Figure 3.

In Vitro and In Vivo Functional Characterization of CDC42 Mutations

(A) CDC42 mutations differentially impact polarized migration and cell proliferation. Wound-healing assays (above) and proliferation assays (below) were performed using NIH 3T3 cells transiently transfected to express wild-type CDC42 or each of the indicated mutants. Mean ± SD densitometry values of three independent experiments are shown. The wound was generated 24 hr after transfection, and migration in the wounded area was evaluated after 4 and 7 hr. Cells expressing exogenous wild-type CDC42 migrate more rapidly into the scratched area than cells transfected with the empty vector (EV). Mutants differentially perturb polarized migration, with CDC42^Ser83Pro and CDC42^Ala159Val overexpression variably enhancing the wound closure ability of transfected cells compared to the wild-type protein, whereas CDC42^Tyr23Cys, CDC42^Arg68Gln, and CDC42^Glu171Lys fail to do that, supporting a gain-of-function and a loss-of-function effect of these mutants, respectively. Cell proliferation was evaluated in transfected cells at the indicated time points and quantified by manual counting using a Neubauer hemocytometer. The trypan blue dye exclusion test was used to consider viable cells only. While the CDC42^Ala159Val and CDC42^Ser83Pro mutants variably enhance proliferation compared to cells expressing wild-type CDC42, no effect on proliferation (CDC42^Glu171Lys) and reduced proliferation (CDC42^Tyr23Cys and CDC42^Arg68Gln) is documented for the other mutants, indicating a loss-of-function and a dominant-negative effect, respectively. Asterisks indicate significant differences compared with wild-type CDC42 (^∗p < 0.05; ^∗∗p < 0.01; Student’s t test).

(B) Consequences of CDC-42 expression on vulval development in C. elegans. Ectopic expression of wild-type CDC-42 at the L2/L3 stage elicits a multivulva (Muv) phenotype (left, upper panel), and CDC-42 overexpression in a LET-23/EGFR hypomorphic background reduces the penetrance of the vulvaless (Vul) phenotype (left, lower panel). Compared to animals expressing wild-type CDC-42, those expressing CDC-42^Ser83Pro and CDC-42^Ala159Val show higher prevalence of the Muv phenotype and lower prevalence of the Vul phenotype, indicating a gain-of-function role on LET-60/RAS signaling. Animals expressing the other tested CDC-42 mutants do not significantly differ from those expressing wild-type CDC-42. Ectopic expression of wild-type CDC-42 at the early L3 stage elicits a protruding vulva (Pvl) phenotype (right, upper panel). Animals expressing CDC-42^Ser83Pro and CDC-42^Ala159Val show a higher prevalence of the phenotype compared to worms expressing wild-type CDC-42, while a less penetrant phenotype was scored for animals expressing CDC-42^Tyr23Cys, CDC-42^Arg68Gln, or CDC-42^Glu171Lys mutants. RNA interference (RNAi) experiments show that the Pvl phenotype associated with overexpression of wild-type CDC-42 is mediated, in part, by WSP-1/WASP (right, lower panel). White and gray bars indicate the penetrance of Pvl in non-interfered and interfered animals, respectively. Error bars indicate SEM of four independent experiments, and asterisks specify significance differences between animals expressing CDC-42 mutants and those expressing wild-type CDC-42 or between interfered and non-interfered nematodes (^∗p < 0.05; ^∗∗p < 0.001; ^∗∗∗p < 0.0001; ^∗∗∗∗p < 0.00005; two-tailed Fisher’s exact test). Comparisons between worms expressing wild-type CDC-42 and control animals are also shown. RNAi was performed by feeding using HT115 E. coli bacteria expressing double stranded wsp-1 RNA (Ahringer’s C. elegans RNAi feeding library) and optimized to overcome lethality. As a control of the efficiency of the modified RNAi protocol, let-60 RNAi experiments were performed on animals carrying the let-60 gain-of-function allele n1046 (p.Gly13Glu), and the prevalence of the Muv phenotype was scored at a dissecting microscope (Table S4).