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Nature Communications Jan 2020Lysosomes are membrane-surrounded cytoplasmic organelles filled with a powerful cocktail of hydrolases. Besides degrading cellular constituents inside the lysosomal...
Lysosomes are membrane-surrounded cytoplasmic organelles filled with a powerful cocktail of hydrolases. Besides degrading cellular constituents inside the lysosomal lumen, lysosomal hydrolases promote tissue remodeling when delivered to the extracellular space and cell death when released to the cytosol. Here, we show that spatially and temporally controlled lysosomal leakage contributes to the accurate chromosome segregation in normal mammalian cell division. One or more chromatin-proximal lysosomes leak in the majority of prometaphases, after which active cathepsin B (CTSB) localizes to the metaphase chromatin and cleaves a small subset of histone H3. Stabilization of lysosomal membranes or inhibition of CTSB activity during mitotic entry results in a significant increase in telomere-related chromosome segregation defects, whereas cells and tissues lacking CTSB and cells expressing CTSB-resistant histone H3 accumulate micronuclei and other nuclear defects. These data suggest that lysosomal leakage and chromatin-associated CTSB contribute to proper chromosome segregation and maintenance of genomic integrity.
Topics: Animals; Cathepsin B; Cell Line; Cell Nucleus; Chromatin; Chromosome Segregation; Female; Gene Silencing; Histones; Humans; Intracellular Membranes; Lysosomes; Metaphase; Mice; Mitosis; Permeability; Telomere
PubMed: 31932607
DOI: 10.1038/s41467-019-14009-0 -
Current Biology : CB Mar 2024The outer corona plays an essential role at the onset of mitosis by expanding to maximize microtubule attachment to kinetochores. The low-density structure of the corona...
The outer corona plays an essential role at the onset of mitosis by expanding to maximize microtubule attachment to kinetochores. The low-density structure of the corona forms through the expansion of unattached kinetochores. It comprises the RZZ complex, the dynein adaptor Spindly, the plus-end directed microtubule motor centromere protein E (CENP-E), and the Mad1/Mad2 spindle-assembly checkpoint proteins. CENP-E specifically associates with unattached kinetochores to facilitate chromosome congression, interacting with BubR1 at the kinetochore through its C-terminal region (2091-2358). We recently showed that CENP-E recruitment to BubR1 at the kinetochores is both rapid and essential for correct chromosome alignment. However, CENP-E is also recruited to the outer corona by a second, slower pathway that is currently undefined. Here, we show that BubR1-independent localization of CENP-E is mediated by a conserved loop that is essential for outer-corona targeting. We provide a structural model of the entire CENP-E kinetochore-targeting domain combining X-ray crystallography and Alphafold2. We reveal that maximal recruitment of CENP-E to unattached kinetochores critically depends on BubR1 and the outer corona, including dynein. Ectopic expression of the CENP-E C-terminal domain recruits the RZZ complex, Mad1, and Spindly, and prevents kinetochore biorientation in cells. We propose that BubR1-recruited CENP-E, in addition to its essential role in chromosome alignment to the metaphase plate, contributes to the recruitment of outer corona proteins through interactions with the CENP-E corona-targeting domain to facilitate the rapid capture of microtubules for efficient chromosome alignment and mitotic progression.
Topics: Humans; Cell Cycle Proteins; Chromosomal Proteins, Non-Histone; Kinetochores; Microtubules; Mad2 Proteins; Mitosis; Dyneins; Spindle Apparatus; HeLa Cells
PubMed: 38354735
DOI: 10.1016/j.cub.2024.01.042 -
The Journal of Cell Biology Jul 2018The two Condensin complexes in human cells are essential for mitotic chromosome structure. We used homozygous genome editing to fluorescently tag Condensin I and II...
The two Condensin complexes in human cells are essential for mitotic chromosome structure. We used homozygous genome editing to fluorescently tag Condensin I and II subunits and mapped their absolute abundance, spacing, and dynamic localization during mitosis by fluorescence correlation spectroscopy (FSC)-calibrated live-cell imaging and superresolution microscopy. Although ∼35,000 Condensin II complexes are stably bound to chromosomes throughout mitosis, ∼195,000 Condensin I complexes dynamically bind in two steps: prometaphase and early anaphase. The two Condensins rarely colocalize at the chromatid axis, where Condensin II is centrally confined, but Condensin I reaches ∼50% of the chromatid diameter from its center. Based on our comprehensive quantitative data, we propose a three-step hierarchical loop model of mitotic chromosome compaction: Condensin II initially fixes loops of a maximum size of ∼450 kb at the chromatid axis, whose size is then reduced by Condensin I binding to ∼90 kb in prometaphase and ∼70 kb in anaphase, achieving maximum chromosome compaction upon sister chromatid segregation.
Topics: Adenosine Triphosphatases; Anaphase; Chromatids; Chromosome Segregation; Chromosomes; DNA-Binding Proteins; Gene Editing; Humans; Mitosis; Multiprotein Complexes
PubMed: 29632028
DOI: 10.1083/jcb.201801048 -
Molecular Biology of the Cell Apr 2010To maintain genomic stability, chromosome architecture needs to be tightly regulated as chromosomes undergo condensation during prophase and separation during anaphase,...
To maintain genomic stability, chromosome architecture needs to be tightly regulated as chromosomes undergo condensation during prophase and separation during anaphase, but the mechanisms remain poorly understood. Here, we show that the Plk1-binding protein PICH and Plk1 kinase coordinately maintain chromosome architecture during prometaphase. PICH knockdown results in a loss of Plk1 from the chromosome arm and an increase in highly disorganized "wavy" chromosomes that exhibit an "open" or "X-shaped" configuration, consistent with a loss of chromosome arm cohesion. Such chromosome disorganization occurs with essentially no change in the localization of condensin or cohesin on chromosomes. Interestingly, the chromosome disorganization could be prevented by treatment with a topoisomerase II inhibitor ICRF-193, suggesting that the PICH-Plk1 complex normally maintains chromosome architecture in a manner that involves topoisomerase II activity. PICH knockdown does not affect initial chromosome compaction at prophase but causes anaphase DNA bridge formation and failed abscission. Our studies suggest that the PICH-Plk1 complex plays a critical role in maintaining prometaphase chromosome architecture.
Topics: Adenosine Triphosphatases; Cell Cycle Proteins; Chromosomal Proteins, Non-Histone; Chromosomes; DNA; DNA Helicases; DNA-Binding Proteins; Diketopiperazines; HeLa Cells; Humans; Immunoprecipitation; Microscopy, Confocal; Mitosis; Models, Biological; Multiprotein Complexes; Piperazines; Prometaphase; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Trypsin; Cohesins; Polo-Like Kinase 1
PubMed: 20130082
DOI: 10.1091/mbc.e09-11-0950 -
The Journal of Cell Biology Feb 2019Aurora kinases create phosphorylation gradients within the spindle during prometaphase and anaphase, thereby locally regulating factors that promote spindle...
Aurora kinases create phosphorylation gradients within the spindle during prometaphase and anaphase, thereby locally regulating factors that promote spindle organization, chromosome condensation and movement, and cytokinesis. We show that one such factor is the kinesin KIF4A, which is present along the chromosome axes throughout mitosis and the central spindle in anaphase. These two pools of KIF4A depend on condensin I and PRC1, respectively. Previous work has shown KIF4A is activated by Aurora B at the anaphase central spindle. However, whether or not chromosome-associated KIF4A bound to condensin I is regulated by Aurora kinases remain unclear. To determine the roles of the two different pools of KIF4A, we generated specific point mutants that are unable to interact with either condensin I or PRC1 or are deficient for Aurora kinase regulation. By analyzing these mutants, we show that Aurora A phosphorylates the condensin I-dependent pool of KIF4A and thus actively promotes chromosome congression from the spindle poles to the metaphase plate.
Topics: Adenosine Triphosphatases; Anaphase; Aurora Kinase A; Cell Line; Cell Line, Tumor; Chromosome Positioning; Chromosome Segregation; Chromosomes; DNA-Binding Proteins; HEK293 Cells; HeLa Cells; Humans; Kinesins; Microtubules; Mitosis; Multiprotein Complexes; Phosphorylation; Spindle Apparatus
PubMed: 31881080
DOI: 10.1083/jcb.201905194 -
Genes & Development Jul 2020During mitosis, transcription of genomic DNA is dramatically reduced, before it is reactivated during nuclear reformation in anaphase/telophase. Many aspects of the...
During mitosis, transcription of genomic DNA is dramatically reduced, before it is reactivated during nuclear reformation in anaphase/telophase. Many aspects of the underlying principles that mediate transcriptional memory and reactivation in the daughter cells remain unclear. Here, we used ChIP-seq on synchronized cells at different stages after mitosis to generate genome-wide maps of histone modifications. Combined with EU-RNA-seq and Hi-C analyses, we found that during prometaphase, promoters, enhancers, and insulators retain H3K4me3 and H3K4me1, while losing H3K27ac. Enhancers globally retaining mitotic H3K4me1 or locally retaining mitotic H3K27ac are associated with cell type-specific genes and their transcription factors for rapid transcriptional activation. As cells exit mitosis, promoters regain H3K27ac, which correlates with transcriptional reactivation. Insulators also gain H3K27ac and CCCTC-binding factor (CTCF) in anaphase/telophase. This increase of H3K27ac in anaphase/telophase is required for posttranscriptional activation and may play a role in the establishment of topologically associating domains (TADs). Together, our results suggest that the genome is reorganized in a sequential order, in which histone methylations occur first in prometaphase, histone acetylation, and CTCF in anaphase/telophase, transcription in cytokinesis, and long-range chromatin interactions in early G1. We thus provide insights into the histone modification landscape that allows faithful reestablishment of the transcriptional program and TADs during cell division.
Topics: Animals; Cell Cycle Checkpoints; Chromatin; Chromosomes; Enhancer Elements, Genetic; Genome; Histone Code; Histones; Humans; Mitosis; Promoter Regions, Genetic; Protein Binding; Protein Processing, Post-Translational; Time Factors; Transcriptional Activation
PubMed: 32499403
DOI: 10.1101/gad.335794.119 -
Nature Oct 2013The most conspicuous event in the cell cycle is the alignment of chromosomes in metaphase. Chromosome alignment fosters faithful segregation through the formation of...
The most conspicuous event in the cell cycle is the alignment of chromosomes in metaphase. Chromosome alignment fosters faithful segregation through the formation of bi-oriented attachments of kinetochores to spindle microtubules. Notably, numerous kinetochore-microtubule (k-MT) attachment errors are present in early mitosis (prometaphase), and the persistence of those errors is the leading cause of chromosome mis-segregation in aneuploid human tumour cells that continually mis-segregate whole chromosomes and display chromosomal instability. How robust error correction is achieved in prometaphase to ensure error-free mitosis remains unknown. Here we show that k-MT attachments in prometaphase cells are considerably less stable than in metaphase cells. The switch to more stable k-MT attachments in metaphase requires the proteasome-dependent destruction of cyclin A in prometaphase. Persistent cyclin A expression prevents k-MT stabilization even in cells with aligned chromosomes. By contrast, k-MTs are prematurely stabilized in cyclin-A-deficient cells. Consequently, cells lacking cyclin A display higher rates of chromosome mis-segregation. Thus, the stability of k-MT attachments increases decisively in a coordinated fashion among all chromosomes as cells transit from prometaphase to metaphase. Cyclin A creates a cellular environment that promotes microtubule detachment from kinetochores in prometaphase to ensure efficient error correction and faithful chromosome segregation.
Topics: Cell Line; Chromosome Segregation; Cyclin A; Enzyme Inhibitors; Gene Expression Regulation; Humans; Kinetochores; Microtubules; Mitosis; Protein Stability; Pyrimidines; Thiones
PubMed: 24013174
DOI: 10.1038/nature12507 -
Current Biology : CB Apr 2010Centrosome separation, critical for bipolar spindle formation and subsequent chromosome segregation during mitosis, occurs via distinct prophase and prometaphase...
Centrosome separation, critical for bipolar spindle formation and subsequent chromosome segregation during mitosis, occurs via distinct prophase and prometaphase pathways. Kinesin-5 (Eg5), a microtubule (MT) motor, pushes centrosomes apart during bipolar spindle assembly; its suppression results in monopolar spindles and mitotic arrest. Forces that antagonize Eg5 in prophase are unknown. Here we identify a new force generating mechanism mediated by the guanine nucleotide exchange factor (GEF) Tiam1, dependent on its ability to activate the GTPase Rac. We reveal that Tiam1 and Rac localize to centrosomes during prophase and prometaphase, and Tiam1, acting through Rac, ordinarily retards centrosome separation. Importantly, both Tiam1-depleted cells in culture and Rac1-deficient epithelial cells in vivo escape the mitotic arrest induced by Eg5 suppression. Moreover, Tiam1-depleted cells transit more slowly through prometaphase and display increased chromosome congression errors. Significantly, Eg5 suppression in Tiam1-depleted cells rectifies not only their increased centrosome separation but also their chromosome congression errors and mitotic delay. These findings identify Tiam1-Rac signaling as the first antagonist of centrosome separation during prophase, demonstrate its requirement in balancing Eg5-induced forces during bipolar spindle assembly in vitro and in vivo, and show that proper centrosome separation in prophase facilitates subsequent chromosome congression.
Topics: Animals; Base Sequence; Cell Line; Centrosome; Chromosome Segregation; Dogs; Guanine Nucleotide Exchange Factors; Kinesins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mitosis; Models, Biological; Neuropeptides; RNA Interference; Signal Transduction; Spindle Apparatus; rac GTP-Binding Proteins; rac1 GTP-Binding Protein
PubMed: 20346677
DOI: 10.1016/j.cub.2010.02.033 -
Current Biology : CB Sep 2010The mitotic checkpoint maintains genomic stability by blocking the metaphase-anaphase transition until all kinetochores attach to spindle microtubules [1, 2]. However,...
The mitotic checkpoint maintains genomic stability by blocking the metaphase-anaphase transition until all kinetochores attach to spindle microtubules [1, 2]. However, some defects are not detected by this checkpoint. With low concentrations of microtubule-targeting agents, the checkpoint eventually becomes satisfied, though the spindles may be short and/or multipolar [3, 4] and the fidelity of chromosome distribution and cleavage completion are compromised. In real life, environmental toxins, radiation, or chemotherapeutic agents may lead to completed but inaccurate mitoses. It has been assumed that once the checkpoint is satisfied and cells divide, the daughter cells would proliferate regardless of prometaphase duration. However, when continuously exposed to microtubule inhibitors, untransformed cells eventually slip out of mitosis after 12-48 hr and arrest in G1 [5-8] (see also [9]). Interestingly, transient but prolonged treatments with nocodazole allow completion of mitosis, but the daughter cells arrest in interphase [10, 11] (see also [9, 12]). Here we characterize the relationship between prometaphase duration and the proliferative capacity of daughter cells. Our results reveal the existence of a mechanism that senses prometaphase duration; if prometaphase lasts >1.5 hr, this mechanism triggers a durable p38- and p53-dependent G1 arrest of the daughter cells despite normal division of their mothers.
Topics: Antineoplastic Agents; Cell Line; Cell Proliferation; Humans; Leupeptins; Mitosis; Nocodazole; Prometaphase
PubMed: 20832310
DOI: 10.1016/j.cub.2010.08.018 -
Cell Cycle (Georgetown, Tex.) Jul 2023Tightly controlled fluctuations in kinase and phosphatase activity play important roles in regulating M-phase transitions. Protein Phosphatase 1 (PP1) is one of these...
Tightly controlled fluctuations in kinase and phosphatase activity play important roles in regulating M-phase transitions. Protein Phosphatase 1 (PP1) is one of these phosphatases, with oscillations in PP1 activity driving mitotic M-phase. Evidence from a variety of experimental systems also points to roles in meiosis. Here, we report that PP1 is important for M-phase transitions through mouse oocyte meiosis. We employed a unique small-molecule approach to inhibit or activate PP1 at distinct phases of mouse oocyte meiosis. These studies show that temporal control of PP1 activity is essential for the G2/M transition, metaphase I/anaphase I transition, and the formation of a normal metaphase II oocyte. Our data also reveal that inappropriate activation of PP1 is more deleterious at the G2/M transition than at prometaphase I-to-metaphase I, and that an active pool of PP1 during prometaphase is vital for metaphase I/anaphase I transition and metaphase II chromosome alignment. Taken together, these results establish that loss of oscillations in PP1 activity causes a range of severe meiotic defects, pointing to essential roles for PP1 in female fertility, and more broadly, M-phase regulation.
Topics: Female; Mice; Animals; Meiosis; Oocytes; Metaphase; Anaphase; Mitosis; Protein Phosphatase 1; Mammals
PubMed: 37340734
DOI: 10.1080/15384101.2023.2225924