Figure 7.

Cdc42 Knockout Melanocytes Form Less Dynamic Adhesions and Show Spreading Defects

All panels show EW7 Cdc42 f/f; ROSA::Cre-ER^T2; CDKN2^−/− melanocytes treated with DMSO (control) or OHT (to delete Cdc42).

(A) Live confocal imaging sequence of control (top) versus OHT-treated cells (bottom) expressing GFP-paxillin, imaged every 4 min. The merge shows each frame in a unique color. Scale bar, 5 μm.

(B–E) Quantification of the rate of assembly and disassembly of adhesions marked with GFP-paxillin (B and C) or mApple-vinculin (D and E) over 30 min; N = 15 cells per condition over three experiments.

(F) EW7 cells treated with OHT transfected with α4 integrin and GFP and stained with anti-α4 integrin or anti-vinculin. Yellow boxes show zoom-in. Yellow arrows indicate α4 integrin-positive adhesions. Scale bars, 10 μm.

(G and H) Length-to-width ratio (G) and roundness (H) of EW7 +OHT cells transfected with pcDNA3.1 control vector or α4 integrin. N = 45 cells per condition over three experiments.

(I and J) Number (I) and size (J) of adhesion complexes in EW7 +OHT cells as in (G) and (H); N = 24–27 cells per condition over three experiments.

(K–M) Area of melanocyte spreading at the indicated times on fibronectin (K), laminin-111 (L), and concanavalin A (M); N = 89–156 cells over three experiments.

Graphs (B–E and G–J) show mean ± SEM. Boxplots (K–M) show mean with minimum and maximum values. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗∗p < 0.0001, t test with Welch’s correction. See also Figure S7.