Academic literature on the topic 'Yeast, Swe1, mitosis, spindle elongation'

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.

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.

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.

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.

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.

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.

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.

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.

Swe1 is the effector kinase of the morphogenesis checkpoint that, in budding yeast, provides a link between cell morphology and entry into mitosis. Although there are some differences due to the particular kind of cell division established by the budding, Swe1 functions and regulators are evolutionarily conserved, indicating that this is an ancient cell cycle control strategy that has been adapted to respond to cytoskeletal signals in vertebrate as well as in S. cerevisiae cells. Swe1 blocks entry into mitosis through inhibitory phosphorylation of the catalytic subunit of the cyclin-dependent kinase Cdk1, Cdc28, and this modification is reversed by the protein phosphatase Mih1. Cdc28 activity is required both for entry into mitosis and for the switch from polar to isotropic bud growth, so when Cdc28 is phosphorylated on Tyr19 (Y19) both these events are inhibited. Timely degradation of Swe1 is important for cell survival in case of DNA replication stress, while it is inhibited by the morphogenesis checkpoint in response to alterations in actin cytoskeleton or septins’ structure. We show here that the lack of the Dma1 and Dma2 ubiquitin ligases, which moderately affects Swe1 localization and degradation during an unperturbed cell cycle with no apparent phenotypic effects, is toxic for cells that are partially defective in Swe1 down-regulation. Interestingly Swe1 is stabilized, but differently from morphogenesis checkpoint activation, restrained at the bud neck and hyperphosphorylated in dma1∆ dma2∆ cells subjected to DNA replication stress, indicating that the mechanism stabilizing Swe1 under these conditions is different from the one triggered by the morphogenesis checkpoint. Finally, the Dma proteins are required for proper Swe1 ubiquitylation. Altogether, our data highlight a previously unknown role of these proteins in the complex regulation of Swe1 degradation and suggest that they might contribute to control, directly or indirectly, Swe1 ubiquitylation. As already said, Swe1 stabilization prevents mitotic entry in response to different problems. In addition, elevated Swe1 levels inhibits mitotic spindle formation and elongation and several data indicate that, apart Cdc28, other Swe1 targets are likely involved in this process. In fact, the expression of Cdc28 alleles that could escape from Swe1 inhibition is not sufficient neither to restore proper spindle elongation nor progression through mitosis of cells that overexpress Swe1. We tried to identify new Swe1 targets acting in mitotic spindle dynamics and progression through mitosis by performing a genetic screen and by analyzing putative candidates among factors known to be involved in these processes. About the genetic screen, we found twelve recessive spontaneous suppressors that are able to restore spindle elongation and viability of SWE1 overespressing cells that lack the APC regulatory subunit CDH1. Interestingly, the suppression phenotype is due to inactivation of the same gene in all the suppressors. We are now trying to identify this gene and to characterize the suppression mechanism. In parallel, we pursued the identification of Swe1 targets by analyzing spindle associated factors and proteins involved in spindle dynamycs. In particular, the MAP Bik1 was found in a proteome chip array as a protein phosphorylated by Swe1. We found that, differently from what is published, high Swe1 levels reduce Bik1 phosphorylation independently of Swe1 inhibitory activity on Cdc28. Further analysis will be required to better understand the molecular details of the indirect Swe1 action on Bik1 phosphorylation. In addition, we found that high levels of the Polo kinase CDC5 partially restore spindle elongation and viability of SWE1 overexpressing cells, but only in the presence of functional Mih1. In particular, high Swe1 levels cause the accumulation of fully phosphorylated Mih1, that is not functional, and CDC5 overexpression in these cells restores the proper balance between Mih1 phosphorylation forms, and so its functionality. Further analysis will be required to better understand the interaction between Swe1, Cdc5 and the protein phosphatase Mih1. Altogether our data shed new light on Swe1 regulation mechanism and put the basis for the identification its new target(s) .