Homologous recombination proteins are associated with centrosomes and are required for mitotic stability

In response to DNA damage, cells need robust repair mechanisms to complete the cell cycle successfully. Severe forms of DNA damage are repaired by homologous recombination (HR), in which the XRCC2 protein plays a vital role. Cells deficient in XRCC2 also show disruption of the centrosome, a key component of the mitotic apparatus. We find that this centrosome disruption is dynamic and when it occurs during mitosis it is linked directly to the onset of mitotic catastrophe in a significant fraction of the XRCC2-deficient cells. However, we also show for the first time that XRCC2 and other HR proteins, including the key recombinase RAD51, co-localize with the centrosome. Co-localization is maintained throughout the cell cycle, except when cells are finishing mitosis when RAD51 accumulates in the midbody between the separating cells. Taken together, these data suggest a tight functional linkage between the centrosome and HR proteins, potentially to coordinate the deployment of a DNA damage response at vulnerable phases of the cell cycle.     

CLIC4 is enriched at cell-cell junctions and colocalizes with AKAP350 at the centrosome and midbody of cultured mammalian cells

CLIC4 is a member of the chloride intracellular channel (CLIC) protein family whose principal cellular functions are poorly understood. Recently, we demonstrated that several CLIC proteins, including CLIC4, interact with AKAP350. AKAP350 is concentrated at the Golgi apparatus, centrosome, and midbody and acts as a scaffolding protein for several protein kinases and phosphatases. In this report, we show that endogenous CLIC4 and AKAP350 colocalize at the centrosome and midbody of cultured cells by immunofluorescence microscopy. Unlike AKAP350, CLIC4 is not enriched in the Golgi apparatus but is enriched in mitochondria, actin-based structures at the cell cortex, and the nuclear matrix, indicating that CLIC4-AKAP350 interactions are regulated at specific subcellular sites in vivo. In addition to the centrosome and midbody, CLIC4 colocalizes with AKAP350 and the tight junction protein ZO-1 in the apical region of polarized epithelial cells, suggesting that CLIC4 may play a role in maintaining apical-basolateral membrane polarity during mitosis and cytokinesis. Biochemical studies show that CLIC4 behaves mainly as a soluble cytosolic protein and can associate with proteins of the microtubule cytoskeleton. The localization of CLIC4 to the cortical actin cytoskeleton and its association with AKAP350 at the centrosome and midbody suggests that CLIC4 may be important for regulating cytoskeletal organization during the cell cycle. These findings lead to the conclusion that CLIC4 and possibly other CLIC proteins have alternate cellular functions that are distinct from their proposed roles as chloride channels.     

EFHC1, a protein mutated in juvenile myoclonic epilepsy, associates with the mitotic spindle through its N-terminus

A novel gene, EFHC1, mutated in juvenile myoclonic epilepsy (JME) encodes a protein with three DM10 domains of unknown function and one putative EF-hand motif. To study the properties of EFHC1, we expressed EGFP-tagged protein in various cell lines. In interphase cells, the fusion protein was present in the cytoplasm and in the nucleus with specific accumulation at the centrosome. During mitosis EGFP-EFHC1 colocalized with the mitotic spindle, especially at spindle poles and with the midbody during cytokinesis. Using a specific antibody, we demonstrated the same distribution of the endogenous protein. Deletion analyses revealed that the N-terminal region of EFHC1 is crucial for the association with the mitotic spindle and the midbody. Our results suggest that EFHC1 could play an important role during cell division.     

PCM-1, A 228-kD centrosome autoantigen with a distinct cell cycle distribution

We report the identification and primary sequence of PCM-1, a 228-kD centrosomal protein that exhibits a distinct cell cycle-dependent association with the centrosome complex. Immunofluorescence microscopy using antibodies against recombinant PCM-1 demonstrated that PCM-1 is tightly associated with the centrosome complex through G1, S, and a portion of G2. However, late in G2, as cells prepare for mitosis, PCM-1 dissociates from the centrosome and then remains dispersed throughout the cell during mitosis before re-associating with the centrosomes in the G1 phase progeny cells. These results demonstrate that the pericentriolar material is a dynamic substance whose composition can fluctuate during the cell cycle.     

Inhibition of Motile and Invasive Properties of Ovarian Cancer Cells by ASODN Against Rho-associated Protein Kinase

p160ROCK, a kinase effector of Rho GTPase mediating RhoA-induced assembly of focal adhesions and stress fibers, plays an important role in the invasive process of various tumor cells. The purpose of this study was to investigate the role of p160ROCK in the invasive behaviors of human ovarian cancer cells and their metastasis. Transfection with a dominant-active form of p160ROCK mutant (p160ROCKΔ 3) enhanced cell migration and invasion of ovarian cancer cells, while antisense oligodeoxynucleotide (ASODN) against p160ROCK inhibited the motile and invasive properties of the cells. Our data suggested that p160ROCK was involved in ovarian cancer cell invasion and metastasis by facilitating cancer cell migration, and that p160ROCK might be a potential new effective target for preventing metastasis of ovarian cancer.     

Inhibition of motile and invasive properties of ovarian cancer cells by ASODN against Rho-associated protein kinase

p160ROCK, a kinase effector of Rho GTPase mediating RhoA-induced assembly of focal adhesions and stress fibers, plays an important role in the invasive process of various tumor cells. The purpose of this study was to investigate the role of p160ROCK in the invasive behaviors of human ovarian cancer cells and their metastasis. Transfection with a dominant-active form of p160ROCK mutant (p160ROCKDelta 3) enhanced cell migration and invasion of ovarian cancer cells, while antisense oligodeoxynucleotide (ASODN) against p160ROCK inhibited the motile and invasive properties of the cells. Our data suggested that p160ROCK was involved in ovarian cancer cell invasion and metastasis by facilitating cancer cell migration, and that p160ROCK might be a potential new effective target for preventing metastasis of ovarian cancer.     

A functional role for p38 MAPK in modulating mitotic transit in the absence of stress

Although p38 MAPK is known to be activated in response to various environmental stresses and to have inhibitory roles in cell proliferation and tumor progression, its role in cell cycle progression in the absence of stress is unknown in most cell types. In the case of G(2)/M cell cycle control, p38 activation has been shown to trigger a rapid G(2)/M cell cycle checkpoint after DNA damage stress and a spindle checkpoint after microtubule disruption. In the course of our studies, we observed that p38 became actively phosphorylated, and its kinase activity increased transiently during G(2)/M cell cycle transition. Using an immunocytochemistry approach, the active form of p38 was found at the centrosome from late G(2) throughout mitosis, which suggests functional relevance for active p38 protein during mitotic entry. A closer examination reveals that p38 inhibition by pharmacologic inhibitors significantly accelerated the timing of mitotic entry. In addition, long term exposure of the inhibitor enhanced Cdc2 activity. These results indicate that p38 activity during G(2)/M may be involved in a mechanism for fine tuning the initiation of mitosis and perhaps transit of mitosis. Consistent with our previous findings, Cdc25B was phosphorylated on serine 309 at the centrosome during G(2)/M when p38 was active at this site; Cdc25B phosphorylation inhibits Cdc25B activity, and this phosphorylation was found to be p38-dependent. Taken together, our findings suggest that p38 regulates the timing of mitotic entry via modulation of Cdc25B activity under normal nonstress conditions.     

Functional relationship among PLK2, PLK4 and ROCK2 to induce centrosome amplification

The presence of more than 2 centrosomes (centrosome amplification) leads to defective mitosis and chromosome segregation errors, is frequently found in a variety of cancer types, and believed to be the major cause of chromosome instability. One mechanism for generation of amplified centrosomes is over-duplication of centrosomes in a single cell cycle, which is expected to occur when cells are temporarily arrested. There are a growing number of kinases that are critical for induction and promotion of centrosome amplification in the cell cycle-arrested cells, including Rho-associated kinase (ROCK2), Polo-like kinase 2 (PLK2) and PLK4. Here, we tested whether these kinases induce centrosome amplification in a linear pathway or parallel pathways. We first confirmed that ROCK2, PLK2 and PLK4 are all essential for centrosomes to re-duplicate in the cells arrested by exposure to DNA synthesis inhibitor. Using the centrosome amplification rescue assay, we found that PLK2 indirectly activates ROCK2 via phosphorylating nucleophosmin (NPM), and PLK4 functions downstream of ROCK2 to drive centrosome amplification in the arrested cells.     

TBCD Links Centriologenesis,Spindle Microtubule Dynamics,and Midbody Abscission in Human Cells

Microtubule-organizing centers recruit α- and β-tubulin polypeptides for microtubule nucleation. Tubulin synthesis is complex, requiring five specific cofactors, designated tubulin cofactors (TBCs) A–E, which contribute to various aspects of microtubule dynamics in vivo. Here, we show that tubulin cofactor D (TBCD) is concentrated at the centrosome and midbody, where it participates in centriologenesis, spindle organization, and cell abscission. TBCD exhibits a cell-cycle-specific pattern, localizing on the daughter centriole at G1 and on procentrioles by S, and disappearing from older centrioles at telophase as the protein is recruited to the midbody. Our data show that TBCD overexpression results in microtubule release from the centrosome and G1 arrest, whereas its depletion produces mitotic aberrations and incomplete microtubule retraction at the midbody during cytokinesis. TBCD is recruited to the centriole replication site at the onset of the centrosome duplication cycle. A role in centriologenesis is further supported in differentiating ciliated cells, where TBCD is organized into “centriolar rosettes”. These data suggest that TBCD participates in both canonical and de novo centriolar assembly pathways.     

A role for the Rho-p160 Rho coiled-coil kinase axis in the chemokine stromal cell-derived factor-1alpha-induced lymphocyte actomyosin and microtubular organization and chemotaxis.

The possible involvement of the Rho-p160ROCK (Rho coiled-coil kinase) pathway in the signaling induced by the chemokine Stromal cell-derived factor (SDF)-1alpha has been studied in human PBL. SDF-1alpha induced activation of RhoA, but not that of Rac. RhoA activation was followed by p160ROCK activation mediated by RhoA, which led to myosin light chain (MLC) phosphorylation, which was dependent on RhoA and p160ROCK activities. The kinetics of MLC activation was similar to that of RhoA and p160ROCK. The role of this cascade in overall cell morphology and functional responses to the chemokine was examined employing different chemical inhibitors. Inhibition of either RhoA or p160ROCK did not block SDF-1alpha-induced short-term actin polymerization, but induced the formation of long spikes arising from the cell body, which were found to be microtubule based. This morphological change was associated with an increase in microtubule instability, which argues for an active microtubule polymerization in the formation of these spikes. Inhibition of the Rho-p160ROCK-MLC kinase signaling cascade at different steps blocked lymphocyte migration and the chemotaxis induced by SDF-1alpha. Our results indicate that the Rho-p160ROCK axis plays a pivotal role in the control of the cell shape as a step before lymphocyte migration toward a chemotactic gradient.     

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.     

p53 dependent centrosome clustering prevents multipolar mitosis in tetraploid cells

Backgroundp53 abnormality and aneuploidy often coexist in human tumors, and tetraploidy is considered as an intermediate between normal diploidy and aneuploidy. The purpose of this study was to investigate whether and how p53 influences the transformation from tetraploidy to aneuploidy.Conclusionsp53 could not prevent tetraploid cells entering mitosis or induce tetraploid cell death. However, p53 abnormality impaired centrosome clustering and lead to multipolar mitosis in tetraploid cells by modulating the RhoA/ROCK signaling pathway.     

Cyclin-like accumulation and loss of the putative kinetochore motor CENP-E results from coupling continuous synthesis with specific degradation at the end of mitosis

CENP-E is a kinesin-like protein that binds to kinetochores through the early stages of mitosis, but after initiation of anaphase, it relocalizes to the overlapping microtubules in the midzone, ultimately concentration in the developing midbody. By immunoblotting of cells separated at various positions in the cell cycle using centrifugal elutriation, we show that CENP-E levels increase progressively across the cycle peaking at approximately 22,000 molecules/cell early in mitosis, followed by an abrupt (> 10 fold) loss at the end of mitosis. Pulse-labeling with [35S]methionine reveals that beyond a twofold increase in synthesis between G1 and G2, interphase accumulation results primarily from stabilization of CENP-E during S and G2. Despite localizing in the midbody during normal cell division, CENP-E loss at the end of mitosis is independent of cytokinesis, since complete blockage of division with cytochalasin has no affect on CENP-E loss at the M/G1 transition. Thus, like mitotic cyclins, CENP-E accumulation peaks before cell division, and it is specifically degraded at the end of mitosis. However, CENP-E degradation kinetically follows proteolysis of cyclin B in anaphase. Combined with cyclin A destruction before the end of metaphase, degradation of as yet unidentified components at the metaphase/anaphase transition, and cyclin B degradation at or after the anaphase transition, CENP-E destruction defines a fourth point in a mitotic cascade of timed proteolysis.     

Cytokinesis and cancer： Polo loves ROCK＇n＇ Rho（A）

Cytokinesis is the last step of the M (mitosis) phase, yet it is crucial for the faithful division of one cell into two. Cytokinesis failure is often associated with cancer. Cytokinesis can be morphologically divided into four steps: cleavage furrow initiation, cleavage furrow ingression, midbody formation and abscission. Molecular studies have revealed that RhoA as well as its regulators and effectors are important players to ensure a successful cytokinesis. At the same time, Polo-like kinase 1 (Plk1) is an important kinase that can target many substrates and carry out different functions during mitosis, including cytokinesis. Recent studies are beginning to unveil a closer tie between Plk1 and RhoA networks. More specifically, Plk1 phosphorylates the centralspindlin complex Cyk4 and MKLP1/CHO1, thus recruiting RhoA guanine nucleotide-exchange factor (GEF) Ect2 through its phosphopeptide-binding BRCT domains. Ect2 itself can be phosphorylated by Plk1 in vitro. Plk1 can also phosphorylate another GEF MyoGEF to regulate RhoA activity. Once activated, RhoA-GTP will activate downstream effectors, including ROCK1 and ROCK2. ROCK2 is among the proteins that associate with Plk1 Polo-binding domain (PBD) in a large proteomic screen, and Plk1 can phosphorylate ROCK2 in vitro. We review current understandings of the interplay between Plk1, RhoA proteins and other proteins (e.g., NudC, MKLP2, PRC1, CEP55) involved in cytokinesis, with particular emphasis of its clinical implications in cancer.     

Cytokinesis and cancer:Polo loves ROCK'n' Rho(A)

Cytokinesis is the last step of the M (mitosis) phase,yet it is crucial for the faithful division of one cell into two.Cytokinesis failure is often associated with cancer.Cytokinesis can be morphologically divided into four steps:cleavage furrow initiation,cleavage furrow ingression,midbody formation and abscission.Molecular studies have revealed that RhoA as well as its regulators and effectors are important players to ensure a successful cytokinesis.At the same time,Polo-like kinase 1 (Plk1) is an important kinase that can target many substrates and carry out different functions during mitosis,including cytokinesis.Recent studies are beginning to unveil a closer tie between Plk1 and RhoA networks.More specifically,Plk1 phosphorylates the centralspindlin complex Cyk4 and MKLP1/CHO1,thus recruiting RhoA guanine nucleotide-exchange factor (GEF) Ect2 through its phosphopeptide-binding BRCT domains.Ect2 itself can be phosphorylated by Plk1 in vitro.Plk1 can also phosphorylate another GEF MyoGEF to regulate RhoA activity.Once activated,RhoA-GTP will activate downstream effectors,including ROCK1 and ROCK2.ROCK2 is among the proteins that associate with Plk1 Polo-binding domain (PBD) in a large proteomic screen,and Plk1 can phosphorylate ROCK2 in vitro.We review current understandings of the interplay between Plk1,RhoA proteins and other proteins (e.g.,NudC,MKLP2,PRC1,CEP55) involved in cytokinesis,with partitular emphasis of its clinical implications in cancer.     

TBCCD1, a new centrosomal protein,is required for centrosome and Golgi apparatus positioning

In animal cells the centrosome is positioned at the cell centre in close association with the nucleus. The mechanisms responsible for this are not completely understood. Here, we report the first characterization of human TBCC‐domain containing 1 (TBCCD1), a protein related to tubulin cofactor C. TBCCD1 localizes at the centrosome and at the spindle midzone, midbody and basal bodies of primary and motile cilia. Knockdown of TBCCD1 in RPE‐1 cells caused the dissociation of the centrosome from the nucleus and disorganization of the Golgi apparatus. TBCCD1‐depleted cells are larger, less efficient in primary cilia assembly and their migration is slower in wound‐healing assays. However, the major microtubule‐nucleating activity of the centrosome is not affected by TBCCD1 silencing. We propose that TBCCD1 is a key regulator of centrosome positioning and consequently of internal cell organization.     

p21-Mediated nuclear retention of cyclin B1-Cdk1 in response to genotoxic stress

G2 arrest of cells suffering DNA damage in S phase is crucial to avoid their entry into mitosis, with the concomitant risks of oncogenic transformation. According to the current model, signals elicited by DNA damage prevent mitosis by inhibiting both activation and nuclear import of cyclin B1-Cdk1, a master mitotic regulator. We now show that normal human fibroblasts use additional mechanisms to block activation of cyclin B1-Cdk1. In these cells, exposure to nonrepairable DNA damage leads to nuclear accumulation of inactive cyclin B1-Cdk1 complexes. This nuclear retention, which strictly depends on association with endogenous p21, prevents activation of cyclin B1-Cdk1 by Cdc25 and Cdk-activating kinase as well as its recruitment to the centrosome. In p21-deficient normal human fibroblasts and immortal cell lines, cyclin B1 fails to accumulate in the nucleus and could be readily detected at the centrosome in response to DNA damage. Therefore, in normal cells, p21 exerts a dual role in mediating DNA damage-induced cell cycle arrest and exit before mitosis. In addition to blocking pRb phosphorylation, p21 directly prevents mitosis by inactivating and maintaining the inactive state of mitotic cyclin-Cdk complexes. This, with subsequent degradation of mitotic cyclins, further contributes to the establishment of a permanent G2 arrest.     

Perturbing integrin function inhibits microtubule growth from centrosomes, spindle assembly, and cytokinesis

In many mammalian cell types, integrin-mediated cell-matrix adhesion is required for the G1-S transition of the cell cycle. As cells approach mitosis, a dramatic remodeling of their cytoskeleton accompanies dynamic changes in matrix adhesion, suggesting a mechanistic link. However, the role of integrins in cell division remains mostly unexplored. Using two cellular systems, we demonstrate that a point mutation in the beta1 cytoplasmic domain (beta1 tail) known to decrease integrin activity supports entry into mitosis but inhibits the assembly of a radial microtubule array focused at the centrosome during interphase, the formation of a bipolar spindle at mitosis and cytokinesis. These events are restored by externally activating the mutant integrin with specific antibodies. This is the first demonstration that the integrin beta1 tail can regulate centrosome function, the assembly of the mitotic spindle, and cytokinesis.     

TAC-1, a regulator of microtubule length in the C. elegans embryo

Regulation of microtubule growth is critical for many cellular processes, including meiosis, mitosis, and nuclear migration. We carried out a genome-wide RNAi screen in Caenorhabditis elegans to identify genes required for pronuclear migration, one of the first events in embryogenesis requiring microtubules. Among these, we identified and characterized tac-1 a new member of the TACC (Transforming Acidic Coiled-Coil) family [1]. tac-1(RNAi) embryos exhibit very short microtubules nucleated from the centrosomes as well as short spindles. TAC-1 is initially enriched at the meiotic spindle poles and is later recruited to the sperm centrosome. TAC-1 localization at the centrosomes is regulated during the cell cycle, with high levels during mitosis and a reduction during interphase, and is dependent on aurora kinase 1 (AIR-1), a protein involved in centrosome maturation. tac-1(RNAi) embryos resemble mutants of zyg-9, which encodes a previously characterized centrosomal protein of the XMAP215 family and was also found in our screen. We show that TAC-1 and ZYG-9 are dependent on one another for their localization at the centrosome, and this dependence suggests that they may function together as a complex. We conclude that TAC-1 is a major regulator of microtubule length in the C. elegans embryo.