Triple deletion of TP53, PCNT, and CEP215 promotes centriole amplification in the M phase.
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ABSTRACT:
SUBMITTER: Jung GI
PROVIDER: S-EPMC8354662 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:An intercentrosomal linker keeps a cell's two centrosomes joined together until it is dissolved at the onset of mitosis. A second connection keeps daughter centrioles engaged to their mothers until they lose their orthogonal arrangement at the end of mitosis. Centriole disengagement is required to license centrioles for duplication. We show that the intercentrosomal linker protein Cep68 is degraded in prometaphase through the SCF(?TrCP) (Skp1-Cul1-F-box protein) ubiquitin ligase complex. Cep68 degradation is initiated by PLK1 phosphorylation of Cep68 on Ser 332, allowing recognition by ?TrCP. We also found that Cep68 forms a complex with Cep215 (also known as Cdk5Rap2) and PCNT (also known as pericentrin), two PCM (pericentriolar material) proteins involved in centriole engagement. Cep68 and PCNT bind to different pools of Cep215. We propose that Cep68 degradation allows Cep215 removal from the peripheral PCM preventing centriole separation following disengagement, whereas PCNT cleavage mediates Cep215 removal from the core of the PCM to inhibit centriole disengagement and duplication.
| S-EPMC4415623 | biostudies-literature
Project description:Centrosomes, composed of centrioles that recruit a pericentriolar material (PCM) matrix assembled from PCNT and CDK5RAP2, catalyze mitotic spindle assembly. Here, we inhibit centriole formation and/or remove PCNT-CDK5RAP2 in RPE1 cells to address their relative contributions to spindle formation. While CDK5RAP2 and PCNT are normally dispensable for spindle formation, they become essential when centrioles are absent. Acentriolar spindle assembly is accompanied by the formation of foci containing PCNT and CDK5RAP2 via a microtubule and Polo-like kinase 1-dependent process. Foci formation and spindle assembly require PCNT-CDK5RAP2-dependent matrix assembly and the ability of CDK5RAP2 to recruit γ-tubulin complexes. Thus, the PCM matrix can self-organize independently of centrioles to generate microtubules for spindle assembly; conversely, an alternative centriole-anchored mechanism supports spindle assembly when the PCM matrix is absent. Extension to three cancer cell lines revealed similar results in HeLa cells, whereas DLD1 and U2OS cells could assemble spindles in the absence of centrioles and PCNT-CDK5RAP2, suggesting cell type variation in spindle assembly mechanisms.
| S-EPMC7658699 | biostudies-literature
Project description:The transcriptional control of primary cilium formation and ciliary motility are beginning to be understood, but little is known about the transcriptional programs that control cilium number and other structural and functional specializations. One of the most intriguing ciliary specializations occurs in multiciliated cells (MCCs), which amplify their centrioles to nucleate hundreds of cilia per cell, instead of the usual monocilium. Here we report that the transcription factor MYB, which promotes S phase and drives cycling of a variety of progenitor cells, is expressed in postmitotic epithelial cells of the mouse airways and ependyma destined to become MCCs. MYB is expressed early in multiciliogenesis, as progenitors exit the cell cycle and amplify their centrioles, then switches off as MCCs mature. Conditional inactivation of Myb in the developing airways blocks or delays centriole amplification and expression of FOXJ1, a transcription factor that controls centriole docking and ciliary motility, and airways fail to become fully ciliated. We provide evidence that MYB acts in a conserved pathway downstream of Notch signaling and multicilin, a protein related to the S-phase regulator geminin, and upstream of FOXJ1. MYB can activate endogenous Foxj1 expression and stimulate a cotransfected Foxj1 reporter in heterologous cells, and it can drive the complete multiciliogenesis program in Xenopus embryonic epidermis. We conclude that MYB has an early, crucial and conserved role in multiciliogenesis, and propose that it promotes a novel S-like phase in which centriole amplification occurs uncoupled from DNA synthesis, and then drives later steps of multiciliogenesis through induction of Foxj1.
| S-EPMC3787764 | biostudies-literature
Project description: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.
| S-EPMC4682042 | biostudies-literature
Project description:Centrosomes are the major microtubule organising centres of animal cells. Deregulation in their number occurs in cancer and was shown to trigger tumorigenesis in mice. However, the incidence, consequence and origins of this abnormality are poorly understood. Here, we screened the NCI-60 panel of human cancer cell lines to systematically analyse centriole number and structure. Our screen shows that centriole amplification is widespread in cancer cell lines and highly prevalent in aggressive breast carcinomas. Moreover, we identify another recurrent feature of cancer cells: centriole size deregulation. Further experiments demonstrate that severe centriole over-elongation can promote amplification through both centriole fragmentation and ectopic procentriole formation. Furthermore, we show that overly long centrioles form over-active centrosomes that nucleate more microtubules, a known cause of invasiveness, and perturb chromosome segregation. Our screen establishes centriole amplification and size deregulation as recurrent features of cancer cells and identifies novel causes and consequences of those abnormalities.
| S-EPMC5871873 | biostudies-literature
Project description:Centrosome duplication is tightly controlled in coordination with DNA replication. The molecular mechanism of centrosome duplication remains unclear. Previous studies found that a fraction of human proline-directed phosphatase Cdc14B associates with centrosomes. However, Cdc14B's involvement in centrosome cycle control has never been explored. Here, we show that depletion of Cdc14B by RNA interference leads to centriole amplification in both HeLa and normal human fibroblast BJ and MRC-5 cells. Induction of Cdc14B expression through a regulatable promoter significantly attenuates centriole amplification in prolonged S phase-arrested cells and proteasome inhibitor Z-L(3)VS-treated cells. This inhibitory function requires centriole-associated Cdc14B catalytic activity. Together, these results suggest a potential function for Cdc14B phosphatase in maintaining the fidelity of centrosome duplication cycle.
| S-EPMC2364701 | biostudies-literature
Project description:Centriole duplication is tightly controlled to occur once per cell cycle, and disruption of this synchrony causes centriole amplification, which is frequently observed in many cancers. Our previous work showed that nuclear distribution gene C (NudC)-like protein 2 (NudCL2) localizes to centrosomes; however, little is known about the role of NudCL2 in the regulation of centrosome function. Here, we find that NudCL2 is required for accurate centriole duplication by stabilizing the E3 ligase HECT domain and RCC1-like domain-containing protein 2 (HERC2). Knockout (KO) of NudCL2 using CRISPR/Cas9-based genome editing or depletion of NudCL2 using small interfering RNA causes significant centriole amplification. Overexpression of NudCL2 significantly suppresses hydroxyurea-induced centriole overduplication. Quantitative proteomic analysis reveals that HERC2 is downregulated in NudCL2 KO cells. NudCL2 is shown to interact with and stabilize HERC2. Depletion of HERC2 leads to the similar defects to that in NudCL2-downregulated cells, and ectopic expression of HERC2 effectively rescues the centriole amplification caused by the loss of NudCL2, whereas the defects induced by HERC2 depletion cannot be reversed by exogenous expression of NudCL2. Either loss of NudCL2 or depletion of HERC2 leads to the accumulation of ubiquitin-specific peptidase 33 (USP33), a centrosomal protein that positively regulates centriole duplication. Moreover, knockdown of USP33 reverses centriole amplification in both NudCL2 KO and HERC2-depleted cells. Taken together, our data suggest that NudCL2 plays an important role in maintaining the fidelity of centriole duplication by stabilizing HERC2 to control USP33 protein levels, providing a previously undescribed mechanism restraining centriole amplification.
| S-EPMC6700069 | biostudies-literature
Project description:Reproductive and respiratory organs, along with brain ventricles, are lined by multiciliated epithelial cells (MCC) that generate cilia-powered fluid flows. MCC hijack the centrosome duplication pathway to form hundreds of centrioles and nucleate motile cilia. In these cells, the large majority of procentrioles are formed associated with partially characterized organelles called deuterosomes. We recently challenged the paradigm that deuterosomes and procentrioles are formed de novo by providing data, in brain MCC, suggesting that they are nucleated from the pre-existing centrosomal younger centriole. However, the origin of deuterosomes and procentrioles is still under debate. Here, we further question centrosome importance for deuterosome and centriole amplification. First, we provide additional data confirming that centriole amplification occurs sequentially from the centrosomal region, and that the first procentriole-loaded deuterosomes are associated with the daughter centriole or in the centrosomal centriole vicinity. Then, to further test the requirement of the centrosome in deuterosome and centriole formation, we depleted centrosomal centrioles using a Plk4 inhibitor. We reveal unexpected limited consequences in deuterosome/centriole number in absence of centrosomal centrioles. Notably, in absence of the daughter centriole only, deuterosomes are not seen associated with the mother centriole. In absence of both centrosomal centrioles, procentrioles are still amplified sequentially and with no apparent structural defects. They seem to arise from a focal region, characterized by microtubule convergence and pericentriolar material (PCM) assembly. The relevance of deuterosome association with the daughter centriole as well as the role of the PCM in the focal and sequential genesis of centrioles in absence of centrosomal centrioles are discussed.
| S-EPMC6736942 | biostudies-literature
Project description:Centriole amplification in multiciliated cells occurs in a pseudo-cell cycle regulated process that typically utilizes a poorly characterized molecularly dense structure called the deuterosome. We identified the centrosomal protein Cep70 as a novel deuterosome-associated protein that forms a complex with other deuterosome proteins, CCDC78 and Deup1. Cep70 dynamically associates with deuterosomes during centriole amplification in the ciliated epithelia of Xenopus embryos. Cep70 is not found in nascent deuterosomes prior to amplification. However, it becomes localized at deuterosomes at the onset of centriole biogenesis and remains there after the completion of centriole amplification. Deuterosome localization requires a conserved C-terminal "Cep70" motif. Depletion of Cep70 using morpholino oligos or CRISPR/Cas9 editing in F0 embryos leads to a severe decrease in centriole formation in both endogenous MCCs, as well as ectopically induced MCCs. Consistent with a decrease in centrioles, endogenous MCCs have defects in the process of radial intercalation. We propose that Cep70 represents a novel regulator of centriole biogenesis in MCCs.
| S-EPMC7856201 | biostudies-literature
Project description:Centrioles are critical for many cellular processes including cell division and cilia assembly. The number of centrioles within a cell is under strict control, and deregulation of centriole copy number is a hallmark of cancer. The molecular mechanisms that halt centriole amplification have not been fully elucidated. Here, we found that centrosomal protein of 76?kDa (Cep76), previously shown to restrain centriole amplification, interacts with cyclin-dependent kinase 2 (CDK2) and is a bona fide substrate of this kinase. Cep76 is preferentially phosphorylated by cyclin A/CDK2 at a single site S83, and this event is crucial to suppress centriole amplification in S phase. A novel Cep76 mutation S83C identified in a cancer patient fails to prevent centriole amplification. Mechanistically, Cep76 phosphorylation inhibits activation of polo-like kinase 1 (Plk1), thereby blocking premature centriole disengagement and subsequent amplification. Cep76 can also be acetylated, and enforced acetylation at K279 dampens the protein's ability to inhibit amplification and precludes S83 phosphorylation. Acetylation of Cep76 normally occurs in G2 phase and correlates with loss of protein function. Our data suggest that temporal changes in post-translational modifications of Cep76 during the cell cycle regulate its capacity to suppress centriole amplification, and its deregulation may contribute to malignancy.
| S-EPMC5125818 | biostudies-literature