Drosophila asterless and vertebrate Cep152 Are orthologs essential for centriole duplication

The centriole is the core structure of centrosome and cilium. Failure to restrict centriole duplication to once per cell cycle has serious consequences and is commonly observed in cancer. Despite its medical importance, the mechanism of centriole formation is poorly understood. Asl was previously reported to be a centrosomal protein essential for centrosome function. Here we identify mecD, a severe loss-of-function allele of the asl gene, and demonstrate that it is required for centriole and cilia formation. Similarly, Cep152, the Asl ortholog in vertebrates, is essential for cilia formation and its function can be partially rescued by the Drosophila Asl. The study of Asl localization suggests that it is closely associated with the centriole wall, but is not part of the centriole structure. By analyzing the biogenesis of centrosomes in cells depleted of Asl, we found that, while pericentriolar material (PCM) function is mildly affected, Asl is essential for daughter centriole formation. The clear absence of several centriolar markers in mecD mutants suggests that Asl is critical early in centriole duplication.     

Clockwise or anticlockwise? Turning the centriole triplets in the right direction!

Centrosomes are small cytoplasmic macromolecular assemblies composed from two major components, centrioles and pericentriolar material, each with its own complex architecture. This organelle is of interest because it plays a role in a number of fundamental cellular processes and defects in these processes have recently been correlated with variety of human disease. Increasingly, what is known about the structure of this organelle has been overshadowed by the increasing wealth of information on its biochemistry. In this short review, we highlight some of the common centriole structural errors found in the literature and define a set of rules that define centriole structure.     

Cartwheel assembly

The cartwheel is a subcentriolar structure consisting of a central hub and nine radially arranged spokes, located at the proximal end of the centriole. It appears at the initial stage of the centriole assembly process as the first ninefold symmetrical structure. The cartwheel was first described more than 50 years ago, but it is only recently that its pivotal role in establishing the ninefold symmetry of the centriole was demonstrated. Significant progress has since been made in understanding its fine structure and assembly mechanism. Most importantly, the central part of the cartwheel, from which the ninefold symmetry originates, is shown to form by self-association of nine dimers of the protein SAS-6. This finding, together with emerging data on other components of the cartwheel, has opened new avenues in centrosome biology.     

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.     

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.     

Higher order structure of the PCM adjacent to the centriole

The centrosome is the major microtubule organizing center in most animal cells. This cytoplasmic organelle consists of two components : a mature centriole (or a pair of centrioles) and a mass of pericentriolar material (PCM). The PCM has been described as either a cloud of material that encases the entire centriole or as a cluster of proteins divided into two subsets, one that adheres to the lateral surface of the centriole and another that extends outward from this region as a cloud of material. In contrast to these protein distribution patterns, we demonstrated in a previous study that a subset of proteins present within the PCM is integrated together to form a tube (PCM tube) with an open and closed end that is duplicated in concert with centrosome duplication. The present study was undertaken to determine if this tubular conformation represents proteins that are confined to the surface of the centriole or if it represents a subset of proteins within the cloud of material that extends outward from the centriole. We document that : (1) the PCM tube represents a portion of the PCM directly associated with the centriole; (2) the PCM tube has a specific and reproducible relationship to the polar structure of the centriole; (3) the tube is a site of cytoplasmic microtubule organization, and has a structure that influences the initial pattern of microtubule assembly within the juxta-centriolar region; and (4) the PCM tube has a structural relationship with respect to the centriole, which allows the simultaneous expression of centriole- and PCM-based functions (e.g., ciliogenesis and cytoplasmic microtubule organization). Based on these findings, we propose a new model of the PCM at the centriole. This model highlights the role played by the proximal end of the centriole in the nucleation and organization of centriole-associated PCM, and indicates that the centrosome has an overall polarity in the region of the centriole.     

Centriole is the pivot co-ordinating dynamic signaling for cell proliferation and organization during early development in the vertebrates

Vertebrates have an elaborate and functionally segmented body. It evolves from a single cell by systematic cell proliferation but attains a complex body structure with exquisite precision. This development requires two cellular events: cell cycle and ciliogenesis. For these events, the dynamic molecular signaling is converged at the centriole. The cell cycle helps in cell proliferation and growth of the body and is a highly regulated and integrated process. Its errors cause malignancies and developmental disorders. The cells newly proliferated are organized during organogenesis. For a cellular organization, dedicated signaling hubs are developed in the cells, and most often cilia are utilized. The cilium is generated from one of the centrioles involved in cell proliferation. The developmental signaling pathways hosted in cilia are essential for the elaboration of the body plan. The cilium's compartmental seclusion is ideal for noise-free molecular signaling and is essential for the precision of the body layout. The dysfunctional centrioles and primary cilia distort the development of body layout that manifest as serious developmental disorders. Thus, centriole has a dual role in the growth and cellular organization. It organizes dynamically expressed molecules of cell cycle and ciliogenesis and plays a balancing act to generate new cells and organize them during development. A putative master molecule may regulate and co-ordinate the dynamic gene expression at the centrioles. The convergence of many critical signaling components at the centriole reiterates the idea that centriole is a major molecular workstation involved in elaborating the structural design and complexity in vertebrates.     

Abnormal centrosomal structure and duplication in Cep135-deficient vertebrate cells

Centrosomes are key microtubule-organizing centers that contain a pair of centrioles, conserved cylindrical, microtubule-based structures. Centrosome duplication occurs once per cell cycle and relies on templated centriole assembly. In many animal cells this process starts with the formation of a radially symmetrical cartwheel structure. The centrosomal protein Cep135 localizes to this cartwheel, but its role in vertebrates is not well understood. Here we examine the involvement of Cep135 in centriole function by disrupting the Cep135 gene in the DT40 chicken B-cell line. DT40 cells that lack Cep135 are viable and show no major defects in centrosome composition or function, although we note a small decrease in centriole numbers and a concomitant increase in the frequency of monopolar spindles. Furthermore, electron microscopy reveals an atypical structure in the lumen of Cep135-deficient centrioles. Centrosome amplification after hydroxyurea treatment increases significantly in Cep135-deficient cells, suggesting an inhibitory role for the protein in centrosome reduplication during S-phase delay. We propose that Cep135 is required for the structural integrity of centrioles in proliferating vertebrate cells, a role that also limits centrosome amplification in S-phase–arrested cells.     

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.     

The centriole and the cell

The endosymbiotic theory of the origin of the centriole is found lacking as no DNA has been found in the structure. The centriole is related to the ribosome in that it is a structure nucleated by RNA. This RNA (morphic) has the property of localizing itself at the site of the old centriole before nucleating a new one. The implication of this property of the localization of RNA is discussed with respect to the way the eukaryotic cell determines its shape.     

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.     

Spermatogenesis and sperm structure of Acerella muscorum, (Ionescu, 1930) (Hexapoda, Protura)

The general organization of the male genital system, the spermatogenesis and the sperm structure of the proturan Acerella muscorum have been described. At the apex of testis apical huge cells are present; their cytoplasm contains a conventional centriole, a large amount of dense material and several less electron-dense masses surrounded by mitochondria. Spermatocytes have normal centrioles and are interconnected by cytoplasmic bridges. Such bridges seem to be absent between spermatid cells and justify the lack of synchronization of cell maturation. Spermatids are almost globular cells with a spheroidal nucleus and a large mass of dense material corresponding to the centriole adjunct. Within this mass a centriole is preserved. Mitochondria of normal structure are located between the nucleus and the plasma membrane. The spermatids are surrounded by a thick membrane. No flagellar structure is formed. Sperm have a compact spheroidal nucleus, a large cap of centriole adjunct material within which a centriole is still visible. A layer of mitochondria is located over the nucleus. The cytoplasm is reduced in comparison to spermatids; many dense bodies are interspersed with sperm in the testicular lumen. The sperm are small, immotile cells of about 2.5-3 μm in diameter.     

Centrioles in the cell cycle. I. Epithelial cells

A study was made of the structure of the centrosome in the cell cycle in a nonsynchronous culture of pig kidney embryo (PE) cells. In the spindle pole of the metaphase cell there are two mutually perpendicular centrioles (mother and daughter) which differ in their ultrastructure. An electron-dense halo, which surrounds only the mother centriole and is the site where spindle microtubules converge, disappears at the end of telophase. In metaphase and anaphase, the mother centriole is situated perpendicular to the spindle axis. At the beginning of the G1 period, pericentriolar satellites are formed on the mother centriole with microtubules attached to them; the two centrioles diverge. The structures of the two centrioles differ throughout interphase; the mother centriole has appendages, the daughter does not. Replication of the centrioles occurs approximately in the middle of the S period. The structure of the procentrioles differs sharply from that of the mature centriole. Elongation of procentrioles is completed in prometaphase, and their structure undergoes a number of successive changes. In the G2 period, pericentriolar satellites disappear and some time later a fibrillar halo is formed on both mother centrioles, i.e., spindle poles begin to form. In the cells that have left the mitotic cycle (G0 period), replication of centrioles does not take place; in many cells, a cilium is formed on the mother centriole. In a small number of cells a cilium is formed in the S and G2 periods, but unlike the cilium in the G0 period it does not reach the surface of the cell. In all cases, it locates on the centriole with appendages. At the beginning of the G1 period, during the G2 period, and in nonciliated cells in the G0 period, one of the centrioles is situated perpendicular to the substrate. On the whole, it takes a mature centriole a cycle and a half to form in PE cells.     

The ultrastructure of primary cilia in the endocrine and excretory duct cells of the pancreas of mice and rats

A primary cilium was frequently observed in the endocrine alpha, beta and delta cells, as well as in the excretory duct cells of the pancreas of normal mice and rats. The characteristic components of the cilium including the basal body, axoneme (shaft), and terminal part were clearly recognizable. The basal body or distal centriole surrounded by Golgi vesicles was perpendicularly oriented to the proximal centriole, and a dense striated band was seen filling the gap between them. The microtubules of the basal body consisted of nine peripheral triplets exhibiting a 9 + 0 pattern, an appearance similar to that of the proximal centriole. Rootlets, basal feet and alar sheets associated with the basal body were occasionally seen. The axoneme usually consisted of a 9 + 0 pattern of microtubule doublets, but other irregular patterns of 7 + 2, 7 + 3, and 8 + 1 were also seen. The microtubules in the terminal part of the cilium became fewer in number and had no peculiar arrangement. The cilium of the endocrine cells always projected into the intercellular canaliculus and was covered by the ciliary sheath, and occasionally, double cilia were visualized in the vicinity of beta cells. In the excretory duct cells, the cilium showed similar features, but it was slightly longer and always projected into the dense secretory content of duct lumen. On the other hand, no primary cilium was ever observed in the acinar cells of mouse and rat pancreas. In conclusion, the present study describes the morphology of primary cilia and its associated components in the endocrine and excretory duct cells of the pancreas of mice and rats. The findings suggest that the primary cilium should be considered as a constant intracellular organelle though its function and significance remain speculative.     

From Zero to Many: Control of Centriole Number in Development and Disease

Centrioles are essential for the formation of microtubule-derived structures, including cilia, flagella and centrosomes. These structures are involved in a variety of functions, from cell motility to division. In most dividing animal cells, centriole formation is coupled to the chromosome cycle. However, this is not the case in certain specialized divisions, such as meiosis, and in some differentiating cells. For example, oocytes loose their centrioles upon differentiation, whereas multiciliated epithelial cells make several of those structures after they exit the cell cycle. Aberrations of centriole number are seen in many cancer cells. Recent studies began to shed light on the molecular control of centriole number, its variations in development, and how centriole number changes in human disease. Here we review the recent developments in this field.     

Molecular characterization of centriole assembly in ciliated epithelial cells

Ciliated epithelial cells have the unique ability to generate hundreds of centrioles during differentiation. We used centrosomal proteins as molecular markers in cultured mouse tracheal epithelial cells to understand this process. Most centrosomal proteins were up-regulated early in ciliogenesis, initially appearing in cytoplasmic foci and then incorporated into centrioles. Three candidate proteins were further characterized. The centrosomal component SAS-6 localized to basal bodies and the proximal region of the ciliary axoneme, and depletion of SAS-6 prevented centriole assembly. The intraflagellar transport component polaris localized to nascent centrioles before incorporation into cilia, and depletion of polaris blocked axoneme formation. The centriolar satellite component PCM-1 colocalized with centrosomal components in cytoplasmic granules surrounding nascent centrioles. Interfering with PCM-1 reduced the amount of centrosomal proteins at basal bodies but did not prevent centriole assembly. This system will help determine the mechanism of centriole formation in mammalian cells and how the limitation on centriole duplication is overcome in ciliated epithelial cells.     

DSas-6 and Ana2 coassemble into tubules to promote centriole duplication and engagement

Centrioles form cilia and centrosomes, organelles whose dysfunction is increasingly linked to human disease. Centriole duplication relies on a few conserved proteins (ZYG-1/Sak/Plk4, SAS-6, SAS-5/Ana2, and SAS-4), and is often initiated by the formation of an inner "cartwheel" structure. Here, we show that overexpressed Drosophila Sas-6 and Ana2 coassemble into extended tubules (SAStubules) that bear a striking structural resemblance to the inner cartwheel of the centriole. SAStubules specifically interact with centriole proximal ends, but extra DSas-6/Ana2 is only recruited onto centrioles when Sak/Plk4 kinase is also overexpressed. This extra centriolar DSas-6/Ana2 induces centriole overduplication and, surprisingly, increased centriole cohesion. Intriguingly, we observe tubules that are structurally similar to SAStubules linking the engaged centrioles in normal wild-type cells. We conclude that DSas-6 and Ana2 normally cooperate to drive the formation of the centriole inner cartwheel and that they promote both centriole duplication and centriole cohesion in a Sak/Plk4-dependent manner.