Fig 2. WSP-1 acts downstream of CDC-42 during invadopodia. (A) A transcriptional reporter (
wsp-1>GFP) revealed
wsp-1 expression in the AC (arrow).(B) Markers for the phospholipid PI(4,5)P2 (left;
cdh3> mCherry::PLC PH) and laminin (center;
lam-1>
lam-1::GFP) are overlaid on DIC image (right). Uterinespecific RNAi against
wsp-1 blocked AC invasion (arrowhead marks intact BM). (C) Ventral views show that prior to invasion GFP::WSP-1 (
cdh3>GFP::
wsp1) is distributed in a punctate manner within the AC and colocalizes with F-actin (
cdh3>mCherry:moeABD) (r = Pearson's correlation coefficient calculated from 12 animals). (D) Knockdown of
cdc-42 (RNAi) decreased the number of GFP::WSP-1 puncta at the invasive membrane (overlaid text reports average number of puncta from 9 animals; p < 0.01, Student's t-test). (E) A ventral time series shows fewer invadopodia (visualized with F-actin) formed in animals lacking
wsp-1 (
wsp-1(
gm324); bottom) relative to wild type (top) (overlaid text reports averages number of invadopodia number per timepoint from 6 wild type animals and 6
wsp-1(
gm324) animals; p < 0.0001, Student's t-test). (F) The distribution of invadopodia lifetimes did not change in animals treated with
wsp-1 RNAi (n = 6 wild type and 6
wsp-1 RNAi treated animals; p = 0.252, Chi-square test) (G) Ventral views showing BM breaches (white dashes outline AC footprint and orange dashes outline breaches).
wsp-1(
gm324) mutants (bottom) show a single BM breach more frequently relative to wild type (overlaid text reporting percentage of animals with two or more holes; n = 17/25 wild type animals with > 2 breaches versus 11/25
wsp-1(
gm324) animals; p<0.01, Fisher's exact test). (H) BM breaching was delayed in
wsp-1(
gm324) mutants (P6.p vulval precursor cell descendant development was used to calibrate timing; n = 25 animals per stage, per genotype; *** p < 0.0001, * p < 0.05, Fisher's exact test; bars represent 95% confidence intervals). Scale bars = 5 um.