FLN-2 docks VHA-5 vesicles on the actin cytoskeleton. (A and B) Confocal fluorescence images (A) and line scan analysis (B) of the epidermis in WT adults co-expressing FLN-2::RFP and the actin probe ABD::GFP. (C–F) Confocal images of the epidermis in WT (C) and fln-2 (D) day 2 adults expressing ABD::GFP. (E) Line scan analyses. (F) Mean fluorescence intensity of ABD::GFP; n ≥ 17 worms per group. (G–J) Merged images of the epidermis in WT day 2 adults co-expressing VHA-5::RFP and ABD::GFP treated with DMSO (G) or LatA (H). (I) Line scan analyses. (J) Mean fluorescence intensity of VHA-5::RFP and ABD::GFP; n = 16 worms per group. (K–S) SIM fluorescence images of epidermis WT adults co-expressing VHA-5::RFP, ABD::GFP, and FLN-2::BFP (K–R). Boxed area in K is magnified in L–R. (S) Three different co-localization patterns of FLN-2, VHA-5, and actin as indicated in R. Yellow, red, and white arrows indicate VHA-5-, FLN-2-, and F-actin–positive structures, respectively. (T and U) Quantification of FLN-2-actin attachment (T) and position of VHA-5 vesicles relative to FLN-2 and actin (U). 10 (T) and 20 (U) different regions were scored in 8 and 15 worms, respectively. F, J, T, and U show mean ± SD. **, P < 0.01; ***, P < 0.001, by unpaired two-tailed Student’s t test. Scale bars, 5 μm (A–A″, C, D, G, and H), 1 μm (K–R).