All human EHD proteins rescue the vacuolated intestinal phenotype in the intestine of C. elegans rme-1 ( b1045 ). (A) Micrograph images of middle intestinal regions of transgenic animals expressing the human EHD proteins. The rme-1(b1045) worms were injected with pVha-6::SL2-GFP (50 ng/μl) or with the same construct containing the human EHD cDNAs along with myo2::GFP (100 ng/μl) as a co-injection marker. Intestinal vacuoles are viewed as spaces devoid of green fluorescence in the rme-1(b1045) mutant (arrows). (B) Intestinal vacuoles were counted in at least 3 independent transgenic lines expressing no vector (rme-1(b1045)), vector alone (Vector), or a vector containing EHD1-4. (C) Basolateral endocytosis assay of the intestinal vacuoles. Adult hermaphrodites were microinjected with 1 mg/mL Texas-Red BSA (TR-BSA) into the pseudocoelom and examined for uptake in intestinal vacuoles. Lack of accumulation of TRed-BSA microinjected into the pseoudocelum in wild-type (WT) worms (N2 Bristol strain) (left). Rapid accumulation of TR-BSA in the enlarged intestinal vacuoles (arrows) in the rme-1(b1045) mutant worms (middle). rme-1(b1045) worms rescued with human EHD4 do not display accumulation of the dye in any intestinal cells (right) similar to WT animals. * – pseudocoelom, ** – gonads. DIC – differential interference contrast microscopy.