The intercentriolar fibers function as docking sites of centriolar satellites for cilia assembly.

Two mother centrioles in an animal cell are linked by intercentriolar fibers that have CROCC/rootletin as their main building block. Here, we investigated the regulatory role of intercentriolar/rootlet fibers in cilia assembly. The cilia formation rates were significantly reduced in the CEP250/C-NAP1 and CROCC/rootletin knockout (KO) cells, irrespective of the departure of the young mother centrioles from the basal bodies. In addition, centriolar satellites were dispersed throughout the cytoplasm in the CEP250 and CROCC KO cells. We observed that PCM1 directly binds to CROCC. Their interaction is critical not only for the accumulation of centriolar satellites near the centrosomes/basal bodies but also for cilia formation. Finally, we observed that the centriolar satellite proteins are localized at the intercentriolar/rootlet fibers in the kidney epithelial cells. Based on these findings, we propose that the intercentriolar/rootlet fibers function as docking sites for centriolar satellites near the centrosomes/basal bodies and facilitate the cilia assembly process.

Generation and Fates of Supernumerary Centrioles in Dividing Cells.

The centrosome is a subcellular organelle from which a cilium assembles. Since centrosomes function as spindle poles during mitosis, they have to be present as a pair in a cell. How the correct number of centrosomes is maintained in a cell has been a major issue in the fields of cell cycle and cancer biology. Centrioles, the core of centrosomes, assemble and segregate in close connection to the cell cycle. Abnormalities in centriole numbers are attributed to decoupling from cell cycle regulation. Interestingly, supernumerary centrioles are commonly observed in cancer cells. In this review, we discuss how supernumerary centrioles are generated in diverse cellular conditions. We also discuss how the cells cope with supernumerary centrioles during the cell cycle.

Triple deletion of TP53, PCNT, and CEP215 promotes centriole amplification in the M phase.

Supernumerary centrioles are frequently observed in diverse types of cancer cells. In this study, we investigated the mechanism underlying the generation of supernumerary centrioles during the M phase. We generated the TP53;PCNT;CEP215 triple knockout (KO) cells and determined the configurations of the centriole during the cell cycle. The triple KO cells exhibited a precocious separation of centrioles and unscheduled centriole assembly in the M phase. Supernumerary centrioles in the triple KO cells were present throughout the cell cycle; however, among all the centrioles, only two maintained an intact composition, including CEP135, CEP192, CEP295 and CEP152. Intact centrioles were formed during the S phase and the rest of the centrioles may be generated during the M phase. M-phase-assembled centrioles lacked the ability to organize microtubules in the interphase; however, a fraction of them may acquire pericentriolar material to organize microtubules during the M phase. Taken together, our work reveals the heterogeneity of the supernumerary centrioles in the triple KO cells. .

E2F1 Mediates the Retinoic Acid-Induced Transcription of Tshz1 during Neuronal Differentiation in a Cell Division-Dependent Manner.

The involvement of cell division in cellular differentiation has long been accepted. Cell division may be required not only for the expansion of a differentiated cell population but also for the execution of differentiation processes. Nonetheless, knowledge regarding how specific differentiation processes are controlled in a cell division-dependent manner is far from complete. Here, we determined the involvement of cell division in neuronal differentiation. We initially confirmed that cell division is an essential event for the neuronal differentiation of P19 embryonic carcinoma cells. We investigated the induction mechanisms of Tshz1, whose expression is induced by retinoic acid (RA) in a cell division-dependent manner. Promoter analysis of Tshz1 revealed a specific region required for RA-dependent transcription. A series of experiments was used to identify E2F1 as the induction factor for the RA-dependent transcription of Tshz1 We propose that E2F1 mediates neuronal differentiation in a cell division-dependent manner.

Phosphorylation of human enhancer filamentation 1 (HEF1) stimulates interaction with Polo-like kinase 1 leading to HEF1 localization to focal adhesions.

Elevated expression of human enhancer filamentation 1 (HEF1; also known as NEDD9 or Cas-L) is an essential stimulus for the metastatic process of various solid tumors. This process requires HEF1 localization to focal adhesions (FAs). Although the association of HEF1 with FAs is considered to play a role in cancer cell migration, the mechanism targeting HEF1 to FAs remains unclear. Moreover, up-regulation of Polo-like kinase 1 (Plk1) positively correlates with human cancer metastasis, yet how Plk1 deregulation promotes metastasis remains elusive. Here, we report that casein kinase 1δ (CK1δ) phosphorylates HEF1 at Ser-780 and Thr-804 and that these phosphorylation events promote a physical interaction between Plk1 and HEF1. We found that this interaction is critical for HEF1 translocation to FAs and for inducing migration of HeLa cells. Plk1-docking phosphoepitopes were mapped/confirmed in HEF1 by various methods, including X-ray crystallography, and mutated for functional analysis in HeLa cells. In summary, our results reveal the role of a phosphorylation-dependent HEF1-Plk1 complex in HEF1 translocation to FAs to induce cell migration. Our findings provide critical mechanistic insights into the HEF1-Plk1 complex-dependent localization of HEF1 to FAs underlying the metastatic process and may therefore contribute to the development of new cancer therapies.

The DNA replication protein Cdc6 inhibits the microtubule-organizing activity of the centrosome.

The centrosome serves as a major microtubule-organizing center (MTOC). The Cdc6 protein is a component of the pre-replicative complex and a licensing factor for the initiation of chromosome replication and localizes to centrosomes during the S and G2 phases of the cfell cycle of human cells. This cell cycle-dependent localization of Cdc6 to the centrosome motivated us to investigate whether Cdc6 negatively regulates MTOC activity and to determine the integral proteins that comprise the pericentriolar material (PCM). Time-lapse live-cell imaging of microtubule regrowth revealed that Cdc6 depletion increased microtubule nucleation at the centrosomes and that expression of Cdc6 in Cdc6-depleted cells reversed this effect. This increase and decrease in microtubule nucleation correlated with the centrosomal intensities of PCM proteins such as γ-tubulin, pericentrin, CDK5 regulatory subunit-associated protein 2 (CDK5RAP2), and centrosomal protein 192 (Cep192). The regulation of microtubule nucleation and the recruitment of PCM proteins to the centrosome required Cdc6 ATPase activity, as well as a centrosomal localization of Cdc6. These results suggest a novel function for Cdc6 in coordinating centrosome assembly and function.

PLK1 regulation of PCNT cleavage ensures fidelity of centriole separation during mitotic exit.

Centrioles are duplicated and segregated in close link to the cell cycle. During mitosis, daughter centrioles are disengaged and eventually separated from mother centrioles. New daughter centrioles may be generated only after centriole separation. Therefore, centriole separation is considered a licensing step for centriole duplication. It was previously known that separase specifically cleaves pericentrin (PCNT) during mitotic exit. Here we report that PCNT has to be phosphorylated by PLK1 to be a suitable substrate of separase. Phospho-resistant mutants of PCNT are not cleaved by separase and eventually inhibit centriole separation. Furthermore, phospho-mimetic PCNT mutants rescue centriole separation even in the presence of a PLK1 inhibitor. On the basis on these results, we propose that PLK1 phosphorylation is a priming step for separase-mediated cleavage of PCNT and eventually for centriole separation. PLK1 phosphorylation of PCNT provides an additional layer of regulatory mechanism to ensure the fidelity of centriole separation during mitotic exit.

Integrity of the Pericentriolar Material Is Essential for Maintaining Centriole Association during M Phase.

A procentriole is assembled next to the mother centriole during S phase and remains associated until M phase. After functioning as a spindle pole during mitosis, the mother centriole and procentriole are separated at the end of mitosis. A close association of the centriole pair is regarded as an intrinsic block to the centriole reduplication. Therefore, deregulation of this process may cause a problem in the centriole number control, resulting in increased genomic instability. Despite its importance for faithful centriole duplication, the mechanism of centriole separation is not fully understood yet. Here, we report that centriole pairs are prematurely separated in cells whose cell cycle is arrested at M phase by STLC. Dispersal of the pericentriolar material (PCM) was accompanied. This phenomenon was independent of the separase activity but needed the PLK1 activity. Nocodazole effectively inhibited centriole scattering in STLC-treated cells, possibly by reducing the microtubule pulling force around centrosomes. Inhibition of PLK1 also reduced the premature separation of centrioles and the PCM dispersal as well. These results revealed the importance of PCM integrity in centriole association. Therefore, we propose that PCM disassembly is one of the driving forces for centriole separation during mitotic exit.

Structural and biochemical insights into the role of testis-expressed gene 14 (TEX14) in forming the stable intercellular bridges of germ cells.

Intercellular bridges are a conserved feature of spermatogenesis in mammalian germ cells and derive from arresting cell abscission at the final stage of cytokinesis. However, it remains to be fully understood how germ cell abscission is arrested in the presence of general cytokinesis components. The TEX14 (testis-expressed gene 14) protein is recruited to the midbody and plays a key role in the inactivation of germ cell abscission. To gain insights into the structural organization of TEX14 at the midbody, we have determined the crystal structures of the EABR [endosomal sorting complex required for transport (ESCRT) and ALIX-binding region] of CEP55 bound to the TEX14 peptide (or its chimeric peptides) and performed functional characterization of the CEP55-TEX14 interaction by multiexperiment analyses. We show that TEX14 interacts with CEP55-EABR via its AxGPPx3Y (Ala793, Gly795, Pro796, Pro797, and Tyr801) and PP (Pro803 and Pro804) sequences, which together form the AxGPPx3YxPP motif. TEX14 competitively binds to CEP55-EABR to prevent the recruitment of ALIX, which is a component of the ESCRT machinery with the AxGPPx3Y motif. We also demonstrate that a high affinity and a low dissociation rate of TEX14 to CEP55, and an increase in the local concentration of TEX14, cooperatively prevent ALIX from recruiting ESCRT complexes to the midbody. The action mechanism of TEX14 suggests a scheme of how to inactivate the abscission of abnormal cells, including cancer cells.

The microtubule nucleation activity of centrobin in both the centrosome and cytoplasm.

Centrobin resides in daughter centriole and play a critical role in centriole duplication. Nucleation and stabilization of microtubules are known biological activities of centrobin. Here, we report a specific localization of centrobin outside the centrosome. Centrobin was associated with the stable microtubules. In hippocampal cells, centrobin formed cytoplasmic dots in addition to the localization at both centrosomes with the mother and daughter centrioles. Such specific localization pattern suggests that cytoplasmic centrobin is not just a reserved pool for centrosomal localization but also has a specific role in the cytoplasm. In fact, centrobin enhanced microtubule formation outside as well as inside the centrosome. These results propose specific roles of the cytoplasmic centrobin for noncentrosomal microtubule formation in specific cell types and during the cell cycle.

NEK2 phosphorylation antagonizes the microtubule stabilizing activity of centrobin.

Centrobin was initially identified as a centrosome protein for centriole duplication. Centrobin is also detected outside the centrosome and involved in other cellular functions, such as spindle assembly. We previously reported that centrobin is a substrate of both NEK2 and PLK1, but it is not clear what functional properties of centrobin are regulated by two kinases. Here, we report that centrobin is involved in cell spreading, migration and microtubule stabilization in interphase cells. The NEK2-depleted cells looked spread with well-developed microtubule networks and migrated faster than the control cells. The microtubule stability in NEK2-depleted cells was higher than the control cells. However, the opposite was the case in centrobin-depleted cells. The opposite outcomes in NEK2- and centrobin-depleted cells suggest that NEK2 antagonizes biological functions of centrobin. We identified NEK2 phosphorylation sites within centrobin, which is distinct from the PLK1 phosphorylation sites. In fact, the phospho-resistant mutant of centrobin against NEK2 stabilized microtubule networks in vivo. Based on the results, we propose that NEK2 phosphorylation antagonizes the microtubule stabilizing activity of centrobin. Centrobin is a novel example that NEK2 and PLK1 independently phosphorylate a substrate and result in opposite outcomes in substrate function.

PLK1 phosphorylation of pericentrin initiates centrosome maturation at the onset of mitosis.

The microtubule-organizing activity of the centrosome oscillates during the cell cycle, reaching its highest level at mitosis. At the onset of mitosis, the centrosome undergoes maturation, which is characterized by a drastic expansion of the pericentriolar matrix (PCM) and a robust increase in microtubule-organizing activity. It is known that PLK1 is critical for the initiation of centrosome maturation. In this paper, we report that pericentrin (PCNT), a PCM protein, was specifically phosphorylated by PLK1 during mitosis. Phosphoresistant point mutants of PCNT did not recruit centrosomal proteins, such as CEP192, GCP-WD (γ-complex protein with WD repeats), γ-tubulin, Aurora A, and PLK1, into the centrosome during mitosis. However, centrosomal recruitment of CEP215 depended on PCNT irrespective of its phosphorylation status. Furthermore, ectopic expression of PLK1-PCNT fusion proteins induced the centrosomal accumulation of CEP192, GCP-WD, and γ-tubulin even in interphase cells, mimicking centrosome maturation. Based on these results, we propose that PLK1-mediated phosphorylation of PCNT initiates centrosome maturation by organizing the spindle pole-specific PCM lattice.

Pierce1, a novel p53 target gene contributing to the ultraviolet-induced DNA damage response.

Retinoblastoma (Rb) and p53 genes are mutated or inactivated in most human cancers and mutually regulate each other. Recently, we reported that expression of diverse genes was altered in Rb-deficient mouse embryonic fibroblasts (MEF). In this study, we found that Pierce1, a novel transcript upregulated in Rb-deficient MEFs, is a transcriptional target of p53. Although Pierce1 promoter did not respond to the ectopic expression of E2F1, it was strongly activated by p53 via 2 cis-elements. Consistently, the expression of Pierce1 was induced by genotoxic stresses that activate p53 but was not detected in p53-deficient MEFs. Pierce1 was posttranslationally stabilized by ultraviolet C (UVC) irradiation, and UVC-activated ATR (ataxia telangiectasia-mutated and Rad3-related) signaling suppressed proteosomal degradation of Pierce1 protein. Furthermore, knockdown of Pierce1 compromised the checkpoint response of wild-type MEFs to UVC irradiation, accompanying the diminished expression of p53 target genes. Together, our data suggest that Pierce1 is an important p53 target gene contributing to normal DNA damage response and may play crucial roles in maintaining genomic integrity against genotoxic stresses, including UVC irradiation.

Centrobin/NIP2 is a microtubule stabilizer whose activity is enhanced by PLK1 phosphorylation during mitosis.

Centrobin/NIP2 is a centrosomal protein that is required for centrosome duplication. It is also critical for microtubule organization in both interphase and mitotic cells. In the present study, we observed that centrobin is phosphorylated in a cell cycle stage-specific manner, reaching its maximum at M phase. PLK1 is a kinase that is responsible for M phase-specific phosphorylation of centrobin. The microtubule forming activity of centrobin was enhanced by PLK1 phosphorylation. Furthermore, mitotic spindles were not assembled properly with the phospho-resistant mutant of centrobin. Based on these results, we propose that centrobin functions as a microtubule stabilizing factor and PLK1 enhances centrobin activity for proper spindle formation during mitosis.

CEP215 is involved in the dynein-dependent accumulation of pericentriolar matrix proteins for spindle pole formation.

CEP 215 is a human orthologue of Drosophila centrosomin which is a core centrosome component for the pericentriolar matrix protein recruitment. Recent investigations revealed that CEP 215 is required for centrosome cohesion, centrosomal attachment of the gamma-TuRC, and microtubule dynamics. However, it remains obscure how CEP 215 functions for recruitment of the centrosomal proteins during the centrosome cycle. Here, we investigated a role of CEP 215 during mitosis. Knockdown of CEP 215 resulted in characteristic mitotic phenotypes, including monopolar spindle formation, a decrease in distance between the spindle pole pair, and detachment of the centrosomes from the spindle poles. We noticed that CEP 215 is critical for centrosomal localization of dynein throughout the cell cycle. As a consequence, the selective centrosomal proteins were not recruited to the centrosome properly. Finally, the centrosomal localization of CEP 215 also depends on the dynein-dynactin complex. Based on the results, we propose that CEP 215 regulates a dynein-dependent transport of the pericentriolar matrix proteins during the centrosome maturation.

Plk1-dependent and -independent roles of an ODF2 splice variant, hCenexin1, at the centrosome of somatic cells.

Outer dense fiber 2 (ODF2) was initially identified as a major component of the sperm tail cytoskeleton, and was later suggested to be localized to somatic centrosomes and required for the formation of primary cilia. Here we show that a splice variant of hODF2 called hCenexin1, but not hODF2 itself, efficiently localizes to somatic centrosomes via a variant-specific C-terminal extension and recruits Plk1 through a Cdc2-dependent phospho-S796 motif within the extension. This interaction and Plk1 activity were important for proper recruitment of pericentrin and gamma-tubulin, and, ultimately, for formation of normal bipolar spindles. Earlier in the cell cycle, hCenexin1, but again not hODF2, also contributed to centrosomal recruitment of ninein and primary cilia formation independent of Plk1 interaction. These findings provide a striking example of how a splice-generated C-terminal extension of a sperm tail-associating protein mediates unanticipated centrosomal events at distinct stages of the somatic cell cycle.

Dazl can bind to dynein motor complex and may play a role in transport of specific mRNAs.

Male germ cell development includes mitotic and meiotic cell divisions that are followed by dramatic morphological changes resulting in the production of spermatozoa. Genetic evidence has indicated that the DAZ family genes are critical for successful male germ cell development in diverse animals as well as humans. In the present study, we investigated the cellular functions of Dazl in the mouse male germ cells. We identified a specific interaction of Dazl with the dynein light chain, a component of the dynein-dynactin motor complex. The subcellular distribution of Dazl was microtubule-dependent and a selected number of Dazl-bound mRNAs could accumulate in the perinuclear area. Based on these results, we propose that Dazl may play a role in transport of specific mRNAs via dynein motor complex. The Dazl-bound mRNAs may be stored at specific sites and would be available for future developmental processes. Our study revealed the presence of an active mRNA transport system in mouse male germ cells.

Hematopoietic malignancies associated with increased Stat5 and Bcl-x(L) expressions in Ink4a/Arf-deficient mice.

The INK4a/ARF locus, which encodes the two distinct proteins p16(INK4a) and p14(ARF), is frequently altered in various hematological malignancies as well as in other types of cancers in humans. In this study, we surveyed tumors that had spontaneously developed in Ink4a/Arf-deficient mice with an inbred FVB/NJ genetic background. We found that an Ink4a/Arf-deficiency exerted more severe effects on the induction of hematopoietic malignancies in mice with an inbred FVB/NJ genetic background than in mice with a mixed genetic background. We also provided the evidence that this prevalence of hematopoietic malignancies in Ink4a/Arf-deficient mice is associated with the upregulated expressions of Stat5 and its transcriptional target, Bcl-x(L), both of which are involved in the regulation of hematopoiesis. These results suggest a possible implication of the Ink4a/Arf locus in the control of hematopoietic pathways by negatively regulating the Stat5-signalling pathways.

Contribution of the PI3K/Akt/PKB signal pathway to maintenance of self-renewal in human embryonic stem cells.

Although basic fibroblast growth factor (FGF2) is generally included in the media for maintenance of human embryonic stem cells (hESCs), the action of FGF2 in these cells has not been well defined. Here, we determined the roles of FGF2 in maintaining hESC self-renewal. Withdrawal of FGF2 from the media led to acquisition of typical differentiated characteristics in hESCs. In the presence of FGF2, which is normally required for proliferation in an undifferentiated state, inhibition of phosphatidylinositol 3-kinase (PI3K)/Akt/PKB signal stimulated differentiation and attenuated the expression of extracellular matrix (ECM) molecules. We suggest that FGF2 maintains hESC self-renewal by supporting stable expression of ECM molecules through activation of the PI3K/Akt/PKB pathway.