A centriole's subdistal appendages: contributions to cell division,ciliogenesis and differentiation

The centrosome is a highly conserved structure composed of two centrioles surrounded by pericentriolar material. The mother, and inherently older, centriole has distal and subdistal appendages, whereas the daughter centriole is devoid of these appendage structures. Both appendages have been primarily linked to functions in cilia formation. However, subdistal appendages present with a variety of potential functions that include spindle placement, chromosome alignment, the final stage of cell division (abscission) and potentially cell differentiation. Subdistal appendages are particularly interesting in that they do not always display a conserved ninefold symmetry in appendage organization on the mother centriole across eukaryotic species, unlike distal appendages. In this review, we aim to differentiate both the morphology and role of the distal and subdistal appendages, with a particular focus on subdistal appendages.     

Is the centrosome a dynamic structure?]

Ca++ effects on the organization of human centrosomes isolated according to Bornens et al., were followed by double immunofluorescence technique. Ca++, at millimolar range, is able to modify the distribution of the pericentriolar material (PCM) and to decrease the intercentriolar distance. In the light of these results, we have slightly modified the centrosome isolation method and shown that centrosomes isolated in the absence of EDTA have several structural differences from the previously described structure. In particular, centriole diameter is decreased by a transverse sliding of microtubule triplets with respect to each other, suggesting the possibility of movements within centrioles themselves.     

Epsilon-tubulin is required for centriole duplication and microtubule organization

Centrosomes nucleate microtubules and serve as poles of the mitotic spindle. Centrioles are a core component of centrosomes and duplicate once per cell cycle. We previously identified epsilon-tubulin as a new member of the tubulin superfamily that localizes asymmetrically to the two centrosomes after duplication. We show that recruitment of epsilon-tubulin to the new centrosome can only occur after exit from S phase and that epsilon-tubulin is associated with the sub-distal appendages of mature centrioles. Xenopus laevis epsilon-tubulin was cloned and shown to be similar to human epsilon-tubulin in both sequence and localization. Depletion of epsilon-tubulin from Xenopus egg extracts blocks centriole duplication in S phase and formation of organized centrosome-independent microtubule asters in M phase. We conclude that epsilon-tubulin is a component of the sub-distal appendages of the centriole, explaining its asymmetric localization to old and new centrosomes, and that epsilon-tubulin is required for centriole duplication and organization of the pericentriolar material.     

Centrosome reduction during gametogenesis and its significance

Animal spermatids and primary oocytes initially have typical centrosomes comprising pairs of centrioles and pericentriolar fibrous centrosomal proteins. These somatic cell-like centrosomes are partially or completely degenerated during gametogenesis. Centrosome reduction during spermiogenesis comprises attenuation of microtubule nucleation function, loss of pericentriolar material, and centriole degeneration. Centrosome reduction during oogenesis is due to complete degeneration of centrioles, which leads to dispersal of the pericentriolar centrosomal proteins, loss of replicating capacity of the spindle poles, and switching to acentrosomal mode of spindle organization. Oocyte centrosome reduction plays an important role in preventing parthenogenetic embryogenesis and balancing centrosome number in the embryonic cells.     

Separate to operate: control of centrosome positioning and separation

The centrosome is the main microtubule (MT)-organizing centre of animal cells. It consists of two centrioles and a multi-layered proteinaceous structure that surrounds the centrioles, the so-called pericentriolar material. Centrosomes promote de novo assembly of MTs and thus play important roles in Golgi organization, cell polarity, cell motility and the organization of the mitotic spindle. To execute these functions, centrosomes have to adopt particular cellular positions. Actin and MT networks and the association of the centrosomes to the nuclear envelope define the correct positioning of the centrosomes. Another important feature of centrosomes is the centrosomal linker that connects the two centrosomes. The centrosome linker assembles in late mitosis/G1 simultaneously with centriole disengagement and is dissolved before or at the beginning of mitosis. Linker dissolution is important for mitotic spindle formation, and its cell cycle timing has profound influences on the execution of mitosis and proficiency of chromosome segregation. In this review, we will focus on the mechanisms of centrosome positioning and separation, and describe their functions and mechanisms in the light of recent findings.     

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     

Structural and chemical characterization of isolated centrosomes

A procedure adapted from that described by Mitchison and Kirschner [Nature 312:232-237, 1984] was used to isolate centrosomes from human lymphoid cells. High yields of homogeneous centrosomes (60% of the theoretical total, assuming one centrosome per cell) were obtained. Centrosomes were isolated as pairs of centrioles, plus their associated pericentriolar material. Ultrastructural investigation revealed: 1) a link between both centrioles in a centrosome formed by the gathering in of a unique bundle of thin filaments surrounding each centriole; 2) a stereotypic organization of the pericentriolar material, including a rim of constant width at the proximal end of each centriole and a disc of nine satellite arms organized according to a ninefold symmetry at the distal end and; 3) an axial hub in the lumen of each centriole at the distal end surrounded by some ill-defined material. The total protein content was 2 to 3 X 10(-2) pg per isolated centrosome, a figure that suggests that the preparations were close to homogeneity. The protein composition was complex but specific, showing proteins ranging from 180 to 300 kD, one prominent band at 130 kD, and a group of proteins between 50 and 65 kD. Actin was also present in centrosome preparations. Functional studies demonstrated that the isolated centrosomes were competent to nucleate microtubules in vitro from purified tubulin in conditions in which spontaneous assembly could not occur. They were also very effective at inducing cleavage when microinjected into unfertilized Xenopus eggs.     

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.     

Kif3a interacts with Dynactin subunit p150Glued to organize centriole subdistal appendages

Formation of cilia, microtubule‐based structures that function in propulsion and sensation, requires Kif3a, a subunit of Kinesin II essential for intraflagellar transport (IFT). We have found that, Kif3a is also required to organize centrioles. In the absence of Kif3a, the subdistal appendages of centrioles are disorganized and lack p150^Glued and Ninein. Consequently, microtubule anchoring, centriole cohesion and basal foot formation are abrogated by loss of Kif3a. Kif3a localizes to the mother centriole and interacts with the Dynactin subunit p150^Glued. Depletion of p150^Glued phenocopies the effects of loss of Kif3a, indicating that Kif3a recruitment of p150^Glued is critical for subdistal appendage formation. The transport functions of Kif3a are dispensable for subdistal appendage organization as mutant forms of Kif3a lacking motor activity or the motor domain can restore p150^Glued localization. Comparison to cells lacking Ift88 reveals that the centriolar functions of Kif3a are independent of IFT. Thus, in addition to its ciliogenic roles, Kif3a recruits p150^Glued to the subdistal appendages of mother centrioles, critical for centrosomes to function as microtubule‐organizing centres.     

Breaking the ties that bind: new advances in centrosome biology

The centrosome, which consists of two centrioles and the surrounding pericentriolar material, is the primary microtubule-organizing center (MTOC) in animal cells. Like chromosomes, centrosomes duplicate once per cell cycle and defects that lead to abnormalities in the number of centrosomes result in genomic instability, a hallmark of most cancer cells. Increasing evidence suggests that the separation of the two centrioles (disengagement) is required for centrosome duplication. After centriole disengagement, a proteinaceous linker is established that still connects the two centrioles. In G2, this linker is resolved (centrosome separation), thereby allowing the centrosomes to separate and form the poles of the bipolar spindle. Recent work has identified new players that regulate these two processes and revealed unexpected mechanisms controlling the centrosome cycle.     

<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.     

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.     

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.     

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.     

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.     

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.     

Detection of the centriole tyr- or acet-tubulin changes in endothelial cells treated with thrombin using microscopic immunocytochemistry

We used electron microscopic immunocytochemistry to examine the pattern of centriolar staining for tyrosinated or acetylated alpha-tubulin in endothelial cells during short-term incubation with thrombin. Endothelial cells isolated from human aorta (HAEC) and those isolated from umbilical vein (HUVEC) displayed an increase in the intensity of centriolar staining for acet-tubulin within 1 min after thrombin addition. A decrease in the intensity of centriolar staining for tyr-tubulin was detected in HUVEC within 1 min after thrombin addition, while in HAEC centriolar staining for tyr-tubulin became less intense only 5 min later. Mother and daughter centrioles of HUVEC cells displayed different intensity of immunostaining for acet-tubulin and showed no significant variation in the number of subdistal appendages after thrombin addition. Differently, HAEC cells had the same staining pattern of mother and daughter centrioles in both thrombin-untreated and thrombin-treated cultures. A sharp increase in the number of subdistal appendages of mother centriole occurred in HAEC within 5 min of incubation with thrombin. Our findings provided the direct evidence for centrosome involvement in the ligand-mediated signaling events and showed for the first time that ligand-dependent centrosome reorganization includes the centriole per se. Furthermore, based on our observations we would like to propose that MT-nucleating/anchoring properties of the centrosome are subject to rapid regulation by external signals such as thrombin.     

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.     

The forkhead-associated domain protein Cep170 interacts with Polo-like kinase 1 and serves as a marker for mature centrioles

We report the characterization of Cep170, a forkhead-associated (FHA) domain protein of previously unknown function. Cep170 was identified in a yeast two-hybrid screen for interactors of Polo-like kinase 1 (Plk1). In human cells, Cep170 is constantly expressed throughout the cell cycle but phosphorylated during mitosis. It interacts with Plk1 in vivo and can be phosphorylated by Plk1 in vitro, suggesting that it is a physiological substrate of this kinase. Both overexpression and small interfering RNA (siRNA)-mediated depletion studies suggest a role for Cep170 in microtuble organization and cell morphology. Cep170 associates with centrosomes during interphase and with spindle microtubules during mitosis. As shown by immunoelectron microscopy, Cep170 associates with subdistal appendages, typical of the mature mother centriole. Thus, anti-Cep170 antibodies stain only one centriole during G1, S, and early G2, but two centrioles during late G2 phase of the cell cycle. We show that Cep170 labeling can be used to discriminate bona fide centriole overduplication from centriole amplification that results from aborted cell division.     

ANKRD26 recruits PIDD1 to centriolar distal appendages to activate the PIDDosome following centrosome amplification

Centriole copy number is tightly maintained by the once‐per‐cycle duplication of these organelles. Centrioles constitute the core of centrosomes, which organize the microtubule cytoskeleton and form the poles of the mitotic spindle. Centrosome amplification is frequently observed in tumors, where it promotes aneuploidy and contributes to invasive phenotypes. In non‐transformed cells, centrosome amplification triggers PIDDosome activation as a protective response to inhibit cell proliferation, but how extra centrosomes activate the PIDDosome remains unclear. Using a genome‐wide screen, we identify centriole distal appendages as critical for PIDDosome activation in cells with extra centrosomes. The distal appendage protein ANKRD26 is found to interact with and recruit the PIDDosome component PIDD1 to centriole distal appendages, and this interaction is required for PIDDosome activation following centrosome amplification. Furthermore, a recurrent ANKRD26 mutation found in human tumors disrupts PIDD1 localization and PIDDosome activation in cells with extra centrosomes. Our data support a model in which ANKRD26 initiates a centriole‐derived signal to limit cell proliferation in response to centrosome amplification.