Figure 9.

Initial rate of AL-induced endocytosis of YFP-HA-ΔN-DAT is higher than that of wild-type YFP-HA-DAT. A, PAE/YFP-HA-DAT (WT13) and PAE/YFP-HA-ΔN-DAT cells were incubated with HA11 for 1 h at 37 °C and then incubated with 20 μM AL4 for 0 to 90 min. After fixation, the cells were immunolabeled as in Figure 8C. Mean values of the Cy3/Cy5 ratio (±SD, n = 5) were calculated as in Figure 8D and plotted against time. The p values are shown for YFP-HA-DAT compared with YFP-HA-ΔN-DAT (difference is significant at 5- and 15-min points). Differences were not significant at other time points (n.s., p > 0.05). Inset shows part of the graph indicated by the rectangle for better demonstration of different initial endocytosis rates of WT YFP-HA-DAT and YFP-HA-ΔN-DAT. The experiment is representative of three independent experiments. B and C, Time-lapse 3D imaging of PAE/YFP-HA-DAT (WT13) and PAE/YFP-HA-ΔN-DAT cells incubated with AL4 (20 μM) at 37 °C was performed using the LLS system. Image acquisition intervals were 15 s. Maximum intensity projections (B) or “volume” 3D-view images (C) of selected 3D images from time-lapse sequences are presented. Scale bars are 10 μm. Insets below images in (B) are high-magnification images of the areas indicated by white rectangles. Scale bars, 5 μm. See corresponding Videos S1 and S2 in Supplemental Information. The experiment is representative of multiple time-lapse imaging experiments where similar kinetics of endocytosis were observed in cells treated with AIM-100, AL3, and AL4, whereas AL8 had a much weaker impact on the endocytic process in PAE/YFP-HA-DAT (WT13 and WT8) and PAE/YFP-HA-ΔN-DAT cells. AL, AIM-100–like; DAT, dopamine transporter; LLS, lattice light sheet; PAE, porcine aortic endothelial; YFP-HA-DAT, YFP- and HA-epitope tagged DAT.