Search

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.

Polo kinase (PLK1 in mammals) is a master cell cycle regulator that is recruited to various subcellular structures, often by its polo-box domain (PBD), which binds to phosphorylated S-pS/pT motifs. Polo/PLK1 kinases have multiple functions at centrioles and centrosomes, and we have previously shown that in Drosophila phosphorylated Sas-4 initiates Polo recruitment to newly formed centrioles, while phosphorylated Spd-2 recruits Polo to the pericentriolar material (PCM) that assembles around mother centrioles in mitosis. Here, we show that Ana1 (Cep295 in humans) also helps to recruit Polo to mother centrioles in Drosophila. If Ana1-dependent Polo recruitment is impaired, mother centrioles can still duplicate, disengage from their daughters and form functional cilia, but they can no longer efficiently assemble mitotic PCM or elongate during G2. We conclude that Ana1 helps recruit Polo to mother centrioles to specifically promote mitotic centrosome assembly and centriole elongation in G2, but not centriole duplication, centriole disengagement or cilia assembly. This article has an associated First Person interview with the first author of the paper., Summary: The C-terminus of Ana1 helps recruit Polo kinase to centrioles. This is key for efficient PCM assembly and centriole elongation, and independent from other roles of Ana1 in centriole maturation.

The primary cilium is a ubiquitous, microtubule-based cellular organelle. Primary cilia dysfunction results in a group of disorders termed ciliopathies. C2 domain containing 3 centriole elongation regulator (C2cd3), encodes a centriolar protein essential for ciliogenesis. Mutations in human C2CD3 are associated with the human ciliopathy Oral-Facial-Digital syndrome type 14 (OFD14). In order to better understand the etiology of ciliopathies including OFD14, we generated numerous murine models targeting C2cd3. Initial analysis revealed several tissue-specific isoforms of C2cd3, and while the loss of C2cd3 has previously been reported to result in exencephaly, tight mesencephalic flexure, pericardial edema, abnormal heart looping and a twisted body axis, further analysis revealed that genetic background may also contribute to phenotypic variation. Additional analyses of a conditional allelic series targeting C-terminal PKC-C2 domains or the N-terminal C2CD3N-C2 domain of C2cd3 revealed a variable degree of phenotypic severity, suggesting that while the N-terminal C2CD3N-C2 domain was critical for early embryonic development as a whole, there was also a craniofacial specific role for the C2CD3N-C2 domains. Together, through generation of novel models and evaluation of C2cd3 expression, these data provide valuable insight into mechanisms of pathology for craniofacial ciliopathies that can be further explored in the future.

Centrosomal protein 120 (CEP120) was originally identified as a daughter centriole-enriched protein that participates in centriole elongation. Recent studies showed that CEP120 gene mutations cause complex ciliopathy phenotypes in humans, including Joubert syndrome and Jeune asphyxiating thoracic dystrophy, suggesting that CEP120 plays an additional role in ciliogenesis. To investigate the potential roles of CEP120 in centriole elongation and cilia formation, we knocked out the CEP120 gene in p53-deficient RPE1 cells using the CRISPR/Cas9 editing system, and performed various analyses. We herein report that loss of CEP120 produces short centrioles with no apparent distal and subdistal appendages. CEP120 knockout was also associated with defective centriole elongation, impaired recruitment of C2CD3 and Talpid3 to the distal ends of centrioles, and consequent defects in centriole appendage assembly and cilia formation. Interestingly, wild-type CEP120 interacts with C2CD3 and Talpid3, whereas a disease-associated CEP120 mutant (I975S) has a low affinity for C2CD3 binding and perturbs cilia assembly. Together, our findings reveal a novel role of CEP120 in ciliogenesis by showing that it interacts with C2CD3 and Talpid3 to assemble centriole appendages and by illuminating the molecular mechanism through which the CEP120 (I975S) mutation causes complex ciliopathies.

After centrosome duplication, centrioles elongate before M phase. To identify genes required for this process and to understand the regulatory mechanism, we investigated the centrioles in Drosophila premeiotic spermatocytes expressing fluorescently tagged centriolar proteins. We demonstrated that an essential microtubule polymerisation factor, Orbit (the Drosophila CLASP orthologue, encoded by chb), accumulated at the distal end of centrioles and was required for the elongation. Conversely, a microtubule-severing factor, Klp10A, shortened the centrioles. Genetic analyses revealed that these two proteins functioned antagonistically to determine centriole length. Furthermore, Cp110 in the distal tip complex was closely associated with the factors involved in centriolar dynamics at the distal end. We observed loss of centriole integrity, including fragmentation of centrioles and earlier separation of the centriole pairs, in Cp110-null mutant cells either overexpressing Orbit or depleted of Klp10A. Excess centriole elongation in the absence of the distal tip complex resulted in the loss of centriole integrity, leading to the formation of multipolar spindle microtubules emanating from centriole fragments, even when they were unpaired. Our findings contribute to understanding the mechanism of centriole integrity, disruption of which leads to chromosome instability in cancer cells., Summary: A microtubule polymerising factor, Orbit, localises on centrioles and determines centriole length by antagonising Klp10A in Drosophila spermatocytes.

Panda et al. find that Rcd4 is required for centriole duplication and ciliogenesis in neurosensory organs but dispensable for spermatogenesis, indicating tissue specific requirements for centriole formation. Loading of Rcd4 and its binding partner Ana3 onto daughter centrioles is a prerequisite for the mitotic conversion of centrioles into centrosomes., 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.

Polo kinase (PLK1) is a master cell cycle regulator that is recruited to various subcellular structures by its Polo-Box domain (PBD), which binds to phosphorylated S-pS/pT motifs. Polo has multiple functions at centrioles and centrosomes, and we previously showed that phosphorylated Sas-4 initiates Polo recruitment to newly formed centrioles, while phosphorylated Spd-2 recruits Polo to the mitotic Pericentriolar Material (PCM) that assembles around mother centrioles. Here, we investigate whether additional proteins recruit Polo to centrioles and/or centrosomes, and find that Ana1 (Cep295 in mammals) helps recruit Polo to mother centrioles. If this function is impaired, mother centrioles can still duplicate and disengage from their daughters, but they can no longer efficiently assemble a mitotic PCM or elongate their centrioles in G2. Thus, Ana1 is part of a sequential phosphorylation cascade that recruits Polo to centrioles to drive mitotic centrosome assembly and centriole elongation in G2, but not centriole duplication or disengagement.

Centrioles are precisely built microtubule-based structures that assemble centrosomes and cilia. Aberrations in centriole structure are common in tumors, yet how these aberrations arise is unknown. Analysis of centriole structure is difficult because it requires demanding electron microscopy. Here we employ expansion microscopy to study the origins of centriole structural aberrations in large populations of human cells. We discover that centrioles do not have an elongation monitoring mechanism, which renders them prone to over-elongation, especially during prolonged mitosis induced by various factors, importantly including supernumerary centrioles. We identify that mitotic centriole over-elongation is dependent on mitotic Polo-like kinase 1, which we uncover as a novel regulator of centriole elongation in human cycling cells. While insufficient Plk1 levels lead to the formation of shorter centrioles lacking a full set of microtubule triplets, its overactivity results in over-elongated and structurally aberrant centrioles. Our data help explain the origin of structurally aberrant centrioles and why centriole numerical and structural defects coexist in tumors.

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.

SUMMARYCentrioles are highly elaborate microtubule-based structures responsible for the formation of centrosomes and cilia. Despite considerable variation across species and tissues, within any given tissue their size is essentially constant [1, 2]. While the diameter of the centriole cylinder is set by the dimensions of the inner scaffolding structure of the cartwheel [3], how centriole length is set so precisely and stably maintained over many cell divisions is not well understood. Cep97 and CP110 are conserved proteins that localize to the distal end of centrioles and have been reported to limit centriole elongation in vertebrates [4, 5]. Here, we examine Cep97 function in Drosophila melanogaster. We show that Cep97 is essential for formation of full-length centrioles in multiple tissues of the fly. We further identify the microtubule deacetylase Sirt2 as a Cep97 proximity interactor. Deletion of Sirt2 likewise affects centriole size. Interestingly, so does deletion of the acetylase Atat1, indicating that loss of stabilizing acetyl marks impairs centriole integrity. Cep97 and CP110 were originally identified as inhibitors of cilia formation in vertebrate cultured cells [6] and loss of CP110 is a widely used marker of basal body maturation. In contrast, in Drosophila Cep97 is only transiently removed from basal bodies and loss of Cep97 strongly impairs ciliogenesis. Collectively, our results support a model whereby Cep97 functions as part of a protective cap that acts together with the microtubule acetylation machinery to maintain centriole stability, essential for proper function in cilium biogenesis.

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.

Most animal cells contain a structure called the centrosome, which plays a vital role in helping cells to divide for producing new cells. Early in the cell division process, cells make a copy of their centrosome. Each centrosome includes two cylindrical structures called centrioles encased in a complex web of other proteins. The centrioles must get longer for the duplication process to work correctly, but it is not clear which proteins help the centrioles to elongate. Previous work suggested that a protein called PPP1R35 might be a centrosome protein. To investigate its role, Sydor et al. performed experiments that reduced the amount of PPP1R35 in cells grown in the laboratory. Cells that contained fewer PPP1R35 proteins also contained fewer centrioles; these centrioles were also shorter and lacked some of the proteins that can elongate them. Super-resolution microscopy found PPP1R35 in the centre of the centrioles, in a region involved in the early stages of elongation. Sydor et al. also found that PPP1R35 interacts with a protein called RTTN, which is linked to centriole elongation. RTTN contributes to a condition called microcephaly, which prevents the brain from developing properly and results in individuals having a small head. Future work that builds on the findings presented by Sydor et al. could therefore help researchers to understand the causes of microcephaly in patients.

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.

Centrosomal P4.1-associated protein (CPAP) is a cell cycle regulated protein fundamental for centrosome assembly and centriole elongation. In humans, the region between residues 897–1338 of CPAP mediates interactions with other proteins and includes a homodimerization domain. CPAP mutations cause primary autosomal recessive microcephaly and Seckel syndrome. Despite of the biological/clinical relevance of CPAP, its mechanistic behavior remains unclear and its C-terminus (the G-box/TCP domain) is the only part whose structure has been solved. This situation is perhaps due in part to the challenges that represent obtaining the protein in a soluble, homogeneous state for structural studies. Our work constitutes a systematic structural analysis on multiple oligomers of HsCPAP897−1338, using single-particle electron microscopy (EM) of negatively stained (NS) samples. Based on image classification into clearly different regular 3D maps (putatively corresponding to dimers and tetramers) and direct observation of individual images representing other complexes of HsCPAP897−1338 (i.e., putative flexible monomers and higher-order multimers), we report a dynamic oligomeric behavior of this protein, where different homo-oligomers coexist in variable proportions. We propose that dimerization of the putative homodimer forms a putative tetramer which could be the structural unit for the scaffold that either tethers the pericentriolar material to centrioles or promotes procentriole elongation. A coarse fitting of atomic models into the NS 3D maps at resolutions around 20 Å is performed only to complement our experimental data, allowing us to hypothesize on the oligomeric composition of the different complexes. In this way, the current EM work represents an initial step toward the structural characterization of different oligomers of CPAP, suggesting further insights to understand how this protein works, contributing to the elucidation of control mechanisms for centriole biogenesis., This work was supported by the Comunidad de Madrid through grant CAM (S2010/BMD- 2305) and the Spanish Ministry of Economy and Competitiveness through Grants AIC-A-2011-0638 and BIO2013-44647-R.

The cilium is an elongated and continuous structure that spans two major subcellular domains. The cytoplasmic domain contains a short centriole, which serves to nucleate the main projection of the cilium. This projection, known as the axoneme, remains separated from the cytoplasm by a specialized gatekeeping complex within a ciliary subdomain called the transition zone. In this way, the axoneme is compartmentalized. Intriguingly, however, this general principle of cilium biology is altered in the sperm cells of many animals, which instead contain a cytoplasmic axoneme domain. Here, we discuss the hypothesis that the formation of specialized sperm giant centrioles and cytoplasmic cilia is mediated by the migration of the transition zone from its typical location as part of a structure known as the annulus, and examine the intrinsic properties of the transition zone that may facilitate its migratory behavior.

Oral–facial–digital syndromes (OFDS) gather rare genetic disorders characterised by facial, oral and digital abnormalities associated with a wide range of additional features (polycystic kidney disease, cerebral malformations and several others) to delineate a growing list of OFDS subtypes. The most frequent, OFD type I, is caused by a heterozygous mutation in theOFD1gene encoding a centrosomal protein. The wide clinical heterogeneity of OFDS suggests the involvement of other ciliary genes. For 15 years, we have aimed to identify the molecular bases of OFDS. This effort has been greatly helped by the recent development of whole-exome sequencing (WES). Here, we present all our published and unpublished results for WES in 24 cases with OFDS. We identified causal variants in five new genes (C2CD3,TMEM107,INTU,KIAA0753andIFT57) and related the clinical spectrum of four genes in other ciliopathies (C5orf42,TMEM138,TMEM231andWDPCP) to OFDS. Mutations were also detected in two genes previously implicated in OFDS. Functional studies revealed the involvement of centriole elongation, transition zone and intraflagellar transport defects in OFDS, thus characterising three ciliary protein modules: the complex KIAA0753-FOPNL-OFD1, a regulator of centriole elongation; the Meckel-Gruber syndrome module, a major component of the transition zone; and the CPLANE complex necessary for IFT-A assembly. OFDS now appear to be a distinct subgroup of ciliopathies with wide heterogeneity, which makes the initial classification obsolete. A clinical classification restricted to the three frequent/well-delineated subtypes could be proposed, and for patients who do not fit one of these three main subtypes, a further classification could be based on the genotype.

Centrioles are cylindrical structures that are usually composed of nine triplets of microtubules (MTs) organized around a cartwheel-shaped structure. Recent studies have proposed a structural model of the SAS-6-based cartwheel, yet we do not know the molecular detail of how the cartwheel participates in centriolar MT assembly. In this study, we demonstrate that the human microcephaly protein, CEP135, directly interacts with hSAS-6 via its carboxyl-terminus and with MTs via its amino-terminus. Unexpectedly, CEP135 also interacts with another microcephaly protein CPAP via its amino terminal domain. Depletion of CEP135 not only perturbed the centriolar localization of CPAP, but also blocked CPAP-induced centriole elongation. Furthermore, CEP135 depletion led to abnormal centriole structures with altered numbers of MT triplets and shorter centrioles. Overexpression of a CEP135 mutant lacking the proper interaction with hSAS-6 had a dominant-negative effect on centriole assembly. We propose that CEP135 may serve as a linker protein that directly connects the central hub protein, hSAS-6, to the outer MTs, and suggest that this interaction stabilizes the proper cartwheel structure for further CPAP-mediated centriole elongation.

CEP120 cooperates with CPAP to promote centriole elongation in a cell cycle– and microtubule-dependent manner., Centriole duplication begins with the formation of a single procentriole next to a preexisting centriole. CPAP (centrosomal protein 4.1–associated protein) was previously reported to participate in centriole elongation. Here, we show that CEP120 is a cell cycle–regulated protein that directly interacts with CPAP and is required for centriole duplication. CEP120 levels increased gradually from early S to G2/M and decreased significantly after mitosis. Forced overexpression of either CEP120 or CPAP not only induced the assembly of overly long centrioles but also produced atypical supernumerary centrioles that grew from these long centrioles. Depletion of CEP120 inhibited CPAP-induced centriole elongation and vice versa, implying that these proteins work together to regulate centriole elongation. Furthermore, CEP120 was found to contain an N-terminal microtubule-binding domain, a C-terminal dimerization domain, and a centriolar localization domain. Overexpression of a microtubule binding–defective CEP120-K76A mutant significantly suppressed the formation of elongated centrioles. Together, our results indicate that CEP120 is a CPAP-interacting protein that positively regulates centriole elongation.

Maintenance of normal levels of CP110 is essential to prevent over-duplication of centrosomes and genome instability; here, a deubiquitinating enzyme, USP33, is shown to stabilize CP110. Centrosomes function as microtubule-organizing centres in cells and coordinate spindle pole formation during mitosis. Centrosome duplication is critical for cell division, and genome instability can result if duplication is not restricted to a single round per cell cycle. This process is controlled by the CP110 protein, and maintenance of normal CP110 levels is essential to prevent over-duplication of centrosomes. This study identifies USP33 (ubiquitin specific protease 33) as a centrosomal deubiquitinating enzyme that acts to stabilize CP110 at centrioles. USP33 positively regulates centrosome duplication by regulating CP110 abundance. These findings raise the possibility that deubiquitinating enzymes like USP33 might be therapeutic targets in cancers associated with centrosome amplification and genomic instability. Centrosome duplication is critical for cell division, and genome instability can result if duplication is not restricted to a single round per cell cycle. Centrosome duplication is controlled in part by CP110, a centriolar protein that positively regulates centriole duplication while restricting centriole elongation and ciliogenesis. Maintenance of normal CP110 levels is essential, as excessive CP110 drives centrosome over-duplication and suppresses ciliogenesis, whereas its depletion inhibits centriole amplification and leads to highly elongated centrioles and aberrant assembly of cilia in growing cells1,2. CP110 levels are tightly controlled, partly through ubiquitination by the ubiquitin ligase complex SCFcyclin F during G2 and M phases of the cell cycle3. Here, using human cells, we report a new mechanism for the regulation of centrosome duplication that requires USP33, a deubiquitinating enzyme that is able to regulate CP110 levels. USP33 interacts with CP110 and localizes to centrioles primarily in S and G2/M phases, the periods during which centrioles duplicate and elongate. USP33 potently and specifically deubiquitinates CP110, but not other cyclin-F substrates. USP33 activity antagonizes SCFcyclin F-mediated ubiquitination and promotes the generation of supernumerary centriolar foci, whereas ablation of USP33 destabilizes CP110 and thereby inhibits centrosome amplification and mitotic defects. To our knowledge, we have identified the first centriolar deubiquitinating enzyme whose expression regulates centrosome homeostasis by countering cyclin-F-mediated destruction of a key substrate. Our results point towards potential therapeutic strategies for inhibiting tumorigenesis associated with centrosome amplification.

International audience; Background: Centrosomes have important roles in many aspects of cell organization, and aberrations in their number and function are associated with various diseases, including cancer. Centrosomes consist of a pair of centrioles surrounded by a pericentriolar matrix (PCM), and their replication is tightly regulated. Here, we investigate the effects of overexpressing the three proteins known to be required for centriole replication in Drosophila-DSas-6, DSas-4, and Sak. Results: By directly observing centriole replication in living Drosophila embryos, we show that the overexpression of GFP-DSas-6 can drive extra rounds of centriole replication within a single cell cycle. Extra centriole-like structures also accumulate in brain cells that overexpress either GFP-DSas-6 or GFP-Sak, but not DSas-4-GFP. No extra centrioles accumulate in spermatocytes that overexpress any of these three proteins. Most remarkably, the overexpression of any one of these three proteins results in the rapid de novo formation of many hundreds of centriole-like structures in unfertilized eggs, which normally do not contain centrioles. Conclusions: Our data suggest that the levels of centriolar DSas-6 determine the number of daughter centrioles formed during centriole replication. Overexpression of either DSas-6 or Sak can induce the formation of extra centrioles in some tissues but not others, suggesting that centriole replication is regulated differently in different tissues. The finding that the overexpression of DSas-4, DSas-6, or Sak can rapidly induce the de novo formation of centriole-like structures in Drosophila eggs suggests that this process results from the stabilization of centriole-precursors that are normally present in the egg.

Centriole duplication is a tightly ordered process during which procentrioles are assembled in G1-S and elongate during S and G2. Here, we show that human CEP295 (Drosophila Ana1) is not essential for initial cartwheel assembly, but is required to build distal half centrioles during S and G2. Using super-resolution and immunogold electron microscopy, we demonstrate that CEP295 is recruited to the proximal end of procentrioles in early S phase, when it is also localized at the centriolar microtubule wall that surrounds the human SAS6 cartwheel hub. Interestingly, depletion of CEP295 not only inhibits the recruitments of POC5 and POC1B to the distal half centrioles in G2, resulting in shorter centrioles, it also blocks the post-translational modification of centriolar microtubules (e.g. acetylation and glutamylation). Importantly, our results indicate that CEP295 directly interacts with microtubules, and that excess CEP295 could induce the assembly of overly long centrioles. Furthermore, exogenous expression of the N-terminal domain of CEP295 exerts a dominant-negative effect on centriole elongation. Collectively, these findings suggest that CEP295 is essential for building the distal half centrioles and for post-translational modification of centriolar microtubules., Highlighted Article: Human CEP295 interacts with microtubules and is essential for building the distal half centrioles and post-translational modification of centriolar microtubules.

CPAP--a gene mutated in primary microcephaly--is required for procentriole formation. Here we show that CPAP degradation and function is controlled by the poly(ADP-ribose) polymerase tankyrase 1. CPAP is PARsylated by tankyrase 1 in vitro and in vivo. Overexpression of tankyrase 1 leads to CPAP proteasomal degradation, preventing centriole duplication, whereas depletion of tankyrase 1 stabilizes CPAP in G1, generating elongated procentrioles and multipolarity. Tankyrase 1 localizes to centrosomes exclusively in G1, coinciding with CPAP degradation. Hence, tankyrase 1-mediated PARsylation regulates CPAP levels during the cell cycle to limit centriole elongation and ensure normal centrosome function.

Centriole duplication involves the growth of a procentriole next to the parental centriole. Mutations in STIL and CPAP/CENPJ cause primary microcephaly (MCPH). Here, we show that human STIL has an asymmetric localization to the daughter centriole and is required for procentriole formation. STIL levels oscillate during the cell cycle. Interestingly, STIL interacts directly with CPAP and forms a complex with hSAS6. A natural mutation of CPAP (E1235V) that causes MCPH in humans leads to significantly lower binding to STIL. Overexpression of STIL induced the formation of multiple procentrioles around the parental centriole. STIL depletion inhibited normal centriole duplication, Plk4-induced centriole amplification, and CPAP-induced centriole elongation, and resulted in a failure to localize hSAS6 and CPAP to the base of the nascent procentriole. Furthermore, hSAS6 depletion hindered STIL targeting to the procentriole, implying that STIL and hSAS6 are mutually dependent for their centriolar localization. Together, our results indicate that the two MCPH-associated proteins STIL and CPAP interact with each other and are required for procentriole formation, implying a central role of centriole biogenesis in MCPH.

Centrobin recruitment to the centriole biogenesis site and its function during elongation and stabilization of centrioles depend on tubulin interaction., Centrobin is a daughter centriole protein that is essential for centrosome duplication. However, the molecular mechanism by which centrobin functions during centriole duplication remains undefined. In this study, we show that centrobin interacts with tubulin directly, and centrobin–tubulin interaction is pivotal for the function of centrobin during centriole duplication. We found that centrobin is recruited to the centriole biogenesis site via its interaction with tubulins during the early stage of centriole biogenesis, and its recruitment is dependent on hSAS-6 but not centrosomal P4.1–associated protein (CPAP) and CP110. The function of centrobin is also required for the elongation of centrioles, which is likely mediated by its interaction with tubulin. Furthermore, disruption of centrobin–tubulin interaction led to destabilization of existing centrioles and the preformed procentriole-like structures induced by CPAP expression, indicating that centrobin–tubulin interaction is critical for the stability of centrioles. Together, our study demonstrates that centrobin facilitates the elongation and stability of centrioles via its interaction with tubulins.

This report shows that inhibition of proteolysis can cause abnormal centriole elongation and identifies several centrosome proteins involved in this process by using a small interfering RNA screen., The centrosome is the major microtubule-organizing center of most mammalian cells and consists of a pair of centrioles embedded in pericentriolar material. Before mitosis, the two centrioles duplicate and two new daughter centrioles form adjacent to each preexisting maternal centriole. After initiation of daughter centriole synthesis, the procentrioles elongate in a process that is poorly understood. Here, we show that inhibition of cellular proteolysis by Z-L3VS or MG132 induces abnormal elongation of daughter centrioles to approximately 4 times their normal length. This activity of Z-L3VS or MG132 was found to correlate with inhibition of intracellular protease-mediated substrate cleavage. Using a small interfering RNA screen, we identified a total of nine gene products that either attenuated (seven) or promoted (two) abnormal Z-L3VS–induced daughter centriole elongation. Our hits included known regulators of centriole length, including CPAP and CP110, but, interestingly, several proteins involved in microtubule stability and anchoring as well as centrosome cohesion. This suggests that nonproteasomal functions, specifically inhibition of cellular proteases, may play an important and underappreciated role in the regulation of centriole elongation. They also highlight the complexity of daughter centriole length control and provide a framework for future studies to dissect the molecular details of this process.

Centrioles are found in the centrosome core and, as basal bodies, at the base of cilia and flagella. Centriole assembly and duplication is controlled by Polo-like-kinase 4 (Plk4): these processes fail if Plk4 is downregulated and are promoted by Plk4 overexpression. Here we show that the centriolar protein Asterless (Asl; human orthologue CEP152) provides a conserved molecular platform, the amino terminus of which interacts with the cryptic Polo box of Plk4 whereas the carboxy terminus interacts with the centriolar protein Sas-4 (CPAP in humans). Drosophila Asl and human CEP152 are required for the centrosomal loading of Plk4 in Drosophila and CPAP in human cells, respectively. Depletion of Asl or CEP152 caused failure of centrosome duplication; their overexpression led to de novo centriole formation in Drosophila eggs, duplication of free centrosomes in Drosophila embryos, and centrosome amplification in cultured Drosophila and human cells. Overexpression of a Plk4-binding-deficient mutant of Asl prevented centriole duplication in cultured cells and embryos. However, this mutant protein was able to promote microtubule organizing centre (MTOC) formation in both embryos and oocytes. Such MTOCs had pericentriolar material and the centriolar protein Sas-4, but no centrioles at their core. Formation of such acentriolar MTOCs could be phenocopied by overexpression of Sas-4 in oocytes or embryos. Our findings identify independent functions for Asl as a scaffold for Plk4 and Sas-4 that facilitates self-assembly and duplication of the centriole and organization of pericentriolar material.

Control of centrosome duplication is tightly linked with the progression of the cell cycle. Recent studies suggest that the fundamental process of centriole duplication is evolutionally conserved. Here, we identified centrosomal P4.1-associated protein (CPAP), a human homologue of SAS-4, as a substrate of PLK2 whose activity oscillates during the cell cycle. PLK2 phosphorylates the S589 and S595 residues of CPAP in vitro and in vivo. This phosphorylation is critical for procentriole formation during the centrosome cycle. PLK4 also phosphorylates S595 of CPAP, but PLK4 phosphorylation is not a critical step in the PLK4 function in procentriole assembly. CPAP is phosphorylated in a cell cycle stage-specific manner, so that its phosphorylation increases at the G1/S transition phase and decreases during the exit of mitosis. Phosphorylated CPAP is preferentially located at the procentriole. Furthermore, overexpression of a phospho-resistant CPAP mutant resulted in the failure to form elongated centrioles. On the basis of these results, we propose that phosphorylated CPAP is involved in procentriole assembly, possibly for centriole elongation. This work demonstrates an example of how procentriole formation is linked to the progression of the cell cycle.

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.

SummaryCPAP is required for centriole elongation during S/G2 phase, but the role of CPAP in mitosis is incompletely understood. Here, we show that CPAP maintains spindle pole integrity through its phosphorylation by Aurora-A during mitosis. Depletion of CPAP induced a prolonged delay in mitosis, pericentriolar material (PCM) dispersion, and multiple mitotic abnormalities. Further studies demonstrated that CPAP directly interacts with and is phosphorylated by Aurora-A at serine 467 during mitosis. Interestingly, the dispersal of the PCM was effectively rescued by ectopic expression of wild-type CPAP or a phospho-mimic CPAP-S467D mutant, but not a non-phosphorylated CPAP-S467A mutant. Finally, we found that CPAP-S467D has a low affinity for microtubule binding but a high affinity for PCM proteins. Together, our results support a model wherein CPAP is required for proper mitotic progression, and phosphorylation of CPAP by Aurora-A is essential for maintaining spindle pole integrity.

Aurora A kinase plays an important role in several aspects of cell division, including centrosome maturation and separation, a crucial step for the correct organization of the bipolar spindle. Although it has long been showed that this kinase accumulates at the centrosome throughout mitosis its precise contribution to centriole biogenesis and structure has until now not been reported. It is not surprising that so little is known, due to the small size of somatic centrioles, where only dramatic structural changes may be identified by careful electron microscopy analysis. Conversely, centrioles of Drosophila primary spermatocytes increase tenfold in length during the first prophase, thus making any change easily detectable. Therefore, we examined the consequence of the pharmacological inhibition of Aurora A by MLN8054 on centriole biogenesis during early Drosophila gametogenesis. Here, we show that depletion of this kinase results in longer centrioles, mainly during transition from prophase to prometaphase of the first meiosis. We also found abnormal ciliogenesis characterized by irregularly growing axonemal doublets. Our results represent the first documentation of a potential requirement of Aurora A in centriole integrity and elongation.

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.

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.

It has been reported that nontransformed mammalian cells become arrested during G1 in the absence of centrioles (Hinchcliffe, E., F. Miller, M. Cham, A. Khodjakov, and G. Sluder. 2001. Science. 291:1547–1550). Here, we show that removal of resident centrioles (by laser ablation or needle microsurgery) does not impede cell cycle progression in HeLa cells. HeLa cells born without centrosomes, later, assemble a variable number of centrioles de novo. Centriole assembly begins with the formation of small centrin aggregates that appear during the S phase. These, initially amorphous “precentrioles” become morphologically recognizable centrioles before mitosis. De novo–assembled centrioles mature (i.e., gain abilities to organize microtubules and replicate) in the next cell cycle. This maturation is not simply a time-dependent phenomenon, because de novo–formed centrioles do not mature if they are assembled in S phase–arrested cells. By selectively ablating only one centriole at a time, we find that the presence of a single centriole inhibits the assembly of additional centrioles, indicating that centrioles have an activity that suppresses the de novo pathway.

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.

Centrioles are microtubule-based, barrel-shaped structures that initiate the assembly of centrosomes and cilia(1,2). How centriole length is precisely set remains elusive. The microcephaly protein CPAP (also known as MCPH6) promotes procentriole growth(3-5), whereas the oral-facial-digital (OFD) syndrome protein OFD1 represses centriole elongation(6,7). Here we uncover a new subtype of OFD with severe microcephaly and cerebral malformations and identify distinct mutations in two affected families in the evolutionarily conserved C2CD3 gene. Concordant with the clinical overlap, C2CD3 colocalizes with OFD1 at the distal end of centrioles, and C2CD3 physically associates with OFD1. However, whereas OFD1 deletion leads to centriole hyperelongation, loss of C2CD3 results in short centrioles without subdistal and distal appendages. Because C2CD3 overexpression triggers centriole hyperelongation and OFD1 antagonizes this activity, we propose that C2CD3 directly promotes centriole elongation and that OFD1 acts as a negative regulator of C2CD3. Our results identify regulation of centriole length as an emerging pathogenic mechanism in ciliopathies.

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.

Centrosomes were microsurgically removed from BSC-1 African green monkey kidney cells before the completion of S phase. Karyoplasts (acentrosomal cells) entered and completed mitosis. However, postmitotic karyoplasts arrested before S phase, whereas adjacent control cells divided repeatedly. Postmitotic karyoplasts assembled a microtubule-organizing center containing γ-tubulin and pericentrin, but did not regenerate centrioles. These observations reveal the existence of an activity associated with core centrosomal structures—distinct from elements of the microtubule-organizing center—that is required for the somatic cell cycle to progress through G 1 into S phase. Once the cell is in S phase, these core structures are not needed for the G 2 -M phase transition.

As an organelle coupling nuclear and cytoplasmic divisions, the centrosome is essential to mitotic fidelity, and its inheritance could be critical to understanding cell transformation. Investigating the behavior of the centrosome in living mitotic cells, we documented a transient and remarkable postanaphase repositioning of this organelle, which apparently controls the release of central microtubules from the midbody and the completion of cell division. We also observed that the absence of the centrosome leads to defects in cytokinesis. Together with recent results in yeasts, our data point to a conserved centrosome-dependent pathway that integrates spatial controls into the decision of completing cell division, which requires the repositioning of the centrosome organelle.

The novel concept of a centrosomal anchoring complex, which is distinct from the gamma-tubulin nucleating complex, has previously been proposed following studies on cochlear epithelial cells. In this investigation we present evidence from two different cell systems which suggests that the centrosomal protein ninein is a strong candidate for the proposed anchoring complex. Ninein has recently been observed in cultured fibroblast cells to localise primarily to the post-mitotic mother centriole, which is the focus for a classic radial microtubule array. We show here by immunoelectron microscopical analyses of centrosomes from mouse L929 cells that ninein concentrates at the appendages surrounding the mother centriole and at the microtubule minus-ends. We further show that localisation of ninein in the cochlear supporting epithelial cells, where the vast majority of the microtubule minus-ends are associated with apical non-centrosomal sites, suggests that it is not directly involved in microtubule nucleation. Ninein seems to play an important role in the positioning and anchorage of the microtubule minus-ends in these epithelial cells. Evidence is presented which suggests that ninein is released from the centrosome, translocated with the microtubules, and is responsible for the anchorage of microtubule minus-ends to the apical sites. We propose that ninein is a non-nucleating microtubule minus-end associated protein which may have a dual role as a minus-end capping and anchoring protein.