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ELife Mar 2018The Anaphase Promoting Complex/Cyclosome (APC/C) is a ubiquitin E3 ligase that functions as the gatekeeper to mitotic exit. APC/C activity is controlled by an interplay...
The Anaphase Promoting Complex/Cyclosome (APC/C) is a ubiquitin E3 ligase that functions as the gatekeeper to mitotic exit. APC/C activity is controlled by an interplay of multiple pathways during mitosis, including the spindle assembly checkpoint (SAC), that are not yet fully understood. Here, we show that sumoylation of the APC4 subunit of the APC/C peaks during mitosis and is critical for timely APC/C activation and anaphase onset. We have also identified a functionally important SUMO interacting motif in the cullin-homology domain of APC2 located near the APC4 sumoylation sites and APC/C catalytic core. Our findings provide evidence of an important regulatory role for SUMO modification and binding in affecting APC/C activation and mitotic exit.
Topics: Anaphase; Anaphase-Promoting Complex-Cyclosome; Apc4 Subunit, Anaphase-Promoting Complex-Cyclosome; Catalytic Domain; Cytoskeletal Proteins; HeLa Cells; Humans; M Phase Cell Cycle Checkpoints; Mitosis; Plasmids; Protein Binding; Protein Conformation; Spindle Apparatus; Sumoylation; Ubiquitin-Protein Ligases; Ubiquitination
PubMed: 29517484
DOI: 10.7554/eLife.29539 -
Molecular Biology of the Cell Jan 2004Mitosis requires the concerted activities of multiple microtubule (MT)-based motor proteins. Here we examined the contribution of the chromokinesin, KLP3A, to mitotic...
Mitosis requires the concerted activities of multiple microtubule (MT)-based motor proteins. Here we examined the contribution of the chromokinesin, KLP3A, to mitotic spindle morphogenesis and chromosome movements in Drosophila embryos and cultured S2 cells. By immunofluorescence, KLP3A associates with nonfibrous punctae that concentrate in nuclei and display MT-dependent associations with spindles. These punctae concentrate in indistinct domains associated with chromosomes and central spindles and form distinct bands associated with telophase midbodies. The functional disruption of KLP3A by antibodies or dominant negative proteins in embryos, or by RNA interference (RNAi) in S2 cells, does not block mitosis but produces defects in mitotic spindles. Time-lapse confocal observations of mitosis in living embryos reveal that KLP3A inhibition disrupts the organization of interpolar (ip) MTs and produces short spindles. Kinetic analysis suggests that KLP3A contributes to spindle pole separation during the prometaphase-to-metaphase transition (when it antagonizes Ncd) and anaphase B, to normal rates of chromatid motility during anaphase A, and to the proper spacing of daughter nuclei during telophase. We propose that KLP3A acts on MTs associated with chromosome arms and the central spindle to organize ipMT bundles, to drive spindle pole separation and to facilitate chromatid motility.
Topics: Anaphase; Animals; Cell Nucleus; Cells, Cultured; Chromatids; Chromosome Positioning; Cloning, Molecular; Drosophila Proteins; Drosophila melanogaster; Embryo, Nonmammalian; Kinesins; Metaphase; Microinjections; Microscopy, Fluorescence; Microtubules; Models, Molecular; Mutation; RNA, Small Interfering; Spindle Apparatus; Telophase
PubMed: 14528012
DOI: 10.1091/mbc.e03-07-0489 -
Cell Proliferation Oct 2011Accurate chromosome segregation relies on activity of the spindle assembly checkpoint, a surveillance mechanism that prevents premature anaphase onset until all... (Review)
Review
Accurate chromosome segregation relies on activity of the spindle assembly checkpoint, a surveillance mechanism that prevents premature anaphase onset until all chromosomes are properly attached to the mitotic spindle apparatus and aligned at the metaphase plate. Defects in this mechanism contribute to chromosome instability and aneuploidy, a hallmark of malignant cells. Here, we review the molecular mechanisms of activation and silencing of the spindle assembly checkpoint and its relationship to tumourigenesis.
Topics: Anaphase; Animals; Chromosome Segregation; Genes, cdc; Humans; Kinetochores; Microtubules; Mitosis; Models, Biological; Neoplasms; Saccharomyces cerevisiae; Signal Transduction; Spindle Apparatus
PubMed: 21951282
DOI: 10.1111/j.1365-2184.2011.00767.x -
Proceedings of the National Academy of... Jun 2019The mitotic protein polo-like kinase 4 (PLK4) plays a critical role in centrosome duplication for cell division. By using immunofluorescence, we confirm that PLK4 is...
The mitotic protein polo-like kinase 4 (PLK4) plays a critical role in centrosome duplication for cell division. By using immunofluorescence, we confirm that PLK4 is localized to centrosomes. In addition, we find that phospho-PLK4 (pPLK4) is cleaved and distributed to kinetochores (metaphase and anaphase), spindle midzone/cleavage furrow (anaphase and telophase), and midbody (cytokinesis) during cell division in immortalized epithelial cells as well as breast, ovarian, and colorectal cancer cells. The distribution of pPLK4 midzone/cleavage furrow and midbody positions pPLK4 to play a functional role in cytokinesis. Indeed, we found that inhibition of PLK4 kinase activity with a small-molecule inhibitor, CFI-400945, prevents translocation to the spindle midzone/cleavage furrow and prevents cellular abscission, leading to the generation of cells with polyploidy, increased numbers of duplicated centrosomes, and vulnerability to anaphase or mitotic catastrophe. The regulatory role of PLK4 in cytokinesis makes it a potential target for therapeutic intervention in appropriately selected cancers.
Topics: Anaphase; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Centrosome; Cytokinesis; HCT116 Cells; HT29 Cells; Humans; Kinetochores; MCF-7 Cells; Mitosis; Protein Serine-Threonine Kinases; Spindle Apparatus
PubMed: 31097597
DOI: 10.1073/pnas.1818820116 -
Cytoskeleton (Hoboken, N.J.) Jun 2016The iconic bipolar structure of the mitotic spindle is of extreme importance to proper spindle function. At best, spindle abnormalities result in a delayed mitosis,...
The iconic bipolar structure of the mitotic spindle is of extreme importance to proper spindle function. At best, spindle abnormalities result in a delayed mitosis, while worse outcomes include cell death or disease. Recent work has uncovered an important role for the actin-based motor protein myosin-10 in the regulation of spindle structure and function. Here we examine the contribution of the myosin tail homology 4 (MyTH4) domain of the myosin-10 tail to the protein's spindle functions. The MyTH4 domain is known to mediate binding to microtubules and we verify the suspicion that this domain contributes to myosin-10's close association with the spindle. More surprisingly, our data demonstrate that some but not all of myosin-10's spindle functions require microtubule binding. In particular, myosin-10's contribution to spindle pole integrity requires microtubule binding, whereas its contribution to normal mitotic progression does not. This is demonstrated by the observation that dominant negative expression of the wild-type MyTH4 domain produces multipolar spindles and an increased mitotic index, whereas overexpression of a version of the MyTH4 domain harboring point mutations that abrogate microtubule binding results in only the mitotic index phenotype. Our data suggest that myosin-10 helps to control the metaphase to anaphase transition in cells independent of microtubule binding. © 2016 Wiley Periodicals, Inc.
Topics: Anaphase; Animals; Metaphase; Myosins; Protein Domains; Spindle Apparatus; Xenopus Proteins; Xenopus laevis
PubMed: 27220038
DOI: 10.1002/cm.21311 -
Bioinformatics (Oxford, England) Jun 2022Lattice light-sheet microscopy (LLSM) is revolutionizing cell biology since it enables fast, high-resolution extended imaging in three dimensions combined with a drastic...
MOTIVATION
Lattice light-sheet microscopy (LLSM) is revolutionizing cell biology since it enables fast, high-resolution extended imaging in three dimensions combined with a drastic reduction in photo-toxicity and bleaching. However, analysis of such datasets still remains a major challenge.
RESULTS
Automated tracking of kinetochores, the protein complex facilitating and controlling microtubule attachment of the chromosomes within the mitotic spindle, provides quantitative assessment of chromosome dynamics in mitosis. Here, we extend existing open-source kinetochore tracking software (KiT) to track (and pair) kinetochores throughout prometaphase to anaphase in LLSM data. One of the key improvements is a regularization term in the objective function to enforce biological information about the number of kinetochores in a human mitotic cell, as well as improved diagnostic tools. This software provides quantitative insights into how kinetochores robustly ensure congression and segregation of chromosomes during mitosis.
AVAILABILITY AND IMPLEMENTATION
KiT is free, open-source software implemented in MATLAB and can be downloaded as a package from https://github.com/cmcb-warwick/KiT. The source repository is available at https://bitbucket.org/jarmond/kit (tag v2.4.0) and under continuing development.
SUPPLEMENTARY INFORMATION
Supplementary data are available at Bioinformatics online.
Topics: Humans; Kinetochores; Spindle Apparatus; Anaphase; Microtubules; Software; Chromosome Segregation
PubMed: 35579370
DOI: 10.1093/bioinformatics/btac330 -
Molecular Biology of the Cell May 2017The spindle assembly checkpoint ensures the faithful inheritance of chromosomes by arresting mitotic progression in the presence of kinetochores that are not attached to...
The spindle assembly checkpoint ensures the faithful inheritance of chromosomes by arresting mitotic progression in the presence of kinetochores that are not attached to spindle microtubules. This is achieved through inhibition of the anaphase-promoting complex/cyclosome by a kinetochore-derived "wait anaphase" signal known as the mitotic checkpoint complex. It remains unclear whether the localization and activity of these inhibitory complexes are restricted to the mitotic spindle compartment or are diffusible throughout the cytoplasm. Here we report that "wait anaphase" signals are indeed able to diffuse outside the confines of the mitotic spindle compartment. Using a cell fusion approach to generate multinucleate cells, we investigate the effects of checkpoint signals derived from one spindle compartment on a neighboring spindle compartment. We find that spindle compartments in close proximity wait for one another to align all chromosomes before entering anaphase synchronously. Synchrony is disrupted in cells with increased interspindle distances and cellular constrictions between spindle compartments. In addition, when mitotic cells are fused with interphase cells, "wait anaphase" signals are diluted, resulting in premature mitotic exit. Overall our studies reveal that anaphase inhibitors are diffusible and active outside the confines of the mitotic spindle from which they are derived.
Topics: Anaphase; Anaphase-Promoting Complex-Cyclosome; Cell Culture Techniques; Cell Cycle Checkpoints; Cell Cycle Proteins; Humans; Kinetochores; M Phase Cell Cycle Checkpoints; Microtubules; Mitosis; Protein Serine-Threonine Kinases; Spindle Apparatus
PubMed: 28298492
DOI: 10.1091/mbc.E17-01-0036 -
Stem Cell Reports Feb 2023During in vitro propagation, human pluripotent stem cells (hPSCs) frequently become aneuploid with incorrect chromosome numbers due to mitotic chromosome segregation...
During in vitro propagation, human pluripotent stem cells (hPSCs) frequently become aneuploid with incorrect chromosome numbers due to mitotic chromosome segregation errors. Yet, it is not understood why hPSCs exhibit a low mitotic fidelity. Here, we investigate the mechanisms responsible for mitotic errors in hPSCs and show that the primary cause is lagging chromosomes in anaphase with improper merotelic microtubule attachments. Accordingly, short-term treatment (<24 h) with small molecules that prolong mitotic duration or destabilize chromosome microtubule attachments reduces merotelic errors and lagging chromosome rates, although hPSCs adapt and lagging chromosome rates rebound upon long-term (>24 h) microtubule destabilization. Strikingly, we also demonstrate that mitotic error rates correlate with developmental potential decreasing or increasing upon loss or gain of pluripotency, respectively. Thus, a low mitotic fidelity is an inherent and conserved phenotype of hPSCs. Moreover, chromosome segregation fidelity depends on developmental state in normal human cells.
Topics: Humans; Chromosome Segregation; Kinetochores; Mitosis; Microtubules; Anaphase; Spindle Apparatus
PubMed: 36638786
DOI: 10.1016/j.stemcr.2022.12.008 -
ELife May 2022Cell mass and composition change with cell cycle progression. Our previous work characterized buoyant mass dynamics in mitosis (Miettinen et al., 2019), but how dry mass...
Cell mass and composition change with cell cycle progression. Our previous work characterized buoyant mass dynamics in mitosis (Miettinen et al., 2019), but how dry mass and cell composition change in mitosis has remained unclear. To better understand mitotic cell growth and compositional changes, we develop a single-cell approach for monitoring dry mass and the density of that dry mass every ~75 s with 1.3% and 0.3% measurement precision, respectively. We find that suspension grown mammalian cells lose dry mass and increase dry mass density following mitotic entry. These changes display large, non-genetic cell-to-cell variability, and the changes are reversed at metaphase-anaphase transition, after which dry mass continues accumulating. The change in dry mass density causes buoyant and dry mass to differ specifically in early mitosis, thus reconciling existing literature on mitotic cell growth. Mechanistically, cells in early mitosis increase lysosomal exocytosis, and inhibition of lysosomal exocytosis decreases the dry mass loss and dry mass density increase in mitosis. Overall, our work provides a new approach for monitoring single-cell dry mass and dry mass density, and reveals that mitosis is coupled to extensive exocytosis-mediated secretion of cellular contents.
Topics: Anaphase; Animals; Cell Cycle; Exocytosis; Mammals; Metaphase; Mitosis
PubMed: 35535854
DOI: 10.7554/eLife.76664 -
EMBO Reports Jul 2021Progression through the cell cycle is driven by cyclin-dependent kinases that control gene expression, orchestration of mitotic spindle, and cell division. To identify...
Progression through the cell cycle is driven by cyclin-dependent kinases that control gene expression, orchestration of mitotic spindle, and cell division. To identify new regulators of the cell cycle, we performed transcriptomic analysis of human non-transformed cells expressing a fluorescent ubiquitination-based cell cycle indicator and identified 701 transcripts differentially expressed in G1 and G2 cells. Family with sequence similarity 110 member A (FAM110A) protein is highly expressed in G2 cells and localized at mitotic spindle and spindle poles during mitosis. Depletion of FAM110A impairs chromosomal alignment, delays metaphase-to-anaphase transition, and affects spindle positioning. Using mass spectrometry and immunoprecipitation, we identified casein kinase I (CK1) in complex with FAM110A during mitosis. CK1 phosphorylates the C-terminal domain of FAM110A in vitro, and inhibition of CK1 reduces phosphorylation of mitotic FAM110A. Wild-type FAM110A, but not the FAM110A-S252-S255A mutant deficient in CK1 phosphorylation, rescues the chromosomal alignment, duration of mitosis, and orientation of the mitotic spindle after depletion of endogenous FAM110A. We propose that CK1 regulates chromosomal alignment by phosphorylating FAM110A and promoting its interaction with mitotic spindle.
Topics: Anaphase; Cell Cycle Proteins; HeLa Cells; Humans; Mitosis; Phosphorylation; Spindle Apparatus
PubMed: 34080749
DOI: 10.15252/embr.202051847