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Centrobin: a novel daughter centriole-associated protein that is required for centriole duplication.

Zou C, Li J, Bai Y, Gunning WT, Wazer DE, Band V, Gao Q - J. Cell Biol. (2005)

Bottom Line: In this study, we have identified centrobin as a centriole-associated protein that asymmetrically localizes to the daughter centriole.The silencing of centrobin expression by small interfering RNA inhibited centriole duplication and resulted in centrosomes with one or no centriole, demonstrating that centrobin is required for centriole duplication.Furthermore, inhibition of centriole duplication by centrobin depletion led to impaired cytokinesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Division of Cancer Biology, Evanston Northwestern Healthcare Research Institute, Northwestern University Feinberg School of Medicine, Evanston, IL 60201, USA.

ABSTRACT
In mammalian cells, the centrosome consists of a pair of centrioles and amorphous pericentriolar material. The pair of centrioles, which are the core components of the centrosome, duplicate once per cell cycle. Centrosomes play a pivotal role in orchestrating the formation of the bipolar spindle during mitosis. Recent studies have linked centrosomal activity on centrioles or centriole-associated structures to cytokinesis and cell cycle progression through G1 into the S phase. In this study, we have identified centrobin as a centriole-associated protein that asymmetrically localizes to the daughter centriole. The silencing of centrobin expression by small interfering RNA inhibited centriole duplication and resulted in centrosomes with one or no centriole, demonstrating that centrobin is required for centriole duplication. Furthermore, inhibition of centriole duplication by centrobin depletion led to impaired cytokinesis.

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Copurification of centrobin with the centrosomes through sucrose gradients. Centrosomes from 293T were purified through a 40–70% sucrose gradient as described (Blomberg-Wirschell and Doxsey, 1998). The resulting fractions were separated on SDS-PAGE gels and blotted with anti-centrobin, anti–γ-tubulin, anti-Lamin B1, and anti-Cbl antibodies.
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fig3: Copurification of centrobin with the centrosomes through sucrose gradients. Centrosomes from 293T were purified through a 40–70% sucrose gradient as described (Blomberg-Wirschell and Doxsey, 1998). The resulting fractions were separated on SDS-PAGE gels and blotted with anti-centrobin, anti–γ-tubulin, anti-Lamin B1, and anti-Cbl antibodies.

Mentions: To corroborate our observation that centrobin localizes to the centrosomes, using immunofluorescence analysis, we biochemically purified the centrosomes from 293T cells using sucrose-gradient sedimentation (Blomberg-Wirschell and Doxsey, 1998) and analyzed their compositions by Western blotting. As shown in Fig. 3, centrobin was found in the fractions that were expected to contain the centrosomes, as confirmed by the presence of γ-tubulin in these fractions (Fig. 3, top two panels). Similar data were obtained using MCF7 cells, which are human breast cancer cells (unpublished data). The centrosomal fractions were shown to be free of nuclear and cytoplasmic contamination by Western blotting using Lamin B1 as a nuclear marker and Cbl as a cytoplasmic marker (Fig. 3, bottom two panels). Collectively, the immunofluorescence and biochemical studies unequivocally demonstrated that centrobin is a centrosomal protein.


Centrobin: a novel daughter centriole-associated protein that is required for centriole duplication.

Zou C, Li J, Bai Y, Gunning WT, Wazer DE, Band V, Gao Q - J. Cell Biol. (2005)

Copurification of centrobin with the centrosomes through sucrose gradients. Centrosomes from 293T were purified through a 40–70% sucrose gradient as described (Blomberg-Wirschell and Doxsey, 1998). The resulting fractions were separated on SDS-PAGE gels and blotted with anti-centrobin, anti–γ-tubulin, anti-Lamin B1, and anti-Cbl antibodies.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171251&req=5

fig3: Copurification of centrobin with the centrosomes through sucrose gradients. Centrosomes from 293T were purified through a 40–70% sucrose gradient as described (Blomberg-Wirschell and Doxsey, 1998). The resulting fractions were separated on SDS-PAGE gels and blotted with anti-centrobin, anti–γ-tubulin, anti-Lamin B1, and anti-Cbl antibodies.
Mentions: To corroborate our observation that centrobin localizes to the centrosomes, using immunofluorescence analysis, we biochemically purified the centrosomes from 293T cells using sucrose-gradient sedimentation (Blomberg-Wirschell and Doxsey, 1998) and analyzed their compositions by Western blotting. As shown in Fig. 3, centrobin was found in the fractions that were expected to contain the centrosomes, as confirmed by the presence of γ-tubulin in these fractions (Fig. 3, top two panels). Similar data were obtained using MCF7 cells, which are human breast cancer cells (unpublished data). The centrosomal fractions were shown to be free of nuclear and cytoplasmic contamination by Western blotting using Lamin B1 as a nuclear marker and Cbl as a cytoplasmic marker (Fig. 3, bottom two panels). Collectively, the immunofluorescence and biochemical studies unequivocally demonstrated that centrobin is a centrosomal protein.

Bottom Line: In this study, we have identified centrobin as a centriole-associated protein that asymmetrically localizes to the daughter centriole.The silencing of centrobin expression by small interfering RNA inhibited centriole duplication and resulted in centrosomes with one or no centriole, demonstrating that centrobin is required for centriole duplication.Furthermore, inhibition of centriole duplication by centrobin depletion led to impaired cytokinesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Division of Cancer Biology, Evanston Northwestern Healthcare Research Institute, Northwestern University Feinberg School of Medicine, Evanston, IL 60201, USA.

ABSTRACT
In mammalian cells, the centrosome consists of a pair of centrioles and amorphous pericentriolar material. The pair of centrioles, which are the core components of the centrosome, duplicate once per cell cycle. Centrosomes play a pivotal role in orchestrating the formation of the bipolar spindle during mitosis. Recent studies have linked centrosomal activity on centrioles or centriole-associated structures to cytokinesis and cell cycle progression through G1 into the S phase. In this study, we have identified centrobin as a centriole-associated protein that asymmetrically localizes to the daughter centriole. The silencing of centrobin expression by small interfering RNA inhibited centriole duplication and resulted in centrosomes with one or no centriole, demonstrating that centrobin is required for centriole duplication. Furthermore, inhibition of centriole duplication by centrobin depletion led to impaired cytokinesis.

Show MeSH
Related in: MedlinePlus