UNC-40 directs F-actin formation at the site of protrusion formation. (A) F-actin effectors (middle), basement membrane (right), and overlay (left) show that GFP::MIG-2 (Rac) and GFP::UNC-34 (Ena/VASP) localize to the initial site of basement membrane breach in wild-type animals (top, arrows) but fail to do so in unc-40 (e271) mutants (bottom, arrows). (B) The spectral representation of the fluorescence intensity of the summation of a 10-min, 40-s time-lapse shows that F-actin (left, viewed with cdh-3 > mCherry::moeABD) overlaps precisely with the stable patch of UNC-40::GFP (middle, arrow; overlay right; see Video 7; similar results were observed in 6/6 animals). (C) A similar summation of a 90-min time-lapse revealed that this dynamic F-actin formation (top, left) was targeted at the site of invasive protrusion formation at the center of the basement membrane gap (middle, viewed with laminin::GFP) in a wild-type animal (see Video 8). In contrast, in unc-40 mutants (bottom), F-actin was not concentrated at the sites of basement membrane breach (see Video 9). Bars, 5 µm.