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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 -
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 -
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 -
Current Biology : CB Feb 2013The centrosome functions as the main microtubule-organizing center of animal cells and is crucial for several fundamental cellular processes. Abnormalities in centrosome...
The centrosome functions as the main microtubule-organizing center of animal cells and is crucial for several fundamental cellular processes. Abnormalities in centrosome number and composition correlate with tumor progression and other diseases. Although proteomic studies have identified many centrosomal proteins, their interactions are incompletely characterized. The lack of information on the precise localization and interaction partners for many centrosomal proteins precludes comprehensive understanding of centrosome biology. Here, we utilize a combination of selective chemical crosslinking and superresolution microscopy to reveal novel functional interactions among a set of 31 centrosomal proteins. We reveal that Cep57, Cep63, and Cep152 are parts of a ring-like complex localizing around the proximal end of centrioles. Furthermore, we identify that STIL, together with HsSAS-6, resides at the proximal end of the procentriole, where the cartwheel is located. Our studies also reveal that the known interactors Cep152 and Plk4 reside in two separable structures, suggesting that the kinase Plk4 contacts its substrate Cep152 only transiently, at the centrosome or within the cytoplasm. Our findings provide novel insights into protein interactions critical for centrosome biology and establish a toolbox for future studies of centrosomal proteins.
Topics: Cell Cycle Proteins; Cell Line, Tumor; Centrioles; Cross-Linking Reagents; HEK293 Cells; Humans; Microtubule-Associated Proteins; Neoplasm Proteins; Nuclear Proteins
PubMed: 23333316
DOI: 10.1016/j.cub.2012.12.030 -
Open Biology Aug 2015Centrioles are short microtubule-based organelles with a conserved ninefold symmetry. They are essential for both centrosome formation and cilium biogenesis in most... (Review)
Review
Centrioles are short microtubule-based organelles with a conserved ninefold symmetry. They are essential for both centrosome formation and cilium biogenesis in most eukaryotes. A core set of five centriolar proteins has been identified and their sequential recruitment to procentrioles has been established. However, structures at atomic resolution for most of the centriolar components were scarce, and the underlying molecular mechanisms for centriole assembly had been a mystery--until recently. In this review, I briefly summarize recent advancements in high-resolution structural characterization of the core centriolar components and discuss perspectives in the field.
Topics: Animals; Cell Cycle Proteins; Centrioles; Chromosomal Proteins, Non-Histone; Humans; Protein Interaction Domains and Motifs; Protein Serine-Threonine Kinases
PubMed: 26269428
DOI: 10.1098/rsob.150082 -
The Journal of Biophysical and... Jun 1961This paper describes the replication of centrioles during spermatogenesis in the Prosobranch snail, Viviparus malleatus Reeve. Sections for electron microscopy were cut...
This paper describes the replication of centrioles during spermatogenesis in the Prosobranch snail, Viviparus malleatus Reeve. Sections for electron microscopy were cut from pieces of testis fixed in OsO(4) and embedded in the polyester resin Vestopal W. Two kinds of spermatocytes are present. These give rise to typical uniflagellate sperm carrying the haploid number of 9 chromosomes, and atypical multiflagellate sperm with only one chromosome. Two centrioles are present in the youngest typical spermatocyte. Each is a hollow cylinder about 160 mmicro in diameter and 330 mmicro long. The wall consists of 9 sets of triplet fibers arranged in a characteristic pattern. Sometime before pachytene an immature centriole, or procentriole as it will be called, appears next to each of the mature centrioles. The procentriole resembles a mature centriole in most respects except length: it is more annular than tubular. The daughter procentriole lies with its axis perpendicular to that of its parent. It presumably grows to full size during the late prophase, although the maturation stages have not been observed with the electron microscope. It is suggested that centrioles possess a constant polarization. The distal end forms the flagellum or other centriole products, while the proximal end represents the procentriole and is concerned with replication. The four centrioles of prophase (two parents and two daughters) are distributed by the two meiotic divisions to the four typical spermatids, in which they function as the basal bodies of the flagella. Atypical spermatocytes at first contain two normal centrioles. Each of these becomes surrounded by a cluster of procentrioles, which progressively elongate during the late prophase. After two aberrant meiotic divisions the centriole clusters give rise to the basal bodies of the multiflagellate sperm. These facts are discussed in the light of the theory, first proposed by Pollister, that the supernumerary centrioles in the atypical cells are derived from the centromeres of degenerating chromosomes.
Topics: Animals; Cell Cycle; Centrioles; Flagella; Helix, Snails; Humans; Male; Microscopy, Electron; Mitosis; Prophase; Snails; Spermatids; Spermatocytes; Spermatogenesis; Spermatozoa; Testis
PubMed: 13703108
DOI: 10.1083/jcb.10.2.163 -
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 -
The Journal of Cell Biology Jul 1971Basal body replication during estrogen-driven ciliogenesis in the rhesus monkey (Macaca mulatta) oviduct has been studied by stereomicroscopy, rotation photography, and...
Basal body replication during estrogen-driven ciliogenesis in the rhesus monkey (Macaca mulatta) oviduct has been studied by stereomicroscopy, rotation photography, and serial section analysis. Two pathways for basal body production are described: acentriolar basal body formation (major pathway) where procentrioles are generated from a spherical aggregate of fibers; and centriolar basal body formation, where procentrioles are generated by the diplosomal centrioles. In both pathways, the first step in procentriole formation is the arrangement of a fibrous granule precursor into an annulus. A cartwheel structure, present within the lumen of the annulus, is composed of a central cylinder with a core, spoke components, and anchor filaments. Tubule formation consists of an initiation and a growth phase. The A tubule of each triplet set first forms within the wall material of the annulus in juxtaposition to a spoke of the cartwheel. After all nine A tubules are initiated, B and C tubules begin to form. The initiation of all three tubules occurs sequentially around the procentriole. Simultaneous with tubule initiation is a nonsequential growth of each tubule. The tubules lengthen and the procentriole is complete when it is about 200 mmicro long. The procentriole increases in length and diameter during its maturation into a basal body. The addition of a basal foot, nine alar sheets, and a rootlet completes the maturation process. Fibrous granules are also closely associated with the formation of these basal body accessory structures.
Topics: Animals; Cilia; Cytoplasmic Granules; Estradiol; Fallopian Tubes; Female; Haplorhini; Macaca; Microscopy, Electron; Models, Structural
PubMed: 4998200
DOI: 10.1083/jcb.50.1.10 -
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 -
The Journal of Cell Biology Jan 2024
PubMed: 38095873
DOI: 10.1083/jcb.20230106912112023c