Fig. 1. tTG is internalized from the cell surface by means of a cholesterol- and dynamin-dependent mechanism. (A) Endocytosis of tTG from the surface of WI-38 fibroblasts. The Fab fragments of mouse mAb 4G3 against tTG were incubated with WI-38 fibroblasts at 4°C. Next, the cells were warmed up to 37°C for 15 or 60 minutes and the Fab fragments remaining on the cell surface were stripped by a low-pH treatment. The internalized tTG–4G3-Fab complexes were detected by immunofluorescence after cell permeabilization. (B) Plasma membrane cholesterol is required for internalization of cell-surface tTG. NIH3T3 fibroblasts expressing exogenous tTG were treated with 10 mM M--CD and then incubated for 3 hours at 37°C in DMEM-FBS without the inhibitor. Cell-surface proteins were biotinylated with membrane-impermeable sulfo-NHS-LC-biotin. Biotinylated proteins were isolated and total cellular and cell-surface fractions were analyzed for tTG by SDS-PAGE and immunoblotting. The numbers beneath the tTG bands display relative intensities compared with a value of 1.0 assigned to untreated cells. Shown is a representative result of three independent experiments. (C) GTPase activity of dynamin-2 is required for endocytosis of cell-surface tTG. MRC-5 fibroblasts were transiently transfected with either wild-type (wt) or a GTPase-deficient dynamin-2 mutant (K44A) with a hemagglutinin (HA) tag. 40 hours after transfection, antibody-uptake assays with the Fab fragments of mAb 4G3 were performed for 15 minutes as described in the legend to panel 1A. After permeabilization, the cells were co-stained for the transfected dynamin-2 or the dynamin-2 K44A mutant with antibody against HA (left panels) and for the tTG–4G3-Fab complexes (right panels). Arrows mark multiple vesicles containing the internalized tTG–4G3-Fab complexes in the cells expressing the endogenous or transfected wild-type dynamin-2, but not its (K44A) mutant. The percentages of cells with positive staining for the internalized tTG–4G3-Fab complexes (mean pixel intensity within the cell area 30) were calculated for the populations of untransfected and wild-type dynamin- or mutant K44A-dynamin-transfected MRC-5 fibroblasts (n=120, lower panel).