Regulation of microtubule dynamics by TOG-domain proteins XMAP215/Dis1 and CLASP

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.     

XMAP215 is a processive microtubule polymerase

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.     

Structural basis of microtubule plus end tracking by XMAP215, CLIP-170, and EB1

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.     

Fission yeast ch-TOG/XMAP215 homologue Alp14 connects mitotic spindles with the kinetochore and is a component of the Mad2-dependent spindle checkpoint.

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.     

Stu2p,the budding yeast member of the conserved Dis1/XMAP215 family of microtubule-associated proteins is a plus end-binding microtubule destabilizer

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.     

XMAP215: a tip tracker that really moves

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.     

Reconstitution of dynamic microtubules with Drosophila XMAP215, EB1, and Sentin

Dynamic microtubules (MTs) are essential for various intracellular events, such as mitosis. In Drosophila melanogaster S2 cells, three MT tip-localizing proteins, Msps/XMAP215, EB1, and Sentin (an EB1 cargo protein), have been identified as being critical for accelerating MT growth and promoting catastrophe events, thus resulting in the formation of dynamic MTs. However, the molecular activity of each protein and the basis of the modulation of MT dynamics by these three factors are unknown. In this paper, we showed in vitro that XMAP215^msps had a potent growth-promoting activity at a wide range of tubulin concentrations, whereas Sentin, when recruited by EB1 to the growing MT tip, accelerated growth and also increased catastrophe frequency. When all three factors were combined, the growth rate was synergistically enhanced, and rescue events were observed most frequently, but frequent catastrophes restrained the lengthening of the MTs. We propose that MT dynamics are promoted by the independent as well as the cooperative action of XMAP215^msps polymerase and the EB1–Sentin duo.     

A microtubule-associated protein from Xenopus eggs that specifically promotes assembly at the plus-end

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.     

XMAP from Xenopus eggs promotes rapid plus end assembly of microtubules and rapid microtubule polymer turnover

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.     

The XMAP215 family drives microtubule polymerization using a structurally diverse TOG array

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.     

XMAP215 promotes microtubule catastrophe by disrupting the growing microtubule end

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.     

SLAIN2 links microtubule plus end-tracking proteins and controls microtubule growth in interphase

The ends of growing microtubules (MTs) accumulate a set of diverse factors known as MT plus end-tracking proteins (+TIPs), which control microtubule dynamics and organization. In this paper, we identify SLAIN2 as a key component of +TIP interaction networks. We showed that the C-terminal part of SLAIN2 bound to end-binding proteins (EBs), cytoplasmic linker proteins (CLIPs), and CLIP-associated proteins and characterized in detail the interaction of SLAIN2 with EB1 and CLIP-170. Furthermore, we found that the N-terminal part of SLAIN2 interacted with ch-TOG, the mammalian homologue of the MT polymerase XMAP215. Through its multiple interactions, SLAIN2 enhanced ch-TOG accumulation at MT plus ends and, as a consequence, strongly stimulated processive MT polymerization in interphase cells. Depletion or disruption of the SLAIN2-ch-TOG complex led to disorganization of the radial MT array. During mitosis, SLAIN2 became highly phosphorylated, and its interaction with EBs and ch-TOG was inhibited. Our study provides new insights into the molecular mechanisms underlying cell cycle-specific regulation of MT polymerization and the organization of the MT network.     

Stu2p binds tubulin and undergoes an open-to-closed conformational change

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.     

Identification of XMAP215 as a microtubule-destabilizing factor in Xenopus egg extract by biochemical purification

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.     

EB1 promotes microtubule dynamics by recruiting Sentin in Drosophila cells

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.     

The interaction of TOGp with microtubules and tubulin

TOGp is the human homolog of XMAP215, a Xenopus microtubule-associated protein that promotes rapid microtubule assembly at plus ends. These proteins are thought to be critical for microtubule assembly and/or mitotic spindle formation. To understand how TOGp interacts with the microtubule lattice, we cloned full-length TOGp and various truncations for expression in a reticulocyte lysate system. Based on microtubule co-pelleting assays, the microtubule binding domain is contained within a basic 600-amino acid region near the N terminus, with critical domains flanking a region homologous to the microtubule binding domain found in the related proteins Stu2p (S. cerevisiae) and Dis1 (S. pombe). Both full-length TOGp and the N-terminal fragment show enhanced binding to microtubule ends. Full-length TOGp also binds altered polymer lattice structures including parallel protofilament sheets, antiparallel protofilament sheets induced with zinc ions, and protofilament rings, suggesting that TOGp binds along the length of individual protofilaments. The C-terminal region of TOGp has a low affinity for microtubule polymer but binds tubulin dimer. We propose a model to explain the microtubule-stabilizing and/or assembly-promoting functions of the XMAP215/TOGp family of microtubule-associated proteins based on the binding properties we have identified.     

TACC3 is a microtubule plus end–tracking protein that promotes axon elongation and also regulates microtubule plus end dynamics in multiple embryonic cell types

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.     

Microtubule binding proteins CLIP-170, EB1, and p150Glued form distinct plus-end complexes

Microtubule plus-end proteins CLIP-170 and EB1 dynamically track the tips of growing microtubules in vivo. Here we examine the association of these proteins with microtubules in vitro. CLIP-170 binds tubulin dimers and co-assembles into growing microtubules. EB1 binds tubulin dimers more weakly, so no co-assembly is observed. However, EB1 binds to CLIP-170, and forms a co-complex with CLIP-170 and tubulin that is recruited to growing microtubule plus ends. The interaction between CLIP-170 and EB1 is competitively inhibited by the related CAP-Gly protein p150Glued, which also localizes to microtubule plus ends in vivo. Based on these observations, we propose a model in which the formation of distinct plus-end complexes may differentially affect microtubule dynamics in vivo.     

TOGp, the human homolog of XMAP215/Dis1, is required for centrosome integrity, spindle pole organization, and bipolar spindle assembly

The XMAP215/Dis1 MAP family is thought to regulate microtubule plus-end assembly in part by antagonizing the catastrophe-promoting function of kin I kinesins, yet XMAP215/Dis1 proteins localize to centrosomes. We probed the mitotic function of TOGp (human homolog of XMAP215/Dis1) using siRNA. Cells lacking TOGp assembled multipolar spindles, confirming results of Gergely et al. (2003. Genes Dev. 17, 336-341). Eg5 motor activity was necessary to maintain the multipolar morphology. Depletion of TOGp decreased microtubule length and density in the spindle by approximately 20%. Depletion of MCAK, a kin I kinesin, increased MT lengths and density by approximately 20%, but did not disrupt spindle morphology. Mitotic cells lacking both TOGp and MCAK formed bipolar and monopolar spindles, indicating that TOGp and MCAK contribute to spindle bipolarity, without major effects on MT stability. TOGp localized to centrosomes in the absence of MTs and depletion of TOGp resulted in centrosome fragmentation. TOGp depletion also disrupted MT minus-end focus at the spindle poles, detected by localizations of NuMA and the p150 component of dynactin. The major functions of TOGp during mitosis are to focus MT minus ends at spindle poles, maintain centrosome integrity, and contribute to spindle bipolarity.     

Spindle-kinetochore attachment requires the combined action of Kin I-like Klp5/6 and Alp14/Dis1-MAPs in fission yeast

Fission yeast Klp5 and Klp6 belong to the microtubule-destabilizing Kin I family. In klp5 mutants, spindle checkpoint proteins Mad2 and Bub1 are recruited to mitotic kinetochores for a prolonged duration, indicating that these kinetochores are unattached. Further analysis shows that there are kinetochores to which only Bub1, but not Mad2, localizes. These kinetochores are likely to have been captured, yet lack tension. Thus Klp5 and Klp6 play a role in a spindle- kinetochore interaction at dual steps, capture and generation of tension. The TOG/XMAP215 family, Alp14 and Dis1 are known to stabilize microtubules and be required for the bivalent attachment of the kinetochore to the spindle. Despite apparent opposing activities towards microtubule stability, Klp5/Klp6 and Alp14/Dis1 share an essential function, as either dis1klp or alp14klp mutants are synthetically lethal, like alp14dis1. Defective phenotypes are similar to each other, characteristic of attachment defects and chromosome mis-segregation. Furthermore Alp14 is of significance for kinetochore localization of Klp5. We propose that Klp5/Klp6 and Alp14/Dis1 play a collaborative role in bipolar spindle formation during prometaphase through producing spindle dynamism.