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Molecular and Cellular Biology Apr 2020Cep57 has been characterized as a component of a pericentriolar complex containing Cep63 and Cep152. Interestingly, Cep63 and Cep152 self-assemble into a pericentriolar...
Cep57 has been characterized as a component of a pericentriolar complex containing Cep63 and Cep152. Interestingly, Cep63 and Cep152 self-assemble into a pericentriolar cylindrical architecture, and this event is critical for the orderly recruitment of Plk4, a key regulator of centriole duplication. However, the way in which Cep57 interacts with the Cep63-Cep152 complex and contributes to the structure and function of Cep63-Cep152 self-assembly remains unknown. We demonstrate that Cep57 interacts with Cep63 through N-terminal motifs and associates with Cep152 via Cep63. Three-dimensional structured illumination microscopy (3D-SIM) analyses suggested that the Cep57-Cep63-Cep152 complex is concentrically arranged around a centriole in a Cep57-in and Cep152-out manner. Cep57 mutant cells defective in Cep63 binding exhibited improper Cep63 and Cep152 localization and impaired Sas6 recruitment for procentriole assembly, proving the significance of the Cep57-Cep63 interaction. Intriguingly, Cep63 fused to a microtubule (MT)-binding domain of Cep57 functioned in concert with Cep152 to assemble around stabilized MTs Thus, Cep57 plays a key role in architecting the Cep63-Cep152 assembly around centriolar MTs and promoting centriole biogenesis. This study may offer a platform to investigate how the organization and function of the pericentriolar architecture are altered by disease-associated mutations found in the Cep57-Cep63-Cep152 complex.
Topics: Cell Cycle Proteins; Cell Line, Tumor; Centrioles; HEK293 Cells; Humans; Microtubule-Associated Proteins; Nuclear Proteins; Protein Interaction Domains and Motifs; Protein Interaction Maps
PubMed: 32152252
DOI: 10.1128/MCB.00535-19 -
The Journal of Biological Chemistry Apr 2020Formation of a single new centriole from a pre-existing centriole is strictly controlled to maintain correct centrosome number and spindle polarity in cells. However,...
Formation of a single new centriole from a pre-existing centriole is strictly controlled to maintain correct centrosome number and spindle polarity in cells. However, the mechanisms that govern this process are incompletely understood. Here, using several human cell lines, immunofluorescence and structured illumination microscopy methods, and ubiquitination assays, we show that the E3 ubiquitin ligase F-box and WD repeat domain-containing 7 (FBXW7), a subunit of the SCF ubiquitin ligase, down-regulates spindle assembly 6 homolog (HsSAS-6), a key protein required for procentriole cartwheel assembly, and thereby regulates centriole duplication. We found that FBXW7 abrogation stabilizes HsSAS-6 and increases its recruitment to the mother centriole at multiple sites, leading to supernumerary centrioles. Ultrastructural analyses revealed that FBXW7 is broadly localized on the mother centriole and that its presence is reduced at the site where the HsSAS-6-containing procentriole is formed. This observation suggested that FBXW7 restricts procentriole assembly to a specific site to generate a single new centriole. In contrast, during HsSAS-6 overexpression, FBXW7 strongly associated with HsSAS-6 at the centriole. We also found that SCF interacts with HsSAS-6 and targets it for ubiquitin-mediated degradation. Further, we identified putative phosphodegron sites in HsSAS-6, whose substitutions rendered it insensitive to FBXW7-mediated degradation and control of centriole number. In summary, SCF targets HsSAS-6 for degradation and thereby controls centriole biogenesis by restraining HsSAS-6 recruitment to the mother centriole, a molecular mechanism that controls supernumerary centrioles/centrosomes and the maintenance of bipolar spindles.
Topics: Amino Acid Motifs; Binding Sites; Cell Cycle Proteins; Cell Line, Tumor; Centrioles; Centrosome; F-Box-WD Repeat-Containing Protein 7; G1 Phase; Gene Duplication; Humans; Protein Binding; RNA Interference; RNA, Small Interfering; S Phase; Substrate Specificity; Ubiquitin; Ubiquitination
PubMed: 32086376
DOI: 10.1074/jbc.AC119.012178 -
The Journal of Cell Biology Feb 2020During centriole duplication, a preprocentriole forms at a single site on the mother centriole through a process that includes the hierarchical recruitment of a...
During centriole duplication, a preprocentriole forms at a single site on the mother centriole through a process that includes the hierarchical recruitment of a conserved set of proteins, including the Polo-like kinase 4 (Plk4), Ana2/STIL, and the cartwheel protein Sas6. Ana2/STIL is critical for procentriole assembly, and its recruitment is controlled by the kinase activity of Plk4, but how this works remains poorly understood. A structural motif called the G-box in the centriole outer wall protein Sas4 interacts with a short region in the N terminus of Ana2/STIL. Here, we show that binding of Ana2 to the Sas4 G-box enables hyperphosphorylation of the Ana2 N terminus by Plk4. Hyperphosphorylation increases the affinity of the Ana2-G-box interaction, and, consequently, promotes the accumulation of Ana2 at the procentriole to induce daughter centriole formation.
Topics: Animals; Cell Cycle; Cell Cycle Proteins; Cell Line; Centrioles; Drosophila Proteins; Drosophila melanogaster; Intracellular Signaling Peptides and Proteins; Microtubule-Associated Proteins; Phosphorylation; Protein Binding; Protein Serine-Threonine Kinases
PubMed: 31841145
DOI: 10.1083/jcb.201905172 -
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 -
Feedback loops in the Plk4-STIL-HsSAS6 network coordinate site selection for procentriole formation.Biology Open Sep 2019Centrioles are duplicated once in every cell cycle, ensuring the bipolarity of the mitotic spindle. How the core components cooperate to achieve high fidelity in...
Centrioles are duplicated once in every cell cycle, ensuring the bipolarity of the mitotic spindle. How the core components cooperate to achieve high fidelity in centriole duplication remains poorly understood. By live-cell imaging of endogenously tagged proteins in human cells throughout the entire cell cycle, we quantitatively tracked the dynamics of the critical duplication factors: Plk4, STIL and HsSAS6. Centriolar Plk4 peaks and then starts decreasing during the late G1 phase, which coincides with the accumulation of STIL at centrioles. Shortly thereafter, the HsSAS6 level increases steeply at the procentriole assembly site. We also show that both STIL and HsSAS6 are necessary for attenuating Plk4 levels. Furthermore, our mathematical modeling and simulation suggest that the STIL-HsSAS6 complex in the cartwheel has a negative feedback effect on centriolar Plk4. Combined, these findings illustrate how the dynamic behavior of and interactions between critical duplication factors coordinate the centriole-duplication process.This article has an associated First Person interview with the first author of the paper.
PubMed: 31533936
DOI: 10.1242/bio.047175 -
Scientific Reports Sep 2019Reproductive and respiratory organs, along with brain ventricles, are lined by multiciliated epithelial cells (MCC) that generate cilia-powered fluid flows. MCC hijack...
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.
Topics: Biomarkers; Brain; Cell Cycle; Centrioles; Centrosome; Cilia; Epithelial Cells; Fluorescent Antibody Technique; Humans; Molecular Imaging; Organelles
PubMed: 31506528
DOI: 10.1038/s41598-019-49416-2 -
ELife May 2019Centrioles play critical roles in organizing the assembly of the mitotic spindle and templating the formation of primary cilia. Centriole duplication occurs once per...
Centrioles play critical roles in organizing the assembly of the mitotic spindle and templating the formation of primary cilia. Centriole duplication occurs once per cell cycle and is regulated by Polo-like kinase 4 (PLK4). Although significant progress has been made in understanding centriole composition, we have limited knowledge of how PLK4 activity controls specific steps in centriole formation. Here, we show that PLK4 phosphorylates its centriole substrate STIL on a conserved site, S428, to promote STIL binding to CPAP. This phospho-dependent binding interaction is conserved in and facilitates the stable incorporation of both STIL and CPAP into the centriole. We propose that procentriole assembly requires PLK4 to phosphorylate STIL in two different regions: phosphorylation of residues in the STAN motif allow STIL to bind SAS6 and initiate cartwheel assembly, while phosphorylation of S428 promotes the binding of STIL to CPAP, linking the cartwheel to microtubules of the centriole wall.
Topics: Animals; Centrioles; Drosophila; Drosophila Proteins; Microtubule-Associated Proteins; Microtubules; Nuclear Proteins; Organelle Biogenesis; Phosphorylation; Protein Binding; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases
PubMed: 31115335
DOI: 10.7554/eLife.46054 -
Nature Communications Apr 2019During centriole duplication, a single daughter centriole is formed next to the mother centriole. The molecular mechanism that determines a single duplication site...
During centriole duplication, a single daughter centriole is formed next to the mother centriole. The molecular mechanism that determines a single duplication site remains a long-standing question. Here, we show that intrinsic self-organization of Plk4 is implicated in symmetry breaking in the process of centriole duplication. We demonstrate that Plk4 has an ability to phase-separate into condensates via an intrinsically disordered linker and that the condensation properties of Plk4 are regulated by autophosphorylation. Consistently, the dissociation dynamics of centriolar Plk4 are controlled by autophosphorylation. We further found that autophosphorylated Plk4 is already distributed as a single focus around the mother centriole before the initiation of procentriole formation, and is subsequently targeted for STIL-HsSAS6 loading. Perturbation of Plk4 self-organization affects the asymmetry of centriolar Plk4 distribution and proper centriole duplication. Overall, we propose that the spatial pattern formation of Plk4 is a determinant of a single duplication site per mother centriole.
Topics: Cell Cycle; Cell Cycle Proteins; Centrioles; HeLa Cells; Humans; Intracellular Signaling Peptides and Proteins; Intravital Microscopy; Microscopy, Confocal; Phosphorylation; Protein Aggregates; Protein Domains; Protein Serine-Threonine Kinases; Pyrimidines; RNA, Small Interfering; Recombinant Proteins; Sulfones
PubMed: 31000710
DOI: 10.1038/s41467-019-09847-x -
EMBO Reports Apr 2019Mammalian epithelial cells use a pair of parental centrioles and numerous deuterosomes as platforms for efficient basal body production during multiciliogenesis. How...
Mammalian epithelial cells use a pair of parental centrioles and numerous deuterosomes as platforms for efficient basal body production during multiciliogenesis. How deuterosomes form and function, however, remain controversial. They are proposed to arise either spontaneously for massive centriole biogenesis or in a daughter centriole-dependent manner as shuttles to carry away procentrioles assembled at the centriole. Here, we show that both parental centrioles are dispensable for deuterosome formation. In both mouse tracheal epithelial and ependymal cells (mTECs and mEPCs), discrete deuterosomes in the cytoplasm are initially procentriole-free. They emerge at widely dispersed positions in the cytoplasm and then enlarge, concomitant with their increased ability to form procentrioles. More importantly, deuterosomes still form efficiently in mEPCs whose daughter centriole or even both parental centrioles are eliminated through shRNA-mediated depletion or drug inhibition of Plk4, a kinase essential to centriole biogenesis in both cycling cells and multiciliated cells. Therefore, deuterosomes can be assembled autonomously to mediate centriole amplification in multiciliated cells.
PubMed: 30833343
DOI: 10.15252/embr.201846735 -
ELife Feb 2019Centriole is an essential structure with multiple functions in cellular processes. Centriole biogenesis and homeostasis is tightly regulated. Using electron...
Centriole is an essential structure with multiple functions in cellular processes. Centriole biogenesis and homeostasis is tightly regulated. Using electron cryo-tomography (cryoET) we present the structure of procentrioles from . We identified a set of non-tubulin components attached to the triplet microtubule (MT), many are at the junctions of tubules likely to reinforce the triplet. We describe structure of the A-C linker that bridges neighboring triplets. The structure infers that POC1 is likely an integral component of A-C linker. Its conserved WD40 β-propeller domain provides attachment sites for other A-C linker components. The twist of A-C linker results in an iris diaphragm-like motion of the triplets in the longitudinal direction of procentriole. Finally, we identified two assembly intermediates at the growing ends of procentriole allowing us to propose a model for the procentriole assembly. Our results provide a comprehensive structural framework for understanding the molecular mechanisms underpinning procentriole biogenesis and assembly.
Topics: Cell Cycle; Cell Cycle Proteins; Centrioles; Chlamydomonas reinhardtii; Cryoelectron Microscopy; Electron Microscope Tomography; Microtubules
PubMed: 30741631
DOI: 10.7554/eLife.43434