Involvement of Girdin in the establishment of cell polarity.

<p>A. Girdin depletion inhibited directional cell migration. Control or Girdin-depleted Vero cells (eight dishes for each group) were scratched to induce cell migration and incubated in medium supplemented with 3% FBS for nine hr. The wound's width was measured in each group at zero, seven, and nine hr after the wounding. Asterisks indicate significant difference compared with control cells (Student's t test; P<0.05). B. Schematic illustration of scoring principle of cell polarity in Vero cells. Monolayers of confluent Vero cells on glass-based dishes were scratched to initiate sheet migration into the wound (left), incubated for eight hrs, fixed, and immunostained for GM130 (a marker of the Golgi apparatus). Polarization of leading cells that face the wound was analyzed by the location of the Golgi apparatus (middle). Cells with the Golgi apparatus locating within one third of the cytoplasm relative to the direction of sheet migration were counted (right). C, D. Depletion of Girdin and Par-3 from Vero cells: effects on cell polarization. Monolayers of control- (far left panel), Girdin- (sh1 and sh7, middle panels), and Par-3 shRNA-expressing Vero cells (far right panel) were scratched to induce migration and the polarization of leading cells was evaluated by immunostaining for GM130 (red). Nuclei and actin filaments were visualized by staining with DAPI (blue) and phalloidin (green), respectively. Properly polarized and non-polarized leading cells are indicated by white and yellow arrowheads, respectively. In D, the graph shows the percentage of leading cells with polarized Golgi apparatus in each group (300 cells from three independent experiments). Inset shows the data from Western blot analysis showing stable depletion of endogenous Par-3 by the infection of a retrovirus expressing Par-3 shRNA. Asterisks indicate statistical significance (Student's t test; P<0.05). E, F, G. Overexpression of Par-3 Δ4N and Δ4N/2 mutants in Vero cells: effects on cell polarization. Shown in E is a schematic illustration of Par-3 Δ4N and Δ4N/2 mutants used in the study. Monolayers of control (myc-GST), Par-3 Δ4N-, and Par-3 Δ4N/2-overexpressing Vero cells were scratched to induce sheet migration. The polarization of leading cells, immunostained for GM130 (F), and the percentage of leading cells with polarized Golgi apparatus was quantified in each group (150 cells each) (G). The asterisk indicates statistical significance (Student's t test; P<0.05).