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

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

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.     

Centriole inheritance

Early cell biologists perceived centrosomes to be permanent cellular structures. Centrosomes were observed to reproduce once each cycle and to orchestrate assembly a transient mitotic apparatus that segregated chromosomes and a centrosome to each daughter at the completion of cell division. Centrosomes are composed of a pair of centrioles buried in a complex pericentriolar matrix. The bulk of microtubules in cells lie with one end buried in the pericentriolar matrix and the other extending outward into the cytoplasm. Centrioles recruit and organize pericentriolar material. As a result, centrioles dominate microtubule organization and spindle assembly in cells born with centrosomes. Centrioles duplicate in concert with chromosomes during the cell cycle. At the onset of mitosis, sibling centrosomes separate and establish a bipolar spindle that partitions a set of chromosomes and a centrosome to each daughter cell at the completion of mitosis and cell division. Centriole inheritance has historically been ascribed to a template mechanism in which the parental centriole contributed to, if not directed, assembly of a single new centriole once each cell cycle. It is now clear that neither centrioles nor centrosomes are essential to cell proliferation. This review examines the recent literature on inheritance of centrioles in animal cells.Key words: centrosome, centriol, spindle, mitosis, microtubule, cell cycle, checkpoints     

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.     

Daughter centrioles assemble preferentially towards the nuclear envelope in Drosophila syncytial embryos

Centrosomes are important organizers of microtubules within animal cells. They comprise a pair of centrioles surrounded by the pericentriolar material, which nucleates and organizes the microtubules. To maintain centrosome numbers, centrioles must duplicate once and only once per cell cycle. During S-phase, a single new ‘daughter’ centriole is built orthogonally on one side of each radially symmetric ‘mother’ centriole. Mis-regulation of duplication can result in the simultaneous formation of multiple daughter centrioles around a single mother centriole, leading to centrosome amplification, a hallmark of cancer. It remains unclear how a single duplication site is established. It also remains unknown whether this site is pre-defined or randomly positioned around the mother centriole. Here, we show that within Drosophila syncytial embryos daughter centrioles preferentially assemble on the side of the mother facing the nuclear envelope, to which the centrosomes are closely attached. This positional preference is established early during duplication and remains stable throughout daughter centriole assembly, but is lost in centrosomes forced to lose their connection to the nuclear envelope. This shows that non-centrosomal cues influence centriole duplication and raises the possibility that these external cues could help establish a single duplication site.     

The Protein Phosphatase 2A regulatory subunit Twins stabilizes Plk4 to induce centriole amplification

Centriole duplication is a tightly regulated process that must occur only once per cell cycle; otherwise, supernumerary centrioles can induce aneuploidy and tumorigenesis. Plk4 (Polo-like kinase 4) activity initiates centriole duplication and is regulated by ubiquitin-mediated proteolysis. Throughout interphase, Plk4 autophosphorylation triggers its degradation, thus preventing centriole amplification. However, Plk4 activity is required during mitosis for proper centriole duplication, but the mechanism stabilizing mitotic Plk4 is unknown. In this paper, we show that PP2A (Protein Phosphatase 2A(Twins)) counteracts Plk4 autophosphorylation, thus stabilizing Plk4 and promoting centriole duplication. Like Plk4, the protein level of PP2A's regulatory subunit, Twins (Tws), peaks during mitosis and is required for centriole duplication. However, untimely Tws expression stabilizes Plk4 inappropriately, inducing centriole amplification. Paradoxically, expression of tumor-promoting simian virus 40 small tumor antigen (ST), a reported PP2A inhibitor, promotes centrosome amplification by an unknown mechanism. We demonstrate that ST actually mimics Tws function in stabilizing Plk4 and inducing centriole amplification.     

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.     

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.     

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.     

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

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

The centrosome duplication cycle in health and disease

Centrioles function in the assembly of centrosomes and cilia. Structural and numerical centrosome aberrations have long been implicated in cancer, and more recent genetic evidence directly links centrosomal proteins to the etiology of ciliopathies, dwarfism and microcephaly. To better understand these disease connections, it will be important to elucidate the biogenesis of centrioles as well as the controls that govern centriole duplication during the cell cycle. Moreover, it remains to be fully understood how these organelles organize a variety of dynamic microtubule-based structures in response to different physiological conditions. In proliferating cells, centrosomes are crucial for the assembly of microtubule arrays, including mitotic spindles, whereas in quiescent cells centrioles function as basal bodies in the formation of ciliary axonemes. In this short review, we briefly introduce the key gene products required for centriole duplication. Then we discuss recent findings on the centriole duplication factor STIL that point to centrosome amplification as a potential root cause for primary microcephaly in humans. We also present recent data on the role of a disease-related centriole-associated protein complex, Cep164-TTBK2, in ciliogenesis.     

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.     

Centrobin-tubulin interaction is required for centriole elongation and stability

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.     

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

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

Centrobin-Centrosomal Protein 4.1-associated Protein (CPAP) Interaction Promotes CPAP Localization to the Centrioles during Centriole Duplication

Centriole duplication is the process by which two new daughter centrioles are generated from the proximal end of preexisting mother centrioles. Accurate centriole duplication is important for many cellular and physiological events, including cell division and ciliogenesis. Centrosomal protein 4.1-associated protein (CPAP), centrosomal protein of 152 kDa (CEP152), and centrobin are known to be essential for centriole duplication. However, the precise mechanism by which they contribute to centriole duplication is not known. In this study, we show that centrobin interacts with CEP152 and CPAP, and the centrobin-CPAP interaction is critical for centriole duplication. Although depletion of centrobin from cells did not have an effect on the centriolar levels of CEP152, it caused the disappearance of CPAP from both the preexisting and newly formed centrioles. Moreover, exogenous expression of the CPAP-binding fragment of centrobin also caused the disappearance of CPAP from both the preexisting and newly synthesized centrioles, possibly in a dominant negative manner, thereby inhibiting centriole duplication and the PLK4 overexpression-mediated centrosome amplification. Interestingly, exogenous overexpression of CPAP in the centrobin-depleted cells did not restore CPAP localization to the centrioles. However, restoration of centrobin expression in the centrobin-depleted cells led to the reappearance of centriolar CPAP. Hence, we conclude that centrobin-CPAP interaction is critical for the recruitment of CPAP to procentrioles to promote the elongation of daughter centrioles and for the persistence of CPAP on preexisting mother centrioles. Our study indicates that regulation of CPAP levels on the centrioles by centrobin is critical for preserving the normal size, shape, and number of centrioles in the cell.     

The C. elegans zyg-1 gene encodes a regulator of centrosome duplication with distinct maternal and paternal roles in the embryo

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.     

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.     

Kendrin is a novel substrate for separase involved in the licensing of centriole duplication

The centrosome, consisting of a pair of centrioles surrounded by pericentriolar material, directs the formation of bipolar spindles during mitosis. Aberrant centrosome number can promote chromosome instability, which is implicated in tumorigenesis. Thus, centrosome duplication needs to be tightly regulated to occur only once per cell cycle. Separase, a cysteine protease that triggers sister chromatid separation, is involved in centriole disengagement, which licenses centrosomes for the next round of duplication. However, at least two questions remain unsolved: what is the substrate relevant to the disengagement, and how does separase, activated at anaphase onset, act on the disengagement that occurs during late mitosis. Here, we show that kendrin, also named pericentrin, is cleaved by activated separase at a consensus site in vivo and in vitro, and this leads to the delayed release of kendrin from the centrosome later in mitosis. Furthermore, we demonstrate that expression of a noncleavable kendrin mutant suppresses centriole disengagement and subsequent centriole duplication. Based on these results, we propose that kendrin is a novel and crucial substrate for separase at the centrosome, protecting the engaged centrioles from premature disengagement and thereby blocking reduplication until the cell passes through mitosis.     

CDK11(p58) is required for centriole duplication and Plk4 recruitment to mitotic centrosomes

Background

CDK11^p58 is a mitotic protein kinase, which has been shown to be required for different mitotic events such as centrosome maturation, chromatid cohesion and cytokinesis.

Methodology/Principal Findings

In addition to these previously described roles, our study shows that CDK11^p58 inhibition induces a failure in the centriole duplication process in different human cell lines. We propose that this effect is mediated by the defective centrosomal recruitment of proteins at the onset of mitosis. Indeed, Plk4 protein kinase and the centrosomal protein Cep192, which are key components of the centriole duplication machinery, showed reduced levels at centrosomes of mitotic CDK11-depleted cells. CDK11^p58, which accumulates only in the vicinity of mitotic centrosomes, directly interacts with the centriole-associated protein kinase Plk4 that regulates centriole number in cells. In addition, we show that centriole from CDK11 defective cells are not able to be over duplicated following Plk4 overexpression.

Conclusion/Significance

We thus propose that CDK11 is required for centriole duplication by two non-mutually-exclusive mechanisms. On one hand, the observed duplication defect could be caused indirectly by a failure of the centrosome to fully maturate during mitosis. On the other hand, CDK11^p58 could also directly regulate key centriole components such as Plk4 during mitosis to trigger essential mitotic centriole modifications, required for centriole duplication during subsequent interphase.