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The Journal of Biological Chemistry Dec 2022Cartwheel assembly is considered the first step in the initiation of procentriole biogenesis; however, the reason for persistence of the assembled human cartwheel...
Cartwheel assembly is considered the first step in the initiation of procentriole biogenesis; however, the reason for persistence of the assembled human cartwheel structure from S phase to late mitosis remains unclear. Here, we demonstrate mainly using cell synchronization, RNA interference, immunofluorescence and time-lapse-microscopy, biochemical analysis, and methods that the cartwheel persistently assembles and maintains centriole engagement and centrosome integrity during S phase to late G2 phase. Blockade of the continuous accumulation of centriolar Sas-6, a major cartwheel protein, after procentriole formation induces premature centriole disengagement and disrupts pericentriolar matrix integrity. Additionally, we determined that during mitosis, CDK1-cyclin B phosphorylates Sas-6 at T495 and S510, disrupting its binding to cartwheel component STIL and pericentriolar component Nedd1 and promoting cartwheel disassembly and centriole disengagement. Perturbation of this phosphorylation maintains the accumulation of centriolar Sas-6 and retains centriole engagement during mitotic exit, which results in the inhibition of centriole reduplication. Collectively, these data demonstrate that persistent cartwheel assembly after procentriole formation maintains centriole engagement and that this configuration is relieved through phosphorylation of Sas-6 by CDK1-cyclin B during mitosis in human cells.
Topics: Humans; CDC2 Protein Kinase; Cell Cycle Proteins; Centrioles; Centrosome; Mitosis; Phosphorylation; Proteins; Cyclin B
PubMed: 36356903
DOI: 10.1016/j.jbc.2022.102658 -
Philosophical Transactions of the Royal... Sep 2014This review discusses some of the history of the fundamental, but not fully solved problem of how the centrosome duplicates from one to only two as the cell prepares for... (Review)
Review
This review discusses some of the history of the fundamental, but not fully solved problem of how the centrosome duplicates from one to only two as the cell prepares for mitosis. We start with some of the early descriptions of the centrosome and the remarkably prescient but then controversial inferences drawn concerning its function in the cell. For more than 100 years, one of the most difficult issues for the concept of the centrosome has been to integrate observations that centrosomes appear to be important for spindle assembly in animal cells yet are not evident in higher plant cells and some animal cells. This stirred debate over the existence of centrosomes and their importance. A parallel debate concerned the role of the centrioles in organizing centrosomes. The relatively recent elucidation of bipolar spindle assembly around chromatin allows a re-examination of the role of centrioles in controlling centrosome duplication in animal cells. The problem of how centrosomes precisely double in preparation for mitosis in animal cells has now moved to the mystery of how only one procentriole is assembled at each mother centriole.
Topics: Animals; Cell Division; Centrioles; Centrosome; Models, Biological; Sea Urchins; Species Specificity; Spindle Apparatus
PubMed: 25047609
DOI: 10.1098/rstb.2013.0455 -
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 -
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 -
PloS One 2008Centrioles are microtubule-based cylindrical structures composed of nine triplet tubules and are required for the formation of the centrosome, flagella and cilia....
BACKGROUND
Centrioles are microtubule-based cylindrical structures composed of nine triplet tubules and are required for the formation of the centrosome, flagella and cilia. Despite theirs importance, centriole biogenesis is poorly understood. Centrosome duplication is initiated at the G1/S transition by the sequential recruitment of a set of conserved proteins under the control of the kinase Plk4. Subsequently, the procentriole is assembled by the polymerization of centriolar tubules via an unknown mechanism involving several tubulin paralogs.
METHODOLOGY/PRINCIPAL FINDINGS
Here, we developed a cellular assay to study centrosome duplication and procentriole stability based on its sensitivity to the microtubule-depolymerizing drug nocodazole. By using RNA interference experiments, we show that the stability of growing procentrioles is regulated by the microtubule-stabilizing protein CAP350, independently of hSAS-6 and CPAP which initiate procentriole growth. Furthermore, our analysis reveals the critical role of centriolar tubule stability for an efficient procentriole growth.
CONCLUSIONS/SIGNIFICANCE
CAP350 belongs to a new class of proteins which associate and stabilize centriolar tubules to control centriole duplication.
Topics: Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Centrioles; Centrosome; Humans; Microtubule Proteins; Microtubules; Protein Serine-Threonine Kinases; RNA Interference
PubMed: 19052644
DOI: 10.1371/journal.pone.0003855 -
Molecular Biology of the Cell Nov 2022DNA replication is normally coupled with centriole duplication in the cell cycle. Trophoblast giant cells (TGCs) of the placenta undergo endocycles resulting in...
DNA replication is normally coupled with centriole duplication in the cell cycle. Trophoblast giant cells (TGCs) of the placenta undergo endocycles resulting in polyploidy but their centriole state is not known. We used a cell culture model for TGC differentiation to examine centriole and centrosome number and properties. Before differentiation, trophoblast stem cells (TSCs) have either two centrioles before duplication or four centrioles after. We find that the average nuclear area increases approximately eight-fold over differentiation, but most TGCs do not have more than four centrioles. However, these centrioles become disengaged, acquire centrosome proteins, and can nucleate microtubules. In addition, some TGCs undergo further duplication and disengagement of centrioles, resulting in substantially higher numbers. Live imaging revealed that disengagement and separation are centriole autonomous and can occur asynchronously. Centriole amplification, when present, occurs by the standard mechanism of one centriole generating one procentriole. PLK4 inhibition blocks centriole formation in differentiating TGCs but does not affect endocycle progression. In summary, centrioles in TGC endocycles undergo disengagement and conversion to centrosomes. This increases centrosome number but to a limited extent compared with DNA reduplication.
Topics: Pregnancy; Female; Humans; Centrioles; Trophoblasts; Centrosome; Cell Cycle Proteins; Giant Cells; Polyploidy; Protein Serine-Threonine Kinases
PubMed: 36001376
DOI: 10.1091/mbc.E22-05-0182 -
Nature Communications Oct 2014Formation of one procentriole next to each pre-existing centriole is essential for centrosome duplication, robust bipolar spindle assembly and maintenance of genome...
Formation of one procentriole next to each pre-existing centriole is essential for centrosome duplication, robust bipolar spindle assembly and maintenance of genome integrity. However, the mechanisms maintaining strict control over centriole copy number are incompletely understood. Here we show that Plk4 and STIL, the key regulators of centriole formation, form a protein complex that provides a scaffold for recruiting HsSAS-6, a major component of the centriolar cartwheel, at the onset of procentriole formation. Furthermore, we demonstrate that phosphorylation of STIL by Plk4 facilitates the STIL/HsSAS-6 interaction and centriolar loading of HsSAS-6. We also provide evidence that negative feedback by centriolar STIL regulates bimodal centriolar distribution of Plk4 and seemingly restricts occurrence of procentriole formation to one site on each parental centriole. Overall, these findings suggest a mechanism whereby coordinated action of three critical factors ensures formation of a single procentriole per parental centriole.
Topics: 3' Untranslated Regions; Amino Acid Motifs; Amino Acid Sequence; Animals; Cell Cycle; Cell Cycle Proteins; Cell Line; Centrioles; HEK293 Cells; Humans; Immunoprecipitation; Intracellular Signaling Peptides and Proteins; Models, Biological; Molecular Sequence Data; Phosphorylation; Protein Binding; Protein Serine-Threonine Kinases; RNA, Small Interfering; Sequence Alignment
PubMed: 25342035
DOI: 10.1038/ncomms6267 -
The Journal of Cell Biology Apr 2009Centrin has been shown to be involved in centrosome biogenesis in a variety of eukaryotes. In this study, we characterize hPOC5, a conserved centrin-binding protein that...
Centrin has been shown to be involved in centrosome biogenesis in a variety of eukaryotes. In this study, we characterize hPOC5, a conserved centrin-binding protein that contains Sfi1p-like repeats. hPOC5 is localized, like centrin, in the distal portion of human centrioles. hPOC5 recruitment to procentrioles occurs during G2/M, a process that continues up to the full maturation of the centriole during the next cell cycle and is correlated with hyperphosphorylation of the protein. In the absence of hPOC5, RPE1 cells arrest in G1 phase, whereas HeLa cells show an extended S phase followed by cell death. We show that hPOC5 is not required for the initiation of procentriole assembly but is essential for building the distal half of centrioles. Interestingly, the hPOC5 family reveals an evolutionary divergence between vertebrates and organisms like Drosophila melanogaster or Caenorhabditis elegans, in which the loss of hPOC5 may correlate with the conspicuous differences in centriolar structure.
Topics: Amino Acid Sequence; Carrier Proteins; Centrioles; Conserved Sequence; Cyclin-Dependent Kinase Inhibitor p21; Flow Cytometry; HeLa Cells; Humans; Phosphoproteins; Phylogeny; Protein Structure, Tertiary; S Phase; Sequence Alignment; Trimethoprim, Sulfamethoxazole Drug Combination; Tumor Suppressor Protein p53
PubMed: 19349582
DOI: 10.1083/jcb.200808082 -
PloS One 2013Centrosomes consist of two centrioles embedded in pericentriolar material and function as the main microtubule organising centres in dividing animal cells. They ensure...
Centrosomes consist of two centrioles embedded in pericentriolar material and function as the main microtubule organising centres in dividing animal cells. They ensure proper formation and orientation of the mitotic spindle and are therefore essential for the maintenance of genome stability. Centrosome function is crucial during embryonic development, highlighted by the discovery of mutations in genes encoding centrosome or spindle pole proteins that cause autosomal recessive primary microcephaly, including Cep63 and Cep152. In this study we show that Cep63 functions to ensure that centriole duplication occurs reliably in dividing mammalian cells. We show that the interaction between Cep63 and Cep152 can occur independently of centrosome localisation and that the two proteins are dependent on one another for centrosomal localisation. Further, both mouse and human Cep63 and Cep152 cooperate to ensure efficient centriole duplication by promoting the accumulation of essential centriole duplication factors upstream of SAS-6 recruitment and procentriole formation. These observations describe the requirement for Cep63 in maintaining centriole number in dividing mammalian cells and further establish the order of events in centriole formation.
Topics: Animals; Cell Cycle Proteins; Cell Line; Centrioles; Centrosome; Chromosomal Proteins, Non-Histone; Humans; Mice; Neoplasm Proteins; Protein Binding; Protein Transport
PubMed: 23936128
DOI: 10.1371/journal.pone.0069986 -
Nature Communications Feb 2021Multicilia are delicate motile machineries, and how they are accurately assembled is poorly understood. Here, we show that fibrogranular materials (FGMs), large arrays...
Multicilia are delicate motile machineries, and how they are accurately assembled is poorly understood. Here, we show that fibrogranular materials (FGMs), large arrays of electron-dense granules specific to multiciliated cells, are essential for their ultrastructural fidelity. Pcm1 forms the granular units that further network into widespread FGMs, which are abundant in spherical FGM cores. FGM cores selectively concentrate multiple important centriole-related proteins as clients, including Cep131 that specifically decorates a foot region of ciliary central pair (CP) microtubules. FGMs also tightly contact deuterosome-procentriole complexes. Disruption of FGMs in mouse cells undergoing multiciliogenesis by Pcm1 RNAi markedly deregulates centriolar targeting of FGM clients, elongates CP-foot, and alters deuterosome size, number, and distribution. Although the multicilia are produced in correct numbers, they display abnormal ultrastructure and motility. Our results suggest that FGMs organize deuterosomes and centriole-related proteins to facilitate the faithful assembly of basal bodies and multiciliary axonemes.
Topics: Animals; Axoneme; Basal Bodies; Cell Cycle Proteins; Epithelial Cells; Mice; Microtubules; Trachea
PubMed: 33627667
DOI: 10.1038/s41467-021-21506-8