Chlamydia trachomatis infection causes mitotic spindle pole defects independently from its effects on centrosome amplification

Chlamydiae are Gram negative, obligate intracellular bacteria, and Chlamydia trachomatis is the etiologic agent of the most commonly reported sexually transmitted disease in the United States. Chlamydiae undergo a biphasic life cycle that takes place inside a parasitophorous vacuole termed an inclusion. Chlamydial infections have been epidemiologically linked to cervical cancer in patients previously infected by human papillomavirus (HPV). The inclusion associates very closely with host cell centrosomes, and this association is dependent upon the host motor protein dynein. We have previously reported that this interaction induces supernumerary centrosomes in infected cells, leading to multipolar mitotic spindles and inhibiting accurate chromosome segregation. Our findings demonstrate that chlamydial infection causes mitotic spindle defects independently of its effects on centrosome amplification. We show that chlamydial infection increases centrosome spread and inhibits the spindle assembly checkpoint delay to disrupt centrosome clustering. These data suggest that chlamydial infection exacerbates the consequences of centrosome amplification by inhibiting the cells' ability to suppress the effects of these defects on mitotic spindle organization. We hypothesize that these combined effects on mitotic spindle architecture identifies a possible mechanism for Chlamydia as a cofactor in cervical cancer formation.     

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 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.     

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.     

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.     

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.     

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.     

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.     

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.     

Generation and Fates of Supernumerary Centrioles in Dividing Cells

The centrosome is a subcellular organelle from which a cilium assembles. Since centrosomes function as spindle poles during mitosis, they have to be present as a pair in a cell. How the correct number of centrosomes is maintained in a cell has been a major issue in the fields of cell cycle and cancer biology. Centrioles, the core of centrosomes, assemble and segregate in close connection to the cell cycle. Abnormalities in centriole numbers are attributed to decoupling from cell cycle regulation. Interestingly, supernumerary centrioles are commonly observed in cancer cells. In this review, we discuss how supernumerary centrioles are generated in diverse cellular conditions. We also discuss how the cells cope with supernumerary centrioles during the cell cycle.     

Centrosome duplication: of rules and licenses

Most microtubule arrays in animal cells, including the bipolar spindle required for cell division, are organized by centrosomes. Thus, strict control of centrosome numbers is crucial for accurate chromosome segregation. Each centrosome comprises two centrioles, which need to be duplicated exactly once in every cell cycle. Recent work has begun to illuminate the mechanisms that regulate centriole duplication. First, genetic and structural studies concur to delineate a centriole assembly pathway in Caenorhabditis elegans. Second, the protease Separase, previously known to trigger sister chromatid separation, has been implicated in a licensing mechanism that restricts centrosome duplication to a single occurrence per cell cycle. Finally, Plk4 (also called Sak), a member of the Polo kinase family, has been identified as a novel positive regulator of centriole formation.     

SAS-4 is essential for centrosome duplication in C elegans and is recruited to daughter centrioles once per cell cycle

The mechanisms governing centrosome duplication remain poorly understood. We identified a gene called sas-4 that is essential for this process in C. elegans. SAS-4 encodes a predicted coiled-coil protein that localizes to a tiny dot in the center of centrosomes throughout the cell cycle. FRAP experiments with GFP-SAS-4 transgenic embryos reveal that SAS-4 is recruited to the centrosome once per cell cycle, at the time of organelle duplication. Additional evidence indicates that SAS-4 is recruited to the daughter centriole or a closely associated structure. These findings identify SAS-4 recruitment as a key step in the centrosome duplication cycle.     

Procentriole assembly revealed by cryo‐electron tomography

Centrosomes are cellular organelles that have a major role in the spatial organisation of the microtubule network. The centrosome is comprised of two centrioles that duplicate only once during the cell cycle, generating a procentriole from each mature centriole. Despite the essential roles of centrosomes, the detailed structural mechanisms involved in centriole duplication remain largely unknown. Here, we describe human procentriole assembly using cryo‐electron tomography. In centrosomes, isolated from human lymphoblasts, we observed that each one of the nine microtubule triplets grows independently around a periodic central structure. The proximal end of the A‐microtubule is capped by a conical structure and the B‐ and C‐microtubules elongate bidirectionally from its wall. These observations suggest that the gamma tubulin ring complex (γ‐TuRC) has a fundamental role in procentriole formation by nucleating the A‐microtubule that acts as a template for B‐microtubule elongation that, in turn, supports C‐microtubule growth. This study provides new insights into the initial structural events involved in procentriole assembly and establishes the basis for determining the molecular mechanisms of centriole duplication on the nanometric scale.     

Regulated expression of the centrosomal protein DdCP224 affects microtubule dynamics and reveals mechanisms for the control of supernumerary centrosome number

The Dictyostelium XMAP215 family member DdCP224 is involved in centrosome duplication and cytokinesis and is concentrated at the centrosome and microtubule tips. Herein, we have created a DdCP224 promoter replacement mutant that allows both over- and underexpression. Overexpression led to supernumerary microtubule-organizing centers and, independently, an increase of the number of multinuclear cells. Electron microscopy demonstrated that supernumerary microtubule-organizing centers represented bona fide centrosomes. Live cell imaging of DdCP224-green fluorescent protein mutants also expressing green fluorescent protein-histone2B as a DNA label revealed that supernumerary centrosomes were also competent of cell cycle-dependent duplication. In contrast, underexpression of DdCP224 inhibited cell growth, reduced the number and length of astral microtubules, and caused nocodazole hypersensitivity. Moreover, microtubule regrowth after nocodazole removal was dependent on DdCP224. Underexpression also resulted in a striking disappearance of supernumerary centrosomes and multinuclear cells caused by previous overexpression. We show for the first time by live cell observation that the number of supernumerary centrosomes can be reduced either by centrosome fusion (coalescence) or by the formation of cytoplasts containing supernumerary centrosomes during 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.     

HOPS is an essential constituent of centrosome assembly

Centrosomes direct microtubule organization during cell division. Aberrant number of centrosomes results from alteration of its components and leads to abnormal mitoses and chromosome instability. HOPS is a newly discovered protein isolated during liver regeneration, implicated in cell proliferation. Here, we provide evidence that HOPS is an integral constituent of centrosomes. HOPS is associated with classical markers of centrosomes and found in cytosolic complexes containing CRM-1, γ-tubulin, eEF-1A and HSP70. These features suggest that HOPS is involved in centrosome assembly and maintenance. HOPS depletion generates supernumerary centrosomes, multinucleated cells and multipolar spindle formation leading to activation of p53 checkpoint and cell cycle arrest. The presence of HOPS in cytosolic complexes supports that centrosome proteins might be preassembled in the cytoplasm to then be rapidly recruited for centrosome duplication. Altogether these data show HOPS implication in the control of cell division. HOPS contribution appears relevant to understand genomic instability and centrosome amplification in cancer.     

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.     

Asymmetric localization of the CDC25B phosphatase to the mother centrosome during interphase

The Cyclin-Dependent Kinase (CDK)-activating phosphatase CDC25B, localises to the centrosomes where its activity is both positively and negatively regulated by several kinases including Aurora A and CHK1. Our recent data also demonstrate a role for CDC25B in the centrosome duplication cycle and microtubule nucleation in interphase that appears to involve the recruitment of γ-tubulin to the centrosomes. In the present study, we report that CDC25B, along with CHK1, CDK1 and WEE1, localise asymmetrically around the mother centrosome from S to G2-phases, and gradually become evenly distributed to the two centrosomes by late G2 phase, concomitant with centrosome maturation. We further demonstrate that siRNA inhibition of CDC25B results in an accumulation of cells in G2 phase with two separated centrosomes, each containing only a single centriole, suggesting a requirement for CDC25B in centriole duplication. We propose that the localisation of key cell cycle regulators to the mother centrosome ensures synchrony between the centrosome duplication and cell division cycles.     

Centrosome-centriole abnormalities are markers for abnormal cell divisions and cancer in the transgenic adenocarcinoma mouse prostate (TRAMP) model

We utilized the transgenic adenocarcinoma mouse prostate (TRAMP) model to study the formation of abnormal mitosis in malignant tumors of the prostate. The results presented here are focused on centrosome and centriole abnormalities and the implications for abnormal cell divisions, genomic instability, and apoptosis. Centrosomes are microtubule organizing organelles which assemble bipolar spindles in normal cells but can organize mono-, tri-, and multipolar mitoses in tumor cells, as shown here with histology and electron microscopy in TRAMP neoplastic tissue. These abnormalities will cause unequal distribution of chromosomes and can initiate imbalanced cell cycles in which checkpoints for cell cycle control are lost. Neoplastic tissue of the TRAMP model is also characterized by numerous apoptotic cells. This may be the result of multipolar mitoses related to aberrant centrosome formations. Our results also reveal that centrosomes at the poles in mitotic cancer cells contain more than the regular perpendicular pair of centrioles which indicates abnormal distribution of centrioles during separation to the mitotic poles. Abnormalities in the centriole-centrosome complex are also seen during interphase where the complex is either closely associated with the nucleus or loosely dispersed in the cytoplasm. An increase in centriole numbers is observed during interphase, which may be the result of increased centriole duplication. Alternatively, these centrioles may be derived from basal bodies that have accumulated in the cell's cytoplasm, after the loss of cell borders. The supernumerary centrioles may participate in the formation of abnormal mitoses during cell division. These results demonstrate multiple abnormalities in the centrosome-centriole complex during prostate cancer that result in abnormal mitoses and may lead to increases in genomic instability and/or apoptosis.