Optically inducible endocytosis. (A) Illustration of optically induced endocytosis. Cells coexpress a plasma membrane anchored LOVpep (CD8–TagRFP657–LOVpep) and a clathrin hook with a PDZb1 tag (PDZb1–β2–mCherry). Upon illumination with blue light, an epitope is exposed to which the PDZ domain can bind, and the clathrin hook is rerouted to the plasma membrane so that endocytosis can occur at that site. (B) Rapid and reversible ePDZb1–mCherry recruitment to CD8–TagRFP657–LOVpep(T406A,T407A,I532A) at the plasma membrane. Brief (<1 s) patterned illumination of plasma membrane areas followed by rapid imaging. Mean pixel density of an ROI at the membrane (purple) compared with one in the cytoplasm (gray) is shown. (C) Endocytosis is activated in a spatiotemporal manner by sustained, patterned light activation within a defined ROI (blue dashed box). For analysis, the number of vesicles formed within the ROI (purple box) is compared with a similar ROI outside the activated region (gray box). (D) Stills from a typical optogenetic hot-wiring experiment. HeLa cell expressing CD8–TagRFP657–LOVpep(T406A,T407A,I532A) and ePDZb1–β2–mCherry. Only the mCherry confocal channel is shown. Time 0 indicates when patterned illumination began. Bar, 10 µm. A movie of this panel is available as Video 9. (E) Plots of mean ± SEM vesicle formation in cells expressing CD8–TagRFP657–LOVpep(T406A,T407A,I532A) and either ePDZb1–β2–mCherry (number of cells = 13 and number of experiments = 4) or ePDZb–β2–mCherry (number of cells = 8 and number of experiments = 3) as indicated. Purple and gray indicate vesicles formed inside and outside the photo-activation zone, respectively.