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Molecular Cell May 2003Degradation of mitotic cyclins is critical for exit from mitosis. Recent studies in budding yeast address the role of cyclin degradation in meiosis. Cyclin stabilization... (Review)
Review
Degradation of mitotic cyclins is critical for exit from mitosis. Recent studies in budding yeast address the role of cyclin degradation in meiosis. Cyclin stabilization in meiosis I interferes with anaphase I spindle disassembly but, surprisingly, does not halt progression into meiosis II.
Topics: Anaphase; Cell Cycle Proteins; Cyclins; Endopeptidases; Fungal Proteins; Meiosis; Microtubule-Associated Proteins; Nuclear Proteins; Protein Tyrosine Phosphatases; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Separase; Spindle Apparatus
PubMed: 12769836
DOI: 10.1016/s1097-2765(03)00194-1 -
Cell Cycle (Georgetown, Tex.) 2014
Topics: Anaphase; Animals; Aurora Kinase B; Chromosome Segregation; Feedback, Physiological; Humans; Nuclear Envelope
PubMed: 25486554
DOI: 10.4161/15384101.2014.959853 -
Nature Reviews. Cancer Jul 2017Ever since initial suggestions that instability at common fragile sites (CFSs) could be responsible for chromosome rearrangements in cancers, CFSs and associated genes... (Review)
Review
Ever since initial suggestions that instability at common fragile sites (CFSs) could be responsible for chromosome rearrangements in cancers, CFSs and associated genes have been the subject of numerous studies, leading to questions and controversies about their role and importance in cancer. It is now clear that CFSs are not frequently involved in translocations or other cancer-associated recurrent gross chromosome rearrangements. However, recent studies have provided new insights into the mechanisms of CFS instability, their effect on genome instability, and their role in generating focal copy number alterations that affect the genomic landscape of many cancers.
Topics: Anaphase; Animals; Chromosomal Instability; Chromosome Breakage; Chromosome Fragile Sites; DNA Breaks, Double-Stranded; DNA Copy Number Variations; DNA Replication; Gene Rearrangement; Humans; Metaphase; Neoplasms; Oncogenes
PubMed: 28740117
DOI: 10.1038/nrc.2017.52 -
Nature Structural & Molecular Biology Nov 2023Substrate polyubiquitination drives a myriad of cellular processes, including the cell cycle, apoptosis and immune responses. Polyubiquitination is highly dynamic, and...
Substrate polyubiquitination drives a myriad of cellular processes, including the cell cycle, apoptosis and immune responses. Polyubiquitination is highly dynamic, and obtaining mechanistic insight has thus far required artificially trapped structures to stabilize specific steps along the enzymatic process. So far, how any ubiquitin ligase builds a proteasomal degradation signal, which is canonically regarded as four or more ubiquitins, remains unclear. Here we present time-resolved cryogenic electron microscopy studies of the 1.2 MDa E3 ubiquitin ligase, known as the anaphase-promoting complex/cyclosome (APC/C), and its E2 co-enzymes (UBE2C/UBCH10 and UBE2S) during substrate polyubiquitination. Using cryoDRGN (Deep Reconstructing Generative Networks), a neural network-based approach, we reconstruct the conformational changes undergone by the human APC/C during polyubiquitination, directly visualize an active E3-E2 pair modifying its substrate, and identify unexpected interactions between multiple ubiquitins with parts of the APC/C machinery, including its coactivator CDH1. Together, we demonstrate how modification of substrates with nascent ubiquitin chains helps to potentiate processive substrate polyubiquitination, allowing us to model how a ubiquitin ligase builds a proteasomal degradation signal.
Topics: Humans; Anaphase-Promoting Complex-Cyclosome; Cryoelectron Microscopy; Anaphase; Ubiquitination; Ubiquitin; Cell Cycle Proteins
PubMed: 37735619
DOI: 10.1038/s41594-023-01105-5 -
Molecular Biology of the Cell Nov 2020How cells regulate microtubule cross-linking activity to control the rate and duration of spindle elongation during anaphase is poorly understood. In this study, we test...
How cells regulate microtubule cross-linking activity to control the rate and duration of spindle elongation during anaphase is poorly understood. In this study, we test the hypothesis that PRC1/Ase1 proteins use distinct microtubule-binding domains to control the spindle elongation rate. Using the budding yeast Ase1, we identify unique contributions for the spectrin and carboxy-terminal domains during different phases of spindle elongation. We show that the spectrin domain uses conserved basic residues to promote the recruitment of Ase1 to the midzone before anaphase onset and slow spindle elongation during early anaphase. In contrast, a partial Ase1 carboxy-terminal truncation fails to form a stable midzone in late anaphase, produces higher elongation rates after early anaphase, and exhibits frequent spindle collapses. We find that the carboxy-terminal domain interacts with the plus-end tracking protein EB1/Bim1 and recruits Bim1 to the midzone to maintain midzone length. Overall, our results suggest that the Ase1 domains provide cells with a modular system to tune midzone activity and control elongation rates.
Topics: Anaphase; Cell Cycle; Cell Cycle Proteins; Chromosome Segregation; Microtubule Proteins; Microtubule-Associated Proteins; Microtubules; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Saccharomycetales; Spindle Apparatus
PubMed: 32997572
DOI: 10.1091/mbc.E20-07-0493-T -
The Journal of Cell Biology Jun 2022Errors in mitosis that cause chromosome missegregation lead to aneuploidy and micronucleus formation, which are associated with cancer. Accurate segregation requires the...
Errors in mitosis that cause chromosome missegregation lead to aneuploidy and micronucleus formation, which are associated with cancer. Accurate segregation requires the alignment of all chromosomes by the mitotic spindle at the metaphase plate, and any misalignment must be corrected before anaphase is triggered. The spindle is situated in a membrane-free "exclusion zone"; beyond this zone, endomembranes (mainly endoplasmic reticulum) are densely packed. We investigated what happens to misaligned chromosomes localized beyond the exclusion zone. Here we show that such chromosomes become ensheathed in multiple layers of endomembranes. Chromosome ensheathing delays mitosis and increases the frequency of chromosome missegregation and micronucleus formation. We use an induced organelle relocalization strategy in live cells to show that clearance of endomembranes allows for the rescue of chromosomes that were destined for missegregation. Our findings indicate that endomembranes promote the missegregation of misaligned chromosomes that are outside the exclusion zone and therefore constitute a risk factor for aneuploidy.
Topics: Anaphase; Aneuploidy; Cell Membrane; Chromosome Segregation; Chromosomes; Endoplasmic Reticulum; Humans; Metaphase; Mitosis; Spindle Apparatus
PubMed: 35486148
DOI: 10.1083/jcb.202203021 -
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 -
Life Science Alliance Dec 2023SiR-DNA/SiR-Hoechst is a far-red fluorescent DNA probe that is routinely used for live-cell imaging of cell nuclei in interphase and chromosomes during mitosis. Despite...
SiR-DNA/SiR-Hoechst is a far-red fluorescent DNA probe that is routinely used for live-cell imaging of cell nuclei in interphase and chromosomes during mitosis. Despite being reported to induce DNA damage, SiR-DNA has been used in more than 300 research articles, covering topics like mitosis, chromatin biology, cancer research, cytoskeletal research, and DNA damage response. Here, we used live-cell imaging to perform a comprehensive analysis of the effects of SiR-DNA on mitosis of four human cell lines (RPE-1, DLD-1, HeLa, and U2OS). We report a dose-, time-, and light-dependent effect of SiR-DNA on chromosome segregation. We found that, upon the exposure to light during imaging, nanomolar concentrations of SiR-DNA induce non-centromeric chromosome entanglement that severely impairs sister chromatid segregation and spindle elongation during anaphase. This causes DNA damage that is passed forward to the following cell cycle, thereby having a detrimental effect on genome integrity. Our findings highlight the drawbacks in using SiR-DNA for investigation of late mitotic events and DNA damage-related topics and urge the use of alternative labeling strategies to study these processes.
Topics: Humans; Anaphase; DNA; Chromatin; Chromosome Segregation; DNA Damage; HeLa Cells
PubMed: 37726128
DOI: 10.26508/lsa.202302260 -
Molecular Cancer Therapeutics Mar 2021Cyclin-dependent kinase 2 (CDK2) antagonism inhibits clustering of excessive centrosomes at mitosis, causing multipolar cell division and apoptotic death. This is called...
Cyclin-dependent kinase 2 (CDK2) antagonism inhibits clustering of excessive centrosomes at mitosis, causing multipolar cell division and apoptotic death. This is called anaphase catastrophe. To establish induced anaphase catastrophe as a clinically tractable antineoplastic mechanism, induced anaphase catastrophe was explored in different aneuploid cancers after treatment with CYC065 (Cyclacel), a CDK2/9 inhibitor. Antineoplastic activity was studied in preclinical models. CYC065 treatment augmented anaphase catastrophe in diverse cancers including lymphoma, lung, colon, and pancreatic cancers, despite oncoprotein expression. Anaphase catastrophe was a broadly active antineoplastic mechanism. Reverse phase protein arrays (RPPAs) revealed that along with known CDK2/9 targets, focal adhesion kinase and Src phosphorylation that regulate metastasis were each repressed by CYC065 treatment. Intriguingly, CYC065 treatment decreased lung cancer metastases in murine models. CYC065 treatment also significantly reduced the rate of lung cancer growth in syngeneic murine and patient-derived xenograft (PDX) models independent of oncoprotein expression. Immunohistochemistry analysis of CYC065-treated lung cancer PDX models confirmed repression of proteins highlighted by RPPAs, implicating them as indicators of CYC065 antitumor response. Phospho-histone H3 staining detected anaphase catastrophe in CYC065-treated PDXs. Thus, induced anaphase catastrophe after CYC065 treatment can combat aneuploid cancers despite oncoprotein expression. These findings should guide future trials of this novel CDK2/9 inhibitor in the cancer clinic.
Topics: Anaphase; Aneuploidy; Animals; Carcinogenesis; Cell Proliferation; Cyclin-Dependent Kinase 2; Humans; Mice; Mice, Nude; Neoplasm Metastasis; Transfection
PubMed: 33277443
DOI: 10.1158/1535-7163.MCT-19-0987 -
ELife Oct 2022Chromosome segregation requires both the separation of sister chromatids and the sustained condensation of chromatids during anaphase. In yeast cells, cohesin is not...
Chromosome segregation requires both the separation of sister chromatids and the sustained condensation of chromatids during anaphase. In yeast cells, cohesin is not only required for sister chromatid cohesion but also plays a major role determining the structure of individual chromatids in metaphase. Separase cleavage is thought to remove all cohesin complexes from chromosomes to initiate anaphase. It is thus not clear how the length and organisation of segregating chromatids is maintained during anaphase in the absence of cohesin. Here, we show that degradation of cohesin at the anaphase onset causes aberrant chromatid segregation. Hi-C analysis on segregating chromatids demonstrates that cohesin depletion causes loss of intrachromatid organisation. Surprisingly, tobacco etch virus (TEV)-mediated cleavage of cohesin does not dramatically disrupt chromatid organisation in anaphase, explaining why bulk segregation is achieved. In addition, we identified a small pool of cohesin complexes bound to telophase chromosomes in wild-type cells and show that they play a role in the organisation of centromeric regions. Our data demonstrates that in yeast cells cohesin function is not over in metaphase, but extends to the anaphase period when chromatids are segregating.
Topics: Anaphase; Chromatids; Chromatin; Saccharomyces cerevisiae; Separase; Chromosomal Proteins, Non-Histone; Cell Cycle Proteins; Saccharomyces cerevisiae Proteins; Cohesins
PubMed: 36196991
DOI: 10.7554/eLife.80147