Fig. 4. PtdIns(4,5)P₂ hydrolysis has a much smaller effect on clathrin assembly at the PM compared to its effect on dissociating AP-2 adaptors. (A) TIRF imaging of YFP-CLC fluorescence shows only a partial loss of CLC at the PM upon iRap-induced translocation of CF-Inp. Scale bars: 5 µm. (B) PtdIns(4,5)P₂ hydrolysis resulted in only an 20% reduction in mean clathrin-puncta intensity at the PM. Mean intensity value for each time point was normalized to the maximum mean intensity value measured in each cell. Error bars represent s.e.m. (C) Comparison of the number of puncta at each time point observed for AP-2-µ2–YFP or YFP-CLC after iRap-induced translocation of CF-Inp. Number of puncta was normalized to the maximum number detected in each cell. PtdIns(4,5)P₂ hydrolysis caused a more than fourfold greater effect on the number of AP-2-µ2–YFP-positive puncta compared with that on YFP-CLC-positive puncta. Errors represent s.e.m. (D) Normalized histogram of the lifetimes of YFP-CLC-positive puncta before and after iRap-induced translocation of CF-Inp. There was no significant reduction in the lifetime of CLC-positive puncta in iRap-treated cells. Errors represent s.e.m.