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Nature Communications May 2018Kinetochores are multi-protein complexes that power chromosome movements by tracking microtubules plus-ends in the mitotic spindle. Human kinetochores bind up to 20...
Kinetochores are multi-protein complexes that power chromosome movements by tracking microtubules plus-ends in the mitotic spindle. Human kinetochores bind up to 20 microtubules, even though single microtubules can generate sufficient force to move chromosomes. Here, we show that high microtubule occupancy at kinetochores ensures robust chromosome segregation by providing a strong mechanical force that favours segregation of merotelic attachments during anaphase. Using low doses of the microtubules-targeting agent BAL27862 we reduce microtubule occupancy and observe that spindle morphology is unaffected and bi-oriented kinetochores can still oscillate with normal intra-kinetochore distances. Inter-kinetochore stretching is, however, dramatically reduced. The reduction in microtubule occupancy and inter-kinetochore stretching does not delay satisfaction of the spindle assembly checkpoint or induce microtubule detachment via Aurora-B kinase, which was so far thought to release microtubules from kinetochores under low stretching. Rather, partial microtubule occupancy slows down anaphase A and increases incidences of lagging chromosomes due to merotelically attached kinetochores.
Topics: Anaphase; Aurora Kinase B; Benzimidazoles; Cell Line; Chromosome Segregation; Humans; Intravital Microscopy; Kinetochores; Microscopy, Electron; Microtubules; Oxadiazoles; Spindle Apparatus
PubMed: 29795284
DOI: 10.1038/s41467-018-04427-x -
The Journal of Cell Biology Dec 2020Anaphase chromosome movement is thought to be mediated by pulling forces generated by end-on attachment of microtubules to the outer face of kinetochores. However, it...
Anaphase chromosome movement is thought to be mediated by pulling forces generated by end-on attachment of microtubules to the outer face of kinetochores. However, it has been suggested that during C. elegans female meiosis, anaphase is mediated by a kinetochore-independent pushing mechanism with microtubules only attached to the inner face of segregating chromosomes. We found that the kinetochore proteins KNL-1 and KNL-3 are required for preanaphase chromosome stretching, suggesting a role in pulling forces. In the absence of KNL-1,3, pairs of homologous chromosomes did not separate and did not move toward a spindle pole. Instead, each homolog pair moved together with the same spindle pole during anaphase B spindle elongation. Two masses of chromatin thus ended up at opposite spindle poles, giving the appearance of successful anaphase.
Topics: Anaphase; Animals; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Kinetochores; Microtubule-Associated Proteins
PubMed: 33064834
DOI: 10.1083/jcb.202005179 -
PloS One 2016Accurate chromosome segregation during cell division is essential to maintain genome stability, and chromosome segregation errors are causally linked to genetic...
Accurate chromosome segregation during cell division is essential to maintain genome stability, and chromosome segregation errors are causally linked to genetic disorders and cancer. An anaphase chromosome bridge is a particular chromosome segregation error observed in cells that enter mitosis with fused chromosomes/sister chromatids. The widely accepted Breakage/Fusion/Bridge cycle model proposes that anaphase chromosome bridges break during mitosis to generate chromosome ends that will fuse during the following cell cycle, thus forming new bridges that will break, and so on. However, various studies have also shown a link between chromosome bridges and aneuploidy and/or polyploidy. In this study, we investigated the behavior and properties of chromosome bridges during mitosis, with the idea to gain insight into the potential mechanism underlying chromosome bridge-induced aneuploidy. We find that only a small number of chromosome bridges break during anaphase, whereas the rest persist through mitosis into the subsequent cell cycle. We also find that the microtubule bundles (k-fibers) bound to bridge kinetochores are not prone to breakage/detachment, thus supporting the conclusion that k-fiber detachment is not the cause of chromosome bridge-induced aneuploidy. Instead, our data suggest that while the microtubules bound to the kinetochores of normally segregating chromosomes shorten substantially during anaphase, the k-fibers bound to bridge kinetochores shorten only slightly, and may even lengthen, during anaphase. This causes some of the bridge kinetochores/chromosomes to lag behind in a position that is proximal to the cell/spindle equator and may cause the bridged chromosomes to be segregated into the same daughter nucleus or to form a micronucleus.
Topics: Anaphase; Cells, Cultured; Chromosome Segregation; Chromosomes, Human; HeLa Cells; Humans; Kinetochores; Mammary Glands, Human; Microtubules; Mitosis
PubMed: 26784746
DOI: 10.1371/journal.pone.0147420 -
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 -
The Journal of Cell Biology Nov 1987Changes in free calcium concentration [( Ca]) have been detected during anaphase in stamen hair cells of Tradescantia. Cells have been injected iontophoretically with...
Changes in free calcium concentration [( Ca]) have been detected during anaphase in stamen hair cells of Tradescantia. Cells have been injected iontophoretically with the calcium sensitive metallochromic dye arsenazo III and changes in differential absorbance have been measured using a spinning wheel microspectrophotometer. The results obtained on single cells progressing from midmetaphase through to cytokinesis show that the free [Ca] first begins in increase after the initial separation of the sister chromosomes marking the onset of anaphase. The increase continues for 10-15 min while the chromosomes move to the poles; thereafter the [Ca] declines with the cell plate appearing about the time that the ion returns to its basal level. The close temporal correlation firstly between the rise in [Ca] and the breakdown of spindle microtubules (MTs) during anaphase and secondly, between the subsequent fall in [Ca] and the emergence of the MT-containing phragmoplast provides evidence consistent with the idea that endogenous fluctuations in [Ca] control the disassembly/assembly of MTs during mitosis.
Topics: Anaphase; Calcium; Metaphase; Mitosis; Plant Cells; Plants
PubMed: 3680374
DOI: 10.1083/jcb.105.5.2137 -
Cell Structure and Function Jul 1984
Review
Topics: Anaphase; Animals; Cell Cycle; Cell Division; Cell Survival; Meiosis; Mitosis; Spindle Apparatus
PubMed: 6383636
DOI: 10.1247/csf.9.supplement_s73 -
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 -
Current Biology : CB Apr 2021The spindle-assembly checkpoint facilitates mitotic fidelity by delaying anaphase onset in response to microtubule vacancy at kinetochores. Following microtubule...
The spindle-assembly checkpoint facilitates mitotic fidelity by delaying anaphase onset in response to microtubule vacancy at kinetochores. Following microtubule attachment, kinetochores receive microtubule-derived force, which causes kinetochores to undergo repetitive cycles of deformation; this phenomenon is referred to as kinetochore stretching. The nature of the forces and the relevance relating this deformation are not well understood. Here, we show that kinetochore stretching occurs within a framework of single end-on attached kinetochores, irrespective of microtubule poleward pulling force. An experimental method to conditionally interfere with the stretching allowed us to determine that kinetochore stretching comprises an essential process of checkpoint silencing by promoting PP1 phosphatase recruitment after the establishment of end-on attachments and removal of the majority of checkpoint-activating kinase Mps1 from kinetochores. Remarkably, we found that a lower frequency of kinetochore stretching largely correlates with a prolonged metaphase in cancer cell lines with chromosomal instability. Perturbation of kinetochore stretching and checkpoint silencing in chromosomally stable cells produced anaphase bridges, which can be alleviated by reducing chromosome-loaded cohesin. These observations indicate that kinetochore stretching-mediated checkpoint silencing provides an unanticipated etiology underlying chromosomal instability and underscores the importance of a rapid metaphase-to-anaphase transition in sustaining mitotic fidelity.
Topics: Anaphase; Cell Line, Tumor; Chromosomal Instability; Chromosome Segregation; Humans; Kinetochores; M Phase Cell Cycle Checkpoints; Microtubules; Spindle Apparatus
PubMed: 33651990
DOI: 10.1016/j.cub.2021.01.062 -
Methods in Molecular Biology (Clifton,... 2017In sexually reproducing organisms, the formation of healthy gametes (sperm and eggs) requires the proper establishment and release of meiotic sister chromatid cohesion...
In sexually reproducing organisms, the formation of healthy gametes (sperm and eggs) requires the proper establishment and release of meiotic sister chromatid cohesion (SCC). SCC tethers replicated sisters from their formation in premeiotic S phase until the stepwise removal of cohesion in anaphase of meiosis I and II allows the separation of homologs and then sisters. Defects in the establishment or release of meiotic cohesion cause chromosome segregation errors that lead to the formation of aneuploid gametes and inviable embryos. The nematode Caenorhabditis elegans is an attractive model for studies of meiotic sister chromatid cohesion due to its genetic tractability and the excellent cytological properties of the hermaphrodite gonad. Moreover, mutants defective in the establishment or maintenance of meiotic SCC nevertheless produce abundant gametes, allowing analysis of the pattern of chromosome segregation. Here I describe two approaches for analysis of meiotic cohesion in C. elegans. The first approach relies on cytology to detect and quantify defects in SCC. The second approach relies on PCR and restriction digests to identify embryos that inherited an incorrect complement of chromosomes due to aberrant meiotic chromosome segregation. Both approaches are sensitive enough to identify rare errors and precise enough to reveal distinctive phenotypes resulting from mutations that perturb meiotic SCC in different ways. The robust, quantitative nature of these assays should strengthen phenotypic comparisons of different meiotic mutants and enhance the reproducibility of data generated by different investigators.
Topics: Anaphase; Animals; Caenorhabditis elegans; Cell Cycle Proteins; Chromatids; Chromosomal Proteins, Non-Histone; Chromosome Segregation; Meiosis; Molecular Biology; Sister Chromatid Exchange; Cohesins
PubMed: 27797074
DOI: 10.1007/978-1-4939-6545-8_5 -
The Journal of Cell Biology May 2021Tight regulation of the APC/C-Cdc20 ubiquitin ligase that targets cyclin B1 for degradation is important for mitotic fidelity. The spindle assembly checkpoint (SAC)...
Tight regulation of the APC/C-Cdc20 ubiquitin ligase that targets cyclin B1 for degradation is important for mitotic fidelity. The spindle assembly checkpoint (SAC) inhibits Cdc20 through the mitotic checkpoint complex (MCC). In addition, phosphorylation of Cdc20 by cyclin B1-Cdk1 independently inhibits APC/C-Cdc20 activation. This creates a conundrum for how Cdc20 is activated before cyclin B1 degradation. Here, we show that the MCC component BubR1 harbors both Cdc20 inhibition and activation activities, allowing for cross-talk between the two Cdc20 inhibition pathways. Specifically, BubR1 acts as a substrate specifier for PP2A-B56 to enable efficient Cdc20 dephosphorylation in the MCC. A mutant Cdc20 mimicking the dephosphorylated state escapes a mitotic checkpoint arrest, arguing that restricting Cdc20 dephosphorylation to the MCC is important. Collectively, our work reveals how Cdc20 can be dephosphorylated in the presence of cyclin B1-Cdk1 activity without causing premature anaphase onset.
Topics: Anaphase; Anaphase-Promoting Complex-Cyclosome; Cdc20 Proteins; Cell Cycle Proteins; Cell Line, Tumor; Cyclin B1; HeLa Cells; Humans; M Phase Cell Cycle Checkpoints; Mitosis; Phosphorylation; Protein Binding; Protein Serine-Threonine Kinases; Spindle Apparatus
PubMed: 33819340
DOI: 10.1083/jcb.202012081