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1.
XMAP215 is a processive microtubule polymerase 总被引:6,自引:0,他引:6
Brouhard GJ Stear JH Noetzel TL Al-Bassam J Kinoshita K Harrison SC Howard J Hyman AA 《Cell》2008,132(1):79-88
Fast growth of microtubules is essential for rapid assembly of the microtubule cytoskeleton during cell proliferation and differentiation. XMAP215 belongs to a conserved family of proteins that promote microtubule growth. To determine how XMAP215 accelerates growth, we developed a single-molecule assay to visualize directly XMAP215-GFP interacting with dynamic microtubules. XMAP215 binds free tubulin in a 1:1 complex that interacts with the microtubule lattice and targets the ends by a diffusion-facilitated mechanism. XMAP215 persists at the plus end for many rounds of tubulin subunit addition in a form of "tip tracking." These results show that XMAP215 is a processive polymerase that directly catalyzes the addition of up to 25 tubulin dimers to the growing plus end. Under some circumstances XMAP215 can also catalyze the reverse reaction, namely microtubule shrinkage. The similarities between XMAP215 and formins, actin polymerases, suggest that processive tip tracking is a common mechanism for stimulating the growth of cytoskeletal polymers. 相似文献
2.
Microtubule plus end binding proteins (+TIPs) localize to the dynamic plus ends of microtubules, where they stimulate microtubule growth and recruit signaling molecules. Three main +TIP classes have been identified (XMAP215, EB1, and CLIP-170), but whether they act upon microtubule plus ends through a similar mechanism has not been resolved. Here, we report crystal structures of the tubulin binding domains of XMAP215 (yeast Stu2p and Drosophila Msps), EB1 (yeast Bim1p and human EB1), and CLIP-170 (human), which reveal diverse tubulin binding interfaces. Functional studies, however, reveal a common property that native or artificial dimerization of tubulin binding domains (including chemically induced heterodimers of EB1 and CLIP-170) induces tubulin nucleation/assembly in vitro and, in most cases, plus end tracking in living cells. We propose that +TIPs, although diverse in structure, share a common property of multimerizing tubulin, thus acting as polymerization chaperones that aid in subunit addition to the microtubule plus end. 相似文献
3.
Popov AV Pozniakovsky A Arnal I Antony C Ashford AJ Kinoshita K Tournebize R Hyman AA Karsenti E 《The EMBO journal》2001,20(3):397-410
XMAP215 belongs to a family of proteins involved in the regulation of microtubule dynamics. In this study we analyze the function of different parts of XMAP215 in vivo and in Xenopus egg extracts. XMAP215 has been divided into three fragments, FrN, FrM and FrC (for N-terminal, middle and C-terminal, respectively). FrN co-localizes with microtubules in egg extracts but not in cells, FrC co- localizes with microtubules and centrosomes both in egg extracts and in cells, while FrM does not co- localize with either centrosomes or microtubules. In Xenopus egg extracts, FrN stimulates microtubule growth at plus-ends by inhibiting catastrophes, while FrM has no effect, and FrC suppresses microtubule growth by promoting catastrophes. Our results suggest that XMAP215 is targeted to centrosomes and microtubules mainly through its C-terminal domain, while the evolutionarily conserved N-terminal domain contains its microtubule-stabilizing activity. 相似文献
4.
Microtubules are essential for various cellular processes including cell division and intracellular organization. Their function depends on their ability to rearrange their distribution at different times and places. Microtubules are dynamic polymers and their behaviour is described as dynamic instability. Rearrangement of the microtubule cytoskeleton is made possible by proteins that modulate the parameters of dynamic instability. Studies using Xenopus egg extracts led to identification of a microtubule-associated protein called XMAP215 as a major regulator of physiological microtubule dynamics. XMAP215 belongs to an evolutionarily conserved protein family present in organisms ranging from yeast to mammals. Together with members of the Kin I family of kinesins, XMAP215 and its orthologues form an essential circuit for generating dynamic microtubules in vivo. 相似文献
5.
The formation of a functional bipolar mitotic spindle is essential for genetic integrity. In human cells, the microtubule polymerase XMAP215/ch-Tog ensures spindle bipolarity by counteracting the activity of the microtubule-depolymerizing kinesin XKCM1/MCAK. Their antagonistic effects on microtubule polymerization confer dynamic instability on microtubules assembled in cell-free systems. It is, however, unclear if a similar interplay governs microtubule behavior in mammalian cells in vivo. Using real-time analysis of spindle assembly, we found that ch-Tog is required to produce or maintain long centrosomal microtubules after nuclear-envelope breakdown. In the absence of ch-Tog, microtubule assembly at centrosomes was impaired and microtubules were nondynamic. Interkinetochore distances and the lengths of kinetochore fibers were also reduced in these cells. Codepleting MCAK with ch-Tog improved kinetochore fiber length and interkinetochore separation but, surprisingly, did not rescue centrosomal microtubule assembly and microtubule dynamics. Our data therefore suggest that ch-Tog has at least two distinct roles in spindle formation. First, it protects kinetochore microtubules from depolymerization by MCAK. Second, ch-Tog plays an essential role in centrosomal microtubule assembly, a function independent of MCAK activity. Thus, the notion that the antagonistic activities of MCAK and ch-Tog determine overall microtubule stability is too simplistic to apply to human cells. 相似文献
6.
XMAP from Xenopus eggs promotes rapid plus end assembly of microtubules and rapid microtubule polymer turnover 总被引:8,自引:1,他引:7
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《The Journal of cell biology》1994,127(4):985-993
We have used video-enhanced DIC microscopy to examine the effects of XMAP, a Mr 215,000 microtubule-associated protein from Xenopus eggs (Gard, D.L., and M. W. Kirschner. 1987. J. Cell Biol. 105:2203-2215), on the dynamic instability of microtubules nucleated from axoneme fragments in vitro. Our results indicate that XMAP substantially alters the parameters of microtubule assembly at plus ends. Specifically, addition of 0.2 microM XMAP resulted in (a) 7-10-fold increase in elongation velocity, (b) approximately threefold increase in shortening velocity, and (c) near elimination of rescue (the switch from rapid shortening to elongation). Thus, addition of XMAP resulted in the assembly of longer, but more dynamic, microtubules from the plus ends of axonemes which upon catastrophe disassembled back to the axoneme nucleation site. In agreement with previous observations (Gard, D.L., and M. W. Kirschner. 1987. J. Cell Biol. 105:2203-2215), the effects of XMAP on the minus end were much less dramatic, with only a 1.5-3-fold increase in elongation velocity. These results indicate that XMAP, unlike brain MAPs, promotes both polymer assembly and turnover, and suggests that the interaction of XMAP with tubulin and the function of XMAP in vivo may differ from previously characterized MAPs. 相似文献
7.
The molecular mechanisms by which microtubule-associated proteins (MAPs) regulate the dynamic properties of microtubules (MTs) are still poorly understood. We review recent advances in our understanding of two conserved families of MAPs, the XMAP215/Dis1 and CLASP family of proteins. In vivo and in vitro studies show that XMAP215 proteins act as microtubule polymerases at MT plus ends to accelerate MT assembly, and CLASP proteins promote MT rescue and suppress MT catastrophe events. These are structurally related proteins that use conserved TOG domains to recruit tubulin dimers to MTs. We discuss models for how these proteins might use these individual tubulin dimers to regulate dynamic behavior of MT plus ends. 相似文献
8.
Jaime C. Fox Amy E. Howard Joshua D. Currie Stephen L. Rogers Kevin C. Slep 《Molecular biology of the cell》2014,25(16):2375-2392
XMAP215 family members are potent microtubule (MT) polymerases, with mutants displaying reduced MT growth rates and aberrant spindle morphologies. XMAP215 proteins contain arrayed tumor overexpressed gene (TOG) domains that bind tubulin. Whether these TOG domains are architecturally equivalent is unknown. Here we present crystal structures of TOG4 from Drosophila Msps and human ch-TOG. These TOG4 structures architecturally depart from the structures of TOG domains 1 and 2, revealing a conserved domain bend that predicts a novel engagement with α-tubulin. In vitro assays show differential tubulin-binding affinities across the TOG array, as well as differential effects on MT polymerization. We used Drosophila S2 cells depleted of endogenous Msps to assess the importance of individual TOG domains. Whereas a TOG1-4 array largely rescues MT polymerization rates, mutating tubulin-binding determinants in any single TOG domain dramatically reduces rescue activity. Our work highlights the structurally diverse yet positionally conserved TOG array that drives MT polymerization. 相似文献
9.
Tournebize R Popov A Kinoshita K Ashford AJ Rybina S Pozniakovsky A Mayer TU Walczak CE Karsenti E Hyman AA 《Nature cell biology》2000,2(1):13-19
Microtubules are dynamic polymers that move stochastically between periods of growth and shrinkage, a property known as dynamic instability. Here, to investigate the mechanisms regulating microtubule dynamics in Xenopus egg extracts, we have cloned the complementary DNA encoding the microtubule-associated protein XMAP215 and investigated the function of the XMAP215 protein. Immunodepletion of XMAP215 indicated that it is a major microtubule-stabilizing factor in Xenopus egg extracts. During interphase, XMAP215 stabilizes microtubules primarily by opposing the activity of the destabilizing factor XKCM1, a member of the kinesin superfamily. These results indicate that microtubule dynamics in Xenopus egg extracts are regulated by a balance between a stabilizing factor, XMAP215, and a destabilizing factor, XKCM1. 相似文献
10.
Microtubules are essential for a variety of fundamental cellular processes such as organelle positioning and control of cell shape. Schizosaccharomyces pombe is an ideal organism for studying the function and organization of microtubules into bundles in interphase cells. Using light microscopy and electron tomography we analyzed the bundle organization of interphase microtubules in S. pombe. We show that cells lacking ase1p and klp2p still contain microtubule bundles. In addition, we show that ase1p is the major determinant of inter-microtubule spacing in interphase bundles since ase1 deleted cells have an inter-microtubule spacing that differs from that observed in wild-type cells. We then identified dis1p, a XMAP215 homologue, as factor that promotes the stabilization of microtubule bundles. In wild-type cells dis1p partially co-localized with ase1p at regions of microtubule overlap. In cells deleted for ase1 and klp2, dis1p accumulated at the overlap regions of interphase microtubule bundles. In cells lacking all three proteins, both microtubule bundling and inter-microtubule spacing were further reduced, suggesting that Dis1p contributes to interphase microtubule bundling. 相似文献
11.
The XMAP215 homologue Stu2 at yeast spindle pole bodies regulates microtubule dynamics and anchorage
The yeast protein Stu2 belongs to the XMAP215 family of conserved microtubule-binding proteins which regulate microtubule plus end dynamics. XMAP215-related proteins also bind to centrosomes and spindle pole bodies (SPBs) through proteins like the mammalian transforming acidic coiled coil protein TACC or the yeast Spc72. We show that yeast Spc72 has two distinct domains involved in microtubule organization. The essential 100 N-terminal amino acids of Spc72 interact directly with the gamma-tubulin complex, and an adjacent non-essential domain of Spc72 mediates binding to Stu2. Through these domains, Spc72 brings Stu2 and the gamma-tubulin complex together into a single complex. Manipulation of Spc72-Stu2 interaction at SPBs compromises the anchorage of astral microtubules at the SPB and surprisingly also influences the dynamics of microtubule plus ends. Permanently tethering Stu2 to SPBs by fusing it to a version of Spc72 that lacks the Stu2-binding site in part complements these defects in a manner which is dependent upon the microtubule-binding domain of Stu2. Thus, the SPB-associated Spc72-Stu2 complex plays a key role in regulating microtubule properties. 相似文献
12.
13.
A comparison of the ability of XMAP215 and tau to inhibit the microtubule destabilizing activity of XKCM1 总被引:3,自引:0,他引:3
Noetzel TL Drechsel DN Hyman AA Kinoshita K 《Philosophical transactions of the Royal Society of London. Series B, Biological sciences》2005,360(1455):591-594
During mitosis, microtubules not only grow fast, but also have a high rate of catastrophe. This is achieved in part by the activity of the MAP, XMAP215, which can stimulate the growth rate of microtubules without fully inhibiting the function of the catastrophe-kinesin XKCM1. We do not know whether this activity is particular to XMAP215, or is a general property of all MAPs. Here, we compare the activities of XMAP215 with the neuronal MAP tau, in opposing the destabilizing activity of the non-conventional kinesin XKCM1. We show that tau is a much more potent inhibitor of XKCM1 than XMAP215. Because tau completely suppresses XKCM1 activity, even at low concentrations, the combination of tau and XKCM1 is unable to generate mitotic microtubule dynamics. 相似文献
14.
Microtubules are essential structures that organize the cytoplasm and form the mitotic spindle. Their number and orientation depend on the rate of nucleation events and their dynamics. Microtubules are often, but not always, nucleated off a single cytoplasmic element, the centrosome. One microtubule-associated protein, XMAP215, is also a resident centrosomal protein. In this study, we have found that XMAP215 is a key component for the microtubule-nucleating activity of centrosomes. We show that depletion of XMAP215 from Xenopus egg extracts impairs their ability to reconstitute the microtubule nucleation potential of salt-stripped centrosomes. We also show that XMAP215 immobilized on polymer beads induces the formation of microtubule asters in egg extracts as well as in solutions of pure tubulin. Formation of asters by XMAP215 beads indicates that this protein is able to anchor nascent microtubules via their minus ends. The aster-forming activity of XMAP215 does not require gamma-tubulin in pure tubulin solutions, but it is gamma-tubulin-dependent in egg extracts. Our results indicate that XMAP215, a resident centrosomal protein, contributes to the microtubule-nucleating activity of centrosomes, suggesting that, in vivo, the formation of asters by centrosomes requires factors additional to gamma-tubulin. 相似文献
15.
Ines Hahn Andre Voelzmann Jill Parkin Judith B. Fülle Paula G. Slater Laura Anne Lowery Natalia Sanchez-Soriano Andreas Prokop 《PLoS genetics》2021,17(7)
The formation and maintenance of microtubules requires their polymerisation, but little is known about how this polymerisation is regulated in cells. Focussing on the essential microtubule bundles in axons of Drosophila and Xenopus neurons, we show that the plus-end scaffold Eb1, the polymerase XMAP215/Msps and the lattice-binder Tau co-operate interdependently to promote microtubule polymerisation and bundle organisation during axon development and maintenance. Eb1 and XMAP215/Msps promote each other’s localisation at polymerising microtubule plus-ends. Tau outcompetes Eb1-binding along microtubule lattices, thus preventing depletion of Eb1 tip pools. The three factors genetically interact and show shared mutant phenotypes: reductions in axon growth, comet sizes, comet numbers and comet velocities, as well as prominent deterioration of parallel microtubule bundles into disorganised curled conformations. This microtubule curling is caused by Eb1 plus-end depletion which impairs spectraplakin-mediated guidance of extending microtubules into parallel bundles. Our demonstration that Eb1, XMAP215/Msps and Tau co-operate during the regulation of microtubule polymerisation and bundle organisation, offers new conceptual explanations for developmental and degenerative axon pathologies. 相似文献
16.
Microtubules are cytoskeletal filaments whose self-assembly occurs by abrupt switching between states of roughly constant growth and shrinkage, a process known as dynamic instability. Understanding the mechanism of dynamic instability offers potential for controlling microtubule-dependent cellular processes such as nerve growth and mitosis. The growth to shrinkage transitions (catastrophes) and the reverse transitions (rescues) that characterize microtubule dynamic instability have been assumed to be random events with first-order kinetics. By direct observation of individual microtubules in vitro and probabilistic analysis of their distribution of growth times, we found that while the slower growing and biologically inactive (minus) ends obeyed first-order catastrophe kinetics, the faster growing and biologically active (plus) ends did not. The non-first-order kinetics at plus ends imply that growing microtubule plus ends have an effective frequency of catastrophe that depends on how long the microtubules have been growing. This frequency is low initially but then rises asymptotically to a limiting value. Our results also suggest that an additional parameter, beyond the four parameters typically used to describe dynamic instability, is needed to account for the observed behavior and that changing this parameter can significantly affect the distribution of microtubule lengths at steady state. 相似文献
17.
Aspergillus nidulans Dis1/XMAP215 protein AlpA localizes to spindle pole bodies and microtubule plus ends and contributes to growth directionality
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The dynamics of cytoplasmic microtubules (MTs) is largely controlled by a protein complex at the MT plus end. In Schizosaccharomyces pombe and in filamentous fungi, MT plus end-associated proteins also determine growth directionality. We have characterized the Dis1/XMAP215 family protein AlpA from Aspergillus nidulans and show that it determines MT dynamics as well as hyphal morphology. Green fluorescent protein-tagged AlpA localized to MT-organizing centers (centrosomes) and to MT plus ends. The latter accumulation occurred independently of conventional kinesin or the Kip2-familiy kinesin KipA. alpA deletion strains were viable and only slightly temperature sensitive. Mitosis, nuclear migration, and nuclear positioning were not affected, but hyphae grew in curves rather than straight, which appeared to be an effect of reduced MT growth and dynamics. 相似文献
18.
Identification of XMAP215 as a microtubule-destabilizing factor in Xenopus egg extract by biochemical purification 总被引:3,自引:0,他引:3
Microtubules (MTs) polymerized with GMPCPP, a slowly hydrolyzable GTP analogue, are stable in buffer but are rapidly depolymerized in Xenopus egg extracts. This depolymerization is independent of three previously identified MT destabilizers (Op18, katanin, and XKCM1/KinI). We purified the factor responsible for this novel depolymerizing activity using biochemical fractionation and a visual activity assay and identified it as XMAP215, previously identified as a prominent MT growth-promoting protein in Xenopus extracts. Consistent with the purification results, we find that XMAP215 is necessary for GMPCPP-MT destabilization in extracts and that recombinant full-length XMAP215 as well as an NH2-terminal fragment have depolymerizing activity in vitro. Stimulation of depolymerization is specific for the MT plus end. These results provide evidence for a robust MT-destabilizing activity intrinsic to this microtubule-associated protein and suggest that destabilization may be part of its essential biochemical functions. We propose that the substrate in our assay, GMPCPP-stabilized MTs, serves as a model for the pause state of MT ends and that the multiple activities of XMAP215 are unified by a mechanism of antagonizing MT pauses. 相似文献
19.
The Dis1/XMAP215 family of microtubule-associated proteins conserved from yeast to mammals is essential for cell division. XMAP215, the Xenopus member of this family, has been shown to stabilize microtubules in vitro, but other members of this family have not been biochemically characterized. Here we investigate the properties of the Saccharomyces cerevisiae homologue Stu2p in vitro. Surprisingly, Stu2p is a microtubule destabilizer that binds preferentially to microtubule plus ends. Quantitative analysis of microtubule dynamics suggests that Stu2p induces microtubule catastrophes by sterically interfering with tubulin addition to microtubule ends. These results reveal both a new biochemical activity for a Dis1/XMAP215 family member and a novel mechanism for microtubule destabilization. 相似文献
20.
Microtubules are dynamic cytoskeletal polymers present in all eukaryotic cells. In animal cells, they are organized by the centrosome, the major microtubule-organizing center. Many centrosomal proteins act coordinately to modulate microtubule assembly and organization. Our previous work has shown that Cep70, a novel centrosomal protein regulates microtubule assembly and organization in mammalian cells. However, the molecular details remain to be investigated. In this study, we investigated the molecular mechanism of how Cep70 regulates microtubule assembly using purified proteins. Our data showed that Cep70 increased the microtubule length without affecting the microtubule number in the purified system. These results demonstrate that Cep70 could directly regulate microtubule assembly by promoting microtubule elongation instead of microtubule nucleation. 相似文献