Journal articles on the topic 'Mitotic delay'
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1
Boronat, Susanna, and Judith L. Campbell. "Mitotic Cdc6 Stabilizes Anaphase-Promoting Complex Substrates by a Partially Cdc28-Independent Mechanism, and This Stabilization Is Suppressed by Deletion of Cdc55." Molecular and Cellular Biology 27, no. 3 (November 27, 2006): 1158–71. http://dx.doi.org/10.1128/mcb.01745-05.
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ABSTRACT Ectopic expression of Cdc6p results in mitotic delay, and this has been attributed to Cdc6p-mediated inhibition of Cdc28 protein kinase and failure to activate the anaphase-promoting complex (APC). Here we show that endogenous Cdc6p delays a specific subset of mitotic events and that Cdc28 inhibition is not sufficient to account for it. The depletion of Cdc6p in G2/M cells reveals that Cdc6p is rate limiting for the degradation of the APC/Cdc20 substrates Pds1p and Clb2p. Conversely, the premature expression of Cdc6p delays the degradation of APC/Cdc20 substrates. Abolishing Cdc6p/Cdc28p interaction does not eliminate the Cdc6-dependent delay of these anaphase events. To identify additional Cdc6-mediated, APC-inhibitory mechanisms, we looked for mutants that reversed the mitotic delay. The deletion of SWE1, RAD24, MAD2, or BUB2 had no effect. However, disrupting CDC55, a PP2A regulatory subunit, suppressed the Cdc6p-dependent delay of Pds1 and Clb2 destruction. A specific role for CDC55 was supported by demonstrating that the lethality of Cdc6 ectopic expression in a cdc16-264 mutant is suppressed by the deletion of CDC55, that endogenous Cdc6p coimmunoprecipitates with the Cdc55 and Tpd3 subunits of PP2A, that Cdc6p/Cdc55p/Tpd3 interaction occurs only during mitosis, and that Cdc6 affects PP2A-Cdc55 activity during anaphase. This demonstrates that the levels and timing of accumulation of Cdc6p in mitosis are appropriate for mediating the modulation of APC/Cdc20.
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Kang, Dongmin, James Chen, Jim Wong, and Guowei Fang. "The checkpoint protein Chfr is a ligase that ubiquitinates Plk1 and inhibits Cdc2 at the G2 to M transition." Journal of Cell Biology 156, no. 2 (January 21, 2002): 249–60. http://dx.doi.org/10.1083/jcb.200108016.
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The checkpoint protein Chfr delays entry into mitosis, in the presence of mitotic stress (Scolnick, D.M., and T.D. Halazonetis. 2000. Nature. 406:430–435). We show here that Chfr is a ubiquitin ligase, both in vitro and in vivo. When transfected into HEK293T cells, Myc–Chfr promotes the formation of high molecular weight ubiquitin conjugates. The ring finger domain in Chfr is required for the ligase activity; this domain auto-ubiquitinates, and mutations of conserved residues in this domain abolish the ligase activity. Using Xenopus cell-free extracts, we demonstrated that Chfr delays the entry into mitosis by negatively regulating the activation of the Cdc2 kinase at the G2–M transition. Specifically, the Chfr pathway prolongs the phosphorylated state of tyrosine 15 in Cdc2. The Chfr-mediated cell cycle delay requires ubiquitin-dependent protein degradation, because inactivating mutations in Chfr, interference with poly-ubiquitination, and inhibition of proteasomes all abolish this delay in mitotic entry. The direct target of the Chfr pathway is Polo-like kinase 1 (Plk1). Ubiquitination of Plk1 by Chfr delays the activation of the Cdc25C phosphatase and the inactivation of the Wee1 kinase, leading to a delay in Cdc2 activation. Thus, the Chfr pathway represents a novel checkpoint pathway that regulates the entry into mitosis by ubiquitin-dependent proteolysis.
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Zachos, George, Michael D. Rainey, and David A. F. Gillespie. "Chk1-Dependent S-M Checkpoint Delay in Vertebrate Cells Is Linked to Maintenance of Viable Replication Structures." Molecular and Cellular Biology 25, no. 2 (January 15, 2005): 563–74. http://dx.doi.org/10.1128/mcb.25.2.563-574.2005.
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ABSTRACT We investigated mitotic delay during replication arrest (the S-M checkpoint) in DT40 B-lymphoma cells deficient in the Chk1 or Chk2 kinase. We show here that cells lacking Chk1, but not those lacking Chk2, enter mitosis with incompletely replicated DNA when DNA synthesis is blocked, but only after an initial delay. This initial delay persists when S-M checkpoint failure is induced in Chk2−/− cells with the Chk1 inhibitor UCN-01, indicating that it does not depend on Chk1 or Chk2 activity. Surprisingly, dephosphorylation of tyrosine 15 did not accompany Cdc2 activation during premature entry to mitosis in Chk1−/− cells, although mitotic phosphorylation of cyclin B2 did occur. Previous studies have shown that Chk1 is required to stabilize stalled replication forks during replication arrest, and strikingly, premature mitosis occurs only in Chk1-deficient cells which have lost the capacity to synthesize DNA as a result of progressive replication fork inactivation. These results suggest that Chk1 maintains the S-M checkpoint indirectly by preserving the viability of replication structures and that it is the continued presence of such structures, rather than the activation of Chk1 per se, which delays mitosis until DNA replication is complete.
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Gong, Delquin, and James E. Ferrell. "The Roles of Cyclin A2, B1, and B2 in Early and Late Mitotic Events." Molecular Biology of the Cell 21, no. 18 (September 15, 2010): 3149–61. http://dx.doi.org/10.1091/mbc.e10-05-0393.
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Here we have used siRNAs and time-lapse epifluorescence microscopy to examine the roles of various candidate mitotic cyclins in chromatin condensation in HeLa cells. Knocking down cyclin A2 resulted in a substantial (∼7 h) delay in chromatin condensation and histone H3 phosphorylation, and expressing an siRNA-resistant form of cyclin A2 partially rescued chromatin condensation. There was no detectable delay in DNA replication in the cyclin A2 knockdowns, arguing that the delay in chromatin condensation is not secondary to a delay in S-phase completion. Cyclin A2 is required for the activation and nuclear accumulation of cyclin B1-Cdk1, raising the possibility that cyclin B1-Cdk1 mediates the effects of cyclin A2. Consistent with this possibility, we found that chromatin condensation was tightly associated temporally with the redistribution of cyclin B1 to the nucleus. Moreover, a constitutively nuclear cyclin B1 rescued chromatin condensation in cyclin A2 knockdown cells. On the other hand, knocking down cyclin B1 delayed chromatin condensation by only about one hour. Our working hypothesis is that active, nuclear cyclin B1-Cdk1 normally cooperates with cyclin A2 to bring about early mitotic events. Because cyclin A2 is present only during the early stages of mitosis, we asked whether cyclin B knockdown might have more dramatic defects on late mitotic events. Consistent with this possibility, we found that cyclin B1- and cyclin B1/B2-knockdown cells had difficulty in maintaining a mitotic arrest in the presence of nocodazole. Taken together, these data suggest that cyclin A2 helps initiate mitosis, in part through its effects on cyclin B1, and that cyclins B1 and B2 are particularly critical for the maintenance of the mitotic state.
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Jelluma, Nannette, Tobias B. Dansen, Tale Sliedrecht, Nicholas P. Kwiatkowski, and Geert J. P. L. Kops. "Release of Mps1 from kinetochores is crucial for timely anaphase onset." Journal of Cell Biology 191, no. 2 (October 11, 2010): 281–90. http://dx.doi.org/10.1083/jcb.201003038.
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Mps1 kinase activity is required for proper chromosome segregation during mitosis through its involvements in microtubule–chromosome attachment error correction and the mitotic checkpoint. Mps1 dynamically exchanges on unattached kinetochores but is largely removed from kinetochores in metaphase. Here we show that Mps1 promotes its own turnover at kinetochores and that removal of Mps1 upon chromosome biorientation is a prerequisite for mitotic checkpoint silencing. Inhibition of Mps1 activity increases its half-time of recovery at unattached kinetochores and causes accumulation of Mps1 protein at these sites. Strikingly, preventing dissociation of active Mps1 from kinetochores delays anaphase onset despite normal chromosome attachment and alignment, and high interkinetochore tension. This delay is marked by continued recruitment of Mad1 and Mad2 to bioriented chromosomes and is attenuated by Mad2 depletion, indicating chronic engagement of the mitotic checkpoint in metaphase. We propose that release of Mps1 from kinetochores is essential for mitotic checkpoint silencing and a fast metaphase-to-anaphase transition.
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Szkotnicki, Lee, John M. Crutchley, Trevin R. Zyla, Elaine S. G. Bardes, and Daniel J. Lew. "The Checkpoint Kinase Hsl1p Is Activated by Elm1p-dependent Phosphorylation." Molecular Biology of the Cell 19, no. 11 (November 2008): 4675–86. http://dx.doi.org/10.1091/mbc.e08-06-0663.
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Saccharomyces cerevisiae cells growing in the outdoor environment must adapt to sudden changes in temperature and other variables. Many such changes trigger stress responses that delay bud emergence until the cells can adapt. In such circumstances, the morphogenesis checkpoint delays mitosis until a bud has been formed. Mitotic delay is due to the Wee1 family mitotic inhibitor Swe1p, whose degradation is linked to bud emergence by the checkpoint kinase Hsl1p. Hsl1p is concentrated at the mother-bud neck through association with septin filaments, and it was reported that Hsl1p activation involved relief of autoinhibition in response to septin interaction. Here we challenge the previous identification of an autoinhibitory domain and show instead that Hsl1p activation involves the phosphorylation of threonine 273, promoted by the septin-associated kinase Elm1p. We identified elm1 mutants in a screen for defects in Swe1p degradation and show that a phosphomimic T273E mutation in HSL1 bypasses the need for Elm1p in this pathway.
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Markossian, Sarine, Subbulakshmi Suresh, Aysha H. Osmani, and Stephen A. Osmani. "Nup2 requires a highly divergent partner, NupA, to fulfill functions at nuclear pore complexes and the mitotic chromatin region." Molecular Biology of the Cell 26, no. 4 (February 15, 2015): 605–21. http://dx.doi.org/10.1091/mbc.e14-09-1359.
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Chromatin and nuclear pore complexes (NPCs) undergo dramatic changes during mitosis, which in vertebrates and Aspergillus nidulans involves movement of Nup2 from NPCs to the chromatin region to fulfill unknown functions. This transition is shown to require the Cdk1 mitotic kinase and be promoted prematurely by ectopic expression of the NIMA kinase. Nup2 localizes with a copurifying partner termed NupA, a highly divergent yet essential NPC protein. NupA and Nup2 locate throughout the chromatin region during prophase but during anaphase move to surround segregating DNA. NupA function is shown to involve targeting Nup2 to its interphase and mitotic locations. Deletion of either Nup2 or NupA causes identical mitotic defects that initiate a spindle assembly checkpoint (SAC)–dependent mitotic delay and also cause defects in karyokinesis. These mitotic problems are not caused by overall defects in mitotic NPC disassembly–reassembly or general nuclear import. However, without Nup2 or NupA, although the SAC protein Mad1 locates to its mitotic locations, it fails to locate to NPCs normally in G1 after mitosis. Collectively the study provides new insight into the roles of Nup2 and NupA during mitosis and in a surveillance mechanism that regulates nucleokinesis when mitotic defects occur after SAC fulfillment.
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Muraoka-Cook, Rebecca S., Laura S. Caskey, Melissa A. Sandahl, Debra M. Hunter, Carty Husted, Karen E. Strunk, Carolyn I. Sartor, et al. "Heregulin-Dependent Delay in Mitotic Progression Requires HER4 and BRCA1." Molecular and Cellular Biology 26, no. 17 (September 1, 2006): 6412–24. http://dx.doi.org/10.1128/mcb.01950-05.
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ABSTRACT HER4 expression in human breast cancers correlates with a positive prognosis. While heregulin inhibits the growth of HER4-positive breast cancer cells, it does so by undefined mechanisms. We demonstrate that heregulin-induced HER4 activity inhibits cell proliferation and delays G2/M progression of breast cancer cells. While investigating pathways of G2/M delay, we noted that heregulin increased the expression of BRCA1 in a HER4-dependent, HER2-independent manner. Induction of BRCA1 by HER4 occurred independently of the cell cycle. Moreover, BRCA1 expression was elevated in HER4-postive human breast cancer specimens. Heregulin stimulated c-Jun N-terminal kinase (JNK), and pharmacologic inhibition of JNK impaired heregulin-enhanced expression of BRCA1 and mitotic delay; inhibition of Erk1/2 did not. Knockdown of BRCA1 with small interfering RNA in a human breast cancer cell line interfered with HER4-mediated mitotic delay. Heregulin/HER4-dependent mitotic delay was examined further with an isogenic pair of mouse mammary epithelial cells (MECs) derived from mice harboring homozygous LoxP sites flanking exon 11 of BRCA1, such that one cell line expressed BRCA1 while the other cell line, after Cre-mediated excision, did not. BRCA1-positive MECs displayed heregulin-dependent mitotic delay; however, the isogenic BRCA1-negative MECs did not. These results suggest that heregulin-mediated growth inhibition in HER4-postive breast cancer cells requires BRCA1.
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Petsalaki, Eleni, and George Zachos. "Chk2 prevents mitotic exit when the majority of kinetochores are unattached." Journal of Cell Biology 205, no. 3 (May 5, 2014): 339–56. http://dx.doi.org/10.1083/jcb.201310071.
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The spindle checkpoint delays exit from mitosis in cells with spindle defects. In this paper, we show that Chk2 is required to delay anaphase onset when microtubules are completely depolymerized but not in the presence of relatively few unattached kinetochores. Mitotic exit in Chk2-deficient cells correlates with reduced levels of Mps1 protein and increased Cdk1–tyrosine 15 inhibitory phosphorylation. Chk2 localizes to kinetochores and is also required for Aurora B–serine 331 phosphorylation in nocodazole or unperturbed early prometaphase. Serine 331 phosphorylation contributed to prometaphase accumulation in nocodazole after partial Mps1 inhibition and was required for spindle checkpoint establishment at the beginning of mitosis. In addition, expression of a phosphomimetic S331E mutant Aurora B rescued chromosome alignment or segregation in Chk2-deficient cells. We propose that Chk2 stabilizes Mps1 and phosphorylates Aurora B–serine 331 to prevent mitotic exit when most kinetochores are unattached. These results highlight mechanisms of an essential function of Chk2 in mitosis.
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Wang, Y., and D. J. Burke. "Checkpoint genes required to delay cell division in response to nocodazole respond to impaired kinetochore function in the yeast Saccharomyces cerevisiae." Molecular and Cellular Biology 15, no. 12 (December 1995): 6838–44. http://dx.doi.org/10.1128/mcb.15.12.6838.
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Inhibition of mitosis by antimitotic drugs is thought to occur by destruction of microtubules, causing cells to arrest through the action of one or more mitotic checkpoints. We have patterned experiments in the yeast Saccharomyces cerevisiae after recent studies in mammalian cells that demonstrate the effectiveness of antimitotic drugs at concentrations that maintain spindle structure. We show that low concentrations of nocodazole delay cell division under the control of the previously identified mitotic checkpoint genes BUB1, BUB3, MAD1, and MAD2 and independently of BUB2. The same genes mediate the cell cycle delay induced in ctf13 mutants, limited for an essential kinetochore component. Our data suggest that a low concentration of nocodazole induces a cell cycle delay through checkpoint control that is sensitive to impaired kinetochore function. The BUB2 gene may be part of a separate checkpoint that responds to abnormal spindle structure.
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Chippalkatti, Rohan, Boris Egger, and Beat Suter. "Mms19 promotes spindle microtubule assembly in Drosophila neural stem cells." PLOS Genetics 16, no. 11 (November 19, 2020): e1008913. http://dx.doi.org/10.1371/journal.pgen.1008913.
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Mitotic divisions depend on the timely assembly and proper orientation of the mitotic spindle. Malfunctioning of these processes can considerably delay mitosis, thereby compromising tissue growth and homeostasis, and leading to chromosomal instability. Loss of functional Mms19 drastically affects the growth and development of mitotic tissues in Drosophila larvae and we now demonstrate that Mms19 is an important factor that promotes spindle and astral microtubule (MT) growth, and MT stability and bundling. Mms19 function is needed for the coordination of mitotic events and for the rapid progression through mitosis that is characteristic of neural stem cells. Surprisingly, Mms19 performs its mitotic activities through two different pathways. By stimulating the mitotic kinase cascade, it triggers the localization of the MT regulatory complex TACC/Msps (Transforming Acidic Coiled Coil/Minispindles, the homolog of human ch-TOG) to the centrosome. This activity of Mms19 can be rescued by stimulating the mitotic kinase cascade. However, other aspects of the Mms19 phenotypes cannot be rescued in this way, pointing to an additional mechanism of Mms19 action. We provide evidence that Mms19 binds directly to MTs and that this stimulates MT stability and bundling.
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O'Regan, Laura, and Andrew M. Fry. "The Nek6 and Nek7 Protein Kinases Are Required for Robust Mitotic Spindle Formation and Cytokinesis." Molecular and Cellular Biology 29, no. 14 (May 4, 2009): 3975–90. http://dx.doi.org/10.1128/mcb.01867-08.
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ABSTRACT Nek6 and Nek7 are members of the NIMA-related serine/threonine kinase family. Previous work showed that they contribute to mitotic progression downstream of another NIMA-related kinase, Nek9, although the roles of these different kinases remain to be defined. Here, we carried out a comprehensive analysis of the regulation and function of Nek6 and Nek7 in human cells. By generating specific antibodies, we show that both Nek6 and Nek7 are activated in mitosis and that interfering with their activity by either depletion or expression of reduced-activity mutants leads to mitotic arrest and apoptosis. Interestingly, while completely inactive mutants and small interfering RNA-mediated depletion delay cells at metaphase with fragile mitotic spindles, hypomorphic mutants or RNA interference treatment combined with a spindle assembly checkpoint inhibitor delays cells at cytokinesis. Importantly, depletion of either Nek6 or Nek7 leads to defective mitotic progression, indicating that although highly similar, they are not redundant. Indeed, while both kinases localize to spindle poles, only Nek6 obviously localizes to spindle microtubules in metaphase and anaphase and to the midbody during cytokinesis. Together, these data lead us to propose that Nek6 and Nek7 play independent roles not only in robust mitotic spindle formation but also potentially in cytokinesis.
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Lin, Chiou-Hong, Chi-Kuo Hu, and Hsiu-Ming Shih. "Clathrin heavy chain mediates TACC3 targeting to mitotic spindles to ensure spindle stability." Journal of Cell Biology 189, no. 7 (June 21, 2010): 1097–105. http://dx.doi.org/10.1083/jcb.200911120.
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Mitotic spindles play essential roles in chromosome congression and segregation during mitosis. Aurora A regulates spindle assembly in part via phosphorylating human TACC3 on S558, which triggers TACC3 relocalization to mitotic spindles and stabilizes microtubules (MTs). In this study, we identified clathrin heavy chain (CHC) as an adaptor protein to recruit S558-phosphorylated TACC3 onto the spindle during mitosis for MT stabilization. CHC binds phospho-S558 TACC3 via its linker domain and first CHC repeat. CHC depletion or mutation on phospho-TACC3 binding abrogates TACC3 spindle relocalization. Depletion of either or both CHC and TACC3 yields similar defective phenotypes: loss of ch-TOG on spindles, disorganized spindles, and chromosome misalignment with comparable mitotic delay. Our findings elucidate the association between aurora A phosphorylation and spindle apparatus and demonstrate that regulation from aurora A is mediated by CHC in recruiting phospho-TACC3 and subsequently ch-TOG to mitotic spindles.
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Silva, Victoria C., and Lynne Cassimeris. "Stathmin and microtubules regulate mitotic entry in HeLa cells by controlling activation of both Aurora kinase A and Plk1." Molecular Biology of the Cell 24, no. 24 (December 15, 2013): 3819–31. http://dx.doi.org/10.1091/mbc.e13-02-0108.
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Depletion of stathmin, a microtubule (MT) destabilizer, delays mitotic entry by ∼4 h in HeLa cells. Stathmin depletion reduced the activity of CDC25 and its upstream activators, Aurora A and Plk1. Chemical inhibition of both Aurora A and Plk1 was sufficient to delay mitotic entry by 4 h, while inhibiting either kinase alone did not cause a delay. Aurora A and Plk1 are likely regulated downstream of stathmin, because the combination of stathmin knockdown and inhibition of Aurora A and Plk1 was not additive and again delayed mitotic entry by 4 h. Aurora A localization to the centrosome required MTs, while stathmin depletion spread its localization beyond that of γ-tubulin, indicating an MT-dependent regulation of Aurora A activation. Plk1 was inhibited by excess stathmin, detected in in vitro assays and cells overexpressing stathmin–cyan fluorescent protein. Recruitment of Plk1 to the centrosome was delayed in stathmin-depleted cells, independent of MTs. It has been shown that depolymerizing MTs with nocodazole abrogates the stathmin-depletion induced cell cycle delay; in this study, depolymerization with nocodazole restored Plk1 activity to near normal levels, demonstrating that MTs also contribute to Plk1 activation. These data demonstrate that stathmin regulates mitotic entry, partially via MTs, to control localization and activation of both Aurora A and Plk1.
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Colin, Didier J., Karolina O. Hain, Lindsey A. Allan, and Paul R. Clarke. "Cellular responses to a prolonged delay in mitosis are determined by a DNA damage response controlled by Bcl-2 family proteins." Open Biology 5, no. 3 (March 2015): 140156. http://dx.doi.org/10.1098/rsob.140156.
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Anti-cancer drugs that disrupt mitosis inhibit cell proliferation and induce apoptosis, although the mechanisms of these responses are poorly understood. Here, we characterize a mitotic stress response that determines cell fate in response to microtubule poisons. We show that mitotic arrest induced by these drugs produces a temporally controlled DNA damage response (DDR) characterized by the caspase-dependent formation of γH2AX foci in non-apoptotic cells. Following exit from a delayed mitosis, this initial response results in activation of DDR protein kinases, phosphorylation of the tumour suppressor p53 and a delay in subsequent cell cycle progression. We show that this response is controlled by Mcl-1, a regulator of caspase activation that becomes degraded during mitotic arrest. Chemical inhibition of Mcl-1 and the related proteins Bcl-2 and Bcl-x L by a BH3 mimetic enhances the mitotic DDR, promotes p53 activation and inhibits subsequent cell cycle progression. We also show that inhibitors of DDR protein kinases as well as BH3 mimetics promote apoptosis synergistically with taxol (paclitaxel) in a variety of cancer cell lines. Our work demonstrates the role of mitotic DNA damage responses in determining cell fate in response to microtubule poisons and BH3 mimetics, providing a rationale for anti-cancer combination chemotherapies.
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Sreenivasan, Aparna, and Douglas Kellogg. "The Elm1 Kinase Functions in a Mitotic Signaling Network in Budding Yeast." Molecular and Cellular Biology 19, no. 12 (December 1, 1999): 7983–94. http://dx.doi.org/10.1128/mcb.19.12.7983.
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ABSTRACT In budding yeast, the Clb2 mitotic cyclin initiates a signaling network that negatively regulates polar bud growth during mitosis. This signaling network appears to require the function of a Clb2-binding protein called Nap1, the Cdc42 GTPase, and two protein kinases called Gin4 and Cla4. In this study, we demonstrate that the Elm1 kinase also plays a role in the control of bud growth during mitosis. Cells carrying a deletion of the ELM1 gene undergo a prolonged mitotic delay, fail to negatively regulate polar bud growth during mitosis, and show defects in septin organization. In addition, Elm1 is required in vivo for the proper regulation of both the Cla4 and Gin4 kinases and interacts genetically with Cla4, Gin4, and the mitotic cyclins. Previous studies have suggested that Elm1 may function to negatively regulate the Swe1 kinase. To further understand the functional relationship between Elm1 and Swe1, we have characterized the phenotype of Δelm1 Δswe1 cells. We found that Δelm1 Δswe1 cells are inviable at 37°C and that a large proportion of Δelm1Δswe1 cells grown at 30°C contain multiple nuclei, suggesting severe defects in cytokinesis. In addition, we found that Elm1 is required for the normal hyperphosphorylation of Swe1 during mitosis. We propose a model in which the Elm1 kinase functions in a mitotic signaling network that controls events required for normal bud growth and cytokinesis, while the Swe1 kinase functions in a checkpoint pathway that delays nuclear division in response to defects in these events.
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Nelson, Scott A., and John A. Cooper. "A Novel Pathway that Coordinates Mitotic Exit with Spindle Position." Molecular Biology of the Cell 18, no. 9 (September 2007): 3440–50. http://dx.doi.org/10.1091/mbc.e07-03-0242.
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In budding yeast, the spindle position checkpoint (SPC) delays mitotic exit until the mitotic spindle moves into the neck between the mother and bud. This checkpoint works by inhibiting the mitotic exit network (MEN), a signaling cascade initiated and controlled by Tem1, a small GTPase. Tem1 is regulated by a putative guanine exchange factor, Lte1, but the function and regulation of Lte1 remains poorly understood. Here, we identify novel components of the checkpoint that operate upstream of Lte1. We present genetic evidence in agreement with existing biochemical evidence for the molecular mechanism of a pathway that links microtubule-cortex interactions with Lte1 and mitotic exit. Each component of this pathway is required for the spindle position checkpoint to delay mitotic exit until the spindle is positioned correctly.
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Carrino, J. J., and T. G. Laffler. "The effect of heat shock on the cell cycle regulation of tubulin expression in Physarum polycephalum." Journal of Cell Biology 100, no. 2 (February 1, 1985): 642–47. http://dx.doi.org/10.1083/jcb.100.2.642.
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In the myxomycete Physarum polycephalum, tubulin synthesis is subject to mitotic cycle control. Virtually all tubulin synthesis is limited to a 2-h period immediately preceding mitosis, and the peak of tubulin protein synthesis is accompanied by a parallel increase in the level of tubulin mRNA. The mechanism by which the accumulation of tubulin mRNA is turned on and off is not clear. To probe the relationship between tubulin regulation and cell cycle controls, we have used heat shocks to delay mitosis and have followed the pattern of tubulin synthesis during these delays. Two peaks of tubulin synthesis are observed after a heat shock. One occurs at a time when synthesis would have occurred without a heat shock, and a second peak immediately precedes the eventual delayed mitosis. These results are clearly due to altered cell cycle regulation. No mitotic activity is detected in delayed plasmodia at the time of the control mitosis, and tubulin behavior is shown to be clearly distinct from that of heat shock proteins. We believe that the tubulin family of proteins is subject to regulation by a thermolabile mitotic control mechanism but that once the cell has been committed to a round of tubulin synthesis the "tubulin clock" runs independently of the heat sensitive system. In delayed plasmodia, the second peak of synthesis may be turned on by a repeat of the commitment event.
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Takeda, Yutaka, Kaho Yamazaki, Kaho Hashimoto, Koki Watanabe, Takumi Chinen, and Daiju Kitagawa. "The centriole protein CEP76 negatively regulates PLK1 activity in the cytoplasm for proper mitotic progression." Journal of Cell Science 133, no. 19 (September 2, 2020): jcs241281. http://dx.doi.org/10.1242/jcs.241281.
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ABSTRACTPolo-like kinase 1 (PLK1) dynamically changes its localization and plays important roles in proper mitotic progression. In particular, strict control of cytoplasmic PLK1 is needed to prevent mitotic defects. However, the regulation of cytoplasmic PLK1 is not fully understood. In this study, we show that CEP76, a centriolar protein, physically interacts with PLK1 and tightly controls the activation of cytoplasmic PLK1 during mitosis in human cells. We found that removal of centrosomes induced ectopic aggregation of PLK1, which is highly phosphorylated, in the cytoplasm during mitosis. Importantly, a targeted RNAi screen revealed that depletion of CEP76 resulted in a similar phenotype. In addition, depletion of CEP76 caused defective spindle orientation and mitotic delay. Moreover, the formation of ectopic PLK1 aggregates and defective spindle orientation were significantly suppressed by the inhibition of PLK1 kinase activity. Overall, these results demonstrate that CEP76 suppresses the aberrant activation of cytoplasmic PLK1 for proper mitotic progression.This article has an associated First Person interview with the first author of the paper.
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Mall, Moritz, Thomas Walter, Mátyás Gorjánácz, Iain F. Davidson, Thi Bach Nga Ly-Hartig, Jan Ellenberg, and Iain W. Mattaj. "Mitotic lamin disassembly is triggered by lipid-mediated signaling." Journal of Cell Biology 198, no. 6 (September 17, 2012): 981–90. http://dx.doi.org/10.1083/jcb.201205103.
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Disassembly of the nuclear lamina is a key step during open mitosis in higher eukaryotes. The activity of several kinases, including CDK1 (cyclin-dependent kinase 1) and protein kinase C (PKC), has been shown to trigger mitotic lamin disassembly, yet their precise contributions are unclear. In this study, we develop a quantitative imaging assay to study mitotic lamin B1 disassembly in living cells. We find that CDK1 and PKC act in concert to mediate phosphorylation-dependent lamin B1 disassembly during mitosis. Using ribonucleic acid interference (RNAi), we showed that diacylglycerol (DAG)-dependent PKCs triggered rate-limiting steps of lamin disassembly. RNAi-mediated depletion or chemical inhibition of lipins, enzymes that produce DAG, delayed lamin disassembly to a similar extent as does PKC inhibition/depletion. Furthermore, the delay of lamin B1 disassembly after lipin depletion could be rescued by the addition of DAG. These findings suggest that lipins activate a PKC-dependent pathway during mitotic lamin disassembly and provide evidence for a lipid-mediated mitotic signaling event.
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Brock, J. A., and K. Bloom. "A chromosome breakage assay to monitor mitotic forces in budding yeast." Journal of Cell Science 107, no. 4 (April 1, 1994): 891–902. http://dx.doi.org/10.1242/jcs.107.4.891.
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During the eukaryotic cell cycle, genetic material must be accurately duplicated and faithfully segregated to each daughter cell. Segregation of chromosomes is dependent on the centromere, a region of the chromosome which interacts with mitotic spindle microtubules during cell division. Centromere function in the budding yeast, Saccharomyces cerevisiae, can be regulated by placing an inducible promotor adjacent to centromere DNA. This conditional centromere can be integrated into chromosome III to generate a conditionally functional dicentric chromosome. Activation of the dicentric chromosome results in a transient mitotic delay followed by the generation of monocentric derivatives. The propagation of viable cells containing these monocentric derivative chromosomes is dependent upon the DNA repair gene RAD52, indicating that double-strand DNA breaks are structural intermediates in the dicentric repair pathway. We have used these conditionally dicentric chromosomes to monitor the exertion of mitotic forces during cell division. Analysis of synchronized cells reveal that lethality in dicentric, rad52 mutant cells occurs during G2/M phase and is concomitant with the transient mitotic delay. the delay is largely dependent upon the cell cycle checkpoint gene RAD9, which is involved in monitoring DNA damage. These data demonstrate that DNA lesions resulting from dicentric activation are responsible for signalling the mitotic delay. Since the delay precedes the decline of p34cdc28 kinase activity, mitotic forces sufficient to result in dicentric chromosome breakage are generated prior to spindle elongation and anaphase onset in yeast.
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Kim, Mijin, Katie Murphy, Fang Liu, Sharon E. Parker, Melissa L. Dowling, Wesley Baff, and Gary D. Kao. "Caspase-Mediated Specific Cleavage of BubR1 Is a Determinant of Mitotic Progression." Molecular and Cellular Biology 25, no. 21 (November 1, 2005): 9232–48. http://dx.doi.org/10.1128/mcb.25.21.9232-9248.2005.
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ABSTRACT The fidelity of chromosomal duplication is monitored by cell cycle checkpoints operational during mitosis. One such cell cycle delay is invoked by microtubule-targeting agents such as nocodazole or paclitaxel (Taxol) and is mediated by mitotic checkpoint proteins that include BubR1. Relatively little is known about the regulation of expression and stability of BubR1 (or other checkpoint proteins) and how these factors dictate the durability of the cell cycle delay. We report here that treatment of HeLa cells with spindle-disrupting agents resulted in caspase activation and precipitated the cleavage of BubR1. This mechanism ultimately leads to reduced levels of full-length protein, which are accompanied by abrogation of the mitotic block; the checkpoint abrogation is substantially accelerated by inhibition of de novo protein synthesis. In contrast, inhibition of caspase activity blocked BubR1 degradation and prolonged mitosis. To confirm a direct link between caspase activity and BubR1 protein expression, we identified by site-directed mutagenesis the specific caspase cleavage sites cleaved after exposure to paclitaxel. Surprisingly, BubR1 has two sites of cleavage: primarily at Asp607/Asp610 and secondarily at Asp576/Asp579. BubR1 mutated at both locations (BubR1Δ579Δ610) was resistant to paclitaxel-induced degradation. Expression of BubR1Δ579Δ610 augmented the mitotic delay induced by spindle disruption in transfected cells as well as in clones engineered to inducibly express the mutant protein upon exposure to doxycycline and ultimately led to increased aneuploidy. Underscoring the importance of these caspase cleavage sites, both tetrapeptide motifs are identified in the amino acid sequences of human, mouse, chicken, and Xenopus BubR1. These results are potentially the first to link the control of the stability of a key mitotic checkpoint protein to caspase activation, a regulatory pathway that may be involved in killing defective cells and that has been evolutionarily conserved.
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Prigozhina, Natalie L., C. Elizabeth Oakley, Amanda M. Lewis, Tania Nayak, Stephen A. Osmani, and Berl R. Oakley. "γ-Tubulin Plays an Essential Role in the Coordination of Mitotic Events." Molecular Biology of the Cell 15, no. 3 (March 2004): 1374–86. http://dx.doi.org/10.1091/mbc.e03-06-0405.
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Recent data from multiple organisms indicate that γ-tubulin has essential, but incompletely defined, functions in addition to nucleating microtubule assembly. To investigate these functions, we examined the phenotype of mipAD159, a cold-sensitive allele of the γ-tubulin gene of Aspergillus nidulans. Immunofluorescence microscopy of synchronized material revealed that at a restrictive temperature mipAD159 does not inhibit mitotic spindle formation. Anaphase A was inhibited in many nuclei, however, and after a slight delay in mitosis (∼6% of the cell cycle period), most nuclei reentered interphase without dividing. In vivo observations of chromosomes at a restrictive temperature revealed that mipAD159 caused a failure of the coordination of late mitotic events (anaphase A, anaphase B, and chromosomal disjunction) and nuclei reentered interphase quickly even though mitosis was not completed successfully. Time-lapse microscopy also revealed that transient mitotic spindle abnormalities, in particular bent spindles, were more prevalent in mipAD159 strains than in controls. In experiments in which microtubules were depolymerized with benomyl, mipAD159 nuclei exited mitosis significantly more quickly (as judged by chromosomal condensation) than nuclei in a control strain. These data reveal that γ-tubulin has an essential role in the coordination of late mitotic events, and a microtubule-independent function in mitotic checkpoint control.
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den Elzen, Nicole, and Jonathon Pines. "Cyclin a Is Destroyed in Prometaphase and Can Delay Chromosome Alignment and Anaphase." Journal of Cell Biology 153, no. 1 (April 2, 2001): 121–36. http://dx.doi.org/10.1083/jcb.153.1.121.
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Mitosis is controlled by the specific and timely degradation of key regulatory proteins, notably the mitotic cyclins that bind and activate the cyclin-dependent kinases (Cdks). In animal cells, cyclin A is always degraded before cyclin B, but the exact timing and the mechanism underlying this are not known. Here we use live cell imaging to show that cyclin A begins to be degraded just after nuclear envelope breakdown. This degradation requires the 26S proteasome, but is not affected by the spindle checkpoint. Neither deletion of its destruction box nor disrupting Cdk binding prevents cyclin A proteolysis, but Cdk binding is necessary for degradation at the correct time. We also show that increasing the levels of cyclin A delays chromosome alignment and sister chromatid segregation. This delay depends on the proteolysis of cyclin A and is not caused by a lag in the bipolar attachment of chromosomes to the mitotic spindle, nor is it mediated via the spindle checkpoint. Thus, proteolysis that is not under the control of the spindle checkpoint is required for chromosome alignment and anaphase.
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Yunes, Sarah A., Jennifer L. S. Willoughby, Julian H. Kwan, Jessica M. Biagi, Niranjana Pokharel, Hang Gyeong Chin, Emily A. York, et al. "Factor quinolinone inhibitors disrupt spindles and multiple LSF (TFCP2)-protein interactions in mitosis, including with microtubule-associated proteins." PLOS ONE 17, no. 6 (June 15, 2022): e0268857. http://dx.doi.org/10.1371/journal.pone.0268857.
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Factor quinolinone inhibitors (FQIs), a first-in-class set of small molecule inhibitors targeted to the transcription factor LSF (TFCP2), exhibit promising cancer chemotherapeutic properties. FQI1, the initial lead compound identified, unexpectedly induced a concentration-dependent delay in mitotic progression. Here, we show that FQI1 can rapidly and reversibly lead to mitotic arrest, even when added directly to mitotic cells, implying that FQI1-mediated mitotic defects are not transcriptionally based. Furthermore, treatment with FQIs resulted in a striking, concentration-dependent diminishment of spindle microtubules, accompanied by a concentration-dependent increase in multi-aster formation. Aberrant γ-tubulin localization was also observed. These phenotypes suggest that perturbation of spindle microtubules is the primary event leading to the mitotic delays upon FQI1 treatment. Previously, FQIs were shown to specifically inhibit not only LSF DNA-binding activity, which requires LSF oligomerization to tetramers, but also other specific LSF-protein interactions. Other transcription factors participate in mitosis through non-transcriptional means, and we recently reported that LSF directly binds α-tubulin and is present in purified cellular tubulin preparations. Consistent with a microtubule role for LSF, here we show that LSF enhanced the rate of tubulin polymerization in vitro, and FQI1 inhibited such polymerization. To probe whether the FQI1-mediated spindle abnormalities could result from inhibition of mitotic LSF-protein interactions, mass spectrometry was performed using as bait an inducible, tagged form of LSF that is biotinylated by endogenous enzymes. The global proteomics analysis yielded expected associations for a transcription factor, notably with RNA processing machinery, but also to nontranscriptional components. In particular, and consistent with spindle disruption due to FQI treatment, mitotic, FQI1-sensitive interactions were identified between the biotinylated LSF and microtubule-associated proteins that regulate spindle assembly, positioning, and dynamics, as well as centrosome-associated proteins. Probing the mitotic LSF interactome using small molecule inhibitors therefore supported a non-transcriptional role for LSF in mediating progression through mitosis.
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Akhter, Shamima, Christopher T. Richie, Jian Min Deng, Eric Brey, Xiaoshan Zhang, Charles Patrick, Richard R. Behringer, and Randy J. Legerski. "Deficiency in SNM1 Abolishes an Early Mitotic Checkpoint Induced by Spindle Stress." Molecular and Cellular Biology 24, no. 23 (December 1, 2004): 10448–55. http://dx.doi.org/10.1128/mcb.24.23.10448-10455.2004.
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ABSTRACT Spindle poisons represent an important class of anticancer drugs that act by interfering with microtubule polymerization and dynamics and thereby induce mitotic checkpoints and apoptosis. Here we show that mammalian SNM1 functions in an early mitotic stress checkpoint that is distinct from the well-characterized spindle checkpoint that regulates the metaphase-to-anaphase transition. Specifically, we found that compared to wild-type cells, Snm1-deficient mouse embryonic fibroblasts exposed to spindle poisons exhibited elevated levels of micronucleus formation, decreased mitotic delay, a failure to arrest in mitosis prior to chromosome condensation, supernumerary centrosomes, and decreased viability. In addition, we show that both Snm1 and 53BP1, previously shown to interact, coimmunoprecipitate with components of the anaphase-promoting complex (APC)/cyclosome. These findings suggest that Snm1 is a component of a mitotic stress checkpoint that negatively targets the APC prior to chromosome condensation.
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Tanudji, Marcel, John Shoemaker, Lawrence L'Italien, Loren Russell, Gregory Chin, and Xiao Min Schebye. "Gene Silencing of CENP-E by Small Interfering RNA in HeLa Cells Leads to Missegregation of Chromosomes after a Mitotic Delay." Molecular Biology of the Cell 15, no. 8 (August 2004): 3771–81. http://dx.doi.org/10.1091/mbc.e03-07-0482.
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Centromeric protein-E (CENP-E) is a kinesin-like motor protein required for chromosome congression at prometaphase. Functional perturbation of CENP-E by various methods results in a consistent phenotype, i.e., unaligned chromosomes during mitosis. One unresolved question from previous studies is whether cells complete mitosis or sustain mitotic arrest in the presence of unaligned chromosomes. Using RNA interference and video-microscopy, we analyzed the dynamic process of mitotic progression of HeLa(H2B)-GFP cells lacking CENP-E. Our results demonstrate that these cells initiated anaphase after a delayed mitotic progression due to the presence of unaligned chromosomes. In some dividing cells, unaligned chromosomes are present during anaphase, causing nondisjunction of some sister chromatids producing aneuploid daughter cells. Unlike in Xenopus extract, the loss of CENP-E in HeLa cells does not impair gross checkpoint activation because cells were arrested in mitosis in response to microtubule-interfering agents. However, the lack of CENP-E at kinetochores reduced the hyperphosphorylation of BubR1 checkpoint protein during mitosis, which may explain the loss of sensitivity of a cell to a few unaligned chromosomes in the absence of CENP-E. We also found that presynchronization with nocodazole sensitizes cells to the depletion of CENP-E, leading to more unaligned chromosomes, longer arrest, and cell death.
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van de Weerdt, Barbara C. M., Marcel A. T. M. van Vugt, Catherine Lindon, Jos J. W. Kauw, Marieke J. Rozendaal, Rob Klompmaker, Rob M. F. Wolthuis, and René H. Medema. "Uncoupling Anaphase-Promoting Complex/Cyclosome Activity from Spindle Assembly Checkpoint Control by Deregulating Polo-Like Kinase 1." Molecular and Cellular Biology 25, no. 5 (March 1, 2005): 2031–44. http://dx.doi.org/10.1128/mcb.25.5.2031-2044.2005.
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ABSTRACT Polo-like kinase 1 (Plk1) plays a role in numerous events in mitosis, but how the multiple functions of Plk1 are separated is poorly understood. We studied regulation of Plk1 through two putative phosphorylation residues, Ser-137 and Thr-210. Using phospho-specific antibodies, we found that Thr-210 phosphorylation precedes Ser-137 phosphorylation in vivo, the latter occurring specifically in late mitosis. We show that expression of two activating mutants of these residues, S137D and T210D, results in distinct mitotic phenotypes. Whereas expression of both phospho-mimicking mutants as well as of the double mutant leads to accelerated mitotic entry, further progression through mitosis is dramatically different: the T210D mutant causes a spindle assembly checkpoint-dependent delay, whereas the expression of the S137D mutant or the double mutant results in untimely activation of the anaphase-promoting complex/cyclosome (APC/C) and frequent mitotic catastrophe. Using nonphosphorylatable Plk1-S137A and Plk1-T210A mutants, we show that both sites contribute to proper mitotic progression. Based on these observations, we propose that Plk1 function is altered at different stages of mitosis through consecutive posttranslational events, e.g., at Ser-137 and Thr-210. Furthermore, our data show that uncontrolled Plk1 activation can uncouple APC/C activity from spindle assembly checkpoint control.
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Shirnekhi, Hazheen K., Erin P. Kelley, Jennifer G. DeLuca, and Jacob A. Herman. "Spindle assembly checkpoint signaling and sister chromatid cohesion are disrupted by HPV E6-mediated transformation." Molecular Biology of the Cell 28, no. 15 (July 15, 2017): 2035–41. http://dx.doi.org/10.1091/mbc.e16-12-0853.
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Aneuploidy, a condition that results from unequal partitioning of chromosomes during mitosis, is a hallmark of many cancers, including those caused by human papillomaviruses (HPVs). E6 and E7 are the primary transforming proteins in HPV that drive tumor progression. In this study, we stably expressed E6 and E7 in noncancerous RPE1 cells and analyzed the specific mitotic defects that contribute to aneuploidy in each cell line. We find that E6 expression results in multiple chromosomes associated with one or both spindle poles, causing a significant mitotic delay. In most cells, the misaligned chromosomes eventually migrated to the spindle equator, leading to mitotic exit. In some cells, however, mitotic exit occurred in the presence of pole-associated chromosomes. We determined that this premature mitotic exit is due to defects in spindle assembly checkpoint (SAC) signaling, such that cells are unable to maintain a prolonged mitotic arrest in the presence of unaligned chromosomes. This SAC defect is caused in part by a loss of kinetochore-associated Mad2 in E6-expressing cells. Our results demonstrate that E6-expressing cells exhibit previously unappreciated mitotic defects that likely contribute to HPV-mediated cancer progression.
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Shiozaki, K., M. Shiozaki, and P. Russell. "Mcs4 mitotic catastrophe suppressor regulates the fission yeast cell cycle through the Wik1-Wis1-Spc1 kinase cascade." Molecular Biology of the Cell 8, no. 3 (March 1997): 409–19. http://dx.doi.org/10.1091/mbc.8.3.409.
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Spc1 in Schizosaccharomyces pombe is a member of the stress-activated protein kinase family, an evolutionary conserved subfamily of mitogen-activated protein kinases (MAPKs). Spc1 is activated by a MAPK kinase homologue, Wis1, and negatively regulated by Pyp1 and Pyp2 tyrosine phosphatases. Mutations in the spc1+ and wis1+ genes cause a G2 cell cycle delay that is exacerbated during stress. Herein, we describe two upstream regulators of the Wis1-Spc1 cascade. wik1+ (Wis1 kinase) was identified from its homology to budding yeast SSK2, which encodes a MAPKK kinase that regulates the HOG1 osmosensing pathway. Delta wik1 cells are impaired in stress-induced activation of Spc1 and show a G2 cell cycle delay and osmosensitive growth. Moreover, overproduction of a constitutively active form of Wik1 induces hyperactivation of Spc1 in wis1(+)-dependent manner, suggesting that Wik1 regulates Spc1 through activation of Wis1. A mutation of mcs4+ (mitotic catastrophe suppressor) was originally isolated as a suppressor of the mitotic catastrophe phenotype of a cdc2-3w wee1-50 double mutant. We have found that mcs4- cells are defective at activation of Spc1 in response to various forms of stress. Epistasis analysis has placed Mcs4-upstream of Wik1 in the Spc1 activation cascade. These results indicate that Mcs4 is part of a sensor system for multiple environmental signals that modulates the timing of entry into mitosis by regulating the Wik1-Wis1-Spc1 kinase cascade. Inactivation of the sensor system delays the onset of mitosis and rescues lethal premature mitosis in cdc2-3w wee1-50 cells.
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Krauss, Sharon Wald, Jeffrey R. Spence, Shirin Bahmanyar, Angela I. M. Barth, Minjoung M. Go, Debra Czerwinski, and Adam J. Meyer. "Downregulation of Protein 4.1R, a Mature Centriole Protein, Disrupts Centrosomes, Alters Cell Cycle Progression, and Perturbs Mitotic Spindles and Anaphase." Molecular and Cellular Biology 28, no. 7 (January 22, 2008): 2283–94. http://dx.doi.org/10.1128/mcb.02021-07.
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ABSTRACT Centrosomes nucleate and organize interphase microtubules and are instrumental in mitotic bipolar spindle assembly, ensuring orderly cell cycle progression with accurate chromosome segregation. We report that the multifunctional structural protein 4.1R localizes at centrosomes to distal/subdistal regions of mature centrioles in a cell cycle-dependent pattern. Significantly, 4.1R-specific depletion mediated by RNA interference perturbs subdistal appendage proteins ninein and outer dense fiber 2/cenexin at mature centrosomes and concomitantly reduces interphase microtubule anchoring and organization. 4.1R depletion causes G1 accumulation in p53-proficient cells, similar to depletion of many other proteins that compromise centrosome integrity. In p53-deficient cells, 4.1R depletion delays S phase, but aberrant ninein distribution is not dependent on the S-phase delay. In 4.1R-depleted mitotic cells, efficient centrosome separation is reduced, resulting in monopolar spindle formation. Multipolar spindles and bipolar spindles with misaligned chromatin are also induced by 4.1R depletion. Notably, all types of defective spindles have mislocalized NuMA (nuclear mitotic apparatus protein), a 4.1R binding partner essential for spindle pole focusing. These disruptions contribute to lagging chromosomes and aberrant microtubule bridges during anaphase/telophase. Our data provide functional evidence that 4.1R makes crucial contributions to the structural integrity of centrosomes and mitotic spindles which normally enable mitosis and anaphase to proceed with the coordinated precision required to avoid pathological events.
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Hixon, Mary L., Ana I. Flores, Mark W. Wagner, and Antonio Gualberto. "Ectopic Expression of cdc2/cdc28 Kinase Subunit Homo sapiens 1 Uncouples Cyclin B Metabolism from the Mitotic Spindle Cell Cycle Checkpoint." Molecular and Cellular Biology 18, no. 11 (November 1, 1998): 6224–37. http://dx.doi.org/10.1128/mcb.18.11.6224.
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ABSTRACT Primary human fibroblasts arrest growth in response to the inhibition of mitosis by mitotic spindle-depolymerizing drugs. We show that the mechanism of mitotic arrest is transient and implicates a decrease in the expression of cdc2/cdc28 kinase subunit Homo sapiens 1 (CKsHs1) and a delay in the metabolism of cyclin B. Primary human fibroblasts infected with a retroviral vector that drives the expression of a mutant p53 protein failed to downregulate CKsHs1 expression, degraded cyclin B despite the absence of chromosomal segregation, and underwent DNA endoreduplication. In addition, ectopic expression of CKsHs1 interfered with the control of cyclin B metabolism by the mitotic spindle cell cycle checkpoint and resulted in a higher tendency to undergo DNA endoreduplication. These results demonstrate that an altered regulation of CKsHs1 and cyclin B in cells that carry mutant p53 undermines the mitotic spindle cell cycle checkpoint and facilitates the development of aneuploidy. These data may contribute to the understanding of the origin of heteroploidy in mutant p53 cells.
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Farr, Katie A., and M. Andrew Hoyt. "Bub1p Kinase Activates the Saccharomyces cerevisiae Spindle Assembly Checkpoint." Molecular and Cellular Biology 18, no. 5 (May 1, 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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Schnerch, Dominik, Julia Felthaus, Monika Engelhardt, and Ralph M. Waesch. "Spindle Checkpoint Insufficiency and Unscheduled APC-Dependent Proteolysis in Acute Myeloid Leukemia." Blood 108, no. 11 (November 1, 2006): 2082. http://dx.doi.org/10.1182/blood.v108.11.2082.2082.
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Abstract Genetic instability including aneuploidy is frequent in most cancers. The spindle assembly checkpoint (SAC) is a mitotic surveillance mechanism responsible for accurate chromosome segregation. Unattached chromosomes or lack of spindle tension are sensed by the SAC. The activated SAC inhibits the ubiquitin-ligase anaphase-promoting complex (APC), which prevents the proteolysis of cell cycle regulators in order to delay progression through mitosis and allow cells to recover from defective mitotic spindle attachment. Spindle checkpoint malfunction proved to favor the generation of aneuploidy. In our recent work we investigated the roles of essential SAC proteins in acute myeloid leukemia (AML). We found the SAC-protein Bub1 to be posttranscriptionally downregulated in all investigated AML cell lines. As a consequence, after exposure to the microtubule disrupting agent nocodazole we observed a defective mitotic delay mechanism in comparison to SAC-competent cell lines and increased apoptosis consistent with the effects of Bub1 downregulation by RNA interference. At the molecular level we found a dramatic decline in mitotic regulator levels such as cyclin B1 and securin despite lasting spindle disruption. Additional data showed that the levels of these regulator proteins can be efficiently restored by exposure to the proteasome inhibitor MG-132 indicating that APC-dependent proteolysis is directly involved in SAC insufficiency. Thus, continuous activation of the APC triggers degradation of essential regulator proteins even in leukemic cells faced to mitotic stress such as complete spindle disruption. Such defects can lead to establishment of aneuploidy in vivo. Our findings emphasize a role of SAC insufficiency and unscheduled proteolysis in rise and progression of AML with complex karyotype.
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Rischitor, Patricia E., Sven Konzack, and Reinhard Fischer. "The Kip3-Like Kinesin KipB Moves along Microtubules and Determines Spindle Position during Synchronized Mitoses in Aspergillus nidulans Hyphae." Eukaryotic Cell 3, no. 3 (June 2004): 632–45. http://dx.doi.org/10.1128/ec.3.3.632-645.2004.
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ABSTRACT Kinesins are motor proteins which are classified into 11 different families. We identified 11 kinesin-like proteins in the genome of the filamentous fungus Aspergillus nidulans. Relatedness analyses based on the motor domains grouped them into nine families. In this paper, we characterize KipB as a member of the Kip3 family of microtubule depolymerases. The closest homologues of KipB are Saccharomyces cerevisiae Kip3 and Schizosaccharomyces pombe Klp5 and Klp6, but sequence similarities outside the motor domain are very low. A disruption of kipB demonstrated that it is not essential for vegetative growth. kipB mutant strains were resistant to high concentrations of the microtubule-destabilizing drug benomyl, suggesting that KipB destabilizes microtubules. kipB mutations caused a failure of spindle positioning in the cell, a delay in mitotic progression, an increased number of bent mitotic spindles, and a decrease in the depolymerization of cytoplasmic microtubules during interphase and mitosis. Meiosis and ascospore formation were not affected. Disruption of the kipB gene was synthetically lethal in combination with the temperature-sensitive mitotic kinesin motor mutation bimC4, suggesting an important but redundant role of KipB in mitosis. KipB localized to cytoplasmic, astral, and mitotic microtubules in a discontinuous pattern, and spots of green fluorescent protein moved along microtubules toward the plus ends.
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Kadauke, Stephan, Amy E. Campbell, Aaron J. Stonestrom, Deepti P. Jain, Ross C. Hardison, and Gerd A. Blobel. "GATA1 and the BET Family Protein Brd3 Form a Mitotic Bookmarking Complex." Blood 120, no. 21 (November 16, 2012): 282. http://dx.doi.org/10.1182/blood.v120.21.282.282.
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Abstract Abstract 282 Erythroid-specific transcription patterns are maintained throughout cell division. During mitosis, transcription is silenced globally. This raises the question whether mechanisms are in place that ensure the spatially and temporally correct reassembly of transcriptional regulators and thus maintain lineage fidelity. We recently found that, in contrast to most nuclear regulators, the master hematopoietic regulator GATA1 remains associated at a subset of its targets within mitotic chromosomes in erythroid cells (Kadauke et al., Cell 2012). GATA1 appears to function by creating an epigenetic “bookmark” to facilitate timely post-mitotic transcription reactivation of its mitotic target genes. GATA1 is acetylated at two lysine-rich domains near its zinc finger domains. We recently discovered that acetylated GATA1 recruits the double bromodomain protein Brd3 to erythroid target genes (Lamonica et al., PNAS 2011). Brd3 interacts with acetylated GATA1 via its first bromodomain, and Brd3 recruitment to GATA1 target sites is critically required for induction of terminal erythroid target genes such as α- and β-globin. Notably, Brd3 belongs to a family of proteins (called the BET family) of which two members (Brd2 and Brd4) are known to be retained on mitotic chromosomes. We now find by immunofluorescence and live cell confocal imaging that Brd3 globally binds to mitotic chromosomes. ChIP-seq experiments demonstrate a high degree of co-localization of Brd3 and GATA1 genome-wide both in interphase and in mitosis. We further demonstrate that GATA1 directly recruits Brd3 to mitotic GATA1 target sites. Transient mitosis-specific disruption of the Brd3-GATA1 interaction using the small molecule BET bromodomain inhibitor JQ1 removed Brd3, but not GATA1, from mitotic binding sites and led to a profound delay in the reactivation of GATA1-bookmarked genes. This suggests that Brd3 is an integral component of GATA1's bookmarking function. In concert, these studies support a requirement of mitotic bookmarking by a GATA1/Brd3 complex for the propagation of lineage-specific transcription programs in dividing erythroid cells. Disclosures: No relevant conflicts of interest to declare.
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Lee, Kyunghee, Alison E. Kenny, and Conly L. Rieder. "P38 Mitogen-activated Protein Kinase Activity Is Required during Mitosis for Timely Satisfaction of the Mitotic Checkpoint But Not for the Fidelity of Chromosome Segregation." Molecular Biology of the Cell 21, no. 13 (July 2010): 2150–60. http://dx.doi.org/10.1091/mbc.e10-02-0125.
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Although p38 activity is reported to be required as cells enter mitosis for proper spindle assembly and checkpoint function, its role during the division process remains controversial in lieu of direct data. We therefore conducted live cell studies to determine the effect on mitosis of inhibiting or depleting p38. We found that in the absence of p38 activity the duration of mitosis is prolonged by ∼40% in nontransformed human RPE-1, ∼80% in PtK2 (rat kangaroo), and ∼25% in mouse cells, and this prolongation leads to an elevated mitotic index. However, under this condition chromatid segregation and cytokinesis are normal. Using Mad2/YFP-expressing cells, we show the prolongation of mitosis in the absence of p38 activity is directly due to a delay in satisfying the mitotic checkpoint. Inhibiting p38 did not affect the rate of chromosome motion; however, it did lead to the formation of significantly (10%) longer metaphase spindles. From these data we conclude that normal p38 activity is required for the timely stable attachment of all kinetochores to spindle microtubules, but not for the fidelity of the mitotic process. We speculate that p38 activity promotes timely checkpoint satisfaction by indirectly influencing those motor proteins (e.g., Klp10, Klp67A) involved in regulating the dynamics of kinetochore microtubule ends.
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Maekawa, Hiromi, Claire Priest, Johannes Lechner, Gislene Pereira, and Elmar Schiebel. "The yeast centrosome translates the positional information of the anaphase spindle into a cell cycle signal." Journal of Cell Biology 179, no. 3 (October 29, 2007): 423–36. http://dx.doi.org/10.1083/jcb.200705197.
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The spindle orientation checkpoint (SPOC) of budding yeast delays mitotic exit when cytoplasmic microtubules (MTs) are defective, causing the spindle to become misaligned. Delay is achieved by maintaining the activity of the Bfa1–Bub2 guanosine triphosphatase–activating protein complex, an inhibitor of mitotic exit. In this study, we show that the spindle pole body (SPB) component Spc72, a transforming acidic coiled coil–like molecule that interacts with the γ-tubulin complex, recruits Kin4 kinase to both SPBs when cytoplasmic MTs are defective. This allows Kin4 to phosphorylate the SPB-associated Bfa1, rendering it resistant to inactivation by Cdc5 polo kinase. Consistently, forced targeting of Kin4 to both SPBs delays mitotic exit even when the anaphase spindle is correctly aligned. Moreover, we present evidence that Spc72 has an additional function in SPOC regulation that is independent of the recruitment of Kin4. Thus, Spc72 provides a missing link between cytoplasmic MT function and components of the SPOC.
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Martineau, S. N., P. R. Andreassen, and R. L. Margolis. "Delay of HeLa cell cleavage into interphase using dihydrocytochalasin B: retention of a postmitotic spindle and telophase disc correlates with synchronous cleavage recovery." Journal of Cell Biology 131, no. 1 (October 1, 1995): 191–205. http://dx.doi.org/10.1083/jcb.131.1.191.
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The molecular signals that determine the position and timing of the cleavage furrow during mammalian cell cytokinesis are presently unknown. We have studied in detail the effect of dihydrocytochalasin B (DCB), a drug that interferes with actin assembly, on specific late mitotic events in synchronous HeLa cells. When cleavage furrow formation is blocked at 10 microM DCB, cells return to interphase by the criteria of reformation of nuclei with lamin borders, degradation of the cyclin B component of p34cdc2 kinase, and loss of mitosis specific MPM-2 antigens. However, the machinery for cell cleavage is retained for up to one hour into G1 when cleavage cannot proceed. The components retained consist prominently of a "postmitotic" spindle and a telophase disc, a structure templated by the mitotic spindle in anaphase that may determine the position and timing of the cleavage furrow. Upon release from DCB block, G1 cells proceed through a rapid and synchronous cleavage. We conclude that the mitotic spindle is not inevitably destroyed at the end of mitosis, but persists as an integral structure with the telophase disc in the absence of cleavage. We also conclude that cell cleavage can occur in G1, and is therefore an event metabolically independent of mitosis. The retained telophase disc may indeed signal the position of furrow formation, as G1 cleavage occurs only in the position where the retained disc underlies the cell cortex. The protocol we describe should now enable development of a model system for the study of mammalian cell cleavage as a synchronous event independent of mitosis.
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Maldonado-Codina, G., S. Llamazares, and D. M. Glover. "Heat shock results in cell cycle delay and synchronisation of mitotic domains in cellularised Drosophila melanogaster embryos." Journal of Cell Science 105, no. 3 (July 1, 1993): 711–20. http://dx.doi.org/10.1242/jcs.105.3.711.
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Cells of Drosophila embryos that are subjected to a 37 degrees C temperature shock whilst undergoing the S-phase of cell cycle 14 arrest with their microtubules in an interphase-like state, and with nuclei showing unusual chromatin condensation. They do not recover from this state within a 30 minute period even though extensive gastrulation movements can occur. Cells of embryos heat shocked in G2-phase are delayed in interphase with high levels of cyclins A and B. Within ten minutes recovery from heat shock, cells enter mitosis throughout the embryo. The degradation of the mitotic cyclins A and B in these synchronised mitotic domains does not follow the normal timing, but is delayed. These findings point to a need for caution when interpreting experiments that use the heat shock promoter to study the expression of cell cycle control genes in Drosophila.
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Kishi, Kazuhiro, Marcel A. T. M. van Vugt, Ken-ichi Okamoto, Yasunori Hayashi, and Michael B. Yaffe. "Functional Dynamics of Polo-Like Kinase 1 at the Centrosome." Molecular and Cellular Biology 29, no. 11 (March 23, 2009): 3134–50. http://dx.doi.org/10.1128/mcb.01663-08.
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ABSTRACT Polo-like kinase 1 (Plk1) functions as a key regulator of mitotic events by phosphorylating substrate proteins on centrosomes, kinetochores, the mitotic spindle, and the midbody. Through mechanisms that are incompletely understood, Plk1 is released from and relocalizes to different mitotic structures as cells proceed through mitosis. We used fluorescence recovery after photobleaching to examine the kinetics of this process in more detail. We observed that Plk1 displayed a range of different recovery rates that differ at each mitotic substructure and depend on both the Polo-box domain and a functional kinase domain. Upon mitotic entry, centrosomal Plk1 becomes more dynamic, a process that is directly enhanced by Plk1 kinase activity. In contrast, Plk1 displays little dynamic exchange at the midbody, a process that again is modulated by the kinase activity of Plk1. Our findings suggest that the intrinsic kinase activity of Plk1 triggers its release from early mitotic structures and its relocalization to late mitotic structures. To assess the importance of Plk1 dynamic relocalization, Plk1 was persistently tethered to the centrosome. This resulted in a G2 delay, followed by a prominent prometaphase arrest, as a consequence of defective spindle formation and activation of the spindle checkpoint. The dynamic release of Plk1 from early mitotic structures is thus crucial for mid- to late-stage mitotic events and demonstrates the importance of a fully dynamic Plk1 at the centrosome for proper cell cycle progression. This dependence on dynamic Plk1 was further observed during the mitotic reentry of cells after a DNA damage G2 checkpoint, as this process was significantly delayed upon centrosomal tethering of Plk1. These results indicate that mitotic progression and control of mitotic reentry after DNA damage resides, at least in part, on the dynamic behavior of Plk1.
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GRAEM, NIELS, and KARIN HELWEG-LARSEN. "MITOTIC ACTIVITY AND DELAY IN FIXATION OF TUMOUR TISSUE." Acta Pathologica Microbiologica Scandinavica Section A Pathology 87A, no. 1-6 (August 15, 2009): 375–78. http://dx.doi.org/10.1111/j.1699-0463.1979.tb00065.x.
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Dewey, Evan B., and Christopher A. Johnston. "Diverse mitotic functions of the cytoskeletal cross-linking protein Shortstop suggest a role in Dynein/Dynactin activity." Molecular Biology of the Cell 28, no. 19 (September 15, 2017): 2555–68. http://dx.doi.org/10.1091/mbc.e17-04-0219.
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Proper assembly and orientation of the bipolar mitotic spindle is critical to the fidelity of cell division. Mitotic precision fundamentally contributes to cell fate specification, tissue development and homeostasis, and chromosome distribution within daughter cells. Defects in these events are thought to contribute to several human diseases. The underlying mechanisms that function in spindle morphogenesis and positioning remain incompletely defined, however. Here we describe diverse roles for the actin-microtubule cross-linker Shortstop (Shot) in mitotic spindle function in Drosophila. Shot localizes to mitotic spindle poles, and its knockdown results in an unfocused spindle pole morphology and a disruption of proper spindle orientation. Loss of Shot also leads to chromosome congression defects, cell cycle progression delay, and defective chromosome segregation during anaphase. These mitotic errors trigger apoptosis in Drosophila epithelial tissue, and blocking this apoptotic response results in a marked induction of the epithelial–mesenchymal transition marker MMP-1. The actin-binding domain of Shot directly interacts with Actin-related protein-1 (Arp-1), a key component of the Dynein/Dynactin complex. Knockdown of Arp-1 phenocopies Shot loss universally, whereas chemical disruption of F-actin does so selectively. Our work highlights novel roles for Shot in mitosis and suggests a mechanism involving Dynein/Dynactin activation.
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Ramírez-Valle, Francisco, Michelle L. Badura, Steve Braunstein, Manisha Narasimhan, and Robert J. Schneider. "Mitotic Raptor Promotes mTORC1 Activity, G2/M Cell Cycle Progression, and Internal Ribosome Entry Site-Mediated mRNA Translation." Molecular and Cellular Biology 30, no. 13 (May 3, 2010): 3151–64. http://dx.doi.org/10.1128/mcb.00322-09.
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ABSTRACT The mTOR signaling complex integrates signals from growth factors and nutrient availability to control cell growth and proliferation, in part through effects on the protein-synthetic machinery. Protein synthesis rates fluctuate throughout the cell cycle but diminish significantly during the G2/M transition. The fate of the mTOR complex and its role in coordinating cell growth and proliferation signals with protein synthesis during mitosis remain unknown. Here we demonstrate that the mTOR complex 1 (mTORC1) pathway, which stimulates protein synthesis, is actually hyperactive during mitosis despite decreased protein synthesis and reduced activity of mTORC1 upstream activators. We describe previously unknown G2/M-specific phosphorylation of a component of mTORC1, the protein raptor, and demonstrate that mitotic raptor phosphorylation alters mTORC1 function during mitosis. Phosphopeptide mapping and mutational analysis demonstrate that mitotic phosphorylation of raptor facilitates cell cycle transit through G2/M. Phosphorylation-deficient mutants of raptor cause cells to delay in G2/M, whereas depletion of raptor causes cells to accumulate in G1. We identify cyclin-dependent kinase 1 (cdk1 [cdc2]) and glycogen synthase kinase 3 (GSK3) pathways as two probable mitosis-regulated protein kinase pathways involved in mitosis-specific raptor phosphorylation and altered mTORC1 activity. In addition, mitotic raptor promotes translation by internal ribosome entry sites (IRES) on mRNA during mitosis and is demonstrated to be associated with rapamycin resistance. These data suggest that this pathway may play a role in increased IRES-dependent mRNA translation during mitosis and in rapamycin insensitivity.
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Jin, P., Y. Gu, and D. O. Morgan. "Role of inhibitory CDC2 phosphorylation in radiation-induced G2 arrest in human cells." Journal of Cell Biology 134, no. 4 (August 15, 1996): 963–70. http://dx.doi.org/10.1083/jcb.134.4.963.
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The activity of the mitosis-promoting kinase CDC2-cyclin B is normally suppressed in S phase and G2 by inhibitory phosphorylation at Thr14 and Tyr15. This work explores the possibility that these phosphorylations are responsible for the G2 arrest that occurs in human cells after DNA damage. HeLa cell lines were established in which CDC2AF, a mutant that cannot be phosphorylated at Thr14 and Tyr15, was expressed from a tetracycline-repressible promoter. Expression of CDC2AF did not induce mitotic events in cells arrested at the beginning of S phase with DNA synthesis inhibitors, but induced low levels of premature chromatin condensation in cells progressing through S phase and G2. Expression of CDC2AF greatly reduced the G2 delay that resulted when cells were X-irradiated in S phase. However, a significant G2 delay was still observed and was accompanied by high CDC2-associated kinase activity. Expression of wild-type CDC2, or the related kinase CDK2AF, had no effect on the radiation-induced delay. Thus, inhibitory phosphorylation of CDC2, as well as additional undefined mechanisms, delay mitosis after DNA damage.
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Althoff, Friederike, Roger E. Karess, and Christian F. Lehner. "Spindle checkpoint–independent inhibition of mitotic chromosome segregation byDrosophilaMps1." Molecular Biology of the Cell 23, no. 12 (June 15, 2012): 2275–91. http://dx.doi.org/10.1091/mbc.e12-02-0117.
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Monopolar spindle 1 (Mps1) is essential for the spindle assembly checkpoint (SAC), which prevents anaphase onset in the presence of misaligned chromosomes. Moreover, Mps1 kinase contributes in a SAC-independent manner to the correction of erroneous initial attachments of chromosomes to the spindle. Our characterization of the Drosophila homologue reveals yet another SAC-independent role. As in yeast, modest overexpression of Drosophila Mps1 is sufficient to delay progression through mitosis during metaphase, even though chromosome congression and metaphase alignment do not appear to be affected. This delay in metaphase depends on the SAC component Mad2. Although Mps1 overexpression in mad2 mutants no longer causes a metaphase delay, it perturbs anaphase. Sister kinetochores barely move apart toward spindle poles. However, kinetochore movements can be restored experimentally by separase-independent resolution of sister chromatid cohesion. We propose therefore that Mps1 inhibits sister chromatid separation in a SAC-independent manner. Moreover, we report unexpected results concerning the requirement of Mps1 dimerization and kinase activity for its kinetochore localization in Drosophila. These findings further expand Mps1's significance for faithful mitotic chromosome segregation and emphasize the importance of its careful regulation.
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Lindqvist, Arne, Helena Källström, Andreas Lundgren, Emad Barsoum, and Christina Karlsson Rosenthal. "Cdc25B cooperates with Cdc25A to induce mitosis but has a unique role in activating cyclin B1–Cdk1 at the centrosome." Journal of Cell Biology 171, no. 1 (October 10, 2005): 35–45. http://dx.doi.org/10.1083/jcb.200503066.
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Cdc25 phosphatases are essential for the activation of mitotic cyclin–Cdks, but the precise roles of the three mammalian isoforms (A, B, and C) are unclear. Using RNA interference to reduce the expression of each Cdc25 isoform in HeLa and HEK293 cells, we observed that Cdc25A and -B are both needed for mitotic entry, whereas Cdc25C alone cannot induce mitosis. We found that the G2 delay caused by small interfering RNA to Cdc25A or -B was accompanied by reduced activities of both cyclin B1–Cdk1 and cyclin A–Cdk2 complexes and a delayed accumulation of cyclin B1 protein. Further, three-dimensional time-lapse microscopy and quantification of Cdk1 phosphorylation versus cyclin B1 levels in individual cells revealed that Cdc25A and -B exert specific functions in the initiation of mitosis: Cdc25A may play a role in chromatin condensation, whereas Cdc25B specifically activates cyclin B1–Cdk1 on centrosomes.
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Carroll, Christopher W., Roger Altman, David Schieltz, John R. Yates, and Douglas Kellogg. "The Septins Are Required for the Mitosis-specific Activation of the Gin4 Kinase." Journal of Cell Biology 143, no. 3 (November 2, 1998): 709–17. http://dx.doi.org/10.1083/jcb.143.3.709.
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In budding yeast, a protein kinase called Gin4 is specifically activated during mitosis and functions in a pathway initiated by the Clb2 cyclin to control bud growth. We have used genetics and biochemistry to identify additional proteins that function with Gin4 in this pathway, and both of these approaches have identified members of the septin family. Loss of septin function produces a phenotype that is very similar to the phenotype caused by loss of Gin4 function, and the septins are required early in mitosis to activate Gin4 kinase activity. Furthermore, septin mutants display a prolonged mitotic delay at the short spindle stage, consistent with a role for the septins in the control of mitotic events. Members of the septin family bind directly to Gin4, demonstrating that the functions of Gin4 and the septins must be closely linked within the cell. These results demonstrate that the septins in budding yeast play an integral role in the mitosis-specific regulation of the Gin4 kinase and that they carry out functions early in mitosis.
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Krause, Sue A., Marie-Louise Loupart, Sharron Vass, Stefan Schoenfelder, Steve Harrison, and Margarete M. S. Heck. "Loss of Cell Cycle Checkpoint Control in Drosophila Rfc4 Mutants." Molecular and Cellular Biology 21, no. 15 (August 1, 2001): 5156–68. http://dx.doi.org/10.1128/mcb.21.15.5156-5168.2001.
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ABSTRACT Two alleles of the Drosophila melanogaster Rfc4(DmRfc4) gene, which encodes subunit 4 of the replication factor C (RFC) complex, cause striking defects in mitotic chromosome cohesion and condensation. These mutations produce larval phenotypes consistent with a role in DNA replication but also result in mitotic chromosomal defects appearing either as premature chromosome condensation-like or precocious sister chromatid separation figures. Though the DmRFC4 protein localizes to all replicating nuclei, it is dispersed from chromatin in mitosis. Thus the mitotic defects appear not to be the result of a direct role for RFC4 in chromosome structure. We also show that the mitotic defects in these twoDmRfc4 alleles are the result of aberrant checkpoint control in response to DNA replication inhibition or damage to chromosomes. Not all surveillance function is compromised in these mutants, as the kinetochore attachment checkpoint is operative. Intriguingly, metaphase delay is frequently observed with the more severe of the two alleles, indicating that subsequent chromosome segregation may be inhibited. This is the first demonstration that subunit 4 of RFC functions in checkpoint control in any organism, and our findings additionally emphasize the conserved nature of RFC's involvement in checkpoint control in multicellular eukaryotes.
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Ruden, D. M., and H. Jackle. "Mitotic delay dependent survival identifies components of cell cycle control in the Drosophila blastoderm." Development 121, no. 1 (January 1, 1995): 63–73. http://dx.doi.org/10.1242/dev.121.1.63.
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The Drosophila body pattern is laid down by maternal and zygotic factors which act during the early phase of embryonic development. During this period, nascent zygotic transcripts longer than about 6 kilobases are aborted between the rapid mitotic cycles. Resurrector1 (Res1) and Godzilla1 (God1), two newly identified dominant zygotic suppressor mutations, and a heterozygous maternal deficiency of the cyclin B locus, complement the partial loss of function of the segmentation gene knirps (kni) by extending the length of mitotic cycles at blastoderm. The mitotic delay caused by Res1 and God1 zygotically and by the deficiency of the cyclin B locus maternally allows the expression of a much longer transcript of a kni cognate gene normally aborted between the short mitotic cycles and consequently allows survival of kni mutant progeny. In addition to the practical benefits of identifying mutations in Drosophila cell cycle regulatory genes as suppressors of kni, our results have evolutionary implications regarding the flexibility of the genome to meet sudden selective pressures by recruiting cognate genes to function.