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Centrosome linker diversity and its function in centrosome clustering and mitotic spindle formation.The EMBO Journal Sep 2023The centrosome linker joins the two interphase centrosomes of a cell into one microtubule organizing center. Despite increasing knowledge on linker components, linker...
The centrosome linker joins the two interphase centrosomes of a cell into one microtubule organizing center. Despite increasing knowledge on linker components, linker diversity in different cell types and their role in cells with supernumerary centrosomes remained unexplored. Here, we identified Ninein as a C-Nap1-anchored centrosome linker component that provides linker function in RPE1 cells while in HCT116 and U2OS cells, Ninein and Rootletin link centrosomes together. In interphase, overamplified centrosomes use the linker for centrosome clustering, where Rootletin gains centrosome linker function in RPE1 cells. Surprisingly, in cells with centrosome overamplification, C-Nap1 loss prolongs metaphase through persistent activation of the spindle assembly checkpoint indicated by BUB1 and MAD1 accumulation at kinetochores. In cells lacking C-Nap1, the reduction of microtubule nucleation at centrosomes and the delay in nuclear envelop rupture in prophase probably cause mitotic defects like multipolar spindle formation and chromosome mis-segregation. These defects are enhanced when the kinesin HSET, which normally clusters multiple centrosomes in mitosis, is partially inhibited indicating a functional interplay between C-Nap1 and centrosome clustering in mitosis.
Topics: Centrosome; Cell Cycle; Cell Cycle Proteins; Interphase; Mitosis; Spindle Apparatus
PubMed: 37401899
DOI: 10.15252/embj.2021109738 -
Mathematical Biosciences May 2024This paper develops a theory for anaphase in cells. After a brief description of microtubules, the mitotic spindle and the centrosome, a mathematical model for anaphase...
This paper develops a theory for anaphase in cells. After a brief description of microtubules, the mitotic spindle and the centrosome, a mathematical model for anaphase is introduced and developed in the context of the cell cytoplasm and liquid crystalline structures. Prophase, prometaphase and metaphase are then briefly described in order to focus on anaphase, which is the main study of this paper. The entities involved are modelled in terms of liquid crystal defects and microtubules are represented as defect flux lines. The mathematical techniques employed make extensive use of energy considerations based on the work that was developed by Dafermos (1970) from the classical Frank-Oseen nematic liquid crystal energy (Frank, 1958; Oseen, 1933). With regard to liquid crystal theory we introduce the concept of regions of influence for defects which it is believed have important implications beyond the subject of this paper. The results of this paper align with observed biochemical phenomena and are explored in application to HeLa cells and Caenorhabditis elegans. This unified approach offers the possibility of gaining insight into various consequences of mitotic abnormalities which may result in Down syndrome, Hodgkin lymphoma, breast, prostate and various other types of cancer.
PubMed: 38795952
DOI: 10.1016/j.mbs.2024.109219 -
The Journal of Cell Biology Nov 2023UHRF1 is an epigenetic coordinator bridging DNA methylation and histone modifications. Additionally, UHRF1 regulates DNA replication and cell cycle, and its deletion...
UHRF1 is an epigenetic coordinator bridging DNA methylation and histone modifications. Additionally, UHRF1 regulates DNA replication and cell cycle, and its deletion induces G1/S or G2/M cell cycle arrest. The roles of UHRF1 in the regulation of G2/M transition remain poorly understood. UHRF1 depletion caused chromosome misalignment, thereby inducing cell cycle arrest at mitotic metaphase, and these cells exhibited the defects of spindle geometry, prominently manifested as shorter spindles. Mechanistically, UHRF1 protein directly interacts with EG5, a kinesin motor protein, during mitosis. Furthermore, UHRF1 induced EG5 polyubiquitination at the site of K1034 and further promoted the interaction of EG5 with spindle assembly factor TPX2, thereby ensuring accurate EG5 distribution to the spindles during metaphase. Our study clarifies a novel UHRF1 function as a nuclear protein catalyzing EG5 polyubiquitination for proper spindle architecture and faithful genomic transmission, which is independent of its roles in epigenetic regulation and DNA damage repair inside the nucleus. These findings revealed a previously unknown mechanism of UHRF1 in controlling mitotic spindle architecture and chromosome behavior and provided mechanistic evidence for UHRF1 deletion-mediated G2/M arrest.
Topics: Apoptosis; Cell Line, Tumor; Epigenesis, Genetic; G2 Phase Cell Cycle Checkpoints; Mitosis; Humans; Ubiquitin-Protein Ligases; Kinesins; Ubiquitination; DNA Damage; Spindle Apparatus; Chromosomes
PubMed: 37728657
DOI: 10.1083/jcb.202210093 -
Nature Communications Dec 2023The centromere components cohesin, CENP-A, and centromeric DNA are essential for biorientation of sister chromatids on the mitotic spindle and accurate sister chromatid...
The centromere components cohesin, CENP-A, and centromeric DNA are essential for biorientation of sister chromatids on the mitotic spindle and accurate sister chromatid segregation. Insight into the 3D organization of centromere components would help resolve how centromeres function on the mitotic spindle. We use ChIP-seq and super-resolution microscopy with single particle averaging to examine the geometry of essential centromeric components on human chromosomes. Both modalities suggest cohesin is enriched at pericentromeric DNA. CENP-A localizes to a subset of the α-satellite DNA, with clusters separated by ~562 nm and a perpendicular intervening ~190 nM wide axis of cohesin in metaphase chromosomes. Differently sized α-satellite arrays achieve a similar core structure. Here we present a working model for a common core configuration of essential centromeric components that includes CENP-A nucleosomes, α-satellite DNA and pericentromeric cohesion. This configuration helps reconcile how centromeres function and serves as a foundation to add components of the chromosome segregation machinery.
Topics: Humans; DNA, Satellite; Centromere Protein A; Centromere; Mitosis; Cell Cycle Proteins; Spindle Apparatus; Chromatids; Chromosome Segregation
PubMed: 38040722
DOI: 10.1038/s41467-023-42980-2 -
The EMBO Journal Oct 2023Two major mechanisms safeguard genome stability during mitosis: the mitotic checkpoint delays mitosis until all chromosomes have attached to microtubules, and the...
Two major mechanisms safeguard genome stability during mitosis: the mitotic checkpoint delays mitosis until all chromosomes have attached to microtubules, and the kinetochore-microtubule error-correction pathway keeps this attachment process free from errors. We demonstrate here that the optimal strength and dynamics of these processes are set by a kinase-phosphatase pair (PLK1-PP2A) that engage in negative feedback from adjacent phospho-binding motifs on the BUB complex. Uncoupling this feedback to skew the balance towards PLK1 produces a strong checkpoint, hypostable microtubule attachments and mitotic delays. Conversely, skewing the balance towards PP2A causes a weak checkpoint, hyperstable microtubule attachments and chromosome segregation errors. These phenotypes are associated with altered BUB complex recruitment to KNL1-MELT motifs, implicating PLK1-PP2A in controlling auto-amplification of MELT phosphorylation. In support, KNL1-BUB disassembly becomes contingent on PLK1 inhibition when KNL1 is engineered to contain excess MELT motifs. This elevates BUB-PLK1/PP2A complex levels on metaphase kinetochores, stabilises kinetochore-microtubule attachments, induces chromosome segregation defects and prevents KNL1-BUB disassembly at anaphase. Together, these data demonstrate how a bifunctional PLK1/PP2A module has evolved together with the MELT motifs to optimise BUB complex dynamics and ensure accurate chromosome segregation.
Topics: Humans; M Phase Cell Cycle Checkpoints; Kinetochores; Protein Serine-Threonine Kinases; Cell Cycle Proteins; Chromosome Segregation; Phosphorylation; Microtubules; Mitosis; HeLa Cells
PubMed: 37712330
DOI: 10.15252/embj.2022112630 -
Journal of Obstetrics and Gynaecology :... Dec 2023FAM64A is a mitotic regulator which promotes cell metaphase-anaphase transition and is highly expressed in a cell-cycle-dependent manner. In this study, we examined the...
FAM64A is a mitotic regulator which promotes cell metaphase-anaphase transition and is highly expressed in a cell-cycle-dependent manner. In this study, we examined the clinicopathological and prognostic significance of mRNA expression in gynecological cancers. We conducted a bioinformatics analysis of mRNA expression using Gene Expression Omnibus (GEO), The Cancer Genome Atlas (TCGA), xiantao, The University of ALabama at Birmingham CANcer data analysis Portal (UALCAN), and Kaplan-Meier (KM) plotter databases. expression was elevated in breast, cervical, endometrial, and ovarian cancers when compared with normal tissue. Expression was positively correlated with white race, low T stages, infiltrating ductal carcinoma, or favourable PAM50 classification in breast cancer patients, and with clinical stage, histological grade and TP53 mutation, and endometrial cancer serous subtype. expression was negatively associated with overall and/or recurrence-free survival rates in breast and endometrial cancer patients, while the opposite was observed in cervical and ovarian cancer patients. functioned as an independent predictor of overall and disease-specific survival in breast cancer patients. -correlated genes were involved in ligand-receptor interactions, and chromosomal, cell cycle, and DNA replication processes in breast, cervical, endometrial and ovarian cancers. Top hub genes primarily included cell cycle-related proteins in breast cancer, mucins and acetylgalactosaminyl transferases in cervical cancer, kinesin family members in endometrial cancer, and synovial sarcoma X and the cancer/testis antigen in ovarian cancer. mRNA expression was positively related to Th2 cell infiltration, but negatively associated with neutrophil and Th17 cell infiltration in breast, cervical, endometrial, and ovarian cancers. expression may be considered a potential biomarker reflecting carcinogenesis, histogenesis, aggressive behaviour, and prognosis in gynecological cancers.Impact statement FAM64A is located in cell nucleolar and nucleoplasmic regions, and during mitosis it putatively controls metaphase-to-anaphase transition. FAM64A appears to regulate different physiological processes, including apoptosis, tumorigenesis, neural differentiation, stress responses, and the cell cycle. expression was up-regulated in breast, cervical, endometrial, and ovarian cancers, and positively correlated with white race, low T stages, infiltrating ductal carcinoma, or favourable PAM50 classification in breast cancer patients, and with clinical stage, histological grade, and TP53 mutation, and a serous subtype in endometrial cancer. expression was negatively associated with overall and/or recurrence-free survival rates in breast and endometrial cancer patients, while the opposite was observed in cervical and ovarian cancer patients. functioned as an independent predictor of overall and disease-specific survival in breast cancer. -correlated genes were involved in ligand-receptor interactions, chromosomal, cell cycle, and DNA replication processes, while mRNA expression was positively related to Th2 cell infiltration but negatively correlated with neutrophil and Th17 cell infiltration in four gynecological cancers. In the future, abnormal mRNA expression may serve as a biomarker of carcinogenesis, histogenesis, aggressiveness, and prognosis in gynecological malignancies.
Topics: Female; Humans; Male; Breast Neoplasms; Carcinogenesis; Carcinoma, Ductal; Computational Biology; Endometrial Neoplasms; Gene Expression Regulation, Neoplastic; Ligands; Ovarian Neoplasms; Prognosis; RNA, Messenger
PubMed: 37227120
DOI: 10.1080/01443615.2023.2216280 -
ELife Jun 2023Human oocyte maturation arrest represents one of the severe conditions for female patients with primary infertility. However, the genetic factors underlying this human...
Human oocyte maturation arrest represents one of the severe conditions for female patients with primary infertility. However, the genetic factors underlying this human disease remain largely unknown. The spindle assembly checkpoint (SAC) is an intricate surveillance mechanism that ensures accurate segregation of chromosomes throughout cell cycles. Once the kinetochores of chromosomes are correctly attached to bipolar spindles and the SAC is satisfied, the MAD2L1BP, best known as p31, binds mitosis arrest deficient 2 (MAD2) and recruits the AAA+-ATPase TRIP13 to disassemble the mitotic checkpoint complex (MCC), leading to the cell-cycle progression. In this study, by whole-exome sequencing (WES), we identified homozygous and compound heterozygous variants in three families with female patients diagnosed with primary infertility owing to oocyte metaphase I (MI) arrest. Functional studies revealed that the protein variants resulting from the C-terminal truncation of MAD2L1BP lost their binding ability to MAD2. cRNA microinjection of full-length or truncated uncovered their discordant roles in driving the extrusion of polar body 1 (PB1) in mouse oocytes. Furthermore, the patient's oocytes carrying the mutated resumed polar body extrusion (PBE) when rescued by microinjection of full-length cRNAs. Together, our studies identified and characterized novel biallelic variants in responsible for human oocyte maturation arrest at MI, and thus prompted new therapeutic avenues for curing female primary infertility.
Topics: Animals; Female; Humans; Mice; Adaptor Proteins, Signal Transducing; Cell Cycle Checkpoints; Cell Cycle Proteins; Exome Sequencing; Infertility, Female; Mad2 Proteins; Nuclear Proteins; Oocytes; Young Adult; Adult; Meiosis
PubMed: 37334967
DOI: 10.7554/eLife.85649 -
The EMBO Journal Mar 2024The efficacy of current antimitotic cancer drugs is limited by toxicity in highly proliferative healthy tissues. A cancer-specific dependency on the microtubule motor...
The efficacy of current antimitotic cancer drugs is limited by toxicity in highly proliferative healthy tissues. A cancer-specific dependency on the microtubule motor protein KIF18A therefore makes it an attractive therapeutic target. Not all cancers require KIF18A, however, and the determinants underlying this distinction remain unclear. Here, we show that KIF18A inhibition drives a modest and widespread increase in spindle assembly checkpoint (SAC) signaling from kinetochores which can result in lethal mitotic delays. Whether cells arrest in mitosis depends on the robustness of the metaphase-to-anaphase transition, and cells predisposed with weak basal anaphase-promoting complex/cyclosome (APC/C) activity and/or persistent SAC signaling through metaphase are uniquely sensitive to KIF18A inhibition. KIF18A-dependent cancer cells exhibit hallmarks of this SAC:APC/C imbalance, including a long metaphase-to-anaphase transition, and slow mitosis overall. Together, our data reveal vulnerabilities in the cell division apparatus of cancer cells that can be exploited for therapeutic benefit.
Topics: Humans; Anaphase-Promoting Complex-Cyclosome; Dyneins; Kinesins; Kinetochores; Mitosis; Neoplasms
PubMed: 38279026
DOI: 10.1038/s44318-024-00031-6 -
European Journal of Medical Research Dec 2023Hepatocellular carcinoma (HCC) is one of the most prevalent forms of cancer and poses a threat to the health and survival of humans. Mitochondrial ribosomal protein L48...
BACKGROUND
Hepatocellular carcinoma (HCC) is one of the most prevalent forms of cancer and poses a threat to the health and survival of humans. Mitochondrial ribosomal protein L48 (MRPL48) belongs to the mitochondrial ribosomal protein family, which participates in energy production. Studies have shown that MRPL48 can predict osteosarcoma incidence and prognosis, as well as promotes colorectal cancer progression. However, the role of MRPL48 in HCC remains unknown.
METHODS
TCGA, GEO, HCCDB, CPTAC, SMART, UALCAN, Kaplan-Meier plotter, cBioPortal, and MethSurv were performed for bioinformatics purposes. Quantitative RT-PCR, immunoblotting, and functional studies were conducted to validate the methodology in vitro.
RESULTS
MRPL48 was greatly overexpressed in HCC tissues, compared with healthy tissue, which was subsequently demonstrated in vitro as well. The survival and regression analyses showed that MRPL48 expression is of significant clinical prognostic value in HCC. The ROC curve and nomogram analysis indicated that MRPL48 is a powerful predictor of HCC. MRPL48 methylation was adversely associated with the expression of MRPL48, and patients with a low level of methylation had poorer overall survival than those with a high level of methylation. GSEA showed that the expression of the MRPL48 was correlated with Resolution of Sister Chromatid Cohesion, Mitotic Prometaphase, Retinoblastoma Gene in Cancer, RHO Gtpases Activate Formins, Mitotic Metaphase and Anaphase, and Cell Cycle Checkpoints. An analysis of immune cell infiltration showed a significant association between MRPL48 and immune cell infiltration subsets, which impacted the survival of HCC patients. Additionally, MRPL48 knockdown reduced HCC cell proliferation, migration, and invasion in vitro.
CONCLUSIONS
We demonstrated that MRPL48 expression may be associated with HCC development and prognosis. These findings may open up new research directions and opportunities for the development of HCC treatments.
Topics: Humans; Prognosis; Carcinoma, Hepatocellular; Liver Neoplasms; Biomarkers; Ribosomal Proteins
PubMed: 38093387
DOI: 10.1186/s40001-023-01571-z -
Cellular and Molecular Life Sciences :... Nov 2023Faithful chromosome segregation requires correct attachment of kinetochores with the spindle microtubules. Erroneously-attached kinetochores recruit proteins to activate...
Faithful chromosome segregation requires correct attachment of kinetochores with the spindle microtubules. Erroneously-attached kinetochores recruit proteins to activate Spindle assembly checkpoint (SAC), which senses the errors and signals cells to delay anaphase progression for error correction. Temporal control of the levels of SAC activating-proteins is critical for checkpoint activation and silencing, but its mechanism is not fully understood. Here, we show that E3 ubiquitin ligase, SCF-FBXW7 targets BubR1 for ubiquitin-mediated degradation and thereby controls SAC in human cells. Depletion of FBXW7 results in prolonged metaphase arrest with increased stabilization of BubR1 at kinetochores. Similar kinetochore stabilization is also observed for BubR1-interacting protein, CENP-E. FBXW7 induced ubiquitination of both BubR1 and the BubR1-interacting kinetochore-targeting domain of CENP-E, but CENP-E domain degradation is dependent on BubR1. Interestingly, Cdk1 inhibition disrupts FBXW7-mediated BubR1 targeting and further, phospho-resistant mutation of Cdk1-targeted phosphorylation site, Thr 620 impairs BubR1-FBXW7 interaction and FBXW7-mediated BubR1 ubiquitination, supporting its role as a phosphodegron for FBXW7. The results demonstrate SCF-FBXW7 as a key regulator of spindle assembly checkpoint that controls stability of BubR1 and its associated CENP-E at kinetochores. They also support that upstream Cdk1 specific BubR1 phosphorylation signals the ligase to activate the process.
Topics: Humans; Cell Cycle Proteins; F-Box-WD Repeat-Containing Protein 7; HeLa Cells; Kinetochores; Mitosis; Protein Serine-Threonine Kinases; Spindle Apparatus; Ubiquitin; Ubiquitin-Protein Ligases
PubMed: 38008853
DOI: 10.1007/s00018-023-05019-9