The Xenopus XMAP215 and Its Human Homologue TOG Proteins Interact with Cyclin B1 to Target p34cdc2 to Microtubules during Mitosis

Cytoskeleton reorganization, leading to mitotic spindle formation, is an M-phase-specific event and is controlled by maturation promoting factor (MPF: p34cdc2–cyclinB1 complex). It has previously been demonstrated that the p34cdc2–cyclin B complex associates with mitotic spindle microtubules and that microtubule-associated proteins (MAPs), in particular MAP4, might be responsible for this interaction. In this study, we report that another ubiquitous MAP, TOG in human and its homologue in Xenopus XMAP215, associates also with p34cdc2 kinase and directs it to the microtubule cytoskeleton. Costaining of Xenopus cells with anti-TOGp and anti-cyclin B1 antibodies demonstrated colocalization in interphase cells and also with microtubules throughout the cell cycle. Cyclin B1, TOG/XMAP215, and p34cdc2 proteins were recovered in microtubule pellets isolated from Xenopus egg extracts and were eluted with the same ionic strength. Cosedimentation of cyclin B1 with in vitro polymerized microtubules was detected only in the presence of purified TOG protein. Using a recombinant C-terminal TOG fragment containing a Pro-rich region, we showed that this domain is sufficient to mediate cosedimentation of cyclin B1 with microtubules. Finally, we demonstrated interaction between TOG/XMAP215 and cyclin B1 by co-immunoprecipitation assays. As XMAP215 was shown to be the only identified assembly promoting MAP which increases the rapid turnover of microtubules, the TOG/XMAP215–cyclin B1 interaction may be important for regulation of microtubule dynamics at mitosis.     

Cyclin B interaction with microtubule-associated protein 4 (MAP4) targets p34cdc2 kinase to microtubules and is a potential regulator of M-phase microtubule dynamics

We previously demonstrated (Ookata et al., 1992, 1993) that the p34cdc2/cyclin B complex associates with microtubules in the mitotic spindle and premeiotic aster in starfish oocytes, and that microtubule- associated proteins (MAPs) might be responsible for this interaction. In this study, we have investigated the mechanism by which p34cdc2 kinase associates with the microtubule cytoskeleton in primate tissue culture cells whose major MAP is known to be MAP4. Double staining of primate cells with anti-cyclin B and anti-MAP4 antibodies demonstrated these two antigens were colocalized on microtubules and copartitioned following two treatments that altered MAP4 distribution. Detergent extraction before fixation removed cyclin B as well as MAP4 from the microtubules. Depolymerization of some of the cellular microtubules with nocodazole preferentially retained the microtubule localization of both cyclin B and MAP4. The association of p34cdc2/cyclin B kinase with microtubules was also shown biochemically to be mediated by MAP4. Cosedimentation of purified p34cdc2/cyclin B with purified microtubule proteins containing MAP4, but not with MAP-free microtubules, as well as binding of MAP4 to GST-cyclin B fusion proteins, demonstrated an interaction between cyclin B and MAP4. Using recombinant MAP4 fragments, we demonstrated that the Pro-rich C-terminal region of MAP4 is sufficient to mediate the cyclin B-MAP4 interaction. Since p34cdc2/cyclin B physically associated with MAP4, we examined the ability of the kinase complex to phosphorylate MAP4. Incubation of a ternary complex of p34cdc2, cyclin B, and the COOH-terminal domain of MAP4, PA4, with ATP resulted in intracomplex phosphorylation of PA4. Finally, we tested the effects of MAP4 phosphorylation on microtubule dynamics. Phosphorylation of MAP4 by p34cdc2 kinase did not prevent its binding to microtubules, but abolished its microtubule stabilizing activity. Thus, the cyclin B/MAP4 interaction we have described may be important in targeting the mitotic kinase to appropriate cytoskeletal substrates, for the regulation of spindle assembly and dynamics.     

Mutations at phosphorylation sites of Xenopus microtubule-associated protein 4 affect its microtubule-binding ability and chromosome movement during mitosis

Microtubule-associated proteins (MAPs) bind to and stabilize microtubules (MTs) both in vitro and in vivo and are thought to regulate MT dynamics during the cell cycle. It is known that p220, a major MAP of Xenopus, is phosphorylated by p34(cdc2) kinase as well as MAP kinase in mitotic cells, and that the phosphorylated p220 loses its MT-binding and -stabilizing abilities in vitro. We cloned a full-length cDNA encoding p220, which identified p220 as a Xenopus homologue of MAP4 (XMAP4). To examine the physiological relevance of XMAP4 phosphorylation in vivo, Xenopus A6 cells were transfected with cDNAs encoding wild-type or various XMAP4 mutants fused with a green fluorescent protein. Mutations of serine and threonine residues at p34(cdc2) kinase-specific phosphorylation sites to alanine interfered with mitosis-associated reduction in MT affinity of XMAP4, and their overexpression affected chromosome movement during anaphase A. These findings indicated that phosphorylation of XMAP4 (probably by p34(cdc2) kinase) is responsible for the decrease in its MT-binding and -stabilizing abilities during mitosis, which are important for chromosome movement during anaphase A.     

Specific association of an M-phase kinase with isolated mitotic spindles and identification of two of its substrates as MAP4 and MAP1B.

Isolated mammalian (Chinese hamster ovary [CHO]) metaphase spindles were found to be enriched in a histone H1 kinase whose activity was mitotic-cycle dependent. Two substrates for the kinase were identified as MAP1B and MAP4. Partially purified spindle kinase retained activity for the spindle microtubule-associated proteins (MAPs) as well as brain and other tissue culture MAPs; on phosphorylation, spindle MAPs exhibited increased immunoreactivity with MPM-2, a monoclonal antibody specific for a subset of mitotic phosphoproteins. Immunofluorescence using an anti-thiophosphoprotein antibody localized in vitro phosphorylated spindle proteins to microtubule fibers, centrosomes, kinetochores, and midbodies. The fractionated spindle kinase was reactive with anti-human p34cdc2 antibodies and with an anti-human cyclin B but not an anti-human cyclin A antibody. We conclude that spindle MAPs undergo mitotic cycle-dependent phosphorylations in vivo and associate with a kinase that remains active on spindle isolation and may be related to p34cdc2.     

Xenopus TACC3/maskin is not required for microtubule stability but is required for anchoring microtubules at the centrosome

Members of the transforming acidic coiled coil (TACC) protein family are emerging as important mitotic spindle assembly proteins in a variety of organisms. The molecular details of how TACC proteins function are unknown, but TACC proteins have been proposed to recruit microtubule-stabilizing proteins of the tumor overexpressed gene (TOG) family to the centrosome and to facilitate their loading onto newly emerging microtubules. Using Xenopus egg extracts and in vitro assays, we show that the Xenopus TACC protein maskin is required for centrosome function beyond recruiting the Xenopus TOG protein XMAP215. The conserved C-terminal TACC domain of maskin is both necessary and sufficient to restore centrosome function in maskin-depleted extracts, and we provide evidence that the N terminus of maskin inhibits the function of the TACC domain. Time-lapse video microscopy reveals that microtubule dynamics in Xenopus egg extracts are unaffected by maskin depletion. Our results provide direct experimental evidence of a role for maskin in centrosome function and suggest that maskin is required for microtubule anchoring at the centrosome.     

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.     

Spindle assembly and the art of regulating microtubule dynamics by MAPs and Stathmin/Op18

The way that microtubules reorganize from their long, stable interphase configuration to form the mitotic spindle remains a challenging and unsolved question. It is now widely recognized that microtubule polymerization during the cell cycle is regulated by a balance between microtubule-stabilizing and-destabilizing factors. Stabilizing factors include a large group of microtubule-associated proteins (MAPs; e.g. MAP4, XMAP215, XMAP230/XMAP4 and XMAP310) and the destabilizing factors are a growing family of proteins (e.g. Stathmin/Op18 and XKCM1). Recent studies have allowed a mechanistic dissection of how these stabilizing and destabilizing factors regulate microtubule dynamics and spindle assembly.     

Relocation and distinct subcellular localization of p34cdc2-cyclin B complex at meiosis reinitiation in starfish oocytes.

M phase promoting factor (MPF) is a major element controlling entry into the M phase of the eukaryotic cell cycle. MPF is composed of two subunits, p34cdc2 and cyclin B. Using indirect immunofluorescence staining with specific antibody against starfish cyclin B, we monitored the dynamics of the subcellular distribution of MPF during meiosis reinitiation in starfish oocytes. We found that all of the cyclin B is already associated with p34cdc2 in immature oocytes arrested at the G2/M border and that this inactive complex is present exclusively in the cytoplasm. After its activation, part of the p34cdc2-cyclin B complex moves into the germinal vesicle before nuclear envelope breakdown, independently of either microtubules or actin filaments. Thereafter, some part of the complex accumulates in the nucleolus and condensed chromosomes. Another portion of the complex accumulates on meiotic asters and spindles, while the rest is still present throughout the cytoplasm. As these patterns of localization are detected in the detergent-extracted oocytes, we propose at least four distinct subcellular states of the p34cdc2-cyclin B complex: freely soluble, microtubule-associated, detergent-resistant cytoskeleton-associated and chromosome-associated. Thus, in addition to the intramolecular modification of p34cdc2-cyclin B complex, its intracellular relocation plays a key role in promoting the M phase.     

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.     

Phosphorylation and activation of human cdc25-C by cdc2--cyclin B and its involvement in the self-amplification of MPF at mitosis.

We have investigated the mechanisms responsible for the sudden activation of the cdc2-cyclin B protein kinase before mitosis. It has been found previously that cdc25 is the tyrosine phosphatase responsible for dephosphorylating and activating cdc2-cyclin B. In Xenopus eggs and early embryos a cdc25 homologue undergoes periodic phosphorylation and activation. Here we show that the catalytic activity of human cdc25-C phosphatase is also activated directly by phosphorylation in mitotic cells. Phosphorylation of cdc25-C in mitotic HeLa extracts or by cdc2-cyclin B increases its catalytic activity. cdc25-C is not a substrate of the cyclin A-associated kinases. cdc25-C is able to activate cdc2-cyclin B1 in Xenopus egg extracts and to induce Xenopus oocyte maturation, but only after stable thiophosphorylation. This demonstrates that phosphorylation of cdc25-C is required for the activation of cdc2-cyclin B and entry into M-phase. Together, these studies offer a plausible explanation for the rapid activation of cdc2-cyclin B at the onset of mitosis and the self-amplification of MPF observed in vivo.     

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.     

Regulation of microtubule organization during interphase and M phase

Microtubule (MT) dynamics and organization change markedly during interphase-M phase transition of the cell cycle. This mini review focuses first on p220, a ubiquitous MT-associated protein of Xenopus. p220 is phosphorylated by p34cdc2 kinase and MAP kinase in M phase, and concomitantly loses its MT-binding and MT-stabilizing activities. A cDNA encoding p220 was cloned, which identified p220 as a Xenopus homolog of MAP4, and p220 was therefore termed XMAP4. To examine the physiological relevance of XMAP4 phosphorylation during mitosis, Xenopus A6 cells were transfected with cDNA encoding wild-type or various XMAP4 mutants fused with a green fluorescent protein (GFP). Mutations of serine and threonine within potential phosphorylation sites for p34cdc2 kinase to nonphosphorylatable alanine interfered with mitosis-associated reduction in MT-affinity of XMAP4 and their overexpression affected chromosome movement during anaphase A. These results indicated that phosphorylation of XMAP4 by p34cdc2 kinase is responsible for the decrease in its MT-binding and MT-stabilizing activities during mitosis which are important for chromosome movement during anaphase A. The second focus is on a novel monoclonal antibody W8C3, which recognizes alpha-tubulin. W8C3 stained spindle MTs but not interphase MTs of Xenopus A6 cells, although tubulin dimers in M phase and interphase were equally recognized by this antibody. The difference in MT staining pattern may be because the W8C3-recognition site on alpha-tubulin is sterically hidden in interphase MTs but not in spindle MTs.     

XMAP215–EB1 Interaction Is Required for Proper Spindle Assembly and Chromosome Segregation in Xenopus Egg Extract

In metaphase Xenopus egg extracts, global microtubule growth is mainly promoted by two unrelated microtubule stabilizers, end-binding protein 1 (EB1) and XMAP215. Here, we explore their role and potential redundancy in the regulation of spindle assembly and function. We find that at physiological expression levels, both proteins are required for proper spindle architecture: Spindles assembled in the absence of EB1 or at decreased XMAP215 levels are short and frequently multipolar. Moreover, the reduced density of microtubules at the equator of ΔEB1 or ΔXMAP215 spindles leads to faulty kinetochore–microtubule attachments. These spindles also display diminished poleward flux rates and, upon anaphase induction, they neither segregate chromosomes nor reorganize into interphasic microtubule arrays. However, EB1 and XMAP215 nonredundantly regulate spindle assembly because an excess of XMAP215 can compensate for the absence of EB1, whereas the overexpression of EB1 cannot substitute for reduced XMAP215 levels. Our data indicate that EB1 could positively regulate XMAP215 by promoting its binding to the microtubules. Finally, we show that disruption of the mitosis-specific XMAP215–EB1 interaction produces a phenotype similar to that of either EB1 or XMAP215 depletion. Therefore, the XMAP215–EB1 interaction is required for proper spindle organization and chromosome segregation in Xenopus egg extracts.     

Survivin phosphorylation and M-phase promoting factor in oral carcinogenesis

Survivin is a recently described inhibitor of apoptosis and mitotic regulator which is selectively over-expressed in human tumors. Its expression rate is predictive of disease progression, early recurrences and resistance to therapy. Up-regulation of survivin in oral pre-malignant lesions (OPL) and in oral squamous cell carcinoma (OSCC) has already been demonstrated in previous studies. A critical step for activation of survivin has been identified in the phosphorylation on Thr34 by the main mitotic kinase p34cdc2-cyclin B1. The aim of this work was to investigate the relationship between survivin, its phosphorylated active form (p-survivin) and M-phase promoting factor (MPF), p34cdc2-cyclin B1 in oral carcinogenesis. 32 OSCCs and 17 OPLs from surgical specimens were studied for cyclin B1, p-survivin, survivin, and p34cdc2 expression by immunohistochemistry. All cases of OSCC expressed survivin and its expression rate was correlated to p-survivin levels (P<0.05). Cyclin B1 was positive in 80% of cases, while p-34cdc2 was over-expressed in all OSCCs. All OPLs associated with OSCC expressed survivin and its levels were correlated to p-survivin levels (P<0.05). Cyclin B1 was positive in 70% of cases, while p-34cdc2 was positive in all OPLs. In conclusion, this study demonstrated that MPF, survivin and p-survivin are expressed during early and late phase of oral carcinogenesis. MPF proteins, which are co-expressed on mitotic apparatus, could represent a potential target for therapies based on manipulation of survivin phosphorylation, which would induce apoptosis in cancer cells.     

Drosophila melanogaster Mini Spindles TOG3 Utilizes Unique Structural Elements to Promote Domain Stability and Maintain a TOG1- and TOG2-like Tubulin-binding Surface

Microtubule-associated proteins regulate microtubule (MT) dynamics spatially and temporally, which is essential for proper formation of the bipolar mitotic spindle. The XMAP215 family is comprised of conserved microtubule-associated proteins that use an array of tubulin-binding tumor overexpressed gene (TOG) domains, consisting of six (A–F) Huntingtin, elongation factor 3, protein phosphatase 2A, target of rapamycin (HEAT) repeats, to robustly increase MT plus-end polymerization rates. Recent work showed that TOG domains have differentially conserved architectures across the array, with implications for position-dependent TOG domain tubulin binding activities and function within the XMAP215 MT polymerization mechanism. Although TOG domains 1, 2, and 4 are well described, structural and mechanistic information characterizing TOG domains 3 and 5 is outstanding. Here, we present the structure and characterization of Drosophila melanogaster Mini spindles (Msps) TOG3. Msps TOG3 has two unique features as follows: the first is a C-terminal tail that stabilizes the ultimate four HEAT repeats (HRs), and the second is a unique architecture in HR B. Structural alignments of TOG3 with other TOG domain structures show that the architecture of TOG3 is most similar to TOG domains 1 and 2 and diverges from TOG4. Docking TOG3 onto recently solved Stu2 TOG1· and TOG2·tubulin complex structures suggests that TOG3 uses similarly conserved tubulin-binding intra-HEAT loop residues to engage α- and β-tubulin. This indicates that TOG3 has maintained a TOG1- and TOG2-like TOG-tubulin binding mode despite structural divergence. The similarity of TOG domains 1–3 and the divergence of TOG4 suggest that a TOG domain array with polarized structural diversity may play a key mechanistic role in XMAP215-dependent MT polymerization activity.     

The Drosophila microtubule-associated protein mini spindles is required for cytoplasmic microtubules in oogenesis

The XMAP215/TOG family of proteins is a closely related set of MAPs (microtubule-associated proteins) found in animals, yeast, and plants . In yeast and animal cells, the XMAP215/TOG proteins are required for both mitosis and meiosis. Although effects of XMAP215/TOG proteins on cytoplasmic microtubules have not previously been shown in animal cells, in plants the Arabidopsis family member MOR1 is required for the organization of cortical microtubule arrays . The Drosophila family member, encoded by the mini spindles (msps) gene, is maternally expressed and loaded into the egg, where it is an essential component of meiotic and mitotic spindles . Here we show that msps is also required during oogenesis for the structure and function of cytoplasmic microtubules. Localization of bicoid (bcd) mRNA in the oocyte is a microtubule-mediated event . We show that bcd RNA localization is defective in msps mutants. We also identify defects in cytoplasmic microtubules in both the germ and follicle cells of mutant ovaries and determine the expression pattern of msps mRNA and protein in developing egg chambers. Our findings reveal a new role for msps in cell patterning and raise the possibility that other family members may perform similar functions.     

The Xenopus TACC homologue, maskin, functions in mitotic spindle assembly

Maskin is the Xenopus homolog of the transforming acidic coiled coil (TACC)-family of microtubule and centrosome-interacting proteins. Members of this family share a approximately 200 amino acid coiled coil motif at their C-termini, but have only limited homology outside of this domain. In all species examined thus far, perturbations of TACC proteins lead to disruptions of cell cycle progression and/or embryonic lethality. In Drosophila, Caenorhabditis elegans, and humans, these disruptions have been attributed to mitotic spindle assembly defects, and the TACC proteins in these organisms are thought to function as structural components of the spindle. In contrast, cell division failure in early Xenopus embryo blastomeres has been attributed to a role of maskin in regulating the translation of, among others, cyclin B1 mRNA. In this study, we show that maskin, like other TACC proteins, plays a direct role in mitotic spindle assembly in Xenopus egg extracts and that this role is independent of cyclin B. Maskin immunodepletion and add-back experiments demonstrate that maskin, or a maskin-associated activity, is required for two distinct steps during spindle assembly in Xenopus egg extracts that can be distinguished by their response to "rescue" experiments. Defects in the "early" step, manifested by greatly reduced aster size during early time points in maskin-depleted extracts, can be rescued by readdition of purified full-length maskin. Moreover, defects in this step can also be rescued by addition of only the TACC-domain of maskin. In contrast, defects in the "late" step during spindle assembly, manifested by abnormal spindles at later time points, cannot be rescued by readdition of maskin. We show that maskin interacts with a number of proteins in egg extracts, including XMAP215, a known modulator of microtubule dynamics, and CPEB, a protein that is involved in translational regulation of important cell cycle regulators. Maskin depletion from egg extracts results in compromised microtubule asters and spindles and the mislocalization of XMAP215, but CPEB localization is unaffected. Together, these data suggest that in addition to its previously reported role as a translational regulator, maskin is also important for mitotic spindle assembly.     

A comparison of the ability of XMAP215 and tau to inhibit the microtubule destabilizing activity of XKCM1

During mitosis, microtubules not only grow fast, but also have a high rate of catastrophe. This is achieved in part by the activity of the MAP, XMAP215, which can stimulate the growth rate of microtubules without fully inhibiting the function of the catastrophe-kinesin XKCM1. We do not know whether this activity is particular to XMAP215, or is a general property of all MAPs. Here, we compare the activities of XMAP215 with the neuronal MAP tau, in opposing the destabilizing activity of the non-conventional kinesin XKCM1. We show that tau is a much more potent inhibitor of XKCM1 than XMAP215. Because tau completely suppresses XKCM1 activity, even at low concentrations, the combination of tau and XKCM1 is unable to generate mitotic microtubule dynamics.     

Microtubule dependency of p34cdc2 inactivation and mitotic exit in mammalian cells

The protein kinase inhibitor 2-aminopurine induces checkpoint override and mitotic exit in BHK cells which have been arrested in mitosis by inhibitors of microtubule function (Andreassen, P. R., and R. L. Margolis. 1991. J. Cell Sci. 100:299-310). Mitotic exit is monitored by loss of MPM-2 antigen, by the reformation of nuclei, and by the extinction of p34cdc2-dependent H1 kinase activity. 2-AP-induced inactivation of p34cdc2 and mitotic exit depend on the assembly state of microtubules. During mitotic arrest generated by the microtubule assembly inhibitor nocodazole, the rate of mitotic exit induced by 2-AP decreases proportionally with increasing nocodazole concentrations. At nocodazole concentrations of 0.12 microgram/ml or greater, 2-AP induces no apparent exit through 75 min of treatment. In contrast, 2-AP brings about a rapid exit (t1/2 = 20 min) from mitotic arrest by taxol, a drug which causes inappropriate overassembly of microtubules. In control mitotic cells, p34cdc2 localizes to kinetochores, centrosomes, and spindle microtubules. We find that efficient exit from mitosis occurs under conditions where p34cdc2 remains associated with centrosomal microtubules, suggesting it must be present on these microtubules in order to be inactivated. Mitotic slippage, the natural reentry of cells into G1 during prolonged mitotic block, is also microtubule dependent. At high nocodazole concentrations slippage is prevented and mitotic arrest approaches 100%. We conclude that essential components of the machinery for exit from mitosis are present on the mitotic spindle, and that normal mitotic exit thereby may be regulated by the microtubule assembly state.     

CDC2 phosphorylation of the fission yeast dis1 ensures accurate chromosome segregation

Shortened kinetochore microtubules take separated chromatids to the opposing spindle poles in anaphase. Fission yeast Dis1 belongs to the Dis1/XMAP215/TOG family that is required for proper microtubule dynamics. Here, we report that Dis1is regulated by Cdc2 phosphorylation and that this mitotic phosphorylation ensures the fidelity of chromosome segregation. Whereas mutants Dis1(6A) and Dis1(6E) that substitute all of the six Cdc2 sites for Ala or Glu, respectively, produce colonies at 22 degrees C-36 degrees C, Dis1(6A) but not Dis1(6E) loses a minichromosome and reveals aberrant chromosome segregation at significant frequencies. Dis1(WT) is recruited to two regions of the mitotic spindle: kinetochores (possibly also kinetochore microtubules) in metaphase and the pole-to-pole microtubule lattice in anaphase. Mutant Dis1(6E) preferentially binds to metaphase kinetochores, whereas Dis1(6A), which is located along microtubules, fails in its accumulation at kinetochores. Dis1(6A) displays synthetic lethality with the mis12-537, which is a mutant that compromises kinetochore function. Dis1(6E) mimics the Cdc2-phosphorylated form of Dis1(WT), whereas Dis1(6A) can partially rescue the phenotype resulting form deletion of Mtc1/Alp14, another XMAP215-like protein. In anaphase, dephosphorylated Dis1 and Dis1(6A), but not Dis1(6E), move to the spindle microtubule lattice near the SPBs. Cdc2 thus directly phosphorylates Dis1, and this phosphorylation regulates Dis1 localization in both metaphase and anaphase and ensures high-fidelity segregation.