Development of an inducible model of clathrin-mediated endocytosis.

Clathrin-mediated endocytosis is the major route of internalisation of a wide range of cargo at the plasma membrane. Despite the high frequency of endocytic events, it is difficult to predict when and where a clathrin-coated pit will form, complicating any analysis of initiation mechanisms. This thesis describes the development of an inducible model of endocytosis that provides spatiotemporal control over initiation. This is achieved through chemical- or light-induced dimerisation of a clathrin-binding protein (hook) to a plasma membrane cargo (anchor), bypassing any preparation steps prior to clathrin recruitment. It was found that this rapidly induced clathrin-mediated endocytosis that could function independently of AP‑2 and without detriment to endogenous CME. This synthetic endocytosis was found to be a useful measure of functional interaction between clathrin and its adaptors, for example β3 adaptin hinge + appendage was non-functional for CME despite previous reports of in vitro clathrin binding. Also, these clathrin hooks can be easily mutated to pinpoint interaction sites. Mutagenesis of a β2 hinge + appendage hook revealed that a low level of endocytic activity could be maintained when either the hinge or appendage site was disrupted, but mutation of both sites or removal of the entire appendage inhibited synthetic endocytosis. Additionally, the ability of clathrin alone to promote clathrin-coated vesicle formation was shown by the success of a GTSE1 hook, a protein not known to interact with endocytic accessory proteins. This technique offers great potential for further examination of molecular level clathrin/adaptor interactions as well as CME mechanics on a cell-wide scale. In particular, the localised initiation of endocytosis demonstrated here using optogenetics is ideally suited to manipulate cells displaying polarised endocytosis