Fig. 1. Dynein and dynactin localize to the leading edge of migrating cells and promote centrosome reorientation, leading-edge protrusion and nuclear migration. (A) Dynein and dynactin concentrate in cortical patches at the leading edge of migrating cells. NIH/3T3 cells were mock transfected, transfected with siRNAs targeting p150^Glued or DHC, or transfected with a scrambled oligonucleotide control, then grown to confluency and the monolayer wounded. Six hours after wounding, cells were fixed and stained for -tubulin and either p150^Glued or DIC. Mock-transfected cells show patches of dynein and dynactin at the leading edge of migrating cells (arrowheads). Inset, overlay of cortical patch of p150^Glued (red) and projecting MTs (green). Scale bar, 10 µm. (B) Western blot of protein lysates from cells that were mock transfected or transfected with siRNAs targeting p150^Glued, DHC or β-catenin consistently show knockdown of target protein levels by 75-95%. Levels of DIC decrease in DHC knockdown cells, suggesting the dynein complex is being destabilized. By contrast, knockdown of β-catenin, which binds to DIC but is not part of the dynein motor complex (Ligon et al., 2001), does not destabilize DIC. (C) Centrosome reorientation is inhibited in dynein and dynactin knockdown cells. Orientation of centrosomes 6 hours after wounding. Centrosome orientation was determined by measuring the position of centrosomes, as determined by -tubulin staining, in relation to Hoechst-stained nuclei. Centrosomes in the forward-facing 120 degree sector (green zone) were scored as reoriented. (D) Leading-edge extension is inhibited in dynein knockdown cells. Displacement of the leading edge over 45 minutes of wound healing was measured (n=15). (E) Tracks of nuclei centroids during migration of fibroblasts over 45 minutes. Paths are oriented so cells are migrating towards the top of the graph, each path starts at (0,0). Axis labels are in µm. (F) Rates of nuclear movement are decreased in dynein and dynactin knockdown cells. Nuclear velocity during migration was decreased in DHC and p150^Glued siRNA cells, compared with mock-transfected cells (n=15). Error bars indicate s.e.m.; ^*P<0.05; ^**P<0.005.