The Caenorhabditis elegans centrosomal protein SPD-2 is required for both pericentriolar material recruitment and centriole duplication

BACKGROUND: The centrosome is composed of a centriole pair and pericentriolar material (PCM). By marking the site of PCM assembly, the centrioles define the number of centrosomes present in the cell. The PCM, in turn, is responsible for the microtubule (MT) nucleation activity of centrosomes. Therefore, in order to assemble a functional bipolar mitotic spindle, a cell needs to control both centriole duplication and PCM recruitment. To date, however, the molecular mechanisms that govern these two processes still remain poorly understood. RESULTS: Here we show that SPD-2 is a novel component of the C. elegans centrosome. SPD-2 localizes to the centriole throughout the cell cycle and accumulates on the PCM during mitosis. We show that SPD-2 requires SPD-5 for its accumulation on the PCM and that in the absence of SPD-2, centrosome assembly fails. We further show that centriole duplication is also defective in spd-2(RNAi) embryos, but not in spd-5(RNAi) embryos, where PCM recruitment is efficiently blocked. CONCLUSIONS: Taken together, our results suggest that SPD-2 may link PCM recruitment and centriole duplication in C. elegans. SPD-2 shares homology with a human centrosome protein, suggesting that this key component of the C. elegans centrosome is evolutionarily conserved.     

The pericentriolar material in Chinese hamster ovary cells nucleates microtubule formation

The structure and function of the centrosomes from Chinese hamster ovary (CHO) cells were investigated by electron microscopy of negatively stained wholemount preparations of cell lysates. Cells were trypsinized from culture dishes, lysed with Triton X-100, sedimented onto ionized, carbon-coated grids, and negatively stained with phosphotungstate. The centrosomes from both interphase and dividing cells consisted of pairs of centrioles, a fibrous pericentriolar material, and a group of virus-like particles which were characteristic of the CHO cells and which served as markers for the pericentriolar material. Interphase centrosomes anchored up to two dozen microtubules when cells were lysed under conditions which preserved native microtubules. When Colcemid-blocked mitotic cells, initially devoid of microtubules, were allowed to recover for 10 min, microtubules formed at the pericentriolar material, but not at the centrioles. When lysates of Colcemid-blocked cells were incubated in vitro with micotubule protein purified from porcine brain tissue, up to 250 microtubules assembled at the centrosomes, similar to the number of microtubules that would normally form at the centrosome during cell division. A few microtubules could also be assembled in vitro onto the ends of isolated centrioles from which the pericentriolar material had been removed, forming characteristic axoneme- like bundles. In addition, microtubules; were assembled onto fragments of densely staining, fibrous material which was tentatively identified as periocentriolar material by its association of CHO can initiate and anchor microtubules both in vivo and in vitro.     

Cep120 is asymmetrically localized to the daughter centriole and is essential for centriole assembly

Centrioles form the core of the centrosome in animal cells and function as basal bodies that nucleate and anchor cilia at the plasma membrane. In this paper, we report that Cep120 (Ccdc100), a protein previously shown to be involved in maintaining the neural progenitor pool in mouse brain, is associated with centriole structure and function. Cep120 is up-regulated sevenfold during differentiation of mouse tracheal epithelial cells (MTECs) and localizes to basal bodies. Cep120 localizes preferentially to the daughter centriole in cycling cells, and this asymmetry between mother and daughter centrioles is relieved coincident with new centriole assembly. Photobleaching recovery analysis identifies two pools of Cep120, differing in their halftime at the centriole. We find that Cep120 is required for centriole duplication in cycling cells, centriole amplification in MTECs, and centriole overduplication in S phase-arrested cells. We propose that Cep120 is required for centriole assembly and that the observed defect in neuronal migration might derive from a defect in this process.     

Neurl4, a novel daughter centriole protein, prevents formation of ectopic microtubule organizing centres

Here we identify Neuralized homologue 4 (Neurl4) as a protein that interacts with CP110, a centrosomal protein that regulates centrosome duplication. Neurl4 uses a Neuralized homology repeat to preferentially localize to procentrioles and daughter centrioles. Neurl4 depletion results in ectopic microtubular organizing centres (MTOCs), leading to accumulation of CP110 and recruitment of a cohort of centrosomal proteins. We show that these ectopic MTOCs persist through mitosis and assemble aberrant mitotic spindles. Interestingly, Neurl4 promotes ubiquitylation of CP110, thereby destabilizing this protein. Our results indicate that Neurl4 counteracts accumulation of CP110, thereby maintaining normal centriolar homeostasis and preventing formation of ectopic MTOCs.     

Tubulin nucleotide status controls Sas-4-dependent pericentriolar material recruitment

Regulated centrosome biogenesis is required for accurate cell division and for maintaining genome integrity. Centrosomes consist of a centriole pair surrounded by a protein network known as pericentriolar material (PCM). PCM assembly is a tightly regulated, critical step that determines the size and capability of centrosomes. Here, we report a role for tubulin in regulating PCM recruitment through the conserved centrosomal protein Sas-4. Tubulin directly binds to Sas-4; together they are components of cytoplasmic complexes of centrosomal proteins. A Sas-4 mutant, which cannot bind tubulin, enhances centrosomal protein complex formation and has abnormally large centrosomes with excessive activity. These results suggest that tubulin negatively regulates PCM recruitment. Whereas tubulin-GTP prevents Sas-4 from forming protein complexes, tubulin-GDP promotes it. Thus, the regulation of PCM recruitment by tubulin depends on its GTP/GDP-bound state. These results identify a role for tubulin in regulating PCM recruitment independent of its well-known role as a building block of microtubules. On the basis of its guanine-bound state, tubulin can act as a molecular switch in PCM recruitment.     

Centrobin: a novel daughter centriole-associated protein that is required for centriole duplication

In mammalian cells, the centrosome consists of a pair of centrioles and amorphous pericentriolar material. The pair of centrioles, which are the core components of the centrosome, duplicate once per cell cycle. Centrosomes play a pivotal role in orchestrating the formation of the bipolar spindle during mitosis. Recent studies have linked centrosomal activity on centrioles or centriole-associated structures to cytokinesis and cell cycle progression through G1 into the S phase. In this study, we have identified centrobin as a centriole-associated protein that asymmetrically localizes to the daughter centriole. The silencing of centrobin expression by small interfering RNA inhibited centriole duplication and resulted in centrosomes with one or no centriole, demonstrating that centrobin is required for centriole duplication. Furthermore, inhibition of centriole duplication by centrobin depletion led to impaired cytokinesis.     

Structured illumination of the interface between centriole and peri-centriolar material

The increase in centrosome size in mitosis was described over a century ago, and yet it is poorly understood how centrioles, which lie at the core of centrosomes, organize the pericentriolar material (PCM) in this process. Now, structured illumination microscopy reveals in Drosophila that, before clouds of PCM appear, its proteins are closely associated with interphase centrioles in two tube-like layers: an inner layer occupied by centriolar microtubules, Sas-4, Spd-2 and Polo kinase; and an outer layer comprising Pericentrin-like protein (Dplp), Asterless (Asl) and Plk4 kinase. Centrosomin (Cnn) and γ-tubulin associate with this outer tube in G2 cells and, upon mitotic entry, Polo activity is required to recruit them together with Spd-2 into PCM clouds. Cnn is required for Spd-2 to expand into the PCM during this maturation process but can itself contribute to PCM independently of Spd-2. By contrast, the centrioles of spermatocytes elongate from a pre-existing proximal unit during the G2 preceding meiosis. Sas-4 is restricted to the microtubule-associated, inner cylinder and Dplp and Cnn to the outer cylinder of this proximal part. γ-Tubulin and Asl associate with the outer cylinder and Spd-2 with the inner cylinder throughout the entire G2 centriole. Although they occupy different spatial compartments on the G2 centriole, Cnn, Spd-2 and γ-tubulin become diminished at the centriole upon entry into meiosis to become part of PCM clouds.     

Microtubule nucleating sites in higher plant cells identified by an auto-antibody against pericentriolar material

Human scleroderma serum 5051, which is known to recognize the amorphous pericentriolar microtubule organizing center material of a variety of vertebrate cells, was found to immunostain spindle poles of meristematic higher plants from pre-prophase to late anaphase. Subsequently, during cytokinesis, staining was redistributed around the reforming telophase nuclei, but was not evident in the cytokinetic phragmoplast. At the transition between telophase and interphase, before the typical cortical interphase microtubule array was established, short microtubules radiated from the nucleus and in such cells the material recognized by 5051 was located around the daughter nuclei and not the cortex. These observations have led us to propose that the perinuclear region, or the nuclear surface, may function as a nucleation center for both spindle and interphase microtubules in higher plant cells.     

Dependence of centriole formation on protein synthesis

Centriole formation was studied after inhibition of protein synthesis for various portions of the cell cycle. Synchronous populations of mitotic L929 (mouse) cells were plated into petri dishes and the course of procentriole formation was monitored by electron microscope analysis. The frequency with which procentrioles were seen in association with mature centrioles normally increased steadily in the interval from 4 to 12 h after mitosis. The formation of procentrioles was abruptly inhibited by the addition of cycloheximide at any time from mitosis until 12 h postmitosis (S phase). This suggested that the formation of procentrioles was dependent upon protein synthesis immediately before their appearance. Prophase-accociated elongation of procentrioles appeared to occur normally in cells treated with cycloheximide for up to 4 h before prophase, though the mitotic index in treated cultures decreased somewhat. Thus, protein synthesis did not appear to be essential for procentriolar elongation to the mature length.     

Cnn dynamics drive centrosome size asymmetry to ensure daughter centriole retention in Drosophila neuroblasts

Centrosomes comprise a pair of centrioles surrounded by an amorphous network of pericentriolar material (PCM). In certain stem cells, the two centrosomes differ in size, and this appears to be important for asymmetric cell division [1, 2]. In some cases, centrosome asymmetry is linked to centriole age because the older, mother centriole always organizes more PCM than the daughter centriole, thus ensuring that the mother centriole is always retained in the stem cell after cell division [3]. This has raised the possibility that an "immortal" mother centriole may help maintain stem cell fate [4, 5]. It is unclear, however, how centrosome size asymmetry is generated in stem cells. Here we provide compelling evidence that centrosome size asymmetry in Drosophila neuroblasts is generated by the differential regulation of Cnn incorporation into the PCM at mother and daughter centrioles. Shortly after centriole separation, mother and daughter centrioles organize similar amounts of PCM, but Cnn incorporation is then rapidly downregulated at the mother centriole, while it is maintained at the daughter centriole. This ensures that the daughter centriole maintains its PCM and so its position at the apical cortex. Thus, the?daughter centriole, rather than an "immortal" mother centriole, is ultimately retained in these stem cells.     

Redistribution of microtubules and pericentriolar material during the development of polarity in mouse blastomeres

The distribution of microtubules and microtubule organizing centers (MTOCs) during the development of cell polarity in eight-cell mouse blastomeres was studied by immunofluorescence and immunoelectron microscopy using monoclonal anti-tubulin antibodies and an anti-pericentriolar material (PCM) serum. In early eight-cell blastomeres microtubules were found mainly around the nucleus and in the cell cortex, whereas PCM foci were observed dispersed in the cytoplasm. During the eight-cell stage, microtubules disappeared from the area adjacent to the zone of intercellular contact and accumulated in the apical part of the cell while their number decreased in the basal domain. The PCM also relocalized to the apical domain of the cell, but this occurred after the redistribution of the microtubules by a mechanism that involved the microtubule network. The possible roles of both MTOCs and microtubules in establishing cell polarity are discussed.     

Immunostaining of spindle components in tipulid spermatocytes using a serum against pericentriolar material

First meiotic division of tipulid (Pales ferruginea) spermatocytes was investigated by double immunostaining with anti-tubulin IgG and scleroderma 5051 serum against pericentriolar material (PCM). PCM-like material became visible in late diakinesis in centrosomal areas as well as in kinetochores. Anti-PCM fluorescence was most pronounced in metaphase and diminished again in anaphase. Displacement of one of the centrosomes from the nucleus at diakinesis in Pales spermatocytes leads to the formation of a bipolar, normally functioning spindle possessing one aster and centriole-free spindle pole (AFP). In about 80% of the AFPs observed there were no traces of anti-PCM staining detectable. This finding supports the assumption based on previous studies that polar PCM is not obligatory for spindle pole formation. The chromosomes seem to be able to induce the organization of a half-spindle. The strong anti-PCM fluorescence of the kinetochores observed here may be taken as further indication of tipulid chromosome autonomy regarding spindle formation.     

Sequential protein recruitment in C. elegans centriole formation

Formation of the microtubule-based centriole is a poorly understood process that is crucial for duplication of the centrosome, the principal microtubule-organizing center of animal cells . Five proteins have been identified as being essential for centriole formation in Caenorhabditis elegans: the kinase ZYG-1, as well as the coiled-coil proteins SAS-4, SAS-5, SAS-6, and SPD-2 . The relationship between these proteins is incompletely understood, limiting understanding of how they contribute to centriole formation. In this study, we established the order in which these five proteins are recruited to centrioles, and we conducted molecular epistasis experiments expanding on earlier work. We find that SPD-2 is loaded first and is needed for the centriolar localization of the four other proteins. ZYG-1 recruitment is required thereafter for the remaining three proteins to localize to centrioles. SAS-5 and SAS-6 are recruited next and are needed for the presence of SAS-4, which is incorporated last. Our results indicate in addition that the presence of SAS-5 and SAS-6 allows diminution of centriolar ZYG-1. Moreover, astral microtubules appear dispensable for the centriolar recruitment of all five proteins. Several of these proteins have homologs in other metazoans, and we expect the assembly pathway that stems from our work to be conserved.     

The iris diaphragm model of centriole and basal body formation

This paper suggests that the formation and structure of the microtubular skeleton of centrioles and basal bodies can be derived from the following simple geometric principle. A closed ring of nine microtubular initiation sites defines (1) a template for the packing of 18 additional microtubular initiation sites, and (2) the shape of nine rigid arms. Upon swivelling of each arm around a point located four initiation sites away on the initial ring, the array unfolds in a manner similar to the opening of an iris diaphragm. As a consequence, the curved shape of the microtubular triplet blades arises together with the clockwise rotational sense of the slanted blades of the centriole or basal body. The final diameter of the centriole (basal body) self-adjusts. Furthermore, the pitch of the triplet blades, the taper of centrioles and basal bodies, and the change of slant of the blades towards the distal end can be derived. In addition, the model points to a method of replication of pro-centrioles (pro-basal bodies). The hypothesis was tested by the fitting of electron microscopical cross sections of centrioles of 3T3 cells to the geometric shapes predicted by the model.     

Cell cycle-dependent, in vitro assembly of microtubules onto pericentriolar material of HeLa cells

A centriolar complex comprising a pair of centrioles and a cloud of pericentriolar materials is located at the point of covergence of the microtubules of the mitotic apparatus. The in vitro assembly of microtubules was observed onto these complexes in the 1,400 g supernatant fraction of colcemid-blocked, mitotic HeLa cells lysed into solutions containing tubulin and Triton X-100. Dark-field microscopy provided a convenient means by which this process could be visualized directly. When this 1,400 g supernate was incubated at 30 degrees C and centrifuged into a discontinuous sucrose gradient, a band containing centriolar complexes and assembled microtubles was obtained at 50-60% sucrose interface. Ultrastructual analysis indicated that the majority of the microtubules assembled predominantly from the pericentriolar material but also onto the centrioles. When cells were synchronized by a double thymide block, the assembly of microtubules onto centriolar complexes was observed only in lysates of mitotic cells; no assembly was seen in lysed material of interphase cells. Microtubule assembly occured onto centriolar complexes in solutions of either 100,000 g brain supernate, 2 X cycled tubulin, or purified tubulin dimers. This study demonstrates that the pericentriolar material becomes competent as a microtubule-organizing center (MTOC) at the time of mitosis. With use of the techniques described, a method for the isolation of centriolar complexes may be developed.     

CEP215 is involved in the dynein-dependent accumulation of pericentriolar matrix proteins for spindle pole formation

CEP215 is a human orthologue of Drosophila centrosomin which is a core centrosome component for the pericentriolar matrix protein recruitment. Recent investigations revealed that CEP215 is required for centrosome cohesion, centrosomal attachment of the g-TuRC, and microtubule dynamics. However, it remains to be obscure how CEP215 functions for recruitment of the centrosomal proteins during the centrosome cycle. Here, we investigated a role of CEP215 during mitosis. Knockdown of CEP215 resulted in characteristic mitotic phenotypes, including monopolar spindle formation, a decrease in distance between the spindle pole pair, and detachment of the centrosomes from the spindle poles. We noticed that CEP215 is critical for centrosomal localization of dynein throughout the cell cycle. As a consequence, the selective centrosomal proteins were not recruited to the centrosome properly. Finally, the centrosomal localization of CEP215 also depends on the dynein-dynactin complex. Based on the results, we propose that CEP215 regulates a dynein-dependent transport of the pericentriolar matrix proteins during the centrosome maturation.