A novel role for the deubiquitinase USP1 in the control of centrosome duplication

Defects in the regulation of centrosome duplication lead to tumorigenesis through abnormal cell division and resulting chromosome missegregation. Therefore, maintenance of accurate centrosome number is critical for cell fate. The deubiquitinating enzyme USP1 plays important roles in DNA repair and cell differentiation. Importantly, increased levels of USP1 are detected in certain types of human cancer, but little is known about the significance of USP1 overexpression in cancer development. Here we show that Usp1 plays a novel role in regulating centrosome duplication. The ectopic expression of wild-type Usp1, but not C90S Usp1 (catalytically inactive mutant form), induced centrosome amplification. Conversely, ablation of Usp1 in mouse embryonic fibroblasts (MEFs) showed a significant delay in centrosome duplication. Moreover, Usp1-induced centrosome amplification caused abnormal mitotic spindles, chromosome missegregation and aneuploidy. Interestingly, loss of inhibitor of DNA binding protein 1 (ID1) suppressed Usp1-induced centrosome amplification. Taken together, our results strongly suggest that Usp1 is involved in the regulation of centrosome duplication, at least in part via ID1, and Usp1 may exert its oncogenic activity, partially through inducing centrosome abnormality.     

Migration of the cell nucleus during the amoebo-flagellate transformation ofPhysarum polycephalum is mediated by an actin-generated force that acts on the centrosome

Summary The mechanism of cell-nuclear migration during the amoebo-flagellate transformation inPhysarum polycephalum was examined by fluorescence microscopy after staining with a tubulinspecific antibody, rhodamine-conjugated phalloidin and 4,6-diamidino-2-phenylindole (DAPI). While the round amoeba cells changed to comma-shaped swarm cells within 20min after suspension in buffer, the cell nuclei moved from the central region of each cell to the periphery, each forming a sharp projection in the direction of movement. A centrosome also migrated from the center of the cell to the cell periphery. Since the centrosome was in close contact with the tip that protruded from the cell nucleus throughout the cellnuclear migration, the migration of the cell nucleus and the centrosome could be recognized as comigration. Then the flagella began to elongate from the centrosome and the cells became slender and polarized, adopting the so-called comma-shape. On the basis of these observations, the transformation process was classified into three steps: cell-nuclear migration, flagella formation and swarm maturation. The comigration of the cell nucleus and the centrosome was not inhibited by the anti-microtubule drug nocodazole (4 M) but it was inhibited by the anti-microfilament drug cytochalasin A (4 M), suggesting that the force of migration is generated by microfilaments. To investigate the role of the centrosome in this comigration in detail, we identified two aberrant strains, defective in swimming ability, from among various laboratory strains. The two strains, TM 4 and J, were found to have defects in cell-nuclear migration. Strain TM 4 had two types of irregular swarm cells: in one, only a part of the cell nucleus projected a thin filamentous structure; and in the other, no cell-nuclear migration occurred. Strain J had two centrosomes per cell and such swarm cells exhibited an attempt of cell-nuclear migration at two sites which corresponded to the position of the centrosome. The characteristics of these two strains indicate that the centrosome is essential for cell-nuclear migration. Our observations suggest that the cell-nuclear migration is mediated by actin-generated forces that act on the centrosome rather than on the cell nucleus itself.Abbreviations FITC fluorescein isothiocyanate - DAP 4,6-diamidino-2-phenylindole - PBS phosphate-buffered saline - KPB potassium phosphate buffer - MTOC microtubule organizing center     

DIXDC1 co-localizes and interacts with γ-tubulin in HEK293 cells

DIXDC1 is a Dishevelled-Axin (DIX) domain-containing protein involved in neural development and Wnt signaling pathway. Besides the DIX domain, DIXDC1 also contains a coiled-coil domain (MTH domain), which is a common feature of centrosomal proteins. We have demonstrated that exogenously expressed GFP-tag fused DIXDC1 co-localize with γ-tubulin both at interphase and mitotic phase in HEK293 cells. By immunostaining with anti-DIXDC1 and anti-γ-tubulin antibody, endogenous DIXDC1 was also co-localized with γ-tubulin at the centrosomes in HEK293 cells. We confirmed this interaction of DIXDC1 with γ-tubulin by co-immunoprecipitation. The findings suggest that DIXDC1 might play an important role in chromosome segregation and cell cycle regulation.     

Involvement of a centrosomal protein kendrin in the maintenance of centrosome cohesion by modulating Nek2A kinase activity

Centrosome cycle is strictly coordinated with chromosome duplication cycle to ensure the faithful segregation of chromosomes. Centrosome duplication occurs from the beginning of S phase, and the duplicated centrosomes are held together by centrosome cohesion to function as a single microtubule organizing center during interphase. At late G2 phase centrosome cohesion is disassembled by Nek2A kinase-mediated phosphorylation and, as a consequence, centrosomes are split and constitute spindle poles in mitosis. It has been reported that depletion of a centrosomal protein kendrin (also named pericentrin) induces premature centrosome splitting in interphase, however, it remains unknown how kendrin contributes to the maintenance of centrosome cohesion. Here we show that kendrin associates with Nek2A kinase, which exhibits considerably low activity. Nek2A kinase activity is inhibited in vitro by addition of the Nek2A-binding region of kendrin in a dose-dependent manner. Furthermore, ectopic expression of the same region decreases the number of the cells with split centrosomes at late G2 phase. Taken together, these results suggest that kendrin anchors Nek2A and suppresses its kinase activity at the centrosomes, and thus, is involved in the mechanism to prevent premature centrosome splitting during interphase.     

The PAM-1 aminopeptidase regulates centrosome positioning to ensure anterior-posterior axis specification in one-cell C. elegans embryos

In the one-cell Caenorhabditis elegans embryo, the anterior-posterior (A-P) axis is established when the sperm donated centrosome contacts the posterior cortex. While this contact appears to be essential for axis polarization, little is known about the mechanisms governing centrosome positioning during this process. pam-1 encodes a puromycin sensitive aminopeptidase that regulates centrosome positioning in the early embryo. Previously we showed that pam-1 mutants fail to polarize the A-P axis. Here we show that PAM-1 can be found in mature sperm and in cytoplasm throughout early embryogenesis where it concentrates around mitotic centrosomes and chromosomes. We provide further evidence that PAM-1 acts early in the polarization process by showing that PAR-1 and PAR-6 do not localize appropriately in pam-1 mutants. Additionally, we tested the hypothesis that PAM-1's role in polarity establishment is to ensure centrosome contact with the posterior cortex. We inactivated the microtubule motor dynein, DHC-1, in pam-1 mutants, in an attempt to prevent centrosome movement from the cortex and restore anterior-posterior polarity. When this was done, the aberrant centrosome movements of pam-1 mutants were not observed and anterior-posterior polarity was properly established, with proper localization of cortical and cytoplasmic determinants. We conclude that PAM-1's role in axis polarization is to prevent premature movement of the centrosome from the posterior cortex, ensuring proper axis establishment in the embryo.     

Cdk1 Activity Is Required for Mitotic Activation of Aurora A during G2/M Transition of Human Cells

In mammalian cells entry into and progression through mitosis are regulated by multiple mitotic kinases. How mitotic kinases interact with each other and coordinately regulate mitosis remains to be fully understood. Here we employed a chemical biology approach using selective small molecule kinase inhibitors to dissect the relationship between Cdk1 and Aurora A kinases during G₂/M transition. We find that activation of Aurora A first occurs at centrosomes at late G₂ and is required for centrosome separation independently of Cdk1 activity. Upon entry into mitosis, Aurora A then becomes fully activated downstream of Cdk1 activation. Inactivation of Aurora A or Plk1 individually during a synchronized cell cycle shows no significant effect on Cdk1 activation and entry into mitosis. However, simultaneous inactivation of both Aurora A and Plk1 markedly delays Cdk1 activation and entry into mitosis, suggesting that Aurora A and Plk1 have redundant functions in the feedback activation of Cdk1. Together, our data suggest that Cdk1, Aurora A, and Plk1 mitotic kinases participate in a feedback activation loop and that activation of Cdk1 initiates the feedback loop activity, leading to rapid and timely entry into mitosis in human cells. In addition, live cell imaging reveals that the nuclear cycle of cells becomes uncoupled from cytokinesis upon inactivation of both Aurora A and Aurora B kinases and continues to oscillate in a Cdk1-dependent manner in the absence of cytokinesis, resulting in multinucleated, polyploidy cells.     

Dynamic association of the mammalian insulator protein CTCF with centrosomes and the midbody

CTCF is a highly conserved, ubiquitously expressed DNA-binding protein that has widespread capabilities in gene regulation. CTCF plays important roles in cell growth regulatory processes and epigenetic functions. Ectopic expression of CTCF results in severe cell growth inhibition at multiple points within the cell cycle, indicating that CTCF levels must be stringently monitored. We have investigated the subcellular localization of CTCF in detail. Interestingly, we observe that CTCF shows a dynamic cell cycle-dependent distribution. Immunofluorescent staining reveals that in interphase CTCF is a nuclear protein, which is mainly excluded from the nucleolus. Strikingly, CTCF is associated with the centrosome during mitosis, especially from metaphase to anaphase. At telophase, CTCF dissociates from the centrosome and localizes to the midbody and the reformed nuclei. The association of CTCF with centrosomes and the midbody is further confirmed by biochemical fractionation. Moreover, subcellular fractions of CTCF show cell cycle and organelle-specific posttranslational modifications, suggesting different roles for CTCF at different stages of the cell cycle.     

Nek2A specifies the centrosomal localization of Erk2

Nek2A is a cell-cycle-regulated protein kinase that localizes to the centrosome and kinetochore. Our recent studies provide a link between Nek2A and spindle checkpoint signaling [J. Biol. Chem. 279 (2004) 20049]. Extracellular signal-regulated kinase 2 (Erk2) is an important kinase, which belongs to mitogen activating protein (MAP) kinase family. Here we demonstrated that Nek2A binds specifically to Erk2. Erk2 interacts with Nek2A via a conserved Erk2 docking site located to the C-terminus of Nek2A. Our studies indicate this docking site is essential and sufficient for a direct Nek2A-Erk2 interaction. In addition, our immunocytochemical studies show that Nek2A and Erk2 are co-localized to centrosome. Significantly, elimination of Nek2A by RNA interference delocalized Erk2 from its centrosomal location, while inhibition of Erk2 kinase activity did not affect the localization of Nek2A in centrosome. We propose that Erk2 links extracellular signaling to centrosome dynamics by Nek2A.