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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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Centrobin depletion did not affect the localization of γ-tubulin or microtubule organization and nucleation. (A) Western blot analysis of HeLa cells transfected with centrobin siRNA or control siRNA. HeLa cells were transfected with control (scrambled or FITC-GFP siRNA) or two-centrobin siRNA. After 72 h, cells were harvested and separated through a 6% SDS-PAGE, then blotted with anti-centrobin and anti–α-tubulin antibodies. (B) Immunostaining of centrobin. HeLa cells transfected with scrambled or centrobin siRNA #1 were stained with anti-centrobin and rhodamine-labeled goat anti–rabbit IgG. DNA was stained with DAPI. (C) Centrobin depletion did not affect the localization of γ-tubulin. HeLa cells transfected with scrambled or centrobin siRNA #1 were stained with anti-centrobin and anti–γ-tubulin and with rhodamine-labeled goat anti–rabbit IgG and FITC-labeled goat anti–mouse IgG. (D) Centrobin depletion did not affect microtubule organization and nucleation. HeLa cells were transfected with scrambled siRNA or centrobin siRNA; after 72 h, the cells were treated with 1 μM nocodazole for 1 h and washed three times with PBS to remove the nocodazole. The cells were harvested for fixation using cold methanol and stained for α-tubulin at 0, 2, 5, 10, and 15 min after the removal of nocodazole.
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fig5: Centrobin depletion did not affect the localization of γ-tubulin or microtubule organization and nucleation. (A) Western blot analysis of HeLa cells transfected with centrobin siRNA or control siRNA. HeLa cells were transfected with control (scrambled or FITC-GFP siRNA) or two-centrobin siRNA. After 72 h, cells were harvested and separated through a 6% SDS-PAGE, then blotted with anti-centrobin and anti–α-tubulin antibodies. (B) Immunostaining of centrobin. HeLa cells transfected with scrambled or centrobin siRNA #1 were stained with anti-centrobin and rhodamine-labeled goat anti–rabbit IgG. DNA was stained with DAPI. (C) Centrobin depletion did not affect the localization of γ-tubulin. HeLa cells transfected with scrambled or centrobin siRNA #1 were stained with anti-centrobin and anti–γ-tubulin and with rhodamine-labeled goat anti–rabbit IgG and FITC-labeled goat anti–mouse IgG. (D) Centrobin depletion did not affect microtubule organization and nucleation. HeLa cells were transfected with scrambled siRNA or centrobin siRNA; after 72 h, the cells were treated with 1 μM nocodazole for 1 h and washed three times with PBS to remove the nocodazole. The cells were harvested for fixation using cold methanol and stained for α-tubulin at 0, 2, 5, 10, and 15 min after the removal of nocodazole.

Mentions: We used 21-nt small interfering RNA (siRNA) targeting the coding region of centrobin to knock down centrobin expression. As shown in Fig. 5 A, the endogenous centrobin level was markedly reduced after centrobin siRNA transfection of HeLa cells, but not after the scrambled siRNA and GFP-siRNA were transfected (Fig. 5 A, top). Transfection of HeLa cells with FITC-labeled GFP-siRNA indicated a transfection efficiency of ∼90%. Densitometry analysis of the Western blots indicated that 80% of centrobin was reproducibly depleted with centrobin RNAi #1, which we used in all subsequent experiments. The reduction of centrobin levels was also confirmed by immunofluorescence analysis (Fig. 5 B). Immunofluorescence analysis with anti–γ-tubulin antibodies revealed that the γ-tubulin staining pattern was not visibly altered in the HeLa cells with undetectable levels of centrobin (Fig. 5 C). Furthermore, no gross abnormalities in microtubule nucleation and organization was observed in centrobin-depleted cells (Fig. 5 D), suggesting that centrobin likely does not substantially contribute to microtubule organization and nucleation, at least in interphase cells.


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)

Centrobin depletion did not affect the localization of γ-tubulin or microtubule organization and nucleation. (A) Western blot analysis of HeLa cells transfected with centrobin siRNA or control siRNA. HeLa cells were transfected with control (scrambled or FITC-GFP siRNA) or two-centrobin siRNA. After 72 h, cells were harvested and separated through a 6% SDS-PAGE, then blotted with anti-centrobin and anti–α-tubulin antibodies. (B) Immunostaining of centrobin. HeLa cells transfected with scrambled or centrobin siRNA #1 were stained with anti-centrobin and rhodamine-labeled goat anti–rabbit IgG. DNA was stained with DAPI. (C) Centrobin depletion did not affect the localization of γ-tubulin. HeLa cells transfected with scrambled or centrobin siRNA #1 were stained with anti-centrobin and anti–γ-tubulin and with rhodamine-labeled goat anti–rabbit IgG and FITC-labeled goat anti–mouse IgG. (D) Centrobin depletion did not affect microtubule organization and nucleation. HeLa cells were transfected with scrambled siRNA or centrobin siRNA; after 72 h, the cells were treated with 1 μM nocodazole for 1 h and washed three times with PBS to remove the nocodazole. The cells were harvested for fixation using cold methanol and stained for α-tubulin at 0, 2, 5, 10, and 15 min after the removal of nocodazole.
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Related In: Results  -  Collection

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fig5: Centrobin depletion did not affect the localization of γ-tubulin or microtubule organization and nucleation. (A) Western blot analysis of HeLa cells transfected with centrobin siRNA or control siRNA. HeLa cells were transfected with control (scrambled or FITC-GFP siRNA) or two-centrobin siRNA. After 72 h, cells were harvested and separated through a 6% SDS-PAGE, then blotted with anti-centrobin and anti–α-tubulin antibodies. (B) Immunostaining of centrobin. HeLa cells transfected with scrambled or centrobin siRNA #1 were stained with anti-centrobin and rhodamine-labeled goat anti–rabbit IgG. DNA was stained with DAPI. (C) Centrobin depletion did not affect the localization of γ-tubulin. HeLa cells transfected with scrambled or centrobin siRNA #1 were stained with anti-centrobin and anti–γ-tubulin and with rhodamine-labeled goat anti–rabbit IgG and FITC-labeled goat anti–mouse IgG. (D) Centrobin depletion did not affect microtubule organization and nucleation. HeLa cells were transfected with scrambled siRNA or centrobin siRNA; after 72 h, the cells were treated with 1 μM nocodazole for 1 h and washed three times with PBS to remove the nocodazole. The cells were harvested for fixation using cold methanol and stained for α-tubulin at 0, 2, 5, 10, and 15 min after the removal of nocodazole.
Mentions: We used 21-nt small interfering RNA (siRNA) targeting the coding region of centrobin to knock down centrobin expression. As shown in Fig. 5 A, the endogenous centrobin level was markedly reduced after centrobin siRNA transfection of HeLa cells, but not after the scrambled siRNA and GFP-siRNA were transfected (Fig. 5 A, top). Transfection of HeLa cells with FITC-labeled GFP-siRNA indicated a transfection efficiency of ∼90%. Densitometry analysis of the Western blots indicated that 80% of centrobin was reproducibly depleted with centrobin RNAi #1, which we used in all subsequent experiments. The reduction of centrobin levels was also confirmed by immunofluorescence analysis (Fig. 5 B). Immunofluorescence analysis with anti–γ-tubulin antibodies revealed that the γ-tubulin staining pattern was not visibly altered in the HeLa cells with undetectable levels of centrobin (Fig. 5 C). Furthermore, no gross abnormalities in microtubule nucleation and organization was observed in centrobin-depleted cells (Fig. 5 D), suggesting that centrobin likely does not substantially contribute to microtubule organization and nucleation, at least in interphase cells.

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