Clathrin-mediated endocytosis (CME) is the major route of receptor internalization at the plasma membrane. Analysis of constitutive CME is complicated by the fact that initiation of endocytic events is unpredictable. When and where a clathrin-coated pit will form and what cargo it will contain are difficult to foresee. Here we describe a series of genetically encoded reporters that allow the initiation of CME on demand. This is achieved by inducibly tethering a clathrin "hook" to a plasma membrane "anchor". Our design incorporates temporal and spatial control of initiation using chemical and optical tools, and the cargo is defined. Since this system bypasses multiple steps in vesicle creation, we term it "hot-wiring". In this paper we use hot-wired endocytosis to define the functional interactions between clathrin and the β2 subunit of the AP2 complex. However, there are numerous applications for this new technology, which we hope will be broadly useful to the field.
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