Figure 3

APP internalization is inhibited in cells transfected with dyn I K44A. A. HEK-695 cells were surface-immunolabeled with 6E10 antibodies for 45 min while on ice. The cells were then incubated at 37°C for varying periods of time, to allow internalization to occur. Prior to warming (0 min), APP immunofluorescence was confined to the cell membrane. Within 5 minutes APP immunoreactivity was located within punctate structures near the cell membrane. By 60 minutes, most of the immunoreactivity had coalesced at a perinuclear site. B. HEK-695 cells were transiently transfected with dyn I K44A. After 48 hours, cells were surface-labeled with 6E10 antibodies, then incubated at 37° for 10 min. In untransfected cells (arrows) APP (green) was internalized within intracellular vesicles within 10 minutes. In dyn I K44A-transfected cells (red), APP immunoreactivity was still largely membrane-associated at this time-point, indicating that internalization was impaired in cells expressing the dynamin mutant. Bar, 10 μm.