Figure 4.

Cdc42 Null Melanoblasts Have Migration Defects and Altered Pseudopod Dynamics

(A) Still images of Ctr (Cdc42 wt/wt; Tyr::CreB+; Z/EG) and Cdc42 f/f (Cdc42 f/f; Tyr::CreB+; Z/EG) skin explants (see Movie S3). Yellow arrows indicate pseudopods; the red arrow indicates a blebby pseudopod; and the orange arrow denotes the “bipolar” phenotype. Scale bars, 10 μm.

(B–F) N = 125 cells per genotype, from 301 control pseudopods and 239 Cdc42 f/f pseudopods.

(B) Cell area.

(C) Number of pseudopods.

(D) Length-to-width ratio.

(E) Pseudopod length in microns.

(F) Pseudopod lifetime in minutes.

(G) Migration speed. Dashed vertical lines indicate average speed. N = 115 cells per genotype.

(H) Representative images of melanoblast F-actin in control Lifeact-GFP lox-stop-lox; Tyr::CreB+ (Ctr) skin explants and Lifeact-GFP lox-stop-lox; Cdc42 f/f; Tyr::CreB+ (Cdc42 f/f) skin explants (see Movie S4). Scale bar, 10 μm.

(I) Number of minutes between actin bursts in Ctr (9.0 ± 0.8 min SEM) and Cdc42 f/f (11.6 ± 0.7 min SEM) pseudopods. N = 100 pseudopods per genotype.

(J) Action of pseudopods within three frames (3 min) following an actin burst. Extension, “protrude”; retraction, “retract”; and static, “remain.” Percentage of pseudopods that protruded, retracted, or remained following an actin burst (within three frames) was a calculated per skin from at least eight bursts from four skins.

(K) Scatterplots showing pseudopod lifetime versus actin bursts. The line of best fit for Ctr has slope 7.2 ± 0.7, R² = 0.55; for Cdc42 f/f, slope 10.8 ± 0.6, R² = 0.77.

N = 5–6 embryos from four different litters, except (J), where N = 20 cells, over three experiments. Boxplots show mean, minimum, and maximum values. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, t test.