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Centrioles are microtubule-based structures involved in cell division and ciliogenesis. Centriole formation is a highly regulated cellular process and aberrations in centriole structure, size or numbers have implications in multiple human pathologies. In this review, we propose that the proteins that control centriole length can be subdivided into two classes based on their antagonistic activities on centriolar microtubules, which we refer to as 'centriole elongation activators' (CEAs) and 'centriole elongation inhibitors' (CEIs). We discuss and illustrate the structure-function relationship of CEAs and CEIs as well as their interaction networks. Based on our current knowledge, we formulate some outstanding open questions in the field and present possible routes for future studies.

Viruses exploit the host cell machinery for their own profit. To evade innate immune sensing and promote viral replication, HIV type 1 (HIV-1) subverts DNA repair regulatory proteins and induces G2/M arrest. The preintegration complex of HIV-1 is known to traffic along microtubules and accumulate near the microtubule-organizing center. The centrosome is the major microtubule-organizing center in most eukaryotic cells, but precisely how HIV-1 impinges on centrosome biology remains poorly understood. We report here that the HIV-1 accessory protein viral protein R (Vpr) localized to the centrosome through binding to DCAF1, forming a complex with the ubiquitin ligase EDD-DYRK2-DDB1DCAF1 and Cep78, a resident centrosomal protein previously shown to inhibit EDD-DYRK2-DDB1DCAF1. Vpr did not affect ubiquitination of Cep78. Rather, it enhanced ubiquitination of an EDD-DYRK2-DDB1DCAF1 substrate, CP110, leading to its degradation, an effect that could be overcome by Cep78 expression. The down-regulation of CP110 and elongation of centrioles provoked by Vpr were independent of G2/M arrest. Infection of T lymphocytes with HIV-1, but not with HIV-1 lacking Vpr, promoted CP110 degradation and centriole elongation. Elongated centrioles recruited more γ-tubulin to the centrosome, resulting in increased microtubule nucleation. Our results suggest that Vpr is targeted to the centrosome where it hijacks a ubiquitin ligase, disrupting organelle homeostasis, which may contribute to HIV-1 pathogenesis.

The centriole, or basal body, is the center of attachment between the sperm head and tail. While the distal end of the centriole templates the cilia, the proximal end associates with the nucleus. Using Drosophila, we identify a centriole-centric mechanism that ensures proper proximal end docking to the nucleus. This mechanism relies on the restriction of Pericentrin-Like Protein (PLP) and the pericentriolar material (PCM) to the proximal end of the centriole. PLP is restricted proximally by limiting its mRNA and protein to the earliest stages of centriole elongation. Ectopic positioning of PLP to more distal portions of the centriole is sufficient to redistribute PCM and microtubules along the entire centriole length. This results in erroneous, lateral centriole docking to the nucleus, leading to spermatid decapitation as a result of a failure to form a stable head-tail linkage.

Microtubule-based centrioles in the centrosome mediate accurate bipolar cell division, spindle orientation, and primary cilia formation. Cellular checkpoints ensure that the centrioles duplicate only once in every cell cycle and achieve precise dimensions, dysregulation of which results in genetic instability and neuro- and ciliopathies. The normal cellular level of centrosomal protein 4.1-associated protein (CPAP), achieved by its degradation at mitosis, is considered as one of the major mechanisms that limits centriole growth at a predetermined length. Here we show that CPAP levels and centriole elongation are regulated by centrobin. Exogenous expression of centrobin causes abnormal elongation of centrioles due to massive accumulation of CPAP in the cell. Conversely, CPAP was undetectable in centrobin-depleted cells, suggesting that it undergoes degradation in the absence of centrobin. Only the reintroduction of full-length centrobin, but not its mutant form that lacks the CPAP binding site, could restore cellular CPAP levels in centrobin-depleted cells, indicating that persistence of CPAP requires its interaction with centrobin. Interestingly, inhibition of the proteasome in centrobin-depleted cells restored the cellular and centriolar CPAP expression, suggesting its ubiquitination and proteasome-mediated degradation when centrobin is absent. Intriguingly, however, centrobin-overexpressing cells also showed proteasome-independent accumulation of ubiquitinated CPAP and abnormal, ubiquitin-positive, elongated centrioles. Overall, our results show that centrobin interacts with ubiquitinated CPAP and prevents its degradation for normal centriole elongation function. Therefore, it appears that loss of centrobin expression destabilizes CPAP and triggers its degradation to restrict the centriole length during biogenesis.

SummaryCentrioles are microtubule-based cylindrical structures that exhibit 9-fold symmetry and facilitate the organization of centrosomes, flagella, and cilia [1]. Abnormalities in centrosome structure and number occur in many cancers [1, 2]. Despite its importance, very little is known about centriole biogenesis. Recent studies in C. elegans have highlighted a group of molecules necessary for centriole assembly [1, 3]. ZYG-1 kinase recruits a complex of two coiled-coil proteins, SAS-6 and SAS-5, which are necessary to form the C. elegans centriolar tube, a scaffold in centriole formation [4, 5]. This complex also recruits SAS-4, which is required for the assembly of the centriolar microtubules that decorate that tube [4, 5]. Here we show that Drosophila SAS-6 is involved in centriole assembly and cohesion. Overexpression of DSAS-6 in syncitial embryos led to the de novo formation of multiple microtubule-organizing centers (MTOCs). Strikingly, the center of these MTOCs did not contain centrioles, as described previously for SAK/PLK4 overexpression [6]. Instead, tube-like structures were present, supporting the idea that centriolar assembly starts with the formation of a tube-like scaffold, dependent on DSAS-6 [5]. In DSAS-6 loss-of-function mutants, centrioles failed to close and to elongate the structure along all axes of the 9-fold symmetry, suggesting modularity in centriole assembly. We propose that the tube is built from nine subunits fitting together laterally and longitudinally in a modular and sequential fashion, like pieces of a layered “hollow” cake.

Summary Centrosome duplication involves the formation of a single procentriole next to each centriole, once per cell cycle. The mechanisms governing procentriole formation and those restricting its occurrence to one event per centriole are poorly understood. Here, we show that HsSAS-6 is necessary for procentriole formation and that it localizes asymmetrically next to the centriole at the onset of procentriole formation. HsSAS-6 levels oscillate during the cell cycle, with the protein being degraded in mitosis and starting to accumulate again at the end of the following G1. Our findings indicate that APC Cdh1 targets HsSAS-6 for degradation by the 26S proteasome. Importantly, we demonstrate that increased HsSAS-6 levels promote formation of more than one procentriole per centriole. Therefore, regulated HsSAS-6 levels normally ensure that each centriole seeds the formation of a single procentriole per cell cycle, thus playing a fundamental role in driving the centrosome duplication cycle and ensuring genome integrity.

Most microtubule arrays in animal cells, including the bipolar spindle required for cell division, are organized by centrosomes. Thus, strict control of centrosome numbers is crucial for accurate chromosome segregation. Each centrosome comprises two centrioles, which need to be duplicated exactly once in every cell cycle. Recent work has begun to illuminate the mechanisms that regulate centriole duplication. First, genetic and structural studies concur to delineate a centriole assembly pathway in Caenorhabditis elegans. Second, the protease Separase, previously known to trigger sister chromatid separation, has been implicated in a licensing mechanism that restricts centrosome duplication to a single occurrence per cell cycle. Finally, Plk4 (also called Sak), a member of the Polo kinase family, has been identified as a novel positive regulator of centriole formation.

SummaryFormation 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 [1]. 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 [2–9]. 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.

Centrioles organize pericentriolar material to form centrosomes and also template the formation of cilia. Despite the importance of centrioles in dividing and differentiated cells, their assembly remains poorly understood at a molecular level. Here, we develop a fluorescence microscopy-based assay for centriole assembly in the 1-cell stage C. elegans embryo. We use this assay to characterize SAS-6, a centriolar protein that we identified based on its requirement for centrosome duplication. We show that SAS-6, a member of a conserved metazoan protein family, is specifically required for new centriole assembly, a result we confirm by electron microscopy. We further use the centriole assembly assay to examine the roles of three pericentriolar material proteins: SPD-5, the kinase aurora-A, and γ-tubulin. Our results suggest that the pericentriolar material promotes daughter centriole formation by concentrating γ-tubulin around the parent centriole. Thus, both centriolar and pericentriolar material proteins contribute to centriole assembly.

Centrosome duplication and separation are linked inextricably to certain cell cycle events, in particular activation of cyclin-dependent kinases (CDKs). However, relatively few CDK targets driving these events have been uncovered. Here, we have performed a screen for CDK substrates and have isolated a target, CP110, which is phosphorylated by CDKs in vitro and in vivo. Human CP110 localizes to centrosomes. Its expression is strongly induced at the G1-to-S phase transition, coincident with the initiation of centrosome duplication. RNAi-mediated depletion of CP110 indicates that this protein plays an essential role in centrosome duplication. Long-term disruption of CP110 phosphorylation leads to unscheduled centrosome separation and overt polyploidy. Our data suggest that CP110 is a physiological centrosomal CDK target that promotes centrosome duplication, and its deregulation may contribute to genomic instability.

Background: Centrosomes are the favored microtubule-organizing framework of eukaryotic cells. Centrosomes contain a pair of centrioles that normally duplicate once during the cell cycle to give rise to two mitotic spindle poles, each containing one old and one new centriole. However, aside from their role as an anchor point for pericentriolar material and as basal bodies of flagella and cilia, the functional attributes of centrioles remain enigmatic. Results: Here, using RNA interference, we demonstrate that "knockdown" of centrin-2, a protein of centrioles, results in failure of centriole duplication during the cell cycle in HeLa cells. Following inhibition of centrin-2 synthesis, the preexisting pair of centrioles separate, and functional bipolar spindles form with only one centriole at each spindle pole. Centriole dilution results from the ensuing cell division, and daughter cells are "born" with only a single centriole. Remarkably, these unicentriolar daughter cells may complete a second and even third bipolar mitosis in which spindle microtubules converge onto unusually broad spindle poles and in which cell division results in daughter cells containing either one or no centrioles at all. Cells thus denuded of the mature or both centrioles fail to undergo cytokinesis in subsequent cell cycles, give rise to multinucleate products, and finally die. Conclusions: These results demonstrate a requirement for centrin in centriole duplication and demonstrate that centrioles play a role in organizing spindle pole morphology and in the completion of cytokinesis.

Centrosomes of animal cells and spindle pole bodies of fungi are the major microtubule nucleating centers. Recent studies indicate that their capacity to organize microtubule arrays rests on elaborate control of the anchoring and release of the nucleated microtubules. Although common molecular mechanisms are likely to be involved in both cases, the centrosome from animal cells shows considerable complexity and flexibility, which contrasts with the simple laminar organization of spindle pole bodies in fungi. The role of the centriole pair in controlling both the structural stability and the activity of the centrosome in animal cells is now becoming clearer. The potential use of the generational asymmetry of centrosomes or spindle pole bodies for controlling cell polarity is also a growing theme.

Background: Centriole duplication is a key step in the cell cycle whose mechanism is completely unknown. Why new centrioles always form next to preexisting ones is a fundamental question. The simplest model is that preexisting centrioles nucleate the assembly of new centrioles, and that although centrioles can in some cases form de novo without this nucleation, the de novo assembly mechanism should be too slow to compete with normal duplication in order to maintain fidelity of centriole duplication. Results: We have measured the rate of de novo centriole assembly in vegetatively dividing cells that normally always contain centrioles. By using mutants of Chlamydomonas that are defective in centriole segregation, we obtained viable centrioleless cells that continue to divide, and find that within a single generation, 50% of these cells reacquire new centrioles by de novo assembly. This suggests that the rate of de novo assembly is approximately half the rate of templated duplication. A mutation in the VFL3 gene causes a complete loss of the templated assembly pathway without eliminating de novo assembly. A mutation in the centrin gene also reduced the rate of templated assembly. Conclusions: These results suggest that there are two pathways for centriole assembly, namely a templated pathway that requires preexisting centrioles to nucleate new centriole assembly, and a de novo assembly pathway that is normally turned off when centrioles are present.

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.