Sufu negatively regulates both initiations of centrosome duplication and DNA replication.

Centrosome duplication and DNA replication are two pivotal events that higher eukaryotic cells use to initiate proliferation. While DNA replication is initiated through origin licensing, centrosome duplication starts with cartwheel assembly and is partly controlled by CP110. However, the upstream coordinator for both events has been, until now, a mystery. Here, we report that suppressor of fused protein (Sufu), a negative regulator of the Hedgehog (Hh) pathway playing a significant role in restricting the trafficking and function of glioma-related (Gli) proteins, acts as an upstream switch by facilitating CP110 phosphorylation by CDK2, promoting intranuclear Cdt1 degradation and excluding prereplication complex (pre-RC) components from chromosomes, independent of its canonical function in the Hh pathway. We found that Sufu localizes to both the centrosome and the nucleus and that knockout of Sufu induces abnormalities including centrosome amplification, increased nuclear size, multipolar spindle formation, and polyploidy. Serum stimulation promotes the elimination of Sufu from the centrosome by vesicle release at the ciliary tip and from the nucleus via protein degradation, which allows centrosome duplication and DNA replication to proceed. Collectively, this work reveals a mechanism through which Sufu negatively regulates the G1-S transition.

14-3-3γ prevents centrosome duplication by inhibiting NPM1 function.

14-3-3 proteins bind to ligands via phospho-serine containing consensus motifs. However, the molecular mechanisms underlying complex formation and dissociation between 14-3-3 proteins and their ligands remain unclear. We identified two conserved acidic residues in the 14-3-3 peptide-binding pocket (D129 and E136) that potentially regulate complex formation and dissociation. Altering these residues to alanine led to opposing effects on centrosome duplication. D129A inhibited centrosome duplication, whereas E136A stimulated centrosome amplification. These results were due to the differing abilities of these mutant proteins to form a complex with NPM1. Inhibiting complex formation between NPM1 and 14-3-3γ led to an increase in centrosome duplication and over-rode the ability of D129A to inhibit centrosome duplication. We identify a novel role of 14-3-3γ in regulating centrosome licensing and a novel mechanism underlying the formation and dissociation of 14-3-3 ligand complexes dictated by conserved residues in the 14-3-3 family.

Dhh1 is a member of the SESA network.

The correct separation of chromosomes during mitosis is necessary to prevent genetic instability and aneuploidy, which are responsible for cancer and other diseases, and it depends on proper centrosome duplication. In a recent study, we found that Smy2 can suppress the essential role of Mps2 in the insertion of yeast centrosome into the nuclear membrane by interacting with Eap1, Scp160, and Asc1 and designated this network as SESA (Smy2, Eap1, Scp160, Asc1). Detailed analysis showed that the SESA network is part of a mechanism which regulates translation of POM34 mRNA. Thus, SESA is a system that suppresses spindle pole body duplication defects by repressing the translation of POM34 mRNA. In this study, we performed a genome-wide screening in order to identify new members of the SESA network and confirmed Dhh1 as a putative member. Dhh1 is a cytoplasmic DEAD-box helicase known to regulate translation. Therefore, we hypothesized that Dhh1 is responsible for the highly selective inhibition of POM34 mRNA by SESA.

Transient window of resilience during early development minimizes teratogenic effects of heat in zebrafish embryos.

BACKGROUND: Transient heat shock during early development is an established experimental paradigm for doubling the genome of the zebrafish zygote, which has practical applications in expedited identification of recessive mutations in genetic screens. Despite the simplicity of the strategy and the genetic tractability of zebrafish, heat shock has not been used for genome doubling since the proof-of-principle experiments done in the 1980s. This is because of poor survival of embryos that ensue from transient heat shocks and gross developmental abnormalities in the few survivors, which is incompatible with phenotype driven screens. RESULTS: We show that heat shocks during early zebrafish development uncouple the second cycle of DNA and centrosome duplication. Interestingly, the developmental time of the heat shock that triggers the dissociation between DNA and centrosome duplication cycles significantly affect the potential of embryos to survive and attain normal morphology. The potential to develop normally after a heat shock alters in a developmental time span of 2 min in zebrafish embryos, a phenomenon that has not been reported in any species. CONCLUSIONS: The existence of heat resilient developmental windows and reduced heat teratogenicity during these windows could be an effective step forward in practical application of transient heat for experimental manipulation of ploidy in zebrafish. More broadly, heat resilience before zygotic genome activation suggests that metazoan embryos may possess innate protective features against heat beyond the canonical heat shock response. Developmental Dynamics 247:992-1004, 2018. © 2018 Wiley Periodicals, Inc.

Therapeutic potential of targeting polo-like kinase 4.

Polo-like kinase 4 (PLK4), a highly conserved serine/threonine kinase, masterfully regulates centriole duplication in a spatiotemporal manner to ensure the fidelity of centrosome duplication and proper mitosis. Abnormal expression of PLK4 contributes to genomic instability and associates with a poor prognosis in cancer. Inhibition of PLK4 is demonstrated to exhibit significant efficacy against various types of human cancers, further highlighting its potential as a promising therapeutic target for cancer treatment. As such, numerous small-molecule inhibitors with distinct chemical scaffolds targeting PLK4 have been extensively investigated for the treatment of different human cancers, with several undergoing clinical evaluation (e.g., CFI-400945). Here, we review the structure, distribution, and biological functions of PLK4, encapsulate its intricate regulatory mechanisms of expression, and highlighting its multifaceted roles in cancer development and metastasis. Moreover, the recent advancements of PLK4 inhibitors in patent or literature are summarized, and their therapeutic potential as monotherapies or combination therapies with other anticancer agents are also discussed.

Centrosome Clustering & Chemotherapy.

Centrosome abnormalities are the hallmark of cancer. How it affects tumorigenesis is still a mystery. However, the presence of more than two centrosomes at the onset of mitosis often leads to chromosomal instability and subsequent tumorigenesis. Unlike normal cells that undergo repair or apoptosis in response to this instability, cancer cells learn to cope with supernumerary centrosomes through various mechanisms and survive. Centrosome clustering is the most prevalent mechanism, allowing the cancer cells to form two daughter cells through a pseudo-bipolar spindle. Since healthy cells are devoid of the mechanisms involved in clustering, the de-clustering of centrosomes can be considered a promising approach to selectively eliminate cells with extra centrosomes. Several proteins such as PARP, KIFC1, Hsp70, Cortical actin, APC/C-CDH1 complex and Eg5 have been discussed in this review which participate in centrosome clustering, and the inhibition of these proteins can facilitate in impeding tumor growth specifically by declustering centrosomes. In this review, we also present the role of the centrosome in the cell cycle, centrosome amplification, clustering mechanism and reported centrosome de-clustering agents to present the current state of work in the field.

Disruption of desmosome function leads to increased centrosome clustering in 14-3-3γ-knockout cells with supernumerary centrosomes.

14-3-3 proteins are conserved, dimeric, acidic proteins that regulate multiple cellular pathways. Loss of either 14-3-3ε or 14-3-3γ leads to centrosome amplification. However, we find that while the knockout of 14-3-3ε leads to multipolar mitoses, the knockout of 14-3-3γ results in centrosome clustering and pseudo-bipolar mitoses. 14-3-3γ knockouts demonstrate compromised desmosome function and a decrease in keratin levels, leading to decreased cell stiffness and an increase in centrosome clustering. Restoration of desmosome function increased multipolar mitoses, whereas knockdown of either plakoglobin or keratin 5 led to decreased cell stiffness and increased pseudo-bipolar mitoses. These results suggest that the ability of the desmosome to anchor keratin filaments maintains cell stiffness, thus inhibiting centrosome clustering, and that phenotypes observed upon 14-3-3 loss reflect the dysregulation of multiple pathways.

ATAD5 suppresses centrosome over-duplication by regulating UAF1 and ID1.

Centrosomes are the primary microtubule-organizing centers that are important for mitotic spindle assembly. Centrosome amplification is commonly observed in human cancer cells and contributes to genomic instability. However, it is not clear how centrosome duplication is dysregulated in cancer cells. Here, we report that ATAD5, a replisome protein that unloads PCNA from chromatin as a replication factor C-like complex (RLC), plays an important role in regulating centrosome duplication. ATAD5 is present at the centrosome, specifically at the base of the mother and daughter centrioles that undergo duplication. UAF1, which interacts with ATAD5 and regulates PCNA deubiquitination as a complex with ubiquitin-specific protease 1, is also localized at the centrosome. Depletion of ATAD5 or UAF1 increases cells with over-duplicated centrosome whereas ATAD5 overexpression reduces such cells. Consistently, the proportion of cells showing the multipolar mode of chromosome segregation is increased among ATAD5-depleted cells. The localization and function of ATAD5 at the centrosomes do not require other RLC subunits. UAF1 interacts and co-localizes with ID1, a protein that increases centrosome amplification upon overexpression. ATAD5 depletion reduces interactions between UAF1 and ID1 and increases ID1 signal at the centrosome, providing a mechanistic framework for understanding the role of ATAD5 in centrosome duplication.

CCDC84 Acetylation Oscillation Regulates Centrosome Duplication by Modulating HsSAS-6 Degradation.

In animal cells, centriole number is strictly controlled in order to guarantee faithful cell division and genetic stability, but the mechanism by which the accuracy of centrosome duplication is maintained is not fully understood. Here, we show that CCDC84 constrains centriole number by modulating APC/CCdh1-mediated HsSAS-6 degradation. More importantly, CCDC84 acetylation oscillates throughout the cell cycle, and the acetylation state of CCDC84 at lysine 31 is regulated by the deacetylase SIRT1 and the acetyltransferase NAT10. Deacetylated CCDC84 is responsible for its centrosome targeting, and acetylated CCDC84 promotes HsSAS-6 ubiquitination by enhancing the binding affinity of HsSAS-6 for Cdh1. Our findings shed new light on the function of (de)acetylation in centriole number regulation as well as refine the established centrosome duplication model.

PLK4 phosphorylation of CP110 is required for efficient centriole assembly.

Centrioles are assembled during S phase and segregated into 2 daughter cells at the end of mitosis. The initiation of centriole assembly is regulated by polo-like kinase 4 (PLK4), the major serine/threonine kinase in centrioles. Despite its importance in centriole duplication, only a few substrates have been identified, and the detailed mechanism of PLK4 has not been fully elucidated. CP110 is a coiled-coil protein that plays roles in centriolar length control and ciliogenesis in mammals. Here, we revealed that PLK4 specifically phosphorylates CP110 at the S98 position. The phospho-resistant CP110 mutant inhibited centriole assembly, whereas the phospho-mimetic CP110 mutant induced centriole assembly, even in PLK4-limited conditions. This finding implies that PLK4 phosphorylation of CP110 is an essential step for centriole assembly. The phospho-mimetic form of CP110 augmented the centrosomal SAS6 level. Based on these results, we propose that the phosphorylated CP110 may be involved in the stabilization of cartwheel SAS6 during centriole assembly.

The Tumor Suppressor Cdkn3 Is Required for Maintaining the Proper Number of Centrosomes by Regulating the Centrosomal Stability of Mps1.

Supernumerary centrosomes promote the assembly of abnormal spindles in many human cancers. The observation that modest changes in the centrosomal levels of Mps1 kinase can cause centrosome overduplication in human cells suggests the existence of a regulatory system that may tightly control its centrosomal stability. Here, we show that Cdkn3, a Cdk-associated phosphatase, prevents Mps1-mediated centrosome overduplication. We identify Cdkn3 as a direct binding partner of Mps1. The interaction between Mps1 and Cdkn3 is required for Mps1 to recruit Cdkn3 to centrosomes. Subsequently, Mps1-bound Cdkn3 forms a regulatory system that controls the centrosomal levels of Mps1 through proteasome-mediated degradation and thereby prevents Mps1-mediated centrosome overduplication. Conversely, knockdown of Cdkn3 stabilizes Mps1 at centrosomes, which promotes centrosome overduplication. We suggest that Mps1 and Cdkn3 form a self-regulated feedback loop at centrosomes to tightly control the centrosomal levels of Mps1, which prevents centrosome overduplication in human cells.

Constitutively active NDR1-PIF kinase functions independent of MST1 and hMOB1 signalling.

The human MST1/hMOB1/NDR1 tumour suppressor cascade regulates important cellular processes, such as centrosome duplication. hMOB1/NDR1 complex formation appears to be essential for NDR1 activation by autophosphorylation on Ser281 and hydrophobic motif (HM) phosphorylation at Thr444 by MST1. To dissect these mechanistic relationships in MST1/hMOB1/NDR signalling, we designed NDR1 variants carrying modifications that mimic HM phosphorylation and/or abolish hMOB1/NDR1 interactions. Significantly, the analyses of these variants revealed that NDR1-PIF, an NDR1 variant containing the PRK2 hydrophobic motif, remains hyperactive independent of hMOB1/NDR1-PIF complex formation. In contrast, as reported for the T444A phospho-acceptor mutant, NDR1 versions carrying single phospho-mimicking mutations at the HM phosphorylation site, namely T444D or T444E, do not display increased kinase activities. Collectively, these observations suggest that in cells Thr444 phosphorylation by MST1 depends on the hMOB1/NDR1 association, while Ser281 autophosphorylation of NDR1 can occur independently. By testing centrosome-targeted NDR1 variants in NDR1- or MST1-depleted cells, we further observed that centrosome-enriched NDR1-PIF requires neither hMOB1 binding nor MST1 signalling to function in centrosome overduplication. Taken together, our biochemical and cell biological characterisation of NDR1 versions provides novel unexpected insights into the regulatory mechanisms of NDR1 and NDR1's role in centrosome duplication.

Radiation-induced mitotic cell death and glioblastoma radioresistance: a new regulating pathway controlled by integrin-linked kinase, hypoxia-inducible factor 1 alpha and survivin in U87 cells.

We have previously shown that integrin-linked kinase (ILK) regulates U87 glioblastoma cell radioresistance by modulating the main radiation-induced cell death mechanism in solid tumours, the mitotic cell death. To decipher the biological pathways involved in these mechanisms, we constructed a U87 glioblastoma cell model expressing an inducible shRNA directed against ILK (U87shILK). We then demonstrated that silencing ILK enhanced radiation-induced centrosome overduplication, leading to radiation-induced mitotic cell death. In this model, ionising radiations induce hypoxia-inducible factor 1 alpha (HIF-1α) stabilisation which is inhibited by silencing ILK. Moreover, silencing HIF-1α in U87 cells reduced the surviving fraction after 2 Gy irradiation by increasing cell sensitivity to radiation-induced mitotic cell death and centrosome amplification. Because it is known that HIF-1α controls survivin expression, we then looked at the ILK silencing effect on survivin expression. We show that survivin expression is decreased in U87shILK cells. Furthermore, treating U87 cells with the specific survivin suppressor YM155 significantly increased the percentage of giant multinucleated cells, centrosomal overduplication and thus U87 cell radiosensitivity. In consequence, we decipher here a new pathway of glioma radioresistance via the regulation of radiation-induced centrosome duplication and therefore mitotic cell death by ILK, HIF-1α and survivin. This work identifies new targets in glioblastoma with the intention of radiosensitising these highly radioresistant tumours.