F-actin–rich AC-invadopodia breach the basement membrane. (A) A schematic diagram depicts the two perspectives used for time-lapse imaging of AC invasion. (top) Wild-type animals lay on their side, allowing AC invasion to be imaged laterally. The basement membrane is visualized with laminin::GFP in magenta and the AC-specific F-actin probe cdh-3 > mCherry::moeABD is shown in green and overlaid on differential interference contrast images. (bottom) rol-6 mutant animals orient randomly, permitting ventral imaging. Confocal slices through the AC–basement membrane interface are shown at magnification of two relative to top panels. Orientation is indicated in all panels. (B) Ventral view time series shows dynamic patches of F-actin–rich invadopodia at the AC–basement membrane interface. To illustrate the rapid rate of turnover, colored spots were assigned to new F-actin structures at 2-min intervals (see Fig. S1 for detailed information on size, lifetime, rate of formation, and number of invadopodia over time). The behavior of these F-actin structures was similar when visualized with Lifeact::GFP and when worms were immobilized by compression (Fig. S1). (C) Before breaching, invadopodia (middle, arrow) appear to depress the basement membrane (right, arrow). (D) At the time of basement membrane breach an invadopodium (middle, arrow) occupies the site of breach (right, arrow). (E) A ventral view time series shows an AC-invadopodium (middle row, arrows) presaging and then occupying a visible basement membrane breach (bottom, arrows; similar results were observed in 8/8 animals). Bars, 5 µm.