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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 inhibited centrosome duplication. (A and B) Centrobin depletion inhibited centrosome duplication in interphase and mitotic HeLa cells. HeLa cells were transfected with scrambled siRNA or centrobin siRNA and, 72 h later, were fixed with cold methanol and stained with anti-centrobin and anti–centrin-2 antibodies. The number of centrioles was counted according to the centrin-2 staining in interphase (A) and mitotic cells (B) with an undetectable level of centrobin. Data presented are the percentages of cells with more than four, four, two, one, or zero centrioles (average from three independent experiments). For interphase cells, 300 cells were counted in every experiment. For mitotic cells, 100 cells were counted in every experiment. (C) Electron microscopic examination of centrosomes from control or centrobin-depleted HeLa cells over consecutive thick sections spanning the entire nuclear–centrosome complexes. A representative control cell with two centrioles and a centrobin-depleted cell with one centriole are shown here. (D and E) Centrobin depletion inhibited centriole duplication in HeLa cells arrested by HU. HeLa cells were transfected with scrambled siRNA or centrobin siRNA. 48 h later the cells were treated with16 mM HU for another 24 h, pulse-labeled with BrdU for 30 min, and fixed and stained with anti-BrdU and anti-centrin. Data presented in D are the percentage of cells with four, two, one, or zero centrioles (average from three independent experiments with 300 cells counted in every experiment). Data presented in E are the percentages of cells with positive or negative BrdU staining (average from three independent experiments with 300 cells counted in every experiment). (F) Centrobin depletion inhibited centrosome overamplification in U2OS cells treated with HU. U2OS cells were either treated with HU for 16 h, then transfected with scrambled siRNA or centrobin siRNA, and incubated for an additional 48 h in the presence of 16 mM HU (HU→siRNA) or transfected with scrambled siRNA or centrobin siRNA, and 8 h later treated with 16 mM HU for an additional 62 h (siRNA→HU). All cells were fixed and stained with anti-centrobin and anti–γ-tubulin antibodies. Data presented are the percentages of cells with more than two centrosomes (average and SD from three independent experiments with 300 cells counted in every experiment). (G) Representative U2OS cells transfected with control or centrobin siRNA.
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fig6: Centrobin depletion inhibited centrosome duplication. (A and B) Centrobin depletion inhibited centrosome duplication in interphase and mitotic HeLa cells. HeLa cells were transfected with scrambled siRNA or centrobin siRNA and, 72 h later, were fixed with cold methanol and stained with anti-centrobin and anti–centrin-2 antibodies. The number of centrioles was counted according to the centrin-2 staining in interphase (A) and mitotic cells (B) with an undetectable level of centrobin. Data presented are the percentages of cells with more than four, four, two, one, or zero centrioles (average from three independent experiments). For interphase cells, 300 cells were counted in every experiment. For mitotic cells, 100 cells were counted in every experiment. (C) Electron microscopic examination of centrosomes from control or centrobin-depleted HeLa cells over consecutive thick sections spanning the entire nuclear–centrosome complexes. A representative control cell with two centrioles and a centrobin-depleted cell with one centriole are shown here. (D and E) Centrobin depletion inhibited centriole duplication in HeLa cells arrested by HU. HeLa cells were transfected with scrambled siRNA or centrobin siRNA. 48 h later the cells were treated with16 mM HU for another 24 h, pulse-labeled with BrdU for 30 min, and fixed and stained with anti-BrdU and anti-centrin. Data presented in D are the percentage of cells with four, two, one, or zero centrioles (average from three independent experiments with 300 cells counted in every experiment). Data presented in E are the percentages of cells with positive or negative BrdU staining (average from three independent experiments with 300 cells counted in every experiment). (F) Centrobin depletion inhibited centrosome overamplification in U2OS cells treated with HU. U2OS cells were either treated with HU for 16 h, then transfected with scrambled siRNA or centrobin siRNA, and incubated for an additional 48 h in the presence of 16 mM HU (HU→siRNA) or transfected with scrambled siRNA or centrobin siRNA, and 8 h later treated with 16 mM HU for an additional 62 h (siRNA→HU). All cells were fixed and stained with anti-centrobin and anti–γ-tubulin antibodies. Data presented are the percentages of cells with more than two centrosomes (average and SD from three independent experiments with 300 cells counted in every experiment). (G) Representative U2OS cells transfected with control or centrobin siRNA.

Mentions: To examine the function of centrobin in centriole duplication, we enumerated the centrioles in control RNAi-treated versus centrobin-depleted HeLa cells using anti–centrin-2 staining. In centrobin-depleted cultures, 21% of interphase cells had four centrioles, 67% had two centrioles, 7% had one centriole, and 2% had no centriole (Fig. 6 A). In contrast, among the control HeLa cells, 47% of the interphase cells had four centrioles, 47% had two centrioles, only 2% of interphase cells had one centriole, and 0.3% had no centriole (Fig. 6 A). Thus, centrobin depletion induced a marked reduction in the proportion of cells with four centrioles and, correspondingly, an increase in the proportion of cells with fewer than four centrioles. An even more pronounced difference was seen in mitotic cells. Essentially all mitotic cells in the control culture had four centrioles; in contrast, only 51% of the centrobin-depleted mitotic cells had four centrioles, whereas 45% had two and 4% had one centriole (Fig. 6 B). Thus, RNAi-mediated centrobin depletion dramatically inhibited centriole duplication. Nevertheless, the cells were still able to progress through the cell cycle, at least once or twice, generating cells with one centriole or none.


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 inhibited centrosome duplication. (A and B) Centrobin depletion inhibited centrosome duplication in interphase and mitotic HeLa cells. HeLa cells were transfected with scrambled siRNA or centrobin siRNA and, 72 h later, were fixed with cold methanol and stained with anti-centrobin and anti–centrin-2 antibodies. The number of centrioles was counted according to the centrin-2 staining in interphase (A) and mitotic cells (B) with an undetectable level of centrobin. Data presented are the percentages of cells with more than four, four, two, one, or zero centrioles (average from three independent experiments). For interphase cells, 300 cells were counted in every experiment. For mitotic cells, 100 cells were counted in every experiment. (C) Electron microscopic examination of centrosomes from control or centrobin-depleted HeLa cells over consecutive thick sections spanning the entire nuclear–centrosome complexes. A representative control cell with two centrioles and a centrobin-depleted cell with one centriole are shown here. (D and E) Centrobin depletion inhibited centriole duplication in HeLa cells arrested by HU. HeLa cells were transfected with scrambled siRNA or centrobin siRNA. 48 h later the cells were treated with16 mM HU for another 24 h, pulse-labeled with BrdU for 30 min, and fixed and stained with anti-BrdU and anti-centrin. Data presented in D are the percentage of cells with four, two, one, or zero centrioles (average from three independent experiments with 300 cells counted in every experiment). Data presented in E are the percentages of cells with positive or negative BrdU staining (average from three independent experiments with 300 cells counted in every experiment). (F) Centrobin depletion inhibited centrosome overamplification in U2OS cells treated with HU. U2OS cells were either treated with HU for 16 h, then transfected with scrambled siRNA or centrobin siRNA, and incubated for an additional 48 h in the presence of 16 mM HU (HU→siRNA) or transfected with scrambled siRNA or centrobin siRNA, and 8 h later treated with 16 mM HU for an additional 62 h (siRNA→HU). All cells were fixed and stained with anti-centrobin and anti–γ-tubulin antibodies. Data presented are the percentages of cells with more than two centrosomes (average and SD from three independent experiments with 300 cells counted in every experiment). (G) Representative U2OS cells transfected with control or centrobin siRNA.
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fig6: Centrobin depletion inhibited centrosome duplication. (A and B) Centrobin depletion inhibited centrosome duplication in interphase and mitotic HeLa cells. HeLa cells were transfected with scrambled siRNA or centrobin siRNA and, 72 h later, were fixed with cold methanol and stained with anti-centrobin and anti–centrin-2 antibodies. The number of centrioles was counted according to the centrin-2 staining in interphase (A) and mitotic cells (B) with an undetectable level of centrobin. Data presented are the percentages of cells with more than four, four, two, one, or zero centrioles (average from three independent experiments). For interphase cells, 300 cells were counted in every experiment. For mitotic cells, 100 cells were counted in every experiment. (C) Electron microscopic examination of centrosomes from control or centrobin-depleted HeLa cells over consecutive thick sections spanning the entire nuclear–centrosome complexes. A representative control cell with two centrioles and a centrobin-depleted cell with one centriole are shown here. (D and E) Centrobin depletion inhibited centriole duplication in HeLa cells arrested by HU. HeLa cells were transfected with scrambled siRNA or centrobin siRNA. 48 h later the cells were treated with16 mM HU for another 24 h, pulse-labeled with BrdU for 30 min, and fixed and stained with anti-BrdU and anti-centrin. Data presented in D are the percentage of cells with four, two, one, or zero centrioles (average from three independent experiments with 300 cells counted in every experiment). Data presented in E are the percentages of cells with positive or negative BrdU staining (average from three independent experiments with 300 cells counted in every experiment). (F) Centrobin depletion inhibited centrosome overamplification in U2OS cells treated with HU. U2OS cells were either treated with HU for 16 h, then transfected with scrambled siRNA or centrobin siRNA, and incubated for an additional 48 h in the presence of 16 mM HU (HU→siRNA) or transfected with scrambled siRNA or centrobin siRNA, and 8 h later treated with 16 mM HU for an additional 62 h (siRNA→HU). All cells were fixed and stained with anti-centrobin and anti–γ-tubulin antibodies. Data presented are the percentages of cells with more than two centrosomes (average and SD from three independent experiments with 300 cells counted in every experiment). (G) Representative U2OS cells transfected with control or centrobin siRNA.
Mentions: To examine the function of centrobin in centriole duplication, we enumerated the centrioles in control RNAi-treated versus centrobin-depleted HeLa cells using anti–centrin-2 staining. In centrobin-depleted cultures, 21% of interphase cells had four centrioles, 67% had two centrioles, 7% had one centriole, and 2% had no centriole (Fig. 6 A). In contrast, among the control HeLa cells, 47% of the interphase cells had four centrioles, 47% had two centrioles, only 2% of interphase cells had one centriole, and 0.3% had no centriole (Fig. 6 A). Thus, centrobin depletion induced a marked reduction in the proportion of cells with four centrioles and, correspondingly, an increase in the proportion of cells with fewer than four centrioles. An even more pronounced difference was seen in mitotic cells. Essentially all mitotic cells in the control culture had four centrioles; in contrast, only 51% of the centrobin-depleted mitotic cells had four centrioles, whereas 45% had two and 4% had one centriole (Fig. 6 B). Thus, RNAi-mediated centrobin depletion dramatically inhibited centriole duplication. Nevertheless, the cells were still able to progress through the cell cycle, at least once or twice, generating cells with one centriole or none.

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