the anterior cortex and the nucleus in the oocyte, where many bacteria appear to contact the nuclear envelope. Following programmed rearrangement of the microtubule network, Wolbachia dissociate from this anterior position and become dispersed throughout the oocyte. This localization pattern is distinct from mitochondria and all known axis determinants. Manipulation of microtubules and cytoplasmic Dynein and Dynactin, but not Kinesin-1, disrupts anterior bacterial localization in the oocyte. In live egg chambers, Wolbachia exhibit movement in nurse cells but not in the oocyte, suggesting that the bacteria are anchored by host factors. In addition, we identify mid-oogenesis as a period in the life cycle of Wolbachia in which bacterial replication occurs. Total bacterial counts show that Wolbachia increase at a significantly higher rate in the oocyte than in the average nurse cell, and that normal Wolbachia levels in the oocyte depend on microtubules. These findings demonstrate that Wolbachia utilize the host microtubule network and associated proteins for their subcellular localization in the Drosophila oocyte. These interactions may also play a role in bacterial motility and replication, ultimately leading to the bacteria's efficient maternal transmission. PMID:16228015

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

    PubMed

    Feizabadi, Mitra Shojania; Rosario, Brandon

    2017-03-04

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

  2. Rimonabant, Gastrointestinal Motility and Obesity

    PubMed Central

    Sun, Yan; Chen, Jiande

    2012-01-01

    Background: Obesity and overweight affect more than half of the US population and are associated with a number of diseases. Rimonabant, a cannabinoid receptor 1 blocker in the endocannabinoid (EC) system, was indicated in Europe for the treatment of obesity and overweight patients with associated risk factors but withdrawn on Jan, 2009 because of side effects. Many studies have reported the effects of rimonabant on gastrointestinal (GI) motility and food intake. The aims of this review are: to review the relationship of EC system with GI motility and food intake;to review the studies of rimonabant on GI motility, food intake and obesity;and to report the tolerance and side effects of rimonabant. Methods: the literature (Pubmed database) was searched using keywords: rimonabant, obesity and GI motility. Results: GI motility is related with appetite, food intake and nutrients absorption. The EC system inhibits GI motility, reduces emesis and increases food intake; Rimonabant accelerates gastric emptying and intestinal transition but decreases energy metabolism and food intake. There is rapid onset of tolerance to the prokinetic effect of rimonabant. The main side effects of rimonabant are depression and GI symptoms. Conclusions: Rimonabant has significant effects on energy metabolism and food intake, probably mediated via its effects on GI motility. PMID:23449551

  3. Mechanical Properties of Doubly Stabilized Microtubule Filaments

    PubMed Central

    Hawkins, Taviare L.; Sept, David; Mogessie, Binyam; Straube, Anne; Ross, Jennifer L.

    2013-01-01

    Microtubules are cytoskeletal filaments responsible for cell morphology and intracellular organization. Their dynamical and mechanical properties are regulated through the nucleotide state of the tubulin dimers and the binding of drugs and/or microtubule-associated proteins. Interestingly, microtubule-stabilizing factors have differential effects on microtubule mechanics, but whether stabilizers have cumulative effects on mechanics or whether one effect dominates another is not clear. This is especially important for the chemotherapeutic drug Taxol, an important anticancer agent and the only known stabilizer that reduces the rigidity of microtubules. First, we ask whether Taxol will combine additively with another stabilizer or whether one stabilizer will dominate another. We call microtubules in the presence of Taxol and another stabilizer, doubly stabilized. Second, since Taxol is often added to a number of cell types for therapeutic purposes, it is important from a biomedical perspective to understand how Taxol added to these systems affects the mechanical properties in treated cells. To address these questions, we use the method of freely fluctuating filaments with our recently developed analysis technique of bootstrapping to determine the distribution of persistence lengths of a large population of microtubules treated with different stabilizers, including Taxol, guanosine-5′ [(α, β)-methyleno] triphosphate, guanosine-5′-O-(3-thiotriphosphate), tau, and MAP4. We find that combinations of these stabilizers have novel effects on the mechanical properties of microtubules. PMID:23561528

  4. 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

  5. Microtubule motors: moving forward on many fronts.

    PubMed

    Allan, Viki

    2009-07-08

    Microtubule motors drive the movement of many different cargoes in eukaryotic cells. A combination of in vitro and in vivo approaches has led to a better understanding of their mechanism of action and function and are also revealing that the microtubule track itself may have an important role to play in directing cargo movement within the cell.

  6. Microtubule stabilization: formins assert their independence.

    PubMed

    DeWard, Aaron D; Alberts, Arthur S

    2008-07-22

    Mammalian Diaphanous-related (mDia) formins are well known for their actin nucleation and filament elongation activities. They have since emerged as microtubule-binding proteins, and a recent study shows that mDia2 stabilizes microtubules independently of its actin nucleation activity.

  7. Mechanical properties of doubly stabilized microtubule filaments.

    PubMed

    Hawkins, Taviare L; Sept, David; Mogessie, Binyam; Straube, Anne; Ross, Jennifer L

    2013-04-02

    Microtubules are cytoskeletal filaments responsible for cell morphology and intracellular organization. Their dynamical and mechanical properties are regulated through the nucleotide state of the tubulin dimers and the binding of drugs and/or microtubule-associated proteins. Interestingly, microtubule-stabilizing factors have differential effects on microtubule mechanics, but whether stabilizers have cumulative effects on mechanics or whether one effect dominates another is not clear. This is especially important for the chemotherapeutic drug Taxol, an important anticancer agent and the only known stabilizer that reduces the rigidity of microtubules. First, we ask whether Taxol will combine additively with another stabilizer or whether one stabilizer will dominate another. We call microtubules in the presence of Taxol and another stabilizer, doubly stabilized. Second, since Taxol is often added to a number of cell types for therapeutic purposes, it is important from a biomedical perspective to understand how Taxol added to these systems affects the mechanical properties in treated cells. To address these questions, we use the method of freely fluctuating filaments with our recently developed analysis technique of bootstrapping to determine the distribution of persistence lengths of a large population of microtubules treated with different stabilizers, including Taxol, guanosine-5' [(α, β)-methyleno] triphosphate, guanosine-5'-O-(3-thiotriphosphate), tau, and MAP4. We find that combinations of these stabilizers have novel effects on the mechanical properties of microtubules.

  8. HEATR2 plays a conserved role in assembly of the ciliary motile apparatus.

    PubMed

    Diggle, Christine P; Moore, Daniel J; Mali, Girish; zur Lage, Petra; Ait-Lounis, Aouatef; Schmidts, Miriam; Shoemark, Amelia; Garcia Munoz, Amaya; Halachev, Mihail R; Gautier, Philippe; Yeyati, Patricia L; Bonthron, David T; Carr, Ian M; Hayward, Bruce; Markham, Alexander F; Hope, Jilly E; von Kriegsheim, Alex; Mitchison, Hannah M; Jackson, Ian J; Durand, Bénédicte; Reith, Walter; Sheridan, Eamonn; Jarman, Andrew P; Mill, Pleasantine

    2014-09-01

    Cilia are highly conserved microtubule-based structures that perform a variety of sensory and motility functions during development and adult homeostasis. In humans, defects specifically affecting motile cilia lead to chronic airway infections, infertility and laterality defects in the genetically heterogeneous disorder Primary Ciliary Dyskinesia (PCD). Using the comparatively simple Drosophila system, in which mechanosensory neurons possess modified motile cilia, we employed a recently elucidated cilia transcriptional RFX-FOX code to identify novel PCD candidate genes. Here, we report characterization of CG31320/HEATR2, which plays a conserved critical role in forming the axonemal dynein arms required for ciliary motility in both flies and humans. Inner and outer arm dyneins are absent from axonemes of CG31320 mutant flies and from PCD individuals with a novel splice-acceptor HEATR2 mutation. Functional conservation of closely arranged RFX-FOX binding sites upstream of HEATR2 orthologues may drive higher cytoplasmic expression of HEATR2 during early motile ciliogenesis. Immunoprecipitation reveals HEATR2 interacts with DNAI2, but not HSP70 or HSP90, distinguishing it from the client/chaperone functions described for other cytoplasmic proteins required for dynein arm assembly such as DNAAF1-4. These data implicate CG31320/HEATR2 in a growing intracellular pre-assembly and transport network that is necessary to deliver functional dynein machinery to the ciliary compartment for integration into the motile axoneme.

  9. Peroxisomes, lipid droplets, and endoplasmic reticulum “hitchhike” on motile early endosomes

    PubMed Central

    Guimaraes, Sofia C.; Schuster, Martin; Bielska, Ewa; Dagdas, Gulay; Kilaru, Sreedhar; Meadows, Ben R.A.; Schrader, Michael

    2015-01-01

    Intracellular transport is mediated by molecular motors that bind cargo to be transported along the cytoskeleton. Here, we report, for the first time, that peroxisomes (POs), lipid droplets (LDs), and the endoplasmic reticulum (ER) rely on early endosomes (EEs) for intracellular movement in a fungal model system. We show that POs undergo kinesin-3– and dynein-dependent transport along microtubules. Surprisingly, kinesin-3 does not colocalize with POs. Instead, the motor moves EEs that drag the POs through the cell. PO motility is abolished when EE motility is blocked in various mutants. Most LD and ER motility also depends on EE motility, whereas mitochondria move independently of EEs. Covisualization studies show that EE-mediated ER motility is not required for PO or LD movement, suggesting that the organelles interact with EEs independently. In the absence of EE motility, POs and LDs cluster at the growing tip, whereas ER is partially retracted to subapical regions. Collectively, our results show that moving EEs interact transiently with other organelles, thereby mediating their directed transport and distribution in the cell. PMID:26620910

  10. 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

  11. 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.

  12. Tubulin bistability and polymorphic dynamics of microtubules.

    PubMed

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

    2010-12-31

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

  13. 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.

  14. Tubulin Bistability and Polymorphic Dynamics of Microtubules

    NASA Astrophysics Data System (ADS)

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

    2010-12-01

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

  15. FLUCTUATING MOTOR FORCES BEND GROWING MICROTUBULES

    PubMed Central

    Shekhar, Nandini; Neelam, Srujana; Wu, Jun; Ladd, Anthony JC; Dickinson, Richard B.; Lele, Tanmay P.

    2013-01-01

    Despite their rigidity, microtubules in living cells bend significantly during polymerization resulting in greater curvature than can be explained by thermal forces alone. However, the source of the non-thermal forces that bend growing microtubules remains obscure. We analyzed the motion of microtubule tips in NIH-3T3 fibroblasts expressing EGFP-EB1, a fluorescent +TIP protein that specifically binds to the growing ends of microtubules. We found that dynein inhibition significantly reduced the deviation of the growing tip from its initial trajectory. Inhibiting myosin modestly reduced tip fluctuations, while simultaneous myosin and dynein inhibition caused no further decrease in fluctuations compared to dynein inhibition alone. Our results can be interpreted with a model in which dynein linkages play a key role in generating and transmitting fluctuating forces that bend growing microtubules. PMID:24039637

  16. 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

  17. Effects of iodide on the coupling between ATP hydrolysis and motile activity in axonemal dynein.

    PubMed

    Nakano, Izumi; Fujiwara, Rin; Wada, Mikiyo; Shingyoji, Chikako

    2011-05-01

    Dynein transduces the chemical energy of ATP hydrolysis into mechanical work through conformational changes. To identify the factors governing the coupling between the ATPase activity and the motile activity of the dynein molecule, we examined the effects of potassium iodide, which can unfold protein tertiary structures, on dynein activity in reactivated sea urchin sperm flagella. The presence of low concentrations of KI (0.05-0.1 M) in the reactivating solution did not influence the stable beating of demembranated flagella at 0.02-1 mM ATP, when the total concentration of potassium was kept at 0.15 M by adding K-acetate. However, double-reciprocal plots of ATP concentration and beat frequency showed a mixed type of inhibition by KI, indicating the possibility that KI inhibits the ATP hydrolysis and decreases the maximum sliding velocity. The ATPase activity of 21S dynein with or without microtubules did not decrease with the KI concentration. In the elastase-treated axonemes, KI decreased the velocity of sliding disintegration, while it increased the frequency of occurrence of axonemes showing no sliding. This may be related to some defect in the coordination of dynein activities. On 21S dynein adsorbed on a glass surface, however, the velocity of microtubule sliding was increased by KI, while KI lowered the dynein-microtubule affinity. The velocity further increased under lower salt conditions enhancing the dynein-microtubule interactions. The results suggest the importance of organized regulation of the dynamic states of dynein-microtubule interactions through the stalk for the coupling between the ATPase activity and the motile activity of dynein in beating flagella.

  18. Gastrointestinal Motility Disorders in Children

    PubMed Central

    Ambartsumyan, Lusine

    2014-01-01

    The most common and challenging gastrointestinal motility disorders in children include gastroesophageal reflux disease (GERD), esophageal achalasia, gastroparesis, chronic intestinal pseudo-obstruction, and constipation. GERD is the most common gastrointestinal motility disorder affecting children and is diagnosed clinically and treated primarily with acid secretion blockade. Esophageal achalasia, a less common disorder in the pediatric patient population, is characterized by dysphagia and treated with pneumatic balloon dilation and/or esophagomyotomy. Gastroparesis and chronic intestinal pseudo-obstruction are poorly characterized in children and are associated with significant morbidity. Constipation is among the most common complaints in children and is associated with significant morbidity as well as poor quality of life. Data on epidemiology and outcomes, clinical trials, and evaluation of new diagnostic techniques are needed to better diagnose and treat gastrointestinal motility disorders in children. We present a review of the conditions and challenges related to these common gastrointestinal motility disorders in children. PMID:24799835

  19. Shape determination in motile cells

    NASA Astrophysics Data System (ADS)

    Mogilner, Alex

    2010-03-01

    Flat, simple shaped, rapidly gliding fish keratocyte cell is the model system of choice to study cell motility. The cell motile appendage, lamellipod, has a characteristic bent-rectangular shape. Recent experiments showed that the lamellipodial geometry is tightly correlated with cell speed and with actin dynamics. These quantitative data combined with computational modeling suggest that a model for robust actin treadmill inside the 'unstretchable membrane bag'. According to this model, a force balance between membrane tension and growing and pushing actin network distributed unevenly along the cell periphery can explain the cell shape and motility. However, when adhesion of the cell to the surface weakens, the actin dynamics become less regular, and myosin-powered contraction starts playing crucial role in stabilizing the cell shape. I will illustrate how the combination of theoretical and experimental approaches helped to unravel the keratocyte motile behavior.

  20. Surface motility of Myxococcus Xanthus

    NASA Astrophysics Data System (ADS)

    Gibiansky, Maxsim; Hu, William; Jin, Fan; Zhao, Kun; Shi, Wenyuan; Wong, Gerard

    2011-03-01

    We examine the surface motility of Myxococcus Xanthus, a bacterium species found in soil that exhibits a broad range of self-organizing behavior, including predatory ``swarms'' and survival-enhancing ``fruiting bodies.'' To quantify the effects of exopolysaccharides (EPS) on surface adhesion and motility, we use modified versions of particle tracking algorithms from colloid physics to analyze bacterial trajectories, and compare the wild type (WT) strain to EPS knockout and EPS overproducer strains. We find that EPS deficiency leads to an increase in the number of ``standing'' bacteria oriented normal to the surface, attached by one end with minimal motility. EPS overproduction, by contrast, suppresses this phenotype. A detailed investigation of the influence of EPS on Myxococcus social motility will be presented.

  1. Microtubule organization by kinesin motors and microtubule crosslinking protein MAP65

    NASA Astrophysics Data System (ADS)

    Pringle, Joshua; Muthukumar, Amutha; Tan, Amanda; Crankshaw, Laura; Conway, Leslie; Ross, Jennifer L.

    2013-09-01

    Microtubules are rigid, proteinaceous filaments required to organize and rearrange the interior of cells. They organize space by two mechanisms, including acting as the tracks for long-distance cargo transporters, such as kinesin-1, and by forming a network that supports the shape of the cell. The microtubule network is composed of microtubules and a bevy of associated proteins and enzymes that self-organize using non-equilibrium dynamic processes. In order to address the effects of self-organization of microtubules, we have utilized the filament-gliding assay with kinesin-1 motors driving microtubule motion. To further enhance the complexity of the system and determine if new patterns are formed, we added the microtubule crosslinking protein MAP65-1. MAP65-1 is a microtubule-associated protein from plants that crosslinks antiparallel microtubules, similar to mammalian PRC1 and fission yeast Ase1. We find that MAP65 can slow and halt the velocity of microtubules in gliding assays, but when pre-formed microtubule bundles are added to gliding assays, kinesin-1 motors can pull apart the bundles and reconstitute cell-like protrusions.

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

    PubMed

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

    1989-05-01

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

  3. Elenoside increases intestinal motility

    PubMed Central

    Navarro, E; Alonso, SJ; Navarro, R; Trujillo, J; Jorge, E

    2006-01-01

    AIM: To study the effects of elenoside, an arylnaph-thalene lignan from Justicia hyssopifolia, on gastro-intestinal motility in vivo and in vitro in rats. METHODS: Routine in vivo experimental assessments were catharsis index, water percentage of boluses, intestinal transit, and codeine antagonism. The groups included were vehicle control (propylene glycol-ethanol-plant oil-tween 80), elenoside (i.p. 25 and 50 mg/kg), cisapride (i.p. 10 mg/kg), and codeine phosphate (intragastric route, 50 mg/kg). In vitro approaches used isolated rat intestinal tissues (duodenum, jejunum, and ileum). The effects of elenoside at concentrations of 3.2 x 10-4, 6.4 x 10-4 and 1.2 x 10-3 mol/L, and cisapride at 10-6 mol/L were investigated. RESULTS: Elenoside in vivo produced an increase in the catharsis index and water percentage of boluses and in the percentage of distance traveled by a suspension of activated charcoal. Codeine phosphate antagonized the effect of 25 mg/kg of elenoside. In vitro, elenoside in duodenum, jejunum and ileum produced an initial decrease in the contraction force followed by an increase. Elenoside resulted in decreased intestinal frequency in duodenum, jejunum, and ileum. The in vitro and in vivo effects of elenoside were similar to those produced by cisapride. CONCLUSION: Elenoside is a lignan with an action similar to that of purgative and prokinetics drugs. Elenoside, could be an alternative to cisapride in treatment of gastrointestinal diseases as well as a preventive therapy for the undesirable gastrointestinal effects produced by opioids used for mild to moderate pain. PMID:17131476

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

    PubMed

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

    2003-06-10

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

  5. Microtubules in viral replication and transport.

    PubMed

    Niehl, Annette; Peña, Eduardo J; Amari, Khalid; Heinlein, Manfred

    2013-07-01

    Viruses use and subvert host cell mechanisms to support their replication and spread between cells, tissues and organisms. Microtubules and associated motor proteins play important roles in these processes in animal systems, and may also play a role in plants. Although transport processes in plants are mostly actin based, studies, in particular with Tobacco mosaic virus (TMV) and its movement protein (MP), indicate direct or indirect roles of microtubules in the cell-to-cell spread of infection. Detailed observations suggest that microtubules participate in the cortical anchorage of viral replication complexes, in guiding their trafficking along the endoplasmic reticulum (ER)/actin network, and also in developing the complexes into virus factories. Microtubules also play a role in the plant-to-plant transmission of Cauliflower mosaic virus (CaMV) by assisting in the development of specific virus-induced inclusions that facilitate viral uptake by aphids. The involvement of microtubules in the formation of virus factories and of other virus-induced inclusions suggests the existence of aggresomal pathways by which plant cells recruit membranes and proteins into localized macromolecular assemblies. Although studies related to the involvement of microtubules in the interaction of viruses with plants focus on specific virus models, a number of observations with other virus species suggest that microtubules may have a widespread role in viral pathogenesis. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

  6. Reovirus Cell Entry Requires Functional Microtubules

    PubMed Central

    Mainou, Bernardo A.; Zamora, Paula F.; Ashbrook, Alison W.; Dorset, Daniel C.; Kim, Kwang S.; Dermody, Terence S.

    2013-01-01

    ABSTRACT Mammalian reovirus binds to cell-surface glycans and junctional adhesion molecule A and enters cells by receptor-mediated endocytosis in a process dependent on β1 integrin. Within the endocytic compartment, reovirus undergoes stepwise disassembly, allowing release of the transcriptionally active viral core into the cytoplasm. To identify cellular mediators of reovirus infectivity, we screened a library of small-molecule inhibitors for the capacity to block virus-induced cytotoxicity. In this screen, reovirus-induced cell killing was dampened by several compounds known to impair microtubule dynamics. Microtubule inhibitors were assessed for blockade of various stages of the reovirus life cycle. While these drugs did not alter reovirus cell attachment or internalization, microtubule inhibitors diminished viral disassembly kinetics with a concomitant decrease in infectivity. Reovirus virions colocalize with microtubules and microtubule motor dynein 1 during cell entry, and depolymerization of microtubules results in intracellular aggregation of viral particles. These data indicate that functional microtubules are required for proper sorting of reovirus virions following internalization and point to a new drug target for pathogens that use the endocytic pathway to invade host cells. PMID:23820395

  7. Profilin connects actin assembly with microtubule dynamics

    PubMed Central

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

    2016-01-01

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

  8. Microtubules regulate disassembly of epithelial apical junctions

    PubMed Central

    Ivanov, Andrei I; McCall, Ingrid C; Babbin, Brian; Samarin, Stanislav N; Nusrat, Asma; Parkos, Charles A

    2006-01-01

    Background Epithelial tight junction (TJ) and adherens junction (AJ) form the apical junctional complex (AJC) which regulates cell-cell adhesion, paracellular permeability and cell polarity. The AJC is anchored on cytoskeletal structures including actin microfilaments and microtubules. Such cytoskeletal interactions are thought to be important for the assembly and remodeling of apical junctions. In the present study, we investigated the role of microtubules in disassembly of the AJC in intestinal epithelial cells using a model of extracellular calcium depletion. Results Calcium depletion resulted in disruption and internalization of epithelial TJs and AJs along with reorganization of perijunctional F-actin into contractile rings. Microtubules reorganized into dense plaques positioned inside such F-actin rings. Depolymerization of microtubules with nocodazole prevented junctional disassembly and F-actin ring formation. Stabilization of microtubules with either docetaxel or pacitaxel blocked contraction of F-actin rings and attenuated internalization of junctional proteins into a subapical cytosolic compartment. Likewise, pharmacological inhibition of microtubule motors, kinesins, prevented contraction of F-actin rings and attenuated disassembly of apical junctions. Kinesin-1 was enriched at the AJC in cultured epithelial cells and it also accumulated at epithelial cell-cell contacts in normal human colonic mucosa. Furthermore, immunoprecipitation experiments demonstrated association of kinesin-1 with the E-cadherin-catenin complex. Conclusion Our data suggest that microtubules play a role in disassembly of the AJC during calcium depletion by regulating formation of contractile F-actin rings and internalization of AJ/TJ proteins. PMID:16509970

  9. Xyloglucan Deficiency Disrupts Microtubule Stability and Cellulose Biosynthesis in Arabidopsis, Altering Cell Growth and Morphogenesis1[OPEN

    PubMed Central

    Xiao, Chaowen; Zhang, Tian; Zheng, Yunzhen

    2016-01-01

    Xyloglucan constitutes most of the hemicellulose in eudicot primary cell walls and functions in cell wall structure and mechanics. Although Arabidopsis (Arabidopsis thaliana) xxt1 xxt2 mutants lacking detectable xyloglucan are viable, they display growth defects that are suggestive of alterations in wall integrity. To probe the mechanisms underlying these defects, we analyzed cellulose arrangement, microtubule patterning and dynamics, microtubule- and wall-integrity-related gene expression, and cellulose biosynthesis in xxt1 xxt2 plants. We found that cellulose is highly aligned in xxt1 xxt2 cell walls, that its three-dimensional distribution is altered, and that microtubule patterning and stability are aberrant in etiolated xxt1 xxt2 hypocotyls. We also found that the expression levels of microtubule-associated genes, such as MAP70-5 and CLASP, and receptor genes, such as HERK1 and WAK1, were changed in xxt1 xxt2 plants and that cellulose synthase motility is reduced in xxt1 xxt2 cells, corresponding with a reduction in cellulose content. Our results indicate that loss of xyloglucan affects both the stability of the microtubule cytoskeleton and the production and patterning of cellulose in primary cell walls. These findings establish, to our knowledge, new links between wall integrity, cytoskeletal dynamics, and wall synthesis in the regulation of plant morphogenesis. PMID:26527657

  10. Structural Basis of Backwards Motion in Kinesin-1-Kinesin-14 Chimera: Implication for Kinesin-14 Motility.

    PubMed

    Yamagishi, Masahiko; Shigematsu, Hideki; Yokoyama, Takeshi; Kikkawa, Masahide; Sugawa, Mitsuhiro; Aoki, Mari; Shirouzu, Mikako; Yajima, Junichiro; Nitta, Ryo

    2016-08-02

    Kinesin-14 is a unique minus-end-directed microtubule-based motor. A swinging motion of a class-specific N-terminal neck helix has been proposed to produce minus-end directionality. However, it is unclear how swinging of the neck helix is driven by ATP hydrolysis utilizing the highly conserved catalytic core among all kinesins. Here, using a motility assay, we show that in addition to the neck helix, the conserved five residues at the C-terminal region in kinesin-14, namely the neck mimic, are necessary to give kinesin-1 an ability to reverse its directionality toward the minus end of microtubules. Our structural analyses further demonstrate that the C-terminal neck mimic, in cooperation with conformational changes in the catalytic core during ATP binding, forms a kinesin-14 bundle with the N-terminal neck helix to swing toward the minus end of microtubules. Thus, the neck mimic plays a crucial role in coupling the chemical ATPase reaction with the mechanical cycle to produce the minus-end-directed motility of kinesin-14. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Centrosome motility is essential for initial axon formation in the neocortex.

    PubMed

    de Anda, Froylan Calderon; Meletis, Konstantinos; Ge, Xuecai; Rei, Damien; Tsai, Li-Huei

    2010-08-04

    The mechanisms underlying the normal development of neuronal morphology remain a fundamental question in neurobiology. Studies in cultured neurons have suggested that the position of the centrosome and the Golgi may predict the site of axon outgrowth. During neuronal migration in the developing cortex, however, the centrosome and Golgi are oriented toward the cortical plate at a time when axons grow toward the ventricular zone. In the current work, we use in situ live imaging to demonstrate that the centrosome and the accompanying polarized cytoplasm exhibit apical translocation in newborn cortical neurons preceding initial axon outgrowth. Disruption of centrosomal activity or downregulation of the centriolar satellite protein PCM-1 affects axon formation. We further show that downregulation of the centrosomal protein Cep120 impairs microtubule organization, resulting in increased centrosome motility. Decreased centrosome motility resulting from microtubule stabilization causes an aberrant centrosomal localization, leading to misplaced axonal outgrowth. Our results reveal the dynamic nature of the centrosome in developing cortical neurons, and implicate centrosome translocation and microtubule organization during the multipolar stage as important determinants of axon formation.

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

    PubMed

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

    2013-12-06

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

  13. 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.

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

    PubMed Central

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

    2015-01-01

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

  15. Dynamic response of axonal microtubules under suddenly applied end forces.

    PubMed

    Manuchehrfar, Farid; Shamloo, Amir; Mehboudi, Nastaran

    2014-01-01

    Axon is a filament in neuronal system and axonal microtubules are bundles in axons. In axons, microtubules are coated with microtubule-associated protein tau, a natively unfolded profuse filamentous protein in the central nervous system. These proteins are responsible for the cross-linked structure of the axonal microtubule bundles. Through complimentary dimerization with other tau proteins, bridges are formed to nearby microtubules to create bundles. The transverse reinforcement of microtubules by cross-linking to the cytoskeleton has been shown to enhance their ability to bear compressive loads. Though microtubules are conventionally regarded as bearing compressive loads, in certain circumstances such as in traumatic stretch injury, they are placed in tension. We employ Standard Linear Solid, a viscoelastic model, to computationally simulate microtubules. This study investigates the dynamic response of two dimensional axonal microtubules under suddenly applied end forces. We obtain the results for steady state behavior of axonal microtubule for different forces.

  16. 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.

  17. 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

  18. Actin filaments connected with the microtubules of lipotubuloids, cytoplasmic domains rich in lipid bodies and microtubules.

    PubMed

    Kwiatkowska, M; Popłońska, K; Stepiński, D

    2005-12-01

    Lipotubuloids, i.e., cytoplasmic domains containing an agglomeration of lipid bodies surrounded by half-unit membrane, entwined and held together by a system of microtubules, have been found in the ovary epidermis of Ornithogalum umbellatum. Ultrastructural studies demonstrated thin filaments in lipotubuloids that are probably actin filaments arranged parallel to microtubules. It is suggested that interaction of actin filaments with the microtubules determines the driving force for the rotary motion characteristic of lipotubuloids, as this movement is sensitive to cytochalasin B.

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

    PubMed

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

    2012-01-01

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

  20. Molecular cloning of the microtubule-associated mechanochemical enzyme dynamin reveals homology with a new family of GTP-binding proteins.

    PubMed

    Obar, R A; Collins, C A; Hammarback, J A; Shpetner, H S; Vallee, R B

    1990-09-20

    A complementary DNA encoding the D100 polypeptide of rat brain dynamin--a force-producing, microtubule-activated nucleotide triphosphatase--has been cloned and sequenced. The predicted amino acid sequence includes a guanine nucleotide-binding domain that is homologous with those of a family of antiviral factors, inducible by interferon and known as Mx proteins, and with the product of the essential yeast vacuolar protein sorting gene VPS1. These relationships imply the existence of a new family of GTPases with physiological roles that may include microtubule-based motility and protein sorting.

  1. 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.

  2. Measuring kinetochore-microtubule interaction in vitro

    PubMed Central

    Driver, Jonathan W.; Powers, Andrew F.; Sarangapani, Krishna K.; Biggins, Sue; Asbury, Charles L.

    2014-01-01

    Many proteins and protein complexes perform sophisticated, regulated functions in vivo. Many of these functions can be recapitulated using in vitro reconstitution, which serves as a means to establish unambiguous cause-effect relationships, for example between a protein and its phosphorylating kinase. Here, we describe a protocol to purify kinetochores, the protein complexes that attach chromosomes to microtubules during mitosis, and quantitatively assay their microtubule binding characteristics. Our assays, based on DIC imaging and laser trapping microscopy, are used to measure the attachment of microtubules to kinetochores and the load-bearing capabilities of those attachments. These assays provide a platform for studying kinase disruption of kinetochore-microtubule attachments, which is believed to be critical for correcting erroneous kinetochore-spindle attachments and thereby avoiding chromosome mis-segregation. The principles of our approach should be extensible to studies of a wide range of force-bearing interactions in biology. PMID:24630115

  3. The microtubule as a breast cancer target.

    PubMed

    Higa, Gerald M

    2011-04-01

    Manifestations of non-equilibrium polarity, random transgressions, and catastrophes are not conditions usually associated with a sense of normalcy. Yet these disquieting features distinguish a utilitarian behavior known as dynamic instability, the signature characteristic of the microtubule. Long known to be a tumor target, disruption of this fragile attribute is associated with some of the most effective agents used to treat breast cancer today. Although the biology of the microtubule is under intense investigation much still remains unknown. As such, our understanding of regulatory molecules and resistance mechanisms are still rudimentary, further compromising our ability to develop novel therapeutic strategies to improve microtubule inhibitors. This review focuses on several classes of anti-microtubule agents and their effects on the functional dynamics of the targeted polymer. The primary objective is to critically examine the molecular mechanisms that contribute to tumor cell death, tumor-resistance, and incident neurotoxicity.

  4. Rigidity of microtubules is increased by stabilizing agents

    PubMed Central

    1995-01-01

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

  5. Reduction of organelle motility by removal of potassium and other solutes

    PubMed Central

    Yin, David; Wolkoff, Allan W.

    2017-01-01

    There are surprisingly few studies that describe how the composition of cell culture medium may affect the trafficking of organelles. Here we utilize time lapse multi-channel fluorescent imaging to show that short term exposure of Huh-7 cells to medium lacking potassium, sodium, or chloride strongly reduces but does not eliminate the characteristic back and forth and cell-traversing movement of fluorescent EGF (FL-EGF) containing organelles. We focused on potassium because of its relatively low abundance in media and serum and its energy requiring accumulation into cells. Upon exposure to potassium free medium, organelle motility declined steadily through 90 min and then persisted at a low level. Reduced motility was confirmed in 5 independent cell lines and for organelles of the endocytic pathway (FL-EGF and Lysotracker), autophagosomes (LC3-GFP), and mitochondria (TMRE). As has been previously established, potassium free medium also inhibited endocytosis. We expected that diminished cellular metabolism would precede loss of organelle motility. However, extracellular flux analysis showed near normal mitochondrial oxygen consumption and only a small decrease in extracellular acidification, the latter suggesting decreased glycolysis or proton efflux. Other energy dependent activities such as the accumulation of Lysotracker, TMRE, DiBAC4(3), and the exclusion of propidium iodide remained intact, as did the microtubule cytoskeleton. We took advantage of cell free in vitro motility assays and found that removal of potassium or sodium from the reconstituted cytosolic medium decreased the movement of endosomes on purified microtubules. The results indicate that although changes in proton homeostasis and cell energetics under solute depletion are not fully understood, potassium as well as sodium appear to be directly required by the motile machinery of organelles for optimal trafficking. PMID:28922372

  6. Sperm-Associated Antigen–17 Gene Is Essential for Motile Cilia Function and Neonatal Survival

    PubMed Central

    Teves, Maria Eugenia; Zhang, Zhibing; Costanzo, Richard M.; Henderson, Scott C.; Corwin, Frank D.; Zweit, Jamal; Sundaresan, Gobalakrishnan; Subler, Mark; Salloum, Fadi N.; Rubin, Bruce K.

    2013-01-01

    Primary ciliary dyskinesia (PCD), resulting from defects in cilia assembly or motility, is caused by mutations in a number of genes encoding axonemal proteins. PCD phenotypes are variable, and include recurrent respiratory tract infections, bronchiectasis, hydrocephaly, situs inversus, and male infertility. We generated knockout mice for the sperm-associated antigen–17 (Spag17) gene, which encodes a central pair (CP) protein present in the axonemes of cells with “9 + 2” motile cilia or flagella. The targeting of Spag17 resulted in a severe phenotype characterized by immotile nasal and tracheal cilia, reduced clearance of nasal mucus, profound respiratory distress associated with lung fluid accumulation and disruption of the alveolar epithelium, cerebral ventricular expansion consistent with emerging hydrocephalus, failure to suckle, and neonatal demise within 12 hours of birth. Ultrastructural analysis revealed the loss of one CP microtubule in approximately one quarter of tracheal cilia axonemes, an absence of a C1 microtubule projection, and other less frequent CP structural abnormalities. SPAG6 and SPAG16 (CP proteins that interact with SPAG17) were increased in tracheal tissue from SPAG17-deficient mice. We conclude that Spag17 plays a critical role in the function and structure of motile cilia, and that neonatal lethality is likely explained by impaired airway mucociliary clearance. PMID:23418344

  7. Microtubules, polarity and vertebrate neural tube morphogenesis

    PubMed Central

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

    2016-01-01

    Microtubules are key cellular components, long known to participate in morphogenetic events that shape the developing embryo. However, the links between the cellular functions of microtubules, their effects on cell shape and polarity and their role in large-scale morphogenesis remain poorly understood. Here, we examine these relationships with respect to two strategies for generating the vertebrate neural tube: bending and closure of the mammalian neural plate, and cavitation of the teleost neural rod. The latter process has been compared to ‘secondary’ neurulation that generates the caudal spinal cord in mammals. Microtubules align along the apico-basal axis of the mammalian neuroepithelium early in neural tube closure, participating functionally in interkinetic nuclear migration which indirectly impacts on cell shape. Whether microtubules play other functional roles in mammalian neurulation remains unclear. In the zebrafish, microtubules are important for defining the neural rod midline prior to its cavitation, both by localizing apical proteins at the tissue midline and by orienting cell division through a mirror-symmetric microtubule apparatus that helps to further define the medial localization of apical polarity proteins. Par proteins have been implicated in centrosome positioning in neuroepithelia as well as in the control of polarized morphogenetic movements in the neural rod. Understanding of microtubule functions during early nervous system development has so far been limited, partly by techniques that fail to distinguish ‘cause’ from ‘effect’. Future developments will likely rely on novel ways to selectively impair microtubule function in order to investigate the roles they play. PMID:27025884

  8. Actin-based motility drives baculovirus transit to the nucleus and cell surface

    PubMed Central

    Ohkawa, Taro; Volkman, Loy E.

    2010-01-01

    Most viruses move intracellularly to and from their sites of replication using microtubule-based mechanisms. In this study, we show that nucleocapsids of the baculovirus Autographa californica multiple nucleopolyhedrovirus undergo intracellular motility driven by actin polymerization. Motility requires the viral P78/83 capsid protein and the host Arp2/3 complex. Surprisingly, the virus directs two sequential and coordinated phases of actin-based motility. Immediately after cell entry, motility enables exploration of the cytoplasm and collision with the nuclear periphery, speeding nuclear entry and the initiation of viral gene expression. Nuclear entry itself requires transit through nuclear pore complexes. Later, after the onset of early gene expression, motility is required for accumulation of a subpopulation of nucleocapsids in the tips of actin-rich surface spikes. Temporal coordination of actin-based nuclear and surface translocation likely enables rapid transmission to neighboring cells during infection in insects and represents a distinctive evolutionary strategy for overcoming host defenses. PMID:20660627

  9. Automated detection of whole-cell mitochondrial motility and its dependence on cytoarchitectural integrity.

    PubMed

    Kandel, Judith; Chou, Philip; Eckmann, David M

    2015-07-01

    Current methodologies used for mitochondrial motility analysis tend to either overlook individual mitochondrial tracks or analyze only peripheral mitochondria instead of mitochondria in all regions of the cell. Furthermore, motility analysis of an individual mitochondrion is usually quantified by establishing an arbitrary threshold for "directed" motion. In this work, we created a custom, publicly available computational algorithm based on a previously published approach (Giedt et al., 2012. Ann Biomed Eng 40:1903-1916) in order to characterize the distribution of mitochondrial movements at the whole-cell level, while still preserving information about single mitochondria. Our technique is easy to use, robust, and computationally inexpensive. Images are first pre-processed for increased resolution, and then individual mitochondria are tracked based on object connectivity in space and time. When our method is applied to microscopy fields encompassing entire cells, we reveal that the mitochondrial net distances in fibroblasts follow a lognormal distribution within a given cell or group of cells. The ability to model whole-cell mitochondrial motility as a lognormal distribution provides a new quantitative paradigm for comparing mitochondrial motility in naïve and treated cells. We further demonstrate that microtubule and microfilament depolymerization shift the lognormal distribution in directions which indicate decreased and increased mitochondrial movement, respectively. These findings advance earlier work on neuronal axons (Morris and Hollenbeck, 1993. J Cell Sci 104:917-927) by relating them to a different cell type, applying them on a global scale, and automating measurement of mitochondrial motility in general.

  10. Kinetochore-microtubule interactions during cell division.

    PubMed

    Maiato, Helder; Sunkel, Claudio E

    2004-01-01

    Proper segregation of chromosomes during cell division is essential for the maintenance of genetic stability. During this process chromosomes must establish stable functional interactions with microtubules through the kinetochore, a specialized protein structure located on the surface of the centromeric heterochromatin. Stable attachment of kinetochores to a number of microtubules results in the formation of a kinetochore fibre that mediates chromosome movement. How the kinetochore fibre is formed and how chromosome motion is produced and regulated remain major questions in cell biology. Here we look at some of the history of research devoted to the study of kinetochore-microtubule interaction and attempt to identify significant advances in the knowledge of the basic processes. Ultrastructural work has provided substantial insights into the structure of the kinetochore and associated microtubules during different stages of mitosis. Also, recent in-vivo studies have probed deep into the dynamics of kinetochore-attached microtubules suggesting possible models for the way in which kinetochores harness the capacity of microtubules to do work and turn it into chromosome motion. Much of the research in recent years suggests that indeed multiple mechanisms are involved in both formation of the k-fibre and chromosome motion. Thus, rather than moving to a unified theory, it has become apparent that most cell types have the capacity to build the spindle using multiple and probably redundant mechanisms.

  11. Harnessing microtubule dynamic instability for nanostructure assembly

    NASA Astrophysics Data System (ADS)

    Bouchard, Ann M.; Warrender, Christina E.; Osbourn, Gordon C.

    2006-10-01

    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.

  12. 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.

  13. Mobility of Taxol in Microtubule Bundles

    NASA Astrophysics Data System (ADS)

    Ross, J.

    2003-06-01

    Mobility of taxol inside microtubules was investigated using fluorescence recovery after photobleaching (FRAP) on flow-aligned bundles. Bundles were made of microtubules with either GMPCPP or GTP at the exchangeable site on the tubulin dimer. Recovery times were sensitive to bundle thickness and packing, indicating that taxol molecules are able to move laterally through the bundle. The density of open binding sites along a microtubule was varied by controlling the concentration of taxol in solution for GMPCPP samples. With > 63% sites occupied, recovery times were independent of taxol concentration and, therefore, inversely proportional to the microscopic dissociation rate, k_{off}. It was found that 10*k_{off} (GMPCPP) ~ k_{off} (GTP), consistent with, but not fully accounting for, the difference in equilibrium constants for taxol on GMPCPP and GTP microtubules. With < 63% sites occupied, recovery times decreased as ~ [Tax]^{-1/5} for both types of microtubules. We conclude that the diffusion of taxol along the microtubule interior is hindered by rebinding events when open sites are within ~7 nm of each other.

  14. Mitosis. Microtubule detyrosination guides chromosomes during mitosis.

    PubMed

    Barisic, Marin; Silva e Sousa, Ricardo; Tripathy, Suvranta K; Magiera, Maria M; Zaytsev, Anatoly V; Pereira, Ana L; Janke, Carsten; Grishchuk, Ekaterina L; Maiato, Helder

    2015-05-15

    Before chromosomes segregate into daughter cells, they align at the mitotic spindle equator, a process known as chromosome congression. Centromere-associated protein E (CENP-E)/Kinesin-7 is a microtubule plus-end-directed kinetochore motor required for congression of pole-proximal chromosomes. Because the plus-ends of many astral microtubules in the spindle point to the cell cortex, it remains unknown how CENP-E guides pole-proximal chromosomes specifically toward the equator. We found that congression of pole-proximal chromosomes depended on specific posttranslational detyrosination of spindle microtubules that point to the equator. In vitro reconstitution experiments demonstrated that CENP-E-dependent transport was strongly enhanced on detyrosinated microtubules. Blocking tubulin tyrosination in cells caused ubiquitous detyrosination of spindle microtubules, and CENP-E transported chromosomes away from spindle poles in random directions. Thus, CENP-E-driven chromosome congression is guided by microtubule detyrosination. Copyright © 2015, American Association for the Advancement of Science.

  15. Esophageal motility disorders: medical therapy.

    PubMed

    Lacy, Brian E; Weiser, Kirsten

    2008-01-01

    Symptoms of chest pain and dysphagia are common in the adult population. Most patients initially undergo an evaluation to exclude anatomic causes (ie, esophagitis, stricture) and cardiovascular disease as the etiology of these symptoms. Patients with persistent symptoms may then be referred for specialized testing of the esophagus, including esophageal manometry. Disorders of esophageal motility, which include achalasia, diffuse esophageal spasm, nutcracker esophagus, hypertensive lower esophageal sphincter, and ineffective motility are often identified in these patients. Unfortunately, the etiology of these disorders has not been well characterized and the treatment has not been standardized. This review will briefly discuss the impact, etiology, and diagnosis of esophageal motility disorders, and then focus on the medical management of these disorders using evidence from well-designed, prospective studies, where available.

  16. Dynein Light Chain 1 (LC8) Association Enhances Microtubule Stability and Promotes Microtubule Bundling*

    PubMed Central

    Asthana, Jayant; Kuchibhatla, Anuradha; Jana, Swadhin Chandra; Ray, Krishanu; Panda, Dulal

    2012-01-01

    Dynein light chain 1 (LC8), a highly conserved protein, is known to bind to a variety of different polypeptides. It functions as a dimer, which is inactivated through phosphorylation at the Ser-88 residue. A loss of LC8 function causes apoptosis in Drosophila embryos, and its overexpression induces malignant transformation of breast cancer cells. Here we show that LC8 binds to tubulin, promotes microtubule assembly, and induces the bundling of reconstituted microtubules in vitro. Furthermore, LC8 decorates microtubules both in Drosophila embryos and in HeLa cells, increases the microtubule stability, and promotes microtubule bundling in these cells. Microtubule stability influences a number of different cellular functions including mitosis and cell differentiation. The LC8 overexpression reduces the susceptibility of microtubules to cold and nocodazole-induced depolymerization in tissue-cultured cells and increases microtubule acetylation, suggesting that LC8 stabilizes microtubules. We also show that LC8 knockdown or transfection with inhibitory peptides destabilizes microtubules and inhibits bipolar spindle assembly in HeLa cells. In addition, LC8 knockdown leads to the mitotic block in HeLa cells. Furthermore, molecular docking analysis using the crystal structures of tubulin and LC8 dimer indicated that the latter may bind at α-β tubulin junction in a protofilament at sites distinct from the kinesin and dynein binding sites. Together, we provide the first evidence of a novel microtubule-associated protein-like function of LC8 that could explain its reported roles in cellular metastasis and differentiation. PMID:23038268

  17. Structural basis for chirality and directional motility of Plasmodium sporozoites

    PubMed Central

    Kudryashev, Mikhail; Münter, Sylvia; Lemgruber, Leandro; Montagna, Georgina; Stahlberg, Henning; Matuschewski, Kai; Meissner, Markus; Cyrklaff, Marek; Frischknecht, Friedrich

    2014-01-01

    Summary Plasmodium sporozoites can move at high speed for several tens of minutes, which is essential for the initial stage of a malaria infection. The crescent-shaped sporozoites move on 2D substrates preferably in the same direction on circular paths giving raise to helical paths in 3D matrices. Here we determined the structural basis that underlies this type of movement. Immature, non-motile sporozoites were found to lack the subpellicular network required for obtaining the crescent parasite shape. In vitro, parasites moving in the favoured direction move faster and more persistent than the few parasites that move in the opposite direction. Photobleaching experiments showed that sporozoites flip their ventral side up when switching the direction of migration. Cryo-electron tomography revealed a polarized arrangement of microtubules and polar rings towards the substrate in Plasmodium sporozoites, but not in the related parasite Toxoplasma gondii. As a consequence, secretory vesicles, which release proteins involved in adhesion, migration and invasion at the front end of the parasite, are delivered towards the substrate. The resulting chiral structure of the parasite appears to determine the unique directionality of movement and could explain how the sporozoite achieves rapid and sustained directional motility in the absence of external stimuli. PMID:22776715

  18. Glycogen synthase kinase 3 phosphorylates kinesin light chains and negatively regulates kinesin-based motility

    NASA Technical Reports Server (NTRS)

    Morfini, Gerardo; Szebenyi, Gyorgyi; Elluru, Ravindhra; Ratner, Nancy; Brady, Scott T.

    2002-01-01

    Membrane-bounded organelles (MBOs) are delivered to different domains in neurons by fast axonal transport. The importance of kinesin for fast antero grade transport is well established, but mechanisms for regulating kinesin-based motility are largely unknown. In this report, we provide biochemical and in vivo evidence that kinesin light chains (KLCs) interact with and are in vivo substrates for glycogen synthase kinase 3 (GSK3). Active GSK3 inhibited anterograde, but not retrograde, transport in squid axoplasm and reduced the amount of kinesin bound to MBOs. Kinesin microtubule binding and microtubule-stimulated ATPase activities were unaffected by GSK3 phosphorylation of KLCs. Active GSK3 was also localized preferentially to regions known to be sites of membrane delivery. These data suggest that GSK3 can regulate fast anterograde axonal transport and targeting of cargos to specific subcellular domains in neurons.

  19. Glycogen synthase kinase 3 phosphorylates kinesin light chains and negatively regulates kinesin-based motility

    NASA Technical Reports Server (NTRS)

    Morfini, Gerardo; Szebenyi, Gyorgyi; Elluru, Ravindhra; Ratner, Nancy; Brady, Scott T.

    2002-01-01

    Membrane-bounded organelles (MBOs) are delivered to different domains in neurons by fast axonal transport. The importance of kinesin for fast antero grade transport is well established, but mechanisms for regulating kinesin-based motility are largely unknown. In this report, we provide biochemical and in vivo evidence that kinesin light chains (KLCs) interact with and are in vivo substrates for glycogen synthase kinase 3 (GSK3). Active GSK3 inhibited anterograde, but not retrograde, transport in squid axoplasm and reduced the amount of kinesin bound to MBOs. Kinesin microtubule binding and microtubule-stimulated ATPase activities were unaffected by GSK3 phosphorylation of KLCs. Active GSK3 was also localized preferentially to regions known to be sites of membrane delivery. These data suggest that GSK3 can regulate fast anterograde axonal transport and targeting of cargos to specific subcellular domains in neurons.

  20. Single file diffusion in microtubules

    NASA Astrophysics Data System (ADS)

    Rutenberg, Andrew; Farrell, Spencer; Brown, Aidan

    2015-03-01

    We investigate the single file diffusion (SFD) of large particles entering a confined tubular geometry, such as luminal diffusion of proteins inside microtubules or flagella. While single-file effects have no effect on particle density, we report significant single-file effects for individually-tracked tracer particle motion. Both exact and approximate ordering statistics of particles entering semi-infinite tubes agree well with our stochastic simulations. Considering initially empty semi-infinite tubes, with particles entering at one end starting from an initial time t = 0 , tracked particles display super-diffusive effective exponents just after they enter the system and trends towards diffusive exponents at later times. Equivalently, if diffusive exponents are assumed the effective diffusivity is reduced at early times and enhanced at later times through a logarithmic factor logN , where N is the number of particles in the tube. When we number each particle from the first (n = 1) to the most recent (n = N), we find good scaling collapse of the effective diffusivity for all n. Techniques that track individual particles, or local groups of particles, such as photo-activation or photobleaching, will exhibit single-file effects.

  1. Microtubule organization is determined by the shape of epithelial cells

    PubMed Central

    Gomez, Juan Manuel; Chumakova, Lyubov; Bulgakova, Natalia A.; Brown, Nicholas H.

    2016-01-01

    Interphase microtubule organization is critical for cell function and tissue architecture. In general, physical mechanisms are sufficient to drive microtubule organization in single cells, whereas cells within tissues are thought to utilize signalling mechanisms. By improving the imaging and quantitation of microtubule alignment within developing Drosophila embryos, here we demonstrate that microtubule alignment underneath the apical surface of epithelial cells follows cell shape. During development, epidermal cell elongation and microtubule alignment occur simultaneously, but by perturbing cell shape, we discover that microtubule organization responds to cell shape, rather than the converse. A simple set of microtubule behaviour rules is sufficient for a computer model to mimic the observed responses to changes in cell surface geometry. Moreover, we show that microtubules colliding with cell boundaries zip-up or depolymerize in an angle-dependent manner, as predicted by the model. Finally, we show microtubule alignment responds to cell shape in diverse epithelia. PMID:27779189

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

    PubMed

    Vorob'ev, V S; Portuganov, V V

    1980-10-01

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

  3. Organization of neuronal microtubules in the nematode Caenorhabditis elegans

    PubMed Central

    1979-01-01

    We have studied the organization of microtubules in neurons of the nematode Caenorhabditis elegans. Six neurons, which we call the microtubule cells, contain bundles of darkly staining microtubules which can be followed easily in serial-section electron micrographs. Reconstruction of individual microtubules in these cells indicate that most, if not all, microtubules are short compared with the length of the cell process. Average microtubule length varies characteristically with cell type. The arrangement of microtubules gives an overall polarity to each bundle: the distal ends of the microtubles are on the outside of the bundle, whereas the proximal ends are preferentially inside. The distal and proximal ends each have a characteristic appearance indicating that these microtubules may have a polarity of their own. Short microtubules in processes of other neurons in C. elegans have also been observed. PMID:479300

  4. 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.

  5. 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

  6. 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.

  7. History-dependent catastrophes regulate axonal microtubule behavior.

    PubMed

    Stepanova, Tatiana; Smal, Ihor; van Haren, Jeffrey; Akinci, Umut; Liu, Zhe; Miedema, Marja; Limpens, Ronald; van Ham, Marco; van der Reijden, Michael; Poot, Raymond; Grosveld, Frank; Mommaas, Mieke; Meijering, Erik; Galjart, Niels

    2010-06-08

    In Chinese hamster ovary cells, microtubules originate at the microtubule organizing center (MTOC) and grow persistently toward the cell edge, where they undergo catastrophe. In axons, microtubule dynamics must be regulated differently because microtubules grow parallel to the plasma membrane and there is no MTOC. GFP-tagged microtubule plus end tracking proteins (+TIPs) mark the ends of growing neuronal microtubules. Their fluorescent "comet-like" pattern reflects turnover of +TIP binding sites. Using GFP-tagged +TIPs and fluorescence-based segmentation and tracking tools, we show that axonal microtubules grow with a constant average velocity and that they undergo catastrophes at random positions, yet in a programmed fashion. Using protein depletion approaches, we find that the +TIPs CLIP-115 and CLIP-170 affect average microtubule growth rate and growth distance in neurons but not the duration of a microtubule growth event. In N1E-115 neuroblastoma cells, we find that EB1, the core +TIP, regulates microtubule growth rate, growth distance, and duration, consistent with in vitro data. Combined, our data suggest that CLIPs influence the axonal microtubule/tubulin ratio, whereas EB1 stimulates microtubule growth and structural transitions at microtubule ends, thereby regulating microtubule catastrophes and the turnover of +TIP binding sites.

  8. Regulation of microtubule motors by tubulin isotypes and post-translational modifications.

    PubMed

    Sirajuddin, Minhajuddin; Rice, Luke M; Vale, Ronald D

    2014-04-01

    The 'tubulin-code' hypothesis proposes that different tubulin genes or post-translational modifications (PTMs), which mainly confer variation in the carboxy-terminal tail (CTT), result in unique interactions with microtubule-associated proteins for specific cellular functions. However, the inability to isolate distinct and homogeneous tubulin species has hindered biochemical testing of this hypothesis. Here, we have engineered 25 α/β-tubulin heterodimers with distinct CTTs and PTMs and tested their interactions with four different molecular motors using single-molecule assays. Our results show that tubulin isotypes and PTMs can govern motor velocity, processivity and microtubule depolymerization rates, with substantial changes conferred by even single amino acid variation. Revealing the importance and specificity of PTMs, we show that kinesin-1 motility on neuronal β-tubulin (TUBB3) is increased by polyglutamylation and that robust kinesin-2 motility requires detyrosination of α-tubulin. Our results also show that different molecular motors recognize distinctive tubulin 'signatures', which supports the premise of the tubulin-code hypothesis.

  9. A Fungal Kinesin Required for Organelle Motility, Hyphal Growth, and Morphogenesis

    PubMed Central

    Wu, Qindong; Sandrock, Tanya M.; Turgeon, B. Gillian; Yoder, Olen C.; Wirsel, Stefan G.; Aist, James R.

    1998-01-01

    A gene (NhKIN1) encoding a kinesin was cloned from Nectria haematococca genomic DNA by polymerase chain reaction amplification, using primers corresponding to conserved regions of known kinesin-encoding genes. Sequence analysis showed that NhKIN1 belongs to the subfamily of conventional kinesins and is distinct from any of the currently designated kinesin-related protein subfamilies. Deletion of NhKIN1 by transformation-mediated homologous recombination caused several dramatic phenotypes: a 50% reduction in colony growth rate, helical or wavy hyphae with reduced diameter, and subcellular abnormalities including withdrawal of mitochondria from the growing hyphal apex and reduction in the size of the Spitzenkörper, an apical aggregate of secretory vesicles. The effects on mitochondria and Spitzenkörper were not due to altered microtubule distribution, as microtubules were abundant throughout the length of hyphal tip cells of the mutant. The rate of spindle elongation during anaphase B of mitosis was reduced 11%, but the rate was not significantly different from that of wild type. This lack of a substantial mitotic phenotype is consistent with the primary role of the conventional kinesins in organelle motility rather than mitosis. Our results provide further evidence that the microtubule-based motility mechanism has a direct role in apical transport of secretory vesicles and the first evidence for its role in apical transport of mitochondria in a filamentous fungus. They also include a unique demonstration that a microtubule-based motor protein is essential for normal positioning of the Spitzenkörper, thus providing a new insight into the cellular basis for the aberrant hyphal morphology. PMID:9436993

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

    PubMed

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

    2016-01-15

    Spatiotemporal cytoskeleton remodeling is pivotal for cell adhesion and migration. Here we investigated the function of Gas2-related protein on chromosome 22 (GAR22β), a poorly characterized protein that interacts with actin and microtubules. Primary and immortalized GAR22β(-/-) Sertoli cells moved faster than wild-type cells. In addition, GAR22β(-/-) cells showed a more prominent focal adhesion turnover. GAR22β overexpression or its reexpression in GAR22β(-/-) cells reduced cell motility and focal adhesion turnover. GAR22β-actin interaction was stronger than GAR22β-microtubule interaction, resulting in GAR22β localization and dynamics that mirrored those of the actin cytoskeleton. Mechanistically, GAR22β interacted with the regulator of microtubule dynamics end-binding protein 1 (EB1) via a novel noncanonical amino acid sequence, and this GAR22β-EB1 interaction was required for the ability of GAR22β to modulate cell motility. We found that GAR22β is highly expressed in mouse testes, and its absence resulted in reduced spermatozoa generation, lower actin levels in testes, and impaired motility and ultrastructural disorganization of spermatozoa. Collectively our findings identify GAR22β as a novel regulator of cell adhesion and migration and provide a foundation for understanding the molecular basis of diverse cytoskeleton-dependent processes. © 2016 Gamper et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  11. TIPsy tour guides: how microtubule plus-end tracking proteins (+TIPs) facilitate axon guidance

    PubMed Central

    Bearce, Elizabeth A.; Erdogan, Burcu; Lowery, Laura Anne

    2015-01-01

    The growth cone is a dynamic cytoskeletal vehicle, which drives the end of a developing axon. It serves to interpret and navigate through the complex landscape and guidance cues of the early nervous system. The growth cone’s distinctive cytoskeletal organization offers a fascinating platform to study how extracellular cues can be translated into mechanical outgrowth and turning behaviors. While many studies of cell motility highlight the importance of actin networks in signaling, adhesion, and propulsion, both seminal and emerging works in the field have highlighted a unique and necessary role for microtubules (MTs) in growth cone navigation. Here, we focus on the role of singular pioneer MTs, which extend into the growth cone periphery and are regulated by a diverse family of microtubule plus-end tracking proteins (+TIPs). These +TIPs accumulate at the dynamic ends of MTs, where they are well-positioned to encounter and respond to key signaling events downstream of guidance receptors, catalyzing immediate changes in microtubule stability and actin cross-talk, that facilitate both axonal outgrowth and turning events. PMID:26175669

  12. Contact formation during fibroblast locomotion: involvement of membrane ruffles and microtubules

    PubMed Central

    1988-01-01

    We have correlated the motility of the leading edge of fibroblasts, monitored by phase-contrast cinematography, with the relative distributions of several cytoskeletal elements (vinculin, tubulin, and actin) as well as with the contact patterns determined by interference reflection microscopy. This analysis has revealed the involvement of both ruffles and microspikes, as well as microtubules in the initiation of focal contact formation. Nascent vinculin sites within the leading edge or at its base, taken as primordial cell-substrate contacts, were invariably colocalized with sites that showed a history of transient, prolonged, or cyclic ruffling activity. Extended microspike structures, often preceded the formation of ruffles. Immunofluorescent labeling indicated that some of these primordial contacts were in close apposition to the ends of microtubules that penetrated into the leading edge. By fluorescence and electron microscopy short bundles of actin filaments found at the base of the leading edge were identified as presumptive, primordial contacts. It is concluded that ruffles and microspikes, either independently or in combination, initiate and mark the sites for future contact. Plaque proteins then accumulate (within 10-30 s) at the contract site and, beneath ruffles, induce localized bundling of actin filaments. We propose that all primordial contacts support traction for leading edge protrusion but that only some persist long enough to nucleate stress fiber assembly. Microtubules are postulated as the elements that select, stabilize, and potentiate the formation of these latter, long-lived contacts. PMID:3126193

  13. Reconstitution of dynein transport to the microtubule plus end by kinesin

    PubMed Central

    Roberts, Anthony J; Goodman, Brian S; Reck-Peterson, Samara L

    2014-01-01

    Cytoplasmic dynein powers intracellular movement of cargo toward the microtubule minus end. The first step in a variety of dynein transport events is the targeting of dynein to the dynamic microtubule plus end, but the molecular mechanism underlying this spatial regulation is not understood. Here, we reconstitute dynein plus-end transport using purified proteins from S. cerevisiae and dissect the mechanism using single-molecule microscopy. We find that two proteins–homologs of Lis1 and Clip170–are sufficient to couple dynein to Kip2, a plus-end-directed kinesin. Dynein is transported to the plus end by Kip2, but is not a passive passenger, resisting its own plus-end-directed motion. Two microtubule-associated proteins, homologs of Clip170 and EB1, act as processivity factors for Kip2, helping it overcome dynein's intrinsic minus-end-directed motility. This reveals how a minimal system of proteins transports a molecular motor to the start of its track. DOI: http://dx.doi.org/10.7554/eLife.02641.001 PMID:24916158

  14. Intracellular proadrenomedullin-derived peptides decorate the microtubules and contribute to cytoskeleton function.

    PubMed

    Sackett, Dan L; Ozbun, Laurent; Zudaire, Enrique; Wessner, Lisa; Chirgwin, John M; Cuttitta, Frank; Martínez, Alfredo

    2008-06-01

    Adrenomedullin (AM) and proadrenomedullin N-terminal 20 peptide (PAMP) are secretory hormones, but it is not unusual to find them in intracellular compartments. Using yeast-2 hybrid technology, we found interactions between AM and several microtubule-associated proteins (MAPs), and between PAMP and tubulin. Expression of fluorescent-tagged AM and PAMP as well as immunofluorescence for the native peptides showed a complete decoration of the microtubules and colocalization with other MAPs. PAMP, but not AM, bound to tubulin in vitro and destabilized tubulin polymerization. Down-regulation of the gene coding for both AM and PAMP through small interfering RNA technology resulted in morphological changes, microtubule stabilization, increase in posttranslational modifications of tubulin such as acetylation and detyrosination, reduction in cell motility, and partial arrest at the G2 phase of the cell cycle, when compared with cells transfected with the same vector carrying a scrambled sequence. These results show that PAMP is a novel MAP, whereas AM may be exerting more subtle effects in regulating cytoskeleton function.

  15. Microtubule-dependent movement of late endocytic vesicles in vitro: requirements for Dynein and Kinesin.

    PubMed

    Bananis, Eustratios; Nath, Sangeeta; Gordon, Kristie; Satir, Peter; Stockert, Richard J; Murray, John W; Wolkoff, Allan W

    2004-08-01

    Our previous studies demonstrated that fluorescent early endocytic vesicles prepared from rat liver after injection of Texas red asialoorosomucoid contain asialoglycoprotein and its receptor and move and undergo fission along microtubules using kinesin I and KIFC2, with Rab4 regulating KIFC2 activity (J. Cell Sci. 116, 2749, 2003). In the current study, procedures to prepare fluorescent late endocytic vesicles were devised. In addition, flow cytometry was utilized to prepare highly purified fluorescent endocytic vesicles, permitting validation of microscopy-based experiments as well as direct biochemical analysis. These studies revealed that late vesicles bound to and moved along microtubules, but in contrast to early vesicles, did not undergo fission. As compared with early vesicles, late vesicles had reduced association with receptor, Rab4, and kinesin I but were highly associated with dynein, Rab7, dynactin, and KIF3A. Dynein and KIF3A antibodies inhibited late vesicle motility, whereas kinesin I and KIFC2 antibodies had no effect. Dynamitin antibodies prevented the association of late vesicles with microtubules. These results indicate that acquisition and exchange of specific motor and regulatory proteins characterizes and may regulate the transition of early to late endocytic vesicles. Flow cytometric purification should ultimately facilitate detailed proteomic analysis and mapping of endocytic vesicle-associated proteins.

  16. 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

  17. Motility of Electric Cable Bacteria.

    PubMed

    Bjerg, Jesper Tataru; Damgaard, Lars Riis; Holm, Simon Agner; Schramm, Andreas; Nielsen, Lars Peter

    2016-07-01

    Cable bacteria are filamentous bacteria that electrically couple sulfide oxidation and oxygen reduction at centimeter distances, and observations in sediment environments have suggested that they are motile. By time-lapse microscopy, we found that cable bacteria used gliding motility on surfaces with a highly variable speed of 0.5 ± 0.3 μm s(-1) (mean ± standard deviation) and time between reversals of 155 ± 108 s. They frequently moved forward in loops, and formation of twisted loops revealed helical rotation of the filaments. Cable bacteria responded to chemical gradients in their environment, and around the oxic-anoxic interface, they curled and piled up, with straight parts connecting back to the source of sulfide. Thus, it appears that motility serves the cable bacteria in establishing and keeping optimal connections between their distant electron donor and acceptors in a dynamic sediment environment. This study reports on the motility of cable bacteria, capable of transmitting electrons over centimeter distances. It gives us a new insight into their behavior in sediments and explains previously puzzling findings. Cable bacteria greatly influence their environment, and this article adds significantly to the body of knowledge about this organism. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  18. Brow motility in mitochondrial myopathy.

    PubMed

    de Castro, Flávia Augusta Attié; Cruz, Antonio Augusto V; Sobreira, Cláudia Ferreira da Rosa

    2010-01-01

    To quantify the range of brow excursion in patients with mitochondrial myopathy and chronic progressive external ophthalmoplegia (CPEO). Comparative case series. Digital image processing techniques were used to quantify the upper eyelid resting position, brow excursion, and monocular eye movements (ductions) in 19 patients with mitochondrial myopathy and CPEO and in 27 healthy control subjects. All patients with CPEO had ptosis ranging from 0.6 to 8 mm. For most patients, eye motility limitation was symmetrical. Elevation was the most affected eye movement. Patient's brow motility was on average 56.7% of the motility seen in the control group, and did not correlate with age or eye motility in any direction. Seventy-six percent of the brows displayed more than 2 mm of excursion. In patients with CPEO, the occipitofrontalis muscle is less affected than the extraocular muscles. Most patients display a useful degree of brow excursion that theoretically can be used to clear the visual axis after a conservative brow suspension.

  19. Motility of Electric Cable Bacteria

    PubMed Central

    Damgaard, Lars Riis; Holm, Simon Agner; Schramm, Andreas; Nielsen, Lars Peter

    2016-01-01

    ABSTRACT Cable bacteria are filamentous bacteria that electrically couple sulfide oxidation and oxygen reduction at centimeter distances, and observations in sediment environments have suggested that they are motile. By time-lapse microscopy, we found that cable bacteria used gliding motility on surfaces with a highly variable speed of 0.5 ± 0.3 μm s−1 (mean ± standard deviation) and time between reversals of 155 ± 108 s. They frequently moved forward in loops, and formation of twisted loops revealed helical rotation of the filaments. Cable bacteria responded to chemical gradients in their environment, and around the oxic-anoxic interface, they curled and piled up, with straight parts connecting back to the source of sulfide. Thus, it appears that motility serves the cable bacteria in establishing and keeping optimal connections between their distant electron donor and acceptors in a dynamic sediment environment. IMPORTANCE This study reports on the motility of cable bacteria, capable of transmitting electrons over centimeter distances. It gives us a new insight into their behavior in sediments and explains previously puzzling findings. Cable bacteria greatly influence their environment, and this article adds significantly to the body of knowledge about this organism. PMID:27084019

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

    PubMed

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

    2013-03-01

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

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

    PubMed

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

    1990-12-01

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

  2. Non-centrosomal nucleation mediated by augmin organizes microtubules in post-mitotic neurons and controls axonal microtubule polarity

    PubMed Central

    Sánchez-Huertas, Carlos; Freixo, Francisco; Viais, Ricardo; Lacasa, Cristina; Soriano, Eduardo; Lüders, Jens

    2016-01-01

    Neurons display a highly polarized microtubule network that mediates trafficking throughout the extensive cytoplasm and is crucial for neuronal differentiation and function. In newborn migrating neurons, the microtubule network is organized by the centrosome. During neuron maturation, however, the centrosome gradually loses this activity, and how microtubules are organized in more mature neurons remains poorly understood. Here, we demonstrate that microtubule organization in post-mitotic neurons strongly depends on non-centrosomal nucleation mediated by augmin and by the nucleator γTuRC. Disruption of either complex not only reduces microtubule density but also microtubule bundling. These microtubule defects impair neurite formation, interfere with axon specification and growth, and disrupt axonal trafficking. In axons augmin does not merely mediate nucleation of microtubules but ensures their uniform plus end-out orientation. Thus, the augmin-γTuRC module, initially identified in mitotic cells, may be commonly used to generate and maintain microtubule configurations with specific polarity. PMID:27405868

  3. Identification of novel microtubule-binding proteins by taxol-mediated microtubule stabilization and mass spectrometry analysis

    PubMed Central

    He, Xianfei; Liu, Zhu; He, Qianqian; Qin, Juan; Liu, Ningning; Zhang, Linlin; Li, Dengwen; Zhou, Jun; Shui, Wenqing; Liu, Min

    2015-01-01

    Microtubule-binding proteins (MBPs) are structurally and functionally diverse regulators of microtubule-mediated cellular processes. Alteration of MBPs has been implicated in the pathogenesis of human diseases, including cancer. MBPs can stabilize or destabilize microtubules or move along microtubules to transport various cargoes. In addition, MBPs can control microtubule dynamics through direct interaction with microtubules or coordination with other proteins. To better understand microtubule structure and function, it is necessary to identify additional MBPs. In this study, we isolated microtubules and MBPs from mammalian cells by a taxol-based method and then profiled a panel of MBPs by mass spectrometry. We discovered a number of previously uncharacterized MBPs, including several membrane-associated proteins and proteins involved in post-translational modifications, in addition to several structural components. These results support the notion that microtubules have a wide range of functions and may undergo more exquisite regulation than previously recognized. PMID:26445615

  4. Esophageal motility in eosinophilic esophagitis.

    PubMed

    Weiss, A H; Iorio, N; Schey, R

    2015-01-01

    Eosinophilic esophagitis (EoE) is characterized by eosinophilic infiltration of the esophagus and is a potential cause of dysphagia and food impaction, most commonly affecting young men. Esophageal manometry findings vary from normal motility to aperistalsis, simultaneous contractions, diffuse esophageal spasm, nutcracker esophagus or hypotonic lower esophageal sphincter (LES). It remains unclear whether esophageal dysmotility plays a significant role in the clinical symptoms of EoE. Our aim is to review the pathogenesis, diagnosis, and effect of treatment on esophageal dysmotility in EoE. A literature search utilizing the PubMed database was performed using keywords: eosinophilic esophagitis, esophageal dysmotility, motility, manometry, impedance planimetry, barium esophagogram, endoscopic ultrasound, and dysphagia. Fifteen studies, totaling 387 patients with eosinophilic esophagitis were identified as keeping in accordance with the aim of this study and included in this review. The occurrence of abnormal esophageal manometry was reported to be between 4 and 87% among patients with EoE. Esophageal motility studies have shown reduced distensibility, abnormal peristalsis, and hypotonicity of the LES in patients with EoE, which may also mimic other esophageal motility disorders such as achalasia or nutcracker esophagus. Studies have shown conflicting results regarding the presence of esophageal dysmotility and symptoms with some reports suggesting a higher rate of food impaction, while others report no correlation between motor function and dysphagia. Motility dysfunction of the esophagus in EoE has not been well reported in the literature and studies have reported conflicting evidence regarding the clinical significance of dysmotility seen in EoE. The correlation between esophageal dysmotility and symptoms of EoE remains unclear. Larger studies are needed to investigate the incidence of esophageal dysmotility, clinical implications, and effect of treatment on

  5. A Hopping Mechanism for Cargo Transport by Molecular Motors on Crowded Microtubules

    NASA Astrophysics Data System (ADS)

    Goldman, Carla

    2010-05-01

    Most models designed to study the bidirectional movement of cargos as they are driven by molecular motors rely on the idea that motors of different polarities can be coordinated by external agents if arranged into a motor-cargo complex to perform the necessary work Gross, Hither and yon: a review of bidirectional microtubule-based transport (Gross in Phys. Biol. 1:R1-R11, 2004). Although these models have provided us with important insights into these phenomena, there are still many unanswered questions regarding the mechanisms through which the movement of the complex takes place on crowded microtubules. For example (i) how does cargo-binding affect motor motility? and in connection with that - (ii) how does the presence of other motors (and also other cargos) on the microtubule affect the motility of the motor-cargo complex? We discuss these questions from a different perspective. The movement of a cargo is conceived here as a hopping process resulting from the transference of cargo between neighboring motors. In the light of this, we examine the conditions under which cargo might display bidirectional movement even if directed by motors of a single polarity. The global properties of the model in the long-time regime are obtained by mapping the dynamics of the collection of interacting motors and cargos into an asymmetric simple exclusion process (ASEP) which can be resolved using the matrix ansatz introduced by Derrida (Derrida and Evans in Nonequilibrium Statistical Mechanics in One Dimension, pp. 277-304, 1997; Derrida et al. in J. Phys. A 26:1493-1517, 1993).

  6. Identification of a MAP 2-like ATP-binding protein associated with axoplasmic vesicles that translocate on isolated microtubules

    PubMed Central

    1986-01-01

    Axoplasmic vesicles were purified and observed to translocate on isolated microtubules in an ATP-dependent, trypsin-sensitive manner, implying that ATP-binding polypeptides essential for force generation were present on the vesicle surface. To identify these proteins [alpha 32P]8-azidoadenosine 5'-triphosphate ([alpha 32P]8-N3ATP), a photoaffinity analogue of ATP, was used. The results presented here identify and characterize a vesicle-associated polypeptide having a relative molecular mass of 292 kD that bound [alpha 32P]8-N3ATP. The incorporation of label is ultraviolet light-dependent and ATP- sensitive. Moreover, the 292-kD polypeptide could be isolated in association with vesicles or microtubules, depending on the conditions used, and the data indicate that the 292-kD polypeptide is similar to mammalian brain microtubule-associated protein 2 (MAP 2) for the following reasons: The 292-kD polypeptide isolated from either squid axoplasm or optic lobe cross-reacts with antiserum to porcine brain MAP 2. Furthermore, it purifies with taxol-stabilized microtubules and is released with salt. Based on these characteristics, the 292-kD polypeptide is distinct from the known force-generating molecules myosin and flagellar dynein, as well as the 110-130-kD kinesin-like polypeptides that have recently been described (Brady, S. T., 1985, Nature (Lond.), 317:73-75; Vale, R. D., T. S. Reese, and M. P. Sheetz, 1985b, Cell, 42:39-50; Scholey, J. M., M. E. Porter, P. M. Grissom, and J. R. McIntosh, 1985, Nature (Lond.), 318:483-486). Because the 292-kD polypeptide binds ATP and is associated with vesicles that translocate on purified MAP-free microtubules in an ATP-dependent fashion, it is therefore believed to be involved in vesicle-microtubule interactions that promote organelle motility. PMID:3091608

  7. Physical basis of large microtubule aster growth

    PubMed Central

    Ishihara, Keisuke; Korolev, Kirill S; Mitchison, Timothy J

    2016-01-01

    Microtubule asters - radial arrays of microtubules organized by centrosomes - play a fundamental role in the spatial coordination of animal cells. The standard model of aster growth assumes a fixed number of microtubules originating from the centrosomes. However, aster morphology in this model does not scale with cell size, and we recently found evidence for non-centrosomal microtubule nucleation. Here, we combine autocatalytic nucleation and polymerization dynamics to develop a biophysical model of aster growth. Our model predicts that asters expand as traveling waves and recapitulates all major aspects of aster growth. With increasing nucleation rate, the model predicts an explosive transition from stationary to growing asters with a discontinuous jump of the aster velocity to a nonzero value. Experiments in frog egg extract confirm the main theoretical predictions. Our results suggest that asters observed in large fish and amphibian eggs are a meshwork of short, unstable microtubules maintained by autocatalytic nucleation and provide a paradigm for the assembly of robust and evolvable polymer networks. DOI: http://dx.doi.org/10.7554/eLife.19145.001 PMID:27892852

  8. Microtubule binding distinguishes dystrophin from utrophin

    PubMed Central

    Belanto, Joseph J.; Mader, Tara L.; Eckhoff, Michael D.; Strandjord, Dana M.; Banks, Glen B.; Gardner, Melissa K.; Lowe, Dawn A.; Ervasti, James M.

    2014-01-01

    Dystrophin and utrophin are highly similar proteins that both link cortical actin filaments with a complex of sarcolemmal glycoproteins, yet localize to different subcellular domains within normal muscle cells. In mdx mice and Duchenne muscular dystrophy patients, dystrophin is lacking and utrophin is consequently up-regulated and redistributed to locations normally occupied by dystrophin. Transgenic overexpression of utrophin has been shown to significantly improve aspects of the disease phenotype in the mdx mouse; therefore, utrophin up-regulation is under intense investigation as a potential therapy for Duchenne muscular dystrophy. Here we biochemically compared the previously documented microtubule binding activity of dystrophin with utrophin and analyzed several transgenic mouse models to identify phenotypes of the mdx mouse that remain despite transgenic utrophin overexpression. Our in vitro analyses revealed that dystrophin binds microtubules with high affinity and pauses microtubule polymerization, whereas utrophin has no activity in either assay. We also found that transgenic utrophin overexpression does not correct subsarcolemmal microtubule lattice disorganization, loss of torque production after in vivo eccentric contractions, or physical inactivity after mild exercise. Finally, our data suggest that exercise-induced inactivity correlates with loss of sarcolemmal neuronal NOS localization in mdx muscle, whereas loss of in vivo torque production after eccentric contraction-induced injury is associated with microtubule lattice disorganization. PMID:24706788

  9. Polyribosome targeting to microtubules: enrichment of specific mRNAs in a reconstituted microtubule preparation from sea urchin embryos

    PubMed Central

    1994-01-01

    A subset of mRNAs, polyribosomes, and poly(A)-binding proteins copurify with microtubules from sea urchin embryos. Several lines of evidence indicate that the interaction of microtubules with ribosomes is specific: a distinct stalk-like structure appears to mediate their association; ribosomes bind to microtubules with a constant stoichiometry through several purification cycles; and the presence of ribosomes in these preparations depends on the presence of intact microtubules. Five specific mRNAs are enriched with the microtubule- bound ribosomes, indicating that translation of specific proteins may occur on the microtubule scaffolding in vivo. PMID:7962079

  10. Rapid intracellular motility and dynamic membrane events in an Antarctic foraminifer.

    PubMed

    Bowser, S S; DeLaca, T E

    1985-10-01

    Some properties of cytoplasmic transport in a cold-adapted (Antarctic) organism are reported for the first time. Phase-contrast light microscopy of Astrammina rara, an arenaceous foraminiferan protozoan, reveals that the saltatory transport of cytoplasmic granules in reticulopods occurs bidirectionally and at rates up to 7.5-micron/s. Extracellularly attached latex microspheres are rapidly translocated on the reticulopodial surface, thus demonstrating membrane fluidity at low (-1.8 degrees C) ambient temperatures. Rapid extension/withdrawal and branching/fusing of pseudopodia further illustrate dynamic plasma membrane activity at subzero temperatures. Immunofluorescence microscopy with an antibody monospecific for tubulin shows that these pseudopods contain microtubules. The motility of this cold-adapted foraminifer therefore appears fully comparable to the motility of allogromiid foraminifers from temperate waters.

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

    PubMed Central

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

    1981-01-01

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

  12. Insights into the structure and function of ciliary and flagellar doublet microtubules: tektins, Ca2+-binding proteins, and stable protofilaments.

    PubMed

    Linck, Richard; Fu, Xiaofeng; Lin, Jianfeng; Ouch, Christna; Schefter, Alexandra; Steffen, Walter; Warren, Peter; Nicastro, Daniela

    2014-06-20

    Cilia and flagella are conserved, motile, and sensory cell organelles involved in signal transduction and human disease. Their scaffold consists of a 9-fold array of remarkably stable doublet microtubules (DMTs), along which motor proteins transmit force for ciliary motility and intraflagellar transport. DMTs possess Ribbons of three to four hyper-stable protofilaments whose location, organization, and specialized functions have been elusive. We performed a comprehensive analysis of the distribution and structural arrangements of Ribbon proteins from sea urchin sperm flagella, using quantitative immunobiochemistry, proteomics, immuno-cryo-electron microscopy, and tomography. Isolated Ribbons contain acetylated α-tubulin, β-tubulin, conserved protein Rib45, >95% of the axonemal tektins, and >95% of the calcium-binding proteins, Rib74 and Rib85.5, whose human homologues are related to the cause of juvenile myoclonic epilepsy. DMTs contain only one type of Ribbon, corresponding to protofilaments A11-12-13-1 of the A-tubule. Rib74 and Rib85.5 are associated with the Ribbon in the lumen of the A-tubule. Ribbons contain a single ∼5-nm wide filament, composed of equimolar tektins A, B, and C, which interact with the nexin-dynein regulatory complex. A summary of findings is presented, and the functions of Ribbon proteins are discussed in terms of the assembly and stability of DMTs, ciliary motility, and other microtubule systems.

  13. CFAP54 is required for proper ciliary motility and assembly of the central pair apparatus in mice

    PubMed Central

    McKenzie, Casey W.; Craige, Branch; Kroeger, Tiffany V.; Finn, Rozzy; Wyatt, Todd A.; Sisson, Joseph H.; Pavlik, Jacqueline A.; Strittmatter, Lara; Hendricks, Gregory M.; Witman, George B.; Lee, Lance

    2015-01-01

    Motile cilia and flagella play critical roles in fluid clearance and cell motility, and dysfunction commonly results in the pediatric syndrome primary ciliary dyskinesia (PCD). CFAP221, also known as PCDP1, is required for ciliary and flagellar function in mice and Chlamydomonas reinhardtii, where it localizes to the C1d projection of the central microtubule apparatus and functions in a complex that regulates flagellar motility in a calcium-dependent manner. We demonstrate that the genes encoding the mouse homologues of the other C. reinhardtii C1d complex members are primarily expressed in motile ciliated tissues, suggesting a conserved function in mammalian motile cilia. The requirement for one of these C1d complex members, CFAP54, was identified in a mouse line with a gene-trapped allele. Homozygous mice have PCD characterized by hydrocephalus, male infertility, and mucus accumulation. The infertility results from defects in spermatogenesis. Motile cilia have a structural defect in the C1d projection, indicating that the C1d assembly mechanism requires CFAP54. This structural defect results in decreased ciliary beat frequency and perturbed cilia-driven flow. This study identifies a critical role for CFAP54 in proper assembly and function of mammalian cilia and flagella and establishes the gene-trapped allele as a new model of PCD. PMID:26224312

  14. Two Microtubule-associated Proteins of Arabidopsis MAP65s Promote Antiparallel Microtubule Bundling

    PubMed Central

    Gaillard, Jérémie; Neumann, Emmanuelle; Van Damme, Daniel; Stoppin-Mellet, Virginie; Ebel, Christine; Barbier, Elodie; Geelen, Danny

    2008-01-01

    The Arabidopsis MAP65s are a protein family with similarity to the microtubule-associated proteins PRC1/Ase1p that accumulate in the spindle midzone during late anaphase in mammals and yeast, respectively. Here we investigate the molecular and functional properties of AtMAP65-5 and improve our understanding of AtMAP65-1 properties. We demonstrate that, in vitro, both proteins promote the formation of a planar network of antiparallel microtubules. In vivo, we show that AtMAP65-5 selectively binds the preprophase band and the prophase spindle microtubule during prophase, whereas AtMAP65-1-GFP selectively binds the preprophase band but does not accumulate at the prophase spindle microtubules that coexists within the same cell. At later stages of mitosis, AtMAP65-1 and AtMAP65-5 differentially label the late spindle and phragmoplast. We present evidence for a mode of action for both proteins that involves the binding of monomeric units to microtubules that “zipper up” antiparallel arranged microtubules through the homodimerization of the N-terminal halves when adjacent microtubules encounter. PMID:18667529

  15. Transport properties of melanosomes along microtubules interpreted by a tug-of-war model with loose mechanical coupling.

    PubMed

    Bouzat, Sebastián; Levi, Valeria; Bruno, Luciana

    2012-01-01

    In this work, we explored theoretically the transport of organelles driven along microtubules by molecular motors of opposed polarities using a stochastic model that considers a Langevin dynamics for the cargo, independent cargo-motor linkers and stepping motion for the motors. It has been recently proposed that the stiffness of the motor plays an important role when multiple motors collectively transport a cargo. Therefore, we considered in our model the recently reported values for the stiffness of the cargo-motor linker determined in living cells (∼0.01 pN/nm,) which is significantly lower than the motor stiffness obtained in in vitro assays and used in previous studies. Our model could reproduce the multimodal velocity distributions and typical trajectory characteristics including the properties of the reversions in the overall direction of motion observed during melanosome transport along microtubules in Xenopus laevis melanophores. Moreover, we explored the contribution of the different motility states of the cargo-motor system to the different modes of the velocity distributions and could identify the microscopic mechanisms of transport leading to trajectories compatible with those observed in living cells. Finally, by changing the attachment and detachment rates, the model could reproduce the different velocity distributions observed during melanosome transport along microtubules in Xenopus laevis melanophores stimulated for aggregation and dispersion. Our analysis suggests that active tug-of-war processes with loose mechanical coupling can account for several aspects of cargo transport along microtubules in living cells.

  16. Transport Properties of Melanosomes along Microtubules Interpreted by a Tug-of-War Model with Loose Mechanical Coupling

    PubMed Central

    Bouzat, Sebastián; Levi, Valeria; Bruno, Luciana

    2012-01-01

    In this work, we explored theoretically the transport of organelles driven along microtubules by molecular motors of opposed polarities using a stochastic model that considers a Langevin dynamics for the cargo, independent cargo-motor linkers and stepping motion for the motors. It has been recently proposed that the stiffness of the motor plays an important role when multiple motors collectively transport a cargo. Therefore, we considered in our model the recently reported values for the stiffness of the cargo-motor linker determined in living cells (∼0.01 pN/nm, [1]) which is significantly lower than the motor stiffness obtained in in vitro assays and used in previous studies. Our model could reproduce the multimodal velocity distributions and typical trajectory characteristics including the properties of the reversions in the overall direction of motion observed during melanosome transport along microtubules in Xenopus laevis melanophores. Moreover, we explored the contribution of the different motility states of the cargo-motor system to the different modes of the velocity distributions and could identify the microscopic mechanisms of transport leading to trajectories compatible with those observed in living cells. Finally, by changing the attachment and detachment rates, the model could reproduce the different velocity distributions observed during melanosome transport along microtubules in Xenopus laevis melanophores stimulated for aggregation and dispersion. Our analysis suggests that active tug-of-war processes with loose mechanical coupling can account for several aspects of cargo transport along microtubules in living cells. PMID:22952716

  17. A novel microtubule-based motor protein (KIF4) for organelle transports, whose expression is regulated developmentally

    PubMed Central

    1994-01-01

    To understand the mechanisms of transport for organelles in the axon, we isolated and sequenced the cDNA encoding KIF4 from murine brain, and characterized the molecule biochemically and immunocytochemically. Complete amino acid sequence analysis of KIF4 and ultrastructural studies of KIF4 molecules expressed in Sf9 cells revealed that the protein contains 1,231 amino acid residues (M(r) 139,550) and that the molecule (116-nm rod with globular heads and tail) consists of three domains: an NH2-terminal globular motor domain, a central alpha-helical stalk domain and a COOH-terminal tail domain. KIF4 protein has the property of nucleotide-dependent binding to microtubules, microtubule- activated ATPase activity, and microtubule plus-end-directed motility. Northern blot analysis and in situ hybridization demonstrated that KIF4 is strongly expressed in juvenile tissues including differentiated young neurons, while its expression is decreased considerably in adult mice except in spleen. Immunocytochemical studies revealed that KIF4 colocalized with membranous organelles both in growth cones of differentiated neurons and in the cytoplasm of cultured fibroblasts. During mitotic phase of cell cycle, KIF4 appears to colocalize with membranous organelles in the mitotic spindle. Hence we conclude that KIF4 is a novel microtubule-associated anterograde motor protein for membranous organelles, the expression of which is regulated developmentally. PMID:7929562

  18. The influence of dynein processivity control, MAPs, and microtubule ends on directional movement of a localising mRNA

    PubMed Central

    Soundararajan, Harish Chandra; Bullock, Simon L

    2014-01-01

    Many cellular constituents travel along microtubules in association with multiple copies of motor proteins. How the activity of these motors is regulated during cargo sorting is poorly understood. In this study, we address this issue using a novel in vitro assay for the motility of localising Drosophila mRNAs bound to native dynein-dynactin complexes. High precision tracking reveals that individual RNPs within a population undergo either diffusive, or highly processive, minus end-directed movements along microtubules. RNA localisation signals stimulate the processive movements, with regulation of dynein-dynactin’s activity rather than its total copy number per RNP, responsible for this effect. Our data support a novel mechanism for multi-motor translocation based on the regulation of dynein processivity by discrete cargo-associated features. Studying the in vitro responses of RNPs to microtubule-associated proteins (MAPs) and microtubule ends provides insights into how an RNA population could navigate the cytoskeletal network and become anchored at its destination in cells. DOI: http://dx.doi.org/10.7554/eLife.01596.001 PMID:24737859

  19. Microtubules move the nucleus to quiescence.

    PubMed

    Laporte, Damien; Sagot, Isabelle

    2014-01-01

    The nucleus is a cellular compartment that hosts several macro-molecular machines displaying a highly complex spatial organization. This tight architectural orchestration determines not only DNA replication and repair but also regulates gene expression. In budding yeast microtubules play a key role in structuring the nucleus since they condition the Rabl arrangement in G1 and chromosome partitioning during mitosis through their attachment to centromeres via the kinetochore proteins. Recently, we have shown that upon quiescence entry, intranuclear microtubules emanating from the spindle pole body elongate to form a highly stable bundle that spans the entire nucleus. Here, we examine some molecular mechanisms that may underlie the formation of this structure. As the intranuclear microtubule bundle causes a profound re-organization of the yeast nucleus and is required for cell survival during quiescence, we discuss the possibility that the assembly of such a structure participates in quiescence establishment.

  20. Mechanical model of kinesin moving on microtubule

    NASA Astrophysics Data System (ADS)

    To, Kiwing; Chou, Ya-Chang; Hsiao, Yi-Feng; Chen, Kuan-Hua

    Kinesins are biomolecules that serve as intercellular motors for carrying cellular cargos along microtubules. Although the mechanism of converting the chemical energy of ATP to mechanical work is not fully understood, the motion of a kinesin on a microtubule has been measured and two different mechanisms, namely the ``hand-over-hand'' and ``inchworm'', has been proposed. The particular shape of kinesin and microtubules suggest a possible mechanism for force generation similar to Brownian ratchet. Using a bead chain connected to two heads that are attracted to a vibrated ratchet plate as a scaled up analog of the kinesinmicrotubule system, we manage to simulate both ``handoverhand'' and ``inchworm'' motion [Chou, et. al., Physica A443, 66 (2015)]. In addition, we find that chain, which play the role of the stalk in a kinesin molecule, can also generate force by interacting with the ratchet plate [Chen, et. al. Phys. Rev. E87, 012711 (2013)].

  1. Orthotropic elastic shell model for buckling of microtubules.

    PubMed

    Wang, C Y; Ru, C Q; Mioduchowski, A

    2006-11-01

    In view of the fact that microtubules exhibit strong anisotropic elastic properties, an orthotropic elastic shell model for microtubules is developed to study buckling behavior of microtubules. The predicted critical pressure is found to agree well with recent unexplained experimental data on pressure-induced buckling of microtubules [Needleman, Phys. Rev. Lett. 93, 198104 (2004); Biophys. J. 89, 3410 (2005)] which are lower than that predicted by the isotropic shell model by four orders of magnitude. General buckling behavior of microtubules under axial compression or radial pressure is studied. The results show that the isotropic shell model greatly overestimates the bucking loads of microtubules, except columnlike axially compressed buckling of long microtubules (of length-to-diameter ratio larger than, say, 150). In particular, the present results also offer a plausible explanation for the length dependency of flexibility of microtubules reported in the literature.

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

    PubMed Central

    Kok, Maurits; Dogterom, Marileen

    2016-01-01

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

  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. Protofilament number in microtubules in cells of two parasitic nematodes.

    PubMed

    Davis, C; Gull, K

    1983-12-01

    The parasitic nematodes, Ascaridia galli and Trichostrongylus colubriformis, were prepared for electron microscopy with fixatives containing tannic acid, which allowed their microtubule protofilament number to be examined. In contrast to many mammalian tissues, the nematodes did not contain microtubules with 13 protofilaments. Ascaridia galli contained microtubules with 11 protofilaments in all tissues examined, including nerve, intestinal, pharyngeal, and hypodermal cells. Trichostrongylus colubriformis contained nerve cells, known as microtubule cells, with bundles of larger microtubules (approximately 30 nm in diameter) with 14 protofilaments. The microtubules in these cells did not appear to be continuous for the entire length of the axon. Other cells examined in T. colubriformis, including nerve, intestinal and pharyngeal cells, contained two distinct types of microtubules, one with 11 protofilaments and an approximate diameter of 25 nm, and one with 12 protofilaments and an approximate diameter of 27 nm. All cell types examined contained both types of microtubules.

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

    PubMed

    Lloyd, Clive

    2011-01-01

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

  6. Structural investigations into microtubule-MAP complexes.

    PubMed

    Hoenger, Andreas; Gross, Heinz

    2008-01-01

    Microtubules interact with a large variety of factors commonly referred to as either molecular motors (kinesins, dyneins) or structural microtubule-associated proteins (MAPs). MAPs do not exhibit motor activity, but regulate microtubule dynamics and their interactions with molecular motors, and organelles such as kinetochores or centrosomes. Structural investigations into microtubule-kinesin motor complexes are quite advanced today and by helical three-dimensional (3-D) analysis reveal a resolution of the motor-tubulin interface at <1.0 nm. However, due to their flexible structure MAPs like tau or MAP2C cannot be visualized in the same straightforward manner. Helical averaging usually reveals only the location of strong binding sites while the overall structure of the MAP remains unsolved. Other MAPs such as EB1 bind very selectively only to some parts of the microtubule lattice such as the lattice seam. Thus, they do not reveal a stoichiometric tubulin:MAP-binding ratio that would allow for a quantitative helical 3-D analysis. Therefore, to get a better view on the structure of microtubule-MAP complexes we often used a strategy that combined cryo-electron microscopy and helical or tomographic 3-D analysis with freeze-drying and high-resolution unidirectional surface shadowing. 3-D analysis of ice-embedded specimens reveals their full 3-D volume. This relies either on a repetitive structure following a helical symmetry that can be used for averaging or suffers from the limited resolution that is currently achievable with cryotomography. Surface metal shadowing exclusively images surface-exposed features at very high contrast, adding highly valuable information to 2-D or 3-D data of vitrified structures.

  7. Evolving tip structures can explain age-dependent microtubule catastrophe.

    PubMed

    Coombes, Courtney E; Yamamoto, Ami; Kenzie, Madeline R; Odde, David J; Gardner, Melissa K

    2013-07-22

    Microtubules are key structural and transport elements in cells. The dynamics at microtubule ends are characterized by periods of slow growth, followed by stochastic switching events termed "catastrophes," in which microtubules suddenly undergo rapid shortening. Growing microtubules are thought to be protected from catastrophe by a GTP-tubulin "cap": GTP-tubulin subunits add to the tips of growing microtubules but are subsequently hydrolyzed to GDP-tubulin subunits once they are incorporated into the microtubule lattice. Loss of the GTP-tubulin cap exposes GDP-tubulin subunits at the microtubule tip, resulting in a catastrophe event. However, the mechanistic basis for sudden loss of the GTP cap, leading to catastrophe, is not known. To investigate microtubule catastrophe events, we performed 3D mechanochemical simulations that account for interactions between neighboring protofilaments. We found that there are two separate factors that contribute to catastrophe events in the 3D simulation: the GTP-tubulin cap size, which settles into a steady-state value that depends on the free tubulin concentration during microtubule growth, and the structure of the microtubule tip. Importantly, 3D simulations predict, and both fluorescence and electron microscopy experiments confirm, that microtubule tips become more tapered as the microtubule grows. This effect destabilizes the tip and ultimately contributes to microtubule catastrophe. Thus, the likelihood of a catastrophe event may be intimately linked to the aging physical structure of the growing microtubule tip. These results have important consequences for catastrophe regulation in cells, as microtubule-associated proteins could promote catastrophe events in part by modifying microtubule tip structures.

  8. Polewards chromosome movement driven by microtubule depolymerization in vitro

    NASA Astrophysics Data System (ADS)

    Koshland, Douglas E.; Mitchison, T. J.; Kirschner, Marc W.

    1988-02-01

    We constructed complexes between isolated chromosomes and microtubules made from purified tubulin to study the movement of chromosomes towards the 'minus' end of microtubules in vitro, a process analogous to the movement of chromosomes towards the pole of the spindle at anaphase of mitosis. Our results show that the energy for this movement is derived solely from microtubule depolymerization, and indicate that anaphase movement of chromosomes is both powered and regulated by microtubule depolymerization at the kinetochore.

  9. Fluorescent microtubules break up under illumination

    PubMed Central

    1988-01-01

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

  10. Colloidal Stabilization of Neurofilaments and Microtubules

    DTIC Science & Technology

    2002-06-01

    Alzheimer’s disease . To address this problem we used a set of biophysical methods, including atomic force microscopy, to investigate interfilament potentials. There are several main conclusions from the work under this award. First, microtubule associated proteins behave as though they are largely unstructured and can give rise to a long range repulsive force that is predominantly entropic in origin. This is an important finding that provides a biophysical mechanism that explains how microtubule spacing is maintained. Second, treating the unstructured proteins domains

  11. Self-organized cell motility

    NASA Astrophysics Data System (ADS)

    Du, Xinxin; Doubrovinski, Konstantin

    2011-03-01

    Cell migration plays a key role in a wide range of biological phenomena, such as morphogenesis, chemotaxis, and wound healing. Cell locomotion relies on the cytoskeleton, a meshwork of filamentous proteins, intrinsically out of thermodynamic equilibrium and cross-linked by molecular motors, proteins that turn chemical energy into mechanical work. In the course of locomotion, cells remain polarized, i.e. they retain a single direction of motion in the absence of external cues. Traditionally, polarization has been attributed to intracellular signaling. However, recent experiments show that polarization may be a consequence of self-organized cytoskeletal dynamics. Our aim is to elucidate the mechanisms by which persistent unidirectional locomotion may arise through simple mechanical interactions of the cytoskeletal proteins. To this end, we develop a simple physical description of cytoskeletal dynamics. We find that the proposed description accounts for a range of phenomena associated with cell motility, including spontaneous polarization, persistent unidirectional motion, and the co-existence of motile and non-motile states.

  12. Bacterial motility: From propulsion to collective behavior

    NASA Astrophysics Data System (ADS)

    Dombrowski, Christopher C.

    This work explores bacterial motility from the mechanisms of propulsion of an individual cell to the complex behavior of collective motility. The shear modulus of bacterial flagella was measured by stretching isolated flagella with an optical trap and by measuring force extension curves of the stretched flagella shedding light onto the mechanics involved in the motility of single micro-organisms. Experiments in concentrated suspensions of bacteria show collective behavior with large scale mixing on a time and length scale greater than can be understood from the standard model of "run and tumble" motility of a single organism are reported. To further understand the transition from individual to collective motility a novel form of motility where an individual bacterium can reverse direction without changing cell orientation is reported here. These experiments further the understanding of bacterial motility.

  13. Microfabricated systems and assays for studying the cytoskeletal organization, micromechanics, and motility patterns of cancerous cells

    SciTech Connect

    Huda, Sabil; Pilans, Didzis; Makurath, Monika; Hermans, Thomas M.; Kandere-Grzybowska, Kristiana; Grzybowski, Bartosz A.

    2014-08-28

    Cell motions are driven by coordinated actions of the intracellular cytoskeleton – actin, microtubules (MTs) and substrate/focal adhesions (FAs). This coordination is altered in metastatic cancer cells resulting in deregulated and increased cellular motility. Microfabrication tools, including photolithography, micromolding, microcontact printing, wet stamping and microfluidic devices have emerged as a powerful set of experimental tools with which to probe and define the differences in cytoskeleton organization/dynamics and cell motility patterns in non-metastatic and metastatic cancer cells. In this paper, we discuss four categories of microfabricated systems: (i) micropatterned substrates for studying of cell motility sub-processes (for example, MT targeting of FAs or cell polarization); (ii) systems for studying cell mechanical properties, (iii) systems for probing overall cell motility patterns within challenging geometric confines relevant to metastasis (for example, linear and ratchet geometries), and (iv) microfluidic devices that incorporate co-cultures of multiple cell types and chemical gradients to mimic in vivo intravasation/extravasation steps of metastasis. Finally, together, these systems allow for creating controlled microenvironments that not only mimic complex soft tissues, but are also compatible with live cell high-resolution imaging and quantitative analysis of single cell behavior.

  14. Microfabricated systems and assays for studying the cytoskeletal organization, micromechanics, and motility patterns of cancerous cells

    DOE PAGES

    Huda, Sabil; Pilans, Didzis; Makurath, Monika; ...

    2014-08-28

    Cell motions are driven by coordinated actions of the intracellular cytoskeleton – actin, microtubules (MTs) and substrate/focal adhesions (FAs). This coordination is altered in metastatic cancer cells resulting in deregulated and increased cellular motility. Microfabrication tools, including photolithography, micromolding, microcontact printing, wet stamping and microfluidic devices have emerged as a powerful set of experimental tools with which to probe and define the differences in cytoskeleton organization/dynamics and cell motility patterns in non-metastatic and metastatic cancer cells. In this paper, we discuss four categories of microfabricated systems: (i) micropatterned substrates for studying of cell motility sub-processes (for example, MT targeting ofmore » FAs or cell polarization); (ii) systems for studying cell mechanical properties, (iii) systems for probing overall cell motility patterns within challenging geometric confines relevant to metastasis (for example, linear and ratchet geometries), and (iv) microfluidic devices that incorporate co-cultures of multiple cell types and chemical gradients to mimic in vivo intravasation/extravasation steps of metastasis. Finally, together, these systems allow for creating controlled microenvironments that not only mimic complex soft tissues, but are also compatible with live cell high-resolution imaging and quantitative analysis of single cell behavior.« less

  15. Microfabricated Systems and Assays for Studying the Cytoskeletal Organization, Micromechanics, and Motility Patterns of Cancerous Cells

    PubMed Central

    Huda, Sabil; Pilans, Didzis; Makurath, Monika; Hermans, Thomas

    2015-01-01

    Cell motions are driven by coordinated actions of the intracellular cytoskeleton – actin, microtubules (MTs) and substrate/focal adhesions (FAs). This coordination is altered in metastatic cancer cells resulting in deregulated and increased cellular motility. Microfabrication tools, including photolithography, micromolding, microcontact printing, wet stamping and microfluidic devices have emerged as a powerful set of experimental tools with which to probe and define the differences in cytoskeleton organization/dynamics and cell motility patterns in non-metastatic and metastatic cancer cells. In this review, we discuss four categories of microfabricated systems: (i) micropatterned substrates for studying of cell motility sub-processes (for example, MT targeting of FAs or cell polarization); (ii) systems for studying cell mechanical properties, (iii) systems for probing overall cell motility patterns within challenging geometric confines relevant to metastasis (for example, linear and ratchet geometries), and (iv) microfluidic devices that incorporate co-cultures of multiple cells types and chemical gradients to mimic in vivo intravasation/extravasation steps of metastasis. Together, these systems allow for creating controlled microenvironments that not only mimic complex soft tissues, but are also compatible with live cell high-resolution imaging and quantitative analysis of single cell behavior. PMID:26900544

  16. Proteomic analysis of endocytic vesicles: Rab1a regulates motility of early endocytic vesicles

    PubMed Central

    Mukhopadhyay, Aparna; Nieves, Edward; Che, Fa-Yun; Wang, Jean; Jin, Lianji; Murray, John W.; Gordon, Kristie; Angeletti, Ruth Hogue; Wolkoff, Allan W.

    2011-01-01

    Texas-Red–asialoorosomucoid (ASOR) fluorescence-sorted early and late endocytic vesicles from rat liver were subjected to proteomic analysis with the aim of identifying functionally important proteins. Several Rab GTPases, including Rab1a, were found. The present study immunolocalized Rab1a to early and late endocytic vesicles and examined its potential role in endocytosis. Huh7 cells with stable knockdown of Rab1a exhibited reduced endocytic processing of ASOR. This correlated with the finding that Rab1a antibody reduced microtubule-based motility of rat-liver-derived early but not late endocytic vesicles in vitro. The inhibitory effect of Rab1a antibody was observed to be specifically towards minus-end-directed motility. Total and minus-end-directed motility was also reduced in early endocytic vesicles prepared from Rab1a-knockdown cells. These results corresponded with virtual absence of the minus-end-directed kinesin Kifc1 from early endocytic vesicles in Rab1a knockdown cells and imply that Rab1a regulates minus-end-directed motility largely by recruiting Kifc1 to early endocytic vesicles. PMID:21303926

  17. Microtubules and the endoplasmic reticulum are highly interdependent structures

    PubMed Central

    1986-01-01

    The interrelationships of the endoplasmic reticulum (ER), microtubules, and intermediate filaments were studied in the peripheral regions of thin, spread fibroblasts, epithelial, and vascular endothelial cells in culture. We combined a fluorescent dye staining technique to localize the ER with immunofluorescence to localize microtubules or intermediate filaments in the same cell. Microtubules and the ER are sparse in the lamellipodia, but intermediate filaments are usually completely absent. These relationships indicate that microtubules and the ER advance into the lamellipodia before intermediate filaments. We observed that microtubules and tubules of the ER have nearly identical distributions in lamellipodia, where new extensions of both are taking place. We perturbed microtubules by nocodazole, cold temperature, or hypotonic shock, and observed the effects on the ER distribution. On the basis of our observations in untreated cells and our experiments with microtubule perturbation, we conclude that microtubules and the ER are highly interdependent in two ways: (a) polymerization of individual microtubules and extension of individual ER tubules occur together at the level of resolution of the fluorescence microscope, and (b) depolymerization of microtubules does not disrupt the ER network in the short term (15 min), but prolonged absence of microtubules (2 h) leads to a slow retraction of the ER network towards the cell center, indicating that over longer periods of time, the extended state of the entire ER network requires the microtubule system. PMID:3533956

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

    PubMed

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

    2010-06-04

    Microtubule growth proceeds through the endwise addition of nucleotide-bound tubulin dimers. The microtubule wall is composed of GDP-tubulin subunits, which are thought to come exclusively from the incorporation of GTP-tubulin complexes at microtubule ends followed by GTP hydrolysis within the polymer. The possibility of a direct GDP-tubulin incorporation into growing polymers is regarded as hardly compatible with recent structural data. Here, we have examined GTP-tubulin and GDP-tubulin incorporation into polymerizing microtubules using a minimal assembly system comprised of nucleotide-bound tubulin dimers, in the absence of free nucleotide. We find that GDP-tubulin complexes can efficiently co-polymerize with GTP-tubulin complexes during microtubule assembly. GDP-tubulin incorporation into microtubules occurs with similar efficiency during bulk microtubule assembly as during microtubule growth from seeds or centrosomes. Microtubules formed from GTP-tubulin/GDP-tubulin mixtures display altered microtubule dynamics, in particular a decreased shrinkage rate, apparently due to intrinsic modifications of the polymer disassembly properties. Thus, although microtubules polymerized from GTP-tubulin/GDP-tubulin mixtures or from homogeneous GTP-tubulin solutions are both composed of GDP-tubulin subunits, they have different dynamic properties, and this may reveal a novel form of microtubule "structural plasticity."

  19. Mmb1p binds mitochondria to dynamic microtubules

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2010-01-01

    Microtubule growth proceeds through the endwise addition of nucleotide-bound tubulin dimers. The microtubule wall is composed of GDP-tubulin subunits, which are thought to come exclusively from the incorporation of GTP-tubulin complexes at microtubule ends followed by GTP hydrolysis within the polymer. The possibility of a direct GDP-tubulin incorporation into growing polymers is regarded as hardly compatible with recent structural data. Here, we have examined GTP-tubulin and GDP-tubulin incorporation into polymerizing microtubules using a minimal assembly system comprised of nucleotide-bound tubulin dimers, in the absence of free nucleotide. We find that GDP-tubulin complexes can efficiently co-polymerize with GTP-tubulin complexes during microtubule assembly. GDP-tubulin incorporation into microtubules occurs with similar efficiency during bulk microtubule assembly as during microtubule growth from seeds or centrosomes. Microtubules formed from GTP-tubulin/GDP-tubulin mixtures display altered microtubule dynamics, in particular a decreased shrinkage rate, apparently due to intrinsic modifications of the polymer disassembly properties. Thus, although microtubules polymerized from GTP-tubulin/GDP-tubulin mixtures or from homogeneous GTP-tubulin solutions are both composed of GDP-tubulin subunits, they have different dynamic properties, and this may reveal a novel form of microtubule “structural plasticity.” PMID:20371874

  1. Motors and MAPs collaborate to size up microtubules.

    PubMed

    Bechstedt, Susanne; Brouhard, Gary J

    2013-07-29

    Midzone microtubules keep chromosomes apart after segregation and provide a platform for cytokinesis factors. Reporting recently in Cell, Subramanian et al. (2013) describe how the motor protein kinesin-4 and the microtubule-associated protein PRC1 work together to mark microtubule ends for incorporation into the midzone in a length-dependent manner.

  2. Fundoplication improves disordered esophageal motility.

    PubMed

    Heider, T Ryan; Behrns, Kevin E; Koruda, Mark J; Shaheen, Nicholas J; Lucktong, Tananchai A; Bradshaw, Barbara; Farrell, Timothy M

    2003-02-01

    Patients with gastroesophageal reflux disease (GERD) and disordered esophageal motility are at risk for postoperative dysphagia, and are often treated with partial (270-degree) fundoplication as a strategy to minimize postoperative swallowing difficulties. Complete (360-degree) fundoplication, however, may provide more effective and durable reflux protection over time. Recently we reported that postfundoplication dysphagia is uncommon, regardless of preoperative manometric status and type of fundoplication. To determine whether esophageal function improves after fundoplication, we measured postoperative motility in patients in whom disordered esophageal motility had been documented before fundoplication. Forty-eight of 262 patients who underwent laparoscopic fundoplication between 1995 and 2000 satisfied preoperative manometric criteria for disordered esophageal motility (distal esophageal peristaltic amplitude < or =30 mm Hg and/or peristaltic frequency < or =80%). Of these, 19 had preoperative manometric assessment at our facility and consented to repeat study. Fifteen (79%) of these patients had a complete fundoplication and four (21%) had a partial fundoplication. Each patient underwent repeat four-channel esophageal manometry 29.5 +/- 18.4 months (mean +/- SD) after fundoplication. Distal esophageal peristaltic amplitude and peristaltic frequency were compared to preoperative data by paired t test. After fundoplication, mean peristaltic amplitude in the distal esophagus increased by 47% (56.8 +/- 30.9 mm Hg to 83.5 +/- 36.5 mm Hg; P < 0.001) and peristaltic frequency improved by 33% (66.4 +/- 28.7% to 87.6 +/- 16.3%; P < 0.01). Normal esophageal motor function was present in 14 patients (74%) after fundoplication, whereas in five patients the esophageal motor function remained abnormal (2 improved, 1 worsened, and 2 remained unchanged). Three patients with preoperative peristaltic frequencies of 0%, 10%, and 20% improved to 84%, 88%, and 50%, respectively

  3. 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.

  4. Microtubule Actin Cross-Linking Factor 1 Regulates Cardiomyocyte Microtubule Distribution and Adaptation to Hemodynamic Overload

    PubMed Central

    Kwak, Dongmin; Wang, Huan; Liu, Xiaoyu; Hu, Xinli; Bache, Robert J.; Chen, Yingjie

    2013-01-01

    Aberrant cardiomyocyte microtubule growth is a feature of pressure overload induced cardiac hypertrophy believed to contribute to left ventricular (LV) dysfunction. Microtubule Actin Cross-linking Factor 1 (MACF1/Acf7) is a 600 kd spectraplakin that stabilizes and guides microtubule growth along actin filaments. MACF1 is expressed in the heart, but its impact on cardiac microtubules, and how this influences cardiac structure, function, and adaptation to hemodynamic overload is unknown. Here we used inducible cardiac-specific MACF1 knockout mice (MACF1 KO) to determine the impact of MACF1 on cardiac microtubules and adaptation to pressure overload (transverse aortic constriction (TAC).In adult mouse hearts, MACF1 expression was low under basal conditions, but increased significantly in response to TAC. While MACF1 KO had no observable effect on heart size or function under basal conditions, MACF1 KO exacerbated TAC induced LV hypertrophy, LV dilation and contractile dysfunction. Interestingly, subcellular fractionation of ventricular lysates revealed that MACF1 KO altered microtubule distribution in response to TAC, so that more tubulin was associated with the cell membrane fraction. Moreover, TAC induced microtubule redistribution into this cell membrane fraction in both WT and MACF1 KO mice correlated strikingly with the level of contractile dysfunction (r2 = 0.786, p<.001). MACF1 disruption also resulted in reduction of membrane caveolin 3 levels, and increased levels of membrane PKCα and β1 integrin after TAC, suggesting MACF1 function is important for spatial regulation of several physiologically relevant signaling proteins during hypertrophy. Together, these data identify for the first time, a role for MACF1 in cardiomyocyte microtubule distribution and in adaptation to hemodynamic overload. PMID:24086300

  5. Microtubule Actin Cross-linking Factor 1 regulates cardiomyocyte microtubule distribution and adaptation to hemodynamic overload.

    PubMed

    Fassett, John T; Xu, Xin; Kwak, Dongmin; Wang, Huan; Liu, Xiaoyu; Hu, Xinli; Bache, Robert J; Chen, Yingjie

    2013-01-01

    Aberrant cardiomyocyte microtubule growth is a feature of pressure overload induced cardiac hypertrophy believed to contribute to left ventricular (LV) dysfunction. Microtubule Actin Cross-linking Factor 1 (MACF1/Acf7) is a 600 kd spectraplakin that stabilizes and guides microtubule growth along actin filaments. MACF1 is expressed in the heart, but its impact on cardiac microtubules, and how this influences cardiac structure, function, and adaptation to hemodynamic overload is unknown. Here we used inducible cardiac-specific MACF1 knockout mice (MACF1 KO) to determine the impact of MACF1 on cardiac microtubules and adaptation to pressure overload (transverse aortic constriction (TAC).In adult mouse hearts, MACF1 expression was low under basal conditions, but increased significantly in response to TAC. While MACF1 KO had no observable effect on heart size or function under basal conditions, MACF1 KO exacerbated TAC induced LV hypertrophy, LV dilation and contractile dysfunction. Interestingly, subcellular fractionation of ventricular lysates revealed that MACF1 KO altered microtubule distribution in response to TAC, so that more tubulin was associated with the cell membrane fraction. Moreover, TAC induced microtubule redistribution into this cell membrane fraction in both WT and MACF1 KO mice correlated strikingly with the level of contractile dysfunction (r(2) = 0.786, p<.001). MACF1 disruption also resulted in reduction of membrane caveolin 3 levels, and increased levels of membrane PKCα and β1 integrin after TAC, suggesting MACF1 function is important for spatial regulation of several physiologically relevant signaling proteins during hypertrophy. Together, these data identify for the first time, a role for MACF1 in cardiomyocyte microtubule distribution and in adaptation to hemodynamic overload.

  6. Vesicle deformation by microtubules: A phase diagram

    NASA Astrophysics Data System (ADS)

    Emsellem, Virginie; Cardoso, Olivier; Tabeling, Patrick

    1998-10-01

    The experimental investigation of vesicles deformed by the growth of encapsulated microtubules shows that the axisymmetric morphologies can be classified into ovals, lemons, φ, cherries, dumbbells, and pearls. A geometrical phase diagram is established. Numerical minimization of the elastic energy of the membrane reproduces satisfactorily well the observed morphologies and the corresponding phase diagram.

  7. Structural and Functional Studies Indicate That Shigella VirA Is Not a Protease and Does Not Directly Destabilize Microtubules

    SciTech Connect

    Germane, Katherine L.; Ohi, Ryoma; Goldberg, Marcia B.; Spiller, Benjamin W.

    2008-11-24

    VirA, an essential virulence factor in Shigella disease pathogenesis, is involved in the uptake, motility, and cell-to-cell spread of Shigella organisms within the human host. These functions have been attributed to a VirA protease activity and a mechanism of microtubule destruction via tubulin degradation [Yoshida, S., et al. (2006) Science 314, 985--989]. We report functional and crystallographic data indicating a novel VirA structure that lacks these activities but highlights the homology to the EspG virulence factor of pathogenic Escherichia coli.

  8. Microtubule Initiation from the Nuclear Surface Controls Cortical Microtubule Growth Polarity and Orientation in Arabidopsis thaliana

    PubMed Central

    Ambrose, Chris; Wasteneys, Geoffrey O.

    2014-01-01

    The nuclear envelope in plant cells has long been known to be a microtubule organizing center (MTOC), but its influence on microtubule organization in the cell cortex has been unclear. Here we show that nuclear MTOC activity favors the formation of longitudinal cortical microtubule (CMT) arrays. We used green fluorescent protein (GFP)-tagged gamma tubulin-complex protein 2 (GCP2) to identify nuclear MTOC activity and GFP-tagged End-Binding Protein 1b (EB1b) to track microtubule growth directions. We found that microtubules initiate from nuclei and enter the cortex in two directions along the long axis of the cell, creating bipolar longitudinal CMT arrays. Such arrays were observed in all cell types showing nuclear MTOC activity, including root hairs, recently divided cells in root tips, and the leaf epidermis. In order to confirm the causal nature of nuclei in bipolar array formation, we displaced nuclei by centrifugation, which generated a corresponding shift in the bipolarity split point. We also found that bipolar CMT arrays were associated with bidirectional trafficking of vesicular components to cell ends. Together, these findings reveal a conserved function of plant nuclear MTOCs and centrosomes/spindle pole bodies in animals and fungi, wherein all structures serve to establish polarities in microtubule growth. PMID:25008974

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

    PubMed

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

    2003-03-27

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

  10. Analysis of microtubule polymerization dynamics in live cells

    PubMed Central

    Gierke, Sarah; Kumar, Praveen; Wittmann, Torsten

    2012-01-01

    Intracellular microtubule polymerization dynamics are spatiotemporally controlled by numerous microtubule-associated proteins and other mechanisms, and this regulation is central to many cell processes. Here, we give an overview and practical guide on how to acquire and analyze time-lapse sequences of dynamic microtubules in live cells by either fluorescently labeling entire microtubules or by utilizing proteins that specifically associate only with growing microtubule ends, and summarize the strengths and weaknesses of different approaches. We give practical recommendations for imaging conditions, and we also discuss important limitations of such analysis that are dictated by the maximal achievable spatial and temporal sampling frequencies. PMID:20719263

  11. Effects of α-Tubulin K40 Acetylation and Detyrosination on Kinesin-1 Motility in a Purified System

    PubMed Central

    Kaul, Neha; Soppina, Virupakshi; Verhey, Kristen J.

    2014-01-01

    Long-range transport in cells is achieved primarily through motor-based transport along a network of microtubule tracks. Targeted transport by kinesin motors can be correlated with posttranslational modifications (PTMs) of the tubulin subunits in specific microtubules. To directly examine the influence of specific PTMs on kinesin-1 motility, we generated tubulin subunits that were either enriched in or lacking acetylation of α-tubulin lysine 40 (K40) or detyrosination of the α-tubulin C-terminal tail. We show that K40 acetylation does not result in significant changes in kinesin-1’s landing rate or motility parameters (velocity and run length) across experimental conditions. In contrast, detyrosination causes a moderate increase in kinesin-1’s landing rate. The fact that the effects of detyrosination are dampened by prior K40 acetylation indicates that the combination of PTMs may be an important aspect of the functional output of microtubule heterogeneity. Importantly, our results indicate that the moderate influences that single PTMs have on kinesin-1 in vitro do not explain the strong correlation between specific PTMs and kinesin-1 transport in cells. Thus, additional mechanisms for regulating kinesin-1 transport in cells must be explored in future work. PMID:24940781

  12. Motor protein and microtubule mechanics: Application of a novel high-resolution optical trapping technique

    NASA Astrophysics Data System (ADS)

    Allersma, Miriam W.

    Using optical tweezers and a novel detection technique (a quadrant photodiode at the back focal plane or, BFP-QD), this thesis investigates two problems in biophysics, ncd motility and microtubule flexural rigidity. We use optically trapped microspheres to probe the samples. The technique detects the displacements of the microspheres relative to the trap center by monitoring the laser intensity shifts in the back focal plane of the microscope condenser. We use a quadrant diode to detect the shifts, which are due to far-field interference between the trapping laser and scattered laser light from the trapped object. The method yields high-resolution (nm-spatial and μsec-temporal), two-dimensional data, which is largely independent of trap position in the field of view. We first studied the motility of ncd, a kinesin-related motor protein. Motor proteins are able to harness the energy of ATP hydrolysis to perform mechanical work for the cell. Many ncd molecules were adsorbed onto silica microspheres and their motions along the microtubule surface lattice were observed with the BFP-QD method. Since the method is two-dimensional, we were able to monitor axial and lateral motions simultaneously. The average axial velocity was 230 +/- 30 nm/sec (average +/- SD). The high temporal resolution allowed us to investigate dynamical parameters. Spectral analysis showed an increase in viscous drag near the surface for ncd-driven microspheres. In addition, we found that the binding of the motors to microtubules in the presence of the nonhydrolyzable nucleotide adenylylimidodiphosphate caused an increase in the motor elastic constraint. Using a dual optical trap configuration in conjunction with the BFP-QD, we also investigated the elastic properties of taxol-stabilized microtubules. Cytoskeletal filaments are responsible for myriad structural cell functions. Our results were not readily interpreted by a standard bent strut treatment because of the finite size of the microspheres

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

    SciTech Connect

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

    2016-04-01

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

  14. The family-specific K-loop influences the microtubule on-rate but not the superprocessivity of kinesin-3 motors

    PubMed Central

    Soppina, Virupakshi; Verhey, Kristen J.

    2014-01-01

    The kinesin-3 family (KIF) is one of the largest among the kinesin superfamily and an important driver of a variety of cellular transport events. Whereas all kinesins contain the highly conserved kinesin motor domain, different families have evolved unique motor features that enable different mechanical and functional outputs. A defining feature of kinesin-3 motors is the presence of a positively charged insert, the K-loop, in loop 12 of their motor domains. However, the mechanical and functional output of the K-loop with respect to processive motility of dimeric kinesin-3 motors is unknown. We find that, surprisingly, the K-loop plays no role in generating the superprocessive motion of dimeric kinesin-3 motors (KIF1, KIF13, and KIF16). Instead, we find that the K-loop provides kinesin-3 motors with a high microtubule affinity in the motor's ADP-bound state, a state that for other kinesins binds only weakly to the microtubule surface. A high microtubule affinity results in a high landing rate of processive kinesin-3 motors on the microtubule surface. We propose that the family-specific K-loop contributes to efficient kinesin-3 cargo transport by enhancing the initial interaction of dimeric motors with the microtubule track. PMID:24850887

  15. The family-specific K-loop influences the microtubule on-rate but not the superprocessivity of kinesin-3 motors.

    PubMed

    Soppina, Virupakshi; Verhey, Kristen J

    2014-07-15

    The kinesin-3 family (KIF) is one of the largest among the kinesin superfamily and an important driver of a variety of cellular transport events. Whereas all kinesins contain the highly conserved kinesin motor domain, different families have evolved unique motor features that enable different mechanical and functional outputs. A defining feature of kinesin-3 motors is the presence of a positively charged insert, the K-loop, in loop 12 of their motor domains. However, the mechanical and functional output of the K-loop with respect to processive motility of dimeric kinesin-3 motors is unknown. We find that, surprisingly, the K-loop plays no role in generating the superprocessive motion of dimeric kinesin-3 motors (KIF1, KIF13, and KIF16). Instead, we find that the K-loop provides kinesin-3 motors with a high microtubule affinity in the motor's ADP-bound state, a state that for other kinesins binds only weakly to the microtubule surface. A high microtubule affinity results in a high landing rate of processive kinesin-3 motors on the microtubule surface. We propose that the family-specific K-loop contributes to efficient kinesin-3 cargo transport by enhancing the initial interaction of dimeric motors with the microtubule track.

  16. Experimental protocols for and studies of the effects of surface passivation and water isotopes on the gliding speed of microtubules propelled by kinesin-1

    NASA Astrophysics Data System (ADS)

    Maloney, Roger Andrew

    This dissertation explores how the kinesin-1 and microtubule system is affected by surface passivation and water isotopes. Surface passivation was found to affect the gliding speed that microtubules exhibit in the gliding motility assay and the lengths of microtubules supported by the passivation. It was also found that gliding speeds of microtubules are very sensitive to temperature changes. Studies changing the water isotope were a first attempt to investigate if changing the solvent changed the osmotic pressure of the solution kinesin and microtubules were in. No osmotic pressure changes were observed, however, the experiments using different isotopes of water did illuminate the possibility that kinesin may be sensitive to viscosity changes in the solvent. This experiment also suggests further experiments that can be specifically designed to probe osmotic pressure changes. This thesis was also the first thesis ever, to the best of the author's knowledge, to be done in a completely open format. All information and notebook entries that are related to it, as well as the thesis itself, can be found on the website OpenWetWare. The thesis can also be found there including all the different versions that went into its editing. The philosophy and process of making data open and accessible to every one is also discussed.

  17. Target molecules of calmodulin on microtubules of Tetrahymena cilia

    SciTech Connect

    Hirano-Ohnishi, Junko; Watanabe, Yoshio )

    1988-09-01

    In the course of an attempt to isolate the calmodulin-binding proteins (CaMBPs) from cilia of Tetrahymena, it was found that some CaMBPs tend to interact with axonemal microtubules. The present study demonstrates this interaction by cosedimentation experiments using in vitro polymerized Tetrahymena axonemal microtubules and Tetrahymena CaMBPs purified from axonemes by calmodulin affinity column chromatography. Analysis by the ({sup 125}I)calmodulin overlay method showed that at least three CaMBPs (M{sub r} 69, 45, and 37 kDa) cosediment with microtubules. Furthermore, without any addition of exogenous CaMBPs, microtubules purified after three cycles of temperature-dependent polymerization and depolymerization included the above CaMBPs and additional CaMBPs which could not cosediment with microtubules. From the results, the authors have classified these microtubule-associated CaMBPs into two groups: (i) CaMBPs which interact with microtubules only during polymerization, and (ii) CaMBPs which interact not only with microtubules during polymerization, but also with polymerized microtubules. These results suggest that the microtubule-associated CaMBPs, especially those of the latter group, are located on the surface of ciliary microtubules, and may become the target molecules of calmodulin at Ca{sup 2+}-triggered ciliary reversal.

  18. Three-dimensional structure of cytoplasmic dynein bound to microtubules

    PubMed Central

    Mizuno, Naoko; Narita, Akihiro; Kon, Takahide; Sutoh, Kazuo; Kikkawa, Masahide

    2007-01-01

    Cytoplasmic dynein is a large, microtubule-dependent molecular motor (1.2 MDa). Although the structure of dynein by itself has been characterized, its conformation in complex with microtubules is still unknown. Here, we used cryoelectron microscopy (cryo-EM) to visualize the interaction between dynein and microtubules. Most dynein molecules in the nucleotide-free state are bound to the microtubule in a defined conformation and orientation. A 3D image reconstruction revealed that dynein's head domain, formed by a ring-like arrangement of AAA+ domains, is located ≈280 Å away from the center of the microtubule. The order of the AAA+ domains in the ring was determined by using recombinant markers. Furthermore, a 3D helical image reconstruction of microtubules with a dynein's microtubule binding domain [dynein stalk (DS)] revealed that the stalk extends perpendicular to the microtubule. By combining the 3D maps of the dynein-microtubule and DS-microtubule complexes, we present a model for how dynein in the nucleotide-free state binds to microtubules and discuss models for dynein's power stroke. PMID:18093913

  19. Loop formation of microtubules during gliding at high density

    NASA Astrophysics Data System (ADS)

    Liu, Lynn; Tüzel, Erkan; Ross, Jennifer L.

    2011-09-01

    The microtubule cytoskeleton, including the associated proteins, forms a complex network essential to multiple cellular processes. Microtubule-associated motor proteins, such as kinesin-1, travel on microtubules to transport membrane bound vesicles across the crowded cell. Other motors, such as cytoplasmic dynein and kinesin-5, are used to organize the cytoskeleton during mitosis. In order to understand the self-organization processes of motors on microtubules, we performed filament-gliding assays with kinesin-1 motors bound to the cover glass with a high density of microtubules on the surface. To observe microtubule organization, 3% of the microtubules were fluorescently labeled to serve as tracers. We find that microtubules in these assays are not confined to two dimensions and can cross one other. This causes microtubules to align locally with a relatively short correlation length. At high density, this local alignment is enough to create 'intersections' of perpendicularly oriented groups of microtubules. These intersections create vortices that cause microtubules to form loops. We characterize the radius of curvature and time duration of the loops. These different behaviors give insight into how crowded conditions, such as those in the cell, might affect motor behavior and cytoskeleton organization.

  20. Expansion and Polarity Sorting in Microtubule-Dynein Bundles

    NASA Astrophysics Data System (ADS)

    Zemel, A.; Mogilner, A.

    Interactions of multiple molecular motors with dynamicpolymers, such as actin and microtubules, form the basis for many processes in the cell cytoskeleton. One example is the active `sorting' of microtubule bundles by dynein molecular motors into aster-like arrays of microtubules; in these bundles dynein motors cross-link and slide neighboring microtubules apart. A number of models have been suggested to quantify the active dynamics of cross-linked bundles of polar filaments. In the case of densely packed bundles, however, a major complication arises from the fact that each microtubule interacts with multiple neighboring filaments. To explicitly take these interactions into account we performed detailed computer simulations in which the equations of motion for all microtubules in the bundle were iteratively solved. Our simulations demonstrate the phenomenon of polarity sorting and reveal the variable-rate of the concurrent bundle expansion and its dependence on the nature of the microtubule-motor interactions.

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

    PubMed

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

    2010-12-01

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

  2. Effects of Tau on Flow-Aligned Microtubule Bundles

    NASA Astrophysics Data System (ADS)

    Ross, Jennifer L.; Kuchnir Fygenson, D.

    2003-03-01

    Microtubules are cylindrical crystals of the protein tubulin with 17nm inner diameter and 25nm outer diameter. Recent structural studies suggest that the microtubule wall may be porous to small molecules. We have investigated the mobility of molecules in bundles of flow aligned microtubules. We find the spacing between the microtubules in the bundle is increased by the addition of tau, a microtubule associated protein. In the absence of tau, flow can be used to make tightly packed bundles of microtubules. Adding tau causes the tight bundles to swell and separate. We use fluorescence recovery after photobleaching (FRAP) to quantify the mobility of a taxol, a small drug that binds to the microtubule interior.

  3. Microtubules search for chromosomes by pivoting around the spindle pole

    NASA Astrophysics Data System (ADS)

    Tolic-Norrelykke, Iva

    2014-03-01

    During cell division, proper segregation of genetic material between the two daughter cells requires that the spindle microtubules attach to the chromosomes via kinetochores, protein complexes on the chromosome. The central question, how microtubules find kinetochores, is still under debate. We observed in fission yeast that kinetochores are captured by microtubules pivoting around the spindle pole body, instead of growing towards the kinetochores. By introducing a theoretical model, we show that the observed angular movement of microtubules is sufficient to explain the process of kinetochore capture. Our theory predicts that the speed of the capture process depends mainly on how fast microtubules pivot. We confirmed this prediction experimentally by speeding up and slowing down microtubule pivoting. Thus, microtubules explore space by pivoting, as they search for intracellular targets such as kinetochores.

  4. Cortical microtubule contacts position the spindle in C. elegans embryos.

    PubMed

    Kozlowski, Cleopatra; Srayko, Martin; Nedelec, Francois

    2007-05-04

    Interactions between microtubules and the cell cortex play a critical role in positioning organelles in a variety of biological contexts. Here we used Caenorhabditis elegans as a model system to study how cortex-microtubule interactions position the mitotic spindle in response to polarity cues. Imaging EBP-2::GFP and YFP::alpha-tubulin revealed that microtubules shrink soon after cortical contact, from which we propose that cortical adaptors mediate microtubule depolymerization energy into pulling forces. We also observe association of dynamic microtubules to form astral fibers that persist, despite the catastrophe events of individual microtubules. Computer simulations show that these effects, which are crucially determined by microtubule dynamics, can explain anaphase spindle oscillations and posterior displacement in 3D.

  5. Dual Role for Microtubules in Regulating Cortical Contractility during Cytokinesis

    PubMed Central

    Murthy, Kausalya; Wadsworth, Patricia

    2008-01-01

    Microtubules stimulate contractile ring formation in the equatorial cortex and simultaneously suppress contractility in the polar cortex; how they accomplish these differing activities is incompletely understood. We measured the behavior of GFP-actin in mammalian cells treated with nocodazole under conditions that either completely eliminate microtubules or selectively disassemble astral microtubules. Selective disassembly of astral microtubules resulted functional contractile rings that were wider than controls and had altered dynamic activity, as measured by FRAP. Complete microtubule disassembly or selective loss of astral microtubules resulted in wave-like contractile behavior of actin in the non-equatorial cortex and mislocalization of myosin II and Rho. FRAP experiments showed that both contractility and actin polymerization contributed to the wave-like behavior of actin. Wave-like, contractile behavior in anaphase cells was Rho-dependent. We conclude that dynamic astral microtubules function to suppress Rho activation in the nonequatorial cortex, limiting the contractile activity of the polar cortex. PMID:18559890

  6. The cytokineplast: purified, stable, and functional motile machinery from human blood polymorphonuclear leukocytes

    PubMed Central

    Malawista, SE; De Boisfleury Chevance, A

    1982-01-01

    We examined the formation of motile, chemotactically active, anucleate fragments from human blood polymorphonuclear leukocytes (PMN, granulocytes), induced by the brief application of heat. These granule-poor fragments are former protopods (leading fronts, lamellipodia) that become uncoupled from the main body of the cell and leave it, at first with a connecting filament that breaks and seals itself. The usual random orientation of such filaments can be controlled by preorientation of cells in a gradient of the chemotactic peptide, N-formylmethionylleucylphenylalanine (F-Met-Leu-Phe) (2x10(-9) M- 1x10(-8)). Cytochalsin B, 2.5-5 μg/ml, prevents fragment formation; colchicine, 10(-5) M, does not. In scanning electron micrographs, fragments are ruffled and the cell body rounded up and rather smooth. In transmission electron micrographs, fragments contain microfilaments but lack centrioles and microtubules. Like intact cells, both bound and free fragments can respond chemotactically to an erythrocyte destroyed by laser microirradiation (necrotaxis); the free, anucleate fragments may do so repeatedly, even after having been held overnight at ambient temperatures. We propse the name cytokineplast for the result of this self-purification of motile apparatus. The exodus of the motile machinery from the granulocyte requires anchoring of the bulk of the cell to glass and uncoupling, which may involve heat-induced dysfunction of the centrosome. In ultrastructural studies of the centrosomal region after heat, centriolar structure remains intact, but pericentriolar osmiophilic material appears condensed, and microtubules are sparse. These changes are found in all three blood cell types examined: PMN, eosinophil, and monocyte. Of these, the first two make fragments under our conditions; the more sluggish monocyte does not. Uncoupling is further linked to centrosomal dysfunction by the observation that colchicines-treated granulocytes (10(-5)M, to destroy the centrosome

  7. Motile properties of the bi-directional kinesin-5 Cin8 are affected by phosphorylation in its motor domain

    PubMed Central

    Shapira, Ofer; Gheber, Larisa

    2016-01-01

    The Saccharomyces cerevisiae kinesin-5 Cin8 performs essential mitotic functions in spindle assembly and anaphase B spindle elongation. Recent work has shown that Cin8 is a bi-directional motor which moves towards the minus-end of microtubules (MTs) under high ionic strength (IS) conditions and changes directionality in low IS conditions and when bound between anti-parallel microtubules. Previous work from our laboratory has also indicated that Cin8 is differentially phosphorylated during late anaphase at cyclin-dependent kinase 1 (Cdk1)-specific sites located in its motor domain. In vivo, such phosphorylation causes Cin8 detachment from spindles and reduces the spindle elongation rate, while maintaining proper spindle morphology. To study the effect of phosphorylation on Cin8 motor function, we examined in vitro motile properties of wild type Cin8, as well as its phosphorylation using phospho-deficient and phospho-mimic variants, in a single molecule fluorescence motility assay. Analysis was performed on whole cell extracts and on purified Cin8 samples. We found that addition of negative charges in the phospho-mimic mutant weakened the MT-motor interaction, increased motor velocity and promoted minus-end-directed motility. These results indicate that phosphorylation in the catalytic domain of Cin8 regulates its motor function. PMID:27216310

  8. Mechanism of Actin-Based Motility

    NASA Astrophysics Data System (ADS)

    Pantaloni, Dominique; Le Clainche, Christophe; Carlier, Marie-France

    2001-05-01

    Spatially controlled polymerization of actin is at the origin of cell motility and is responsible for the formation of cellular protrusions like lamellipodia. The pathogens Listeria monocytogenes and Shigella flexneri, which undergo actin-based propulsion, are acknowledged models of the leading edge of lamellipodia. Actin-based motility of the bacteria or of functionalized microspheres can be reconstituted in vitro from only five pure proteins. Movement results from the regulated site-directed treadmilling of actin filaments, consistent with observations of actin dynamics in living motile cells and with the biochemical properties of the components of the synthetic motility medium.

  9. 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

  10. Reversal of axonal growth defects in an extraocular fibrosis model by engineering the kinesin–microtubule interface

    PubMed Central

    Minoura, Itsushi; Takazaki, Hiroko; Ayukawa, Rie; Saruta, Chihiro; Hachikubo, You; Uchimura, Seiichi; Hida, Tomonobu; Kamiguchi, Hiroyuki; Shimogori, Tomomi; Muto, Etsuko

    2016-01-01

    Mutations in human β3-tubulin (TUBB3) cause an ocular motility disorder termed congenital fibrosis of the extraocular muscles type 3 (CFEOM3). In CFEOM3, the oculomotor nervous system develops abnormally due to impaired axon guidance and maintenance; however, the underlying mechanism linking TUBB3 mutations to axonal growth defects remains unclear. Here, we investigate microtubule (MT)-based motility in vitro using MTs formed with recombinant TUBB3. We find that the disease-associated TUBB3 mutations R262H and R262A impair the motility and ATPase activity of the kinesin motor. Engineering a mutation in the L12 loop of kinesin surprisingly restores a normal level of motility and ATPase activity on MTs carrying the R262A mutation. Moreover, in a CFEOM3 mouse model expressing the same mutation, overexpressing the suppressor mutant kinesin restores axonal growth in vivo. Collectively, these findings establish the critical role of the TUBB3-R262 residue for mediating kinesin interaction, which in turn is required for normal axonal growth and brain development. PMID:26775887

  11. Saccharin enhances neurite extension by regulating organization of the microtubules.

    PubMed

    Yamashita, Hiroo; Muroi, Yoshikage; Ishii, Toshiaki

    2013-11-06

    In the present study, we found that saccharin, an artificial calorie-free sweetener, promotes neurite extension in the cultured neuronal cells. The purposes of this study are to characterize the effect of saccharine on neurite extension and to determine how saccharin enhances neurite extension. The analyses were performed using mouse neuroblastoma N1E-115 cells and rat pheochromocytoma PC12 cells. Neurite extension was evaluated by counting the cells bearing neurites and measuring the length of neurites. Formation, severing and transportation of the microtubules were evaluated by immunostaining and western blotting analysis. Deprivation of glucose increased the number of N1E-115 cells bearing long processes. And the effect was inhibited by addition of glucose. Saccharin increased the number of these cells bearing long processes in a dose-dependent manner and total neurite length and longest neurite length in each cell. Saccharin also had a similar effect on NGF-treated PC12 cells. Saccharin increased the amount of the microtubules reconstructed after treatment with nocodazole, a disruptor of microtubules. The effect of saccharin on microtubule reconstruction was not influenced by dihydrocytochalasin B, an inhibitor of actin polymerization, indicating that saccharin enhances microtubule formation without requiring actin dynamics. In the cells treated with vinblastine, an inhibitor of microtubule polymerization, after microtubule reorganization, filamentous microtubules were observed more distantly from the centrosome in saccharin-treated cells, indicating that saccharin enhances microtubule severing and/or transportation. These results suggest that saccharin enhances neurite extension by promoting microtubule organization. © 2013.

  12. 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

  13. 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

  14. Microtubule Stabilization Leads to Growth Reorientation in Arabidopsis Trichomes

    PubMed Central

    Mathur, Jaideep; Chua, Nam-Hai

    2000-01-01

    The single-cell trichomes in wild-type Arabidopsis are either unbranched or have two to five branches. Using transgenic Arabidopsis plants expressing a green fluorescent protein–microtubule-associated protein4 fusion protein, which decorates the microtubular cytoskeleton, we observed that during trichome branching, microtubules reorient with respect to the longitudinal growth axis. Considering branching to be a localized microtubule-dependent growth reorientation event, we investigated the effects of microtubule-interacting drugs on branch induction in trichomes. In unbranched trichomes of the mutant stichel, a change in growth directionality, closely simulating branch initiation, could be elicited by a short treatment with paclitaxel, a microtubule-stabilizing drug, but not with microtubule-disrupting drugs. The growth reorientation appeared to be linked to increased microtubule stabilization and to aster formation in the treated trichomes. Taxol-induced microtubule stabilization also led to the initiation of new branch points in the zwichel mutant of Arabidopsis, which is defective in a kinesin-like microtubule motor protein and possesses trichomes that are less branched. Our observations suggest that trichome cell branching in Arabidopsis might be mediated by transiently stabilized microtubular structures, which may form a component of a multiprotein complex required to reorient freshly polymerizing microtubules into new growth directions. PMID:10760237

  15. Microtubules Growth Rate Alteration in Human Endothelial Cells

    PubMed Central

    Alieva, Irina B.; Zemskov, Evgeny A.; Kireev, Igor I.; Gorshkov, Boris A.; Wiseman, Dean A.; Black, Stephen M.; Verin, Alexander D.

    2010-01-01

    To understand how microtubules contribute to the dynamic reorganization of the endothelial cell (EC) cytoskeleton, we established an EC model expressing EB3-GFP, a protein that marks microtubule plus-ends. Using this model, we were able to measure microtubule growth rate at the centrosome region and near the cell periphery of a single human EC and in the EC monolayer. We demonstrate that the majority of microtubules in EC are dynamic, the growth rate of their plus-ends is highest in the internal cytoplasm, in the region of the centrosome. Growth rate of microtubule plus-ends decreases from the cell center toward the periphery. Our data suggest the existing mechanism(s) of local regulation of microtubule plus-ends growth in EC. Microtubule growth rate in the internal cytoplasm of EC in the monolayer is lower than that of single EC suggesting the regulatory effect of cell-cell contacts. Centrosomal microtubule growth rate distribution in single EC indicated the presence of two subpopulations of microtubules with “normal” (similar to those in monolayer EC) and “fast” (three times as much) growth rates. Our results indicate functional interactions between cell-cell contacts and microtubules. PMID:20445745

  16. Microtubules growth rate alteration in human endothelial cells.

    PubMed

    Alieva, Irina B; Zemskov, Evgeny A; Kireev, Igor I; Gorshkov, Boris A; Wiseman, Dean A; Black, Stephen M; Verin, Alexander D

    2010-01-01

    To understand how microtubules contribute to the dynamic reorganization of the endothelial cell (EC) cytoskeleton, we established an EC model expressing EB3-GFP, a protein that marks microtubule plus-ends. Using this model, we were able to measure microtubule growth rate at the centrosome region and near the cell periphery of a single human EC and in the EC monolayer. We demonstrate that the majority of microtubules in EC are dynamic, the growth rate of their plus-ends is highest in the internal cytoplasm, in the region of the centrosome. Growth rate of microtubule plus-ends decreases from the cell center toward the periphery. Our data suggest the existing mechanism(s) of local regulation of microtubule plus-ends growth in EC. Microtubule growth rate in the internal cytoplasm of EC in the monolayer is lower than that of single EC suggesting the regulatory effect of cell-cell contacts. Centrosomal microtubule growth rate distribution in single EC indicated the presence of two subpopulations of microtubules with "normal" (similar to those in monolayer EC) and "fast" (three times as much) growth rates. Our results indicate functional interactions between cell-cell contacts and microtubules.

  17. Tubulin acetylation protects long-lived microtubules against mechanical ageing.

    PubMed

    Portran, Didier; Schaedel, Laura; Xu, Zhenjie; Théry, Manuel; Nachury, Maxence V

    2017-04-01

    Long-lived microtubules endow the eukaryotic cell with long-range transport abilities. While long-lived microtubules are acetylated on Lys40 of α-tubulin (αK40), acetylation takes place after stabilization and does not protect against depolymerization. Instead, αK40 acetylation has been proposed to mechanically stabilize microtubules. Yet how modification of αK40, a residue exposed to the microtubule lumen and inaccessible to microtubule-associated proteins and motors, could affect microtubule mechanics remains an open question. Here we develop FRET-based assays that report on the lateral interactions between protofilaments and find that αK40 acetylation directly weakens inter-protofilament interactions. Congruently, αK40 acetylation affects two processes largely governed by inter-protofilament interactions, reducing the nucleation frequency and accelerating the shrinkage rate. Most relevant to the biological function of acetylation, microfluidics manipulations demonstrate that αK40 acetylation enhances flexibility and confers resilience against repeated mechanical stresses. Thus, unlike deacetylated microtubules that accumulate damage when subjected to repeated stresses, long-lived microtubules are protected from mechanical ageing through their acquisition of αK40 acetylation. In contrast to other tubulin post-translational modifications that act through microtubule-associated proteins, motors and severing enzymes, intraluminal acetylation directly tunes the compliance and resilience of microtubules.

  18. 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.

  19. Mitochondria drive autophagy pathology via microtubule disassembly

    PubMed Central

    Arduíno, Daniela M.; Esteves, A. Raquel; Cardoso, Sandra Morais

    2013-01-01

    Neurons are exquisitely dependent on quality control systems to maintain a healthy intracellular environment. A permanent assessment of protein and organelle “quality” allows a coordinated action between repair and clearance of damage proteins and dysfunctional organelles. Impairments in the intracellular clearance mechanisms in long-lived postmitotic cells, like neurons, result in the progressive accumulation of damaged organelles and aggregates of aberrant proteins. Using cells bearing Parkinson disease (PD) patients’ mitochondria, we demonstrated that aberrant accumulation of autophagosomes in PD, commonly interpreted as an abnormal induction of autophagy, is instead due to defective autophagic clearance. This defect is a consequence of alterations in the microtubule network driven by mitochondrial dysfunction that hinder mitochondria and autophagosome trafficking. We uncover mitochondria and microtubule-directed traffic as main players in the regulation of autophagy in PD. PMID:23075854

  20. Mechanical splitting of microtubules into protofilament bundles by surface-bound kinesin-1

    DOE PAGES

    VanDelinder, Virginia; Adams, Peter G.; Bachand, George D.

    2016-12-21

    The fundamental biophysics of gliding microtubule (MT) motility by surface-tethered kinesin-1 motor proteins has been widely studied, as well as applied to capture and transport analytes in bioanalytical microdevices. In these systems, phenomena such as molecular wear and fracture into shorter MTs have been reported due the mechanical forces applied on the MT during transport. In the present work, we show that MTs can be split longitudinally into protofilament bundles (PFBs) by the work performed by surface-bound kinesin motors. We examine the properties of these PFBs using several techniques (e.g., fluorescence microscopy, SEM, AFM), and show that the PFBs continuemore » to be mobile on the surface and display very high curvature compared to MT. Further, higher surface density of kinesin motors and shorter kinesin-surface tethers promote PFB formation, whereas modifying MT with GMPCPP or higher paclitaxel concentrations did not affect PFB formation.« less

  1. Live Cell Imaging Reveals Structural Associations between the Actin and Microtubule Cytoskeleton in Arabidopsis [W] [OA

    PubMed Central

    Sampathkumar, Arun; Lindeboom, Jelmer J.; Debolt, Seth; Gutierrez, Ryan; Ehrhardt, David W.; Ketelaar, Tijs; Persson, Staffan

    2011-01-01

    In eukaryotic cells, the actin and microtubule (MT) cytoskeletal networks are dynamic structures that organize intracellular processes and facilitate their rapid reorganization. In plant cells, actin filaments (AFs) and MTs are essential for cell growth and morphogenesis. However, dynamic interactions between these two essential components in live cells have not been explored. Here, we use spinning-disc confocal microscopy to dissect interaction and cooperation between cortical AFs and MTs in Arabidopsis thaliana, utilizing fluorescent reporter constructs for both components. Quantitative analyses revealed altered AF dynamics associated with the positions and orientations of cortical MTs. Reorganization and reassembly of the AF array was dependent on the MTs following drug-induced depolymerization, whereby short AFs initially appeared colocalized with MTs, and displayed motility along MTs. We also observed that light-induced reorganization of MTs occurred in concert with changes in AF behavior. Our results indicate dynamic interaction between the cortical actin and MT cytoskeletons in interphase plant cells. PMID:21693695

  2. Mechanical splitting of microtubules into protofilament bundles by surface-bound kinesin-1

    SciTech Connect

    VanDelinder, Virginia; Adams, Peter G.; Bachand, George D.

    2016-12-21

    The fundamental biophysics of gliding microtubule (MT) motility by surface-tethered kinesin-1 motor proteins has been widely studied, as well as applied to capture and transport analytes in bioanalytical microdevices. In these systems, phenomena such as molecular wear and fracture into shorter MTs have been reported due the mechanical forces applied on the MT during transport. In the present work, we show that MTs can be split longitudinally into protofilament bundles (PFBs) by the work performed by surface-bound kinesin motors. We examine the properties of these PFBs using several techniques (e.g., fluorescence microscopy, SEM, AFM), and show that the PFBs continue to be mobile on the surface and display very high curvature compared to MT. Further, higher surface density of kinesin motors and shorter kinesin-surface tethers promote PFB formation, whereas modifying MT with GMPCPP or higher paclitaxel concentrations did not affect PFB formation.

  3. Mechanical splitting of microtubules into protofilament bundles by surface-bound kinesin-1

    PubMed Central

    VanDelinder, Virginia; Adams, Peter G.; Bachand, George D.

    2016-01-01

    The fundamental biophysics of gliding microtubule (MT) motility by surface-tethered kinesin-1 motor proteins has been widely studied, as well as applied to capture and transport analytes in bioanalytical microdevices. In these systems, phenomena such as molecular wear and fracture into shorter MTs have been reported due the mechanical forces applied on the MT during transport. In the present work, we show that MTs can be split longitudinally into protofilament bundles (PFBs) by the work performed by surface-bound kinesin motors. We examine the properties of these PFBs using several techniques (e.g., fluorescence microscopy, SEM, AFM), and show that the PFBs continue to be mobile on the surface and display very high curvature compared to MT. Further, higher surface density of kinesin motors and shorter kinesin-surface tethers promote PFB formation, whereas modifying MT with GMPCPP or higher paclitaxel concentrations did not affect PFB formation. PMID:28000714

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

    PubMed

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

    2006-10-01

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

  5. Kinesin motors in plants: from subcellular dynamics to motility regulation.

    PubMed

    Lee, Yuh-Ru Julie; Qiu, Weihong; Liu, Bo

    2015-12-01

    Plants produce enormous forms of the microtubule (MT)-based motor kinesins that have been inspiring plant cell biologists to uncover their functions in relation to plant growth and development. Subcellular localization of kinesin proteins detected through live-cell imaging or immunofluorescence microscopy has provided great insights into the functions of these motors. Dozens of mitotic kinesins exhibit particularly splendid localization patterns from chromosomes and kinetochores to MT arrays like the preprophase band, spindle poles, the spindle midzone, phragmoplast distal ends, and the phragmoplast midzone. Different subcellular localizations indicate distinct functions of these motors that are yet to be characterized. The localization difference between plant kinesins and their animal counterparts implies mechanistic differences in mitosis and cytokinesis between the two kingdoms. When many forms of kinesins are present simultaneously, it becomes critical that their motility is differentially regulated with spatial and temporal precision. Insights into regulatory mechanisms of motors can often be brought about by in vitro single-molecule biophysical studies. Significant advances are expected in this area in the coming years owing to rapid technological advances that are being brought to various model plants. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. 21 CFR 876.1725 - Gastrointestinal motility monitoring system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Gastrointestinal motility monitoring system. 876... Gastrointestinal motility monitoring system. (a) Identification. A gastrointestinal motility monitoring system is a... esophageal motility monitor and tube, the gastrointestinal motility (electrical) system, and...

  7. 21 CFR 876.1725 - Gastrointestinal motility monitoring system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Gastrointestinal motility monitoring system. 876... Gastrointestinal motility monitoring system. (a) Identification. A gastrointestinal motility monitoring system is a... esophageal motility monitor and tube, the gastrointestinal motility (electrical) system, and...

  8. 21 CFR 876.1725 - Gastrointestinal motility monitoring system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Gastrointestinal motility monitoring system. 876... Gastrointestinal motility monitoring system. (a) Identification. A gastrointestinal motility monitoring system is a... esophageal motility monitor and tube, the gastrointestinal motility (electrical) system, and...

  9. 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.

  10. Biological Information Processing in Single Microtubules

    DTIC Science & Technology

    2012-02-15

    radio wave to the proteins in a heat bath. Experiment: We felt three years back in 2008, that it was not sufficient just to try to build the...solution individually or as a combination, produced microtubules were isolated for single nanowire measurement of resonance levels. Ultrasound power...varied between pico-watt to femto-watt, if Mg2+ is not added, effect of ultrasound pumping is not observed, so Mg2+ is essential, GTP is not. Phase

  11. Microtubule Actin Cross-Linking Factor (Macf)

    PubMed Central

    Leung, Conrad L.; Sun, Dongming; Zheng, Min; Knowles, David R.; Liem, Ronald K.H.

    1999-01-01

    We cloned and characterized a full-length cDNA of mouse actin cross-linking family 7 (mACF7) by sequential rapid amplification of cDNA ends–PCR. The completed mACF7 cDNA is 17 kb and codes for a 608-kD protein. The closest relative of mACF7 is the Drosophila protein Kakapo, which shares similar architecture with mACF7. mACF7 contains a putative actin-binding domain and a plakin-like domain that are highly homologous to dystonin (BPAG1-n) at its NH2 terminus. However, unlike dystonin, mACF7 does not contain a coiled–coil rod domain; instead, the rod domain of mACF7 is made up of 23 dystrophin-like spectrin repeats. At its COOH terminus, mACF7 contains two putative EF-hand calcium-binding motifs and a segment homologous to the growth arrest–specific protein, Gas2. In this paper, we demonstrate that the NH2-terminal actin-binding domain of mACF7 is functional both in vivo and in vitro. More importantly, we found that the COOH-terminal domain of mACF7 interacts with and stabilizes microtubules. In transfected cells full-length mACF7 can associate not only with actin but also with microtubules. Hence, we suggest a modified name: MACF (microtubule actin cross-linking factor). The properties of MACF are consistent with the observation that mutations in kakapo cause disorganization of microtubules in epidermal muscle attachment cells and some sensory neurons. PMID:10601340

  12. Molecular Communication: Simulation of Microtubule Topology

    NASA Astrophysics Data System (ADS)

    Moore, Michael J.; Enomoto, Akihiro; Nakano, Tadashi; Kayasuga, Atsushi; Kojima, Hiroaki; Sakakibara, Hitoshi; Oiwa, Kazuhiro; Suda, Tatsuya

    Molecular communication is one method for communication among biological nanomachines. Nanomachines are artificial or biological nano-scale devices that perform simple computation, sensing, or actuation. Future applications using nanomachines may require various communication mechanisms. For example, broadcast is one primitive communication for transmission from one sender to many receivers. In this paper, we discuss preliminary work on designing a molecular communication system that is adapted from the molecular motor transport mechanism existing in biological cells. In the proposed molecular motor mechanism, a sender releases information molecules, and molecular motors transport the information molecules along microtubule filaments to receiver nanomachines up to hundreds of micrometers away. This paper describes some possible arrangements for microtubule filaments and simulations to evaluate sending of one information molecule to many receivers. The simulation results indicate that the proposed molecular motor system transports simulated information molecules (100nm radius spheres) more quickly than a diffusion-only communication and that placement of receivers at the plus-end of microtubules results in lower propagation delay.

  13. Mechanics of microtubules and viral capsids

    NASA Astrophysics Data System (ADS)

    Schmidt, Christoph F.

    2004-03-01

    Polymeric macromolecular assemblies play crucial roles in biology, from DNA to the cytoskeleton or the cell membrane. I will report on recent measurements of the elastic properties of two types of 2D-crystalline protein shells which we have probed at the nanometer scale by indentation with a scanning force microscope (SFM) tip. Microtubules are cylindrical shells and we find a linear elastic regime that can be described by both thin-shell theory and finite element methods, in which microtubules are modeled as homogeneous hollow tubes. We also find a non-linear regime and catastrophic collapse of the microtubules under large loads. The main physics of protein shells at the nanometer scale shows simultaneously aspects of continuum elasticity in their linear response, as well as molecular graininess in their non-linear behavior. Bacteriophages use highly ordered proteinaceous shells to protect their genome from the environment and, interestingly, also to store elastic energy for the injection process. We have studied empty and filled bacteriophage Phi29 shells, again by SFM indentation. These shells are approximately ellipsoidal. We again find a regime of linear elastic response, followed by non-linear response and break-down. The linear regime can again be described by thin shell theory, assuming a homogeneous material, but we observe, already in the linear regime, signatures of the substructure of the shells.

  14. 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'.

  15. Microtubule self-organisation depends upon gravity

    NASA Astrophysics Data System (ADS)

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

    2001-01-01

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

  16. Validation of microtubule-associated Tobacco mosaic virus RNA movement and involvement of microtubule-aligned particle trafficking.

    PubMed

    Boyko, Vitaly; Hu, Quanan; Seemanpillai, Mark; Ashby, Jamie; Heinlein, Manfred

    2007-08-01

    Functional studies of Tobacco mosaic virus (TMV) infection using virus derivatives expressing functional, dysfunctional, and temperature-sensitive movement protein (MP) mutants indicated that the cell-to-cell transport of TMV RNA is functionally correlated with the association of MP with microtubules. However, the role of microtubules in the movement process during early infection remains unclear, since MP accumulates on microtubules rather late in infection and treatment of plants with microtubule-disrupting agents fails to strongly interfere with cell-to-cell movement of TMV RNA. To further test the role of microtubules in TMV cell-to-cell movement, we investigated TMV strain Ni2519, which is temperature-sensitive for movement. We demonstrate that the temperature-sensitive defect in movement is correlated with temperature-sensitive changes in the localization of MP to microtubules. Furthermore, we show that during early phases of recovery from non-permissive conditions, the MP localizes to microtubule-associated particles. Similar particles are found in cells at the leading front of spreading TMV infection sites. Initially mobile, the particles become immobile when MP starts to accumulate along the length of the particle-associated microtubules. Our observations confirm a role for microtubules in the spread of TMV infection and associate this role with microtubule-associated trafficking of MP-containing particles in cells engaged in the cell-to-cell movement of the TMV genome.

  17. A novel microtubule-binding motif identified in a high molecular weight microtubule-associated protein from Trypanosoma brucei

    PubMed Central

    1992-01-01

    The major component of the cytoskeleton of the parasitic hemoflagellate Trypanosoma brucei is a membrane skeleton which consists of a single layer of tightly spaced microtubules. This array encloses the entire cell body, and it is apposed to, and connected with, the overlying cell membrane. The microtubules of this array contain numerous microtubule- associated proteins. Prominent among those is a family of high molecular weight, repetitive proteins which consist to a large extent of tandemly arranged 38-amino acid repeat units. The binding of one of these proteins, MARP-1, to microtubules has now been characterized in vitro and in vivo. MARP-1 binds to microtubules via tubulin domains other than the COOH-termini used by microtubule-associated proteins from mammalian brain, e.g., MAP2 or Tau. In vitro binding assays using recombinant protein, as well as transfection of mammalian cell lines, have established that the repetitive 38-amino acid repeat units represent a novel microtubule-binding motif. This motif is very similar in length to those of the mammalian microtubule-associated proteins Tau, MAP2, and MAP-U, but both its sequence and charge are different. The observation that the microtubule-binding motifs both of the neural and the trypanosomal proteins are of similar length may reflect the fact that both mediate binding to the same repetitive surface, the microtubule, while their sequence and charge differences are in agreement with the observation that they interact with different domains of the tubulins. PMID:1348252

  18. A novel microtubule-binding motif identified in a high molecular weight microtubule-associated protein from Trypanosoma brucei.

    PubMed

    Hemphill, A; Affolter, M; Seebeck, T

    1992-04-01

    The major component of the cytoskeleton of the parasitic hemoflagellate Trypanosoma brucei is a membrane skeleton which consists of a single layer of tightly spaced microtubules. This array encloses the entire cell body, and it is apposed to, and connected with, the overlying cell membrane. The microtubules of this array contain numerous microtubule-associated proteins. Prominent among those is a family of high molecular weight, repetitive proteins which consist to a large extent of tandemly arranged 38-amino acid repeat units. The binding of one of these proteins, MARP-1, to microtubules has now been characterized in vitro and in vivo. MARP-1 binds to microtubules via tubulin domains other than the COOH-termini used by microtubule-associated proteins from mammalian brain, e.g., MAP2 or Tau. In vitro binding assays using recombinant protein, as well as transfection of mammalian cell lines, have established that the repetitive 38-amino acid repeat units represent a novel microtubule-binding motif. This motif is very similar in length to those of the mammalian microtubule-associated proteins Tau, MAP2, and MAP-U, but both its sequence and charge are different. The observation that the microtubule-binding motifs both of the neural and the trypanosomal proteins are of similar length may reflect the fact that both mediate binding to the same repetitive surface, the microtubule, while their sequence and charge differences are in agreement with the observation that they interact with different domains of the tubulins.

  19. Gaslike model of social motility.

    PubMed

    Parravano, A; Reyes, L M

    2008-08-01

    We propose a model to represent the motility of social elements. The model is completely deterministic, possesses a small number of parameters, and exhibits a series of properties that are reminiscent of the behavior of communities in social-ecological competition; these are (i) similar individuals attract each other; (ii) individuals can form stable groups; (iii) a group of similar individuals breaks into subgroups if it reaches a critical size; (iv) interaction between groups can modify the distribution of the elements as a result of fusion, fission, or pursuit; (v) individuals can change their internal state by interaction with their neighbors. The simplicity of the model and its richness of emergent behaviors, such as, for example, pursuit between groups, make it a useful toy model to explore a diversity of situations by changing the rule by which the internal state of individuals is modified by the interactions with the environment.

  20. Astral Microtubule Dynamics in Yeast: A Microtubule-based Searching Mechanism for Spindle Orientation and Nuclear Migration into the Bud

    PubMed Central

    Shaw, Sidney L.; Yeh, Elaine; Maddox, Paul; Salmon, E.D.; Bloom, Kerry

    1997-01-01

    Localization of dynein–green fluorescent protein (GFP) to cytoplasmic microtubules allowed us to obtain one of the first views of the dynamic properties of astral microtubules in live budding yeast. Several novel aspects of microtubule function were revealed by time-lapse, three-dimensional fluorescence microscopy. Astral microtubules, about four to six in number for each pole, exhibited asynchronous dynamic instability throughout the cell cycle, growing at ≅0.3–1.5 μm/min toward the cell surface then switching to shortening at similar velocities back to the spindle pole body (SPB). During interphase, a conical array of microtubules trailed the SPB as the nucleus traversed the cytoplasm. Microtubule disassembly by nocodozole inhibited these movements, indicating that the nucleus was pushed around the interior of the cell via dynamic astral microtubules. These forays were evident in unbudded G1 cells, as well as in late telophase cells after spindle disassembly. Nuclear movement and orientation to the bud neck in S/G2 or G2/M was dependent on dynamic astral microtubules growing into the bud. The SPB and nucleus were then pulled toward the bud neck, and further microtubule growth from that SPB was mainly oriented toward the bud. After SPB separation and central spindle formation, a temporal delay in the acquisition of cytoplasmic dynein at one of the spindle poles was evident. Stable microtubule interactions with the cell cortex were rarely observed during anaphase, and did not appear to contribute significantly to spindle alignment or elongation into the bud. Alterations of microtubule dynamics, as observed in cells overexpressing dynein-GFP, resulted in eventual spindle misalignment. These studies provide the first mechanistic basis for understanding how spindle orientation and nuclear positioning are established and are indicative of a microtubule-based searching mechanism that requires dynamic microtubules for nuclear migration into the bud. PMID:9362516

  1. 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

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

    PubMed

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

    2013-11-01

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

  3. Cell cycle regulation of dynein association with membranes modulates microtubule-based organelle transport.

    PubMed

    Niclas, J; Allan, V J; Vale, R D

    1996-05-01

    Cytoplasmic dynein is a minus end-directed microtubule motor that performs distinct functions in interphase and mitosis. In interphase, dynein transports organelles along microtubules, whereas in metaphase this motor has been implicated in mitotic spindle formation and orientation as well as chromosome segregation. The manner in which dynein activity is regulated during the cell cycle, however, has not been resolved. In this study, we have examined the mechanism by which organelle transport is controlled by the cell cycle in extracts of Xenopus laevis eggs. Here, we show that photocleavage of the dynein heavy chain dramatically inhibits minus end-directed organelle transport and that purified dynein restores this motility, indicating that dynein is the predominant minus end-directed membrane motor in Xenopus egg extracts. By measuring the amount of dynein associated with isolated membranes, we find that cytoplasmic dynein and its activator dynactin detach from the membrane surface in metaphase extracts. The sevenfold decrease in membrane-associated dynein correlated well with the eightfold reduction in minus end-directed membrane transport observed in metaphase versus interphase extracts. Although dynein heavy or intermediate chain phosphorylation did not change in a cell cycle-dependent manner, the dynein light intermediate chain incorporated approximately 12-fold more radiolabeled phosphate in metaphase than in interphase extracts. These studies suggest that cell cycle-dependent phosphorylation of cytoplasmic dynein may regulate organelle transport by modulating the association of this motor with membranes.

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed

    Quarmby, L

    2000-08-01

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

  6. 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.

  7. 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

  8. 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.

  9. Active gel model of amoeboid cell motility

    NASA Astrophysics Data System (ADS)

    Callan-Jones, A. C.; Voituriez, R.

    2013-02-01

    We develop a model of amoeboid cell motility based on active gel theory. Modeling the motile apparatus of a eukaryotic cell as a confined layer of finite length of poroelastic active gel permeated by a solvent, we first show that, due to active stress and gel turnover, an initially static and homogeneous layer can undergo a contractile-type instability to a polarized moving state in which the rear is enriched in gel polymer. This agrees qualitatively with motile cells containing an actomyosin-rich uropod at their rear. We find that the gel layer settles into a steadily moving, inhomogeneous state at long times, sustained by a balance between contractility and filament turnover. In addition, our model predicts an optimal value of the gel-substrate adhesion leading to maximum layer speed, in agreement with cell motility assays. The model may be relevant to motility of cells translocating in complex, confining environments that can be mimicked experimentally by cell migration through microchannels.

  10. Sperm motility under conditions of weightlessness.

    PubMed

    Engelmann, U; Krassnigg, F; Schill, W B

    1992-01-01

    The aim of this study was to determine the differences in motility of frozen and thawed bull spermatozoa under conditions of weightlessness compared with ground conditions. The tests were performed within a series of scientific and technologic experiments under microgravity using sounding rockets in the Technologische Experimente unter Schwerelosigkeit (TEXUS) program launched in Kiruna, North Sweden. Using a computerized sperm motility analyzer, significant differences were found in sperm motility under microgravity compared with sperm under gravitational conditions on earth. Computer analysis showed alterations in straight line and curvilinear velocity, as well as in linearity values. The amount of progressively motile spermatozoa, including all spermatozoa with a velocity > 20 microns/second, increased significantly from 24% +/- 9.5% in the reference test to 49% +/- 7.6% in the microgravity test. In conclusion, there is strong evidence that gravity influences sperm motility.

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

    PubMed

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

    2013-03-01

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

  12. 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.

  13. High EGFR_1 Inside-Out Activated Inflammation-Induced Motility through SLC2A1-CCNB2-HMMR-KIF11-NUSAP1-PRC1-UBE2C.

    PubMed

    Zhou, Huilei; Wang, Lin; Huang, Juxiang; Jiang, Minghu; Zhang, Xiaoyu; Zhang, Liyuan; Wang, Yangming; Jiang, Zhenfu; Zhang, Zhongjie

    2015-01-01

    48 different Pearson mutual-positive-correlation epidermal growth factor receptor (EGFR_1)-activatory molecular feedback, up- and down-stream network was constructed from 171 overlapping of 366 GRNInfer and 223 Pearson under EGFR_1 CC ≥0.25 in high lung adenocarcinoma compared with low human normal adjacent tissues. Our identified EGFR_1 inside-out upstream activated molecular network showed SLC2A1 (solute carrier family 2 (facilitated glucose transporter) member 1), CCNB2 (cyclin B2), HMMR (hyaluronan-mediated motility receptor (RHAMM)), KIF11 (kinesin family member 11), NUSAP1 (nucleolar and spindle associated protein 1), PRC1 (protein regulator of cytokinesis 1), UBE2C (ubiquitin-conjugating enzyme E2C) in high lung adenocarcinoma. EGFR_1 inside-out upstream activated terms network includes intracellular, membrane fraction, cytoplasm, plasma membrane, integral to membrane, basolateral plasma membrane, transmembrane transport, nucleus, cytosol, cell surface; T cell homeostasis, inflammation; microtubule cytoskeleton, embryonic development (sensu Mammalia), cell cycle, mitosis, thymus development, cell division, regulation of cell cycle, Contributed--cellular process--Hs cell cycle KEGG, cytokinesis, M phase, M phase of mitotic cell cycle, estrogen-responsive protein Efp controls cell cycle and breast tumors growth, cell motility, locomotion, locomotory behavior, neoplasm metastasis, spindle pole, spindle microtubule, microtubule motor activity, microtubule-based movement, mitotic spindle organization and biogenesis, mitotic centrosome separation, spindle pole body organization and biogenesis, microtubule-based process, microtubule, cytokinesis after mitosis, mitotic chromosome condensation, establishment of mitotic spindle localization, positive regulation of mitosis, mitotic spindle elongation, spindle organization and biogenesis, positive regulation of exit from mitosis, regulation of cell proliferation, positive regulation of cell proliferation based on

  14. Sunday Driver/JIP3 binds kinesin heavy chain directly and enhances its motility

    PubMed Central

    Sun, Faneng; Zhu, Chuanmei; Dixit, Ram; Cavalli, Valeria

    2011-01-01

    Neuronal development, function and repair critically depend on axonal transport of vesicles and protein complexes, which is mediated in part by the molecular motor kinesin-1. Adaptor proteins recruit kinesin-1 to vesicles via direct association with kinesin heavy chain (KHC), the force-generating component, or via the accessory light chain (KLC). Binding of adaptors to the motor is believed to engage the motor for microtubule-based transport. We report that the adaptor protein Sunday Driver (syd, also known as JIP3 or JSAP1) interacts directly with KHC, in addition to and independently of its known interaction with KLC. Using an in vitro motility assay, we show that syd activates KHC for transport and enhances its motility, increasing both KHC velocity and run length. syd binding to KHC is functional in neurons, as syd mutants that bind KHC but not KLC are transported to axons and dendrites similarly to wild-type syd. This transport does not rely on syd oligomerization with itself or other JIP family members. These results establish syd as a positive regulator of kinesin activity and motility. PMID:21750526

  15. Motor-driven motility of fungal nuclear pores organizes chromosomes and fosters nucleocytoplasmic transport.

    PubMed

    Steinberg, Gero; Schuster, Martin; Theisen, Ulrike; Kilaru, Sreedhar; Forge, Andrew; Martin-Urdiroz, Magdalena

    2012-08-06

    Exchange between the nucleus and the cytoplasm is controlled by nuclear pore complexes (NPCs). In animals, NPCs are anchored by the nuclear lamina, which ensures their even distribution and proper organization of chromosomes. Fungi do not possess a lamina and how they arrange their chromosomes and NPCs is unknown. Here, we show that motor-driven motility of NPCs organizes the fungal nucleus. In Ustilago maydis, Aspergillus nidulans, and Saccharomyces cerevisiae fluorescently labeled NPCs showed ATP-dependent movements at ~1.0 µm/s. In S. cerevisiae and U. maydis, NPC motility prevented NPCs from clustering. In budding yeast, NPC motility required F-actin, whereas in U. maydis, microtubules, kinesin-1, and dynein drove pore movements. In the latter, pore clustering resulted in chromatin organization defects and led to a significant reduction in both import and export of GFP reporter proteins. This suggests that fungi constantly rearrange their NPCs and corresponding chromosomes to ensure efficient nuclear transport and thereby overcome the need for a structural lamina.

  16. Motility, morphology and phylogeny of the plasmodial worm, Ceratomyxa vermiformis n. sp. (Cnidaria: Myxozoa: Myxosporea).

    PubMed

    Adriano, E A; Okamura, B

    2017-02-01

    The Myxozoa demonstrate extensive morphological simplification and miniaturization relative to their free-living cnidarian ancestors. This is particularly pronounced in the highly derived myxosporeans, which develop as plasmodia and pseudoplasmodia. To date, motility in these stages has been linked with membrane deformation (e.g. as pseudopodia and mobile folds). Here we illustrate a motile, elongate plasmodium that undergoes coordinated undulatory locomotion, revealing remarkable convergence to a functional worm at the cellular level. Ultrastructural and confocal analyses of these plasmodia identify a highly differentiated external layer containing an actin-rich network, long tubular mitochondria, abundant microtubules, a secreted glycocalyx layer, and an internal region where sporogony occurs and which contains homogeneously distributed granular/fibrillar material. We consider how some of these features may support motility. We also describe the species based on spore morphology and SSU rDNA sequence data, undertake molecular phylogenetic analysis to place it within an early-diverging clade of the ceratomyxids, and evaluate the resultant implications for classification (validity of the genus Meglitschia) and for inferring early host environments (freshwater) of ceratomyxids.

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

    NASA Astrophysics Data System (ADS)

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

    2009-03-01

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

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

    PubMed

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

    2017-08-29

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

  19. 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

  20. 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

  1. Mechanically cut mitotic spindles: clean cuts and stable microtubules.

    PubMed

    Nicklas, R B; Lee, G M; Rieder, C L; Rupp, G

    1989-11-01

    We have discovered an easy way to cut through the mitotic spindle at any desired place. Spindles of demembranated cricket or grasshopper spermatocytes were severed with a microneedle between the chromosomes and one pole, and the cut-off polar piece was swept away. Spindle structure and microtubule dynamics in cut spindles were studied by anti-tubulin immunostaining and electron microscopy. The cut is clean: all microtubules are severed and only a few extend beyond the others. This provides the basis for a clear test of whether traction fibers pull chromosomes to the pole in anaphase, because the putative traction fiber is cleanly severed. Cutting creates new plus ends on microtubules in the cut-off polar piece and new minus ends on microtubules in the main spindle body. The microtubules with new plus ends are unstable, as expected from the dynamic instability of microtubules. However, the microtubules with new minus ends are as stable as uncut microtubules in the same spindle. Our mechanical method of cutting microtubules very likely creates native, reactive ends, and therefore the surprising stability of new minus ends is genuinely interesting, not an artifact of cutting.

  2. Producing Conditional Mutants for Studying Plant Microtubule Function

    SciTech Connect

    Richard Cyr

    2009-09-29

    The cytoskeleton, and in particular its microtubule component, participates in several processes that directly affect growth and development in higher plants. Normal cytoskeletal function requires the precise and orderly arrangement of microtubules into several cell cycle and developmentally specific arrays. One of these, the cortical array, is notable for its role in directing the deposition of cellulose (the most prominent polymer in the biosphere). An understanding of how these arrays form, and the molecular interactions that contribute to their function, is incomplete. To gain a better understanding of how microtubules work, we have been working to characterize mutants in critical cytoskeletal genes. This characterization is being carried out at the subcellular level using vital microtubule gene constructs. In the last year of funding colleagues have discovered that gamma-tubulin complexes form along the lengths of cortical microtubules where they act to spawn new microtubules at a characteristic 40 deg angle. This finding complements nicely the finding from our lab (which was funded by the DOE) showing that microtubule encounters are angle dependent; high angles encounters results in catastrophic collisions while low angle encounters result in favorable zippering. The finding of a 40 deg spawn of new microtubules from extant microtubule, together with aforementioned rules of encounters, insures favorable co-alignment in the array. I was invited to write a New and Views essay on this topic and a PDF is attached (News and Views policy does not permit funding acknowledgments and so I was not allowed to acknowledge support from the DOE).

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

    NASA Astrophysics Data System (ADS)

    Sept, David; Mackintosh, Fred C.

    2010-01-01

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

  4. The parallel lives of microtubules and cellulose microfibrils.

    PubMed

    Lloyd, Clive; Chan, Jordi

    2008-12-01

    A major breakthrough was the recent discovery that cellulose synthases really do move along the plasma membrane upon tracks provided by the underlying cortical microtubules. It emphasized the cytoplasmic contribution to cell wall organization. A growing number of microtubule-associated proteins has been identified and shown to affect the way that microtubules are ordered, with downstream effects on the pattern of growth. The dynamic properties of microtubules turn out to be key in understanding the behaviour of the global array and good progress has been made in deciphering the rules by which the array is self-organized.

  5. INSIGHTS INTO ANTI-PARALLEL MICROTUBULE CROSSLINKING BY PRC1, A CONSERVED NON-MOTOR MICROTUBULE BINDING PROTEIN

    PubMed Central

    Subramanian, Radhika; Wilson-Kubalek, Elizabeth M.; Arthur, Christopher P.; Bick, Matthew J.; Campbell, Elizabeth A.; Darst, Seth A.; Milligan, Ronald A.; Kapoor, Tarun M.

    2010-01-01

    SUMMARY Formation of microtubule architectures, required for cell shape maintenance in yeast, directional cell expansion in plants and cytokinesis in eukaryotes, depends on antiparallel microtubule crosslinking by the conserved MAP65 protein family. Here, we combine structural and single molecule fluorescence methods to examine how PRC1, the human MAP65, crosslinks antiparallel microtubules. We find that PRC1's microtubule binding is mediated by a structured domain with a spectrin-fold and an unstructured Lys/Arg-rich domain. These two domains, at each end of a homodimer, are connected by a linkage that is flexible on single microtubules, but forms well-defined crossbridges between antiparallel filaments. Further, we show that PRC1 crosslinks do not substantially resist filament sliding by motor proteins in vitro. Together, our data show how MAP65s, by combining structural flexibility and rigidity, tune microtubule associations to establish compliant crosslinks that selectively `mark' antiparallel overlap in dynamic cytoskeletal networks. PMID:20691902

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

    PubMed

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

    2015-10-20

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

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

    NASA Astrophysics Data System (ADS)

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

    1991-05-01

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

  8. Effects of pharmacological agents on gastrointestinal motility.

    PubMed

    Gerring, E L

    1989-08-01

    The control mechanisms of gastrointestinal motility are complex. Extrinsic neurohormonal effects modulate an intrinsic system, often called the "gut brain," composed of nervous and neuropeptide components. To exert pharmacologic influence on GI motility, use is made of agents that mimic the external control system. Agents that stimulate opioid receptors, block adrenoceptors, block or facilitate acetylcholine action, or antagonize the action of prostaglandins are used to effect changes in GI motility. The major indications for pharmacologic intervention are to increase motility in constipation, to reduce it in most cases of diarrhea, and to restore propulsive coordination in postoperative ileus. In cases of clinical colic the primary requirement is control of pain. Agents used for this purpose may adversely affect motility, and choice requires knowledge of their actions in this respect. In addition, drugs used for other purposes, anthelmintics for instance, may also influence gut motility. A synopsis of the actions of the agents commonly employed in GI motility control and some associated drugs are displayed in Table 3. Recent advances in the understanding of drug action on the gut should help in the selection of drugs for clinical use.

  9. Regulation of flagellar motility during biofilm formation

    PubMed Central

    Guttenplan, Sarah B.; Kearns, Daniel B.

    2013-01-01

    Many bacteria swim in liquid or swarm over solid surfaces by synthesizing rotary flagella. The same bacteria that are motile also commonly form non-motile multicellular aggregates held together by an extracellular matrix called biofilms. Biofilms are an important part of the lifestyle of pathogenic bacteria and it is assumed that there is a motility-to-biofilm transition wherein the inhibition of motility promotes biofilm formation. The transition is largely inferred from regulatory mutants that reveal the opposite regulation of the two phenotypes. Here we review the regulation of motility during biofilm formation in Bacillus, Pseudomonas, Vibrio, and Escherichia, and we conclude that the motility-to-biofilm transition, if necessary, likely involves two steps. In the short term, flagella are functionally regulated to either inhibit rotation or modulate the basal flagellar reversal frequency. Over the long term, flagellar gene transcription is inhibited and in the absence of de novo synthesis, flagella are likely diluted to extinction through growth. Both short term and long term control is likely important to the motility-to-biofilm transition to stabilize aggregates and optimize resource investment. We emphasize the newly discovered classes of flagellar functional regulators and speculate that others await discovery in the context of biofilm formation. PMID:23480406

  10. Control of microtubule nucleation and stability in Madin-Darby canine kidney cells: the occurrence of noncentrosomal, stable detyrosinated microtubules

    PubMed Central

    1987-01-01

    The microtubule-nucleating activity of centrosomes was analyzed in fibroblastic (Vero) and in epithelial cells (PtK2, Madin-Darby canine kidney [MDCK]) by double-immunofluorescence labeling with anti- centrosome and antitubulin antibodies. Most of the microtubules emanated from the centrosomes in Vero cells, whereas the microtubule network of MDCK cells appeared to be noncentrosome nucleated and randomly organized. The pattern of microtubule organization in PtK2 cells was intermediate to the patterns observed in the typical fibroblastic and epithelial cells. The two centriole cylinders were tightly associated and located close to the nucleus in Vero and PtK2 cells. In MDCK cells, however, they were clearly separated and electron microscopy revealed that they nucleated only a few microtubules. The stability of centrosomal and noncentrosomal microtubules was examined by treatment of these different cell lines with various concentrations of nocodazole. 1.6 microM nocodazole induced an almost complete depolymerization of microtubules in Vero cells; some centrosome nucleated microtubules remained in PtK2 cells, while many noncentrosomal microtubules resisted that treatment in MDCK cells. Centrosomal and noncentrosomal microtubules regrew in MDCK cells with similar kinetics after release from complete disassembly by high concentrations of nocodazole (33 microM). During regrowth, centrosomal microtubules became resistant to 1.6 microM nocodazole before the noncentrosomal ones, although the latter eventually predominate. We suggest that in MDCK cells, microtubules grow and shrink as proposed by the dynamic instability model but the presence of factors prevents them from complete depolymerization. This creates seeds for reelongation that compete with nucleation off the centrosome. By using specific antibodies, we have shown that the abundant subset of nocodazole- resistant microtubules in MDCK cells contained detyrosinated alpha- tubulin (glu tubulin). On the other hand

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

    PubMed

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

    2017-01-01

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

  12. The microtubule plus-end tracking protein ARMADILLO-REPEAT KINESIN1 promotes microtubule catastrophe in Arabidopsis.

    PubMed

    Eng, Ryan Christopher; Wasteneys, Geoffrey O

    2014-08-01

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

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

    PubMed

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

    2004-06-01

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

  14. Effects of fluoro-doxorubicin (ME2303) on microtubules: influence of different classes of microtubule-associated proteins.

    PubMed

    Fromes, Y; Gounon, P; Tapiero, H; Fellous, A

    1996-08-01

    Anthracyclines are among the most useful agents for the treatment of neoplastic disease, but their clinical use is limited by progressive cardiomyopathy. A few studies have suggested the role of microtubules for the understanding of this toxicity. By using kinetic and structural studies, we demonstrate the disorganizing action of fluoro-doxorubicin, a novel anthracycline, on the microtubule system. Microtubules have a rich and complex composition in relation to their numerous functions in cells. In the present study, we investigate the role of two major microtubule-associated protein (MAP) families, Tau and MAP2. Both MAP families are responsible for the properties of different classes of microtubules. We show the differential effect of fluoro-doxorubicin on these two classes of microtubules. Furthermore, we show that fluoro-doxorubicin is able to affect the capacity of purified tubulin to form normal microtubules. This study confirms that anthracyclines may interfer with the microtubule organization. We suggest that some classes of microtubules, with regard to their MAP composition, may be affected more specifically in cardiac myocytes.

  15. Modulation of mammalian sperm motility by quercetin.

    PubMed

    Nass-Arden, L; Breitbart, H

    1990-04-01

    The flavonoid quercetin inhibits collective motility of ejaculated ram spermatozoa in the first 2 hr of incubation; during the next 3-4 hr motility is stimulated. To explain this interesting effect, we followed the influence of quercetin on sperm glycolysis, extracellular pH, ATP content, mitochondrial respiration, and lipid peroxidation. The collective motility of untreated cells is decreased to about 40% of the original motility during two hours of incubation. During this time, the rate of glycolysis is constant, respiration rate is increasing, there is no change in ATP content, the rate of lipid peroxidation is very slow, and the extracellular pH became very acidic (pH 5.5). It is concluded that motility is decreased due to this acidification. This acidification is prevented to some extent by quercetin, which indirectly inhibits glycolysis. Quercetin inhibits motility due to the inhibition of the plasma membrane calcium pump, as we showed previously (Breitbart et al., J Biol Chem 260:11548-11553, 1985). The motility of untreated cells is arrested after 3.5 hr of incubation, whereas quercetin-treated cells show high motility, which continues for additional 2-3 hr. After 3.5 hr, the control cells show no glycolytic activity, ATP content and respiration rates are decreased, and rate of lipid peroxidation is highly increased. At this time, quercetin-treated cells show no glycolytic activity, only a small decrease in ATP content and respiratory rate, and a very low rate of lipid peroxidation. Based on these data it is concluded that sperm motility after 3.5 hr of incubation is dependent mainly on mitochondrial respiration.(ABSTRACT TRUNCATED AT 250 WORDS)

  16. MARK4 is a novel microtubule-associated proteins/microtubule affinity-regulating kinase that binds to the cellular microtubule network and to centrosomes.

    PubMed

    Trinczek, Bernhard; Brajenovic, Miro; Ebneth, Andreas; Drewes, Gerard

    2004-02-13

    The MARK protein kinases were originally identified by their ability to phosphorylate a serine motif in the microtubule-binding domain of tau that is critical for microtubule binding. Here, we report the cloning and expression of a novel human paralog, MARK4, which shares 75% overall homology with MARK1-3 and is predominantly expressed in brain. Homology is most pronounced in the catalytic domain (90%), and MARK4 readily phosphorylates tau and the related microtubule-associated protein 2 (MAP2) and MAP4. In contrast to the three paralogs that all exhibit uniform cytoplasmic localization, MARK4 colocalizes with the centrosome and with microtubules in cultured cells. Overexpression of MARK4 causes thinning out of the microtubule network, concomitant with a reorganization of microtubules into bundles. In line with these findings, we show that a tandem affinity-purified MARK4 protein complex contains alpha-, beta-, and gamma-tubulin. In differentiated neuroblastoma cells, MARK4 is localized prominently at the tips of neurite-like processes. We suggest that although the four MARK/PAR-1 kinases might play multiple cellular roles in concert with different targets, MARK4 is likely to be directly involved in microtubule organization in neuronal cells and may contribute to the pathological phosphorylation of tau in Alzheimer's disease.

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

    PubMed

    Qin, Tao; Li, Jiejie; Yuan, Ming; Mao, Tonglin

    2012-07-01

    Regulation of cell elongation is important for plant morphogenesis. Many studies have shown that cortical microtubules play crucial roles during cell elongation and that microtubule stability, organization, and dynamics are regulated by microtubule regulatory proteins. Recently, we reported that a novel protein from Arabidopsis, termed microtubule-destabilizing protein 25 (MDP25), functions as a negative regulator of hypocotyl cell elongation. MDP25 destabilizes microtubules and exerts its effect on microtubules as a result of transient elevation of cytosolic calcium levels.

  18. Collective behavior of minus-ended motors in mitotic microtubule asters gliding toward DNA

    NASA Astrophysics Data System (ADS)

    Athale, Chaitanya A.; Dinarina, Ana; Nedelec, Francois; Karsenti, Eric

    2014-02-01

    Microtubules (MTs) nucleated by centrosomes form star-shaped structures referred to as asters. Aster motility and dynamics is vital for genome stability, cell division, polarization and differentiation. Asters move either toward the cell center or away from it. Here, we focus on the centering mechanism in a membrane independent system of Xenopus cytoplasmic egg extracts. Using live microscopy and single particle tracking, we find that asters move toward chromatinized DNA structures. The velocity and directionality profiles suggest a random-walk with drift directed toward DNA. We have developed a theoretical model that can explain this movement as a result of a gradient of MT length dynamics and MT gliding on immobilized dynein motors. In simulations, the antagonistic action of the motor species on the radial array of MTs leads to a tug-of-war purely due to geometric considerations and aster motility resembles a directed random-walk. Additionally, our model predicts that aster velocities do not change greatly with varying initial distance from DNA. The movement of asymmetric asters becomes increasingly super-diffusive with increasing motor density, but for symmetric asters it becomes less super-diffusive. The transition of symmetric asters from superdiffusive to diffusive mobility is the result of number fluctuations in bound motors in the tug-of-war. Overall, our model is in good agreement with experimental data in Xenopus cytoplasmic extracts and predicts novel features of the collective effects of motor-MT interactions.

  19. Collective behavior of minus-ended motors in mitotic microtubule asters gliding toward DNA.

    PubMed

    Athale, Chaitanya A; Dinarina, Ana; Nedelec, Francois; Karsenti, Eric

    2014-02-01

    Microtubules (MTs) nucleated by centrosomes form star-shaped structures referred to as asters. Aster motility and dynamics is vital for genome stability, cell division, polarization and differentiation. Asters move either toward the cell center or away from it. Here, we focus on the centering mechanism in a membrane independent system of Xenopus cytoplasmic egg extracts. Using live microscopy and single particle tracking, we find that asters move toward chromatinized DNA structures. The velocity and directionality profiles suggest a random-walk with drift directed toward DNA. We have developed a theoretical model that can explain this movement as a result of a gradient of MT length dynamics and MT gliding on immobilized dynein motors. In simulations, the antagonistic action of the motor species on the radial array of MTs leads to a tug-of-war purely due to geometric considerations and aster motility resembles a directed random-walk. Additionally, our model predicts that aster velocities do not change greatly with varying initial distance from DNA. The movement of asymmetric asters becomes increasingly super-diffusive with increasing motor density, but for symmetric asters it becomes less super-diffusive. The transition of symmetric asters from superdiffusive to diffusive mobility is the result of number fluctuations in bound motors in the tug-of-war. Overall, our model is in good agreement with experimental data in Xenopus cytoplasmic extracts and predicts novel features of the collective effects of motor-MT interactions.

  20. Direct measurement of conformational strain energy in protofilaments curling outward from disassembling microtubule tips

    PubMed Central

    Driver, Jonathan W; Geyer, Elisabeth A; Bailey, Megan E; Rice, Luke M; Asbury, Charles L

    2017-01-01

    Disassembling microtubules can generate movement independently of motor enzymes, especially at kinetochores where they drive chromosome motility. A popular explanation is the ‘conformational wave’ model, in which protofilaments pull on the kinetochore as they curl outward from a disassembling tip. But whether protofilaments can work efficiently via this spring-like mechanism has been unclear. By modifying a previous assay to use recombinant tubulin and feedback-controlled laser trapping, we directly demonstrate the spring-like elasticity of curling protofilaments. Measuring their mechanical work output suggests they carry ~25% of the energy of GTP hydrolysis as bending strain, enabling them to drive movement with efficiency similar to conventional motors. Surprisingly, a β-tubulin mutant that dramatically slows disassembly has no effect on work output, indicating an uncoupling of disassembly speed from protofilament strain. These results show the wave mechanism can make a major contribution to kinetochore motility and establish a direct approach for measuring tubulin mechano-chemistry. DOI: http://dx.doi.org/10.7554/eLife.28433.001 PMID:28628007

  1. Microtubules Are Essential for Mitochondrial Dynamics–Fission, Fusion, and Motility–in Dictyostelium discoideum

    PubMed Central

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

    2016-01-01

    Mitochondrial function is dependent upon mitochondrial structure which is in turn dependent upon mitochondrial dynamics, including fission, fusion, and motility. Here we examined the relationship between mitochondrial dynamics and the cytoskeleton in Dictyostelium discoideum. Using time-lapse analysis, we quantified mitochondrial fission, fusion, and motility in the presence of cytoskeleton disrupting pharmaceuticals and the absence of the potential mitochondria-cytoskeleton linker protein, CluA. Our results indicate that microtubules are essential for mitochondrial movement, as well as fission and fusion; actin plays a less significant role, perhaps selecting the mitochondria for transport. We also suggest that CluA is not a linker protein but plays an unidentified role in mitochondrial fission and fusion. The significance of our work is to gain further insight into the role the cytoskeleton plays in mitochondrial dynamics and function. By better understanding these processes we can better appreciate the underlying mitochondrial contributions to many neurological disorders characterized by altered mitochondrial dynamics, structure, and/or function. PMID:27047941

  2. Active transport of oil droplets along oriented microtubules by kinesin molecular motors.

    PubMed

    Bottier, Céline; Fattaccioli, Jacques; Tarhan, Mehmet C; Yokokawa, Ryuji; Morin, Fabrice O; Kim, Beomjoon; Collard, Dominique; Fujita, Hiroyuki

    2009-06-21

    We demonstrate the active transport of liquid cargos in the form of oil-in-water emulsion droplets loaded on kinesin motor proteins moving along oriented microtubules. We analyze the motility properties of the kinesin motors (velocity and run length) and find that the liquid cargo in the form of oil droplets does not alter the motor function of the kinesin molecules. This work provides a novel method for handling only a few molecules/particles encapsulated inside the oil droplets and represents a key finding for the integration of kinesin-based active transport into nanoscale lab-on-a-chip devices. We also investigate the effect of the diameter of the droplets on the motility properties of the kinesin motors. The velocity is approximately constant irrespective of the diameter of the droplets whereas we highlight a strong increase of the run length when the diameter of the droplets increases. We correlate these results with the number of kinesin motors involved in the transport process and find an excellent agreement between our experimental result and a theoretical model.

  3. Syntaxin 1C, a soluble form of syntaxin, attenuates membrane recycling by destabilizing microtubules.

    PubMed

    Nakayama, Takahiro; Kamiguchi, Hiroyuki; Akagawa, Kimio

    2012-02-15

    Syntaxin 1C (STX1C), produced by alternative splicing of the stx1A gene, is a soluble syntaxin lacking a SNARE domain and a transmembrane domain. It is unclear how soluble syntaxin can control intracellular membrane trafficking. We found that STX1C affected microtubule (MT) dynamics through its tubulin-binding domain (TBD) and regulated recycling of intracellular vesicles carrying glucose transporter-1 (GLUT1). We demonstrated that the amino acid sequence VRSK of the TBD was important for the interaction between STX1C and tubulin and that wild-type STX1C (STX1C-WT), but not the TBD mutant, reduced the V(max) of glucose transport and GLUT1 translocation to the plasma membrane in FRSK cells. Moreover, by time-lapse analysis, we revealed that STX1C-WT suppressed MT stability and vesicle-transport motility in cells expressing GFP-α-tubulin, whereas TBD mutants had no effect. We also identified that GLUT1 was recycled in the 45 minutes after endocytosis and that GLUT1 vesicles moved along with MTs. Finally, we showed, by a recycling assay and FCM analysis, that STX1C-WT delayed the recycling phase of GLUT1 to PM, without affecting the endocytotic process of GLUT1. These data indicate that STX1C delays the GLUT1 recycling phase by suppressing MT stability and vesicle-transport motility through its TBD, providing the first insight into how soluble syntaxin controls membrane trafficking.

  4. Distinct Interaction Modes of the Kinesin-13 Motor Domain with the Microtubule

    PubMed Central

    Chatterjee, Chandrima; Benoit, Matthieu P.M.H.; DePaoli, Vania; Diaz-Valencia, Juan D.; Asenjo, Ana B.; Gerfen, Gary J.; Sharp, David J.; Sosa, Hernando

    2016-01-01

    Kinesins-13s are members of the kinesin superfamily of motor proteins that depolymerize microtubules (MTs) and have no motile activity. Instead of generating unidirectional movement over the MT lattice, like most other kinesins, kinesins-13s undergo one-dimensional diffusion (ODD) and induce depolymerization at the MT ends. To understand the mechanism of ODD and the origin of the distinct kinesin-13 functionality, we used ensemble and single-molecule fluorescence polarization microscopy to analyze the behavior and conformation of Drosophila melanogaster kinesin-13 KLP10A protein constructs bound to the MT lattice. We found that KLP10A interacts with the MT in two coexisting modes: one in which the motor domain binds with a specific orientation to the MT lattice and another where the motor domain is very mobile and able to undergo ODD. By comparing the orientation and dynamic behavior of mutated and deletion constructs we conclude that 1) the Kinesin-13 class specific neck domain and loop-2 help orienting the motor domain relative to the MT. 2) During ODD the KLP10A motor-domain changes orientation rapidly (rocks or tumbles). 3) The motor domain alone is capable of undergoing ODD. 4) A second tubulin binding site in the KLP10A motor domain is not critical for ODD. 5) The neck domain is not the element preventing KLP10A from binding to the MT lattice like motile kinesins. PMID:27074684

  5. Isolation of microtubule-based motor proteins by ATP release from paclitaxel-stabilized microtubules.

    PubMed

    Sloboda, Roger D

    2015-02-02

    The α-β-tubulin heterodimer is asymmetric, and when asymmetric subunits assemble in a head-to-tail fashion, they produce a polymer that is itself asymmetric. Microtubules are therefore polar polymers having a head (or plus) end and a tail (or minus) end. Both ends can be distinguished kinetically because they add and lose subunits at different rates. Because of this inherent asymmetry, translocation of a particle along a microtubule from the head to the tail is a different molecular event than is translocation from the minus to the plus end. Currently, two classes of microtubule-dependent motor proteins are recognized: Those that are plus-end-directed (i.e., kinesin-like) and those that are minus-end-directed (dynein-like). The kinesin family of proteins in humans contains at least 14 classes of kinesins, a grouping based on tertiary and quaternary structure considerations, as well as on enzymatic activity. The dyneins are organized into two groups: Axonemal dyneins and cytoplasmic dyneins. This protocol provides methods for the enrichment of kinesin or cytoplasmic dynein, based on the differential interactions of each type of motor protein with microtubules in the presence of different nucleotides. For a cleaner preparation of motor proteins, the protocol includes steps for the further separation of kinesin and dynein from one another by sucrose gradient centrifugation.

  6. Microtubules in protozoan cells. III. Ultrastructural changes during disintegration and reformation of heliozoan microtubules.

    PubMed

    Toyohara, A; Shigenaka, Y; Mohri, H

    1978-08-01

    In the heliozoan, Echinosphaerium nucleofilum strain MA, cold temperature (2 degrees C) induced axopodial retraction to about 36% of the initial length after 3 h. By electron microscopy, it was found that such axopodial shortening is accompanied by degradation of axonemal microtubules (25 nm in diameter), followed by the appearance of macrotubles (37 nm in diameter) and filamentous structures (14 nm in diameter) of a tubular and twisted appearance. All of these structures (microtubules, macrotubules and filamentous structures) were found to be depolymerized completely by applying 10 mM colchicine for 1-2 h, and to be replaced by regions with low electron density. Axopodial re-extension was induced rapidly by returning the cold-treated organisms to room temperature (20 degrees C). At a very early stage of axopodial re-extension, the filamentous structures were often observed to be continuous with the macrotubules. At a late stage of axopodial re-extension, the reforming axoneme was composed only of normal microtubules, while the macrotubules and filamentous structures had disappeared. On the basis of these results, the processes of disintegration and reformation of microtubules are discussed.

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

    NASA Astrophysics Data System (ADS)

    Salmon, E. D.

    1991-05-01

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

  8. Motility-Induced Phase Separation

    NASA Astrophysics Data System (ADS)

    Cates, Michael E.; Tailleur, Julien

    2015-03-01

    Self-propelled particles include both self-phoretic synthetic colloids and various microorganisms. By continually consuming energy, they bypass the laws of equilibrium thermodynamics. These laws enforce the Boltzmann distribution in thermal equilibrium: The steady state is then independent of kinetic parameters. In contrast, self-propelled particles tend to accumulate where they move more slowly. They may also slow down at high density for either biochemical or steric reasons. This creates positive feedback, which can lead to motility-induced phase separation (MIPS) between dense and dilute fluid phases. At leading order in gradients, a mapping relates variable-speed, self-propelled particles to passive particles with attractions. This deep link to equilibrium phase separation is confirmed by simulations but generally breaks down at higher order in gradients: New effects, with no equilibrium counterpart, then emerge. We give a selective overview of the fast-developing field of MIPS, focusing on theory and simulation but including a brief speculative survey of its experimental implications.

  9. Methanogens, Methane and Gastrointestinal Motility

    PubMed Central

    Triantafyllou, Konstantinos; Chang, Christopher

    2014-01-01

    Anaerobic fermentation of the undigested polysaccharide fraction of carbohydrates produces hydrogen in the intestine which is the substrate for methane production by intestinal methanogens. Hydrogen and methane are excreted in the flatus and in breath giving the opportunity to indirectly measure their production using breath testing. Although methane is detected in 30%-50% of the healthy adult population worldwide, its production has been epidemiologically and clinically associated with constipation related diseases, like constipation predominant irritable bowel syndrome and chronic constipation. While a causative relation is not proven yet, there is strong evidence from animal studies that methane delays intestinal transit, possibly acting as a neuromuscular transmitter. This evidence is further supported by the universal finding that methane production (measured by breath test) is associated with delayed transit time in clinical studies. There is also preliminary evidence that antibiotic reduction of methanogens (as evidenced by reduced methane production) predicts the clinical response in terms of symptomatic improvement in patients with constipation predominant irritable bowel syndrome. However, we have not identified yet the mechanism of action of methane on intestinal motility, and since methane production does not account for all constipation associated cases, there is need for high quality clinical trials to examine methane as a biomarker for the diagnosis or as a biomarker that predicts antibiotic treatment response in patients with constipation related disorders. PMID:24466443

  10. Deterministic patterns in cell motility

    NASA Astrophysics Data System (ADS)

    Lavi, Ido; Piel, Matthieu; Lennon-Duménil, Ana-Maria; Voituriez, Raphaël; Gov, Nir S.

    2016-12-01

    Cell migration paths are generally described as random walks, associated with both intrinsic and extrinsic noise. However, complex cell locomotion is not merely related to such fluctuations, but is often determined by the underlying machinery. Cell motility is driven mechanically by actin and myosin, two molecular components that generate contractile forces. Other cell functions make use of the same components and, therefore, will compete with the migratory apparatus. Here, we propose a physical model of such a competitive system, namely dendritic cells whose antigen capture function and migratory ability are coupled by myosin II. The model predicts that this coupling gives rise to a dynamic instability, whereby cells switch from persistent migration to unidirectional self-oscillation, through a Hopf bifurcation. Cells can then switch to periodic polarity reversals through a homoclinic bifurcation. These predicted dynamic regimes are characterized by robust features that we identify through in vitro trajectories of dendritic cells over long timescales and distances. We expect that competition for limited resources in other migrating cell types can lead to similar deterministic migration modes.

  11. Building the Microtubule Cytoskeleton Piece by Piece*

    PubMed Central

    Alfaro-Aco, Ray; Petry, Sabine

    2015-01-01

    The microtubule (MT) cytoskeleton gives cells their shape, organizes the cellular interior, and segregates chromosomes. These functions rely on the precise arrangement of MTs, which is achieved by the coordinated action of MT-associated proteins (MAPs). We highlight the first and most important examples of how different MAP activities are combined in vitro to create an ensemble function that exceeds the simple addition of their individual activities, and how the Xenopus laevis egg extract system has been utilized as a powerful intermediate between cellular and purified systems to uncover the design principles of self-organized MT networks in the cell. PMID:25957410

  12. Self-organization of microtubules and motors.

    SciTech Connect

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

    2006-01-01

    Here we introduce a model for spatio-temporal self-organization of an ensemble of microtubules interacting via molecular motors. Starting from a generic stochastic model of inelastic polar rods with an anisotropic interaction kernel we derive 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. The corresponding phase diagram of vortexasters transitions is in qualitative agreement with experiment.

  13. 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.

  14. Structural insights into microtubule doublet interactions inaxonemes

    SciTech Connect

    Downing, Kenneth H.; Sui, Haixin

    2007-06-06

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

  15. TCTEX1D4, a novel protein phosphatase 1 interactor: connecting the phosphatase to the microtubule network

    PubMed Central

    Korrodi-Gregório, Luís; Vieira, Sandra I.; Esteves, Sara L. C.; Silva, Joana V.; Freitas, Maria João; Brauns, Ann-Kristin; Luers, Georg; Abrantes, Joana; Esteves, Pedro J.; da Cruz e Silva, Odete A. B.; Fardilha, Margarida; da Cruz e Silva, Edgar F.

    2013-01-01

    Summary Reversible phosphorylation plays an important role as a mechanism of intracellular control in eukaryotes. PPP1, a major eukaryotic Ser/Thr-protein phosphatase, acquires its specificity by interacting with different protein regulators, also known as PPP1 interacting proteins (PIPs). In the present work we characterized a physiologically relevant PIP in testis. Using a yeast two-hybrid screen with a human testis cDNA library, we identified a novel PIP of PPP1CC2 isoform, the T-complex testis expressed protein 1 domain containing 4 (TCTEX1D4) that has recently been described as a Tctex1 dynein light chain family member. The overlay assays confirm that TCTEX1D4 interacts with the different spliced isoforms of PPP1CC. Also, the binding domain occurs in the N-terminus, where a consensus PPP1 binding motif (PPP1BM) RVSF is present. The distribution of TCTEX1D4 in testis suggests its involvement in distinct functions, such as TGFβ signaling at the blood–testis barrier and acrosome cap formation. Immunofluorescence in human ejaculated sperm shows that TCTEX1D4 is present in the flagellum and in the acrosome region of the head. Moreover, TCTEX1D4 and PPP1 co-localize in the microtubule organizing center (MTOC) and microtubules in cell cultures. Importantly, the TCTEX1D4 PPP1BM seems to be relevant for complex formation, for PPP1 retention in the MTOC and movement along microtubules. These novel results open new avenues to possible roles of this dynein, together with PPP1. In essence TCTEX1D4/PPP1C complex appears to be involved in microtubule dynamics, sperm motility, acrosome reaction and in the regulation of the blood–testis barrier. PMID:23789093

  16. RHAMM Promotes Interphase Microtubule Instability and Mitotic Spindle Integrity through MEK1/ERK1/2 Activity*

    PubMed Central

    Tolg, Cornelia; Hamilton, Sara R.; Morningstar, Lyndsey; Zhang, Jing; Zhang, S.; Esguerra, Kenneth V.; Telmer, Patrick G.; Luyt, Len G.; Harrison, Rene; McCarthy, James B.; Turley, Eva A.

    2010-01-01

    An oncogenic form of RHAMM (receptor for hyaluronan-mediated motility, mouse, amino acids 163–794 termed RHAMMΔ163) is a cell surface hyaluronan receptor and mitotic spindle protein that is highly expressed in aggressive human cancers. Its regulation of mitotic spindle integrity is thought to contribute to tumor progression, but the molecular mechanisms underlying this function have not previously been defined. Here, we report that intracellular RHAMMΔ163 modifies the stability of interphase and mitotic spindle microtubules through ERK1/2 activity. RHAMM−/− mouse embryonic fibroblasts exhibit strongly acetylated interphase microtubules, multi-pole mitotic spindles, aberrant chromosome segregation, and inappropriate cytokinesis during mitosis. These defects are rescued by either expression of RHAMM or mutant active MEK1. Mutational analyses show that RHAMMΔ163 binds to α- and β-tubulin protein via a carboxyl-terminal leucine zipper, but in vitro analyses indicate this interaction does not directly contribute to tubulin polymerization/stability. Co-immunoprecipitation and pulldown assays reveal complexes of RHAMMΔ163, ERK1/2-MEK1, and α- and β-tubulin and demonstrate direct binding of RHAMMΔ163 to ERK1 via a D-site motif. In vitro kinase analyses, expression of mutant RHAMMΔ163 defective in ERK1 binding in mouse embryonic fibroblasts, and blocking MEK1 activity collectively confirm that the effect of RHAMMΔ163 on interphase and mitotic spindle microtubules is mediated by ERK1/2 activity. Our results suggest a model wherein intracellular RHAMMΔ163 functions as an adaptor protein to control microtubule polymerization during interphase and mitosis as a result of localizing ERK1/2-MEK1 complexes to their tubulin-associated substrates. PMID:20558733

  17. Microtubule stabilising peptides rescue tau phenotypes in-vivo

    PubMed Central

    Quraishe, Shmma; Sealey, Megan; Cranfield, Louise; Mudher, Amritpal

    2016-01-01

    The microtubule cytoskeleton is a highly dynamic, filamentous network underpinning cellular structure and function. In Alzheimer’s disease, the microtubule cytoskeleton is compromised, leading to neuronal dysfunction and eventually cell death. There are currently no disease-modifying therapies to slow down or halt disease progression. However, microtubule stabilisation is a promising therapeutic strategy that is being explored. We previously investigated the disease-modifying potential of a microtubule-stabilising peptide NAP (NAPVSIPQ) in a well-established Drosophila model of tauopathy characterised by microtubule breakdown and axonal transport deficits. NAP prevented as well as reversed these phenotypes even after they had become established. In this study, we investigate the neuroprotective capabilities of an analogous peptide SAL (SALLRSIPA). We found that SAL mimicked NAP’s protective effects, by preventing axonal transport disruption and improving behavioural deficits, suggesting both NAP and SAL may act via a common mechanism. Both peptides contain a putative ‘SIP’ (Ser-Ile-Pro) domain that is important for interactions with microtubule end-binding proteins. Our data suggests this domain may be central to the microtubule stabilising function of both peptides and the mechanism by which they rescue phenotypes in this model of tauopathy. Our observations support microtubule stabilisation as a promising disease-modifying therapeutic strategy for tauopathies like Alzheimer’s disease. PMID:27910888

  18. Microtubule-severing enzymes at the cutting edge

    PubMed Central

    Sharp, David J.; Ross, Jennifer L.

    2012-01-01

    ATP-dependent severing of microtubules was first reported in Xenopus laevis egg extracts in 1991. Two years later this observation led to the purification of the first known microtubule-severing enzyme, katanin. Katanin homologs have now been identified throughout the animal kingdom and in plants. Moreover, members of two closely related enzyme subfamilies, spastin and fidgetin, have been found to sever microtubules and might act alongside katanins in some contexts (Roll-Mecak and McNally, 2010; Yu et al., 2008; Zhang et al., 2007). Over the past few years, it has become clear that microtubule-severing enzymes contribute to a wide range of cellular activities including mitosis and meiosis, morphogenesis, cilia biogenesis and disassembly, and migration. Thus, this group of enzymes is revealing itself to be among the most important of the microtubule regulators. This Commentary focuses on our growing understanding of how microtubule-severing enzymes contribute to the organization and dynamics of diverse microtubule arrays, as well as the structural and biophysical characteristics that afford them the unique capacity to catalyze the removal of tubulin from the interior microtubule lattice. Our goal is to provide a broader perspective, focusing on a limited number of particularly informative, representative and/or timely findings. PMID:22595526

  19. Microtubule distribution in gravitropic protonemata of the moss Ceratodon

    NASA Technical Reports Server (NTRS)

    Schwuchow, J.; Sack, F. D.; Hartmann, E.

    1990-01-01

    Tip cells of dark-grown protonemata of the moss Ceratodon purpureus are negatively gravitropic (grow upward). They possess a unique longitudinal zonation: (1) a tip group of amylochloroplasts in the apical dome, (2) a plastid-free zone, (3) a zone of significant plastid sedimentation, and (4) a zone of mostly non-sedimenting plastids. Immunofluorescence of vertical cells showed microtubules distributed throughout the cytoplasm in a mostly axial orientation extending through all zones. Optical sectioning revealed a close spatial association between microtubules and plastids. A majority (two thirds) of protonemata gravistimulated for > 20 min had a higher density of microtubules near the lower flank compared to the upper flank in the plastid-free zone. This apparent enrichment of microtubules occurred just proximal to sedimented plastids and near the part of the tip that presumably elongates more to produce curvature. Fewer than 5% of gravistimulated protonemata had an enrichment in microtubules near the upper flank, whereas 14% of vertical protonemata were enriched near one of the side walls. Oryzalin and amiprophos-methyl (APM) disrupted microtubules, gravitropism, and normal tip growth and zonation, but did not prevent plastid sedimentation. We hypothesize that a microtubule redistribution plays a role in gravitropism in this protonema. This appears to be the first report of an effect of gravity on microtubule distribution in plants.

  20. Microtubules: dynamically unstable stochastic phase-switching polymers

    NASA Astrophysics Data System (ADS)

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

    2016-08-01

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

  1. Buckling of microtubules on elastic media via breakable bonds.

    PubMed

    Afrin, Tanjina; Kabir, Arif Md Rashedul; Sada, Kazuki; Kakugo, Akira; Nitta, Takahiro

    2016-11-04

    Buckling of microtubules observed in cells has been reconstructed on a two-dimensional elastic medium consisting of kinesins grafted over compressible substrates, enabling precise control of experimental conditions and quantitative analysis. However, interpretations of the observations have ambiguities due to inevitable experimental difficulties. In this study, with computer simulations, we investigated importance of the mode of interaction of microtubule with elastic medium in the buckling behavior of microtubule. By taking into consideration of forced-induced detachments of kinesins from microtubules, our simulations reproduced the previous experimental results, and showed deviations from predictions of the elastic foundation model. On the other hand, with hypothetical linkers permanently bound to microtubules, our simulation reproduced the predictions of the elastic foundation model. By analyzing the results of the simulations, we investigated as to why the difference arose. These findings indicate the importance of the mode of interaction of microtubule with the medium in the buckling behavior of microtubule. Our findings would bring new insights on buckling of microtubules in living cells.

  2. Microtubule-severing enzymes at the cutting edge.

    PubMed

    Sharp, David J; Ross, Jennifer L

    2012-06-01

    ATP-dependent severing of microtubules was first reported in Xenopus laevis egg extracts in 1991. Two years later this observation led to the purification of the first known microtubule-severing enzyme, katanin. Katanin homologs have now been identified throughout the animal kingdom and in plants. Moreover, members of two closely related enzyme subfamilies, spastin and fidgetin, have been found to sever microtubules and might act alongside katanins in some contexts (Roll-Mecak and McNally, 2010; Yu et al., 2008; Zhang et al., 2007). Over the past few years, it has become clear that microtubule-severing enzymes contribute to a wide range of cellular activities including mitosis and meiosis, morphogenesis, cilia biogenesis and disassembly, and migration. Thus, this group of enzymes is revealing itself to be among the most important of the microtubule regulators. This Commentary focuses on our growing understanding of how microtubule-severing enzymes contribute to the organization and dynamics of diverse microtubule arrays, as well as the structural and biophysical characteristics that afford them the unique capacity to catalyze the removal of tubulin from the interior microtubule lattice. Our goal is to provide a broader perspective, focusing on a limited number of particularly informative, representative and/or timely findings.

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

    PubMed

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

    2002-12-01

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

  4. 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.

  5. Microtubule distribution in gravitropic protonemata of the moss Ceratodon

    NASA Technical Reports Server (NTRS)

    Schwuchow, J.; Sack, F. D.; Hartmann, E.

    1990-01-01

    Tip cells of dark-grown protonemata of the moss Ceratodon purpureus are negatively gravitropic (grow upward). They possess a unique longitudinal zonation: (1) a tip group of amylochloroplasts in the apical dome, (2) a plastid-free zone, (3) a zone of significant plastid sedimentation, and (4) a zone of mostly non-sedimenting plastids. Immunofluorescence of vertical cells showed microtubules distributed throughout the cytoplasm in a mostly axial orientation extending through all zones. Optical sectioning revealed a close spatial association between microtubules and plastids. A majority (two thirds) of protonemata gravistimulated for > 20 min had a higher density of microtubules near the lower flank compared to the upper flank in the plastid-free zone. This apparent enrichment of microtubules occurred just proximal to sedimented plastids and near the part of the tip that presumably elongates more to produce curvature. Fewer than 5% of gravistimulated protonemata had an enrichment in microtubules near the upper flank, whereas 14% of vertical protonemata were enriched near one of the side walls. Oryzalin and amiprophos-methyl (APM) disrupted microtubules, gravitropism, and normal tip growth and zonation, but did not prevent plastid sedimentation. We hypothesize that a microtubule redistribution plays a role in gravitropism in this protonema. This appears to be the first report of an effect of gravity on microtubule distribution in plants.

  6. Association of Lis1 with outer arm dynein is modulated in response to alterations in flagellar motility

    PubMed Central

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

    2012-01-01

    The cytoplasmic dynein regulatory factor Lis1, which induces a persistent tight binding to microtubules and allows for transport of cargoes under high-load conditions, is also present in motile cilia/flagella. We observed that Lis1 levels in flagella of Chlamydomonas strains that exhibit defective motility due to mutation of various axonemal substructures were greatly enhanced compared with wild type; this increase was absolutely dependent on the presence within the flagellum of the outer arm dynein α heavy chain/light chain 5 thioredoxin unit. To assess whether cells might interpret defective motility as a “high-load environment,” we reduced the flagellar beat frequency of wild-type cells through enhanced viscous load and by reductive stress; both treatments resulted in increased levels of flagellar Lis1, which altered the intrinsic beat frequency of the trans flagellum. Differential extraction of Lis1 from wild-type and mutant axonemes suggests that the affinity of outer arm dynein for Lis1 is directly modulated. In cytoplasm, Lis1 localized to two punctate structures, one of which was located near the base of the flagella. These data reveal that the cell actively monitors motility and dynamically modulates flagellar levels of the dynein regulatory factor Lis1 in response to imposed alterations in beat parameters. PMID:22855525

  7. Mammalian Sperm Motility: Observation and Theory

    NASA Astrophysics Data System (ADS)

    Gaffney, E. A.; Gadêlha, H.; Smith, D. J.; Blake, J. R.; Kirkman-Brown, J. C.

    2011-01-01

    Mammalian spermatozoa motility is a subject of growing importance because of rising human infertility and the possibility of improving animal breeding. We highlight opportunities for fluid and continuum dynamics to provide novel insights concerning the mechanics of these specialized cells, especially during their remarkable journey to the egg. The biological structure of the motile sperm appendage, the flagellum, is described and placed in the context of the mechanics underlying the migration of mammalian sperm through the numerous environments of the female reproductive tract. This process demands certain specific changes to flagellar movement and motility for which further mechanical insight would be valuable, although this requires improved modeling capabilities, particularly to increase our understanding of sperm progression in vivo. We summarize current theoretical studies, highlighting the synergistic combination of imaging and theory in exploring sperm motility, and discuss the challenges for future observational and theoretical studies in understanding the underlying mechanics.

  8. The influence of prostaglandins on sperm motility.

    PubMed

    Schlegel, W; Rotermund, S; Färber, G; Nieschlag, E

    1981-01-01

    Prostaglandin E2 and F2 alpha were measured in ejaculates from 10 fertile and 55 infertile men. Prostaglandin F2 alpha was negatively correlated with motility (r = 0.77; p less than 0.01) in normal men. In patients with disturbed fertility, prostaglandin F2 alpha was always higher than in the controls, while prostaglandin E2 was elevated only in patients with persisting varicocele and in those with very low sperm counts and severely impaired motility. There was neither de novo synthesis of prostaglandins in spermatozoa nor were binding sites for prostaglandin E2 and F2 alpha detectable. Inactivation of seminal prostaglandins by incubation with prostaglandin 15-hydroxydehydrogenase resulted in a dramatic fall in motility. The results suggest that prostaglandin F2 alpha act on motility, but the action is not mediated by receptors.

  9. Motility in the epsilon-proteobacteria.

    PubMed

    Beeby, Morgan

    2015-12-01

    The epsilon-proteobacteria are a widespread group of flagellated bacteria frequently associated with either animal digestive tracts or hydrothermal vents, with well-studied examples in the human pathogens of Helicobacter and Campylobacter genera. Flagellated motility is important to both pathogens and hydrothermal vent members, and a number of curious differences between the epsilon-proteobacterial and enteric bacterial motility paradigms make them worthy of further study. The epsilon-proteobacteria have evolved to swim at high speed and through viscous media that immobilize enterics, a phenotype that may be accounted for by the molecular architecture of the unusually large epsilon-proteobacterial flagellar motor. This review summarizes what is known about epsilon-proteobacterial motility and focuses on a number of recent discoveries that rationalize the differences with enteric flagellar motility. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Mitotic Regulation of the Stability of Microtubule Plus-end Tracking Protein EB3 by Ubiquitin Ligase SIAH-1 and Aurora Mitotic Kinases*

    PubMed Central

    Ban, Reiko; Matsuzaki, Hideki; Akashi, Tomohiro; Sakashita, Gyosuke; Taniguchi, Hisaaki; Park, Sam-Yong; Tanaka, Hirofumi; Furukawa, Koichi; Urano, Takeshi

    2009-01-01

    Microtubule plus-end tracking proteins (+TIPs) control microtubule dynamics in fundamental processes such as cell cycle, intracellular transport, and cell motility, but how +TIPs are regulated during mitosis remains largely unclear. Here we show that the endogenous end-binding protein family EB3 is stable during mitosis, facilitates cell cycle progression at prometaphase, and then is down-regulated during the transition to G1 phase. The ubiquitin-protein isopeptide ligase SIAH-1 facilitates EB3 polyubiquitination and subsequent proteasome-mediated degradation, whereas SIAH-1 knockdown increases EB3 stability and steady-state levels. Two mitotic kinases, Aurora-A and Aurora-B, phosphorylate endogenous EB3 at Ser-176, and the phosphorylation triggers disruption of the EB3-SIAH-1 complex, resulting in EB3 stabilization during mitosis. Our results provide new insight into a regulatory mechanism of +TIPs in cell cycle transition. PMID:19696028

  11. Select Acetophenones Modulate Flagellar Motility in Chlamydomonas

    PubMed Central

    Evans, Shakila K.; Pearce, Austin A.; Ibezim, Prudence K.; Primm, Todd P.; Gaillard, Anne R.

    2009-01-01

    Acetophenones were screened for activity against positive phototaxis of Chlamydomonas cells, a process that requires coordinated flagellar motility. The structure-activity relationships of a series of acetophenones are reported, including acetophenones that affect flagellar motility and cell viability. Notably, 4-methoxyacetophenone, 3,4-dimethoxyacetophenone, and 4-hydroxyacetophenone induced negative phototaxis in Chlamydomonas, suggesting interference with activity of flagellar proteins and control of flagellar dominance. PMID:20659114

  12. ATPases, ion exchangers and human sperm motility.

    PubMed

    Peralta-Arias, Rubén D; Vívenes, Carmen Y; Camejo, María I; Piñero, Sandy; Proverbio, Teresa; Martínez, Elizabeth; Marín, Reinaldo; Proverbio, Fulgencio

    2015-05-01

    Human sperm has several mechanisms to control its ionic milieu, such as the Na,K-ATPase (NKA), the Ca-ATPase of the plasma membrane (PMCA), the Na(+)/Ca(2) (+)-exchanger (NCX) and the Na(+)/H(+)-exchanger (NHE). On the other hand, the dynein-ATPase is the intracellular motor for sperm motility. In this work, we evaluated NKA, PMCA, NHE, NCX and dynein-ATPase activities in human sperm and investigated their correlation with sperm motility. Sperm motility was measured by Computer Assisted Semen Analysis. It was found that the NKA activity is inhibited by ouabain with two Ki (7.9 × 10(-9) and 9.8 × 10(-5) M), which is consistent with the presence of two isoforms of α subunit of the NKA in the sperm plasma membranes (α1 and α4), being α4 more sensitive to ouabain. The decrease in NKA activity is associated with a reduction in sperm motility. In addition, sperm motility was evaluated in the presence of known inhibitors of NHE, PMCA and NCX, such as amiloride, eosin, and KB-R7943, respectively, as well as in the presence of nigericin after incubation with ouabain. Amiloride, eosin and KB-R7943 significantly reduced sperm motility. Nigericin reversed the effect of ouabain and amiloride on sperm motility. Dynein-ATPase activity was inhibited by acidic pH and micromolar concentrations of Ca(2) (+). We explain our results in terms of inhibition of the dynein-ATPase in the presence of higher cytosolic H(+) and Ca(2) (+), and therefore inhibition of sperm motility. © 2015 Society for Reproduction and Fertility.

  13. The Ndc80 kinetochore complex forms oligomeric arrays along microtubules

    PubMed Central

    Alushin, Gregory M.; Ramey, Vincent H.; Pasqualato, Sebastiano; Ball, David A.; Grigorieff, Nikolaus; Musacchio, Andrea; Nogales, Eva

    2010-01-01

    The Ndc80 complex is a key site of regulated kinetochore-microtubule attachment, but the molecular mechanism underlying its function remains unknown. Here we present a subnanometer resolution cryo-electron microscopy reconstruction of the human Ndc80 complex bound to microtubules, sufficient for precise docking of crystal structures of the component proteins. We find that Ndc80 binds the microtubule with a tubulin monomer repeat, recognizing α- and β-tubulin at both intra- and inter-dimer interfaces in a manner that is sensitive to tubulin conformation. Furthermore, Ndc80 complexes self-associate along protofilaments via interactions mediated by the amino-terminal tail of the Ndc80 protein, the site of phospho-regulation by the Aurora B kinase. Ndc80's mode of interaction with the microtubule and its oligomerization suggest a mechanism by which Aurora B could regulate the stability of load-bearing Ndc80-microtubule attachments. PMID:20944740

  14. Multimodal microtubule binding by the Ndc80 kinetochore complex.

    PubMed

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

    2012-11-01

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

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

    PubMed

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

    2013-02-01

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

  16. 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.

  17. Dynamic shape changes of cytoplasmic organelles translocating along microtubules

    PubMed Central

    1987-01-01

    Transient shape changes of organelles translocating along microtubules are directly visualized in thinly spread cytoplasmic processes of the marine foraminifer. Allogromia laticollaris, by a combination of high- resolution video-enhanced microscopy and fast-freezing electron microscopy. The interacting side of the organelle flattens upon binding to a microtubule, as if to maximize contact with it. Organelles typically assume a teardrop shape while moving, as if they were dragged through a viscous medium. Associated microtubules bend around attachments of the teardrop-shaped organelles, suggesting that they too are acted on by the forces deforming the organelles. An 18-nm gap between the organelles and the microtubules is periodically bridged by 10-nm-thick cross-bridge structures that may be responsible for the binding and motive forces deforming organelles and microtubules. PMID:3654751

  18. Dynamic shape changes of cytoplasmic organelles translocating along microtubules.

    PubMed

    Kachar, B; Bridgman, P C; Reese, T S

    1987-09-01

    Transient shape changes of organelles translocating along microtubules are directly visualized in thinly spread cytoplasmic processes of the marine foraminifer. Allogromia laticollaris, by a combination of high-resolution video-enhanced microscopy and fast-freezing electron microscopy. The interacting side of the organelle flattens upon binding to a microtubule, as if to maximize contact with it. Organelles typically assume a teardrop shape while moving, as if they were dragged through a viscous medium. Associated microtubules bend around attachments of the teardrop-shaped organelles, suggesting that they too are acted on by the forces deforming the organelles. An 18-nm gap between the organelles and the microtubules is periodically bridged by 10-nm-thick cross-bridge structures that may be responsible for the binding and motive forces deforming organelles and microtubules.

  19. Role of microtubule cytoskeleton in regulation of endothelial barrier function.

    PubMed

    Alieva, I B

    2014-09-01

    Cytoplasmic microtubules are an obligatory component of the cytoskeleton of all types of cells. Microtubules are involved in many cellular processes including directed transport of vesicles and signaling molecules and changes in cell shape during its spreading, polarization, and movement. The intracellular organization of the system of microtubules and their dynamic properties are different in different types of cells because they play a key role in the implementation of a variety of cell and tissue functions, including the regulation of the endothelial barrier function. This review presents an overview of current studies on the properties of endothelial microtubules, their interaction with other components of the cytoskeleton and cell adhesion structures, and the role of microtubules in the regulation of the endothelial barrier function.

  20. Developmental neurotoxicity of methylmercury: the role of microtubules

    SciTech Connect

    Sager, P.R.

    1982-01-01

    The purpose of this research was to investigate the interaction of methylmercury with microtubules as a possible mechanism for methylmercury-caused developmental neurotoxicity. Methylmercury effects on developing cerebellar cortex, an area of rapid proliferation, were examined. This model was used to test the hypothesis that microtubules of the mitotic spindle are sensitive to methylmercury in vivo as well as in cultured cells. The effect of methylmercury on non-spindle microtubules was studied in cultured cells. Cellular levels of methylmercury were determined and were used to construct a dose-response relationship. The direct effects of methylmercury on microtubule assembly in vitro were also documented. The data from these three systems have been integrated to form a hypothesis for the role of microtubules in developmental neurotoxicity caused by methylmercury. 159 references, 23 figures, 16 tables.

  1. SEROLOGICAL SIMILARITY OF FLAGELLAR AND MITOTIC MICROTUBULES

    PubMed Central

    Fulton, Chandler; Kane, R. E.; Stephens, R. E.

    1971-01-01

    An antiserum to flagellar axonemes from sperm of Arbacia punctulata contains antibodies which react both with intact flagellar outer fibers and with purified tubulin from the outer fibers. Immunodiffusion tests indicate the presence of similar antigenic determinants on outer-fiber tubulins from sperm flagella of five species of sea urchins and a sand dollar, but not a starfish. The antibodies also react with extracts containing tubulins from different classes of microtubules, including central-pair fibers and both A- and B-subfibers from outer fibers of sperm flagella, an extract from unfertilized eggs, mitotic apparatuses from first cleavage embryos, and cilia from later embryos. Though most tubulins tested share similar antigenic determinants, some clear differences have been detected, even, in Pseudoboletia indiana, between the outer-fiber tubulins of sperm flagella and blastular cilia. Though tubulins are "actin-like" proteins, antitubulin serum does not react with actin from sea urchin lantern muscle. On the basis of these observations, we suggest that various echinoid microtubules are built of similar, but not identical, tubulins. PMID:4106543

  2. Multifunctional Microtubule-Associated Proteins in Plants

    PubMed Central

    Krtková, Jana; Benáková, Martina; Schwarzerová, Kateřina

    2016-01-01

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

  3. Tumor invasion as dysregulated cell motility.

    PubMed

    Kassis, J; Lauffenburger, D A; Turner, T; Wells, A

    2001-04-01

    Investigations across a range of disciplines over the past decade have brought the study of cell motility and its role in invasion to an exciting threshold. The biophysical forces proximally involved in generating cell locomotion, as well as the underlying signaling and genomic regulatory processes, are gradually becoming elucidated. We now appreciate the intricacies of the many cellular and extracellular events that modulate cell migration. This has enabled the demonstration of a causal role of cell motility in tumor progression, with various points of 'dysregulation' of motility being responsible for promoting invasion. In this paper, we describe key fundamental principles governing cell motility and branch out to describe the essence of the data that describe these principles. It has become evident that many proposed models may indeed be converging into a tightly-woven tapestry of coordinated events which employ various growth factors and their receptors, adhesion receptors (integrins), downstream molecules, cytoskeletal components, and altered genomic regulation to accomplish cell motility. Tumor invasion occurs in response to dysregulation of many of these modulatory points; specific examples include increased signaling from the EGF receptor and through PLC gamma, altered localization and expression of integrins, changes in actin modifying proteins and increased transcription from specific promoter sites. This diversity of alterations all leading to tumor invasion point to the difficulty of correcting causal events leading to tumor invasion and rather suggest that the underlying common processes required for motility be targeted for therapeutic intervention.

  4. Flagellar motility in eukaryotic human parasites.

    PubMed

    Krüger, Timothy; Engstler, Markus

    2015-10-01

    A huge variety of protists rely on one or more motile flagella to either move themselves or move fluids and substances around them. Many of these flagellates have evolved a symbiotic or parasitic lifestyle. Several of the parasites have adapted to human hosts, and include agents of prevalent and serious diseases. These unicellular parasites have become specialised in colonising a wide range of biological niches within humans. They usually have diverse transmission cycles, and frequently manifest a variety of distinct morphological stages. The motility of the single or multiple flagella plays important but understudied roles in parasite transmission, host invasion, dispersal, survival, proliferation and pathology. In this review we provide an overview of the important human pathogens that possess a motile flagellum for at least part of their life cycle. We highlight recently published studies that aim to elucidate motility mechanisms, and their relevance for human disease. We then bring the physics of swimming at the microscale into context, emphasising the importance of interdisciplinary approaches for a full understanding of flagellate motility - especially in light of the parasites' microenvironments and population dynamics. Finally, we summarise some important technological aspects, describing challenges for the field and possibilities for motility analyses in the future.

  5. Toward the reconstitution of synthetic cell motility

    PubMed Central

    Siton-Mendelson, Orit; Bernheim-Groswasser, Anne

    2016-01-01

    ABSTRACT Cellular motility is a fundamental process essential for embryonic development, wound healing, immune responses, and tissues development. Cells are mostly moving by