Modulation of microtubule dynamics during the cell cycle

23

Modulation of microtubule dynamics during the cell cycle Francis J McNally Microtubule dynamics change dramatically during the cell cycle, but the mechanisms by which these changes occur are unknown. Recent progress has been made in four areas: firstly, in the determination of changes in microtubule

turnover and net tubulin polymer levels in vivo; secondly, in the elucidation of mechanisms of regulation of microtubule dynamics by microtubule-associated protein 4; thirdly, in the determination of the mechanisms by which Xenopus microtubule-associated protein regulates microtubule dynamics; and fourthly, in the elucidation of the structural basis of microtubule nucleation by 7 tubulin. Address Section of Molecular and Cellular Biology, 2310 Storer Hall, University of California, Davis, Davis, CA 95616, USA; e-mail: [email protected] Current Opinion in Cell Biology 1996, 8:23-29 © Current Biology Lid ISSN 0955-0674 Abbreviations yTuRC y tubulin ring complex MAP microtubule-associatedprotein NEB nuclearenvelope breakdown XMAP XenopusMAP

Introduction

Microtubules are dynamic polymers that grow by the addition of etl3 tubulin heterodimers and shrink by the release of tubulin heterodimers from both ends [1,21. Solutions of pure tubulin exhibit dynamic instability, a state in which polymers continue to polymerize and depolymerize even when polymer mass has reached a steady state [3-5]. This instability is thought to be driven by a cycle of G T P hydrolysis and exchange. GTP-bound tubulin dimers preferentially polymerize, and do not depolymerize from microtubule ends until bound GTP is hydrolyzed to GDP [3,6,7]. Microtubule dynamics can be described by a number of measurable parameters. T h e total number of microtubules depends on the number of nucleation sites in vivo, as microtubules grow only from specific sites in cells, usually from centrosomes [8]. T h e 'growth rate' and 'shrinkage rate' are defined by the rate of addition and loss, respectively, of tubulin dimers at the ends of individual microtubules. 'Catastrophe rate' is the frequency of transitions of individual microtubule ends from a growing state to a shrinking state. 'Rescue rate' is the frequency of transitions of individual microtubule ends from a shrinking state to a growing state [5]. Given a constant number of nucleation sites, the four rates (i.e. growth, shrinkage, catastrophe and rescue) determine two other measurable parameters: the fraction of tubulin present as polymers

versus the fraction present as the base unit heterodimers (i.e. the net polymer level) and the rate of turnover, or exchange of tubulin dimers between polymer and dimer pools [9,10,11"']. T h e finding that microtubule turnover increases dramatically in mitosis relative to its level in interphase [12] has led to the hypothesis that the regulation of microtubule dynamics is important in mitotic spindle assembly and function. Microtubule-associated

protein 2 and tau

Ideas about how microtubule dynamics may be regulated in vivo have been greatly influenced by in vitro studies of the neuronal microtubule-associated proteins (MAPs), MAP2 and tau. When either of these proteins is added to solutions of pure tubulin, growth rate is increased, catastrophe rate is reduced and rescue frequency is increased [13-16]. T h e net effect is an increased polymer level and decreased turnover compared with solutions containing only pure tubulin. These effects are eliminated when tau or MAP2 is phosphorylated [13,17], suggesting that phosphorylation and dephosphorylation of tau and MAP2 (or of similar MAPs) could be used to regulate microtubule dynamics in vivo. In this scenario, microtubules would be stabilized by unphosphorylated MAPs during interphase. At this time, polymer levels would be high and tubulin turnover would be low. Upon entry into mitosis, phosphorylation of MAPs would result in decreased microtubule polymer level and increased tubulin turnover. In this model, the high level of turnover in mitosis should be similar to that observed in solutions of pure tubulin. There are two problems with this model. First, microtubules in vivo [18-20] and in cell extracts [21,22] exhibit higher turnover than microtubules in solutions of pure tubulin. Even during interphase, although turnover is low, it is still higher than that of pure tubulin. It has been demonstrated that this increased turnover in cell extracts is not due to buffer components but rather to factors that are greater than 30kDa in size [21]. This leads to the conclusion that microtubule dynamics in vivo are driven by proteins that increase transition frequencies, rather than by proteins such as tau and MAP2 which decrease transition frequencies and turnover. T h e second problem is that MAP2 and tau are neuronal proteins and, as such, are not found in dividing cells. Until recently, detailed studies of non-neuronal MAPs have been limited. Here, I review recent papers that firstly demonstrate that the relationship between turnover and polymer levels in vivo is not always as predicted by in vitro studies of tau and MAP2, secondly characterize the unique effects of two non-neuronal MAPs, MAP4 and Xenopus MAP,

24

Cytoskeleton

on microtubule dynamics, and thirdly demonstrate the structural basis for microtubule nucleation by 7 tubulin. The

G2-~M

Borisy, personal communication). T h e correspondence of increased turnover and decreased polymer level at NEB is thus consistent with a model in which stabilizing MAPs are inactivated by phosphorylation at NEB. T h e surprising result was that polymer levels recovered during prometaphase, and returned to the levels found during interphase by the time metaphase was reached, even though turnover remained high from NEB through to metaphase (Y Zhai, G Borisy, personal communication). Thus, from prometaphase through to metaphase, turnover and polymer levels do not correlate in the same way as at NEB (see Table 1).

transition

Although it has been known for some time that the turnover of tubulin between soluble (dimer) and polymer pools increases dramatically in mitosis relative to interphase in living cells [12], observations of individual microtubules in living mitotic cells have thus far been impossible. Observations of individual microtubules have, however, been carried out in Xenopus egg extracts, which can be converted to a 'mitosis-like' state by addition of cyclins or active p34cdcZ protein kinase [23]. In these experiments, activation of p34cdc2 induced an increased catastrophe rate [21], suggesting that this increase in catastrophe rate might explain the increased turnover observed in vivo during mitosis.

MAP4

Recent work on MAP4 could help explain the rapid increase in turnover and decrease in polymer levels that occur precisely at NEB. Unlike MAP2 and tau, MAP4 is a ubiquitous protein found from humans [25] to Drosophila [26], in both neuronal and non-neuronal cells [25]. MAP4 remains associated with microtubules throughout the cell cycle in vivo [27"']. In vitro, unphosphorylated MAP4 stabilizes microtubules by increasing the rescue frequency. MAP4 also forms complexes between microtubules and cyclin B-p34cdc2, resulting in association of active p34cdc2 with microtubules. This complex formation occurs as a result of specific binding of MAP4 with cyclin B [27"°], which is the cyclin responsible for achieving the GZ---)M transition [23]. MAP4 is phosphorylated by p34cdcZ in these complexes. The phosphorylated MAP4 does

From the perspective of in vitro studies of MAP2 and tau, it would be expected that the increased turnover and catastrophe upon entry into mitosis would result in a reduced polymer level. This expectation has recently been confirmed with a technique that allows quantification of tubulin polymer and dimer pools in individual cells [24°]. A precipitous drop in polymer level was observed precisely at the time of 'nuclear envelope breakdown' (NEB) (see Table 1). Accurately timed measurements of turnover rate revealed that the GZ---)M increase in microtubule turnover also occurred precisely at NEB (Y Zhai, G

Table 1 C h a n g e s In m l c r o t u b u l e d y n a m i c s during t h e cell cycle, Mitotic stage

Total microtubules

Non-kinetochore microtubules

Kinetochore microtubules

Interphase

PreNEB

PostNEB

Prometaphase

Turnover (t 1/2) (min)*

4.5-5.?5

-

0.58_+0.3

.

Anaphase

Telophase

Polymer level (oh)*

68.4_+7.5

61.9+9

23.4+_3.8

47+_7.8

59+_5

62,8_+4.6

Flux (wn rain-l)

0t

.

Turnover (t 1/2) (rain)*

4.5-5.?5

-

0.58_+0.3

-

0.68-0.9

1.0+-0.5

-

Polymer level (%)*

68.4+7.5

61.9+-9

23.4-+3.8

-

42

33

-

Flux (pmmin "1)

Ot

-

-

-

ltt

-

-

Turnover (tl/2) (min)*

NA

NA

NA

-

4.7-5.3

22.4+_4.8

-

Polymer level (oh)*

NA

NA

NA

-

15

26

-

Flux (ltrn min1)

NA

NA

NA

-

0.39-0.46t

0.2-0.21 t

-

.

.

.

.

Metaphase .

.

56.4_+6.9 .

.

*Most values are from PtK1 and/or LLC-PK cells at 37"C ([24",38"]; Y Zhai, GG Borisy, personal communication), t Values for poleward flux are from PtK1 and LLC-PK cells at 30"C [38"]. t t Value for poleward flux in asters formed in mitotic Xenopusegg extracts [59°]. NA, not applicable.

NEB, nuclear envelope breakdown.

Modulation of microtubule dynamics during the cell cycle McNally

not affect microtubule dynamics even though it still binds to microtubules [27°°1. In vivo, cyclin B-p34cdcZ is transported into the nucleus just before NEB. At NEB, cyclin B-p34cdc2 associates with microtubules of the nascent mitotic spindle, presumably as a result of MAP4 interaction [27°',28] (see Fig. 1). These data suggest that unphosphorylated MAP4 could reduce turnover of microtubules during interphase, and that release of cyclin B-p34cdc2 from the nucleus at NEB would result in cyclin B-p34cdc2 association with, and phosphorylation of, MAP4, with consequent increased microtubule turnover and decreased polymer levels. In light of this model, it is interesting that inactivation of p34cdc2 at the metaphase---)anaphase transition requires an intact microtubule cytoskeleton [29]. Thus, MAP4 may mediate the inactivation of p34 cdc2 at the onset of anaphase, in addition to mediating the effects of p34cdcZ on the microtubule cytoskeleton at NEB. T h e association of protein phosphatase 2A (PP2A) with microtubules [30] may also be important in this regulation, as PP2A is a negative regulator of p34cdc2 [31]. XMAP

Although MAP4 is an attractive candidate for a protein to regulate microtubule dynamics at the G2---~M transition, it does not allow turnover at rates-approaching even the relatively slow turnover of interphase microtubules in vivo. Recent work on a Xenopus egg protein called Xenopus MAP (XMAP) demonstrates that it can dramatically increase microtubule turnover [11=°]. XMAP was originally described as a protein that increased polymer level by

95

increasing the growth rate at microtubule plus (or faster growing) ends specifically [32]. More recent work demonstrates that XMAP increases turnover by decreasing rescue frequency and increasing both growth and shrinkage rates. The increase in growth rate is sufficient to compensate for the reduced rescue and increased shrinkage rates, and hence promotes net assembly [11°']. This is an important result for two reasons. First, XMAP is the first protein to be characterized that increases turnover. All other characterized MAPs reduce turnover by increasing rescue. The existence of proteins such as XMAP could explain why turnover in vivo is so much higher than turnover in solutions of pure tubulin. Second, XMAP promotes net assembly at the same time that it increases turnover. T h e action of XMAP or a similar protein could partially explain how net polymer levels in interphase and metaphase are the same even though turnover is much higher in metaphase (see Table 1). Although there is no evidence for cell cycle regulation of XMAP, MAP4 or a related MAP might regulate XMAPdriven dynamic instability in a cell cycle dependent manner. A major drawback for this idea is that XMAP does not increase catastrophe [11°°], which is more frequent in v/vo than in solutions of pure tubulin [18-20]. Thus, it is likely that more MAPs remain to be found. The metaphase-~anaphase

transition

Dramatic changes in microtubule dynamics also occur upon inactivation of p34cdc2 at the metaphase---)anaphase transition. T h e situation is more complicated because of

Figure 1

NEB 0

FactorX ~]

FactorX (active)

Decreased transition frequencies

I p34CdC2-cyclinB MAP4-~ (inactive)

Promotion of transition frequencies

/1\

"

I-(~ - phosphorylation O- spindlepolebody I

© 1996 Current Opinion in Cell Biology

Model of the G2-~M transition in microtubule dynamics. Before NEB, active cyclin B-p34 cdc2 is sequestered into the nucleus. Unphosphorylated MAP4 reduces the transition frequencies of microtubule dynamics that are promoted by unknown factors (Factor X). At NEB, cyclin B-p34 cdc2 is released from the nucleus and associates with, and phosphorylates, MAP4. The resulting inactivation of MAP4 allows Factor X to promote increased transition frequencies, and a consequent increase in turnover and decrease in polymer levels occurs.

26

Cytoskeleton

the presence of different classes of microtubules that did not differentiate at the GZ--)M transition: kinetochore microtubules that are attached at their plus ends in the kinetochore; non-kinetochore spindle microtubules; astral microtubules; and interzone microtubules. It has been known for some time that kinetochore microtubules turn over more slowly than non-kinetochore microtubules [33,341. Recent work reveals that the dynamics of these classes respond differently at the point of the metaphase---)anaphase transition. Turnover of kinetochore microtubules has been shown to decrease in anaphase relative to metaphase [35-37], and recently this decrease has been more accurately quantified, demonstrating that a fivefold decrease in turnover of kinetochore microtubules occurs upon entry into anaphase (see Table 1) [38"*]. Kinetochore microtubules also exhibit a property called poleward flux [39], which is characterized by net depolymerization of microtubule minus (or slower growing) ends at the centrosome. Recent work shows that the velocity of poleward flux decreases at the metaphase---)anaphase transition (see Table 1) [38",40], and that poleward flux does not occur at all in interphase cells [38",41]. In contrast to kinetochore microtubules, turnover of nonkinetochore spindle microtubules stays constant throughout the metaphase---)anaphase transition [38"']. Two conclusions can be made from this observation. First, the disassembly and reduced turnover of kinetochore microtubules at anaphase is likely to be driven by specific catalysts at kinetochores [42,43] rather than by a global destabilization of microtubules. Second, the inactivation of p34cdc2 at anaphase onset (see above) is not sufficient to return non-kinetochore spindle microtubules to their interphase state of slow turnover. Recent experiments have also demonstrated that there is no net change in polymer level in anaphase relative to metaphase [24"], and that the number of non-kinetochore spindle microtubules decreases during anaphase [38"°]. Thus, the net depolymerization of kinetochore [35] and non-kinetochore [38"'] spindle microtubules during anaphase must be compensated for by net assembly of astral and interzone microtubules. It remains to be determined how the net polymer level of non-kinetochore spindle microtubules is reduced during anaphase without any change in turnover.

The structural basis of microtubule nucleation by y tubulin In addition to the regulation of the dynamics of growth and shrinkage, the number of microtubule nucleation sites at the centrosome also appears to be regulated, with an increase in the number of nucleation sites occurring during mitosis [44,451. It has been assumed for some time that the nucleation sites in the centrosome are, at least in part, composed of 3' tubulin. This assumption has been made on the basis of two sets of data. First, immunolocalization of 3' tubulin shows that it is concentrated at centrosomes

and other sites of microtubule nucleation [46--48]. Second, inhibition of 3' tubulin in vivo [49,50] and in vitro [51,52] results in a loss of microtubule nucleation activity at centrosomes. Recently, the structural basis of nucleation by 3' tubulin has been elucidated. A 'g-tubulin-containing complex, ~TuRC (3' tubulin ring complex), has been purified from Xenopus eggs. This complex is a ring structure with a diameter close to that of a microtubule (i.e. 25nm). Each ring complex has at least 10 ~tubulin molecules in addition to a number of other proteins. T h e fact that each complex contains roughly the same number of T-tubulin subunits as the number of ctl~-tubulin dimers around the circumference of a microtubule suggests that 0tl~-tubulin subunits might add directly to a ring of 3'-tubulin subunits in order to nucleate a microtubule. T h e 3'TuRC does indeed have potent microtubule-nucleating activity, and the complex can be observed to be attached to the minus ends of microtubules nucleated by the 3r['uRCs (Y Zheng, ML Wong, B Alberts, T Mitchison, personal communication; see Note added in proof). In complementary work, it has been demonstrated that isolated centrosomes contain hundreds of ring complexes of the same diameter as yTuRCs, that microtubules grown from centrosomes initiate at these rings, and that these rings contain multiple molecules of 3' tubulin (M Moritz, MB Braunfeld, JW Sedat, B Alberts, DA Agard, personal communication; see Note added in proof). These new data provide solid evidence that the microtubule nucleation sites in centrosomes consist of rings composed of~tubulin subunits and associated proteins (3'TuRCs). T h e remaining question, then, is whether the nucleating activity of the •~tFuRCs is regulated by the number of 3'TuRCs in the centrosome, a number which might increase during mitosis by recruitment, or whether nucleating activity is regulated by phosphorylation instead.

Microtubule severing Microtubule severing could regulate the number of nucleation sites, and could also provide another method of regulating microtubule number and length. Microtubulesevering activity increases upon activation of p34cdc2 in Xenopus egg extracts, suggesting that microtubule severing might contribute to cell cycle regulation of microtubule dynamics [53,54]. Three microtubule-severing proteins have been isolated (reviewed in [55]): p56 [54], katanin [56] and EFlct [57]. A number of problems limit our understanding of the role of microtubule severing in vivo. First, direct in vivo observations of microtubule severing have not been reported, making it difficult to guess what role severing may play. Second, p56 and katanin have so far been found only in eggs and it is not clear if they play a general role in all cells. It also remains to be seen precisely what effect severing has on microtubule dynamics. Severing might stimulate catastrophe by exposing unstable (GDP-bound) tubulin ends and thereby decreasing polymer levels. Alternatively,

Modulation of microtubule dynamics during the cell cycle McNally

severing might increase polymer level by increasing the number of nucleation sites.

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • ••

Conclusions Recent work demonstrates that the relationship between microtubule turnover and net polymer level is not as simple as was predicted by early work on MAPs. Kinetochore microtubules exhibit both slow turnover and net depolymerization in anaphase. In contrast, non-kinetochore microtubules exhibit rapid turnover in metaphase but are maintained at a high net polymer level. Regulation by MAP4 might explain the changes in microtubule dynamics at the GZ---)M transition, but cannot explain the high turnover and high levels of polymer found in metaphase. Metaphase dynamics might be explained instead by the action of XMAP, the first protein that promotes both high turnover and high polymer level to be described. Thus, the behavior of microtubules in cells is likely to be affected by the simultaneous activity of several proteins with overlapping activities, the existence of which presumably explains why disruption of the tau gene in mice has only a slight phenotypic effect [58]. Future experiments will have to examine the in vitro effects of combinations of proteins on microtubule dynamics, and the in vivo effects of inhibiting multiple proteins. Understanding the regulation of individual MAPs will require examination of the in vivo effects of constitutively phosphorylated and constitutively dephosphorylated proteins. T h e major limitation to these experiments is that microtubule dynamics are not generally studied in genetically tractable organisms. In addition, the search is on for as yet undiscovered proteins that may, for example, increase catastrophe frequency.

Note added in proof T h e paper referred to in the text as Y Zheng, ML Wong, B Alberts, T Mitchison, personal communication has now been accepted for publication [60]. T h e paper referred to in the text as M Moritz, MB Braunfeld, JW Sedat, B Alberts, DA Agard, personal communication has now been accepted for publication [61]. A kinesin-like protein, XKCM1, has recently been identified as a factor that increases the catastrophe rate of microtubule dynamics. Immunodepletion of XKCM1 from mitotic Xenopus egg extracts results in a significant decrease in catastrophe frequency [62•]. Thus, XKCM1, like XMAP, may be a component of Factor X (see Fig. 1).

Acknowledgements I thank G Borisy, M Mofitz and Y Zheng for communication of results prior to publication.

27

of special interest of outstanding interest

1.

Mandelkow EM, Mandelkow E, Milligan RA: Microtubule dynamics and mlcrotubule caps: a time-resolved cryo-electron microscopy study. J Cell Biol 1991, 114:977-991.

2.

Chretien D, Fuller SD, Karsenti E: Structure of growing microtubule ends: two-dimensional sheets close Into tubes st vadabie rates. J Cell Biol 1995, 129:1311-1328.

3.

Mitchison T, Kirechner M: Dynamic Instability of mlcrotubule growth. Nature 1984, 312:237-242.

4.

Horio T, Hotani H: Visualization of the dynamic Instability of Individual mlcrotubules by dark-field microscopy. Nature 1986, 321:605-607.

5.

Walker RA, O'Brien ET, Pryer NK, Soboeiro MF, Voter WA, Erickson HP, Salmon ED: Dynamic Instability of Individual mlcrotubles analyzed by video light microscopy:, rate constants and transition frequencies. J Cell Biol 1988, 107:1437-1448.

8.

Hyrnan AA, Salser AS, Drechsel DN, Unwin N, Mitchison TJ: Role of GTP hydrolysis in mlcrotubule dynamics: information from a slowly hydrolyzabie analog, GMPCPR Mol Biol Cell 1992, 3:1155-1167.

7.

Mitchison TJ: Localization of an exchangeable GTP binding site at the plus end of mlcrotubules- Science 1993, 261:1044-1047.

8.

Karsenti E, Kobayashi S, Mitchison T, Kirechner M: Role of the centrosome in organizing the Interphase microtubule array: properties of cytoplasts containing or lacking centrosomes- J Cell Biol 1984, 98:1763-1776.

9.

Gliksman NR, Skibbens RV, Salmon ED: How the transition frequencies of mtcrotubule dynamic instability (nucleation, catastrophe, and rescue) regulate mlcrotubule dynamics In Interphese and mitosis: analysis using a Monte Cado computer simulation. Mol Biol Cell 1993, 4:1035-1050.

10.

Toso RJ, Jordan MA, Farrell KW, Matsumoto B, Wilson L: Kinetic stabilization of mlcrotubule dynamic instability in vitro by vlnblasUne. Biochemistry 1993, 32:1285-1293.

11. •.

Vasquez RJ, Gard DL, Cassimeris L: XMAP from Xenopus eggs promotes rapid plus end assembly of microtubules and rapid microtubule polymer turnover. J Cell Bio/1994, 127:985-993. The first report of a protein that increases, rather than decreases, tubulin turnover. XMAP decreases rescue frequency and increases shrinkage rate, resulting in higher turnover. Remarkably, XMAP effects a compensating increase in growth rate, so that XMAP increases net polymer levels at the same time that it increases turnover. This situation is similar to that observed in vitro. 12.

Saxton WM, Stemple DL, Leslie RJ, Salmon ED, Zavortink M, Mclntosh RJ: Tubulin dynamics in cultured mammalian ceils. J Cell Bio11984, 99:2175-2186.

13.

Drechsal DN, Hyman AA, Cobb MH, Kirschner MW: Modulation of dynamic instability of tubulln assembly by the microtubuleassociated protein tau. Mol Biol Ceil 1992, 3:1141-1154.

14.

Pryer NK, Walker RA, Skeen VP, Bourns BD, Soboeiro MF, Salmon ED: Brain mlcrotubule-essoclated proteins modulate microtubule dynamic instability in vitro. Real time observations using video microscopy. J Cell Sci 1992, 103:965-976.

15.

Kowalski RJ, Williams RC: MIcrotubule-aseoclated protein 2 aRere the dynamic properties of mlcrotubule assembly and disassembly. J Biol Chern 1993, 268:9847-9855.

16.

Itoh TJ, Hotani H: Microtubule-stebllizlng activity of mk:rotubuieassociated proteins (MAPs) Is due to Increase in frequency of rescue In dynamic Instability:. shortening length decreases wRh binding of MAPs onto microtubules- Cell Struct Funct 1994, 19:279-290.

17.

Hoehi M, Ohta K, Gotoh Y, Mori A, Murofushi H, Sakai H, Nishida E: MRngen-octivated-proteln-ldnase-cstalyzed phosphorylation of microtubule-sssoclated proteins, micretubule-assoclated protein 2 and mlcrotubule-assoclated protein 4, induces an alteration In their function. Eur J Biochem 1992, 203:43-52.

28

Cytoskeleton

18.

Cassimeris L, Pryer NK, Salmon ED: Real-time observations of microtubule dynamic Instability In living cells. J Cell Bio11988, 107:2223-2231.

37.

19.

Sammak PJ, Borisy GG: Direct observation of mlorotubule dynamics In living cells. Nature 1988, 332:724-726.

38. *-

20.

Sheldon E, Wadsworth P: Observation and quantification of individual mlcrotubule behavior in vivo: mlcrotubule dynamics are cell-type specific. J Cell Biol 1993, 120:935-945.

21.

Belmont A, Hyman A, Sawin K, Mitchison T: Real-time visualization of cell cycle dependent changes in mlcrotubule dynamics In cytoplasmic extracts. Cell 1990, 62:579-589.

22.

Simon JR, Parsons SF, Salmon ED: Buffer conditions and non-tubulln factors critically affect the mlcrotubule dynamic instability of sea urchin egg tubulln. Cell Motll Cytoskeleton 1992, 21:1-14.

23.

Murray M, Kirschner M: Cyclin synthesis ddves the eady embryonic cell cycle. Nature 1989, 339:275-280.

24. •

Zhal Y, Borisy GG: Quantitative determination of the proportion of mlcrotubule polymer present dudng the mitosls-lnterphase transition. J Cell Sci 1994, 107:881-890. Demonstration that total microtubule polymer levels remain constant throughout the metaphass-~anaphass transition. 25.

26.

Bulinski JC, Borisy GG: Widespread distdbution of a 210,000 mol wt mlcrotubule-assoclated protein In cells and tissue of primate. J Cell Biol 1980, 87:802-808. Goldstein LSB, Laymon RA, Mclntosh JR: A microtubuleassociated protein In Drosophila melenogeste~. Identification, characterization, and isolation of coding sequences. J Cell Biol 1986, 107:2076-2087.

2?. •*

Ookata K, Hisanaga S, Bulinski JC, Murofuahi H, Aizawa H, Itoh TJ, Hotani H, Okumura E, Tachibana K, Kishimoto T: Cydln B interaction with mlcrotubule-assodsted protein 4 (MAP4) targets p34cdc2 klnese to mlcrotubules and Is a potential regulator of M-phase mlcrotubule dynamics. J Ca//Bio/1995, 128:849-862. Unphosphorylated MAP4 binds to microtubules and reduces turnover of tubulin. The results described here demonstrate that phosphorylation of MAP4 by p34cdc2 kinase results in a loss of any effect on tubulin turnover, without affecting microtubule binding. Results also demonstrate the direct binding of MAP4 to cyclin B, suggesting that MAP4 mediates the localization of cyclin B to the spindle after nuclear envelope breakdown.

Gorbsky G J, Boriay GG: Mlcrotubules of the Idnetochore fiber turn over In metaphase but not In anaphasa. J Cell Biol 1989, 109:653-662.

Zhal Y, Kronebusch PJ, Boriay GG: Klnetochore microtubule dynamics and the metaphase-anephasa transition. J Cell Biol 1995, in press. The most comprehensive analysis of turnover and flux of spindle microtubules during metaphase and anaphase. 39.

Mitchison TJ: Polewards mlcrotubule flux in the mltoUc spindle: evidence from photcectlvatlon of fluorescence. J Cell Biol 1989, 109:637-652.

40.

Mitchison TJ, Salmon ED: Poleward klnetochore fiber movement occurs during both metaphase end anephasa A In newt lung cell mitosis. J Cell Biol 1992, 119:569-582.

41.

Rodionov VI, Lim SS, Gelfand VI, Borisy GG: Mlcrotubule dynamics in fish melanophores. J Ceil Biol 1994, 126:1455-1464.

42.

Hyman AA, Mitchison TJ: ModulaUon of mlcrotubule stability by kinatochores in vitro. J Cell Biol 1990, 110:1607-1616.

43.

Lombillo VA, Nislow C, Yen TJ, Gelfand VI, Mclntosh JR: Antibodies to the klnesln motor domain and CENP-E Inhibit microtubule depolymerlzation-dependent motion of chromosomes in vitro. J Cell Bio/1995, 128:107-115.

44.

Kuriyama R, Boris), GG: Microtubule-nucleating activity of centrosomes In C l i o cells Is independent of the centdole cycle, but coupled to the mitoUc cycle. J Cell Bio11981, 91:822-826.

45.

Buendia B, Draetta G, Kamenti E: Regulation of the microtubule nucleating activity in Xenopus egg extracts: role of cyclln Aassociated protein klnase. J Cell Biol 1992, 116:1431-1442.

46.

Stearns T, Evans L, Kirschner M: Gamma-tubulln Is a highly conserved component of the centrosome. Cell 1991, 65:825-836.

47.

Zheng YX, Jung MK, Oakley BR: Gamma-tubuIin is present In Drosophila melanogester and Homo sapiens and is associated with the centrosome. Cell 1991, 65:817-823.

48.

Lajoie-Mazenc I, Tollon Y, Detrevea C, Julian M, Moisand A, GuethHallonet C, Debec A, Sallss-Passador I, Puget A, Mazarguil H e t al.: Recruitment of anUgenic gamme-tubulln during mitosis In animal cells: presence of 9amma-tubulJn In the mitetic spindle. J Cell Sci 1994, 107:2825-2837.

28.

Pines J, Hunter T: Human cycllns A and B1 are differentially located in the cell and undergo cell cycle-dependent nuclear transport. J Cell Biol 1991, 115:1-17.

49.

29.

Andreassen PR, Margolis RL: Mlcrotubule dependency of p34cdc2 inactivation and mitotic exit In mammalian cells. J Cell B/o/1994, 127:789-802.

Joshi HC, Palsclos MJ, McNamara L, Cleveland DW: 7-tubulln Is a centrosomal protein required for cell cycle-dependent mlcrotubule nucleation. Nature 1992, 358:80-83.

50.

Sontag E, Nunbhakdi-Creig V, Bloom GS, Mumby MC: A novel pool of protein phosphatase 2A Is assodated with microtubules and Is regulated during the cell cycle. J Cell Biol 1995, 128:1131-1144.

Sunkel CE, Gomes R, Sampalo P, Perdigao J, Gonzalez C: Gamma-tubulin is required for the structure end function of the microtubule organizing centre In Drosophila neuroblasts. EMBO J 1995, 14:28-36.

51.

Lee TH, Solomon MJ, Mumby MC, Kirschner MW: INH, a negative regulator of MPF, Is a form of protein phosphatase 2A. Cell 1991, 64:415-423.

Stearns T, Kirschner M: In vitro reconstitution of centrosome assembly and function: the central role of 7-tubulln. Cell 1994, 76:623-637.

52.

Gard DL, Kirschner MW: A mlorotubule-essociated protein from Xenopus eggs that specifically promotes assembly at the plus end. J Cell Bio11987, 105:2203-2215.

Felix MA, Antony C, Wright M, Maro B: Centrosome assembly in vitro: role of ~-tubulln recruitment in Xenopus sperm aster formation. J Cell Bio11994, 124:19-31.

53.

Cassimeris L, Rieder CL, Rupp G, Salmon ED: Stability of mlcrotubule attachment to metaphase kinetochores In PtK1 cells. J Cell Sci 1990, 96:9-15.

Vale R: Severing of stable microtubules by a mitotically activated protein in Xenopus egg extracts. Cell 1991, 64:827-839.

54.

34.

Mitchison TJ, Evans L, Schulze E, Kirschner MW: Sites of mlcrotubule assembly and disassembly in the mitotic spindle. Cell 1986, 45:515-527.

Shiina N, Gotoh Y, Nishida E: A novel homo-oligomedc protein responsible for an MPF-dependent mlcrotubule-sevedng activity. EMBO J 1992, 11:4723-4731.

55.

35.

Gorbsky G J, Sammak PJ, Borisy GG: Chromosomes move poleward In anaphasa along stationary mlcrotubules that coordinately disassemble from their klnatochore ends. J Cell Bio11987, 104:9-18.

Shiina N, Gotoh Y, Nishida E: Microtubule-sevorlng In M phase. Trends Cell Biol 1995, 5:283-286.

56.

McNally F, Vale R: Identification of katanln, an ATPase that severs end disassembles stable mlcrotubules. Cell 1993, 75:419-429.

57.

Shiina N, Gotoh Y, Kubomura J, Iwamatsu A, Nishida E: Mlcrotubule sevedng by elongation factor-l(x. Science 1994, 266:282-285.

30.

31.

32.

33.

36.

Gorbsky G J, Sammak PJ, Borisy GG: Mlcrotubule dynamics and chromosome moUon visualized In living anephase cells. J Cell Bio11988, 106:1185-1192.

Modulation of microtubule dynamics during the cell cycle McNally

58.

Harada A, Oguchi K, Okabe S, Kuno J, Tarada S, Ohshima T, Sato-Yoshitake R, Takei Y, Node T, Hirokawa N: Altered microtubule organization In small-calibre axons of mice lacking teu protein. Nature 1994, 369:488-491.

59. •

Sawin KE, Mitchison TJ: Microtubule flux In mitosis is independent of chromosomes, centrosomes, and antlparallel microtubules. Mol Bio/Cell 1994, 5:217-226. The only demonstration of poleward flux in non-kinetochore microtubules. 60.

Zheng Y, Wong ML, Alberts B, Mitchison T: A 3' tubulln dng complex purified from the unfertilized egg of Xenopus leevis can nucleate microfubule activity in vitro. Nature 1996, in press.

61.

62. •

29

Moritz M, Braunfeld MB, Sedat JW, Alberta B, Agard DA: Microtubule-nudeatlng sites In the csntrosome are ring structures that contain 3' tubulln. Nature 1996, in press.

Walczak CE, Mitchison TJ, Desai A: XKCM1: a Xenopus klneslnrelated protein that regulates mlcrotubule dynamics dudng mitotic spindle assembly. Cell 1996, in press. A kinesin-like protein, XKCM1, has recently been identified as a fact(x that incrass~ the _~__~r0pherate of ~ l e dynem~ Immunodeple~ of XKCM1 from mitotic Xenopus egg extracts results in a significant decrease in