Centrosome-associated Chk1 prevents premature activation of cyclin-B-Cdk1 kinase

Entry into mitosis occurs after activation of Cdk1, resulting in chromosome condensation in the nucleus and centrosome separation, as well as increased microtubule nucleation activity in the cytoplasm. The active cyclin-B1-Cdk1 complex first appears at the centrosome, suggesting that the centrosome may facilitate the activation of mitotic regulators required for the commitment of cells to mitosis. However, the signalling pathways involved in controlling the initial activation of Cdk1 at the centrosome remain largely unknown. Here, we show that human Chk1 kinase localizes to interphase, but not mitotic, centrosomes. Chemical inhibition of Chk1 resulted in premature centrosome separation and activation of centrosome-associated Cdk1. Forced immobilization of kinase-inactive Chk1 to centrosomes also resulted in premature Cdk1 activation. Conversely, under such conditions wild-type Chk1 impaired activation of centrosome-associated Cdk1, thereby resulting in DNA endoreplication and centrosome amplification. Activation of centrosomal Cdk1 in late prophase seemed to be mediated by cytoplasmic Cdc25B, whose activity is controlled by centrosome-associated Chk1. These results suggest that centrosome-associated Chk1 shields centrosomal Cdk1 from unscheduled activation by cytoplasmic Cdc25B, thereby contributing to proper timing of the initial steps of cell division, including mitotic spindle formation.     

The PTEN-Akt pathway impacts the integrity and composition of mitotic centrosomes

Loss of the tumor suppressor PTEN is observed in many human cancers that display increased chromosome instability and aneuploidy. The subcellular fractions of PTEN are associated with different functions that regulate cell growth, invasion and chromosome stability. In this study, we show a novel role for PTEN in regulating mitotic centrosomes. PTEN localization at mitotic centrosomes peaks between prophase and metaphase, paralleling the centrosomal localization of PLK-1 and γ-tubulin and coinciding with the time frame of centrosome maturation. In primary keratinocytes, knockdown of PTEN increased whole-cell levels of γ-tubulin and PLK-1 in an Akt-dependent manner and had little effect on recruitment of either protein to mitotic centrosomes. Conversely, knockdown of PTEN reduced centrosomal levels of pericentrin in an Akt-independent manner. Inhibition of Akt activation with MK2206 reduced the whole-cell and centrosome levels of PLK-1 and γ-tubulin and also prevented the recruitment of PTEN to mitotic centrosomes. This reduction in centrosome-associated proteins upon inhibition of Akt activity may contribute to the increase in defects in centrosome number and separation observed in metaphase cells. Concomitant PTEN knockdown and Akt inhibition reduced the frequency of metaphase cells with centrosome defects when compared with MK2206 treatment alone, indicating that both PTEN and pAkt are required to properly regulate centrosome composition during mitosis. The findings presented in this study demonstrate a novel role for PTEN and Akt in controlling centrosome composition and integrity during mitosis and provide insight into how PTEN functions as a multifaceted tumor suppressor.     

Identification of novel centrosomal proteins in Dictyostelium discoideum by comparative proteomic approaches

The centrosome functions as the main microtubule-organization center of the cell and is of importance for all microtubule-dependent processes such as organelle transport and directionality of cell migration. One of the major model organisms in centrosome research is the slime mold Dictyostelium discoideum. Since only 10 centrosomal proteins are known so far in Dictyostelium discoideum, the elucidation of new centrosomal components may give a more comprehensive understanding of centrosomal function. To distinguish between centrosomal and contaminating proteins we established different separation and relative quantification strategies including techniques such as iTRAQ and DIGE. In this work, we present the identification of several known components as well as more than 70 new candidates--currently subject of further investigations--for the protein inventory of the Dictyostelium centrosome. Among these protein identifications, 44% represent hypothetical proteins of still unknown function associated with the centrosome.     

The primary cilium: keeper of the key to cell division

Assembly of the nonmotile primary cilium of vertebrate cells requires one of the centrioles of the centrosome. A cluster of new studies, including one in this issue of Cell by Pugacheva et al. (2007), reveal that ciliary assembly proteins influence cell-cycle progression and that a centrosomal "mitotic kinase" promotes ciliary disassembly. The link between the cell cycle and the primary cilium may reflect a requirement for liberation of the ciliary centriole to allow the centrosome to form the mitotic spindle.     

Dictyostelium centrin-related protein (DdCrp), the most divergent member of the centrin family, possesses only two EF hands and dissociates from the centrosome during mitosis.

We have identified a Dictyostelium discoideum cDNA sequence with homology to centrins. The derived protein, Dictyostelium discoideum centrinn-related protein (DdCrp), is the most divergent member of the centrin family. Most strikingly it lacks the first two EF-hand consensus motifs, whereas a number of other centrin-specific sequence features are conserved. Southern and Northern blot analysis and the data presently available from the Dictyostelium genome and cDNA projects suggest that DdCrp is the only centrin isoform present in Dictyostelium. Immunofluorescence analysis with anti-DdCrp antibodies revealed that the protein is localized to the centrosome, to a second, centrosome-associated structure close to the nucleus and to the nucleus itself. Confocal microscopy resolved that the centrosomal label is confined to the corona surrounding the centrosome core. Unlike for other centrins the localization of DdCrp is cell cycle-dependent. Both the centrosomal and the centrosome-associated label disappear during prometaphase, most likely in concert with the dissociation of the corona at this stage. The striking differences of DdCrp to all other centrins may be related to the distinct structure and duplication mode of the Dictyostelium centrosome.     

Involvement of poly(ADP-Ribose) polymerase 1 and poly(ADP-Ribosyl)ation in regulation of centrosome function

The regulatory mechanism of centrosome function is crucial to the accurate transmission of chromosomes to the daughter cells in mitosis. Recent findings on the posttranslational modifications of many centrosomal proteins led us to speculate that these modifications might be involved in centrosome behavior. Poly(ADP-ribose) polymerase 1 (PARP-1) catalyzes poly(ADP-ribosyl)ation to various proteins. We show here that PARP-1 localizes to centrosomes and catalyzes poly(ADP-ribosyl)ation of centrosomal proteins. Moreover, centrosome hyperamplification is frequently observed with PARP inhibitor, as well as in PARP-1-null cells. Thus, it is possible that chromosomal instability known in PARP-1-null cells can be attributed to the centrosomal dysfunction. P53 tumor suppressor protein has been also shown to be localized at centrosomes and to be involved in the regulation of centrosome duplication and monitoring of the chromosomal stability. We found that centrosomal p53 is poly(ADP-ribosyl)ated in vivo and centrosomal PARP-1 directly catalyzes poly(ADP-ribosyl)ation of p53 in vitro. These results indicate that PARP-1 and PARP-1-mediated poly(ADP-ribosyl)ation of centrosomal proteins are involved in the regulation of centrosome function.     

Proteomic and functional analysis of the mitotic Drosophila centrosome

Regulation of centrosome structure, duplication and segregation is integrated into cellular pathways that control cell cycle progression and growth. As part of these pathways, numerous proteins with well‐established non‐centrosomal localization and function associate with the centrosome to fulfill regulatory functions. In turn, classical centrosomal components take up functional and structural roles as part of other cellular organelles and compartments. Thus, although a comprehensive inventory of centrosome components is missing, emerging evidence indicates that its molecular composition reflects the complexity of its functions. We analysed the Drosophila embryonic centrosomal proteome using immunoisolation in combination with mass spectrometry. The 251 identified components were functionally characterized by RNA interference. Among those, a core group of 11 proteins was critical for centrosome structure maintenance. Depletion of any of these proteins in Drosophila SL2 cells resulted in centrosome disintegration, revealing a molecular dependency of centrosome structure on components of the protein translation machinery, actin‐ and RNA‐binding proteins. In total, we assigned novel centrosome‐related functions to 24 proteins and confirmed 13 of these in human cells.     

Loss of centrosome integrity induces p38-p53-p21-dependent G1-S arrest

Centrosomes organize the microtubule cytoskeleton for both interphase and mitotic functions. They are implicated in cell-cycle progression but the mechanism is unknown. Here, we show that depletion of 14 out of 15 centrosome proteins arrests human diploid cells in G1 with reduced Cdk2-cyclin A activity and that expression of a centrosome-disrupting dominant-negative construct gives similar results. Cell-cycle arrest is always accompanied by defects in centrosome structure and function (for example, duplication and primary cilia assembly). The arrest occurs from within G1, excluding contributions from mitosis and cytokinesis. The arrest requires p38, p53 and p21, and is preceded by p38-dependent activation and centrosomal recruitment of p53. p53-deficient cells fail to arrest, leading to centrosome and spindle dysfunction and aneuploidy. We propose that loss of centrosome integrity activates a checkpoint that inhibits G1-S progression. This model satisfies the definition of a checkpoint in having three elements: a perturbation that is sensed, a transducer (p53) and a receiver (p21).     

The sudden recruitment of gamma-tubulin to the centrosome at the onset of mitosis and its dynamic exchange throughout the cell cycle, do not require microtubules.

gamma-Tubulin is a centrosomal component involved in microtubule nucleation. To determine how this molecule behaves during the cell cycle, we have established several vertebrate somatic cell lines that constitutively express a gamma-tubulin/green fluorescent protein fusion protein. Near simultaneous fluorescence and DIC light microscopy reveals that the amount of gamma-tubulin associated with the centrosome remains relatively constant throughout interphase, suddenly increases during prophase, and then decreases to interphase levels as the cell exits mitosis. This mitosis-specific recruitment of gamma-tubulin does not require microtubules. Fluorescence recovery after photobleaching (FRAP) studies reveal that the centrosome possesses two populations of gamma-tubulin: one that turns over rapidly and another that is more tightly bound. The dynamic exchange of centrosome-associated gamma-tubulin occurs throughout the cell cycle, including mitosis, and it does not require microtubules. These data are the first to characterize the dynamics of centrosome-associated gamma-tubulin in vertebrate cells in vivo and to demonstrate the microtubule-independent nature of these dynamics. They reveal that the additional gamma-tubulin required for spindle formation does not accumulate progressively at the centrosome during interphase. Rather, at the onset of mitosis, the centrosome suddenly gains the ability to bind greater than three times the amount of gamma-tubulin than during interphase.     

The centrosome in vertebrates: more than a microtubule-organizing center

The somatic cells of all higher animals contain a single minute organelle called the centrosome. For years, the functions of the centrosome were thought to revolve around its ability to nucleate and organize the various microtubule arrays seen in interphase and mitosis. But the centrosome is more than just a microtubule-organizing center. Recent work reveals that this organelle is essential for cell-cycle progression and that this requirement is independent of its ability to organize microtubules. Here, we review the various functions attributed to the centrosome and ask which are essential for the survival and reproduction of the cell, the organism, or both.     

Functional role of centrosomes in spindle assembly and organization

The centrosome is the main MT organizing center in animal cells, and has traditionally been regarded as essential for organization of the bipolar spindle that facilitates chromosome segregation during mitosis. Centrosomes are associated with the poles of the mitotic spindle, and several cell types require these organelles for spindle formation. However, most plant cells and some female meiotic systems get along without this organelle, and centrosome‐independent spindle assembly has now been identified within some centrosome containing cells. How can such observations, which point to mutually incompatible conclusions regarding the requirement of centrosomes in spindle formation, be interpreted? With emphasis on the functional role of centrosomes, this article summarizes the current models of spindle formation, and outlines how observations obtained from spindle assembly assays in vitro may reconcile conflicting opinions about the mechanism of spindle assembly. It is further described how Drosophila mutants are used to address the functional interrelationships between individual centrosomal proteins and spindle formation in vivo. © 2004 Wiley‐Liss, Inc.     

Identification of the tumor metastasis suppressor Nm23-H1/Nm23-R1 as a constituent of the centrosome

Processes like cell proliferation, differentiation, and tumor metastasis require a flexible adaptation of cell shape and cell plasticity. A regulator of cell structure and shape is the centrosome and its associated microtubules. Recently, oncogenes like p53, pRB, and the tumor suppressor BRCA1 have been characterized as members of the centrosome. In this communication, we identified rat Nm23-R1/NDPKbeta, a homologue of the human tumor metastasis suppressor Nm23-H1 and a regulator of cell proliferation and differentiation, as a component of the centrosomal complex. We used confocal laser scanning microscopy on different cell types and biochemical analysis of purified centrosomes to demonstrate that Nm23-R1 is located in the centrosome of dividing and nondividing cells. We also showed that the centrosomal enzyme is catalytically active and able to transfer the gamma-phosphate from a nucleoside triphosphate to a nucleoside diphosphate. In addition, Nm23-R1 coimmunoprecipitated with gamma-tubulin, a core centrosomal protein essential for microtubule nucleation. In addition, human Nm23-R1/-H1 was also shown to be present in the centrosome of different human and rat cell types, demonstrating that the presence of Nm23-H1 homologues in the latter organelle is a general event.     

Coiled-Coil Proteins Facilitated the Functional Expansion of the Centrosome

Repurposing existing proteins for new cellular functions is recognized as a main mechanism of evolutionary innovation, but its role in organelle evolution is unclear. Here, we explore the mechanisms that led to the evolution of the centrosome, an ancestral eukaryotic organelle that expanded its functional repertoire through the course of evolution. We developed a refined sequence alignment technique that is more sensitive to coiled coil proteins, which are abundant in the centrosome. For proteins with high coiled-coil content, our algorithm identified 17% more reciprocal best hits than BLAST. Analyzing 108 eukaryotic genomes, we traced the evolutionary history of centrosome proteins. In order to assess how these proteins formed the centrosome and adopted new functions, we computationally emulated evolution by iteratively removing the most recently evolved proteins from the centrosomal protein interaction network. Coiled-coil proteins that first appeared in the animal–fungi ancestor act as scaffolds and recruit ancestral eukaryotic proteins such as kinases and phosphatases to the centrosome. This process created a signaling hub that is crucial for multicellular development. Our results demonstrate how ancient proteins can be co-opted to different cellular localizations, thereby becoming involved in novel functions.     

Cell cycle progression and de novo centriole assembly after centrosomal removal in untransformed human cells

How centrosome removal or perturbations of centrosomal proteins leads to G1 arrest in untransformed mammalian cells has been a mystery. We use microsurgery and laser ablation to remove the centrosome from two types of normal human cells. First, we find that the cells assemble centrioles de novo after centrosome removal; thus, this phenomenon is not restricted to transformed cells. Second, normal cells can progress through G1 in its entirety without centrioles. Therefore, the centrosome is not a necessary, integral part of the mechanisms that drive the cell cycle through G1 into S phase. Third, we provide evidence that centrosome loss is, functionally, a stress that can act additively with other stresses to arrest cells in G1 in a p38-dependent fashion.     

Human papillomavirus type 16 E7 oncoprotein associates with the centrosomal component gamma-tubulin

Expression of a high-risk human papillomavirus (HPV) E7 oncoprotein is sufficient to induce aberrant centrosome duplication in primary human cells. The resulting centrosome-associated mitotic abnormalities have been linked to the development of aneuploidy. HPV type 16 (HPV16) E7 induces supernumerary centrosomes through a mechanism that is at least in part independent of the inactivation of the retinoblastoma tumor suppressor pRb and is dependent on cyclin-dependent kinase 2 activity. Here, we show that HPV16 E7 can concentrate around mitotic spindle poles and that a small pool of HPV16 E7 is associated with centrosome fractions isolated by sucrose density gradient centrifugation. The targeting of HPV16 E7 to the centrosome, however, was not sufficient for centrosome overduplication. Nonetheless, we found that HPV16 E7 can associate with the centrosomal regulator γ-tubulin and that the recruitment of γ-tubulin to the centrosome is altered in HPV16 E7-expressing cells. Since the association of HPV16 E7 with γ-tubulin is independent of pRb, p107, and p130, our results suggest that the association with γ-tubulin contributes to the pRb/p107/p130-independent ability of HPV16 E7 to subvert centrosome homeostasis.     

Functional Analysis of Centrosomal Kinase Substrates in Drosophila melanogaster Reveals a New Function of the Nuclear Envelope Component Otefin in Cell Cycle Progression

Phosphorylation is one of the key mechanisms that regulate centrosome biogenesis, spindle assembly, and cell cycle progression. However, little is known about centrosome-specific phosphorylation sites and their functional relevance. Here, we identified phosphoproteins of intact Drosophila melanogaster centrosomes and found previously unknown phosphorylation sites in known and unexpected centrosomal components. We functionally characterized phosphoproteins and integrated them into regulatory signaling networks with the 3 important mitotic kinases, cdc2, polo, and aur, as well as the kinase CkIIβ. Using a combinatorial RNA interference (RNAi) strategy, we demonstrated novel functions for P granule, nuclear envelope (NE), and nuclear proteins in centrosome duplication, maturation, and separation. Peptide microarrays confirmed phosphorylation of identified residues by centrosome-associated kinases. For a subset of phosphoproteins, we identified previously unknown centrosome and/or spindle localization via expression of tagged fusion proteins in Drosophila SL2 cells. Among those was otefin (Ote), an NE protein that we found to localize to centrosomes. Furthermore, we provide evidence that it is phosphorylated in vitro at threonine 63 (T63) through Aurora-A kinase. We propose that phosphorylation of this site plays a dual role in controlling mitotic exit when phosphorylated while dephosphorylation promotes G(2)/M transition in Drosophila SL2 cells.     

Hook2 localizes to the centrosome, binds directly to centriolin/CEP110 and contributes to centrosomal function

Centrosomes serve as microtubule-organizing centers. However, centrosome function depends on microtubule organization and protein transport because the formation, positioning and maintenance of centrosomes require microtubule-dependent retrograde transport. Linker proteins that associate with the motor protein dynein, organelles and microtubules facilitate loading of cargos for retrograde transport and thus contribute to the composition and placement of the centrosome and other juxtanuclear protein complexes. Members of the hook family of proteins may function as adaptors to link various organelle cargos to dynein for transport and have also been implicated directly in centrosome positioning. Here, we show that mammalian hook2, a previously uncharacterized member of the hook family, localizes to the centrosome through all phases of the cell cycle, the C-terminal domain of hook2 directly binds to centriolin/CEP110, the expression of the C-terminal domain of centriolin/CEP110 alters the distribution of endogenous hook2 and mislocalized wild-type or mutant hook2 proteins perturb endogenous centrosomal and pericentrosomal proteins in cultured mammalian cells. In addition, interference with hook2 function results in the loss of the radial organization of microtubules and a defect in regrowth of microtubules following their nocodazole-induced depolymerization. Thus, we propose that hook2 contributes to the establishment and maintenance of centrosomal structure and function.     

A potential role for NEDD1 and the centrosome in senescence of mouse embryonic fibroblasts

Mouse embryonic fibroblasts (MEFs) are commonly grown in cell culture and are known to enter senescence after a low number of passages as a result of oxidative stress. Oxidative stress has also been suggested to promote centrosome disruption; however, the contribution of this organelle to senescence is poorly understood. Therefore, this study aimed to assess the role of the centrosome in oxidative stress induced-senescence using MEFs as a model. We demonstrate here that coincident with the entry of late-passage MEFs into senescence, there was an increase in supernumerary centrosomes, most likely due to centrosome fragmentation. In addition, disrupting the centrosome in early-passage MEFs by depletion of neural precursor cell expressed developmentally downregulated gene 1 (NEDD1) also resulted in centrosomal fragmentation and subsequent premature entry into senescence. These data show that a loss of centrosomal integrity may contribute to the entry of MEFs into senescence in culture, and that centrosomal disruption can cause senescence.     

Dynamic association of a tumor amplified kinase,Aurora-A,with the centrosome and mitotic spindle

Aurora-A kinase, also known as STK15/BTAK kinase, is a member of a serine/threonine kinase superfamily that includes the prototypic yeast Ipl1 and Drosophila aurora kinases as well as other mammalian and non-mammalian aurora kinases involved in the regulation of centrosomes and chromosome segregation. The Aurora-A gene is amplified and overexpressed in a wide variety of human tumors. Aurora-A is centrosome-associated during interphase, and binds the poles and half-spindle during mitosis; its over-expression has been associated with centrosome amplification and multipolar spindles. GFP-Aurora-A was used to mark centrosomes and spindles, and monitor their movements in living cells. Centrosome pairs labeled with GFP-Aurora-A are motile throughout interphase undergoing oscillations and tumbling motions requiring intact microtubules and ATP. Fluorescence recovery after photobleaching (FRAP) was used to examine the relative molecular mobility of GFP-Aurora-A, and GFP-labeled alpha-tubulin, gamma-tubulin, and NuMA. GFP-Aurora-A rapidly exchanges in and out of the centrosome and mitotic spindle (t(1/2) approximately 3 sec); in contrast, both tubulins are relatively immobile indicative of a structural role. GFP-NuMA mobility was intermediate in both interphase nuclei and at the mitotic spindle (t(1/2) approximately 23-30 sec). Deletion mapping identifies a central domain of Aurora-A as essential for its centrosomal localization that is augmented by both the amino and the carboxyl terminal ends of the protein. Interestingly, amino or carboxy terminal deletion mutants that maintained centrosomal targeting exhibited significantly slower molecular exchange. Collectively, these studies contrast the relative cellular dynamics of Aurora-A with other cytoskeletal proteins that share its micro-domains, and identify essential regions required for targeting and dynamics.     

The centriolar satellite proteins Cep72 and Cep290 interact and are required for recruitment of BBS proteins to the cilium

Defects in centrosome and cilium function are associated with phenotypically related syndromes called ciliopathies. Centriolar satellites are centrosome-associated structures, defined by the protein PCM1, that are implicated in centrosomal protein trafficking. We identify Cep72 as a PCM1-interacting protein required for recruitment of the ciliopathy-associated protein Cep290 to centriolar satellites. Loss of centriolar satellites by depletion of PCM1 causes relocalization of Cep72 and Cep290 from satellites to the centrosome, suggesting that their association with centriolar satellites normally restricts their centrosomal localization. We identify interactions between PCM1, Cep72, and Cep290 and find that disruption of centriolar satellites by overexpression of Cep72 results in specific aggregation of these proteins and the BBSome component BBS4. During ciliogenesis, BBS4 relocalizes from centriolar satellites to the primary cilium. This relocalization occurs normally in the absence of centriolar satellites (PCM1 depletion) but is impaired by depletion of Cep290 or Cep72, resulting in defective ciliary recruitment of the BBSome subunit BBS8. We propose that Cep290 and Cep72 in centriolar satellites regulate the ciliary localization of BBS4, which in turn affects assembly and recruitment of the BBSome. Finally, we show that loss of centriolar satellites in zebrafish leads to phenotypes consistent with cilium dysfunction and analogous to those observed in human ciliopathies.