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IScience Oct 2018Centrioles, the cores of centrosomes and cilia, duplicate every cell cycle to ensure their faithful inheritance. How only a single procentriole is produced on each...
Centrioles, the cores of centrosomes and cilia, duplicate every cell cycle to ensure their faithful inheritance. How only a single procentriole is produced on each mother centriole remains enigmatic. We propose the first mechanistic biophysical model for procentriole initiation which posits that interactions between kinase PLK4 and its activator-substrate STIL are central for procentriole initiation. The model recapitulates the transition from a uniform "ring" of PLK4 surrounding the mother centriole to a single PLK4 "spot" that initiates procentriole assembly. This symmetry breaking requires autocatalytic activation of PLK4 and enhanced centriolar anchoring of PLK4 by phosphorylated STIL. We find that in situ degradation of active PLK4 cannot break symmetry. The model predicts that competition between transient PLK4 activity maxima for PLK4-STIL complexes destabilizes the PLK4 ring and produces instead a single PLK4 spot. Weakening of competition by overexpression of PLK4 and STIL causes progressive addition of supernumerary procentrioles, as observed experimentally.
PubMed: 30340068
DOI: 10.1016/j.isci.2018.10.003 -
Biology Open Aug 2018The centrosome is the organizing center of microtubules in the cell, the basis for the origin of cilia and flagella and a site for the concentration of a regulatory...
The centrosome is the organizing center of microtubules in the cell, the basis for the origin of cilia and flagella and a site for the concentration of a regulatory proteins multitude. The centrosome comprises two centrioles surrounded by pericentriolar material. Centrioles in the cells of different organisms can contain nine triplets, doublets or singlets of microtubules. Here, we show that in somatic cells of male wasp larvae , centrioles do not contain microtubules and are composed of nine electron-dense prongs, which together form a cogwheel structure. These microtubule-free centrioles can be the platform for procentriole formation and form microtubule-free cilia-like structures. In nymph and imago cells centrioles have a microtubule triplet structure. Our study describes how centriole structure differs in a development-stage-dependent and a cell-type-dependent manner. The discovery of a centriole without microtubules casts a new light on the centriole formation process and the evolution of this organelle.
PubMed: 29997243
DOI: 10.1242/bio.036012 -
Cell Reports Jun 2018The number of centrioles is tightly controlled to ensure bipolar spindle assembly, which is a prerequisite to maintain genome integrity. However, our understanding of...
The number of centrioles is tightly controlled to ensure bipolar spindle assembly, which is a prerequisite to maintain genome integrity. However, our understanding of the fundamental principle that governs the formation of a single procentriole per parental centriole is incomplete. Here, we show that the local restriction of Plk4, a master regulator of the procentriole formation, is achieved by a bimodal interaction of STIL with Plk4. We demonstrate that the conserved short coiled-coil region of STIL binds to and protects Plk4 from protein degradation at the site of procentriole formation. On the other hand, the conserved C-terminal region of STIL named truncated in microcephaly (TIM) domain promotes autophosphorylation and degradation of adjacent Plk4 by the direct interaction. Thus, we propose that positive and negative regulation based on the bimodal binding of Plk4 and STIL ensures the formation of a single procentriole per parental centriole.
Topics: 3' Untranslated Regions; Amino Acid Motifs; Animals; Cell Line; Centrioles; HEK293 Cells; Humans; Intracellular Signaling Peptides and Proteins; Phosphorylation; Protein Binding; Protein Domains; Protein Serine-Threonine Kinases; RNA Interference; RNA, Small Interfering; Sequence Alignment
PubMed: 29898389
DOI: 10.1016/j.celrep.2018.05.030 -
PLoS Biology Apr 2018The centrosome is a non-membrane-bound cellular compartment consisting of 2 centrioles surrounded by a protein coat termed the pericentriolar material (PCM). Centrioles...
The centrosome is a non-membrane-bound cellular compartment consisting of 2 centrioles surrounded by a protein coat termed the pericentriolar material (PCM). Centrioles generally remain physically associated together (a phenomenon called centrosome cohesion), yet how this occurs in the absence of a bounding lipid membrane is unclear. One model posits that pericentriolar fibres formed from rootletin protein directly link centrioles, yet little is known about the structure, biophysical properties, or assembly kinetics of such fibres. Here, I combine live-cell imaging of endogenously tagged rootletin with cell fusion and find previously unrecognised plasticity in centrosome cohesion. Rootletin forms large, diffusionally stable bifurcating fibres, which amass slowly on mature centrioles over many hours from anaphase. Nascent centrioles (procentrioles), in contrast, do not form roots and must be licensed to do so through polo-like kinase 1 (PLK1) activity. Transient separation of roots accompanies centriolar repositioning during the interphase, suggesting that centrioles organize as independent units, each containing discrete roots. Indeed, forced induction of duplicate centriole pairs allows independent reshuffling of individual centrioles between the pairs. Therefore collectively, these findings suggest that progressively nucleated polymers mediate the dynamic association of centrioles as either 1 or 2 interphase centrosomes, with implications for the understanding of how non-membrane-bound organelles self-organise.
Topics: Anaphase; Cell Cycle Proteins; Cell Fusion; Cell Line, Tumor; Centrosome; Cytoskeletal Proteins; Epithelial Cells; Gene Expression Regulation; HeLa Cells; Humans; Interphase; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Signal Transduction; Time-Lapse Imaging; Polo-Like Kinase 1
PubMed: 29649211
DOI: 10.1371/journal.pbio.2003998 -
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 -
The Journal of Cell Biology Apr 2018Polo-like kinase 4 (Plk4) initiates an early step in centriole assembly by phosphorylating Ana2/STIL, a structural component of the procentriole. Here, we show that Plk4...
Polo-like kinase 4 (Plk4) initiates an early step in centriole assembly by phosphorylating Ana2/STIL, a structural component of the procentriole. Here, we show that Plk4 binding to the central coiled-coil (CC) of Ana2 is a conserved event involving Polo-box 3 and a previously unidentified putative CC located adjacent to the kinase domain. Ana2 is then phosphorylated along its length. Previous studies showed that Plk4 phosphorylates the C-terminal STil/ANa2 (STAN) domain of Ana2/STIL, triggering binding and recruitment of the cartwheel protein Sas6 to the procentriole assembly site. However, the physiological relevance of N-terminal phosphorylation was unknown. We found that Plk4 first phosphorylates the extreme N terminus of Ana2, which is critical for subsequent STAN domain modification. Phosphorylation of the central region then breaks the Plk4-Ana2 interaction. This phosphorylation pattern is important for centriole assembly and integrity because replacement of endogenous Ana2 with phospho-Ana2 mutants disrupts distinct steps in Ana2 function and inhibits centriole duplication.
Topics: Animals; Cell Cycle; Cell Cycle Proteins; Cell Line; Centrioles; Drosophila Proteins; Drosophila melanogaster; Microtubule-Associated Proteins; Mutation; Phosphorylation; Protein Binding; Protein Interaction Domains and Motifs; Protein Serine-Threonine Kinases; Protein Transport; Signal Transduction
PubMed: 29496738
DOI: 10.1083/jcb.201605106 -
Open Biology Dec 2017The conserved process of centriole duplication requires Plk4 kinase to recruit and promote interactions between Sas6 and Sas5/Ana2/STIL (respective nomenclature of...
The conserved process of centriole duplication requires Plk4 kinase to recruit and promote interactions between Sas6 and Sas5/Ana2/STIL (respective nomenclature of worms/flies/humans). Plk4-mediated phosphorylation of Ana2/STIL in its conserved STAN motif has been shown to promote its interaction with Sas6. However, STAN motif phosphorylation is not required for recruitment of Ana2 to the centriole. Here we show that in , Ana2 loads onto the site of procentriole formation ahead of Sas6 in a process that also requires Plk4. However, whereas Plk4 is first recruited to multiple sites around the ring of zone II at the periphery of the centriole, Ana2 is recruited to a single site in telophase before Plk4 becomes finally restricted to this same single site. When we over-ride the auto-destruction of Plk4, it remains localized to multiple sites in the outer ring of the centriole and, if catalytically active, recruits Ana2 to these sites. Thus, it is the active form of Plk4 that promotes Ana2's recruitment to the centriole. We now show that Plk4 phosphorylates Ana2 at a site other than the STAN motif, which lies in a conserved region we term the ANST (ANa2-STil) motif. Mutation of this site, S38, to a non-phosphorylatable residue prevents the procentriole loading of Ana2 and blocks centriole duplication. Thus the initiation of procentriole formation requires Plk4 to first phosphorylate a single serine residue in the ANST motif to promote Ana2's recruitment and, secondly, to phosphorylate four residues in the STAN motif enabling Ana2 to recruit Sas6. We discuss these findings in light of the multiple Plk4 phosphorylation sites on Ana2.
Topics: Animals; Cell Cycle Proteins; Cell Line; Centrioles; Drosophila Proteins; Drosophila melanogaster; Microtubule-Associated Proteins; Phosphorylation; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases
PubMed: 29263250
DOI: 10.1098/rsob.170247 -
Human microcephaly protein RTTN interacts with STIL and is required to build full-length centrioles.Nature Communications Aug 2017Mutations in many centriolar protein-encoding genes cause primary microcephaly. Using super-resolution and electron microscopy, we find that the human microcephaly...
Mutations in many centriolar protein-encoding genes cause primary microcephaly. Using super-resolution and electron microscopy, we find that the human microcephaly protein, RTTN, is recruited to the proximal end of the procentriole at early S phase, and is located at the inner luminal walls of centrioles. Further studies demonstrate that RTTN directly interacts with STIL and acts downstream of STIL-mediated centriole assembly. CRISPR/Cas9-mediated RTTN gene knockout in p53-deficient cells induce amplification of primitive procentriole bodies that lack the distal-half centriolar proteins, POC5 and POC1B. Additional analyses show that RTTN serves as an upstream effector of CEP295, which mediates the loading of POC1B and POC5 to the distal-half centrioles. Interestingly, the naturally occurring microcephaly-associated mutant, RTTN (A578P), shows a low affinity for STIL binding and blocks centriole assembly. These findings reveal that RTTN contributes to building full-length centrioles and illuminate the molecular mechanism through which the RTTN (A578P) mutation causes primary microcephaly.Mutations in many centriolar protein-encoding genes cause primary microcephaly. Here the authors show that human microcephaly protein RTTN directly interacts with STIL and acts downstream of STIL-mediated centriole assembly, contributing to building full-length centrioles.
Topics: Carrier Proteins; Cell Cycle Proteins; Centrioles; Humans; Intracellular Signaling Peptides and Proteins; Mutation; Protein Binding
PubMed: 28811500
DOI: 10.1038/s41467-017-00305-0 -
Molecular Biology of the Cell Jul 2017The decision to commit to the cell cycle is made during G1 through the concerted action of various cyclin-CDK complexes. Not only DNA replication, but also centriole...
The decision to commit to the cell cycle is made during G1 through the concerted action of various cyclin-CDK complexes. Not only DNA replication, but also centriole duplication is initiated as cells enter the S-phase. The NIMA-related kinase NEK7 is one of many factors required for proper centriole duplication, as well as for timely cell cycle progression. However, its specific roles in these events are poorly understood. In this study, we find that depletion of NEK7 inhibits progression through the G1 phase in human U2OS cells via down-regulation of various cyclins and CDKs and also inhibits the earliest stages of procentriole formation. Depletion of NEK7 also induces formation of primary cilia in human RPE1 cells, suggesting that NEK7 acts at least before the restriction point during G1. G1-arrested cells in the absence of NEK7 exhibit abnormal accumulation of the APC/C cofactor Cdh1 at the vicinity of centrioles. Furthermore, the ubiquitin ligase APC/C continuously degrades the centriolar protein STIL in these cells, thus inhibiting centriole assembly. Collectively our results demonstrate that NEK7 is involved in the timely regulation of G1 progression, S-phase entry, and procentriole formation.
Topics: Cell Cycle Proteins; Cell Division; Cells, Cultured; Centrioles; Cilia; Cyclin-Dependent Kinases; Cyclins; DNA Replication; G1 Phase; G1 Phase Cell Cycle Checkpoints; Humans; NIMA-Related Kinases; Phosphorylation; S Phase
PubMed: 28539406
DOI: 10.1091/mbc.E16-09-0643 -
Frontiers in Molecular Biosciences 2017Centrosomal P4.1-associated protein (CPAP) is a cell cycle regulated protein fundamental for centrosome assembly and centriole elongation. In humans, the region between...
Centrosomal P4.1-associated protein (CPAP) is a cell cycle regulated protein fundamental for centrosome assembly and centriole elongation. In humans, the region between residues 897-1338 of CPAP mediates interactions with other proteins and includes a homodimerization domain. CPAP mutations cause primary autosomal recessive microcephaly and Seckel syndrome. Despite of the biological/clinical relevance of CPAP, its mechanistic behavior remains unclear and its C-terminus (the G-box/TCP domain) is the only part whose structure has been solved. This situation is perhaps due in part to the challenges that represent obtaining the protein in a soluble, homogeneous state for structural studies. Our work constitutes a systematic structural analysis on multiple oligomers of , using single-particle electron microscopy (EM) of negatively stained (NS) samples. Based on image classification into clearly different regular 3D maps (putatively corresponding to dimers and tetramers) and direct observation of individual images representing other complexes of CPAP (i.e., putative flexible monomers and higher-order multimers), we report a dynamic oligomeric behavior of this protein, where different homo-oligomers coexist in variable proportions. We propose that dimerization of the putative homodimer forms a putative tetramer which could be the structural unit for the scaffold that either tethers the pericentriolar material to centrioles or promotes procentriole elongation. A coarse fitting of atomic models into the NS 3D maps at resolutions around 20 Å is performed only to complement our experimental data, allowing us to hypothesize on the oligomeric composition of the different complexes. In this way, the current EM work represents an initial step toward the structural characterization of different oligomers of CPAP, suggesting further insights to understand how this protein works, contributing to the elucidation of control mechanisms for centriole biogenesis.
PubMed: 28396859
DOI: 10.3389/fmolb.2017.00017