Fig 1. CDC-42 promotes invadopodia formation, BM breaching and invasion. (A) Diagram depicting anchor cell (AC) invasion through the basementmembrane (BM) into the vulval epithelium in C. elegans. AC invasion is coordinated with the divisions of the P6.p vulval precursor cell descendants. At the P6.p two-cell stage, invadopodia dynamically form and turn over before breaching the BM. Following BM breach a single invasive protrusion forms, which displaces BM underneath the AC and creates a large hole in the BM. In
unc-40(
e271) mutants, the invasive protrusion does not form and the AC invades inefficiently by creating many BM breaches solely with invadopodia. In our genome-wide screen, enhancers of the
unc-40(
e271) invasion defect wereidentified as potential regulators of invadopodia. (B) Markers for F-actin (left;
cdh3>mCherry::moeABD) and BM laminin (center;
lam-1>
lam-1::GFP) overlaid on DIC image (right). Top panels show wild type AC invasion and bottom panels show blocked AC invasion in a homozygous
cdc-42(
gk388) mutant(arrowhead marks intact BM) at the P6.p 4-cell stage. (C, D) Images of a functional GFP::CDC-42 GFP fusion protein acquired at the same developmentalstage prior to invasion. (C) Lateral views of the GFP::CDC-42 reporter driven by the endogenous
cdc-42 promoter (
cdc-42>GFP::
cdc-42) show that
cdc-42is expressed in the vulval and uterine cells, including the AC (arrow). (D) Ventral views showing that AC-specific GFP::CDC-42 (
cdh3>GFP::
cdc-42) is distributed in a punctate manner at the AC invasive surface and colocalizes with F-actin (r = Pearson's correlation coefficient calculated from 5 animals). (E) A ventral time series shows dynamic formation of invadopodia (visualized with F-actin probe) in a wild type animal (top) and in an animal with reduced
cdc-42 (RNAi; bottom). The average number of invadopodia formed was decreased in animals treated with
cdc-42 RNAi (overlaid text reports average number of invadopodia per timepoint from 6 wild type animals and 10
cdc-42 RNAi treated animals; p < 0.0001, Tukey's multiple comparisons test). (F) The distribution of invadopodia lifetimes did not change in animals treated with
cdc-42 RNAi (n = 6 wild type and 10
cdc-42 RNAi treated animals p = 0.178, Chi-square test) (G) Ventral views showing BM breaches mediated by invadopodia (white dashes outline AC footprint and orange dashes outline breaches). Wild type (top) animals typically breached through several small holes whereas animals with reduced
cdc-42 (RNAi; bottom) more frequently had a single breach (overlaid text reports percentage of animals with two or more holes; n = 17/25 wild type animals with > 2 breaches versus 6/25 animals treated with
cdc-42 RNAi; p<0.001, Fisher's exact test. (H) BM breaching was delayed in
cdc-42(
gk388) mutants relative to wild type animals (P6.p vulval precursor cell development was used to calibrate time; n = 25 animals per stage, per genotype; *** p < 0001, * p < 0.05, Fisher's exact test; bars represent 95% confidence intervals). Scale bars = 5 um.