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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.

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.

Over the past 100 years, the centrosome has risen in status from an enigmatic organelle, located at the focus of microtubules, to a key player in cell-cycle progression and cellular control. A growing body of evidence indicates that centrosomes might not be essential for spindle assembly, whereas recent data indicate that they might be important for initiating S phase and completing cytokinesis. Molecules that regulate centrosome duplication have been identified, and the expanding list of intriguing centrosome-anchored activities, the functions of which have yet to be determined, promises continued discovery.

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.

The abnormally high number of centrosomes found in many human tumor cells can lead directly to aneuploidy and genomic instability through the formation of multipolar mitotic spindles. To facilitate investigation of the mechanisms that control centrosome reproduction, a frog egg extract arrested in S phase of the cell cycle that supported repeated assembly of daughter centrosomes was developed. Multiple rounds of centrosome reproduction were blocked by selective inactivation of cyclin-dependent kinase 2–cyclin E (Cdk2-E) and were restored by addition of purified Cdk2-E. Confocal immunomicroscopy revealed that cyclin E was localized at the centrosome. These results demonstrate that Cdk2-E activity is required for centrosome duplication during S phase and suggest a mechanism that could coordinate centrosome reproduction with cycles of DNA synthesis and mitosis.

Glutamylation is the major posttranslational modification of neuronal and axonemal tubulin and is restricted predominantly to centrioles in nonneuronal cells (Bobinnec, Y., M. Moudjou, J.P. Fouquet, E. Desbruyères, B. Eddé, and M. Bornens. 1998. Cell Motil. Cytoskel. 39:223–232). To investigate a possible relationship between the exceptional stability of centriole microtubules and the compartmentalization of glutamylated isoforms, we loaded HeLa cells with the monoclonal antibody GT335, which specifically reacts with polyglutamylated tubulin. The total disappearance of the centriole pair was observed after 12 h, as judged both by immunofluorescence labeling with specific antibodies and electron microscopic observation of cells after complete thick serial sectioning. Strikingly, we also observed a scattering of the pericentriolar material (PCM) within the cytoplasm and a parallel disappearance of the centrosome as a defined organelle. However, centriole disappearance was transient, as centrioles and discrete centrosomes ultimately reappeared in the cell population.During the acentriolar period, a large proportion of monopolar half-spindles or of bipolar spindles with abnormal distribution of PCM and NuMA were observed. However, as judged by a quasinormal increase in cell number, these cells likely were not blocked in mitosis.Our results suggest that a posttranslational modification of tubulin is critical for long-term stability of centriolar microtubules. They further demonstrate that in animal cells, centrioles are instrumental in organizing centrosomal components into a structurally stable organelle.

Autosomal recessive primary microcephaly is a potential model in which to research genes involved in human brain growth. We show that two forms of the disorder result from homozygous mutations in the genes CDK5RAP2 and CENPJ. We found neuroepithelial expression of the genes during prenatal neurogenesis and protein localization to the spindle poles of mitotic cells, suggesting that a centrosomal mechanism controls neuron number in the developing mammalian brain.

The centrosome plays a vital role in mitotic fidelity, ensuring establishment of bipolar spindles and balanced chromosome segregation. Centrosome duplication occurs only once during the cell cycle and is therefore highly regulated. Here, it is shown that in mouse embryonic fibroblasts (MEFs) lacking the p53 tumor suppressor protein, multiple copies of functionally competent centrosomes are generated during a single cell cycle. In contrast, MEFs prepared from normal mice or mice deficient in the retinoblastoma tumor suppressor gene product do not display these abnormalities. The abnormally amplified centrosomes profoundly affect mitotic fidelity, resulting in unequal segregation of chromosomes. These observations implicate p53 in the regulation of centrosome duplication and suggest one possible mechanism by which the loss of p53 may cause genetic instability.

Relatively little is known about the mechanisms used by somatic cells to regulate the replication of the centrosome complex. Centrosome doubling was studied in CHO cells by electron microscopy and immunofluorescence microscopy using human autoimmune anticentrosome antiserum, and by Northern blotting using the cDNA encoding portion of the centrosome autoantigen pericentriolar material (PCM)-1. Centrosome doubling could be dissociated from cycles of DNA synthesis and mitotic division by arresting cells at the G1/S boundary of the cell cycle using either hydroxyurea or aphidicolin. Immunofluorescence micros-copy using SPJ human autoimmune anticentrosome antiserum demonstrated that arrested cells were able to undergo numerous rounds of centrosome replication in the absence of cycles of DNA synthesis and mitosis. Northern blot analysis demonstrated that the synthesis and degradation of the mRNA encoding PCM-1 occurred in a cell cycle-dependent fashion in CHO cells with peak levels of PCM-1 mRNA being present in G1 and S phase cells before mRNA amounts dropped to undetectable levels in G2 and M phases. Conversely, cells arrested at the G1/S boundary of the cell cycle maintained PCM-1 mRNA at artificially elevated levels, providing a possible molecular mechanism for explaining the multiple rounds of centrosome replication that occurred in CHO cells during prolonged hydroxyurea-induced arrest. The capacity to replicate centrosomes could be abolished in hydroxyurea-arrested CHO cells by culturing the cells in dialyzed serum. However, the ability to replicate centrosomes and to synthesize PCM-1 mRNA could be re-initiated by adding EGF to the dialyzed serum. This experimental system should be useful for investigating the positive and negative molecular mechanisms used by somatic cells to regulate the replication of centrosomes and for studying and the methods used by somatic cells for coordinating centrosome duplication with other cell cycle progression events.

THE STRUCTURE OF THE CENTROSOME . . . . . . . . . . . . . . . . . . . . . . . . 641 The Centriole Is a Complicated Structure of Unknown Function .. . . . .. . . . 642 The Centrosome Undergoes Characteristic Structural Changes during the Cell Cycle .. ..... .... ...... ..... ... ..... ... . ... . 643 The Animal Cell Centrosome and the Spindle Pole Body of Yeast Are Structurally Distinct, but Functionally Similar . . . . . . . . . . . . . 643

Klp10A is a kinesin-13 of Drosophila melanogaster that depolymerizes cytoplasmic microtubules [1]. In interphase, it promotes microtubule catastrophe [2, 3, 4]; in mitosis, it contributes to anaphase chromosome movement by enabling tubulin flux [1, 5]. Here we show that Klp10A also acts as a microtubule depolymerase on centriolar microtubules to regulate centriole length. Thus, in both cultured cell lines and the testes, absence of Klp10A leads to longer centrioles that show incomplete 9-fold symmetry at their ends. These structures and associated pericentriolar material undergo fragmentation. We also show that in contrast to mammalian cells where depletion of CP110 leads to centriole elongation [6], in Drosophila cells it results in centriole length diminution that is overcome by codepletion of Klp10A to give longer centrioles than usual. We discuss how loss of centriole capping by CP110 might have different consequences for centriole length in mammalian [6, 7, 8] and insect cells and also relate these findings to the functional interactions between mammalian CP110 and another kinesin-13, Kif24, that in mammalian cells regulates cilium formation.

SUMMARYCentrosomes and their component centrioles represent the principal microtubule organizing centers of animal cells. Here we show that the gene underlying Orofaciodigital Syndrome 1, Ofd1, is a component of the distal centriole that controls centriole length. In the absence of Ofd1, distal regions of centrioles, but not procentrioles, elongate abnormally. These long centrioles are structurally similar to normal centrioles, but contain destabilized microtubules with abnormal post-translational modifications. Ofd1 is also important for centriole distal appendage formation and centriolar recruitment of the intraflagellar transport protein Ift88. To model OFD1 Syndrome in embryonic stem cells, we replaced the Ofd1 gene with missense alleles from human OFD1 patients. Distinct disease-associated mutations cause different degrees of excessive or decreased centriole elongation, all of which are associated with diminished ciliogenesis. Our results indicate that Ofd1 acts at the distal centriole to build distal appendages, recruit Ift88, and stabilize centriolar microtubules at a defined length.

SummaryThe centrosome is the principal microtubule organizing center (MTOC) of animal cells [1]. Accurate centrosome duplication is fundamental for genome integrity and entails the formation of one procentriole next to each existing centriole, once per cell cycle. The procentriole then elongates to eventually reach the same size as the centriole. The mechanisms that govern elongation of the centriolar cylinder and their potential relevance for cell division are not known. Here, we show that the SAS-4-related protein CPAP [2] is required for centrosome duplication in cycling human cells. Furthermore, we demonstrate that CPAP overexpression results in the formation of abnormally long centrioles. This also promotes formation of more than one procentriole in the vicinity of such overly long centrioles, eventually resulting in the presence of supernumerary MTOCs. This in turn leads to multipolar spindle assembly and cytokinesis defects. Overall, our findings suggest that centriole length must be carefully regulated to restrict procentriole number and thus ensure accurate cell division.

In addition to their role in centrosome organization, the centrioles have another distinct function as basal bodies for the formation of cilia and flagella. Centriole duplication has been reported to require two alternate assembly pathways: template or de novo. Since spermiogenesis in the termite Mastotermes darwiniensis lead to the formation of multiflagellate sperm, this process represents a useful model system in which to follow basal body formation and flagella assembly. We present evidence of a possible de novo pathway for basal body formation in the differentiating germ cell. This cell also contains typical centrosomal proteins, such as centrosomin, pericentrin-like protein, γ-tubulin, that undergo redistribution as spermatid differentiation proceeds. The spermatid centrioles are long structures formed by nine doublet rather than triplet microtubules provided with short projections extending towards the surrounding cytoplasm and with links between doublets. The sperm basal bodies are aligned in parallel beneath the nucleus. They consist of long regions close to the nucleus showing nine doublets in a cartwheel array devoid of any projections; on the contrary, the short region close to the plasma membrane, where the sperm flagella emerge, is characterized by projections similar to those observed in the centrioles linking the basal body to the plasma membrane. It is hypothesized that this appearance is in connection with the centriole elongation and further with the flagellar axonemal organization. Microtubule doublets of sperm flagellar axonemes are provided with outer dynein arms, while inner arms are rarely visible. Cell Motil. Cytoskeleton 2009. © 2009 Wiley-Liss, Inc.

We previously identified a novel centrosomal protein CPAP, which carries a 112-residue motif that is essential for microtubule destabilization. In this report, we define both the microtubule (MT) binding and destabilizing domains in human CPAP and analyze the mutations that affect its MT-destabilizing activity. Analysis of a series of CPAP truncated proteins showed that the MT-binding domain (MBD; residues 423-607) of CPAP is located next to its MT-destabilizing domain (MDD; residues 311-422). Site-specific mutagenesis revealed that the mutations that either disrupt the alpha-helical structure (Y341P, I346P, L348P, and triple-P) or alter the charge property (KR377EE) of the MDD significantly affect its MT-destabilizing ability. The activity for binding to a tubulin heterodimer was also significantly reduced in KR377EE mutant. Furthermore, we have analyzed the putative function of Drosophila d-SAS-4, a distant relative of human CPAP, which shares a conserved approximately 20-aa sequence with the MDD of CPAP. Our results show that mutations in this conserved sequence also eliminate d-SAS-4's MT-destabilizing activity, suggesting that d-SAS-4 and CPAP may play similar roles within cells.

Centrioles are surrounded by pericentriolar material (PCM), which is proposed to promote new centriole assembly by concentrating γ-tubulin. Here, we quantitatively monitor new centriole assembly in living Caenorhabditis elegans embryos, focusing on the conserved components SAS-4 and SAS-6. We show that SAS-4 and SAS-6 are coordinately recruited to the site of new centriole assembly and reach their maximum levels during S phase. Centriolar SAS-6 is subsequently reduced by a mechanism intrinsic to the early assembly pathway that does not require progression into mitosis. Centriolar SAS-4 remains in dynamic equilibrium with the cytoplasmic pool until late prophase, when it is stably incorporated in a step that requires γ-tubulin and microtubule assembly. These results indicate that γ-tubulin in the PCM stabilizes the nascent daughter centriole by promoting microtubule addition to its outer wall. Such a mechanism may help restrict new centriole assembly to the vicinity of preexisting parent centrioles that recruit PCM.

The centrosome has evolved in multicellular organisms from the basal body/axoneme of the unicellular ancestor ([Azimzadeh and Bornens, 2004][1]). It plays a major role in organizing the microtubule cytoskeleton in animal cells. During interphase, the centrosome organizes an astral array of

Centrioles duplicate once in each cell division cycle through so-called templated or canonical duplication. SAK, also called PLK4 (SAK/PLK4), a kinase implicated in tumor development, is an upstream regulator of canonical biogenesis necessary for centriole formation. We found that overexpression of SAK/PLK4 could induce amplification of centrioles in Drosophila embryos and their de novo formation in unfertilized eggs. Both processes required the activity of DSAS-6 and DSAS-4, two molecules required for canonical duplication. Thus, centriole biogenesis is a template-free self-assembly process triggered and regulated by molecules that ordinarily associate with the existing centriole. The mother centriole is not a bona fide template but a platform for a set of regulatory molecules that catalyzes and regulates daughter centriole assembly.

We show that overexpression of Polo-like kinase 4 (Plk4) in human cells induces centrosome amplification through the simultaneous generation of multiple procentrioles adjoining each parental centriole. This provided an opportunity for dissecting centriole assembly and characterizing assembly intermediates. Critical components were identified and ordered into an assembly pathway through siRNA and localized through immunoelectron microscopy. Plk4, hSas-6, CPAP, Cep135, gamma-tubulin, and CP110 were required at different stages of procentriole formation and in association with different centriolar structures. Remarkably, hSas-6 associated only transiently with nascent procentrioles, whereas Cep135 and CPAP formed a core structure within the proximal lumen of both parental and nascent centrioles. Finally, CP110 was recruited early and then associated with the growing distal tips, indicating that centrioles elongate through insertion of alpha-/beta-tubulin underneath a CP110 cap. Collectively, these data afford a comprehensive view of the assembly pathway underlying centriole biogenesis in human cells.

Cilia formation starts at the mother centriole and requires the activity of the microtubule affinity regulator, MARK4, which promotes axoneme extension during the initial phase of ciliogenesis. The defective axoneme extension in MARK4 depleted cells could be in part rescued by co-depletion of the inhibitory complex CP110/Cep97. Whether MARK4 only influences CP110/Cep97 or influences additional inhibitory components is not known. Interestingly, MARK4 has been recently shown to regulate the position and movement of autophagic vesicles within the cell. Recent studies also revealed that autophagy promotes ciliogenesis by inducing the selective degradation of the centriolar satellite pool of OFD1, an inhibitor of centriole elongation and axoneme extension. Here, we investigated whether MARK4 is functionally linked to autophagy to promote cilia formation. Analysis of RPE1 cells stably expressing YFP-LC3 shows that autophagosomes exhibit a perinuclear clustering upon MARK4 depletion. Quantitative immunofluorescence analysis shows that in MARK4 depleted cells, OFD1 levels at the centriolar satellites are higher than in control cells. This could be reverted by partial knock down of OFD1. By co-depleting MARK4 and OFD1, the cilia loss phenotype of MARK4 knockdown was partially reverted. Our results suggest that MARK4 acts in ciliogenesis by regulating the movement and position of the autophagosomes that degrade OFD1 at the centriolar satelites.

Centrioles are necessary for flagella and cilia formation, cytokinesis, cell-cycle control and centrosome organization/spindle assembly. They duplicate once per cell cycle, but the mechanisms underlying their duplication remain unclear. Here we show using electron tomography of staged C. elegans one-cell embryos that daughter centriole assembly begins with the formation and elongation of a central tube followed by the peripheral assembly of nine singlet microtubules. Tube formation and elongation is dependent on the SAS-5 and SAS-6 proteins, whereas the assembly of singlet microtubules onto the central tube depends on SAS-4. We further show that centriole assembly is triggered by an upstream signal mediated by SPD-2 and ZYG-1. These results define a structural pathway for the assembly of a daughter centriole and should have general relevance for future studies on centriole assembly in other organisms.

The centrosome is duplicated just once per cell cycle so that there is one centrosome at the beginning of the cell cycle and two in mitosis. Defects in this process can cause genome instability and even cancer. An in vitro study points to a strikingly similarity between the way cells control centrosome duplication and chromosome segregation. The pair of centrioles in the centrosome disengage at the end of mitosis, a process requiring separase, a protease involved in sister chromatid separation at mitosis. The separated centrioles are a substrate for duplication in the next cell cycle. Newly duplicated centrioles are paired, so they cannot duplicate again until passage through mitosis, when they are freed to duplicate by separase-mediated disengagement. This work reveals a common mechanism behind the two cellular processes that result in exact duplication once per cycle. Centrosome duplication needs to be tightly controlled to occur once and only once per cell cycle. The regulated timing of centriole disengagement in late mitosis is shown to allow one round of centrosome duplication in the next cycle. The centrosome organizes the microtubule cytoskeleton and consists of a pair of centrioles surrounded by pericentriolar material. Cells begin the cell cycle with a single centrosome, which duplicates once before mitosis. During duplication, new centrioles grow orthogonally to existing ones and remain engaged (tightly opposed) with those centrioles until late mitosis or early G1 phase, when they become disengaged1. The relationship between centriole engagement/disengagement and centriole duplication potential is not understood, and the mechanisms that control these processes are not known. Here we show that centriole disengagement requires the protease separase2 at anaphase, and that this disengagement licences centriole duplication in the next cell cycle. We describe an in vitro system using Xenopus egg extract and purified centrioles in which both centriole disengagement and centriole growth occur. Centriole disengagement at anaphase is independent of mitotic exit and Cdk2/cyclin E activity, but requires the anaphase-promoting complex and separase. In contrast to disengagement, new centriole growth occurs in interphase, is dependent on Cdk2/cyclin E, and requires previously disengaged centrioles. This suggests that re-duplication of centrioles within a cell cycle is prevented by centriole engagement itself. We propose that the ‘once-only’ control of centrosome duplication is achieved by temporally separating licensing in anaphase from growth of new centrioles during S phase. The involvement of separase in both centriole disengagement and sister chromatid separation would prevent premature centriole disengagement before anaphase onset, which can lead to multipolar spindles and genomic instability3,4.

Centrioles and centrosomes have an important role in animal cell organization, but it is uncertain to what extent they are essential for animal development. The Drosophila protein DSas-4 is related to the human microcephaly protein CenpJ and the C. elegans centriolar protein Sas-4. We show that DSas-4 is essential for centriole replication in flies. DSas-4 mutants start to lose centrioles during embryonic development, and, by third-instar larval stages, no centrioles or centrosomes are detectable. Mitotic spindle assembly is slow in mutant cells, and approximately 30% of the asymmetric divisions of larval neuroblasts are abnormal. Nevertheless, mutant flies develop with near normal timing into morphologically normal adults. These flies, however, have no cilia or flagella and die shortly after birth because their sensory neurons lack cilia. Thus, centrioles are essential for the formation of centrosomes, cilia, and flagella, but, remarkably, they are not essential for most aspects of Drosophila development.

Outer dense fibre 2 (Odf2; also known as cenexin) was initially identified as a main component of the sperm tail cytoskeleton, but was later shown to be a general scaffold protein that is specifically localized at the distal/subdistal appendages of mother centrioles. Here we show that Odf2 expression is suppressed in mouse F9 cells when both alleles of Odf2 genes are deleted. Unexpectedly, the cell cycle of Odf2(-/-) cells does not seem to be affected. Immunofluorescence and ultrathin-section electron microscopy reveals that in Odf2(-/-) cells, distal/subdistal appendages disappear from mother centrioles, making it difficult to distinguish mother from daughter centrioles. In Odf2(-/-) cells, however, the formation of primary cilia is completely suppressed, although approximately 25% of wild-type F9 cells are ciliated under the steady-state cell cycle. The loss of primary cilia in Odf2(-/-) F9 cells can be rescued by exogenous Odf2 expression. These findings indicate that Odf2 is indispensable for the formation of distal/subdistal appendages and the generation of primary cilia, but not for other cell-cycle-related centriolar functions.

Summary Background SAK/PLK4 is a distinct member of the polo-like kinase family. SAK −/− mice die during embryogenesis, whereas SAK +/− mice develop liver and lung tumors and SAK +/− MEFs show mitotic abnormalities. However, the mechanism underlying these phenotypes is still not known. Results Here, we show that downregulation of SAK in Drosophila cells, by mutation or RNAi, leads to loss of centrioles, the core structures of centrosomes. Such cells are able to undergo repeated rounds of cell division, but display broad disorganized mitotic spindle poles. We also show that SAK mutants lose their centrioles during the mitotic divisions preceding male meiosis but still produce cysts of 16 primary spermatocytes as in the wild-type. Mathematical modeling of the stereotyped cell divisions of spermatogenesis can account for such loss by defective centriole duplication. The majority of spermatids in SAK mutants lack centrioles and so are unable to make sperm axonemes. Finally, we show that depletion of SAK in human cells also prevents centriole duplication and gives rise to mitotic abnormalities. Conclusions SAK/PLK4 is necessary for centriole duplication both in Drosophila and human cells. Drosophila cells tolerate the lack of centrioles and undertake mitosis but cannot form basal bodies and hence flagella. Human cells depleted of SAK show error-prone mitosis, likely to underlie its tumor-suppressor role.

How do cells regulate centrosome number? A canonical duplication cycle generates two centrosomes from one in most proliferating cells. Centrioles are key to this process, and molecules such as centrins, SAS-4 and ZYG-1 govern daughter centriole formation. Cdk2 activity probably couples centrosome duplication with the S phase, and a licensing mechanism appears to limit centrosome duplication to once per cell cycle. However, such mechanisms must be altered in some cells – for example, spermatocytes – in which centrosome duplication and DNA replication are uncoupled. There are also alternative pathways of centrosome biogenesis. For example, one centrosome is reconstituted from two gametes at fertilization; in this case, the most common strategy involves differential contributions of centrioles and pericentriolar material (PCM) from each gamete. Furthermore, centrioles can sometimes form de novo from no apparent template. This occurs, for instance, in the early mouse embryo and in parthenogenetic species and might rely on a pre-existing seed that resides within PCM but is not visible by ultrastructural analysis.

We have previously identified a new centrosomal protein, centrosomal protein 4.1-associated protein (CPAP), which is associated with the γ-tubulin complex. Here, we report that CPAP carries a novel microtubule-destabilizing motif that not only inhibits microtubule nucleation from the centrosome but also depolymerizes taxol-stabilized microtubules. Deletion mapping and functional analyses have defined a 112-residue CPAP that is necessary and sufficient for microtubule destabilization. This 112-residue CPAP directly recognizes the plus end of a microtubule and inhibits microtubule nucleation from the centrosome. Biochemical and functional analyses revealed that this 112-residue CPAP also binds to tubulin dimers, resulting in the destabilization of microtubules. Using the tetracycline-controlled system (tet-off), we observed that overexpression of this 112-residue CPAP inhibits cell proliferation and induces apoptosis after G2/M arrest. The possible mechanisms of how this 112-residue motif in CPAP that inhibits microtubule nucleation from the centrosome and disassembles preformed microtubules are discussed.

Many human tumours show centrosome aberrations, indicating an underlying deregulation of centrosome structure, duplication or segregation. Centrosomes organize microtubule arrays throughout the cell cycle, thereby influencing both tissue architecture and the accuracy of chromosome segregation. But what are the origins of centrosomal abnormalities in tumours, and what impact do they have on the generation of invasive, genetically unbalanced cells during cancer progression?

Centrosome duplication is a critical step in assembly of the bipolar mitotic spindle, but the molecular mechanisms regulating this process during the cell cycle and during animal development are poorly understood. Here, we report that the zyg-1 gene of Caenorhabditis elegans is an essential regulator of centrosome duplication. ZYG-1 is a protein kinase specifically required for daughter centriole formation that localizes transiently to centrosomes and acts at least one cell cycle prior to each spindle assembly event. In the embryo, ZYG-1 participates in a unique regulatory scheme whereby paternal ZYG-1 regulates duplication and bipolar spindle assembly during the first cell cycle, and maternal ZYG-1 regulates these processes thereafter. ZYG-1 is therefore a key molecular component of the centrosome/centriole duplication process.

The essence of successful mitosis is the generation of two genetically identical daughter cells. This requires the assembly of a strictly bipolar mitotic apparatus that will ensure that all daughter chromosomes are segregated to opposite sides of the cell before the completion of mitosis. The agent of the higher animal cell for the establishment of this essential “twoness” is the pair of centrosomes that organize the spindle poles during mitosis (Mazia 1987). Before the cell enters mitosis, the single interphase centrosome duplicates or reproduces exactly once, and the daughter centrosomes form the two poles of the spindle after breakdown of the nuclear envelope. Failure of the cell to precisely control this duplication of the centrosome can have disastrous consequences. If the centrosome fails to duplicate before the onset of mitosis, most somatic cells eventually return to interphase without dividing, which causes polyploidy. If the centrosome duplicates more than once in a cell cycle, then a multipolar spindle may be assembled and the chromosomes may be unequally distributed to the daughter cells, leading to genetic imbalances that produce cells with aggressive growth characteristics (for reviews, see Orr-Weaver and Weinberg 1998; Brinkley 2001). In fact, the cells of many lethal human tumors are genetically unstable and have abnormally high numbers of centrosomes (Lingle et al. 1998; Pihan et al. 1998; Carroll et al. 1999). Extra centrosomes are a problem because the condition is not remediated; cells do not have a checkpoint that aborts mitosis in response to extra spindle poles (Sluder et al. 1997). In this review, we focus on the mechanisms that control centrosome duplication and coordinate it with nuclear events during the cell cycle.

Using a yeast two-hybrid system, we isolated a novel human centrosomal protein, CPAP (centrosomal P4.1-associated protein), which specifically interacts with the head domain of the 135-kDa protein 4.1R isoform (4.1R-135). Sequence analysis revealed that the carboxyl terminus of CPAP has 31.3% amino acid identity with human Tcp-10 (a t-complex responder gene product). Interestingly, most of the sequence identity is restricted to two conserved regions. One carries a leucine zipper, which may form a series of heptad repeats involved in coiled-coil formation; the other contains unusual glycine repeats with unknown function. Immunofluorescence analysis revealed that CPAP and gamma-tubulin are localized within the centrosome throughout the cell cycle. CPAP cosediments with gamma-tubulin in sucrose gradients and coimmunoprecipitates with gamma-tubulin, indicating that CPAP is a part of the gamma-tubulin complex. Furthermore, functional analysis revealed that CPAP is localized within the center of microtubule asters and may participate in microtubule nucleation. The formation of microtubule asters was significantly inhibited by anti-CPAP antibody. Together, these observations indicate that CPAP may play an important role in cell division and centrosome function.

We have generated several stable cell lines expressing GFP-labeled centrin. This fusion protein becomes concentrated in the lumen of both centrioles, making them clearly visible in the living cell. Time-lapse fluorescence microscopy reveals that the centriole pair inherited after mitosis splits during or just after telophase. At this time the mother centriole remains near the cell center while the daughter migrates extensively throughout the cytoplasm. This differential behavior is not related to the presence of a nucleus because it is also observed in enucleated cells. The characteristic motions of the daughter centriole persist in the absence of microtubules (Mts). or actin, but are arrested when both Mts and actin filaments are disrupted. As the centrioles replicate at the G1/S transition the movements exhibited by the original daughter become progressively attenuated, and by the onset of mitosis its behavior is indistinguishable from that of the mother centriole. While both centrioles possess associated gamma-tubulin, and nucleate similar number of Mts in Mt repolymerization experiments. during G1 and S only the mother centriole is located at the focus of the Mt array. A model, based on differences in Mt anchoring and release by the mother and daughter centrioles, is proposed to explain these results.

Centrosome duplication is indispensable for the formation of the bipolar mitotic spindle. Surprisingly, even if DNA replication or mitosis is inhibited, centrosome duplication can still occur [1] [2] [3] [4] [5]. Thus, it remains unknown how centrosome duplication is coordinated with the cell cycle. Here, we show that centrosome duplication requires cyclin-dependent kinase 2 (Cdk2) in mammalian cells. We have found that in Chinese hamster ovary (CHO) cells, whereas centrosome duplication is not inhibited by hydroxyurea (HU) treatment, which arrests the cells in S phase, it is inhibited by mimosine treatment, which arrests the cells in late G1 phase. Cdk2 activity was higher in HU-treated cells than in mimosine-treated cells. Remarkably, inhibition of the Cdk2 activity in HU-treated cells with butyrolactone I or roscovitine [6], or by expression of the Cdk inhibitor p21(Waf1/Cip1), blocked the continued centrosome duplication. Moreover, overexpression of Cdk2 reversed the inhibition of centrosome duplication by mimosine treatment. These results indicate a requirement of Cdk2 activity for centrosome duplication and therefore suggest an underlying mechanism for the coordination of centrosome duplication with the cell cycle.

Centrosomes nucleate microtubules and duplicate once per cell cycle. This duplication and subsequent segregation in mitosis results in maintenance of the one centrosome/cell ratio. Centrosome duplication occurs during the G 1 /S transition in somatic cells and must be coupled to the events of the nuclear cell cycle; failure to coordinate duplication and mitosis results in abnormal numbers of centrosomes and aberrant mitoses. Using both in vivo and in vitro assays, we show that centrosome duplication in Xenopus laevis embryos requires cyclin/cdk2 kinase activity. Injection of the cdk (cyclin-dependent kinase) inhibitor p21 into one blastomere of a dividing embryo blocks centrosome duplication in that blastomere; the related cdk inhibitor p27 has a similar effect. An in vitro system using Xenopus extracts carries out separation of the paired centrioles within the centrosome. This centriole separation activity is dependent on cyclin/cdk2 activity; depletion of either cdk2 or of the two activating cyclins, cyclin A and cyclin E, eliminates centriole separation activity. In addition, centriole separation is inhibited by the mitotic state, suggesting a mechanism of linking the cell cycle to periodic duplication of the centrosome.

Pericentrin and gamma-tubulin are integral centrosome proteins that play a role in microtubule nucleation and organization. In this study, we examined the relationship between these proteins in the cytoplasm and at the centrosome. In extracts prepared from Xenopus eggs, the proteins were part of a large complex as demonstrated by sucrose gradient sedimentation, gel filtration and coimmunoprecipitation analysis. The pericentrin-gamma-tubulin complex was distinct from the previously described gamma-tubulin ring complex (gamma-TuRC) as purified gamma-TuRC fractions did not contain detectable pericentrin. When assembled at the centrosome, the two proteins remained in close proximity as shown by fluorescence resonance energy transfer. The three- dimensional organization of the centrosome-associated fraction of these proteins was determined using an improved immunofluorescence method. This analysis revealed a novel reticular lattice that was conserved from mammals to amphibians, and was organized independent of centrioles. The lattice changed dramatically during the cell cycle, enlarging from G1 until mitosis, then rapidly disassembling as cells exited mitosis. In cells colabeled to detect centrosomes and nucleated microtubules, lattice elements appeared to contact the minus ends of nucleated microtubules. Our results indicate that pericentrin and gamma-tubulin assemble into a unique centrosome lattice that represents the higher-order organization of microtubule nucleating sites at the centrosome.

The absence of detailedin vitrostudies leaves the molecular events involved in the centrosome cycle poorly characterized. Most earlier studies have employed electron microscopy of thin or thick sections of cells. Here we have analyzed the structure of centrosomes isolated from nonsynchronized human lymphoblastic KE37 cells using cryoelectron microscopy of vitrified specimens. The centrosomes were classified into five categories depending on the number of centrioles (one or two), the respective orientation of the two centrioles in a pair (orthogonal or disoriented), and the presence or absence of appendages at the distal extremity of the centrioles (referred to as mature and immature, respectively). A detailed analysis of the centriole dimensions in these categories allowed us to reconstruct the centrosome cycle in KE37 cells. Our results suggest that centriole assembly is completed only when the mother centriole of an immature orthogonal pair separates from its daughter in preparation to centrosome duplication. Our study shows that anin vitroapproach based on cryoelectron microscopy of vitrified specimens can be used to obtain detailed structural information on the centrosome cycle.

The centrosome plays a major role in the spatial organization of the microtubular network and has a controlled cycle of duplication, the two duplicated centrosomes functioning as mitotic poles during subsequent cell division. However, a comprehensive description of the overall organization of the centrosome in animal cells is lacking. In order to integrate the various pieces contributing to the centrosome structure and to optimize the quality of the data, we have undertaken an extensive ultrastructural study of centrosomes isolated from human lymphoblasts, which involved (i) orientation of centrosomes by sedimentation before embedding and sectioning, (ii) ultrathin serial sectioning, (iii) digitalization of micrographs to obtain quantitative data, and finally, (iv) comparison between two methods of isolation, which differ by the presence or absence of EDTA. Using this strategy, we have unambiguously described the pericentriolar organization of two distinct sets of appendages (distal and subdistal) about the so-called parental centriole. New structures have been also observed in association with the microtubule sets in this study: (i) external columns, which are dense structures localized at the basis of the subdistal appendages and (ii) internal columns, which are made of globular subunits integrated in a more luminal and probably helical structure. We have also observed that removal of divalent cations by the EDTA during the isolation procedure could affect the centrosomal structure at different levels (subdistal appendages, internal and external columns, pericentriolar matrix), including a significant variation in centriole diameter. A scheme of the overall organization of the centrosome from animal cells and of its modulation by divalent cations can be drawn from this study. Our data gives a view of the centrosome as an organelle displaying a complex and possibly dynamic structural organization.

Centriole stability and distribution during the mammalian cell cycle was studied by microinjecting biotinylated tubulin into early G1 cells and analyzing the pattern of incorporation into centrioles. Cells were extracted and cold treated to depolymerize labile microtubules, allowing the fluorescent microscopic visualization of the stable centrioles. The ability to detect single centrioles was confirmed by use of correlative electron microscopy. Indirect hapten and immunofluorescent labeling of biotinylated and total tubulin permitted us to distinguish newly formed from preexisting centrioles. Daughter centrioles incorporated biotinylated tubulin, and at mitosis each cell received a centrosome containing one new and one old centriole. We conclude that in each cell cycle tubulin incorporation into centrioles is conservative, and centriole distribution is semiconservative.

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 relationship between centriole formation and DNA synthesis was investigated by examining the effect of taxol on the centriole cycle and the initiation of DNA synthesis in synchronized cells. The centriole cycle was monitored by electron microscopy of whole-mount preparations [Kuriyama and Borisy, J. Cell Biol., 1981, 91:814-821]. A short daughter centriole appeared in perpendicular orientation to each parent during late G1 or early S and elongated slowly during S to G2. Addition of 5-20 micrograms/ml taxol to a synchronous population of cells in S phase did not inhibit centriole elongation; rather, elongation was accelerated. In contrast, when taxol was added to M phase or early G1 cells, centriole duplication was completely inhibited. The taxol block was reversible since nucleation and elongation of centrioles resumed as soon as the drug was removed. Cells exposed to taxol progressed through the cell cycle and became blocked in mitosis, as indicated by an increase in the mitotic index, but eventually the mitotic arrest was overcome, resulting in formation of multinucleated cells. A peak in mitotic index was seen in the following generation, indicating that chromosomes duplicated in the presence of taxol. Incorporation of 3H-thymidine followed by autoradiography confirmed that DNA synthesis was initiated in the presence of taxol even though formation of daughter centrioles was inhibited. It seems, therefore, that centriole duplication is not a prerequisite for entry into S phase. Since DNA synthesis has already been demonstrated not to be necessary for centriole duplication, these two events, normally coordinated in time, appear to be independent of each other.

Progression of the HeLa cell through its life cycle is accompanied by centriolar replication and pericentriolar changes that are in synchrony with DNA synthesis and mitosis. The first signs of preparation for replication occur during G1 at which time the two orthogonal centrioles separate. Replication by budding begins at/or near the initiation of DNA synthesis and is completed by G2. Pericentriolar changes which probably are causally related to spindle tubule formation occur at this time and include the appearance of vesicles, electron-opaque bodies, and an amorphous pericentriolar halo. These phenomena begin to disappear by late prophase, and the remainder of mitosis manifests decreasing centriolar and pericentriolar activity.

SummaryCentrosomes organize microtubule (MT) arrays and are comprised of centrioles surrounded by ordered pericentriolar proteins [1–4]. Centrioles are barrel-shaped structures composed of MTs, and as basal bodies they template the formation of cilia/flagella. Defects in centriole assembly can lead to ciliopathies and genome instability [5]. The assembly of procentrioles requires a set of conserved proteins [6–8]. It is initiated at the G1-to-S transition by PLK4 and CEP152 [9, 10], which help recruit SASS6 and STIL to the vicinity of the mother centriole to organize the cartwheel [11–14]. Subsequently, CPAP promotes centriolar MT assembly and elongation in G2 [15–17]. While centriole integrity is maintained by CEP135 and POC1 through MT stabilization [18–20], centriole elongation requires POC5 and is restricted by CP110 and CEP97 [21, 22]. How strict control of centriole length is achieved remains unclear. Here, we show that CEP120 and SPICE1 are required to localize CEP135 (but not SASS6, STIL, or CPAP) to procentrioles. CEP120 associates with SPICE1 and CPAP, and depletion of any of these proteins results in short procentrioles. Furthermore, CEP120 or CPAP overexpression results in excessive centriole elongation, a process dependent on CEP120, SPICE1, and CPAP. Our findings identify a shared function for these proteins in centriole length control.

The mechanisms governing centrosome duplication remain poorly understood. We identified a gene called sas-4 that is essential for this process in C. elegans. SAS-4 encodes a predicted coiled-coil protein that localizes to a tiny dot in the center of centrosomes throughout the cell cycle. FRAP experiments with GFP-SAS-4 transgenic embryos reveal that SAS-4 is recruited to the centrosome once per cell cycle, at the time of organelle duplication. Additional evidence indicates that SAS-4 is recruited to the daughter centriole or a closely associated structure. These findings identify SAS-4 recruitment as a key step in the centrosome duplication cycle.

Centrioles are barrel-shaped structures that are essential for the formation of centrosomes, cilia, and flagella. Here we review recent advances in our understanding of the function and biogenesis of these organelles, and we emphasize their connection to human disease. Deregulation of centrosome numbers has long been proposed to contribute to genome instability and tumor formation, whereas mutations in centrosomal proteins have recently been genetically linked to microcephaly and dwarfism. Finally, structural or functional centriole aberrations contribute to ciliopathies, a variety of complex diseases that stem from the absence or dysfunction of cilia.