Chemically inducible endocytosis. (A) Illustration of chemically inducible endocytosis. Normally, clathrin recognizes the appendage and hinge of the β2 subunit of the AP2 complex after it has engaged cargo and membrane. In chemically induced endocytosis, cells coexpress a plasma membrane anchor (CD8–mCherry–FRB) and a clathrin hook (FKBP–β2–GFP). Rapamycin (200 nM) is added, which causes heterodimerization of FKBP and FRB domains. The clathrin hook is rerouted to the plasma membrane. Clathrin recognizes the clathrin hook, and the plasma membrane anchor is internalized. (B) Chemically inducible endocytosis in live cells. Cells expressing CD8–mCherry–FRB with either FKBP–β2–GFP or GFP–FKBP. Stills from live-cell confocal imaging experiments are shown: the frame before rerouting occurs (top) and 133 frames (665 s) later (bottom). See Videos 1 and 2. Insets show a 2× zoom of the boxed region; intense orange bar indicates rapamycin application. Bars: (main) 10 µm; (insets) 2 µm. (C) Example plots to show chemically inducible endocytosis. Colored traces indicate the total area of bright GFP puncta that form in cells after rerouting. Gray traces show the rerouting of the clathrin hook from the cytoplasm to the plasma membrane (see Materials and methods).