Audhya Lab
Clathrin-mediated endocytosis
The clathrin coat functions at multiple intracellular locations to package a wide range of cargoes for transport. However, clathrin does not engage substrates directly nor does it exhibit the ability to associate with membranes. Instead, recruitment of clathrin is mediated by specific adaptor proteins found on different organelles, which interact with cargoes and lipid bilayers. At the plasma membrane, several endocytic adaptors coordinately regulate clathrin accumulation. The best characterized is AP-2, a heterotetrameric adaptor complex that can simultaneously bind to clathrin, sorting signals on cargoes and phosphatidylinositol 4,5-bisphosphate, a lipid specifically enriched on the cell surface. Additionally, AP-2 associates directly with other endocytic adaptors including the Eps15 homology (EH) domain containing proteins Eps15 and intersectin, members of the muniscin family such as FCHO1, and the cargo-binding factors Dab2 and Epsin. Thus, the AP-2 complex is a multifunctional scaffold that promotes the formation of cargo-laden, clathrin-coated subdomains on the plasma membrane.

Despite its central role in clathrin-mediated endocytosis, the AP-2 complex is unlikely to be the sole initiator of the process. Studies in C. elegans have demonstrated that cells lacking components of AP-2 continue to perform clathrin-mediated endocytosis, indicating that alternative pathways exist to recruit clathrin to the cell surface. In particular, the FCHO proteins, Eps15 and intersectin exhibit highly similar recruitment kinetics as compared to AP-2 and clathrin and may function independently of AP-2. Current models suggest that the FCHO proteins initiate membrane bending and cargo clustering via their F-BAR and μ-homology domains, respectively. Simultaneously, Eps15 and intersectin recruit downstream adaptors and accessory proteins, which collectively drive maturation of the nascent endocytic pit.

A large number of cell surface molecules undergo internalization in a clathrin-dependent fashion. This process requires multiple endocytic adaptors to recognize largely distinct cargoes in a manner that relies on short signal sequences or post-translational modifications found within substrates. To understand how such an array of macromolecules can be efficiently internalized, we take advantage of genetic, biochemical, and microscopy-based approaches to dissect the mechanisms underlying clathrin recruitment to the plasma membrane.