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1.
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
2.
XMAP215 is a microtubule plus-end binding protein implicated in modulating microtubule dynamics. In this issue, Brouhard et al. (2008) propose a new mechanism to explain how XMAP215 promotes microtubule growth. They report that XMAP215 moves with the growing microtubule plus ends where it catalyzes the addition of tubulin subunits. 相似文献
3.
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
4.
The GTP-tubulin cap is widely accepted to protect microtubules against catastrophe. The GTP-cap size is thought to increase with the microtubule growth rate, presumably endowing fast-growing microtubules with enhanced stability. It is unknown what GTP-cap properties permit frequent microtubule catastrophe despite fast growth. Here, we investigate microtubules growing in the presence and absence of the polymerase XMAP215. Using EB1 as a GTP-cap marker, we find that GTP-cap size increases regardless of whether growth acceleration is achieved by increasing tubulin concentration or by XMAP215. Despite increased mean GTP-cap size, microtubules grown with XMAP215 display increased catastrophe frequency, in contrast to microtubules grown with more tubulin, for which catastrophe is abolished. However, microtubules polymerized with XMAP215 have large fluctuations in growth rate; display tapered and curled ends; and undergo catastrophe at faster growth rates and with higher EB1 end-localization. Our results suggest that structural perturbations induced by XMAP215 override the protective effects of the GTP-cap, ultimately driving microtubule catastrophe. 相似文献
5.
Belinda U. Nwagbara Anna E. Faris Elizabeth A. Bearce Burcu Erdogan Patrick T. Ebbert Matthew F. Evans Erin L. Rutherford Tiffany B. Enzenbacher Laura Anne Lowery 《Molecular biology of the cell》2014,25(21):3350-3362
Microtubule plus end dynamics are regulated by a conserved family of proteins called plus end–tracking proteins (+TIPs). It is unclear how various +TIPs interact with each other and with plus ends to control microtubule behavior. The centrosome-associated protein TACC3, a member of the transforming acidic coiled-coil (TACC) domain family, has been implicated in regulating several aspects of microtubule dynamics. However, TACC3 has not been shown to function as a +TIP in vertebrates. Here we show that TACC3 promotes axon outgrowth and regulates microtubule dynamics by increasing microtubule plus end velocities in vivo. We also demonstrate that TACC3 acts as a +TIP in multiple embryonic cell types and that this requires the conserved C-terminal TACC domain. Using high-resolution live-imaging data on tagged +TIPs, we show that TACC3 localizes to the extreme microtubule plus end, where it lies distal to the microtubule polymerization marker EB1 and directly overlaps with the microtubule polymerase XMAP215. TACC3 also plays a role in regulating XMAP215 stability and localizing XMAP215 to microtubule plus ends. Taken together, our results implicate TACC3 as a +TIP that functions with XMAP215 to regulate microtubule plus end dynamics. 相似文献
6.
A microtubule-associated protein from Xenopus eggs that specifically promotes assembly at the plus-end 总被引:13,自引:9,他引:4
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We have isolated a protein factor from Xenopus eggs that promotes microtubule assembly in vitro. Assembly promotion was associated with a 215-kD protein after a 1,000-3,000-fold enrichment of activity. The 215-kD protein, termed Xenopus microtubule assembly protein (XMAP), binds to microtubules with a stoichiometry of 0.06 mol/mol tubulin dimer. XMAP is immunologically distinct from the Xenopus homologues to mammalian brain microtubule-associated proteins; however, protein species immunologically related to XMAP with different molecular masses are found in Xenopus neuronal tissues and testis. XMAP is unusual in that it specifically promotes microtubule assembly at the plus-end. At a molar ratio of 0.01 mol XMAP/mol tubulin the assembly rate of the microtubule plus-end is accelerated 8-fold while the assembly rate of the minus-end is increased only 1.8-fold. Under these conditions XMAP promotes a 10-fold increase in the on-rate constant (from 1.4 s-1.microM-1 for microtubules assembled from pure tubulin to 15 s-1.microM-1), and a 10-fold decrease in off-rate constant (from 340 to 34 s-1). Given its stoichiometry in vivo, XMAP must be the major microtubule assembly factor in the Xenopus egg. XMAP is phosphorylated during M-phase of both meiotic and mitotic cycles, suggesting that its activity may be regulated during the cell cycle. 相似文献
7.
Li W Miki T Watanabe T Kakeno M Sugiyama I Kaibuchi K Goshima G 《The Journal of cell biology》2011,193(6):973-983
Highly conserved EB1 family proteins bind to the growing ends of microtubules, recruit multiple cargo proteins, and are critical for making dynamic microtubules in vivo. However, it is unclear how these master regulators of microtubule plus ends promote microtubule dynamics. In this paper, we identify a novel EB1 cargo protein, Sentin. Sentin depletion in Drosophila melanogaster S2 cells, similar to EB1 depletion, resulted in an increase in microtubule pausing and led to the formation of shorter spindles, without displacing EB1 from growing microtubules. We demonstrate that Sentin's association with EB1 was critical for its plus end localization and function. Furthermore, the EB1 phenotype was rescued by expressing an EBN-Sentin fusion protein in which the C-terminal cargo-binding region of EB1 is replaced with Sentin. Knockdown of Sentin attenuated plus end accumulation of Msps (mini spindles), the orthologue of XMAP215 microtubule polymerase. These results indicate that EB1 promotes dynamic microtubule behavior by recruiting the cargo protein Sentin and possibly also a microtubule polymerase to the microtubule tip. 相似文献
8.
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. 相似文献
9.
Plus-end-tracking proteins (+TIPs) are localized at the fast-growing, or plus end, of microtubules, and link microtubule ends to cellular structures. One of the best studied +TIPs is EB1, which forms comet-like structures at the tips of growing microtubules. The molecular mechanisms by which EB1 recognizes and tracks growing microtubule ends are largely unknown. However, one clue is that EB1 can bind directly to a microtubule end in the absence of other proteins. Here we use an in vitro assay for dynamic microtubule growth with two-color total-internal-reflection-fluorescence imaging to investigate binding of mammalian EB1 to both stabilized and dynamic microtubules. We find that under conditions of microtubule growth, EB1 not only tip tracks, as previously shown, but also preferentially recognizes the GMPCPP microtubule lattice as opposed to the GDP lattice. The interaction of EB1 with the GMPCPP microtubule lattice depends on the E-hook of tubulin, as well as the amount of salt in solution. The ability to distinguish different nucleotide states of tubulin in microtubule lattice may contribute to the end-tracking mechanism of EB1. 相似文献
10.
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
11.
12.
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
13.
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
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.
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
16.
The TOG/XMAP215-related proteins play a role in microtubule dynamics at its plus end. Fission yeast Alp14, a newly identified TOG/XMAP215 family protein, is essential for proper chromosome segregation in concert with a second homologue Dis1. We show that the alp14 mutant fails to progress towards normal bipolar spindle formation. Intriguingly, Alp14 itself is a component of the Mad2-dependent spindle checkpoint cascade, as upon addition of microtubule-destabilizing drugs the alp14 mutant is incapable of maintaining high H1 kinase activity, which results in securin destruction and premature chromosome separation. Live imaging of Alp14-green fluorescent protein shows that during mitosis, Alp14 is associated with the peripheral region of the kinetochores as well as with the spindle poles. This is supported by ChIP (chromatin immunoprecipitation) and overlapping localization with the kinetochore marker Mis6. An intact spindle is required for Alp14 localization to the kinetochore periphery, but not to the poles. These results indicate that the TOG/XMAP215 family may play a central role as a bridge between the kinetochores and the plus end of pole to chromosome microtubules. 相似文献
17.
On and Around Microtubules: An Overview 总被引:1,自引:0,他引:1
Richard H. Wade 《Molecular biotechnology》2009,43(2):177-191
Microtubules are hollow tubes some 25 nm in diameter participating in the eukaryotic cytoskeleton. They are built from αβ-tubulin heterodimers that associate to form protofilaments running lengthwise along the microtubule wall with the β-tubulin subunit facing the microtubule plus end conferring a structural polarity. The α- and β-tubulins are highly conserved. A third member of the tubulin family, γ-tubulin, plays a role in microtubule nucleation and assembly. Other members of the tubulin family appear to be involved in microtubule nucleation. Microtubule assembly is accompanied by hydrolysis of GTP associated with β-tubulin so that microtubules consist principally of ‘GDP-tubulin’ stabilized at the plus end by a short ‘cap’. An important property of microtubules is dynamic instability characterized by growth randomly interrupted by pauses and shrinkage. Many proteins interact with microtubules within the cell and are involved in essential functions such as microtubule growth, stabilization, destabilization, and interactions with chromosomes during cell division. The motor proteins kinesin and dynein use microtubules as pathways for transport and are also involved in cell division. Crystallography and electron microscopy are providing a structural basis for understanding the interactions of microtubules with antimitotic drugs, with motor proteins and with plus end tracking proteins. 相似文献
18.
Al-Bassam J Kim H Flor-Parra I Lal N Velji H Chang F 《Molecular biology of the cell》2012,23(15):2878-2890
XMAP215/Dis1 proteins are conserved tubulin-binding TOG-domain proteins that regulate microtubule (MT) plus-end dynamics. Here we show that Alp14, a XMAP215 orthologue in fission yeast, Schizosaccharomyces pombe, has properties of a MT polymerase. In vivo, Alp14 localizes to growing MT plus ends in a manner independent of Mal3 (EB1). alp14-null mutants display short interphase MTs with twofold slower assembly rate and frequent pauses. Alp14 is a homodimer that binds a single tubulin dimer. In vitro, purified Alp14 molecules track growing MT plus ends and accelerate MT assembly threefold. TOG-domain mutants demonstrate that tubulin binding is critical for function and plus end localization. Overexpression of Alp14 or only its TOG domains causes complete MT loss in vivo, and high Alp14 concentration inhibits MT assembly in vitro. These inhibitory effects may arise from Alp14 sequestration of tubulin and effects on the MT. Our studies suggest that Alp14 regulates the polymerization state of tubulin by cycling between a tubulin dimer-bound cytoplasmic state and a MT polymerase state that promotes rapid MT assembly. 相似文献
19.
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
20.
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