Fig 2: Chemically inducible endocytosis. (A) Illustration of chemically inducible endocytosis. Normally, clathrin recognizes the appendage and hinge of the ß2 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–ß2–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–ß2–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).

Fig 3: Chemically induced endocytosis is inhibited during mitosis. Live-cell confocal imaging experiment, mitotic cell expressing CD8–mCherry–FRB with FKBP–ß2–GFP. Rapamycin (200 nM) was applied during metaphase. Clear rerouting occurs, but no puncta form in the cytoplasm until after cytokinesis. This cell is shown in Video 5. Bar, 10 µm.

Fig 4: 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–ß2–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.

Fig 5: The ß2 appendage is crucial for hot-wired clathrin-mediated endocytosis Schematic diagram of hot-wired endocytosis. Under normal conditions the AP2 complex engages with cargo and PI(4,5)P2 at the plasma membrane (PM) and the ß2 hinge and appendage become available for clathrin engagement. AP3 acts analogously at the early endosome (EE). In hot-wired endocytosis a clathrin hook, e.g. ß2 hinge and appendage (FKBP-ß2HA-GFP) is attached to a plasma membrane anchor CD8-mCherry-FRB inducibly by rapamycin application. Endocytosis is measured by uptake of a fluorescent antibody that binds the plasma membrane anchor.Representative confocal micrographs of cells expressing the plasma membrane anchor (CD8-mCherry-FRB) and the indicated hooks (green). Cells were incubated with anti-CD8-Alexa647 (red) and treated with rapamycin (200 nM, 15 min). Endocytic vesicles coinciding in both green and red channels (yellow in merge) were quantified in B. Scale bar, 10 µm.Scatter dot plot shows the number of intracellular GFP-positive vesicles that contained anti-CD8 Alexa647 per cell, bars indicate mean ± SD. Number of experiments = 3. P-values from Dunnett’s post hoc test that were < 0.1 are shown above.