-
Frontiers in Cell and Developmental... 2023Accurate chromosome segregation is vital for cell and organismal viability. The mitotic spindle, a bipolar macromolecular machine composed largely of dynamic... (Review)
Review
Accurate chromosome segregation is vital for cell and organismal viability. The mitotic spindle, a bipolar macromolecular machine composed largely of dynamic microtubules, is responsible for chromosome segregation during each cell replication cycle. Prior to anaphase, a bipolar metaphase spindle must be formed in which each pair of chromatids is attached to microtubules from opposite spindle poles. In this bipolar configuration pulling forces from the dynamic microtubules can generate tension across the sister kinetochores. The tension status acts as a signal that can destabilize aberrant kinetochore-microtubule attachments and reinforces correct, bipolar connections. Historically it has been challenging to isolate the specific role of tension in mitotic processes due to the interdependency of attachment and tension status at kinetochores. Recent technical and experimental advances have revealed new insights into how tension functions during mitosis. Here we summarize the evidence that tension serves as a biophysical signal that unifies multiple aspects of kinetochore and centromere function to ensure accurate chromosome segregation.
PubMed: 36755973
DOI: 10.3389/fcell.2023.1096333 -
The Plant Journal : For Cell and... Aug 2023Mitosis and cytokinesis are fundamental processes through which somatic cells increase their numbers and allow plant growth and development. Here, we analyzed the...
Mitosis and cytokinesis are fundamental processes through which somatic cells increase their numbers and allow plant growth and development. Here, we analyzed the organization and dynamics of mitotic chromosomes, nucleoli, and microtubules in living cells of barley root primary meristems using a series of newly developed stable fluorescent protein translational fusion lines and time-lapse confocal microscopy. The median duration of mitosis from prophase until the end of telophase was 65.2 and 78.2 min until the end of cytokinesis. We showed that barley chromosomes frequently start condensation before mitotic pre-prophase as defined by the organization of microtubules and maintain it even after entering into the new interphase. Furthermore, we found that the process of chromosome condensation does not finish at metaphase, but gradually continues until the end of mitosis. In summary, our study features resources for in vivo analysis of barley nuclei and chromosomes and their dynamics during mitotic cell cycle.
Topics: Hordeum; Mitosis; Chromosomes; Microtubules; Cell Nucleus; Prophase
PubMed: 37326283
DOI: 10.1111/tpj.16355 -
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 -
PLoS Biology Jul 2022Precise spatiotemporal control of microtubule nucleation and organization is critical for faithful segregation of cytoplasmic and genetic material during cell division...
Precise spatiotemporal control of microtubule nucleation and organization is critical for faithful segregation of cytoplasmic and genetic material during cell division and signaling via the primary cilium in quiescent cells. Microtubule-associated proteins (MAPs) govern assembly, maintenance, and remodeling of diverse microtubule arrays. While a set of conserved MAPs are only active during cell division, an emerging group of MAPs acts as dual regulators in dividing and nondividing cells. Here, we elucidated the nonciliary functions and molecular mechanism of action of the ciliopathy-linked protein CCDC66, which we previously characterized as a regulator of ciliogenesis in quiescent cells. We showed that CCDC66 dynamically localizes to the centrosomes, the bipolar spindle, the spindle midzone, the central spindle, and the midbody in dividing cells and interacts with the core machinery of centrosome maturation and MAPs involved in cell division. Loss-of-function experiments revealed its functions during mitotic progression and cytokinesis. Specifically, CCDC66 depletion resulted in defective spindle assembly and orientation, kinetochore fiber stability, chromosome alignment in metaphase as well as central spindle and midbody assembly and organization in anaphase and cytokinesis. Notably, CCDC66 regulates mitotic microtubule nucleation via noncentrosomal and centrosomal pathways via recruitment of gamma-tubulin to the centrosomes and the spindle. Additionally, CCDC66 bundles microtubules in vitro and in cells by its C-terminal microtubule-binding domain. Phenotypic rescue experiments showed that the microtubule and centrosome-associated pools of CCDC66 individually or cooperatively mediate its mitotic and cytokinetic functions. Collectively, our findings identify CCDC66 as a multifaceted regulator of the nucleation and organization of the diverse mitotic and cytokinetic microtubule arrays and provide new insight into nonciliary defects that underlie ciliopathies.
Topics: Anaphase; Centrosome; Ciliopathies; Cytokinesis; Eye Proteins; Humans; Microtubule-Associated Proteins; Microtubules; Mitosis; Spindle Apparatus
PubMed: 35849559
DOI: 10.1371/journal.pbio.3001708 -
Methods in Molecular Biology (Clifton,... 2023After DNAs are damaged, DNA repair proteins accumulate and are activated at the DNA damaged site. These accumulated proteins are visualized as foci by fluorescent...
After DNAs are damaged, DNA repair proteins accumulate and are activated at the DNA damaged site. These accumulated proteins are visualized as foci by fluorescent immunocytochemistry technique. This allows the DNA damage responses in interphase nuclei to be detected; it was earlier times difficult to analyze DNA damage in situ. In order to analyze DNA damage in interphase cells, either DNA is extracted to assay breaks biochemically, or premature chromosome condensation is conducted to observe as chromatin breaks. Although DNA damage-induced foci are typically analyzed in interphase cells, these foci can be also visualized on mitotic chromosomes. The foci where the repair proteins accumulate at the damage site is observed as mitotic chromosome break site. Since mitotic cells attach loosely or not attached to cell culture vessels, it is difficult to analyze foci on chromosomes in culture vessels under a microscope, so metaphase chromosome spread must be prepared for accurate analysis. The cytocentrifuge system is an ideal method to adhere mitotic cells to microscope slides for the fluorescent immunocytochemistry. This chapter introduces cytocentrifuge method to prepare metaphase spread for DNA damage foci analysis.
Topics: Chromosomes; DNA; DNA Damage; Interphase; Metaphase
PubMed: 36066713
DOI: 10.1007/978-1-0716-2433-3_10 -
Methods in Molecular Biology (Clifton,... 2023In plants, the segregation of genetic material is achieved by an acentrosomal, mitotic spindle. This macromolecular machinery consists of different microtubule... (Review)
Review
In plants, the segregation of genetic material is achieved by an acentrosomal, mitotic spindle. This macromolecular machinery consists of different microtubule subpopulations and interacting proteins. The majority of what we know about the assembly and shape control of the mitotic spindle arose from vertebrate model systems. The dynamic properties of the individual tubulin polymers are crucial for the accurate assembly of the spindle array and are modulated by microtubule-associated motor and non-motor proteins. The mitotic spindle relies on a phenomenon called poleward microtubule flux that is critical to establish spindle shape, chromosome alignment, and segregation. This flux is under control of the non-motor microtubule-associated proteins and force-generating motors. Despite the large number of (plant-specific) kinesin motor proteins expressed during mitosis, their mitotic roles remain largely elusive. Moreover, reports on mitotic spindle formation and shape control in higher plants are scarce. In this chapter, an overview of the basic principles and methods concerning live imaging of prometa- and metaphase spindles and the analysis of spindle microtubule flux using fluorescence recovery after photobleaching is provided.
Topics: Kinesins; Microtubule-Associated Proteins; Microtubules; Mitosis; Spindle Apparatus; Tubulin
PubMed: 36773229
DOI: 10.1007/978-1-0716-2867-6_9 -
Cell Death & Disease Jun 2022CCAR2 (cell cycle and apoptosis regulator 2) is a multifaceted protein involved in cell survival and death following cytotoxic stress. However, little is known about the...
CCAR2 (cell cycle and apoptosis regulator 2) is a multifaceted protein involved in cell survival and death following cytotoxic stress. However, little is known about the physiological functions of CCAR2 in regulating cell proliferation in the absence of external stimuli. The present study shows that CCAR2-deficient cells possess multilobulated nuclei, suggesting a defect in cell division. In particular, the duration of mitotic phase was perturbed. This disturbance of mitotic progression resulted from premature loss of cohesion with the centromere, and inactivation of the spindle assembly checkpoint during prometaphase and metaphase. It resulted in the formation of lagging chromosomes during anaphase, leading ultimately to the activation of the abscission checkpoint to halt cytokinesis. The CCAR2-dependent mitotic progression was related to spatiotemporal regulation of active Aurora B. In conclusion, the results suggest that CCAR2 governs mitotic events, including proper chromosome segregation and cytokinetic division, to maintain chromosomal stability.
Topics: Aurora Kinase B; Cell Cycle Proteins; Centromere; Chromosome Segregation; Mitosis; Protein Serine-Threonine Kinases; Spindle Apparatus
PubMed: 35672287
DOI: 10.1038/s41419-022-04990-8 -
Frontiers in Cell and Developmental... 2022Depletion of the Anaphase-Promoting Complex/Cyclosome (APC/C) activator Cdc20 arrests cells in metaphase with high levels of the mitotic cyclin (Cyclin B) and the...
Depletion of the Anaphase-Promoting Complex/Cyclosome (APC/C) activator Cdc20 arrests cells in metaphase with high levels of the mitotic cyclin (Cyclin B) and the Separase inhibitor Securin. In mammalian cells this arrest has been exploited for the treatment of cancer with drugs that engage the spindle assembly checkpoint and, recently, with chemical inhibitors of the APC/C. While most cells arrested in mitosis for prolonged periods undergo apoptosis, others skip cytokinesis and enter G1 with unsegregated chromosomes. This process, known as mitotic slippage, generates aneuploidy and increases genomic instability in the cancer cell. Here, we analyze the behavior of fission yeast cells arrested in mitosis through the transcriptional silencing of the Cdc20 homolog . While depletion of readily halts cells in metaphase, this arrest is only transient and a majority of cells eventually undergo cytokinesis and show steady mitotic dephosphorylation. Notably, this occurs in the absence of Cyclin B (Cdc13) degradation. We investigate the involvement of phosphatase activity in these events and demonstrate that PP2A-B55 is required to prevent septation and, during the arrest, its CDK-mediated inhibition facilitates the induction of cytokinesis. In contrast, deletion of PP2A-B56 completely abrogates septation. We show that this effect is partly due to this mutant entering mitosis with reduced CDK activity. Interestingly, both PP2A-B55 and PP2A-B56, as well as Clp1 (the homolog of the budding yeast mitotic phosphatase Cdc14) are required for the dephosphorylation of mitotic substrates during the escape. Finally, we show that the mitotic transcriptional wave controlled by the RFX transcription factor Sak1 facilitates the induction of cytokinesis and also requires the activity of PP2A-B56 in a mechanism independent of CDK.
PubMed: 35923846
DOI: 10.3389/fcell.2022.876810 -
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 -
Journal of Cell Science Jun 2023The budding yeast Saccharomyces cerevisiae has a closed mitosis in which the mitotic spindle and the cytoplasmic microtubules (MTs), both of which generate forces to...
The budding yeast Saccharomyces cerevisiae has a closed mitosis in which the mitotic spindle and the cytoplasmic microtubules (MTs), both of which generate forces to faithfully segregate chromosomes, remain separated by the nuclear envelope throughout the cell cycle. Kar3, the yeast kinesin-14, has distinct functions on MTs in each compartment. Here, we show that two proteins, Cik1 and Vik1, which form heterodimers with Kar3, regulate its localization and function within the cell, and along MTs in a cell cycle-dependent manner. Using a yeast MT dynamics reconstitution assay in lysates from cell cycle-synchronized cells, we found that Kar3-Vik1 induces MT catastrophes in S phase and metaphase, and limits MT polymerization in G1 and anaphase. In contrast, Kar3-Cik1 promotes catastrophes and pauses in G1, while increasing catastrophes in metaphase and anaphase. Adapting this assay to track MT motor protein motility, we observed that Cik1 is necessary for Kar3 to track MT plus-ends in S phase and metaphase but, surprisingly, not during anaphase. These experiments demonstrate how the binding partners of Kar3 modulate its diverse functions both spatially and temporally.
Topics: Kinesins; Saccharomyces cerevisiae; Cell Cycle; Anaphase; Metaphase
PubMed: 37305999
DOI: 10.1242/jcs.260621