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Cell Apr 2024Centriole biogenesis, as in most organelle assemblies, involves the sequential recruitment of sub-structural elements that will support its function. To uncover this...
Centriole biogenesis, as in most organelle assemblies, involves the sequential recruitment of sub-structural elements that will support its function. To uncover this process, we correlated the spatial location of 24 centriolar proteins with structural features using expansion microscopy. A time-series reconstruction of protein distributions throughout human procentriole assembly unveiled the molecular architecture of the centriole biogenesis steps. We found that the process initiates with the formation of a naked cartwheel devoid of microtubules. Next, the bloom phase progresses with microtubule blade assembly, concomitantly with radial separation and rapid cartwheel growth. In the subsequent elongation phase, the tubulin backbone grows linearly with the recruitment of the A-C linker, followed by proteins of the inner scaffold (IS). By following six structural modules, we modeled 4D assembly of the human centriole. Collectively, this work provides a framework to investigate the spatial and temporal assembly of large macromolecules.
Topics: Centrioles; Humans; Microtubules; Tubulin; Cell Cycle Proteins
PubMed: 38604175
DOI: 10.1016/j.cell.2024.03.025 -
The Journal of Cell Biology Dec 2023Centriole duplication is a high-fidelity process driven by Polo-like kinase 4 (Plk4) and a few conserved initiators. Dissecting how Plk4 and its receptors organize...
Centriole duplication is a high-fidelity process driven by Polo-like kinase 4 (Plk4) and a few conserved initiators. Dissecting how Plk4 and its receptors organize within centrosomes is critical to understand the centriole duplication process and biochemical and architectural differences between centrosomes of different species. Here, at nanoscale resolution, we dissect centrosomal localization of Plk4 in G1 and S phase in its catalytically active and inhibited state during centriole duplication and amplification. We build a precise distribution map of Plk4 and its receptor Cep152, as well as Cep44, Cep192, and Cep152-anchoring factors Cep57 and Cep63. We find that Cep57, Cep63, Cep44, and Cep192 localize in ninefold symmetry. However, during centriole maturation, Cep152, which we suggest is the major Plk4 receptor, develops a more complex pattern. We propose that the molecular arrangement of Cep152 creates flexibility for Plk4 and procentriole placement during centriole initiation. As a result, procentrioles form at variable positions in relation to the mother centriole microtubule triplets.
Topics: Cell Cycle; Centrioles; Centrosome; Microtubules; S Phase; Humans; Cell Cycle Proteins; Protein Serine-Threonine Kinases
PubMed: 37707473
DOI: 10.1083/jcb.202301092 -
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 -
Communications Biology Jul 2023Proper organization of intracellular assemblies is fundamental for efficient promotion of biochemical processes and optimal assembly functionality. Although advances in...
Proper organization of intracellular assemblies is fundamental for efficient promotion of biochemical processes and optimal assembly functionality. Although advances in imaging technologies have shed light on how the centrosome is organized, how its constituent proteins are coherently architected to elicit downstream events remains poorly understood. Using multidisciplinary approaches, we showed that two long coiled-coil proteins, Cep63 and Cep152, form a heterotetrameric building block that undergoes a stepwise formation into higher molecular weight complexes, ultimately generating a cylindrical architecture around a centriole. Mutants defective in Cep63•Cep152 heterotetramer formation displayed crippled pericentriolar Cep152 organization, polo-like kinase 4 (Plk4) relocalization to the procentriole assembly site, and Plk4-mediated centriole duplication. Given that the organization of pericentriolar materials (PCM) is evolutionarily conserved, this work could serve as a model for investigating the structure and function of PCM in other species, while offering a new direction in probing the organizational defects of PCM-related human diseases.
Topics: Humans; Cell Cycle; Centrioles; Centrosome; Molecular Weight; Protein Domains; Protein Serine-Threonine Kinases
PubMed: 37433832
DOI: 10.1038/s42003-023-05067-8 -
The Journal of Cell Biology Dec 2023Polo-like kinase 4 (PLK4) is a key regulator of centriole biogenesis, but how PLK4 selects a single site for procentriole assembly remains unclear. Using ultrastructure...
Polo-like kinase 4 (PLK4) is a key regulator of centriole biogenesis, but how PLK4 selects a single site for procentriole assembly remains unclear. Using ultrastructure expansion microscopy, we show that PLK4 localizes to discrete sites along the wall of parent centrioles. While there is variation in the number of sites PLK4 occupies on the parent centriole, most PLK4 localize at a dominant site that directs procentriole assembly. Inhibition of PLK4 activity leads to stable binding of PLK4 to the centriole and increases occupancy to a maximum of nine sites. We show that self-phosphorylation of an unstructured linker promotes the release of active PLK4 from the centriole to drive the selection of a single site for procentriole assembly. Preventing linker phosphorylation blocks PLK4 turnover, leading to supernumerary sites of PLK4 localization and centriole amplification. Therefore, self-phosphorylation is a major driver of the spatial patterning of PLK4 at the centriole and plays a critical role in selecting a single centriole duplication site.
Topics: Cell Cycle; Cell Cycle Proteins; Centrioles; Phosphorylation; Humans; Protein Serine-Threonine Kinases
PubMed: 37773039
DOI: 10.1083/jcb.202301069 -
PLoS Biology Sep 2022Uncovering organizing principles of organelle assembly is a fundamental pursuit in the life sciences. Caenorhabditis elegans was key in identifying evolutionary...
Uncovering organizing principles of organelle assembly is a fundamental pursuit in the life sciences. Caenorhabditis elegans was key in identifying evolutionary conserved components governing assembly of the centriole organelle. However, localizing these components with high precision has been hampered by the minute size of the worm centriole, thus impeding understanding of underlying assembly mechanisms. Here, we used Ultrastructure Expansion coupled with STimulated Emission Depletion (U-Ex-STED) microscopy, as well as electron microscopy (EM) and electron tomography (ET), to decipher the molecular architecture of the worm centriole. Achieving an effective lateral resolution of approximately 14 nm, we localize centriolar and PeriCentriolar Material (PCM) components in a comprehensive manner with utmost spatial precision. We found that all 12 components analysed exhibit a ring-like distribution with distinct diameters and often with a 9-fold radial symmetry. Moreover, we uncovered that the procentriole assembles at a location on the centriole margin where SPD-2 and ZYG-1 also accumulate. Moreover, SAS-6 and SAS-5 were found to be present in the nascent procentriole, with SAS-4 and microtubules recruited thereafter. We registered U-Ex-STED and EM data using the radial array of microtubules, thus allowing us to map each centriolar and PCM protein to a specific ultrastructural compartment. Importantly, we discovered that SAS-6 and SAS-4 exhibit a radial symmetry that is offset relative to microtubules, leading to a chiral centriole ensemble. Furthermore, we established that the centriole is surrounded by a region from which ribosomes are excluded and to which SAS-7 localizes. Overall, our work uncovers the molecular architecture of the C. elegans centriole in unprecedented detail and establishes a comprehensive framework for understanding mechanisms of organelle biogenesis and function.
Topics: Animals; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Cell Cycle Proteins; Centrioles; Centrosome; Protein Kinases
PubMed: 36107993
DOI: 10.1371/journal.pbio.3001784 -
Cells Jun 2020Centrioles are microtubule-based cellular structures present in most human cells that build centrosomes and cilia. Proliferating cells have only two centrosomes and this... (Review)
Review
Centrioles are microtubule-based cellular structures present in most human cells that build centrosomes and cilia. Proliferating cells have only two centrosomes and this number is stringently maintained through the temporally and spatially controlled processes of centriole assembly and segregation. The assembly of new centrioles begins in early S phase and ends in the third G1 phase from their initiation. This lengthy process of centriole assembly from their initiation to their maturation is characterized by numerous structural and still poorly understood biochemical changes, which occur in synchrony with the progression of cells through three consecutive cell cycles. As a result, proliferating cells contain three structurally, biochemically, and functionally distinct types of centrioles: procentrioles, daughter centrioles, and mother centrioles. This age difference is critical for proper centrosome and cilia function. Here we discuss the centriole assembly process as it occurs in somatic cycling human cells with a focus on the structural, biochemical, and functional characteristics of centrioles of different ages.
Topics: Aging; Centrioles; Centrosome; Humans
PubMed: 32526902
DOI: 10.3390/cells9061429 -
The EMBO Journal Nov 2022Over the course of evolution, the centrosome function has been conserved in most eukaryotes, but its core architecture has evolved differently in some clades, with the...
Over the course of evolution, the centrosome function has been conserved in most eukaryotes, but its core architecture has evolved differently in some clades, with the presence of centrioles in humans and a spindle pole body (SPB) in yeast. Similarly, the composition of these two core elements has diverged, with the exception of Centrin and SFI1, which form a complex in yeast to initiate SPB duplication. However, it remains unclear whether this complex exists at centrioles and whether its function has been conserved. Here, using expansion microscopy, we demonstrate that human SFI1 is a centriolar protein that associates with a pool of Centrin at the distal end of the centriole. We also find that both proteins are recruited early during procentriole assembly and that depletion of SFI1 results in the loss of the distal pool of Centrin, without altering centriole duplication. Instead, we show that SFI1/Centrin complex is essential for centriolar architecture, CEP164 distribution, and CP110 removal during ciliogenesis. Together, our work reveals a conserved SFI1/Centrin module displaying divergent functions between mammals and yeast.
Topics: Animals; Humans; Cell Cycle Proteins; Centrioles; Repressor Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Spindle Pole Bodies; Calcium-Binding Proteins
PubMed: 36125182
DOI: 10.15252/embj.2022112107 -
Nature Communications Mar 2024HIV-1 infection elevates the risk of developing various cancers, including T-cell lymphoma. Whether HIV-1-encoded proteins directly contribute to oncogenesis remains...
HIV-1 infection elevates the risk of developing various cancers, including T-cell lymphoma. Whether HIV-1-encoded proteins directly contribute to oncogenesis remains unknown. We observe that approximately 1-5% of CD4 T cells from the blood of people living with HIV-1 exhibit over-duplicated centrioles, suggesting that centrosome amplification underlies the development of HIV-1-associated cancers by driving aneuploidy. Through affinity purification, biochemical, and cellular analyses, we discover that Vpr, an accessory protein of HIV-1, hijacks the centriole duplication machinery and induces centrosome amplification and aneuploidy. Mechanistically, Vpr forms a cooperative ternary complex with an E3 ligase subunit, VprBP, and polo-like kinase 4 (Plk4). Unexpectedly, however, the complex enhances Plk4's functionality by promoting its relocalization to the procentriole assembly and induces centrosome amplification. Loss of either Vpr's C-terminal 17 residues or VprBP acidic region, the two elements required for binding to Plk4 cryptic polo-box, abrogates Vpr's capacity to induce these events. Furthermore, HIV-1 WT, but not its Vpr mutant, induces multiple centrosomes and aneuploidy in human primary CD4 T cells. We propose that the Vpr•VprBP•Plk4 complex serves as a molecular link that connects HIV-1 infection to oncogenesis and that inhibiting the Vpr C-terminal motif may reduce the occurrence of HIV-1-associated cancers.
Topics: Humans; T-Lymphocytes; HIV-1; Centrosome; Carcinogenesis; Cell Transformation, Neoplastic; Aneuploidy; CD4-Positive T-Lymphocytes
PubMed: 38443376
DOI: 10.1038/s41467-024-46306-8 -
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