Assembly of centrosomal proteins and microtubule organization depends on PCM-1

The protein PCM-1 localizes to cytoplasmic granules known as "centriolar satellites" that are partly enriched around the centrosome. We inhibited PCM-1 function using a variety of approaches: microinjection of antibodies into cultured cells, overexpression of a PCM-1 deletion mutant, and specific depletion of PCM-1 by siRNA. All approaches led to reduced targeting of centrin, pericentrin, and ninein to the centrosome. Similar effects were seen upon inhibition of dynactin by dynamitin, and after prolonged treatment of cells with the microtubule inhibitor nocodazole. Inhibition or depletion of PCM-1 function further disrupted the radial organization of microtubules without affecting microtubule nucleation. Loss of microtubule organization was also observed after centrin or ninein depletion. Our data suggest that PCM-1-containing centriolar satellites are involved in the microtubule- and dynactin-dependent recruitment of proteins to the centrosome, of which centrin and ninein are required for interphase microtubule organization.     

Control of centrin stability by Aurora A

Aurora A is an oncogenic serine/threonine kinase which can cause cell transformation and centrosome amplification when over-expressed. Human breast tumors show excess Aurora A and phospho-centrin in amplified centrosomes. Here, we show that Aurora A mediates the phosphorylation of and localizes with centrin at the centrosome, with both proteins reaching maximum abundance from prophase through metaphase, followed by their precipitous loss in late stages of mitosis. Over-expression of Aurora A results in excess phospho-centrin and centrosome amplification. In contrast, centrosome amplification is not seen in cells over-expressing Aurora A in the presence of a recombinant centrin mutant lacking the serine phosphorylation site at residue 170. Expression of a kinase dead Aurora A results in a decrease in mitotic index and abrogation of centrin phosphorylation. Finally, a recombinant centrin mutation that mimics centrin phosphorylation increases centrin's stability against APC/C-mediated proteasomal degradation. Taken together, these results suggest that the stability of centrin is regulated in part by Aurora A, and that excess phosphorylated centrin may promote centrosome amplification in cancer.     

CDC25B Overexpression Stabilises Centrin 2 and Promotes the Formation of Excess Centriolar Foci

CDK-cyclin complexes regulate centriole duplication and microtubule nucleation at specific cell cycle stages, although their exact roles in these processes remain unclear. As the activities of CDK-cyclins are themselves positively regulated by CDC25 phosphatases, we investigated the role of centrosomal CDC25B during interphase. We report that overexpression of CDC25B, as is commonly found in human cancer, results in a significant increase in centrin 2 at the centrosomes of interphase cells. Conversely, CDC25B depletion causes a loss of centrin 2 from the centrosome, which can be rescued by treatment with the proteasome inhibitor MG132. CDC25B overexpression also promotes the formation of excess centrin 2 “foci”. These foci can accumulate other centrosome proteins, including γ-tubulin and PCM-1, and can function as microtubule organising centres, indicating that these represent functional centrosomes. Formation of centrin 2 foci can be blocked by specific inhibition of CDK2 but not CDK1. CDK2-mediated phosphorylation of Monopolar spindle 1 (Mps1) at the G1/S transition is essential for the initiation of centrosome duplication, and Mps1 is reported to phosphorylate centrin 2. Overexpression of wild-type or non-degradable Mps1 exacerbated the formation of excess centrin 2 foci induced by CDC25B overexpression, while kinase-dead Mps1 has a protective effect. Together, our data suggest that CDC25B, through activation of a centrosomal pool of CDK2, stabilises the local pool of Mps1 which in turn regulates the level of centrin 2 at the centrosome. Overexpression of CDC25B may therefore contribute to tumourigenesis by perturbing the natural turnover of centrosome proteins such as Mps1 and centrin 2, thus resulting in the de novo assembly of extra-numerary centrosomes and potentiating chromosome instability.     

Characterization of microsome-associated tobacco BY-2 centrins

Centrin - higher plants - MTOCs - microtubules nucleation In most eukaryotic cells, the Ca(2+)-binding protein centrin is associated with structured microtubule-organizing centers (MTOCs) such as centrosomes. In these cells, centrin either forms centrosome-associated contractile fibers, or is involved in centrosome biogenesis. Our aim was to investigate the functions of centrin in higher plant cells which do not contain centrosome-like MTOCs. We have cloned two tobacco BY-2 centrin cDNAs and we show that higher plant centrins define a phylogenetic group of proteins distinct from centrosome-associated centrins. In addition, tobacco centrins were found primarily associated with microsomes and did not colocalize with gamma-tubulin, a known MTOC marker. While the overall level of centrin did not vary during the cell cycle, centrin was prominently detected at the cell plate during telophase. Our results suggest that in tobacco, the major portion of centrin is not MTOC-associated and could be involved in the formation of the cell plate during cytokinesis.     

Remodeling of Centrosomes in Intraspecies and Interspecies Nuclear Transfer Porcine Embryos

Remodeling of donor cell centrosomes and the centrosome-associated cytoskeleton is crucially important for nuclear cloning as centrosomes are the main microtubule organizing centers that play a significant role in cell division and embryo development. Centrosome dysfunctions have been implicated in various diseases including cancer and metabolic disorders and may also play a role in developmental abnormalities that are frequently seen in cloned animals. In the present studies we investigated microtubule organization and the reorganization and fate of the integral centrosome protein γ-tubulin and the centrosome-associated protein centrin in intraspecies (pig oocytes; pig fetal fibroblast cells) and interspecies (pig oocytes; mouse fibroblast cells) reconstructed embryos by using antibodies to γ-tubulin or GFP-centrin transfected mouse fibroblasts as donor cells. Microtubules were stained with antibodies to α-tubulin. In-vitro-fertilized oocytes and nuclear transfer (NT) reconstructed oocytes were sequentially analyzed at different developmental stages. Epi-fluorescence results revealed mitotic spindle abnormalities in NT embryos during the first cell cycle (39.4%, 13/33) which were significantly higher than those in IVF embryos (17.0%, 7/41). The abnormalities in IVF embryos are due to polyspermy while the abnormalities in NT embryos are due to donor cell centrosome dysfunctions. In the NT embryos with abnormal microtubule and centrosome organization, γ-tubulin staining revealed multipolar centrosome foci while DAPI staining showed misalignment of chromosomes. In intraspecies and interspecies embryos the GFP-centrin signal was detected until 3 hrs after fusion. GFP-centrin was not detected at 8 hrs after NT which is consistent with previous results using anti-centrin antibody staining in intraspecies NT porcine embryos. These data indicate that 1) abnormalities in microtubule and centrosome organization are associated with nuclear cloning at a higher rate than observed in IVF embryos; 2) centrosome and cytoskeletal abnormalities in IVF embryos are due to polyspermy while centrosome and cytoskeletal abnormalities in NT embryos are due to donor cell centrosome dysfunctions; and 3) GFP-centrin of the donor cell centrosome provides a reliable marker to follow its fate in intraspecies reconstructed embryos.     

Defective nucleotide excision repair with normal centrosome structures and functions in the absence of all vertebrate centrins

The principal microtubule-organizing center in animal cells, the centrosome, contains centrin, a small, conserved calcium-binding protein unique to eukaryotes. Several centrin isoforms exist and have been implicated in various cellular processes including nuclear export and deoxyribonucleic acid (DNA) repair. Although centrins are required for centriole/basal body duplication in lower eukaryotes, centrin functions in vertebrate centrosome duplication are less clear. To define these roles, we used gene targeting in the hyperrecombinogenic chicken DT40 cell line to delete all three centrin genes in individual clones. Unexpectedly, centrin-deficient cells underwent normal cellular division with no detectable cell cycle defects. Light and electron microscopy analyses revealed no significant difference in centrosome composition or ultrastructure. However, centrin deficiency made DT40 cells highly sensitive to ultraviolet (UV) irradiation, with Cetn3 deficiency exacerbating the sensitivity of Cetn4/Cetn2 double mutants. DNA damage checkpoints were intact, but repair of UV-induced DNA damage was delayed in centrin nulls. These data demonstrate a role for vertebrate centrin in nucleotide excision repair.     

Binding of human centrin 2 to the centrosomal protein hSfi1

hSfi1, a human centrosomal protein with homologs in other eukaryotic organisms, includes 23 repeats, each of 23 amino acids, separated by 10 residue linkers. The main molecular partner in the centrosome is a small, calcium-binding EF-hand protein, the human centrin 2. Using isothermal titration calorimetry experiments, we characterized the centrin-binding capacity of three isolated hSfi1 repeats, two exhibiting the general consensus motif and the third being the unique Pro-containing human repeat. The two standard peptides bind human centrin 2 and its isolated C-terminal domain with high affinity (approximately 10(7) M(-1)) by an enthalpy-driven mechanism, with a moderate Ca2+ dependence. The Pro-containing repeat shows a binding affinity that is two orders of magnitude lower. The target binding site is localized within the C-terminal domain of human centrin 2. Fluorescence titration and NMR spectroscopy show that the well-conserved Trp residue situated in the C-terminus of each repeat is deeply embedded in a protein hydrophobic cavity, indicating that the peptide direction is reversed relative to previously studied centrin targets. The present results suggest that almost all of the repeats of the Sfi1 protein may independently bind centrin molecules. On the basis of this hypothesis and previous studies on centrin self-assembly, we propose a working model for the role of centrin-Sfi1 interactions in the dynamic structure of centrosome-associated contractile fibers.     

CP110 cooperates with two calcium-binding proteins to regulate cytokinesis and genome stability

The centrosome is an integral component of the eukaryotic cell cycle machinery, yet very few centrosomal proteins have been fully characterized to date. We have undertaken a series of biochemical and RNA interference (RNAi) studies to elucidate a role for CP110 in the centrosome cycle. Using a combination of yeast two-hybrid screens and biochemical analyses, we report that CP110 interacts with two different Ca2+-binding proteins, calmodulin (CaM) and centrin, in vivo. In vitro binding experiments reveal a direct, robust interaction between CP110 and CaM and the existence of multiple high-affinity CaM-binding domains in CP110. Native CP110 exists in large (approximately 300 kDa to 3 MDa) complexes that contain both centrin and CaM. We investigated a role for CP110 in CaM-mediated events using RNAi and show that its depletion leads to a failure at a late stage of cytokinesis and the formation of binucleate cells, mirroring the defects resulting from ablation of either CaM or centrin function. Importantly, expression of a CP110 mutant unable to bind CaM also promotes cytokinesis failure and binucleate cell formation. Taken together, our data demonstrate a functional role for CaM binding to CP110 and suggest that CP110 cooperates with CaM and centrin to regulate progression through cytokinesis.     

Centrin depletion causes cyst formation and other ciliopathy-related phenotypes in zebrafish

Most bona fide centrosome proteins, including centrins, small calcium-binding proteins, participate in spindle function during mitosis and play a role in cilia assembly in non-cycling cells. Although the basic cellular functions of centrins have been studied in lower eukaryotes and vertebrate cells in culture, phenotypes associated with centrin depletion in vertebrates in vivo has not been directly addressed. To test this, we depleted centrin2 in zebrafish and found that it leads to ciliopathy phenotypes, including enlarged pronephric tubules and pronephric cysts. Consistent with the ciliopathy phenotypes, cilia defects were observed in differentiated epithelial cells of ciliated organs, such as the olfactory bulb and pronephric duct. The organ phenotypes were also accompanied by cell cycle deregulation, namely, mitotic delay resulting from mitotic defects. Overall, this work demonstrates that centrin2 depletion causes cilia-related disorders in zebrafish. Moreover, given the presence of both cilia and mitotic defects in the affected organs, it suggests that cilia disorders may arise from a combination of these defects.Key words: centrosome, cilia, centrin, mitosis, cystogenesis, ciliopathies, zebrafish     

The cloning and expression characterization of the centrosome protein genes family (centrin genes) in rat testis

Centrins are members of the centrosome protein family, which is highly conserved during revolution. The homologous genes of centrin in many organisms had been cloned, but the sequences of the rat centrin genes were not reported yet in GenBank. We cloned the cDNA fragments of centrin-1, -2 and -3 from the rat testis by RT-PCR, and analyzed the homology of the deduced amino acid sequences. The expression characterization of centrin genes in rat spermatogenesis was carried out by semi-quantitative RT-PCR. The results show that the homology of the corresponding centrin proteins in human, mouse and rat is high. The expression of centrin-1 is testis-specific, spermatogenic cell-specific and developmental stage-related. Centrin-1 begins to be transcribed when the meiosis occurs, and its mRNA level reaches the peak in round spermatids. Centrin-2 and centrin-3 are highly expressed in spermatogonia and their mRNA level decreases markedly when meiosis occurs. These results suggest that centrin-1 may play roles in meiosis and spermiogenesis, and centrin-2 and centrin-3 may be related to mitosis.     

A Highlights from MBoC Selection: Centrin 3 is an inhibitor of centrosomal Mps1 and antagonizes centrin 2 function

Centrins are a family of small, calcium-binding proteins with diverse cellular functions that play an important role in centrosome biology. We previously identified centrin 2 and centrin 3 (Cetn2 and Cetn3) as substrates of the protein kinase Mps1. However, although Mps1 phosphorylation sites control the function of Cetn2 in centriole assembly and promote centriole overproduction, Cetn2 and Cetn3 are not functionally interchangeable, and we show here that Cetn3 is both a biochemical inhibitor of Mps1 catalytic activity and a biological inhibitor of centrosome duplication. In vitro, Cetn3 inhibits Mps1 autophosphorylation at Thr-676, a known site of T-loop autoactivation, and interferes with Mps1-dependent phosphorylation of Cetn2. The cellular overexpression of Cetn3 attenuates the incorporation of Cetn2 into centrioles and centrosome reduplication, whereas depletion of Cetn3 generates extra centrioles. Finally, overexpression of Cetn3 reduces Mps1 Thr-676 phosphorylation at centrosomes, and mimicking Mps1-dependent phosphorylation of Cetn2 bypasses the inhibitory effect of Cetn3, suggesting that the biological effects of Cetn3 are due to the inhibition of Mps1 function at centrosomes.     

Expression of a mutant form of Leishmania donovani centrin reduces the growth of the parasite.

Leishmania donovani, a protozoan parasite, causes visceral disease in humans. To identify genes that control growth, we have isolated for the first time in the order Kinetoplastida a gene encoding for centrin from L. donovani. Centrin is a calcium-binding cytoskeletal protein essential for centrosome duplication or segregation. Protein sequence similarity and immunoreactivity confirmed that Leishmania centrin is a homolog of human centrin 2. Immunofluorescence analysis localized the protein in the basal body. Calcium binding analysis revealed that its C-terminal Ca(2+) binding domain binds 16-fold more calcium than the N-terminal domain. Electrophoretic mobility shift of centrin treated with EGTA and abrogation of the shift in its mutants lacking a Ca(2+) binding site suggest that Ca(2+) binding to these regions may have a role in the protein conformation. The levels of centrin mRNA and protein were high during the exponential growth of the parasite in culture and declined to a low level in the stationary phase. Expression of N-terminal-deleted centrin in the parasite significantly reduces its growth rate, and it was found that significantly more cells are arrested in the G(2)/M stage than in control cells. These studies indicate that centrin may have a functional role in Leishmania growth.     

Localization of centrins in the hypotrich ciliate Paraurostyla weissei

Centrins are ubiquitous cytoskeletal proteins that are generally associated with the centrosome and form large cytoskeletal networks in protists. To obtain more data on the respective role of different centrin proteins, we studied their distribution and behavior in one ciliate species, Paraurostyla weissei, using specific antibodies. In this species, only two major proteins of 21 and 24 kDa corresponding to centrins, were identified by 1D and 2D electrophoresis. Immunofluorescence analysis showed that these two proteins displayed non-overlapping localization in the interphase cell and during morphogenesis. Both centrin proteins localize on the fibrous network linking the oral basal bodies in the interphase cell and in the form of marginal dots, which correspond to the proximal ends of the striated rootlets; the 21 kDa centrin was also detected within the basal bodies, whereas the 24 kDa centrin allowed identifying new structures, the frontal dashes. During morphogenesis, the 21 kDa centrin locates at the basal bodies, while the 24 kDa centrin is detected along the striated rootlets and in close association with the basal bodies pairs. These data are discussed in terms of the potential roles of the two centrins in different cellular functions.     

Centrosomes: Sfi1p and centrin unravel a structural riddle

The discovery of Sfi1p as a novel binding partner for the Ca2+-binding protein centrin has provided new insight into the dynamic behavior of centrosomes. Sfi1 binds to multiple centrin molecules along a series of internal repeats, and the complex forms Ca2+-sensitive contractile fibers that function to reorient centrioles and alter centrosome structure.     

Calcium-Binding Capacity of Centrin2 Is Required for Linear POC5 Assembly but Not for Nucleotide Excision Repair

Centrosomes, the principal microtubule-organising centres in animal cells, contain centrins, small, conserved calcium-binding proteins unique to eukaryotes. Centrin2 binds to xeroderma pigmentosum group C protein (XPC), stabilising it, and its presence slightly increases nucleotide excision repair (NER) activity in vitro. In previous work, we deleted all three centrin isoforms present in chicken DT40 cells and observed delayed repair of UV-induced DNA lesions, but no centrosome abnormalities. Here, we explore how centrin2 controls NER. In the centrin null cells, we expressed centrin2 mutants that cannot bind calcium or that lack sites for phosphorylation by regulatory kinases. Expression of any of these mutants restored the UV sensitivity of centrin null cells to normal as effectively as expression of wild-type centrin. However, calcium-binding-deficient and T118A mutants showed greatly compromised localisation to centrosomes. XPC recruitment to laser-induced UV-like lesions was only slightly slower in centrin-deficient cells than in controls, and levels of XPC and its partner HRAD23B were unaffected by centrin deficiency. Interestingly, we found that overexpression of the centrin interactor POC5 leads to the assembly of linear, centrin-dependent structures that recruit other centrosomal proteins such as PCM-1 and NEDD1. Together, these observations suggest that assembly of centrins into complex structures requires calcium binding capacity, but that such assembly is not required for centrin activity in NER.     

Dynamin 2 binds gamma-tubulin and participates in centrosome cohesion

Dynamin 2 (Dyn2) is a large GTPase involved in vesicle formation and actin reorganization. In this study, we report a novel role for Dyn2 as a component of the centrosome that is involved in centrosome cohesion. By light microscopy, Dyn2 localized aside centrin and colocalized with gamma-tubulin at the centrosome; by immunoelectron microscopy, however, Dyn2 was detected in the pericentriolar material as well as on centrioles. Exogenously expressed green fluorescent protein (GFP)-tagged Dyn2 also localized to the centrosome, whereas glutathione S-transferase (GST)-tagged Dyn2 pulled down a protein complex(es) containing actin, alpha-tubulin and gamma-tubulin from liver homogenate. Furthermore, gel overlay and immunoprecipitation indicated a direct interaction between gamma-tubulin and a 219-amino-acid middle domain of Dyn2. Reduction of Dyn2 protein levels with small-interfering RNA (siRNA) resulted in centrosome splitting, whereas microtubule nucleation from centrosomes was not affected, suggesting a role for Dyn2 in centrosome cohesion. Finally, fluorescence recovery after photobleaching (FRAP) analysis of a GFP-tagged Dyn2 middle domain indicated that Dyn2 is a dynamic exchangeable component of the centrosome. These findings suggest a novel function for Dyn2 as a participant in centrosome cohesion.     

Phosphorylation of centrin during the cell cycle and its role in centriole separation preceding centrosome duplication

Once during each cell cycle, mitotic spindle poles arise by separation of newly duplicated centrosomes. We report here the involvement of phosphorylation of the centrosomal protein centrin in this process. We show that centrin is phosphorylated at serine residue 170 during the G(2)/M phase of the cell cycle. Indirect immunofluorescence staining of HeLa cells using a phosphocentrin-specific antibody reveals intense labeling of mitotic spindle poles during prophase and metaphase of the cell division cycle, with diminished staining of anaphase and no staining of telophase and interphase centrosomes. Cultured cells undergo a dramatic increase in centrin phosphorylation following the experimental elevation of PKA activity, suggesting that this kinase can phosphorylate centrin in vivo. Surprisingly, elevated PKA activity also resulted intense phosphocentrin antibody labeling of interphase centrosomes and in the concurrent movement of individual centrioles apart from one another. Taken together, these results suggest that centrin phosphorylation signals the separation of centrosomes at prophase and implicates centrin phosphorylation in centriole separation that normally precedes centrosome duplication.     

Centrins, cell cycle regulation proteins in human malaria parasite Plasmodium falciparum

Molecules and cellular mechanisms that regulate the process of cell division in malaria parasites remain poorly understood. In this study we isolate and characterize the four Plasmodium falciparum centrins (PfCENs) and, by growth complementation studies, provide evidence for their involvement in cell division. Centrins are cytoskeleton proteins with key roles in cell division, including centrosome duplication, and possess four Ca(2+)-binding EF hand domains. By means of phylogenetic analysis, we were able to decipher the evolutionary history of centrins in eukaryotes with particular emphasis on the situation in apicomplexans and other alveolates. Plasmodium possesses orthologs of four distinct centrin paralogs traceable to the ancestral alveolate, including two that are unique to alveolates. By real time PCR and/or immunofluorescence, we determined the expression of PfCEN mRNA or protein in sporozoites, asexual blood forms, gametocytes, and in the oocysts developing inside mosquito mid-gut. Immunoelectron microscopy studies showed that centrin is expressed in close proximity with the nucleus of sporozoites and asexual schizonts. Furthermore, confocal and widefield microscopy using the double staining with alpha-tubulin and centrin antibodies strongly suggested that centrin is associated with the parasite centrosome. Following the episomal expression of the four PfCENs in a centrin knock-out Leishmania donovani parasite line that exhibited a severe growth defect, one of the PfCENs was able to partially restore Leishmania growth rate and overcome the defect in cytokinesis in such mutant cell line. To our knowledge, this study is the first characterization of a Plasmodium molecule that is involved in the process of cell division. These results provide the opportunity to further explore the role of centrins in cell division in malaria parasites and suggest novel targets to construct genetically modified, live attenuated malaria vaccines.