Chemically inducible internalization: using a clathrin hook of nonendocytic origin. (A) Colocalization of clathrin with bright green puncta formed by chemically induced internalization with a GTSE1 clathrin hook. Cells were coexpressing CD8–dCherry–FRB, mCherry–LCa, and FKBP–GTSE1–GFP. (B) Scatterplot to indicate variability in chemically induced endocytosis. The total normalized area above threshold at 10 min after rerouting for each cell analyzed is shown (spots). Bars: (main) 10 µm; (insets) 2 µm. Bars indicate mean ± SD. Number of cells = 35 and number of experiments = 10. (C) Antibody uptake after hot-wiring using a GTSE1 clathrin hook. Cells coexpressing CD8–mCherry–FRB and FKBP–GTSE1–GFP were incubated with anti-CD8–Alexa 647 to label the membrane anchor extracellularly, and rapamycin (200 nM) was applied. In A and C, stills from live-cell confocal imaging experiments are shown: (top) the frame before rerouting occurs and (bottom) 180 frames (15 min) later; empty and filled orange bars indicate before and after rapamycin addition. Bars: (main) 10 µm; (insets) 2 µm. (D) Table to show the rate of puncta appearance for GTSE1 compared with other clathrin hooks. Slope is the coefficient of a line fit to the averaged puncta data, postrapamycin; units are puncta per second.