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Journal articles on the topic 'Microtubule stabilization'
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1
Sato, Hiroshi, Toshio Nagai, Dhandapani Kuppuswamy, et al. "Microtubule Stabilization in Pressure Overload Cardiac Hypertrophy." Journal of Cell Biology 139, no. 4 (1997): 963–73. http://dx.doi.org/10.1083/jcb.139.4.963.
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Increased microtubule density, for which microtubule stabilization is one potential mechanism, causes contractile dysfunction in cardiac hypertrophy. After microtubule assembly, α-tubulin undergoes two, likely sequential, time-dependent posttranslational changes: reversible carboxy-terminal detyrosination (Tyr-tubulin ↔ Glu-tubulin) and then irreversible deglutamination (Glu-tubulin → Δ2-tubulin), such that Glu- and Δ2-tubulin are markers for long-lived, stable microtubules. Therefore, we generated antibodies for Tyr-, Glu-, and Δ2-tubulin and used them for staining of right and left ventricular cardiocytes from control cats and cats with right ventricular hypertrophy. Tyr- tubulin microtubule staining was equal in right and left ventricular cardiocytes of control cats, but Glu-tubulin and Δ2-tubulin staining were insignificant, i.e., the microtubules were labile. However, Glu- and Δ2-tubulin were conspicuous in microtubules of right ventricular cardiocytes from pressure overloaded cats, i.e., the microtubules were stable. This finding was confirmed in terms of increased microtubule drug and cold stability in the hypertrophied cells. In further studies, we found an increase in a microtubule binding protein, microtubule-associated protein 4, on both mRNA and protein levels in pressure-hypertrophied myocardium. Thus, microtubule stabilization, likely facilitated by binding of a microtubule-associated protein, may be a mechanism for the increased microtubule density characteristic of pressure overload cardiac hypertrophy.
2
Witte, Harald, Dorothee Neukirchen, and Frank Bradke. "Microtubule stabilization specifies initial neuronal polarization." Journal of Cell Biology 180, no. 3 (2008): 619–32. http://dx.doi.org/10.1083/jcb.200707042.
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Axon formation is the initial step in establishing neuronal polarity. We examine here the role of microtubule dynamics in neuronal polarization using hippocampal neurons in culture. We see increased microtubule stability along the shaft in a single neurite before axon formation and in the axon of morphologically polarized cells. Loss of polarity or formation of multiple axons after manipulation of neuronal polarity regulators, synapses of amphids defective (SAD) kinases, and glycogen synthase kinase-3β correlates with characteristic changes in microtubule turnover. Consistently, changing the microtubule dynamics is sufficient to alter neuronal polarization. Application of low doses of the microtubule-destabilizing drug nocodazole selectively reduces the formation of future dendrites. Conversely, low doses of the microtubule-stabilizing drug taxol shift polymerizing microtubules from neurite shafts to process tips and lead to the formation of multiple axons. Finally, local stabilization of microtubules using a photoactivatable analogue of taxol induces axon formation from the activated area. Thus, local microtubule stabilization in one neurite is a physiological signal specifying neuronal polarization.
3
Job, D., M. Pabion, and R. L. Margolis. "Generation of microtubule stability subclasses by microtubule-associated proteins: implications for the microtubule "dynamic instability" model." Journal of Cell Biology 101, no. 5 (1985): 1680–89. http://dx.doi.org/10.1083/jcb.101.5.1680.
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We have developed a method to distinguish microtubule associated protein (MAP)-containing regions from MAP-free regions within a microtubule, or within microtubule sub-populations. In this method, we measure the MAP-dependent stabilization of microtubule regions to dilution-induced disassembly of the polymer. The appropriate microtubule regions are identified by assembly in the presence of [3H]GTP, and assayed by filter trapping and quantitation of microtubule regions that contain label. We find that MAPs bind very rapidly to polymer binding sites and that they do not exchange from these sites measurably once bound. Also, very low concentrations of MAPs yield measurable stabilization of local microtubule regions. Unlike the stable tubule only polypeptide (STOP) proteins, MAPs do not exhibit any sliding behavior under our assay conditions. These results predict the presence of different stability subclasses of microtubules when MAPs are present in less than saturating amounts. The data can readily account for the observed "dynamic instability" of microtubules through unequal MAP distributions. Further, we report that MAP dependent stabilization is quantitatively reversed by MAP phosphorylation, but that calmodulin, in large excess, has no specific influence on MAP protein activity when MAPs are on microtubules.
4
Takemura, R., S. Okabe, T. Umeyama, Y. Kanai, N. J. Cowan, and N. Hirokawa. "Increased microtubule stability and alpha tubulin acetylation in cells transfected with microtubule-associated proteins MAP1B, MAP2 or tau." Journal of Cell Science 103, no. 4 (1992): 953–64. http://dx.doi.org/10.1242/jcs.103.4.953.
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We previously transfected MAP2, tau and MAP1B cDNA into fibroblasts and have studied the effect of expression of these microtubule-associated proteins on microtubule organization. In this study, we examined some additional characteristics of microtubule bundles and arrays formed in fibroblasts transfected with these microtubule-associated proteins. It was found that microtubule bundles formed in MAP2c- or tau-transfected cells were stabilized against microtubule depolymerizing reagents and were enriched in acetylated alpha tubulin. When mouse MAP1B cDNA was expressed following transfection into COS cells, MAP1B was localized along microtubule arrays, but no extensive reorganization of microtubules such as bundle formation was observed, in agreement with our previous finding using HeLa and 3T3 cells. However, stabilization of microtubules was indicated: (a) microtubules in MAP1B-transfected cells were stabilized against a microtubule depolymerizing reagent, although stabilization was less efficient than that seen in MAP2c- or tau-transfected cells, and (b) microtubules in MAP1B-transfected cells were enriched in acetylated alpha tubulin. These results suggest that neuronal microtubule-associated proteins introduced into fibroblasts by cDNA transfection stabilize microtubules and affect the state of post-translational modification of tubulin.
5
Dixit, Ram, Eric Chang, and Richard Cyr. "Establishment of Polarity during Organization of the Acentrosomal Plant Cortical Microtubule Array." Molecular Biology of the Cell 17, no. 3 (2006): 1298–305. http://dx.doi.org/10.1091/mbc.e05-09-0864.
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The plant cortical microtubule array is a unique acentrosomal array that is essential for plant morphogenesis. To understand how this array is organized, we exploited the microtubule (+)-end tracking activity of two Arabidopsis EB1 proteins in combination with FRAP (fluorescence recovery after photobleaching) experiments of GFP-tubulin to examine the relationship between cortical microtubule array organization and polarity. Significantly, our observations show that the majority of cortical microtubules in ordered arrays, within a particular cell, face the same direction in both Arabidopsis plants and cultured tobacco cells. We determined that this polar microtubule coalignment is at least partially due to a selective stabilization of microtubules, and not due to a change in microtubule polymerization rates. Finally, we show that polar microtubule coalignment occurs in conjunction with parallel grouping of cortical microtubules and that cortical array polarity is progressively enhanced during array organization. These observations reveal a novel aspect of plant cortical microtubule array organization and suggest that selective stabilization of dynamic cortical microtubules plays a predominant role in the self-organization of cortical arrays.
6
Guillaud, Laurent, Christophe Bosc, Anne Fourest-Lieuvin, et al. "STOP Proteins are Responsible for the High Degree of Microtubule Stabilization Observed in Neuronal Cells." Journal of Cell Biology 142, no. 1 (1998): 167–79. http://dx.doi.org/10.1083/jcb.142.1.167.
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Neuronal differentiation and function require extensive stabilization of the microtubule cytoskeleton. Neurons contain a large proportion of microtubules that resist the cold and depolymerizing drugs and exhibit slow subunit turnover. The origin of this stabilization is unclear. Here we have examined the role of STOP, a calmodulin-regulated protein previously isolated from cold-stable brain microtubules. We find that neuronal cells express increasing levels of STOP and of STOP variants during differentiation. These STOP proteins are associated with a large proportion of microtubules in neuronal cells, and are concentrated on cold-stable, drug-resistant, and long-lived polymers. STOP inhibition abolishes microtubule cold and drug stability in established neurites and impairs neurite formation. Thus, STOP proteins are responsible for microtubule stabilization in neurons, and are apparently required for normal neurite formation.
7
Saoudi, Y., I. Paintrand, L. Multigner, and D. Job. "Stabilization and bundling of subtilisin-treated microtubules induced by microtubule associated proteins." Journal of Cell Science 108, no. 1 (1995): 357–67. http://dx.doi.org/10.1242/jcs.108.1.357.
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The acidic carboxy-terminal regions of alpha- and beta-tubulin subunits are currently thought to be centrally involved in microtubule stability and in microtubule association with a variety of proteins (MAPs) such as MAP2 and tau proteins. Here, pure tubulin microtubules were exposed to subtilisin to produce polymers composed of cleaved tubulin subunits lacking carboxy termini. Polymer exposure to subtilisin was achieved in buffer conditions compatible with further tests of microtubule stability. Microtubules composed of normal alpha-tubulin and cleaved beta-tubulin were indistinguishable from control microtubules with regard to resistance to dilution-induced disassembly, to cold temperature-induced disassembly and to Ca(2+)-induced disassembly. Microtubules composed of cleaved alpha- and beta-tubulins showed normal sensitivity to dilution-induced disassembly and to low temperature-induced disassembly, but marked resistance to Ca(2+)-induced disassembly. Polymers composed of normal alpha-tubulin and cleaved beta-tubulin or of cleaved alpha- and beta-tubulins were stabilized in the presence of added MAP2, myelin basic protein and histone H1. Cleavage of tubulin carboxy termini greatly potentiated microtubule stabilization by tau proteins. We show that this potentiation of polymer stabilization can be ascribed to tau-induced microtubule bundling. In our working conditions, such bundling upon association with tau proteins occurred only in the case of microtubules composed of cleaved alpha- and beta-tubulins and triggered apparent microtubule cross-stabilization among the bundled polymers. These results, as well as immunofluorescence analysis, which directly showed interactions between subtilisin-treated microtubules and MAPs, suggest that the carboxy termini of alpha- and beta-tubulins are not primarily involved in the binding of MAPs onto microtubules. However, interactions between tubulin carboxy termini and MAPs remain possible and might be involved in the regulation of MAP-induced microtubule bundling.
8
Podkowa, Monika, Xin Zhao, Chi-Wing Chow, Eleanor T. Coffey, Roger J. Davis, and Liliana Attisano. "Microtubule Stabilization by Bone Morphogenetic Protein Receptor-Mediated Scaffolding of c-Jun N-Terminal Kinase Promotes Dendrite Formation." Molecular and Cellular Biology 30, no. 9 (2010): 2241–50. http://dx.doi.org/10.1128/mcb.01166-09.
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ABSTRACT Neuronal outgrowth occurs via coordinated remodeling of the cytoskeleton involving both actin and microtubules. Microtubule stabilization drives the extending neurite, yet little is known of the molecular mechanisms whereby extracellular cues regulate microtubule dynamics. Bone morphogenetic proteins (BMPs) play an important role in neuronal differentiation and morphogenesis, and BMP7 in particular induces the formation of dendrites. Here, we show that BMP7 induces stabilization of microtubules in both a MAP2-dependent neuronal cell culture model and in dendrites of primary cortical neurons. BMP7 rapidly activates c-Jun N-terminal kinases (JNKs), known regulators of microtubule dynamics, and we show that JNKs associate with the carboxy terminus of the BMP receptor, BMPRII. Activation and binding of JNKs to BMPRII is required for BMP7-induced microtubule stabilization and for BMP7-mediated dendrite formation in primary cortical neurons. These data indicate that BMPRII acts as a scaffold to localize and coordinate cytoskeletal remodeling and thereby provides an efficient means for extracellular cues, such as BMPs, to control neuronal dendritogenesis.
9
Elliott, Gillian, and Peter O’Hare. "Herpes Simplex Virus Type 1 Tegument Protein VP22 Induces the Stabilization and Hyperacetylation of Microtubules." Journal of Virology 72, no. 8 (1998): 6448–55. http://dx.doi.org/10.1128/jvi.72.8.6448-6455.1998.
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ABSTRACT The role of the herpes simplex virus type 1 tegument protein VP22 during infection is as yet undefined. We have previously shown that VP22 has the unusual property of efficient intercellular transport, such that the protein spreads from single expressing cells into large numbers of surrounding cells. We also noted that in cells expressing VP22 by transient transfection, the protein localizes in a distinctive cytoplasmic filamentous pattern. Here we show that this pattern represents a colocalization between VP22 and cellular microtubules. Moreover, we show that VP22 reorganizes microtubules into thick bundles which are easily distinguishable from nonbundled microtubules. These bundles are highly resistant to microtubule-depolymerizing agents such as nocodazole and incubation at 4°C, suggesting that VP22 has the capacity to stabilize the microtubule network. In addition, we show that the microtubules contained in these bundles are modified by acetylation, a marker for microtubule stability. Analysis of infected cells by both immunofluorescence and measurement of microtubule acetylation further showed that colocalization between VP22 and microtubules, and induction of microtubule acetylation, also occurs during infection. Taken together, these results suggest that VP22 exhibits the properties of a classical microtubule-associated protein (MAP) during both transfection and infection. This is the first demonstration of a MAP encoded by an animal virus.
10
Dogterom, M., M. A. Félix, C. C. Guet, and S. Leibler. "Influence of M-phase chromatin on the anisotropy of microtubule asters." Journal of Cell Biology 133, no. 1 (1996): 125–40. http://dx.doi.org/10.1083/jcb.133.1.125.
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In many eukaryotic cells going through M-phase, a bipolar spindle is formed by microtubules nucleated from centrosomes. These microtubules, in addition to being "captured" by kinetochores, may be stabilized by chromatin in two different ways: short-range stabilization effects may affect microtubules in close contact with the chromatin, while long-range stabilization effects may "guide" microtubule growth towards the chromatin (e.g., by introducing a diffusive gradient of an enzymatic activity that affects microtubule assembly). Here, we use both meiotic and mitotic extracts from Xenopus laevis eggs to study microtubule aster formation and microtubule dynamics in the presence of chromatin. In "low-speed" meiotic extracts, in the presence of salmon sperm chromatin, we find that short-range stabilization effects lead to a strong anisotropy of the microtubule asters. Analysis of the dynamic parameters of microtubule growth show that this anisotropy arises from a decrease in the catastrophe frequency, an increase in the rescue frequency and a decrease in the growth velocity. In this system we also find evidence for long-range "guidance" effects, which lead to a weak anisotropy of the asters. Statistically relevant results on these long-range effects are obtained in "high-speed" mitotic extracts in the presence of artificially constructed chromatin stripes. We find that aster anisotropy is biased in the direction of the chromatin and that the catastrophe frequency is reduced in its vicinity. In this system we also find a surprising dependence of the catastrophe and the rescue frequencies on the length of microtubules nucleated from centrosomes: the catastrophe frequency increase and the rescue frequency decreases with microtubule length.
11
Reilein, Amy, Soichiro Yamada, and W. James Nelson. "Self-organization of an acentrosomal microtubule network at the basal cortex of polarized epithelial cells." Journal of Cell Biology 171, no. 5 (2005): 845–55. http://dx.doi.org/10.1083/jcb.200505071.
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Mechanisms underlying the organization of centrosome-derived microtubule arrays are well understood, but less is known about how acentrosomal microtubule networks are formed. The basal cortex of polarized epithelial cells contains a microtubule network of mixed polarity. We examined how this network is organized by imaging microtubule dynamics in acentrosomal basal cytoplasts derived from these cells. We show that the steady-state microtubule network appears to form by a combination of microtubule–microtubule and microtubule–cortex interactions, both of which increase microtubule stability. We used computational modeling to determine whether these microtubule parameters are sufficient to generate a steady-state acentrosomal microtubule network. Microtubules undergoing dynamic instability without any stabilization points continuously remodel their organization without reaching a steady-state network. However, the addition of increased microtubule stabilization at microtubule–microtubule and microtubule–cortex interactions results in the rapid assembly of a steady-state microtubule network in silico that is remarkably similar to networks formed in situ. These results define minimal parameters for the self-organization of an acentrosomal microtubule network.
12
Tirnauer, Jennifer S., Sonia Grego, E. D. Salmon, and Timothy J. Mitchison. "EB1–Microtubule Interactions in Xenopus Egg Extracts: Role of EB1 in Microtubule Stabilization and Mechanisms of Targeting to Microtubules." Molecular Biology of the Cell 13, no. 10 (2002): 3614–26. http://dx.doi.org/10.1091/mbc.e02-04-0210.
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EB1 targets to polymerizing microtubule ends, where it is favorably positioned to regulate microtubule polymerization and confer molecular recognition of the microtubule end. In this study, we focus on two aspects of the EB1–microtubule interaction: regulation of microtubule dynamics by EB1 and the mechanism of EB1 association with microtubules. Immunodepletion of EB1 from cytostatic factor-arrested M-phaseXenopus egg extracts dramatically reduced microtubule length; this was complemented by readdition of EB1. By time-lapse microscopy, EB1 increased the frequency of microtubule rescues and decreased catastrophes, resulting in increased polymerization and decreased depolymerization and pausing. Imaging of EB1 fluorescence revealed a novel structure: filamentous extensions on microtubule plus ends that appeared during microtubule pauses; loss of these extensions correlated with the abrupt onset of polymerization. Fluorescent EB1 localized to comets at the polymerizing plus ends of microtubules in cytostatic factor extracts and uniformly along the lengths of microtubules in interphase extracts. The temporal decay of EB1 fluorescence from polymerizing microtubule plus ends predicted a dissociation half-life of seconds. Fluorescence recovery after photobleaching also revealed dissociation and rebinding of EB1 to the microtubule wall with a similar half-life. EB1 targeting to microtubules is thus described by a combination of higher affinity binding to polymerizing ends and lower affinity binding along the wall, with continuous dissociation. The latter is likely to be attenuated in interphase. The highly conserved effect of EB1 on microtubule dynamics suggests it belongs to a core set of regulatory factors conserved in higher organisms, and the complex pattern of EB1 targeting to microtubules could be exploited by the cell for coordinating microtubule behaviors.
13
Kaverina, Irina, Klemens Rottner, and J. Victor Small. "Targeting, Capture, and Stabilization of Microtubules at Early Focal Adhesions." Journal of Cell Biology 142, no. 1 (1998): 181–90. http://dx.doi.org/10.1083/jcb.142.1.181.
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By co-injecting fluorescent tubulin and vinculin into fish fibroblasts we have revealed a “cross talk” between microtubules and early sites of substrate contact. This mutuality was first indicated by the targeting of vinculin-rich foci by microtubules during their growth towards the cell periphery. In addition to passing directly over contact sites, the ends of single microtubules could be observed to target several contacts in succession or the same contact repetitively, with intermittent withdrawals. Targeting sometimes involved side-stepping, or the major re-routing of a microtubule, indicative of a guided, rather than a random process. The paths that microtubules followed into contacts were unrelated to the orientation of stress fiber assemblies and targeting occurred also in mouse fibroblasts that lacked a system of intermediate filaments. Further experiments with microtubule inhibitors showed that adhesion foci can: (a) capture microtubules and stabilize them against disassembly by nocodazole; and (b), act as preferred sites of microtubule polymerization, during either early recovery from nocodazole, or brief treatment with taxol. From these and other findings we speculate that microtubules are guided into substrate contact sites and through the motor-dependent delivery of signaling molecules serve to modulate their development. It is further proposed this modulation provides the route whereby microtubules exert their influence on cell shape and polarity.
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Panda, Dulal, Keith DeLuca, Daniel Williams, Mary Ann Jordan, and Leslie Wilson. "Antiproliferative mechanism of action of cryptophycin-52: Kinetic stabilization of microtubule dynamics by high-affinity binding to microtubule ends." Proceedings of the National Academy of Sciences 95, no. 16 (1998): 9313–18. http://dx.doi.org/10.1073/pnas.95.16.9313.
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Cryptophycin-52 (LY355703) is a new synthetic member of the cryptophycin family of antimitotic antitumor agents that is currently undergoing clinical evaluation. At high concentrations (≥10 times the IC50), cryptophycin-52 blocked HeLa cell proliferation at mitosis by depolymerizing spindle microtubules and disrupting chromosome organization. However, low concentrations of cryptophycin-52 inhibited cell proliferation at mitosis (IC50 = 11 pM) without significantly altering spindle microtubule mass or organization. Cryptophycin-52 appears to be the most potent suppressor of microtubule dynamics found thus far. It suppressed the dynamic instability behavior of individual microtubules in vitro (IC50 = 20 nM), reducing the rate and extent of shortening and growing without significantly reducing polymer mass or mean microtubule length. Using [3H]cryptophycin-52, we found that the compound bound to microtubule ends in vitro with high affinity (Kd, 47 nM, maximum of ≈19.5 cryptophycin-52 molecules per microtubule). By analyzing the effects of cryptophycin-52 on dynamics in relation to its binding to microtubules, we determined that ≈5–6 molecules of cryptophycin-52 bound to a microtubule were sufficient to decrease dynamicity by 50%. Cryptophycin-52 became concentrated in cells 730-fold, and the resulting intracellular cryptophycin-52 concentration was similar to that required to stabilize microtubule dynamics in vitro. The data suggest that cryptophycin-52 potently perturbs kinetic events at microtubule ends that are required for microtubule function during mitosis and that it acts by forming a reversible cryptophycin-52-tubulin stabilizing cap at microtubule ends.
15
Lee, G., and S. L. Rook. "Expression of tau protein in non-neuronal cells: microtubule binding and stabilization." Journal of Cell Science 102, no. 2 (1992): 227–37. http://dx.doi.org/10.1242/jcs.102.2.227.
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The microtubule-associated protein tau is a developmentally regulated family of neuronal phosphoproteins that promotes the assembly and stabilization of microtubules. The carboxy-terminal half of the protein contains three copies of an imperfectly repeated sequence; this region has been found to bind microtubules in vitro. In addition, a fourth copy of the repeat has been found in adult-specific forms of tau protein. To examine the structure and function of tau protein in vivo, we have transiently expressed fetal and adult forms of tau protein and tau protein fragments in tissue culture cells. Biochemical analysis reveals full-length products with heterogeneity in post-translational modification synthesized in the cells. Immunofluorescent staining of transfected cells shows that, under our conditions, sequences on both sides of the repeat region are required for in vivo microtubule co-localization. These additional regions may be required either for enhancing microtubule contacts or for proper protein folding in the cell. In our expression system, the bundling of cellular microtubules occurs only in transfections using four-repeat tau constructs; any four-repeat construct capable of binding is also able to induce bundling. Our data suggest that the presence of bundles is correlated with enhanced microtubule stability; factors that increase stability such as higher levels of tau protein expression or the presence of the fourth repeat, increase the fraction of transfected cells showing bundles. Finally, the presence of tau protein in the cell allows all interphase microtubules to become acetylated, a post-translational modification usually reserved for a subset of stable cellular microtubules.
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Castle, Brian T., Seth McCubbin, Louis S. Prahl, Jordan N. Bernens, David Sept, and David J. Odde. "Mechanisms of kinetic stabilization by the drugs paclitaxel and vinblastine." Molecular Biology of the Cell 28, no. 9 (2017): 1238–57. http://dx.doi.org/10.1091/mbc.e16-08-0567.
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Microtubule-targeting agents (MTAs), widely used as biological probes and chemotherapeutic drugs, bind directly to tubulin subunits and “kinetically stabilize” microtubules, suppressing the characteristic self-assembly process of dynamic instability. However, the molecular-level mechanisms of kinetic stabilization are unclear, and the fundamental thermodynamic and kinetic requirements for dynamic instability and its elimination by MTAs have yet to be defined. Here we integrate a computational model for microtubule assembly with nanometer-scale fluorescence microscopy measurements to identify the kinetic and thermodynamic basis of kinetic stabilization by the MTAs paclitaxel, an assembly promoter, and vinblastine, a disassembly promoter. We identify two distinct modes of kinetic stabilization in live cells, one that truly suppresses on-off kinetics, characteristic of vinblastine, and the other a “pseudo” kinetic stabilization, characteristic of paclitaxel, that nearly eliminates the energy difference between the GTP- and GDP-tubulin thermodynamic states. By either mechanism, the main effect of both MTAs is to effectively stabilize the microtubule against disassembly in the absence of a robust GTP cap.
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Delphin, Christian, Denis Bouvier, Maxime Seggio, et al. "MAP6-F Is a Temperature Sensor That Directly Binds to and Protects Microtubules from Cold-induced Depolymerization." Journal of Biological Chemistry 287, no. 42 (2012): 35127–38. http://dx.doi.org/10.1074/jbc.m112.398339.
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Microtubules are dynamic structures that present the peculiar characteristic to be ice-cold labile in vitro. In vivo, microtubules are protected from ice-cold induced depolymerization by the widely expressed MAP6/STOP family of proteins. However, the mechanism by which MAP6 stabilizes microtubules at 4 °C has not been identified. Moreover, the microtubule cold sensitivity and therefore the needs for microtubule stabilization in the wide range of temperatures between 4 and 37 °C are unknown. This is of importance as body temperatures of animals can drop during hibernation or torpor covering a large range of temperatures. Here, we show that in the absence of MAP6, microtubules in cells below 20 °C rapidly depolymerize in a temperature-dependent manner whereas they are stabilized in the presence of MAP6. We further show that in cells, MAP6-F binding to and stabilization of microtubules is temperature- dependent and very dynamic, suggesting a direct effect of the temperature on the formation of microtubule/MAP6 complex. We also demonstrate using purified proteins that MAP6-F binds directly to microtubules through its Mc domain. This binding is temperature-dependent and coincides with progressive conformational changes of the Mc domain as revealed by circular dichroism. Thus, MAP6 might serve as a temperature sensor adapting its conformation according to the temperature to maintain the cellular microtubule network in organisms exposed to temperature decrease.
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Häussinger, Dieter, Barbara Stoll, Stephan vom Dahl, et al. "Effect of hepatocyte swelling on microtubule stability and tubulin mRNA levels." Biochemistry and Cell Biology 72, no. 1-2 (1994): 12–19. http://dx.doi.org/10.1139/o94-003.
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Incubation of isolated rat hepatocytes under conditions known to induce cell swelling caused several alterations in microtubule physiology. As shown by immunofluorescence microscopy experiments in the absence and presence of triethyllead or colchicine (two well-established microtubule inhibitors), an apparent stabilization of the microtubule network became evident in hepatocytes exposed to hypotonic (190 mosmol/L) conditions. A similar stabilizing effect was also observed upon cell swelling induced by addition of insulin (100 nmol/L) or glutamine (10 mmol/L). The differential microtubule stabilities were not attributed to a differential incorporation of the antimicrotubular agents into hepatocytes as shown by [3H]colchicine-uptake experiments. The swelling-induced alterations of microtubules may contribute to the swelling-induced changes of liver cell function: in perfused rat liver it was found that the established inhibitory effect of hypotonic cell swelling on hepatic proteolysis was largely abolished in presence of colchicine. Tubulin mRNA levels increased by 1.9-, 2.1- and 2.7-fold in isolated hepatocytes being exposed for 120 min to hypotonic medium, insulin, or glutamine, respectively. The results suggest an involvement of microtubular structures in the regulation of liver metabolism in response to alterations of the cellular hydration state.Key words: microtubules, cell swelling, glutamine, gene expression, proteolysis.
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Gundersen, G. G., S. Khawaja, and J. C. Bulinski. "Postpolymerization detyrosination of alpha-tubulin: a mechanism for subcellular differentiation of microtubules." Journal of Cell Biology 105, no. 1 (1987): 251–64. http://dx.doi.org/10.1083/jcb.105.1.251.
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Tyrosinated (Tyr) and detyrosinated (Glu) alpha-tubulin, species interconverted by posttranslational modification, are largely segregated in separate populations of microtubules in interphase cultured cells. We sought to understand how distinct Tyr and Glu microtubules are generated in vivo, by examining time-dependent alterations in Tyr and Glu tubulin levels (by immunoblots probed with antibodies specific for each species) and distributions (by immunofluorescence) after microtubule regrowth and stabilization. When microtubules were allowed to regrow after complete depolymerization by microtubule antagonists, Glu microtubules reappeared with a delay of approximately 25 min after the complete array of Tyr microtubules had regrown. In these experiments, Tyr tubulin immunofluorescence first appeared as an aster of distinct microtubules, while Glu tubulin staining first appeared as a grainy pattern that was not altered by detergent extraction, suggesting that Glu microtubules were created by detyrosination of Tyr microtubules. Treatments with taxol, azide, or vinblastine, to stabilize polymeric tubulin, all resulted in time-dependent increases in polymeric Glu tubulin levels, further supporting the hypothesis of postpolymerization detyrosination. Analysis of monomer and polymer fractions during microtubule regrowth and in microtubule stabilization experiments were also consistent with postpolymerization detyrosination; in each case, Glu polymer levels increased in the absence of detectable Glu monomer. The low level of Glu monomer in untreated or nocodazole-treated cells (we estimate that Glu tubulin comprises less than 2% of the monomer pool) also suggested that Glu tubulin entering the monomer pool is efficiently retyrosinated. Taken together these results demonstrate that microtubules are polymerized from Tyr tubulin and are then rapidly converted to Glu microtubules. When Glu microtubules depolymerize, the resulting Glu monomer is retyrosinated. This cycle generates structurally, and perhaps functionally, distinct microtubules.
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Krylova, Olga, Marcus J. Messenger, and Patricia C. Salinas. "Dishevelled-1 Regulates Microtubule Stability." Journal of Cell Biology 151, no. 1 (2000): 83–94. http://dx.doi.org/10.1083/jcb.151.1.83.
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Dishevelled has been implicated in the regulation of cell fate decisions, cell polarity, and neuronal function. However, the mechanism of Dishevelled action remains poorly understood. Here we examine the cellular localization and function of the mouse Dishevelled protein, DVL-1. Endogenous DVL-1 colocalizes with axonal microtubules and sediments with brain microtubules. Expression of DVL-1 protects stable microtubules from depolymerization by nocodazole in both dividing cells and differentiated neuroblastoma cells. Deletion analyses reveal that the PDZ domain, but not the DEP domain, of DVL-1 is required for microtubule stabilization. The microtubule stabilizing function of DVL-1 is mimicked by lithium-mediated inhibition of glycogen synthase kinase-3β (GSK-3β) and blocked by expression of GSK-3β. These findings suggest that DVL-1, through GSK-3β, can regulate microtubule dynamics. This new function of DVL-1 in controlling microtubule stability may have important implications for Dishevelled proteins in regulating cell polarity.
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Logan, Caitlin M., and A. Sue Menko. "Microtubules: Evolving roles and critical cellular interactions." Experimental Biology and Medicine 244, no. 15 (2019): 1240–54. http://dx.doi.org/10.1177/1535370219867296.
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Microtubules are cytoskeletal elements known as drivers of directed cell migration, vesicle and organelle trafficking, and mitosis. In this review, we discuss new research in the lens that has shed light into further roles for stable microtubules in the process of development and morphogenesis. In the lens, as well as other systems, distinct roles for characteristically dynamic microtubules and stabilized populations are coming to light. Understanding the mechanisms of microtubule stabilization and the associated microtubule post-translational modifications is an evolving field of study. Appropriate cellular homeostasis relies on not only one cytoskeletal element, but also rather an interaction between cytoskeletal proteins as well as other cellular regulators. Microtubules are key integrators with actin and intermediate filaments, as well as cell–cell junctional proteins and other cellular regulators including myosin and RhoGTPases to maintain this balance. Impact statement The role of microtubules in cellular functioning is constantly expanding. In this review, we examine new and exciting fields of discovery for microtubule’s involvement in morphogenesis, highlight our evolving understanding of differential roles for stabilized versus dynamic subpopulations, and further understanding of microtubules as a cellular integrator.
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Nabi, Ivan R., Ginette Guay, and Danièle Simard. "AMF-R Tubules Concentrate in a Pericentriolar Microtubule Domain After MSV Transformation of Epithelial MDCK Cells." Journal of Histochemistry & Cytochemistry 45, no. 10 (1997): 1351–63. http://dx.doi.org/10.1177/002215549704501004.
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Autocrine motility factor receptor (AMF-R) is localized to an intracellular microtubule-associated membranous organelle, the AMF-R tubule. In well-spread untrans-formed MDCK epithelial cells, the microtubules originate from a broad perinuclear region and AMF-R tubules extend throughout the cytoplasm of the cells. In Moloney sarcoma virus (mos)-transformed MDCK (MSV-MDCK) cells, microtubules accumulate around the centrosome, forming a microtubule domain rich in stabilized detyrosinated microtubules. AMF-R tubules are quantitatively associated with this pericentriolar microtubule domain and the rough endoplasmic reticulum and lysosomes also co-distribute with the pericentriolar mass of microtubules. The Golgi apparatus is closely associated with the microtubule organizing center (MTOC) within the juxtanuclear mass of AMF-R tubules, and no co-localization of AMF-R tubules with the Golgi marker β-COP could be detected by confocal microscopy. After nocodazole treatment and washout, microtubule nucleation occurs exclusively at the centrosome of MSV-MDCK cells, and only after microtubule extension to the cell periphery does the microtubule cytoskeleton reorganize to generate the pericentriolar microtubule domain after 30–60 min. AMF-R tubules dispersed by nocodazole treatment concentrate in the pericentriolar region in parallel with the reorganization of the microtubule cytoskeleton. MSV transformation of epithelial MDCK cells results in the stabilization of a pericentriolar microtubule domain responsible for the concentration and polarized distribution of AMF-R tubules.
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Vemu, Annapurna, Ewa Szczesna, Elena A. Zehr, et al. "Severing enzymes amplify microtubule arrays through lattice GTP-tubulin incorporation." Science 361, no. 6404 (2018): eaau1504. http://dx.doi.org/10.1126/science.aau1504.
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Spastin and katanin sever and destabilize microtubules. Paradoxically, despite their destructive activity they increase microtubule mass in vivo. We combined single-molecule total internal reflection fluorescence microscopy and electron microscopy to show that the elemental step in microtubule severing is the generation of nanoscale damage throughout the microtubule by active extraction of tubulin heterodimers. These damage sites are repaired spontaneously by guanosine triphosphate (GTP)–tubulin incorporation, which rejuvenates and stabilizes the microtubule shaft. Consequently, spastin and katanin increase microtubule rescue rates. Furthermore, newly severed ends emerge with a high density of GTP-tubulin that protects them against depolymerization. The stabilization of the newly severed plus ends and the higher rescue frequency synergize to amplify microtubule number and mass. Thus, severing enzymes regulate microtubule architecture and dynamics by promoting GTP-tubulin incorporation within the microtubule shaft.
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Ray, Shayoni, Joseph A. Fanti, Diego P. Macedo, and Melinda Larsen. "LIM kinase regulation of cytoskeletal dynamics is required for salivary gland branching morphogenesis." Molecular Biology of the Cell 25, no. 16 (2014): 2393–407. http://dx.doi.org/10.1091/mbc.e14-02-0705.
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Coordinated actin microfilament and microtubule dynamics is required for salivary gland development, although the mechanisms by which they contribute to branching morphogenesis are not defined. Because LIM kinase (LIMK) regulates both actin and microtubule organization, we investigated the role of LIMK signaling in mouse embryonic submandibular salivary glands using ex vivo organ cultures. Both LIMK 1 and 2 were necessary for branching morphogenesis and functioned to promote epithelial early- and late-stage cleft progression through regulation of both microfilaments and microtubules. LIMK-dependent regulation of these cytoskeletal systems was required to control focal adhesion protein–dependent fibronectin assembly and integrin β1 activation, involving the LIMK effectors cofilin and TPPP/p25, for assembly of the actin- and tubulin-based cytoskeletal systems, respectively. We demonstrate that LIMK regulates the early stages of cleft formation—cleft initiation, stabilization, and progression—via establishment of actin stability. Further, we reveal a novel role for the microtubule assembly factor p25 in regulating stabilization and elongation of late-stage progressing clefts. This study demonstrates the existence of multiple actin- and microtubule-dependent stabilization steps that are controlled by LIMK and are required in cleft progression during branching morphogenesis.
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Burakov, Anton V., Olga N. Zhapparova, Olga V. Kovalenko, et al. "Ste20-related Protein Kinase LOSK (SLK) Controls Microtubule Radial Array in Interphase." Molecular Biology of the Cell 19, no. 5 (2008): 1952–61. http://dx.doi.org/10.1091/mbc.e06-12-1156.
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Interphase microtubules are organized into a radial array with centrosome in the center. This organization is a subject of cellular regulation that can be driven by protein phosphorylation. Only few protein kinases that regulate microtubule array in interphase cells have been described. Ste20-like protein kinase LOSK (SLK) was identified as a microtubule and centrosome-associated protein. In this study we have shown that the inhibition of LOSK activity by dominant-negative mutant K63R-ΔT or by LOSK depletion with RNAi leads to unfocused microtubule arrangement. Microtubule disorganization is prominent in Vero, CV-1, and CHO-K1 cells but less distinct in HeLa cells. The effect is a result neither of microtubule stabilization nor of centrosome disruption. In cells with suppressed LOSK activity centrosomes are unable to anchor or to cap microtubules, though they keep nucleating microtubules. These centrosomes are depleted of dynactin. Vero cells overexpressing K63R-ΔT have normal dynactin “comets” at microtubule ends and unaltered morphology of Golgi complex but are unable to polarize it at the wound edge. We conclude that protein kinase LOSK is required for radial microtubule organization and for the proper localization of Golgi complex in various cell types.
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Fassett, John T., Xinli Hu, Xin Xu, et al. "AMPK attenuates microtubule proliferation in cardiac hypertrophy." American Journal of Physiology-Heart and Circulatory Physiology 304, no. 5 (2013): H749—H758. http://dx.doi.org/10.1152/ajpheart.00935.2011.
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Cell hypertrophy requires increased protein synthesis and expansion of the cytoskeletal networks that support cell enlargement. AMPK limits anabolic processes, such as protein synthesis, when energy supply is insufficient, but its role in cytoskeletal remodeling is not known. Here, we examined the influence of AMPK in cytoskeletal remodeling during cardiomyocyte hypertrophy, a clinically relevant condition in which cardiomyocytes enlarge but do not divide. In neonatal cardiomyocytes, activation of AMPK with 5-aminoimidazole carboxamide ribonucleotide (AICAR) or expression of constitutively active AMPK (CA-AMPK) attenuated cell area increase by hypertrophic stimuli (phenylephrine). AMPK activation had little effect on intermediate filaments or myofilaments but dramatically reduced microtubule stability, as measured by detyrosinated tubulin levels and cytoskeletal tubulin accumulation. Importantly, low-level AMPK activation limited cell expansion and microtubule growth independent of mTORC1 or protein synthesis repression, identifying a new mechanism by which AMPK regulates cell growth. Mechanistically, AICAR treatment increased Ser-915 phosphorylation of microtubule-associated protein 4 (MAP4), which reduces affinity for tubulin and prevents stabilization of microtubules (MTs). RNAi knockdown of MAP4 confirmed its critical role in cardiomyocyte MT stabilization. In support of a pathophysiological role for AMPK regulation of cardiac microtubules, AMPK α2 KO mice exposed to pressure overload (transverse aortic constriction; TAC) demonstrated reduced MAP4 phosphorylation and increased microtubule accumulation that correlated with the severity of contractile dysfunction. Together, our data identify the microtubule cytoskeleton as a sensitive target of AMPK activity, and the data suggest a novel role for AMPK in limiting accumulation and densification of microtubules that occurs in response to hypertrophic stress.
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Leger, J. G., R. Brandt, and G. Lee. "Identification of tau protein regions required for process formation in PC12 cells." Journal of Cell Science 107, no. 12 (1994): 3403–12. http://dx.doi.org/10.1242/jcs.107.12.3403.
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Tau is a neuronal microtubule-associated protein that is required for the development and maintenance of neuronal cell polarity. It promotes microtubule assembly in vitro and we have recently reported that a specific tau region, which spans amino acid residues 154–172 of human fetal tau, is not required for growth of existing microtubules, but is required for nucleation of new microtubules. These residues also confer stronger microtubule binding activity in 3T3 cells. The aim of this study was to investigate the functional organization of tau in relation to its role in promoting process formation in a neuronal model system. We transfected undifferentiated PC12 cells with vectors expressing tau fragments and treated the expressing cells with cytochalasin B to allow process extension. We found that deletion of amino acid residues 154–172 greatly reduced the percentage of transfected cells bearing processes compared to that of cells transfected with full-length tau or with an amino-terminally deleted tau fragment containing residues 154–172. These differences do not appear to result from a quantitative difference in protein expression, as shown by immunoblot analysis of transfected cells. We also observed that while the presence of tau fragments increases acetylation of microtubules, the pattern of acetylation in cells transfected with the fragment missing residues 154–172 is less extensive, suggesting that it does not result in the same level of stabilization as the longer tau fragments. Taxol promoted process outgrowth in cells treated with cytochalasin and restored process outgrowth to cells transfected with the tau fragment lacking this activity. Therefore, process formation involves primarily the stabilization and nucleation of microtubules. We conclude that the residues necessary for conferring microtubule nucleation activity of tau in vitro are important for process formation in vivo. It is likely that these residues influence the binding affinity and therefore the stabilization activity of tau.
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Li, J., Z. Sun, Z. Lv, and D. Shi. "Microtubule stabilization potentiates cartilage regeneration." Osteoarthritis and Cartilage 29 (April 2021): S196. http://dx.doi.org/10.1016/j.joca.2021.02.265.
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Scaife, Robin M., Didier Job, and Wallace Y. Langdon. "Rapid Microtubule-dependent Induction of Neurite-like Extensions in NIH 3T3 Fibroblasts by Inhibition of ROCK and Cbl." Molecular Biology of the Cell 14, no. 11 (2003): 4605–17. http://dx.doi.org/10.1091/mbc.e02-11-0739.
Full textAbstract:
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.
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Preuss, U., J. Biernat, E. M. Mandelkow, and E. Mandelkow. "The ‘jaws’ model of tau-microtubule interaction examined in CHO cells." Journal of Cell Science 110, no. 6 (1997): 789–800. http://dx.doi.org/10.1242/jcs.110.6.789.
Full textAbstract:
Tau is a neuronal microtubule-associated protein which promotes microtubule assembly. The C-terminal half of the protein contains three or four tandem repeats that are often considered to be the microtubule binding domain. This view is in conflict with in vitro data showing that the repeat domain binds only weakly to microtubules while the domains flanking the repeats bind strongly, even in the absence of the repeats. This has lead us to propose a ‘jaws’ model of tau whereby the regions flanking the repeats are considered as targetting domains, responsible for positioning tau on the microtubule surface, and the repeats which act as catalytic domains for microtubule assembly. To examine whether this model is appropriate in vivo we generated recombinant tau isoforms and microinjected them into CHO cells. Immunofluorescence microscopy of microtubules and tau shows that binding to microtubules, stabilization of microtubules and formation of bundles is not achieved by tau constructs comprising individual domains, but requires the combination of the flanking regions and the repeat domain. The results show that the jaws model describes the interactions between tau and microtubules in living cells. Since the targetting and catalytic domains are affected differently by phosphorylation the model provides a basis for studying the regulation of the interaction between microtubules and tau or other microtubule-associated proteins.
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Tonami, Kazuo, Yukiko Kurihara, Hiroyuki Aburatani, Yasunobu Uchijima, Tomoichiro Asano, and Hiroki Kurihara. "Calpain 6 Is Involved in Microtubule Stabilization and Cytoskeletal Organization." Molecular and Cellular Biology 27, no. 7 (2007): 2548–61. http://dx.doi.org/10.1128/mcb.00992-06.
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ABSTRACT The calpains are a family of Ca2+-dependent cysteine proteases implicated in various biological processes. In this family, calpain 6 (Capn6) is unique in that it lacks the active-site cysteine residues requisite for protease activity. During the search for genes downstream of the endothelin 1 (ET-1) signaling in pharyngeal-arch development, we identified Capn6. After confirming that the expression of Capn6 in pharyngeal arches is downregulated in ET-1-null embryos by in situ hybridization, we investigated its function. In Capn6-transfected cells, cytokinesis was retarded and was often aborted to yield multinucleated cells. Capn6 overexpression also caused the formation of microtubule bundles rich in acetylated α-tubulin and resistant to the depolymerizing activity of nocodazole. Green fluorescent protein-Capn6 overexpression, immunostaining for endogenous Capn6, and biochemical analysis demonstrated interaction between Capn6 and microtubules, which appeared to be mainly mediated by domain III. Furthermore, RNA interference-mediated Capn6 inactivation caused microtubule instability with a loss of acetylated α-tubulin and induced actin reorganization, resulting in lamellipodium formation with membrane ruffling. Taken together, these results indicate that Capn6 is a microtubule-stabilizing protein expressed in embryonic tissues that may be involved in the regulation of microtubule dynamics and cytoskeletal organization.
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Asrih, Mohamed, Corinne Pellieux, Irène Papageorgiou, René Lerch, and Christophe Montessuit. "Role of ERK1/2 activation in microtubule stabilization and glucose transport in cardiomyocytes." American Journal of Physiology-Endocrinology and Metabolism 301, no. 5 (2011): E836—E843. http://dx.doi.org/10.1152/ajpendo.00160.2011.
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We previously demonstrated that microtubule disruption impairs stimulation of glucose uptake in cardiomyocytes and that 9-cis retinoic acid (9cRA) treatment preserved both microtubule integrity and stimulated glucose transport. Herein we investigated whether 1) activation of the extracellular signal-regulated kinases (ERK1/2) is responsible for microtubule destabilization and 2) ERK1/2 inactivation may explain the positive effects of 9cRA on glucose uptake and microtubule stabilization. Adult rat cardiomyocytes in primary culture showed increased basal ERK1/2 phosphorylation. Cardiomyocytes exposed to inhibitors of the ERK1/2 kinase mitogen/extracellular signal-regulated kinase (MEK) 1/2 had preserved microtubular scaffold, including microtubule-organizing centers (MTOC), together with increased insulin and metabolic stress-stimulated glucose transport as well as signaling, thus replicating the effects of 9cRA treatment. Although 9cRA treatment did not significantly reduce global ERK1/2 activation, it markedly reduced perinuclear-activated ERK1/2 at the location of MTOC. 9cRA also triggered relocation of the ERK1/2 phosphatase mitogen-activated protein kinase phosphatase-3 from the cytosol to the nucleus. These results indicate that, in cardiomyocytes, microtubule destabilization, leading to impaired stimulation of glucose transport, is mediated by ERK1/2 activation, impacting on the MTOC. 9cRA acid restores stimulated glucose transport indirectly through compartmentalized inactivation of ERK1/2.
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Kremer, Brandon E., Timothy Haystead, and Ian G. Macara. "Mammalian Septins Regulate Microtubule Stability through Interaction with the Microtubule-binding Protein MAP4." Molecular Biology of the Cell 16, no. 10 (2005): 4648–59. http://dx.doi.org/10.1091/mbc.e05-03-0267.
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Mammalian septins constitute a family of at least 12 GTP-binding proteins that can form hetero-oligomers and that are sometimes found in association with actin or microtubule filaments. However, their functions are not understood. Using RNA interference, we found that suppression of septin expression in HeLa cells caused a pronounced increase in microtubule stability. Mass spectroscopic analysis of proteins coprecipitating with Sept6 identified the microtubule-associated protein MAP4 as a septin binding partner. A small, proline-rich region in the C-terminal half of MAP4 bound directly to a Sept 2:6:7 heterotrimer, and to the Sept2 monomer. The trimer blocked the ability of this MAP4 fragment to bind and bundle microtubules in vitro. In intact cells, MAP4 was required for the stabilization of microtubules induced by septin depletion. Moreover, septin depletion increased the number of cells with abnormal nuclei, and this effect was blocked by gene silencing of MAP4. These data identify a novel molecular function for septins in mammalian cells: the modulation of microtubule dynamics through interaction with MAP4.
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Rodionov, V. I., S. S. Lim, V. I. Gelfand, and G. G. Borisy. "Microtubule dynamics in fish melanophores." Journal of Cell Biology 126, no. 6 (1994): 1455–64. http://dx.doi.org/10.1083/jcb.126.6.1455.
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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.
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Nguyen, H. L., D. Gruber, T. Mcgraw, M. P. Sheetz, and J. C. Bulinski. "Stabilization and Functional Modulation of Microtubules by Microtubule-Associated Protein 4." Biological Bulletin 194, no. 3 (1998): 354–57. http://dx.doi.org/10.2307/1543111.
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Horníková, Lenka, Kateřina Bruštíková, Boris Ryabchenko, et al. "The Major Capsid Protein, VP1, of the Mouse Polyomavirus Stimulates the Activity of Tubulin Acetyltransferase 1 by Microtubule Stabilization." Viruses 12, no. 2 (2020): 227. http://dx.doi.org/10.3390/v12020227.
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Viruses have evolved mechanisms to manipulate microtubules (MTs) for the efficient realization of their replication programs. Studying the mechanisms of replication of mouse polyomavirus (MPyV), we observed previously that in the late phase of infection, a considerable amount of the main structural protein, VP1, remains in the cytoplasm associated with hyperacetylated microtubules. VP1–microtubule interactions resulted in blocking the cell cycle in the G2/M phase. We are interested in the mechanism leading to microtubule hyperacetylation and stabilization and the roles of tubulin acetyltransferase 1 (αTAT1) and deacetylase histone deacetylase 6 (HDAC6) and VP1 in this mechanism. Therefore, HDAC6 inhibition assays, αTAT1 knock out cell infections, in situ cell fractionation, and confocal and TIRF microscopy were used. The experiments revealed that the direct interaction of isolated microtubules and VP1 results in MT stabilization and a restriction of their dynamics. VP1 leads to an increase in polymerized tubulin in cells, thus favoring αTAT1 activity. The acetylation status of MTs did not affect MPyV infection. However, the stabilization of MTs by VP1 in the late phase of infection may compensate for the previously described cytoskeleton destabilization by MPyV early gene products and is important for the observed inhibition of the G2→M transition of infected cells to prolong the S phase.
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Shannon, Katie B., Julie C. Canman, C. Ben Moree, Jennifer S. Tirnauer, and E. D. Salmon. "Taxol-stabilized Microtubules Can Position the Cytokinetic Furrow in Mammalian Cells." Molecular Biology of the Cell 16, no. 9 (2005): 4423–36. http://dx.doi.org/10.1091/mbc.e04-11-0974.
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How microtubules act to position the plane of cell division during cytokinesis is a topic of much debate. Recently, we showed that a subpopulation of stable microtubules extends past chromosomes and interacts with the cell cortex at the site of furrowing, suggesting that these stabilized microtubules may stimulate contractility. To test the hypothesis that stable microtubules can position furrows, we used taxol to rapidly suppress microtubule dynamics during various stages of mitosis in PtK1 cells. Cells with stabilized prometaphase or metaphase microtubule arrays were able to initiate furrowing when induced into anaphase by inhibition of the spindle checkpoint. In these cells, few microtubules contacted the cortex. Furrows formed later than usual, were often aberrant, and did not progress to completion. Images showed that furrowing correlated with the presence of one or a few stable spindle microtubule plus ends at the cortex. Actin, myosin II, and anillin were all concentrated in these furrows, demonstrating that components of the contractile ring can be localized by stable microtubules. Inner centromere protein (INCENP) was not found in these ingressions, confirming that INCENP is dispensable for furrow positioning. Taxol-stabilization of the numerous microtubule-cortex interactions after anaphase onset delayed furrow initiation but did not perturb furrow positioning. We conclude that taxol-stabilized microtubules can act to position the furrow and that loss of microtubule dynamics delays the timing of furrow onset and prevents completion. We discuss our findings relative to models for cleavage stimulation.
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Kim, Yumi, John E. Heuser, Clare M. Waterman, and Don W. Cleveland. "CENP-E combines a slow, processive motor and a flexible coiled coil to produce an essential motile kinetochore tether." Journal of Cell Biology 181, no. 3 (2008): 411–19. http://dx.doi.org/10.1083/jcb.200802189.
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The mitotic kinesin centromere protein E (CENP-E) is an essential kinetochore component that directly contributes to the capture and stabilization of spindle microtubules by kinetochores. Although reduction in CENP-E leads to high rates of whole chromosome missegregation, neither its properties as a microtubule-dependent motor nor how it contributes to the dynamic linkage between kinetochores and microtubules is known. Using single-molecule assays, we demonstrate that CENP-E is a very slow, highly processive motor that maintains microtubule attachment for long periods. Direct visualization of full-length Xenopus laevis CENP-E reveals a highly flexible 230-nm coiled coil separating its kinetochore-binding and motor domains. We also show that full-length CENP-E is a slow plus end–directed motor whose activity is essential for metaphase chromosome alignment. We propose that the highly processive microtubule-dependent motor activity of CENP-E serves to power chromosome congression and provides a flexible, motile tether linking kinetochores to dynamic spindle microtubules.
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Moores, Carolyn A., Mylène Perderiset, Fiona Francis, Jamel Chelly, Anne Houdusse, and Ronald A. Milligan. "Mechanism of Microtubule Stabilization by Doublecortin." Molecular Cell 14, no. 6 (2004): 833–39. http://dx.doi.org/10.1016/j.molcel.2004.06.009.
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DeWard, Aaron D., and Arthur S. Alberts. "Microtubule Stabilization: Formins Assert Their Independence." Current Biology 18, no. 14 (2008): R605—R608. http://dx.doi.org/10.1016/j.cub.2008.06.001.
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Manka, Szymon W., and Carolyn A. Moores. "Microtubule Nucleation and Stabilization by Doublecortin." Biophysical Journal 116, no. 3 (2019): 256a. http://dx.doi.org/10.1016/j.bpj.2018.11.1394.
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Ruthel, Gordon, Gretchen L. Demmin, George Kallstrom, et al. "Association of Ebola Virus Matrix Protein VP40 with Microtubules." Journal of Virology 79, no. 8 (2005): 4709–19. http://dx.doi.org/10.1128/jvi.79.8.4709-4719.2005.
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ABSTRACT Viruses exploit a variety of cellular components to complete their life cycles, and it has become increasingly clear that use of host cell microtubules is a vital part of the infection process for many viruses. A variety of viral proteins have been identified that interact with microtubules, either directly or via a microtubule-associated motor protein. Here, we report that Ebola virus associates with microtubules via the matrix protein VP40. When transfected into mammalian cells, a fraction of VP40 colocalized with microtubule bundles and VP40 coimmunoprecipitated with tubulin. The degree of colocalization and microtubule bundling in cells was markedly intensified by truncation of the C terminus to a length of 317 amino acids. Further truncation to 308 or fewer amino acids abolished the association with microtubules. Both the full-length and the 317-amino-acid truncation mutant stabilized microtubules against depolymerization with nocodazole. Direct physical interaction between purified VP40 and tubulin proteins was demonstrated in vitro. A region of moderate homology to the tubulin binding motif of the microtubule-associated protein MAP2 was identified in VP40. Deleting this region resulted in loss of microtubule stabilization against drug-induced depolymerization. The presence of VP40-associated microtubules in cells continuously treated with nocodazole suggested that VP40 promotes tubulin polymerization. Using an in vitro polymerization assay, we demonstrated that VP40 directly enhances tubulin polymerization without any cellular mediators. These results suggest that microtubules may play an important role in the Ebola virus life cycle and potentially provide a novel target for therapeutic intervention against this highly pathogenic virus.
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Bré, M. H., R. Pepperkok, A. M. Hill, et al. "Regulation of microtubule dynamics and nucleation during polarization in MDCK II cells." Journal of Cell Biology 111, no. 6 (1990): 3013–21. http://dx.doi.org/10.1083/jcb.111.6.3013.
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MDCK cells form a polarized epithelium when they reach confluence in tissue culture. We have previously shown that concomitantly with the establishment of intercellular junctions, centrioles separate and microtubules lose their radial organization (Bacallao, R., C. Antony, C. Dotti, E. Karsenti, E.H.K. Stelzer, and K. Simons. 1989. J. Cell Biol. 109:2817-2832. Buendia, B., M.H. Bré, G. Griffiths, and E. Karsenti. 1990. 110:1123-1136). In this work, we have examined the pattern of microtubule nucleation before and after the establishment of intercellular contacts. We analyzed the elongation rate and stability of microtubules in single and confluent cells. This was achieved by microinjection of Paramecium axonemal tubulin and detection of the newly incorporated subunits by an antibody directed specifically against the Paramecium axonemal tubulin. The determination of newly nucleated microtubule localization has been made possible by the use of advanced double-immunofluorescence confocal microscopy. We have shown that in single cells, newly nucleated microtubules originate from several sites concentrated in a region localized close to the nucleus and not from a single spot that could correspond to a pair of centrioles. In confluent cells, newly nucleated microtubules were still more dispersed. The microtubule elongation rate of individual microtubules was not different in single and confluent cells (4 microns/min). However, in confluent cells, the population of long lived microtubules was strongly increased. In single or subconfluent cells most microtubules showed a t1/2 of less than 30 min, whereas in confluent monolayers, a large population of microtubules had a t1/2 of greater than 2 h. These results, together with previous observations cited above, indicate that during the establishment of polarity in MDCK cells, microtubule reorganization involves both a relocalization of microtubule-nucleating activity and increased microtubule stabilization.
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Zhou, Qian, Chi Hang Wong, Cecilia Pik Yuk Lau, et al. "Enhanced Antitumor Activity with Combining Effect of mTOR Inhibition and Microtubule Stabilization in Hepatocellular Carcinoma." International Journal of Hepatology 2013 (2013): 1–10. http://dx.doi.org/10.1155/2013/103830.
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Mammalian target of rapamycin (mTOR) and the microtubules are shown to be potential targets for treating hepatocellular carcinoma (HCC). PI3K/Akt/mTOR activation is associated with resistance to microtubule inhibitors. Here, we evaluated the antitumor activity by cotargeting of the mTOR (using allosteric mTOR inhibitor everolimus) and the microtubules (using novel microtubule-stabilizing agent patupilone) in HCC models.In vitrostudies showed that either targeting mTOR signaling with everolimus or targeting microtubules with patupilone was able to suppress HCC cell growth in a dose-dependent manner. Cotargeting of the mTOR (by everolimus) and the microtubules (by patupilone, at low nM) resulted in enhanced growth inhibition in HCC cells (achieving maximal growth inhibition of 60–87%), demonstrating potent antitumor activity of this combination.In vivostudies showed that everolimus treatment alone for two weeks was able to inhibit the growth of Hep3B xenografts. Strikingly, the everolimus/patupilone combination induced a more significant antitumor activity. Mechanistic study demonstrated that this enhanced antitumor effect was accompanied by marked cell apoptosis induction and antiangiogenic activity, which were more significant than single-agent treatments. Our findings demonstrated that the everolimus/patupilone combination, which had potent antitumor activity, was a potential therapeutic strategy for HCC.
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Bouguenina, Habib, Danièle Salaun, Aurélie Mangon, et al. "EB1-binding–myomegalin protein complex promotes centrosomal microtubules functions." Proceedings of the National Academy of Sciences 114, no. 50 (2017): E10687—E10696. http://dx.doi.org/10.1073/pnas.1705682114.
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Control of microtubule dynamics underlies several fundamental processes such as cell polarity, cell division, and cell motility. To gain insights into the mechanisms that control microtubule dynamics during cell motility, we investigated the interactome of the microtubule plus-end–binding protein end-binding 1 (EB1). Via molecular mapping and cross-linking mass spectrometry we identified and characterized a large complex associating a specific isoform of myomegalin termed “SMYLE” (for short myomegalin-like EB1 binding protein), the PKA scaffolding protein AKAP9, and the pericentrosomal protein CDK5RAP2. SMYLE was associated through an evolutionarily conserved N-terminal domain with AKAP9, which in turn was anchored at the centrosome via CDK5RAP2. SMYLE connected the pericentrosomal complex to the microtubule-nucleating complex (γ-TuRC) via Galectin-3–binding protein. SMYLE associated with nascent centrosomal microtubules to promote microtubule assembly and acetylation. Disruption of SMYLE interaction with EB1 or AKAP9 prevented microtubule nucleation and their stabilization at the leading edge of migrating cells. In addition, SMYLE depletion led to defective astral microtubules and abnormal orientation of the mitotic spindle and triggered G1 cell-cycle arrest, which might be due to defective centrosome integrity. As a consequence, SMYLE loss of function had a profound impact on tumor cell motility and proliferation, suggesting that SMYLE might be an important player in tumor progression.
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Pearson, Chad G., Paul S. Maddox, Ted R. Zarzar, E. D. Salmon, and Kerry Bloom. "Yeast Kinetochores Do Not Stabilize Stu2p-dependent Spindle Microtubule Dynamics." Molecular Biology of the Cell 14, no. 10 (2003): 4181–95. http://dx.doi.org/10.1091/mbc.e03-03-0180.
Full textAbstract:
The interaction of kinetochores with dynamic microtubules during mitosis is essential for proper centromere motility, congression to the metaphase plate, and subsequent anaphase chromosome segregation. Budding yeast has been critical in the discovery of proteins necessary for this interaction. However, the molecular mechanism for microtubule–kinetochore interactions remains poorly understood. Using live cell imaging and mutations affecting microtubule binding proteins and kinetochore function, we identify a regulatory mechanism for spindle microtubule dynamics involving Stu2p and the core kinetochore component, Ndc10p. Depleting cells of the microtubule binding protein Stu2p reduces kinetochore microtubule dynamics. Centromeres remain under tension but lack motility. Thus, normal microtubule dynamics are not required to maintain tension at the centromere. Loss of the kinetochore (ndc10-1, ndc10-2, and ctf13-30) does not drastically affect spindle microtubule turnover, indicating that Stu2p, not the kinetochore, is the foremost governor of microtubule dynamics. Disruption of kinetochore function with ndc10-1 does not affect the decrease in microtubule turnover in stu2 mutants, suggesting that the kinetochore is not required for microtubule stabilization. Remarkably, a partial kinetochore defect (ndc10-2) suppresses the decreased spindle microtubule turnover in the absence of Stu2p. These results indicate that Stu2p and Ndc10p differentially function in controlling kinetochore microtubule dynamics necessary for centromere movements.
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Casanova, Claudia M., Sofia Rybina, Hideki Yokoyama, Eric Karsenti, and Iain W. Mattaj. "Hepatoma Up-Regulated Protein Is Required for Chromatin-induced Microtubule Assembly Independently of TPX2." Molecular Biology of the Cell 19, no. 11 (2008): 4900–4908. http://dx.doi.org/10.1091/mbc.e08-06-0624.
Full textAbstract:
The production of RanGTP around chromosomes is crucial for spindle microtubule assembly in mitosis. Previous work has shown that hepatoma up-regulated protein (HURP) is a Ran target, required for microtubule stabilization and spindle organization. Here we report a detailed analysis of HURP function in Xenopus laevis mitotic egg extracts. HURP depletion severely impairs bipolar spindle assembly around chromosomes: the few spindles that do form show a significant decrease in microtubule density at the spindle midzone. HURP depletion does not interfere with microtubule growth from purified centrosomes, but completely abolishes microtubule assembly induced by chromatin beads or RanGTP. Simultaneous depletion of the microtubule destabilizer MCAK with HURP does not rescue the phenotype, demonstrating that the effect of HURP is not to antagonize the destabilization activity of MCAK. Although the phenotype of HURP depletion closely resembles that reported for TPX2 depletion, we find no evidence that TPX2 and HURP physically interact or that they influence each other in their effects on spindle microtubules. Our data indicate that HURP and TPX2 have nonredundant functions essential for chromatin-induced microtubule assembly.
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Ferhat, L., A. Represa, A. Bernard, Y. Ben-Ari, and M. Khrestchatisky. "MAP2d promotes bundling and stabilization of both microtubules and microfilaments." Journal of Cell Science 109, no. 5 (1996): 1095–103. http://dx.doi.org/10.1242/jcs.109.5.1095.
Full textAbstract:
Two low molecular weight MAP2 variants have been described, MAP2c and MAP2d. These variants are produced from a single gene by alternative splicing, and in their C-terminal regions contain, respectively, 3 and 4 tandem repeats, some of which are known to be involved in binding to microtubules. Substantial differences in the developmental expression pattern of MAP2c and MAP2d suggest they have different functions in neural cells. In order to investigate the respective roles of these MAP2 variants, we have analyzed the effects of MAP2c and MAP2d expression on microtubule and microfilament organization in transiently transfected cells. Our results show that both variants stabilize microtubules, but only MAP2d stabilizes microfilaments.
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Chapin, Steven J., and Jeannette Chlo� Bulinski. "Microtubule stabilization by assembly-promoting microtubule-associated proteins: A repeat performance." Cell Motility and the Cytoskeleton 23, no. 4 (1992): 236–43. http://dx.doi.org/10.1002/cm.970230403.
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Al-Bassam, Jawdat, Mark van Breugel, Stephen C. Harrison, and Anthony Hyman. "Stu2p binds tubulin and undergoes an open-to-closed conformational change." Journal of Cell Biology 172, no. 7 (2006): 1009–22. http://dx.doi.org/10.1083/jcb.200511010.
Full textAbstract:
Stu2p from budding yeast belongs to the conserved Dis1/XMAP215 family of microtubule-associated proteins (MAPs). The common feature of proteins in this family is the presence of HEAT repeat–containing TOG domains near the NH2 terminus. We have investigated the functions of the two TOG domains of Stu2p in vivo and in vitro. Our data suggest that Stu2p regulates microtubule dynamics through two separate activities. First, Stu2p binds to a single free tubulin heterodimer through its first TOG domain. A large conformational transition in homodimeric Stu2p from an open structure to a closed one accompanies the capture of a single free tubulin heterodimer. Second, Stu2p has the capacity to associate directly with microtubule ends, at least in part, through its second TOG domain. These two properties lead to the stabilization of microtubules in vivo, perhaps by the loading of tubulin dimers at microtubule ends. We suggest that this mechanism of microtubule regulation is a conserved feature of the Dis1/XMAP215 family of MAPs.