- Email: [email protected]
Microtubule dynamics and tubulin interacting proteins
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Microtubule dynamics and tubulin interacting proteins Claire E Walczak Microtubule dynamics are crucial in generation of the mitotic spindle. During the transition from interphase to mitosis, there is an increase in the frequency of microtubule catastrophes. Recent work has identified two proteins, Op18/stathmin and XKCM1, which can promote microtubule catastrophes in vitro and in cells or extracts. Although both of these proteins share the ability to bind tubulin dimers, their mechanisms of action in destabilizing microtubules are distinct.
This review focuses on these tubulin binding proteins and how they influence microtubule dynamics. The past year has provided us with several studies that begin to elucidate the biochemical mechanism by which these proteins act on microtubules. My goal is to present these studies and to try to place them in the overall context of how microtubules are regulated in cells.
Op18 regulates microtubule dynamics Addresses Medical Science, Indiana University, 1001 East 3rd Street, Jordan Hall 104, Bloomington, IN 47405, USA; e-mail: [email protected] Current Opinion in Cell Biology 2000, 12:52–56 0955-0674/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviation MAP microtubule-associated protein
Introduction Microtubules play diverse roles in eukaryotic cells. During interphase, they serve as tracks on which motor proteins transport vesicles and other components throughout the cell. As the cell enters mitosis, microtubules become the key structural component of the mitotic spindle on which the chromosomes are segregated to the two daughter cells. Microtubules are polar polymers composed of α/β tubulin heterodimers. They are inherently-dynamic polymers that transduce energy derived from nucleotide hydrolysis accompanying their polymerization into polymer dynamics [1–3]. This property — known as dynamic instability — allows both polymerizing and depolymerizing microtubules to exist in the same population and to infrequently interconvert between these two states [4,5]. A transition from a state of growth to one of shrinkage is called a catastrophe, whereas a transition from a state of shrinkage back to growth is termed a rescue [6]. Microtubule polymerization dynamics are of fundamental importance to the intracellular functions of the microtubule cytoskeleton and are therefore highly regulated [7]. Proteins that regulate microtubule dynamics fall into two main classes: proteins that stabilize microtubules and proteins that destabilize microtubules. The former class of proteins is exemplified by the classic microtubule-associated proteins (MAPs), which are thought to bind along the length of the microtubule polymer and enhance their stability. More recently, two proteins Op18 and XKCM1 have been identified that act as potent destabilizers of microtubules [8 ••]. Interestingly, both of these proteins have the ability to bind tubulin dimers in vitro.
Op18, also referred to as Stathmin, is a small heat-stable protein originally identified because it is highly expressed in some types of tumor cell and is multiply phosphorylated [9]. More recently, Op18 was purified as a protein that destabilized microtubules in vitro and was shown to regulate microtubule polymer levels both in Xenopus egg extracts and in tissue culture cells [10–12]. Op18 is highly phosphorylated and its activity is regulated throughout the cell cycle but appears to be more active in interphase cells [12–14]. Op18 was also proposed to be important in mediating the local changes in microtubule dynamics that occur in the region of chromatin during spindle assembly in Xenopus egg extracts [15].
Op18: catastrophe promoter or tubulin sequestering protein? Op18 has been shown to cause microtubule destabilization both in Xenopus egg extracts and in tissue culture cells [10–12] but the mechanism of this destabilization has been controversial. Originally, purified Op18 was shown to destabilize microtubules in vitro primarily by increasing the catastrophe frequency [10]. In this study, it was also observed that Op18 exists in a complex with tubulin dimers, indicating that Op18 could act as a tubulin sequestering protein. This would allow Op18 to increase the catastrophe frequency by essentially lowering the amount of tubulin available for polymerization. Purified tubulin assembled at lower concentrations polymerize more and have an increased catastrophe frequency [6]. Belmont and Mitchison [11] argued that this was not the primary mechanism by which Op18 acted because the catastrophe frequency was increased 3–6 fold in the presence of Op18 when compared to pure tubulin polymerized at the same growth rate; however, further in vitro studies showed that Op18 exists in a tight complex with tubulin, and that it does not promote microtubule catastrophes but rather slows the growth rate by sequestering tubulin [16,17]. Recent in vitro experimentswork may have solved this apparent dilemma of whether Op18 is a catastrophe promoter or a sequestering protein. It is now possible to dissociate the tubulin-sequestering and microtubule catastrophe-promoting activities of Op18. Howell and colleagues [18••] showed that the differences in Op18
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Figure 1 Two models for destabilization of microtubules. (a) Op18 (black oval) is proposed to bind transiently to the end of a microtubule and perhaps induce GTP hydroylsis of the microtubule, which induces microtubule depolymerization. (b) XKCM1 induces a conformational change in the microtubule lattice without affecting the nucleotide state of the GTP-bound tubulin. This conformational change is sufficient to cause microtubule depolymerization.
(a)
Op18 induces GTP hydrolysis of tubulin GTP GDP + Pi
(b) XKCM1 induces a conformational change in the microtubule
Op18
function can be controlled by the pH at which the in vitro experiments are performed. At pH 6.8, Op18 acts mainly by a mechanism consistent with that of tubulin sequestration but at pH 7.5 Op18 acted primarily as a catastrophe promoter. To further substantiate their claims, these authors have shown that the two activities could be physically separated within the Op18 protein. The carboxyl terminus of Op18 is required for tight binding to tubulin, and the amino terminus of Op18 is required for catastrophe promotion. Thus, Op18 appears to be a bifunctional protein in vitro but the question remains as to the mechanism of microtubule destabilization in vivo. In the past year studies have shown that Op18 destabilizes microtubules in vivo by a mechanism that does not involve tubulin sequestration. The effects of several Op18 mutants were examined by transfection into tissue culture cells [19•]. Mutation of the DNA encoding a putative coiled-coil motif in Op18 had only a small effect on the ability of Op18 to interact with tubulin dimers in vivo but greatly diminished the ability of Op18 to cause a loss of microtubule polymer in cells. On the basis of these in vitro studies, which demonstrated that Op18 could induce catastrophes, it is tempting to suggest that Op18 also promotes catastrophes in vivo, but this has not been shown with transfected Op18 mutants. In a separate study, both microinjection of anti-Op18 antibodies and antisense inhibition of Op18 in newt lung epithelial cells result in a decreased catastrophe frequency without affecting the growth rate of microtubules, suggesting that in vivo Op18 can induce microtubule catastrophes without sequestering tubulin [20•]. More recently, a series of Op18 deletion mutants have been analyzed both in vitro and in vivo [21••]. Mutants analyzed
XKCM1
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for their ability to bind pure tubulin dimers and affect GTP hydrolysis were also tested functionally in cells via overexpression or microinjection of purified proteins. Consistent with the results of [18••], deletion of the carboxy-terminal domain of Op18 causes a marked suppression of tubulin binding in extracts from transfected cells — and yet this truncated protein was nearly as effective as wild-type protein in inducing microtubule depolymerization in cells. Perhaps even more convincing is the observation that truncated derivatives of Op18 that induce microtubule depolymerization in cells do not compete with the endogenous Op18 for tubulin dimer. Taken together, these results suggest that, at least in some cell types, Op18 induces microtubule depolymerization by a mechanism that does not involve tubulin sequestration.
Mechanism of Op18 action in microtubule destabilization On the basis of the accepted model of tubulin polymerization dynamics, one can envision several mechanisms whereby a protein could induce microtubule catastrophes. One possibility is that a protein could induce hydrolysis of the GTP cap of the microtubule, which would cause that microtubule to become unstable and depolymerize (Figure 1a). Alternatively, a protein could somehow interact with the microtubule and cause a conformational change that results in microtubule depolymerization (Figure 1b). Recent data suggest that Op18 may act by affecting the GTP state of the microtubule. Purified Op18 was capable of inducing microtubule catastrophes at the plus ends of GTP microtubules; however, it was not capable of inducing depolymerization of microtubules that had been capped with the slowly hydrolyzable GTP analog guanylyl (α,B)-methylene diphosphonate (GMPCPP)
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used a pH6.8 buffer, in which Op18 acts primarily by tubulin sequestration. It would be interesting to compare the effect of Op18 on GTPase activity of microtubules and tubulin in a pH7.5 buffer, where Op18 acts primarily by promoting microtubule catastrophes.
Figure 2 Interphase
Mitosis
Regulation of Op18 activity
(a) Catastrophe activity low
(b)
Catastrophe activity high
MAPs off
MAPs on
P
P
MAP
P
XKCM1 Current Opinion in Cell Biology
Two models for regulation of microtubule dynamics during interphase and mitosis. Interphase microtubule arrays contain long, stable microtubules, whereas mitotic microtubule arrays have short dynamic microtubules. (a) In the first model, catastrophe factor activity is turned up during mitosis to increase dynamics. (b) In the second model, MAPs bind tightly to microtubules during interphase, but the hyperphosphorylation of MAPs during mitosis causes them to release from the microtubules. XKCM1, a known catastrophe promoter, would have similar levels of activity in interphase and mitosis but it would cause catastrophes more readily during mitosis because it would have access to microtubules that were no longer stabilized by MAPs. Op18 is not shown here for simplification, but its activity is high in interphase and lower during mitosis so it is not thought to contribute substantially to the increase in microtubule dynamics during mitosis.
[18••]. In addition, it was shown that stathmin/Op18 had no effect on the steady state GTPase of microtubules [17]. An analysis of domains of Op18 that could contribute to regulation of the tubulin GTPase suggest that the Op18 activity toward tubulin is complex [21••]. Op18 contains distinct regions that are capable of inhibiting GTP exchange and inhibiting or stimulating GTPase activity. Because of the magnitude of these effects, the authors [21••] are in favor of the idea that Op18 acts to stimulate GTP hydrolysis specifically at the ends of microtubules. Clearly this is an enticing idea, but more experiments are required to substantiate this model. It should be noted that in both studies that looked directly at the modulation of GTPase activity of microtubules or tubulin, the authors
It was shown that during the transition from interphase to mitosis, there is an increase in microtubule dynamics that occurs primarily through an increase in the catastrophe frequency [22–24]. It was originally proposed that Op18 might represent the catastrophe factor, the activity of which was turned on during this transition [10]. But this idea appears inconsistent with data that showing that phosphorylation of Op18 by mitotic cyclin-dependent kinase turns off the activity of Op18 as a microtubule-destabilizing protein [12,25], which suggests that Op18 activity is decreased during the transition to mitosis. Phosphorylation-dependent regulation of Op18 is a complex issue as it is multiply phosphorylated by a combination of kinases. Phosphorylation site mutants have been generated which showed that multiple phosphorylations resulted in decreased microtubule destabilizing activity [26•,27,28]. The multiple levels of phosphorylation of Op18 by many different kinases might allow Op18 to be a central player in the rearrangement of the microtubule cytoskeleton in response to many different signaling pathways. In addition, Op18 phosphorylation specifically in the region of chromatin might make an important contribution to the localized stabilization of microtubules near chromosomes, which is essential for spindle assembly [15].
XKCM1 is a microtubule destabilizing kinesin A second microtubule destabilizing protein XKCM1 is a kinesin-related protein isolated from Xenopus eggs [29]. Immunodepletion experiments in Xenopus egg extracts showed that XKCM1 is a major catastrophe promoter in this system [29]. Loss of XKCM1 function in extracts inhibited mitotic spindle formation and resulted in large microtubule asters. Purified XKCM1 protein was able to induce growing microtubules to undergo catastrophes in vitro, consistent with its proposed role in regulating microtubule dynamics [30••]. In addition, XKIF2, a homolog of XKCM1, was also able to destabilize microtubules suggesting that multiple members of this Kin I subfamily might be microtubule destabilizing enzymes.
Mechanism of XKCM1-induced destabilization It was originally proposed that XKCM1 acted as a conventional microtubule motor protein that translocated along microtubules and, upon reaching the end of the microtubule, induced a microtubule catastrophe [29]. More recent work has shown that XKCM1 does not act as a conventional motor protein but rather induces microtubule depolymerization by a mechanism that does not involve directed motility [30••]. Unlike Op18, XKCM1 can depolymerize microtubules that have been polymerized with GMPCPP. XKCM1 appears to act by inducing a
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conformational change in the microtubule lattice (Figure 1b). XKCM1 was found to bind to both ends of the microtubule and binding was sufficient to induce a conformational change in the microtubule that resulted in protofilament peeling. This conformational change mimics the conformational change induced by GTP hydrolysis [31] and destabilizes the microtubule so depolymerization is favored, resulting in the release of many tubulin dimers and a small number of tubulin dimer/XKCM1 complexes. ATP hydrolysis by XKCM1 appears to be necessary to recycle the protein for an additional round of depolymerization, thus making its action catalytic. It is not known how many XKCM1 molecules are needed to induce microtubule depolymerization. It was also shown that the Kin I kinesin XKIF2 could bind to tubulin dimers in the presence of the nonhydrolyzable AMP–PNP but not in the presence of ATP. This suggests that these destabilizing kinesins have the ability to interact with tubulin dimers as well as tubulin polymer. The stoichiometry of this binding to tubulin dimers is not known. Because the tubulin binding activity of XKIF2 is ATP-dependent, it is unlikely that these Kin I kinesins act as tubulin-sequestering proteins in vivo but this has not been demonstrated.
Regulation of XKCM1 activity Given that Op18 activity is turned down and not up during the transition from interphase to mitosis, the question remained about which proteins are responsible for the increased catastrophes that occur at this time. XKCM1 appears to contribute to the majority of the catastrophepromoting activity in mitotic extracts [10,32••]. However, recent evidence suggests that it is not the activity of catastrophe factors that is regulated during this transition, but rather that the microtubules become more dynamic because of loss of the stabilizing influence of MAPs due to their mitotic hyperphosphorylation (Figure 2). In Xenopus egg extracts it was shown that XKCM1 is active during interphase and mitosis; however, XKCM1 does not stimulate catastrophes during interphase because of the stabilizing effects of the Xenopus MAP, XMAP215 [32••]. Reconstitution of microtubules, MAPs and catastrophe factors might serve as a further test of this model. In addition, it will be important to establish whether the same mechanism of regulation of dynamics occurs in cells.
expressing Op18, there might be sufficient Op18 present to sequester a significant amount of the soluble tubulin pool. This will be an interesting area for future research. The Kin I kinesins XKCM1 and XKIF2 have been shown to be nonconventional kinesins that act to destabilize microtubules via a mechanism that does not involve classical motility. This raises the question of whether other kinesins possess nonmotor functions that have an impact on microtubule dynamics. In addition, it has been shown that these Kin I kinesins can form an ATP-dependent complex with tubulin dimer, but whether this complex exists within the cell and has any influence on microtubule dynamics is unknown. We are now beginning to get a handle on the molecular components that regulate microtubule dynamics, specifically those that affect microtubule catastrophes. Perhaps the biggest question that remains is how the activities of these proteins are regulated within a cell. To answer this question will require a detailed analysis of the many regulators of microtubule dynamics that exist in the cell and a careful study of how their activities are integrated to generate the intricate microtubule dynamics that facilitate the multiple functions of this cytoskeletal system.
Acknowledgements I would like to thank Lynne Cassimeris, Martin Gullberg and Rebecca Heald for thoughtful discussions and for sharing unpublished data. I would also like to thank Rebecca Heald and Susan Kline-Smith for critical comments on the manuscript.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest 1.
Mitchison TJ: Self-organization of polymer-motor systems in the cytoskeleton. Phil Trans R Soc Lond B 1992, 336:99-106.
2.
Desai A, Mitchison TJ: Microtubule polymerization dynamics. Annu Rev Cell Dev Biol 1997, 13:83-117.
3.
Erickson HP, O’Brien ET: Microtubule dynamic instability and GTP hydrolysis. Annu Rev Biophys Biolmol Struct 1992, 21:145-166.
4.
Mitchison TJ, Kirschner MW: Dynamic instability of microtubule growth. Nature 1984, 312:237-242.
5.
Horio T, Hotani H: Visualization of the dynamic instability of individual microtubules by dark-field microscopy. Nature 1986, 321:605-607.
6.
Walker RA, O’Brien ET, Pryer NK, Sobeiro MF, Voter WA, Erickson HP, Salmon ED: Dynamic instability of individual microtubules analysed by video light microscopy: rate constants and transition frequencies. J Cell Biol 1988, 107:1437-1448.
7.
Kirschner MW, Mitchison TJ: Beyond self assembly: from microtubules to morphogenesis. Cell 1986, 45:329-342.
Conclusions and future directions The past year has seen significant advances in our understanding of how two proteins that stimulate microtubule catastrophes act mechanistically as well as how their activities may be regulated. It appears that Op18 acts to induce catastrophes primarily through a mechanism that may involve the alteration of the GTP state of the microtubule, although it can also sequester tubulin dimers and stimulate catastrophes by a reduction of the tubulin dimer pool. In the cell types tested, sequestration does not appear to be the primary mechanism of action, although it can not be ruled out that tubulin sequestration will have a major impact in other cell types. For instance, in a cell type over-
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8. McNally FJ: Microtubule dynamics: controlling split ends. Curr Biol •• 1999, 9:R274-R276. This is an excellent review that summarizes models of how Op18 and XKCM1 regulate microtubule dynamics. The author provides a model to explain how distinct mechanisms of catastrophe promotion can affect either one or both ends of a microtubule.
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9.
Sobel A: Stathmin: a relay phosphoprotein for multiple signal transduction? Trends in Biol Sci 1991, 16:301-305.
10. Belmont LD, Mitchison TJ: Identification of a protein that interacts with tubulin dimers and increases the catastrophe rate of microtubules. Cell 1996, 84:623-631. 11. Belmont L, Deacon HW, Mitchison TJ: Catastrophic revelations about Op18/stathmin. Trends Biochem Sci 1996, 21:197-198. 12. Marklund U, Larsson N, Melander H, Gullberg M: Oncoprotein 18 is a phosphorylation-responsive regulator of microtubule dynamics. EMBO J 1996, 15:290-298. 13. Marklund U, Brattsand G, Shingler V, Gullberg M: Serine 25 of oncoprotein 18 is a major cytosolic target for the mitogenactivated protein kinase. J Biol Chem 1993, 268:15039-15047. 14. Marklund U, Osterman O, Melander H, Bergh A, Gullberg M: The phenotype of a ‘Cdc2 kinase target site-deficient’ mutant of oncoprotein 18 reveals a role of this protein in cell cycle control. J Biol Chem 1994, 269:30626-30635. 15. Andersen SSL, Ashford AJ, Tournebize R, Gavet OAS, Hyman AA, Karsenti E: Mitotic chromatin regulates phosphorylation of Stathmin/Op18. Nature 1997, 389:640-643. 16. Curmi PA, Andersen SS, Lachkar S, Gavet O, Karsenti E, Knossow M, Sobel A: The stathmin/tubulin interaction in vitro. J Biol Chem 1997, 272:25029-25036. 17.
Jourdain L, Curmi P, Sobel A, Pantaloni D, Carlier MF: Stathmin: a tubulin-sequestering protein which forms a ternary T2S complex with two tubulin molecules. Biochemistry 1997, 36:10817-10821.
18. Howell B, Larsson N, Gullberg M, Cassimeris L: Dissociation of the •• tubulin-sequestering and microtubule catastrophe- promoting activities of oncoprotein 18/stathmin. Mol Biol Cell 1999, 10:105-118. This paper describes an in vitro study in which the authors are able to dissect the catastrophe-promoting activity of Op18 from its tubulin sequestering activity. They show that, at pH 6.8, Op18 acted primarily via tubulin sequestering but, at pH 7.5, it was primarily a catastrophe factor. In addition, they showed that truncation of the carboxy-terminal domain caused a loss of tubulin sequestering activity, whereas truncation of the aminoterminal domain resulted in a protein that was unable to cause catastrophes at pH 7.5. 19. Larsson N, Segerman B, Gradin HM, Wandzioch E, Cassimeris L, • Gulberg M: Mutations of oncoprotein 18/stathmin identify tubulindirected regulatory activities distinct from tubulin association. Mol Cell Biol 1999, 19:2242-2250. These authors generated a series of mutations of the gene encoding Op18. The mutant proteins were altered either in the putative coiled-coil domain or at all four phosphorylation sites. Both had only a slightly decreased tubulin binding activity. In addition, the coiled-coil mutant had a marked effect on microtubule destabilizing activity, demonstrating that tubulin binding and microtubule destabilization activity could be separated. 20. Howell B, Deacon H, Cassimeris L: Decreasing oncoprotein • 18/stathmin levels reduces microtubule catastrophes and increases microtubule polymer in vivo. J Cell Sci 1999, 112:3713-3722. Op18 antibodies were injected into interphase newt lung cells. The authors found that inhibition of Op18 via microinjection resulted in an increase in microtubule polymer levels and a reduction in the catastrophe frequency of individual microtubules. Treatment of cells with Op18 antisense oligonucleotides also reduced the catastrophe frequency of microtubules.
21. Larsson N, Segerman B, Howell B, Fridell K, Cassimeris L, •• Gullberg M: Op18/stathmin mediates multiple region-specific tubulin and microtubule regulating activities. J Cell Biol 1999, 146:1286-1302. These authors carried out deletion analysis of Op18 to determine its effect on tubulin binding, GTP hydrolysis and exchange, and microtubule destabilization activity. Deletion mutants, in which either the amino terminus or the carboxyl terminus was removed, were unable to complex with tubulin in cells, yet they were able to affect the microtubule cytoskeleton. The deletion analysis also revealed that Op18 contains domains that can either inhibit nucleotide exchange, stimulate GTP hydrolysis, or inhibit GTP hydrolysis. 22. Saxton WM, Stemple DL, Leslie RJ, Salmon ED, Zavortink M, McIntosh JR: Tubulin dynamics in cultured mammalian cells. J Cell Biol 1984, 99:2175-2186. 23. Belmont LD, Hyman AA, Sawin KE, Mitchison TJ: Real-time visualization of cell cycle dependent changes in microtubule dynamics in cytoplasmic extracts. Cell 1990, 62:579-589. 24. Verde F, Dogterom M, Stelzer E, Karsenti E, Leibler S: Control of microtubule dynamics and length by cyclin A- and cyclin Bdependent kinases in Xenopus egg extracts. J Cell Biol 1992, 118:1097-1108. 25. Larsson N, Marklund U, Gradin HM, Brattsand G, Gullberg M: Control of microtubule dynamics by oncoprotein 18: dissection of the regulatory role of multisite phosphorylation during mitosis. Mol Cell Biol 1997, 17:5530-5539. 26. Melander Gradin H, Larsson N, Marklund U, Gullberg M: Regulation • of microtubule dynamics by extracellular signals: cAMPdependent protein kinase switches off the activity of oncoprotein 18 in intact cells. J Cell Biol 1998, 140:131-141. This study shows that phosphorylation of Op18 on Ser16 and Ser63 by protein kinase A results in downregulation of Op18 activity toward microtubules both in vitro with purified tubulin and by transfection into tissue culture cells. 27.
Melander Gradin H, Marklund U, Larsson N, Chatila TA, Gullberg M: Regulation of microtubule dynamics by Ca2+/calmodulindependent kinase IV/Gr-dependent phosphorylation of oncoprotein 18. Mol Cell Biol 1997, 17:3459-3467.
28. Gavet O, Ozon S, Manceau V, Lawler S, Curmi P, Sobel A: The stathmin phosphoprotein family: intracellular localization and effects on the microtubule network. J Cell Sci 1998, 111:3333-3346. 29. Walczak CE, Mitchison TJ, Desai A: XKCM1: a Xenopus kinesinrelated protein that regulates microtubule dynamics during mitotic spindle assembly. Cell 1996, 84:37-47. 30. Desai A, Verma S, Mitchison TJ, Walczak CE: Kin I kinesins are •• microtubule-destabilizing enzymes. Cell 1999, 96:69-78. The authors used a series of in vitro assays to demonstrate that two members of the Kin I subfamily of kinesins act exclusively as microtubule-destabilizing enzymes. The mechanism of microtubule destabilization is distinct from the mechanisms of motility established for other kinesins. 31. Mandelkow EM, Mandelkow E, Milligan RA: Microtubule dynamics and microtubule caps: a time-resolved cryo-electron microscopy study. J Cell Biol 1991, 114:977-991. 32. Tournebize R, Popov A, Kinoshita K, Ashford AJ, Rybina S, Mayer T, •• Walczak CE, Karsenti E, Hyman AA: Control of microtubule dynamics requires the antagonistic activities of XMAP215 and XKCM1. Nat Cell Biol 2000, in press. The authors use the Xenopus egg extract system to explore the relationship between the stabilizing effects of MAPs versus the destabilizing effects of the catastrophe promoter XKCM1. They find that XKCM1 is active as a catastrophe promoter during interphase but that it does not stimulate catastrophes because of the stabilizing influence of MAPs.
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Microtubule dynamics and tubulin interacting proteins Claire E Walczak Microtubule dynamics are crucial in generation of the mitotic spindle. During the transition from interphase to mitosis, there is an increase in the frequency of microtubule catastrophes. Recent work has identified two proteins, Op18/stathmin and XKCM1, which can promote microtubule catastrophes in vitro and in cells or extracts. Although both of these proteins share the ability to bind tubulin dimers, their mechanisms of action in destabilizing microtubules are distinct.
This review focuses on these tubulin binding proteins and how they influence microtubule dynamics. The past year has provided us with several studies that begin to elucidate the biochemical mechanism by which these proteins act on microtubules. My goal is to present these studies and to try to place them in the overall context of how microtubules are regulated in cells.
Op18 regulates microtubule dynamics Addresses Medical Science, Indiana University, 1001 East 3rd Street, Jordan Hall 104, Bloomington, IN 47405, USA; e-mail: [email protected] Current Opinion in Cell Biology 2000, 12:52–56 0955-0674/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviation MAP microtubule-associated protein
Introduction Microtubules play diverse roles in eukaryotic cells. During interphase, they serve as tracks on which motor proteins transport vesicles and other components throughout the cell. As the cell enters mitosis, microtubules become the key structural component of the mitotic spindle on which the chromosomes are segregated to the two daughter cells. Microtubules are polar polymers composed of α/β tubulin heterodimers. They are inherently-dynamic polymers that transduce energy derived from nucleotide hydrolysis accompanying their polymerization into polymer dynamics [1–3]. This property — known as dynamic instability — allows both polymerizing and depolymerizing microtubules to exist in the same population and to infrequently interconvert between these two states [4,5]. A transition from a state of growth to one of shrinkage is called a catastrophe, whereas a transition from a state of shrinkage back to growth is termed a rescue [6]. Microtubule polymerization dynamics are of fundamental importance to the intracellular functions of the microtubule cytoskeleton and are therefore highly regulated [7]. Proteins that regulate microtubule dynamics fall into two main classes: proteins that stabilize microtubules and proteins that destabilize microtubules. The former class of proteins is exemplified by the classic microtubule-associated proteins (MAPs), which are thought to bind along the length of the microtubule polymer and enhance their stability. More recently, two proteins Op18 and XKCM1 have been identified that act as potent destabilizers of microtubules [8 ••]. Interestingly, both of these proteins have the ability to bind tubulin dimers in vitro.
Op18, also referred to as Stathmin, is a small heat-stable protein originally identified because it is highly expressed in some types of tumor cell and is multiply phosphorylated [9]. More recently, Op18 was purified as a protein that destabilized microtubules in vitro and was shown to regulate microtubule polymer levels both in Xenopus egg extracts and in tissue culture cells [10–12]. Op18 is highly phosphorylated and its activity is regulated throughout the cell cycle but appears to be more active in interphase cells [12–14]. Op18 was also proposed to be important in mediating the local changes in microtubule dynamics that occur in the region of chromatin during spindle assembly in Xenopus egg extracts [15].
Op18: catastrophe promoter or tubulin sequestering protein? Op18 has been shown to cause microtubule destabilization both in Xenopus egg extracts and in tissue culture cells [10–12] but the mechanism of this destabilization has been controversial. Originally, purified Op18 was shown to destabilize microtubules in vitro primarily by increasing the catastrophe frequency [10]. In this study, it was also observed that Op18 exists in a complex with tubulin dimers, indicating that Op18 could act as a tubulin sequestering protein. This would allow Op18 to increase the catastrophe frequency by essentially lowering the amount of tubulin available for polymerization. Purified tubulin assembled at lower concentrations polymerize more and have an increased catastrophe frequency [6]. Belmont and Mitchison [11] argued that this was not the primary mechanism by which Op18 acted because the catastrophe frequency was increased 3–6 fold in the presence of Op18 when compared to pure tubulin polymerized at the same growth rate; however, further in vitro studies showed that Op18 exists in a tight complex with tubulin, and that it does not promote microtubule catastrophes but rather slows the growth rate by sequestering tubulin [16,17]. Recent in vitro experimentswork may have solved this apparent dilemma of whether Op18 is a catastrophe promoter or a sequestering protein. It is now possible to dissociate the tubulin-sequestering and microtubule catastrophe-promoting activities of Op18. Howell and colleagues [18••] showed that the differences in Op18
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Figure 1 Two models for destabilization of microtubules. (a) Op18 (black oval) is proposed to bind transiently to the end of a microtubule and perhaps induce GTP hydroylsis of the microtubule, which induces microtubule depolymerization. (b) XKCM1 induces a conformational change in the microtubule lattice without affecting the nucleotide state of the GTP-bound tubulin. This conformational change is sufficient to cause microtubule depolymerization.
(a)
Op18 induces GTP hydrolysis of tubulin GTP GDP + Pi
(b) XKCM1 induces a conformational change in the microtubule
Op18
function can be controlled by the pH at which the in vitro experiments are performed. At pH 6.8, Op18 acts mainly by a mechanism consistent with that of tubulin sequestration but at pH 7.5 Op18 acted primarily as a catastrophe promoter. To further substantiate their claims, these authors have shown that the two activities could be physically separated within the Op18 protein. The carboxyl terminus of Op18 is required for tight binding to tubulin, and the amino terminus of Op18 is required for catastrophe promotion. Thus, Op18 appears to be a bifunctional protein in vitro but the question remains as to the mechanism of microtubule destabilization in vivo. In the past year studies have shown that Op18 destabilizes microtubules in vivo by a mechanism that does not involve tubulin sequestration. The effects of several Op18 mutants were examined by transfection into tissue culture cells [19•]. Mutation of the DNA encoding a putative coiled-coil motif in Op18 had only a small effect on the ability of Op18 to interact with tubulin dimers in vivo but greatly diminished the ability of Op18 to cause a loss of microtubule polymer in cells. On the basis of these in vitro studies, which demonstrated that Op18 could induce catastrophes, it is tempting to suggest that Op18 also promotes catastrophes in vivo, but this has not been shown with transfected Op18 mutants. In a separate study, both microinjection of anti-Op18 antibodies and antisense inhibition of Op18 in newt lung epithelial cells result in a decreased catastrophe frequency without affecting the growth rate of microtubules, suggesting that in vivo Op18 can induce microtubule catastrophes without sequestering tubulin [20•]. More recently, a series of Op18 deletion mutants have been analyzed both in vitro and in vivo [21••]. Mutants analyzed
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Current Opinion in Cell Biology
for their ability to bind pure tubulin dimers and affect GTP hydrolysis were also tested functionally in cells via overexpression or microinjection of purified proteins. Consistent with the results of [18••], deletion of the carboxy-terminal domain of Op18 causes a marked suppression of tubulin binding in extracts from transfected cells — and yet this truncated protein was nearly as effective as wild-type protein in inducing microtubule depolymerization in cells. Perhaps even more convincing is the observation that truncated derivatives of Op18 that induce microtubule depolymerization in cells do not compete with the endogenous Op18 for tubulin dimer. Taken together, these results suggest that, at least in some cell types, Op18 induces microtubule depolymerization by a mechanism that does not involve tubulin sequestration.
Mechanism of Op18 action in microtubule destabilization On the basis of the accepted model of tubulin polymerization dynamics, one can envision several mechanisms whereby a protein could induce microtubule catastrophes. One possibility is that a protein could induce hydrolysis of the GTP cap of the microtubule, which would cause that microtubule to become unstable and depolymerize (Figure 1a). Alternatively, a protein could somehow interact with the microtubule and cause a conformational change that results in microtubule depolymerization (Figure 1b). Recent data suggest that Op18 may act by affecting the GTP state of the microtubule. Purified Op18 was capable of inducing microtubule catastrophes at the plus ends of GTP microtubules; however, it was not capable of inducing depolymerization of microtubules that had been capped with the slowly hydrolyzable GTP analog guanylyl (α,B)-methylene diphosphonate (GMPCPP)
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used a pH6.8 buffer, in which Op18 acts primarily by tubulin sequestration. It would be interesting to compare the effect of Op18 on GTPase activity of microtubules and tubulin in a pH7.5 buffer, where Op18 acts primarily by promoting microtubule catastrophes.
Figure 2 Interphase
Mitosis
Regulation of Op18 activity
(a) Catastrophe activity low
(b)
Catastrophe activity high
MAPs off
MAPs on
P
P
MAP
P
XKCM1 Current Opinion in Cell Biology
Two models for regulation of microtubule dynamics during interphase and mitosis. Interphase microtubule arrays contain long, stable microtubules, whereas mitotic microtubule arrays have short dynamic microtubules. (a) In the first model, catastrophe factor activity is turned up during mitosis to increase dynamics. (b) In the second model, MAPs bind tightly to microtubules during interphase, but the hyperphosphorylation of MAPs during mitosis causes them to release from the microtubules. XKCM1, a known catastrophe promoter, would have similar levels of activity in interphase and mitosis but it would cause catastrophes more readily during mitosis because it would have access to microtubules that were no longer stabilized by MAPs. Op18 is not shown here for simplification, but its activity is high in interphase and lower during mitosis so it is not thought to contribute substantially to the increase in microtubule dynamics during mitosis.
[18••]. In addition, it was shown that stathmin/Op18 had no effect on the steady state GTPase of microtubules [17]. An analysis of domains of Op18 that could contribute to regulation of the tubulin GTPase suggest that the Op18 activity toward tubulin is complex [21••]. Op18 contains distinct regions that are capable of inhibiting GTP exchange and inhibiting or stimulating GTPase activity. Because of the magnitude of these effects, the authors [21••] are in favor of the idea that Op18 acts to stimulate GTP hydrolysis specifically at the ends of microtubules. Clearly this is an enticing idea, but more experiments are required to substantiate this model. It should be noted that in both studies that looked directly at the modulation of GTPase activity of microtubules or tubulin, the authors
It was shown that during the transition from interphase to mitosis, there is an increase in microtubule dynamics that occurs primarily through an increase in the catastrophe frequency [22–24]. It was originally proposed that Op18 might represent the catastrophe factor, the activity of which was turned on during this transition [10]. But this idea appears inconsistent with data that showing that phosphorylation of Op18 by mitotic cyclin-dependent kinase turns off the activity of Op18 as a microtubule-destabilizing protein [12,25], which suggests that Op18 activity is decreased during the transition to mitosis. Phosphorylation-dependent regulation of Op18 is a complex issue as it is multiply phosphorylated by a combination of kinases. Phosphorylation site mutants have been generated which showed that multiple phosphorylations resulted in decreased microtubule destabilizing activity [26•,27,28]. The multiple levels of phosphorylation of Op18 by many different kinases might allow Op18 to be a central player in the rearrangement of the microtubule cytoskeleton in response to many different signaling pathways. In addition, Op18 phosphorylation specifically in the region of chromatin might make an important contribution to the localized stabilization of microtubules near chromosomes, which is essential for spindle assembly [15].
XKCM1 is a microtubule destabilizing kinesin A second microtubule destabilizing protein XKCM1 is a kinesin-related protein isolated from Xenopus eggs [29]. Immunodepletion experiments in Xenopus egg extracts showed that XKCM1 is a major catastrophe promoter in this system [29]. Loss of XKCM1 function in extracts inhibited mitotic spindle formation and resulted in large microtubule asters. Purified XKCM1 protein was able to induce growing microtubules to undergo catastrophes in vitro, consistent with its proposed role in regulating microtubule dynamics [30••]. In addition, XKIF2, a homolog of XKCM1, was also able to destabilize microtubules suggesting that multiple members of this Kin I subfamily might be microtubule destabilizing enzymes.
Mechanism of XKCM1-induced destabilization It was originally proposed that XKCM1 acted as a conventional microtubule motor protein that translocated along microtubules and, upon reaching the end of the microtubule, induced a microtubule catastrophe [29]. More recent work has shown that XKCM1 does not act as a conventional motor protein but rather induces microtubule depolymerization by a mechanism that does not involve directed motility [30••]. Unlike Op18, XKCM1 can depolymerize microtubules that have been polymerized with GMPCPP. XKCM1 appears to act by inducing a
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conformational change in the microtubule lattice (Figure 1b). XKCM1 was found to bind to both ends of the microtubule and binding was sufficient to induce a conformational change in the microtubule that resulted in protofilament peeling. This conformational change mimics the conformational change induced by GTP hydrolysis [31] and destabilizes the microtubule so depolymerization is favored, resulting in the release of many tubulin dimers and a small number of tubulin dimer/XKCM1 complexes. ATP hydrolysis by XKCM1 appears to be necessary to recycle the protein for an additional round of depolymerization, thus making its action catalytic. It is not known how many XKCM1 molecules are needed to induce microtubule depolymerization. It was also shown that the Kin I kinesin XKIF2 could bind to tubulin dimers in the presence of the nonhydrolyzable AMP–PNP but not in the presence of ATP. This suggests that these destabilizing kinesins have the ability to interact with tubulin dimers as well as tubulin polymer. The stoichiometry of this binding to tubulin dimers is not known. Because the tubulin binding activity of XKIF2 is ATP-dependent, it is unlikely that these Kin I kinesins act as tubulin-sequestering proteins in vivo but this has not been demonstrated.
Regulation of XKCM1 activity Given that Op18 activity is turned down and not up during the transition from interphase to mitosis, the question remained about which proteins are responsible for the increased catastrophes that occur at this time. XKCM1 appears to contribute to the majority of the catastrophepromoting activity in mitotic extracts [10,32••]. However, recent evidence suggests that it is not the activity of catastrophe factors that is regulated during this transition, but rather that the microtubules become more dynamic because of loss of the stabilizing influence of MAPs due to their mitotic hyperphosphorylation (Figure 2). In Xenopus egg extracts it was shown that XKCM1 is active during interphase and mitosis; however, XKCM1 does not stimulate catastrophes during interphase because of the stabilizing effects of the Xenopus MAP, XMAP215 [32••]. Reconstitution of microtubules, MAPs and catastrophe factors might serve as a further test of this model. In addition, it will be important to establish whether the same mechanism of regulation of dynamics occurs in cells.
expressing Op18, there might be sufficient Op18 present to sequester a significant amount of the soluble tubulin pool. This will be an interesting area for future research. The Kin I kinesins XKCM1 and XKIF2 have been shown to be nonconventional kinesins that act to destabilize microtubules via a mechanism that does not involve classical motility. This raises the question of whether other kinesins possess nonmotor functions that have an impact on microtubule dynamics. In addition, it has been shown that these Kin I kinesins can form an ATP-dependent complex with tubulin dimer, but whether this complex exists within the cell and has any influence on microtubule dynamics is unknown. We are now beginning to get a handle on the molecular components that regulate microtubule dynamics, specifically those that affect microtubule catastrophes. Perhaps the biggest question that remains is how the activities of these proteins are regulated within a cell. To answer this question will require a detailed analysis of the many regulators of microtubule dynamics that exist in the cell and a careful study of how their activities are integrated to generate the intricate microtubule dynamics that facilitate the multiple functions of this cytoskeletal system.
Acknowledgements I would like to thank Lynne Cassimeris, Martin Gullberg and Rebecca Heald for thoughtful discussions and for sharing unpublished data. I would also like to thank Rebecca Heald and Susan Kline-Smith for critical comments on the manuscript.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
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Kirschner MW, Mitchison TJ: Beyond self assembly: from microtubules to morphogenesis. Cell 1986, 45:329-342.
Conclusions and future directions The past year has seen significant advances in our understanding of how two proteins that stimulate microtubule catastrophes act mechanistically as well as how their activities may be regulated. It appears that Op18 acts to induce catastrophes primarily through a mechanism that may involve the alteration of the GTP state of the microtubule, although it can also sequester tubulin dimers and stimulate catastrophes by a reduction of the tubulin dimer pool. In the cell types tested, sequestration does not appear to be the primary mechanism of action, although it can not be ruled out that tubulin sequestration will have a major impact in other cell types. For instance, in a cell type over-
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8. McNally FJ: Microtubule dynamics: controlling split ends. Curr Biol •• 1999, 9:R274-R276. This is an excellent review that summarizes models of how Op18 and XKCM1 regulate microtubule dynamics. The author provides a model to explain how distinct mechanisms of catastrophe promotion can affect either one or both ends of a microtubule.
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Sobel A: Stathmin: a relay phosphoprotein for multiple signal transduction? Trends in Biol Sci 1991, 16:301-305.
10. Belmont LD, Mitchison TJ: Identification of a protein that interacts with tubulin dimers and increases the catastrophe rate of microtubules. Cell 1996, 84:623-631. 11. Belmont L, Deacon HW, Mitchison TJ: Catastrophic revelations about Op18/stathmin. Trends Biochem Sci 1996, 21:197-198. 12. Marklund U, Larsson N, Melander H, Gullberg M: Oncoprotein 18 is a phosphorylation-responsive regulator of microtubule dynamics. EMBO J 1996, 15:290-298. 13. Marklund U, Brattsand G, Shingler V, Gullberg M: Serine 25 of oncoprotein 18 is a major cytosolic target for the mitogenactivated protein kinase. J Biol Chem 1993, 268:15039-15047. 14. Marklund U, Osterman O, Melander H, Bergh A, Gullberg M: The phenotype of a ‘Cdc2 kinase target site-deficient’ mutant of oncoprotein 18 reveals a role of this protein in cell cycle control. J Biol Chem 1994, 269:30626-30635. 15. Andersen SSL, Ashford AJ, Tournebize R, Gavet OAS, Hyman AA, Karsenti E: Mitotic chromatin regulates phosphorylation of Stathmin/Op18. Nature 1997, 389:640-643. 16. Curmi PA, Andersen SS, Lachkar S, Gavet O, Karsenti E, Knossow M, Sobel A: The stathmin/tubulin interaction in vitro. J Biol Chem 1997, 272:25029-25036. 17.
Jourdain L, Curmi P, Sobel A, Pantaloni D, Carlier MF: Stathmin: a tubulin-sequestering protein which forms a ternary T2S complex with two tubulin molecules. Biochemistry 1997, 36:10817-10821.
18. Howell B, Larsson N, Gullberg M, Cassimeris L: Dissociation of the •• tubulin-sequestering and microtubule catastrophe- promoting activities of oncoprotein 18/stathmin. Mol Biol Cell 1999, 10:105-118. This paper describes an in vitro study in which the authors are able to dissect the catastrophe-promoting activity of Op18 from its tubulin sequestering activity. They show that, at pH 6.8, Op18 acted primarily via tubulin sequestering but, at pH 7.5, it was primarily a catastrophe factor. In addition, they showed that truncation of the carboxy-terminal domain caused a loss of tubulin sequestering activity, whereas truncation of the aminoterminal domain resulted in a protein that was unable to cause catastrophes at pH 7.5. 19. Larsson N, Segerman B, Gradin HM, Wandzioch E, Cassimeris L, • Gulberg M: Mutations of oncoprotein 18/stathmin identify tubulindirected regulatory activities distinct from tubulin association. Mol Cell Biol 1999, 19:2242-2250. These authors generated a series of mutations of the gene encoding Op18. The mutant proteins were altered either in the putative coiled-coil domain or at all four phosphorylation sites. Both had only a slightly decreased tubulin binding activity. In addition, the coiled-coil mutant had a marked effect on microtubule destabilizing activity, demonstrating that tubulin binding and microtubule destabilization activity could be separated. 20. Howell B, Deacon H, Cassimeris L: Decreasing oncoprotein • 18/stathmin levels reduces microtubule catastrophes and increases microtubule polymer in vivo. J Cell Sci 1999, 112:3713-3722. Op18 antibodies were injected into interphase newt lung cells. The authors found that inhibition of Op18 via microinjection resulted in an increase in microtubule polymer levels and a reduction in the catastrophe frequency of individual microtubules. Treatment of cells with Op18 antisense oligonucleotides also reduced the catastrophe frequency of microtubules.
21. Larsson N, Segerman B, Howell B, Fridell K, Cassimeris L, •• Gullberg M: Op18/stathmin mediates multiple region-specific tubulin and microtubule regulating activities. J Cell Biol 1999, 146:1286-1302. These authors carried out deletion analysis of Op18 to determine its effect on tubulin binding, GTP hydrolysis and exchange, and microtubule destabilization activity. Deletion mutants, in which either the amino terminus or the carboxyl terminus was removed, were unable to complex with tubulin in cells, yet they were able to affect the microtubule cytoskeleton. The deletion analysis also revealed that Op18 contains domains that can either inhibit nucleotide exchange, stimulate GTP hydrolysis, or inhibit GTP hydrolysis. 22. Saxton WM, Stemple DL, Leslie RJ, Salmon ED, Zavortink M, McIntosh JR: Tubulin dynamics in cultured mammalian cells. J Cell Biol 1984, 99:2175-2186. 23. Belmont LD, Hyman AA, Sawin KE, Mitchison TJ: Real-time visualization of cell cycle dependent changes in microtubule dynamics in cytoplasmic extracts. Cell 1990, 62:579-589. 24. Verde F, Dogterom M, Stelzer E, Karsenti E, Leibler S: Control of microtubule dynamics and length by cyclin A- and cyclin Bdependent kinases in Xenopus egg extracts. J Cell Biol 1992, 118:1097-1108. 25. Larsson N, Marklund U, Gradin HM, Brattsand G, Gullberg M: Control of microtubule dynamics by oncoprotein 18: dissection of the regulatory role of multisite phosphorylation during mitosis. Mol Cell Biol 1997, 17:5530-5539. 26. Melander Gradin H, Larsson N, Marklund U, Gullberg M: Regulation • of microtubule dynamics by extracellular signals: cAMPdependent protein kinase switches off the activity of oncoprotein 18 in intact cells. J Cell Biol 1998, 140:131-141. This study shows that phosphorylation of Op18 on Ser16 and Ser63 by protein kinase A results in downregulation of Op18 activity toward microtubules both in vitro with purified tubulin and by transfection into tissue culture cells. 27.
Melander Gradin H, Marklund U, Larsson N, Chatila TA, Gullberg M: Regulation of microtubule dynamics by Ca2+/calmodulindependent kinase IV/Gr-dependent phosphorylation of oncoprotein 18. Mol Cell Biol 1997, 17:3459-3467.
28. Gavet O, Ozon S, Manceau V, Lawler S, Curmi P, Sobel A: The stathmin phosphoprotein family: intracellular localization and effects on the microtubule network. J Cell Sci 1998, 111:3333-3346. 29. Walczak CE, Mitchison TJ, Desai A: XKCM1: a Xenopus kinesinrelated protein that regulates microtubule dynamics during mitotic spindle assembly. Cell 1996, 84:37-47. 30. Desai A, Verma S, Mitchison TJ, Walczak CE: Kin I kinesins are •• microtubule-destabilizing enzymes. Cell 1999, 96:69-78. The authors used a series of in vitro assays to demonstrate that two members of the Kin I subfamily of kinesins act exclusively as microtubule-destabilizing enzymes. The mechanism of microtubule destabilization is distinct from the mechanisms of motility established for other kinesins. 31. Mandelkow EM, Mandelkow E, Milligan RA: Microtubule dynamics and microtubule caps: a time-resolved cryo-electron microscopy study. J Cell Biol 1991, 114:977-991. 32. Tournebize R, Popov A, Kinoshita K, Ashford AJ, Rybina S, Mayer T, •• Walczak CE, Karsenti E, Hyman AA: Control of microtubule dynamics requires the antagonistic activities of XMAP215 and XKCM1. Nat Cell Biol 2000, in press. The authors use the Xenopus egg extract system to explore the relationship between the stabilizing effects of MAPs versus the destabilizing effects of the catastrophe promoter XKCM1. They find that XKCM1 is active as a catastrophe promoter during interphase but that it does not stimulate catastrophes because of the stabilizing influence of MAPs.