Requirement of NME in CCP upstream of dynamin and auxillin steps to allow optimal clathrin-coated vesicle budding. (A) Still image and kymographs of simultaneous two-color TIRF-M time series of mock- or siRNA-treated BSC-1 cells overexpressing mRFP-LCa and GFP-auxilin (1 image/s). In mock-treated cells, vertical arrowhead points to a burst of GFP-auxilin coincident with mRFP-LCa signal disappearing. In siRNA-treated cells, arrowhead points to mRFP-LCa signal disappearing without associating GFP-auxilin. (B) Percentage of disappearing mRFP-LCa CCPs ending with a burst of GFP-auxilin in mock- and siRNA-treated BSC-1 cells. At least 300 events were analyzed for each condition.