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Current Biology : CB May 2002Anaphase, mitotic exit, and cytokinesis proceed in rapid succession, and while mitotic exit is a requirement for cytokinesis in yeast, it may not be a direct requirement...
Anaphase, mitotic exit, and cytokinesis proceed in rapid succession, and while mitotic exit is a requirement for cytokinesis in yeast, it may not be a direct requirement for furrow initiation in animal cells. In this report, we physically manipulated the proximity of the mitotic apparatus (MA) to the cell cortex in combination with microinjection of effectors of the spindle checkpoint and CDK1 activity to determine how the initiation of cytokinesis is coupled to the onset of anaphase and mitotic exit. Whereas precocious contact between the MA and the cell surface advanced the onset of cytokinesis into early anaphase A, furrowing could not be advanced prior to the metaphase-anaphase transition. Additionally, while cells arrested in anaphase could be induced to initiate cleavage furrows, cells arrested in metaphase could not. Finally, activation of the mitotic checkpoint in one spindle of a binucleate cell failed to arrest cytokinesis induced by the control spindle but did inhibit the formation of furrows between the arrested MA and the control, nonarrested MA. Our experiments suggest that the competence of the mitotic apparatus to initiate cytokinesis is not dependent on cyclin degradation but does require anaphase-promoting complex (APC) activity and, thus, inactivation of the mitotic checkpoint.
Topics: Anaphase; Anaphase-Promoting Complex-Cyclosome; Animals; Blastomeres; Calcium-Binding Proteins; Carrier Proteins; Cell Cycle Proteins; Cell Division; Cyclin B; Embryo, Nonmammalian; Fungal Proteins; Ligases; Metaphase; Mitosis; Nuclear Proteins; Sea Urchins; Spindle Apparatus; Time Factors; Ubiquitin-Protein Ligase Complexes
PubMed: 12015124
DOI: 10.1016/s0960-9822(02)00838-2 -
Cancer Biology & Therapy Jul 2013To develop new therapies for inflammatory breast cancer (IBC) we have compared the effects of two hydroxamic acid-based histone deacetylase (HDAC) inhibitors, CG-1521... (Comparative Study)
Comparative Study
To develop new therapies for inflammatory breast cancer (IBC) we have compared the effects of two hydroxamic acid-based histone deacetylase (HDAC) inhibitors, CG-1521 and Trichostatin A (TSA) on the biology of two IBC cell lines: SUM149PT and SUM190PT. CG-1521 and TSA induce dose (0-10 µM) and time-dependent (0-96 h) increases in the proportion of cells undergoing cell cycle arrest and apoptosis in the presence or absence of 17β-estradiol. In SUM 149PT cells, both CG-1521 and TSA increase the levels of acetylated α-tubulin; however the morphological effects are different: CG-1521 blocks mitotic spindle formation and prevents abscission during cytokinesis while TSA results in an increase in cell size. In SUM190PT cells CG-1521 does not cause an increase in acetylated-α-tubulin and even though TSA significantly increases the levels of acetylated tubulin, neither inhibitor alters the morphology of the cells. Microarray analysis demonstrates that CG-1521 modulates the expression of 876 mRNAs and 63 miRNAs in SUM149PT cells, and 1227 mRNAs and 35 miRNAs in SUM190PT cells. Only 9% of the genes are commonly modulated in both cell lines, suggesting that CG-1521 and TSA target different biological processes in the two cell lines most likely though the inhibition of different HDACs in these cell lines. Gene ontology (GO) analysis reveals that CG-1521 affects the expression of mRNAs that encode proteins associated with the spindle assembly checkpoint, chromosome segregation, and microtubule-based processes in both cell lines and has cell-type specific effects on lipid biosynthesis, response to DNA damage, and cell death.
Topics: Apoptosis; Cell Line, Tumor; Female; Gene Expression; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Inflammatory Breast Neoplasms; Metaphase; MicroRNAs; Middle Aged; RNA, Messenger
PubMed: 23792638
DOI: 10.4161/cbt.25088 -
Cells Feb 2020The microtubule-associated protein TPX2 is a key mitotic regulator that contributes through distinct pathways to spindle assembly. A well-characterised function of TPX2...
The microtubule-associated protein TPX2 is a key mitotic regulator that contributes through distinct pathways to spindle assembly. A well-characterised function of TPX2 is the activation, stabilisation and spindle localisation of the Aurora-A kinase. High levels of TPX2 are reported in tumours and the effects of its overexpression have been investigated in cancer cell lines, while little is known in non-transformed cells. Here we studied TPX2 overexpression in hTERT RPE-1 cells, using either the full length TPX2 or a truncated form unable to bind Aurora-A, to identify effects that are dependent-or independent-on its interaction with the kinase. We observe significant defects in mitotic spindle assembly and progression through mitosis that are more severe when overexpressed TPX2 is able to interact with Aurora-A. Furthermore, we describe a peculiar, and Aurora-A-interaction-independent, phenotype in telophase cells, with aberrantly stable microtubules interfering with nuclear reconstitution and the assembly of a continuous lamin B1 network, resulting in daughter cells displaying doughnut-shaped nuclei. Our results using non-transformed cells thus reveal a previously uncharacterised consequence of abnormally high TPX2 levels on the correct microtubule cytoskeleton remodelling and G1 nuclei reformation, at the mitosis-to-interphase transition.
Topics: Aurora Kinase A; Cell Cycle Proteins; Cell Line; Cell Nucleus; Chromatin; Cytoskeleton; Golgi Apparatus; Humans; Lamin Type B; Metaphase; Microtubule-Associated Proteins; Microtubules; Mitosis; Protein Binding; Telophase
PubMed: 32041138
DOI: 10.3390/cells9020374 -
Genes To Cells : Devoted To Molecular &... Aug 1999The 20S cyclosome/APC complex promotes metaphase-anaphase transition by ubiquitinating its specific substrates such as mitotic cyclins and anaphase inhibitor...
BACKGROUND
The 20S cyclosome/APC complex promotes metaphase-anaphase transition by ubiquitinating its specific substrates such as mitotic cyclins and anaphase inhibitor Cut2/Pds1/securin. The complex has been shown to contain more than 10 proteins in budding yeast and frog. In fission yeast, however, only five (Cut4, Cut9, Nuc2, Apc10, Hcn1) have been identified.
RESULTS
More than five hundred temperature-sensitive mutants were screened for identifying those defective in mitotic anaphase. Fifty-five showed the cut (cell untimely torn) phenotype or metaphase-arrest phenotypes, 27 of them locating at new loci. Their extracts were run in sucrose gradient centrifugation, and four showed alterations in the sedimentation profiles. The gene products of cut20+ and cut23+ were thus identified. Phenotypes of cut20-100 mutant highly resemble cut4-533 in many ways: they are hypersensitive to canavanine and CdCl2, and suppressed by PKA-inactivating regulators, cAMP-dependent phosphodiesterase and PKA regulatory subunits. Cut20 interacts closely with Cut4 in the assembly process of cyclosome. But cut20 mutant differs from cut4, as a novel gene stw1+ suppresses cut20 mutant but not cut4. cut23-194 mutant cells are sterile and blocked at metaphase, but does not show sensitivity to the stress and cAMP. TPR repeat-containing Cut23 may not be the stable component of APC/cyclosome, and its level significantly fluctuates during cell cycle. Cut23 may be ubiquitinated and degraded in a cell cycle dependent fashion.
CONCLUSIONS
We identified two new subunits of fission yeast cyclosome/APC complex. Our observations indicate that cyclosome components are divided into several subgroups with distinctly different roles.
Topics: Adenomatous Polyposis Coli Protein; Amino Acid Sequence; Anaphase; Anaphase-Promoting Complex-Cyclosome; Cadmium; Canavanine; Cell Cycle; Cell Cycle Proteins; Conserved Sequence; Cyclic AMP-Dependent Protein Kinases; Cytoskeletal Proteins; Fungal Proteins; Ligases; Metaphase; Models, Genetic; Molecular Sequence Data; Phenotype; Plasmids; Saccharomyces cerevisiae Proteins; Schizosaccharomyces; Suppression, Genetic; Temperature; Time Factors; Ubiquitin-Protein Ligase Complexes; Ubiquitin-Protein Ligases; Ubiquitins
PubMed: 10526233
DOI: 10.1046/j.1365-2443.1999.00274.x -
Molecular Human Reproduction Sep 2020As the age of child-bearing increases and correlates with infertility, cryopreservation of female gametes is becoming common-place in ART. However, the developmental...
As the age of child-bearing increases and correlates with infertility, cryopreservation of female gametes is becoming common-place in ART. However, the developmental competence of vitrified oocytes has remained low. The underlying mechanisms responsible for reduced oocyte quality post-vitrification are largely unknown. Mouse cumulus-oocyte complexes were vitrified using a cryoloop technique and a mixture of dimethylsulphoxide, ethylene glycol and trehalose as cryoprotectants. Fresh and vitrified/thawed oocytes were compared for chromosome alignment, spindle morphology, kinetochore-microtubule attachments, spindle assembly checkpoint (SAC) and aneuploidy. Although the majority of vitrified oocytes extruded the first polar body (PB), they had a significant increase of chromosome misalignment, abnormal spindle formation and aneuploidy at metaphase II. In contrast to controls, vitrified oocytes extruded the first PB in the presence of nocodazole and etoposide, which should induce metaphase I arrest in a SAC-dependent manner. The fluorescence intensity of mitotic arrest deficient 2 (MAD2), an essential SAC protein, at kinetochores was reduced in vitrified oocytes, indicating that the SAC is weakened after vitrification/thawing. Furthermore, we found that vitrification-associated stress disrupted lysosomal function and stimulated cathepsin B activity, with a subsequent activation of caspase 3. MAD2 localization and SAC function in vitrified oocytes were restored upon treatment with a cathepsin B or a caspase 3 inhibitor. This study was conducted using mouse oocytes, therefore confirming these results in human oocytes is a prerequisite before applying these findings in IVF clinics. Here, we uncovered underlying molecular pathways that contribute to an understanding of how vitrification compromises oocyte quality. Regulating these pathways will be a step toward improving oocyte quality post vitrification and potentially increasing the efficiency of the vitrification program.
Topics: Animals; Cathepsin B; Cryopreservation; Cryoprotective Agents; Female; Freezing; Lysosomes; M Phase Cell Cycle Checkpoints; Meiosis; Metaphase; Mice; Oocytes; Spindle Apparatus; Vitrification
PubMed: 32634244
DOI: 10.1093/molehr/gaaa051 -
Cell Cycle (Georgetown, Tex.) 2017Checkpoint 1 (Chk1), as an important member of DNA replication checkpoint and DNA damage response, has an important role during the G2/M stage of mitosis. In this study,...
Checkpoint 1 (Chk1), as an important member of DNA replication checkpoint and DNA damage response, has an important role during the G2/M stage of mitosis. In this study, we used porcine oocyte as a model to investigate the function of Chk1 during porcine oocyte maturation. Chk1 was expressed from germinal vesicle (GV) to metaphase II (MII) stages, mainly localized in the cytoplasm at GV stage and moved to the spindle after germinal vesicle breakdown (GVBD). Chk1 depletion not only induced oocytes to be arrested at MI stage with abnormal chromosomes arrangement, but also inhibited the degradation of Cyclin B1 and decreased the expression of Mitotic Arrest Deficient 2-Like 1 (Mad2L1), one of spindle assembly checkpoint (SAC) proteins, and cadherin 1 (Cdh1), one of coactivation for anaphase-promoting complex/cyclosome (APC/C). Moreover, Chk1 overexpression delayed GVBD. These results demonstrated that Chk1 facilitated the timely degradation of Cyclin B1 at anaphase I (AI) and maintained the expression of Mad2L1 and Cdh1, which ensured that all chromosomes were accurately located in a line, and then oocytes passed metaphase I (MI) and AI and exited from the first meiotic division successfully. In addition, we proved that Chk1 had not function on GVBD of porcine oocytes, which suggested that maturation of porcine oocytes did not need the DNA damage checkpoint, which was different from the mouse oocyte maturation.
Topics: Animals; Checkpoint Kinase 1; Meiosis; Metaphase; Oocytes; Swine
PubMed: 28933982
DOI: 10.1080/15384101.2017.1373221 -
Developmental Cell Sep 2020Mesoscale macromolecular complexes and organelles, tens to hundreds of nanometers in size, crowd the eukaryotic cytoplasm. It is therefore unclear how mesoscale...
Mesoscale macromolecular complexes and organelles, tens to hundreds of nanometers in size, crowd the eukaryotic cytoplasm. It is therefore unclear how mesoscale particles remain sufficiently mobile to regulate dynamic processes such as cell division. Here, we study mobility across dividing cells that contain densely packed, dynamic microtubules, comprising the metaphase spindle. In dividing human cells, we tracked 40 nm genetically encoded multimeric nanoparticles (GEMs), whose sizes are commensurate with the inter-filament spacing in metaphase spindles. Unexpectedly, the effective diffusivity of GEMs was similar inside the dense metaphase spindle and the surrounding cytoplasm. Eliminating microtubules or perturbing their polymerization dynamics decreased diffusivity by ~30%, suggesting that microtubule polymerization enhances random displacements to amplify diffusive-like motion. Our results suggest that microtubules effectively fluidize the mitotic cytoplasm to equalize mesoscale mobility across a densely packed, dynamic, non-uniform environment, thus spatially maintaining a key biophysical parameter that impacts biochemistry, ranging from metabolism to the nucleation of cytoskeletal filaments.
Topics: Cell Division; Cytoplasm; Humans; Metaphase; Microtubules; Nanoparticles; Organelles; Spindle Apparatus
PubMed: 32818469
DOI: 10.1016/j.devcel.2020.07.020 -
Human chromokinesins promote chromosome congression and spindle microtubule dynamics during mitosis.The Journal of Cell Biology Sep 2012Chromokinesins are microtubule plus end-directed motor proteins that bind to chromosome arms. In Xenopus egg cell-free extracts, Xkid and Xklp1 are essential for bipolar...
Chromokinesins are microtubule plus end-directed motor proteins that bind to chromosome arms. In Xenopus egg cell-free extracts, Xkid and Xklp1 are essential for bipolar spindle formation but the functions of the human homologues, hKID (KIF22) and KIF4A, are poorly understood. By using RNAi-mediated protein knockdown in human cells, we find that only co-depletion delayed progression through mitosis in a Mad2-dependent manner. Depletion of hKID caused abnormal chromosome arm orientation, delayed chromosome congression, and sensitized cells to nocodazole. Knockdown of KIF4A increased the number and length of microtubules, altered kinetochore oscillations, and decreased kinetochore microtubule flux. These changes were associated with failures in establishing a tight metaphase plate and an increase in anaphase lagging chromosomes. Co-depletion of both chromokinesins aggravated chromosome attachment failures, which led to mitotic arrest. Thus, hKID and KIF4A contribute independently to the rapid and correct attachment of chromosomes by controlling the positioning of chromosome arms and the dynamics of microtubules, respectively.
Topics: Anaphase; Animals; Calcium-Binding Proteins; Cell Cycle Checkpoints; Cell Cycle Proteins; Cell Line; Cell Line, Tumor; Chromosome Segregation; DNA-Binding Proteins; HCT116 Cells; HEK293 Cells; HeLa Cells; Humans; Kinesins; Kinetochores; Mad2 Proteins; Metaphase; Microtubules; Mitosis; Nuclear Proteins; Repressor Proteins; Spindle Apparatus; Xenopus; Xenopus Proteins
PubMed: 22945934
DOI: 10.1083/jcb.201110060 -
Folia Biologica 2017This study was aimed at elucidating the fate of three important nuclear envelope components - lamins B and A/C and nucleoporin Nup160, during meiotic maturation of mouse...
This study was aimed at elucidating the fate of three important nuclear envelope components - lamins B and A/C and nucleoporin Nup160, during meiotic maturation of mouse oocytes. These proteins were localized by epifluorescence and confocal microscopy using specific antibodies in oocytes at different stages from prophase I (germinal vesicle) to metaphase II. In immature germinal vesicle oocytes, all three proteins were detected at the nuclear periphery. In metaphase I and metaphase II, lamin B co-localized with the meiotic spindle, lamin A/C was found in a diffuse halo surrounding the spindle and to a lesser degree throughout the cytoplasm, and Nup160 was concentrated to the spindle poles. To our knowledge, this is the first report on nucleoporin localization in mammalian oocytes and the first successful detection of lamins in mature oocytes. While the distribution patterns of both lamins closely paralleled the respective stages of mitosis, Nup160 localization in metaphase oocytes corresponded to that in mitotic prometaphase rather than metaphase. The peculiar distribution of this nucleoporin in oocytes may reflect its role in meiosis-specific mechanisms of spindle assembly and its regulation.
Topics: Animals; Cell Differentiation; Female; Lamins; Meiosis; Metaphase; Mice, Inbred BALB C; Microscopy, Confocal; Nuclear Proteins; Oocytes
PubMed: 28374669
DOI: No ID Found -
Cell Cycle (Georgetown, Tex.) Apr 2013The effect of UV irradiation on replicating cells during interphase has been studied extensively. However, how the mitotic cell responds to UV irradiation is less well...
The effect of UV irradiation on replicating cells during interphase has been studied extensively. However, how the mitotic cell responds to UV irradiation is less well defined. Herein, we found that UV-C irradiation (254 nm) increases recruitment of the spindle checkpoint proteins Mps1 and Mad2 to the kinetochore during metaphase, suggesting that the spindle assembly checkpoint (SAC) is reactivated. In accordance with this, cells exposed to UV-C showed delayed mitotic progression, characterized by a prolonged chromosomal alignment during metaphase. UV-C irradiation also induced the DNA damage response and caused a significant accumulation of γ-H2AX on mitotic chromosomes. Unexpectedly, the mitotic delay upon UV-C irradiation is not due to the DNA damage response but to the relocation of Mps1 to the kinetochore. Further, we found that UV-C irradiation activates Aurora B kinase. Importantly, the kinase activity of Aurora B is indispensable for full recruitment of Mps1 to the kinetochore during both prometaphase and metaphase. Taking these findings together, we propose that UV irradiation delays mitotic progression by evoking the Aurora B-Mps1 signaling cascade, which exerts its role through promoting the association of Mps1 with the kinetochore in metaphase.
Topics: Blotting, Western; Cell Cycle Proteins; Cell Line, Tumor; Fluorescent Antibody Technique; Humans; Kinetochores; Metaphase; Mitosis; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; RNA, Small Interfering; Signal Transduction; Time-Lapse Imaging; Ultraviolet Rays
PubMed: 23531678
DOI: 10.4161/cc.24403