Mitotic spindle poles are organized by structural and motor proteins in addition to centrosomes

The focusing of microtubules into mitotic spindle poles in vertebrate somatic cells has been assumed to be the consequence of their nucleation from centrosomes. Contrary to this simple view, in this article we show that an antibody recognizing the light intermediate chain of cytoplasmic dynein (70.1) disrupts both the focused organization of microtubule minus ends and the localization of the nuclear mitotic apparatus protein at spindle poles when injected into cultured cells during metaphase, despite the presence of centrosomes. Examination of the effects of this dynein-specific antibody both in vitro using a cell-free system for mitotic aster assembly and in vivo after injection into cultured cells reveals that in addition to its direct effect on cytoplasmic dynein this antibody reduces the efficiency with which dynactin associates with microtubules, indicating that the antibody perturbs the cooperative binding of dynein and dynactin to microtubules during spindle/aster assembly. These results indicate that microtubule minus ends are focused into spindle poles in vertebrate somatic cells through a mechanism that involves contributions from both centrosomes and structural and microtubule motor proteins. Furthermore, these findings, together with the recent observation that cytoplasmic dynein is required for the formation and maintenance of acentrosomal spindle poles in extracts prepared from Xenopus eggs (Heald, R., R. Tournebize, T. Blank, R. Sandaltzopoulos, P. Becker, A. Hyman, and E. Karsenti. 1996. Nature (Lond.). 382: 420-425) demonstrate that there is a common mechanism for focusing free microtubule minus ends in both centrosomal and acentrosomal spindles. We discuss these observations in the context of a search-capture-focus model for spindle assembly.     

XCTK2: a kinesin-related protein that promotes mitotic spindle assembly in Xenopus laevis egg extracts

We used a peptide antibody to a conserved sequence in the motor domain of kinesins to screen a Xenopus ovary cDNA expression library. Among the clones isolated were two that encoded a protein we named XCTK2 for Xenopus COOH-terminal kinesin 2. XCTK2 contains an NH2-terminal globular domain, a central alpha-helical stalk, and a COOH-terminal motor domain. XCTK2 is similar to CTKs in other organisms and is most homologous to CHO2. Antibodies raised against XCTK2 recognize a 75-kD protein in Xenopus egg extracts that cosediments with microtubules. In Xenopus tissue culture cells, the anti-XCTK2 antibodies stain mitotic spindles as well as a subset of interphase nuclei. To probe the function of XCTK2, we have used an in vitro assay for spindle assembly in Xenopus egg extracts. Addition of antibodies to cytostatic factor-arrested extracts causes a 70% reduction in the percentage of bipolar spindles formed. XCTK2 is not required for maintenance of bipolar spindles, as antibody addition to preformed spindles has no effect. To further evaluate the function of XCTK2, we expressed XCTK2 in insect Sf-9 cells using the baculovirus expression system. When purified (recombinant XCTK2 is added to Xenopus egg extracts at a fivefold excess over endogenous levels) there is a stimulation in both the rate and extent of bipolar spindle formation. XCTK2 exists in a large complex in extracts and can be coimmunoprecipitated with two other proteins from extracts. XCTK2 likely plays an important role in the establishment and structural integrity of mitotic spindles.     

Stepwise reconstitution of interphase microtubule dynamics in permeabilized cells and comparison to dynamic mechanisms in intact cells

Microtubules in permeabilized cells are devoid of dynamic activity and are insensitive to depolymerizing drugs such as nocodazole. Using this model system we have established conditions for stepwise reconstitution of microtubule dynamics in permeabilized interphase cells when supplemented with various cell extracts. When permeabilized cells are supplemented with mammalian cell extracts in the presence of protein phosphatase inhibitors, microtubules become sensitive to nocodazole. Depolymerization induced by nocodazole proceeds from microtubule plus ends, whereas microtubule minus ends remain inactive. Such nocodazole-sensitive microtubules do not exhibit subunit turnover. By contrast, when permeabilized cells are supplemented with Xenopus egg extracts, microtubules actively turn over. This involves continuous creation of free microtubule minus ends through microtubule fragmentation. Newly created minus ends apparently serve as sites of microtubule depolymerization, while net microtubule polymerization occurs at microtubule plus ends. We provide evidence that similar microtubule fragmentation and minus end-directed disassembly occur at the whole-cell level in intact cells. These data suggest that microtubule dynamics resembling dynamics observed in vivo can be reconstituted in permeabilized cells. This model system should provide means for in vitro assays to identify molecules important in regulating microtubule dynamics. Furthermore, our data support recent work suggesting that microtubule treadmilling is an important mechanism of microtubule turnover.     

Microtubule flux in mitosis is independent of chromosomes, centrosomes, and antiparallel microtubules

We investigated the mechanism of poleward microtubule flux in the mitotic spindle by generating spindle subassemblies in Xenopus egg extracts in vitro and assaying their ability to flux by photoactivation of fluorescence and low-light multichannel fluorescence video-microscopy. We find that monopolar intermediates of in vitro spindle assembly (half-spindles) exhibit normal poleward flux, as do astral microtubule arrays induced by the addition of dimethyl sulfoxide to egg extracts in the absence of both chromosomes and conventional centrosomes. Immunodepletion of the kinesin-related microtubule motor protein Eg5, a candidate flux motor, suggests that Eg5 is not required for flux. These results suggest that poleward flux is a basic element of microtubule behavior exhibited by even simple self-organized microtubule arrays and presumably underlies the most elementary levels of spindle morphogenesis.     

Mitotic chromatin regulates phosphorylation of Stathmin/Op18

Meiotic and mitotic spindles are required for the even segregation of duplicated chromosomes to the two daughter cells. The mechanism of spindle assembly is not fully understood, but two have been proposed that are not mutually exclusive. The 'search and capture' model suggests that dynamic microtubules become progressively captured and stabilized by the kinetochores on chromosomes, leading to spindle assembly. The 'local stabilization' model proposes that chromosomes change the state of the cytoplasm around them, making it more favourable to microtubule polymerization. It has been shown that Stathmin/Op18 inhibits microtubule polymerization in vitro by interaction with tubulin, and that overexpression in tissue culture cells of non-phosphorylatable mutants of Stathmin/Op18 prevents the assembly of mitotic spindles. We have used Xenopus egg extracts and magnetic chromatin beads to show that mitotic chromatin induces phosphorylation of Stathmin/Op18. We have also shown that Stathmin/Op18 is one of the factors regulated by mitotic chromatin that governs preferential microtubule growth around chromosomes during spindle assembly.     

Enhanced regional deformation at the anterior papillary muscle insertion site after chordal transsection

Cytoplasmic dynein is a minus end-directed microtubule motor that performs distinct functions in interphase and mitosis. In interphase, dynein transports organelles along microtubules, whereas in metaphase this motor has been implicated in mitotic spindle formation and orientation as well as chromosome segregation. The manner in which dynein activity is regulated during the cell cycle, however, has not been resolved. In this study, we have examined the mechanism by which organelle transport is controlled by the cell cycle in extracts of Xenopus laevis eggs. Here, we show that photocleavage of the dynein heavy chain dramatically inhibits minus end-directed organelle transport and that purified dynein restores this motility, indicating that dynein is the predominant minus end-directed membrane motor in Xenopus egg extracts. By measuring the amount of dynein associated with isolated membranes, we find that cytoplasmic dynein and its activator dynactin detach from the membrane surface in metaphase extracts. The sevenfold decrease in membrane-associated dynein correlated well with the eightfold reduction in minus end-directed membrane transport observed in metaphase versus interphase extracts. Although dynein heavy or intermediate chain phosphorylation did not change in a cell cycle-dependent manner, the dynein light intermediate chain incorporated approximately 12-fold more radiolabeled phosphate in metaphase than in interphase extracts. These studies suggest that cell cycle-dependent phosphorylation of cytoplasmic dynein may regulate organelle transport by modulating the association of this motor with membranes.     

Microtubule nucleation by gamma-tubulin-containing rings in the centrosome

The microtubule cytoskeleton of animal cells does not assemble spontaneously, but instead requires the centrosome. This organelle consists of a pair of centrioles surrounded by a complex collection of proteins known as the pericentriolar material (PCM). The PCM is required for microtubule nucleation. The minus, or slow-growing, ends of microtubules are embedded in the PCM and the plus, or fast-growing, ends project outwards into the cytoplasm during interphase, or into the spindle apparatus during mitosis. gamma-Tubulin is the only component of the PCM that is so far implicated in microtubule nucleation. Here we use immuno-electron microscopic tomography to show that gamma-tubulin is localized in ring structures in the PCM of purified centrosomes without microtubules. When these centrosomes are used to nucleate microtubule growth, gamma-tubulin is localized at the minus ends of the microtubules. We conclude that microtubule-nucleating sites within the PCM are ring-shaped templates that contain multiple copies of gamma-tubulin.     

Anastral meiotic spindle morphogenesis: role of the non-claret disjunctional kinesin-like protein

We have used time-lapse laser scanning confocal microscopy to directly examine microtubule reorganization during meiotic spindle assembly in living Drosophila oocytes. These studies indicate that the bipolarity of the meiosis I spindle is not the result of a duplication and separation of centrosomal microtubule organizing centers (MTOCs). Instead, microtubules first associate with a tight chromatin mass, and then bundle to form a bipolar spindle that lacks asters. Analysis of mutant oocytes indicates that the Non-Claret Disjunctional (NCD) kinesin-like protein is required for normal spindle assembly kinetics and stabilization of the spindle during metaphase arrest. Immunolocalization analyses demonstrate that NCD is associated with spindle microtubules, and that the centrosomal components gamma-tubulin, CP-190, and CP-60 are not concentrated at the meiotic spindle poles. Based on these observations, we propose that microtubule bundling by the NCD kinesin-like protein promotes assembly of a stable bipolar spindle in the absence of typical MTOCs.     

Bipolar meiotic spindle formation without chromatin

Establishing a bipolar spindle is an early event of mitosis or meiosis. In somatic cells, the bipolarity of the spindle is predetermined by the presence of two centrosomes in prophase. Interactions between the microtubules nucleated by centrosomes and the chromosomal kinetochores enable the formation of the spindle. Non-specific chromatin is sufficient, however, to promote spindle assembly in Xenopus cell-free extracts that contain centrosomes [1,2]. The mouse oocyte represents an excellent model system in which to study the mechanism of meiotic spindle formation because of its size, transparency and slow development. These cells have no centrioles, and their multiple microtubule-organizing centers (MTOCs) are composed of foci of pericentriolar material [3,4]. The bipolarity of the meiotic spindle emerges from the reorganization of these randomly distributed MTOCs [4]. Regardless of the mechanisms involved in this reorganization, the chromosomes seem to have a major role during spindle formation in promoting microtubule polymerization and directing the appropriate rearrangement of MTOCs to form the two poles [5]. Here, we examined spindle formation in chromosome-free mouse oocyte fragments. We found that a bipolar spindle can form in vivo in the absence of any chromatin due to the establishment of interactions between microtubule asters that are progressively stabilized by an increase in the number of microtubules involved, demonstrating that spindle formation is an intrinsic property of the microtubule network.     

Influence of M-phase chromatin on the anisotropy of microtubule asters

In many eukaryotic cells going through M-phase, a bipolar spindle is formed by microtubules nucleated from centrosomes. These microtubules, in addition to being "captured" by kinetochores, may be stabilized by chromatin in two different ways: short-range stabilization effects may affect microtubules in close contact with the chromatin, while long-range stabilization effects may "guide" microtubule growth towards the chromatin (e.g., by introducing a diffusive gradient of an enzymatic activity that affects microtubule assembly). Here, we use both meiotic and mitotic extracts from Xenopus laevis eggs to study microtubule aster formation and microtubule dynamics in the presence of chromatin. In "low-speed" meiotic extracts, in the presence of salmon sperm chromatin, we find that short-range stabilization effects lead to a strong anisotropy of the microtubule asters. Analysis of the dynamic parameters of microtubule growth show that this anisotropy arises from a decrease in the catastrophe frequency, an increase in the rescue frequency and a decrease in the growth velocity. In this system we also find evidence for long-range "guidance" effects, which lead to a weak anisotropy of the asters. Statistically relevant results on these long-range effects are obtained in "high-speed" mitotic extracts in the presence of artificially constructed chromatin stripes. We find that aster anisotropy is biased in the direction of the chromatin and that the catastrophe frequency is reduced in its vicinity. In this system we also find a surprising dependence of the catastrophe and the rescue frequencies on the length of microtubules nucleated from centrosomes: the catastrophe frequency increase and the rescue frequency decreases with microtubule length.     

Expression of the mitotic motor protein Eg5 in postmitotic neurons: implications for neuronal development

It is well established that the microtubules of the mitotic spindle are organized by a variety of motor proteins, and it appears that the same motors or closely related variants organize microtubules in the postmitotic neuron. Specifically, cytoplasmic dynein and the kinesin-related motor known as CHO1/MKLP1 are used within the mitotic spindle, and recent studies suggest that they are also essential for the establishment of the axonal and dendritic microtubule arrays of the neuron. Other motors are required to tightly regulate microtubule behaviors in the mitotic spindle, and it is attractive to speculate that these motors might also help to regulate microtubule behaviors in the neuron. Here we show that a homolog of the mitotic kinesin-related motor known as Eg5 continues to be expressed in rodent neurons well after their terminal mitotic division. In neurons, Eg5 is directly associated with the microtubule array and is enriched within the distal regions of developing processes. This distal enrichment is transient, and typically lost after a process has been clearly defined as an axon or a dendrite. Strong expression can resume later in development, and if so, the protein concentrates within newly forming sprouts at the distal tips of dendrites. We suggest that Eg5 generates forces that help to regulate microtubule behaviors within the distal tips of developing axons and dendrites.     

Fizzy is required for activation of the APC/cyclosome in Xenopus egg extracts

The Xenopus homologue of Drosophila Fizzy and budding yeast CDC20 has been characterized. The encoded protein (X-FZY) is a component of a high molecular weight complex distinct from the APC/cyclosome. Antibodies directed against FZY were produced and shown to prevent calmodulin-dependent protein kinase II (CaMKII) from inducing the metaphase to anaphase transition of spindles assembled in vitro in Xenopus egg extracts, and this was associated with suppression of the degradation of mitotic cyclins. The same antibodies suppressed M phase-promoting factor (MPF)-dependent activation of the APC/cyclosome in interphase egg extracts, although they did not appear to alter the pattern or extent of MPF-dependent phosphorylation of APC/cyclosome subunits. As these phosphorylations are thought to be essential for APC/cyclosome activation in eggs and early embryos, we conclude that at least two events are required for MPF to activate the APC/cyclosome, allowing both chromatid segregation and full degradation of mitotic cyclins. The first one, which does not require FZY function, is the phosphorylation of APC/cyclosome subunits. The second one, that requires FZY function (even in the absence of MAD2 protein and when the spindle assembly checkpoint is not activated) is not yet understood at its molecular level.     

Activation of the protein kinase p38 in the spindle assembly checkpoint and mitotic arrest

The mitogen-activated protein kinase (MAPK) superfamily comprises classical MAPK (also called ERK), c-Jun amino-terminal or stress-activated protein kinase (JNK or SAPK), and p38. Although MAPK is essential for meiotic processes in Xenopus oocytes and the spindle assembly checkpoint in Xenopus egg extracts, the role of members of the MAPK superfamily in M phase or the spindle assembly checkpoint during somatic cell cycles has not been elucidated. The kinase p38, but not MAPK or JNK, was activated in mammalian cultured cells when the cells were arrested in M phase by disruption of the spindle with nocodazole. Addition of activated recombinant p38 to Xenopus cell-free extracts caused arrest of the extracts in M phase, and injection of activated p38 into cleaving embryos induced mitotic arrest. Treatment of NIH 3T3 cells with a specific inhibitor of p38 suppressed activation of the checkpoint by nocodazole. Thus, p38 functions as a component of the spindle assembly checkpoint in somatic cell cycles.     

Kinetochores distinguish GTP from GDP forms of the microtubule lattice

During prometaphase in mitotic cell division, chromosomes attach to the walls of microtubules and subsequently move to microtubule ends, where they stay throughout mitosis. This end-attachment seems to be essential for correct chromosome segregating. However, the mechanism by which kinetochores, the multiprotein complexes that link chromosomes to the microtubules of the mitotic spindle, recognize and stay attached to microtubule ends is not understood. One clue comes from the hydrolysis of GTP that occurs during microtubule polymerization. Although tubulin dimers must contain GTP to polymerize, this GTP is rapidly hydrolysed following the addition of dimers to a growing polymer. This creates a microtubule consisting largely of GDP-tubulin, with a small cap of GTP-tubulin at the end. It is possible that kinetochores distinguish the different structural states of a GTP- versus a GDP-microtubule lattice. We have examined this question in vitro using reconstituted kinetochores from the yeast Saccharomyces cerevisiae. We found that kinetochores in vitro bind preferentially to GTP- rather than GDP-microtubules, and to the plus-end preferentially over the lattice. Our results could explain how kinetochores stay at microtubule ends and thus segregate chromosomes correctly during mitosis in vivo. This result demonstrates that proteins exist that can distinguish the GTP conformation of the microtubule lattice.     

Purification of a WD repeat protein, EMAP, that promotes microtubule dynamics through an inhibition of rescue

The major microtubule-associated protein in echinoderms is a 77-kDa, WD repeat protein, called EMAP. EMAP-related proteins have been identified in sea urchins, starfish, sanddollars, and humans. We describe the purification of sea urchin EMAP and demonstrate that EMAP binding to microtubules is saturable at a molar ratio of 1 mol of EMAP to 3 mol of tubulin dimer. Unlike MAP-2, MAP-4, or tau proteins, EMAP binding to microtubules is not lost by cleavage of tubulin with subtilisin. In addition to binding to the microtubule polymer, EMAP binds to tubulin dimers in a 1:1 molar ratio. The abundance of EMAP in the egg suggests that it could function to regulate microtubule assembly. To test this hypothesis, we examined the effects of EMAP on the dynamic instability of microtubules nucleated from axoneme fragments as monitored by video-enhanced differential interference contrast microscopy. Addition of 2.2 microM EMAP to 21 microM tubulin results in a slight increase in the elongation and shortening velocities at the microtubule plus ends but not at the minus ends. Significantly, EMAP inhibits the frequency of rescue 8-fold without producing a change in the frequency of catastrophe. These results indicate that EMAP, unlike brain microtubule-associated proteins, promotes microtubule dynamics.     

The effect of the increase in the calcium concentration induced by action of the ionophore A-23187 on mitosis in cultured ESK cells

After addition of 20 mM calcium ionophore A23187 to cultured PK (pig kidney embryo) cells, [Ca++] in cytosol increased by more than 10 times. The maximum [Ca++] concentration was observed 1-2 min after drug introduction. Later on [Ca++] gradually decreased, and after 30 min of incubation with A23187 [Ca++] its concentration was 3-5 times higher than in the norm. 1 min after introduction of the calcium ionophore, mitotic spindles shortened for 1/3 and the angle of divergence of spindle microtubules from the centrosome extended. These changes remained for 5 min of treatment. After nocodazole treatment the length of the mitotic spindle reduced (2 min), then mitotic spindle and the metaphase plate were disrupted. The rate of mitotic spindle shortening after addition of the ionophore is about the same as after addition of nocodazole, but after ionophore treatment the metaphase plate remained for more than 5 min. Based on the results obtained we suggest that the maximum distance between spindle poles at metaphase is in the intact cell, and after any perturbation of normal microtubule dynamics the spindle may rapidly collapse. The collapsed mitotic spindle becomes more stable and its size is determined predominantly by kinetochore fibers. The metaphase spindle is completely and rapidly destroyed when the microtubule growth is prohibited, but it is preserved when this growth is restricted.     

Xe-p9, a Xenopus Suc1/Cks protein, is essential for the Cdc2-dependent phosphorylation of the anaphase- promoting complex at mitosis

Degradation of mitotic cyclins on exit from M phase occurs by ubiquitin-mediated proteolysis. The ubiquitination of mitotic cyclins is regulated by the anaphase-promoting complex (APC) or cyclosome. Xe-p9, the Xenopus homolog of the Suc1/Cks protein, is required for some step in mitotic cyclin destruction in Xenopus egg extracts. Specifically, if p9 is removed from interphase egg extracts, these p9-depleted extracts are unable to carry out the proteolysis of cyclin B after entry into mitosis and thus remain arrested in M phase. To explore the molecular basis of this defect, we depleted p9 from extracts that had already entered M phase and thus contained an active APC. We found that ubiquitin-mediated proteolysis of cyclin B was not compromised under these circumstances, suggesting that p9 is not directly required for ubiquitination or proteolysis. Further analysis of extracts from which p9 had been removed during interphase showed that, at the beginning of mitosis, these extracts are unable to carry out the hyperphosphorylation of the Cdc27 component of the APC, which coincides with the initial activation of the APC. p9 can be found in a complex with a small fraction of the Cdc27 protein during M phase but not interphase. The phosphorylation of the Cdc27 protein (either associated with the APC or in an isolated, bacterially expressed form) by recombinant Cdc2/cyclin B is strongly enhanced by p9. Our results indicate that p9 directly regulates the phosphorylation of the APC by Cdc2/cyclin B. These studies indicate that the Suc1/Cks protein modulates substrate recognition by a cyclin-dependent kinase.     

Pericentrin, a highly conserved centrosome protein involved in microtubule organization

Antisera from scleroderma patients that react widely with centrosomes in plants and animals were used to isolate cDNAs encoding a novel centrosomal protein. The nucleotide sequence is consistent with a 7 kb mRNA and contains an open reading frame encoding a protein with a putative large coiled-coil domain flanked by noncoiled ends. Antisera recognize a 220 kd protein and stain centrosomes and acentriolar microtubule-organizing centers, where the protein is localized to the pericentriolar material (hence, the name pericentrin). Anti-pericentrin antibodies disrupt mitotic and meiotic divisions in vivo and block microtubule aster formation in Xenopus extracts, but do not block gamma-tubulin assembly or microtubule nucleation from mature centrosomes. These results suggest that pericentrin is a conserved integral component of the filamentous matrix of the centrosome involved in the initial establishment of organized microtubule arrays.     

Antiproliferative mechanism of action of cryptophycin-52: kinetic stabilization of microtubule dynamics by high-affinity binding to microtubule ends

Cryptophycin-52 (LY355703) is a new synthetic member of the cryptophycin family of antimitotic antitumor agents that is currently undergoing clinical evaluation. At high concentrations (>/=10 times the IC50), cryptophycin-52 blocked HeLa cell proliferation at mitosis by depolymerizing spindle microtubules and disrupting chromosome organization. However, low concentrations of cryptophycin-52 inhibited cell proliferation at mitosis (IC50 = 11 pM) without significantly altering spindle microtubule mass or organization. Cryptophycin-52 appears to be the most potent suppressor of microtubule dynamics found thus far. It suppressed the dynamic instability behavior of individual microtubules in vitro (IC50 = 20 nM), reducing the rate and extent of shortening and growing without significantly reducing polymer mass or mean microtubule length. Using [3H]cryptophycin-52, we found that the compound bound to microtubule ends in vitro with high affinity (Kd, 47 nM, maximum of approximately 19.5 cryptophycin-52 molecules per microtubule). By analyzing the effects of cryptophycin-52 on dynamics in relation to its binding to microtubules, we determined that approximately 5-6 molecules of cryptophycin-52 bound to a microtubule were sufficient to decrease dynamicity by 50%. Cryptophycin-52 became concentrated in cells 730-fold, and the resulting intracellular cryptophycin-52 concentration was similar to that required to stabilize microtubule dynamics in vitro. The data suggest that cryptophycin-52 potently perturbs kinetic events at microtubule ends that are required for microtubule function during mitosis and that it acts by forming a reversible cryptophycin-52-tubulin stabilizing cap at microtubule ends.     

MHP1, an essential gene in Saccharomyces cerevisiae required for microtubule function

The gene for a microtubule-associated protein (MAP), termed MHP1 (MAP-Homologous Protein 1), was isolated from Saccharomyces cerevisiae by expression cloning using antibodies specific for the Drosophila 205K MAP. MHP1 encodes an essential protein of 1,398 amino acids that contains near its COOH-terminal end a sequence homologous to the microtubule-binding domain of MAP2, MAP4, and tau. While total disruptions are lethal, NH2-terminal deletion mutations of MHP1 are viable, and the expression of the COOH-terminal two-thirds of the protein is sufficient for vegetative growth. Nonviable deletion-disruption mutations of MHP1 can be partially complemented by the expression of the Drosophila 205K MAP. Mhp1p binds to microtubules in vitro, and it is the COOH-terminal region containing the tau-homologous motif that mediates microtubule binding. Antibodies directed against a COOH-terminal peptide of Mhp1p decorate cytoplasmic microtubules and mitotic spindles as revealed by immunofluorescence microscopy. The overexpression of an NH2-terminal deletion mutation of MHP1 results in an accumulation of large-budded cells with short spindles and disturbed nuclear migration. In asynchronously growing cells that overexpress MHP1 from a multicopy plasmid, the length and number of cytoplasmic microtubules is increased and the proportion of mitotic cells is decreased, while haploid cells in which the expression of MHP1 has been silenced exhibit few microtubules. These results suggest that MHP1 is essential for the formation and/or stabilization of microtubules.