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The mitotic spindle of the budding yeast Saccharomyces cerevisiae will probably be the first such organelle to be understood in molecular detail. Here we describe the mitotic spindle cycle of budding yeast using electron-microscope-derived structures and dynamic live-cell imaging. Recent work has revealed that many general aspects of mitosis are conserved, making budding yeast an excellent model for the study of mitosis. 相似文献
3.
Mihoko Tame Jonne Raaijmakers Bram van den Broek Arne Lindqvist Kees Jalink 《Cell cycle (Georgetown, Tex.)》2014,13(7):1162-1170
Cytoplasmic dynein is recruited to the cell cortex in early mitosis, where it can generate pulling forces on astral microtubules to position the mitotic spindle. Recent work has shown that dynein displays a dynamic asymmetric cortical localization, and that dynein recruitment is negatively regulated by spindle pole-proximity. This results in oscillating dynein recruitment to opposite sides of the cortex to center the mitotic spindle. However, although the centrosome-derived signal that promotes displacement of dynein has been identified, it is currently unknown how dynein is re-recruited to the cortex once it has been displaced. Here we show that re-recruitment of cortical dynein requires astral microtubules. We find that microtubules are necessary for the sustained localized enrichment of dynein at the cortex. Furthermore, we show that stabilization of astral microtubules causes spindle misorientation, followed by mispositioning of dynein at the cortex. Thus, our results demonstrate the importance of astral microtubules in the dynamic regulation of cortical dynein recruitment in mitosis. 相似文献
4.
gamma-Tubulin is essential for microtubule nucleation in yeast and other organisms; whether this protein is regulated in vivo has not been explored. We show that the budding yeast gamma-tubulin (Tub4p) is phosphorylated in vivo. Hyperphosphorylated Tub4p isoforms are restricted to G1. A conserved tyrosine near the carboxy terminus (Tyr445) is required for phosphorylation in vivo. A point mutation, Tyr445 to Asp, causes cells to arrest prior to anaphase. The frequency of new microtubules appearing in the SPB region and the number of microtubules are increased in tub4-Y445D cells, suggesting this mutation promotes microtubule assembly. These data suggest that modification of gamma-tubulin is important for controlling microtubule number, thereby influencing microtubule organization and function during the yeast cell cycle. 相似文献
5.
In the February 21 issue of Cell, demonstrate that asymmetrical loading of Kar9 onto astral microtubules (MTs) emanating from the bud-ward-directed spindle pole ensures delivery of this spindle pole to the bud. Kar9 mediates alignment of the spindle with the cell polarity axis through a Myo2-dependent mechanism that reorients astral MTs toward the bud. 相似文献
6.
A mechanism for nuclear positioning in fission yeast based on microtubule pushing 总被引:1,自引:0,他引:1
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The correct positioning of the nucleus is often important in defining the spatial organization of the cell, for example, in determining the cell division plane. In interphase Schizosaccharomyces pombe cells, the nucleus is positioned in the middle of the cylindrical cell in an active microtubule (MT)-dependent process. Here, we used green fluorescent protein markers to examine the dynamics of MTs, spindle pole body, and the nuclear envelope in living cells. We find that interphase MTs are organized in three to four antiparallel MT bundles arranged along the long axis of the cell, with MT plus ends facing both the cell tips and minus ends near the middle of the cell. The MT bundles are organized from medial MT-organizing centers that may function as nuclear attachment sites. When MTs grow to the cell tips, they exert transient forces produced by plus end MT polymerization that push the nucleus. After an average of 1.5 min of growth at the cell tip, MT plus ends exhibit catastrophe and shrink back to the nuclear region before growing back to the cell tip. Computer modeling suggests that a balance of these pushing MT forces can provide a mechanism to position the nucleus at the middle of the cell. 相似文献
7.
Aberrantly segregating centromeres activate the spindle assembly checkpoint in budding yeast 总被引:16,自引:5,他引:11
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《The Journal of cell biology》1996,133(1):75-84
The spindle assembly checkpoint is the mechanism or set of mechanisms that prevents cells with defects in chromosome alignment or spindle assembly from passing through mitosis. We have investigated the effects of mini-chromosomes on this checkpoint in budding yeast by performing pedigree analysis. This method allowed us to observe the frequency and duration of cell cycle delays in individual cells. Short, centromeric linear mini-chromosomes, which have a low fidelity of segregation, cause frequent delays in mitosis. Their circular counterparts and longer linear mini-chromosomes, which segregate more efficiently, show a much lower frequency of mitotic delays, but these delays occur much more frequently in divisions where the mini-chromosome segregates to only one of the two daughter cells. Using a conditional centromere to increase the copy number of a circular mini-chromosome greatly increases the frequency of delayed divisions. In all cases the division delays are completely abolished by the mad mutants that inactivate the spindle assembly checkpoint, demonstrating that the Mad gene products are required to detect the subtle defects in chromosome behavior that have been observed to arrest higher eukaryotic cells in mitosis. 相似文献
8.
M Knop G Pereira E Schiebel 《Biology of the cell / under the auspices of the European Cell Biology Organization》1999,91(4-5):291-304
In budding yeast microtubule organizing functions are provided by the spindle pole body (SPB), a multi-layered structure that is embedded in the nuclear envelope throughout the cell cycle. The SPB organizes the nuclear and cytoplasmic microtubules which are spatially and functionally distinct. Microtubule formation in yeast requires the Tub4p-complex, containing the gamma-tubulin Tub4p, and two additional proteins, the SPB components Spc97p and Spc98p. The Tub4p complex assembles in the cytoplasm and is then anchored to the sides of the SPB which organize microtubules. This is achieved by the binding of Spc97p and Spc98p to so-called gamma-tubulin complex binding proteins (GTBPs) at the SPB. Spc72p is the yeast GTBP at the cytoplasmic side of the SPB, while Spc110p is the nuclear GTBP. Both GTBPs control the number of Tub4p complexes associated with the SPB and thereby the number of microtubules formed. In addition, the GTBPs may regulate the activity of the Tub4p complex. Homologues of Spc97p and Spc98p have been identified from yeast to mammalian cells and these are also part of gamma-tubulin complexes, suggesting that these related proteins may also interact with GTBPs at the centrosome. Candidates for GTBPs have been identified in mammalian and insect cells. 相似文献
9.
Sprague BL Pearson CG Maddox PS Bloom KS Salmon ED Odde DJ 《Biophysical journal》2003,84(6):3529-3546
It has been hypothesized that spatial gradients in kMT dynamic instability facilitate mitotic spindle formation and chromosome movement. To test this hypothesis requires the analysis of kMT dynamics, which have not been resolved at the single kMT level in living cells. The budding yeast spindle offers an attractive system in which to study kMT dynamics because, in contrast to animal cells, there is only one kMT per kinetochore. To visualize metaphase kMT plus-end dynamics in yeast, a strain containing a green fluorescent protein fusion to the kinetochore protein, Cse4, was imaged by fluorescence microscopy. Although individual kinetochores were not resolvable, we found that models of kMT dynamics could be evaluated by simulating the stochastic kMT dynamics and then simulating the fluorescence imaging of kMT plus-end-associated kinetochores. Statistical comparison of model-predicted images to experimentally observed images demonstrated that a pure dynamic instability model for kMT dynamics in the yeast metaphase spindle was unacceptable. However, when a temporally stable spatial gradient in the catastrophe or rescue frequency was added to the model, there was reasonable agreement between the model and the experiment. These results provide the first evidence of temporally stable spatial gradients of kMT catastrophe and/or rescue frequency in living cells. 相似文献
10.
Abnormal kinetochore structure activates the spindle assembly checkpoint in budding yeast. 总被引:12,自引:1,他引:11
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Saccharomyces cerevisiae cells containing one or more abnormal kinetochores delay anaphase entry. The delay can be produced by using centromere DNA mutations present in single-copy or kinetochore protein mutations. This observation is strikingly similar to the preanaphase delay or arrest exhibited in animal cells that experience spontaneous or induced failures in bipolar attachment of one or more chromosomes and may reveal the existence of a conserved surveillance pathway that monitors the state of chromosome attachment to the spindle before anaphase. We find that three genes (MAD2, BUB1, and BUB2) that are required for the spindle assembly checkpoint in budding yeast (defined by antimicrotubule drug-induced arrest or delay) are also required in the establishment and/or maintenance of kinetochore-induced delays. This was tested in strains in which the delays were generated by limited function of a mutant kinetochore protein (ctf13-30) or by the presence of a single-copy centromere DNA mutation (CDEII delta 31). Whereas the MAD2 and BUB1 genes were absolutely required for delay, loss of BUB2 function resulted in a partial delay defect, and we suggest that BUB2 is required for delay maintenance. The inability of mad2-1 and bub1 delta mutants to execute kinetochore-induced delay is correlated with striking increases in chromosome missegregation, indicating that the delay does indeed have a role in chromosome transmission fidelity. Our results also indicated that the yeast RAD9 gene, necessary for DNA damage-induced arrest, had no role in the kinetochore-induced delays. We conclude that abnormal kinetochore structures induce preanaphase delay by activating the same functions that have defined the spindle assembly checkpoint in budding yeast. 相似文献
11.
The spindle checkpoint delays the onset of anaphase until all pairs of sister chromatids are attached to the mitotic spindle. The checkpoint could monitor the attachment of microtubules to kinetochores, the tension that results from the two sister chromatids attaching to opposite spindle poles, or both. We tested the role of tension by allowing cells to enter mitosis without a prior round of DNA replication. The unreplicated chromatids are attached to spindle microtubules but are not under tension since they lack a sister chromatid that could attach to the opposite pole. Because the spindle checkpoint is activated in these cells, we conclude that the absence of tension at the yeast kinetochore is sufficient to activate the spindle checkpoint in mitosis. 相似文献
12.
The spindle checkpoint arrests cells in mitosis in response to defects in the assembly of the mitotic spindle or errors in chromosome alignment. We determined which spindle defects the checkpoint can detect by examining the interaction of mutations that compromise the checkpoint (mad1, mad2, and mad3) with those that damage various structural components of the spindle. Defects in microtubule polymerization, spindle pole body duplication, microtubule motors, and kinetochore components all activate the MAD-dependent checkpoint. In contrast, the cell cycle arrest caused by mutations that induce DNA damage (cdc13), inactivate the cyclin proteolysis machinery (cdc16 and cdc23), or arrest cells in anaphase (cdc15) is independent of the spindle checkpoint. 相似文献
13.
Park CJ Park JE Karpova TS Soung NK Yu LR Song S Lee KH Xia X Kang E Dabanoglu I Oh DY Zhang JY Kang YH Wincovitch S Huffaker TC Veenstra TD McNally JG Lee KS 《Eukaryotic cell》2008,7(3):444-453
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. 相似文献
14.
Stephen L. Rogers 《The EMBO journal》2014,33(2):96-98
Proteins residing at the plus and minus ends of microtubules have been thought not to communicate with each other, but recent findings on bona fide nucleation factors also regulating microtubule dynamics have challenged this notion. New work by Bouissou et al ( 2014 ) in The EMBO Journal now reveals that interplay between the nucleation factor γ‐TuRC and the plus‐end tracking protein EB1 controls mitotic spindle positioning by affecting the stability and dynamics of astral microtubules. 相似文献
15.
Microtubule assembly in Saccharomyces cerevisiae is initiated from sites within spindle pole bodies (SPBs) in the nuclear envelope. Microtubule plus ends are thought to be organized distal to the SPBs, while minus ends are proximal. Several hypotheses for the function of microtubule motor proteins in force generation and regulation of microtubule assembly propose that assembly and disassembly occur at minus ends as well as at plus ends. Here we analyse microtubule assembly relative to the SPBs in haploid yeast cells expressing green fluorescent protein fused to alpha-tubulin, a microtubule subunit. Throughout the cell cycle, analysis of fluorescent speckle marks on cytoplasmic astral microtubules reveals that there is no detectable assembly or disassembly at minus ends. After laser-photobleaching, metaphase spindles recover about 63% of the bleached fluorescence, with a half-life of about 1 minute. After anaphase onset, photobleached marks in the interpolar spindle are persistent and do not move relative to the SPBs. In late anaphase, the elongated spindles disassemble at the microtubule plus ends. These results show for astral and anaphase interpolar spindle microtubules, and possibly for metaphase spindle microtubules, that microtubule assembly and disassembly occur at plus, and not minus, ends. 相似文献
16.
ABSTRACT : Mitotic exit and cytokinesis must be tightly coupled to nuclear division both in time and space in order to preserve genome stability and to ensure that daughter cells inherit the right set of chromosomes after cell division. This is achieved in budding yeast through control over a signal transduction cascade, the mitotic exit network (MEN), which is required for mitotic CDK inactivation in telophase and for cytokinesis. Current models of MEN activation emphasize on the bud as the place where most control is exerted. This review focuses on recent data that instead point to the mother cell as being the residence of key regulators of late mitotic events. 相似文献
17.
Centrosome duplication must be tightly controlled so that duplication occurs only once each cell cycle. Accumulation of multiple centrosomes can result in the assembly of a multipolar spindle and lead to chromosome mis-segregation and genomic instability. In metazoans, a centrosome-intrinsic mechanism prevents reduplication until centriole disengagement. Mitotic cyclin/cyclin-dependent kinases (CDKs) prevent reduplication of the budding yeast centrosome, called a spindle pole body (SPB), in late S-phase and G2/M, but the mechanism remains unclear. How SPB reduplication is prevented early in the cell cycle is also not understood. Here we show that, similar to metazoans, an SPB-intrinsic mechanism prevents reduplication early in the cell cycle. We also show that mitotic cyclins can inhibit SPB duplication when expressed before satellite assembly in early G1, but not later in G1, after the satellite had assembled. Moreover, electron microscopy revealed that SPBs do not assemble a satellite in cells expressing Clb2 in early G1. Finally, we demonstrate that Clb2 must localize to the cytoplasm in order to inhibit SPB duplication, suggesting the possibility for direct CDK inhibition of satellite components. These two mechanisms, intrinsic and extrinsic control by CDK, evoke two-step system that prevents SPB reduplication throughout the cell cycle. 相似文献
18.
Coordinated spindle assembly and orientation requires Clb5p-dependent kinase in budding yeast
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Segal M Clarke DJ Maddox P Salmon ED Bloom K Reed SI 《The Journal of cell biology》2000,148(3):441-452
The orientation of the mitotic spindle along a polarity axis is critical in asymmetric cell divisions. In the budding yeast, Saccharomyces cerevisiae, loss of the S-phase B-type cyclin Clb5p under conditions of limited cyclin-dependent kinase activity (cdc28-4 clb5Delta cells) causes a spindle positioning defect that results in an undivided nucleus entering the bud. Based on time-lapse digital imaging microscopy of microtubules labeled with green fluorescent protein fusions to either tubulin or dynein, we observed that the asymmetric behavior of the spindle pole bodies during spindle assembly was lost in the cdc28-4 clb5Delta cells. As soon as a spindle formed, both poles were equally likely to interact with the bud cell cortex. Persistent dynamic interactions with the bud ultimately led to spindle translocation across the bud neck. Thus, the mutant failed to assign one spindle pole body the task of organizing astral microtubules towards the mother cell. Our data suggest that Clb5p-associated kinase is required to confer mother-bound behavior to one pole in order to establish correct spindle polarity. In contrast, B-type cyclins, Clb3p and Clb4p, though partially redundant with Clb5p for an early role in spindle morphogenesis, preferentially promote spindle assembly. 相似文献
19.
Emmanuel T. Nsamba Abesh Bera Michael Costanzo Charles Boone Mohan L. Gupta Jr. 《The Journal of cell biology》2021,220(12)
Microtubules are dynamic cytoskeleton filaments that are essential for a wide range of cellular processes. They are polymerized from tubulin, a heterodimer of α- and β-subunits. Most eukaryotic organisms express multiple isotypes of α- and β-tubulin, yet their functional relevance in any organism remains largely obscure. The two α-tubulin isotypes in budding yeast, Tub1 and Tub3, are proposed to be functionally interchangeable, yet their individual functions have not been rigorously interrogated. Here, we develop otherwise isogenic yeast strains expressing single tubulin isotypes at levels comparable to total tubulin in WT cells. Using genome-wide screening, we uncover unique interactions between the isotypes and the two major mitotic spindle positioning mechanisms. We further exploit these cells to demonstrate that Tub1 and Tub3 optimize spindle positioning by differentially recruiting key components of the Dyn1- and Kar9-dependent mechanisms, respectively. Our results provide novel mechanistic insights into how tubulin isotypes allow highly conserved microtubules to function in diverse cellular processes. 相似文献
20.
CENP-A exceeds microtubule attachment sites in centromere clusters of both budding and fission yeast
The stoichiometries of kinetochores and their constituent proteins in yeast and vertebrate cells were determined using the histone H3 variant CENP-A, known as Cse4 in budding yeast, as a counting standard. One Cse4-containing nucleosome exists in the centromere (CEN) of each chromosome, so it has been assumed that each anaphase CEN/kinetochore cluster contains 32 Cse4 molecules. We report that anaphase CEN clusters instead contained approximately fourfold more Cse4 in Saccharomyces cerevisiae and ~40-fold more CENP-A (Cnp1) in Schizosaccharomyces pombe than predicted. These results suggest that the number of CENP-A molecules exceeds the number of kinetochore-microtubule (MT) attachment sites on each chromosome and that CENP-A is not the sole determinant of kinetochore assembly sites in either yeast. In addition, we show that fission yeast has enough Dam1-DASH complex for ring formation around attached MTs. The results of this study suggest the need for significant revision of existing CEN/kinetochore architectural models. 相似文献