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Sample records for arsenic promotes centrosome

  1. Arsenic promotes centrosome abnormalities and cell colony formation in p53 compromised human lung cells

    SciTech Connect

    Liao Weiting; Lin Pinpin; Cheng, T.-S.; Yu, H.-S.; Chang, Louis W.

    2007-12-01

    Epidemiological evidence indicated that residents, especially cigarette smokers, in arseniasis areas had significantly higher lung cancer risk than those living in non-arseniasis areas. Thus, an interaction between arsenic and cigarette smoking in lung carcinogenesis was suspected. p53 dysfunction or mutation in lung epithelial cells was frequently observed in cigarette smokers. Our present study was to explore the differential effects by arsenic on H1355 cells (human lung adenocarcinoma cell line with mutation in p53), BEAS-2B (immortalized lung epithelial cell with functional p53) and pifithrin-{alpha}-treated BEAS-2B cells (p53-inhibited cells). These cells were treated with different doses of sodium arsenite (0, 0.1, 1, 5 and 10 {mu}M) for 48 h. A greater reduction in cell viability was observed in the BEAS-2B cells vs. p53 compromised cells (H1355 or p53-inhibited BEAS-2B). Similar observation was also made on 7-day cell survival (growth) study. TUNEL analysis confirmed that there was indeed a significantly reduced arsenite-induced apoptosis found in p53-compromised cells. Centrosomal abnormality has been attributed to eventual chromosomal missegregation, aneuploidy and tumorigenesis. In our present study, reduced p21 and Gadd45a expressions and increased centrosomal abnormality (atopic and multiple centrosomes) were observed in both arsenite-treated H1355 and p53-inhibited BEAS-2B cells as compared with similarly treated BEAS-2B cells. Increased anchorage-independent growth (colony formation) of BEAS-2B cells co-treated with pifithrin-{alpha} and 5 {mu}M sodium arsenite was also observed in soft agar. Our present investigation demonstrated that arsenic would act specifically on p53 compromised cells (either with p53 dysfunction or inhibited) to induce centrosomal abnormality and colony formation. These findings provided strong evidence on the carcinogenic promotional role of arsenic, especially under the condition of p53 dysfunction.

  2. Centrosome-Kinase Fusions Promote Oncogenic Signaling and Disrupt Centrosome Function in Myeloproliferative Neoplasms

    PubMed Central

    Lee, Joanna Y.; Hong, Wan-Jen; Majeti, Ravindra; Stearns, Tim

    2014-01-01

    Chromosomal translocations observed in myeloproliferative neoplasms (MPNs) frequently fuse genes that encode centrosome proteins and tyrosine kinases. This causes constitutive activation of the kinase resulting in aberrant, proliferative signaling. The function of centrosome proteins in these fusions is not well understood. Among others, kinase centrosome localization and constitutive kinase dimerization are possible consequences of centrosome protein-kinase fusions. To test the relative contributions of localization and dimerization on kinase signaling, we targeted inducibly dimerizable FGFR1 to the centrosome and other subcellular locations and generated a mutant of the FOP-FGFR1 MPN fusion defective in centrosome localization. Expression in mammalian cells followed by western blot analysis revealed a significant decrease in kinase signaling upon loss of FOP-FGFR1 centrosome localization. Kinase dimerization alone resulted in phosphorylation of the FGFR1 signaling target PLCγ, however levels comparable to FOP-FGFR1 required subcellular targeting in addition to kinase dimerization. Expression of MPN fusion proteins also resulted in centrosome disruption in epithelial cells and transformed patient cells. Primary human MPN cells showed masses of modified tubulin that colocalized with centrin, Smoothened (Smo), IFT88, and Arl13b. This is distinct from acute myeloid leukemia (AML) cells, which are not associated with centrosome-kinase fusions and had normal centrosomes. Our results suggest that effective proliferative MPN signaling requires both subcellular localization and dimerization of MPN kinases, both of which may be provided by centrosome protein fusion partners. Furthermore, centrosome disruption may contribute to the MPN transformation phenotype. PMID:24658090

  3. Nek5 promotes centrosome integrity in interphase and loss of centrosome cohesion in mitosis

    PubMed Central

    Sahota, Navdeep K.; Pelletier, Laurence; Morrison, Ciaran G.

    2015-01-01

    Nek5 is a poorly characterized member of the NIMA-related kinase family, other members of which play roles in cell cycle progression and primary cilia function. Here, we show that Nek5, similar to Nek2, localizes to the proximal ends of centrioles. Depletion of Nek5 or overexpression of kinase-inactive Nek5 caused unscheduled separation of centrosomes in interphase, a phenotype also observed upon overexpression of active Nek2. However, separated centrosomes that resulted from Nek5 depletion remained relatively close together, exhibited excess recruitment of the centrosome linker protein rootletin, and had reduced levels of Nek2. In addition, Nek5 depletion led to loss of PCM components, including γ-tubulin, pericentrin, and Cdk5Rap2, with centrosomes exhibiting reduced microtubule nucleation. Upon mitotic entry, Nek5-depleted cells inappropriately retained centrosome linker components and exhibited delayed centrosome separation and defective chromosome segregation. Hence, Nek5 is required for the loss of centrosome linker proteins and enhanced microtubule nucleation that lead to timely centrosome separation and bipolar spindle formation in mitosis. PMID:25963817

  4. Nek5 promotes centrosome integrity in interphase and loss of centrosome cohesion in mitosis.

    PubMed

    Prosser, Suzanna L; Sahota, Navdeep K; Pelletier, Laurence; Morrison, Ciaran G; Fry, Andrew M

    2015-05-11

    Nek5 is a poorly characterized member of the NIMA-related kinase family, other members of which play roles in cell cycle progression and primary cilia function. Here, we show that Nek5, similar to Nek2, localizes to the proximal ends of centrioles. Depletion of Nek5 or overexpression of kinase-inactive Nek5 caused unscheduled separation of centrosomes in interphase, a phenotype also observed upon overexpression of active Nek2. However, separated centrosomes that resulted from Nek5 depletion remained relatively close together, exhibited excess recruitment of the centrosome linker protein rootletin, and had reduced levels of Nek2. In addition, Nek5 depletion led to loss of PCM components, including γ-tubulin, pericentrin, and Cdk5Rap2, with centrosomes exhibiting reduced microtubule nucleation. Upon mitotic entry, Nek5-depleted cells inappropriately retained centrosome linker components and exhibited delayed centrosome separation and defective chromosome segregation. Hence, Nek5 is required for the loss of centrosome linker proteins and enhanced microtubule nucleation that lead to timely centrosome separation and bipolar spindle formation in mitosis. PMID:25963817

  5. EGF Induced Centrosome Separation Promotes Mitotic Progression and Cell Survival

    PubMed Central

    Mardin, Balca R.; Isokane, Mayumi; Cosenza, Marco R.; Krämer, Alwin; Ellenberg, Jan; Fry, Andrew M.; Schiebel, Elmar

    2014-01-01

    Summary Timely and accurate assembly of the mitotic spindle is critical for the faithful segregation of chromosomes and centrosome separation is a key step in this process. The timing of centrosome separation varies dramatically between cell types; however, the mechanisms responsible for these differences and its significance are unclear. Here, we show that activation of epidermal growth factor receptor (EGFR) signaling determines the timing of centrosome separation. Premature separation of centrosomes decreases the requirement for the major mitotic kinesin Eg5 for spindle assembly, accelerates mitosis and decreases the rate of chromosome missegregation. Importantly, EGF stimulation impacts upon centrosome separation and mitotic progression to different degrees in different cell lines. Cells with high EGFR levels fail to arrest in mitosis upon Eg5 inhibition. This has important implications for cancer therapy since cells with high centrosomal response to EGF are more susceptible to combinatorial inhibition of EGFR and Eg5. PMID:23643362

  6. Centriolar satellites assemble centrosomal microcephaly proteins to recruit CDK2 and promote centriole duplication.

    PubMed

    Kodani, Andrew; Yu, Timothy W; Johnson, Jeffrey R; Jayaraman, Divya; Johnson, Tasha L; Al-Gazali, Lihadh; Sztriha, Lāszló; Partlow, Jennifer N; Kim, Hanjun; Krup, Alexis L; Dammermann, Alexander; Krogan, Nevan J; Walsh, Christopher A; Reiter, Jeremy F

    2015-01-01

    Primary microcephaly (MCPH) associated proteins CDK5RAP2, CEP152, WDR62 and CEP63 colocalize at the centrosome. We found that they interact to promote centriole duplication and form a hierarchy in which each is required to localize another to the centrosome, with CDK5RAP2 at the apex, and CEP152, WDR62 and CEP63 at sequentially lower positions. MCPH proteins interact with distinct centriolar satellite proteins; CDK5RAP2 interacts with SPAG5 and CEP72, CEP152 with CEP131, WDR62 with MOONRAKER, and CEP63 with CEP90 and CCDC14. These satellite proteins localize their cognate MCPH interactors to centrosomes and also promote centriole duplication. Consistent with a role for satellites in microcephaly, homozygous mutations in one satellite gene, CEP90, may cause MCPH. The satellite proteins, with the exception of CCDC14, and MCPH proteins promote centriole duplication by recruiting CDK2 to the centrosome. Thus, centriolar satellites build a MCPH complex critical for human neurodevelopment that promotes CDK2 centrosomal localization and centriole duplication. PMID:26297806

  7. Centriolar satellites assemble centrosomal microcephaly proteins to recruit CDK2 and promote centriole duplication

    PubMed Central

    Kodani, Andrew; Yu, Timothy W; Johnson, Jeffrey R; Jayaraman, Divya; Johnson, Tasha L; Al-Gazali, Lihadh; Sztriha, Lāszló; Partlow, Jennifer N; Kim, Hanjun; Krup, Alexis L; Dammermann, Alexander; Krogan, Nevan J; Walsh, Christopher A; Reiter, Jeremy F

    2015-01-01

    Primary microcephaly (MCPH) associated proteins CDK5RAP2, CEP152, WDR62 and CEP63 colocalize at the centrosome. We found that they interact to promote centriole duplication and form a hierarchy in which each is required to localize another to the centrosome, with CDK5RAP2 at the apex, and CEP152, WDR62 and CEP63 at sequentially lower positions. MCPH proteins interact with distinct centriolar satellite proteins; CDK5RAP2 interacts with SPAG5 and CEP72, CEP152 with CEP131, WDR62 with MOONRAKER, and CEP63 with CEP90 and CCDC14. These satellite proteins localize their cognate MCPH interactors to centrosomes and also promote centriole duplication. Consistent with a role for satellites in microcephaly, homozygous mutations in one satellite gene, CEP90, may cause MCPH. The satellite proteins, with the exception of CCDC14, and MCPH proteins promote centriole duplication by recruiting CDK2 to the centrosome. Thus, centriolar satellites build a MCPH complex critical for human neurodevelopment that promotes CDK2 centrosomal localization and centriole duplication. DOI: http://dx.doi.org/10.7554/eLife.07519.001 PMID:26297806

  8. Clathrin regulates centrosome positioning by promoting acto-myosin cortical tension in C. elegans embryos.

    PubMed

    Spiró, Zoltán; Thyagarajan, Kalyani; De Simone, Alessandro; Träger, Sylvain; Afshar, Katayoun; Gönczy, Pierre

    2014-07-01

    Regulation of centrosome and spindle positioning is crucial for spatial cell division control. The one-cell Caenorhabditis elegans embryo has proven attractive for dissecting the mechanisms underlying centrosome and spindle positioning in a metazoan organism. Previous work revealed that these processes rely on an evolutionarily conserved force generator complex located at the cell cortex. This complex anchors the motor protein dynein, thus allowing cortical pulling forces to be exerted on astral microtubules emanating from microtubule organizing centers (MTOCs). Here, we report that the clathrin heavy chain CHC-1 negatively regulates pulling forces acting on centrosomes during interphase and on spindle poles during mitosis in one-cell C. elegans embryos. We establish a similar role for the cytokinesis/apoptosis/RNA-binding protein CAR-1 and uncover that CAR-1 is needed to maintain proper levels of CHC-1. We demonstrate that CHC-1 is necessary for normal organization of the cortical acto-myosin network and for full cortical tension. Furthermore, we establish that the centrosome positioning phenotype of embryos depleted of CHC-1 is alleviated by stabilizing the acto-myosin network. Conversely, we demonstrate that slight perturbations of the acto-myosin network in otherwise wild-type embryos results in excess centrosome movements resembling those in chc-1(RNAi) embryos. We developed a 2D computational model to simulate cortical rigidity-dependent pulling forces, which recapitulates the experimental data and further demonstrates that excess centrosome movements are produced at medium cortical rigidity values. Overall, our findings lead us to propose that clathrin plays a critical role in centrosome positioning by promoting acto-myosin cortical tension. PMID:24961801

  9. Induction of Aneuploidy, Centrosome Abnormality, Multipolar Spindle, and Multipolar Division in Cultured Mammalian Cells Exposed to an Arsenic Metabolite, Dimethylarsinate.

    PubMed

    Ochi, Takafumi

    2016-01-01

    Toxicological studies of arsenic compounds were conducted in cultured mammalian cells to investigate the effects of glutathione (GSH) depletion. Dimethylarsinate DMA(V) was not cytotoxic in cells depleted of GSH, but was found to be cytotoxic when GSH was present outside the cells. The results suggested that a reactive form of DMA(V) was generated through interaction with GSH. Dimethylarsine iodide DMI(III) was used as a model compound of DMA(III), and the biological effects were investigated. DMI(III) was about 10000 times more toxic to the cells than DMA(V). Chromosome structural aberrations and numerical changes, such as aneuploidy, were induced by DMI(III). DMA(V) induced multiple foci of the centrosome protein, γ-tubulin, which were colocalized with multipolar spindles in mitotic cells. The multiple foci coalesced into a single dot on disruption of the microtubules (MT). However, reorganization of the MT caused multiple foci of γ-tubulin, suggesting that the induction of centrosome abnormalities by DMA(V) required intact MT. Inhibition of the MT-dependent motor, kinesin, prevented formation of multiple foci of γ-tubulin, which pointed to the involvement of the MT-dependent mitotic motor, kinesin, in the maintenance of centrosome abnormalities. DMI(III) caused abnormal cytokinesis (multipolar division). In addition, DMI(III) caused morphological transformation in Syrian hamster embryo cells. Consideration of the overall process following the centrosome abnormalities caused by DMA(V) suggested a mode of cytotoxicity in which the mitotic centrosome is a critical target. PMID:27252065

  10. Phosphorylation of the centrosomal protein, Cep169, by Cdk1 promotes its dissociation from centrosomes in mitosis.

    PubMed

    Mori, Yusuke; Inoue, Yoko; Taniyama, Yuki; Tanaka, Sayori; Terada, Yasuhiko

    2015-12-25

    Cep169 is a centrosomal protein conserved among vertebrates. In our previous reports, we showed that mammalian Cep169 interacts and collaborates with CDK5RAP2 to regulate microtubule (MT) dynamics and stabilization. Although Cep169 is required for MT regulation, its precise cellular function remains largely elusive. Here we show that Cep169 associates with centrosomes during interphase, but dissociates from these structures from the onset of mitosis, although CDK5RAP2 (Cep215) is continuously located at the centrosomes throughout cell cycle. Interestingly, treatment with purvalanol A, a Cdk1 inhibitor, nearly completely blocked the dissociation of Cep169 from centrosomes during mitosis. In addition, mass spectrometry analyses identified 7 phosphorylated residues of Cep169 corresponding to consensus phosphorylation sequence for Cdk1. These data suggest that the dissociation of Cep169 from centrosomes is controlled by Cdk1/Cyclin B during mitosis, and that Cep169 might regulate MT dynamics of mitotic spindle. PMID:26549230

  11. Oscillation of APC/C activity during cell cycle arrest promotes centrosome amplification

    PubMed Central

    Prosser, Suzanna L.; Samant, Mugdha D.; Baxter, Joanne E.; Morrison, Ciaran G.; Fry, Andrew M.

    2014-01-01

    Centrosome duplication is licensed by the disengagement, or ‘uncoupling’, of centrioles during late mitosis. However, arrest of cells in G2 can trigger premature centriole disengagement. Here, we show that premature disengagement results from untimely activation of the APC/C leading to securin degradation and release of active separase. APC/C activation during G2 arrest is dependent on Plk1-mediated degradation of the APC/C inhibitor, Emi1, but Plk1 also has a second APC/C-independent role in promoting disengagement. Importantly, APC/C and Plk1 activity also stimulate centriole disengagement in response to hydroxyurea or DNA damage-induced cell cycle arrest and this leads to centrosome amplification. However, the re-duplication of disengaged centrioles is dependent on Cdk2 activity and Cdk2 activation coincides with a subsequent inactivation of the APC/C and re-accumulation of cyclin A. Release from these arrests leads to mitotic entry but, due to the presence of disengaged and/or amplified centrosomes, formation of abnormal mitotic spindles that lead to chromosome missegregation. Thus, oscillation of APC/C activity during cell cycle arrest promotes both centrosome amplification and genome instability. PMID:22956538

  12. APC2 and Axin promote mitotic fidelity by facilitating centrosome separation and cytoskeletal regulation.

    PubMed

    Poulton, John S; Mu, Frank W; Roberts, David M; Peifer, Mark

    2013-10-01

    To ensure the accurate transmission of genetic material, chromosome segregation must occur with extremely high fidelity. Segregation errors lead to chromosomal instability (CIN), with deleterious consequences. Mutations in the tumor suppressor adenomatous polyposis coli (APC) initiate most colon cancers and have also been suggested to promote disease progression through increased CIN, but the mechanistic role of APC in preventing CIN remains controversial. Using fly embryos as a model, we investigated the role of APC proteins in CIN. Our findings suggest that APC2 loss leads to increased rates of chromosome segregation error. This occurs through a cascade of events beginning with incomplete centrosome separation leading to failure to inhibit formation of ectopic cleavage furrows, which result in mitotic defects and DNA damage. We test several hypotheses related to the mechanism of action of APC2, revealing that APC2 functions at the embryonic cortex with several protein partners, including Axin, to promote mitotic fidelity. Our in vivo data demonstrate that APC2 protects genome stability by modulating mitotic fidelity through regulation of the cytoskeleton. PMID:24026117

  13. Centrosome amplification and cancer: Branching out

    PubMed Central

    Godinho, Susana A

    2015-01-01

    Despite being a common feature of human cancer, the role of supernumerary centrosomes in tumourigenesis is still poorly understood. We have recently described a novel role for centrosome amplification in promoting cell invasion that could impact tumor progression.

  14. Mechanisms of Metal-Induced Centrosome Amplification

    PubMed Central

    Holmes, Amie L.; Wise, John Pierce

    2014-01-01

    Exposure to toxic and carcinogenic metals is widespread; however, their mechanisms of action remain largely unknown. One potential mechanism for metal-induced carcinogenicity and toxicity is centrosome amplification. Here, we review the mechanisms for metal-induced centrosome amplification, including arsenic, chromium, mercury and nano-titanium dioxide. PMID:21118148

  15. BRCA1 and FancJ cooperatively promote interstrand crosslinker induced centrosome amplification through the activation of polo-like kinase 1

    PubMed Central

    Zou, Jianqiu; Zhang, Deli; Qin, Guang; Chen, Xiangming; Wang, Hongmin; Zhang, Dong

    2014-01-01

    DNA damage response (DDR) and the centrosome cycle are 2 of the most critical cellular processes affecting the genome stability in animal cells. Yet the cross-talks between DDR and the centrosome are poorly understood. Here we showed that deficiency of the breast cancer 1, early onset gene (BRCA1) induces centrosome amplification in non-stressed cells as previously reported while attenuating DNA damage-induced centrosome amplification (DDICA) in cells experiencing prolonged genotoxic stress. Mechanistically, the function of BRCA1 in promoting DDICA is through binding and recruiting polo-like kinase 1 (PLK1) to the centrosome. In a recent study, we showed that FancJ also suppresses centrosome amplification in non-stressed cells while promoting DDICA in both hydroxyurea and mitomycin C treated cells. FancJ is a key component of the BRCA1 B-complex. Here, we further demonstrated that, in coordination with BRCA1, FancJ promotes DDICA by recruiting both BRCA1 and PLK1 to the centrosome in the DNA damaged cells. Thus, we have uncovered a novel role of BRCA1 and FancJ in the regulation of DDICA. Dysregulation of DDR or centrosome cycle leads to aneuploidy, which is frequently seen in both solid and hematological cancers. BRCA1 and FancJ are known tumor suppressors and have well-recognized functions in DNA damage checkpoint and DNA repair. Together with our recent findings, we demonstrated here that BRCA1 and FancJ also play an important role in centrosome cycle especially in DDICA. DDICA is thought to be an alternative fail-safe mechanism to prevent cells experiencing severe DNA damage from becoming carcinogenic. Therefore, BRCA1 and FancJ are potential liaisons linking early DDR with the DDICA. We propose that together with their functions in DDR, the role of BRCA1 and FancJ in the activation of DDICA is also crucial for their tumor suppression functions in vivo. PMID:25483079

  16. Non-centrosomal epidermal microtubules act in parallel to LET-502/ROCK to promote C. elegans elongation.

    PubMed

    Quintin, Sophie; Wang, Shahoe; Pontabry, Julien; Bender, Ambre; Robin, François; Hyenne, Vincent; Landmann, Frédéric; Gally, Christelle; Oegema, Karen; Labouesse, Michel

    2016-01-01

    C. elegans embryonic elongation is a morphogenetic event driven by actomyosin contractility and muscle-induced tension transmitted through hemidesmosomes. A role for the microtubule cytoskeleton has also been proposed, but its contribution remains poorly characterized. Here, we investigate the organization of the non-centrosomal microtubule arrays present in the epidermis and assess their function in elongation. We show that the microtubule regulators γ-tubulin and NOCA-1 are recruited to hemidesmosomes and adherens junctions early in elongation. Several parallel approaches suggest that microtubule nucleation occurs from these sites. Disrupting the epidermal microtubule array by overexpressing the microtubule-severing protein Spastin or by inhibiting the C. elegans ninein homolog NOCA-1 in the epidermis mildly affected elongation. However, microtubules were essential for elongation when hemidesmosomes or the activity of the Rho kinase LET-502/ROCK were partially compromised. Imaging of junctional components and genetic analyses suggest that epidermal microtubules function together with Rho kinase to promote the transport of E-cadherin to adherens junctions and myotactin to hemidesmosomes. Our results indicate that the role of LET-502 in junctional remodeling is likely to be independent of its established function as a myosin II activator, but requires a microtubule-dependent pathway involving the syntaxin SYX-5. Hence, we propose that non-centrosomal microtubules organized by epidermal junctions contribute to elongation by transporting junction remodeling factors, rather than having a mechanical role. PMID:26586219

  17. Chronic centrosome amplification without tumorigenesis

    PubMed Central

    Vitre, Benjamin; Holland, Andrew J.; Kulukian, Anita; Shoshani, Ofer; Hirai, Maretoshi; Wang, Yin; Maldonado, Marcus; Cho, Thomas; Boubaker, Jihane; Swing, Deborah A.; Tessarollo, Lino; Evans, Sylvia M.; Fuchs, Elaine; Cleveland, Don W.

    2015-01-01

    Centrosomes are microtubule-organizing centers that facilitate bipolar mitotic spindle assembly and chromosome segregation. Recognizing that centrosome amplification is a common feature of aneuploid cancer cells, we tested whether supernumerary centrosomes are sufficient to drive tumor development. To do this, we constructed and analyzed mice in which centrosome amplification can be induced by a Cre-recombinase–mediated increase in expression of Polo-like kinase 4 (Plk4). Elevated Plk4 in mouse fibroblasts produced supernumerary centrosomes and enhanced the expected mitotic errors, but proliferation continued only after inactivation of the p53 tumor suppressor. Increasing Plk4 levels in mice with functional p53 produced centrosome amplification in liver and skin, but this did not promote spontaneous tumor development in these tissues or enhance the growth of chemically induced skin tumors. In the absence of p53, Plk4 overexpression generated widespread centrosome amplification, but did not drive additional tumors or affect development of the fatal thymic lymphomas that arise in animals lacking p53. We conclude that, independent of p53 status, supernumerary centrosomes are not sufficient to drive tumor formation. PMID:26578792

  18. CEP63 deficiency promotes p53-dependent microcephaly and reveals a role for the centrosome in meiotic recombination

    PubMed Central

    Marjanović, Marko; Sánchez-Huertas, Carlos; Terré, Berta; Gómez, Rocío; Scheel, Jan Frederik; Pacheco, Sarai; Knobel, Philip A.; Martínez-Marchal, Ana; Aivio, Suvi; Palenzuela, Lluís; Wolfrum, Uwe; McKinnon, Peter J.; Suja, José A.; Roig, Ignasi; Costanzo, Vincenzo; Lüders, Jens; Stracker, Travis H.

    2015-01-01

    CEP63 is a centrosomal protein that facilitates centriole duplication and is regulated by the DNA damage response. Mutations in CEP63 cause Seckel syndrome, a human disease characterized by microcephaly and dwarfism. Here we demonstrate that Cep63 deficient mice recapitulate Seckel syndrome pathology. The attrition of neural progenitor cells involves p53-dependent cell death and brain size is rescued by the deletion of p53. Cell death is not the result of an aberrant DNA damage response but is triggered by centrosome-based mitotic errors. In addition, Cep63 loss severely impairs meiotic recombination, leading to profound male infertility. Cep63 deficient spermatocytes display numerical and structural centrosome aberrations, chromosome entanglements and defective telomere clustering, suggesting that a reduction in centrosome-mediated chromosome movements underlies recombination failure. Our results provide novel insight into the molecular pathology of microcephaly and establish a role for the centrosome in meiotic recombination. PMID:26158450

  19. Exposure to Bisphenol A Correlates with Early-Onset Prostate Cancer and Promotes Centrosome Amplification and Anchorage-Independent Growth In Vitro

    PubMed Central

    Tarapore, Pheruza; Burke, Barbara; Bracken, Bruce; Ho, Shuk-Mei

    2014-01-01

    Human exposure to bisphenol A (BPA) is ubiquitous. Animal studies found that BPA contributes to development of prostate cancer, but human data are scarce. Our study examined the association between urinary BPA levels and Prostate cancer and assessed the effects of BPA on induction of centrosome abnormalities as an underlying mechanism promoting prostate carcinogenesis. The study, involving 60 urology patients, found higher levels of urinary BPA (creatinine-adjusted) in Prostate cancer patients (5.74 µg/g [95% CI; 2.63, 12.51]) than in non-Prostate cancer patients (1.43 µg/g [95% CI; 0.70, 2.88]) (p = 0.012). The difference was even more significant in patients <65 years old. A trend toward a negative association between urinary BPA and serum PSA was observed in Prostate cancer patients but not in non-Prostate cancer patients. In vitro studies examined centrosomal abnormalities, microtubule nucleation, and anchorage-independent growth in four Prostate cancer cell lines (LNCaP, C4-2, 22Rv1, PC-3) and two immortalized normal prostate epithelial cell lines (NPrEC and RWPE-1). Exposure to low doses (0.01–100 nM) of BPA increased the percentage of cells with centrosome amplification two- to eight-fold. Dose responses either peaked or reached the plateaus with 0.1 nM BPA exposure. This low dose also promoted microtubule nucleation and regrowth at centrosomes in RWPE-1 and enhanced anchorage-independent growth in C4-2. These findings suggest that urinary BPA level is an independent prognostic marker in Prostate cancer and that BPA exposure may lower serum PSA levels in Prostate cancer patients. Moreover, disruption of the centrosome duplication cycle by low-dose BPA may contribute to neoplastic transformation of the prostate. PMID:24594937

  20. A CEP215-HSET complex links centrosomes with spindle poles and drives centrosome clustering in cancer.

    PubMed

    Chavali, Pavithra L; Chandrasekaran, Gayathri; Barr, Alexis R; Tátrai, Péter; Taylor, Chris; Papachristou, Evaggelia K; Woods, C Geoffrey; Chavali, Sreenivas; Gergely, Fanni

    2016-01-01

    Numerical centrosome aberrations underlie certain developmental abnormalities and may promote cancer. A cell maintains normal centrosome numbers by coupling centrosome duplication with segregation, which is achieved through sustained association of each centrosome with a mitotic spindle pole. Although the microcephaly- and primordial dwarfism-linked centrosomal protein CEP215 has been implicated in this process, the molecular mechanism responsible remains unclear. Here, using proteomic profiling, we identify the minus end-directed microtubule motor protein HSET as a direct binding partner of CEP215. Targeted deletion of the HSET-binding domain of CEP215 in vertebrate cells causes centrosome detachment and results in HSET depletion at centrosomes, a phenotype also observed in CEP215-deficient patient-derived cells. Moreover, in cancer cells with centrosome amplification, the CEP215-HSET complex promotes the clustering of extra centrosomes into pseudo-bipolar spindles, thereby ensuring viable cell division. Therefore, stabilization of the centrosome-spindle pole interface by the CEP215-HSET complex could promote survival of cancer cells containing supernumerary centrosomes. PMID:26987684

  1. Causes and consequences of centrosome abnormalities in cancer

    PubMed Central

    Godinho, S. A.; Pellman, D.

    2014-01-01

    Centrosome amplification is a hallmark of cancer. However, despite significant progress in recent years, we are still far from understanding how centrosome amplification affects tumorigenesis. Boveri's hypothesis formulated more than 100 years ago was that aneuploidy induced by centrosome amplification promoted tumorigenesis. Although the hypothesis remains appealing 100 years later, it is also clear that the role of centrosome amplification in cancer is more complex than initially thought. Here, we review how centrosome abnormalities are generated in cancer and the mechanisms cells employ to adapt to centrosome amplification, in particular centrosome clustering. We discuss the different mechanisms by which centrosome amplification could contribute to tumour progression and the new advances in the development of therapies that target cells with extra centrosomes. PMID:25047621

  2. Preventing the Degradation of Mps1 at Centrosomes Is Sufficient to Cause Centrosome Reduplication in Human Cells

    PubMed Central

    Kasbek, Christopher; Yang, Ching-Hui; Yusof, Adlina Mohd; Chapman, Heather M.; Winey, Mark

    2007-01-01

    Supernumerary centrosomes promote the assembly of abnormal mitotic spindles in many human tumors. In human cells, overexpression of the cyclin-dependent kinase (Cdk)2 partner cyclin A during a prolonged S phase produces extra centrosomes, called centrosome reduplication. Cdk2 activity protects the Mps1 protein kinase from proteasome-mediated degradation, and we demonstrate here that Mps1 mediates cyclin A-dependent centrosome reduplication. Overexpression of cyclin A or a brief proteasome inhibition increases the centrosomal levels of Mps1, whereas depletion of Cdk2 leads to the proteasome-dependent loss of Mps1 from centrosomes only. When a Cdk2 phosphorylation site within Mps1 (T468) is mutated to alanine, Mps1 cannot accumulate at centrosomes or participate in centrosome duplication. In contrast, phosphomimetic mutations at T468 or deletion of the region surrounding T468 prevent the proteasome-dependent removal of Mps1 from centrosomes in the absence of Cdk2 activity. Moreover, cyclin A-dependent centrosome reduplication requires Mps1, and these stabilizing Mps1 mutations cause centrosome reduplication, bypassing cyclin A. Together, our data demonstrate that the region surrounding T468 contains a motif that regulates the accumulation of Mps1 at centrosomes. We suggest that phosphorylation of T468 attenuates the degradation of Mps1 at centrosomes and that preventing this degradation is necessary and sufficient to cause centrosome reduplication in human cells. PMID:17804818

  3. Cep192 Controls the Balance of Centrosome and Non-Centrosomal Microtubules during Interphase

    PubMed Central

    O’Rourke, Brian P.; Gomez-Ferreria, Maria Ana; Berk, Robin H.; Hackl, Alexandra M. U.; Nicholas, Matthew P.; O’Rourke, Sean C.; Pelletier, Laurence; Sharp, David J.

    2014-01-01

    Cep192 is a centrosomal protein that contributes to the formation and function of the mitotic spindle in mammalian cells. Cep192’s mitotic activities stem largely from its role in the recruitment to the centrosome of numerous additional proteins such as gamma-tubulin and Pericentrin. Here, we examine Cep192’s function in interphase cells. Our data indicate that, as in mitosis, Cep192 stimulates the nucleation of centrosomal microtubules thereby regulating the morphology of interphase microtubule arrays. Interestingly, however, cells lacking Cep192 remain capable of generating normal levels of MTs as the loss of centrosomal microtubules is augmented by MT nucleation from other sites, most notably the Golgi apparatus. The depletion of Cep192 results in a significant decrease in the level of centrosome-associated gamma-tubulin, likely explaining its impact on centrosome microtubule nucleation. However, in stark contrast to mitosis, Cep192 appears to maintain an antagonistic relationship with Pericentrin at interphase centrosomes. Interphase cells depleted of Cep192 display significantly higher levels of centrosome-associated Pericentrin while overexpression of Cep192 reduces the levels of centrosomal Pericentrin. Conversely, depletion of Pericentrin results in elevated levels of centrosomal Cep192 and enhances microtubule nucleation at centrosomes, at least during interphase. Finally, we show that depletion of Cep192 negatively impacts cell motility and alters normal cell polarization. Our current working hypothesis is that the microtubule nucleating capacity of the interphase centrosome is determined by an antagonistic balance of Cep192, which promotes nucleation, and Pericentrin, which inhibits nucleation. This in turn determines the relative abundance of centrosomal and non-centrosomal microtubules that tune cell movement and shape. PMID:24971877

  4. Arsenite promotes centrosome abnormalities under a p53 compromised status induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)

    SciTech Connect

    Liao, W.-T.; Yu, H.-S.; Lin Pinpin; Chang, Louis W.

    2010-02-15

    Epidemiological evidence indicated that residents, especially cigarette smokers, in arseniasis areas had significantly higher lung cancer risk than those living in non-arseniasis areas. Thus an interaction between arsenite and cigarette smoking in lung carcinogenesis was suspected. In the present study, we investigated the interactions of a tobacco-specific carcinogen 4- (methylnitrosamino)-1-(3-pyridyl)-1-butanone (nicotine-derived nitrosamine ketone, NNK) and arsenite on lung cell transformation. BEAS-2B, an immortalized human lung epithelial cell line, was selected to test the centrosomal abnormalities and colony formation by NNK and arsenite. We found that NNK, alone, could enhance BEAS-2B cell growth at 1-5 muM. Under NNK exposure, arsenite was able to increase centrosomal abnormality as compared with NNK or arsenite treatment alone. NNK treatment could also reduce arsenite-induced G2/M cell cycle arrest and apoptosis, these cellular effects were found to be correlated with p53 dysfunction. Increased anchorage-independent growth (colony formation) of BEAS-2B cells cotreated with NNK and arsenite was also observed in soft agar. Our present investigation demonstrated that NNK could provide a p53 compromised status. Arsenite would act specifically on this p53 compromised status to induce centrosomal abnormality and colony formation. These findings provided strong evidence on the carcinogenic promotional role of arsenite under tobacco-specific carcinogen co-exposure.

  5. Arsenic

    MedlinePlus

    Arsenic is a natural element found in soil and minerals. Arsenic compounds are used to preserve wood, as pesticides, and in some industries. Arsenic can get into air, water, and the ground from wind- ...

  6. Arsenic

    MedlinePlus

    ... and minerals. Arsenic compounds are used to preserve wood, as pesticides, and in some industries. Arsenic can ... Breathing sawdust or burning smoke from arsenic-treated wood Living in an area with high levels of ...

  7. The Centrosome-Specific Phosphorylation of Cnn by Polo/Plk1 Drives Cnn Scaffold Assembly and Centrosome Maturation

    PubMed Central

    Conduit, Paul T.; Feng, Zhe; Richens, Jennifer H.; Baumbach, Janina; Wainman, Alan; Bakshi, Suruchi D.; Dobbelaere, Jeroen; Johnson, Steven; Lea, Susan M.; Raff, Jordan W.

    2014-01-01

    Summary Centrosomes are important cell organizers. They consist of a pair of centrioles surrounded by pericentriolar material (PCM) that expands dramatically during mitosis—a process termed centrosome maturation. How centrosomes mature remains mysterious. Here, we identify a domain in Drosophila Cnn that appears to be phosphorylated by Polo/Plk1 specifically at centrosomes during mitosis. The phosphorylation promotes the assembly of a Cnn scaffold around the centrioles that is in constant flux, with Cnn molecules recruited continuously around the centrioles as the scaffold spreads slowly outward. Mutations that block Cnn phosphorylation strongly inhibit scaffold assembly and centrosome maturation, whereas phosphomimicking mutations allow Cnn to multimerize in vitro and to spontaneously form cytoplasmic scaffolds in vivo that organize microtubules independently of centrosomes. We conclude that Polo/Plk1 initiates the phosphorylation-dependent assembly of a Cnn scaffold around centrioles that is essential for efficient centrosome maturation in flies. PMID:24656740

  8. A CEP215–HSET complex links centrosomes with spindle poles and drives centrosome clustering in cancer

    PubMed Central

    Chavali, Pavithra L.; Chandrasekaran, Gayathri; Barr, Alexis R.; Tátrai, Péter; Taylor, Chris; Papachristou, Evaggelia K.; Woods, C. Geoffrey; Chavali, Sreenivas; Gergely, Fanni

    2016-01-01

    Numerical centrosome aberrations underlie certain developmental abnormalities and may promote cancer. A cell maintains normal centrosome numbers by coupling centrosome duplication with segregation, which is achieved through sustained association of each centrosome with a mitotic spindle pole. Although the microcephaly- and primordial dwarfism-linked centrosomal protein CEP215 has been implicated in this process, the molecular mechanism responsible remains unclear. Here, using proteomic profiling, we identify the minus end-directed microtubule motor protein HSET as a direct binding partner of CEP215. Targeted deletion of the HSET-binding domain of CEP215 in vertebrate cells causes centrosome detachment and results in HSET depletion at centrosomes, a phenotype also observed in CEP215-deficient patient-derived cells. Moreover, in cancer cells with centrosome amplification, the CEP215–HSET complex promotes the clustering of extra centrosomes into pseudo-bipolar spindles, thereby ensuring viable cell division. Therefore, stabilization of the centrosome–spindle pole interface by the CEP215–HSET complex could promote survival of cancer cells containing supernumerary centrosomes. PMID:26987684

  9. The centrosome is an actin-organizing center

    PubMed Central

    Farina, Francesca; Gaillard, Jérémie; Guérin, Christophe; Couté, Yohann; Sillibourne, James; Blanchoin, Laurent; Théry, Manuel

    2016-01-01

    Microtubules and actin filaments are the two main cytoskeleton networks supporting intracellular architecture and cell polarity. The centrosome nucleates and anchors microtubules and is therefore considered to be the main microtubule-organizing center. However, recurring, yet unexplained, observations have pointed towards a connection between the centrosome and actin filaments. Here we have used isolated centrosomes to demonstrate that the centrosome can directly promote actin filament assembly. A cloud of centrosome-associated actin filaments could be identified in living cells as well. Actin-filament nucleation at the centrosome was mediated by the nucleation promoting factor WASH in combination with the Arp2/3 complex. Pericentriolar material 1 (PCM1) appeared to modulate the centrosomal actin network by regulating Arp2/3 complex and WASH recruitment to the centrosome. Hence our results reveal an additional facet of the centrosome as an intracellular organizer and provide mechanistic insights into how the centrosome can function as an actin filament-organizing center. PMID:26655833

  10. Interphase centrosome organization by the PLP-Cnn scaffold is required for centrosome function

    PubMed Central

    Lerit, Dorothy A.; Jordan, Holly A.; Poulton, John S.; Fagerstrom, Carey J.; Galletta, Brian J.; Peifer, Mark

    2015-01-01

    Pericentriolar material (PCM) mediates the microtubule (MT) nucleation and anchoring activity of centrosomes. A scaffold organized by Centrosomin (Cnn) serves to ensure proper PCM architecture and functional changes in centrosome activity with each cell cycle. Here, we investigate the mechanisms that spatially restrict and temporally coordinate centrosome scaffold formation. Focusing on the mitotic-to-interphase transition in Drosophila melanogaster embryos, we show that the elaboration of the interphase Cnn scaffold defines a major structural rearrangement of the centrosome. We identify an unprecedented role for Pericentrin-like protein (PLP), which localizes to the tips of extended Cnn flares, to maintain robust interphase centrosome activity and promote the formation of interphase MT asters required for normal nuclear spacing, centrosome segregation, and compartmentalization of the syncytial embryo. Our data reveal that Cnn and PLP directly interact at two defined sites to coordinate the cell cycle–dependent rearrangement and scaffolding activity of the centrosome to permit normal centrosome organization, cell division, and embryonic viability. PMID:26150390

  11. Interphase centrosome organization by the PLP-Cnn scaffold is required for centrosome function.

    PubMed

    Lerit, Dorothy A; Jordan, Holly A; Poulton, John S; Fagerstrom, Carey J; Galletta, Brian J; Peifer, Mark; Rusan, Nasser M

    2015-07-01

    Pericentriolar material (PCM) mediates the microtubule (MT) nucleation and anchoring activity of centrosomes. A scaffold organized by Centrosomin (Cnn) serves to ensure proper PCM architecture and functional changes in centrosome activity with each cell cycle. Here, we investigate the mechanisms that spatially restrict and temporally coordinate centrosome scaffold formation. Focusing on the mitotic-to-interphase transition in Drosophila melanogaster embryos, we show that the elaboration of the interphase Cnn scaffold defines a major structural rearrangement of the centrosome. We identify an unprecedented role for Pericentrin-like protein (PLP), which localizes to the tips of extended Cnn flares, to maintain robust interphase centrosome activity and promote the formation of interphase MT asters required for normal nuclear spacing, centrosome segregation, and compartmentalization of the syncytial embryo. Our data reveal that Cnn and PLP directly interact at two defined sites to coordinate the cell cycle-dependent rearrangement and scaffolding activity of the centrosome to permit normal centrosome organization, cell division, and embryonic viability. PMID:26150390

  12. Oncogene-like induction of cellular invasion from centrosome amplification

    PubMed Central

    Godinho, Susana A.; Picone, Remigio; Burute, Mithila; Dagher, Regina; Su, Ying; Leung, Cheuk T.; Polyak, Kornelia; Brugge, Joan S.; Thery, Manuel; Pellman, David

    2014-01-01

    Centrosome amplification has long been recognized as a feature of human tumors, however its role in tumorigenesis remains unclear1. Centrosome amplification is poorly tolerated by non-transformed cells, and, in the absence of selection, extra centrosomes are spontaneously lost2. Thus, the high frequency of centrosome amplification, particularly in more aggressive tumors3, raises the possibility that extra centrosomes could, in some contexts, confer advantageous characteristics that promote tumor progression. Using a three-dimensional model system and other approaches to culture human mammary epithelial cells, we find that centrosome amplification triggers cell invasion. This invasive behavior is similar to that induced by overexpression of the breast cancer oncogene ErbB24 and indeed enhances invasiveness triggered by ErbB2. We show that, through increased centrosomal microtubule nucleation, centrosome amplification increases Rac1 activity, which disrupts normal cell-cell adhesion and promotes invasion. These findings demonstrate that centrosome amplification, a structural alteration of the cytoskeleton, can promote features of malignant transformation. PMID:24739973

  13. Low doses of arsenic, via perturbing p53, promotes tumorigenesis.

    PubMed

    Ganapathy, Suthakar; Li, Ping; Fagman, Johan; Yu, Tianqi; Lafontant, Jean; Zhang, Guojun; Chen, Changyan

    2016-09-01

    In drinking water and in workplace or living environments, low doses of arsenic can exist and operate as a potent carcinogen. Due to insufficient understanding and information on the pervasiveness of environmental exposures to arsenic, there is an urgent need to elucidate the underlying molecular mechanisms of arsenic regarding its carcinogenic effect on human health. In this study, we demonstrate that low doses of arsenic exposure mitigate or mask p53 function and further perturb intracellular redox state, which triggers persistent endoplasmic reticulum (ER) stress and activates UPR (unfolded protein response), leading to transformation or tumorigenesis. Thus, the results suggest that low doses of arsenic exposure, through attenuating p53-regulated tumor suppressive function, change the state of intracellular redox and create a microenvironment for tumorigenesis. Our study also provides the information for designing more effective strategies to prevent or treat human cancers initiated by arsenic exposure. PMID:27425828

  14. A Microtubule-Independent Role for Centrosomes and Aurora A in Nuclear Envelope Breakdown

    PubMed Central

    Portier, Nathan; Audhya, Anjon; Maddox, Paul S.; Green, Rebecca A.; Dammermann, Alexander; Desai, Arshad; Oegema, Karen

    2010-01-01

    SUMMARY Aurora A kinase localizes to centrosomes and is required for centrosome maturation and spindle assembly. Here, we describe a microtubule-independent role for aurora A and centrosomes in nuclear envelope breakdown (NEBD) during the first mitotic division of the C. elegans embryo. Aurora A depletion does not alter the onset or kinetics of chromosome condensation, but dramatically lengthens the interval between the completion of condensation and NEBD. Inhibiting centrosome assembly by other means also lengthens this interval, albeit to a lesser extent than aurora A depletion. By contrast, centrosomally-nucleated microtubules and the nuclear envelope-associated motor dynein are not required for timely NEBD. These results indicate that mitotic centrosomes generate a diffusible factor, which we propose is activated aurora A, that promotes NEBD. A positive feedback loop, in which an aurora A-dependent increase in centrosome size promotes aurora A activation, may temporally couple centrosome maturation to NEBD during mitotic entry. PMID:17419991

  15. Low level arsenic promotes progressive inflammatory angiogenesis and liver blood vessel remodeling in mice

    SciTech Connect

    Straub, Adam C.; Stolz, Donna B.; Vin, Harina; Ross, Mark A.; Soucy, Nicole V.; Klei, Linda R.; Barchowsky, Aaron

    2007-08-01

    The vascular effects of arsenic in drinking water are global health concerns contributing to human disease worldwide. Arsenic targets the endothelial cells lining blood vessels, and endothelial cell activation or dysfunction may underlie the pathogenesis of both arsenic-induced vascular diseases and arsenic-enhanced tumorigenesis. The purpose of the current studies was to demonstrate that exposing mice to drinking water containing environmentally relevant levels of arsenic promoted endothelial cell dysfunction and pathologic vascular remodeling. Increased angiogenesis, neovascularization, and inflammatory cell infiltration were observed in Matrigel plugs implanted in C57BL/6 mice following 5-week exposures to 5-500 ppb arsenic [Soucy, N.V., Mayka, D., Klei, L.R., Nemec, A.A., Bauer, J.A., Barchowsky, A., 2005. Neovascularization and angiogenic gene expression following chronic arsenic exposure in mice. Cardiovasc.Toxicol 5, 29-42]. Therefore, functional in vivo effects of arsenic on endothelial cell function and vessel remodeling in an endogenous vascular bed were investigated in the liver. Liver sinusoidal endothelial cells (LSEC) became progressively defenestrated and underwent capillarization to decrease vessel porosity following exposure to 250 ppb arsenic for 2 weeks. Sinusoidal expression of PECAM-1 and laminin-1 proteins, a hallmark of capillarization, was also increased by 2 weeks of exposure. LSEC caveolin-1 protein and caveolae expression were induced after 2 weeks of exposure indicating a compensatory change. Likewise, CD45/CD68-positive inflammatory cells did not accumulate in the livers until after LSEC porosity was decreased, indicating that inflammation is a consequence and not a cause of the arsenic-induced LSEC phenotype. The data demonstrate that the liver vasculature is an early target of pathogenic arsenic effects and that the mouse liver vasculature is a sensitive model for investigating vascular health effects of arsenic.

  16. Characterization of BRCA1 Protein Targeting, Dynamics, and Function at the Centrosome

    PubMed Central

    Brodie, Kirsty M.; Henderson, Beric R.

    2012-01-01

    BRCA1 is a DNA damage response protein and functions in the nucleus to stimulate DNA repair and at the centrosome to inhibit centrosome overduplication in response to DNA damage. The loss or mutation of BRCA1 causes centrosome amplification and abnormal mitotic spindle assembly in breast cancer cells. The BRCA1-BARD1 heterodimer binds and ubiquitinates γ-tubulin to inhibit centrosome amplification and promote microtubule nucleation; however regulation of BRCA1 targeting and function at the centrosome is poorly understood. Here we show that both N and C termini of BRCA1 are required for its centrosomal localization and that BRCA1 moves to the centrosome independently of BARD1 and γ-tubulin. Mutations in the C-terminal phosphoprotein-binding BRCT domain of BRCA1 prevented localization to centrosomes. Photobleaching experiments identified dynamic (60%) and immobilized (40%) pools of ectopic BRCA1 at the centrosome, and these are regulated by the nuclear export receptor CRM1 (chromosome region maintenance 1) and BARD1. CRM1 mediates nuclear export of BRCA1, and mutation of the export sequence blocked BRCA1 regulation of centrosome amplification in irradiated cells. CRM1 binds to undimerized BRCA1 and is displaced by BARD1. Photobleaching assays implicate CRM1 in driving undimerized BRCA1 to the centrosome and revealed that when BRCA1 subsequently binds to BARD1, it is less well retained at centrosomes, suggesting a mechanism to accelerate BRCA1 release after formation of the active heterodimer. Moreover, Aurora A binding and phosphorylation of BRCA1 enhanced its centrosomal retention and regulation of centrosome amplification. Thus, CRM1, BARD1 and Aurora A promote the targeting and function of BRCA1 at centrosomes. PMID:22262852

  17. Arsenic

    MedlinePlus

    ... mainly found in its less toxic organic form. Industrial processes Arsenic is used industrially as an alloying ... are also required to reduce occupational exposure from industrial processes. Education and community engagement are key factors ...

  18. Arsenic induces structural and compositional colonic microbiome change and promotes host nitrogen and amino acid metabolism.

    PubMed

    Dheer, Rishu; Patterson, Jena; Dudash, Mark; Stachler, Elyse N; Bibby, Kyle J; Stolz, Donna B; Shiva, Sruti; Wang, Zeneng; Hazen, Stanley L; Barchowsky, Aaron; Stolz, John F

    2015-12-15

    Chronic exposure to arsenic in drinking water causes cancer and non-cancer diseases. However, mechanisms for chronic arsenic-induced pathogenesis, especially in response to lower exposure levels, are unclear. In addition, the importance of health impacts from xeniobiotic-promoted microbiome changes is just being realized and effects of arsenic on the microbiome with relation to disease promotion are unknown. To investigate impact of arsenic exposure on both microbiome and host metabolism, the stucture and composition of colonic microbiota, their metabolic phenotype, and host tissue and plasma metabolite levels were compared in mice exposed for 2, 5, or 10weeks to 0, 10 (low) or 250 (high) ppb arsenite (As(III)). Genotyping of colonic bacteria revealed time and arsenic concentration dependent shifts in community composition, particularly the Bacteroidetes and Firmicutes, relative to those seen in the time-matched controls. Arsenic-induced erosion of bacterial biofilms adjacent to the mucosal lining and changes in the diversity and abundance of morphologically distinct species indicated changes in microbial community structure. Bacterical spores increased in abundance and intracellular inclusions decreased with high dose arsenic. Interestingly, expression of arsenate reductase (arsA) and the As(III) exporter arsB, remained unchanged, while the dissimilatory nitrite reductase (nrfA) gene expression increased. In keeping with the change in nitrogen metabolism, colonic and liver nitrite and nitrate levels and ratios changed with time. In addition, there was a concomitant increase in pathogenic arginine metabolites in the mouse circulation. These data suggest that arsenic exposure impacts the microbiome and microbiome/host nitrogen metabolism to support disease enhancing pathogenic phenotypes. PMID:26529668

  19. CentrosomeDB: a human centrosomal proteins database

    PubMed Central

    Nogales-Cadenas, Rubén; Abascal, Federico; Díez-Pérez, Javier; Carazo, José María; Pascual-Montano, Alberto

    2009-01-01

    Active research on the biology of the centrosome during the past decades has allowed the identification and characterization of many centrosomal proteins. Unfortunately, the accumulated data is still dispersed among heterogeneous sources of information. Here we present centrosome:db, which intends to compile and integrate relevant information related to the human centrosome. We have compiled a set of 383 likely human centrosomal genes and recorded the associated supporting evidences. Centrosome:db offers several perspectives to study the human centrosome including evolution, function and structure. The database contains information on the orthology relationships with other species, including fungi, nematodes, arthropods, urochordates and vertebrates. Predictions of the domain organization of centrosome:db proteins are graphically represented at different sections of the database, including sets of alternative protein isoforms, interacting proteins, groups of orthologs and the homologs identified with blast. Centrosome:db also contains information related to function, gene–disease associations, SNPs and the 3D structure of proteins. Apart from important differences in the coverage of the set of centrosomal genes, our database differentiates from other similar initiatives in the way information is treated and analyzed. Centrosome:db is publicly available at http://centrosome.dacya.ucm.es. PMID:18971254

  20. Centrosome-intrinsic mechanisms modulate centrosome integrity during fever

    PubMed Central

    Vertii, Anastassiia; Zimmerman, Wendy; Ivshina, Maria; Doxsey, Stephen

    2015-01-01

    The centrosome is critical for cell division, ciliogenesis, membrane trafficking, and immunological synapse function. The immunological synapse is part of the immune response, which is often accompanied by fever/heat stress (HS). Here we provide evidence that HS causes deconstruction of all centrosome substructures primarily through degradation by centrosome-associated proteasomes. This renders the centrosome nonfunctional. Heat-activated degradation is centrosome selective, as other nonmembranous organelles (midbody, kinetochore) and membrane-bounded organelles (mitochondria) remain largely intact. Heat-induced centrosome inactivation was rescued by targeting Hsp70 to the centrosome. In contrast, Hsp70 excluded from the centrosome via targeting to membranes failed to rescue, as did chaperone inactivation. This indicates that there is a balance between degradation and chaperone rescue at the centrosome after HS. This novel mechanism of centrosome regulation during fever contributes to immunological synapse formation. Heat-induced centrosome inactivation is a physiologically relevant event, as centrosomes in leukocytes of febrile patients are disrupted. PMID:26269579

  1. Excess centrosomes perturb dynamic endothelial cell repolarization during blood vessel formation

    PubMed Central

    Kushner, Erich J.; Ferro, Luke S.; Yu, Zhixian; Bautch, Victoria L.

    2016-01-01

    Blood vessel formation requires dynamic movements of endothelial cells (ECs) within sprouts. The cytoskeleton regulates migratory polarity, and centrosomes organize the microtubule cytoskeleton. However, it is not well understood how excess centrosomes, commonly found in tumor stromal cells, affect microtubule dynamics and interphase cell polarity. Here we find that ECs dynamically repolarize during sprouting angiogenesis, and excess centrosomes block repolarization and reduce migration and sprouting. ECs with excess centrosomes initially had more centrosome-derived microtubules but, paradoxically, fewer steady-state microtubules. ECs with excess centrosomes had elevated Rac1 activity, and repolarization was rescued by blockade of Rac1 or actomyosin blockers, consistent with Rac1 activity promoting cortical retrograde actin flow and actomyosin contractility, which precludes cortical microtubule engagement necessary for dynamic repolarization. Thus normal centrosome numbers are required for dynamic repolarization and migration of sprouting ECs that contribute to blood vessel formation. PMID:27099371

  2. Stimulatory Effects of Arsenic-Tolerant Soil Fungi on Plant Growth Promotion and Soil Properties

    PubMed Central

    Srivastava, Pankaj Kumar; Shenoy, Belle Damodara; Gupta, Manjul; Vaish, Aradhana; Mannan, Shivee; Singh, Nandita; Tewari, Shri Krishna; Tripathi, Rudra Deo

    2012-01-01

    Fifteen fungi were obtained from arsenic-contaminated agricultural fields in West Bengal, India and examined for their arsenic tolerance and removal ability in our previous study. Of these, the four best arsenic-remediating isolates were tested for plant growth promotion effects on rice and pea in the present study. A greenhouse-based pot experiment was conducted using soil inocula of individual fungi. The results indicated a significant (P<0.05) increase in plant growth and improvement of soil properties in inoculated soils compared to the control. A significant increase in plant growth was recorded in treated soils and varied from 16–293%. Soil chemical and enzymatic properties varied from 20–222% and 34–760%, respectively, in inoculated soil. Plants inoculated with inocula of Westerdykella and Trichoderma showed better stimulatory effects on plant growth and soil nutrient availability than Rhizopus and Lasiodiplodia. These fungi improved soil nutrient content and enhanced plant growth. These fungi may be used as bioinoculants for plant growth promotion and improved soil properties in arsenic-contaminated agricultural soils. PMID:23047145

  3. Licensing of yeast centrosome duplication requires phosphoregulation of sfi1.

    PubMed

    Avena, Jennifer S; Burns, Shannon; Yu, Zulin; Ebmeier, Christopher C; Old, William M; Jaspersen, Sue L; Winey, Mark

    2014-10-01

    Duplication of centrosomes once per cell cycle is essential for bipolar spindle formation and genome maintenance and is controlled in part by cyclin-dependent kinases (Cdks). Our study identifies Sfi1, a conserved component of centrosomes, as the first Cdk substrate required to restrict centrosome duplication to once per cell cycle. We found that reducing Cdk1 phosphorylation by changing Sfi1 phosphorylation sites to nonphosphorylatable residues leads to defects in separation of duplicated spindle pole bodies (SPBs, yeast centrosomes) and to inappropriate SPB reduplication during mitosis. These cells also display defects in bipolar spindle assembly, chromosome segregation, and growth. Our findings lead to a model whereby phosphoregulation of Sfi1 by Cdk1 has the dual function of promoting SPB separation for spindle formation and preventing premature SPB duplication. In addition, we provide evidence that the protein phosphatase Cdc14 has the converse role of activating licensing, likely via dephosphorylation of Sfi1. PMID:25340401

  4. Licensing of Yeast Centrosome Duplication Requires Phosphoregulation of Sfi1

    PubMed Central

    Avena, Jennifer S.; Burns, Shannon; Yu, Zulin; Ebmeier, Christopher C.; Old, William M.; Jaspersen, Sue L.; Winey, Mark

    2014-01-01

    Duplication of centrosomes once per cell cycle is essential for bipolar spindle formation and genome maintenance and is controlled in part by cyclin-dependent kinases (Cdks). Our study identifies Sfi1, a conserved component of centrosomes, as the first Cdk substrate required to restrict centrosome duplication to once per cell cycle. We found that reducing Cdk1 phosphorylation by changing Sfi1 phosphorylation sites to nonphosphorylatable residues leads to defects in separation of duplicated spindle pole bodies (SPBs, yeast centrosomes) and to inappropriate SPB reduplication during mitosis. These cells also display defects in bipolar spindle assembly, chromosome segregation, and growth. Our findings lead to a model whereby phosphoregulation of Sfi1 by Cdk1 has the dual function of promoting SPB separation for spindle formation and preventing premature SPB duplication. In addition, we provide evidence that the protein phosphatase Cdc14 has the converse role of activating licensing, likely via dephosphorylation of Sfi1. PMID:25340401

  5. Murine CENP-F Regulates Centrosomal Microtubule Nucleation and Interacts with Hook2 at the Centrosome

    PubMed Central

    Moynihan, Katherine L.; Pooley, Ryan; Miller, Paul M.; Kaverina, Irina

    2009-01-01

    The microtubule (MT) network is essential in a broad spectrum of cellular functions. Many studies have linked CENP-F to MT-based activities as disruption of this protein leads to major changes in MT structure and function. Still, the basis of CENP-F regulation of the MT network remains elusive. Here, our studies reveal a novel and critical localization and role for CENP-F at the centrosome, the major MT organizing center (MTOC) of the cell. Using a yeast two-hybrid screen, we identify Hook2, a linker protein that is essential for regulation of the MT network at the centrosome, as a binding partner of CENP-F. With recently developed immunochemical reagents, we confirm this interaction and reveal the novel localization of CENP-F at the centrosome. Importantly, in this first report of CENP-F−/− cells, we demonstrate that ablation of CENP-F protein function eliminates MT repolymerization after standard nocodazole treatment. This inhibition of MT regrowth is centrosome specific because MT repolymerization is readily observed from the Golgi in CENP-F−/− cells. The centrosome-specific function of CENP-F in the regulation of MT growth is confirmed by expression of truncated CENP-F containing only the Hook2-binding domain. Furthermore, analysis of partially reconstituted MTOC asters in cells that escape complete repolymerization block shows that disruption of CENP-F function impacts MT nucleation and anchoring rather than promoting catastrophe. Our study reveals a major new localization and function of CENP-F at the centrosome that is likely to impact a broad array of MT-based actions in the cell. PMID:19793914

  6. Arsenic-induced Aurora-A activation contributes to chromosome instability and tumorigenesis

    NASA Astrophysics Data System (ADS)

    Wu, Chin-Han; Tseng, Ya-Shih; Yang, Chao-Chun; Kao, Yu-Ting; Sheu, Hamm-Ming; Liu, Hsiao-Sheng

    2013-11-01

    Arsenic may cause serious environmental pollution and is a serious industrial problem. Depending on the dosage, arsenic may trigger the cells undergoing either proliferation or apoptosis-related cell death. Because of lack of the proper animal model to study arsenic induced tumorigenesis, the accurate risk level of arsenic exposure has not been determined. Arsenic shows genotoxic effect on human beings who uptake water contaminated by arsenic. Chromosome aberration is frequently detected in arsenic exposure-related diseases and is associated with increased oxidative stress and decreased DNA repairing activity, but the underlying mechanism remains elusive. Aurora-A is a mitotic kinase, over-expression of Aurora-A leads to centrosome amplification, chromosomal instability and cell transformation. We revealed that Aurora-A is over-expressed in the skin and bladder cancer patients from blackfoot-disease endemic areas. Our cell line studies reveal that arsenic exposure between 0.5 μM and 1 μM for 2-7 days are able to induce Aurora-A expression and activation based on promoter activity, RNA and protein analysis. Aurora-A overexpression further increases the frequency of unsymmetrical chromosome segregation through centrosome amplification followed by cell population accumulated at S phase in immortalized keratinocyte (HaCaT) and uroepithelial cells (E7). Furthermore, Aurora-A over-expression was sustained for 1-4 weeks by chronic treatment of immortalized bladder and skin cells with NaAsO2. Aurora-A promoter methylation and gene amplification was not detected in the long-term arsenic treated E7 cells. Furthermore, the expression level of E2F1 transcription factor (E2F1) is increased in the presence of arsenic, and arsenic-related Aurora-A over-expression is transcriptionally regulated by E2F1. We further demonstrated that overexpression of Aurora-A and mutant Ha-ras or Aurora-A and mutant p53 may act additively to trigger arsenic-related bladder and skin cancer

  7. Centrosome positioning in vertebrate development

    PubMed Central

    Tang, Nan; Marshall, Wallace F.

    2012-01-01

    Summary The centrosome, a major organizer of microtubules, has important functions in regulating cell shape, polarity, cilia formation and intracellular transport as well as the position of cellular structures, including the mitotic spindle. By means of these activities, centrosomes have important roles during animal development by regulating polarized cell behaviors, such as cell migration or neurite outgrowth, as well as mitotic spindle orientation. In recent years, the pace of discovery regarding the structure and composition of centrosomes has continuously accelerated. At the same time, functional studies have revealed the importance of centrosomes in controlling both morphogenesis and cell fate decision during tissue and organ development. Here, we review examples of centrosome and centriole positioning with a particular emphasis on vertebrate developmental systems, and discuss the roles of centrosome positioning, the cues that determine positioning and the mechanisms by which centrosomes respond to these cues. The studies reviewed here suggest that centrosome functions extend to the development of tissues and organs in vertebrates. PMID:23277534

  8. Regulation of the centrosome cycle

    PubMed Central

    Fujita, Hiroki; Yoshino, Yuki; Chiba, Natsuko

    2016-01-01

    ABSTRACT The centrosome, consisting of mother and daughter centrioles surrounded by the pericentriolar matrix (PCM), functions primarily as a microtubule organizing center (MTOC) in most animal cells. In dividing cells the centrosome duplicates once per cell cycle and its number and structure are highly regulated during each cell cycle to organize an effective bipolar spindle in the mitotic phase. Defects in the regulation of centrosome duplication lead to a variety of human diseases, including cancer, through abnormal cell division and inappropriate chromosome segregation. At the end of mitosis the daughter centriole disengages from the mother centriole. This centriole disengagement is an important licensing step for centrosome duplication. In S phase, one new daughter centriole forms perpendicular to each centriole. The centrosome recruits further PCM proteins in the late G2 phase and the two centrosomes separate at mitotic entry to form a bipolar spindle. Here, we summarize research findings in the field of centrosome biology, focusing on the mechanisms of regulation of the centrosome cycle in human cells. PMID:27308597

  9. Arsenic promotes angiogenesis in vitro via a heme oxygenase-1-dependent mechanism

    SciTech Connect

    Meng Dan; Wang Xin; Chang Qingshan; Hitron, Andrew; Zhang Zhuo; Xu Mei; Chen Gang; Luo Jia; Jiang Binghua; Fang Jing; Shi Xianglin

    2010-05-01

    Angiogenesis and vessel remodeling are fundamental to the pathogenesis of a number of diseases caused by environmental arsenic exposure, including tumorigenesis and cardiovascular diseases. Arsenic (AsIII) has been shown to stimulate angiogenesis and vascular remodeling in vivo. However, the exact molecular mechanisms accounting for arsenic-induced angiogenesis are not clear. The present study investigates the role of heme oxygenase-1 (HO-1) in sodium arsenite-mediated angiogenesis in vitro. Transwell assay, three-dimensional Matrigel assay, RT-PCR, ELISA and immunoblotting were used to determine cell migration, vascular tube formation, mRNA and protein expression. Chromatin immunoprecipitation and luciferase assay were applied to examine the DNA binding with protein and HO-1 transcriptional activity. Here, we report that low concentrations of arsenite (0.1-1 muM) stimulated cell migration and vascular tube formation in human microvascular endothelial cells (HMVEC). Arsenite induced HO-1 mRNA and protein expression. Knock down of HO-1 expression decreased arsenite-induced VEGF expression, cell migration, and tube formation. We showed that arsenite promoted dissociation of Bach1 (a transcriptional repressor) from the HO-1 enhancers and increased Nrf2 binding to these elements. Site directed mutagenesis assay identified that Bach1 cysteine residues 557 and 574 were essential for the induction of HO-1 gene in response to arsenite. These findings demonstrate a role for HO-1 in arsenite-mediated angiogenesis in vitro.

  10. Centrosome Dysfunction Contributes to Chromosome Instability, Chromoanagenesis, and Genome Reprograming in Cancer

    PubMed Central

    Pihan, German A.

    2013-01-01

    The unique ability of centrosomes to nucleate and organize microtubules makes them unrivaled conductors of important interphase processes, such as intracellular payload traffic, cell polarity, cell locomotion, and organization of the immunologic synapse. But it is in mitosis that centrosomes loom large, for they orchestrate, with clockmaker’s precision, the assembly and functioning of the mitotic spindle, ensuring the equal partitioning of the replicated genome into daughter cells. Centrosome dysfunction is inextricably linked to aneuploidy and chromosome instability, both hallmarks of cancer cells. Several aspects of centrosome function in normal and cancer cells have been molecularly characterized during the last two decades, greatly enhancing our mechanistic understanding of this tiny organelle. Whether centrosome defects alone can cause cancer, remains unanswered. Until recently, the aggregate of the evidence had suggested that centrosome dysfunction, by deregulating the fidelity of chromosome segregation, promotes and accelerates the characteristic Darwinian evolution of the cancer genome enabled by increased mutational load and/or decreased DNA repair. Very recent experimental work has shown that missegregated chromosomes resulting from centrosome dysfunction may experience extensive DNA damage, suggesting additional dimensions to the role of centrosomes in cancer. Centrosome dysfunction is particularly prevalent in tumors in which the genome has undergone extensive structural rearrangements and chromosome domain reshuffling. Ongoing gene reshuffling reprograms the genome for continuous growth, survival, and evasion of the immune system. Manipulation of molecular networks controlling centrosome function may soon become a viable target for specific therapeutic intervention in cancer, particularly since normal cells, which lack centrosome alterations, may be spared the toxicity of such therapies. PMID:24282781

  11. Pattern formation in centrosome assembly.

    PubMed

    Mahen, Robert; Venkitaraman, Ashok R

    2012-02-01

    A striking but poorly explained feature of cell division is the ability to assemble and maintain organelles not bounded by membranes, from freely diffusing components in the cytosol. This process is driven by information transfer across biological scales such that interactions at the molecular scale allow pattern formation at the scale of the organelle. One important example of such an organelle is the centrosome, which is the main microtubule organising centre in the cell. Centrosomes consist of two centrioles surrounded by a cloud of proteins termed the pericentriolar material (PCM). Profound structural and proteomic transitions occur in the centrosome during specific cell cycle stages, underlying events such as centrosome maturation during mitosis, in which the PCM increases in size and microtubule nucleating capacity. Here we use recent insights into the spatio-temporal behaviour of key regulators of centrosomal maturation, including Polo-like kinase 1, CDK5RAP2 and Aurora-A, to propose a model for the assembly and maintenance of the PCM through the mobility and local interactions of its constituent proteins. We argue that PCM structure emerges as a pattern from decentralised self-organisation through a reaction-diffusion mechanism, with or without an underlying template, rather than being assembled from a central structural template alone. Self-organisation of this kind may have broad implications for the maintenance of mitotic structures, which, like the centrosome, exist stably as supramolecular assemblies on the micron scale, based on molecular interactions at the nanometer scale. PMID:22245706

  12. Loss of KLF14 triggers centrosome amplification and tumorigenesis

    PubMed Central

    Fan, Guangjian; Sun, Lianhui; Shan, Peipei; Zhang, Xianying; Huan, Jinliang; Zhang, Xiaohong; Li, Dali; Wang, Tingting; Wei, Tingting; Zhang, Xiaohong; Gu, Xiaoyang; Yao, Liangfang; Xuan, Yang; Hou, Zhaoyuan; Cui, Yongping; Cao, Liu; Li, Xiaotao; Zhang, Shengping; Wang, Chuangui

    2015-01-01

    Centrosome amplification is frequent in cancer, but the underlying mechanisms remain unclear. Here we report that disruption of the Kruppel-like factor 14 (KLF14) gene in mice causes centrosome amplification, aneuploidy and spontaneous tumorigenesis. Molecularly, KLF14 functions as a transcriptional repressor of Plk4, a polo-like kinase whose overexpression induces centrosome overduplication. Transient knockdown of KLF14 is sufficient to induce Plk4-directed centrosome amplification. Clinically, KLF14 transcription is significantly downregulated, whereas Plk4 transcription is upregulated in multiple types of cancers, and there exists an inverse correlation between KLF14 and Plk4 protein expression in human breast and colon cancers. Moreover, KLF14 depletion promotes AOM/DSS-induced colon tumorigenesis. Our findings reveal that KLF14 reduction serves as a mechanism leading to centrosome amplification and tumorigenesis. On the other hand, forced expression of KLF14 leads to mitotic catastrophe. Collectively, our findings identify KLF14 as a tumour suppressor and highlight its potential as biomarker and therapeutic target for cancer. PMID:26439168

  13. Loss of KLF14 triggers centrosome amplification and tumorigenesis.

    PubMed

    Fan, Guangjian; Sun, Lianhui; Shan, Peipei; Zhang, Xianying; Huan, Jinliang; Zhang, Xiaohong; Li, Dali; Wang, Tingting; Wei, Tingting; Zhang, Xiaohong; Gu, Xiaoyang; Yao, Liangfang; Xuan, Yang; Hou, Zhaoyuan; Cui, Yongping; Cao, Liu; Li, Xiaotao; Zhang, Shengping; Wang, Chuangui

    2015-01-01

    Centrosome amplification is frequent in cancer, but the underlying mechanisms remain unclear. Here we report that disruption of the Kruppel-like factor 14 (KLF14) gene in mice causes centrosome amplification, aneuploidy and spontaneous tumorigenesis. Molecularly, KLF14 functions as a transcriptional repressor of Plk4, a polo-like kinase whose overexpression induces centrosome overduplication. Transient knockdown of KLF14 is sufficient to induce Plk4-directed centrosome amplification. Clinically, KLF14 transcription is significantly downregulated, whereas Plk4 transcription is upregulated in multiple types of cancers, and there exists an inverse correlation between KLF14 and Plk4 protein expression in human breast and colon cancers. Moreover, KLF14 depletion promotes AOM/DSS-induced colon tumorigenesis. Our findings reveal that KLF14 reduction serves as a mechanism leading to centrosome amplification and tumorigenesis. On the other hand, forced expression of KLF14 leads to mitotic catastrophe. Collectively, our findings identify KLF14 as a tumour suppressor and highlight its potential as biomarker and therapeutic target for cancer. PMID:26439168

  14. Activity and regulation of the centrosome-associated proteasome.

    PubMed

    Fabunmi, R P; Wigley, W C; Thomas, P J; DeMartino, G N

    2000-01-01

    Regulated proteolysis is important for maintaining appropriate cellular levels of many proteins. The bulk of intracellular protein degradation is catalyzed by the proteasome. Recently, the centrosome was identified as a novel site for concentration of the proteasome and associated regulatory proteins (Wigley, W. C., Fabunmi, R. P., Lee, M. G., Marino, C. R., Muallem, S., DeMartino, G. N., and Thomas, P. J. (1999) J. Cell Biol. 145, 481-490). Here we provide evidence that centrosomes contain the active 26 S proteasome that degrades ubiquitinated-protein and proteasome-specific peptide substrates. Moreover, the centrosomes contain an ubiquitin isopeptidase activity. The proteolytic activity is ATP-dependent and is inhibited by proteasome inhibitors. Notably, treatment of cells with inhibitors of proteasome activity promotes redistribution of the proteasome and associated regulatory proteins to the centrosome independent of an intact microtubule system. These data provide biochemical evidence for active proteasomal complexes at the centrosome, highlighting a novel function for this organizing structure. PMID:10617632

  15. PPP1R42, a PP1 binding protein, regulates centrosome dynamics in ARPE-19 cells

    PubMed Central

    DeVaul, Nicole; Wang, Rong; Sperry, Ann O.

    2013-01-01

    centrosomes per cell is consistent with our model that PPP1R42 is a positive regulator of PP1. PPP1R42 depletion reduces the activity of PP1 leading to activation of NEK2, the kinase responsible for phosphorylation of centrosomal linker proteins promoting centrosome separation. This work identifies a new molecule localized to the centrosome and basal body with a role in the complex signaling network responsible for controlling centrosome activities. PMID:23718219

  16. Developmental alterations in centrosome integrity contribute to the post-mitotic state of mammalian cardiomyocytes.

    PubMed

    Zebrowski, David C; Vergarajauregui, Silvia; Wu, Chi-Chung; Piatkowski, Tanja; Becker, Robert; Leone, Marina; Hirth, Sofia; Ricciardi, Filomena; Falk, Nathalie; Giessl, Andreas; Just, Steffen; Braun, Thomas; Weidinger, Gilbert; Engel, Felix B

    2015-01-01

    Mammalian cardiomyocytes become post-mitotic shortly after birth. Understanding how this occurs is highly relevant to cardiac regenerative therapy. Yet, how cardiomyocytes achieve and maintain a post-mitotic state is unknown. Here, we show that cardiomyocyte centrosome integrity is lost shortly after birth. This is coupled with relocalization of various centrosome proteins to the nuclear envelope. Consequently, postnatal cardiomyocytes are unable to undergo ciliogenesis and the nuclear envelope adopts the function as cellular microtubule organizing center. Loss of centrosome integrity is associated with, and can promote, cardiomyocyte G0/G1 cell cycle arrest suggesting that centrosome disassembly is developmentally utilized to achieve the post-mitotic state in mammalian cardiomyocytes. Adult cardiomyocytes of zebrafish and newt, which are able to proliferate, maintain centrosome integrity. Collectively, our data provide a novel mechanism underlying the post-mitotic state of mammalian cardiomyocytes as well as a potential explanation for why zebrafish and newts, but not mammals, can regenerate their heart. PMID:26247711

  17. Arsenic-tolerant plant-growth-promoting bacteria isolated from arsenic-polluted soils in South Korea.

    PubMed

    Shagol, Charlotte C; Krishnamoorthy, Ramasamy; Kim, Kiyoon; Sundaram, Subbiah; Sa, Tongmin

    2014-01-01

    The Janghang smelter in Chungnam, South Korea started in 1936 was subsequently shutdown in 1989 due to heavy metal (loid) pollution concerns in the vicinity. Thus, there is a need for the soil in the area to be remediated to make it usable again especially for agricultural purposes. The present study was conducted to exploit the potential of arsenic (As)-tolerant bacteria thriving in the vicinity of the smelter-polluted soils to enhance phytoremediation of hazardous As. We studied the genetic and taxonomic diversity of 21 As-tolerant bacteria isolated from soils nearer to and away from the smelter. These isolates belonging to the genera Brevibacterium, Pseudomonas, Microbacterium, Rhodococcus, Rahnella, and Paenibacillus, could tolerate high concentrations of arsenite (As(III)) and arsenate (As(V)) with the minimum inhibitory concentration ranging from 3 to >20 mM for NaAsO2 and 140 to 310 mM NaH2AsO4 · 7H2O, respectively. All isolates exhibited As(V) reduction except Pseudomonas koreensis JS123, which exhibited both oxidation and reduction of As. Moreover, all the 21 isolates produced indole acetic acid (IAA), 13 isolates exhibited 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity, 12 produced siderophore, 17 solubilized phosphate, and 13 were putative nitrogen fixers under in vitro conditions. Particularly, Rhodococcus aetherivorans JS2210, P. koreensis JS2214, and Pseudomonas sp. JS238 consistently increased root length of maize in the presence of 100 and 200 μM As(V). Possible utilization of these As-tolerant plant-growth-promoting bacteria can be a potential strategy in increasing the efficiency of phytoremediation in As-polluted soils. PMID:24737020

  18. Brevundimonas diminuta mediated alleviation of arsenic toxicity and plant growth promotion in Oryza sativa L.

    PubMed

    Singh, Namrata; Marwa, Naina; Mishra, Shashank K; Mishra, Jyoti; Verma, Praveen C; Rathaur, Sushma; Singh, Nandita

    2016-03-01

    Arsenic (As), a toxic metalloid adversely affects plant growth in polluted areas. In the present study, we investigated the possibility of improving phytostablization of arsenic through application of new isolated strain Brevundimonas diminuta (NBRI012) in rice plant [Oryza sativa (L.) Var. Sarju 52] at two different concentrations [10ppm (low toxic) and 50ppm (high toxic)] of As. The plant growth promoting traits of bacterial strains revealed the inherent ability of siderophores, phosphate solubilisation, indole acetic acid (IAA), 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase production which may be associated with increased biomass, chlorophyll and MDA content of rice and thereby promoting plant growth. The study also revealed the As accumulation property of NBRI012 strain which could play an important role in As removal from contaminated soil. Furthermore, NBRI012 inoculation significantly restored the hampered root epidermal and cortical cell growth of rice plant and root hair elimination. Altogether our study highlights the multifarious role of B. diminuta in mediating stress tolerance and modulating translocation of As in edible part of rice plant. PMID:26650422

  19. Evidence of arsenic release promoted by disinfection by-products within drinking-water distribution systems.

    PubMed

    Andra, Syam S; Makris, Konstantinos C; Botsaris, George; Charisiadis, Pantelis; Kalyvas, Harris; Costa, Costas N

    2014-02-15

    Changes in disinfectant type could trigger a cascade of reactions releasing pipe-anchored metals/metalloids into finished water. However, the effect of pre-formed disinfection by-products on the release of sorbed contaminants (arsenic-As in particular) from drinking water distribution system pipe scales remains unexplored. A bench-scale study using a factorial experimental design was performed to evaluate the independent and interaction effects of trihalomethanes (TTHM) and haloacetic acids (HAA) on arsenic (As) release from either scales-only or scale-biofilm conglomerates (SBC) both anchored on asbestos/cement pipe coupons. A model biofilm (Pseudomonas aeruginosa) was allowed to grow on select pipe coupons prior experimentation. Either TTHM or HAA individual dosing did not promote As release from either scales only or SBC, detecting <6 μg AsL(-1) in finished water. In the case of scales-only coupons, the combination of the highest spike level of TTHM and HAA significantly (p<0.001) increased dissolved and total As concentrations to levels up to 16 and 95 μg L(-1), respectively. Similar treatments in the presence of biofilm (SBC) resulted in significant (p<0.001) increase in dissolved and total recoverable As up to 20 and 47 μg L(-1), respectively, exceeding the regulatory As limit. Whether or not, our laboratory-based results truly represent mechanisms operating in disinfected finished water in pipe networks remains to be investigated in the field. PMID:24365518

  20. Epithelial to mesenchymal transition in arsenic-transformed cells promotes angiogenesis through activating β-catenin–vascular endothelial growth factor pathway

    SciTech Connect

    Wang, Zhishan; Humphries, Brock; Xiao, Hua; Jiang, Yiguo; Yang, Chengfeng

    2013-08-15

    Arsenic exposure represents a major health concern increasing cancer risks, yet the mechanism of arsenic carcinogenesis has not been elucidated. We and others recently reported that cell malignant transformation by arsenic is accompanied by epithelial to mesenchymal transition (EMT). However, the role of EMT in arsenic carcinogenesis is not well understood. Although previous studies showed that short term exposure of endothelial cells to arsenic stimulated angiogenesis, it remains to be determined whether cells that were malignantly transformed by long term arsenic exposure have a pro-angiogenic effect. The objective of this study was to investigate the effect of arsenic-transformed human bronchial epithelial cells that underwent EMT on angiogenesis and the underlying mechanism. It was found that the conditioned medium from arsenic-transformed cells strongly stimulated tube formation by human umbilical vein endothelial cells (HUVECs). Moreover, enhanced angiogenesis was detected in mouse xenograft tumor tissues resulting from inoculation of arsenic-transformed cells. Mechanistic studies revealed that β-catenin was activated in arsenic-transformed cells up-regulating its target gene expression including angiogenic-stimulating vascular endothelial growth factor (VEGF). Stably expressing microRNA-200b in arsenic-transformed cells that reversed EMT inhibited β-catenin activation, decreased VEGF expression and reduced tube formation by HUVECs. SiRNA knockdown β-catenin decreased VEGF expression. Adding a VEGF neutralizing antibody into the conditioned medium from arsenic-transformed cells impaired tube formation by HUVECs. Reverse transcriptase-PCR analysis revealed that the mRNA levels of canonical Wnt ligands were not increased in arsenic-transformed cells. These findings suggest that EMT in arsenic-transformed cells promotes angiogenesis through activating β-catenin–VEGF pathway. - Highlights: • Arsenic-transformed cells that underwent EMT displayed a pro

  1. Centrosome Positioning in 1D Cell Migration

    NASA Astrophysics Data System (ADS)

    Adlerz, Katrina; Aranda-Espinoza, Helim

    During cell migration, the positioning of the centrosome and nucleus define a cell's polarity. For a cell migrating on a two-dimensional substrate the centrosome is positioned in front of the nucleus. Under one-dimensional confinement, however, the centrosome is positioned behind the nucleus in 60% of cells. It is known that the centrosome is positioned by CDC42 and dynein for cells moving on a 2D substrate in a wound-healing assay. It is currently unknown, however, if this is also true for cells moving under 1D confinement, where the centrosome position is often reversed. Therefore, centrosome positioning was studied in cells migrating under 1D confinement, which mimics cells migrating through 3D matrices. 3 to 5 μm fibronectin lines were stamped onto a glass substrate and cells with fluorescently labeled nuclei and centrosomes migrated on the lines. Our results show that when a cell changes directions the centrosome position is maintained. That is, when the centrosome is between the nucleus and the cell's trailing edge and the cell changes direction, the centrosome will be translocated across the nucleus to the back of the cell again. A dynein inhibitor did have an influence on centrosome positioning in 1D migration and change of directions.

  2. Multisite phosphorylation of C-Nap1 releases it from Cep135 to trigger centrosome disjunction

    PubMed Central

    Hardy, Tara; Lee, Miseon; Hames, Rebecca S.; Prosser, Suzanna L.; Cheary, Donna-Marie; Samant, Mugdha D.; Schultz, Francisca; Baxter, Joanne E.; Rhee, Kunsoo; Fry, Andrew M.

    2014-01-01

    ABSTRACT During mitotic entry, centrosomes separate to establish the bipolar spindle. Delays in centrosome separation can perturb chromosome segregation and promote genetic instability. However, interphase centrosomes are physically tethered by a proteinaceous linker composed of C-Nap1 (also known as CEP250) and the filamentous protein rootletin. Linker disassembly occurs at the onset of mitosis in a process known as centrosome disjunction and is triggered by the Nek2-dependent phosphorylation of C-Nap1. However, the mechanistic consequences of C-Nap1 phosphorylation are unknown. Here, we demonstrate that Nek2 phosphorylates multiple residues within the C-terminal domain of C-Nap1 and, collectively, these phosphorylation events lead to loss of oligomerization and centrosome association. Mutations in non-phosphorylatable residues that make the domain more acidic are sufficient to release C-Nap1 from the centrosome, suggesting that it is an increase in overall negative charge that is required for this process. Importantly, phosphorylation of C-Nap1 also perturbs interaction with the core centriolar protein, Cep135, and interaction of endogenous C-Nap1 and Cep135 proteins is specifically lost in mitosis. We therefore propose that multisite phosphorylation of C-Nap1 by Nek2 perturbs both oligomerization and Cep135 interaction, and this precipitates centrosome disjunction at the onset of mitosis. PMID:24695856

  3. Drosophila Ana1 is required for centrosome assembly and centriole elongation.

    PubMed

    Saurya, Saroj; Roque, Hélio; Novak, Zsofia A; Wainman, Alan; Aydogan, Mustafa G; Volanakis, Adam; Sieber, Boris; Pinto, David Miguel Susano; Raff, Jordan W

    2016-07-01

    Centrioles organise centrosomes and cilia, and these organelles have an important role in many cell processes. In flies, the centriole protein Ana1 is required for the assembly of functional centrosomes and cilia. It has recently been shown that Cep135 (also known as Bld10) initially recruits Ana1 to newly formed centrioles, and that Ana1 then recruits Asl (known as Cep152 in mammals) to promote the conversion of these centrioles into centrosomes. Here, we show that ana1 mutants lack detectable centrosomes in vivo, that Ana1 is irreversibly incorporated into centrioles during their assembly and appears to play a more important role in maintaining Asl at centrioles than in initially recruiting Asl to centrioles. Unexpectedly, we also find that Ana1 promotes centriole elongation in a dose-dependent manner: centrioles are shorter when Ana1 dosage is reduced and are longer when Ana1 is overexpressed. This latter function of Ana1 appears to be distinct from its role in centrosome and cilium function, as a GFP-Ana1 fusion lacking the N-terminal 639 amino acids of the protein can support centrosome assembly and cilium function but cannot promote centriole over-elongation when overexpressed. PMID:27206860

  4. Drosophila Ana1 is required for centrosome assembly and centriole elongation

    PubMed Central

    Saurya, Saroj; Roque, Hélio; Novak, Zsofia A.; Wainman, Alan; Aydogan, Mustafa G.; Volanakis, Adam; Sieber, Boris; Pinto, David Miguel Susano

    2016-01-01

    ABSTRACT Centrioles organise centrosomes and cilia, and these organelles have an important role in many cell processes. In flies, the centriole protein Ana1 is required for the assembly of functional centrosomes and cilia. It has recently been shown that Cep135 (also known as Bld10) initially recruits Ana1 to newly formed centrioles, and that Ana1 then recruits Asl (known as Cep152 in mammals) to promote the conversion of these centrioles into centrosomes. Here, we show that ana1 mutants lack detectable centrosomes in vivo, that Ana1 is irreversibly incorporated into centrioles during their assembly and appears to play a more important role in maintaining Asl at centrioles than in initially recruiting Asl to centrioles. Unexpectedly, we also find that Ana1 promotes centriole elongation in a dose-dependent manner: centrioles are shorter when Ana1 dosage is reduced and are longer when Ana1 is overexpressed. This latter function of Ana1 appears to be distinct from its role in centrosome and cilium function, as a GFP–Ana1 fusion lacking the N-terminal 639 amino acids of the protein can support centrosome assembly and cilium function but cannot promote centriole over-elongation when overexpressed. PMID:27206860

  5. Centriolar satellites: busy orbits around the centrosome.

    PubMed

    Bärenz, Felix; Mayilo, Dmytro; Gruss, Oliver J

    2011-12-01

    Since its first description by Theodor Boveri in 1888, the centrosome has been studied intensely, and it revealed detailed information about its structure, molecular composition and its various functions. The centrosome consists of two centrioles, which generally appear in electron microscopy as barrel-shaped structures usually composed of nine microtubule triplets. An amorphous mass of pericentriolar material surrounds the centrioles and accumulates many proteins important for the integrity and function of centrosomes, such as the γ-tubulin ring complex (γ-TuRC) that mediates microtubule nucleation and capping. In animal somatic cells, the centrosome generally accounts for the major microtubule organizing center, and the duplicated pair of centrosomes determines the poles of the microtubule-based mitotic spindle. Despite detailed insights into the centrosome's structure and function, it has been a complete mystery until a few years ago how centrosomes duplicate and assemble. Moreover, it is still largely unclear if and how centrosomal proteins or protein complexes are exchanged, replaced or qualitatively altered. Previously identified cytoplasmic granules, named "pericentriolar" or "centriolar satellites", might fulfil such functions in protein targeting and exchange, and communication between the centrosomes and the cytoplasm. In this review, we summarize current knowledge about the structure, molecular composition and possible roles of the satellites that seem to surround the core of the centrosome in most animal cells. PMID:21945726

  6. Arsenic in private well water part 2 of 3: Who benefits the most from traditional testing promotion?

    PubMed

    Flanagan, Sara V; Spayd, Steven E; Procopio, Nicholas A; Chillrud, Steven N; Ross, James; Braman, Stuart; Zheng, Yan

    2016-08-15

    Arsenic, a toxic element naturally found in groundwater, is a public health concern for households drinking from wells. Private well water is not regulated to meet the federal drinking water arsenic Maximum Contaminant Level (MCL) of 10μg/L, or the more protective 5μg/L New Jersey (NJ) state MCL. In the absence of consistent private well regulation, public health efforts have relied on promoting testing in affected communities to various degrees of success. Few interventions publish results, and more often focus on the outcome of tested wells rather than who completed a test, and more importantly, who did not. Through our survey of randomly selected addresses (n=670) in 17 NJ towns we find higher rates of arsenic testing in areas with a history of testing promotion. However, we also see a stronger correlation of testing behavior with income and education in high promotion areas, suggesting that community engagement activities may be exacerbating socioeconomic status (SES) testing disparities. Well owners with a bachelor's degree had ten times greater odds of participating in our direct mail testing intervention than those with less education when tests cost $40. After all households (n=255) were offered free tests to overcome many of the usual testing barriers - awareness, convenience, and cost - only 47% participated and those who chose to return water samples were of higher income and education than those who did not. Our findings highlight that while efforts to promote and provide arsenic testing succeed in testing more wells, community testing interventions risk increasing SES disparities if those with more education and resources are more likely to take advantage of testing programs. Therefore, testing interventions can benefit by better targeting socially vulnerable populations in an effort to overcome SES-patterned self-selection when individuals are left alone with the responsibility of managing their drinking water quality. PMID:27142115

  7. Centrosomes. Regulated assembly of a supramolecular centrosome scaffold in vitro.

    PubMed

    Woodruff, Jeffrey B; Wueseke, Oliver; Viscardi, Valeria; Mahamid, Julia; Ochoa, Stacy D; Bunkenborg, Jakob; Widlund, Per O; Pozniakovsky, Andrei; Zanin, Esther; Bahmanyar, Shirin; Zinke, Andrea; Hong, Sun Hae; Decker, Marcus; Baumeister, Wolfgang; Andersen, Jens S; Oegema, Karen; Hyman, Anthony A

    2015-05-15

    The centrosome organizes microtubule arrays within animal cells and comprises two centrioles surrounded by an amorphous protein mass called the pericentriolar material (PCM). Despite the importance of centrosomes as microtubule-organizing centers, the mechanism and regulation of PCM assembly are not well understood. In Caenorhabditis elegans, PCM assembly requires the coiled-coil protein SPD-5. We found that recombinant SPD-5 could polymerize to form micrometer-sized porous networks in vitro. Network assembly was accelerated by two conserved regulators that control PCM assembly in vivo, Polo-like kinase-1 and SPD-2/Cep192. Only the assembled SPD-5 networks, and not unassembled SPD-5 protein, functioned as a scaffold for other PCM proteins. Thus, PCM size and binding capacity emerge from the regulated polymerization of one coiled-coil protein to form a porous network. PMID:25977552

  8. Exploring the evolutionary history of centrosomes

    PubMed Central

    Azimzadeh, Juliette

    2014-01-01

    The centrosome is the main organizer of the microtubule cytoskeleton in animals, higher fungi and several other eukaryotic lineages. Centrosomes are usually located at the centre of cell in tight association with the nuclear envelope and duplicate at each cell cycle. Despite a great structural diversity between the different types of centrosomes, they are functionally equivalent and share at least some of their molecular components. In this paper, we explore the evolutionary origin of the different centrosomes, in an attempt to understand whether they are derived from an ancestral centrosome or evolved independently from the motile apparatus of distinct flagellated ancestors. We then discuss the evolution of centrosome structure and function within the animal lineage. PMID:25047607

  9. HSET overexpression fuels tumor progression via centrosome clustering-independent mechanisms in breast cancer patients

    PubMed Central

    Pannu, Vaishali; Rida, Padmashree C.G.; Ogden, Angela; Turaga, Ravi Chakra; Donthamsetty, Shashikiran; Bowen, Nathan J.; Rudd, Katie; Gupta, Meenakshi V.; Reid, Michelle D.; Cantuaria, Guilherme; Walczak, Claire E.; Aneja, Ritu

    2015-01-01

    Human breast tumors harbor supernumerary centrosomes in almost 80% of tumor cells. Although amplified centrosomes compromise cell viability via multipolar spindles resulting in death-inducing aneuploidy, cancer cells tend to cluster extra centrosomes during mitosis. As a result cancer cells display bipolar spindle phenotypes to maintain a tolerable level of aneuploidy, an edge to their survival. HSET/KifC1, a kinesin-like minus-end directed microtubule motor has recently found fame as a crucial centrosome clustering molecule. Here we show that HSET promotes tumor progression via mechanisms independent of centrosome clustering. We found that HSET is overexpressed in breast carcinomas wherein nuclear HSET accumulation correlated with histological grade and predicted poor progression-free and overall survival. In addition, deregulated HSET protein expression was associated with gene amplification and/or translocation. Our data provide compelling evidence that HSET overexpression is pro-proliferative, promotes clonogenic-survival and enhances cell-cycle kinetics through G2 and M-phases. Importantly, HSET co-immunoprecipitates with survivin, and its overexpression protects survivin from proteasome-mediated degradation, resulting in its increased steady-state levels. We provide the first evidence of centrosome clustering-independent activities of HSET that fuel tumor progression and firmly establish that HSET can serve both as a potential prognostic biomarker and as a valuable cancer-selective therapeutic target. PMID:25788277

  10. HSET overexpression fuels tumor progression via centrosome clustering-independent mechanisms in breast cancer patients.

    PubMed

    Pannu, Vaishali; Rida, Padmashree C G; Ogden, Angela; Turaga, Ravi Chakra; Donthamsetty, Shashikiran; Bowen, Nathan J; Rudd, Katie; Gupta, Meenakshi V; Reid, Michelle D; Cantuaria, Guilherme; Walczak, Claire E; Aneja, Ritu

    2015-03-20

    Human breast tumors harbor supernumerary centrosomes in almost 80% of tumor cells. Although amplified centrosomes compromise cell viability via multipolar spindles resulting in death-inducing aneuploidy, cancer cells tend to cluster extra centrosomes during mitosis. As a result cancer cells display bipolar spindle phenotypes to maintain a tolerable level of aneuploidy, an edge to their survival. HSET/KifC1, a kinesin-like minus-end directed microtubule motor has recently found fame as a crucial centrosome clustering molecule. Here we show that HSET promotes tumor progression via mechanisms independent of centrosome clustering. We found that HSET is overexpressed in breast carcinomas wherein nuclear HSET accumulation correlated with histological grade and predicted poor progression-free and overall survival. In addition, deregulated HSET protein expression was associated with gene amplification and/or translocation. Our data provide compelling evidence that HSET overexpression is pro-proliferative, promotes clonogenic-survival and enhances cell-cycle kinetics through G2 and M-phases. Importantly, HSET co-immunoprecipitates with survivin, and its overexpression protects survivin from proteasome-mediated degradation, resulting in its increased steady-state levels. We provide the first evidence of centrosome clustering-independent activities of HSET that fuel tumor progression and firmly establish that HSET can serve both as a potential prognostic biomarker and as a valuable cancer-selective therapeutic target. PMID:25788277

  11. Communication, the centrosome and the immunological synapse

    PubMed Central

    Stinchcombe, Jane C.; Griffiths, Gillian M.

    2014-01-01

    Recent findings on the behaviour of the centrosome at the immunological synapse suggest a critical role for centrosome polarization in controlling the communication between immune cells required to generate an effective immune response. The features observed at the immunological synapse show parallels to centrosome (basal body) polarization seen in cilia and flagella, and the cellular communication that is now known to occur at all of these sites. PMID:25047617

  12. The mammalian centrosome and its functional significance

    PubMed Central

    2008-01-01

    Primarily known for its role as major microtubule organizing center, the centrosome is increasingly being recognized for its functional significance in key cell cycle regulating events. We are now at the beginning of understanding the centrosome’s functional complexities and its major impact on directing complex interactions and signal transduction cascades important for cell cycle regulation. The centrosome orchestrates entry into mitosis, anaphase onset, cytokinesis, G1/S transition, and monitors DNA damage. Recently, the centrosome has also been recognized as major docking station where regulatory complexes accumulate including kinases and phosphatases as well as numerous other cell cycle regulators that utilize the centrosome as platform to coordinate multiple cell cycle-specific functions. Vesicles that are translocated along microtubules to and away from centrosomes may also carry enzymes or substrates that use centrosomes as main docking station. The centrosome’s role in various diseases has been recognized and a wealth of data has been accumulated linking dysfunctional centrosomes to cancer, Alstrom syndrome, various neurological disorders, and others. Centrosome abnormalities and dysfunctions have been associated with several types of infertility. The present review highlights the centrosome’s significant roles in cell cycle events in somatic and reproductive cells and discusses centrosome abnormalities and implications in disease. PMID:18437411

  13. Heading off with the herd: How cancer cells might maneuver supernumerary centrosomes for directional migration

    PubMed Central

    Ogden, Angela; Rida, Padmashree CG; Aneja, Ritu

    2012-01-01

    The complicity of centrosomes in carcinogenesis is unmistakable. Mounting evidence clearly implicates a robust correlation between centrosome amplification (CA) and malignant transformation in diverse tissue types. Furthermore, CA has been suggested as a marker of cancer aggressiveness, in particular the invasive phenotype, in breast and prostate cancers. One means by which CA promotes malignancy is through induction of transient spindle multipolarity during mitosis, which predisposes the cell to karyotypic changes arising from low-grade chromosome mis-segregation. It is well recognized that during cell migration in interphase, centrosome-mediated nucleation of a radial microtubule array is crucial for establishing a polarized Golgi apparatus, without which directionality is precluded. The question of how cancer cells maneuver their supernumerary centrosomes to achieve directionality during cell migration is virtually uncharted territory. Given CA is a hallmark of cancers and has been correlated with cancer aggressiveness, malignant cells are presumably competent in managing their centrosome surfeit during directional migration, although the cellular logistics of this process remain unexplored. Thus, another key angle worth pondering is whether an overabundance of centrosomes confers some advantage on cancer cells in terms of their migratory and invasive capabilities. Recent studies have uncovered a remarkable strategy that cancer cells employ to deal with the problem of excess centrosomes and ensure bipolar mitoses, viz., centrosome clustering. This review aims to change the narrative by exploring how an increased centrosome complement may, via aneuploidy-independent modulation of the microtubule cytoskeleton, enhance directional migration and invasion of malignant cells. We postulate that CA imbues cancer cells with cytoskeletal advantages that enhance cell polarization, Golgi-dependent vesicular trafficking, stromal invasion and other aspects of metastatic

  14. Tethering of an E3 ligase by PCM1 regulates the abundance of centrosomal KIAA0586/Talpid3 and promotes ciliogenesis.

    PubMed

    Wang, Lei; Lee, Kwanwoo; Malonis, Ryan; Sanchez, Irma; Dynlacht, Brian D

    2016-01-01

    To elucidate the role of centriolar satellites in ciliogenesis, we deleted the gene encoding the PCM1 protein, an integral component of satellites. PCM1 null human cells show marked defects in ciliogenesis, precipitated by the loss of specific proteins from satellites and their relocation to centrioles. We find that an amino-terminal domain of PCM1 can restore ciliogenesis and satellite localization of certain proteins, but not others, pinpointing unique roles for PCM1 and a group of satellite proteins in cilium assembly. Remarkably, we find that PCM1 is essential for tethering the E3 ligase, Mindbomb1 (Mib1), to satellites. In the absence of PCM1, Mib1 destabilizes Talpid3 through poly-ubiquitylation and suppresses cilium assembly. Loss of PCM1 blocks ciliogenesis by abrogating recruitment of ciliary vesicles associated with the Talpid3-binding protein, Rab8, which can be reversed by inactivating Mib1. Thus, PCM1 promotes ciliogenesis by tethering a key E3 ligase to satellites and restricting it from centrioles. PMID:27146717

  15. Tethering of an E3 ligase by PCM1 regulates the abundance of centrosomal KIAA0586/Talpid3 and promotes ciliogenesis

    PubMed Central

    Wang, Lei; Lee, Kwanwoo; Malonis, Ryan; Sanchez, Irma; Dynlacht, Brian D

    2016-01-01

    To elucidate the role of centriolar satellites in ciliogenesis, we deleted the gene encoding the PCM1 protein, an integral component of satellites. PCM1 null human cells show marked defects in ciliogenesis, precipitated by the loss of specific proteins from satellites and their relocation to centrioles. We find that an amino-terminal domain of PCM1 can restore ciliogenesis and satellite localization of certain proteins, but not others, pinpointing unique roles for PCM1 and a group of satellite proteins in cilium assembly. Remarkably, we find that PCM1 is essential for tethering the E3 ligase, Mindbomb1 (Mib1), to satellites. In the absence of PCM1, Mib1 destabilizes Talpid3 through poly-ubiquitylation and suppresses cilium assembly. Loss of PCM1 blocks ciliogenesis by abrogating recruitment of ciliary vesicles associated with the Talpid3-binding protein, Rab8, which can be reversed by inactivating Mib1. Thus, PCM1 promotes ciliogenesis by tethering a key E3 ligase to satellites and restricting it from centrioles. DOI: http://dx.doi.org/10.7554/eLife.12950.001 PMID:27146717

  16. Characterization of Cep85 – a new antagonist of Nek2A that is involved in the regulation of centrosome disjunction

    PubMed Central

    Chen, Canhe; Tian, Fang; Lu, Lin; Wang, Yun; Xiao, Zhe; Yu, Chengtao; Yu, Xianwen

    2015-01-01

    ABSTRACT Nek2 has been implicated in centrosome disjunction at the onset of mitosis to promote bipolar spindle formation, and hyperactivation of Nek2 leads to the premature centrosome separation. Its activity, therefore, needs to be strictly regulated. In this study, we report that Cep85, an uncharacterized centrosomal protein, acts as a binding partner of Nek2A. It colocalizes with isoform A of Nek2 (Nek2A) at centrosomes and forms a granule meshwork enveloping the proximal ends of centrioles. Opposite to the effects of Nek2A, overexpression of Cep85 in conjunction with inhibition of the motor protein Eg5 (also known as KIF11) leads to the failure of centrosome disjunction. By contrast, depletion of Cep85 results in the precocious centrosome separation. We also define the Nek2A binding and centrosome localization domains within Cep85. Although the Nek2A-binding domain alone is sufficient to inhibit Nek2A kinase activity in vitro, both domains are indispensable for full suppression of centrosome disjunction in cells. Thus, we propose that Cep85 is a bona fide Nek2A-binding partner that surrounds the proximal ends of centrioles where it cooperates with PP1γ (also known as PPP1CC) to antagonize Nek2A activity in order to maintain the centrosome integrity in interphase in mammalian cells. PMID:26220856

  17. The Centrosomal Linker and Microtubules Provide Dual Levels of Spatial Coordination of Centrosomes

    PubMed Central

    Panic, Marko; Hata, Shoji; Neuner, Annett; Schiebel, Elmar

    2015-01-01

    The centrosome is the principal microtubule organizing center in most animal cells. It consists of a pair of centrioles surrounded by pericentriolar material. The centrosome, like DNA, duplicates exactly once per cell cycle. During interphase duplicated centrosomes remain closely linked by a proteinaceous linker. This centrosomal linker is composed of rootletin filaments that are anchored to the centrioles via the protein C-Nap1. At the onset of mitosis the linker is dissolved by Nek2A kinase to support the formation of the bipolar mitotic spindle. The importance of the centrosomal linker for cell function during interphase awaits characterization. Here we assessed the phenotype of human RPE1 C-Nap1 knockout (KO) cells. The absence of the linker led to a modest increase in the average centrosome separation from 1 to 2.5 μm. This small impact on the degree of separation is indicative of a second level of spatial organization of centrosomes. Microtubule depolymerisation or stabilization in C-Nap1 KO cells dramatically increased the inter-centrosomal separation (> 8 μm). Thus, microtubules position centrosomes relatively close to one another in the absence of linker function. C-Nap1 KO cells had a Golgi organization defect with a two-fold expansion of the area occupied by the Golgi. When the centrosomes of C-Nap1 KO cells showed considerable separation, two spatially distinct Golgi stacks could be observed. Furthermore, migration of C-Nap1 KO cells was slower than their wild type RPE1 counterparts. These data show that the spatial organization of centrosomes is modulated by a combination of centrosomal cohesion and microtubule forces. Furthermore a modest increase in centrosome separation has major impact on Golgi organization and cell migration. PMID:26001056

  18. Promotion of well-switching to mitigate the current arsenic crisis in Bangladesh.

    PubMed Central

    Van Geen, Alexander; Ahsan, Habibul; Horneman, Allan H.; Dhar, Ratan K.; Zheng, Yan; Hussain, Iftikhhar; Ahmed, Kazi Matin; Gelman, Andrew; Stute, Martin; Simpson, H. James; Wallace, Sean; Small, Christopher; Parvez, Faruque; Slavkovich, Vesna; Loiacono, Nancy J.; Becker, Marck; Cheng, Zhongqi; Momotaj, Hassina; Shahnewaz, Mohammad; Seddique, Ashraf Ali; Graziano, Joseph H.

    2002-01-01

    OBJECTIVE: To survey tube wells and households in Araihazar upazila, Bangladesh, to set the stage for a long-term epidemiological study of the consequences of chronic arsenic exposure. METHODS: Water samples and household data were collected over a period of 4 months in 2000 from 4997 contiguous tube wells serving a population of 55000, the position of each well being determined to within +/- 30 m using Global Positioning System receivers. Arsenic concentrations were determined by graphite-furnace atomic-absorption spectrometry. In addition, groundwater samples collected every 2 weeks for an entire year from six tube wells were analysed for arsenic by high-resolution inductively coupled plasma-mass spectrometry. FINDINGS: Half of the wells surveyed in Araihazar had been installed in the previous 5 years; 94% were privately owned. Only about 48% of the surveyed wells supplied water with an arsenic content below 50 micro g/l, the current Bangladesh standard for drinking-water. Similar to other regions of Bangladesh and West Bengal, India, the distribution of arsenic in Araihazar is spatially highly variable (range: 5-860 micro g/l) and therefore difficult to predict. Because of this variability, however, close to 90% of the inhabitants live within 100 m of a safe well. Monitoring of six tube wells currently meeting the 50 micro g/l standard showed no indication of a seasonal cycle in arsenic concentrations coupled to the hydrological cycle. This suggests that well-switching is a viable option in Araihazar, at least for the short term. CONCLUSIONS: Well-switching should be more systematically encouraged in Araihazar and many other parts of Bangladesh and West Bengal, India. Social barriers to well-switching need to be better understood and, if possible, overcome. PMID:12378292

  19. Screening of plant growth-promoting traits in arsenic-resistant bacteria isolated from agricultural soil and their potential implication for arsenic bioremediation.

    PubMed

    Das, Suvendu; Jean, Jiin-Shuh; Kar, Sandeep; Chou, Mon-Lin; Chen, Chien-Yen

    2014-05-15

    Twelve arsenic (As)-resistant bacteria (minimum inhibitory concentration ranging from 10 to 30mM and 150 to 320mM for As(III) and As(V), respectively) were isolated from the agricultural soil of the Chianan Plain in southwestern Taiwan using enrichment techniques. Eight isolates capable of oxidizing As(III) (rate of oxidation from 0.029 to 0.059μMh(-1) 10(-9) cell) and exhibiting As(III)-oxidase enzyme activity belong to Pseudomonas, Acinetobacter, Klebsiella and Comamonas genera, whereas four isolates that did not show As(III)-oxidizing activity belong to Geobacillus, Bacillus, Paenibacillus, and Enterobacter genera. Assessment of the parameters of plant growth promotion revealed that Pseudomonas sp. ASR1, ASR2 and ASR3, Geobacillus sp. ASR4, Bacillus sp. ASR5, Paenibacillus sp. ASR6, Enterobacter sp. ASR10 and Comamonas sp. ASR11, and ASR12 possessed some or all of the studied plant growth-promoting traits, including phosphate-solubilization, siderophore, IAA-like molecules and ACC deaminase production. In addition, the ability of As-resistant isolates to grow over wide ranges of pH and temperatures signify their potential application for sustainable bioremediation of As in the environment. PMID:24685527

  20. TRIM28 Is an E3 Ligase for ARF-Mediated NPM1/B23 SUMOylation That Represses Centrosome Amplification

    PubMed Central

    Neo, Shu Hui; Itahana, Yoko; Alagu, Jennifer; Kitagawa, Mayumi; Guo, Alvin Kunyao; Lee, Sang Hyun; Tang, Kai

    2015-01-01

    The tumor suppressor ARF enhances the SUMOylation of target proteins; however, the physiological function of ARF-mediated SUMOylation has been unclear due to the lack of a known, associated E3 SUMO ligase. Here we uncover TRIM28/KAP1 as a novel ARF-binding protein and SUMO E3 ligase for NPM1/B23. ARF and TRIM28 cooperate to SUMOylate NPM1, a nucleolar protein that regulates centrosome duplication and genomic stability. ARF-mediated SUMOylation of NPM1 was attenuated by TRIM28 depletion and enhanced by TRIM28 overexpression. Coexpression of ARF and TRIM28 promoted NPM1 centrosomal localization by enhancing its SUMOylation and suppressed centrosome amplification; these functions required the E3 ligase activity of TRIM28. Conversely, depletion of ARF or TRIM28 increased centrosome amplification. ARF also counteracted oncogenic Ras-induced centrosome amplification. Centrosome amplification is often induced by oncogenic insults, leading to genomic instability. However, the mechanisms employed by tumor suppressors to protect the genome are poorly understood. Our findings suggest a novel role for ARF in maintaining genome integrity by facilitating TRIM28-mediated SUMOylation of NPM1, thus preventing centrosome amplification. PMID:26055329

  1. Centrosome positioning in non-dividing cells.

    PubMed

    Barker, Amy R; McIntosh, Kate V; Dawe, Helen R

    2016-07-01

    Centrioles and centrosomes are found in almost all eukaryotic cells, where they are important for organising the microtubule cytoskeleton in both dividing and non-dividing cells. The spatial location of centrioles and centrosomes is tightly controlled and, in non-dividing cells, plays an important part in cell migration, ciliogenesis and immune cell functions. Here, we examine some of the ways that centrosomes are connected to other organelles and how this impacts on cilium formation, cell migration and immune cell function in metazoan cells. PMID:26319517

  2. Centrosome movement in the early divisions of Caenorhabditis elegans: A cortical site determining centrosome position

    SciTech Connect

    Hyman, A.A. )

    1989-09-01

    In Caenorhabditis elegans embryos, early blastomeres of the P cell lineage divide successively on the same axis. This axis is a consequence of the specific rotational movement of the pair of centrosomes and nucleus. A laser has been used to perturb the centrosome movements that determine the pattern of early embryonic divisions. The results support a previously proposed model in which a centrosome rotates towards its correct position by shortening of connections, possibly microtubules, between a centrosome and a defined site on the cortex of the embryo.

  3. CentrosomeDB: a new generation of the centrosomal proteins database for Human and Drosophila melanogaster

    PubMed Central

    Alves-Cruzeiro, Joao Miguel da Conceiçao; Nogales-Cadenas, Rubén; Pascual-Montano, Alberto Domingo

    2014-01-01

    We present the second generation of centrosomeDB, available online at http://centrosome.cnb.csic.es, with a significant expansion of 1357 human and drosophila centrosomal genes and their corresponding information. The centrosome of animal cells takes part in important biological processes such as the organization of the interphase microtubule cytoskeleton and the assembly of the mitotic spindle. The active research done during the past decades has produced lots of data related to centrosomal proteins. Unfortunately, the accumulated data are dispersed among diverse and heterogeneous sources of information. We believe that the availability of a repository collecting curated evidences of centrosomal proteins would constitute a key resource for the scientific community. This was our first motivation to introduce CentrosomeDB in NAR database issue in 2009, collecting a set of human centrosomal proteins that were reported in the literature and other sources. The intensive use of this resource during these years has encouraged us to present this new expanded version. Using our database, the researcher is offered the possibility to study the evolution, function and structure of the centrosome. We have compiled information from many sources, including Gene Ontology, disease-association, single nucleotide polymorphisms and associated gene expression experiments. Special interest has been paid to protein–protein interaction. PMID:24270791

  4. Nucleoporin Nup62 maintains centrosome homeostasis

    PubMed Central

    Hashizume, Chieko; Moyori, Akane; Kobayashi, Akiko; Yamakoshi, Nana; Endo, Aoi; Wong, Richard W

    2013-01-01

    Centrosomes are comprised of 2 orthogonally arranged centrioles surrounded by the pericentriolar material (PCM), which serves as the main microtubule organizing center of the animal cell. More importantly, centrosomes also control spindle polarity and orientation during mitosis. Recently, we and other investigators discovered that several nucleoporins play critical roles during cell division. Here, we show that nucleoporin Nup62 plays a novel role in centrosome integrity. Knockdown of Nup62 induced mitotic arrest in G2/M phases and mitotic cell death. Depletion of Nup62 using RNA interference results in defective centrosome segregation and centriole maturation during the G2 phase. Moreover, Nup62 depletion in human cells leads to the appearance of multinucleated cells and induces the formation of multipolar centrosomes, centriole synthesis defects, dramatic spindle orientation defects, and centrosome component rearrangements that impair cell bi-polarity. Our results also point to a potential role of Nup62 in targeting gamma-tubulin and SAS-6 to the centrioles. PMID:24107630

  5. Where are the limits of the centrosome?

    PubMed

    Alieva, Irina B; Uzbekov, Rustem E

    2016-05-01

    The centrosome is a key component of the cell is involved in the processes of cell division, cell motility, intracellular transport, organization of the microtubules (MT) network and the production of cilia and flagella. The peculiarity of this organelle is that its boundaries are not clearly defined, the centrioles at the center of the centrosome are surrounded by electron dense pericentriolar material, the size and protein composition of this centrosome component experiences significant transformation during the cell cycle. It has been shown in this study that within the centrosome different proteins occupy different areas corresponding to: MT nucleation region (defined as gamma-tubulin-stained area), regulatory region (defined as kinase pEg2-stained area) and motor proteins region (kinesin-like motor XlEg5-stained area). The boundary of pEg2 is near 1.3 times greater while XlEg5 is 3.0 times greater than that of gamma-tubulin. Thus, the size of the centrosome, determined according to the structural electron microscopy (EM) analysis (about 1 µm) corresponds to the regulatory proteins area, but the actual functional centrosome size defined at the motor proteins region, is more than twice the size. PMID:27093502

  6. Centrin 3 is an inhibitor of centrosomal Mps1 and antagonizes centrin 2 function

    PubMed Central

    Sawant, Dwitiya B.; Majumder, Shubhra; Perkins, Jennifer L.; Yang, Ching-Hui; Eyers, Patrick A.; Fisk, Harold A.

    2015-01-01

    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. PMID:26354417

  7. Pten regulates spindle pole movement through Dlg1-mediated recruitment of Eg5 to centrosomes

    PubMed Central

    van Ree, Janine H.; Nam, Hyun-Ja; Jeganathan, Karthik B.; Kanakkanthara, Arun; van Deursen, Jan M.

    2016-01-01

    Phosphatase and tensin homologue (Pten) suppresses neoplastic growth by negatively regulating PI(3)K signalling through its phosphatase activity1. To gain insight into the actions of non-catalytic Pten domains in normal physiological processes and tumorigenesis2,3, we engineered mice lacking the PDZ-binding domain (PDZ-BD). Here, we show that the PDZ-BD regulates centrosome movement and that its heterozygous or homozygous deletion promotes aneuploidy and tumour formation. We found that Pten is recruited to pre-mitotic centrosomes in a Plk1-dependent fashion to create a docking site for protein complexes containing the PDZ-domain-containing protein Dlg1 (also known as Sap97) and Eg5 (also known as Kif11), a kinesin essential for centrosome movement and bipolar spindle formation4. Docking of Dlg1–Eg5 complexes to Pten depended on Eg5 phosphorylation by the Nek9–Nek6 mitotic kinase cascade and Cdk1. PDZ-BD deletion or Dlg1 ablation impaired loading of Eg5 onto centrosomes and spindle pole motility, yielding asymmetrical spindles that are prone to chromosome missegregation. Collectively, these data demonstrate that Pten, through the Dlg1-binding ability of its PDZ-BD, accumulates phosphorylated Eg5 at duplicated centrosomes to establish symmetrical bipolar spindles that properly segregate chromosomes, and suggest that this function contributes to tumour suppression. PMID:27240320

  8. Developmental alterations in centrosome integrity contribute to the post-mitotic state of mammalian cardiomyocytes

    PubMed Central

    Zebrowski, David C; Vergarajauregui, Silvia; Wu, Chi-Chung; Piatkowski, Tanja; Becker, Robert; Leone, Marina; Hirth, Sofia; Ricciardi, Filomena; Falk, Nathalie; Giessl, Andreas; Just, Steffen; Braun, Thomas; Weidinger, Gilbert; Engel, Felix B

    2015-01-01

    Mammalian cardiomyocytes become post-mitotic shortly after birth. Understanding how this occurs is highly relevant to cardiac regenerative therapy. Yet, how cardiomyocytes achieve and maintain a post-mitotic state is unknown. Here, we show that cardiomyocyte centrosome integrity is lost shortly after birth. This is coupled with relocalization of various centrosome proteins to the nuclear envelope. Consequently, postnatal cardiomyocytes are unable to undergo ciliogenesis and the nuclear envelope adopts the function as cellular microtubule organizing center. Loss of centrosome integrity is associated with, and can promote, cardiomyocyte G0/G1 cell cycle arrest suggesting that centrosome disassembly is developmentally utilized to achieve the post-mitotic state in mammalian cardiomyocytes. Adult cardiomyocytes of zebrafish and newt, which are able to proliferate, maintain centrosome integrity. Collectively, our data provide a novel mechanism underlying the post-mitotic state of mammalian cardiomyocytes as well as a potential explanation for why zebrafish and newts, but not mammals, can regenerate their heart. DOI: http://dx.doi.org/10.7554/eLife.05563.001 PMID:26247711

  9. Physical association between a novel plasma-membrane structure and centrosome orients cell division.

    PubMed

    Negishi, Takefumi; Miyazaki, Naoyuki; Murata, Kazuyoshi; Yasuo, Hitoyoshi; Ueno, Naoto

    2016-01-01

    In the last mitotic division of the epidermal lineage in the ascidian embryo, the cells divide stereotypically along the anterior-posterior axis. During interphase, we found that a unique membrane structure invaginates from the posterior to the centre of the cell, in a microtubule-dependent manner. The invagination projects toward centrioles on the apical side of the nucleus and associates with one of them. Further, a cilium forms on the posterior side of the cell and its basal body remains associated with the invagination. A laser ablation experiment suggests that the invagination is under tensile force and promotes the posterior positioning of the centrosome. Finally, we showed that the orientation of the invaginations is coupled with the polarized dynamics of centrosome movements and the orientation of cell division. Based on these findings, we propose a model whereby this novel membrane structure orchestrates centrosome positioning and thus the orientation of cell division axis. PMID:27502556

  10. Activation of maternal centrosomes in unfertilized sea urchin eggs

    NASA Technical Reports Server (NTRS)

    Schatten, H.; Walter, M.; Biessmann, H.; Schatten, G.

    1992-01-01

    Centrosomes are undetectable in unfertilized sea urchin eggs, and normally the sperm introduces the cell's microtubule-organizing center (MTOC) at fertilization. However, artificial activation or parthenogenesis triggers microtubule assembly in the unfertilized egg, and this study explores the reappearance and behavior of the maternal centrosome. During activation with A23187 or ammonia, microtubules appear first at the cortex; centrosomal antigen is detected diffusely throughout the entire cytoplasm. Later, the centrosome becomes more distinct and organizes a radial microtubule shell, and eventually a compact centrosome at the egg center organizes a monaster. In these activated eggs, centrosomes undergo cycles of compaction and decompaction in synchrony with the chromatin, which also undergoes cycles of condensation and decondensation. Parthenogenetic activation with heavy water (50% D2O) or the microtubule-stabilizing drug taxol (10 microM) induces numerous centrosomal foci in the unfertilized sea urchin egg. Within 15 min after incubation in D2O, numerous fine centrosomal foci are detected, and they organize a connected network of numerous asters which fill the entire egg. Taxol induces over 100 centrosomal foci by 15 min after treatment, which organize a corresponding number of asters. The centrosomal material in either D2O- or taxol-treated eggs aggregates with time to form fewer but denser foci, resulting in fewer and larger asters. Fertilization of eggs pretreated with either D2O or taxol shows that the paternal centrosome is dominant over the maternal centrosome. The centrosomal material gradually becomes associated with the enlarged sperm aster. These experiments demonstrate that maternal centrosomal material is present in the unfertilized egg, likely as dispersed undetectable material, which can be activated without paternal contributions. At fertilization, paternal centrosomes become dominant over the maternal centrosomal material.

  11. The Seckel syndrome and centrosomal protein Ninein localizes asymmetrically to stem cell centrosomes but is not required for normal development, behavior, or DNA damage response in Drosophila.

    PubMed

    Zheng, Yiming; Mennella, Vito; Marks, Steven; Wildonger, Jill; Elnagdi, Esraa; Agard, David; Megraw, Timothy L

    2016-06-01

    Ninein (Nin) is a centrosomal protein whose gene is mutated in Seckel syndrome (SCKL, MIM 210600), an inherited recessive disease that results in primordial dwarfism, cognitive deficiencies, and increased sensitivity to genotoxic stress. Nin regulates neural stem cell self-renewal, interkinetic nuclear migration, and microtubule assembly in mammals. Nin is evolutionarily conserved, yet its role in cell division and development has not been investigated in a model organism. Here we characterize the single Nin orthologue in Drosophila Drosophila Nin localizes to the periphery of the centrosome but not at centriolar structures as in mammals. However, Nin shares the property of its mammalian orthologue of promoting microtubule assembly. In neural and germline stem cells, Nin localizes asymmetrically to the younger (daughter) centrosome, yet it is not required for the asymmetric division of stem cells. In wing epithelia and muscle, Nin localizes to noncentrosomal microtubule-organizing centers. Surprisingly, loss of nin expression from a nin mutant does not significantly affect embryonic and brain development, fertility, or locomotor performance of mutant flies or their survival upon exposure to DNA-damaging agents. Although it is not essential, our data suggest that Nin plays a supportive role in centrosomal and extracentrosomal microtubule organization and asymmetric stem cell division. PMID:27053665

  12. The Seckel syndrome and centrosomal protein Ninein localizes asymmetrically to stem cell centrosomes but is not required for normal development, behavior, or DNA damage response in Drosophila

    PubMed Central

    Zheng, Yiming; Mennella, Vito; Marks, Steven; Wildonger, Jill; Elnagdi, Esraa; Agard, David; Megraw, Timothy L.

    2016-01-01

    Ninein (Nin) is a centrosomal protein whose gene is mutated in Seckel syndrome (SCKL, MIM 210600), an inherited recessive disease that results in primordial dwarfism, cognitive deficiencies, and increased sensitivity to genotoxic stress. Nin regulates neural stem cell self-renewal, interkinetic nuclear migration, and microtubule assembly in mammals. Nin is evolutionarily conserved, yet its role in cell division and development has not been investigated in a model organism. Here we characterize the single Nin orthologue in Drosophila. Drosophila Nin localizes to the periphery of the centrosome but not at centriolar structures as in mammals. However, Nin shares the property of its mammalian orthologue of promoting microtubule assembly. In neural and germline stem cells, Nin localizes asymmetrically to the younger (daughter) centrosome, yet it is not required for the asymmetric division of stem cells. In wing epithelia and muscle, Nin localizes to noncentrosomal microtubule-organizing centers. Surprisingly, loss of nin expression from a nin mutant does not significantly affect embryonic and brain development, fertility, or locomotor performance of mutant flies or their survival upon exposure to DNA-damaging agents. Although it is not essential, our data suggest that Nin plays a supportive role in centrosomal and extracentrosomal microtubule organization and asymmetric stem cell division. PMID:27053665

  13. Localization of MLH3 at the Centrosomes

    PubMed Central

    Roesner, Lennart M.; Mielke, Christian; Faehnrich, Silke; Merkhoffer, Yvonne; Dittmar, Kurt E. J.; Drexler, Hans G.; Dirks, Wilhelm G.

    2014-01-01

    Mutations in human DNA mismatch repair (MMR) genes are commonly associated with hereditary nonpolyposis colorectal cancer (HNPCC). MLH1 protein heterodimerizes with PMS2, PMS1, and MLH3 to form MutLα, MutLβ, and MutLγ, respectively. We reported recently stable expression of GFP-linked MLH3 in human cell lines. Monitoring these cell lines during the cell cycle using live cell imaging combined with confocal microscopy, we detected accumulation of MLH3 at the centrosomes. Fluorescence recovery after photobleaching (FRAP) revealed high mobility and fast exchange rates at the centrosomes as it has been reported for other DNA repair proteins. MLH3 may have a role in combination with other repair proteins in the control of centrosome numbers. PMID:25116689

  14. Actin nucleation at the centrosome controls lymphocyte polarity.

    PubMed

    Obino, Dorian; Farina, Francesca; Malbec, Odile; Sáez, Pablo J; Maurin, Mathieu; Gaillard, Jérémie; Dingli, Florent; Loew, Damarys; Gautreau, Alexis; Yuseff, Maria-Isabel; Blanchoin, Laurent; Théry, Manuel; Lennon-Duménil, Ana-Maria

    2016-01-01

    Cell polarity is required for the functional specialization of many cell types including lymphocytes. A hallmark of cell polarity is the reorientation of the centrosome that allows repositioning of organelles and vesicles in an asymmetric fashion. The mechanisms underlying centrosome polarization are not fully understood. Here we found that in resting lymphocytes, centrosome-associated Arp2/3 locally nucleates F-actin, which is needed for centrosome tethering to the nucleus via the LINC complex. Upon lymphocyte activation, Arp2/3 is partially depleted from the centrosome as a result of its recruitment to the immune synapse. This leads to a reduction in F-actin nucleation at the centrosome and thereby allows its detachment from the nucleus and polarization to the synapse. Therefore, F-actin nucleation at the centrosome-regulated by the availability of the Arp2/3 complex-determines its capacity to polarize in response to external stimuli. PMID:26987298

  15. Arsenic trioxide disrupts glioma stem cells via promoting PML degradation to inhibit tumor growth

    PubMed Central

    Zhou, Wenchao; Cheng, Lin; Shi, Yu; Ke, Susan Q.; Huang, Zhi; Fang, Xiaoguang; Chu, Cheng-wei; Xie, Qi; Bian, Xiu-wu; Rich, Jeremy N.; Bao, Shideng

    2015-01-01

    Glioblastoma multiforme (GBM) is the most lethal brain tumor. Tumor relapse in GBM is inevitable despite maximal therapeutic interventions. Glioma stem cells (GSCs) have been found to be critical players in therapeutic resistance and tumor recurrence. Therapeutic drugs targeting GSCs may significantly improve GBM treatment. In this study, we demonstrated that arsenic trioxide (As2O3) effectively disrupted GSCs and inhibited tumor growth in the GSC-derived orthotopic xenografts by targeting the promyelocytic leukaemia (PML). As2O3 treatment induced rapid degradation of PML protein along with severe apoptosis in GSCs. Disruption of the endogenous PML recapitulated the inhibitory effects of As2O3 treatment on GSCs both in vitro and in orthotopic tumors. Importantly, As2O3 treatment dramatically reduced GSC population in the intracranial GBM xenografts and increased the survival of mice bearing the tumors. In addition, As2O3 treatment preferentially inhibited cell growth of GSCs but not matched non-stem tumor cells (NSTCs). Furthermore, As2O3 treatment or PML disruption potently diminished c-Myc protein levels through increased poly-ubiquitination and proteasome degradation of c-Myc. Our study indicated a potential implication of As2O3 in GBM treatment and highlighted the important role of PML/c-Myc axis in the maintenance of GSCs. PMID:26510911

  16. Cep70 overexpression stimulates pancreatic cancer by inducing centrosome abnormality and microtubule disorganization

    PubMed Central

    Xie, Songbo; Qin, Juan; Liu, Shiyu; Zhang, Yijun; Wang, Jun; Shi, Xingjuan; Li, Dengwen; Zhou, Jun; Liu, Min

    2016-01-01

    The centrosome is crucial for mitotic fidelity, and centrosome aberrations are associated with genomic instability and tumorigenesis. The centrosomal protein Cep70 has been reported to play a role in various cellular activities. However, whether this protein is involved in pathological processes remains unknown. In this study, we demonstrate that Cep70 is highly expressed in pancreatic cancer tissues. Cep70 expression correlates with clinicopathological parameters of pancreatic cancer, including histological grade, pathological tumor node metastasis stage, lymph node metastasis, and carbohydrate antigen 19-9 level. Depletion of Cep70 significantly suppresses pancreatic cancer cell proliferation and promotes apoptotic cell death, and exogenous expression of Cep70 can rescue the above effects. Cep70 also stimulates colony formation in soft agar and enhances tumor growth in mice. Our data further show that ectopic expression of Cep70 in pancreatic cancer cells results in the mislocalization of centrosomal proteins, including γ-tubulin and pericentrin, and the formation of intracellular aggregates. In addition, Cep70 overexpression leads to microtubule disorganization and the formation of multipolar spindles during mitosis. Our study thus unravels a critical role for Cep70 in pancreatic cancer and suggests Cep70 as a potential biomarker and therapeutic target for this deadly disease. PMID:26893288

  17. Rampant centrosome amplification underlies more aggressive disease course of triple negative breast cancers

    PubMed Central

    Pannu, Vaishali; Mittal, Karuna; Cantuaria, Guilherme; Reid, Michelle D.; Li, Xiaoxian; Donthamsetty, Shashikiran; McBride, Michelle; Klimov, Sergey; Osan, Remus; Gupta, Meenakshi V.; Rida, Padmashree C.G.; Aneja, Ritu

    2015-01-01

    Centrosome amplification (CA), a cell-biological trait, characterizes pre-neoplastic and pre-invasive lesions and is associated with tumor aggressiveness. Recent studies suggest that CA leads to malignant transformation and promotes invasion in mammary epithelial cells. Triple negative breast cancer (TNBC), a histologically-aggressive subtype shows high recurrence, metastases, and mortality rates. Since TNBC and non-TNBC follow variable kinetics of metastatic progression, they constitute a novel test bed to explore if severity and nature of CA can distinguish them apart. We quantitatively assessed structural and numerical centrosomal aberrations for each patient sample in a large-cohort of grade-matched TNBC (n = 30) and non-TNBC (n = 98) cases employing multi-color confocal imaging. Our data establish differences in incidence and severity of CA between TNBC and non-TNBC cell lines and clinical specimens. We found strong correlation between CA and aggressiveness markers associated with metastasis in 20 pairs of grade-matched TNBC and non-TNBC specimens (p < 0.02). Time-lapse imaging of MDA-MB-231 cells harboring amplified centrosomes demonstrated enhanced migratory ability. Our study bridges a vital knowledge gap by pinpointing that CA underlies breast cancer aggressiveness. This previously unrecognized organellar inequality at the centrosome level may allow early-risk prediction and explain higher tumor aggressiveness and mortality rates in TNBC patients. PMID:25868856

  18. CDK5RAP2 functions in centrosome to spindle pole attachment and DNA damage response.

    PubMed

    Barr, Alexis R; Kilmartin, John V; Gergely, Fanni

    2010-04-01

    The centrosomal protein, CDK5RAP2, is mutated in primary microcephaly, a neurodevelopmental disorder characterized by reduced brain size. The Drosophila melanogaster homologue of CDK5RAP2, centrosomin (Cnn), maintains the pericentriolar matrix (PCM) around centrioles during mitosis. In this study, we demonstrate a similar role for CDK5RAP2 in vertebrate cells. By disrupting two evolutionarily conserved domains of CDK5RAP2, CNN1 and CNN2, in the avian B cell line DT40, we find that both domains are essential for linking centrosomes to mitotic spindle poles. Although structurally intact, centrosomes lacking the CNN1 domain fail to recruit specific PCM components that mediate attachment to spindle poles. Furthermore, we show that the CNN1 domain enforces cohesion between parental centrioles during interphase and promotes efficient DNA damage-induced G2 cell cycle arrest. Because mitotic spindle positioning, asymmetric centrosome inheritance, and DNA damage signaling have all been implicated in cell fate determination during neurogenesis, our findings provide novel insight into how impaired CDK5RAP2 function could cause premature depletion of neural stem cells and thereby microcephaly. PMID:20368616

  19. Effect of arsenic on tolerance mechanisms of two plant growth-promoting bacteria used as biological inoculants.

    PubMed

    Armendariz, Ana L; Talano, Melina A; Wevar Oller, Ana L; Medina, María I; Agostini, Elizabeth

    2015-07-01

    Bacterial ability to colonize the rhizosphere of plants in arsenic (As) contaminated soils is highly important for symbiotic and free-living plant growth-promoting rhizobacteria (PGPR) used as inoculants, since they can contribute to enhance plant As tolerance and limit metalloid uptake by plants. The aim of this work was to study the effect of As on growth, exopolysaccharide (EPS) production, biofilm formation and motility of two strains used as soybean inoculants, Bradyrhizobium japonicum E109 and Azospirillum brasilense Az39. The metabolism of arsenate (As(V)) and arsenite (As(III)) and their removal and/or possible accumulation were also evaluated. The behavior of both bacteria under As treatment was compared and discussed in relation to their potential for colonizing plant rhizosphere with high content of the metalloid. B. japonicum E109 growth was reduced with As(III) concentration from 10 μM while A. brasilense Az39 showed a reduction of growth with As(III) from 500 μM. EPS and biofilm production increased significantly under 25 μM As(III) for both strains. Moreover, this was more notorious for Azospirillum under 500 μM As(III), where motility was seriously affected. Both bacterial strains showed a similar ability to reduce As(V). However, Azospirillum was able to oxidize more As(III) (around 53%) than Bradyrhizobium (17%). In addition, both strains accumulated As in cell biomass. The behavior of Azospirillum under As treatments suggests that this strain would be able to colonize efficiently As contaminated soils. In this way, inoculation with A. brasilense Az39 would positively contribute to promoting growth of different plant species under As treatment. PMID:26141894

  20. Arsenic-induced promoter hypomethylation and over-expression of ERCC2 reduces DNA repair capacity in humans by non-disjunction of the ERCC2-Cdk7 complex.

    PubMed

    Paul, Somnath; Banerjee, Nilanjana; Chatterjee, Aditi; Sau, Tanmoy J; Das, Jayanta K; Mishra, Prafulla K; Chakrabarti, Partha; Bandyopadhyay, Arun; Giri, Ashok K

    2014-04-01

    Arsenic in drinking water is of critical concern in West Bengal, India, as it results in several physiological symptoms including dermatological lesions and cancers. Impairment of the DNA repair mechanism has been associated with arsenic-induced genetic damage as well as with several cancers. ERCC2 (Excision Repair Cross-Complementing rodent repair, complementation group 2), mediates DNA-repair by interacting with Cdk-activating kinase (CAK) complex, which helps in DNA proof-reading during transcription. Arsenic metabolism alters epigenetic regulation; we tried to elucidate the regulation of ERCC2 in arsenic-exposed humans. Water, urine, nails, hair and blood samples from one hundred and fifty seven exposed and eighty eight unexposed individuals were collected. Dose dependent validation was done in vitro using HepG2 and HEK-293. Arsenic content in the biological samples was higher in the exposed individuals compared with the content in unexposed individuals (p < 0.001). Bisulfite-modified methylation specific PCR showed a significant (p < 0.0001) hypomethylation of the ERCC2 promoter in the arsenic-exposed individuals. Densitometric analysis of immunoblots showed a nearly two-fold increase in expression of ERCC2 in exposed individuals, but there was an enhanced genotoxic insult as measured by micronuclei frequency. Immuno-precipitation and western blotting revealed an increased (p < 0.001) association of Cdk7 with ERCC2 in highly arsenic exposed individuals. The decrease in CAK activity was determined by observing the intensity of Ser(392) phosphorylation in p53, in vitro, which decreased with an increase in arsenic dose. Thus we infer that arsenic biotransformation leads to promoter hypomethylation of ERCC2, which in turn inhibits the normal functioning of the CAK-complex, thus affecting DNA-repair; this effect was highest among the arsenic exposed individuals with dermatological lesions. PMID:24473091

  1. Actin nucleation at the centrosome controls lymphocyte polarity

    PubMed Central

    Obino, Dorian; Farina, Francesca; Malbec, Odile; Sáez, Pablo J.; Maurin, Mathieu; Gaillard, Jérémie; Dingli, Florent; Loew, Damarys; Gautreau, Alexis; Yuseff, Maria-Isabel; Blanchoin, Laurent; Théry, Manuel; Lennon-Duménil, Ana-Maria

    2016-01-01

    Cell polarity is required for the functional specialization of many cell types including lymphocytes. A hallmark of cell polarity is the reorientation of the centrosome that allows repositioning of organelles and vesicles in an asymmetric fashion. The mechanisms underlying centrosome polarization are not fully understood. Here we found that in resting lymphocytes, centrosome-associated Arp2/3 locally nucleates F-actin, which is needed for centrosome tethering to the nucleus via the LINC complex. Upon lymphocyte activation, Arp2/3 is partially depleted from the centrosome as a result of its recruitment to the immune synapse. This leads to a reduction in F-actin nucleation at the centrosome and thereby allows its detachment from the nucleus and polarization to the synapse. Therefore, F-actin nucleation at the centrosome—regulated by the availability of the Arp2/3 complex—determines its capacity to polarize in response to external stimuli. PMID:26987298

  2. Centrosome/Cell Cycle Uncoupling and Elimination in the Endoreduplicating Intestinal Cells of C. elegans

    PubMed Central

    Lu, Yu; Roy, Richard

    2014-01-01

    The centrosome cycle is most often coordinated with mitotic cell division through the activity of various essential cell cycle regulators, consequently ensuring that the centriole is duplicated once, and only once, per cell cycle. However, this coupling can be altered in specific developmental contexts; for example, multi-ciliated cells generate hundreds of centrioles without any S-phase requirement for their biogenesis, while Drosophila follicle cells eliminate their centrosomes as they begin to endoreduplicate. In order to better understand how the centrosome cycle and the cell cycle are coordinated in a developmental context we use the endoreduplicating intestinal cell lineage of C. elegans to address how novel variations of the cell cycle impact this important process. In C. elegans, the larval intestinal cells undergo one nuclear division without subsequent cytokinesis, followed by four endocycles that are characterized by successive rounds of S-phase. We monitored the levels of centriolar/centrosomal markers and found that centrosomes lose their pericentriolar material following the nuclear division that occurs during the L1 stage and is thereafter never re-gained. The centrioles then become refractory to S phase regulators that would normally promote duplication during the first endocycle, after which they are eliminated during the L2 stage. Furthermore, we show that SPD-2 plays a central role in the numeral regulation of centrioles as a potential target of CDK activity. On the other hand, the phosphorylation on SPD-2 by Polo-like kinase, the transcriptional regulation of genes that affect centriole biogenesis, and the ubiquitin/proteasome degradation pathway, contribute collectively to the final elimination of the centrioles during the L2 stage. PMID:25360893

  3. Promotion of arsenic phytoextraction efficiency in the fern Pteris vittata by the inoculation of As-resistant bacteria: a soil bioremediation perspective

    PubMed Central

    Lampis, Silvia; Santi, Chiara; Ciurli, Adriana; Andreolli, Marco; Vallini, Giovanni

    2015-01-01

    A greenhouse pot experiment was carried out to evaluate the efficiency of arsenic phytoextraction by the fern Pteris vittata growing in arsenic-contaminated soil, with or without the addition of selected rhizobacteria isolated from the polluted site. The bacterial strains were selected for arsenic resistance, the ability to reduce arsenate to arsenite, and the ability to promote plant growth. P. vittata plants were cultivated for 4 months in a contaminated substrate consisting of arsenopyrite cinders and mature compost. Four different experimental conditions were tested: (i) non-inoculated plants; (ii) plants inoculated with the siderophore-producing and arsenate-reducing bacteria Pseudomonas sp. P1III2 and Delftia sp. P2III5 (A); (iii) plants inoculated with the siderophore and indoleacetic acid-producing bacteria Bacillus sp. MPV12, Variovorax sp. P4III4, and Pseudoxanthomonas sp. P4V6 (B), and (iv) plants inoculated with all five bacterial strains (AB). The presence of growth-promoting rhizobacteria increased plant biomass by up to 45% and increased As removal efficiency from 13% without bacteria to 35% in the presence of the mixed inoculum. Molecular analysis confirmed the persistence of the introduced bacterial strains in the soil and resulted in a significant impact on the structure of the bacterial community. PMID:25741356

  4. Promotion of arsenic phytoextraction efficiency in the fern Pteris vittata by the inoculation of As-resistant bacteria: a soil bioremediation perspective.

    PubMed

    Lampis, Silvia; Santi, Chiara; Ciurli, Adriana; Andreolli, Marco; Vallini, Giovanni

    2015-01-01

    A greenhouse pot experiment was carried out to evaluate the efficiency of arsenic phytoextraction by the fern Pteris vittata growing in arsenic-contaminated soil, with or without the addition of selected rhizobacteria isolated from the polluted site. The bacterial strains were selected for arsenic resistance, the ability to reduce arsenate to arsenite, and the ability to promote plant growth. P. vittata plants were cultivated for 4 months in a contaminated substrate consisting of arsenopyrite cinders and mature compost. Four different experimental conditions were tested: (i) non-inoculated plants; (ii) plants inoculated with the siderophore-producing and arsenate-reducing bacteria Pseudomonas sp. P1III2 and Delftia sp. P2III5 (A); (iii) plants inoculated with the siderophore and indoleacetic acid-producing bacteria Bacillus sp. MPV12, Variovorax sp. P4III4, and Pseudoxanthomonas sp. P4V6 (B), and (iv) plants inoculated with all five bacterial strains (AB). The presence of growth-promoting rhizobacteria increased plant biomass by up to 45% and increased As removal efficiency from 13% without bacteria to 35% in the presence of the mixed inoculum. Molecular analysis confirmed the persistence of the introduced bacterial strains in the soil and resulted in a significant impact on the structure of the bacterial community. PMID:25741356

  5. GABARAP activates ULK1 and traffics from the centrosome dependent on Golgi partners WAC and GOLGA2/GM130

    PubMed Central

    Joachim, Justin; Tooze, Sharon A.

    2016-01-01

    ABSTRACT WAC and GOLGA2/GM130 are 2 Golgi proteins that affect autophagy; however, their mechanism of action was unknown. We have shown that WAC binding to GOLGA2 at the Golgi displaces GABARAP from GOLGA2 to allow the maintenance of a nonlipidated centrosomal GABARAP pool. Centrosomal GABARAP can traffic to autophagic structures during starvation. In addition GABARAP specifically promotes ULK1 activation and this is independent of GABARAP lipidation but likely requires a LIR-mediated GABARAP-ULK1 interaction. PMID:26986052

  6. GT-repeat polymorphism in the heme oxygenase-1 gene promoter is associated with cardiovascular mortality risk in an arsenic-exposed population in northeastern Taiwan

    SciTech Connect

    Wu, Meei-Maan; Chiou, Hung-Yi; Chen, Chi-Ling; Wang, Yuan-Hung; Hsieh, Yi-Chen; Lien, Li-Ming; Lee, Te-Chang; Chen, Chien-Jen

    2010-11-01

    Inorganic arsenic has been associated with increased risk of atherosclerotic vascular disease and mortality in humans. A functional GT-repeat polymorphism in the heme oxygenase-1 (HO-1) gene promoter is inversely correlated with the development of coronary artery disease and restenosis after clinical angioplasty. The relationship of HO-1 genotype with arsenic-associated cardiovascular disease has not been studied. In this study, we evaluated the relationship between the HO-1 GT-repeat polymorphism and cardiovascular mortality in an arsenic-exposed population. A total of 504 study participants were followed up for a median of 10.7 years for occurrence of cardiovascular deaths (coronary heart disease, cerebrovascular disease, and peripheral arterial disease). Cardiovascular risk factors and DNA samples for determination of HO-1 GT repeats were obtained at recruitment. GT repeats variants were grouped into the S (< 27 repeats) or L allele ({>=} 27 repeats). Relative mortality risk was estimated using Cox regression analysis, adjusted for competing risk of cancer and other causes. For the L/L, L/S, and S/S genotype groups, the crude mortalities for cardiovascular disease were 8.42, 3.10, and 2.85 cases/1000 person-years, respectively. After adjusting for conventional cardiovascular risk factors and competing risk of cancer and other causes, carriers with class S allele (L/S or S/S genotypes) had a significantly reduced risk of cardiovascular mortality compared to non-carriers (L/L genotype) [OR, 0.38; 95% CI, 0.16-0.90]. In contrast, no significant association was observed between HO-1 genotype and cancer mortality or mortality from other causes. Shorter (GT)n repeats in the HO-1 gene promoter may confer protective effects against cardiovascular mortality related to arsenic exposure.

  7. Centrosomes are autocatalytic droplets of pericentriolar material organized by centrioles

    PubMed Central

    Zwicker, David; Decker, Markus; Jaensch, Steffen; Hyman, Anthony A.; Jülicher, Frank

    2014-01-01

    Centrosomes are highly dynamic, spherical organelles without a membrane. Their physical nature and their assembly are not understood. Using the concept of phase separation, we propose a theoretical description of centrosomes as liquid droplets. In our model, centrosome material occurs in a form soluble in the cytosol and a form that tends to undergo phase separation from the cytosol. We show that an autocatalytic chemical transition between these forms accounts for the temporal evolution observed in experiments. Interestingly, the nucleation of centrosomes can be controlled by an enzymatic activity of the centrioles, which are present at the core of all centrosomes. This nonequilibrium feature also allows for multiple stable centrosomes, a situation that is unstable in equilibrium phase separation. Our theory explains the growth dynamics of centrosomes for all cell sizes down to the eight-cell stage of the Caenorhabditis elegans embryo, and it also accounts for data acquired in experiments with aberrant numbers of centrosomes and altered cell volumes. Furthermore, the model can describe unequal centrosome sizes observed in cells with perturbed centrioles. We also propose an interpretation of the molecular details of the involved proteins in the case of C. elegans. Our example suggests a general picture of the organization of membraneless organelles. PMID:24979791

  8. A fraction of Crm1 locates at centrosomes by its CRIME domain and regulates the centrosomal localization of pericentrin

    SciTech Connect

    Liu, Qinying; Jiang, Qing; Zhang, Chuanmao

    2009-07-03

    Crm1 plays a role in exporting proteins containing nuclear export signals (NESs) from the nucleus to the cytoplasm. Some proteins that are capable of interacting with Ran/Crm1 were reported to be localized at centrosomes and to function as centrosome checkpoints. But it remains unclear how Crm1 locates at centrosomes. In this study, we found that a fraction of Crm1 is located at centrosomes through its N-terminal CRM1, importin {beta} etc. (CRIME) domain, which is responsible for interacting with RanGTP, suggesting that Crm1 might target to centrosomes through binding centrosomal RanGTP. Moreover, overexpression of the CRIME domain, which is free of NES binding domain, resulted in the dissociation of pericentrin and {gamma}-tubulin complex from centrosomes and the disruption of microtubule nucleation. Deficiency of Crm1 provoked by RNAi also decreased the spindle poles localization of pericentrin and {gamma}-tubulin complex, coupled with mitotic defects. Since pericentrin was sensitive to Crm1 specific inhibitor leptomycin B, we propose that the centrosomal Crm1 might interact with pericentrin and regulate the localization and function of pericentrin at centrosomes.

  9. Apicomplexan cell cycle flexibility: centrosome controls the clutch

    PubMed Central

    Chen, Chun-Ti; Gubbels, Marc-Jan

    2015-01-01

    The centrosome serves as a central hub coordinating multiple cellular events in eukaryotes. A recent study in Toxoplasma gondii revealed a unique bipartite structure of the centrosome, which coordinates the nuclear cycle (S-phase and mitosis) and budding cycle (cytokinesis) of the parasite, and deciphers the principle behind flexible apicomplexan cell division modes. PMID:25899747

  10. APC/C is an essential regulator of centrosome clustering.

    PubMed

    Drosopoulos, Konstantinos; Tang, Chan; Chao, William C H; Linardopoulos, Spiros

    2014-01-01

    Centrosome amplification has been extensively associated with cancer. Cancer cells with extra centrosomes have the ability to cluster the extra centrosomes and divide in a bipolar fashion. Although a number of proteins have been shown to be involved in centrosome clustering, a mechanistic understanding of how this process is coordinated is not yet well defined. Here, to reveal regulators of centrosome clustering, we perform small interfering RNA (siRNA) screens with multiple assay readouts in a human isogenic cellular model. We find that APC/C activity is essential for centrosome clustering. We show that the motor kinesin Eg5 is a substrate of APC/C-CDH1, and that inhibition of APC/C results in stabilization of Eg5. Increased Eg5 protein levels disturb the balance of forces on the spindle and prevent centrosome clustering. This process is completely reversed after a short treatment with the Eg5 inhibitor, monastrol. These data advance our understanding of the regulation of centrosome clustering. PMID:24751481

  11. Sensors at Centrosomes Reveal Determinants of Local Separase Activity

    PubMed Central

    Agircan, Fikret Gurkan; Schiebel, Elmar

    2014-01-01

    Separase is best known for its function in sister chromatid separation at the metaphase-anaphase transition. It also has a role in centriole disengagement in late mitosis/G1. To gain insight into the activity of separase at centrosomes, we developed two separase activity sensors: mCherry-Scc1(142-467)-ΔNLS-eGFP-PACT and mCherry-kendrin(2059-2398)-eGFP-PACT. Both localize to the centrosomes and enabled us to monitor local separase activity at the centrosome in real time. Both centrosomal sensors were cleaved by separase before anaphase onset, earlier than the corresponding H2B-mCherry-Scc1(142-467)-eGFP sensor at chromosomes. This indicates that substrate cleavage by separase is not synchronous in the cells. Depletion of the proteins astrin or Aki1, which have been described as inhibitors of centrosomal separase, did not led to a significant activation of separase at centrosomes, emphasizing the importance of direct separase activity measurements at the centrosomes. Inhibition of polo-like kinase Plk1, on the other hand, decreased the separase activity towards the Scc1 but not the kendrin reporter. Together these findings indicate that Plk1 regulates separase activity at the level of substrate affinity at centrosomes and may explain in part the role of Plk1 in centriole disengagement. PMID:25299182

  12. Centrosomes are autocatalytic droplets of pericentriolar material organized by centrioles

    NASA Astrophysics Data System (ADS)

    Zwicker, David; Decker, Markus; Jaensch, Steffen; Hyman, Anthony A.; Jülicher, Frank

    2014-03-01

    We propose a physical description of the centrosome, a membrane-less organelle involved in cell division. In our model, centrosome material occurs in a soluble form in the cytosol and a form that tends to undergo phase separation from the cytosol. We find that an autocatalytic chemical transition between these forms accounts for the temporal evolution observed in experiments. Interestingly, the nucleation of centrosomes can be controlled by an enzymatic activity of the centrioles, which are present at the core of all centrosomes. This non-equilibrium feature also allows for multiple stable centrosomes, a situation which is unstable in equilibrium phase separation. Our theory explains the growth dynamics of centrosomes for all cell sizes down to the eight-cell stage of the C. elegans embryo. It also accounts for data acquired in experiments with aberrant numbers of centrosomes and altered cell volumes. Furthermore, our model can describe unequal centrosome sizes observed in cells with disturbed centrioles. Our example suggests a general picture of the organization of membrane-less organelles.

  13. Cold-treated centrosome: isolation of centrosomes from mitotic sea urchin eggs, production of an anticentrosomal antibody, and novel ultrastructural imaging.

    PubMed

    Thompson-Coffe, C; Coffe, G; Schatten, H; Mazia, D; Schatten, G

    1996-01-01

    A novel isolation of centrosomes is described and it was used to both generate a centrosome-specific monoclonal antibody and to image with high-resolution low-voltage scanning electron microscopy the surface details of the isolated centrosome. At first mitotic prometaphase, sea urchin zygotes are chilled on ice overnight. While most of the microtubules disassemble, the mitotic centrosomes collapse into aggregated masses. These centrosomes have been isolated, and used to generate a monoclonal antibody, designated 4D2, which is reactive with interphase and mitotic centrosomes. 4D2 staining of centrosomes is similar, but not identical, to that of other centrosomal antibodies like Ah6 and 5051. Centrosomal material is detected as a compact sphere after cold treatment; upon recovery the sphere expands and undergoes the shape changes previously described [Mazia et al., 1987: J. Cell Biol. 105:206a] to eventually reorganize a normal mitotic apparatus. PMID:8674139

  14. The centrosome cycle: Centriole biogenesis, duplication and inherent asymmetries

    PubMed Central

    Nigg, Erich A.; Stearns, Tim

    2013-01-01

    Centrosomes are microtubule-organizing centres of animal cells. They influence the morphology of the microtubule cytoskeleton, function as the base for the primary cilium and serve as a nexus for important signalling pathways. At the core of a typical centrosome are two cylindrical microtubule-based structures termed centrioles, which recruit a matrix of associated pericentriolar material. Cells begin the cell cycle with exactly one centrosome, and the duplication of centrioles is constrained such that it occurs only once per cell cycle and at a specific site in the cell. As a result of this duplication mechanism, the two centrioles differ in age and maturity, and thus have different functions; for example, the older of the two centrioles can initiate the formation of a ciliary axoneme. We discuss spatial aspects of the centrosome duplication cycle, the mechanism of centriole assembly and the possible consequences of the inherent asymmetry of centrioles and centrosomes. PMID:21968988

  15. Centrosomal clustering contributes to chromosomal instability and cancer.

    PubMed

    Milunović-Jevtić, A; Mooney, P; Sulerud, T; Bisht, J; Gatlin, J C

    2016-08-01

    Cells assemble mitotic spindles during each round of division to insure accurate segregation of their duplicated genome. In animal cells, stereotypical spindles have two poles, each containing one centrosome, from which microtubules are nucleated. By contrast, many cancer cells often contain more than two centrosomes and form transient multipolar spindle structures with more than two poles. In order to divide and produce viable progeny, the multipolar spindle intermediate must be reshaped into a pseudo-bipolar structure via a process called centrosomal clustering. Pseudo-bipolar spindles appear to function normally during mitosis, but they occasionally give rise to aneuploid and transformed daughter cells. Agents that inhibit centrosomal clustering might therefore work as a potential cancer therapy, specifically targeting mitosis in supernumerary centrosome-containing cells. PMID:27046071

  16. Behavior of centrosomes during fertilization and cell division in mouse oocytes and in sea urchin eggs

    NASA Technical Reports Server (NTRS)

    Schatten, Heide; Schatten, Gerald; Balczon, Ron; Simerly, Calvin; Mazia, Daniel

    1986-01-01

    The behavior of centrosomes during the stages of fertilization and cell division in mouse oocytes and in sea urchin eggs was monitored in an immunofluorescence microscope, using autoimmune centrosomal antiserum derived from a patient with scleroderma to label the centrosomal material. These observations showed that centrosomes reproduce during the interphase and aggregate and separate during cell mitosis. Results supported the hypothesis of Mazia (1984), who proposed that centrosomes are 'flexible bodies'. It was also found that, while the sea urchin centrosomes are paternally inherited as was initially proposed by Bovery (1904), the mouse centrosomes are of maternal origin.

  17. Centrosome-Dependent Bypass of the DNA Damage Checkpoint by the Polo Kinase Cdc5.

    PubMed

    Ratsima, Hery; Serrano, Diego; Pascariu, Mirela; D'Amours, Damien

    2016-02-16

    Cell-cycle checkpoints are essential feedback mechanisms that promote genome integrity. However, in the face of unrepairable DNA lesions, bypass mechanisms can suppress checkpoint activity and allow cells to resume proliferation. The molecular mechanisms underlying this biological response are currently not understood. Taking advantage of unique separation-of-function mutants, we show that the Polo-like kinase (PLK) Cdc5 uses a phosphopriming-based interaction mechanism to suppress G2/M checkpoint arrest by targeting Polo kinase activity to centrosomes. We also show that key subunits of the evolutionarily conserved RSC complex are critical downstream effectors of Cdc5 activity in checkpoint suppression. Importantly, the lethality and checkpoint defects associated with loss of Cdc5 Polo box activity can be fully rescued by artificially anchoring Cdc5 kinase domain to yeast centrosomes. Collectively, our results highlight a previously unappreciated role for centrosomes as key signaling centers for the suppression of cell-cycle arrest induced by persistent or unrepairable DNA damage. PMID:26832404

  18. Physical association between a novel plasma-membrane structure and centrosome orients cell division

    PubMed Central

    Negishi, Takefumi; Miyazaki, Naoyuki; Murata, Kazuyoshi; Yasuo, Hitoyoshi; Ueno, Naoto

    2016-01-01

    In the last mitotic division of the epidermal lineage in the ascidian embryo, the cells divide stereotypically along the anterior-posterior axis. During interphase, we found that a unique membrane structure invaginates from the posterior to the centre of the cell, in a microtubule-dependent manner. The invagination projects toward centrioles on the apical side of the nucleus and associates with one of them. Further, a cilium forms on the posterior side of the cell and its basal body remains associated with the invagination. A laser ablation experiment suggests that the invagination is under tensile force and promotes the posterior positioning of the centrosome. Finally, we showed that the orientation of the invaginations is coupled with the polarized dynamics of centrosome movements and the orientation of cell division. Based on these findings, we propose a model whereby this novel membrane structure orchestrates centrosome positioning and thus the orientation of cell division axis. DOI: http://dx.doi.org/10.7554/eLife.16550.001 PMID:27502556

  19. Cohesin protein SMC1 is a centrosomal protein.

    PubMed

    Guan, Jikui; Ekwurtzel, Emelie; Kvist, Ulrik; Yuan, Li

    2008-08-01

    Structural maintenance of chromosome protein 1 (SMC1) is well known for its roles in sister chromatid cohesion and DNA repair. In this study, we report a novel centrosomal localization of SMC1 within the cytoplasm in a variety of mammalian cell lines. We showed that SMC1 localized to centrosomes throughout the cell cycle in a microtubule-independent manner. Biochemically, SMC1 was cofractionated with the centrosomal protein gamma-tubulin in centrosomal preparation. Immunohistochemistry and immunoelectron microscopy performed on mouse tissue sections revealed that SMC1 antibody strongly labeled the base of cilia in ciliated epithelia, where basal bodies were located. Furthermore, we showed that SMC1 was associated with both centrioles of a centrosome at G0/G1 stage of the cell cycle. These results demonstrate that SMC1 is a centrosomal protein, suggesting possible involvement of SMC1 in centrosome/basal body-related functions, such as organization of dynamic arrays of microtubules and ciliary formation. PMID:18515072

  20. VHZ is a novel centrosomal phosphatase associated with cell growth and human primary cancers

    PubMed Central

    2010-01-01

    Background VHZ is a VH1-like (member Z) dual specific protein phosphatase encoded by DUSP23 gene. Some of the dual specific protein phosphatases (DSPs) play an important role in cell cycle control and have shown to be associated with carcinogenesis. Here, the expression of VHZ associated with cell growth and human cancers was investigated. Results We generated a mouse monoclonal antibody (mAb clone#209) and rabbit polyclonal antibodies (rAb) against VHZ. We performed cell proliferation assay to learn how VHZ is associated with cell cycle by retroviral transduction to express VHZ, VHZ(C95S), and control vector in MCF-7 cells. Overexpression of VHZ [but not VHZ(C95S)] in MCF-7 cells promoted cell proliferation compared to control cells. shRNA-mediated knockdown of VHZ in MCF-7 cells showed that reduction of VHZ resulted in increased G1 but decreased S phase cell populations. Using indirect immunofluorescence, we showed that both exogenous and endogenous VHZ protein was localized at the centrosome in addition to its cytoplasmic distribution. Furthermore, using immunohistochemistry, we revealed that VHZ protein was overexpressed either in enlarged centrosomes (VHZ-centrosomal-stain) of some invasive ductal carcinomas (IDC) Stage I (8/65 cases) or in entire cytoplasm (VHZ-cytosol-stain) of invasive epithelia of some IDC Stage II/III (11/47 cases) of breast cancers examined. More importantly, upregulation of VHZ protein is also associated with numerous types of human cancer, in particular breast cancer. VHZ mAb may be useful as a reagent in clinical diagnosis for assessing VHZ positive tumors. Conclusions We generated a VHZ-specific mAb to reveal that VHZ has a novel subcellular localization, namely the centrosome. VHZ is able to facilitate G1/S cell cycle transition in a PTP activity-dependent manner. The upregulation of its protein levels in primary human cancers supports the clinical relevance of the protein in cancers. PMID:20509867

  1. A Par-1-Par-3-Centrosome Cell Polarity Pathway and Its Tuning for Isotropic Cell Adhesion.

    PubMed

    Jiang, Tao; McKinley, R F Andrew; McGill, Melanie A; Angers, Stephane; Harris, Tony J C

    2015-10-19

    To form regulated barriers between body compartments, epithelial cells polarize into apical and basolateral domains and assemble adherens junctions (AJs). Despite close links with polarity networks that generate single polarized domains, AJs distribute isotropically around the cell circumference for adhesion with all neighboring cells [1-3]. How AJs avoid the influence of polarity networks to maintain their isotropy has been unclear. In established epithelia, trans cadherin interactions could maintain AJ isotropy [4], but AJs are dynamic during epithelial development and remodeling [5, 6], and thus specific mechanisms may control their isotropy. In Drosophila, aPKC prevents hyper-polarization of junctions as epithelia develop from cellularization to gastrulation [7]. Here, we show that aPKC does so by inhibiting a positive feedback loop between Bazooka (Baz)/Par-3, a junctional organizer [5, 8-10], and centrosomes. Without aPKC, Baz and centrosomes lose their isotropic distributions and recruit each other to single plasma membrane (PM) domains. Surprisingly, our loss- and gain-of-function analyses show that the Baz-centrosome positive feedback loop is driven by Par-1, a kinase known to phosphorylate Baz and inhibit its basolateral localization [8, 11, 12]. We find that Par-1 promotes the positive feedback loop through both centrosome microtubule effects and Baz phosphorylation. Normally, aPKC attenuates the circuit by expelling Par-1 from the apical domain at gastrulation. The combination of local activation and global inhibition is a common polarization strategy [13-16]. Par-1 seems to couple both effects for a potent Baz polarization mechanism that is regulated for the isotropy of Baz and AJs around the cell circumference. PMID:26455305

  2. Arsenic Methyltransferase

    EPA Science Inventory

    The metalloid arsenic enters the environment by natural processes (volcanic activity, weathering of rocks) and by human activity (mining, smelting, herbicides and pesticides). Although arsenic has been exploited for homicidal and suicidal purposes since antiquity, its significan...

  3. The SCF-FBXW5 E3-ubiquitin ligase is regulated by PLK4 and targets HsSAS-6 to control centrosome duplication.

    PubMed

    Puklowski, Anja; Homsi, Yahya; Keller, Debora; May, Martin; Chauhan, Sangeeta; Kossatz, Uta; Grünwald, Viktor; Kubicka, Stefan; Pich, Andreas; Manns, Michael P; Hoffmann, Ingrid; Gönczy, Pierre; Malek, Nisar P

    2011-08-01

    Deregulated centrosome duplication can result in genetic instability and contribute to tumorigenesis. Here, we show that centrosome duplication is regulated by the activity of an E3-ubiquitin ligase that employs the F-box protein FBXW5 (ref. 3) as its targeting subunit. Depletion of endogenous FBXW5 or overexpression of an F-box-deleted mutant version results in centrosome overduplication and formation of multipolar spindles. We identify the centriolar protein HsSAS-6 (refs 4,5) as a critical substrate of the SCF-FBXW5 complex. FBXW5 binds HsSAS-6 and promotes its ubiquitylation in vivo. The activity of SCF-FBXW5 is in turn negatively regulated by Polo-like kinase 4 (PLK4), which phosphorylates FBXW5 at Ser 151 to suppress its ability to ubiquitylate HsSAS-6. FBXW5 is a cell-cycle-regulated protein with expression levels peaking at the G1/S transition. We show that FBXW5 levels are controlled by the anaphase-promoting (APC/C) complex, which targets FBXW5 for degradation during mitosis and G1, thereby helping to reset the centrosome duplication machinery. In summary, we show that a cell-cycle-regulated SCF complex is regulated by the kinase PLK4, and that this in turn restricts centrosome re-duplication through degradation of the centriolar protein HsSAS-6. PMID:21725316

  4. ARSENIC REMOVAL

    EPA Science Inventory

    Presentation covered five topics; arsenic chemistry, best available technology (BAT), surface water technology, ground water technology and case studies of arsenic removal. The discussion on arsenic chemistry focused on the need and method of speciation for AsIII and AsV. BAT me...

  5. Targeting of Fzr/Cdh1 for timely activation of the APC/C at the centrosome during mitotic exit

    PubMed Central

    Meghini, Francesco; Martins, Torcato; Tait, Xavier; Fujimitsu, Kazuyuki; Yamano, Hiroyuki; Glover, David M.; Kimata, Yuu

    2016-01-01

    A multi-subunit ubiquitin ligase, the anaphase-promoting complex/cyclosome (APC/C), regulates critical cellular processes including the cell cycle. To accomplish its diverse functions, APC/C activity must be precisely regulated in time and space. The interphase APC/C activator Fizzy-related (Fzr or Cdh1) is localized at centrosomes in animal cells. However, neither the mechanism of its localization nor its importance is clear. Here we identify the centrosome component Spd2 as a major partner of Fzr in Drosophila. The localization of Fzr to the centriole during interphase depends on direct interaction with Spd2. By generating Spd2 mutants unable to bind Fzr, we show that centrosomal localization of Fzr is essential for optimal APC/C activation towards its centrosomal substrate Aurora A. Finally, we show that Spd2 is also a novel APC/CFzr substrate. Our study is the first to demonstrate the critical importance of distinct subcellular pools of APC/C activators in the spatiotemporal control of APC/C activity. PMID:27558644

  6. Structure and Non-Structure of Centrosomal Proteins

    PubMed Central

    Bertero, Michela G.; Boutin, Maïlys; Guarín, Nayibe; Méndez-Giraldez, Raúl; Nuñez, Alfonso; Pedrero, Juan G.; Redondo, Pilar; Sanz, María; Speroni, Silvia; Teichert, Florian; Bruix, Marta; Carazo, José M.; Gonzalez, Cayetano; Reina, José; Valpuesta, José M.; Vernos, Isabelle; Zabala, Juan C.; Montoya, Guillermo; Coll, Miquel; Bastolla, Ugo; Serrano, Luis

    2013-01-01

    Here we perform a large-scale study of the structural properties and the expression of proteins that constitute the human Centrosome. Centrosomal proteins tend to be larger than generic human proteins (control set), since their genes contain in average more exons (20.3 versus 14.6). They are rich in predicted disordered regions, which cover 57% of their length, compared to 39% in the general human proteome. They also contain several regions that are dually predicted to be disordered and coiled-coil at the same time: 55 proteins (15%) contain disordered and coiled-coil fragments that cover more than 20% of their length. Helices prevail over strands in regions homologous to known structures (47% predicted helical residues against 17% predicted as strands), and even more in the whole centrosomal proteome (52% against 7%), while for control human proteins 34.5% of the residues are predicted as helical and 12.8% are predicted as strands. This difference is mainly due to residues predicted as disordered and helical (30% in centrosomal and 9.4% in control proteins), which may correspond to alpha-helix forming molecular recognition features (α-MoRFs). We performed expression assays for 120 full-length centrosomal proteins and 72 domain constructs that we have predicted to be globular. These full-length proteins are often insoluble: Only 39 out of 120 expressed proteins (32%) and 19 out of 72 domains (26%) were soluble. We built or retrieved structural models for 277 out of 361 human proteins whose centrosomal localization has been experimentally verified. We could not find any suitable structural template with more than 20% sequence identity for 84 centrosomal proteins (23%), for which around 74% of the residues are predicted to be disordered or coiled-coils. The three-dimensional models that we built are available at http://ub.cbm.uam.es/centrosome/models/index.php. PMID:23671615

  7. Structural protein 4.1 is located in mammalian centrosomes

    SciTech Connect

    Krauss, S.W.; Chasis, J.A.; Rogers, C.; Mohandas, N.; Krockmalnic, G.; Penman, S.

    1997-07-01

    Structural protein 4.1 was first characterized as an important 80-kDa protein in the mature red cell membrane skeleton. It is now known to be a member of a family of protein isoforms detected at diverse intracellular sites in many nucleated mammalian cells. We recently reported that protein 4.1 isoforms are present at interphase in nuclear matrix and are rearranged during the cell cycle. Here we report that protein 4.1 epitopes are present in centrosomes of human and murine cells and are detected by using affinity-purified antibodies specific for 80-kDa red cell 4.1 and for 4.1 peptides. Immunofluorescence, by both conventional and confocal microscopy, showed that protein 4.1 epitopes localized in the pericentriolar region. Protein 4.1 epitopes remained in centrosomes after extraction of cells with detergent, salt, and DNase. Higher resolution electron microscopy of detergent-extracted cell whole mounts showed centrosomal protein 4.1 epitopes distributed along centriolar cylinders and on pericentriolar fibers, at least some of which constitute the filamentous network surrounding each centriole. Double-label electron microscopy showed that protein 4.1 epitopes were predominantly localized in regions also occupied by epitopes for centrosome-specific autoimmune serum 5051 but were not found on microtubules. Our results suggest that protein 4.1 is an integral component of centrosome structure, in which it may play an important role in centrosome function during cell division and organization of cellular architecture.

  8. Centrosomal nucleolin is required for microtubule network organization.

    PubMed

    Gaume, Xavier; Tassin, Anne-Marie; Ugrinova, Iva; Mongelard, Fabien; Monier, Karine; Bouvet, Philippe

    2015-01-01

    Nucleolin is a pleiotropic protein involved in a variety of cellular processes. Although multipolar spindle formation has been observed after nucleolin depletion, the roles of nucleolin in centrosome regulation and functions have not been addressed. Here we report using immunofluorescence and biochemically purified centrosomes that nucleolin co-localized only with one of the centrioles during interphase which was further identified as the mature centriole. Upon nucleolin depletion, cells exhibited an amplification of immature centriole markers surrounded by irregular pericentrin staining; these structures were exempt from maturation markers and unable to nucleate microtubules. Furthermore, the microtubule network was disorganized in these cells, exhibiting frequent non-centrosomal microtubules. At the mature centriole a reduced kinetics in the centrosomal microtubule nucleation phase was observed in live silenced cells, as well as a perturbation of microtubule anchoring. Immunoprecipitation experiments showed that nucleolin belongs to protein complexes containing 2 key centrosomal proteins, γ-tubulin and ninein, involved in microtubule nucleation and anchoring steps. Altogether, our study uncovered a new role for nucleolin in restricting microtubule nucleation and anchoring at centrosomes in interphase cells. PMID:25590348

  9. A molecular mechanism of mitotic centrosome assembly in Drosophila

    PubMed Central

    Conduit, Paul T; Richens, Jennifer H; Wainman, Alan; Holder, James; Vicente, Catarina C; Pratt, Metta B; Dix, Carly I; Novak, Zsofia A; Dobbie, Ian M; Schermelleh, Lothar; Raff, Jordan W

    2014-01-01

    Centrosomes comprise a pair of centrioles surrounded by pericentriolar material (PCM). The PCM expands dramatically as cells enter mitosis, but it is unclear how this occurs. In this study, we show that the centriole protein Asl initiates the recruitment of DSpd-2 and Cnn to mother centrioles; both proteins then assemble into co-dependent scaffold-like structures that spread outwards from the mother centriole and recruit most, if not all, other PCM components. In the absence of either DSpd-2 or Cnn, mitotic PCM assembly is diminished; in the absence of both proteins, it appears to be abolished. We show that DSpd-2 helps incorporate Cnn into the PCM and that Cnn then helps maintain DSpd-2 within the PCM, creating a positive feedback loop that promotes robust PCM expansion around the mother centriole during mitosis. These observations suggest a surprisingly simple mechanism of mitotic PCM assembly in flies. DOI: http://dx.doi.org/10.7554/eLife.03399.001 PMID:25149451

  10. Fanconi anemia complementation group A (FANCA) localizes to centrosomes and functions in the maintenance of centrosome integrity.

    PubMed

    Kim, Sunshin; Hwang, Soo Kyung; Lee, Mihee; Kwak, Heejin; Son, Kook; Yang, Jiha; Kim, Sung Hak; Lee, Chang-Hun

    2013-09-01

    Fanconi anemia (FA) proteins are known to play roles in the cellular response to DNA interstrand cross-linking lesions; however, several reports have suggested that FA proteins play additional roles. To elucidate novel functions of FA proteins, we used yeast two-hybrid screening to identify binding partners of the Fanconi anemia complementation group A (FANCA) protein. The candidate proteins included never-in-mitosis-gene A (NIMA)-related kinase 2 (Nek2), which functions in the maintenance of centrosome integrity. The interaction of FANCA and Nek2 was confirmed in human embryonic kidney (HEK) 293T cells. Furthermore, FANCA interacted with γ-tubulin and localized to centrosomes, most notably during the mitotic phase, confirming that FANCA is a centrosomal protein. Knockdown of FANCA increased the frequency of centrosomal abnormalities and enhanced the sensitivity of U2OS osteosarcoma cells to nocodazole, a microtubule-interfering agent. In vitro kinase assays indicated that Nek2 can phosphorylate FANCA at threonine-351 (T351), and analysis with a phospho-specific antibody confirmed that this phosphorylation occurred in response to nocodazole treatment. Furthermore, U2OS cells overexpressing the phosphorylation-defective T351A FANCA mutant showed numerical centrosomal abnormalities, aberrant mitotic arrest, and enhanced nocodazole sensitivity, implying that the Nek2-mediated T351 phosphorylation of FANCA is important for the maintenance of centrosomal integrity. Taken together, this study revealed that FANCA localizes to centrosomes and is required for the maintenance of centrosome integrity, possibly through its phosphorylation at T351 by Nek2. PMID:23806870

  11. The nucleoporin Nup205/NPP-3 is lost near centrosomes at mitotic onset and can modulate the timing of this process in Caenorhabditis elegans embryos

    PubMed Central

    Hachet, Virginie; Busso, Coralie; Toya, Mika; Sugimoto, Asako; Askjaer, Peter; Gönczy, Pierre

    2012-01-01

    Regulation of mitosis in time and space is critical for proper cell division. We conducted an RNA interference–based modifier screen to identify novel regulators of mitosis in Caenorhabditis elegans embryos. Of particular interest, this screen revealed that the Nup205 nucleoporin NPP-3 can negatively modulate the timing of mitotic onset. Furthermore, we discovered that NPP-3 and nucleoporins that are associated with it are lost from the nuclear envelope (NE) in the vicinity of centrosomes at the onset of mitosis. We demonstrate that centrosomes are both necessary and sufficient for NPP-3 local loss, which also requires the activity of the Aurora-A kinase AIR-1. Our findings taken together support a model in which centrosomes and AIR-1 promote timely onset of mitosis by locally removing NPP-3 and associated nucleoporins from the NE. PMID:22740626

  12. E2F Activators Signal and Maintain Centrosome Amplification in Breast Cancer Cells

    PubMed Central

    Lee, Mi-Young; Moreno, Carlos S.

    2014-01-01

    Centrosomes ensure accurate chromosome segregation by directing spindle bipolarity. Loss of centrosome regulation results in centrosome amplification, multipolar mitosis and aneuploidy. Since centrosome amplification is common in premalignant lesions and breast tumors, it is proposed to play a central role in breast tumorigenesis, a hypothesis that remains to be tested. The coordination between the cell and centrosome cycles is of paramount importance to maintain normal centrosome numbers, and the E2Fs may be responsible for regulating these cycles. However, the role of E2F activators in centrosome amplification is unclear. Because E2Fs are deregulated in Her2+ cells displaying centrosome amplification, we addressed whether they signal this abnormal process. Knockdown of E2F1 or E2F3 in Her2+ cells decreased centrosome amplification without significantly affecting cell cycle progression, whereas the overexpression of E2F1, E2F2, or E2F3 increased centrosome amplification in MCF10A mammary epithelial cells. Our results revealed that E2Fs affect the expression of proteins, including Nek2 and Plk4, known to influence the cell/centrosome cycles and mitosis. Downregulation of E2F3 resulted in cell death and delays/blocks in cytokinesis, which was reversed by Nek2 overexpression. Nek2 overexpression enhanced centrosome amplification in Her2+ breast cancer cells silenced for E2F3, revealing a role for the E2F activators in maintaining centrosome amplification in part through Nek2. PMID:24797070

  13. Cyclin G2 is a centrosome-associated nucleocytoplasmic shuttling protein that influences microtubule stability and induces a p53-dependent cell cycle arrest

    SciTech Connect

    Arachchige Don, Aruni S.; Dallapiazza, Robert F.; Bennin, David A.; Brake, Tiffany; Cowan, Colleen E.; Horne, Mary C. . E-mail: mary-horne@uiowa.edu

    2006-12-10

    Cyclin G2 is an atypical cyclin that associates with active protein phosphatase 2A. Cyclin G2 gene expression correlates with cell cycle inhibition; it is significantly upregulated in response to DNA damage and diverse growth inhibitory stimuli, but repressed by mitogenic signals. Ectopic expression of cyclin G2 promotes cell cycle arrest, cyclin dependent kinase 2 inhibition and the formation of aberrant nuclei [Bennin, D. A., Don, A. S., Brake, T., McKenzie, J. L., Rosenbaum, H., Ortiz, L., DePaoli-Roach, A. A., and Horne, M. C. (2002). Cyclin G2 associates with protein phosphatase 2A catalytic and regulatory B' subunits in active complexes and induces nuclear aberrations and a G{sub 1}/S-phase cell cycle arrest. J Biol Chem 277, 27449-67]. Here we report that endogenous cyclin G2 copurifies with centrosomes and microtubules (MT) and that ectopic G2 expression alters microtubule stability. We find exogenous and endogenous cyclin G2 present at microtubule organizing centers (MTOCs) where it colocalizes with centrosomal markers in a variety of cell lines. We previously reported that cyclin G2 forms complexes with active protein phosphatase 2A (PP2A) and colocalizes with PP2A in a detergent-resistant compartment. We now show that cyclin G2 and PP2A colocalize at MTOCs in transfected cells and that the endogenous proteins copurify with isolated centrosomes. Displacement of the endogenous centrosomal scaffolding protein AKAP450 that anchors PP2A at the centrosome resulted in the depletion of centrosomal cyclin G2. We find that ectopic expression of cyclin G2 induces microtubule bundling and resistance to depolymerization, inhibition of polymer regrowth from MTOCs and a p53-dependent cell cycle arrest. Furthermore, we determined that a 100 amino acid carboxy-terminal region of cyclin G2 is sufficient to both direct GFP localization to centrosomes and induce cell cycle inhibition. Colocalization of endogenous cyclin G2 with only one of two GFP-centrin-tagged centrioles

  14. Clinical implication of centrosome amplification and expression of centrosomal functional genes in multiple myeloma

    PubMed Central

    2013-01-01

    Background Multiple myeloma (MM) is a low proliferative tumor of postgerminal center plasma cell (PC). Centrosome amplification (CA) is supposed to be one of the mechanisms leading to chromosomal instability. Also, CA is associated with deregulation of cell cycle, mitosis, DNA repair and proliferation. The aim of our study was to evaluate the prognostic significance and possible role of CA in pathogenesis and analysis of mitotic genes as mitotic disruption markers. Design and methods A total of 173 patients were evaluated for this study. CD138+ cells were separated by MACS. Immunofluorescent labeling of centrin was used for evaluation of centrosome amplification in PCs. Interphase FISH with cytoplasmic immunoglobulin light chain staining (cIg FISH) and qRT-PCR were performed on PCs. Results Based on the immunofluorescent staining results, all patients were divided into two groups: CA positive (38.2%) and CA negative (61.8%). Among the newly diagnosed patients, worse overall survival was indicated in the CA negative group (44/74) in comparison to the CA positive group (30/74) (P = 0.019). Gene expression was significantly down-regulated in the CA positive group in comparison to CA negative in the following genes: AURKB, PLK4, TUBG1 (P < 0.05). Gene expression was significantly down-regulated in newly diagnosed in comparison to relapsed patients in the following genes: AURKA, AURKB, CCNB1, CCNB2, CETN2, HMMR, PLK4, PCNT, and TACC3 (P < 0.05). Conclusions Our findings indicate better prognosis for CA positive newly diagnosed patients. Considering revealed clinical and gene expression heterogeneity between CA negative and CA positive patients, there is a possibility to characterize centrosome amplification as a notable event in multiple myeloma pathogenesis. PMID:23522059

  15. Linking Microbial Activity with Arsenic Fate during Cow Dung Disposal of Arsenic-Bearing Wastes

    NASA Astrophysics Data System (ADS)

    Clancy, T. M.; Reddy, R.; Tan, J.; Hayes, K. F.; Raskin, L.

    2014-12-01

    To address widespread arsenic contamination of drinking water sources numerous technologies have been developed to remove arsenic. All technologies result in the production of an arsenic-bearing waste that must be evaluated and disposed in a manner to limit the potential for environmental release and human exposure. One disposal option that is commonly recommended for areas without access to landfills is the mixing of arsenic-bearing wastes with cow dung. These recommendations are made based on the ability of microorganisms to create volatile arsenic species (including mono-, di-, and tri-methylarsine gases) to be diluted in the atmosphere. However, most studies of environmental microbial communities have found only a small fraction (<0.1 %) of the total arsenic present in soils or rice paddies is released via volatilization. Additionally, past studies often have not monitored arsenic release in the aqueous phase. Two main pathways for microbial arsenic volatilization are known and include methylation of arsenic during methanogenesis and methylation by arsenite S-adenosylmethionine methyltransferase. In this study, we compare the roles of these two pathways in arsenic volatilization and aqueous mobilization through mesocosm experiments with cow dung and arsenic-bearing wastes produced during drinking water treatment in West Bengal, India. Arsenic in gaseous, aqueous, and solid phases was measured. Consistent with previous reports, less than 0.02% of the total arsenic present was volatilized. A much higher amount (~5%) of the total arsenic was mobilized into the liquid phase. Through the application of molecular tools, including 16S rRNA sequencing and quantification of gene transcripts involved in methanogenesis, this study links microbial community activity with arsenic fate in potential disposal environments. These results illustrate that disposal of arsenic-bearing wastes by mixing with cow dung does not achieve its end goal of promoting arsenic volatilization

  16. The centrosome is a dynamic structure that ejects PCM flares.

    PubMed

    Megraw, Timothy L; Kilaru, Sandhya; Turner, F Rudolf; Kaufman, Thomas C

    2002-12-01

    The Drosophila Centrosomin (Cnn) protein is an essential core component of centrosomes in the early embryo. We have expressed a Cnn-GFP fusion construct in cleavage stage embryos, which rescues the maternal effect lethality of cnn mutant animals. The localization patterns seen with GFP-Cnn are identical to the patterns we see by immunofluorescent staining with anti-Cnn antibodies. Live imaging of centrosomes with Cnn-GFP reveals surprisingly dynamic features of the centrosome. Extracentrosomal particles of Cnn move radially from the centrosome and frequently change their direction. D-TACC colocalized with Cnn at these particles. We have named these extrusions 'flares'. Flares are dependent on microtubules, since disruption of the microtubule array severs the movement of these particles. Movement of flare particles is cleavage-cycle-dependent and appears to be attributed mostly to their association with dynamic astral microtubules. Flare activity decreases at metaphase, then increases at telophase and remains at this higher level of activity until the next metaphase. Flares appear to be similar to vertebrate PCM-1-containing 'centriolar satellites' in their behavior. By injecting rhodamine-actin, we observed that flares extend no farther than the actin cage. Additionally, disruption of the microfilament array increased the extent of flare movement. These observations indicate that centrosomes eject particles of Cnn-containing pericentriolar material that move on dynamic astral microtubules at a rate that varies with the cell cycle. We propose that flare particles play a role in organizing the actin cytoskeleton during syncytial cleavage. PMID:12415014

  17. Dynamic Association of Proteasomal Machinery with the Centrosome

    PubMed Central

    Christian Wigley, W.; Fabunmi, Rosalind P.; Lee, Min Goo; Marino, Christopher R.; Muallem, Shmuel; DeMartino, George N.; Thomas, Philip J.

    1999-01-01

    Although the number of pathologies known to arise from the inappropriate folding of proteins continues to grow, mechanisms underlying the recognition and ultimate disposition of misfolded polypeptides remain obscure. For example, how and where such substrates are identified and processed is unknown. We report here the identification of a specific subcellular structure in which, under basal conditions, the 20S proteasome, the PA700 and PA28 (700- and 180-kD proteasome activator complexes, respectively), ubiquitin, Hsp70 and Hsp90 (70- and 90-kD heat shock protein, respectively) concentrate in HEK 293 and HeLa cells. The structure is perinuclear, surrounded by endoplasmic reticulum, adjacent to the Golgi, and colocalizes with γ-tubulin, an established centrosomal marker. Density gradient fractions containing purified centrosomes are enriched in proteasomal components and cell stress chaperones. The centrosome-associated structure enlarges in response to inhibition of proteasome activity and the level of misfolded proteins. For example, folding mutants of CFTR form large inclusions which arise from the centrosome upon inhibition of proteasome activity. At high levels of misfolded protein, the structure not only expands but also extensively recruits the cytosolic pools of ubiquitin, Hsp70, PA700, PA28, and the 20S proteasome. Thus, the centrosome may act as a scaffold, which concentrates and recruits the systems which act as censors and modulators of the balance between folding, aggregation, and degradation. PMID:10225950

  18. Dynamic association of proteasomal machinery with the centrosome.

    PubMed

    Wigley, W C; Fabunmi, R P; Lee, M G; Marino, C R; Muallem, S; DeMartino, G N; Thomas, P J

    1999-05-01

    Although the number of pathologies known to arise from the inappropriate folding of proteins continues to grow, mechanisms underlying the recognition and ultimate disposition of misfolded polypeptides remain obscure. For example, how and where such substrates are identified and processed is unknown. We report here the identification of a specific subcellular structure in which, under basal conditions, the 20S proteasome, the PA700 and PA28 (700- and 180-kD proteasome activator complexes, respectively), ubiquitin, Hsp70 and Hsp90 (70- and 90-kD heat shock protein, respectively) concentrate in HEK 293 and HeLa cells. The structure is perinuclear, surrounded by endoplasmic reticulum, adjacent to the Golgi, and colocalizes with gamma-tubulin, an established centrosomal marker. Density gradient fractions containing purified centrosomes are enriched in proteasomal components and cell stress chaperones. The centrosome-associated structure enlarges in response to inhibition of proteasome activity and the level of misfolded proteins. For example, folding mutants of CFTR form large inclusions which arise from the centrosome upon inhibition of proteasome activity. At high levels of misfolded protein, the structure not only expands but also extensively recruits the cytosolic pools of ubiquitin, Hsp70, PA700, PA28, and the 20S proteasome. Thus, the centrosome may act as a scaffold, which concentrates and recruits the systems which act as censors and modulators of the balance between folding, aggregation, and degradation. PMID:10225950

  19. Tackling centrosome biology through gene targeting in chicken B cells.

    PubMed

    Chavali, Pavithra L; Gergely, Fanni

    2015-01-01

    The centrosome proteome comprises hundreds of proteins whose function at the organelle and in the cellular context is unknown. Loss-of-function studies present a powerful tool to probe the roles of these individual constituents and hence improve our insight into key questions of centrosome biology such as how centrosomes are built, how they duplicate, and which cellular processes they partake in. In cultured cells ribonucleic acid (RNA) interference remains the most widely used method to achieve protein depletion, but due to the remarkable stability of many centrosome components depletion is often incomplete. In such instances genome editing provides a viable alternative. The exceptionally high homologous recombination rate of chicken DT40 cells makes this lymphocytic cell line ideal for genetic manipulation. Here we describe methods for the design and generation of knockouts and in situ tagging of genes in these cells. Furthermore, we report an optimized technique that allows isolation of centrosomes from DT40 cells for use in in vitro functional assays and proteomic analysis. Gene editing by CRISPR-Cas9 technology is fast replacing RNA interference as a method of choice for loss-of-function studies, but the combination of the fast cell cycle, the robustness in culture and ease of gene targeting, will continue to make DT40 cells a useful model system for studies of vertebrate protein function. PMID:26175435

  20. Cargo transport by cytoplasmic Dynein can center embryonic centrosomes.

    PubMed

    Longoria, Rafael A; Shubeita, George T

    2013-01-01

    To complete meiosis II in animal cells, the male DNA material needs to meet the female DNA material contained in the female pronucleus at the egg center, but it is not known how the male pronucleus, deposited by the sperm at the periphery of the cell, finds the cell center in large eggs. Pronucleus centering is an active process that appears to involve microtubules and molecular motors. For small and medium-sized cells, the force required to move the centrosome can arise from either microtubule pushing on the cortex, or cortically-attached dynein pulling on microtubules. However, in large cells, such as the fertilized Xenopus laevis embryo, where microtubules are too long to support pushing forces or they do not reach all boundaries before centrosome centering begins, a different force generating mechanism must exist. Here, we present a centrosome positioning model in which the cytosolic drag experienced by cargoes hauled by cytoplasmic dynein on the sperm aster microtubules can move the centrosome towards the cell's center. We find that small, fast cargoes (diameter ∼100 nm, cargo velocity ∼2 µm/s) are sufficient to move the centrosome in the geometry of the Xenopus laevis embryo within the experimentally observed length and time scales. PMID:23840877

  1. Proximity interactions among centrosome components identify regulators of centriole duplication.

    PubMed

    Firat-Karalar, Elif Nur; Rauniyar, Navin; Yates, John R; Stearns, Tim

    2014-03-17

    The centrosome consists of a pair of centrioles and surrounding pericentriolar material (PCM). Many vertebrate cells also have an array of granules, termed centriolar satellites, that localize around the centrosome and are associated with centrosome and cilium function. Centriole duplication occurs once per cell cycle and is effected by a set of proteins including PLK4, CEP192, CEP152, CEP63, and CPAP. Information on the relationships between these components is limited due to the difficulty in assaying interactions in the context of the centrosome. Here, we used proximity-dependent biotin identification (BioID) to identify proximity interactions among centriole duplication proteins. PLK4, CEP192, and CEP152 BioID identified known physically interacting proteins and a new interaction between CEP152 and CDK5RAP2 consistent with a function of CEP152 in PCM recruitment. BioID for CEP63 and its paralog CCDC67 revealed extensive proximity interactions with centriolar satellite proteins. Focusing on these satellite proteins identified two new regulators of centriole duplication, CCDC14 and KIAA0753. Both proteins colocalize with CEP63 to satellites, bind to CEP63, and identify other satellite proteins by BioID. KIAA0753 positively regulates centriole duplication and CEP63 centrosome localization, whereas CCDC14 negatively regulates both processes. These results suggest that centriolar satellites have a previously unappreciated function in regulating centriole duplication. PMID:24613305

  2. A Phytoremediation Strategy for Arsenic

    SciTech Connect

    Meagher, Richard B.

    2005-06-01

    . Phytochelatins bind diverse thiol-reactive elements like As(III) and are synthesized from amino acids in a three-step enzymatic pathway utilizing three enzymes: ECS = gamma-glutamylcysteine synthetase; GS = GSH synthetase; and PS = phytochelatin synthase. We cloned each of the genes that encode these enzymes and used at least two different plant promoters to express them in transgenic Arabidopsis. We have shown that all three confer significant resistance to arsenic and allow rapid growth on a concentration of arsenic (300 micromolar) that kills wild-type seeds and plants.

  3. Applying carbon dioxide, plant growth-promoting rhizobacterium and EDTA can enhance the phytoremediation efficiency of ryegrass in a soil polluted with zinc, arsenic, cadmium and lead.

    PubMed

    Guo, Junkang; Feng, Renwei; Ding, Yongzhen; Wang, Ruigang

    2014-08-01

    This study was conducted to investigate the use of elevated carbon dioxide (CO2), plant growth-promoting rhizobacterium Burkholderia sp. D54 (PGPR) and ethylenediaminetetraacetic acid (EDTA) to enhance the phytoextraction efficiency of ryegrass in response to multiple heavy metal (or metalloid)-polluted soil containing zinc (Zn), arsenic (As), cadmium (Cd) and lead (Pb). All of the single or combined CO2, PGPR and EDTA treatments promoted ryegrass growth. The stimulation of ryegrass growth by CO2 and PGPR could primarily be attributed to the regulation of photosynthesis rather than decreased levels of Zn, As and Cd in the shoots. Most treatments seemed to reduce the Zn, As and Cd contents in the shoots, which might be associated with enhanced shoot biomass, thus causing a "dilution effect" regarding their levels. The combined treatments seemed to perform better than single treatments in removing Zn, As, Cd and Pb from soil, judging from the larger biomass and relatively higher total amounts (TAs) of Zn, As, Cd and Pb in both the shoots and roots. Therefore, we suggest that the CO2 plus PGPR treatment will be suitable for removing Zn, As, Cd and Pb from heavy metal (or metalloid)-polluted soils using ryegrass as a phytoremediation material. PMID:24762567

  4. Cell confinement controls centrosome positioning and lumen initiation during epithelial morphogenesis

    PubMed Central

    Rodríguez-Fraticelli, Alejo E.; Auzan, Muriel; Alonso, Miguel A.; Bornens, Michel

    2012-01-01

    Epithelial organ morphogenesis involves sequential acquisition of apicobasal polarity by epithelial cells and development of a functional lumen. In vivo, cells perceive signals from components of the extracellular matrix (ECM), such as laminin and collagens, as well as sense physical conditions, such as matrix stiffness and cell confinement. Alteration of the mechanical properties of the ECM has been shown to promote cell migration and invasion in cancer cells, but the effects on epithelial morphogenesis have not been characterized. We analyzed the effects of cell confinement on lumen morphogenesis using a novel, micropatterned, three-dimensional (3D) Madin-Darby canine kidney cell culture method. We show that cell confinement, by controlling cell spreading, limits peripheral actin contractility and promotes centrosome positioning and lumen initiation after the first cell division. In addition, peripheral actin contractility is mediated by master kinase Par-4/LKB1 via the RhoA–Rho kinase–myosin II pathway, and inhibition of this pathway restores lumen initiation in minimally confined cells. We conclude that cell confinement controls nuclear–centrosomal orientation and lumen initiation during 3D epithelial morphogenesis. PMID:22965908

  5. Cortical interneurons migrating on a pure substrate of N-cadherin exhibit fast synchronous centrosomal and nuclear movements and reduced ciliogenesis

    PubMed Central

    Luccardini, Camilla; Leclech, Claire; Viou, Lucie; Rio, Jean-Paul; Métin, Christine

    2015-01-01

    The embryonic development of the cortex involves a phase of long distance migration of interneurons born in the basal telencephalon. Interneurons first migrate tangentially and then reorient their trajectories radially to enter the developing cortex. We have shown that migrating interneurons can assemble a primary cilium, which maintains the centrosome to the plasma membrane and processes signals to control interneuron trajectory (Baudoin et al., 2012). In the developing cortex, N-cadherin is expressed by migrating interneurons and by cells in their migratory pathway. N-cadherin promotes the motility and maintains the polarity of tangentially migrating interneurons (Luccardini et al., 2013). Because N-cadherin is an important factor that regulates the migration of medial ganglionic eminence (MGE) cells in vivo, we further characterized the motility and polarity of MGE cells on a substrate that only comprises this protein. MGE cells migrating on a N-cadherin substrate were seven times faster than on a laminin substrate and two times faster than on a substrate of cortical cells. A primary cilium was much less frequently observed on MGE cells migrating on N-cadherin than on laminin. Nevertheless, the mature centriole (MC) frequently docked to the plasma membrane in MGE cells migrating on N-cadherin, suggesting that plasma membrane docking is a basic feature of the centrosome in migrating MGE cells. On the N-cadherin substrate, centrosomal and nuclear movements were remarkably synchronous and the centrosome remained near the nucleus. Interestingly, MGE cells with cadherin invalidation presented centrosomal movements no longer coordinated with nuclear movements. In summary, MGE cells migrating on a pure substrate of N-cadherin show fast, coordinated nuclear and centrosomal movements, and rarely present a primary cilium. PMID:26283922

  6. Cilium, Centrosome and Cell Cycle Regulation in Polycystic Kidneys Disease

    PubMed Central

    Lee, Kyung; Battini, Lorenzo; Gusella, G. Luca

    2011-01-01

    Polycystic kidney disease is the defining condition of a group of common life-threatening genetic disorders characterized by the bilateral formation and progressive expansion of renal cysts that lead to end stage kidney disease. Although a large body of information has been acquired in the past years about the cellular functions that characterize the cystic cells, the mechanism(s) triggering the cystogenic conversion are just starting to emerge. Recent findings link defects in ciliary functions, planar cell polarity pathway, and centrosome integrity in early cystic development. Many of the signals dysregulated during cystogenesis may converge on the centrosome for its central function as a structural support for cilia formation and a coordinator of protein trafficking, polarity, and cell division. Here, we will discuss the contribution of proliferation, cilium and planar cell polarity to the cystic signal and will analyze in particular the possible role that the basal bodies/centrosome may play in the cystogenetic mechanisms. PMID:21376807

  7. Arsenic trioxide promotes mitochondrial DNA mutation and cell apoptosis in primary APL cells and NB4 cell line.

    PubMed

    Meng, Ran; Zhou, Jin; Sui, Meng; Li, ZhiYong; Feng, GuoSheng; Yang, BaoFeng

    2010-01-01

    This study aimed to investigate the effects of arsenic trioxide (As(2)O(3)) on the mitochondrial DNA (mtDNA) of acute promyelocytic leukemia (APL) cells. The NB4 cell line was treated with 2.0 micromol/L As(2)O(3) in vitro, and the primary APL cells were treated with 2.0 micromol/L As(2)O(3) in vitro and 0.16 mg kg(-1) d(-1) As(2)O(3) in vivo. The mitochondrial DNA of all the cells above was amplified by PCR, directly sequenced and analyzed by Sequence Navigatore and Factura software. The apoptosis rates were assayed by flow cytometry. Mitochondrial DNA mutation in the D-loop region was found in NB4 and APL cells before As(2)O(3) use, but the mutation spots were remarkably increased after As(2)O(3) treatment, which was positively correlated to the rates of cellular apoptosis, the correlation coefficient: r (NB4-As2O3)=0.973818, and r (APL-As2O3)=0.934703. The mutation types include transition, transversion, codon insertion or deletion, and the mutation spots in all samples were not constant and regular. It is revealed that As(2)O(3) aggravates mtDNA mutation in the D-loop region of acute promyelocytic leukemia cells both in vitro and in vivo. Mitochondrial DNA might be one of the targets of As(2)O(3) in APL treatment. PMID:20596959

  8. Arsenic Trioxide Activate Transcription of Heme Oxygenase-1 by Promoting Nuclear Translocation of NFE2L2

    PubMed Central

    Yue, Zhen; Zhong, Lingzhi; Mou, Yan; Wang, Xiaotong; Zhang, Haiying; Wang, Yang; Xia, Jianxin; Li, Ronggui; Wang, Zonggui

    2015-01-01

    In a previous study, we found that induced expression of Heme Oxygenase-1 (HO-1) is responsible for the resistance of human osteosarcoma MG63 cells to the chemotherapeutic agent arsenic trioxide (ATO). The present study was aimed at investigating the molecular mechanisms underlying the induction of HO-1 that occurs after exposure of MG63 cells to ATO. First, using RT-QPCT and Western-blot, we found that ATO strongly induced the expression of heme oxygenase-1 (HO-1) in these human osteosarcoma cells. Then by analyzing HO-1 mRNA of MG63 cells exposed to ATO in the presence and absence of a transcription inhibitor Actinomycin-D (Act-D), we demonstrated that ATO activates HO-1 expression in MG63 cells by regulating the transcription of the gene. Finally, through the analysis of the NFE2L2 protein levels among the total cellular and nuclear proteins by Western-blot and Immunocytochemical staning, we determined that ATO enhanced the nuclear translocation of nuclear factor erythroid 2-like 2 (NFE2L2), also known as Nrf2. From these results we have concluded that transcription activation of HO-1 resulting from the nuclear translocation of NFE2L2 is the underlying molecular mechanism for its high induction, which, in turn, is responsible for the resistance of human osteosarcoma cells to ATO treatment. PMID:26283888

  9. 14-3-3γ Prevents Centrosome Amplification and Neoplastic Progression

    PubMed Central

    Mukhopadhyay, Amitabha; Sehgal, Lalit; Bose, Arunabha; Gulvady, Anushree; Senapati, Parijat; Thorat, Rahul; Basu, Srikanta; Bhatt, Khyati; Hosing, Amol S.; Balyan, Renu; Borde, Lalit; Kundu, Tapas K.; Dalal, Sorab N.

    2016-01-01

    More than 80% of malignant tumors show centrosome amplification and clustering. Centrosome amplification results from aberrations in the centrosome duplication cycle, which is strictly coordinated with DNA-replication-cycle. However, the relationship between cell-cycle regulators and centrosome duplicating factors is not well understood. This report demonstrates that 14-3-3γ localizes to the centrosome and 14-3-3γ loss leads to centrosome amplification. Loss of 14-3-3γ results in the phosphorylation of NPM1 at Thr-199, causing early centriole disjunction and centrosome hyper-duplication. The centrosome amplification led to aneuploidy and increased tumor formation in mice. Importantly, an increase in passage of the 14-3-3γ-knockdown cells led to an increase in the number of cells containing clustered centrosomes leading to the generation of pseudo-bipolar spindles. The increase in pseudo-bipolar spindles was reversed and an increase in the number of multi-polar spindles was observed upon expression of a constitutively active 14-3-3-binding-defective-mutant of cdc25C (S216A) in the 14-3-3γ knockdown cells. The increase in multi-polar spindle formation was associated with decreased cell viability and a decrease in tumor growth. Our findings uncover the molecular basis of regulation of centrosome duplication by 14-3-3γ and inhibition of tumor growth by premature activation of the mitotic program and the disruption of centrosome clustering. PMID:27253419

  10. Centrosome movements in vivo correlate with specific neurite formation downstream of LIM homeodomain transcription factor activity.

    PubMed

    Andersen, Erica F; Halloran, Mary C

    2012-10-01

    Neurons must develop complex structure to form proper connections in the nervous system. The initiation of axons in defined locations on the cell body and their extension to synaptic targets are critical steps in neuronal morphogenesis, yet the mechanisms controlling axon formation in vivo are poorly understood. The centrosome has been implicated in multiple aspects of neuronal morphogenesis; however, its function in axon development is under debate. Conflicting results from studies of centrosome function in axonogenesis suggest that its role is context dependent and underscore the importance of studying centrosome function as neurons develop in their natural environment. Using live imaging of zebrafish Rohon-Beard (RB) sensory neurons in vivo, we discovered a spatiotemporal relationship between centrosome position and the formation of RB peripheral, but not central, axons. We tested centrosome function by laser ablation and found that centrosome disruption inhibited peripheral axon outgrowth. In addition, we show that centrosome position and motility are regulated by LIM homeodomain transcription factor activity, which is specifically required for the development of RB peripheral axons. Furthermore, we show a correlation between centrosome mislocalization and ectopic axon formation in bashful (laminin alpha 1) mutants. Thus, both intrinsic transcription factor activity and extracellular cues can influence centrosome position and axon formation in vivo. This study presents the first positive association between the centrosome and axon formation in vivo and suggests that the centrosome is important for differential neurite formation in neurons with complex axonal morphologies. PMID:22899847

  11. 14-3-3γ Prevents Centrosome Amplification and Neoplastic Progression.

    PubMed

    Mukhopadhyay, Amitabha; Sehgal, Lalit; Bose, Arunabha; Gulvady, Anushree; Senapati, Parijat; Thorat, Rahul; Basu, Srikanta; Bhatt, Khyati; Hosing, Amol S; Balyan, Renu; Borde, Lalit; Kundu, Tapas K; Dalal, Sorab N

    2016-01-01

    More than 80% of malignant tumors show centrosome amplification and clustering. Centrosome amplification results from aberrations in the centrosome duplication cycle, which is strictly coordinated with DNA-replication-cycle. However, the relationship between cell-cycle regulators and centrosome duplicating factors is not well understood. This report demonstrates that 14-3-3γ localizes to the centrosome and 14-3-3γ loss leads to centrosome amplification. Loss of 14-3-3γ results in the phosphorylation of NPM1 at Thr-199, causing early centriole disjunction and centrosome hyper-duplication. The centrosome amplification led to aneuploidy and increased tumor formation in mice. Importantly, an increase in passage of the 14-3-3γ-knockdown cells led to an increase in the number of cells containing clustered centrosomes leading to the generation of pseudo-bipolar spindles. The increase in pseudo-bipolar spindles was reversed and an increase in the number of multi-polar spindles was observed upon expression of a constitutively active 14-3-3-binding-defective-mutant of cdc25C (S216A) in the 14-3-3γ knockdown cells. The increase in multi-polar spindle formation was associated with decreased cell viability and a decrease in tumor growth. Our findings uncover the molecular basis of regulation of centrosome duplication by 14-3-3γ and inhibition of tumor growth by premature activation of the mitotic program and the disruption of centrosome clustering. PMID:27253419

  12. A Genome-Wide RNAi Screen to Dissect Centriole Duplication and Centrosome Maturation in Drosophila

    PubMed Central

    Dobbelaere, Jeroen; Josué, Filipe; Suijkerbuijk, Saskia; Baum, Buzz; Tapon, Nicolas; Raff, Jordan

    2008-01-01

    Centrosomes comprise a pair of centrioles surrounded by an amorphous pericentriolar material (PCM). Here, we have performed a microscopy-based genome-wide RNA interference (RNAi) screen in Drosophila cells to identify proteins required for centriole duplication and mitotic PCM recruitment. We analysed 92% of the Drosophila genome (13,059 genes) and identified 32 genes involved in centrosome function. An extensive series of secondary screens classified these genes into four categories: (1) nine are required for centriole duplication, (2) 11 are required for centrosome maturation, (3) nine are required for both functions, and (4) three genes regulate centrosome separation. These 32 hits include several new centrosomal components, some of which have human homologs. In addition, we find that the individual depletion of only two proteins, Polo and Centrosomin (Cnn) can completely block centrosome maturation. Cnn is phosphorylated during mitosis in a Polo-dependent manner, suggesting that the Polo-dependent phosphorylation of Cnn initiates centrosome maturation in flies. PMID:18798690

  13. Biochar in co-contaminated soil manipulates arsenic solubility and microbiological community structure, and promotes organochlorine degradation.

    PubMed

    Gregory, Samuel J; Anderson, Christopher W N; Camps-Arbestain, Marta; Biggs, Patrick J; Ganley, Austen R D; O'Sullivan, Justin M; McManus, Michael T

    2015-01-01

    We examined the effect of biochar on the water-soluble arsenic (As) concentration and the extent of organochlorine degradation in a co-contaminated historic sheep-dip soil during a 180-d glasshouse incubation experiment. Soil microbial activity, bacterial community and structure diversity were also investigated. Biochar made from willow feedstock (Salix sp) was pyrolysed at 350 or 550°C and added to soil at rates of 10 g kg-1 and 20 g kg-1 (representing 30 t ha-1 and 60 t ha-1). The isomers of hexachlorocyclohexane (HCH) alpha-HCH and gamma-HCH (lindane), underwent 10-fold and 4-fold reductions in concentration as a function of biochar treatment. Biochar also resulted in a significant reduction in soil DDT levels (P < 0.01), and increased the DDE:DDT ratio. Soil microbial activity was significantly increased (P < 0.01) under all biochar treatments after 60 days of treatment compared to the control. 16S amplicon sequencing revealed that biochar-amended soil contained more members of the Chryseobacterium, Flavobacterium, Dyadobacter and Pseudomonadaceae which are known bioremediators of hydrocarbons. We hypothesise that a recorded short-term reduction in the soluble As concentration due to biochar amendment allowed native soil microbial communities to overcome As-related stress. We propose that increased microbiological activity (dehydrogenase activity) due to biochar amendment was responsible for enhanced degradation of organochlorines in the soil. Biochar therefore partially overcame the co-contaminant effect of As, allowing for enhanced natural attenuation of organochlorines in soil. PMID:25923541

  14. Biochar in Co-Contaminated Soil Manipulates Arsenic Solubility and Microbiological Community Structure, and Promotes Organochlorine Degradation

    PubMed Central

    Gregory, Samuel J.; Anderson, Christopher W. N.; Camps-Arbestain, Marta; Biggs, Patrick J.; Ganley, Austen R. D.; O’Sullivan, Justin M.; McManus, Michael T.

    2015-01-01

    We examined the effect of biochar on the water-soluble arsenic (As) concentration and the extent of organochlorine degradation in a co-contaminated historic sheep-dip soil during a 180-d glasshouse incubation experiment. Soil microbial activity, bacterial community and structure diversity were also investigated. Biochar made from willow feedstock (Salix sp) was pyrolysed at 350 or 550°C and added to soil at rates of 10 g kg-1 and 20 g kg-1 (representing 30 t ha-1 and 60 t ha-1). The isomers of hexachlorocyclohexane (HCH) alpha-HCH and gamma-HCH (lindane), underwent 10-fold and 4-fold reductions in concentration as a function of biochar treatment. Biochar also resulted in a significant reduction in soil DDT levels (P < 0.01), and increased the DDE:DDT ratio. Soil microbial activity was significantly increased (P < 0.01) under all biochar treatments after 60 days of treatment compared to the control. 16S amplicon sequencing revealed that biochar-amended soil contained more members of the Chryseobacterium, Flavobacterium, Dyadobacter and Pseudomonadaceae which are known bioremediators of hydrocarbons. We hypothesise that a recorded short-term reduction in the soluble As concentration due to biochar amendment allowed native soil microbial communities to overcome As-related stress. We propose that increased microbiological activity (dehydrogenase activity) due to biochar amendment was responsible for enhanced degradation of organochlorines in the soil. Biochar therefore partially overcame the co-contaminant effect of As, allowing for enhanced natural attenuation of organochlorines in soil. PMID:25923541

  15. Aurora-A recruitment and centrosomal maturation are regulated by a Golgi-activated pool of Src during G2

    PubMed Central

    Barretta, Maria Luisa; Spano, Daniela; D'Ambrosio, Chiara; Cervigni, Romina Ines; Scaloni, Andrea; Corda, Daniela; Colanzi, Antonino

    2016-01-01

    The Golgi apparatus is composed of stacks of cisternae laterally connected by tubules to form a ribbon-like structure. At the onset of mitosis, the Golgi ribbon is broken down into discrete stacks, which then undergo further fragmentation. This ribbon cleavage is required for G2/M transition, which thus indicates that a ‘Golgi mitotic checkpoint' couples Golgi inheritance with cell cycle transition. We previously showed that the Golgi-checkpoint regulates the centrosomal recruitment of the mitotic kinase Aurora-A; however, how the Golgi unlinking regulates this recruitment was unknown. Here we show that, in G2, Aurora-A recruitment is promoted by activated Src at the Golgi. Our data provide evidence that Src and Aurora-A interact upon Golgi ribbon fragmentation; Src phosphorylates Aurora-A at tyrosine 148 and this specific phosphorylation is required for Aurora-A localization at the centrosomes. This process, pivotal for centrosome maturation, is a fundamental prerequisite for proper spindle formation and chromosome segregation. PMID:27242098

  16. Arsenic Induced Phytate Exudation, and Promoted FeAsO4 Dissolution and Plant Growth in As-Hyperaccumulator Pteris vittata.

    PubMed

    Liu, Xue; Fu, Jing-Wei; Guan, Dong-Xing; Cao, Yue; Luo, Jun; Rathinasabapathi, Bala; Chen, Yanshan; Ma, Lena Q

    2016-09-01

    Arsenic hyperaccumulator Pteris vittata (PV) is efficient in taking up As and nutrients from As-contaminated soils. We evaluated the mechanisms used by PV to mobilize As and Fe by examining the impacts of As and root exudates on FeAsO4 solubilization, and As and Fe uptake in four plants: As-hyperaccumulators PV and Pteris multifida (PM), nonhyperaccumulator Pteris ensiformis (PE), and angiosperm plant tomato (Solanum lycopersicum). Phytate and oxalate were dominant in fern plants (>93%), which were 50-83, 15-42, and 0-32 mg kg(-1) phytate and 10-15, 7-26, and 4-12 mg kg(-1) oxalate for PV, PM, and PE respectively, with higher As inducing greater phytate exudation and no phytate being detected in tomato exudates. PV treated with phytate+FeAsO4 had higher As and Fe contents and larger biomass than phytate or FeAsO4 treatment, which were 340 vs 20 and 130 mg kg(-1) As in the fronds and 7900 vs 1600 and 4100 mg kg(-1) Fe in the roots. We hypothesized that As-induced phytate exudation helped PV to take up Fe and As from insoluble FeAsO4 and promoted PV growth. Our study suggests that phytate exudation may be special to fern plants, which may play an important role in enhancing As and nutrient uptake by plants, thereby increasing their efficiency in phytoremediation of As-contaminated soils. PMID:27483027

  17. A Clinical Overview of Centrosome Amplification in Human Cancers

    PubMed Central

    Chan, Jason Yongsheng

    2011-01-01

    The turn of the 21st century had witnessed a surge of interest in the centrosome and its causal relation to human cancer development - a postulate that has existed for almost a century. Centrosome amplification (CA) is frequently detected in a growing list of human cancers, both solid and haematological, and is a candidate "hallmark" of cancer cells. Several lines of evidence support the progressive involvement of CA in the transition from early to advanced stages of carcinogenesis, being also found in pre-neoplastic lesions and even in histopathologically-normal tissue. CA constitutes the major mechanism leading to chromosomal instability and aneuploidy, via the formation of multipolar spindles and chromosomal missegregation. Clinically, CA may translate to a greater risk for initiation of malignant transformation, tumour progression, chemoresistance and ultimately, poor patient prognosis. As mechanisms underlying CA are progressively being unravelled, the centrosome has emerged as a novel candidate target for cancer treatment. This Review summarizes mainly the clinical studies performed to date focusing on the mechanisms underlying CA in human neoplasia, and highlights the potential utility of centrosomes in the diagnosis, prognosis and treatment of human cancers. PMID:22043171

  18. Conserved molecular interactions in centriole-to-centrosome conversion.

    PubMed

    Fu, Jingyan; Lipinszki, Zoltan; Rangone, Hélène; Min, Mingwei; Mykura, Charlotte; Chao-Chu, Jennifer; Schneider, Sandra; Dzhindzhev, Nikola S; Gottardo, Marco; Riparbelli, Maria Giovanna; Callaini, Giuliano; Glover, David M

    2016-01-01

    Centrioles are required to assemble centrosomes for cell division and cilia for motility and signalling. New centrioles assemble perpendicularly to pre-existing ones in G1-S and elongate throughout S and G2. Fully elongated daughter centrioles are converted into centrosomes during mitosis to be able to duplicate and organize pericentriolar material in the next cell cycle. Here we show that centriole-to-centrosome conversion requires sequential loading of Cep135, Ana1 (Cep295) and Asterless (Cep152) onto daughter centrioles during mitotic progression in both Drosophila melanogaster and human. This generates a molecular network spanning from the inner- to outermost parts of the centriole. Ana1 forms a molecular strut within the network, and its essential role can be substituted by an engineered fragment providing an alternative linkage between Asterless and Cep135. This conserved architectural framework is essential for loading Asterless or Cep152, the partner of the master regulator of centriole duplication, Plk4. Our study thus uncovers the molecular basis for centriole-to-centrosome conversion that renders daughter centrioles competent for motherhood. PMID:26595382

  19. Conserved Molecular Interactions in Centriole-to-Centrosome Conversion

    PubMed Central

    Fu, Jingyan; Lipinszki, Zoltan; Rangone, Hélène; Min, Mingwei; Mykura, Charlotte; Chao-Chu, Jennifer; Schneider, Sandra; Dzhindzhev, Nikola S.; Gottardo, Marco; Riparbelli, Maria Giovanna; Callaini, Giuliano; Glover, David M.

    2015-01-01

    Centrioles are required to assemble centrosomes for cell division and cilia for motility and signaling. New centrioles assemble perpendicularly to pre-existing ones in G1-S and elongate throughout S and G2. Fully-elongated daughter centrioles are converted into centrosomes during mitosis to be able to duplicate and organize pericentriolar material in the next cell cycle. Here we show that centriole-to-centrosome conversion requires sequential loading of Cep135, Ana1:Cep295 and Asterless:Cep152 onto daughter centrioles during mitotic progression. This generates a molecular network spanning from inner- to outer-most parts of the centriole. Ana1 forms a molecular strut within the network and its essential role can be substituted by an engineered fragment providing an alternative linkage between Asterless and Cep135. This conserved architectural framework is essential for loading Asterless:Cep152, partner of the master regulator of centriole duplication, Plk4. Our study thus uncovers the molecular basis for centriole-to-centrosome conversion that renders daughter centrioles competent for motherhood. PMID:26595382

  20. Centrosomal amplification and aneuploidy induced by the antiretroviral drug AZT in hamster and human cells

    PubMed Central

    Borojerdi, Jennifer P.; Ming, Jessica; Cooch, Catherine; Ward, Yvona; Semino-Mora, Cristina; Yu, Mia; Braun, Hannan M.; Taylor, Barbara J.; Poirier, Miriam C.; Olivero, Ofelia A.

    2009-01-01

    The centrosome directs chromosomal migration by a complex process of tubulin-chromatin binding. In this contribution centrosomal abnormalities, including centrosomal amplification, were explored in Chinese Hamster Ovary (CHO) and Normal Human Mammary Epithelial (NHMEC) cells exposed to the antiretroviral drug zidovudine (3’-azido-3’-deoxythymidine, AZT). Centrosomal amplification/fragmentation was observed in both cell types and kinetochore positive micronuclei were found in AZT-exposed CHO cells in correlation with dose. Normal human mammary epithelial cell (NMHEC), strain M99005, previously identified as a strain that incorporates high levels of AZT into DNA (High incorporator, HI), showed greater centrosomal amplification when compared with a second strain, NHMEC M98040, which did not incorporate AZT into DNA (Low incorporator, LI). Additionally, an abnormal tubulin distribution was observed in AZT-exposed HI cells bearing multiple centrosomes. Immunofluorescent staining of human cells with Aurora A, a kinase involved in the maturation of the centrosome, confirmed the induction of centrosomal amplification and revealed multipolar mitotic figures. Flow cytometric studies revealed that cells bearing abnormal numbers of centrosomes and abnormal tubulin distribution had similar S-phase percentages suggesting that cells bearing unbalanced chromosomal segregation could divide. Therefore, AZT induces genomic instability and clastogenicity as well as alterations in proteins involved in centrosomal activation, all of which may contribute to the carcinogenic properties of this compound. PMID:19427513

  1. Centrosomal abnormalities characterize human and rodent cystic cholangiocytes and are associated with Cdc25A overexpression.

    PubMed

    Masyuk, Tatyana V; Lee, Seung-Ok; Radtke, Brynn N; Stroope, Angela J; Huang, Bing; Banales, Jesús M; Masyuk, Anatoliy I; Splinter, Patrick L; Gradilone, Sergio A; Gajdos, Gabriella B; LaRusso, Nicholas F

    2014-01-01

    Hepatic cystogenesis in polycystic liver diseases is associated with abnormalities of cholangiocyte cilia. Given the crucial association between cilia and centrosomes, we tested the hypothesis that centrosomal defects occur in cystic cholangiocytes of rodents (Pkd2(WS25/-) mice and PCK rats) and of patients with polycystic liver diseases, contributing to disturbed ciliogenesis and cyst formation. We examined centrosomal cytoarchitecture in control and cystic cholangiocytes, the effects of centrosomal abnormalities on ciliogenesis, and the role of the cell-cycle regulator Cdc25A in centrosomal defects by depleting cholangiocytes of Cdc25A in vitro and in vivo and evaluating centrosome morphology, cell-cycle progression, proliferation, ciliogenesis, and cystogenesis. The cystic cholangiocytes had atypical centrosome positioning, supernumerary centrosomes, multipolar spindles, and extra cilia. Structurally aberrant cilia were present in cystic cholangiocytes during ciliogenesis. Depletion of Cdc25A resulted in i) a decreased number of centrosomes and multiciliated cholangiocytes, ii) an increased fraction of ciliated cholangiocytes with longer cilia, iii) a decreased proportion of cholangiocytes in G1/G0 and S phases of the cell cycle, iv) decreased cell proliferation, and v) reduced cyst growth in vitro and in vivo. Our data support the hypothesis that centrosomal abnormalities in cholangiocytes are associated with aberrant ciliogenesis and that accelerated cystogenesis is likely due to overexpression of Cdc25A, providing additional evidence that pharmacological targeting of Cdc25A has therapeutic potential in polycystic liver diseases. PMID:24211536

  2. Dishevelled is a NEK2 kinase substrate controlling dynamics of centrosomal linker proteins.

    PubMed

    Cervenka, Igor; Valnohova, Jana; Bernatik, Ondrej; Harnos, Jakub; Radsetoulal, Matej; Sedova, Katerina; Hanakova, Katerina; Potesil, David; Sedlackova, Miroslava; Salasova, Alena; Steinhart, Zachary; Angers, Stephane; Schulte, Gunnar; Hampl, Ales; Zdrahal, Zbynek; Bryja, Vitezslav

    2016-08-16

    Dishevelled (DVL) is a key scaffolding protein and a branching point in Wnt signaling pathways. Here, we present conclusive evidence that DVL regulates the centrosomal cycle. We demonstrate that DVL dishevelled and axin (DIX) domain, but not DIX domain-mediated multimerization, is essential for DVL's centrosomal localization. DVL accumulates during the cell cycle and associates with NIMA-related kinase 2 (NEK2), which is able to phosphorylate DVL at a multitude of residues, as detected by a set of novel phospho-specific antibodies. This creates interfaces for efficient binding to CDK5 regulatory subunit-associated protein 2 (CDK5RAP2) and centrosomal Nek2-associated protein 1 (C-NAP1), two proteins of the centrosomal linker. Displacement of DVL from the centrosome and its release into the cytoplasm on NEK2 phosphorylation is coupled to the removal of linker proteins, an event necessary for centrosomal separation and proper formation of the mitotic spindle. Lack of DVL prevents NEK2-controlled dissolution of loose centrosomal linker and subsequent centrosomal separation. Increased DVL levels, in contrast, sequester centrosomal NEK2 and mimic monopolar spindle defects induced by a dominant negative version of this kinase. Our study thus uncovers molecular crosstalk between centrosome and Wnt signaling. PMID:27486244

  3. Arsenic transformation and plant growth promotion characteristics of As-resistant endophytic bacteria from As-hyperaccumulator Pteris vittata.

    PubMed

    Xu, Jia-Yi; Han, Yong-He; Chen, Yanshan; Zhu, Ling-Jia; Ma, Lena Q

    2016-02-01

    The ability of As-resistant endophytic bacteria in As transformation and plant growth promotion was determined. The endophytes were isolated from As-hyperaccumulator Pteris vittata (PV) after growing for 60 d in a soil containing 200 mg kg(-1) arsenate (AsV). They were isolated in presence of 10 mM AsV from PV roots, stems, and leaflets, representing 4 phyla and 17 genera. All endophytes showed at least one plant growth promoting characteristics including IAA synthesis, siderophore production and P solubilization. The root endophytes had higher P solubilization ability than the leaflet (60.0 vs. 18.3 mg L(-1)). In presence of 10 mM AsV, 6 endophytes had greater growth than the control, suggesting As-stimulated growth. Furthermore, root endophytes were more resistant to AsV while the leaflet endophytes were more tolerant to arsenite (AsIII), which corresponded to the dominant As species in PV tissues. Bacterial As resistance was positively correlated to their ability in AsV reduction but not AsIII oxidation. The roles of those endophytes in promoting plant growth and As resistance in P. vittata warrant further investigation. PMID:26469935

  4. Increased centrosome amplification in aged stem cells of the Drosophila midgut

    SciTech Connect

    Park, Joung-Sun; Pyo, Jung-Hoon; Na, Hyun-Jin; Jeon, Ho-Jun; Kim, Young-Shin; Arking, Robert; Yoo, Mi-Ae

    2014-07-25

    Highlights: • Increased centrosome amplification in ISCs of aged Drosophila midguts. • Increased centrosome amplification in ISCs of oxidative stressed Drosophila midguts. • Increased centrosome amplification in ISCs by overexpression of PVR, EGFR, and AKT. • Supernumerary centrosomes can be responsible for abnormal ISC polyploid cells. • Supernumerary centrosomes can be a useful marker for aging stem cells. - Abstract: Age-related changes in long-lived tissue-resident stem cells may be tightly linked to aging and age-related diseases such as cancer. Centrosomes play key roles in cell proliferation, differentiation and migration. Supernumerary centrosomes are known to be an early event in tumorigenesis and senescence. However, the age-related changes of centrosome duplication in tissue-resident stem cells in vivo remain unknown. Here, using anti-γ-tubulin and anti-PH3, we analyzed mitotic intestinal stem cells with supernumerary centrosomes in the adult Drosophila midgut, which may be a versatile model system for stem cell biology. The results showed increased centrosome amplification in intestinal stem cells of aged and oxidatively stressed Drosophila midguts. Increased centrosome amplification was detected by overexpression of PVR, EGFR, and AKT in intestinal stem cells/enteroblasts, known to mimic age-related changes including hyperproliferation of intestinal stem cells and hyperplasia in the midgut. Our data show the first direct evidence for the age-related increase of centrosome amplification in intestinal stem cells and suggest that the Drosophila midgut is an excellent model for studying molecular mechanisms underlying centrosome amplification in aging adult stem cells in vivo.

  5. Inhibition of Proteasome Activity Impairs Centrosome-dependent Microtubule Nucleation and Organization

    PubMed Central

    Didier, Christine; Merdes, Andreas; Gairin, Jean-Edouard

    2008-01-01

    Centrosomes are dynamic organelles that consist of a pair of cylindrical centrioles, surrounded by pericentriolar material. The pericentriolar material contains factors that are involved in microtubule nucleation and organization, and its recruitment varies during the cell cycle. We report here that proteasome inhibition in HeLa cells induces the accumulation of several proteins at the pericentriolar material, including gamma-tubulin, GCP4, NEDD1, ninein, pericentrin, dynactin, and PCM-1. The effect of proteasome inhibition on centrosome proteins does not require intact microtubules and is reversed after removal of proteasome inhibitors. This accrual of centrosome proteins is paralleled by accumulation of ubiquitin in the same area and increased polyubiquitylation of nonsoluble gamma-tubulin. Cells that have accumulated centrosome proteins in response to proteasome inhibition are impaired in microtubule aster formation. Our data point toward a role of the proteasome in the turnover of centrosome proteins, to maintain proper centrosome function. PMID:18094058

  6. Direct Microtubule-Binding by Myosin-10 Orients Centrosomes toward Retraction Fibers and Subcortical Actin Clouds.

    PubMed

    Kwon, Mijung; Bagonis, Maria; Danuser, Gaudenz; Pellman, David

    2015-08-10

    Positioning of centrosomes is vital for cell division and development. In metazoan cells, spindle positioning is controlled by a dynamic pool of subcortical actin that organizes in response to the position of retraction fibers. These actin "clouds" are proposed to generate pulling forces on centrosomes and mediate spindle orientation. However, the motors that pull astral microtubules toward these actin structures are not known. Here, we report that the unconventional myosin, Myo10, couples actin-dependent forces from retraction fibers and subcortical actin clouds to centrosomes. Myo10-mediated centrosome positioning requires its direct microtubule binding. Computational image analysis of large microtubule populations reveals a direct effect of Myo10 on microtubule dynamics and microtubule-cortex interactions. Myo10's role in centrosome positioning is distinct from, but overlaps with, that of dynein. Thus, Myo10 plays a key role in integrating the actin and microtubule cytoskeletons to position centrosomes and mitotic spindles. PMID:26235048

  7. The Centrosome Undergoes Plk1-Independent Interphase Maturation during Inflammation and Mediates Cytokine Release.

    PubMed

    Vertii, Anastassiia; Ivshina, Maria; Zimmerman, Wendy; Hehnly, Heidi; Kant, Shashi; Doxsey, Stephen

    2016-05-23

    Cytokine production is a necessary event in the immune response during inflammation and is associated with mortality during sepsis, autoimmune disorders, cancer, and diabetes. Stress-activated MAP kinase signaling cascades that mediate cytokine synthesis are well established. However, the downstream fate of cytokines before they are secreted remains elusive. We report that pro-inflammatory stimuli lead to recruitment of pericentriolar material, specifically pericentrin and γ-tubulin, to the centrosome. This is accompanied by enhanced microtubule nucleation and enrichment of the recycling endosome component FIP3, all of which are hallmarks of centrosome maturation during mitosis. Intriguingly, centrosome maturation occurs during interphase in an MLK-dependent manner, independent of the classic mitotic kinase, Plk1. Centrosome disruption by chemical prevention of centriole assembly or genetic ablation of pericentrin attenuated interleukin-6, interleukin-10, and MCP1 secretion, suggesting that the centrosome is critical for cytokine production. Our results reveal a function of the centrosome in innate immunity. PMID:27219065

  8. Centrosomes and spindles in ascidian embryos and eggs.

    PubMed

    McDougall, Alex; Chenevert, Janet; Pruliere, Gerard; Costache, Vlad; Hebras, Celine; Salez, Gregory; Dumollard, Remi

    2015-01-01

    During embryonic development and maternal meiotic maturation, positioning of the mitotic/meiotic spindle is subject to control mechanisms that meet the needs of the particular cell type. Here we review the methods, molecular tools, and the ascidian model we use to study three different ways in which centrosomes or spindles are positioned in three different cellular contexts. First, we review unequal cleavage in the ascidian germ lineage. In the germ cell precursors, a large macromolecular structure termed the centrosome-attracting body causes three successive rounds of unequal cleavage from the 8- to the 64-cell stage. Next, we discuss spindle positioning underlying the invariant cleavage pattern. Ascidian embryos display an invariant cleavage pattern whereby the mitotic spindle aligns in a predetermined orientation in every blastomere up to the gastrula stage (composed of 112 cells). Finally, we review methods and approaches to study meiotic spindle positioning in eggs. PMID:26175446

  9. Automated tracking and analysis of centrosomes in early Caenorhabditis elegans embryos

    PubMed Central

    Jaensch, Steffen; Decker, Markus; Hyman, Anthony A.; Myers, Eugene W.

    2010-01-01

    Motivation: The centrosome is a dynamic structure in animal cells that serves as a microtubule organizing center during mitosis and also regulates cell-cycle progression and sets polarity cues. Automated and reliable tracking of centrosomes is essential for genetic screens that study the process of centrosome assembly and maturation in the nematode Caenorhabditis elegans. Results: We have developed a fully automatic system for tracking and measuring fluorescently labeled centrosomes in 3D time-lapse images of early C.elegans embryos. Using a spinning disc microscope, we monitor the centrosome cycle in living embryos from the 1- up to the 16-cell stage at imaging intervals between 30 and 50 s. After establishing the centrosome trajectories with a novel method involving two layers of inference, we also automatically detect the nuclear envelope breakdown in each cell division and recognize the identities of the centrosomes based on the invariant cell lineage of C.elegans. To date, we have tracked centrosomes in over 500 wild type and mutant embryos with almost no manual correction required. Availability: The centrosome tracking software along with test data is freely available at http://publications.mpi-cbg.de/itemPublication.html?documentId=4082 Contact: jaensch@mpi-cbg.de PMID:20529897

  10. Chronic Exposure to Particulate Chromate Induces Premature Centrosome Separation and Centriole Disengagement in Human Lung Cells.

    PubMed

    Martino, Julieta; Holmes, Amie L; Xie, Hong; Wise, Sandra S; Wise, John Pierce

    2015-10-01

    Particulate hexavalent chromium (Cr(VI)) is a well-established human lung carcinogen. Lung tumors are characterized by structural and numerical chromosome instability. Centrosome amplification is a phenotype commonly found in solid tumors, including lung tumors, which strongly correlates with chromosome instability. Human lung cells exposed to Cr(VI) exhibit centrosome amplification but the underlying phenotypes and mechanisms remain unknown. In this study, we further characterize the phenotypes of Cr(VI)-induced centrosome abnormalities. We show that Cr(VI)-induced centrosome amplification correlates with numerical chromosome instability. We also show chronic exposure to particulate Cr(VI) induces centrosomes with supernumerary centrioles and acentriolar centrosomes in human lung cells. Moreover, chronic exposure to particulate Cr(VI) affects the timing of important centriolar events. Specifically, chronic exposure to particulate Cr(VI) causes premature centriole disengagement in S and G2 phase cells. It also induces premature centrosome separation in interphase. Altogether, our data suggest that chronic exposure to particulate Cr(VI) targets the protein linkers that hold centrioles together. These centriolar linkers are important for key events of the centrosome cycle and their premature disruption might underlie Cr(VI)-induced centrosome amplification. PMID:26293554

  11. Centrosomes in the DNA damage response--the hub outside the centre.

    PubMed

    Mullee, Lisa I; Morrison, Ciaran G

    2016-01-01

    Here, we review how DNA damage affects the centrosome and how centrosomes communicate with the DNA damage response (DDR) apparatus. We discuss how several proteins of the DDR are found at centrosomes, including the ATM, ATR, CHK1 and CHK2 kinases, the BRCA1 ubiquitin ligase complex and several members of the poly(ADP-ribose) polymerase family. Stereotypical centrosome organisation, in which two centriole barrels are orthogonally arranged in a roughly toroidal pericentriolar material (PCM), is strongly affected by exposure to DNA-damaging agents. We describe the genetic dependencies and mechanisms for how the centrioles lose their close association, and the PCM both expands and distorts after DNA damage. Another consequence of genotoxic stress is that centrosomes undergo duplication outside the normal cell cycle stage, meaning that centrosome amplification is commonly seen after DNA damage. We discuss several potential mechanisms for how centrosome numbers become dysregulated after DNA damage and explore the links between the DDR and the PLK1- and separase-dependent mechanisms that drive centriole separation and reduplication. We also describe how centrosome components, such as centrin2, are directly involved in responding to DNA damage. This review outlines current questions on the involvement of centrosomes in the DDR. PMID:26614090

  12. Arsenic, inorganic

    Integrated Risk Information System (IRIS)

    Arsenic , inorganic ; CASRN 7440 - 38 - 2 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinoge

  13. Lamins position the nuclear pores and centrosomes by modulating dynein

    PubMed Central

    Guo, Yuxuan; Zheng, Yixian

    2015-01-01

    Lamins, the type V nuclear intermediate filament proteins, are reported to function in both interphase and mitosis. For example, lamin deletion in various cell types can lead to an uneven distribution of the nuclear pore complexes (NPCs) in the interphase nuclear envelope, whereas deletion of B-type lamins results in spindle orientation defects in mitotic neural progenitor cells. How lamins regulate these functions is unknown. Using mouse cells deleted of different combinations or all lamins, we show that lamins are required to prevent the aggregation of NPCs in the nuclear envelope near centrosomes in late G2 and prophase. This asymmetric NPC distribution in the absence of lamins is caused by dynein forces acting on NPCs via the dynein adaptor BICD2. We further show that asymmetric NPC distribution upon lamin depletion disrupts the distribution of BICD2 and p150 dynactin on the nuclear envelope at prophase, which results in inefficient dynein-driven centrosome separation during prophase. Therefore lamins regulate microtubule-based motor forces in vivo to ensure proper NPC distribution in interphase and centrosome separation in the mitotic prophase. PMID:26246603

  14. APC functions at the centrosome to stimulate microtubule growth.

    PubMed

    Lui, Christina; Ashton, Cahora; Sharma, Manisha; Brocardo, Mariana G; Henderson, Beric R

    2016-01-01

    The adenomatous polyposis coli (APC) tumor suppressor is multi-functional. APC is known to localize at the centrosome, and in mitotic cells contributes to formation of the mitotic spindle. To test whether APC contributes to nascent microtubule (MT) growth at interphase centrosomes, we employed MT regrowth assays in U2OS cells to measure MT assembly before and after nocodazole treatment and release. We showed that siRNA knockdown of full-length APC delayed both initial MT aster formation and MT elongation/regrowth. In contrast, APC-mutant SW480 cancer cells displayed a defect in MT regrowth that was unaffected by APC knockdown, but which was rescued by reconstitution of full-length APC. Our findings identify APC as a positive regulator of centrosome MT initial assembly and suggest that this process is disrupted by cancer mutations. We confirmed that full-length APC associates with the MT-nucleation factor γ-tubulin, and found that the APC cancer-truncated form (1-1309) also bound to γ-tubulin through APC amino acids 1-453. While binding to γ-tubulin may help target APC to the site of MT nucleation complexes, additional C-terminal sequences of APC are required to stimulate and stabilize MT growth. PMID:26556314

  15. Cilium, centrosome and cell cycle regulation in polycystic kidney disease.

    PubMed

    Lee, Kyung; Battini, Lorenzo; Gusella, G Luca

    2011-10-01

    Polycystic kidney disease is the defining condition of a group of common life-threatening genetic disorders characterized by the bilateral formation and progressive expansion of renal cysts that lead to end stage kidney disease. Although a large body of information has been acquired in the past years about the cellular functions that characterize the cystic cells, the mechanisms triggering the cystogenic conversion are just starting to emerge. Recent findings link defects in ciliary functions, planar cell polarity pathway, and centrosome integrity in early cystic development. Many of the signals dysregulated during cystogenesis may converge on the centrosome for its central function as a structural support for cilia formation and a coordinator of protein trafficking, polarity, and cell division. Here, we will discuss the contribution of proliferation, cilium and planar cell polarity to the cystic signal and will analyze in particular the possible role that the basal bodies/centrosome may play in the cystogenetic mechanisms. This article is part of a Special Issue entitled: Polycystic Kidney Disease. PMID:21376807

  16. LEGOs® and legacies of centrioles and centrosomes

    PubMed Central

    Schatten, Gerald; Simerly, Calvin

    2015-01-01

    Centriole construction, now revealed by crystallography, proteomics, and imaging to be a sophisticated assembly of interlocking bricks, resembles LEGOs—albeit centrioles have remarkable dynamic capabilities, including self-assembly and dis-assembly, kinases and post-translational modifications, self-replication, and still mysterious mechanisms for transmission through each cell cycle and via the gametes during development. Centrioles are created by core proteins that aggregate to form unique ninefold-symmetrical paracrystalline cylinders. The centrosome then coalesces as a cloud of pericentriolar material (PCM) around the centriole. Together they comprise the cell’s microtubule organizing center (MTOC), which governs the shape, functions, and dynamics of the cell’s microtubule (MT) arrays. This includes the meiotic and mitotic spindle apparatus for chromosome segregation, the accuracy of which is crucial for avoiding aneuploidies and resulting cancer, birth defects, or infertility. Centrioles’ replication and transmission mechanisms—and reduplication blocks—across cell cycles and generations, are only now becoming tractable to molecular analysis, which allows research to address questions about spindle assembly with neither centrioles nor centrosomes or de novo centriole formation. Here we discuss the latest insights into centriole and centrosome assembly and function and their transgenerational inheritance. PMID:26249334

  17. Developing and testing theory-based and evidence-based interventions to promote switching to arsenic-safe wells in Bangladesh.

    PubMed

    Inauen, Jennifer; Mosler, Hans-Joachim

    2014-12-01

    Millions of people in Bangladesh drink arsenic-contaminated water despite increased awareness of consequences to health. Theory-based and evidence-based interventions are likely to have greater impact on people switching to existing arsenic-safe wells than providing information alone. To test this assumption, we first developed interventions based on an empirical test of the Risk, Attitudes, Norms, Abilities and Self-regulation (RANAS) model of behaviour change. In the second part of this study, a cluster-randomised controlled trial revealed that in accordance with our hypotheses, information alone showed smaller increases in switching to arsenic-safe wells than information with reminders or information with reminders and implementation intentions. PMID:23864069

  18. Centrosome detection in sea urchin eggs with a monoclonal antibody against Drosophila intermediate filament proteins: characterization of stages of the division cycle of centrosomes.

    PubMed

    Schatten, H; Walter, M; Mazia, D; Biessmann, H; Paweletz, N; Coffe, G; Schatten, G

    1987-12-01

    A mouse monoclonal antibody generated against Drosophila intermediate filament proteins (designated Ah6/5/9 and referred to herein as Ah6) is found to cross-react specifically with centrosomes in sea urchin eggs and with a 68-kDa antigen in eggs and isolated mitotic apparatus. When preparations stained with Ah6 are counterstained with a human autoimmune serum whose anti-centrosome activity has been established, the immunofluorescence images superimpose exactly. A more severe test of the specificity of the antibody demands that it display all of the stages of the centrosome cycle in the cell cycle: the flattening and spreading of the compact centrosomes followed by their division and the establishment of two compact poles. The test was made by an experimental design that uses a period of exposure of the eggs to 2-mercaptoethanol. This treatment allows observation of the stages of the centrosome cycle--separation, division, and bipolarization--while the chromosomes are arrested in metaphase. Mitosis is arrested in the presence of 0.1 M 2-mercaptoethanol. Chromosomes remain in a metaphase configuration while the centrosomes divide, producing four poles perpendicular to the original spindle axis. Microtubules are still present in the mitotic apparatus, as indicated by immunofluorescence and transmission electron microscopy. When 2-mercaptoethanol is removed, the chromosomes reorient to the poles of a tetrapolar (sometimes tripolar) mitotic apparatus. During the following cycle, the blastomeres form a monopolar mitotic apparatus. The observations of the centrosome cycle with the Ah6 antibody display very clearly all the stages that have been seen or deduced from work with other probes. The 68-kDa antigen that reacts with the Ah6 monoclonal antibody to Drosophila intermediate filament proteins must be a constant component of sea urchin centrosomes because it is present at all stages of the centrosome cycle. PMID:3120191

  19. Environmentally relevant concentration of arsenic trioxide and humic acid promoted tumor progression of human cervical cancer cells: In vivo and in vitro studies.

    PubMed

    Tsai, Min-Ling; Yen, Cheng-Chieh; Lu, Fung-Jou; Ting, Hung-Chih; Chang, Horng-Rong

    2016-09-01

    In a previous study, treatment at higher concentrations of arsenic trioxide or co-exposure to arsenic trioxide and humic acid was found to be inhibited cell growth of cervical cancer cells (SiHa cells) by reactive oxygen species generation. However, treatment at lower concentrations slightly increased cell viability. Here, we investigate the enhancement of progression effects of environmentally relevant concentration of humic acid and arsenic trioxide in SiHa cell lines in vitro and in vivo by measuring cell proliferation, migration, invasion, and the carcinogenesis-related protein (MMP-2, MMP-9, and VEGF-A) expressions. SiHa cells treated with low concentrations of humic acid and arsenic trioxide alone or in co-exposure significantly increased reactive oxygen species, glutathione levels, cell proliferation, scratch wound-healing activities, migration abilities, and MMP-2 expression as compared to the untreated control. In vivo the tumor volume of either single drug (humic acid or arsenic trioxide) or combined drug-treated group was significantly larger than that of the control for an additional 45 days after tumor cell injection on the back of NOD/SCID mice. Levels of MMP-2, MMP-9, and VEGF-A, also significantly increased compared to the control. Histopathologic effects of all tumor cells appeared round in cell shape with high mitosis, focal hyperkeratosis and epidermal hyperplasia in the skin, and some tumor growth in the muscle were observed. Our results may indicate that exposure to low concentrations of arsenic trioxide and humic acid is associated with the progression of cervical cancer. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1121-1132, 2016. PMID:25728215

  20. Actin and Arp2/3 localize at the centrosome of interphase cells

    SciTech Connect

    Hubert, Thomas; Vandekerckhove, Joel; Gettemans, Jan

    2011-01-07

    Research highlights: {yields} Actin was detected at the centrosome with the anti-actin antibody 1C7 that recognizes antiparallel ('lower dimer') actin dimers. {yields} Centrosomal actin was found in interphase but not mitotic MDA-MB-231 cells. {yields} Neither the anti-actin antibody C4 that binds to globular, monomer actin, nor the anti-actin antibody 2G2 that recognizes the nuclear conformation of actin detect actin at the centrosome. {yields} The Arp2/3 complex transiently localizes at the pericentriolar matrix but not at the centrioles of interphase HEK 293T cells. -- Abstract: Although many actin binding proteins such as cortactin and the Arp2/3 activator WASH localize at the centrosome, the presence and conformation of actin at the centrosome has remained elusive. Here, we report the localization of actin at the centrosome in interphase but not in mitotic MDA-MB-231 cells. Centrosomal actin was detected with the anti-actin antibody 1C7 that recognizes antiparallel ('lower dimer') actin dimers. In addition, we report the transient presence of the Arp2/3 complex at the pericentriolar matrix but not at the centrioles of interphase HEK 293T cells. Overexpression of an Arp2/3 component resulted in expansion of the pericentriolar matrix and selective accumulation of the Arp2/3 component in the pericentriolar matrix. Altogether, we hypothesize that the centrosome transiently recruits Arp2/3 to perform processes such as centrosome separation prior to mitotic entry, whereas the observed constitutive centrosomal actin staining in interphase cells reinforces the current model of actin-based centrosome reorientation toward the leading edge in migrating cells.

  1. Conserved motif of CDK5RAP2 mediates its localization to centrosomes and the Golgi complex.

    PubMed

    Wang, Zhe; Wu, Tao; Shi, Lin; Zhang, Lin; Zheng, Wei; Qu, Jianan Y; Niu, Ruifang; Qi, Robert Z

    2010-07-16

    As the primary microtubule-organizing centers, centrosomes require gamma-tubulin for microtubule nucleation and organization. Located in close vicinity to centrosomes, the Golgi complex is another microtubule-organizing organelle in interphase cells. CDK5RAP2 is a gamma-tubulin complex-binding protein and functions in gamma-tubulin attachment to centrosomes. In this study, we find that CDK5RAP2 localizes to the Golgi complex in an ATP- and centrosome-dependent manner and associates with Golgi membranes independently of microtubules. CDK5RAP2 contains a centrosome-targeting domain with its core region highly homologous to the Motif 2 (CM2) of centrosomin, a functionally related protein in Drosophila. This sequence, referred to as the CM2-like motif, is also conserved in related proteins in chicken and zebrafish. Therefore, CDK5RAP2 may undertake a conserved mechanism for centrosomal localization. Using a mutational approach, we demonstrate that the CM2-like motif plays a crucial role in the centrosomal and Golgi localization of CDK5RAP2. Furthermore, the CM2-like motif is essential for the association of the centrosome-targeting domain to pericentrin and AKAP450. The binding with pericentrin is required for the centrosomal and Golgi localization of CDK5RAP2, whereas the binding with AKAP450 is required for the Golgi localization. Although the CM2-like motif possesses the activity of Ca(2+)-independent calmodulin binding, binding of calmodulin to this sequence is dispensable for centrosomal and Golgi association. Altogether, CDK5RAP2 may represent a novel mechanism for centrosomal and Golgi localization. PMID:20466722

  2. Cdc6 localizes to S- and G2-phase centrosomes in a cell cycle-dependent manner

    SciTech Connect

    Kim, Gwang Su; Kang, Jeeheon; Bang, Sung Woong; Hwang, Deog Su

    2015-01-16

    Highlights: • Cdc6 protein is a component of the pre-replicative complex required for chromosomal replication initiation. • Cdc6 localized to centrosomes of S and G2 phases in a cell cycle-dependent manner. • The centrosomal localization was governed by centrosomal localization signal sequences of Cdc6. • Deletions or substitution mutations on the centrosomal localization signal interfered with centrosomal localization of the Cdc6 proteins. - Abstract: The Cdc6 protein has been primarily investigated as a component of the pre-replicative complex for the initiation of chromosome replication, which contributes to maintenance of chromosomal integrity. Here, we show that Cdc6 localized to the centrosomes during S and G2 phases of the cell cycle. The centrosomal localization was mediated by Cdc6 amino acid residues 311–366, which are conserved within other Cdc6 homologues and contains a putative nuclear export signal. Deletions or substitutions of the amino acid residues did not allow the proteins to localize to centrosomes. In contrast, DsRed tag fused to the amino acid residues localized to centrosomes. These results indicated that a centrosome localization signal is contained within amino acid residues 311–366. The cell cycle-dependent centrosomal localization of Cdc6 in S and G2 phases suggest a novel function of Cdc6 in centrosomes.

  3. Centrosome amplification, chromosomal instability and cancer: mechanistic, clinical and therapeutic issues.

    PubMed

    Cosenza, Marco Raffaele; Krämer, Alwin

    2016-01-01

    Centrosomes, the main microtubule-organizing centers in most animal cells, are of crucial importance for the assembly of a bipolar mitotic spindle and subsequent faithful segregation of chromosomes into two daughter cells. Centrosome abnormalities can be found in virtually all cancer types and have been linked to chromosomal instability (CIN) and tumorigenesis. Although our knowledge on centrosome structure, replication, and amplification has greatly increased within recent years, still only very little is known on nature, causes, and consequences of centrosome aberrations in primary tumor tissues. In this review, we summarize our current insights into the mechanistic link between centrosome aberrations, aneuploidy, CIN and tumorigenesis. Mechanisms of induction and cellular consequences of aneuploidy, tetraploidization and CIN, as well as origin and effects of supernumerary centrosomes will be discussed. In addition, animal models for both CIN and centrosome amplification will be outlined. Finally, we describe approaches to exploit centrosome amplification, aneuploidy and CIN for novel and specific anticancer treatment strategies based on the modulation of chromosome missegregation rates. PMID:26645976

  4. One to only two: a short history of the centrosome and its duplication

    PubMed Central

    Sluder, Greenfield

    2014-01-01

    This review discusses some of the history of the fundamental, but not fully solved problem of how the centrosome duplicates from one to only two as the cell prepares for mitosis. We start with some of the early descriptions of the centrosome and the remarkably prescient but then controversial inferences drawn concerning its function in the cell. For more than 100 years, one of the most difficult issues for the concept of the centrosome has been to integrate observations that centrosomes appear to be important for spindle assembly in animal cells yet are not evident in higher plant cells and some animal cells. This stirred debate over the existence of centrosomes and their importance. A parallel debate concerned the role of the centrioles in organizing centrosomes. The relatively recent elucidation of bipolar spindle assembly around chromatin allows a re-examination of the role of centrioles in controlling centrosome duplication in animal cells. The problem of how centrosomes precisely double in preparation for mitosis in animal cells has now moved to the mystery of how only one procentriole is assembled at each mother centriole. PMID:25047609

  5. Centrosomal Localization of Cyclin E-Cdk2 is Required for Initiation of DNA Synthesis

    PubMed Central

    Ferguson, Rebecca L.; Maller, James L.

    2010-01-01

    Summary Cyclin E-Cdk2 is known to regulate both DNA replication and centrosome duplication during the G1-S transition in the cell cycle [1–4], and disruption of centrosomes results in a G1 arrest in some cell types [5–7]. Localization of cyclin E on centrosomes is mediated by a 20 amino acid domain termed the centrosomal localization sequence (CLS), and expression of the GFP-tagged CLS displaces both cyclin E and cyclin A from the centrosome [8]. In asynchronous cells CLS expression inhibits the incorporation of bromodeoxyuridine (BrdU) into DNA, an effect proposed to reflect a G1 arrest. Here we show in synchronized cells that the reduction in BrdU incorporation reflects not a G1 arrest but rather direct inhibition of the initiation of DNA replication in S phase. The loading of essential DNA replication factors such as Cdc45 and PCNA onto chromatin is blocked by CLS expression, but DNA synthesis can be rescued by retargeting active cyclin E-Cdk2 to the centrosome. These results suggest that initial steps of DNA replication require centrosomally localized Cdk activity and link the nuclear cycle with the centrosome cycle at the G1-S transition. PMID:20399658

  6. Dynamic recruitment of CDK5RAP2 to centrosomes requires its association with dynein.

    PubMed

    Jia, Yue; Fong, Ka-Wing; Choi, Yuk-Kwan; See, Siu-San; Qi, Robert Z

    2013-01-01

    CDK5RAP2 is a centrosomal protein known to be involved in the regulation of the γ-tubulin ring complex and thus the organization of microtubule arrays. However, the mechanism by which CDK5RAP2 is itself recruited to centrosomes is poorly understood. We report here that CDK5RAP2 displays highly dynamic attachment to centrosomes in a microtubule-dependent manner. CDK5RAP2 associates with the retrograde transporter dynein-dynactin and contains a sequence motif that binds to dynein light chain 8. Significantly, disruption of cellular dynein-dynactin function reduces the centrosomal level of CDK5RAP2. These results reveal a key role of the dynein-dynactin complex in the dynamic recruitment of CDK5RAP2 to centrosomes. PMID:23874654

  7. Arsenic surveillance program

    NASA Technical Reports Server (NTRS)

    1993-01-01

    Background information about arsenic is presented including forms, common sources, and clinical symptoms of arsenic exposure. The purpose of the Arsenic Surveillance Program and LeRC is outlined, and the specifics of the Medical Surveillance Program for Arsenic Exposure at LeRC are discussed.

  8. Centrosome and spindle pole body dynamics. Review and abstracts of the EMBO/EMBL Conference on Centrosomes and Spindle Pole Bodies, Heidelberg, September 13-17, 2002.

    PubMed

    2003-02-01

    Five years after the first meeting held on Centrosomes and Spindle Pole Bodies, a second meeting was organized by Tano Gonzalez, Eric Karsenti, Kip Sluder, and Mark Winey in Heidelberg, Germany. Sponsored by the gracious European community (EMBO/EMBL), the meeting was both spectacular and exhausting. The wealth of information delivered, the plethora of model systems and unique approaches described, and the free exchange of information by a cooperative and excited community of scientists overwhelmed all participants. Even the best prepared scholars could not have anticipated the avalanche of data and insights that poured from the presentations from beginning to end. Daily posters by young and senior scientists added dimension to round out the well-planned series of presentations. The meeting began with opening remarks by Eric Karsenti and Michel Bornens who reminded participants of the historical questions of the field. Where does the centrosome come from? What are the mechanisms that control centrosome assembly and duplication? How is duplication coordinated with the cell cycle? Why do some cells have centrosomes, while others do not? What are the components of the centrosome? Does the centrosome play an important role in disease? PMID:12529860

  9. Earth Abides Arsenic Biotransformations

    PubMed Central

    Zhu, Yong-Guan; Yoshinaga, Masafumi; Zhao, Fang-Jie; Rosen, Barry P.

    2015-01-01

    Arsenic is the most prevalent environmental toxic element and causes health problems throughout the world. The toxicity, mobility, and fate of arsenic in the environment are largely determined by its speciation, and arsenic speciation changes are driven, at least to some extent, by biological processes. In this article, biotransformation of arsenic is reviewed from the perspective of the formation of Earth and the evolution of life, and the connection between arsenic geochemistry and biology is described. The article provides a comprehensive overview of molecular mechanisms of arsenic redox and methylation cycles as well as other arsenic biotransformations. It also discusses the implications of arsenic biotransformation in environmental remediation and food safety, with particular emphasis on groundwater arsenic contamination and arsenic accumulation in rice. PMID:26778863

  10. Earth Abides Arsenic Biotransformations

    NASA Astrophysics Data System (ADS)

    Zhu, Yong-Guan; Yoshinaga, Masafumi; Zhao, Fang-Jie; Rosen, Barry P.

    2014-05-01

    Arsenic is the most prevalent environmental toxic element and causes health problems throughout the world. The toxicity, mobility, and fate of arsenic in the environment are largely determined by its speciation, and arsenic speciation changes are driven, at least to some extent, by biological processes. In this article, biotransformation of arsenic is reviewed from the perspective of the formation of Earth and the evolution of life, and the connection between arsenic geochemistry and biology is described. The article provides a comprehensive overview of molecular mechanisms of arsenic redox and methylation cycles as well as other arsenic biotransformations. It also discusses the implications of arsenic biotransformation in environmental remediation and food safety, with particular emphasis on groundwater arsenic contamination and arsenic accumulation in rice.

  11. Transplacental arsenic carcinogenesis in mice

    SciTech Connect

    Waalkes, Michael P. Liu, Jie; Diwan, Bhalchandra A.

    2007-08-01

    Our work has focused on the carcinogenic effects of in utero arsenic exposure in mice. Our data show that a short period of maternal exposure to inorganic arsenic in the drinking water is an effective, multi-tissue carcinogen in the adult offspring. These studies have been reproduced in three temporally separate studies using two different mouse strains. In these studies pregnant mice were treated with drinking water containing sodium arsenite at up to 85 ppm arsenic from days 8 to 18 of gestation, and the offspring were observed for up to 2 years. The doses used in all these studies were well tolerated by both the dam and offspring. In C3H mice, two separate studies show male offspring exposed to arsenic in utero developed liver carcinoma and adrenal cortical adenoma in a dose-related fashion during adulthood. Prenatally exposed female C3H offspring show dose-related increases in ovarian tumors and lung carcinoma and in proliferative lesions (tumors plus preneoplastic hyperplasia) of the uterus and oviduct. In addition, prenatal arsenic plus postnatal exposure to the tumor promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA) in C3H mice produces excess lung tumors in both sexes and liver tumors in females. Male CD1 mice treated with arsenic in utero develop tumors of the liver and adrenal and renal hyperplasia while females develop tumors of urogenital system, ovary, uterus and adrenal and hyperplasia of the oviduct. Additional postnatal treatment with diethylstilbestrol or tamoxifen after prenatal arsenic in CD1 mice induces urinary bladder transitional cell proliferative lesions, including carcinoma and papilloma, and enhances the carcinogenic response in the liver of both sexes. Overall this model has provided convincing evidence that arsenic is a transplacental carcinogen in mice with the ability to target tissues of potential human relevance, such as the urinary bladder, lung and liver. Transplacental carcinogenesis clearly occurs with other agents in humans

  12. Interference with BRCA2, which localizes to the centrosome during S and early M phase, leads to abnormal nuclear division

    SciTech Connect

    Nakanishi, Akira; Han, Xiangzi; Saito, Hiroko; Taguchi, Keiko; Ohta, Yoshiyasu; Imajoh-Ohmi, Shinobu; Miki, Yoshio; E-mail: miki.mgen@mri.tmd.ac.jp

    2007-03-30

    BRCA2 is responsible for familial breast and ovarian cancer, and its gene product is linked to DNA repair and transcriptional regulation. The BRCA2 protein exists mainly in the nucleus. Here, we show that BRCA2 has a centrosomal localization signal (CLS), localizes also to centrosomes during S and early M phases, and may regulate duplication and separation of the centrosomes. Green fluorescent protein (GFP) fused to the CLS peptides from BRCA2 (GFP-CLS) localizes to centrosomes and prevents endogenous BRCA2 from localizing to centrosomes. In addition, expression of GFP-CLS in cells leads to the abnormal duplication and positioning of centrosomes, resulting in the generation of multinuclear cells. These results thus implicate BRCA2 in the regulation of the centrosome cycle, and provide new insight into the aneuploid nature of many breast cancers.

  13. THE CELLUAR METABOLISM OF ARSENIC

    EPA Science Inventory

    Because the methylation of arsenic produces intermediates and terminal products that exceed inorganic arsenic in potency as enzyme inhibitors, cytotoxins, and genotoxins, the methylation of arsenic is properly regarded as an activation process. The methylation of arsenic is an e...

  14. Promotion

    PubMed Central

    Alam, Hasan B.

    2013-01-01

    This article gives an overview of the promotion process in an academic medical center. A description of different promotional tracks, tenure and endowed chairs, and the process of submitting an application is provided. Finally, some practical advice about developing skills and attributes that can help with academic growth and promotion is dispensed. PMID:24436683

  15. Chem I Supplement: Arsenic and Old Myths.

    ERIC Educational Resources Information Center

    Sarquis, Mickey

    1979-01-01

    Describes the history of arsenic, the properties of arsenic, production and uses of arsenicals, arsenic in the environment; toxic levels of arsenic, arsenic in the human body, and the Marsh Test. (BT)

  16. Abnormal centrosomal structure and duplication in Cep135-deficient vertebrate cells

    PubMed Central

    Inanç, Burcu; Pütz, Monika; Lalor, Pierce; Dockery, Peter; Kuriyama, Ryoko; Gergely, Fanni; Morrison, Ciaran G.

    2013-01-01

    Centrosomes are key microtubule-organizing centers that contain a pair of centrioles, conserved cylindrical, microtubule-based structures. Centrosome duplication occurs once per cell cycle and relies on templated centriole assembly. In many animal cells this process starts with the formation of a radially symmetrical cartwheel structure. The centrosomal protein Cep135 localizes to this cartwheel, but its role in vertebrates is not well understood. Here we examine the involvement of Cep135 in centriole function by disrupting the Cep135 gene in the DT40 chicken B-cell line. DT40 cells that lack Cep135 are viable and show no major defects in centrosome composition or function, although we note a small decrease in centriole numbers and a concomitant increase in the frequency of monopolar spindles. Furthermore, electron microscopy reveals an atypical structure in the lumen of Cep135-deficient centrioles. Centrosome amplification after hydroxyurea treatment increases significantly in Cep135-deficient cells, suggesting an inhibitory role for the protein in centrosome reduplication during S-phase delay. We propose that Cep135 is required for the structural integrity of centrioles in proliferating vertebrate cells, a role that also limits centrosome amplification in S-phase–arrested cells. PMID:23864714

  17. The centriolar protein Bld10/Cep135 is required to establish centrosome asymmetry in Drosophila neuroblasts.

    PubMed

    Singh, Priyanka; Ramdas Nair, Anjana; Cabernard, Clemens

    2014-07-01

    Centrosome asymmetry has been implicated in stem cell fate maintenance in both flies and vertebrates [1, 2]. Drosophila neuroblasts, the neural precursors of the fly's central nervous system [3], contain molecularly and physically asymmetric centrosomes, established through differences in pericentriolar matrix (PCM) retention [4-7]. For instance, the daughter centriole maintains PCM and thus microtubule-organizing center (MTOC) activity through Polo-mediated phosphorylation of Centrobin (Cnb) [7, 8]. The mother centriole, however, quickly downregulates PCM and moves away from the apical cortex, randomly migrating through the cytoplasm until maturation sets in at prophase [4-6, 8]. How PCM downregulation is molecularly controlled is currently unknown, but it involves Pericentrin (PCNT)-like protein (PLP) to prevent premature Polo localization and thus MTOC activity [9]. Here, we report that the centriolar protein Bld10, the fly ortholog of Cep135, is required to establish centrosome asymmetry in Drosophila neuroblasts through shedding of Polo from the mother centrosome. bld10 mutants fail to downregulate Polo and PCM, generating two active, improperly positioned MTOCs. Failure to shed Polo and PCM causes spindle alignment and centrosome segregation defects, resulting in neuroblasts incorrectly retaining the older mother centrosome. Since Cep135 is implicated in primary microcephaly, we speculate that perturbed centrosome asymmetry could contribute to this rare neurodevelopmental disease. PMID:24954048

  18. Single Centrosome Manipulation Reveals Its Electric Charge and Associated Dynamic Structure

    PubMed Central

    Hormeño, S.; Ibarra, B.; Chichón, F.J.; Habermann, K.; Lange, B.M.H.; Valpuesta, J.M.; Carrascosa, J.L.; Arias-Gonzalez, J.R.

    2009-01-01

    Abstract The centrosome is the major microtubule-organizing center in animal cells and consists of a pair of centrioles surrounded by a pericentriolar material. We demonstrate laser manipulation of individual early Drosophila embryo centrosomes in between two microelectrodes to reveal that it is a net negatively charged organelle with a very low isoelectric region (3.1 ± 0.1). From this single-organelle electrophoresis, we infer an effective charge smaller than or on the order of 103 electrons, which corresponds to a surface-charge density significantly smaller than that of microtubules. We show, however, that the charge of the centrosome has a remarkable influence over its own structure. Specifically, we investigate the hydrodynamic behavior of the centrosome by measuring its size by both Stokes law and thermal-fluctuation spectral analysis of force. We find, on the one hand, that the hydrodynamic size of the centrosome is 60% larger than its electron microscopy diameter, and on the other hand, that this physiological expansion is produced by the electric field that drains to the centrosome, a self-effect that modulates its structural behavior via environmental pH. This methodology further proves useful for studying the action of different environmental conditions, such as the presence of Ca2+, over the thermally induced dynamic structure of the centrosome. PMID:19686649

  19. Chloroquine alleviates etoposide-induced centrosome amplification by inhibiting CDK2 in adrenocortical tumor cells

    PubMed Central

    Chen, T-Y; Syu, J-S; Lin, T-C; Cheng, H-l; Lu, F-l; Wang, C-Y

    2015-01-01

    The antitumor drug etoposide (ETO) is widely used in treating several cancers, including adrenocortical tumor (ACT). However, when used at sublethal doses, tumor cells still survive and are more susceptible to the recurring tumor due to centrosome amplification. Here, we checked the effect of sublethal dose of ETO in ACT cells. Sublethal dose of ETO treatment did not induce cell death but arrested the ACT cells in G2/M phase. This resulted in centrosome amplification and aberrant mitotic spindle formation leading to genomic instability and cellular senescence. Under such conditions, Chk2, cyclin A/CDK2 and ERK1/2 were aberrantly activated. Pharmacological inactivation of Chk2, CDK2 or ERK1/2 or depletion of CDK2 or Chk2 inhibited the centrosome amplification in ETO-treated ACT cells. In addition, autophagy was activated by ETO and was required for ACT cell survival. Chloroquine, the autophagy inhibitor, reduced ACT cell growth and inhibited ETO-induced centrosome amplification. Chloroquine alleviated CDK2 and ERK, but not Chk2, activation and thus inhibited centrosome amplification in either ETO- or hydroxyurea-treated ACT cells. In addition, chloroquine also inhibited centrosome amplification in osteosarcoma U2OS cell lines when treated with ETO or hydroxyurea. In summary, we have demonstrated that chloroquine inhibited ACT cell growth and alleviated DNA damage-induced centrosome amplification by inhibiting CDK2 and ERK activity, thus preventing genomic instability and recurrence of ACT. PMID:26690546

  20. Generation and Characterization of a Tissue-Specific Centrosome Indicator Mouse Line.

    PubMed

    Hirai, Maretoshi; Chen, Ju; Evans, Sylvia M

    2016-05-01

    Centrosomes are major microtubule organizing centers (MTOCs) that play an important role in chromosome segregation during cell division. Centrosomes provide a stable anchor for microtubules, constituting the centers of the spindle poles in mitotic cells, and determining the orientation of cell division. However, visualization of centrosomes is challenging because of their small size. Especially in mouse tissues, it has been extremely challenging to observe centrosomes belonging to a specific cell type of interest among multiple comingled cell types. To overcome this obstacle, we generated a tissue-specific centrosome indicator. In this mouse line, a construct containing a floxed neomyocin resistance gene with a triplicate polyA sequence followed by an EGFP-Centrin1 fusion cassette was knocked into the Rosa locus. Upon Cre-mediated excision, EGFP-Centrin1 was expressed under the control of the Rosa locus. Experiments utilizing mouse embryo fibroblasts (MEFs) demonstrated the feasibility of real-time imaging, and showed that EGFP-Centrin1 expression mirrored the endogenous centrosome cycle, undergoing precisely one round of duplication through the cell cycle. Moreover, experiments using embryo and adult mouse tissues demonstrated that EGFP-Centrin1 specifically mirrors the localization of endogenous centrosomes. genesis 54:286-296, 2016. © 2016 The Authors. Genesis Published by Wiley Periodicals, Inc. PMID:26990996

  1. Kaede-Centrin1 labeling of mother and daughter centrosomes in mammalian neocortical neural progenitors

    PubMed Central

    Imai, Janice H.; Wang, Xiaoqun; Shi, Song-Hai

    2010-01-01

    The importance of the centrosome in regulating basic cellular processes and cell fate decisions has become increasingly evident from recent studies tracing the etiology of developmental disorders to mutations in genes encoding centrosomal proteins (Nigg and Raff, 2009). This unit details a protocol for a fluorescence-based pulse-labeling of centrioles of neural progenitor cells in the developing neocortex of mice. In utero electroporation of Kaede-Centrin1 followed by in utero or ex vivo photoconversion allows a direct monitoring of the inheritance of centrosomes containing centrioles of different ages in dividing neocortical neural progenitors (i.e., radial glial cells). This is achieved by combining the irreversible photoconversion capacity of Kaede protein from green to red fluorescence with the faithful duplication of the centrosome during each cell cycle. After two mitotic divisions following photoconversion, mother centrosomes containing the original labeled centriole appear in both red and green fluorescence, and can be distinguished from daughter centrosomes which appear in green fluorescence only. This facilitates the study of the inheritance and behavior of the mother and daughter centrosomes in asymmetric cell divisions in the developing mammalian neocortex. PMID:20938915

  2. The distribution of centrosomes in endothelial cells of non-wounded and wounded aortic organ cultures.

    PubMed

    Rogers, K A; Boden, P; Kalnins, V I; Gotlieb, A I

    1986-01-01

    The distribution of centrosomes in porcine vascular endothelial cells of the thoracic aorta maintained in organ culture was determined in en face preparations using immunofluorescence. Rectangular pieces of aorta that had the distal half (with respect to the heart) of their endothelial surface gently denuded with a scalpel blade and pieces with intact endothelium were cultured for up to 96 h. At time 0, centrosomes were found to be preferentially oriented toward the heart, both in the cells of intact monolayers and in cells at the wound edge. This distribution was maintained in the intact monolayers for at least 24 h, but by 72 h the number of centrosomes in the center of the cells exceeded the number oriented toward the heart as the cells changed from a fusiform to a polygonal shape. The centrosomes of most endothelial cells at the wound edge began to redistribute themselves within the first 24 h in culture, moving from a position toward the heart to a position either in the center of the cell or away from the heart. By 72 h, the majority of centrosomes in endothelial cells at the wound edge were oriented away from the heart toward the denuded region. It is concluded that the centrosomes in the endothelial cells maintained in organ culture respond to injury in a manner similar to those grown in monolayer cell culture except that the reorientation of centrosomes occurs more slowly. PMID:3948234

  3. Epidermal development, growth control, and homeostasis in the face of centrosome amplification

    PubMed Central

    Kulukian, Anita; Holland, Andrew J.; Vitre, Benjamin; Naik, Shruti; Cleveland, Don W.; Fuchs, Elaine

    2015-01-01

    As nucleators of the mitotic spindle and primary cilium, centrosomes play crucial roles in equal segregation of DNA content to daughter cells, coordination of growth and differentiation, and transduction of homeostatic cues. Whereas the majority of mammalian cells carry no more than two centrosomes per cell, exceptions to this rule apply in certain specialized tissues and in select disease states, including cancer. Centrosome amplification, or the condition of having more than two centrosomes per cell, has been suggested to contribute to instability of chromosomes, imbalance in asymmetric divisions, and reorganization of tissue architecture; however, the degree to which these conditions are a direct cause of or simply a consequence of human disease is poorly understood. Here we addressed this issue by generating a mouse model inducing centrosome amplification in a naturally proliferative epithelial tissue by elevating Polo-like kinase 4 (Plk4) expression in the skin epidermis. By altering centrosome numbers, we observed multiciliated cells, spindle orientation errors, and chromosome segregation defects within developing epidermis. None of these defects was sufficient to impart a proliferative advantage within the tissue, however. Rather, impaired mitoses led to p53-mediated cell death and contributed to defective growth and stratification. Despite these abnormalities, mice remained viable and healthy, although epidermal cells with centrosome amplification were still appreciable. Moreover, these abnormalities were insufficient to disrupt homeostasis and initiate or enhance tumorigenesis, underscoring the powerful surveillance mechanisms in the skin. PMID:26578791

  4. BRCA2 mediates centrosome cohesion via an interaction with cytoplasmic dynein

    PubMed Central

    Malik, Sadiya; Saito, Hiroko; Takaoka, Miho; Miki, Yoshio; Nakanishi, Akira

    2016-01-01

    BRCA2 is responsible for familial breast and ovarian cancer and has been linked to DNA repair and centrosome duplication. Here we analyzed the mechanism by which the centrosomal localization signal (CLS) of BRCA2 interacts with cytoplasmic dynein 1 to localize BRCA2 to the centrosome. In vitro pull-down assays demonstrated that BRCA2 directly binds to the cytoplasmic dynein 1 light intermediate chain 2. A dominant-negative HA-CLS-DsRed fusion protein, the depletion of dynein by siRNA, and the inactivation of dynein by EHNA, inhibited the localization of BRCA2 at centrosomes and caused the separation of centrosome pairs during the S-phase. The double depletion of BRCA2 and C-Nap1 caused a larger dispersion of centrosome distances than the silencing of C-Nap1. These results suggest that cytoplasmic dynein 1 binds to BRCA2 through the latter's CLS and BRCA2 mediates the cohesion between centrosomes during the S phase, potentially serving as a cell-cycle checkpoint. PMID:27433848

  5. Cep68 and Cep215 (Cdk5rap2) are required for centrosome cohesion.

    PubMed

    Graser, Susanne; Stierhof, York-Dieter; Nigg, Erich A

    2007-12-15

    The centrosome duplicates during the cell cycle but functions as a single microtubule-organising centre until shortly before mitosis. This raises the question of how centrosome cohesion is maintained throughout interphase. One dynamic model proposes that parental centrioles are held together through centriole-associated, entangling filaments. Central to this model are C-Nap1, a putative centriolar docking protein and rootletin, a fibrous component. Here we identify two novel proteins, Cep68 and Cep215, as required for centrosome cohesion. Similar to rootletin, Cep68 decorates fibres emanating from the proximal ends of centrioles and dissociates from centrosomes during mitosis. Furthermore, Cep68 and rootletin depend both on each other and on C-Nap1 for centriole association. Unlike rootletin, overexpression of Cep68 does not induce extensive fibre formation, but Cep68 is readily recruited to ectopic rootletin fibres. These data suggest that Cep68 cooperates with rootletin and C-Nap1 in centrosome cohesion. By contrast, Cep215 associates with centrosomes throughout the cell cycle and does not appear to interact with Cep68, rootletin or C-Nap1. Instead, our data suggest that Cep215 functionally interacts with pericentrin, suggesting that both proteins influence centrosome cohesion through an indirect mechanism related to cytoskeletal dynamics. PMID:18042621

  6. Plk1-dependent recruitment of gamma-tubulin complexes to mitotic centrosomes involves multiple PCM components.

    PubMed

    Haren, Laurence; Stearns, Tim; Lüders, Jens

    2009-01-01

    The nucleation of microtubules requires protein complexes containing gamma-tubulin, which are present in the cytoplasm and associate with the centrosome and with the mitotic spindle. We have previously shown that these interactions require the gamma-tubulin targeting factor GCP-WD/NEDD1, which has an essential role in spindle formation. The recruitment of additional gamma-tubulin to the centrosomes occurs during centrosome maturation at the G2/M transition and is regulated by the mitotic kinase Plk1. However, the molecular details of this important pathway are unknown and a Plk1 substrate that controls gamma-tubulin recruitment has not been identified. Here we show that Plk1 associates with GCP-WD in mitosis and Plk1 activity contributes to phosphorylation of GCP-WD. Plk1 depletion or inhibition prevents accumulation of GCP-WD at mitotic centrosomes, but GCP-WD mutants that are defective in Plk1-binding and -phosphorylation still accumulate at mitotic centrosomes and recruit gamma-tubulin. Moreover, Plk1 also controls the recruitment of other PCM proteins implicated in centrosomal gamma-tubulin attachment (Cep192/hSPD2, pericentrin, Cep215/Cdk5Rap2). Our results support a model in which Plk1-dependent recruitment of gamma-tubulin to mitotic centrosomes is regulated upstream of GCP-WD, involves multiple PCM proteins and therefore potentially multiple Plk1 substrates. PMID:19543530

  7. Lats1 suppresses centrosome overduplication by modulating the stability of Cdc25B

    PubMed Central

    Mukai, Satomi; Yabuta, Norikazu; Yoshida, Kaori; Okamoto, Ayumi; Miura, Daisaku; Furuta, Yasuhide; Abe, Takaya; Nojima, Hiroshi

    2015-01-01

    Numerical aberration of the centrosome results in chromosome missegregation, eventually leading to chromosomal instability, a hallmark of human tumor malignancy. Large tumor suppressors 1 and 2 (Lats1 and Lats2) are central kinases in the Hippo pathway and regulate development and tumorigenesis by coordinating the balance between cell proliferation and apoptosis. Importantly, Lats1 and Lats2 also play pivotal roles in cell cycle checkpoint and mitosis. The Lats proteins localize at centrosomes, but their centrosomal functions remain elusive. Here, we generated Lats1-null knockout (Lats1−/−) mice and established Lats1-null mouse embryonic fibroblasts (MEFs). In Lats1−/− MEFs, centrosomes were markedly overduplicated, leading to severe mitotic defects such as chromosome missegregation and cytokinesis failure. We also found that Lats1 physically interacts with Cdc25B phosphatase that localizes both at the centrosome and in the nucleus and regulates the linkage between the centrosome cycle and mitotic progression. Although Lats1 did not phosphorylate Cdc25B, loss of Lats1 in MEFs caused abnormal accumulation of Cdc25B protein and hyperactivation of Cdk2 toward nucleophosmin (NPM/B23), one of the licensing factors involved in centriole duplication. Taken together, these data suggest that Lats1 regulates Cdc25B protein level and subsequent Cdk2 activity, thereby suppressing centrosome overduplication during interphase. PMID:26530630

  8. Force-balance model of suppression of multipolar division in cancer cells with extra centrosomes

    NASA Astrophysics Data System (ADS)

    Zhu, Jie

    2013-03-01

    Cancer cells often possess extra centrosomes which have the potential to cause cell death due to catastrophic multipolar division. Many cancer cells, however, are able to escape multipolar mitosis by clustering the extra centrosomes to form bipolar spindles. The mechanism of centrosome clustering is therefore of great interest to the development of anti-cancer drugs because the de-clustering of extra centrosomes provides an appealing way to eliminate cancer cells while keeping healthy cells intact. We present a physical model assuming 1) dynamic centrosomal microtubules interact with chromosomes by both pushing on chromosome arms and pulling along kinetochores; 2) these microtubules interact with force generators associated with actin/adhesion structures at the cell boundary; and 3) motors act on anti-parallel microtubules from different centrosomes. We find via computer simulations that chromosomes tend to aggregate near the cell center while centrosomes can be either clustered to form bipolar spindles or scattered to form multipolar spindles, depending on the strengths of relative forces, cell shape and adhesion geometry. The model predictions agree with data from cells plated on adhesive micropatterns and from biochemically or genetically perturbed cells. Furthermore, our model is able to explain various microtubule distributions in interphase cells on patterned substrates. This work was supported by NSF

  9. A FRET-based study reveals site-specific regulation of spindle position checkpoint proteins at yeast centrosomes.

    PubMed

    Gryaznova, Yuliya; Koca Caydasi, Ayse; Malengo, Gabriele; Sourjik, Victor; Pereira, Gislene

    2016-01-01

    The spindle position checkpoint (SPOC) is a spindle pole body (SPB, equivalent of mammalian centrosome) associated surveillance mechanism that halts mitotic exit upon spindle mis-orientation. Here, we monitored the interaction between SPB proteins and the SPOC component Bfa1 by FRET microscopy. We show that Bfa1 binds to the scaffold-protein Nud1 and the γ-tubulin receptor Spc72. Spindle misalignment specifically disrupts Bfa1-Spc72 interaction by a mechanism that requires the 14-3-3-family protein Bmh1 and the MARK/PAR-kinase Kin4. Dissociation of Bfa1 from Spc72 prevents the inhibitory phosphorylation of Bfa1 by the polo-like kinase Cdc5. We propose Spc72 as a regulatory hub that coordinates the activity of Kin4 and Cdc5 towards Bfa1. In addition, analysis of spc72∆ cells shows that a mitotic-exit-promoting dominant signal, which is triggered upon elongation of the spindle into the bud, overrides the SPOC. Our data reinforce the importance of daughter-cell-associated factors and centrosome-based regulations in mitotic exit and SPOC control. PMID:27159239

  10. A FRET-based study reveals site-specific regulation of spindle position checkpoint proteins at yeast centrosomes

    PubMed Central

    Gryaznova, Yuliya; Caydasi, Ayse Koca; Malengo, Gabriele; Sourjik, Victor; Pereira, Gislene

    2016-01-01

    The spindle position checkpoint (SPOC) is a spindle pole body (SPB, equivalent of mammalian centrosome) associated surveillance mechanism that halts mitotic exit upon spindle mis-orientation. Here, we monitored the interaction between SPB proteins and the SPOC component Bfa1 by FRET microscopy. We show that Bfa1 binds to the scaffold-protein Nud1 and the γ-tubulin receptor Spc72. Spindle misalignment specifically disrupts Bfa1-Spc72 interaction by a mechanism that requires the 14-3-3-family protein Bmh1 and the MARK/PAR-kinase Kin4. Dissociation of Bfa1 from Spc72 prevents the inhibitory phosphorylation of Bfa1 by the polo-like kinase Cdc5. We propose Spc72 as a regulatory hub that coordinates the activity of Kin4 and Cdc5 towards Bfa1. In addition, analysis of spc72∆ cells shows that a mitotic-exit-promoting dominant signal, which is triggered upon elongation of the spindle into the bud, overrides the SPOC. Our data reinforce the importance of daughter-cell-associated factors and centrosome-based regulations in mitotic exit and SPOC control. DOI: http://dx.doi.org/10.7554/eLife.14029.001 PMID:27159239

  11. Massive acute arsenic poisonings.

    PubMed

    Lech, Teresa; Trela, Franciszek

    2005-07-16

    Arsenic poisonings are still important in the field of toxicology, though they are not as frequent as about 20-30 years ago. In this paper, the arsenic concentrations in ante- and post-mortem materials, and also forensic and anatomo-pathological aspects in three cases of massive acute poisoning with arsenic(III) oxide (two of them with unexplained criminalistic background, in which arsenic was taken for amphetamine and one suicide), are presented. Ante-mortem blood and urine arsenic concentrations ranged from 2.3 to 6.7 microg/ml, respectively. Post-mortem tissue total arsenic concentrations were also detected in large concentrations. In case 3, the contents of the duodenum contained as much as 30.1% arsenic(III) oxide. The high concentrations of arsenic detected in blood and tissues in all presented cases are particularly noteworthy in that they are very rarely detected at these concentrations in fatal arsenic poisonings. PMID:15939162

  12. Induction of centrosome amplification by formaldehyde, but not hydroquinone, in human lymphoblastoid TK6 cells.

    PubMed

    Ji, Zhiying; McHale, Cliona M; Bersonda, Jessica; Tung, Judy; Smith, Martyn T; Zhang, Luoping

    2015-07-01

    Benzene and formaldehyde (FA) are important industrial chemicals and environmental pollutants that cause leukemia by inducing DNA damage and chromosome aberrations in hematopoietic stem cells (HSC), the target cells for leukemia. Our previous studies showed that workers exposed to benzene and FA exhibit increased levels of aneuploidy in their blood cells. As centrosome amplification is a common phenomenon in human cancers, including leukemia, and is associated with aneuploidy in carcinogenesis, we hypothesized that benzene and FA would induce centrosome amplification in vitro. We treated human lymphoblastoid TK6 cells with a range of concentrations of hydroquinone (HQ, a benzene metabolite) or FA for 24 h, allowed the cells to recover in fresh medium for 24 h, and examined centrosome amplification; chromosomal gain, loss, and breakage; and cytotoxicity. We included melphalan and etoposide, chemotherapeutic drugs that cause therapy-related acute myeloid leukemia and that have been shown to induce centrosome amplification as well as chromosomal aneuploidy and breakage, as positive controls. Melphalan and etoposide induced centrosome amplification and chromosome gain and breakage in a dose-dependent manner, at cytotoxic concentrations. HQ, though cytotoxic, did not induce centrosome amplification or any chromosomal aberration. FA-induced centrosome amplification and cytotoxicity, but did not induce chromosomal aberrations. Our data suggest, for the first time, that centrosome amplification is a potential mechanism underlying FA-induced leukemogenesis, but not benzene-induced leukemogenesis, as mediated through HQ. Future studies are needed to delineate the mechanisms of centrosome amplification and its association with DNA damage, chromosomal aneuploidy and carcinogenesis, following exposure to FA. PMID:25821186

  13. Centrosomal protein CP110 controls maturation of the mother centriole during cilia biogenesis

    PubMed Central

    Yadav, Sharda Prasad; Sharma, Neel Kamal; Liu, Chunqiao; Dong, Lijin; Li, Tiansen; Swaroop, Anand

    2016-01-01

    ABSTRACT Defects in cilia centrosomal genes cause pleiotropic clinical phenotypes, collectively called ciliopathies. Cilia biogenesis is initiated by the interaction of positive and negative regulators. Centriolar coiled coil protein 110 (CP110) caps the distal end of the mother centriole and is known to act as a suppressor to control the timing of ciliogenesis. Here, we demonstrate that CP110 promotes cilia formation in vivo, in contrast to findings in cultured cells. Cp110−/− mice die shortly after birth owing to organogenesis defects as in ciliopathies. Shh signaling is impaired in null embryos and primary cilia are reduced in multiple tissues. We show that CP110 is required for anchoring of basal bodies to the membrane during cilia formation. CP110 loss resulted in an abnormal distribution of core components of subdistal appendages (SDAs) and of recycling endosomes, which may be associated with premature extension of axonemal microtubules. Our data implicate CP110 in SDA assembly and ciliary vesicle docking, two requisite early steps in cilia formation. We suggest that CP110 has unique context-dependent functions, acting as both a suppressor and a promoter of ciliogenesis. PMID:26965371

  14. The Arsenic Removal From Molten Steel

    NASA Astrophysics Data System (ADS)

    Wang, JianJun; Luo, Lingen; Kong, Hui; Zhou, Li

    2011-04-01

    Arsenic removal from molten steel by the calcium iron alloy and rare earth alloy (48% Ce, mass percentage) has been exploringly studied at 1853 K. It is found that the addition of rare earth alloy makes the arsenic removal more effective. This phenomenon may originate from the facts that the addition of Ce lowers the activity coefficient of sulfur, and the low activity of sulfur restrains the reaction of calcium iron alloy and sulfur, which promotes the arsenic removal reaction. Thus more rare earth alloy addition results in the higher arsenic removal ratio. However, due to the high cost of rare earth alloy, increasing the quantity of calcium iron alloy may be a choice to improve the arsenic removal effect in molten steel. It is found that when the weight ratio of rare earth alloy/steel is fixed at 5%, the arsenic removal ratio increases with the calcium iron alloy amount increasing. When the weight ratio of calcium iron alloy/steel is 18 %, the arsenic removal ratio is 50 %. This result may be acceptable for the industrial purpose.

  15. Nuclear envelope-associated dynein cooperates with Eg5 to drive prophase centrosome separation

    PubMed Central

    van Heesbeen, Roy G.H.P.; Raaijmakers, Jonne A.; Tanenbaum, Marvin E.; Medema, René H.

    2013-01-01

    Eg5 (kinesin-5) is a highly conserved microtubule motor protein, essential for centrosome separation and bipolar spindle assembly in human cells. Using an “in vitro” evolution approach, we generated human cancer cells that can grow in the complete absence of Eg5 activity. Characterization of these Eg5-independent cells (EICs) led to the identification of a novel pathway for prophase centrosome separation, which depends on nuclear envelope (NE)-associated dynein. Here, we discuss our recent findings and elaborate on the mechanism by which dynein drives centrosome separation. PMID:23713137

  16. LIN-41 inactivation leads to delayed centrosome elimination and abnormal chromosome behavior during female meiosis in Caenorhabditis elegans

    PubMed Central

    Matsuura, Rieko; Ashikawa, Tomoko; Nozaki, Yuka; Kitagawa, Daiju

    2016-01-01

    During oogenesis, two successive meiotic cell divisions occur without functional centrosomes because of the inactivation and subsequent elimination of maternal centrosomes during the diplotene stage of meiosis I. Despite being a conserved phenomenon in most metazoans, the means by which this centrosome behavior is controlled during female meiosis remain elusive. Here, we conducted a targeted RNAi screening in the Caenorhabditis elegans gonad to identify novel regulators of centrosome behavior during oogenesis. We screened 513 genes known to be essential for embryo production and directly visualized GFP–γ-tubulin to monitor centrosome behavior at all stages of oogenesis. In the screening, we found that RNAi-mediated inactivation of 33 genes delayed the elimination of GFP–γ-tubulin at centrosomes during oogenesis, whereas inactivation of nine genes accelerated the process. Depletion of the TRIM-NHL protein LIN-41 led to a significant delay in centrosome elimination and to the separation and reactivation of centrosomes during oogenesis. Upon LIN-41 depletion, meiotic chromosomes were abnormally condensed and pulled toward one of the two spindle poles around late pachytene even though the spindle microtubules emanated from both centrosomes. Overall, our work provides new insights into the regulation of centrosome behavior to ensure critical meiotic events and the generation of intact oocytes. PMID:26764090

  17. Arsenic Efflux from Microcystis aeruginosa under Different Phosphate Regimes

    PubMed Central

    Yan, Changzhou; Wang, Zhenhong; Luo, Zhuanxi

    2014-01-01

    Phytoplankton plays an important role in arsenic speciation, distribution, and cycling in freshwater environments. Little information, however, is available on arsenic efflux from the cyanobacteria Microcystis aeruginosa under different phosphate regimes. This study investigated M. aeruginosa arsenic efflux and speciation by pre-exposing it to 10 µM arsenate or arsenite for 24 h during limited (12 h) and extended (13 d) depuration periods under phosphate enriched (+P) and phosphate depleted (−P) treatments. Arsenate was the predominant species detected in algal cells throughout the depuration period while arsenite only accounted for no greater than 45% of intracellular arsenic. During the limited depuration period, arsenic efflux occurred rapidly and only arsenate was detected in solutions. During the extended depuration period, however, arsenate and dimethylarsinic acid (DMA) were found to be the two predominant arsenic species detected in solutions under −P treatments, but arsenate was the only species detected under +P treatments. Experimental results also suggest that phosphorus has a significant effect in accelerating arsenic efflux and promoting arsenite bio-oxidation in M. aeruginosa. Furthermore, phosphorus depletion can reduce arsenic efflux from algal cells as well as accelerate arsenic reduction and methylation. These findings can contribute to our understanding of arsenic biogeochemistry in aquatic environments and its potential environmental risks under different phosphorus levels. PMID:25549253

  18. Toxic Substances Portal- Arsenic

    MedlinePlus

    ... industrial applications. Organic arsenic compounds are used as pesticides, primarily on cotton fields and orchards. top What ... as copper or lead smelting, wood treating, or pesticide application. top How can arsenic affect my health? ...

  19. Arsenic Trioxide Injection

    MedlinePlus

    Arsenic trioxide is used to treat acute promyelocytic leukemia (APL; a type of cancer in which there ... worsened following treatment with other types of chemotherapy. Arsenic trioxide is in a class of medications called ...

  20. Drosophila parthenogenesis: A tool to decipher centrosomal vs acentrosomal spindle assembly pathways

    SciTech Connect

    Riparbelli, Maria Giovanna; Callaini, Giuliano

    2008-04-15

    Development of unfertilized eggs in the parthenogenetic strain K23-O-im of Drosophila mercatorum requires the stochastic interactions of self-assembled centrosomes with the female chromatin. In a portion of the unfertilized eggs that do not assemble centrosomes, microtubules organize a bipolar anastral mitotic spindle around the chromatin like the one formed during the first female meiosis, suggesting that similar pathways may be operative. In the cytoplasm of eggs in which centrosomes do form, monastral and biastral spindles are found. Analysis by laser scanning confocal microscopy suggests that these spindles are derived from the stochastic interaction of astral microtubules directly with kinetochore regions or indirectly with kinetochore microtubules. Our findings are consistent with the idea that mitotic spindle assembly requires both acentrosomal and centrosomal pathways, strengthening the hypothesis that astral microtubules can dictate the organization of the spindle by capturing kinetochore microtubules.

  1. The microtubule nucleation activity of centrobin in both the centrosome and cytoplasm

    PubMed Central

    Shin, Wonjung; Yu, Nam-Kyung; Kaang, Bong-Kiun; Rhee, Kunsoo

    2015-01-01

    Centrobin resides in daughter centriole and play a critical role in centriole duplication. Nucleation and stabilization of microtubules are known biological activities of centrobin. Here, we report a specific localization of centrobin outside the centrosome. Centrobin was associated with the stable microtubules. In hippocampal cells, centrobin formed cytoplasmic dots in addition to the localization at both centrosomes with the mother and daughter centrioles. Such specific localization pattern suggests that cytoplasmic centrobin is not just a reserved pool for centrosomal localization but also has a specific role in the cytoplasm. In fact, centrobin enhanced microtubule formation outside as well as inside the centrosome. These results propose specific roles of the cytoplasmic centrobin for noncentrosomal microtubule formation in specific cell types and during the cell cycle. PMID:26083938

  2. Centrosome and microtubule instability in aging Drosophila cells

    NASA Technical Reports Server (NTRS)

    Schatten, H.; Chakrabarti, A.; Hedrick, J.

    1999-01-01

    Several cytoskeletal changes are associated with aging which includes alterations in muscle structure leading to muscular atrophy, and weakening of the microtubule network which affects cellular secretion and maintenance of cell shape. Weakening of the microtubule network during meiosis in aging oocytes can result in aneuploidy or trisomic zygotes with increasing maternal age. Imbalances of cytoskeletal organization can lead to disease such as Alzheimer's, muscular disorders, and cancer. Because many cytoskeletal diseases are related to age we investigated the effects of aging on microtubule organization in cell cultures of the Drosophila cell model system (Schneider S-1 and Kc23 cell lines). This cell model is increasingly being used as an alternative system to mammalian cell cultures. Drosophila cells are amenable to genetic manipulations and can be used to identify and manipulate genes which are involved in the aging processes. Immunofluorescence, scanning, and transmission electron microscopy were employed for the analysis of microtubule organizing centers (centrosomes) and microtubules at various times after subculturing cells in fresh medium. Our results reveal that centrosomes and the microtubule network becomes significantly affected in aging cells after 5 days of subculture. At 5-14 days of subculture, 1% abnormal out of 3% mitoses were noted which were clearly distinguishable from freshly subcultured control cells in which 3% of cells undergo normal mitosis with bipolar configurations. Microtubules are also affected in the midbody during cell division. The midbody in aging cells becomes up to 10 times longer when compared with midbodies in freshly subcultured cells. During interphase, microtubules are often disrupted and disorganized, which may indicate improper function related to transport of cell organelles along microtubules. These results are likely to help explain some cytoskeletal disorders and diseases related to aging.

  3. ARSENIC REMOVAL TECHNOLOGY

    EPA Science Inventory

    Presentation will discuss the state-of-art technology for removal of arsenic from drinking water. Presentation includes results of several EPA field studies on removal of arsenic from existing arsenic removal plants and key results from several EPA sponsored research studies. T...

  4. The carcinogenicity of arsenic.

    PubMed Central

    Pershagen, G

    1981-01-01

    A carcinogenic role of inorganic arsenic has been suspected for nearly a century. Exposure to inorganic arsenic compounds occurs in some occupational groups, e.g., among smelter workers and workers engaged in the production and use of arsenic containing pesticides. Substantial exposure can also result from drinking water in certain areas and the use of some drugs. Tobacco and wine have had high As concentrations due to the use of arsenic containing pesticides. Inorganic arsenic compounds interfere with DNA repair mechanisms and an increased frequency of chromosomal aberrations have been observed among exposed workers and patients. Epidemiological data show that inorganic arsenic exposure can cause cancer of the lung and skin. The evidence of an etiologic role of arsenic for angiosarcoma of the liver is highly suggestive; however, the association between arsenic and cancer of other sites needs further investigation. No epidemiological data are available on exposure to organic arsenic compounds and cancer. Animal carcinogenicity studies involving exposure to various inorganic and organic arsenic compounds by different routes have been negative, with the possible exception of some preliminary data regarding lung cancer and leukemia. Some studies have indicated an increased mortality from lung cancer in populations living near point emission sources of arsenic into the air. The role of arsenic cannot be evaluated due to lack of exposure data. Epidemiological data suggest that the present WHO standard for drinking water (50 micrograms As/l.) provides only a small safety margin with regard to skin cancer. PMID:7023936

  5. NEVADA ARSENIC STUDY

    EPA Science Inventory

    The effects of exposure to arsenic in U.S. drinking water at low levels are difficult to assess. In particular, studies of sufficient sample size on US populations exposed to arsenic in drinking water are few. Churchill County, NV (population 25000) has arsenic levels in drinki...

  6. ARSENIC SOURCES AND ASSESSMENT

    EPA Science Inventory

    Recent research has identified a number of potential and current links between environmental arsenic releases and the management of operational and abandoned landfills. Many landfills will receive an increasing arsenic load due to the disposal of arsenic-bearing solid residuals ...

  7. Arsenic in Food

    MedlinePlus

    ... inorganic forms. The FDA has been measuring total arsenic concentrations in foods, including rice and juices, through its Total Diet Study program ... readily take up much arsenic from the ground, rice is different because it takes ... has high levels of less toxic organic arsenic. Do organic foods ...

  8. DDA3 targets Cep290 into the centrosome to regulate spindle positioning.

    PubMed

    Song, Haiyu; Park, Ji Eun; Jang, Chang-Young

    The centrosome is an important cellular organelle which nucleates microtubules (MTs) to form the cytoskeleton during interphase and the mitotic spindle during mitosis. The Cep290 is one of the centrosomal proteins and functions in cilia formation. Even-though it is in the centrosome, the function of Cep290 in mitosis had not yet been evaluated. In this study, we report a novel function of Cep290 that is involved in spindle positioning. Cep290 was identified as an interacting partner of DDA3, and we confirmed that Cep290 specifically localizes in the mitotic centrosome. Depletion of Cep290 caused a reduction of the astral spindle, leading to misorientation of the mitotic spindle. MT polymerization also decreased in Cep290-depleted cells, suggesting that Cep290 is involved in spindle nucleation. Furthermore, DDA3 stabilizes and transports Cep290 to the centrosome. Therefore, we concluded that DDA3 controls astral spindle formation and spindle positioning by targeting Cep290 to the centrosome. PMID:25998387

  9. CENP-32 is required to maintain centrosomal dominance in bipolar spindle assembly.

    PubMed

    Ohta, Shinya; Wood, Laura; Toramoto, Iyo; Yagyu, Ken-Ichi; Fukagawa, Tatsuo; Earnshaw, William C

    2015-04-01

    Centrosomes nucleate spindle formation, direct spindle pole positioning, and are important for proper chromosome segregation during mitosis in most animal cells. We previously reported that centromere protein 32 (CENP-32) is required for centrosome association with spindle poles during metaphase. In this study, we show that CENP-32 depletion seems to release centrosomes from bipolar spindles whose assembly they had previously initiated. Remarkably, the resulting anastral spindles function normally, aligning the chromosomes to a metaphase plate and entering anaphase without detectable interference from the free centrosomes, which appear to behave as free asters in these cells. The free asters, which contain reduced but significant levels of CDK5RAP2, show weak interactions with spindle microtubules but do not seem to make productive attachments to kinetochores. Thus CENP-32 appears to be required for centrosomes to integrate into a fully functional spindle that not only nucleates astral microtubules, but also is able to nucleate and bind to kinetochore and central spindle microtubules. Additional data suggest that NuMA tethers microtubules at the anastral spindle poles and that augmin is required for centrosome detachment after CENP-32 depletion, possibly due to an imbalance of forces within the spindle. PMID:25657325

  10. Formation of bipolar spindles with two centrosomes in tetraploid cells established from normal human fibroblasts.

    PubMed

    Ohshima, Susumu; Seyama, Atsushi

    2012-09-01

    Tetraploid cells with unstable chromosomes frequently arise as an early step in tumorigenesis and lead to the formation of aneuploid cells. The mechanisms responsible for the chromosome instability of polyploid cells are not fully understood, although the supernumerary centrosomes in polyploid cells have been considered the major cause of chromosomal instability. The aim of this study was to examine the integrity of mitotic spindles and centrosomes in proliferative polyploid cells established from normal human fibroblasts. TIG-1 human fibroblasts were treated with demecolcine (DC) for 4 days to induce polyploidy, and the change in DNA content was monitored. Localization of centrosomes and mitotic spindles in polyploid mitotic cells was examined by immunohistochemistry and laser scanning cytometry. TIG-1 cells treated with DC became almost completely tetraploid at 2 weeks after treatment and grew at the same rate as untreated diploid cells. Most mitotic cells with 8C DNA content had only two centrosomes with bipolar spindles in established tetraploid cells, although they had four or more centrosomes with multipolar spindles at 3 days after DC treatment. The frequency of aneuploid cells increased as established tetraploid cells were propagated. These results indicate that tetraploid cells that form bipolar spindles with two centrosomes in mitosis can proliferate as diploid cells. These cells may serve as a useful model for studying the chromosome instability of polyploid cells. PMID:22696268

  11. CENP-32 is required to maintain centrosomal dominance in bipolar spindle assembly

    PubMed Central

    Ohta, Shinya; Wood, Laura; Toramoto, Iyo; Yagyu, Ken-Ichi; Fukagawa, Tatsuo; Earnshaw, William C.

    2015-01-01

    Centrosomes nucleate spindle formation, direct spindle pole positioning, and are important for proper chromosome segregation during mitosis in most animal cells. We previously reported that centromere protein 32 (CENP-32) is required for centrosome association with spindle poles during metaphase. In this study, we show that CENP-32 depletion seems to release centrosomes from bipolar spindles whose assembly they had previously initiated. Remarkably, the resulting anastral spindles function normally, aligning the chromosomes to a metaphase plate and entering anaphase without detectable interference from the free centrosomes, which appear to behave as free asters in these cells. The free asters, which contain reduced but significant levels of CDK5RAP2, show weak interactions with spindle microtubules but do not seem to make productive attachments to kinetochores. Thus CENP-32 appears to be required for centrosomes to integrate into a fully functional spindle that not only nucleates astral microtubules, but also is able to nucleate and bind to kinetochore and central spindle microtubules. Additional data suggest that NuMA tethers microtubules at the anastral spindle poles and that augmin is required for centrosome detachment after CENP-32 depletion, possibly due to an imbalance of forces within the spindle. PMID:25657325

  12. A Unique CaMKIIβ Signaling Pathway at the Centrosome Regulates Dendrite Patterning in the Brain

    PubMed Central

    Puram, Sidharth V.; Kim, Albert H.; Ikeuchi, Yoshiho; Wilson-Grady, Joshua T.; Merdes, Andreas; Gygi, Steven P.; Bonni, Azad

    2012-01-01

    The protein kinase calcium/calmodulin-dependent kinase II (CaMKII) predominantly consists of the α and β isoforms in the brain. Although CaMKIIα functions have been elucidated, the unique catalytic functions of CaMKIIβ have remained unknown. Using knockdown analyses in primary neurons and in the rat cerebellar cortex in vivo, we report that CaMKIIβ operates at the centrosome in a CaMKIIα-independent manner to drive dendrite retraction and pruning. We also find that the targeting protein PCM1 localizes CaMKIIβ at the centrosome. Finally, we uncover the E3 ubiquitin ligase Cdc20-APC as a novel centrosomal substrate of CaMKIIβ. CaMKIIβ phosphorylates Cdc20 at Ser51, which induces Cdc20 dispersion from the centrosome, thereby inhibiting centrosomal Cdc20-APC activity and triggering the transition from growth to retraction of dendrites. Our findings define a novel isoform-specific function for CaMKIIβ that regulates ubiquitin signaling at the centrosome and thereby orchestrates dendrite patterning, with important implications for neuronal connectivity in the brain. PMID:21725312

  13. A centrosome interactome provides insight into organelle assembly and reveals a non-duplication role for Plk4.

    PubMed

    Galletta, Brian J; Fagerstrom, Carey J; Schoborg, Todd A; McLamarrah, Tiffany A; Ryniawec, John M; Buster, Daniel W; Slep, Kevin C; Rogers, Gregory C; Rusan, Nasser M

    2016-01-01

    The centrosome is the major microtubule-organizing centre of many cells, best known for its role in mitotic spindle organization. How the proteins of the centrosome are accurately assembled to carry out its many functions remains poorly understood. The non-membrane-bound nature of the centrosome dictates that protein-protein interactions drive its assembly and functions. To investigate this massive macromolecular organelle, we generated a 'domain-level' centrosome interactome using direct protein-protein interaction data from a focused yeast two-hybrid screen. We then used biochemistry, cell biology and the model organism Drosophila to provide insight into the protein organization and kinase regulatory machinery required for centrosome assembly. Finally, we identified a novel role for Plk4, the master regulator of centriole duplication. We show that Plk4 phosphorylates Cep135 to properly position the essential centriole component Asterless. This interaction landscape affords a critical framework for research of normal and aberrant centrosomes. PMID:27558293

  14. A centrosome interactome provides insight into organelle assembly and reveals a non-duplication role for Plk4

    PubMed Central

    Galletta, Brian J.; Fagerstrom, Carey J.; Schoborg, Todd A.; McLamarrah, Tiffany A.; Ryniawec, John M.; Buster, Daniel W.; Slep, Kevin C.; Rogers, Gregory C.; Rusan, Nasser M.

    2016-01-01

    The centrosome is the major microtubule-organizing centre of many cells, best known for its role in mitotic spindle organization. How the proteins of the centrosome are accurately assembled to carry out its many functions remains poorly understood. The non-membrane-bound nature of the centrosome dictates that protein–protein interactions drive its assembly and functions. To investigate this massive macromolecular organelle, we generated a ‘domain-level' centrosome interactome using direct protein–protein interaction data from a focused yeast two-hybrid screen. We then used biochemistry, cell biology and the model organism Drosophila to provide insight into the protein organization and kinase regulatory machinery required for centrosome assembly. Finally, we identified a novel role for Plk4, the master regulator of centriole duplication. We show that Plk4 phosphorylates Cep135 to properly position the essential centriole component Asterless. This interaction landscape affords a critical framework for research of normal and aberrant centrosomes. PMID:27558293

  15. Arsenic pollution sources.

    PubMed

    Garelick, Hemda; Jones, Huw; Dybowska, Agnieszka; Valsami-Jones, Eugenia

    2008-01-01

    Arsenic is a widely dispersed element in the Earth's crust and exists at an average concentration of approximately 5 mg/kg. There are many possible routes of human exposure to arsenic from both natural and anthropogenic sources. Arsenic occurs as a constituent in more than 200 minerals, although it primarily exists as arsenopyrite and as a constituent in several other sulfide minerals. The introduction of arsenic into drinking water can occur as a result of its natural geological presence in local bedrock. Arsenic-containing bedrock formations of this sort are known in Bangladesh, West Bengal (India), and regions of China, and many cases of endemic contamination by arsenic with serious consequences to human health are known from these areas. Significant natural contamination of surface waters and soil can arise when arsenic-rich geothermal fluids come into contact with surface waters. When humans are implicated in causing or exacerbating arsenic pollution, the cause can almost always be traced to mining or mining-related activities. Arsenic exists in many oxidation states, with arsenic (III) and (V) being the most common forms. Similar to many metalloids, the prevalence of particular species of arsenic depends greatly on the pH and redox conditions of the matrix in which it exists. Speciation is also important in determining the toxicity of arsenic. Arsenic minerals exist in the environment principally as sulfides, oxides, and phosphates. In igneous rocks, only those of volcanic origin are implicated in high aqueous arsenic concentrations. Sedimentary rocks tend not to bear high arsenic loads, and common matrices such as sands and sandstones contain lower concentrations owing to the dominance of quartz and feldspars. Groundwater contamination by arsenic arises from sources of arsenopyrite, base metal sulfides, realgar and orpiment, arsenic-rich pyrite, and iron oxyhydroxide. Mechanisms by which arsenic is released from minerals are varied and are accounted for by

  16. Centrosome attachment to the C. elegans male pronucleus is dependent on the surface area of the nuclear envelope

    PubMed Central

    Meyerzon, Marina; Gao, Zhizhen; Liu, Jin; Wu, Jui-Ching; Malone, Christian J.; Starr, Daniel A.

    2009-01-01

    A close association must be maintained between the male pronucleus and the centrosomes during pronuclear migration. In C. elegans, simultaneous depletion of inner nuclear membrane LEM proteins EMR-1 and LEM-2, depletion of the nuclear lamina proteins LMN-1 or BAF-1, or the depletion of nuclear import components leads to embryonic lethality with small pronuclei. Here, a novel centrosome detachment phenotype in C. elegans zygotes is described. Zygotes with defects in the nuclear envelope had small pronuclei with a single centrosome detached from the male pronucleus. ZYG-12, SUN-1, and LIS-1, which function at the nuclear envelope with dynein to attach centrosomes, were observed at normal concentrations on the nuclear envelope of pronuclei with detached centrosomes. Analysis of time-lapse images showed that as mutant pronuclei grew in surface area, they captured detached centrosomes. Larger tetraploid or smaller histone::mCherry pronuclei suppressed or enhanced the centrosome detachment phenotype respectively. In embryos fertilized with anucleated sperm, only one centrosome was captured by small female pronuclei, suggesting the mechanism of capture is dependent on the surface area of the outer nuclear membrane available to interact with aster microtubules. We propose that the limiting factor for centrosome attachment to the surface of abnormally small pronuclei is dynein. PMID:19162001

  17. CDK5RAP2 is a pericentriolar protein that functions in centrosomal attachment of the gamma-tubulin ring complex.

    PubMed

    Fong, Ka-Wing; Choi, Yuk-Kwan; Rattner, Jerome B; Qi, Robert Z

    2008-01-01

    Microtubule nucleation and organization by the centrosome require gamma-tubulin, a protein that exists in a macromolecular complex called the gamma-tubulin ring complex (gammaTuRC). We report characterization of CDK5RAP2, a novel centrosomal protein whose mutations have been linked to autosomal recessive primary microcephaly. In somatic cells, CDK5RAP2 localizes throughout the pericentriolar material in all stages of the cell cycle. When overexpressed, CDK5RAP2 assembled a subset of centrosomal proteins including gamma-tubulin onto the centrosomes or under the microtubule-disrupting conditions into microtubule-nucleating clusters in the cytoplasm. CDK5RAP2 associates with the gammaTuRC via a short conserved sequence present in several related proteins found in a range of organisms from fungi to mammals. The binding of CDK5RAP2 is required for gammaTuRC attachment to the centrosome but not for gammaTuRC assembly. Perturbing CDK5RAP2 function delocalized gamma-tubulin from the centrosomes and inhibited centrosomal microtubule nucleation, thus leading to disorganization of interphase microtubule arrays and formation of anastral mitotic spindles. Together, CDK5RAP2 is a pericentriolar structural component that functions in gammaTuRC attachment and therefore in the microtubule organizing function of the centrosome. Our findings suggest that centrosome malfunction due to the CDK5RAP2 mutations may underlie autosomal recessive primary microcephaly. PMID:17959831

  18. Arsenic removal from water

    DOEpatents

    Moore, Robert C.; Anderson, D. Richard

    2007-07-24

    Methods for removing arsenic from water by addition of inexpensive and commonly available magnesium oxide, magnesium hydroxide, calcium oxide, or calcium hydroxide to the water. The hydroxide has a strong chemical affinity for arsenic and rapidly adsorbs arsenic, even in the presence of carbonate in the water. Simple and commercially available mechanical methods for removal of magnesium hydroxide particles with adsorbed arsenic from drinking water can be used, including filtration, dissolved air flotation, vortex separation, or centrifugal separation. A method for continuous removal of arsenic from water is provided. Also provided is a method for concentrating arsenic in a water sample to facilitate quantification of arsenic, by means of magnesium or calcium hydroxide adsorption.

  19. Arsenic: homicidal intoxication

    SciTech Connect

    Massey, E.W.; Wold, D.; Heyman, A.

    1984-07-01

    Arsenic-induced deaths have been known to occur from accidental poisoning, as a result of medical therapy, and from intentional poisonings in homicide and suicide. Twenty-eight arsenic deaths in North Carolina from 1972 to 1982 included 14 homicides and seven suicides. In addition, 56 hospitalized victims of arsenic poisoning were identified at Duke Medical Center from 1970 to 1980. Four case histories of arsenic poisoning in North Carolina are presented and clinical manifestations are discussed. In view of the continued widespread use of arsenic in industry and agriculture, and its ubiquity in the environment, arsenic poisoning will continue to occur. A need for knowledge of its toxicity and of the clinical manifestations of acute and chronic arsenic poisoning will also continue.

  20. ARSENIC (+3 OXIDATION STATE) METHYLTRANSFERASE AND THE METHYLATION OF ARSENICALS

    EPA Science Inventory

    Metabolic conversion of inorganic arsenic into methylated products is a multistep process that yields mono, di, and trimethylated arsenicals. In recent years, it has become apparent that formation of methylated metabolites of inorganic arsenic is not necessarily a detoxification...

  1. Reduction and Coordination of Arsenic in Indian Mustard1

    PubMed Central

    Pickering, Ingrid J.; Prince, Roger C.; George, Martin J.; Smith, Robert D.; George, Graham N.; Salt, David E.

    2000-01-01

    The bioaccumulation of arsenic by plants may provide a means of removing this element from contaminated soils and waters. However, to optimize this process it is important to understand the biological mechanisms involved. Using a combination of techniques, including x-ray absorption spectroscopy, we have established the biochemical fate of arsenic taken up by Indian mustard (Brassica juncea). After arsenate uptake by the roots, possibly via the phosphate transport mechanism, a small fraction is exported to the shoot via the xylem as the oxyanions arsenate and arsenite. Once in the shoot, the arsenic is stored as an AsIII-tris-thiolate complex. The majority of the arsenic remains in the roots as an AsIII-tris-thiolate complex, which is indistinguishable from that found in the shoots and from AsIII-tris-glutathione. The thiolate donors are thus probably either glutathione or phytochelatins. The addition of the dithiol arsenic chelator dimercaptosuccinate to the hydroponic culture medium caused a 5-fold-increased arsenic level in the leaves, although the total arsenic accumulation was only marginally increased. This suggests that the addition of dimercaptosuccinate to arsenic-contaminated soils may provide a way to promote arsenic bioaccumulation in plant shoots, a process that will be essential for the development of an efficient phytoremediation strategy for this element. PMID:10759512

  2. The centrosomin protein is required for centrosome assembly and function during cleavage in Drosophila.

    PubMed

    Megraw, T L; Li, K; Kao, L R; Kaufman, T C

    1999-07-01

    Centrosomin is a 150 kDa centrosomal protein of Drosophila melanogaster. To study the function of Centrosomin in the centrosome, we have recovered mutations that are viable but male and female sterile (cnnmfs). We have shown that these alleles (1, 2, 3, 7, 8 and hk21) induce a maternal effect on early embryogenesis and result in the accumulation of low or undetectable levels of Centrosomin in the centrosomes of cleavage stage embryos. Hemizygous cnn females produce embryos that show dramatic defects in chromosome segregation and spindle organization during the syncytial cleavage divisions. In these embryos the syncytial divisions proceed as far as the twelfth cycle, and embryos fail to cellularize. Aberrant divisions and nuclear fusions occur in the early cycles of the nuclear divisions, and become more prominent at later stages. Giant nuclei are seen in late stage embryos. The spindles that form in mutant embryos exhibit multiple anomalies. There is a high occurrence of apparently linked spindles that share poles, indicating that Centrosomin is required for the proper spacing and separation of mitotic spindles within the syncytium. Spindle poles in the mutants contain little or no detectable amounts of the centrosomal proteins CP60, CP190 and (gamma)-tubulin and late stage embryos often do not have astral microtubules at their spindle poles. Spindle morphology and centrosomal composition suggest that the primary cause of these division defects in mutant embryos is centrosomal malfunction. These results suggest that Centrosomin is required for the assembly and function of centrosomes during the syncytial cleavage divisions. PMID:10357928

  3. Assays to Study Mitotic Centrosome and Spindle Pole Assembly and Regulation.

    PubMed

    Joukov, Vladimir; Walter, Johannes C; De Nicolo, Arcangela

    2016-01-01

    Faithful chromosome segregation during cell division requires proper bipolar spindle assembly and critically depends on spindle pole integrity. In most animal cells, spindle poles form as the result of the concerted action of various factors operating in two independent pathways of microtubule assembly mediated by chromatin/RanGTP and by centrosomes. Mutation or deregulation of a number of spindle pole-organizing proteins has been linked to human diseases, including cancer and microcephaly. Our knowledge on how the spindle pole-organizing factors function at the molecular level and cooperate with one another is still quite limited. As the list of these factors expands, so does the need for the development of experimental approaches to study their function. Cell-free extracts from Xenopus laevis eggs have played an instrumental role in the dissection of the mechanisms of bipolar spindle assembly and have recently allowed the reconstitution of the key steps of the centrosome-driven microtubule nucleation pathway (Joukov et al., Mol Cell 55:578-591, 2014). Here we describe assays to study both centrosome-dependent and centrosome-independent spindle pole formation in Xenopus egg extracts. We also provide experimental procedures for the use of artificial centrosomes, such as microbeads coated with an anti-Aurora A antibody or a recombinant fragment of the Cep192 protein, to model and study centrosome maturation in egg extract. In addition, we detail the protocol for a microtubule regrowth assay that allows assessment of the centrosome-driven spindle microtubule assembly in mammalian cells. PMID:27193852

  4. COMMONALITIES IN METABOLISM OF ARSENICALS

    EPA Science Inventory

    Elucidating the pathway of inorganic arsenic metabolism shows that some of methylated arsenicals formed as intermediates and products are reactive and toxic species. Hence, methylated arsenicals likely mediate at least some of the toxic and carcinogenic effects associated with e...

  5. Knockdown of TWIST1 enhances arsenic trioxide- and ionizing radiation-induced cell death in lung cancer cells by promoting mitochondrial dysfunction

    SciTech Connect

    Seo, Sung-Keum; Kim, Jae-Hee; Choi, Ha-Na; Choe, Tae-Boo; Hong, Seok-Il; Yi, Jae-Youn; Hwang, Sang-Gu; Lee, Hyun-Gyu; Lee, Yun-Han; Park, In-Chul

    2014-07-11

    Highlights: • Knockdown of TWIST1 enhanced ATO- and IR-induced cell death in NSCLCs. • Intracellular ROS levels were increased in cells treated with TWIST1 siRNA. • TWIST1 siRNA induced MMP loss and mitochondrial fragmentation. • TWIST1 siRNA upregulated the fission-related proteins FIS1 and DRP1. - Abstract: TWIST1 is implicated in the process of epithelial mesenchymal transition, metastasis, stemness, and drug resistance in cancer cells, and therefore is a potential target for cancer therapy. In the present study, we found that knockdown of TWIST1 by small interfering RNA (siRNA) enhanced arsenic trioxide (ATO)- and ionizing radiation (IR)-induced cell death in non-small-cell lung cancer cells. Interestingly, intracellular reactive oxygen species levels were increased in cells treated with TWIST1 siRNA and further increased by co-treatment with ATO or IR. Pretreatment of lung cancer cells with the antioxidant N-acetyl-cysteine markedly suppressed the cell death induced by combined treatment with TWIST1 siRNA and ATO or IR. Moreover, treatment of cells with TWIST1 siRNA induced mitochondrial membrane depolarization and significantly increased mitochondrial fragmentation (fission) and upregulated the fission-related proteins FIS1 and DRP1. Collectively, our results demonstrate that siRNA-mediated TWIST1 knockdown induces mitochondrial dysfunction and enhances IR- and ATO-induced cell death in lung cancer cells.

  6. High LET Radiation Amplifies Centrosome Overduplication Through a Pathway of γ-Tubulin Monoubiquitination

    SciTech Connect

    Shimada, Mikio; Hirayama, Ryoichi; Komatsu, Kenshi

    2013-06-01

    Purpose: Radiation induces centrosome overduplication, leading to mitotic catastrophe and tumorigenesis. Because mitotic catastrophe is one of the major tumor cell killing factors in high linear energy transfer (LET) radiation therapy and long-term survivors from such treatment have a potential risk of secondary tumors, we investigated LET dependence of radiation-induced centrosome overduplication and the underlying mechanism. Methods and Materials: Carbon and iron ion beams (13-200 keV/μm) and γ-rays (0.5 keV/μm) were used as radiation sources. To count centrosomes after IR exposure, human U2OS and mouse NIH3T3 cells were immunostained with antibodies of γ-tubulin and centrin 2. Similarly, Nbs1-, Brca1-, Ku70-, and DNA-PKcs-deficient mouse cells and their counterpart wild-type cells were used for measurement of centrosome overduplication. Results: The number of excess centrosome-containing cells at interphase and the resulting multipolar spindle at mitosis were amplified with increased LET, reaching a maximum level of 100 keV/μm, followed by sharp decrease in frequency. Interestingly, Ku70 and DNA-PKcs deficiencies marginally affected the induction of centrosome overduplication, whereas the cell killings were significantly enhanced. This was in contrast to observation that high LET radiation significantly enhanced frequencies of centrosome overduplication in Nbs1- and Brca1-deficient cells. Because NBS1/BRCA1 is implicated in monoubiquitination of γ-tubulin, we subsequently tested whether it is affected by high LET radiation. As a result, monoubiquitination of γ-tubulin was abolished in 48 to 72 hours after exposure to high LET radiation, although γ-ray exposure slightly decreased it 48 hours postirradiation and was restored to a normal level at 72 hours. Conclusions: High LET radiation significantly reduces NBS1/BRCA1-mediated monoubiquitination of γ-tubulin and amplifies centrosome overduplication with a peak at 100 keV/μm. In contrast, Ku70 and DNA

  7. Arsenic and chromium in drinking water promote tumorigenesis in a mouse colitis-associated colorectal cancer model and the potential mechanism is ROS-mediated Wnt/β-catenin signaling pathway

    SciTech Connect

    Wang, Xin; Mandal, Ardhendu K.; Saito, Hiroshi; Pulliam, Joseph F.; Lee, Eun Y.; Ke, Zun-Ji; Lu, Jian; Ding, Songze; Li, Li; Shelton, Brent J.; Tucker, Thomas; Evers, B. Mark; Zhang, Zhuo; Shi, Xianglin

    2012-07-01

    Exposure to carcinogenic metals, such as trivalent arsenic [As(III)] and hexavalent chromium [Cr(VI)], through drinking water is a major global public health problem and is associated with various cancers. However, the mechanism of their carcinogenicity remains unclear. In this study, we used azoxymethane/dextran sodium sulfate (AOM/DSS)-induced mouse colitis-associated colorectal cancer model to investigate their tumorigenesis. Our results demonstrate that exposure to As(III) or Cr(VI), alone or in combination, together with AOM/DSS pretreatment has a promotion effect, increasing the colorectal tumor incidence, multiplicity, size, and grade, as well as cell inflammatory response. Two-dimensional differential gel electrophoresis coupled with mass spectrometry revealed that As(III) or Cr(VI) treatment alone significantly changed the density of proteins. The expression of β-catenin and phospho-GSK was increased by treatment of carcinogenic metals alone. Concomitantly, the expression of NADPH oxidase1 (NOX1) and the level of 8-OHdG were also increased by treatment of carcinogenic metals alone. Antioxidant enzymes, such as superoxide dismutase (SOD) and catalase, were decreased. Similarly, in an in vitro system, exposure of CRL-1807 to carcinogenic metals increased reactive oxygen species (ROS) generation, the expression of β-catenin, phospho-GSK, and NOX1. Inhibition of ROS generation by addition of SOD or catalase inhibited β-catenin expression and activity. Our study provides a new animal model to study the carcinogenicity of As(III) and Cr(VI) and suggests that As(III) and Cr(VI) promote colorectal cancer tumorigenesis, at least partly, through ROS-mediated Wnt/β-catenin signaling pathway. -- Highlights: ► Carcinogenic metals in drinking water promote colorectal tumor formation in vivo. ► Carcinogenic metals induce β-catenin activation in vivo and in vitro. ► ROS generation induced by carcinogenic metals mediated β-catenin activation.

  8. Characterization of Adenomatous Polyposis Coli Protein Dynamics and Localization at the Centrosome.

    PubMed

    Lui, Christina; Mok, Myth T S; Henderson, Beric R

    2016-01-01

    The adenomatous polyposis coli (APC) tumor suppressor is a multifunctional regulator of Wnt signaling and acts as a mobile scaffold at different cellular sites. APC was recently found to stimulate microtubule (MT) growth at the interphase centrosome; however, little is known about its dynamics and localization at this site. To address this, we analysed APC dynamics in fixed and live cells by fluorescence microscopy. In detergent-extracted cells, we discovered that APC was only weakly retained at the centrosome during interphase suggesting a rapid rate of exchange. This was confirmed in living cells by fluorescence recovery after photobleaching (FRAP), which identified two pools of green fluorescent protein (GFP)-APC: a major rapidly exchanging pool (~86%) and minor retained pool (~14%). The dynamic exchange rate of APC was unaffected by C-terminal truncations implicating a targeting role for the N-terminus. Indeed, we mapped centrosome localization to N-terminal armadillo repeat (ARM) domain amino acids 334-625. Interestingly, the rate of APC movement to the centrosome was stimulated by intact MTs, and APC dynamics slowed when MTs were disrupted by nocodazole treatment or knockdown of γ-tubulin. Thus, the rate of APC recycling at the centrosome is enhanced by MT growth, suggesting a positive feedback to stimulate its role in MT growth. PMID:27144584

  9. Intensity-based signal separation algorithm for accuratequantification of clustered centrosomes in tissue sections

    SciTech Connect

    Fleisch, Markus C.; Maxell, Christopher A.; Kuper, Claudia K.; Brown, Erika T.; Parvin, Bahram; Barcellos-Hoff, Mary-Helen; Costes,Sylvain V.

    2006-03-08

    Centrosomes are small organelles that organize the mitoticspindle during cell division and are also involved in cell shape andpolarity. Within epithelial tumors, such as breast cancer, and somehematological tumors, centrosome abnormalities (CA) are common, occurearly in disease etiology, and correlate with chromosomal instability anddisease stage. In situ quantification of CA by optical microscopy ishampered by overlap and clustering of these organelles, which appear asfocal structures. CA has been frequently associated with Tp53 status inpremalignant lesions and tumors. Here we describe an approach toaccurately quantify centrosomes in tissue sections and tumors.Considering proliferation and baseline amplification rate the resultingpopulation based ratio of centrosomes per nucleus allow the approximationof the proportion of cells with CA. Using this technique we show that20-30 percent of cells have amplified centrosomes in Tp53 null mammarytumors. Combining fluorescence detection, deconvolution microscopy and amathematical algorithm applied to a maximum intensity projection we showthat this approach is superior to traditional investigator based visualanalysis or threshold-based techniques.

  10. New insights into centrosome imaging in Drosophila and mouse neuroepithelial tissues.

    PubMed

    Rujano, Maria A; Basto, Renata; Marthiens, Véronique

    2015-01-01

    The centrosome is the main microtubule-organizing center in animal cells. It participates in the assembly of a bipolar spindle that ensures accurate segregation of chromosomes during mitosis. Recently, mutations in centrosome genes have been identified in patients affected by neurodevelopmental disorders. In fact, the etiology of several neurodevelopmental pathologies seems to be linked to defects in the assembly of the mitotic spindle in the neural stem cell compartment during neurogenesis. Therefore, getting better insights into the structure and function/dysfunction of the mitotic spindle apparatus in an intact tissue environment is of utmost importance. However, imaging nanometer-scale structures like centrosomes and microtubule bundles within the depth of a tissue is still challenging. Here we describe two procedures to acquire high-resolution images on fixed tissues and to perform live imaging of microtubule-based structures in the neuroepithelia of the Drosophila brain and of the mouse neocortex. We take advantage of the accumulation of centrosomes and mitotic figures at the apical surface of these polarized tissues to improve the quality of staining and imaging. Both Drosophila and mouse models with centrosome dysfunction showed abnormalities in the neuroepithelium reminiscent of the ones described in brains of human patients. These observations have highlighted their value as model organisms to study the etiology of human neurodevelopmental pathologies. PMID:26175441