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1.
Incomplete mitotic spindle disassembly causes lethality in budding yeast. To determine why spindle disassembly is required for cell viability, we used live-cell microscopy to analyze a double mutant strain containing a conditional mutant and a deletion mutant compromised for the kinesin-8 and anaphase-promoting complex-driven spindle-disassembly pathways (td-kip3 and doc1Δ, respectively). Under nonpermissive conditions, spindles in td-kip3 doc1Δ cells could break apart but could not disassemble completely. These cells could exit mitosis and undergo cell division. However, the daughter cells could not assemble functional, bipolar spindles in the ensuing mitosis. During the formation of these dysfunctional spindles, centrosome duplication and separation, as well as recruitment of key midzone-stabilizing proteins all appeared normal, but microtubule polymerization was nevertheless impaired and these spindles often collapsed. Introduction of free tubulin through episomal expression of α- and β-tubulin or introduction of a brief pulse of the microtubule-depolymerizing drug nocodazole allowed spindle assembly in these td-kip3 doc1Δ mutants. Therefore we propose that spindle disassembly is essential for regeneration of the intracellular pool of assembly-competent tubulin required for efficient spindle assembly during subsequent mitoses of daughter cells.  相似文献   

2.
We describe a new Drosophila gene, mini spindles (msps) identified in a cytological screen for mitotic mutant. Mutation in msps disrupts the structural integrity of the mitotic spindle, resulting in the formation of one or more small additional spindles in diploid cells. Nucleation of microtubules from centrosomes, metaphase alignment of chromosomes, or the focusing of spindle poles appears much less affected. The msps gene encodes a 227-kD protein with high similarity to the vertebrate microtubule-associated proteins (MAPs), human TOGp and Xenopus XMAP215, and with limited similarity to the Dis1 and STU2 proteins from fission yeast and budding yeast. Consistent with their sequence similarity, Msps protein also associates with microtubules in vitro. In the embryonic division cycles, Msps protein localizes to centrosomal regions at all mitotic stages, and spreads over the spindles during metaphase and anaphase. The absence of centrosomal staining in interphase of the cellularized embryos suggests that the interactions between Msps protein and microtubules or centrosomes may be regulated during the cell cycle.  相似文献   

3.
Summary Spindle formation and nuclear division of budding and irradiated yeast cells (Saccharomyces uvarum) was investigated by fluorescence microscopy of protoplasted cells. Protoplasts were treated with antitubulin antibodies and DAPI, a fluorescent dye staining DNA. In budding yeast cells, duplication of spindle pole bodies as well as formation of complete 1-µm spindles and elongated 8-µm spindles were documented. In X-irradiated cells, spindle pole bodies were duplicated as well, forming the complete 1-µm spindle. Nuclei of giant cells have lost the elongation ability and remain in a normal G2-phase state, thus preventing nuclear as well as cellular division.  相似文献   

4.
During anaphase, mitotic spindles elongate up to five times their metaphase length. This process, known as anaphase B, is essential for correct segregation of chromosomes. Here, we examine the control of spindle length during anaphase in the budding yeast Saccharomyces cerevisiae. We show that microtubule stabilization during anaphase requires the microtubule-associated protein Stu2. We further show that the activity of Stu2 is opposed by the activity of the kinesin-related protein Kip3. Reexamination of the kinesin homology tree suggests that KIP3 is the S. cerevisiae orthologue of the microtubule-destabilizing subfamily of kinesins (Kin I). We conclude that a balance of activity between evolutionally conserved microtubule-stabilizing and microtubule-destabilizing factors is essential for correct spindle elongation during anaphase B.  相似文献   

5.
The mitotic spindle is a complex and dynamic structure. Although much has been learned about how spindles assemble and mediate chromosome segregation, how spindles rapidly and irreversibly disassemble during telophase is less clear. We used synthetic lethal screens in budding yeast to identify mutants defective in spindle disassembly. Real-time, live cell imaging analysis of spindle disassembly was performed on nine mutants defective in this process. Results of this analysis suggest that spindle disassembly is achieved by mechanistically distinct but functionally overlapping subprocesses: disengagement of the spindle halves, arrest of spindle elongation, and initiation of interpolar microtubule depolymerization. These subprocesses are largely governed by the anaphase-promoting complex, Aurora B kinase, and kinesin-8. Combinatorial inhibition of these subprocesses yielded cells with hyperstable spindle remnants and dramatic defects in cell cycle progression, establishing that rapid spindle disassembly is crucial for cell proliferation.  相似文献   

6.
In budding yeast, the essential roles of microtubules include segregating chromosomes and positioning the nucleus during mitosis. Defects in these functions can lead to aneuploidy and cell death. To ensure proper mitotic spindle and cytoplasmic microtubule formation, the cell must maintain appropriate stoichiometries of alpha- and beta-tubulin, the basic subunits of microtubules. The experiments described here investigate the minimal levels of tubulin heterodimers needed for mitotic function. We have found a triple-mutant strain, pac10Delta plp1Delta yap4Delta, which has only 20% of wild-type tubulin heterodimer levels due to synthesis and folding defects. The anaphase spindles in these cells are approximately 64% the length of wild-type spindles. The mutant cells are viable and accurately segregate chromosomes in mitosis, but they do have specific defects in mitosis such as abnormal nuclear positioning. The results establish that cells with 20% of wild-type levels of tubulin heterodimers can perform essential cellular functions with a short spindle, but require higher tubulin heterodimer concentrations to attain normal spindle length and prevent mitotic defects.  相似文献   

7.
Cells coordinate spindle formation with DNA repair and morphological modifications to chromosomes prior to their segregation to prevent cell division with damaged chromosomes. Here we uncover a novel and unexpected role for Aurora kinase in preventing the formation of spindles by Clb5-CDK (S-CDK) during meiotic prophase I and when the DDR is active in budding yeast. This is critical since S-CDK is essential for replication during premeiotic S-phase as well as double-strand break induction that facilitates meiotic recombination and, ultimately, chromosome segregation. Furthermore, we find that depletion of Cdc5 polo kinase activity delays spindle formation in DDR-arrested cells and that ectopic expression of Cdc5 in prophase I enhances spindle formation, when Ipl1 is depleted. Our findings establish a new paradigm for Aurora kinase function in both negative and positive regulation of spindle dynamics.  相似文献   

8.
Meiotic chromosome segregation leads to the production of haploid germ cells. During meiosis I (MI), the paired homologous chromosomes are separated. Meiosis II (MII) segregation leads to the separation of paired sister chromatids. In the budding yeast Saccharomyces cerevisiae, both of these divisions take place in a single nucleus, giving rise to the four-spored ascus. We have modeled the microtubules in 20 MI and 15 MII spindles by using reconstruction from electron micrographs of serially sectioned meiotic cells. Meiotic spindles contain more microtubules than their mitotic counterparts, with the highest number in MI spindles. It is possible to differentiate between MI versus MII spindles based on microtubule numbers and organization. Similar to mitotic spindles, kinetochores in either MI or MII are attached by a single microtubule. The models indicate that the kinetochores of paired homologous chromosomes in MI or sister chromatids in MII are separated at metaphase, similar to mitotic cells. Examination of both MI and MII spindles reveals that anaphase A likely occurs in addition to anaphase B and that these movements are concurrent. This analysis offers a structural basis for considering meiotic segregation in yeast and for the analysis of mutants defective in this process.  相似文献   

9.
Centromere position in budding yeast: evidence for anaphase A.   总被引:8,自引:2,他引:6       下载免费PDF全文
Although general features of chromosome movement during the cell cycle are conserved among all eukaryotic cells, particular aspects vary between organisms. Understanding the basis for these variations should provide significant insight into the mechanism of chromosome movement. In this context, establishing the types of chromosome movement in the budding yeast Saccharomyces cerevisiae is important since the complexes that mediate chromosome movement (microtubule organizing centers, spindles, and kinetochores) appear much simpler in this organism than in many other eukaryotic cells. We have used fluorescence in situ hybridization to begin an analysis of chromosome movement in budding yeast. Our results demonstrate that the position of yeast centromeres changes as a function of the cell cycle in a manner similar to other eukaryotes. Centromeres are skewed to the side of the nucleus containing the spindle pole in G1; away from the poles in mid-M and clustered near the poles in anaphase and telophase. The change in position of the centromeres relative to the spindle poles supports the existence of anaphase A in budding yeast. In addition, an anaphase A-like activity independent of anaphase B was demonstrated by following the change in centromere position in telophase-arrested cells upon depolymerization and subsequent repolymerization of microtubules. The roles of anaphase A activity and G1 centromere positioning in the segregation of budding yeast chromosomes are discussed. The fluorescence in situ hybridization methodology and experimental strategies described in this study provide powerful new tools to analyze mutants defective in specific kinesin-like molecules, spindle components, and centromere factors, thereby elucidating the mechanism of chromosome movement.  相似文献   

10.
At the metaphase to anaphase transition, chromosome segregation is initiated by the splitting of sister chromatids. Subsequently, spindles elongate, separating the sister chromosomes into two sets. Here, we investigate the cell cycle requirements for spindle elongation in budding yeast using mutants affecting sister chromatid cohesion or DNA replication. We show that separation of sister chromatids is not sufficient for proper spindle integrity during elongation. Rather, successful spindle elongation and stability require both sister chromatid separation and anaphase-promoting complex activation. Spindle integrity during elongation is dependent on proteolysis of the securin Pds1 but not on the activity of the separase Esp1. Our data suggest that stabilization of the elongating spindle at the metaphase to anaphase transition involves Pds1-dependent targets other than Esp1.  相似文献   

11.
Mitotic cell division involves the equal segregation of all chromosomes during anaphase. The presence of ribosomal DNA (rDNA) repeats on the right arm of chromosome XII makes it the longest in the budding yeast genome. Previously, we identified a stage during yeast anaphase when rDNA is stretched across the mother and daughter cells. Here, we show that resolution of sister rDNAs is achieved by unzipping of the locus from its centromere-proximal to centromere-distal regions. We then demonstrate that during this stretched stage sister rDNA arrays are neither compacted nor segregated despite being largely resolved from each other. Surprisingly, we find that rDNA segregation after this period no longer requires spindles but instead involves Cdc14-dependent rDNA axial compaction. These results demonstrate that chromosome resolution is not simply a consequence of compacting chromosome arms and that overall rDNA compaction is necessary to mediate the segregation of the long arm of chromosome XII.  相似文献   

12.
Mitotic spindles were isolated from a cell division cycle mutant of the budding yeast Saccharomyces cerevisiae by the lysis of sphateroplasts on an air:buffer interface and were negatively stained with 1% gold thioglucose. Isolated spindles were incubated under conditions which promoted the sliding disintegration of parallel preparations of Tetrahymena axonemes, namely the addition of ATP to 20 microM. In no experiment was a corresponding change in microtubule organization of the spindle observed even when spindles were first pretreated with either 1-10 microgram/ml trypsin or 0.2-2% Triton X-100. During these experiments a number of spindles were isolated from cells that had passed through the imposed temperature block, and from the images obtained a detailed model of spindle formation and elongation has been constructed. Two sets of microtubules, one from each spindle pole body (SPB), completely interdigitate to form a continuous bundle, and a series of discontinuous microtubules are then nucleated by each SPB. As the spindle elongates, the number of microtubules continuous between the two SPBs decreases until, at a length of 4 micrometer, only one remains. The spindle, composed of only one microtubule, continues to elongate until it reaches the maximal nuclear dimension of 8 micrometer. The data obtained from negatively stained preparations have been verified in thin sections of wild-type cells. We suggest that, as in the later stages of mitosis only one microtubule is involved in the separation of the spindle poles, the microtubular spindle in S. cerevisiae is not a force-generating system but rather acts as a regulatory mechanism controlling the rate of separation.  相似文献   

13.
The distribution of actin and tubulin during the cell cycle of the budding yeast Saccharomyces was mapped by immunofluorescence using fixed cells from which the walls had been removed by digestion. The intranuclear mitotic spindle was shown clearly by staining with a monoclonal antitubulin; the presence of extensive bundles of cytoplasmic microtubules is reported. In cells containing short spindles still entirely within the mother cells, one of the bundles of cytoplasmic microtubules nearly always extended to (or into) the bud. Two independent reagents (anti-yeast actin and fluorescent phalloidin) revealed an unusual distribution of actin: it was present as a set of cortical dots or patches and also as distinct fibers that were presumably bundles of actin filaments. Double labeling showed that at no stage in the cell cycle do the distributions of actin and tubulin coincide for any significant length, and, in particular, that the mitotic spindle did not stain detectably for actin. However, both microtubule and actin staining patterns change in a characteristic way during the cell cycle. In particular, the actin dots clustered in rings about the bases of very small buds and at the sites on unbudded cells at which bud emergence was apparently imminent. Later in the budding cycle, the actin dots were present largely in the buds and, in many strains, primarily at the tips of these buds. At about the time of cytokinesis the actin dots clustered in the neck region between the separating cells. These aspects of actin distribution suggest that it may have a role in the localized deposition of new cell wall material.  相似文献   

14.
Posttranslational modification by the ubiquitin-like protein SUMO (small ubiquitin-like modifier) is emerging as an important regulator in many cellular processes, including genome integrity. In this study, we show that the kinetochore proteins Ndc10, Bir1, Ndc80, and Cep3, which mediate the attachment of chromosomes to spindle microtubules, are sumoylated substrates in budding yeast. Furthermore, we show that Ndc10, Bir1, and Cep3 but not Ndc80 are desumoylated upon exposure to nocodazole, highlighting the possibility of distinct roles for sumoylation in modulating kinetochore protein function and of a potential link between the sumoylation of kinetochore proteins and mitotic checkpoint function. We find that lysine to arginine mutations that eliminate the sumoylation of Ndc10 cause chromosome instability, mislocalization of Ndc10 from the mitotic spindle, abnormal anaphase spindles, and a loss of Bir1 sumoylation. These data suggest that sumoylation of Ndc10 and other kinetochore proteins play a critical role during the mitotic process.  相似文献   

15.
Although budding yeast cell biology and genetics provided a powerful system to isolate S-phase checkpoint mutants, initial studies relied on a defect not likely to be relevant in higher eukaryotes. The first mutants were isolated for their inability to restrain mitotic spindle elongation in S-phase. Since most eukaryotes do not assemble spindles until prometaphase the validity of this approach might have been questioned. However, these early studies were designed with a highly valid assumption in mind; that checkpoints have a variety of targets, but comprise conserved kinase cascades that make up these signaling pathways. The task that lies ahead is to determine targets of the S-phase checkpoint relevant to mammals. One step forward might be the realization that the budding yeast S-phase checkpoint prevents loss of sister chromatid cohesion while DNA replication is ongoing. If this mechanism is conserved in mammals, it could prove vital for chromosome segregation fidelity.  相似文献   

16.
A quantitative analysis of spindles and spindle-related EEG activity was performed in C57BL/6 mice. The hypothesis that spindles are involved in sleep regulatory mechanisms was tested by investigating their occurrence during 24 h and after 6 h sleep deprivation (SD; n = 7). In the frontal derivation distinct spindle events were characterized as EEG oscillations with a dominant frequency approximately at 11 Hz. Spindles were most prominent during NREM sleep and increased before NREM-REM sleep transitions. Whereas spindles increased concomitantly with slow wave activity (SWA, EEG power between 0.5 and 4.0 Hz) at the beginning of the NREM sleep episode, these measures showed an opposite evolution prior to the transition to REM sleep. The 24-h time course of spindles showed a maximum at the end of the 12-h light period, and was a mirror image of SWA in NREM sleep. After 6 h SD the spindles in NREM sleep were initially suppressed, and showed a delayed rebound. In contrast, spindles occurring immediately before the transition to REM sleep were enhanced during the first 2 h of recovery. The data suggest that spindles in NREM sleep may be involved in sleep maintenance, while spindles heralding the transition to REM sleep may be related to mechanisms of REM sleep initiation.  相似文献   

17.
The kinetochore is a protein complex that assembles on centromeric DNA to mediate chromosome–microtubule interaction. Most eukaryotic cells form the spindle and establish kinetochore–microtubule interaction during mitosis, but budding yeast cells finish these processes in S-phase. It has long been noticed that the S-phase spindle in budding yeast is shorter than that in metaphase, but the biological significance of this short S-phase spindle structure remains unclear. We addressed this issue by using ask1-3, a temperature-sensitive kinetochore mutant that exhibits partially elongated spindles at permissive temperature in the presence of hydroxyurea (HU), a DNA synthesis inhibitor. After exposure to and removal of HU, ask1-3 cells show a delayed anaphase entry. This delay depends on the spindle checkpoint, which monitors kinetochore–microtubule interaction defects. Overproduction of microtubule-associated protein Ase1 or Cin8 also induces spindle elongation in HU-arrested cells. The spindle checkpoint-dependent anaphase entry delay is also observed after ASE1 or CIN8 overexpression in HU-arrested cells. Therefore, the shorter spindle in S-phase cells is likely to facilitate proper chromosome–microtubule interaction.  相似文献   

18.
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.  相似文献   

19.
Wang Y  Hu F  Elledge SJ 《Current biology : CB》2000,10(21):1379-1382
At the end of the cell cycle, cyclin-dependent kinase (CDK) activity is inactivated to allow mitotic exit [1]. A protein phosphatase, Cdc14, plays a key role during mitotic exit in budding yeast by activating the Cdh1 component of the anaphase-promoting complex to degrade cyclin B (Clb) and inducing the CDK inhibitor Sic1 to inactivate Cdk1 [2]. To prevent mitotic exit when the cell cycle is arrested at G2/M, cells must prevent CDK inactivation. In the spindle checkpoint pathway, this is accomplished through Bfa1/Bub2, a heteromeric GTPase-activating protein (GAP) that inhibits Clb degradation by keeping the G protein Tem1 inactive [3-5]. Tem1 is required for Cdc14 activation. Here we show that in budding yeast, BUB2 and BFA1 are also required for the maintenance of G2/M arrest in response to DNA damage and to spindle misorientation. cdc13-1 bub2 and cdc13-1 bfa1 but not cdc13-1 mad2 double mutants rebud and reduplicate their DNA at the restrictive temperature. We also found that the delay in mitotic exit in mutants with misoriented spindles depended on BUB2 and BFA1, but not on MAD2. We propose that Bfa1/Bub2 checkpoint pathway functions as a universal checkpoint in G2/M that prevents CDK inactivation in response to cell-cycle delay in G2/M.  相似文献   

20.
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.  相似文献   

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