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Molecular and Cellular Biology Apr 2009Proper mitotic progression is crucial for maintenance of genomic integrity in proliferating cells and is regulated through an intricate series of events, including...
Proper mitotic progression is crucial for maintenance of genomic integrity in proliferating cells and is regulated through an intricate series of events, including protein phosphorylation governed by a complex network of protein kinases. One kinase family implicated in the regulation of mitotic progression is protein kinase CK2, a small family of enzymes that is overexpressed in cancer and induces transformation in mice and cultured fibroblasts. CK2alpha, one isoform of the catalytic subunits of CK2, is maximally phosphorylated at four sites in nocodazole-treated cells. To investigate the effects of CK2alpha phosphorylation on mitotic progression, we generated phosphospecific antibodies against its mitotic phosphorylation sites. In U2OS cells released from S-phase arrest, these antibodies reveal that CK2alpha is most highly phosphorylated in prophase and metaphase. Phosphorylation gradually decreases during anaphase and becomes undetectable during telophase and cytokinesis. Stable expression of phosphomimetic CK2alpha (CK2alpha-4D, CK2alpha-4E) results in aberrant centrosome amplification and chromosomal segregation defects and loss of mitotic cells through mitotic catastrophe. Conversely, cells expressing nonphosphorylatable CK2alpha (CK2alpha-4A) show a decreased ability to arrest in mitosis following nocodazole treatment, suggesting involvement in the spindle assembly checkpoint. Collectively, these studies indicate that reversible phosphorylation of CK2alpha requires precise regulation to allow proper mitotic progression.
Topics: Animals; Binding Sites; Casein Kinase II; Cell Line; Cell Nucleus Division; Centrosome; Chromosome Segregation; Cytokinesis; Mice; Mitosis; Phosphorylation; Spindle Apparatus
PubMed: 19188443
DOI: 10.1128/MCB.01563-08 -
Cell Cycle (Georgetown, Tex.) 2018We have shown that cytoplasmic actin isoforms play different roles in neoplastic cell transformation. β-Cytoplasmic actin acts as a tumor suppressor, affecting...
We have shown that cytoplasmic actin isoforms play different roles in neoplastic cell transformation. β-Cytoplasmic actin acts as a tumor suppressor, affecting epithelial differentiation, cell growth, cell invasion and tumor growth of colon and lung carcinoma cells. In contrast, γ-cytoplasmic actin enhances malignant features of tumor cells whose actin network regulation is carried out the γ-actin isoform. The goal of this study was to describe the role of cytoplasmic actins in cell cycle regulation of breast cancer cell lines MCF-7 and MDA-MB-231. The distinct roles of each cytoplasmic actin in the cell cycle driving were observed. β-Actin as well as γ-actin down-regulation inhibited proliferation of breast cancer cells, but only down-regulation of β-actin induced a significant decrease in diploid cell population and accumulation of tetraploid cells. Down-regulation of β-actin stimulated cyclin A2, B1 and D3 expression, whereas down-regulation of γ-actin reduced expression of these cyclins in both cell lines. Moreover, cyclin B1 and γ-actin were co-localized in mitotic control and β-actin-deficient cells. In mitotic MCF-7 cells down-regulation of β-actin caused an enrichment of prophase/metaphase population compared with control. γ-Actin down-regulation induced telophase enrichment. ERK1/2 and γ-actin co-localization and possible selective binding were revealed in MCF7 cells. β-Actin down-regulation induced ERK1/2 activation, while γ-actin down-regulation led to reduction of p-ERK1/2. A direct interaction of ERK1/2 with γ-actin and cyclin A2 in the same protein complex was also discovered. We suggest that γ-actin down-regulation leads to decrease of cyclin A2 level, inhibits ERK1/2 signaling and deceleration of breast cancer cells proliferation.
PubMed: 30516087
DOI: 10.1080/15384101.2018.1553337 -
IScience Dec 2023The tumor-treating fields (TTFields) technology has revolutionized the management of recurrent and newly diagnosed glioblastoma (GBM) cases. To ameliorate this treatment...
The tumor-treating fields (TTFields) technology has revolutionized the management of recurrent and newly diagnosed glioblastoma (GBM) cases. To ameliorate this treatment modality for GBM and other oncological conditions, it is necessary to understand the biophysical principles of TTFields better. In this study, we further analyzed the mechanism of the electromagnetic exposure with varying frequencies and electric field strengths on cells in mitosis, specifically in telophase. In reference to previous studies, an intuitive finite element model of the mitotic cell was built for electromagnetic simulations, predicting a local increase in the cleavage furrow region, which may help explain TTFields' anti-proliferative effects. Cell experiments confirmed that the reduction in proliferation and migration of glioma cell by TTFields was in a frequency- and field-strength-dependent manner. This work provides unique insights into the selection of frequencies in the anti-proliferative effect of TTFields on tumors, which could improve the application of TTFields.
PubMed: 38125027
DOI: 10.1016/j.isci.2023.108575 -
The Journal of Biological Chemistry Aug 2005The p53-related p73 proteins regulate developmental processes, cell growth, and DNA damage response. p73 function is regulated by post-translational modifications and...
The p53-related p73 proteins regulate developmental processes, cell growth, and DNA damage response. p73 function is regulated by post-translational modifications and protein-protein interactions. At the G2/M transition, p73 is phosphorylated at Thr-86 by the p34cdc2/cyclin B complex; this is associated with its exclusion from condensed chromosomes and loss of DNA binding and transcriptional activation ability. Here we showed that p73 hypo-phosphorylated species reappear during mitotic exit, concomitant with p73 relocalization to telophase nuclei and recovered ability to activate transcription. Functional knock-out of p73 gene expression by small interfering RNAs (siRNAs) alters mitotic progression, yielding an increase of ana-telophase cells, the accumulation of aberrant late mitotic figures, and the appearance of abnormalities in the subsequent interphase. This p73 activity at the M-to-G1 transition is mediated by its transactivating function because expression of the transcription dominant negative mutant p73DD induces the same mitotic exit phenotype. We also found that the cyclin-dependent kinase inhibitor Kip2/p57 gene is a specific target of p73 regulation during mitotic exit and re-entry into G1. Both knock-out of p73 gene expression by siRNAs and abrogation of p73-dependent transcription by the p73DD mutant abrogate Kip2/p57 increase at the M-to-G1 transition. Moreover, similar abnormalities (e.g. delay in late mitotic stages with the accumulation of aberrant ana-telophase figures, and abnormalities in the following interphase) are observed in cultures in which the expression of Kip2/p57 is abrogated by siRNAs. These results identify a novel p73-Kip2/p57 pathway that coordinates mitotic exit and transition to G1.
Topics: Anaphase; CDC2 Protein Kinase; Cell Cycle; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Cell Separation; Chromosomes; Cyclin B; Cyclin-Dependent Kinase Inhibitor p57; DNA Damage; DNA-Binding Proteins; Flow Cytometry; G1 Phase; Genes, Dominant; Genes, Tumor Suppressor; Glioma; Humans; Mitosis; Mutation; Nuclear Proteins; Phenotype; Phosphorylation; Plasmids; Protein Structure, Tertiary; RNA; RNA, Small Interfering; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; Telophase; Threonine; Time Factors; Transcription, Genetic; Transcriptional Activation; Tumor Protein p73; Tumor Suppressor Protein p53; Tumor Suppressor Proteins
PubMed: 15985436
DOI: 10.1074/jbc.M500635200 -
PLoS Genetics Mar 2009The assembly of the mitotic centromere has been extensively studied in recent years, revealing the sequence and regulation of protein loading to this chromosome domain....
The assembly of the mitotic centromere has been extensively studied in recent years, revealing the sequence and regulation of protein loading to this chromosome domain. However, few studies have analyzed centromere assembly during mammalian meiosis. This study specifically targets this approach on mouse spermatocytes. We have found that during prophase I, the proteins of the chromosomal passenger complex Borealin, INCENP, and Aurora-B load sequentially to the inner centromere before Shugoshin 2 and MCAK. The last proteins to be assembled are the outer kinetochore proteins BubR1 and CENP-E. All these proteins are not detected at the centromere during anaphase/telophase I and are then reloaded during interkinesis. The loading sequence of the analyzed proteins is similar during prophase I and interkinesis. These findings demonstrate that the interkinesis stage, regularly overlooked, is essential for centromere and kinetochore maturation and reorganization previous to the second meiotic division. We also demonstrate that Shugoshin 2 is necessary for the loading of MCAK at the inner centromere, but is dispensable for the loading of the outer kinetochore proteins BubR1 and CENP-E.
Topics: Animals; Cell Cycle Proteins; Centromere; Chromosomal Proteins, Non-Histone; Kinesins; Kinetochores; Male; Meiosis; Meiotic Prophase I; Mice; Protein Serine-Threonine Kinases; Spermatocytes
PubMed: 19283064
DOI: 10.1371/journal.pgen.1000417 -
Cell Reports Mar 2019Aurora kinase B (AURKB), a central regulator of chromosome segregation and cytokinesis, is aberrantly expressed in various cancer cells. However, the relationship of...
Aurora kinase B (AURKB), a central regulator of chromosome segregation and cytokinesis, is aberrantly expressed in various cancer cells. However, the relationship of AURKB and oncogenic viruses in cancer progression remains unclear. Here, we reveal that N-cleaved isoforms of AURKB exist in several oncovirus-associated tumor cells and patient cancer tissues, including Kaposi's sarcoma-associated herpesvirus (KSHV), Epstein-Barr virus (EBV), and human papillomavirus virus (HPV). Mechanistically, in KSHV-infected tumor cells, the latent viral antigen LANA cleaves AURKB at Asp in a serine protease-dependent manner. The N'-AURKB relocalizes to the spindle pole and promotes the metaphase-to-telophase transition in mitotic cells. Introduction of N'-AURKB but not C'-AURKB promotes colony formation and malignant growth of tumor cells in vitro and in vivo using a murine xenograft model. Altogether, our findings uncover a proteolytic cleavage mechanism by which oncoviruses induce cancer cell segregation and tumorigenesis.
Topics: Adult; Aged; Animals; Aurora Kinase B; Carcinogenesis; Chromosome Segregation; Female; HEK293 Cells; HeLa Cells; Herpesvirus 8, Human; Humans; Male; Mice; Mice, Inbred NOD; Middle Aged; Mitosis; Neoplasms; Oncogenic Viruses; Xenograft Model Antitumor Assays; Young Adult
PubMed: 30917319
DOI: 10.1016/j.celrep.2019.02.106 -
Cell Cycle (Georgetown, Tex.) 2014Kinesin-14 motor proteins play a variety of roles during metaphase and anaphase. However, it is not known whether members of this family of motors also participate in...
Kinesin-14 motor proteins play a variety of roles during metaphase and anaphase. However, it is not known whether members of this family of motors also participate in the dramatic changes in mitotic spindle organization during the transition from telophase to cytokinesis. We have identified the minus-end-directed motor, KIFC3, as an important contributor to central bridge morphology at this stage. KIFC3's unique motor-dependent localization at the central bridge allows it to congress microtubules, promoting efficient progress through cytokinesis. Conversely, when KIFC3 function is perturbed, abscission is delayed, and the central bridge is both widened and extended. Examination of KIFC3 on growing microtubules in interphase indicates that it caps microtubules released from the centrosome, both in the region of the centrosome and in the cell periphery. In line with other kinesin-14 family members, KIFC3 may guide free microtubules to their destination at the bridge and/or may slide and crosslink central bridge microtubules in order to stage the cells for abscission.
Topics: Anaphase; Animals; Centrosome; Cytokinesis; Dogs; HeLa Cells; Humans; Kinesins; Madin Darby Canine Kidney Cells; Metaphase; Microtubules; Spindle Apparatus
PubMed: 24275865
DOI: 10.4161/cc.27266 -
Nature Communications Apr 2019During mitosis, tension develops across the centromere as a result of spindle-based forces. Metaphase tension may be critical in preventing mitotic chromosome...
During mitosis, tension develops across the centromere as a result of spindle-based forces. Metaphase tension may be critical in preventing mitotic chromosome segregation errors, however, the nature of force transmission at the centromere and the role of centromere mechanics in controlling metaphase tension remains unknown. We combined quantitative, biophysical microscopy with computational analysis to elucidate the mechanics of the centromere in unperturbed, mitotic human cells. We discovered that the mechanical stiffness of the human centromere matures during mitotic progression, which leads to amplified centromere tension specifically at metaphase. Centromere mechanical maturation is disrupted across multiple aneuploid cell lines, leading to a weak metaphase tension signal. Further, increasing deficiencies in centromere mechanical maturation are correlated with rising frequencies of lagging, merotelic chromosomes in anaphase, leading to segregation defects at telophase. Thus, we reveal a centromere maturation process that may be critical to the fidelity of chromosome segregation during mitosis.
Topics: Aneuploidy; Cell Line, Tumor; Centromere; Chromosome Segregation; HeLa Cells; Humans; Metaphase; Mitosis; Models, Biological; Spindle Apparatus
PubMed: 30988289
DOI: 10.1038/s41467-019-09578-z -
The Journal of Cell Biology Jun 1979Indirect immunofluorescence was used to determine the distribution of calmodulin in the mitotic apparatus of rat kangaroo PtK2 and Chinese hamster ovary (CHO) cells. The...
Indirect immunofluorescence was used to determine the distribution of calmodulin in the mitotic apparatus of rat kangaroo PtK2 and Chinese hamster ovary (CHO) cells. The distribution of calmodulin in PtK2 cells was compared to the distribution of tubulin, also as revealed by indirect immunofluorescence. During mitosis, calmodulin was found to be a dynamic component of the mitotic apparatus. Calmodulin first appeared in association with the forming mitotic apparatus during midprophase. In metaphase and anaphase, calmodulin was found between the spindle poles and the chromosomes. While tubulin was found in the interzonal region throughout anaphase, calmodulin appeared in the interzone region only at late anaphase. The interzonal calmodulin of late anaphase condensed during telophase into two small regions, one on each side of the midbody. Calmodulin was not detected in the cleavage furrow. In view of the differences in the localization of calmodulin, tubulin, and actin in the mitotic apparatus, experiments were designed to determine the effects of various antimitotic drugs on calmodulin localization. Cytochalasin B, an inhibitor of actin microfilaments, had no apparent effect on calmodulin or tubulin localization in the mitotic apparatus of CHO cells. Microtubule inhibitors, such as colcemid and N2O, altered the appearance of tubulin- and calmodulin-specific fluorescence in mitotic CHO cells. Cold temperature (0 degrees C) altered tubulin-specific fluorescence of metaphase PtK2 cells but did not alter calmodulin-specific fluorescence. From these studies, it is concluded that calmodulin is more closely associated with the kinetichore-to-pole microtubules than other components of the mitotic apparatus.
Topics: Animals; Calmodulin; Carrier Proteins; Cell Cycle; Cells, Cultured; Cricetinae; Cricetulus; Cytochalasin B; Cytoplasm; Cytoskeleton; Demecolcine; Female; Fluorescent Antibody Technique; Glycoproteins; Macropodidae; Microtubules; Mitosis; Nitrous Oxide; Ovary; Tubulin
PubMed: 379022
DOI: 10.1083/jcb.81.3.624 -
Molecular Biology of the Cell Apr 2009During open mitosis several nuclear pore complex (NPC) proteins have mitotic specific localizations and functions. We find that the Aspergillus nidulans Mlp1 NPC protein...
During open mitosis several nuclear pore complex (NPC) proteins have mitotic specific localizations and functions. We find that the Aspergillus nidulans Mlp1 NPC protein has previously unrealized mitotic roles involving spatial regulation of spindle assembly checkpoint (SAC) proteins. In interphase, An-Mlp1 tethers the An-Mad1 and An-Mad2 SAC proteins to NPCs. During a normal mitosis, An-Mlp1, An-Mad1, and An-Mad2 localize similarly on, and around, kinetochores until telophase when they transiently localize near the spindle but not at kinetochores. During SAC activation, An-Mlp1 remains associated with kinetochores in a manner similar to An-Mad1 and An-Mad2. Although An-Mlp1 is not required for An-Mad1 kinetochore localization during early mitosis, it is essential to maintain An-Mad1 in the extended region around kinetochores in early mitosis and near the spindle in telophase. Our data are consistent with An-Mlp1 being part of a mitotic spindle matrix similar to its Drosophila orthologue and demonstrate that this matrix localizes SAC proteins. By maintaining SAC proteins near the mitotic apparatus, An-Mlp1 may help monitor mitotic progression and coordinate efficient mitotic exit. Consistent with this possibility, An-Mad1 and An-Mlp1 redistribute from the telophase matrix and associate with segregated kinetochores when mitotic exit is prevented by expression of nondegradable cyclin B.
Topics: Aspergillus nidulans; Cyclin B; Fungal Proteins; Kinetochores; Mitosis; Nuclear Pore; Protein Binding; Protein Processing, Post-Translational; Protein Transport; Spindle Apparatus; Telophase; Time Factors
PubMed: 19225157
DOI: 10.1091/mbc.e08-08-0878