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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 -
Journal of Ovarian Research Nov 2023The oocyte and its surrounding cumulus cells (CCs) exist as an inseparable entity. The maturation of the oocyte relies on communication between the oocyte and the... (Review)
Review
BACKGROUND
The oocyte and its surrounding cumulus cells (CCs) exist as an inseparable entity. The maturation of the oocyte relies on communication between the oocyte and the surrounding CCs. However, oocyte evaluation is primarily based on morphological parameters currently, which offer limited insight into the quality and competence of the oocyte. Here, we conducted transcriptomic profiling of oocytes and their CCs from 47 patients undergoing preimplantation genetic testing for aneuploidy (PGT-A). We aimed to investigate the molecular events occurring between oocytes and CCs at different stages of oocyte maturation (germinal vesicle [GV], metaphase I [MI], and metaphase II [MII]). Our goal is to provide new insights into in vitro oocyte maturation (IVM).
RESULTS
Our findings indicate that oocyte maturation is a complex and dynamic process and that MI oocytes can be further classified into two distinct subtypes: GV-like-MI oocytes and MII-like-MI oocytes. Human oocytes and cumulus cells at three different stages of maturation were analyzed using RNA-seq, which revealed unique transcriptional machinery, stage-specific genes and pathways, and transcription factor networks that displayed developmental stage-specific expression patterns. We have also identified that both lipid and cholesterol metabolism in cumulus cells is active during the late stage of oocyte maturation. Lipids may serve as a more efficient energy source for oocytes and even embryogenesis.
CONCLUSIONS
Overall, our study provides a relatively comprehensive overview of the transcriptional characteristics and potential interactions between human oocytes and cumulus cells at various stages of maturation before ovulation. This study may offer novel perspectives on IVM and provide a reliable reference data set for understanding the transcriptional regulation of follicular maturation.
Topics: Female; Humans; Metaphase; Transcriptome; Cumulus Cells; Oocytes; In Vitro Oocyte Maturation Techniques; Ovulation
PubMed: 37993893
DOI: 10.1186/s13048-023-01291-2 -
IScience Feb 2024The subnuclear distribution of centromeres is cooperatively regulated by condensin II and the linker of nucleoskeleton and cytoskeleton (LINC) complex. However, other...
The subnuclear distribution of centromeres is cooperatively regulated by condensin II and the linker of nucleoskeleton and cytoskeleton (LINC) complex. However, other nuclear membrane structures and nuclear proteins are probably involved in centromere dynamics and distribution. Here, we focused on the nuclear pore complex (NPC), which is known to regulate gene expression, transcription memory, and chromatin structure in addition to transport between the cytoplasm and nucleoplasm. We report here that some nucleoporins (Nups), including Nup85, Nup133, CG1, Nup93b, and NUA, are involved in centromere scattering in . In addition, the centromere dynamics after metaphase in mutants were found to be similar to that of the condensin II mutant. Furthermore, both biochemical and genetic approaches showed that the Nups interact with the LINC complex. These results suggest that Nups regulate centromere scattering cooperatively with condensin II and the LINC complex.
PubMed: 38318384
DOI: 10.1016/j.isci.2024.108855 -
Oncotarget Dec 2020Centrosomes amplification is a hallmark of cancer. We hypothesize that 2-methoxyestradiol (2-ME) sensitizes breast cancer (BC) cells to taxanes by targeting amplified...
Centrosomes amplification is a hallmark of cancer. We hypothesize that 2-methoxyestradiol (2-ME) sensitizes breast cancer (BC) cells to taxanes by targeting amplified centrosomes. We assessed the extent by which 2-ME together with paclitaxel (PTX) induces centrosome alterations with subsequent mitotic catastrophe in different BC subtypes. 2-ME induced a significant reduction in PTX IC values in all cells tested ranging from 28-44% ( < 0.05). Treatment with both PTX and 2-ME significantly increased the number of misaligned metaphases compared to PTX alone (34%, 100% and 52% for MCF7, MDA-MB231 and SUM149, respectively; < 0.05). The number of cells with multipolar spindle formation was significantly increased (81%, 220% and 285% for MCF7, MDA-MB231 and SUM 149, respectively; < 0.05). PTX and 2-ME treatment significantly increased interphase declustering in cancer cells (56% for MCF7, 208% for MDA-MB231 and 218% for SUM149, respectively; < 0.05) and metaphase declustering (1.4-fold, 1.56-fold and 2.48-fold increase for MCF7, MDA-MB231 and SUM149, respectively; < 0.05). This report is the first to document centrosome declustering as a mechanism of action of 2-ME and provides a potential approach for reducing taxane toxicity in cancer treated patients.
PubMed: 33400733
DOI: 10.18632/oncotarget.27810 -
Nature Communications Mar 2023Spindle formation in male meiosis relies on the canonical centrosome system, which is distinct from acentrosomal oocyte meiosis, but its specific regulatory mechanisms...
Spindle formation in male meiosis relies on the canonical centrosome system, which is distinct from acentrosomal oocyte meiosis, but its specific regulatory mechanisms remain unknown. Herein, we report that DYNLRB2 (Dynein light chain roadblock-type-2) is a male meiosis-upregulated dynein light chain that is indispensable for spindle formation in meiosis I. In Dynlrb2 KO mouse testes, meiosis progression is arrested in metaphase I due to the formation of multipolar spindles with fragmented pericentriolar material (PCM). DYNLRB2 inhibits PCM fragmentation through two distinct pathways; suppressing premature centriole disengagement and targeting NuMA (nuclear mitotic apparatus) to spindle poles. The ubiquitously expressed mitotic counterpart, DYNLRB1, has similar roles in mitotic cells and maintains spindle bipolarity by targeting NuMA and suppressing centriole overduplication. Our work demonstrates that two distinct dynein complexes containing DYNLRB1 or DYNLRB2 are separately used in mitotic and meiotic spindle formations, respectively, and that both have NuMA as a common target.
Topics: Mice; Animals; Male; Dyneins; Spindle Apparatus; Centrosome; Meiosis; Metaphase
PubMed: 36973253
DOI: 10.1038/s41467-023-37370-7 -
Developmental Biology Feb 2021The early stages of development involve complex sequences of morphological changes that are both reproducible from embryo to embryo and often robust to environmental...
The early stages of development involve complex sequences of morphological changes that are both reproducible from embryo to embryo and often robust to environmental variability. To investigate the relationship between reproducibility and robustness we examined cell cycle progression in early Drosophila embryos at different temperatures. Our experiments show that while the subdivision of cell cycle steps is conserved across a wide range of temperatures (5-35 °C), the relative duration of individual steps varies with temperature. We find that the transition into prometaphase is delayed at lower temperatures relative to other cell cycle events, arguing that it has a different mechanism of regulation. Using an in vivo biosensor, we quantified the ratio of activities of the major mitotic kinase, Cdk1 and one of the major mitotic phosphatases PP1. Comparing activation profile with cell cycle transition times at different temperatures indicates that in early fly embryos activation of Cdk1 drives entry into prometaphase but is not required for earlier cell cycle events. In fact, chromosome condensation can still occur when Cdk1 activity is inhibited pharmacologically. These results demonstrate that different kinases are rate-limiting for different steps of mitosis, arguing that robust inter-regulation may be needed for rapid and ordered mitosis.
Topics: Animals; CDC2 Protein Kinase; Cell Cycle; Cell Cycle Checkpoints; Cyclin B; Drosophila Proteins; Drosophila melanogaster; Embryo, Nonmammalian; Enzyme Activation; Metaphase; Mitosis; Prometaphase; Prophase; Protein Phosphatase 1; Temperature
PubMed: 33278404
DOI: 10.1016/j.ydbio.2020.11.010 -
International Journal of Molecular... Feb 2021The importance of fluorescence light microscopy for understanding cellular and sub-cellular structures and functions is undeniable. However, the resolution is limited by... (Comparative Study)
Comparative Study
The importance of fluorescence light microscopy for understanding cellular and sub-cellular structures and functions is undeniable. However, the resolution is limited by light diffraction (~200-250 nm laterally, ~500-700 nm axially). Meanwhile, super-resolution microscopy, such as structured illumination microscopy (SIM), is being applied more and more to overcome this restriction. Instead, super-resolution by stimulated emission depletion (STED) microscopy achieving a resolution of ~50 nm laterally and ~130 nm axially has not yet frequently been applied in plant cell research due to the required specific sample preparation and stable dye staining. Single-molecule localization microscopy (SMLM) including photoactivated localization microscopy (PALM) has not yet been widely used, although this nanoscopic technique allows even the detection of single molecules. In this study, we compared protein imaging within metaphase chromosomes of barley via conventional wide-field and confocal microscopy, and the sub-diffraction methods SIM, STED, and SMLM. The chromosomes were labeled by DAPI (4',6-diamidino-2-phenylindol), a DNA-specific dye, and with antibodies against topoisomerase IIα (Topo II), a protein important for correct chromatin condensation. Compared to the diffraction-limited methods, the combination of the three different super-resolution imaging techniques delivered tremendous additional insights into the plant chromosome architecture through the achieved increased resolution.
Topics: Chromosomes, Plant; DNA Topoisomerases, Type II; Fluorescent Dyes; Hordeum; Indoles; Metaphase; Microscopy, Confocal; Microscopy, Fluorescence; Reproducibility of Results; Single Molecule Imaging
PubMed: 33672992
DOI: 10.3390/ijms22041903 -
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 -
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 -
PLoS Genetics Nov 2023The centromere is an epigenetic mark that is a loading site for the kinetochore during meiosis and mitosis. This mark is characterized by the H3 variant CENP-A, known as...
The centromere is an epigenetic mark that is a loading site for the kinetochore during meiosis and mitosis. This mark is characterized by the H3 variant CENP-A, known as CID in Drosophila. In Drosophila, CENP-C is critical for maintaining CID at the centromeres and directly recruits outer kinetochore proteins after nuclear envelope break down. These two functions, however, happen at different times in the cell cycle. Furthermore, in Drosophila and many other metazoan oocytes, centromere maintenance and kinetochore assembly are separated by an extended prophase. We have investigated the dynamics of function of CENP-C during the extended meiotic prophase of Drosophila oocytes and found that maintaining high levels of CENP-C for metaphase I requires expression during prophase. In contrast, CID is relatively stable and does not need to be expressed during prophase to remain at high levels in metaphase I of meiosis. Expression of CID during prophase can even be deleterious, causing ectopic localization to non-centromeric chromatin, abnormal meiosis and sterility. CENP-C prophase loading is required for multiple meiotic functions. In early meiotic prophase, CENP-C loading is required for sister centromere cohesion and centromere clustering. In late meiotic prophase, CENP-C loading is required to recruit kinetochore proteins. CENP-C is one of the few proteins identified in which expression during prophase is required for meiotic chromosome segregation. An implication of these results is that the failure to maintain recruitment of CENP-C during the extended prophase in oocytes would result in chromosome segregation errors in oocytes.
Topics: Animals; Meiosis; Chromosome Segregation; Drosophila Proteins; Prophase; Centromere; Drosophila; Mitosis; Kinetochores; Centromere Protein A; Chromosomal Proteins, Non-Histone
PubMed: 38019881
DOI: 10.1371/journal.pgen.1011066