-
Phase separation of Polo-like kinase 4 by autoactivation and clustering drives centriole biogenesis.Nature Communications Oct 2019Tight control of centriole duplication is critical for normal chromosome segregation and the maintenance of genomic stability. Polo-like kinase 4 (Plk4) is a key...
Tight control of centriole duplication is critical for normal chromosome segregation and the maintenance of genomic stability. Polo-like kinase 4 (Plk4) is a key regulator of centriole biogenesis. How Plk4 dynamically promotes its symmetry-breaking relocalization and achieves its procentriole-assembly state remains unknown. Here we show that Plk4 is a unique kinase that utilizes its autophosphorylated noncatalytic cryptic polo-box (CPB) to phase separate and generate a nanoscale spherical condensate. Analyses of the crystal structure of a phospho-mimicking, condensation-proficient CPB mutant reveal that a disordered loop at the CPB PB2-tip region is critically required for Plk4 to generate condensates and induce procentriole assembly. CPB phosphorylation also promotes Plk4's dissociation from the Cep152 tether while binding to downstream STIL, thus allowing Plk4 condensate to serve as an assembling body for centriole biogenesis. This study uncovers the mechanism underlying Plk4 activation and may offer strategies for anti-Plk4 intervention against genomic instability and cancer.
Topics: Animals; Cell Cycle Proteins; Cell Line, Tumor; Centrioles; HEK293 Cells; Humans; Imaging, Three-Dimensional; Intracellular Signaling Peptides and Proteins; Microscopy, Confocal; Organelle Biogenesis; Phosphorylation; Protein Binding; Protein Serine-Threonine Kinases; Sf9 Cells; Spodoptera
PubMed: 31672968
DOI: 10.1038/s41467-019-12619-2 -
Nature Communications Mar 2018Centrosomes are the major microtubule organising centres of animal cells. Deregulation in their number occurs in cancer and was shown to trigger tumorigenesis in mice....
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.
Topics: Automation; Breast Neoplasms; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Centrioles; Centrosome; Chromosomes; Humans; Microscopy, Electron, Transmission; Microtubule-Associated Proteins; Microtubules; Mitosis; Neoplasms; Ploidies; Tumor Suppressor Protein p53
PubMed: 29593297
DOI: 10.1038/s41467-018-03641-x -
PLoS Biology Sep 2022In metazoa, cilia assembly is a cellular process that starts with centriole to basal body maturation, migration to the cell surface, and docking to the plasma membrane....
In metazoa, cilia assembly is a cellular process that starts with centriole to basal body maturation, migration to the cell surface, and docking to the plasma membrane. Basal body docking involves the interaction of both the distal end of the basal body and the transition fibers/distal appendages, with the plasma membrane. Mutations in numerous genes involved in basal body docking and transition zone assembly are associated with the most severe ciliopathies, highlighting the importance of these events in cilium biogenesis. In this context, the ciliate Paramecium has been widely used as a model system to study basal body and cilia assembly. However, despite the evolutionary conservation of cilia assembly events across phyla, whether the same molecular players are functionally conserved, is not fully known. Here, we demonstrated that CEP90, FOPNL, and OFD1 are evolutionary conserved proteins crucial for ciliogenesis. Using ultrastructure expansion microscopy, we unveiled that these proteins localize at the distal end of both centrioles/basal bodies in Paramecium and mammalian cells. Moreover, we found that these proteins are recruited early during centriole duplication on the external surface of the procentriole. Functional analysis performed both in Paramecium and mammalian cells demonstrate the requirement of these proteins for distal appendage assembly and basal body docking. Finally, we show that mammalian centrioles require another component, Moonraker (MNR), to recruit OFD1, FOPNL, and CEP90, which will then recruit the distal appendage proteins CEP83, CEP89, and CEP164. Altogether, we propose that this OFD1, FOPNL, and CEP90 functional module is required to determine in mammalian cells the future position of distal appendage proteins.
Topics: Animals; Cell Membrane; Centrioles; Cilia; Mammals; Paramecium
PubMed: 36070319
DOI: 10.1371/journal.pbio.3001782 -
Cell Biology International Dec 2010In preparation for mitosis, the centrosome doubles once and only once to provide the two poles of the mitotic spindle. The presence of more than two centrosomes... (Review)
Review
In preparation for mitosis, the centrosome doubles once and only once to provide the two poles of the mitotic spindle. The presence of more than two centrosomes increases the chances that mitosis will be multipolar, and chromosomes will be distributed unequally. Since the number of mother-daughter centriole pairs determines the number of centrosomes, it is important that only one daughter centriole is assembled at, but slightly separated from, the proximal end of each mother centriole. This numerical and spatial specificity has led to the belief that a 'template' on the mother centriole provides a unique site for procentriole assembly. We review observations that are leading to the demise of this intuitively attractive idea. In its place, we are left with the notion that pericentriolar material at the wall of the mother centriole provides a local environment that promotes the assembly of a macromolecular complex that seeds the daughter centriole. Even though the system normally behaves in a digital fashion to go from zero to just one daughter centriole per mother, this behaviour appears to be based in the precise analogue control of multiple proteins, their activities, and the structure provided by the mother centriole.
Topics: Animals; Cell Physiological Phenomena; Centrioles; Centrosome; Humans; Mitosis; Models, Biological; Spindle Apparatus
PubMed: 21067522
DOI: 10.1042/CBI20100612 -
Journal of Cell Science Jul 2016Centriole duplication is a tightly ordered process during which procentrioles are assembled in G1-S and elongate during S and G2. Here, we show that human CEP295...
Centriole duplication is a tightly ordered process during which procentrioles are assembled in G1-S and elongate during S and G2. Here, we show that human CEP295 (Drosophila Ana1) is not essential for initial cartwheel assembly, but is required to build distal half centrioles during S and G2. Using super-resolution and immunogold electron microscopy, we demonstrate that CEP295 is recruited to the proximal end of procentrioles in early S phase, when it is also localized at the centriolar microtubule wall that surrounds the human SAS6 cartwheel hub. Interestingly, depletion of CEP295 not only inhibits the recruitments of POC5 and POC1B to the distal half centrioles in G2, resulting in shorter centrioles, it also blocks the post-translational modification of centriolar microtubules (e.g. acetylation and glutamylation). Importantly, our results indicate that CEP295 directly interacts with microtubules, and that excess CEP295 could induce the assembly of overly long centrioles. Furthermore, exogenous expression of the N-terminal domain of CEP295 exerts a dominant-negative effect on centriole elongation. Collectively, these findings suggest that CEP295 is essential for building the distal half centrioles and for post-translational modification of centriolar microtubules.
Topics: Animals; Carrier Proteins; Cell Cycle; Cell Cycle Proteins; Centrioles; Centrosome; Gene Expression Regulation, Neoplastic; HeLa Cells; Humans; Microscopy, Electron; Microtubule-Associated Proteins; Microtubules; Protein Binding; Protein Processing, Post-Translational
PubMed: 27185865
DOI: 10.1242/jcs.186338 -
Current Biology : CB May 2016Centrioles are essential for the assembly of both centrosomes and cilia. Centriole biogenesis occurs once and only once per cell cycle and is temporally coordinated with...
Centrioles are essential for the assembly of both centrosomes and cilia. Centriole biogenesis occurs once and only once per cell cycle and is temporally coordinated with cell-cycle progression, ensuring the formation of the right number of centrioles at the right time. The formation of new daughter centrioles is guided by a pre-existing, mother centriole. The proximity between mother and daughter centrioles was proposed to restrict new centriole formation until they separate beyond a critical distance. Paradoxically, mother and daughter centrioles overcome this distance in early mitosis, at a time when triggers for centriole biogenesis Polo-like kinase 4 (PLK4) and its substrate STIL are abundant. Here we show that in mitosis, the mitotic kinase CDK1-CyclinB binds STIL and prevents formation of the PLK4-STIL complex and STIL phosphorylation by PLK4, thus inhibiting untimely onset of centriole biogenesis. After CDK1-CyclinB inactivation upon mitotic exit, PLK4 can bind and phosphorylate STIL in G1, allowing pro-centriole assembly in the subsequent S phase. Our work shows that complementary mechanisms, such as mother-daughter centriole proximity and CDK1-CyclinB interaction with centriolar components, ensure that centriole biogenesis occurs once and only once per cell cycle, raising parallels to the cell-cycle regulation of DNA replication and centromere formation.
Topics: Animals; CDC2 Protein Kinase; Cell Cycle; Centrioles; Cloning, Molecular; Gene Expression Regulation, Enzymologic; HeLa Cells; Humans; Intracellular Signaling Peptides and Proteins; Protein Serine-Threonine Kinases; Xenopus
PubMed: 27112295
DOI: 10.1016/j.cub.2016.03.055 -
Proceedings of the National Academy of... Nov 1991Heterologous centrosomes from diversed species including humans promote egg cleavage when injected into metaphase-arrested Xenopus eggs. We have recently isolated...
Heterologous centrosomes from diversed species including humans promote egg cleavage when injected into metaphase-arrested Xenopus eggs. We have recently isolated centrosomes from calf thymocytes and shown that they were unable to induce egg cleavage, an inability that was apparently correlated with the peculiar structure of these centrosomes rather than with a lack of microtubule-nucleating activity: the two centrioles were associated in a colinear orientation by their proximal ends. To promote cleavage, a heterologous centrosome probably is required to duplicate, although this has not yet been demonstrated. Therefore, we designed an in vitro assay that would enable us to directly observe the duplication process. We show that competent centrosomes from KE37 cells synchronized in G1 phase initiate procentriole budding in interphasic extracts from Xenopus eggs in the absence of protein synthesis, whereas calf thymocyte centrosomes do not. Since calf thymocyte centrosomes do not support parthenogenesis, the present results suggest that duplication of the foreign centrosome is required for centrosome-induced parthenogenesis. Furthermore, procentriole budding takes place in the absence of protein synthesis in egg extracts arrested in S phase. This in vitro assay should contribute to the identification of molecular mechanisms involved in the initiation of centrosome duplication.
Topics: Animals; Cell Cycle; Cell Line; Cell-Free System; Centrioles; Female; Fluorescent Antibody Technique; Interphase; Kinetics; Microscopy, Electron; Oocytes; Parthenogenesis; Xenopus laevis
PubMed: 1946461
DOI: 10.1073/pnas.88.22.9929 -
Nature Structural & Molecular Biology Aug 2014Polo-like kinase 4 (Plk4) is a key regulator of centriole duplication, an event critical for the maintenance of genomic integrity. We show that Plk4 relocalizes from the...
Polo-like kinase 4 (Plk4) is a key regulator of centriole duplication, an event critical for the maintenance of genomic integrity. We show that Plk4 relocalizes from the inner Cep192 ring to the outer Cep152 ring as newly recruited Cep152 assembles around the Cep192-encircled daughter centriole. Crystal-structure analyses revealed that Cep192- and Cep152-derived peptides bind the cryptic polo box (CPB) of Plk4 in opposite orientations and in a mutually exclusive manner. The Cep152 peptide bound to the CPB markedly better than did the Cep192 peptide and effectively 'snatched' the CPB away from a preformed CPB-Cep192 peptide complex. A cancer-associated Cep152 mutation impairing the Plk4 interaction induced defects in procentriole assembly and chromosome segregation. Thus, Plk4 is intricately regulated in time and space through ordered interactions with two distinct scaffolds, Cep192 and Cep152, and a failure in this process may lead to human cancer.
Topics: Amino Acid Sequence; Cell Cycle Proteins; Centrioles; Chromosomal Proteins, Non-Histone; Crystallography, X-Ray; HEK293 Cells; HeLa Cells; Humans; Hydrogen Bonding; Models, Molecular; Molecular Sequence Data; Mutation, Missense; Neoplasms; Protein Binding; Protein Interaction Domains and Motifs; Protein Serine-Threonine Kinases; Protein Structure, Quaternary; Protein Structure, Secondary
PubMed: 24997597
DOI: 10.1038/nsmb.2846 -
Molecular Biology of the Cell Oct 2021Control of centrosome assembly is critical for cell division, intracellular trafficking, and cilia. Regulation of centrosome number occurs through the precise...
Control of centrosome assembly is critical for cell division, intracellular trafficking, and cilia. Regulation of centrosome number occurs through the precise duplication of centrioles that reside in centrosomes. Here we explored transcriptional control of centriole assembly and find that the RNA splicing factor SON is specifically required for completing procentriole assembly. Whole genome mRNA sequencing identified genes whose splicing and expression are affected by the reduction of SON, with an enrichment in genes involved in the microtubule (MT) cytoskeleton, centrosome, and centriolar satellites. SON is required for the proper splicing and expression of , which encodes a major centriolar satellite protein and is required to organize the trafficking and MT network around the centrosomes. This study highlights the importance of the distinct MT trafficking network that is intimately associated with nascent centrioles and is responsible for procentriole development and efficient ciliogenesis.
Topics: Cell Cycle Proteins; Cell Line; Centrioles; Centrosome; Cilia; Cytoskeletal Proteins; DNA-Binding Proteins; Gene Expression; Humans; Microtubules; Minor Histocompatibility Antigens; Protein Transport; RNA; RNA Splicing Factors
PubMed: 34406792
DOI: 10.1091/mbc.E21-06-0305 -
Current Biology : CB Jul 2013Centrosomes organize microtubule (MT) arrays and are comprised of centrioles surrounded by ordered pericentriolar proteins. Centrioles are barrel-shaped structures...
Centrosomes organize microtubule (MT) arrays and are comprised of centrioles surrounded by ordered pericentriolar proteins. Centrioles are barrel-shaped structures composed of MTs, and as basal bodies they template the formation of cilia/flagella. Defects in centriole assembly can lead to ciliopathies and genome instability. The assembly of procentrioles requires a set of conserved proteins. It is initiated at the G1-to-S transition by PLK4 and CEP152, which help recruit SASS6 and STIL to the vicinity of the mother centriole to organize the cartwheel. Subsequently, CPAP promotes centriolar MT assembly and elongation in G2. While centriole integrity is maintained by CEP135 and POC1 through MT stabilization, centriole elongation requires POC5 and is restricted by CP110 and CEP97. How strict control of centriole length is achieved remains unclear. Here, we show that CEP120 and SPICE1 are required to localize CEP135 (but not SASS6, STIL, or CPAP) to procentrioles. CEP120 associates with SPICE1 and CPAP, and depletion of any of these proteins results in short procentrioles. Furthermore, CEP120 or CPAP overexpression results in excessive centriole elongation, a process dependent on CEP120, SPICE1, and CPAP. Our findings identify a shared function for these proteins in centriole length control.
Topics: Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Centrioles; HeLa Cells; Humans; Microscopy, Immunoelectron; Microtubule-Associated Proteins; Microtubules
PubMed: 23810536
DOI: 10.1016/j.cub.2013.06.002