Centriole Biogenesis Research Articles

Multicilin drives centriole biogenesis via E2f proteins.

Multiciliate cells employ hundreds of motile cilia to produce fluid flow, which they nucleate and extend by first assembling hundreds of centrioles. In most cells, entry into the cell cycle allows centrioles to undergo a single round of duplication, but in differentiating multiciliate cells, massive centriole assembly occurs in G0 by a process initiated by a small coiled-coil protein, Multicilin. Here we show that Multicilin acts by forming a ternary complex with E2f4 or E2f5 and Dp1 that binds and activates most of the genes required for centriole biogenesis, while other cell cycle genes remain off. This complex also promotes the deuterosome pathway of centriole biogenesis by activating the expression of deup1 but not its paralog, cep63. Finally, we show that this complex is disabled by mutations in human Multicilin that cause a severe congenital mucociliary clearance disorder due to reduced generation of multiple cilia. By coopting the E2f regulation of cell cycle genes, Multicilin drives massive centriole assembly in epithelial progenitors in a manner required for multiciliate cell differentiation.

Type Iγ phosphatidylinositol phosphate kinase targets to the centrosome and restrains centriole duplication

Centriole biogenesis depends on the polo-like kinase (PLK4) and a small group of structural proteins. The spatiotemporal regulation of these proteins at pre-existing centrioles is essential to ensure that centriole duplication occurs once per cell cycle. Here, we report that phosphatidylinositol 4-phosphate 5-kinase type-1 gamma (PIP5K1C, hereafter referred to as PIPKIγ) plays an important role in centriole fidelity. PIPKIγ localized in a ring-like pattern in the intermediate pericentriolar materials around the proximal end of the centriole in G1, S and G2 phases, but not in M phase. This localization was dependent upon an association with centrosomal protein of 152 KDa (CEP152). Without detaining cells in S or M phase, the depletion of PIPKIγ led to centriole amplification in a manner that was dependent upon PLK4 and spindle assembly abnormal protein 6 homolog (SAS6). The expression of exogenous PIPKIγ reduced centriole amplification that occurred as a result of endogenous PIPKIγ depletion, hydroxyurea treatment or PLK4 overexpression, suggesting that PIPKIγ is likely to function at the PLK4 level to restrain centriole duplication. Importantly, we found that PIPKIγ bound to the cryptic polo-box domain of PLK4 and that this binding reduced the kinase activity of PLK4. Together, our findings suggest that PIPKIγ is a novel negative regulator of centriole duplication, which acts by modulating the homeostasis of PLK4 activity.

Loss of centrioles causes chromosomal instability in vertebrate somatic cells.

Most animal cells contain a centrosome, which comprises a pair of centrioles surrounded by an ordered pericentriolar matrix (PCM). Although the role of this organelle in organizing the mitotic spindle poles is well established, its precise contribution to cell division and cell survival remains a subject of debate. By genetically ablating key components of centriole biogenesis in chicken DT40 B cells, we generated multiple cell lines that lack centrioles. PCM components accumulated in acentriolar microtubule (MT)-organizing centers but failed to adopt a higher-order structure, as shown by three-dimensional structured illumination microscopy. Cells without centrioles exhibited both a delay in bipolar spindle assembly and a high rate of chromosomal instability. Collectively, our results expose a vital role for centrosomes in establishing a mitotic spindle geometry that facilitates correct kinetochore-MT attachments. We propose that centrosomes are essential in organisms in which rapid segregation of a large number of chromosomes needs to be attained with fidelity.

Centriole biogenesis in multiciliated cells

Centrioles duplicate only once per cell cycle in proliferating cells, whereas in multiciliated cells, hundreds of centrioles form almost simultaneously. The molecular control mechanisms that govern centriole amplification in multiciliated cells are largely unknown. Two studies highlight Deup1 and CCDC78 as key players in this process.

Hierarchical recruitment of Plk4 and regulation of centriole biogenesis by two centrosomal scaffolds, Cep192 and Cep152

Centrosomes play an important role in various cellular processes, including spindle formation and chromosome segregation. They are composed of two orthogonally arranged centrioles, whose duplication occurs only once per cell cycle. Accurate control of centriole numbers is essential for the maintenance of genomic integrity. Although it is well appreciated that polo-like kinase 4 (Plk4) plays a central role in centriole biogenesis, how it is recruited to centrosomes and whether this step is necessary for centriole biogenesis remain largely elusive. Here we showed that Plk4 localizes to distinct subcentrosomal regions in a temporally and spatially regulated manner, and that Cep192 and Cep152 serve as two distinct scaffolds that recruit Plk4 to centrosomes in a hierarchical order. Interestingly, Cep192 and Cep152 competitively interacted with the cryptic polo box of Plk4 through their homologous N-terminal sequences containing acidic-α-helix and N/Q-rich motifs. Consistent with these observations, the expression of either one of these N-terminal fragments was sufficient to delocalize Plk4 from centrosomes. Furthermore, loss of the Cep192- or Cep152-dependent interaction with Plk4 resulted in impaired centriole duplication that led to delayed cell proliferation. Thus, the spatiotemporal regulation of Plk4 localization by two hierarchical scaffolds, Cep192 and Cep152, is critical for centriole biogenesis.

The Cep63 paralogue Deup1 enables massive de novo centriole biogenesis for vertebrate multiciliogenesis

Dense multicilia in higher vertebrates are important for luminal flow and the removal of thick mucus. To generate hundreds of basal bodies for multiciliogenesis, specialized terminally differentiated epithelial cells undergo massive centriole amplification. In proliferating cells, however, centriole duplication occurs only once per cell cycle. How cells ensure proper regulation of centriole biogenesis in different contexts is poorly understood. We report that the centriole amplification is controlled by two duplicated genes, Cep63 and Deup1. Cep63 regulates mother-centriole-dependent centriole duplication. Deup1 governs deuterosome assembly to mediate large-scale de novo centriole biogenesis. Similarly to Cep63, Deup1 binds to Cep152 and then recruits Plk4 to activate centriole biogenesis. Phylogenetic analyses suggest that Deup1 diverged from Cep63 in a certain ancestor of lobe-finned fishes during vertebrate evolution and was subsequently adopted by tetrapods. Thus, the Cep63 gene duplication has enabled mother-centriole-independent assembly of the centriole duplication machinery to satisfy different requirements for centriole number.

Regulation of Autophosphorylation Controls PLK4 Self-Destruction and Centriole Number

Polo-like kinase 4 (PLK4) is a major player in centriole biogenesis: in its absence centrioles fail to form, while in excess leads to centriole amplification. The SCF-Slimb/βTrCP-E3 ubiquitin ligase controls PLK4 levels through recognition of a conserved phosphodegron. SCF-Slimb/βTrCP substrate binding and targeting for degradation is normally regulated by phosphorylation cascades, controlling complex processes, such as circadian clocks and morphogenesis. Here, we show that PLK4 is a suicide kinase, autophosphorylating in residues that are critical for SCF-Slimb/βTrCP binding. We demonstrate a multisite trans-autophosphorylation mechanism, likely to ensure that both a threshold of PLK4 concentration is attained and a sequence of events is observed before PLK4 can autodestruct. First, we show that PLK4 trans-autophosphorylates other PLK4 molecules on both Ser293 and Thr297 within the degron and that these residues contribute differently for PLK4 degradation, the first being critical and the second maximizing auto-destruction. Second, PLK4 trans-autophosphorylates a phospho-cluster outside the degron, which regulates Thr297 phosphorylation, PLK4 degradation, and centriole number. Finally, we show the importance of PLK4-Slimb/βTrCP regulation as it operates in both soma and germline. As βTrCP, PLK4, and centriole number are deregulated in several cancers, our work provides novel links between centriole number control and tumorigenesis.

Deuterosome-Mediated Centriole Biogenesis

The ability of cells to faithfully duplicate their two centrioles once per cell cycle is critical for proper mitotic progression and chromosome segregation. Multiciliated cells represent an interesting variation of centriole duplication in that these cells generate greater than 100 centrioles, which form the basal bodies of their motile cilia. This centriole amplification is proposed to require a structure termed the deuterosome, thought to be capable of promoting de novo centriole biogenesis. Here, we begin to molecularly characterize the deuterosome and identify it as a site for the localization of Cep152, Plk4, andSAS6. Additionally we identify CCDC78 as a centriole-associated and deuterosome protein that is essential for centriole amplification. Overexpression of Cep152, but not Plk4, SAS6, or CCDC78, drives overamplification of centrioles. However, in CCDC78 morphants, Cep152 fails to localize to the deuterosome and centriole biogenesis is impaired, indicating that CCDC78-mediated recruitment of Cep152 is required for deuterosome-mediated centriole biogenesis.

Crystal structures of the CPAP/STIL complex reveal its role in centriole assembly and human microcephaly.

Centrioles organise centrosomes and template cilia and flagella. Several centriole and centrosome proteins have been linked to microcephaly (MCPH), a neuro-developmental disease associated with small brain size. CPAP (MCPH6) and STIL (MCPH7) are required for centriole assembly, but it is unclear how mutations in them lead to microcephaly. We show that the TCP domain of CPAP constitutes a novel proline recognition domain that forms a 1:1 complex with a short, highly conserved target motif in STIL. Crystal structures of this complex reveal an unusual, all-β structure adopted by the TCP domain and explain how a microcephaly mutation in CPAP compromises complex formation. Through point mutations, we demonstrate that complex formation is essential for centriole duplication in vivo. Our studies provide the first structural insight into how the malfunction of centriole proteins results in human disease and also reveal that the CPAP-STIL interaction constitutes a conserved key step in centriole biogenesis. DOI:http://dx.doi.org/10.7554/eLife.01071.001.

Discovering Regulators of Centriole Biogenesis through siRNA-Based Functional Genomics in Human Cells

Centrioles are essential for forming cilia, flagella, and centrosomes and are thus critical for a range of fundamental cellular processes. Despite their importance, the mechanisms governing centriole biogenesis remain incompletely understood. We performed a high-content genome-wide small-interfering-RNA-based screen to identify genes regulating centriole formation in human cells. We designed an algorithm to automatically detect GFP-Centrin foci that, combined with subsequent manual analysis, allowed us to identify 44 genes required for centriole formation and 32 genes needed for restricting centriole number. Detailed follow-up characterization uncovered that the C2 domain protein C2CD3 is required for distal centriole formation and suggests that it functions in the basal body to template primary cilia. Moreover, we found that the E3 ubiquitin ligase TRIM37 prevents centriole reduplication events. We developed a dynamic web interface containing all images and numerical features as a powerful resource to investigate facets of centrosome biology.

A SAS-6-Like Protein Suggests that the Toxoplasma Conoid Complex Evolved from Flagellar Components

SAS-6 is required for centriole biogenesis in diverse eukaryotes. Here, we describe a novel family of SAS-6-like (SAS6L) proteins that share an N-terminal domain with SAS-6 but lack coiled-coil tails. SAS6L proteins are found in a subset of eukaryotes that contain SAS-6, including diverse protozoa and green algae. In the apicomplexan parasite Toxoplasma gondii, SAS-6 localizes to the centriole but SAS6L is found above the conoid, an enigmatic tubulin-containing structure found at the apex of a subset of alveolate organisms. Loss of SAS6L causes reduced fitness in Toxoplasma. The Trypanosoma brucei homolog of SAS6L localizes to the basal-plate region, the site in the axoneme where the central-pair microtubules are nucleated. When endogenous SAS6L is overexpressed in Toxoplasma tachyzoites or Trypanosoma trypomastigotes, it forms prominent filaments that extend through the cell cytoplasm, indicating that it retains a capacity to form higher-order structures despite lacking a coiled-coil domain. We conclude that although SAS6L proteins share a conserved domain with SAS-6, they are a functionally distinct family that predates the last common ancestor of eukaryotes. Moreover, the distinct localization of the SAS6L protein in Trypanosoma and Toxoplasma adds weight to the hypothesis that the conoid complex evolved from flagellar components.

Polo-like kinases

Polo-like kinases

CEP proteins: the knights of centrosome dynasty

Centrosome forms the backbone of cell cycle progression mechanism. Recent debates have occurred regarding the essentiality of centrosome in cell cycle regulation. CEP family protein is the active component of centrosome and plays a vital role in centriole biogenesis and cell cycle progression control. A total of 31 proteins have been categorized into CEP family protein category and many more are under candidate evaluation. Furthermore, by the recent advancements in genomics and proteomics researches, several new CEP proteins have also been characterized. Here we have summarized the importance of CEP family proteins and their regulation mechanism involved in proper cell cycle progression. Further, we have reviewed the detailed molecular mechanism behind the associated pathological phenotypes and the possible therapeutic approaches. Proteins such as CEP57, CEP63, CEP152, CEP164, and CEP215 have been extensively studied with a detailed description of their molecular mechanisms, which are among the primary targets for drug discovery. Moreover, CEP27, CEP55, CEP70, CEP110, CEP120, CEP135, CEP192, CEP250, CEP290, and CEP350 also seem promising for future drug discovery approaches. Since the overview implicates that the overall researches on CEP proteins are not yet able to present significant details required for effective therapeutics development, thus, it is timely to discuss the importance of future investigations in this field.

LGALS3BP regulates centriole biogenesis and centrosome hypertrophy in cancer cells

Centrosome morphology and number are frequently deregulated in cancer cells. Here, to identify factors that are functionally relevant for centrosome abnormalities in cancer cells, we established a protein-interaction network around 23 centrosomal and cell-cycle regulatory proteins, selecting the interacting proteins that are deregulated in cancer for further studies. One of these components, LGALS3BP, is a centriole- and basal body-associated protein with a dual role, triggering centrosome hypertrophy when overexpressed and causing accumulation of centriolar substructures when downregulated. The cancer cell line SK-BR-3 that overexpresses LGALS3BP exhibits hypertrophic centrosomes, whereas in seminoma tissues with low expression of LGALS3BP, supernumerary centriole-like structures are present. Centrosome hypertrophy is reversed by depleting LGALS3BP in cells endogenously overexpressing this protein, supporting a direct role in centrosome aberration. We propose that LGALS3BP suppresses assembly of centriolar substructures, and when depleted, causes accumulation of centriolar complexes comprising CPAP, acetylated tubulin and centrin.

Steroidogenic Factor 1 (NR5A1) Maintains Centrosome Homeostasis in Steroidogenic Cells by Restricting Centrosomal DNA-Dependent Protein Kinase Activation

Steroidogenic factor 1 (SF-1 or NR5A1) is a nuclear receptor that controls adrenogenital cell growth and differentiation. Adrenogenital primordial cells from SF-1 knockout mice die of apoptosis, but the mechanism by which SF-1 regulates cell survival is not entirely clear. Besides functioning in the nucleus, SF-1 also resides in the centrosome and controls centrosome homeostasis. Here, we show that SF-1 restricts centrosome overduplication by inhibiting aberrant activation of DNA-dependent protein kinase (DNA-PK) in the centrosome. SF-1 was found to be associated with Ku70/Ku80 only in the centrosome, sequestering them from the catalytic subunit of DNA-PK (DNA-PKcs). In the absence of SF-1, DNA-PKcs was recruited to the centrosome and activated, causing aberrant activation of centrosomal Akt and cyclin-dependent kinase 2 (CDK2)/cyclin A and leading to centrosome overduplication. Centrosome overduplication caused by SF-1 depletion was averted by the elimination of DNA-PKcs, Ku70/80, or cyclin A or by the inhibition of CDK2 or Akt. In the nucleus, SF-1 did not interact with Ku70/80, and SF-1 depletion did not activate a nuclear DNA damage response. Centriole biogenesis was also unaffected. Thus, centrosomal DNA-PK signaling triggers centrosome overduplication, and this centrosomal event, but not the nuclear DNA damage response, is controlled by SF-1.

Establishment and mitotic characterization of newDrosophilaacentriolar cell lines fromDSas-4mutant

SummaryIn animal cells the centrosome is commonly viewed as the main cellular structure driving microtubule (MT) assembly into the mitotic spindle apparatus. However, additional pathways, such as those mediated by chromatin and augmin, are involved in the establishment of functional spindles. The molecular mechanisms involved in these pathways remain poorly understood, mostly due to limitations inherent to current experimental systems available. To overcome these limitations we have developed six new Drosophila cell lines derived from Drosophila homozygous mutants for DSas-4, a protein essential for centriole biogenesis. These cells lack detectable centrosomal structures, astral MT, with dispersed pericentriolar proteins D-PLP, Centrosomin and γ-tubulin. They show poorly focused spindle poles that reach the plasma membrane. Despite being compromised for functional centrosome, these cells could successfully undergo mitosis.Live-cell imaging analysis of acentriolar spindle assembly revealed that nascent MTs are nucleated from multiple points in the vicinity of chromosomes. These nascent MTs then grow away from kinetochores allowing the expansion of fibers that will be part of the future acentriolar spindle. MT repolymerization assays illustrate that acentriolar spindle assembly occurs “inside-out” from the chromosomes. Colchicine-mediated depolymerization of MTs further revealed the presence of a functional Spindle Assembly Checkpoint (SAC) in the acentriolar cells. Finally, pilot RNAi experiments open the potential use of these cell lines for the molecular dissection of anastral pathways in spindle and centrosome assembly.

CAND1 Promotes PLK4-Mediated Centriole Overduplication and Is Frequently Disrupted in Prostate Cancer

Centrosomes play a crucial role in the maintenance of genome stability by orchestrating bipolar mitotic spindle formation. The centrosome normally duplicates precisely once before mitosis in a process that is extensively regulated by protein degradation including SKP1-Cullin 1 (CUL1)-F-box (SCF) E3 ubiquitin ligase activity. The core SCF component CUL1 has recently been found to be required to suppress the formation of supernumerary centrosomes and centrioles, the core-forming units of centrosomes. Here, we identify the CUL1-interacting protein cullin-associated and neddylation-dissociated 1 (CAND1) as a novel centrosomal protein with a role in centriole duplication control. CAND1 was found to synergize with Polo-like kinase 4 (PLK4), a master regulator of centriole biogenesis, in the induction of centriole overduplication. We provide evidence that CAND1 functions in this process by increasing PLK4 protein stability. Furthermore, mutants of CUL1 that lack the ability to interact with CAND1 and are unable to assemble functional E3 ubiquitin ligase complexes were impaired in their ability to restrain aberrant daughter centriole synthesis. To corroborate a role of CAND1 in human carcinogenesis, we analyzed a series of prostate adenocarcinomas and found altered expression of CAND1 on the mRNA or protein level in 52.9% and 40.8%, respectively, of the tumor samples analyzed. These results highlight the role of altered SCF components in cancer in general and encourage further studies to explore the SCF-CAND1 axis for the development of novel predictive biomarkers and therapeutic approaches in prostate cancer.

Show me your license, please: deregulation of centriole duplication mechanisms that promote amplification.

Centrosomes are organelles involved in generating and organizing the interphase microtubule cytoskeleton, mitotic spindles and cilia. At the centrosome core are a pair of centrioles, structures that act as the duplicating elements of this organelle. Centrioles function to recruit and organize pericentriolar material which nucleates microtubules. While centrioles are relatively simple in construction, the mechanics of centriole biogenesis remain an important yet poorly understood process. More mysterious still are the regulatory mechanisms that oversee centriole assembly. The fidelity of centriole duplication is critical as defects in either the assembly or number of centrioles promote aneuploidy, primary microcephaly, birth defects, ciliopathies and tumorigenesis. In addition, some pathogens employ mechanisms to promote centriole overduplication to the detriment of the host cell. This review summarizes our current understanding of this important topic, highlighting the need for further study if new therapeutics are to be developed to treat diseases arising from defects of centrosome duplication.

BLD10/CEP135 Is a Microtubule-Associated Protein that Controls the Formation of the Flagellum Central Microtubule Pair

Cilia and flagella are involved in a variety of processes and human diseases, including ciliopathies and sterility. Their motility is often controlled bya central microtubule (MT) pair localized within the ciliary MT-based skeleton, the axoneme. We characterized the formation of the motility apparatus in detail in Drosophila spermatogenesis. We show that assembly of the central MT pair starts prior to the meiotic divisions, with nucleation of a singlet MT within the basal body of a small cilium, and that the second MT of the pair only assembles much later, upon flagella formation. BLD10/CEP135, a conserved player in centriole and flagella biogenesis, can bind and stabilize MTs and is required for the early steps of central MT pair formation. This work describes a genetically tractable system to study motile cilia formation and provides an explanation for BLD10/CEP135's role in assembling highly stable MT-based structures, such as motile axonemes and centrioles.

Tankyrase 1 regulates centrosome function by controlling CPAP stability

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