FLN-2 and actin cytoskeleton are required for MVB formation. (A–D) Confocal fluorescence images of the epidermis in WT (A–A″) and fln-2 (B–C″) co-expressing VHA-5::RFP and VHA-13::GFP at adult day 2. Average fluorescence intensity of VHA-5 and VHA-13 is quantified in D. At least 17 animals were scored in each strain. (E–G) The protein levels of VHA-5::RFP and VHA-8::GFP are unaltered in fln-2. Three independent immunoblot experiments were performed; E shows a representative result. (H–N) Confocal fluorescence images of the epidermis in WT animals expressing HGRS-1::GFP with the indicated RNAi treatments at adult day 2. The number of HGRS-1–positive vesicles per unit area (238 μm²) was quantified and shown in N. At least 14 animals were scored in each strain. (O–R) Merged confocal fluorescence images of the epidermis in WT day 2 adults co-expressing VHA-5::RFP and VHA-13::GFP (O and P) or VHA-8::GFP (Q and R) treated with either DMSO or LatA. (S and T) Mean fluorescence intensity and number of VHA-5–, VHA-13–, or VHA-8–positive vesicles per unit area (134 μm²) under the indicated treatments. 15 animals were scored in each treatment condition. (U–X) Representative TEM images of MVBs in the epidermis of WT day 2 adults treated with DMSO (U) or LatA (V and W). The number of MVBs per µm² of epidermis in the indicated treatments is shown in X. 23 and 32 cross sections were quantified under DMSO and LatA treatment, respectively. In D, F, G, N, S, T, and X, data are shown as mean ± SD. Unpaired two-tailed Student’s t test was performed to compare mutant datasets with WT or control treatment or datasets that are linked by lines. *, P < 0.05; ***, P < 0.001. Scale bars, 5 μm in A–C″, H–M, and O–R; 500 nm in U–W. Source data are available for this figure: SourceData FS2.