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1
Raspelli, Erica, Corinne Cassani, Elena Chiroli et Roberta Fraschini. « Budding Yeast Swe1 Is Involved in the Control of Mitotic Spindle Elongation and Is Regulated by Cdc14 Phosphatase during Mitosis ». Journal of Biological Chemistry 290, no 1 (18 novembre 2014) : 1–12. http://dx.doi.org/10.1074/jbc.m114.590984.
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Raspelli, Erica, Corinne Cassani, Elena Chiroli et Roberta Fraschini. « Budding yeast Swe1 is involved in the control of mitotic spindle elongation and is regulated by Cdc14 phosphatase during mitosis. » Journal of Biological Chemistry 290, no 10 (5 mars 2015) : 6006. http://dx.doi.org/10.1074/jbc.a114.590984.
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Pearson, Chad G., Paul S. Maddox, E. D. Salmon et Kerry Bloom. « Budding Yeast Chromosome Structure and Dynamics during Mitosis ». Journal of Cell Biology 152, no 6 (19 mars 2001) : 1255–66. http://dx.doi.org/10.1083/jcb.152.6.1255.
Texte intégralRésumé :
Using green fluorescent protein probes and rapid acquisition of high-resolution fluorescence images, sister centromeres in budding yeast are found to be separated and oscillate between spindle poles before anaphase B spindle elongation. The rates of movement during these oscillations are similar to those of microtubule plus end dynamics. The degree of preanaphase separation varies widely, with infrequent centromere reassociations observed before anaphase. Centromeres are in a metaphase-like conformation, whereas chromosome arms are neither aligned nor separated before anaphase. Upon spindle elongation, centromere to pole movement (anaphase A) was synchronous for all centromeres and occurred coincident with or immediately after spindle pole separation (anaphase B). Chromatin proximal to the centromere is stretched poleward before and during anaphase onset. The stretched chromatin was observed to segregate to the spindle pole bodies at rates greater than centromere to pole movement, indicative of rapid elastic recoil between the chromosome arm and the centromere. These results indicate that the elastic properties of DNA play an as of yet undiscovered role in the poleward movement of chromosome arms.
4
Tanaka, K., et T. Kanbe. « Mitosis in the fission yeast Schizosaccharomyces pombe as revealed by freeze-substitution electron microscopy ». Journal of Cell Science 80, no 1 (1 février 1986) : 253–68. http://dx.doi.org/10.1242/jcs.80.1.253.
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Nuclear division in Schizosaccharomyces pombe has been studied in transmission electron micrographs of sections of cells fixed by a method of freeze-substitution. We have found cytoplasmic microtubules in the vicinity of the spindle pole bodies and two kinds of microtubules, short discontinuous ones and long, parallel ones in the intranuclear mitotic spindle. For most of the time taken by nuclear division the spindle pole bodies face each other squarely across the nuclear space but early in mitosis they briefly appear twisted out of alignment with each other, thereby imparting a sigmoidal shape to the bundle of spindle microtubules extending between them. This configuration is interpreted as indicating active participation of the spindle in the initial elongation of the dividing nucleus. It is proposed that mitosis is accompanied by the shortening of chromosomal microtubules simultaneously with the elongation of the central pole-to-pole bundle of microtubules of the intranuclear spindle. Daughter nuclei are separated by the sliding apart of interdigitating microtubules of the spindle at telophase. Some of the latter bear dense knobs at their ends.
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Cid, Victor J., Mark J. Shulewitz, Kent L. McDonald et Jeremy Thorner. « Dynamic Localization of the Swe1 Regulator Hsl7 During theSaccharomyces cerevisiae Cell Cycle ». Molecular Biology of the Cell 12, no 6 (juin 2001) : 1645–69. http://dx.doi.org/10.1091/mbc.12.6.1645.
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In Saccharomyces cerevisiae, entry into mitosis requires activation of the cyclin-dependent kinase Cdc28 in its cyclin B (Clb)-associated form. Clb-bound Cdc28 is susceptible to inhibitory tyrosine phosphorylation by Swe1 protein kinase. Swe1 is itself negatively regulated by Hsl1, a Nim1-related protein kinase, and by Hsl7, a presumptive protein-arginine methyltransferase. In vivo all three proteins localize to the bud neck in a septin-dependent manner, consistent with our previous proposal that formation of Hsl1-Hsl7-Swe1 complexes constitutes a checkpoint that monitors septin assembly. We show here that Hsl7 is phosphorylated by Hsl1 in immune-complex kinase assays and can physically associate in vitro with either Hsl1 or Swe1 in the absence of any other yeast proteins. With the use of both the two-hybrid method and in vitro binding assays, we found that Hsl7 contains distinct binding sites for Hsl1 and Swe1. A differential interaction trap approach was used to isolate four single-site substitution mutations in Hsl7, which cluster within a discrete region of its N-terminal domain, that are specifically defective in binding Hsl1. When expressed in hsl7Δ cells, each of these Hsl7 point mutants is unable to localize at the bud neck and cannot mediate down-regulation of Swe1, but retains other functions of Hsl7, including oligomerization and association with Swe1. GFP-fusions of these Hsl1-binding defective Hsl7 proteins localize as a bright perinuclear dot, but never localize to the bud neck; likewise, inhsl1Δ cells, a GFP-fusion to wild-type Hsl7 or native Hsl7 localizes to this dot. Cell synchronization studies showed that, normally, Hsl7 localizes to the dot, but only in cells in the G1 phase of the cell cycle. Immunofluorescence analysis and immunoelectron microscopy established that the dot corresponds to the outer plaque of the spindle pole body (SPB). These data demonstrate that association between Hsl1 and Hsl7 at the bud neck is required to alleviate Swe1-imposed G2-M delay. Hsl7 localization at the SPB during G1 may play some additional role in fine-tuning the coordination between nuclear and cortical events before mitosis.
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Rozelle, Daniel K., Scott D. Hansen et Kenneth B. Kaplan. « Chromosome passenger complexes control anaphase duration and spindle elongation via a kinesin-5 brake ». Journal of Cell Biology 193, no 2 (11 avril 2011) : 285–94. http://dx.doi.org/10.1083/jcb.201011002.
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During mitosis, chromosome passenger complexes (CPCs) exhibit a well-conserved association with the anaphase spindle and have been implicated in spindle stability. However, their precise effect on the spindle is not clear. In this paper, we show, in budding yeast, that a CPC consisting of CBF3, Bir1, and Sli15, but not Ipl1, is required for normal spindle elongation. CPC mutants slow spindle elongation through the action of the bipolar kinesins Cin8 and Kip1. The same CPC mutants that slow spindle elongation also result in the enrichment of Cin8 and Kip1 at the spindle midzone. Together, these findings argue that CPCs function to organize the spindle midzone and potentially switch motors between force generators and molecular brakes. We also find that slowing spindle elongation delays the mitotic exit network (MEN)–dependent release of Cdc14, thus delaying spindle breakdown until a minimal spindle size is reached. We propose that these CPC- and MEN-dependent mechanisms are important for coordinating chromosome segregation with spindle breakdown and mitotic exit.
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Lim, H. H., P. Y. Goh et U. Surana. « Spindle pole body separation in Saccharomyces cerevisiae requires dephosphorylation of the tyrosine 19 residue of Cdc28. » Molecular and Cellular Biology 16, no 11 (novembre 1996) : 6385–97. http://dx.doi.org/10.1128/mcb.16.11.6385.
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In eukaryotes, mitosis requires the activation of cdc2 kinase via association with cyclin B and dephosphorylation of the threonine 14 and tyrosine 15 residues. It is known that in the budding yeast Saccharomyces cerevisiae, a homologous kinase, Cdc28, mediates the progression through M phase, but it is not clear what specific mitotic function its activation by the dephosphorylation of an equivalent tyrosine (Tyr-19) serves. We report here that cells expressing cdc28-E19 (in which Tyr-19 is replaced by glutamic acid) perform Start-related functions, complete DNA synthesis, and exhibit high levels of Clb2-associated kinase activity but are unable to form bipolar spindles. The failure of these cells to form mitotic spindles is due to their inability to segregate duplicated spindle pole bodies (SPBs), a phenotype strikingly similar to that exhibited by a previously reported mutant defective in both kinesin-like motor proteins Cin8 and Kip1. We also find that the overexpression of SWE1, the budding-yeast homolog of wee1, also leads to a failure to segregate SPBs. These results imply that dephosphorylation of Tyr-19 is required for the segregation of SPBs. The requirement of Tyr-19 dephosphorylation for spindle assembly is also observed under conditions in which spindle formation is independent of mitosis, suggesting that the involvement of Cdc28/Clb kinase in SPB separation is direct. On the basis of these results, we propose that one of the roles of Tyr-19 dephosphorylation is to promote SPB separation.
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Shulewitz, Mark J., Carla J. Inouye et Jeremy Thorner. « Hsl7 Localizes to a Septin Ring and Serves as an Adapter in a Regulatory Pathway That Relieves Tyrosine Phosphorylation of Cdc28 Protein Kinase inSaccharomyces cerevisiae ». Molecular and Cellular Biology 19, no 10 (1 octobre 1999) : 7123–37. http://dx.doi.org/10.1128/mcb.19.10.7123.
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ABSTRACT Successful mitosis requires faithful DNA replication, spindle assembly, chromosome segregation, and cell division. In the budding yeast Saccharomyces cerevisiae, the G2-to-M transition requires activation of Clb-bound forms of the protein kinase, Cdc28. These complexes are held in an inactive state via phosphorylation of Tyr19 in the ATP-binding loop of Cdc28 by the Swe1 protein kinase. The HSL1 and HSL7 gene products act as negative regulators of Swe1. Hsl1 is a large (1,518-residue) protein kinase with an N-terminal catalytic domain and a very long C-terminal extension. Hsl1 localizes to the incipient site of cytokinesis in the bud neck in a septin-dependent manner; however, the function of Hsl7 was not previously known. Using both indirect immunofluorescence with anti-Hsl7 antibodies and a fusion of Hsl7 to green fluorescent protein, we found that Hsl7 also localizes to the bud neck, congruent with the septin ring that faces the daughter cell. Both Swe1 and a segment of the C terminus of Hsl1 (which has no sequence counterpart in two Hsl1-related protein kinases, Gin4 and Kcc4) were identified as gene products that interact with Hsl7 in a two-hybrid screen of a random S. cerevisiae cDNA library. Hsl7 plus Swe1 and Hsl7 plus Hsl1 can be coimmunoprecipitated from extracts of cells overexpressing these proteins, confirming that Hsl7 physically associates with both partners. Also consistent with the two-hybrid results, Hsl7 coimmunoprecipitates with full-length Hsl1 less efficiently than with a C-terminal fragment of Hsl1. Moreover, Hsl7 does not localize to the bud neck in an hsl1Δ mutant, whereas Hsl1 is localized normally in an hsl7Δ mutant. Phosphorylation and ubiquitinylation of Swe1, preludes to its destruction, are severely reduced in cells lacking either Hsl1 or Hsl7 (or both), as judged by an electrophoretic mobility shift assay. Collectively, these data suggest that formation of the septin rings provides sites for docking Hsl1, exposing its C terminus and thereby permitting recruitment of Hsl7. Hsl7, in turn, presents its cargo of bound Swe1, allowing phosphorylation by Hsl1. Thus, Hsl1 and Hsl7 promote proper timing of cell cycle progression by coupling septin ring assembly to alleviation of Swe1-dependent inhibition of Cdc28. Furthermore, like septins and Hsl1, homologs of Hsl7 are found in fission yeast, flies, worms, and humans, suggesting that its function in this control mechanism may be conserved in all eukaryotes.
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Frieders, Elizabeth M., et David J. McLaughlln. « Mitosis in the yeast phase of Agaricostilbum pulcherrimum and its evolutionary significance ». Canadian Journal of Botany 74, no 9 (1 septembre 1996) : 1392–406. http://dx.doi.org/10.1139/b96-169.
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Agaricostilbum pulcherrimum is an anomaly and is difficult to place systematically. It possesses a yeast phase, and as in most basidiomycetous yeasts, mitosis has not been investigated cytoiogically. Yeast cells of A. pulcherrimum were prepared for immunofluorescence and transmission electron microscopy by a freeze-substitution method. A cladistic analysis of cell cycle characters among A. pulcherrimum and two ascomycetous and two basidiomycetous yeasts, performed with phylogenetic analysis using parsimony, revealed that A. pulcherrimum is basal within these basidiomycetes. Spindle pole bodies are multilayered discs and appear to be intranuclear during early division, similar to meiotic division. Spindle initiation and early elongation occur in the parent, a situation unreported in basidiomycetous yeasts. The site of spindle initiation, the position of the nucleus during division, and the pattern of astral microtubules demonstrate that the mode of nuclear division in A. pulcherrimum is intermediate between those of the investigated ascomycetous and basidiomycetous yeasts. Keywords: basidiomycete, cell cycle, cytoskeleton, immunofluorescence, phylogeny, spindle pole body.
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Farr, Katie A., et M. Andrew Hoyt. « Bub1p Kinase Activates the Saccharomyces cerevisiae Spindle Assembly Checkpoint ». Molecular and Cellular Biology 18, no 5 (1 mai 1998) : 2738–47. http://dx.doi.org/10.1128/mcb.18.5.2738.
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ABSTRACT Saccharomyces cerevisiae BUB1 encodes a protein kinase required for spindle assembly checkpoint function. In the presence of spindle damage, BUB1 is required to prevent cell cycle progression into anaphase. We have identified a dominantly actingBUB1 allele that appears to activate the spindle assembly checkpoint pathway in cells with undamaged spindles. High-level expression of BUB1-5 did not cause detectable spindle damage, yet it delayed yeast cells in mitosis at a stage following bipolar spindle assembly but prior to anaphase spindle elongation. Delayed cells possessed a G2 DNA content and elevated Clb2p mitotic cyclin levels. Unlike cells delayed in mitosis by spindle damage or MPS1 kinase overexpression, hyperphosphorylated forms of the Mad1p checkpoint protein did not accumulate. Similar to cells overexpressing MPS1, the BUB1-5 delay was dependent upon the functions of the other checkpoint genes, includingBUB2 and BUB3 and MAD1,MAD2, and MAD3. We found that the mitotic delay caused by BUB1-5 or MPS1 overexpression was interdependent upon the function of the other. This suggests that the Bub1p and Mps1p kinases act together at an early step in generating the spindle damage signal.
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Yang, Sam S., Elaine Yeh, E. D. Salmon et Kerry Bloom. « Identification of a Mid-anaphase Checkpoint in Budding Yeast ». Journal of Cell Biology 136, no 2 (27 janvier 1997) : 345–54. http://dx.doi.org/10.1083/jcb.136.2.345.
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Activation of a facultative, dicentric chromosome provides a unique opportunity to introduce a double strand DNA break into a chromosome at mitosis. Time lapse video enhanced-differential interference contrast analysis of the cellular response upon dicentric activation reveals that the majority of cells initiates anaphase B, characterized by pole–pole separation, and pauses in mid-anaphase for 30–120 min with spindles spanning the neck of the bud before completing spindle elongation and cytokinesis. The length of the spindle at the delay point (3–4 μm) is not dependent on the physical distance between the two centromeres, indicating that the arrest represents surveillance of a dicentric induced aberration. No mid-anaphase delay is observed in the absence of the RAD9 checkpoint gene, which prevents cell cycle progression in the presence of damaged DNA. These observations reveal RAD9- dependent events well past the G2/M boundary and have considerable implications in understanding how chromosome integrity and the position and state of the mitotic spindle are monitored before cytokinesis.
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West, Robert R., Terra Malmstrom et J. Richard McIntosh. « Kinesinsklp5+ andklp6+ are required for normal chromosome movement in mitosis ». Journal of Cell Science 115, no 5 (1 mars 2002) : 931–40. http://dx.doi.org/10.1242/jcs.115.5.931.
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Proper mitotic chromosome segregation requires dynamic interactions between spindle microtubules and kinetochores. Here we demonstrate that two related fission yeast kinesins, klp5+ and klp6+, are required for normal chromosome segregation in mitosis. Null mutants frequently lack a normal metaphase chromosome alignment. Chromosome pairs move back and forth along the spindle for an extended period prior to sister chromatid separation, a phenotype reminiscent of the loss of CENP-E in metazoans. Ultimately, sister chromatids segregate, regardless of chromosome position along the spindle, and viable daughter cells are usually produced. The initiation of anaphase B is sometimes delayed, but the rate of spindle elongation is similar to wildtype. Despite a delay, anaphase B often begins before anaphase A is completed. The klp5Δ and klp6Δ null mutants are synthetically lethal with a deletion of the spindle assembly checkpoint gene, bub1+, several mutants in components of the anaphase promoting complex, and a cold sensitive allele of the kinetochore and microtubule-binding protein, Dis1p. Klp5p-GFP and Klp6p-GFP localize to kinetochores from prophase to the onset of anaphase A, but relocalize to the spindle midzone during anaphase B. These data indicate that Klp5p and Klp6p are kinetochore kinesins required for normal chromosome movement in prometaphase.
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Fitch, I., C. Dahmann, U. Surana, A. Amon, K. Nasmyth, L. Goetsch, B. Byers et B. Futcher. « Characterization of four B-type cyclin genes of the budding yeast Saccharomyces cerevisiae. » Molecular Biology of the Cell 3, no 7 (juillet 1992) : 805–18. http://dx.doi.org/10.1091/mbc.3.7.805.
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The previously described CLB1 and CLB2 genes encode a closely related pair of B-type cyclins. Here we present the sequences of another related pair of B-type cyclin genes, which we term CLB3 and CLB4. Although CLB1 and CLB2 mRNAs rise in abundance at the time of nuclear division, CLB3 and CLB4 are turned on earlier, rising early in S phase and declining near the end of nuclear division. When all possible single and multiple deletion mutants were constructed, some multiple mutations were lethal, whereas all single mutants were viable. All lethal combinations included the clb2 deletion, whereas the clb1 clb3 clb4 triple mutant was viable, suggesting a key role for CLB2. The inviable multiple clb mutants appeared to have a defect in mitosis. Conditional clb mutants arrested as large budded cells with a G2 DNA content but without any mitotic spindle. Electron microscopy showed that the spindle pole bodies had duplicated but not separated, and no spindle had formed. This suggests that the Clb/Cdc28 kinase may have a relatively direct role in spindle formation. The two groups of Clbs may have distinct roles in spindle formation and elongation.
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Hagan, I. M., et J. S. Hyams. « The use of cell division cycle mutants to investigate the control of microtubule distribution in the fission yeast Schizosaccharomyces pombe ». Journal of Cell Science 89, no 3 (1 mars 1988) : 343–57. http://dx.doi.org/10.1242/jcs.89.3.343.
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We have characterized the changes in microtubule organization that occur through the cell division cycle of the fission yeast Schizosaccharomyces pombe by indirect immunofluorescence microscopy. During interphase, groups of cytoplasmic microtubules, independent of the spindle pole body (SPB), form an array extending between the cell tips. These microtubules are involved in positioning the nucleus at the cell equator and in the establishment of cell polarity. At mitosis, the interphase array disappears and is replaced by an intranuclear spindle extending between the now duplicated SPBs. Elongation of the spindle sees the appearance of astral microtubules emanating from the cytoplasmic face of the SPBs. These persist until the end of anaphase whereupon the spindle microtubules depolymerize and two microtubule organizing centres (MTOCs) at the cell equator re-establish the interphase array. We have used the unique properties of various cell division cycle mutants to investigate further the function of these different microtubule arrays and their temporal and positional control.
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Khmelinskii, Anton, Clare Lawrence, Johanna Roostalu et Elmar Schiebel. « Cdc14-regulated midzone assembly controls anaphase B ». Journal of Cell Biology 177, no 6 (11 juin 2007) : 981–93. http://dx.doi.org/10.1083/jcb.200702145.
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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.
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Troxell, Cynthia L., Mark A. Sweezy, Robert R. West, Karen D. Reed, Bryan D. Carson, Alison L. Pidoux, W. Zacheus Cande et J. Richard McIntosh. « pkl1 +andklp2 + : Two Kinesins of the Kar3 Subfamily in Fission Yeast Perform Different Functions in Both Mitosis and Meiosis ». Molecular Biology of the Cell 12, no 11 (novembre 2001) : 3476–88. http://dx.doi.org/10.1091/mbc.12.11.3476.
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We have identified Klp2p, a new kinesin-like protein (KLP) of the KAR3 subfamily in fission yeast. The motor domain of this protein is 61% identical and 71% similar to Pkl1p, another fission yeast KAR3 protein, yet the two enzymes are different in behavior and function. Pkl1p is nuclear throughout the cell cycle, whereas Klp2p is cytoplasmic during interphase. During mitosis Klp2p enters the nucleus where it forms about six chromatin-associated dots. In metaphase-arrested cells these migrate back and forth across the nucleus. During early anaphase they segregate with the chromosomes into two sets of about three, fade, and are replaced by other dots that form on the spindle interzone. Neitherklp2 + norpkl1 + is essential, and the double deletion is also wild type for both vegetative and sexual reproduction. Each deletion rescues different alleles ofcut7 ts , a KLP that contributes to spindle formation and elongation. When either or both deletions are combined with a dynein deletion, vegetative growth is normal, but sexual reproduction fails: klp2Δ,dhc1-d1 in karyogamy, pkl1Δ,dhc1-d1 in multiple phases of meiosis, and the triple deletion in both. Deletion of Klp2p elongates a metaphase-arrested spindle, but pkl1Δshortens it. The anaphase spindle of klp2Δ becomes longer than the cell, leading it to curl around the cell's ends. Apparently, Klp2p promotes spindle disassembly and contributes to the behavior of mitotic chromosomes.
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Jones, Michele Haltiner, Eileen T. O’Toole, Amy S. Fabritius, Eric G. Muller, Janet B. Meehl, Sue L. Jaspersen et Mark Winey. « Key phosphorylation events in Spc29 and Spc42 guide multiple steps of yeast centrosome duplication ». Molecular Biology of the Cell 29, no 19 (15 septembre 2018) : 2280–91. http://dx.doi.org/10.1091/mbc.e18-05-0296.
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Phosphorylation modulates many cellular processes during cell cycle progression. The yeast centrosome (called the spindle pole body, SPB) is regulated by the protein kinases Mps1 and Cdc28/Cdk1 as it nucleates microtubules to separate chromosomes during mitosis. Previously we completed an SPB phosphoproteome, identifying 297 sites on 17 of the 18 SPB components. Here we describe mutagenic analysis of phosphorylation events on Spc29 and Spc42, two SPB core components that were shown in the phosphoproteome to be heavily phosphorylated. Mutagenesis at multiple sites in Spc29 and Spc42 suggests that much of the phosphorylation on these two proteins is not essential but enhances several steps of mitosis. Of the 65 sites examined on both proteins, phosphorylation of the Mps1 sites Spc29-T18 and Spc29-T240 was shown to be critical for function. Interestingly, these two sites primarily influence distinct successive steps; Spc29-T240 is important for the interaction of Spc29 with Spc42, likely during satellite formation, and Spc29-T18 facilitates insertion of the new SPB into the nuclear envelope and promotes anaphase spindle elongation. Phosphorylation sites within Cdk1 motifs affect function to varying degrees, but mutations only have significant effects in the presence of an MPS1 mutation, supporting a theme of coregulation by these two kinases.
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Hagan, I. M., P. N. Riddle et J. S. Hyams. « Intramitotic controls in the fission yeast Schizosaccharomyces pombe : the effect of cell size on spindle length and the timing of mitotic events. » Journal of Cell Biology 110, no 5 (1 mai 1990) : 1617–21. http://dx.doi.org/10.1083/jcb.110.5.1617.
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We have used a new cinemicroscopy technique in combination with antitubulin immunofluorescence microscopy to investigate the timing of mitotic events in cells of the fission yeast Schizosaccharomyces pombe having lengths at division between 7 and 60 microns. Wild-type fission yeast cells divide at a length of 14 microns. Separation of daughter nuclei (anaphase B) proceeds at a rate of 1.6 +/- 0.2 microns min-1, until the spindle extends the length of the cell. Coincident with spindle depolymerization, the nuclei reverse direction and take up positions that will become the center of the two daughter cells. This post-mitotic nuclear migration occurs at a rate of 1.4 +/- 0.5 microns-1. In cells in which the weel+ gene is overexpressed fivefold and that have an average length at mitosis of 28 microns, the rate of nuclear separation was only slightly reduced but, as spindles in these cells measure 20-22 microns, the duration of anaphase B was extended by approximately 40%. By contrast, in the mutant weel.50, which divides at 7 microns, both the rate and duration of anaphase B were indistinguishable from wild type. Nuclei reach the ends of these cells earlier but remain there until a point corresponding to the time of postmitotic nuclear migration in wild type. Thus, the events of mitosis can be extended but not abbreviated. These results are discussed in terms of a mitotic termination control that monitors many different events, one of which is spindle elongation.
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Rogers, Stephen L., Gregory C. Rogers, David J. Sharp et Ronald D. Vale. « Drosophila EB1 is important for proper assembly, dynamics, and positioning of the mitotic spindle ». Journal of Cell Biology 158, no 5 (2 septembre 2002) : 873–84. http://dx.doi.org/10.1083/jcb.200202032.
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EB1 is an evolutionarily conserved protein that localizes to the plus ends of growing microtubules. In yeast, the EB1 homologue (BIM1) has been shown to modulate microtubule dynamics and link microtubules to the cortex, but the functions of metazoan EB1 proteins remain unknown. Using a novel preparation of the Drosophila S2 cell line that promotes cell attachment and spreading, we visualized dynamics of single microtubules in real time and found that depletion of EB1 by RNA-mediated inhibition (RNAi) in interphase cells causes a dramatic increase in nondynamic microtubules (neither growing nor shrinking), but does not alter overall microtubule organization. In contrast, several defects in microtubule organization are observed in RNAi-treated mitotic cells, including a drastic reduction in astral microtubules, malformed mitotic spindles, defocused spindle poles, and mispositioning of spindles away from the cell center. Similar phenotypes were observed in mitotic spindles of Drosophila embryos that were microinjected with anti-EB1 antibodies. In addition, live cell imaging of mitosis in Drosophila embryos reveals defective spindle elongation and chromosomal segregation during anaphase after antibody injection. Our results reveal crucial roles for EB1 in mitosis, which we postulate involves its ability to promote the growth and interactions of microtubules within the central spindle and at the cell cortex.
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Vernarecci, Stefano, Prisca Ornaghi, AnaCristina Bâgu, Enrico Cundari, Paola Ballario et Patrizia Filetici. « Gcn5p Plays an Important Role in Centromere Kinetochore Function in Budding Yeast ». Molecular and Cellular Biology 28, no 3 (26 novembre 2007) : 988–96. http://dx.doi.org/10.1128/mcb.01366-07.
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ABSTRACT We report that the histone acetyltransferase Gcn5p is involved in cell cycle progression, whereas its absence induces several mitotic defects, including inefficient nuclear division, chromosome loss, delayed G2 progression, and spindle elongation. The fidelity of chromosome segregation is finely regulated by the close interplay between the centromere and the kinetochore, a protein complex hierarchically assembled in the centromeric DNA region, while disruption of GCN5 in mutants of inner components results in sick phenotype. These synthetic interactions involving the ADA complex lay the genetic basis for the critical role of Gcn5p in kinetochore assembly and function. We found that Gcn5p is, in fact, physically linked to the centromere, where it affects the structure of the variant centromeric nucleosome. Our findings offer a key insight into a Gcn5p-dependent epigenetic regulation at centromere/kinetochore in mitosis.
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Ma, Lina, Jennifer McQueen, Lara Cuschieri, Jackie Vogel et Vivien Measday. « Spc24 and Stu2 Promote Spindle Integrity When DNA Replication Is Stalled ». Molecular Biology of the Cell 18, no 8 (août 2007) : 2805–16. http://dx.doi.org/10.1091/mbc.e06-09-0882.
Texte intégralRésumé :
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.
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Thakur, Jitendra, et Kaustuv Sanyal. « The Essentiality of the Fungus-Specific Dam1 Complex Is Correlated with a One-Kinetochore-One-Microtubule Interaction Present throughout the Cell Cycle, Independent of the Nature of a Centromere ». Eukaryotic Cell 10, no 10 (13 mai 2011) : 1295–305. http://dx.doi.org/10.1128/ec.05093-11.
Texte intégralRésumé :
ABSTRACTA fungus-specific outer kinetochore complex, the Dam1 complex, is essential inSaccharomyces cerevisiae, nonessential in fission yeast, and absent from metazoans. The reason for the reductive evolution of the functionality of this complex remains unknown. BothCandida albicansandSchizosaccharomyces pombehave regional centromeres as opposed to the short-point centromeres ofS. cerevisiae. The interaction of one microtubule per kinetochore is established both inS. cerevisiaeandC. albicansearly during the cell cycle, which is in contrast to the multiple microtubules that bind to a kinetochore only during mitosis inS. pombe. Moreover, the Dam1 complex is associated with the kinetochore throughout the cell cycle inS. cerevisiaeandC. albicansbut only during mitosis inS. pombe. Here, we show that the Dam1 complex is essential for viability and indispensable for proper mitotic chromosome segregation inC. albicans. The kinetochore localization of the Dam1 complex is independent of the kinetochore-microtubule interaction, but the function of this complex is monitored by a spindle assembly checkpoint. Strikingly, the Dam1 complex is required to prevent precocious spindle elongation in premitotic phases. Thus, constitutive kinetochore localization associated with a one-microtubule-one kinetochore type of interaction, but not the length of a centromere, is correlated with the essentiality of the Dam1 complex.
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Yukawa, Masashi, Tomoki Kawakami, Masaki Okazaki, Kazunori Kume, Ngang Heok Tang et Takashi Toda. « A microtubule polymerase cooperates with the kinesin-6 motor and a microtubule cross-linker to promote bipolar spindle assembly in the absence of kinesin-5 and kinesin-14 in fission yeast ». Molecular Biology of the Cell 28, no 25 (décembre 2017) : 3647–59. http://dx.doi.org/10.1091/mbc.e17-08-0497.
Texte intégralRésumé :
Accurate chromosome segregation relies on the bipolar mitotic spindle. In many eukaryotes, spindle formation is driven by the plus-end–directed motor kinesin-5 that generates outward force to establish spindle bipolarity. Its inhibition leads to the emergence of monopolar spindles with mitotic arrest. Intriguingly, simultaneous inactivation of the minus-end–directed motor kinesin-14 restores spindle bipolarity in many systems. Here we show that in fission yeast, three independent pathways contribute to spindle bipolarity in the absence of kinesin-5/Cut7 and kinesin-14/Pkl1. One is kinesin-6/Klp9 that engages with spindle elongation once short bipolar spindles assemble. Klp9 also ensures the medial positioning of anaphase spindles to prevent unequal chromosome segregation. Another is the Alp7/TACC-Alp14/TOG microtubule polymerase complex. Temperature-sensitive alp7cut7pkl1 mutants are arrested with either monopolar or very short spindles. Forced targeting of Alp14 to the spindle pole body is sufficient to render alp7cut7pkl1 triply deleted cells viable and promote spindle assembly, indicating that Alp14-mediated microtubule polymerization from the nuclear face of the spindle pole body could generate outward force in place of Cut7 during early mitosis. The third pathway involves the Ase1/PRC1 microtubule cross-linker that stabilizes antiparallel microtubules. Our study, therefore, unveils multifaceted interplay among kinesin-dependent and -independent pathways leading to mitotic bipolar spindle assembly.
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van der Vaart, Babet, Josef Fischböck, Christine Mieck, Peter Pichler, Karl Mechtler, René H. Medema et Stefan Westermann. « TORC1 signaling exerts spatial control over microtubule dynamics by promoting nuclear export of Stu2 ». Journal of Cell Biology 216, no 11 (29 septembre 2017) : 3471–84. http://dx.doi.org/10.1083/jcb.201606080.
Texte intégralRésumé :
The target of rapamycin complex 1 (TORC1) is a highly conserved multiprotein complex that functions in many cellular processes, including cell growth and cell cycle progression. In this study, we define a novel role for TORC1 as a critical regulator of nuclear microtubule (MT) dynamics in the budding yeast Saccharomyces cerevisiae. This activity requires interactions between EB1 and CLIP-170 plus end–tracking protein (+TIP) family members with the TORC1 subunit Kog1/Raptor, which in turn allow the TORC1 proximal kinase Sch9/S6K1 to regulate the MT polymerase Stu2/XMAP215. Sch9-dependent phosphorylation of Stu2 adjacent to a nuclear export signal prevents nuclear accumulation of Stu2 before cells enter mitosis. Mutants impaired in +TIP–TORC1 interactions or Stu2 nuclear export show increased nuclear but not cytoplasmic MT length and display nuclear fusion, spindle positioning, and elongation kinetics defects. Our results reveal key mechanisms by which TORC1 signaling controls Stu2 localization and thereby contributes to proper MT cytoskeletal organization in interphase and mitosis.
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Kanbe, T., Y. Hiraoka, K. Tanaka et M. Yanagida. « The transition of cells of the fission yeast beta-tubulin mutant nda3-311 as seen by freeze-substitution electron microscopy. Requirement of functional tubulin for spindle pole body duplication ». Journal of Cell Science 96, no 2 (1 juin 1990) : 275–82. http://dx.doi.org/10.1242/jcs.96.2.275.
Texte intégralRésumé :
A previous fluorescence light-microscopic study showed that the fission yeast cold-sensitive beta-tubulin mutant nda3-311 was arrested with rod-like condensed chromosomes in a mitotic state at the restrictive temperature. Upon transfer to the permissive temperature, a spindle was formed and the nucleus was divided. In the present study, we employed freeze-substitution electron microscopy to examine the ultrastructure of arrested and released nda3-311 cells. In arrested cells, a single, displaced nucleus was seen with a single spindle pole body. Therefore, spindle pole body duplication seemed to require functional beta-tubulin. The nuclear membrane was highly deformed with a leaf-like profile in cross-section, possibly due to an interaction with the rod-like, condensed chromosomes. Upon transfer to the permissive temperature, the spindle pole duplicated and the daughter spindle pole bodies rapidly migrated to the opposite ends of the nucleus, accompanied by the formation of the mitotic spindle. Elongation of the nuclear envelope occurred with concomitant spindle extension, as in a wild-type mitosis. The deformed nuclear membrane became smooth and described a convex curve. The numerous vacuoles that are seen in the arrested cells decreased in number and increased in size. Septation was completed, leaving the two divided nuclei in one half of the cell. Hexagonally arranged microtubules, apparently forming the mitotic spindle, were observed in a cross-section of a cell after return to the permissive conditions.
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Tsui, Kochung, Lee Simon et David Norris. « Progression Into the First Meiotic Division Is Sensitive to Histone HN-HZB Dimer Concentration in Saccharomyces cerevisiae ». Genetics 145, no 3 (1 mars 1997) : 647–59. http://dx.doi.org/10.1093/genetics/145.3.647.
Texte intégralRésumé :
The yeast Saccharomyces cerevisiae contains two genes for histone H2A and two for histone H2B located in two divergently transcribed gene pairs: HTA1-HTB1 and HTA2-HTB2. Diploid strains lacking HTA1-HTB1 (hta1-htb1Δ/hta1-htb1Δ, HTA2-HTB2/HTA2-HTB2) grow vegetatively, but will not sporulate. This sporulation phenotype results from a partial depletion of H2A-H2B dimers. Since the expression patterns of HTA1-HTB1 and HTA2-HTB2 are similar in mitosis and meiosis, the sporulation pathway is therefore more sensitive than the mitotic cycle to depletion of H2A-H2B dimers. After completing premeiotic DNA replication, commitment to meiotic recombination, and chiasma resolution, the hta1-htb1Δ/hta1-htb1Δ, HTA2-HTB2/HTA2-HTB2 mutant arrests before the first meiotic division. The arrest is not due to any obvious disruptions in spindle pole bodies or microtubules. The meiotic block is not bypassed in backgrounds homozygous for spo13, rad50Δ, or rad9Δ mutations, but is bypassed in the presence of hydroxyurea, a drug known to inhibit DNA chain elongation. We hypothesize that the deposition of H2A-H2B dimers in the mutant is unable to keep pace with the replication fork, thereby leading to a disruption in chromosome structure that interferes with the meiotic divisions.
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Corellou, F., S. R. Bisgrove, D. L. Kropf, L. Meijer, B. Kloareg et F. Y. Bouget. « A S/M DNA replication checkpoint prevents nuclear and cytoplasmic events of cell division including centrosomal axis alignment and inhibits activation of cyclin-dependent kinase-like proteins in fucoid zygotes ». Development 127, no 8 (15 avril 2000) : 1651–60. http://dx.doi.org/10.1242/dev.127.8.1651.
Texte intégralRésumé :
S/M checkpoints prevent various aspects of cell division when DNA has not been replicated. Such checkpoints are stringent in yeast and animal somatic cells but are usually partial or not present in animal embryos. Because little is known about S/M checkpoints in plant cells and embryos, we have investigated the effect of aphidicolin, a specific inhibitor of DNA polymerases (alpha) and (delta), on cell division and morphogenesis in Fucus and Pelvetia zygotes. Both DNA replication and cell division were inhibited by aphidicolin, indicating the presence, in fucoid zygotes, of a S/M checkpoint. This checkpoint prevents chromatin condensation, spindle formation, centrosomal alignment with the growth axis and cytokinesis but has no effect on germination or rhizoid elongation. This S/M checkpoint also prevents tyrosine dephosphorylation of cyclin-dependent kinase-like proteins at the onset of mitosis. The kinase activity is restored in extracts upon incubation with cdc25A phosphatase. When added in S phase, olomoucine, a specific inhibitor of cyclin-dependent kinases, has similar effects as aphidicolin on cell division although alignment of the centrosomal axis still occurs. We propose a model involving the inactivation of CDK-like proteins to account for the S/M DNA replication checkpoint in fucoid zygotes and embryos.
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Krüger, Lara Katharina, Jérémie-Luc Sanchez, Anne Paoletti et Phong Thanh Tran. « Kinesin-6 regulates cell-size-dependent spindle elongation velocity to keep mitosis duration constant in fission yeast ». eLife 8 (26 février 2019). http://dx.doi.org/10.7554/elife.42182.
Texte intégralRésumé :
The length of the mitotic spindle scales with cell size in a wide range of organisms during embryonic development. Interestingly, in C. elegans embryos, this goes along with temporal regulation: larger cells speed up spindle assembly and elongation. We demonstrate that, similarly in fission yeast, spindle length and spindle dynamics adjust to cell size, which allows to keep mitosis duration constant. Since prolongation of mitosis was shown to affect cell viability, this may resemble a mechanism to regulate mitosis duration. We further reveal how the velocity of spindle elongation is regulated: coupled to cell size, the amount of kinesin-6 Klp9 molecules increases, resulting in an acceleration of spindle elongation in anaphase B. In addition, the number of Klp9 binding sites to microtubules increases overproportionally to Klp9 molecules, suggesting that molecular crowding inversely correlates to cell size and might have an impact on spindle elongation velocity control.
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Shoukat, Irsa, Corey Frazer et John S. Allingham. « Kinesin-5 Is Dispensable for Bipolar Spindle Formation and Elongation in Candida albicans, but Simultaneous Loss of Kinesin-14 Activity Is Lethal ». mSphere 4, no 6 (13 novembre 2019). http://dx.doi.org/10.1128/msphere.00610-19.
Texte intégralRésumé :
ABSTRACT Mitotic spindles assume a bipolar architecture through the concerted actions of microtubules, motors, and cross-linking proteins. In most eukaryotes, kinesin-5 motors are essential to this process, and cells will fail to form a bipolar spindle without kinesin-5 activity. Remarkably, inactivation of kinesin-14 motors can rescue this kinesin-5 deficiency by reestablishing the balance of antagonistic forces needed to drive spindle pole separation and spindle assembly. We show that the yeast form of the opportunistic fungus Candida albicans assembles bipolar spindles in the absence of its sole kinesin-5, CaKip1, even though this motor exhibits stereotypical cell-cycle-dependent localization patterns within the mitotic spindle. However, cells lacking CaKip1 function have shorter metaphase spindles and longer and more numerous astral microtubules. They also show defective hyphal development. Interestingly, a small population of CaKip1-deficient spindles break apart and reform two bipolar spindles in a single nucleus. These spindles then separate, dividing the nucleus, and then elongate simultaneously in the mother and bud or across the bud neck, resulting in multinucleate cells. These data suggest that kinesin-5-independent mechanisms drive assembly and elongation of the mitotic spindle in C. albicans and that CaKip1 is important for bipolar spindle integrity. We also found that simultaneous loss of kinesin-5 and kinesin-14 (CaKar3Cik1) activity is lethal. This implies a divergence from the antagonistic force paradigm that has been ascribed to these motors, which could be linked to the high mitotic error rate that C. albicans experiences and often exploits as a generator of diversity. IMPORTANCE Candida albicans is one of the most prevalent fungal pathogens of humans and can infect a broad range of niches within its host. This organism frequently acquires resistance to antifungal agents through rapid generation of genetic diversity, with aneuploidy serving as a particularly important adaptive mechanism. This paper describes an investigation of the sole kinesin-5 in C. albicans, which is a major regulator of chromosome segregation. Contrary to other eukaryotes studied thus far, C. albicans does not require kinesin-5 function for bipolar spindle assembly or spindle elongation. Rather, this motor protein associates with the spindle throughout mitosis to maintain spindle integrity. Furthermore, kinesin-5 loss is synthetically lethal with loss of kinesin-14—canonically an opposing force producer to kinesin-5 in spindle assembly and anaphase. These results suggest a significant evolutionary rewiring of microtubule motor functions in the C. albicans mitotic spindle, which may have implications in the genetic instability of this pathogen.