Figure 1. UNC-60A (ADF/cofilin) localizes to invadopodia and regulates invasion. (A) Diagram depicts normal anchor cell (AC) invasion (BM, basementmembrane; VPCs, vulval precursor cells). AC invasion is precisely timed with underlying VPC development, and initiates breach of the basement membraneduring the P6.p two-cell stage and completes basement membrane clearance by the P6.p four-cell stage (Hagedorn et al., 2013). AC invasion is initiatedby F-actin and membrane-based invadopodia that breach the basement membrane and transform into a protrusion. (B) Fluorescence overlaid on DIC (left);grayscale of basement membrane fluorescence (right, visualized with laminin::GFP). Lateral-view images show a wild-type anchor cell (top, visualizedwith mCherry::moeABD) that has breached the basement membrane (arrowhead). Bottom panels show blocked AC invasion (arrowhead, intact basementmembrane)
unc-60a RNAi treatment. (C)
unc-60 > GFP::UNC-60A (top) is up-regulated in the AC (arrow). AC-specific expression of the C. briggsae orthologueof ADF/cofilin (bottom) rescued invasion in animals depleted of the C. elegans
unc-60a transcript (break in the phase-dense basement membraneline under the AC, arrowhead). (D) GFP::UNC-60A (overlay, left) colocalized with F-actin (mCherry::moeABD) at AC invadopodia (arrowheads, basementmembrane position, dotted orange line). r-value reports mean Pearson's correlation coefficient for colocalization of UNC-60A and F-actin within the AC ±SEM. (E) UNC-60A localized to the initial basement membrane breach (arrowhead). (F) RNAi-mediated knockdown of the -integrin subunit
pat-3 resultedin a loss of UNC-60A localization (bracket), which in wild-type animals is concentrated at the invasive cell membrane (arrowhead). The graph shows fold enrichment of GFP::UNC-60A at the invasive membrane. (P < 0.001, Student's t test; n = 10 animals for each treatment, error bars report ±SEM). Bars, 5 μm.