Figure 5

MPG-EGFP uptake partially depends on the GTPase activity of dynamins. A. A diagram depicting the experimental scheme of B. B. A mutant form of dynamin I which lacks the GTPase activity (Dyn^K44A, 1 μg) was transfected into HeLa cells and MPG-EGFP was added at 40 μg/ml for 1 hr. Transferrin was added to cells as a marker of dynamin-dependent endocytic pathway. After washing, cells were fixed and subjected to the immunofluorescence staining with anti-dynamin I antibody. Control, control transfection of 1 μg pEGFP-N1 plasmid showing a high efficiency of transfection. Arrows indicate that transfected cells show normal transferrin localization. Dyn + transferrin, transferrin cannot enter Dyn^K44A-expressing cells (shown in green), showing efficient blockade of clathrin-mediated endocytosis by overexpression of this mutant form. Dyn + MPG-EGFP, MPG-EGFP was added at 40 μg/ml for 1 hr to Dyn^K44A-transfected cells. Note the reduced MPG-EGFP localization (arrowheads) in Dyn^K44A-expressing cells (shown in red). Dyn + MPG-EGFP + amiloride, 30 min pre-treatment of amiloride effectively blocked overall MPG-EGFP uptake. Photomicrographs are shown at 80X.