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The Journal of Cell Biology Mar 2018The mitotic spindle checkpoint delays anaphase onset in the presence of unattached kinetochores, and efficient checkpoint signaling requires kinetochore localization of...
The mitotic spindle checkpoint delays anaphase onset in the presence of unattached kinetochores, and efficient checkpoint signaling requires kinetochore localization of the Rod-ZW10-Zwilch (RZZ) complex. In the present study, we show that human Chmp4c, a protein involved in membrane remodeling, localizes to kinetochores in prometaphase but is reduced in chromosomes aligned at the metaphase plate. Chmp4c promotes stable kinetochore-microtubule attachments and is required for proper mitotic progression, faithful chromosome alignment, and segregation. Depletion of Chmp4c diminishes localization of RZZ and Mad1-Mad2 checkpoint proteins to prometaphase kinetochores and impairs mitotic arrest when microtubules are depolymerized by nocodazole. Furthermore, Chmp4c binds to ZW10 through a small C-terminal region, and constitutive Chmp4c kinetochore targeting causes a ZW10-dependent checkpoint metaphase arrest. In addition, Chmp4c spindle functions do not require endosomal sorting complex required for transport-dependent membrane remodeling. These results show that Chmp4c regulates the mitotic spindle checkpoint by promoting localization of the RZZ complex to unattached kinetochores.
Topics: Cell Cycle Checkpoints; Cell Cycle Proteins; Endosomal Sorting Complexes Required for Transport; HeLa Cells; Humans; Kinetochores; Mad2 Proteins; Nuclear Proteins; Signal Transduction; Spindle Apparatus
PubMed: 29362225
DOI: 10.1083/jcb.201709005 -
Genome Research Feb 2019CCCTC-binding factor (CTCF) plays a key role in the formation of topologically associating domains (TADs) and loops in interphase. During mitosis TADs are absent, but...
CCCTC-binding factor (CTCF) plays a key role in the formation of topologically associating domains (TADs) and loops in interphase. During mitosis TADs are absent, but how TAD formation is dynamically controlled during the cell cycle is not known. Several contradicting observations have been made regarding CTCF binding to mitotic chromatin using both genomics- and microscopy-based techniques. Here, we have used four different assays to address this debate. First, using 5C, we confirmed that TADs and CTCF loops are readily detected in interphase, but absent during prometaphase. Second, ATAC-seq analysis showed that CTCF sites display greatly reduced accessibility and lose the CTCF footprint in prometaphase, suggesting loss of CTCF binding and rearrangement of the nucleosomal array around the binding motif. In contrast, transcription start sites remain accessible in prometaphase, although adjacent nucleosomes can also become repositioned and occupy at least a subset of start sites during mitosis. Third, loss of site-specific CTCF binding was directly demonstrated using CUT&RUN. Histone modifications and histone variants are maintained in mitosis, suggesting a role in bookmarking of active CTCF sites. Finally, live-cell imaging, fluorescence recovery after photobleaching, and single molecule tracking showed that almost all CTCF chromatin binding is lost in prometaphase. Combined, our results demonstrate loss of CTCF binding to CTCF sites during prometaphase and rearrangement of the chromatin landscape around CTCF motifs. This, combined with loss of cohesin, would contribute to the observed loss of TADs and CTCF loops during mitosis and reveals that CTCF sites, key architectural -elements, display cell cycle stage-dependent dynamics in factor binding and nucleosome positioning.
Topics: Binding Sites; CCCTC-Binding Factor; Cell Cycle; Cells, Cultured; Chromatin; HeLa Cells; Histone Code; Humans; Interphase; Mitosis; Nucleosomes; Nucleotide Motifs; Prometaphase; Transcription Initiation Site
PubMed: 30655336
DOI: 10.1101/gr.241547.118 -
Archives of Biochemistry and Biophysics Jun 2011At mitosis, cells undergo drastic alterations in morphology and cytoskeletal organization including cell rounding during prophase, mitotic spindle assembly during... (Review)
Review
At mitosis, cells undergo drastic alterations in morphology and cytoskeletal organization including cell rounding during prophase, mitotic spindle assembly during prometaphase and metaphase, chromatid segregation in anaphase, and cytokinesis during telophase. It is well established that myosin II is a motor responsible for cytokinesis. Recent reports have indicated that myosin II is also involved in spindle assembly and karyokinesis. In this review, we summarize current understanding of the functions of myosin II in mitosis and cytokinesis of higher eukaryotes, and discuss the roles of possible upstream molecules that control myosin II in these mitotic events.
Topics: Animals; Cytokinesis; Humans; Mitosis; Myosin Type II; Myosin-Light-Chain Kinase; Myosin-Light-Chain Phosphatase
PubMed: 21396909
DOI: 10.1016/j.abb.2011.03.002 -
Cell Reports Jul 2021Stable transmission of genetic material during cell division requires accurate chromosome segregation. PLK1 dynamics at kinetochores control establishment of correct...
Stable transmission of genetic material during cell division requires accurate chromosome segregation. PLK1 dynamics at kinetochores control establishment of correct kinetochore-microtubule attachments and subsequent silencing of the spindle checkpoint. However, the regulatory mechanism responsible for PLK1 activity in prometaphase has not yet been affirmatively identified. Here we identify Apolo1, which tunes PLK1 activity for accurate kinetochore-microtubule attachments. Apolo1 localizes to kinetochores during early mitosis, and suppression of Apolo1 results in misaligned chromosomes. Using the fluorescence resonance energy transfer (FRET)-based PLK1 activity reporter, we found that Apolo1 sustains PLK1 kinase activity at kinetochores for accurate attachment during prometaphase. Apolo1 is a cognate substrate of PLK1, and the phosphorylation enables PP1γ to inactivate PLK1 by dephosphorylation. Mechanistically, Apolo1 constitutes a bridge between kinase and phosphatase, which governs PLK1 activity in prometaphase. These findings define a previously uncharacterized feedback loop by which Apolo1 provides fine-tuning for PLK1 to guide chromosome segregation in mitosis.
Topics: Amino Acid Motifs; Amino Acid Sequence; Cell Cycle Proteins; Chromosome Segregation; Feedback, Physiological; HEK293 Cells; HeLa Cells; Humans; Kinetochores; Mitosis; Phosphoprotein Phosphatases; Phosphorylation; Phosphoserine; Protein Binding; Protein Serine-Threonine Kinases; Proteins; Proto-Oncogene Proteins; Polo-Like Kinase 1
PubMed: 34260926
DOI: 10.1016/j.celrep.2021.109343 -
The Journal of Cell Biology Oct 2017Cyclin A2 is a crucial mitotic Cdk regulatory partner that coordinates entry into mitosis and is then destroyed in prometaphase within minutes of nuclear envelope...
Cyclin A2 is a crucial mitotic Cdk regulatory partner that coordinates entry into mitosis and is then destroyed in prometaphase within minutes of nuclear envelope breakdown. The role of cyclin A2 in female meiosis and its dynamics during the transition from meiosis I (MI) to meiosis II (MII) remain unclear. We found that cyclin A2 decreases in prometaphase I but recovers after the first meiotic division and persists, uniquely for metaphase, in MII-arrested oocytes. Conditional deletion of cyclin A2 from mouse oocytes has no discernible effect on MI but leads to disrupted MII spindles and increased merotelic attachments. On stimulation of exit from MII, there is a dramatic increase in lagging chromosomes and an inhibition of cytokinesis. These defects are associated with an increase in microtubule stability in MII spindles, suggesting that cyclin A2 mediates the fidelity of MII by maintaining microtubule dynamics during the rapid formation of the MII spindle.
Topics: Animals; Chromosomes, Mammalian; Cyclin A2; Kinetochores; Meiosis; Mice; Mice, Knockout; Microtubules
PubMed: 28819014
DOI: 10.1083/jcb.201607111 -
The Journal of Cell Biology Jan 2010We tested the classical hypothesis that astral, prometaphase bipolar mitotic spindles are maintained by balanced outward and inward forces exerted on spindle poles by...
We tested the classical hypothesis that astral, prometaphase bipolar mitotic spindles are maintained by balanced outward and inward forces exerted on spindle poles by kinesin-5 and -14 using modeling of in vitro and in vivo data from Drosophila melanogaster embryos. Throughout prometaphase, puncta of both motors aligned on interpolar microtubules (MTs [ipMTs]), and motor perturbation changed spindle length, as predicted. Competitive motility of purified kinesin-5 and -14 was well described by a stochastic, opposing power stroke model incorporating motor kinetics and load-dependent detachment. Motor parameters from this model were applied to a new stochastic force-balance model for prometaphase spindles, providing a good fit to data from embryos. Maintenance of virtual spindles required dynamic ipMTs and a narrow range of kinesin-5 to kinesin-14 ratios matching that found in embryos. Functional perturbation and modeling suggest that this range can be extended significantly by a disassembling lamin-B envelope that surrounds the prometaphase spindle and augments the finely tuned, antagonistic kinesin force balance to maintain robust prometaphase spindles as MTs assemble and chromosomes are pushed to the equator.
Topics: Animals; Biophysical Phenomena; Drosophila Proteins; Drosophila melanogaster; Kinesins; Lamin Type B; Microtubule-Associated Proteins; Models, Biological; Prometaphase; Spindle Apparatus
PubMed: 20065089
DOI: 10.1083/jcb.200908150 -
The Journal of Cell Biology Apr 2001Mitosis is controlled by the specific and timely degradation of key regulatory proteins, notably the mitotic cyclins that bind and activate the cyclin-dependent kinases...
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.
Topics: Anaphase; Biomarkers; Chromosomes, Human; Cyclin A; Cyclin-Dependent Kinases; Green Fluorescent Proteins; HeLa Cells; Humans; Luminescent Proteins; Metaphase; Mitosis; Recombinant Fusion Proteins
PubMed: 11285279
DOI: 10.1083/jcb.153.1.121 -
EMBO Reports Jun 2020The anaphase-promoting complex (APC/C) is the key E3 ubiquitin ligase which directs mitotic progression and exit by catalysing the sequential ubiquitination of specific...
The anaphase-promoting complex (APC/C) is the key E3 ubiquitin ligase which directs mitotic progression and exit by catalysing the sequential ubiquitination of specific substrates. The activity of the APC/C in mitosis is restrained by the spindle assembly checkpoint (SAC), which coordinates chromosome segregation with the assembly of the mitotic spindle. The SAC effector is the mitotic checkpoint complex (MCC), which binds and inhibits the APC/C. It is incompletely understood how the APC/C switches substrate specificity in a cell cycle-specific manner. For instance, it is unclear how in prometaphase, when APC/C activity towards cyclin B and securin is repressed by the MCC, the kinase Nek2A is ubiquitinated. Here, we combine biochemical and structural analysis with functional studies in cells to show that Nek2A is a conformational-specific binder of the APC/C-MCC complex (APC/C ) and that, in contrast to cyclin A, Nek2A can be ubiquitinated efficiently by the APC/C in conjunction with both the E2 enzymes UbcH10 and UbcH5. We propose that these special features of Nek2A allow its prometaphase-specific ubiquitination.
Topics: Anaphase-Promoting Complex-Cyclosome; Cdc20 Proteins; Cell Cycle Proteins; HeLa Cells; Humans; M Phase Cell Cycle Checkpoints; Mitosis; Prometaphase; Spindle Apparatus; Ubiquitination
PubMed: 32307883
DOI: 10.15252/embr.201949831 -
Proceedings of the National Academy of... Jun 2020Topoisomerase IIα (TOP2A) is a core component of mitotic chromosomes and important for establishing mitotic chromosome condensation. The primary roles of TOP2A in...
Topoisomerase IIα (TOP2A) is a core component of mitotic chromosomes and important for establishing mitotic chromosome condensation. The primary roles of TOP2A in mitosis have been difficult to decipher due to its multiple functions across the cell cycle. To more precisely understand the role of TOP2A in mitosis, we used the auxin-inducible degron (AID) system to rapidly degrade the protein at different stages of the human cell cycle. Removal of TOP2A prior to mitosis does not affect prophase timing or the initiation of chromosome condensation. Instead, it prevents chromatin condensation in prometaphase, extends the length of prometaphase, and ultimately causes cells to exit mitosis without chromosome segregation occurring. Surprisingly, we find that removal of TOP2A from cells arrested in prometaphase or metaphase cause dramatic loss of compacted mitotic chromosome structure and conclude that TOP2A is crucial for maintenance of mitotic chromosomes. Treatments with drugs used to poison/inhibit TOP2A function, such as etoposide and ICRF-193, do not phenocopy the effects on chromosome structure of TOP2A degradation by AID. Our data point to a role for TOP2A as a structural chromosome maintenance enzyme locking in condensation states once sufficient compaction is achieved.
Topics: Chromosome Segregation; Chromosome Structures; Chromosomes, Human; Cytokinesis; DNA Topoisomerases, Type II; HCT116 Cells; Heterochromatin; Humans; Metaphase; Mitosis
PubMed: 32414923
DOI: 10.1073/pnas.2001760117 -
Cytoskeleton (Hoboken, N.J.) Nov 2019Chromosome segregation is mediated by spindle microtubules that attach to the kinetochore via dynamic protein complexes, such as Ndc80, Ska, Cdt1 and ch-TOG during...
Chromosome segregation is mediated by spindle microtubules that attach to the kinetochore via dynamic protein complexes, such as Ndc80, Ska, Cdt1 and ch-TOG during mitotic metaphase. While experimental studies have previously shown that these proteins and protein complexes are all essential for maintaining a stable kinetochore-microtubule (kMT) interface, their exact roles in the mitotic metaphase remains elusive. In this study, we employed experimental and computational methods in order to characterize how these proteins can strengthen kMT attachments in both nonload-bearing and load-bearing conditions, typical of prometaphase and metaphase, respectively. Immunofluorescence staining of HeLa cells showed that the levels of Ska and Cdt1 significantly increased from prometaphase to metaphase, while levels of the Ndc80 complex remained unchanged. Our new computational model showed that by incorporating binding and unbinding of each protein complex coupled with a biased diffusion mechanism, the displacement of a possible complex formed by Ndc80-Ska-Cdt1 is significantly higher than that of Ndc80 alone or Ndc80-Ska. In addition, when we incorporate Ndc80/ch-TOG in the model, rupture force and time of attachment of the kMT interface increases. These results support the hypothesis that Ndc80-associated proteins strengthen kMT attachments, and that the interplay between kMT protein complexes in metaphase ensures stable attachments.
Topics: Cell Cycle Proteins; Chromosomal Proteins, Non-Histone; Computer Simulation; Cytoskeletal Proteins; HeLa Cells; Humans; Kinetochores; Metaphase; Microtubules; Mitosis; Protein Binding
PubMed: 31525284
DOI: 10.1002/cm.21562