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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 -
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 -
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 -
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 -
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 -
Cell Cycle (Georgetown, Tex.) Dec 2020Polo-like kinase 4 (Plk4) is a key regulator of centriole biogenesis. Studies have shown that Plk4 undergoes dynamic relocalization from a ring-like pattern around a...
Polo-like kinase 4 (Plk4) is a key regulator of centriole biogenesis. Studies have shown that Plk4 undergoes dynamic relocalization from a ring-like pattern around a centriole to a dot-like morphology at the procentriole assembly site and this event is central for inducing centriole biogenesis. However, the detailed mechanisms underlying Plk4's capacity to drive its symmetry-breaking ring-to-dot relocalization remain largely unknown. Here, we showed that Plk4 self-initiates this process in an autophosphorylation-dependent manner and that STIL, its downstream target, is not required for this event. Time-dependent analyses with mEOS-fused photoconvertible Plk4 revealed that a portion of ring-state Plk4 acquires a capacity, presumably through autophosphorylation, to linger around a centriole, ultimately generating a dot-state morphology. Interestingly, Plk4 WT, but not its catalytically inactive mutant, showed the ability to form a nanoscale spherical assembly in the cytosol of human cells or heterologous , demonstrating its autophosphorylation-dependent self-organizing capacity. At the biochemical level, Plk4 - unlike its N-terminal βTrCP degron motif - robustly autophosphorylated the PC3 SSTT motif within its C-terminal cryptic polo-box, an event critical for inducing its physical clustering. Additional experiments showed that although STIL was not required for Plk4's initial ring-to-dot conversion, coexpressed STIL greatly enhanced Plk4's ability to generate a spherical condensate and recruit Sas6, a major component of the centriolar cartwheel structure. We propose that Plk4's autophosphorylation-induced clustering is sufficient to induce its ring-to-dot localization conversion and that subsequently recruited STIL potentiates this process to generate a procentriole assembly body critical for Plk4-dependent centriole biogenesis.
Topics: Biocatalysis; Cell Cycle; Cell Line, Tumor; Centrioles; Cytosol; Escherichia coli; Escherichia coli Proteins; HEK293 Cells; Humans; Intracellular Signaling Peptides and Proteins; Osteosarcoma; Phosphorylation; Proteasome Endopeptidase Complex; Protein Binding; Protein Domains; Protein Serine-Threonine Kinases; Proteolysis; RNA Interference; Signal Transduction; Transfection
PubMed: 33323015
DOI: 10.1080/15384101.2020.1843772 -
Open Biology Nov 2020The centriole is a ninefold symmetrical structure found at the core of centrosomes and, as a basal body, at the base of cilia, whose conserved duplication is regulated...
The centriole is a ninefold symmetrical structure found at the core of centrosomes and, as a basal body, at the base of cilia, whose conserved duplication is regulated by Plk4 kinase. Plk4 phosphorylates a single serine residue at the N-terminus of Ana2 to promote Ana2's loading to the site of procentriole formation. Four conserved serines in Ana2's STAN motif are then phosphorylated by Plk4, enabling Sas6 recruitment. Crystallographic data indicate that the coiled-coil domain of Ana2 forms a tetramer but the structure of full-length Ana2 has not been solved. Here, we have employed hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) to uncover the conformational dynamics of Ana2, revealing the high flexibility of this protein with one rigid region. To determine the elusive nature of the interaction surfaces between Ana2 and Sas6, we have confirmed complex formation between the phosphomimetic form of Ana2 (Ana2-4D) and Sas6 and . Analysis of this complex by HDX-MS identifies short critical regions required for this interaction, which lie in the C-terminal parts of both proteins. Mutational studies confirmed the relevance of these regions for the Ana2-Sas6 interaction. The Sas6 site required for Ana2 binding is distinct from the site required for Sas6 to bind Gorab and Sas6 is able to bind both these protein partners simultaneously.
Topics: Amino Acid Sequence; Cell Cycle Proteins; Drosophila Proteins; Mass Spectrometry; Protein Binding; Protein Interaction Domains and Motifs; Protein Interaction Mapping; Protein Interaction Maps
PubMed: 33171067
DOI: 10.1098/rsob.200221 -
The Journal of Cell Biology Sep 2020Rcd4 is a poorly characterized Drosophila centriole component whose mammalian counterpart, PPP1R35, is suggested to function in centriole elongation and conversion to...
Rcd4 is a poorly characterized Drosophila centriole component whose mammalian counterpart, PPP1R35, is suggested to function in centriole elongation and conversion to centrosomes. Here, we show that rcd4 mutants exhibit fewer centrioles, aberrant mitoses, and reduced basal bodies in sensory organs. Rcd4 interacts with the C-terminal part of Ana3, which loads onto the procentriole during interphase, ahead of Rcd4 and before mitosis. Accordingly, depletion of Ana3 prevents Rcd4 recruitment but not vice versa. We find that neither Ana3 nor Rcd4 participates directly in the mitotic conversion of centrioles to centrosomes, but both are required to load Ana1, which is essential for such conversion. Whereas ana3 mutants are male sterile, reflecting a requirement for Ana3 for centriole development in the male germ line, rcd4 mutants are fertile and have male germ line centrioles of normal length. Thus, Rcd4 is essential in somatic cells but is not absolutely required in spermatogenesis, indicating tissue-specific roles in centriole and basal body formation.
Topics: Animals; Axoneme; Centrioles; Cilia; Drosophila Proteins; Female; Male; Mutation; Organelle Biogenesis; Protein Binding; Spermatogenesis
PubMed: 32543652
DOI: 10.1083/jcb.201912154 -
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 -
Current Biology : CB Jun 2020Centrioles are essential components of centrosome, the main microtubule-organizing center of animal cells required for robust spindle bipolarity [1, 2]. They are...
Centrioles are essential components of centrosome, the main microtubule-organizing center of animal cells required for robust spindle bipolarity [1, 2]. They are duplicated once during the cell cycle [3], and the duplication involves assembly of a cartwheel on the pre-existing centriole followed by assembly of triplet microtubules around the cartwheel [4, 5]. Although the molecular details of cartwheel formation are understood [6-13], the mechanisms initiating the formation of centriolar microtubules are not known. Here, we show that the central component of cartwheel, HsSAS-6 plays a crucial role in the formation of centriolar microtubules by interacting with the microtubule nucleation machinery, γ-tubulin ring complex (γ-TuRC) in human cells. The globular N terminus and the central coiled-coil domain of SAS-6 are required for formation of the cartwheel [7, 14], whereas the function of its C-terminal outer cartwheel region in centriole duplication remains unclear. We find that deletion of HsSAS-6 C terminus disrupts microtubule formation in daughter centriole, and as a result, cells fail to form the new centriole. Consequently, this results in mitotic cells having only two centrioles localized at a single site. Detailed molecular analyses showed that HsSAS-6 interacts with the γ-TuRC proteins and associates with the γ-TuRC at the centrosome, and furthermore, the C terminus is essential for this association. High-resolution microscopy revealed localization of the γ-TuRC protein, γ-tubulin as multiple lobes surrounding the HsSAS-6-containing central hub in the centriole. Together, the results indicate that HsSAS-6 regulates centriolar microtubule assembly by anchoring γ-TuRCs to the pro-centriole at the onset of daughter centriole formation.
Topics: Cell Cycle Proteins; Centrioles; HEK293 Cells; HeLa Cells; Humans; Microtubule-Associated Proteins; Organelle Biogenesis
PubMed: 32442461
DOI: 10.1016/j.cub.2020.04.036