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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 -
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 -
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 -
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 Proteomics : MCP Aug 2016The mitotic spindle is required for chromosome congression and subsequent equal segregation of sister chromatids. These processes involve a complex network of signaling...
The mitotic spindle is required for chromosome congression and subsequent equal segregation of sister chromatids. These processes involve a complex network of signaling molecules located at the spindle. The endocytic protein, clathrin, has a "moonlighting" role during mitosis, whereby it stabilizes the mitotic spindle. The signaling pathways that clathrin participates in to achieve mitotic spindle stability are unknown. Here, we assessed the mitotic spindle proteome and phosphoproteome in clathrin-depleted cells using quantitative MS/MS (data are available via ProteomeXchange with identifier PXD001603). We report a spindle proteome that consists of 3046 proteins and a spindle phosphoproteome consisting of 5157 phosphosites in 1641 phosphoproteins. Of these, 2908 (95.4%) proteins and 1636 (99.7%) phosphoproteins are known or predicted spindle-associated proteins. Clathrin-depletion from spindles resulted in dysregulation of 121 proteins and perturbed signaling to 47 phosphosites. The majority of these proteins increased in mitotic spindle abundance and six of these were validated by immunofluorescence microscopy. Functional pathway analysis confirmed the reported role of clathrin in mitotic spindle stabilization for chromosome alignment and highlighted possible new mechanisms of clathrin action. The data also revealed a novel second mitotic role for clathrin in bipolar spindle formation.
Topics: Chromatography, Liquid; Clathrin; HeLa Cells; Humans; Metaphase; Phosphorylation; Protein Binding; Protein Interaction Maps; Proteome; Proteomics; Signal Transduction; Spindle Apparatus; Tandem Mass Spectrometry
PubMed: 27174698
DOI: 10.1074/mcp.M115.054809 -
Folia Biologica 2021The effects of prostaglandin F2α on the cytoskeleton and membrane organelles of oocytes was investigated by culturing ovulated mouse oocytes in its presence (50 or 100...
The effects of prostaglandin F2α on the cytoskeleton and membrane organelles of oocytes was investigated by culturing ovulated mouse oocytes in its presence (50 or 100 ng/ml) for 3 h. Tubulin, fibrillar actin, membranes and chromatin were visualized by specific antibodies, phalloidin, lipophilic dye DiOC6 and Hoechst 33342, respectively. Control oocytes were characterized by a meiotic spindle with chromosomes aligned at its equator, and a cortical layer of microfilaments with an actin cap. Intracellular membranes were localized mostly in the central region in metaphase I and in a broader volume, but still excluding the cell periphery, in metaphase II, and were slightly concentrated around the chromosomes. In oocytes treated with 50 ng/ml prostaglandin, cortical actin staining was diminished, the membrane distribution was clustered, and chromosomes showed signs of misalignment despite the apparently preserved spindle. In cells treated with 100 ng/ml prostaglandin, both the spindle and the actin cortex had degenerated or disappeared as microscopic objects. Metaphase plates were on average broader and more disorganized than in the 50 ng/ml group, and the distribution of membrane organelles had become uniform. These effects, to our knowledge observed for the first time, did not require presence of the cumulus during the incubation. They could be regarded as acceleration of the oocyte postovulatory aging, in which cytoskeletal deterioration seemed to have a leading role.
Topics: Actins; Animals; Dinoprost; Meiosis; Metaphase; Mice; Oocytes; Spindle Apparatus
PubMed: 35439854
DOI: No ID Found -
Science Advances Jul 2022Since the ancestors of modern humans separated from those of Neanderthals, around 100 amino acid substitutions spread to essentially all modern humans. The biological...
Since the ancestors of modern humans separated from those of Neanderthals, around 100 amino acid substitutions spread to essentially all modern humans. The biological significance of these changes is largely unknown. Here, we examine all six such amino acid substitutions in three proteins known to have key roles in kinetochore function and chromosome segregation and to be highly expressed in the stem cells of the developing neocortex. When we introduce these modern human-specific substitutions in mice, three substitutions in two of these proteins, KIF18a and KNL1, cause metaphase prolongation and fewer chromosome segregation errors in apical progenitors of the developing neocortex. Conversely, the ancestral substitutions cause shorter metaphase length and more chromosome segregation errors in human brain organoids, similar to what we find in chimpanzee organoids. These results imply that the fidelity of chromosome segregation during neocortex development improved in modern humans after their divergence from Neanderthals.
Topics: Animals; Brain; Chromosome Segregation; Hominidae; Humans; Kinesins; Metaphase; Mice; Neanderthals
PubMed: 35905187
DOI: 10.1126/sciadv.abn7702 -
Scientific Reports Sep 2023The aim of our study was to evaluate the feasibility and efficiency of delayed ovarian stimulation and metaphase II oocyte banking for fertility preservation after...
Feasibility and efficiency of delayed ovarian stimulation and metaphase II oocyte banking for fertility preservation and childbearing desire after fertility-impairing treatment.
The aim of our study was to evaluate the feasibility and efficiency of delayed ovarian stimulation and metaphase II oocyte banking for fertility preservation after fertility-impairing treatment regardless of the initial disease. We conducted a cohort study based on population of women < 40 years of age with diminished ovarian reserve caused by fertility-impairing treatment (n = 129). Three groups of women were compared according to the type of initial disease: hematological malignancies, solid tumors, and benign diseases. The primary endpoint was the number of metaphase II oocytes collected per woman. We studied the cumulative live-birth rate per cycle with fertilized metaphase II oocyte, for women who wanted to conceive. We studied 245 delayed controlled ovarian stimulation cycles in 129 women: 201 for fertility preservation and 44 for in vitro fertilization and fresh embryo transfers. The number of metaphase II oocytes collected per woman after banking was similar in the three groups, with a mean of 10.7 ± 4.6, 12.3 ± 9.1, and 10.1 ± 7.6 metaphase II oocytes (p = 0.46), respectively. In the subgroup of women who wanted to conceive, the cumulative live birth rate per woman was 38%, with 8 live births for these 21 women. After fertility-impairing treatment, practitioners should discuss a fertility preservation procedure for banking metaphase II oocytes.
Topics: Female; Humans; Fertility Preservation; Metaphase; Cohort Studies; Feasibility Studies; Oocytes; Ovulation Induction
PubMed: 37730827
DOI: 10.1038/s41598-023-42583-3 -
Nature Reviews. Cancer Jul 2017Ever since initial suggestions that instability at common fragile sites (CFSs) could be responsible for chromosome rearrangements in cancers, CFSs and associated genes... (Review)
Review
Ever since initial suggestions that instability at common fragile sites (CFSs) could be responsible for chromosome rearrangements in cancers, CFSs and associated genes have been the subject of numerous studies, leading to questions and controversies about their role and importance in cancer. It is now clear that CFSs are not frequently involved in translocations or other cancer-associated recurrent gross chromosome rearrangements. However, recent studies have provided new insights into the mechanisms of CFS instability, their effect on genome instability, and their role in generating focal copy number alterations that affect the genomic landscape of many cancers.
Topics: Anaphase; Animals; Chromosomal Instability; Chromosome Breakage; Chromosome Fragile Sites; DNA Breaks, Double-Stranded; DNA Copy Number Variations; DNA Replication; Gene Rearrangement; Humans; Metaphase; Neoplasms; Oncogenes
PubMed: 28740117
DOI: 10.1038/nrc.2017.52 -
Annals of Laboratory Medicine May 2017Telomere shortening is thought to be involved in the pathophysiology of myeloid malignancies, but telomere lengths (TL) during interphase and metaphase in hematopoietic...
BACKGROUND
Telomere shortening is thought to be involved in the pathophysiology of myeloid malignancies, but telomere lengths (TL) during interphase and metaphase in hematopoietic malignancies have not been analyzed. We aimed to assess the TLs of interphase and metaphase cells of MDS and telomerase activity (TA) and to find out prognostic significances of TL and TA.
METHODS
The prognostic significance of TA by quantitative PCR and TL by quantitative fluorescence in situ hybridization (QFISH) of interphase nuclei and metaphase chromosome arms of bone marrow cells from patients with MDS were evaluated.
RESULTS
MDS patients had shorter interphase TL than normal healthy donors (P<0.001). Average interphase and metaphase TL were inversely correlated (P=0.013, p arm; P=0.029, q arm), but there was no statistically significant correlation between TA and TL (P=0.258). The progression free survival was significantly shorter in patients with high TA, but the overall survival was not different according to average TA or interphase TL groups. Multivariable Cox analysis showed that old age, higher International Prognostic Scoring System (IPSS) subtypes, transformation to AML, no history of hematopoietic stem cell transplantation and short average interphase TL (<433 TL) as independent prognostic factors for poorer survival (P=0.003, 0.001, 0.005, 0.005, and 0.013, respectively).
CONCLUSIONS
The lack of correlation between age and TL, TA, and TL, and the inverse relationship between TL and TA in MDS patients reflect the dysregulation of telomere status and proliferation. As a prognostic marker for leukemia progression, TA may be considered, and since interphase TL has the advantage of automated measurement by QFISH, it may be used as a prognostic marker for survival in MDS.
Topics: Adult; Aged; Aged, 80 and over; Bone Marrow Cells; Disease-Free Survival; Female; Humans; In Situ Hybridization, Fluorescence; Interphase; Kaplan-Meier Estimate; Karyotyping; Male; Metaphase; Middle Aged; Myelodysplastic Syndromes; Prognosis; Proportional Hazards Models; Telomerase; Telomere; Telomere Shortening
PubMed: 28224765
DOI: 10.3343/alm.2017.37.3.195