Figure 4.

Inhibition of Golgi fragmentation blocks Aur-A recruitment to the centrosomes in HeLa cells. Cells were grown on coverslips and arrested in S phase using the double-thymidine block and processed for immunofluorescence under confocal microscopy. (A) Representative images of cells at the indicated cell cycle stages, labeled with antibodies against γ-tubulin (green, arrow) and with DRAQ5 (blue) for cell cycle phase, and labeled with antibodies against Aur-A (red, arrow). (B) Quantification of cells as described in A, with percentages of Aur-A positive cells calculated as percentages of cells with Aur-A at the centrosome (dark gray bars) and the spindle (light gray bars), at the indicated times. (C) Representative images of cells 8 h after thymidine washout, labeled with antibodies against giantin (green) for the Golgi complex, and with DRAQ5 (blue) for cell cycle phase, and labeled with antibodies against Aur-A (red, arrow). (D and E) The cells were either nonmicroinjected or microinjected 1 h after thymidine washout with recombinant GST (GST-inj; 8 mg/ml), recombinant SBD (SBD-inj; 8–12 mg/ml), or recombinant GRASP65 (GR65-inj; 8–10 mg/ml), and with FITC-conjugated dextran as microinjection marker. For the noninjected cells, 8 h after thymidine washout they were treated with vehicle (−) or 0.25 μM MLN8054 (MLN). The cells were then processed 8 h, 10 h (SBD-injected cells only) and 12 h (all of the cells) after thymidine washout, for immunofluorescence under confocal microscopy with antibodies against Aur-A and with DRAQ5 (for cell cycle phase). (D) Representative images of noninjected and SBD-injected cells. (E) The relative percentages of Aur-A–positive cells were calculated according to the relevant nonmicroinjected cells on the same coverslip (see Materials and Methods), or to cells treated with vehicle (−). All images were acquired at maximal resolution, with fixed imaging conditions. Bars, 5 μm. Quantification data are means ± SD from two (B) and three (E) independent experiments, each carried out in duplicate. More than 200 cells were microinjected for each condition. (F) Representative images of cells 12 h after thymidine washout, labeled with antibodies against pericentrin (green) as a centrosome marker, and with Aurora-A (red). Insets are higher magnification views of representative centrosomes. Equal areas were used to select the centrosome regions (circle) and a noncentrosome region with a similar background (dashed circle). (G) Fluorescence intensity (a.u., arbitrary units) of centrosome-associated Aur-A per cell was quantified by using LSM 710 ZEN 2008 SP1 (see Materials and Methods) in cells that had been treated as described in A and injected with FITC-dextran alone (Dx-inj) or in the presence of SBD (SBD-inj) or GRASP65 (GR65-inj). One data set representative of three independent experiments is shown as a scatter plot. More than 250 cells where injected and analyzed. (H) Fluorescence intensity ±SEM of the samples reported in (G) that showed above-background fluorescence levels of centrosome-associated Aur-A. Two-tailed Student's t tests were applied to the data (*p < 0.005; **p < 0.001). Bar, 5 μm.