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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 localized to the daughter centriole. (A) Localization of centrobin during different phases of the cell cycle in 76NTert cells. 76NTert cells were synchronized by mitotic shake-off and replating. Cells were harvested, extracted, fixed with cold methanol, and stained with anti-centrobin (red) and anti–centrin-2 (green) antibodies. (B) Centrobin localization in interphase NIH-3T3 cells. The cells were fixed with cold methanol, and then stained with anti-acetylated α-tubulin (green) and anti-centrobin (red) antibodies. (C) Centrobin localization in U2OS cells treated with HU. U2OS cells were treated with 16 mM HU for 72 h, fixed with cold methanol, and stained with anti-centrobin (red) and anti–centrin-2 (green) antibodies. (D) Immunogold electron microscopic localization of centrobin on the daughter centrioles. 10-nm gold particles were detected on daughter centrioles (D) but not on mother centrioles (M).
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fig4: Centrobin localized to the daughter centriole. (A) Localization of centrobin during different phases of the cell cycle in 76NTert cells. 76NTert cells were synchronized by mitotic shake-off and replating. Cells were harvested, extracted, fixed with cold methanol, and stained with anti-centrobin (red) and anti–centrin-2 (green) antibodies. (B) Centrobin localization in interphase NIH-3T3 cells. The cells were fixed with cold methanol, and then stained with anti-acetylated α-tubulin (green) and anti-centrobin (red) antibodies. (C) Centrobin localization in U2OS cells treated with HU. U2OS cells were treated with 16 mM HU for 72 h, fixed with cold methanol, and stained with anti-centrobin (red) and anti–centrin-2 (green) antibodies. (D) Immunogold electron microscopic localization of centrobin on the daughter centrioles. 10-nm gold particles were detected on daughter centrioles (D) but not on mother centrioles (M).

Mentions: During our initial immunolocalization analyses we noted that the staining pattern of centrobin differed in cells that appeared to be at different phases of the cell cycle, suggesting the possibility that centrobin may differentially localize in either the mother or daughter centrioles. To further explore this possibility, we performed centrobin localization experiments in synchronized 76NTert cells (an hTert immortalized cell line derived from normal human mammary epithelial 76N cells) in which the two centrioles were typically located farther apart from each other than they are in other cells. Synchronization was achieved by mitotic shake-off. A majority of cells at G0/G1 exhibited a strongly stained centriolar dot, with the other centriole stained weakly or not at all with anti-centrobin antibody. Superimposing centrin and centrobin staining essentially demonstrated complete correspondence of weak centrin staining (daughter centriole) with strong centrobin staining, and vice versa (Fig. 4 A). In the majority of G1/S, S, and G2/M phase cells, there are usually two strongly stained centrobin dots, correlating with the two newly synthesized daughter centrioles, as indicated by the weaker centrin-2 staining. In some cells, three or four centrobin dots can also be found, including one or two dots with weaker centrobin staining, which are likely to correlate with the original daughter centrioles (mother centrioles, in the current duplication cycle). These findings indicate that centrobin is preferentially incorporated into the newly assembled daughter centriole during centriole assembly at the late G1 or early S phase and that centrobin remains in the daughter centrioles throughout the cell cycle. At the next cycle of centriole duplication, the amount of centrobin on the original daughter centriole eventually decreases, as shown in the G1/S and S phase cells (Fig. 4 A).


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 localized to the daughter centriole. (A) Localization of centrobin during different phases of the cell cycle in 76NTert cells. 76NTert cells were synchronized by mitotic shake-off and replating. Cells were harvested, extracted, fixed with cold methanol, and stained with anti-centrobin (red) and anti–centrin-2 (green) antibodies. (B) Centrobin localization in interphase NIH-3T3 cells. The cells were fixed with cold methanol, and then stained with anti-acetylated α-tubulin (green) and anti-centrobin (red) antibodies. (C) Centrobin localization in U2OS cells treated with HU. U2OS cells were treated with 16 mM HU for 72 h, fixed with cold methanol, and stained with anti-centrobin (red) and anti–centrin-2 (green) antibodies. (D) Immunogold electron microscopic localization of centrobin on the daughter centrioles. 10-nm gold particles were detected on daughter centrioles (D) but not on mother centrioles (M).
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fig4: Centrobin localized to the daughter centriole. (A) Localization of centrobin during different phases of the cell cycle in 76NTert cells. 76NTert cells were synchronized by mitotic shake-off and replating. Cells were harvested, extracted, fixed with cold methanol, and stained with anti-centrobin (red) and anti–centrin-2 (green) antibodies. (B) Centrobin localization in interphase NIH-3T3 cells. The cells were fixed with cold methanol, and then stained with anti-acetylated α-tubulin (green) and anti-centrobin (red) antibodies. (C) Centrobin localization in U2OS cells treated with HU. U2OS cells were treated with 16 mM HU for 72 h, fixed with cold methanol, and stained with anti-centrobin (red) and anti–centrin-2 (green) antibodies. (D) Immunogold electron microscopic localization of centrobin on the daughter centrioles. 10-nm gold particles were detected on daughter centrioles (D) but not on mother centrioles (M).
Mentions: During our initial immunolocalization analyses we noted that the staining pattern of centrobin differed in cells that appeared to be at different phases of the cell cycle, suggesting the possibility that centrobin may differentially localize in either the mother or daughter centrioles. To further explore this possibility, we performed centrobin localization experiments in synchronized 76NTert cells (an hTert immortalized cell line derived from normal human mammary epithelial 76N cells) in which the two centrioles were typically located farther apart from each other than they are in other cells. Synchronization was achieved by mitotic shake-off. A majority of cells at G0/G1 exhibited a strongly stained centriolar dot, with the other centriole stained weakly or not at all with anti-centrobin antibody. Superimposing centrin and centrobin staining essentially demonstrated complete correspondence of weak centrin staining (daughter centriole) with strong centrobin staining, and vice versa (Fig. 4 A). In the majority of G1/S, S, and G2/M phase cells, there are usually two strongly stained centrobin dots, correlating with the two newly synthesized daughter centrioles, as indicated by the weaker centrin-2 staining. In some cells, three or four centrobin dots can also be found, including one or two dots with weaker centrobin staining, which are likely to correlate with the original daughter centrioles (mother centrioles, in the current duplication cycle). These findings indicate that centrobin is preferentially incorporated into the newly assembled daughter centriole during centriole assembly at the late G1 or early S phase and that centrobin remains in the daughter centrioles throughout the cell cycle. At the next cycle of centriole duplication, the amount of centrobin on the original daughter centriole eventually decreases, as shown in the G1/S and S phase cells (Fig. 4 A).

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