Figure 7.

PI3K-C2α knockdown prevents the Ca²⁺-dependent increase in PtdIns(3)P production on neurosecretory vesicles. PC12 cells were pretransfected (48 h earlier) with control siRNA or PI3K-C2α siRNA (805, 807) and transfected with 2xFYVE-EGFP for 15–24 h before live-cell imaging. Cells were examined by time-lapse confocal microscopy at two frames/min before (left) and after (right) nicotine (100 μM) treatment. Initial 4D movie was used to obtain a 3D projection image. Surface plot analysis of cells before and during nicotine treatment shows the change of fluorescence intensity of intracellular 2xFYVE-EGFP–positive vesicles (as indicated in the figure). Bar graphs show the variation of vesicular fluorescence intensity at the peak of nicotine action in the indicated experimental conditions. Data shown are representative of 4 independent experiments. The fluorescence intensity changes on identified vesicles (control siRNA, n = 11; siRNA 805, n = 21; siRNA 807, n = 27) are expressed as mean ± SEM (Student's t test, **p < 0.01). Bar, 5 μm.