Clathrin-mediated endocytosis (CME) is the main mechanism by which eukaryotic cells internalize and recycle most membrane proteins. It is driven by different Adaptor Proteins, which directly link the clathrin scaffold to cargos, and modulated by accessory proteins that temporarily regulate clathrin coated vesicles (CCVs) assembly and disassembly. Mutations affecting endocytosis have been directly linked to cancer and other diseases such as Alzheimer1 and Stiff-man2 diseases. In vivo, most clathrin adaptors solely interacts with the inner leaflet of the cell membrane, which is enriched in phosphatidyl inositol 4,5-diphosphate (PIP2). Indeed this negatively charged lipid, together with 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS), is essential for the interaction of adaptor and modulator proteins to the membrane. Therefore, the main approach exploited in this wok, so far, has been to build simple, in vitro, lipid models to mimic the inner leaflet of the cell membrane and investigate the orientation and physical structures formed by CALM and FCHo2 on association to cargo-embedded lipid monolayers enriched in PIP2 or POPS (for FCHo2 and its truncated version interaction). Simplifying the system down to its core elements (specific adaptor proteins and modulators and specific lipid mixture) enables to describe the system using ellipsometry, pressure – area isotherms and Neutron Reflectometry. Exploiting the isotopic contrast with Neutron Reflectometry (i.e. using subphases with different scattering length density), this approach allowed us to understand which is the orientation of both CALM and the truncated version of FCHo2. Owning the crystal structure of CALM (PDB: 3zym3), we obtained the 3D orientation which best fits the NR data. Regarding truncated-FCHo2, whose flexible linkers prevent the crystallization, we built a 4-layers-model that can well fit the data, showing the general orientation of the protein with respect to the membrane and the position of the linkers.

Endocytosis across scales: From molecular structures to a functional process

ACS Spring Meeting 2021

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