Centrobin expression, amino acid sequence, and structure. (A and B) A tissue blot with 2 μg of polyA mRNA per lane (A) and a blot with 20 μg of total mRNA from the indicated breast cell lines (B) were probed with a ³²P-labeled centrobin probe followed by autoradiography. Hybridization with the 36B4 probe was used as a loading control. (C) The predicted amino acid sequence of centrobin-α. (D) The extra 22 residues presented only in centrobin-β. (E) Coiled–coil regions of centrobin, ninein, and pericentrin as predicted by DNASTAR. Boxes indicate the coiled–coil region; lines indicate noncoiled regions. Centrobin-C, the COOH-terminal fragment of centrobin that was isolated from the yeast two-hybrid screen. (F) Centrobin protein expression. Cell lysate from the indicated cell lines or 293T cells transfected with pCR3.1 vector, Myc-centrobin, or GFP-centrobin constructs were fractionated by a 6% SDS-PAGE and blotted with affinity-purified anti-centrobin or anti-Myc antibodies.

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).

Copurification of centrobin with the centrosomes through sucrose gradients. Centrosomes from 293T were purified through a 40–70% sucrose gradient as described (Blomberg-Wirschell and Doxsey, 1998). The resulting fractions were separated on SDS-PAGE gels and blotted with anti-centrobin, anti–γ-tubulin, anti-Lamin B1, and anti-Cbl antibodies.

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).

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.

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.

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.