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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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Localization of centrobin to the centrosomes. (A) Endogenous centrobin localized to the centrosomes. T47D, MCF-7, 76N, and Capan-1 cells were grown on coverslips, fixed with cold methanol, stained with affinity-purified anti-centrobin (1 μg/ml) and anti–α-tubulin (1:500) or anti–γ-tubulin (1:400), and stained with rhodamine-labeled goat anti–rabbit IgG and FITC-labeled goat anti–mouse IgG. DNA was stained with DAPI. (B) Localization of GFP-tagged centrobin to the centrosomes. 76NTert cells were grown on coverslips, transfected with pEGFP-Myc-centrobin, fixed with cold methanol, stained with anti–γ-tubulin (1:400), and stained again with rhodamine-labeled goat anti–mouse IgG. DNA was stained with DAPI. (C) Localization of Myc-tagged centrobin to the centrosomes. 76NTert cells were transfected with pCR3.1-Myc–centrobin, fixed with cold methanol, and stained with anti-Myc antibody 9E10 (1 μg/ml) and anti–γ-tubulin (1:400), and then with rhodamine-labeled goat anti–mouse IgG and FITC-labeled goat anti–rabbit IgG. DNA was stained with DAPI. (D) Localization of Myc-tagged centrobin-C to the centrosomes. 76NTert cells were grown on coverslips, transfected with pSG5-Myc–centrobin-C, fixed with cold methanol, and stained with anti-Myc and anti–γ-tubulin antibodies (1:400).
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fig2: Localization of centrobin to the centrosomes. (A) Endogenous centrobin localized to the centrosomes. T47D, MCF-7, 76N, and Capan-1 cells were grown on coverslips, fixed with cold methanol, stained with affinity-purified anti-centrobin (1 μg/ml) and anti–α-tubulin (1:500) or anti–γ-tubulin (1:400), and stained with rhodamine-labeled goat anti–rabbit IgG and FITC-labeled goat anti–mouse IgG. DNA was stained with DAPI. (B) Localization of GFP-tagged centrobin to the centrosomes. 76NTert cells were grown on coverslips, transfected with pEGFP-Myc-centrobin, fixed with cold methanol, stained with anti–γ-tubulin (1:400), and stained again with rhodamine-labeled goat anti–mouse IgG. DNA was stained with DAPI. (C) Localization of Myc-tagged centrobin to the centrosomes. 76NTert cells were transfected with pCR3.1-Myc–centrobin, fixed with cold methanol, and stained with anti-Myc antibody 9E10 (1 μg/ml) and anti–γ-tubulin (1:400), and then with rhodamine-labeled goat anti–mouse IgG and FITC-labeled goat anti–rabbit IgG. DNA was stained with DAPI. (D) Localization of Myc-tagged centrobin-C to the centrosomes. 76NTert cells were grown on coverslips, transfected with pSG5-Myc–centrobin-C, fixed with cold methanol, and stained with anti-Myc and anti–γ-tubulin antibodies (1:400).

Mentions: The anti-centrobin antibody characterized in Fig. 1 F was used to examine the localization of endogenous centrobin in a normal human mammary epithelial cell line (76N) and several cancer cell lines (T47D, MCF-7, and Capan-1). A typical centrosomal staining pattern was observed in all the cell lines tested, with one or two perinuclear dots in the interphase cells (Fig. 2 A, g, o, and s) and a single focus at the end of each mitotic spindle in mitotic cells (Fig. 2 A, c and k). The centrobin staining pattern was similar to that of γ-tubulin, a protein known to specifically localize to centrosomes (Fig. 2 A, d, h, p, and t). An identical centrosomal staining pattern was observed in MCF10A, HeLa, COS-7, and 293T cells (unpublished data). The centrosomal staining was observed under three different fixation conditions (3.7% formaldehyde, 100% methanol, or 0.5% glutaraldehyde) and also when cells were extracted with 0.5% Triton X-100 in 80 mM Pipes, 1 mM MgCl2, and 5 mM EGTA, pH 6.8, before fixation. Furthermore, the centrosomal localization of centrobin was not affected by treatment with nocodazole (unpublished data). These findings strongly indicated that centrobin is likely to be a bona fide core component of the centrosomes (Oegema et al., 1995). Importantly, GFP-centrobin and Myc-centrobin also localized to the centrosomes in the transfected cells when they were expressed at a very low level (Fig. 2, B and C). It is notable that both GFP-centrobin and Myc-centrobin formed bundle-like structures when expressed at high levels, which is probably an artifact of high-level expression because the endogenous centrobin is expressed at very low levels and is found mainly on centrosomes (unpublished data). A truncated Myc-tagged centrobin (centrobin-C; encoding the COOH-terminal 539 aa) also localized to the centrosomes when expressed at low levels (Fig. 2 D), indicating that the COOH-terminal 539 aa of centrobin is sufficient for centrosomal localization. It is noteworthy that when Myc–centrobin-C was expressed at a high level it decorated the microtubules, which is also probably an artifact of high-level expression (unpublished data).


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)

Localization of centrobin to the centrosomes. (A) Endogenous centrobin localized to the centrosomes. T47D, MCF-7, 76N, and Capan-1 cells were grown on coverslips, fixed with cold methanol, stained with affinity-purified anti-centrobin (1 μg/ml) and anti–α-tubulin (1:500) or anti–γ-tubulin (1:400), and stained with rhodamine-labeled goat anti–rabbit IgG and FITC-labeled goat anti–mouse IgG. DNA was stained with DAPI. (B) Localization of GFP-tagged centrobin to the centrosomes. 76NTert cells were grown on coverslips, transfected with pEGFP-Myc-centrobin, fixed with cold methanol, stained with anti–γ-tubulin (1:400), and stained again with rhodamine-labeled goat anti–mouse IgG. DNA was stained with DAPI. (C) Localization of Myc-tagged centrobin to the centrosomes. 76NTert cells were transfected with pCR3.1-Myc–centrobin, fixed with cold methanol, and stained with anti-Myc antibody 9E10 (1 μg/ml) and anti–γ-tubulin (1:400), and then with rhodamine-labeled goat anti–mouse IgG and FITC-labeled goat anti–rabbit IgG. DNA was stained with DAPI. (D) Localization of Myc-tagged centrobin-C to the centrosomes. 76NTert cells were grown on coverslips, transfected with pSG5-Myc–centrobin-C, fixed with cold methanol, and stained with anti-Myc and anti–γ-tubulin antibodies (1:400).
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Related In: Results  -  Collection

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fig2: Localization of centrobin to the centrosomes. (A) Endogenous centrobin localized to the centrosomes. T47D, MCF-7, 76N, and Capan-1 cells were grown on coverslips, fixed with cold methanol, stained with affinity-purified anti-centrobin (1 μg/ml) and anti–α-tubulin (1:500) or anti–γ-tubulin (1:400), and stained with rhodamine-labeled goat anti–rabbit IgG and FITC-labeled goat anti–mouse IgG. DNA was stained with DAPI. (B) Localization of GFP-tagged centrobin to the centrosomes. 76NTert cells were grown on coverslips, transfected with pEGFP-Myc-centrobin, fixed with cold methanol, stained with anti–γ-tubulin (1:400), and stained again with rhodamine-labeled goat anti–mouse IgG. DNA was stained with DAPI. (C) Localization of Myc-tagged centrobin to the centrosomes. 76NTert cells were transfected with pCR3.1-Myc–centrobin, fixed with cold methanol, and stained with anti-Myc antibody 9E10 (1 μg/ml) and anti–γ-tubulin (1:400), and then with rhodamine-labeled goat anti–mouse IgG and FITC-labeled goat anti–rabbit IgG. DNA was stained with DAPI. (D) Localization of Myc-tagged centrobin-C to the centrosomes. 76NTert cells were grown on coverslips, transfected with pSG5-Myc–centrobin-C, fixed with cold methanol, and stained with anti-Myc and anti–γ-tubulin antibodies (1:400).
Mentions: The anti-centrobin antibody characterized in Fig. 1 F was used to examine the localization of endogenous centrobin in a normal human mammary epithelial cell line (76N) and several cancer cell lines (T47D, MCF-7, and Capan-1). A typical centrosomal staining pattern was observed in all the cell lines tested, with one or two perinuclear dots in the interphase cells (Fig. 2 A, g, o, and s) and a single focus at the end of each mitotic spindle in mitotic cells (Fig. 2 A, c and k). The centrobin staining pattern was similar to that of γ-tubulin, a protein known to specifically localize to centrosomes (Fig. 2 A, d, h, p, and t). An identical centrosomal staining pattern was observed in MCF10A, HeLa, COS-7, and 293T cells (unpublished data). The centrosomal staining was observed under three different fixation conditions (3.7% formaldehyde, 100% methanol, or 0.5% glutaraldehyde) and also when cells were extracted with 0.5% Triton X-100 in 80 mM Pipes, 1 mM MgCl2, and 5 mM EGTA, pH 6.8, before fixation. Furthermore, the centrosomal localization of centrobin was not affected by treatment with nocodazole (unpublished data). These findings strongly indicated that centrobin is likely to be a bona fide core component of the centrosomes (Oegema et al., 1995). Importantly, GFP-centrobin and Myc-centrobin also localized to the centrosomes in the transfected cells when they were expressed at a very low level (Fig. 2, B and C). It is notable that both GFP-centrobin and Myc-centrobin formed bundle-like structures when expressed at high levels, which is probably an artifact of high-level expression because the endogenous centrobin is expressed at very low levels and is found mainly on centrosomes (unpublished data). A truncated Myc-tagged centrobin (centrobin-C; encoding the COOH-terminal 539 aa) also localized to the centrosomes when expressed at low levels (Fig. 2 D), indicating that the COOH-terminal 539 aa of centrobin is sufficient for centrosomal localization. It is noteworthy that when Myc–centrobin-C was expressed at a high level it decorated the microtubules, which is also probably an artifact of high-level expression (unpublished data).

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