FIGURE 4:

WAVE2 is required for actin reassembly. (A) Staining for WAVE2, Arp3, and F-actin during reassembly of the junctional actin cytoskeleton. Caco-2 monolayers were examined after treatment with dimethyl sulfoxide (DMSO) alone (Control), with latrunculin A (LatA; 1 μM, 2 h), or 1 h after washout of latrunculin (LatA W/O). WAVE2, Arp3, and F-actin were visualized by confocal immunofluorescence imaging. WAVE2 and Arp3 colocalized with LatA-resistant F-actin puncta (middle row, arrows). Within 60 min of recovery, WAVE2 and Arp3 had redistributed along the apical actin rings as they reformed, and the F-actin staining pattern was indistinguishable from that of control cells. (B, C) WAVE2 KD impairs junctional actin recovery after latrunculin washout. Representative confocal images of junctional F-actin (B) and quantitation of junctional fluorescence intensity (C) in control and WAVE2 KD cells. Data are n = 60 contacts for control and n = 58 contacts for WAVE2 KD from three independent experiments. Data are means ± SEM, normalized to control DMSO; *p < 0.0001. Scale bars, 10 μm.