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Cellular and Molecular Life Sciences :... Mar 2021Chromosomal fragile sites are described as areas within the tightly packed mitotic chromatin that appear as breaks or gaps mostly tracing back to a loosened structure... (Review)
Review
Chromosomal fragile sites are described as areas within the tightly packed mitotic chromatin that appear as breaks or gaps mostly tracing back to a loosened structure and not a real nicked break within the DNA molecule. Most facts about fragile sites result from studies in mitotic cells, mainly during metaphase and mainly in lymphocytes. Here, we synthesize facts about the genomic regions that are prone to form gaps and breaks on metaphase chromosomes in the context of interphase. We conclude that nuclear architecture shapes the activity profile of the cell, i.e. replication timing and transcriptional activity, thereby influencing genomic integrity during interphase with the potential to cause fragility in mitosis. We further propose fragile sites as examples of regions specifically positioned in the interphase nucleus with putative anchoring points at the nuclear lamina to enable a tightly regulated replication-transcription profile and diverse signalling functions in the cell. Consequently, fragility starts before the actual display as chromosomal breakage in metaphase to balance the initial contradiction of cellular overgrowth or malfunctioning and maintaining diversity in molecular evolution.
Topics: Animals; Cell Nucleus; Chromosomal Instability; Chromosome Fragile Sites; DNA; DNA Replication; Genome, Human; Humans; Interphase; Mitosis
PubMed: 33219838
DOI: 10.1007/s00018-020-03698-2 -
Cells Feb 2020Meiosis is required to reduce to haploid the diploid genome content of a cell, generating gametes-oocytes and sperm-with the correct number of chromosomes. To achieve... (Review)
Review
Meiosis is required to reduce to haploid the diploid genome content of a cell, generating gametes-oocytes and sperm-with the correct number of chromosomes. To achieve this goal, two specialized cell divisions without intermediate S-phase are executed in a time-controlled manner. In mammalian female meiosis, these divisions are error-prone. Human oocytes have an exceptionally high error rate that further increases with age, with significant consequences for human fertility. To understand why errors in chromosome segregation occur at such high rates in oocytes, it is essential to understand the molecular players at work controlling these divisions. In this review, we look at the interplay of kinase and phosphatase activities at the transition from metaphase-to-anaphase for correct segregation of chromosomes. We focus on the activity of PP2A-B56, a key phosphatase for anaphase onset in both mitosis and meiosis. We start by introducing multiple roles PP2A-B56 occupies for progression through mitosis, before laying out whether or not the same principles may apply to the first meiotic division in oocytes, and describing the known meiosis-specific roles of PP2A-B56 and discrepancies with mitotic cell cycle regulation.
Topics: Animals; Kinetochores; Meiosis; Mice; Microtubules; Oocytes; Protein Phosphatase 2; Spindle Apparatus
PubMed: 32046180
DOI: 10.3390/cells9020390 -
Molecular & Cellular Oncology 2021At the metaphase-to-anaphase transition, phosphatase activity feeds back to reverse early mitotic phosphorylation events. Our recent work indicates that the pseudokinase...
At the metaphase-to-anaphase transition, phosphatase activity feeds back to reverse early mitotic phosphorylation events. Our recent work indicates that the pseudokinase domain of the spindle checkpoint protein BUB1 (Budding Uninhibited by Benzimidazoles 1) mitotic checkpoint serine/threonine kinase B (BUB1B, BUBR1) maintains kinase-phosphatase balance at the outer kinetochore during mitotic exit.
PubMed: 33860079
DOI: 10.1080/23723556.2021.1876511 -
Current Biology : CB Mar 2022Chromosome segregation is accomplished by the mitotic spindle, a bipolar micromachine built primarily from microtubules. Different microtubule populations contribute to...
Chromosome segregation is accomplished by the mitotic spindle, a bipolar micromachine built primarily from microtubules. Different microtubule populations contribute to spindle function: kinetochore microtubules attach and transmit forces to chromosomes, antiparallel interpolar microtubules support spindle structure, and astral microtubules connect spindle poles to the cell cortex. In mammalian cells, end-binding (EB) proteins associate with all growing microtubule plus ends throughout the cell cycle and serve as adaptors for diverse +TIPs that control microtubule dynamics and interactions with other intracellular structures. Because binding of many +TIPs to EB1 and thus microtubule-end association is switched off by mitotic phosphorylation, the mitotic function of EBs remains poorly understood. To analyze how EB1 and associated +TIPs on different spindle microtubule populations contribute to mitotic spindle dynamics, we use a light-sensitive EB1 variant, π-EB1, that allows local, acute, and reversible inactivation of +TIP association with growing microtubule ends in live cells. We find that acute π-EB1 photoinactivation results in rapid and reversible metaphase spindle shortening and transient relaxation of tension across the central spindle. However, in contrast to interphase, π-EB1 photoinactivation does not inhibit microtubule growth in metaphase but instead increases astral microtubule length and number. Yet in the absence of EB1 activity, astral microtubules fail to engage the cortical dynein/dynactin machinery, and spindle poles move away from regions of π-EB1 photoinactivation. In conclusion, our optogenetic approach reveals mitotic EB1 functions that remain hidden in genetic experiments, likely due to compensatory molecular systems regulating vertebrate spindle dynamics.
Topics: Animals; Mammals; Metaphase; Microtubule-Associated Proteins; Microtubules; Optogenetics; Spindle Apparatus
PubMed: 35090591
DOI: 10.1016/j.cub.2022.01.017 -
BioEssays : News and Reviews in... Jul 2015How eukaryotic genomes are packaged into compact cylindrical chromosomes in preparation for cell divisions has remained one of the major unsolved questions of cell... (Review)
Review
How eukaryotic genomes are packaged into compact cylindrical chromosomes in preparation for cell divisions has remained one of the major unsolved questions of cell biology. Novel approaches to study the topology of DNA helices inside the nuclei of intact cells, paired with computational modeling and precise biomechanical measurements of isolated chromosomes, have advanced our understanding of mitotic chromosome architecture. In this Review Essay, we discuss - in light of these recent insights - the role of chromatin architecture and the functions and possible mechanisms of SMC protein complexes and other molecular machines in the formation of mitotic chromosomes. Based on the information available, we propose a stepwise model of mitotic chromosome condensation that envisions the sequential generation of intra-chromosomal linkages by condensin complexes in the context of cohesin-mediated inter-chromosomal linkages, assisted by topoisomerase II. The described scenario results in rod-shaped metaphase chromosomes ready for their segregation to the cell poles.
Topics: Adenosine Triphosphatases; Animals; Chromatin Assembly and Disassembly; Chromosomes; DNA-Binding Proteins; Humans; Mitosis; Multiprotein Complexes; Nucleosomes
PubMed: 25988527
DOI: 10.1002/bies.201500020 -
PLoS Genetics Jan 2021Although kinetochores normally play a key role in sister chromatid separation and segregation, chromosome fragments lacking kinetochores (acentrics) can in some cases...
Although kinetochores normally play a key role in sister chromatid separation and segregation, chromosome fragments lacking kinetochores (acentrics) can in some cases separate and segregate successfully. In Drosophila neuroblasts, acentric chromosomes undergo delayed, but otherwise normal sister separation, revealing the existence of kinetochore- independent mechanisms driving sister chromosome separation. Bulk cohesin removal from the acentric is not delayed, suggesting factors other than cohesin are responsible for the delay in acentric sister separation. In contrast to intact kinetochore-bearing chromosomes, we discovered that acentrics align parallel as well as perpendicular to the mitotic spindle. In addition, sister acentrics undergo unconventional patterns of separation. For example, rather than the simultaneous separation of sisters, acentrics oriented parallel to the spindle often slide past one another toward opposing poles. To identify the mechanisms driving acentric separation, we screened 117 RNAi gene knockdowns for synthetic lethality with acentric chromosome fragments. In addition to well-established DNA repair and checkpoint mutants, this candidate screen identified synthetic lethality with X-chromosome-derived acentric fragments in knockdowns of Greatwall (cell cycle kinase), EB1 (microtubule plus-end tracking protein), and Map205 (microtubule-stabilizing protein). Additional image-based screening revealed that reductions in Topoisomerase II levels disrupted sister acentric separation. Intriguingly, live imaging revealed that knockdowns of EB1, Map205, and Greatwall preferentially disrupted the sliding mode of sister acentric separation. Based on our analysis of EB1 localization and knockdown phenotypes, we propose that in the absence of a kinetochore, microtubule plus-end dynamics provide the force to resolve DNA catenations required for sister separation.
Topics: Animals; Cell Cycle Proteins; Chromatids; Chromosomal Proteins, Non-Histone; Chromosome Segregation; DNA Topoisomerases, Type II; Drosophila melanogaster; Kinetochores; Larva; Metaphase; Microtubules; Mitosis; Spindle Apparatus; Cohesins
PubMed: 33513180
DOI: 10.1371/journal.pgen.1009304 -
IScience Feb 2022Unsatisfied kinetochore-microtubule attachment activates the spindle assembly checkpoint to inhibit the metaphase-anaphase transition. However, some cells eventually...
Unsatisfied kinetochore-microtubule attachment activates the spindle assembly checkpoint to inhibit the metaphase-anaphase transition. However, some cells eventually override mitotic arrest by mitotic slippage. Here, we show that inactivation of TORC1 kinase elicits mitotic slippage in budding yeast and human cells. Yeast mitotic slippage was accompanied with aberrant aspects, such as degradation of the nucleolar protein Net1, release of phosphatase Cdc14, and anaphase-promoting complex/cyclosome (APC/C)-Cdh1-dependent degradation of securin and cyclin B in metaphase. This mitotic slippage caused chromosome instability. In human cells, mammalian TORC1 (mTORC1) inactivation also invoked mitotic slippage, indicating that TORC1 inactivation-induced mitotic slippage is conserved from yeast to mammalian cells. However, the invoked mitotic slippage in human cells was not dependent on APC/C-Cdh1. This study revealed an unexpected involvement of TORC1 in mitosis and provides information on undesirable side effects of the use of TORC1 inhibitors as immunosuppressants and anti-tumor drugs.
PubMed: 35141499
DOI: 10.1016/j.isci.2021.103675 -
Cell Reports Jan 2021During mitotic chromosome segregation, the protease separase severs cohesin between sister chromatids. A probe for separase activity has shown that separase undergoes...
During mitotic chromosome segregation, the protease separase severs cohesin between sister chromatids. A probe for separase activity has shown that separase undergoes abrupt activation shortly before anaphase onset, after being suppressed throughout metaphase; however, the relevance of this control remains unclear. Here, we report that separase activates precociously, with respect to anaphase onset, during prolonged metaphase in multiple types of cancer cell lines. The artificial extension of metaphase in chromosomally stable diploid cells leads to precocious activation and, subsequently, to chromosomal bridges in anaphase, which seems to be attributable to incomplete cohesin removal. Conversely, shortening back of a prolonged metaphase restores the activation of separase and ameliorates anaphase bridge formation. These observations suggest that retarded metaphase progression affects the separase activation profile and its enzymatic proficiency. Our findings provide an unanticipated etiology for chromosomal instability in cancers and underscore the relevance of swift mitotic transitions for fail-safe chromosome segregation.
Topics: Animals; Chromosome Segregation; Humans; Mice; Mitosis; Rabbits; Separase
PubMed: 33472072
DOI: 10.1016/j.celrep.2020.108652 -
EMBO Reports Apr 2024Stabilization of microtubule plus end-directed kinesin CENP-E at the metaphase kinetochores is important for chromosome alignment, but its mechanism remains unclear....
Stabilization of microtubule plus end-directed kinesin CENP-E at the metaphase kinetochores is important for chromosome alignment, but its mechanism remains unclear. Here, we show that CKAP5, a conserved microtubule plus tip protein, regulates CENP-E at kinetochores in human cells. Depletion of CKAP5 impairs CENP-E localization at kinetochores at the metaphase plate and results in increased kinetochore-microtubule stability and attachment errors. Erroneous attachments are also supported by computational modeling. Analysis of CKAP5 knockout cancer cells of multiple tissue origins shows that CKAP5 is preferentially essential in aneuploid, chromosomally unstable cells, and the sensitivity to CKAP5 depletion is correlated to that of CENP-E depletion. CKAP5 depletion leads to reduction in CENP-E-BubR1 interaction and the interaction is rescued by TOG4-TOG5 domain of CKAP5. The same domain can rescue CKAP5 depletion-induced CENP-E removal from the kinetochores. Interestingly, CKAP5 depletion facilitates recruitment of PP1 to the kinetochores and furthermore, a PP1 target site-specific CENP-E phospho-mimicking mutant gets stabilized at kinetochores in the CKAP5-depleted cells. Together, the results support a model in which CKAP5 controls mitotic chromosome attachment errors by stabilizing CENP-E at kinetochores and by regulating stability of the kinetochore-attached microtubules.
Topics: Humans; Kinetochores; Chromosomal Proteins, Non-Histone; Microtubules; Metaphase; Kinesins; HeLa Cells; Mitosis; Chromosome Segregation; Microtubule-Associated Proteins
PubMed: 38424231
DOI: 10.1038/s44319-024-00106-9 -
BioRxiv : the Preprint Server For... May 2024The eukaryotic cell division machinery must rapidly and reproducibly duplicate and partition the cell's chromosomes in a carefully coordinated process. However,...
The eukaryotic cell division machinery must rapidly and reproducibly duplicate and partition the cell's chromosomes in a carefully coordinated process. However, chromosome number varies dramatically between genomes, even on short evolutionary timescales. We sought to understand how the mitotic machinery senses and responds to karyotypic changes by using a series of budding yeast strains in which the native chromosomes have been successively fused. Using a combination of cell biological profiling, genetic engineering, and experimental evolution, we show that chromosome fusions are well tolerated up until a critical point. Cells with fewer than five centromeres lack the necessary number of kinetochore-microtubule attachments needed to counter outward forces in the metaphase spindle, triggering the spindle assembly checkpoint and prolonging metaphase. Our findings demonstrate that spindle architecture is a constraining factor for karyotype evolution.
PubMed: 37961714
DOI: 10.1101/2023.10.25.563899