Fig. 1.

Dynamin is required for internalization of NGF and TrkA. a, TS-dynamin PC12 cells were stimulated with biotin-tagged NGF for 2 hr at the indicated temperatures, then fixed and permeabilized (Permeabilized) or not. Accessible biotin was visualized with streptavidin-Cy3. Scale bar, 50 μm. b, TS-dynamin PC12 cells were stimulated with¹²⁵I-NGF alone or with excess unlabeled NGF for 2 hr at the indicated temperatures. Specific internalized counts (¹²⁵I-NGF internalized minus ¹²⁵I-NGF internalized in the presence of 200 ng/ml NGF) were similarly decreased at the nonpermissive temperature and when internalization was blocked at 4°C. c, TS-dynamin PC12 cells were stimulated with NGF (NGF+) or vehicle control (NGF−) for 15 min at the indicated temperature, then fixed and permeabilized (Permeabilized) or not. Cells were immunostained with anti-TrkA, followed by biotinylated secondary antibody and streptavidin-Cy3. Scale bar, 50 μm. d, Intensity of TrkA immunostaining was measured at 33 and 39°C for TS-dynamin or WT-dynamin PC12 cells 15 min after stimulation with NGF (+) or vehicle control (−). Values are mean surface TrkA per cell (±SEM) for each condition, normalized to total TrkA per cell. NGF induces a decrease in the percentage of TrkA at the surface at both temperatures in WT-dynamin cells. In TS-dynamin cells, NGF induces a decrease in surface TrkA at the permissive but not at the nonpermissive temperature (p < 0.001). Paired t test was used to compare surface TrkA at the permissive and nonpermissive temperatures.