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Scientific Reports Sep 2017Mitotic spindles, which consist of microtubules (MTs) and associated proteins, play critical roles in controlling cell division and maintaining tissue homeostasis. The...
Mitotic spindles, which consist of microtubules (MTs) and associated proteins, play critical roles in controlling cell division and maintaining tissue homeostasis. The orientation of the mitotic spindle is closely related with the duration of mitosis. However, the molecular mechanism in regulating the orientation of the mitotic spindles is largely undefined. In this study, we found that Palladin is a novel MT-associated protein and regulator of spindle orientation, which maintains proper spindle orientation by stabilizing astral MTs. Palladin depletion distorted spindle orientation, prolonged the metaphase, and impaired proliferation of HeLa cells. Results showed that Palladin depletion-induced spindle misorientation and astral MT instability could be rescued by constitutively active AKT1 or dominant negative GSK3β. Our findings revealed that Palladin regulates spindle orientation and mitotic progression mainly through the AKT1-GSK3β pathway.
Topics: Cytoskeletal Proteins; Glycogen Synthase Kinase 3 beta; HeLa Cells; Humans; Metaphase; Microtubule-Associated Proteins; Microtubules; Phosphoproteins; Proto-Oncogene Proteins c-akt; Signal Transduction; Spindle Apparatus
PubMed: 28924223
DOI: 10.1038/s41598-017-12051-w -
The Journal of Cell Biology Jun 2013Cdk1 drives both mitotic entry and the metaphase-to-anaphase transition. Past work has shown that Wee1 inhibition of Cdk1 blocks mitotic entry. Here we show that the...
Cdk1 drives both mitotic entry and the metaphase-to-anaphase transition. Past work has shown that Wee1 inhibition of Cdk1 blocks mitotic entry. Here we show that the budding yeast Wee1 kinase, Swe1, also restrains the metaphase-to-anaphase transition by preventing Cdk1 phosphorylation and activation of the mitotic form of the anaphase-promoting complex/cyclosome (APC(Cdc20)). Deletion of SWE1 or its opposing phosphatase MIH1 (the budding yeast cdc25(+)) altered the timing of anaphase onset, and activation of the Swe1-dependent morphogenesis checkpoint or overexpression of Swe1 blocked cells in metaphase with reduced APC activity in vivo and in vitro. The morphogenesis checkpoint also depended on Cdc55, a regulatory subunit of protein phosphatase 2A (PP2A). cdc55Δ checkpoint defects were rescued by mutating 12 Cdk1 phosphorylation sites on the APC, demonstrating that the APC is a target of this checkpoint. These data suggest a model in which stepwise activation of Cdk1 and inhibition of PP2A(Cdc55) triggers anaphase onset.
Topics: Anaphase; CDC2 Protein Kinase; Cell Cycle Proteins; Genes, cdc; Metaphase; Phosphorylation; Protein Phosphatase 2; Protein-Tyrosine Kinases; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Ubiquitination; ras-GRF1
PubMed: 23751495
DOI: 10.1083/jcb.201212038 -
Journal of Cell Science Jan 2021Errors in mitotic chromosome segregation can lead to DNA damage and aneuploidy, both hallmarks of cancer. To achieve synchronous error-free segregation, mitotic...
Errors in mitotic chromosome segregation can lead to DNA damage and aneuploidy, both hallmarks of cancer. To achieve synchronous error-free segregation, mitotic chromosomes must align at the metaphase plate with stable amphitelic attachments to microtubules emanating from opposing spindle poles. The astrin-kinastrin (astrin is also known as SPAG5 and kinastrin as SKAP) complex, also containing DYNLL1 and MYCBP, is a spindle and kinetochore protein complex with important roles in bipolar spindle formation, chromosome alignment and microtubule-kinetochore attachment. However, the molecular mechanisms by which astrin-kinastrin fulfils these diverse roles are not fully understood. Here, we characterise a direct interaction between astrin and the mitotic kinase Plk1. We identify the Plk1-binding site on astrin as well as four Plk1 phosphorylation sites on astrin. Regulation of astrin by Plk1 is dispensable for bipolar spindle formation and bulk chromosome congression, but promotes stable microtubule-kinetochore attachments and metaphase plate maintenance. It is known that Plk1 activity is required for effective microtubule-kinetochore attachment formation, and we suggest that astrin phosphorylation by Plk1 contributes to this process.
Topics: Alcian Blue; Cell Cycle Proteins; Chromosome Segregation; HeLa Cells; Humans; Kinetochores; Metaphase; Microtubule-Associated Proteins; Microtubules; Mitosis; Phenazines; Phenothiazines; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Resorcinols; Spindle Apparatus; Polo-Like Kinase 1
PubMed: 33288550
DOI: 10.1242/jcs.251025 -
Cells Jul 2021Glioblastoma multiforme (GBM) is characterized by several genetic abnormalities, leading to cell cycle deregulation and abnormal mitosis caused by a defective...
BACKGROUND
Glioblastoma multiforme (GBM) is characterized by several genetic abnormalities, leading to cell cycle deregulation and abnormal mitosis caused by a defective checkpoint. We previously demonstrated that arecaidine propargyl ester (APE), an orthosteric agonist of M2 muscarinic acetylcholine receptors (mAChRs), arrests the cell cycle of glioblastoma (GB) cells, reducing their survival. The aim of this work was to better characterize the molecular mechanisms responsible for this cell cycle arrest.
METHODS
The arrest of cell proliferation was evaluated by flow cytometry analysis. Using immunocytochemistry and time-lapse analysis, the percentage of abnormal mitosis and aberrant mitotic spindles were assessed in both cell lines. Western blot analysis was used to evaluate the modulation of Sirtuin2 and acetylated tubulin-factors involved in the control of cell cycle progression.
RESULTS
APE treatment caused arrest in the M phase, as indicated by the increase in p-HH3 (ser10)-positive cells. By immunocytochemistry, we found a significant increase in abnormal mitoses and multipolar mitotic spindle formation after APE treatment. Time-lapse analysis confirmed that the APE-treated GB cells were unable to correctly complete the mitosis. The modulated expression of SIRT2 and acetylated tubulin in APE-treated cells provides new insights into the mechanisms of altered mitotic progression in both GB cell lines.
CONCLUSIONS
Our data show that the M2 agonist increases aberrant mitosis in GB cell lines. These results strengthen the idea of considering M2 acetylcholine receptors a novel promising therapeutic target for the glioblastoma treatment.
Topics: Acetylation; Arecoline; Cell Line, Tumor; Cell Nucleus; Glioblastoma; Humans; Metaphase; Mitosis; Receptor, Muscarinic M2; Sirtuin 2; Spindle Apparatus; Time-Lapse Imaging; Tubulin
PubMed: 34359896
DOI: 10.3390/cells10071727 -
Science (New York, N.Y.) Nov 2013Mitotic chromosomes are among the most recognizable structures in the cell, yet for over a century their internal organization remains largely unsolved. We applied...
Mitotic chromosomes are among the most recognizable structures in the cell, yet for over a century their internal organization remains largely unsolved. We applied chromosome conformation capture methods, 5C and Hi-C, across the cell cycle and revealed two distinct three-dimensional folding states of the human genome. We show that the highly compartmentalized and cell type-specific organization described previously for nonsynchronous cells is restricted to interphase. In metaphase, we identified a homogenous folding state that is locus-independent, common to all chromosomes, and consistent among cell types, suggesting a general principle of metaphase chromosome organization. Using polymer simulations, we found that metaphase Hi-C data are inconsistent with classic hierarchical models and are instead best described by a linearly organized longitudinally compressed array of consecutive chromatin loops.
Topics: Biopolymers; Cell Cycle; Chromatin; Chromosomes, Human, Pair 21; HeLa Cells; Humans; Metaphase; Mitosis; Models, Chemical
PubMed: 24200812
DOI: 10.1126/science.1236083 -
ELife Jun 2022The chromokinesin KIF22 generates forces that contribute to mitotic chromosome congression and alignment. Mutations in the α2 helix of the motor domain of KIF22 have...
The chromokinesin KIF22 generates forces that contribute to mitotic chromosome congression and alignment. Mutations in the α2 helix of the motor domain of KIF22 have been identified in patients with abnormal skeletal development, and we report the identification of a patient with a novel mutation in the KIF22 tail. We demonstrate that pathogenic mutations do not result in a loss of KIF22's functions in early mitosis. Instead, mutations disrupt chromosome segregation in anaphase, resulting in reduced proliferation, abnormal daughter cell nuclear morphology, and, in a subset of cells, cytokinesis failure. This phenotype could be explained by a failure of KIF22 to inactivate in anaphase. Consistent with this model, constitutive activation of the motor via a known site of phosphoregulation in the tail phenocopied the effects of pathogenic mutations. These results suggest that the motor domain α2 helix may be an important site for regulation of KIF22 activity at the metaphase to anaphase transition. In support of this conclusion, mimicking phosphorylation of α2 helix residue T158 also prevents inactivation of KIF22 in anaphase. These findings demonstrate the importance of both the head and tail of the motor in regulating the activity of KIF22 and offer insight into the cellular consequences of preventing KIF22 inactivation and disrupting force balance in anaphase.
Topics: Anaphase; Chromosome Segregation; DNA-Binding Proteins; Kinesins; Metaphase; Mitosis; Mutation; Nuclear Proteins; Spindle Apparatus
PubMed: 35730929
DOI: 10.7554/eLife.78653 -
The Plant Cell Mar 2021The bipolar mitotic spindle is a highly conserved structure among eukaryotes that mediates chromosome alignment and segregation. Spindle assembly and size control are...
The bipolar mitotic spindle is a highly conserved structure among eukaryotes that mediates chromosome alignment and segregation. Spindle assembly and size control are facilitated by force-generating microtubule-dependent motor proteins known as kinesins. In animals, kinesin-12 cooperates with kinesin-5 to produce outward-directed forces necessary for spindle assembly. In plants, the relevant molecular mechanisms for spindle formation are poorly defined. While an Arabidopsis thaliana kinesin-5 ortholog has been identified, the kinesin-12 ortholog in plants remains elusive. In this study, we provide experimental evidence for the function of Arabidopsis KINESIN-12E in spindle assembly. In kinesin-12e mutants, a delay in spindle assembly is accompanied by the reduction of spindle size, demonstrating that KINESIN-12E contributes to mitotic spindle architecture. Kinesin-12E localization is mitosis-stage specific, beginning with its perinuclear accumulation during prophase. Upon nuclear envelope breakdown, KINESIN-12E decorates subpopulations of microtubules in the spindle and becomes progressively enriched in the spindle midzone. Furthermore, during cytokinesis, KINESIN-12E shares its localization at the phragmoplast midzone with several functionally diversified Arabidopsis KINESIN-12 members. Changes in the kinetochore and in prophase and metaphase spindle dynamics occur in the absence of KINESIN-12E, suggest it might play an evolutionarily conserved role during spindle formation similar to its spindle-localized animal kinesin-12 orthologs.
Topics: Arabidopsis; Kinesins; Kinetochores; Metaphase; Microtubules; Prophase
PubMed: 33751090
DOI: 10.1093/plcell/koaa003 -
Molecular Biology of the Cell Jun 2019The endoplasmic reticulum (ER) is extensively remodeled during metazoan open mitosis. However, whether the ER becomes more tubular or more cisternal during mitosis is...
The endoplasmic reticulum (ER) is extensively remodeled during metazoan open mitosis. However, whether the ER becomes more tubular or more cisternal during mitosis is controversial, and dedicated factors governing the morphology of the mitotic ER have remained elusive. Here, we describe the ER membrane proteins REEP3 and REEP4 as major determinants of ER morphology in metaphase cells. REEP3/4 are specifically required for generating the high-curvature morphology of mitotic ER and promote ER tubulation through their reticulon homology domains (RHDs). This ER-shaping activity of REEP3/4 is distinct from their previously described function to clear ER from metaphase chromatin. We further show that related REEP proteins do not contribute to mitotic ER shaping and provide evidence that the REEP3/4 carboxyterminus mediates regulation of the proteins. These findings confirm that ER converts to higher curvature during mitosis, identify REEP3/4 as specific and crucial morphogenic factors mediating ER tubulation during mitosis, and define the first cell cycle-specific role for RHD proteins.
Topics: Amino Acid Sequence; Chromatin; Endoplasmic Reticulum; HeLa Cells; Humans; Membrane Transport Proteins; Metaphase; Mitosis; Protein Domains
PubMed: 30995177
DOI: 10.1091/mbc.E18-11-0698 -
Cell Cycle (Georgetown, Tex.) Jun 2021ATP metabolism during mitosis needs to be coordinated with numerous energy-demanding activities, especially in cancer cells whose metabolic pathways are reprogramed to...
ATP metabolism during mitosis needs to be coordinated with numerous energy-demanding activities, especially in cancer cells whose metabolic pathways are reprogramed to sustain rapid proliferation in a nutrient-deficient environment. Although strategies targeting the energy metabolic pathways have shown therapeutic efficacy in preclinical cancer models, how normal cells and cancer cells differentially respond to energy shortage is unclear. In this study, using time-lapse microscopy, we found that cancer cells displayed unique mitotic phenotypes in a dose-dependent manner upon decreasing ATP (i.e. energy) supply. When reduction in ATP concentration was moderate, chromosome movements in mitosis were barely affected, while the metaphase-anaphase transition was significantly prolonged due to reduced tension between the sister-kinetochores, which delayed the satisfaction of the spindle assembly checkpoint. Further reduction in ATP concentration led to a decreased level of Aurora-B at the centromere, resulting in increased chromosome mis-segregation after metaphase delay. In contrast to cancer cells, ATP restriction in non-transformed cells induced cell cycle arrest in interphase, rather than causing mitotic defects. In addition, data mining of cancer patient database showed a correlation between signatures of energy production and chromosomal instability possibly resulted from mitotic defects. Together, these results reveal that energy restriction induces differential responses in normal and cancer cells, with chromosome mis-segregation only observed in cancer cells. This points to targeting energy metabolism as a potentially cancer-selective therapeutic strategy.
Topics: Adenosine Triphosphate; Anaphase; Aurora Kinase B; Chromosome Segregation; Energy Metabolism; Female; HeLa Cells; Humans; Interphase; Kinetochores; Metaphase; Microscopy; NAD; Signal Transduction; Spindle Apparatus; Time-Lapse Imaging; Uterine Cervical Neoplasms
PubMed: 34048314
DOI: 10.1080/15384101.2021.1930679 -
Cell Reports Aug 2022Chromosome alignment at the spindle equator promotes proper chromosome segregation and depends on pulling forces exerted at kinetochore fiber tips together with polar...
Chromosome alignment at the spindle equator promotes proper chromosome segregation and depends on pulling forces exerted at kinetochore fiber tips together with polar ejection forces. However, kinetochore fibers are also subjected to forces driving their poleward flux. Here we introduce a flux-driven centering model that relies on flux generated by forces within the overlaps of bridging and kinetochore fibers. This centering mechanism works so that the longer kinetochore fiber fluxes faster than the shorter one, moving the kinetochores toward the center. We develop speckle microscopy in human spindles and confirm the key prediction that kinetochore fiber flux is length dependent. Kinetochores are better centered when overlaps are shorter and the kinetochore fiber flux slower than the bridging fiber flux. We identify Kif18A and Kif4A as overlap and flux regulators and NuMA as a fiber coupler. Thus, length-dependent sliding forces exerted by the bridging fiber onto kinetochore fibers support chromosome alignment.
Topics: Anaphase; Cell Cycle Proteins; Chromosome Segregation; Chromosomes; Humans; Kinesins; Kinetochores; Metaphase; Microtubules; Spindle Apparatus
PubMed: 35926461
DOI: 10.1016/j.celrep.2022.111169