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Greenfield Sluder – 1st expert on this subject based on the ideXlab platform
mitosis in vertebrate somatic cells with two spindles implications for the metaphase Anaphase transition checkpoint and cleavageProceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Conly L Rieder, Alexey Khodjakov, Leocadia V Paliulis, Tina M Fortier, Richard W Cole, Greenfield SluderAbstract:
During mitosis an inhibitory activity associated with unattached kinetochores prevents PtK1 cells from entering Anaphase until all kinetochores become attached to the spindle. To gain a better understanding of how unattached kinetochores block the metaphase/Anaphase transition we followed mitosis in PtK1 cells containing two independent spindles in a common cytoplasm. We found that unattached kinetochores on one spindle did not block Anaphase onset in a neighboring mature metaphase spindle 20 μm away that lacked unattached kinetochores. As in cells containing a single spindle, Anaphase onset occurred in the mature spindles x = 24 min after the last kinetochore attached regardless of whether the adjacent immature spindle contained one or more unattached kinetochores. These findings reveal that the inhibitory activity associated with an unattached kinetochore is functionally limited to the vicinity of the spindle containing the unattached kinetochore. We also found that once a mature spindle entered Anaphase the neighboring spindle also entered Anaphase x = 9 min later regardless of whether it contained monooriented chromosomes. Thus, Anaphase onset in the mature spindle catalyzes a “start Anaphase” reaction that spreads globally throughout the cytoplasm and overrides the inhibitory signal produced by unattached kinetochores in an adjacent spindle. Finally, we found that cleavage furrows often formed between the two independent spindles. This reveals that the presence of chromosomes and/or a spindle between two centrosomes is not a prerequisite for cleavage in vertebrate somatic cells.
the checkpoint delaying Anaphase in response to chromosome monoorientation is mediated by an inhibitory signal produced by unattached kinetochoresJournal of Cell Biology, 1995Co-Authors: Conly L Rieder, Alexey Khodjakov, Richard W Cole, Greenfield SluderAbstract:
During mitosis in Ptk1 cells Anaphase is not initiated until, on average, 23 +/- 1 min after the last monooriented chromosome acquires a bipolar attachment to the spindle–an event that may require 3 h (Rieder, C. L., A. Schultz, R. W. Cole, and G. Sluder. 1994. J. Cell Biol. 127:1301-1310). To determine the nature of this cell-cycle checkpoint signal, and its site of production, we followed PtK1 cells by video microscopy prior to and after destroying specific chromosomal regions by laser irradiation. The checkpoint was relieved, and cells entered Anaphase, 17 +/- 1 min after the centromere (and both of its associated sister kinetochores) was destroyed on the last monooriented chromosome. Thus, the checkpoint mechanism monitors an inhibitor of Anaphase produced in the centromere of monooriented chromosomes. Next, in the presence of one monooriented chromosome, we destroyed one kinetochore on a bioriented chromosome to create a second monooriented chromosome lacking an unattached kinetochore. Under this condition Anaphase began in the presence of the experimentally created monooriented chromosome 24 +/- 1.5 min after the nonirradiated monooriented chromosome bioriented. This result reveals that the checkpoint signal is not generated by the attached kinetochore of a monooriented chromosome or throughout the centromere volume. Finally, we selectively destroyed the unattached kinetochore on the last monooriented chromosome. Under this condition cells entered Anaphase 20 +/- 2.5 min after the operation, without congressing the irradiated chromosome. Correlative light microscopy/elctron microscopy of these cells in Anaphase confirmed the absence of a kinetochore on the unattached chromatid. Together, our data reveal that molecules in or near the unattached kinetochore of a monooriented PtK1 chromosome inhibit the metaphase-Anaphase transition.
Anaphase onset in vertebrate somatic cells is controlled by a checkpoint that monitors sister kinetochore attachment to the spindleJournal of Cell Biology, 1994Co-Authors: Conly L Rieder, Richard W Cole, A Schultz, Greenfield SluderAbstract:
To test the popular but unproven assumption that the metaphase-Anaphase transition in vertebrate somatic cells is subject to a checkpoint that monitors chromosome (i.e., kinetochore) attachment to the spindle, we filmed mitosis in 126 PtK1 cells. We found that the time from nuclear envelope breakdown to Anaphase onset is linearly related (r2 = 0.85) to the duration the cell has unattached kinetochores, and that even a single unattached kinetochore delays Anaphase onset. We also found that Anaphase is initiated at a relatively constant 23-min average interval after the last kinetochore attaches, regardless of how long the cell possessed unattached kinetochores. From these results we conclude that vertebrate somatic cells possess a metaphase-Anaphase checkpoint control that monitors sister kinetochore attachment to the spindle. We also found that some cells treated with 0.3-0.75 nM Taxol, after the last kinetochore attached to the spindle, entered Anaphase and completed normal poleward chromosome motion (Anaphase A) up to 3 h after the treatment–well beyond the 9-48-min range exhibited by untreated cells. The fact that spindle bipolarity and the metaphase alignment of kinetochores are maintained in these cells, and that the chromosomes move poleward during Anaphase, suggests that the checkpoint monitors more than just the attachment of microtubules at sister kinetochores or the metaphase alignment of chromosomes. Our data are most consistent with the hypothesis that the checkpoint monitors an increase in tension between kinetochores and their associated microtubules as biorientation occurs.
Kim Nasmyth – 2nd expert on this subject based on the ideXlab platform
separating sister chromatidsTrends in Biochemical Sciences, 1999Co-Authors: Kim NasmythAbstract:
Abstract Loss of cohesion between sister chromatids triggers their segregation during Anaphase. Recent work has identified both a cohesin complex that holds sisters together and a sister-separating protein, separin, that destroys cohesion. Separins are bound by inhibitory proteins whose proteolysis at the metaphase–Anaphase transition is mediated by the Anaphase-promoting complex and its activator protein CDC20 (APC CDC20 ). When chromosomes are misaligned, a surveillance mechanism (checkpoint) blocks sister separation by inhibiting APC CDC20 . Defects in this apparatus are implicated in causing aneuploidy in human cells.
genes involved in sister chromatid separation are needed for b type cyclin proteolysis in budding yeastCell, 1995Co-Authors: Stefan Irniger, Simonetta Piatti, Christine Michaelis, Kim NasmythAbstract:
Abstract 13-type cyclin destruction is necessary for exit from mitosis and the initiation of a new cell cycle. Through the isolation of mutants, we have identified three essential yeast genes, CDC16, CDC23 , and CSE1 , which are required for proteolysis of the B-type cyclin CLB2 but not of other unstable proteins. cdc23-1 mutants are defective in both entering and exiting Anaphase. Their failure to exit Anaphase can be explained by defective cyclin proteolysis. CDC23 is required at the metaphase/Anaphase transition to separate sister chromatids, and we speculate that it might promote proteolysis of proteins that hold sister chromatids together. Proteolysis of CLB2 is initiated in early Anaphase, but a fraction of CLB2 remains stable until Anaphase is complete.
destruction of the cdc28 clb mitotic kinase is not required for the metaphase to Anaphase transition in budding yeastThe EMBO Journal, 1993Co-Authors: Uttam Surana, Angelika Amon, C Dowzer, J Mcgrew, B Byers, Kim NasmythAbstract:
It is widely assumed that degradation of mitotic cyclins causes a decrease in mitotic cdc2/CDC28 kinase activity and thereby triggers the metaphase to Anaphase transition. Two observations made on the budding yeast Saccharomyces cerevisiae are inconsistent with this scenario: (i) Anaphase occurs in the presence of high levels of kinase in cdc15 mutants and (ii) overproduction of a B-type mitotic cyclin causes arrest not in metaphase as previously reported but in telophase. Kinase destruction is therefore implicated in the exit from mitosis rather than the entry into Anaphase. The behaviour of esp1 mutants shows in addition that kinase destruction can occur in the absence of Anaphase completion. The execution of Anaphase and the destruction of CDC28 kinase activity therefore appear to take place independently of one another.
Frank Uhlmann – 3rd expert on this subject based on the ideXlab platform
stabilization of microtubule dynamics at Anaphase onset promotes chromosome segregationNature, 2005Co-Authors: Toru Higuchi, Frank UhlmannAbstract:
Microtubules of the mitotic spindle form the structural basis for chromosome segregation. In metaphase, microtubules show high dynamic instability, which is thought to aid the ‘search and capture’ of chromosomes for bipolar alignment on the spindle. Microtubules suddenly become more stable at the onset of Anaphase, but how this change in microtubule behaviour is regulated and how important it is for the ensuing chromosome segregation are unknown1,2,3,4. Here we show that in the budding yeast Saccharomyces cerevisiae, activation of the phosphatase Cdc14 at Anaphase onset is both necessary and sufficient for silencing microtubule dynamics. Cdc14 is activated by separase, the protease that triggers sister chromatid separation, linking the onset of Anaphase to microtubule stabilization5,6. If sister chromatids separate in the absence of Cdc14 activity, microtubules maintain high dynamic instability; this correlates with defects in both the movement of chromosomes to the spindle poles (Anaphase A) and the elongation of the Anaphase spindle (Anaphase B). Cdc14 promotes localization of microtubule-stabilizing proteins to the Anaphase spindle, and dephosphorylation of the kinetochore component Ask1 contributes to both the silencing of microtubule turnover and successful Anaphase A.
Orchestrating Anaphase and mitotic exit: separase cleavage and localization of Slk19Nature Cell Biology, 2001Co-Authors: Matt Sullivan, Christine Lehane, Frank UhlmannAbstract:
Anaphase in budding yeast is triggered by cleavage of the central subunit, Scc1, of the chromosomal cohesin complex by the protease separase. Here we show that separase also cleaves the kinetochore-associated protein Slk19 at Anaphase onset. Separase activity is also required for the proper localization of a stable Slk19 cleavage product to the spindle midzone in Anaphase. The cleavage and localization of Slk19 are necessary to stabilize the Anaphase spindle, and we show that a stable spindle is a prerequisite for timely exit from mitosis. This demonstrates the cleavage of targets other than cohesin by separase in the orchestration of high-fidelity Anaphase.
characterization of fission yeast cohesin essential Anaphase proteolysis of rad21 phosphorylated in the s phaseGenes & Development, 2000Co-Authors: Takeshi Tomonaga, Koji Nagao, Yosuke Kawasaki, Kanji Furuya, Akiko Murakami, Jun Morishita, Tatsuro Yuasa, Takashi Sutani, Stephen E Kearsey, Frank UhlmannAbstract:
Cohesin complex acts in the formation and maintenance of sister chromatid cohesion during and after S phase. Budding yeast Scc1p/Mcd1p, an essential subunit, is cleaved and dissociates from chromosomes in Anaphase, leading to sister chromatid separation. Most cohesin in higher eukaryotes, in contrast, is dissociated from chromosomes well before Anaphase. The universal role of cohesin during Anaphase thus remains to be determined. We report here initial characterization of four putative cohesin subunits, Psm1, Psm3, Rad21, and Psc3, in fission yeast. They are essential for sister chromatid cohesion. Immunoprecipitation demonstrates stable complex formation of Rad21 with Psm1 and Psm3 but not with Psc3. Chromatin immunoprecipitation shows that cohesin subunits are enriched in broad centromere regions and that the level of centromere-associated Rad21 did not change from metaphase to Anaphase, very different from budding yeast. In contrast, Rad21 containing similar cleavage sites to those of Scc1p/Mcd1p is cleaved specifically in Anaphase. This cleavage is essential, although the amount of cleaved product is very small (<5%). Mis4, another sister chromatid cohesion protein, plays an essential role for loading Rad21 on chromatin. A simple model is presented to explain the specific behavior of fission yeast cohesin and why only a tiny fraction of Rad21 is sufficient to be cleaved for normal Anaphase.