CCVs generated by hot-wired endocytosis are trafficked and recycled similarly to normal endocytic cargo. (A–C) Stills from typical live-cell confocal imaging experiments. Vesicles in HeLa cells expressing CD8–dCherry–FRB, FKBP–β2–mRuby2 (top), or CD8–YAAL (bottom) together with the indicated GFP-tagged construct (A, Rab5; B, EEA1; C, Rab4). CD8 was labeled using extracellularly applied anti-CD8/Alexa 647. Endocytosis was triggered by rapamycin addition (200 nM) in the case of hot-wiring, and cells were imaged at 0.2 Hz. Bars, 1 µm. (D) Summary of imaging experiments to delineate the paths taken by hot-wired cargo or by CD8–YAAL. See also Fig. S4.