The roles of fission yeast ase1 in mitotic cell division, meiotic nuclear oscillation, and cytokinesis checkpoint signaling

The Ase1/Prc1 proteins constitute a conserved microtubule-associated protein family that is implicated in central spindle formation and cytokinesis. Here we characterize a role for fission yeast Ase1. Ase1 localizes to microtubule overlapping zones and displays dynamic alterations of localization during the cell cycle. In particular, its spindle localization during metaphase is reduced substantially, followed by robust appearance at the spindle midzone in anaphase. ase1 deletions are viable but defective in nuclear and septum positioning and completion of cytokinesis, which leads to diploidization and chromosome loss. Time-lapse imaging shows that elongating spindles collapse abruptly in the middle of anaphase B. Either absence or overproduction of Ase1 results in profound defects on microtubule bundling in an opposed manner, indicating that Ase1 is a dose-dependent microtubule-bundling factor. In contrast microtubule nucleating activities are not noticeably compromised in ase1 mutants. During meiosis astral microtubules are not bundled and oscillatory nuclear movement is impaired significantly. The Aurora kinase does not correctly localize to central spindles in the absence of Ase1. Finally Ase1 acts as a regulatory component in the cytokinesis checkpoint that operates to inhibit nuclear division when the cytokinesis apparatus is perturbed. Ase1, therefore, couples anaphase completion with cytokinesis upon cell division.     

Cdc14-regulated midzone assembly controls anaphase B

Spindle elongation in anaphase of mitosis is a cell cycle-regulated process that requires coordination between polymerization, cross-linking, and sliding of microtubules (MTs). Proteins that assemble at the spindle midzone may be important for this process. In this study, we show that Ase1 and the separase-Slk19 complex drive midzone assembly in yeast. Whereas the conserved MT-bundling protein Ase1 establishes a midzone, separase-Slk19 is required to focus and center midzone components. An important step leading to spindle midzone assembly is the dephosphorylation of Ase1 by the protein phosphatase Cdc14 at the beginning of anaphase. Failure to dephosphorylate Ase1 delocalizes midzone proteins and delays the second, slower phase of anaphase B. In contrast, in cells expressing nonphosphorylated Ase1, anaphase spindle extension is faster, and spindles frequently break. Cdc14 also controls the separase-Slk19 complex indirectly via the Aurora B kinase. Thus, Cdc14 regulates spindle midzone assembly and function directly through Ase1 and indirectly via the separase-Slk19 complex.     

Feo, the Drosophila homolog of PRC1, is required for central-spindle formation and cytokinesis

We performed a functional analysis of fascetto (feo), a Drosophila gene that encodes a protein homologous to the Ase1p/PRC1/MAP65 conserved family of microtubule-associated proteins (MAPs). These MAPs are enriched at the spindle midzone in yeast and mammals and at the fragmoplast in plants, and are essential for the organization and function of these microtubule arrays. Here we show that the Feo protein is specifically enriched at the central-spindle midzone and that its depletion either by mutation or by RNAi results in aberrant central spindles. In Feo-depleted cells, late anaphases showed normal overlap of the antiparallel MTs at the cell equator, but telophases displayed thin MT bundles of uniform width instead of robust hourglass-shaped central spindles. These thin central spindles exhibited diffuse localizations of both the Pav and Asp proteins, suggesting that these spindles comprise improperly oriented MTs. Feo-depleted cells also displayed defects in the contractile apparatus that correlated with those in the central spindle; late anaphase cells formed regular contractile structures, but these structures did not constrict during telophase, leading to failures in cytokinesis. The phenotype of Feo-depleted telophases suggests that Feo interacts with the plus ends of central spindle MTs so as to maintain their precise interdigitation during anaphase-telophase MT elongation and antiparallel sliding.     

A TOGL domain specifically targets yeast CLASP to kinetochores to stabilize kinetochore microtubules

Cytoplasmic linker–associated proteins (CLASPs) are proposed to function in cell division based on their ability to bind tubulin via arrayed tumor overexpressed gene (TOG)–like (TOGL) domains. Structure predictions suggest that CLASPs have at least two TOGL domains. We show that only TOGL2 of Saccharomyces cerevisiae CLASP Stu1 binds to tubulin and is required for polymerization of spindle microtubules (MTs) in vivo. In contrast, TOGL1 recruits Stu1 to kinetochores (KTs), where it is essential for the stability and tension-dependent regulation of KT MTs. Stu1 is also recruited to spindle MTs by different mechanisms depending on the mitotic phase: in metaphase, Stu1 binds directly to the MT lattice, whereas in anaphase, it is localized indirectly to the spindle midzone. In both phases, the activity of TOGL2 is essential for interpolar MT stability, whereas TOGL1 is not involved. Thus, the two TOGL domains of yeast CLASP have different activities and execute distinct mitotic functions.     

The molecular function of Ase1p: evidence for a MAP-dependent midzone-specific spindle matrix. Microtubule-associated proteins

The midzone is the domain of the mitotic spindle that maintains spindle bipolarity during anaphase and generates forces required for spindle elongation (anaphase B). Although there is a clear role for microtubule (MT) motor proteins at the spindle midzone, less is known about how microtubule-associated proteins (MAPs) contribute to midzone organization and function. Here, we report that budding yeast Ase1p is a member of a conserved family of midzone-specific MAPs. By size exclusion chromatography and velocity sedimentation, both Ase1p in extracts and purified Ase1p behaved as a homodimer. Ase1p bound and bundled MTs in vitro. By live cell microscopy, loss of Ase1p resulted in a specific defect: premature spindle disassembly in mid-anaphase. Furthermore, when overexpressed, Ase1p was sufficient to trigger spindle elongation in S phase-arrested cells. FRAP revealed that Ase1p has both a very slow rate of turnover within the midzone and limited lateral diffusion along spindle MTs. We propose that Ase1p functions as an MT cross-bridge that imparts matrix-like characteristics to the midzone. MT-dependent networks of spindle midzone MAPs may be one molecular basis for the postulated spindle matrix.     

A computational model predicts Xenopus meiotic spindle organization

The metaphase spindle is a dynamic bipolar structure crucial for proper chromosome segregation, but how microtubules (MTs) are organized within the bipolar architecture remains controversial. To explore MT organization along the pole-to-pole axis, we simulated meiotic spindle assembly in two dimensions using dynamic MTs, a MT cross-linking force, and a kinesin-5-like motor. The bipolar structures that form consist of antiparallel fluxing MTs, but spindle pole formation requires the addition of a NuMA-like minus-end cross-linker and directed transport of MT depolymerization activity toward minus ends. Dynamic instability and minus-end depolymerization generate realistic MT lifetimes and a truncated exponential MT length distribution. Keeping the number of MTs in the simulation constant, we explored the influence of two different MT nucleation pathways on spindle organization. When nucleation occurs throughout the spindle, the simulation quantitatively reproduces features of meiotic spindles assembled in Xenopus egg extracts.     

ISWI is a RanGTP-dependent MAP required for chromosome segregation

Production of RanGTP around chromosomes induces spindle assembly by activating nuclear localization signal (NLS)–containing factors. Here, we show that the NLS protein ISWI, a known chromatin-remodeling ATPase, is a RanGTP-dependent microtubule (MT)-associated protein. Recombinant ISWI induces MT nucleation, stabilization, and bundling in vitro. In Xenopus culture cells and egg extract, ISWI localizes within the nucleus in interphase and on spindles during mitosis. Depletion of ISWI in egg extracts does not affect spindle assembly, but in anaphase spindle MTs disappear and chromosomes do not segregate. We show directly that ISWI is required for the RanGTP-dependent stabilization of MTs during anaphase independently of its effect on chromosomes. ISWI depletion in Drosophila S2 cells induces defects in spindle MTs and chromosome segregation in anaphase, and the cells eventually stop growing. Our results demonstrate that distinctly from its role in spindle assembly, RanGTP maintains spindle MTs in anaphase through the local activation of ISWI and that this is essential for proper chromosome segregation.     

Verteilung der Mikrotubuli in Metaphase- und Anaphase-Spindeln der Spermatocyten von Pales ferruginea

One metaphase I spindle, seven anaphase I spindles of different stages, and one metaphase II spindle were sectioned in series. The ultrastructure of chromosomes was examined and microtubules (MTs) were counted. The main results of the study are summarized as follows: 1. The autosomes move at the periphery of the continuous MTs during anaphase while the sex chromosomes move more or less within this group of MTs. 2. In metaphase the antosomes have few coarse surface projections, in anaphase many, but more delicate projections of irregular shape which seem to transform into regular radial lamellae at the end of movement. 3. In metaphase continuous MTs have no contact with the chromosomal surface, while during anaphase movement continuous MTs lie closer to the chromosomes, and finally arrange themselves between the radial surface lamellae. There they show lateral filamentous connections with the chromosomal surface. 4. The MT distribution profiles of metaphase and anaphase are different. While the highest density of MTs is observed in the middle region of the spindle in metaphase, there are two density zones during autosomal movement, each in one half spindle in front of the autosomes. After the autosomes have reached the poles the distribution profile is again similar to the metaphase condition. The MT distribution in metaphase II is the same as in metaphase I. Possible explanations for these observations are discussed in detail. 5. There is an overall decrease in MT content during anaphase. 6. With the onset of anaphase MTs are seen within the spindle mantle, closely associated with mitochondria. — Several theoretical aspects of anaphase mechanism are briefly discussed.     

A pathway containing the Ipl1/aurora protein kinase and the spindle midzone protein Ase1 regulates yeast spindle assembly

It is critical to elucidate the pathways that mediate spindle assembly and therefore ensure accurate chromosome segregation during cell division. Our studies of a unique allele of the budding yeast Ipl1/Aurora protein kinase revealed that it is required for centrosome-mediated spindle assembly in the absence of the BimC motor protein Cin8. In addition, we found that the Ase1 spindle midzone-associated protein is required for bipolar spindle assembly. The cin8 ipl1 and cin8 ase1 double mutant cells exhibit similar defects, and Ase1 overexpression completely restores spindle assembly in cin8 ipl1 strains. Consistent with the possibility that Ipl1 regulates Ase1, an ase1 mutant lacking the Ipl1 consensus phosphorylation sites cannot assemble spindles in the absence of Cin8. In addition, Ase1 phosphorylation and localization were altered in an ipl1 mutant. We therefore propose that Ipl1/Aurora and Ase1 constitute a previously unidentified spindle assembly pathway that becomes essential in the absence of Cin8.     

The microtubule-based motor Kar3 and plus end-binding protein Bim1 provide structural support for the anaphase spindle

In budding yeast, the mitotic spindle is comprised of 32 kinetochore microtubules (kMTs) and ~8 interpolar MTs (ipMTs). Upon anaphase onset, kMTs shorten to the pole, whereas ipMTs increase in length. Overlapping MTs are responsible for the maintenance of spindle integrity during anaphase. To dissect the requirements for anaphase spindle stability, we introduced a conditionally functional dicentric chromosome into yeast. When centromeres from the same sister chromatid attach to opposite poles, anaphase spindle elongation is delayed and a DNA breakage-fusion-bridge cycle ensues that is dependent on DNA repair proteins. We find that cell survival after dicentric chromosome activation requires the MT-binding proteins Kar3p, Bim1p, and Ase1p. In their absence, anaphase spindles are prone to collapse and buckle in the presence of a dicentric chromosome. Our analysis reveals the importance of Bim1p in maintaining a stable ipMT overlap zone by promoting polymerization of ipMTs during anaphase, whereas Kar3p contributes to spindle stability by cross-linking spindle MTs.     

Microtubule-driven multimerization recruits ase1p onto overlapping microtubules

Microtubule (MT) crosslinking proteins of the ase1p/PRC1/Map65 family play a major role in the construction of MT networks such as the mitotic spindle. Most homologs in this family have been shown to localize with a remarkable specificity to sets of MTs that overlap with an antiparallel relative orientation [1-4]. Regulatory proteins bind to ase1p/PRC1/Map65 and appear to use the localization to set up precise spatial signals [5-10]. Here, we present evidence for a mechanism of localized protein multimerization underlying the specific targeting of ase1p, the fision yeast homolog. In controlled in vitro experiments, dimers of ase1-GFP diffused along the surface of single MTs and, at concentrations above a certain threshold, assembled into static multimeric structures. We observed that this threshold was significantly lower on overlapping MTs. We also observed diffusion and multimerization of ase1-GFP on MTs inside living cells, suggesting that a multimerization-driven localization mechanism is relevant in vivo. The domains responsible for MT binding and multimerization were identified via a series of ase1p truncations. Our findings show that cells use a finely tuned cooperative localization mechanism that exploits differences in the geometry and concentration of ase1p binding sites along single and overlapping MTs.     

Caenorhabditis elegans Aurora A kinase is required for the formation of spindle microtubules in female meiosis

In many animals, female meiotic spindles are assembled in the absence of centrosomes, the major microtubule (MT)-organizing centers. How MTs are formed and organized into meiotic spindles is poorly understood. Here we report that, in Caenorhabditis elegans, Aurora A kinase/AIR-1 is required for the formation of spindle microtubules during female meiosis. When AIR-1 was depleted or its kinase activity was inhibited in C. elegans oocytes, although MTs were formed around chromosomes at germinal vesicle breakdown (GVBD), they were decreased during meiotic prometaphase and failed to form a bipolar spindle, and chromosomes were not separated into two masses. Whereas AIR-1 protein was detected on and around meiotic spindles, its kinase-active form was concentrated on chromosomes at prometaphase and on interchromosomal MTs during late anaphase and telophase. We also found that AIR-1 is involved in the assembly of short, dynamic MTs in the meiotic cytoplasm, and these short MTs were actively incorporated into meiotic spindles. Collectively our results suggest that, after GVBD, the kinase activity of AIR-1 is continuously required for the assembly and/or stabilization of female meiotic spindle MTs.     

Requirement for the budding yeast polo kinase Cdc5 in proper microtubule growth and dynamics

In many organisms, polo kinases appear to play multiple roles during M-phase progression. To provide new insights into the function of the budding yeast polo kinase Cdc5, we generated novel temperature-sensitive cdc5 mutants by mutagenizing the C-terminal noncatalytic polo box domain, a region that is critical for proper subcellular localization. One of these mutants, cdc5-11, exhibited a temperature-sensitive growth defect with an abnormal spindle morphology. Strikingly, provision of a moderate level of benomyl, a microtubule-depolymerizing drug, permitted cdc5-11 cells to grow significantly better than the isogenic CDC5 wild type in a FEAR (cdc Fourteen Early Anaphase Release)-independent manner. In addition, cdc5-11 required MAD2 for both cell growth and the benomyl-remedial phenotype. These results suggest that cdc5-11 is defective in proper spindle function. Consistent with this view, cdc5-11 exhibited abnormal spindle morphology, shorter spindle length, and delayed microtubule regrowth at the nonpermissive temperature. Overexpression of CDC5 moderately rescued the spc98-2 growth defect. Interestingly, both Cdc28 and Cdc5 were required for the proper modification of the spindle pole body components Nud1, Slk19, and Stu2 in vivo. They also phosphorylated these three proteins in vitro. Taken together, these observations suggest that concerted action of Cdc28 and Cdc5 on Nud1, Slk19, and Stu2 is important for proper spindle functions.     

Spc24 and Stu2 promote spindle integrity when DNA replication is stalled

The kinetochore, a protein complex that links chromosomes to microtubules (MTs), is required to prevent spindle expansion during S phase in budding yeast, but the mechanism of how the kinetochore maintains integrity of the bipolar spindle before mitosis is not well understood. Here, we demonstrate that a mutation of Spc24, a component of the conserved Ndc80 kinetochore complex, causes lethality when cells are exposed to the DNA replication inhibitor hydroxyurea (HU) due to premature spindle expansion and segregation of incompletely replicated DNA. Overexpression of Stu1, a CLASP-related MT-associated protein or a truncated form of the XMAP215 orthologue Stu2 rescues spc24-9 HU lethality and prevents spindle expansion. Truncated Stu2 likely acts in a dominant-negative manner, because overexpression of full-length STU2 does not rescue spc24-9 HU lethality, and spindle expansion in spc24-9 HU-treated cells requires active Stu2. Stu1 and Stu2 localize to the kinetochore early in the cell cycle and Stu2 kinetochore localization depends on Spc24. We propose that mislocalization of Stu2 results in premature spindle expansion in S phase stalled spc24-9 mutants. Identifying factors that restrain spindle expansion upon inhibition of DNA replication is likely applicable to the mechanism by which spindle elongation is regulated during a normal cell cycle.     

RanGTP and CLASP1 cooperate to position the mitotic spindle

Accurate positioning of the mitotic spindle is critical to ensure proper distribution of chromosomes during cell division. The small GTPase Ran, which regulates a variety of processes throughout the cell cycle, including interphase nucleocytoplasmic transport and mitotic spindle assembly, was recently shown to also control spindle alignment. Ran is required for the correct cortical localization of LGN and nuclear-mitotic apparatus protein (NuMA), proteins that generate pulling forces on astral microtubules (MTs) through cytoplasmic dynein. Here we use importazole, a small-molecule inhibitor of RanGTP/importin-β function, to study the role of Ran in spindle positioning in human cells. We find that importazole treatment results in defects in astral MT dynamics, as well as in mislocalization of LGN and NuMA, leading to misoriented spindles. Of interest, importazole-induced spindle-centering defects can be rescued by nocodazole treatment, which depolymerizes astral MTs, or by overexpression of CLASP1, which does not restore proper LGN and NuMA localization but stabilizes astral MT interactions with the cortex. Together our data suggest a model for mitotic spindle positioning in which RanGTP and CLASP1 cooperate to align the spindle along the long axis of the dividing cell.     

Three-dimensional structure of the central mitotic spindle of Diatoma vulgare

Central mitotic spindles in Diatoma vulgare have been investigated using serial sections and electron microscopy. Spindles at both early stages (before metaphase) and later stages of mitosis (metaphase to telophase) have been analyzed. We have used computer graphics technology to facilitate the analysis and to produce stereo images of the central spindle reconstructed in three dimensions. We find that at prometaphase, when the nuclear envelope is dissassembling, the spindle is constructed from two sets of polar microtubules (MTs) that interdigitate to form a zone of overlap. As the chromosomes become organized into the metaphase configuration, the polar MTs, the spindle, and the zone of overlap all elongate, while the number of MTs in the central spindle decreases from greater than 700 to approximately 250. Most of the tubules lost are short ones that reside near the spindle poles. The previously described decrease in the length of the zone of overlap during anaphase central spindle elongation is clearly demonstrated in stereo images. In addition, we have used our three- dimensional data to determine the lengths of the spindle MTs at various times during mitotis. The distribution of lengths is bimodal during prometaphase, but the short tubules disappear and the long tubules elongate as mitosis proceeds. The distributions of MT lengths are compared to the length distributions of MTs polymerized in vitro, and a model is presented to account for our findings about both MT length changes and microtubule movements.     

The microtubule-destabilizing kinesin XKCM1 regulates microtubule dynamic instability in cells

The dynamic activities of cellular microtubules (MTs) are tightly regulated by a balance between MT-stabilizing and -destabilizing proteins. Studies in Xenopus egg extracts have shown that the major MT destabilizer during interphase and mitosis is the kinesin-related protein XKCM1, which depolymerizes MT ends in an ATP-dependent manner. Herein, we examine the effects of both overexpression and inhibition of XKCM1 on the regulation of MT dynamics in vertebrate somatic cells. We found that XKCM1 is a MT-destabilizing enzyme in PtK2 cells and that XKCM1 modulates cellular MT dynamics. Our results indicate that perturbation of XKCM1 levels alters the catastrophe frequency and the rescue frequency of cellular MTs. In addition, we found that overexpression of XKCM1 or inhibition of KCM1 during mitosis leads to the formation of aberrant spindles and a mitotic delay. The predominant spindle defects from excess XKCM1 included monoastral and monopolar spindles, as well as small prometaphase-like spindles with improper chromosomal attachments. Inhibition of KCM1 during mitosis led to prometaphase spindles with excessively long MTs and spindles with partially separated poles and a radial MT array. These results show that KCM1 plays a critical role in regulating both interphase and mitotic MT dynamics in mammalian cells.     

Augmin-dependent microtubule nucleation at microtubule walls in the spindle

The formation of a functional spindle requires microtubule (MT) nucleation from within the spindle, which depends on augmin. How augmin contributes to MT formation and organization is not known because augmin-dependent MTs have never been specifically visualized. In this paper, we identify augmin-dependent MTs and their connections to other MTs by electron tomography and 3D modeling. In metaphase spindles of human cells, the minus ends of MTs were located both around the centriole and in the body of the spindle. When augmin was knocked down, the latter population of MTs was significantly reduced. In control cells, we identified connections between the wall of one MT and the minus end of a neighboring MT. Interestingly, the connected MTs were nearly parallel, unlike other examples of end–wall connections between cytoskeletal polymers. Our observations support the concept of augmin-dependent MT nucleation at the walls of existing spindle MTs. Furthermore, they suggest a mechanism for maintaining polarized MT organization, even when noncentrosomal MT initiation is widespread.     

The WD40 Repeat Protein NEDD1 Functions in Microtubule Organization during Cell Division in Arabidopsis thaliana

Although cells of flowering plants lack a structurally defined microtubule-organizing center like the centrosome, organization of the spindles and phragmoplasts in mitosis is known to involve the evolutionarily conserved γ-tubulin complex. We have investigated the function of Arabidopsis thaliana NEDD1, a WD40 repeat protein related to the animal NEDD1/GCP-WD protein, which interacts with the γ-tubulin complex. The NEDD1 protein decorates spindle microtubules (MTs) preferentially toward spindle poles and phragmoplast MTs toward their minus ends. A T-DNA insertional allele of the single NEDD1 gene was isolated and maintained in heterozygous sporophytes, and NEDD1''s function in cell division was analyzed in haploid microspores produced by the heterozygote. In approximately half of the dividing microspores exhibiting aberrant MT organization, spindles were no longer restricted to the cell periphery and became abnormally elongated. After mitosis, MTs aggregated between reforming nuclei but failed to appear in a bipolar configuration. Consequently, defective microspores did not form a continuous cell plate, and two identical nuclei were produced with no differentiation into generative and vegetative cells. Our results support the notion that the plant NEDD1 homolog plays a critical role in MT organization during mitosis, and its function is likely linked to that of the γ-tubulin complex.     

Microcephaly protein Asp focuses the minus ends of spindle microtubules at the pole and within the spindle

Depletion of Drosophila melanogaster Asp, an orthologue of microcephaly protein ASPM, causes spindle pole unfocusing during mitosis. However, it remains unclear how Asp contributes to pole focusing, a process that also requires the kinesin-14 motor Ncd. We show that Asp localizes to the minus ends of spindle microtubule (MT) bundles and focuses them to make the pole independent of Ncd. We identified a critical domain in Asp exhibiting MT cross-linking activity in vitro. Asp was also localized to, and focuses the minus ends of, intraspindle MTs that were nucleated in an augmin-dependent manner and translocated toward the poles by spindle MT flux. Ncd, in contrast, functioned as a global spindle coalescence factor not limited to MT ends. We propose a revised molecular model for spindle pole focusing in which Asp at the minus ends cross-links MTs at the pole and within the spindle. Additionally, this study provides new insight into the dynamics of intraspindle MTs by using Asp as a minus end marker.