Actin binding is required for the efficient sorting of receptors. (A–C) HeLa cells were transfected with EGFR and EGFR actin binding mutant YLIP/AAAA (1,016–1,019) coupled to paGFP and mCherry-Rab4. GFP was activated in Rab4-positive endosomes by exposure to 405-nm laser light, and the rate of recycling was measured by quantifying the decrease in paGFP fluorescence from the endosome. Pretreatment with 100 µM primaquine (Prim) to block receptor recycling was used as a negative control. (A) Representative activation in Rab4-positive endosomes (taken from Video 3; a Gaussian blur has been added). Bars, 10 µm. (B) Representative traces from an individual experiment (>10 cells per experiment per condition; four independent experiments). (C) MDA–MB-231 with paCherry fusion MT1-MMP constructs WT or LLY/AAA (571–573) and GFP-EEA1. paCherry constructs were activated in GFP-EEA1–positive endosomes and quantified as above (graph mean traces over three individual experiments; >20 cells total). Error bars indicate SEM.