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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 led to impaired cytokinesis. (A) Centrobin depletion led to cells with multiple nuclei. HeLa cells were transfected with scrambled siRNA or centrobin siRNA and, 72 h later, stained with anti-centrobin and DAPI (blue). The percentage of cells with two or more nuclei was enumerated. Data presented are averages and SDs from three independent experiments with 300 cells counted in every experiment. (B) Representative HeLa cells with two or four nuclei. (C) Centrobin depletion led to impaired cytokinesis. HeLa cells were transfected with scrambled siRNA or centrobin siRNA. 8 h after transfection, time-lapse phase-contrast microscopy was initiated and continued for 48 h. All the cells that underwent cytokinesis were followed to determine the fate and duration of cell division (from cell roundup until two daughter cells separate). The data presented were compiled from two experiments. The durations of cell division for the 23 control cells are 50, 55, 65, 65, 70, 75, 80, 80, 80, 85, 90, 100,100, 105, 110, 140, 160, 165, 170, 180, 200, 220, and 450 min. The durations of cell division for the 11 centrobin-siRNA–transfected cells are 85, 115, 135, 135, 220, 270, 335, 600, 640, 645, and 665 min. Four centrobin-siRNA–transfected cells exited mitosis without finishing cytokinesis at 175, 190, 210, and 260 min. (D) Two representative HeLa cells, one transfected with centrobin siRNA and one with control siRNA that underwent mitosis. The centrobin–siRNA-transfected cell was in mitosis for >260 min and exited without finishing mitosis. The control siRNA transfected cells took <120 min to finish mitosis.
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fig7: Centrobin depletion led to impaired cytokinesis. (A) Centrobin depletion led to cells with multiple nuclei. HeLa cells were transfected with scrambled siRNA or centrobin siRNA and, 72 h later, stained with anti-centrobin and DAPI (blue). The percentage of cells with two or more nuclei was enumerated. Data presented are averages and SDs from three independent experiments with 300 cells counted in every experiment. (B) Representative HeLa cells with two or four nuclei. (C) Centrobin depletion led to impaired cytokinesis. HeLa cells were transfected with scrambled siRNA or centrobin siRNA. 8 h after transfection, time-lapse phase-contrast microscopy was initiated and continued for 48 h. All the cells that underwent cytokinesis were followed to determine the fate and duration of cell division (from cell roundup until two daughter cells separate). The data presented were compiled from two experiments. The durations of cell division for the 23 control cells are 50, 55, 65, 65, 70, 75, 80, 80, 80, 85, 90, 100,100, 105, 110, 140, 160, 165, 170, 180, 200, 220, and 450 min. The durations of cell division for the 11 centrobin-siRNA–transfected cells are 85, 115, 135, 135, 220, 270, 335, 600, 640, 645, and 665 min. Four centrobin-siRNA–transfected cells exited mitosis without finishing cytokinesis at 175, 190, 210, and 260 min. (D) Two representative HeLa cells, one transfected with centrobin siRNA and one with control siRNA that underwent mitosis. The centrobin–siRNA-transfected cell was in mitosis for >260 min and exited without finishing mitosis. The control siRNA transfected cells took <120 min to finish mitosis.

Mentions: When centrobin was depleted in HeLa cells by the use of siRNA, we observed that the percentage of cells with two or more nuclei increased significantly (from 3% in control siRNA-transfected cells to 20% in the centrobin-depleted cells), indicating a failure of cytokinesis in a proportion of the centrobin-depleted cells (Fig. 7, A and B). To further explore this finding, we directly examined the progression of cell division with time-lapse microscopy. For this purpose, HeLa cells were transfected with control or centrobin siRNA. Phase-contrast time-lapse microscopy was initiated 8 h later and continued for 48 h (Videos 1 and 2, available at http://www.jcb.org/cgi/content/full/jcb.200506185/DC1). In the control cultures, 15 of the 23 observed cells completed mitosis within 2 h, and 22 of the 23 cells completed mitosis within 4 h; no cell failed to complete cytokinesis (Fig. 7, C and D). In contrast, out of the centrobin siRNA-transfected cells that went into mitosis, only 2 of 15 observed cells completed mitosis within 2 h, and only 5 of 15 cells completed mitosis within 4 h; 6 of 15 cells failed to do so within 4 h and 4 of 15 cells exited mitosis without finishing cytokinesis. This finding indicates that centrobin depletion impairs cytokinesis.


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 led to impaired cytokinesis. (A) Centrobin depletion led to cells with multiple nuclei. HeLa cells were transfected with scrambled siRNA or centrobin siRNA and, 72 h later, stained with anti-centrobin and DAPI (blue). The percentage of cells with two or more nuclei was enumerated. Data presented are averages and SDs from three independent experiments with 300 cells counted in every experiment. (B) Representative HeLa cells with two or four nuclei. (C) Centrobin depletion led to impaired cytokinesis. HeLa cells were transfected with scrambled siRNA or centrobin siRNA. 8 h after transfection, time-lapse phase-contrast microscopy was initiated and continued for 48 h. All the cells that underwent cytokinesis were followed to determine the fate and duration of cell division (from cell roundup until two daughter cells separate). The data presented were compiled from two experiments. The durations of cell division for the 23 control cells are 50, 55, 65, 65, 70, 75, 80, 80, 80, 85, 90, 100,100, 105, 110, 140, 160, 165, 170, 180, 200, 220, and 450 min. The durations of cell division for the 11 centrobin-siRNA–transfected cells are 85, 115, 135, 135, 220, 270, 335, 600, 640, 645, and 665 min. Four centrobin-siRNA–transfected cells exited mitosis without finishing cytokinesis at 175, 190, 210, and 260 min. (D) Two representative HeLa cells, one transfected with centrobin siRNA and one with control siRNA that underwent mitosis. The centrobin–siRNA-transfected cell was in mitosis for >260 min and exited without finishing mitosis. The control siRNA transfected cells took <120 min to finish mitosis.
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Related In: Results  -  Collection

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fig7: Centrobin depletion led to impaired cytokinesis. (A) Centrobin depletion led to cells with multiple nuclei. HeLa cells were transfected with scrambled siRNA or centrobin siRNA and, 72 h later, stained with anti-centrobin and DAPI (blue). The percentage of cells with two or more nuclei was enumerated. Data presented are averages and SDs from three independent experiments with 300 cells counted in every experiment. (B) Representative HeLa cells with two or four nuclei. (C) Centrobin depletion led to impaired cytokinesis. HeLa cells were transfected with scrambled siRNA or centrobin siRNA. 8 h after transfection, time-lapse phase-contrast microscopy was initiated and continued for 48 h. All the cells that underwent cytokinesis were followed to determine the fate and duration of cell division (from cell roundup until two daughter cells separate). The data presented were compiled from two experiments. The durations of cell division for the 23 control cells are 50, 55, 65, 65, 70, 75, 80, 80, 80, 85, 90, 100,100, 105, 110, 140, 160, 165, 170, 180, 200, 220, and 450 min. The durations of cell division for the 11 centrobin-siRNA–transfected cells are 85, 115, 135, 135, 220, 270, 335, 600, 640, 645, and 665 min. Four centrobin-siRNA–transfected cells exited mitosis without finishing cytokinesis at 175, 190, 210, and 260 min. (D) Two representative HeLa cells, one transfected with centrobin siRNA and one with control siRNA that underwent mitosis. The centrobin–siRNA-transfected cell was in mitosis for >260 min and exited without finishing mitosis. The control siRNA transfected cells took <120 min to finish mitosis.
Mentions: When centrobin was depleted in HeLa cells by the use of siRNA, we observed that the percentage of cells with two or more nuclei increased significantly (from 3% in control siRNA-transfected cells to 20% in the centrobin-depleted cells), indicating a failure of cytokinesis in a proportion of the centrobin-depleted cells (Fig. 7, A and B). To further explore this finding, we directly examined the progression of cell division with time-lapse microscopy. For this purpose, HeLa cells were transfected with control or centrobin siRNA. Phase-contrast time-lapse microscopy was initiated 8 h later and continued for 48 h (Videos 1 and 2, available at http://www.jcb.org/cgi/content/full/jcb.200506185/DC1). In the control cultures, 15 of the 23 observed cells completed mitosis within 2 h, and 22 of the 23 cells completed mitosis within 4 h; no cell failed to complete cytokinesis (Fig. 7, C and D). In contrast, out of the centrobin siRNA-transfected cells that went into mitosis, only 2 of 15 observed cells completed mitosis within 2 h, and only 5 of 15 cells completed mitosis within 4 h; 6 of 15 cells failed to do so within 4 h and 4 of 15 cells exited mitosis without finishing cytokinesis. This finding indicates that centrobin depletion impairs cytokinesis.

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