MADD-2 promotes UNC-40 clustering and polarization toward UNC-6. Anterior is left; ventral is down. (A) MADD-2::GFP was colocalized with F-actin (visualized with mCherry::moeABD) at the AC’s invasive cell membrane in wild-type animals and in ectopic F-actin patches in unc-6 mutants, locations where UNC-40 resides (arrowheads; the location of basement membrane [BM] is indicated with broken lines; colocalization graphs on the right were measured along the yellow lines in the magnified insets below). (B) The dominant UNC-40–mediated F-actin patch polarized in random sections of the plasma membrane in the ACs of madd-2; unc-6 mutants (P > 0.1, χ² test, n = 24 animals observed; see Fig. 4 C). (C) F-actin cluster formation (arrowheads) was slower before disassembly in a madd-2; unc-6 mutant (shown over a 79-min time lapse). (D) The volume of F-actin patch formation (red line, dominant patch) in the madd-2; unc-6 animal shown in C. (E) Similar analysis of three additional madd-2; unc-6 mutants. (F) UNC-40::GFP and F-actin colocalized (arrowheads) in madd-2; unc-6 mutants (top) and in animals with RNAi-induced loss of madd-2 (bottom). UNC-40::GFP and F-actin were mispolarized after loss of madd-2 (arrowheads, bottom). (G) Quantification of UNC-40 polarity in wild-type and madd-2 (RNAi)-treated animals (n > 20 for each stage per genotype; ***, P < 0.001, Student’s t test). Bars: (main panels) 5 µm; (magnified insets) 1 µm.