Daughter Centriole Elongation Is Controlled by Proteolysis

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.     

Cep152 acts as a scaffold for recruitment of Plk4 and CPAP to the centrosome

Both gain and loss of function studies have identified the Polo-like kinase Plk4/Sak as a crucial regulator of centriole biogenesis, but the mechanisms governing centrosome duplication are incompletely understood. In this study, we show that the pericentriolar material protein, Cep152, interacts with the distinctive cryptic Polo-box of Plk4 via its N-terminal domain and is required for Plk4-induced centriole overduplication. Reduction of endogenous Cep152 levels results in a failure in centriole duplication, loss of centrioles, and formation of monopolar mitotic spindles. Interfering with Cep152 function prevents recruitment of Plk4 to the centrosome and promotes loss of CPAP, a protein required for the control of centriole length in Plk4-regulated centriole biogenesis. Our results suggest that Cep152 recruits Plk4 and CPAP to the centrosome to ensure a faithful centrosome duplication process.     

Polo-like kinase 4 kinase activity limits centrosome overduplication by autoregulating its own stability

Accurate control of the number of centrosomes, the major microtubule-organizing centers of animal cells, is critical for the maintenance of genome integrity. Abnormalities in centrosome number can promote errors in spindle formation that lead to subsequent chromosome missegregation, and extra centrosomes are found in many cancers. Centrosomes are comprised of a pair of centrioles surrounded by amorphous pericentriolar material, and centrosome duplication is controlled by centriole replication. Polo-like kinase 4 (Plk4) plays a key role in initiating centriole duplication, and overexpression of Plk4 promotes centriole overduplication and the formation of extra centrosomes. Using chemical genetics, we show that kinase-active Plk4 is inherently unstable and targeted for degradation. Plk4 is shown to multiply self-phosphorylate within a 24–amino acid phosphodegron. Phosphorylation of multiple sites is required for Plk4 instability, indicating a requirement for a threshold level of Plk4 kinase activity to promote its own destruction. We propose that kinase-mediated, autoregulated instability of Plk4 self-limits Plk4 activity so as to prevent centrosome amplification.     

Plk2 regulated centriole duplication is dependent on its localization to the centrosome and a functional polo-box domain

In mammalian cells, the centrosome consists of a pair of centrioles and amorphous pericentriolar material. The centrosome duplicates once per cell cycle. Polo like kinases (Plks) perform crucial functions in cell-cycle progression and during mitosis. The polo-like kinase-2, Plk2, is activated near the G1/S phase transition, and plays an important role in the reproduction of centrosomes. In this study, we show that the polo-box of Plk2 is required both for association to the centrosome and centriole duplication. Mutation of critical sites in the Plk2 polo-box prevents centrosomal localization and impairs centriole duplication. Plk2 is localized to centrosomes during early G1 phase where it only associates to the mother centriole and then distributes equally to both mother and daughter centrioles at the onset of S phase. Furthermore, our results imply that Plk2 mediated centriole duplication is dependent on Plk4 function. In addition, we find that siRNA-mediated down-regulation of Plk2 leads to the formation of abnormal mitotic spindles confirming that Plk2 may have a function in the reproduction of centrioles.     

PLK4-phosphorylated NEDD1 facilitates cartwheel assembly and centriole biogenesis initiations

Centrosome duplication occurs under strict spatiotemporal regulation once per cell cycle, and it begins with cartwheel assembly and daughter centriole biogenesis at the lateral sites of the mother centrioles. However, although much of this process is understood, how centrosome duplication is initiated remains unclear. Here, we show that cartwheel assembly followed by daughter centriole biogenesis is initiated on the NEDD1-containing layer of the pericentriolar material (PCM) by the recruitment of SAS-6 to the mother centriole under the regulation of PLK4. We found that PLK4-mediated phosphorylation of NEDD1 at its S325 amino acid residue directly promotes both NEDD1 binding to SAS-6 and recruiting SAS-6 to the centrosome. Overexpression of phosphomimicking NEDD1 mutant S325E promoted cartwheel assembly and daughter centriole biogenesis initiations, whereas overexpression of nonphosphorylatable NEDD1 mutant S325A abolished the initiations. Collectively, our results demonstrate that PLK4-regulated NEDD1 facilitates initiation of the cartwheel assembly and of daughter centriole biogenesis in mammals.     

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

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

Identification and proteomic analysis of distinct UBE3A/E6AP protein complexes

The E6AP ubiquitin ligase catalyzes the high-risk human papillomaviruses' E6-mediated ubiquitylation of p53, contributing to the neoplastic progression of cells infected by these viruses. Defects in the activity and the dosage of E6AP are linked to Angelman syndrome and to autism spectrum disorders, respectively, highlighting the need for precise control of the enzyme. With the exception of HERC2, which modulates the ubiquitin ligase activity of E6AP, little is known about the regulation or function of E6AP normally. Using a proteomic approach, we have identified and validated several new E6AP-interacting proteins, including HIF1AN, NEURL4, and mitogen-activated protein kinase 6 (MAPK6). E6AP exists as part of several different protein complexes, including the proteasome and an independent high-molecular-weight complex containing HERC2, NEURL4, and MAPK6. In examining the functional consequence of its interaction with the proteasome, we found that UBE3C (another proteasome-associated ubiquitin ligase), but not E6AP, contributes to proteasomal processivity in mammalian cells. We also found that E6 associates with the HERC2-containing high-molecular-weight complex through its binding to E6AP. These proteomic studies reveal a level of complexity for E6AP that has not been previously appreciated and identify a number of new cellular proteins through which E6AP may be regulated or functioning.     

Klp10A, a microtubule-depolymerizing kinesin-13, cooperates with CP110 to control Drosophila centriole length

Klp10A is a kinesin-13 of Drosophila melanogaster that depolymerizes cytoplasmic microtubules. In interphase, it promotes microtubule catastrophe; in mitosis, it contributes to anaphase chromosome movement by enabling tubulin flux. 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, 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 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.     

Localization of Golgi 58K protein (formiminotransferase cyclodeaminase) to the centrosome

In vertebrate cells, the centrosome consists of a pair of centrioles and surrounding pericentriolar material. Using anti-Golgi 58K protein antibodies that recognize formiminotransferase cyclodeaminase (FTCD), we investigated its localization to the centrosome in various cultured cells and human oviductal secretory cells by immunohistochemistry. In addition to the Golgi apparatus, FTCD was localized to the centrosome, more abundantly around the mother centriole. The centrosome localization of FTCD continued throughout the cell cycle and was not disrupted after Golgi fragmentation, which was induced by colcemid and brefeldin A. Centriole microtubules are polyglutamylated and stable against tubulin depolymerizing drugs. FTCD in the centrosome may be associated with polyglutamylated residues of centriole microtubules and may play a role in providing centrioles with glutamate produced by cyclodeaminase domains of FTCD.     

Centrobin regulates centrosome function in interphase cells by limiting pericentriolar matrix recruitment

The amount of pericentriolar matrix at the centrosome is tightly linked to both microtubule nucleation and centriole duplication, although the exact mechanism by which pericentriolar matrix levels are regulated is unclear. Here we show that Centrobin, a centrosomal protein, is involved in regulating these levels. Interphase microtubule arrays in Centrobin-depleted cells are more focused around the centrosome and are less stable than the arrays in control cells. Centrobin-depleted cells initiate microtubule nucleation more rapidly than control cells and exhibit an increase in the number of growing microtubule ends emanating from the centrosome, while the parameters of microtubule plus end dynamics around the centrosome are not significantly altered. Finally, we show that Centrobin depletion results in the increased recruitment of pericentriolar matrix proteins to the centrosome, including γ-tubulin, AKAP450, Kendrin and PCM-1. We propose that Centrobin might regulate microtubule nucleation and organization by controlling the amount of pericentriolar matrix.     

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

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

Centrosome biogenesis: centrosomin sizes things up!

Centrosomes are composed of a centriole pair surrounded by a proteinaceous matrix of pericentriolar material that contains protein complexes required for microtubule nucleation and anchoring. Recent work reveals an intriguing link between centrioles and pericentriolar material that is involved in modulating centrosome size.     

Centrosome abnormalities during a Chlamydia trachomatis infection are caused by dysregulation of the normal duplication pathway

The presence of supernumerary centrosomes in cells infected with Chlamydia trachomatis may provide a mechanism to explain the association of C. trachomatis genital infection with cervical cancer. We show that the amplified centrosomal foci induced during a chlamydial infection contain both centriolar and pericentriolar matrix markers, demonstrating that they are bona fide centrosomes. As there were multiple immature centrioles but approximately one mature centriole per cell, aborted cytokinesis alone cannot account for centrosome amplification during a chlamydial infection. Production of supernumerary centrosomes required the kinase activities of Cdk2 and Plk4, which are known regulators of centrosome duplication, and progression through S-phase, which is the stage in the cell cycle when duplication of the centrosome occurs. These requirements indicate that centrosome amplification during a chlamydial infection depends on the host centrosome duplication pathway, which normally produces a single procentriole from each template centriole. However, C. trachomatis induces a loss of numerical control so that multiple procentrioles are formed per template.     

Plk4-induced centriole biogenesis in human cells

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.     

SAS-4 is a C. elegans centriolar protein that controls centrosome size

Centrosomes consist of a centriole pair surrounded by pericentriolar material (PCM). Previous work suggested that centrioles are required to organize PCM to form a structurally stable organelle. Here, we characterize SAS-4, a centriole component in Caenorhabditis elegans. Like tubulin, SAS-4 is incorporated into centrioles during their duplication and remains stably associated thereafter. In the absence of SAS-4, centriole duplication fails. Partial depletion of SAS-4 results in structurally defective centrioles that contain reduced levels of SAS-4 and organize proportionally less PCM. Thus, SAS-4 is a centriole-associated component whose amount dictates centrosome size. These results provide novel insight into the poorly understood role of centrioles as centrosomal organizers.     

Cep63 and Cep152 Cooperate to Ensure Centriole Duplication

Centrosomes consist of two centrioles embedded in pericentriolar material and function as the main microtubule organising centres in dividing animal cells. They ensure proper formation and orientation of the mitotic spindle and are therefore essential for the maintenance of genome stability. Centrosome function is crucial during embryonic development, highlighted by the discovery of mutations in genes encoding centrosome or spindle pole proteins that cause autosomal recessive primary microcephaly, including Cep63 and Cep152. In this study we show that Cep63 functions to ensure that centriole duplication occurs reliably in dividing mammalian cells. We show that the interaction between Cep63 and Cep152 can occur independently of centrosome localisation and that the two proteins are dependent on one another for centrosomal localisation. Further, both mouse and human Cep63 and Cep152 cooperate to ensure efficient centriole duplication by promoting the accumulation of essential centriole duplication factors upstream of SAS-6 recruitment and procentriole formation. These observations describe the requirement for Cep63 in maintaining centriole number in dividing mammalian cells and further establish the order of events in centriole formation.     

PLK4 phosphorylation of CP110 is required for efficient centriole assembly

Centrioles are assembled during S phase and segregated into 2 daughter cells at the end of mitosis. The initiation of centriole assembly is regulated by polo-like kinase 4 (PLK4), the major serine/threonine kinase in centrioles. Despite its importance in centriole duplication, only a few substrates have been identified, and the detailed mechanism of PLK4 has not been fully elucidated. CP110 is a coiled-coil protein that plays roles in centriolar length control and ciliogenesis in mammals. Here, we revealed that PLK4 specifically phosphorylates CP110 at the S98 position. The phospho-resistant CP110 mutant inhibited centriole assembly, whereas the phospho-mimetic CP110 mutant induced centriole assembly, even in PLK4-limited conditions. This finding implies that PLK4 phosphorylation of CP110 is an essential step for centriole assembly. The phospho-mimetic form of CP110 augmented the centrosomal SAS6 level. Based on these results, we propose that the phosphorylated CP110 may be involved in the stabilization of cartwheel SAS6 during centriole assembly.     

<Emphasis Type="Italic">Ab ovo or de novo?</Emphasis> Mechanisms of centriole duplication

The centrosome, an organelle comprising centrioles and associated pericentriolar material, is the major microtubule organizing center in animal cells. For the cell to form a bipolar mitotic spindle and ensure proper chromosome segregation at the end of each cell cycle, it is paramount that the cell contains two and only two centrosomes. Because the number of centrosomes in the cell is determined by the number of centrioles, cells have evolved elaborate mechanisms to control centriole biogenesis and to tightly coordinate this process with DNA replication. Here we review key proteins involved in centriole assembly, compare two major modes of centriole biogenesis, and discuss the mechanisms that ensure stringency of centriole number.     

The mammalian SPD-2 ortholog Cep192 regulates centrosome biogenesis

Centrosomes are the major microtubule-organizing centers of mammalian cells. They are composed of a centriole pair and surrounding microtubule-nucleating material termed pericentriolar material (PCM). Bipolar mitotic spindle assembly relies on two intertwined processes: centriole duplication and centrosome maturation. In the first process, the single interphase centrosome duplicates in a tightly regulated manner so that two centrosomes are present in mitosis. In the second process, the two centrosomes increase in size and microtubule nucleation capacity through PCM recruitment, a process referred to as centrosome maturation. Failure to properly orchestrate centrosome duplication and maturation is inevitably linked to spindle defects, which can result in aneuploidy and promote cancer progression. It has been proposed that centriole assembly during duplication relies on both PCM and centriole proteins, raising the possibility that centriole duplication depends on PCM recruitment. In support of this model, C. elegans SPD-2 and mammalian NEDD-1 (GCP-WD) are key regulators of both these processes. SPD-2 protein sequence homologs have been identified in flies, mice, and humans, but their roles in centrosome biogenesis until now have remained unclear. Here, we show that Cep192, the human homolog of C. elegans and D. melanogaster SPD-2, is a major regulator of PCM recruitment, centrosome maturation, and centriole duplication in mammalian cells. We propose a model in which Cep192 and Pericentrin are mutually dependent for their localization to mitotic centrosomes during centrosome maturation. Both proteins are then required for NEDD-1 recruitment and the subsequent assembly of gamma-TuRCs and other factors into fully functional centrosomes.     

A genome-wide RNAi screen to dissect centriole duplication and centrosome maturation in Drosophila

Centrosomes comprise a pair of centrioles surrounded by an amorphous pericentriolar material (PCM). Here, we have performed a microscopy-based genome-wide RNA interference (RNAi) screen in Drosophila cells to identify proteins required for centriole duplication and mitotic PCM recruitment. We analysed 92% of the Drosophila genome (13,059 genes) and identified 32 genes involved in centrosome function. An extensive series of secondary screens classified these genes into four categories: (1) nine are required for centriole duplication, (2) 11 are required for centrosome maturation, (3) nine are required for both functions, and (4) three genes regulate centrosome separation. These 32 hits include several new centrosomal components, some of which have human homologs. In addition, we find that the individual depletion of only two proteins, Polo and Centrosomin (Cnn) can completely block centrosome maturation. Cnn is phosphorylated during mitosis in a Polo-dependent manner, suggesting that the Polo-dependent phosphorylation of Cnn initiates centrosome maturation in flies.