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Genes & Development Mar 2019Formation of individualized sister chromatids is essential for their accurate segregation. In budding yeast, while most of the genome segregates at the metaphase to...
Formation of individualized sister chromatids is essential for their accurate segregation. In budding yeast, while most of the genome segregates at the metaphase to anaphase transition, resolution of the ribosomal DNA (rDNA) repeats is delayed. The timing and mechanism in human cells is unknown. Here we show that resolution of human rDNA occurs in anaphase after the bulk of the genome, dependent on tankyrase 1, condensin II, and topoisomerase IIα. Defective resolution leads to rDNA bridges, rDNA damage, and aneuploidy of an rDNA-containing acrocentric chromosome. Thus, temporal regulation of rDNA segregation is conserved between yeast and man and is essential for genome integrity.
Topics: Adenosine Triphosphatases; Anaphase; Aneuploidy; Chromosome Segregation; DNA Damage; DNA Topoisomerases, Type II; DNA, Ribosomal; DNA-Binding Proteins; Humans; Multiprotein Complexes; Saccharomyces cerevisiae; Tankyrases
PubMed: 30804226
DOI: 10.1101/gad.321836.118 -
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 -
Cell Cycle (Georgetown, Tex.) Oct 2021To ensure genome stability, chromosomes need to undergo proper condensation into two linked sister chromatids from prophase to prometaphase, followed by equal...
To ensure genome stability, chromosomes need to undergo proper condensation into two linked sister chromatids from prophase to prometaphase, followed by equal segregation at anaphase. Emerging evidence has shown that persistent DNA entanglements connecting the sister chromatids lead to the formation of ultrafine anaphase bridges (UFBs). If UFBs are not resolved soon after anaphase, they can induce chromosome missegregation. PICH (PLK1-interacting checkpoint helicase) is a DNA translocase that localizes on chromosome arms, centromeres and UFBs. It plays multiple essential roles in mitotic chromosome organization and segregation. PICH also recruits other associated proteins to UFBs, and together they mediate UFB resolution. Here, the proposed mechanism behind PICH's functions in chromosome organization and UFB resolution will be discussed. We summarize the regulation of PICH action at chromosome arms and centromeres, how PICH recognizes UFBs and recruits other UFB-associated factors, and finally how PICH promotes UFB resolution together with other DNA processing enzymes.
Topics: Anaphase; Centromere; Chromatids; Chromosome Segregation; Genomic Instability; Humans; Mitosis
PubMed: 34530686
DOI: 10.1080/15384101.2021.1970877 -
Cellular and Molecular Life Sciences :... Jul 2010One of the most extraordinary events in the lifetime of a cell is the coordinated separation of sister chromatids during cell division. This is truly the essence of the... (Review)
Review
One of the most extraordinary events in the lifetime of a cell is the coordinated separation of sister chromatids during cell division. This is truly the essence of the entire mitotic process and the reason for the most profound morphological changes in cytoskeleton and nuclear organization that a cell may ever experience. It all occurs within a very short time window known as "anaphase", as if the cell had spent the rest of its existence getting ready for this moment in an ultimate act of survival. And there is a good reason for this: no space for mistakes. Problems in the distribution of chromosomes during cell division have been correlated with aneuploidy, a common feature observed in cancers and several birth defects, and the main cause of spontaneous abortion in humans. In this paper, we critically review the mechanisms of anaphase chromosome motion that resisted the scrutiny of more than 100 years of research, as part of a tribute to the pioneering work of Miguel Mota.
Topics: Anaphase; Animals; Chromatids; Chromosomes; Drosophila; Dyneins; Kinetochores
PubMed: 20306325
DOI: 10.1007/s00018-010-0327-5 -
Cell Dec 2000
Review
Topics: Anaphase; Animals; Calcium-Binding Proteins; Carrier Proteins; Cell Cycle Proteins; Fungal Proteins; Humans; Nuclear Proteins; Spindle Apparatus
PubMed: 11163175
DOI: 10.1016/s0092-8674(00)00202-6 -
Nature Communications Sep 2021The RAD51 recombinase plays critical roles in safeguarding genome integrity, which is fundamentally important for all living cells. While interphase functions of RAD51...
The RAD51 recombinase plays critical roles in safeguarding genome integrity, which is fundamentally important for all living cells. While interphase functions of RAD51 in maintaining genome stability are well-characterised, its role in mitosis remains contentious. In this study, we show that RAD51 protects under-replicated DNA in mitotic human cells and, in this way, promotes mitotic DNA synthesis (MiDAS) and successful chromosome segregation. In cells experiencing mild replication stress, MiDAS was detected irrespective of mitotically generated DNA damage. MiDAS broadly required de novo RAD51 recruitment to single-stranded DNA, which was supported by the phosphorylation of RAD51 by the key mitotic regulator Polo-like kinase 1. Importantly, acute inhibition of MiDAS delayed anaphase onset and induced centromere fragility, suggesting a mechanism that prevents the satisfaction of the spindle assembly checkpoint while chromosomal replication remains incomplete. This study hence identifies an unexpected function of RAD51 in promoting genomic stability in mitosis.
Topics: Anaphase; Cell Cycle Proteins; Cell Line; Chromatin; Chromosome Segregation; DNA; DNA Damage; DNA Repair; DNA Replication; Genomic Instability; Humans; Intravital Microscopy; M Phase Cell Cycle Checkpoints; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Rad51 Recombinase; Polo-Like Kinase 1
PubMed: 34508092
DOI: 10.1038/s41467-021-25643-y -
PLoS Biology Aug 2023The target of rapamycin (TOR) signalling pathway plays a key role in the coordination between cellular growth and the cell cycle machinery in eukaryotes. The underlying...
The target of rapamycin (TOR) signalling pathway plays a key role in the coordination between cellular growth and the cell cycle machinery in eukaryotes. The underlying molecular mechanisms by which TOR might regulate events after anaphase remain unknown. We show for the first time that one of the 2 TOR complexes in budding yeast, TORC1, blocks the separation of cells following cytokinesis by phosphorylation of a member of the NDR (nuclear Dbf2-related) protein-kinase family, the protein Cbk1. We observe that TORC1 alters the phosphorylation pattern of Cbk1 and we identify a residue within Cbk1 activation loop, T574, for which a phosphomimetic substitution makes Cbk1 catalytically inactive and, indeed, reproduces TORC1 control over cell separation. In addition, we identify the exocyst component Sec3 as a key substrate of Cbk1, since Sec3 activates the SNARE complex to promote membrane fusion. TORC1 activity ultimately compromises the interaction between Sec3 and a t-SNARE component. Our data indicate that TORC1 negatively regulates cell separation in budding yeast by participating in Cbk1 phosphorylation, which in turn controls the fusion of secretory vesicles transporting hydrolase at the site of division.
Topics: Saccharomycetales; Phosphorylation; Anaphase; Cell Separation; Mechanistic Target of Rapamycin Complex 1
PubMed: 37647291
DOI: 10.1371/journal.pbio.3002263 -
Yeast (Chichester, England) Oct 2006During the cell cycle of the fission yeast Schizosaccharomyces pombe, striking changes in the organization of the cytoplasmic microtubule cytoskeleton take place. These... (Review)
Review
During the cell cycle of the fission yeast Schizosaccharomyces pombe, striking changes in the organization of the cytoplasmic microtubule cytoskeleton take place. These may serve as a model for understanding the different modes of microtubule organization that are often characteristic of differentiated higher eukaryotic cells. In the last few years, considerable progress has been made in our understanding of the organization and behaviour of fission yeast cytoplasmic microtubules, not only in the identification of the genes and proteins involved but also in the physiological analysis of function using fluorescently-tagged proteins in vivo. In this review we discuss the state of our knowledge in three areas: microtubule nucleation, regulation of microtubule dynamics and the organization and polarity of microtubule bundles. Advances in these areas provide a solid framework for a more detailed understanding of cytoplasmic microtubule organization.
Topics: Anaphase; Cell Polarity; Cytoplasm; Cytoskeleton; Kinetics; Microtubule-Organizing Center; Microtubules; Schizosaccharomyces; Tubulin
PubMed: 17072892
DOI: 10.1002/yea.1404 -
Current Biology : CB Jul 2019In anaphase spindles, antiparallel microtubules associate to form tight midzone bundles, as required for functional spindle architecture and correct chromosome...
In anaphase spindles, antiparallel microtubules associate to form tight midzone bundles, as required for functional spindle architecture and correct chromosome segregation. Several proteins selectively bind to these overlaps to control cytokinesis. How midzone bundles assemble is poorly understood. Here, using an in vitro reconstitution approach, we demonstrate that minimal midzone bundles can reliably self-organize in solution from dynamic microtubules, the microtubule crosslinker PRC1, and the motor protein KIF4A. The length of the central antiparallel overlaps in these microtubule bundles is similar to that observed in cells and is controlled by the PRC1/KIF4A ratio. Experiments and computer simulations demonstrate that minimal midzone bundle formation results from promoting antiparallel microtubule crosslinking, stopping microtubule plus-end dynamicity, and motor-driven midzone compaction and alignment. The robustness of this process suggests that a similar self-organization mechanism may contribute to the reorganization of the spindle architecture during the metaphase to anaphase transition in cells.
Topics: Anaphase; Escherichia coli; Microtubules; Spindle Apparatus
PubMed: 31231047
DOI: 10.1016/j.cub.2019.05.049 -
Proceedings of the National Academy of... Nov 2022How cells adjust their growth to the spatial and mechanical constraints of their surrounding environment is central to many aspects of biology. Here, we examined how...
How cells adjust their growth to the spatial and mechanical constraints of their surrounding environment is central to many aspects of biology. Here, we examined how extracellular matrix (ECM) rigidity affects cell division. We found that cells divide more rapidly when cultured on rigid substrates. While we observed no effect of ECM rigidity on rounding or postmitotic spreading duration, we found that changes in matrix stiffness impact mitosis progression. We noticed that ECM elasticity up-regulates the expression of the linker of nucleoskeleton and cytoskeleton (LINC) complex component SUN2, which in turn promotes metaphase-to-anaphase transition by acting on mitotic spindle formation, whereas when cells adhere to soft ECM, low levels of SUN2 expression perturb astral microtubule organization and delay the onset of anaphase.
Topics: Nuclear Matrix; Cytoskeleton; Microtubules; Mitosis; Extracellular Matrix; Spindle Apparatus; Anaphase
PubMed: 36322767
DOI: 10.1073/pnas.2116167119