BPS2026 – Flat and curved clathrin lattices at HeLa plasma membranes by cryo-electron tomography

Clathrin-mediated endocytosis is a major cell internalization pathway for a broad range of proteins, receptors, small molecules, and pathogens. During this process, adaptor proteins such as AP2 bind cargo receptors and recruit clathrin triskelia, which assemble into a polyhedral lattice coat. This flexible, interlaced arrangement of triskelia helps bend the membrane to encapsulate cargo into a budding vesicle. Clathrin lattices are known to adopt varying curvature from flat to highly curved, capable of forming an empty cage as small as 70 nm in diameter. High-resolution detail of how clathrin-clathrin and clathrin-adaptor interactions vary across this wide range of curvature will improve our understanding of this vital endocytic pathway. Cryo-electron microscopy and tomography have been used to capture such detail of clathrin interactions, primarily using high-curvature purified cages or reconstituted vesicles. Capturing clathrin, particularly low-curvature lattices, on the plasma membrane for cryoET remains a challenge. We have developed a cryo-electron tomography pipeline capable of producing plasma membrane samples for high-resolution cryoET. Mammalian plasma membranes are observed to contain clathrin coats in a variety of endocytic stages. We apply subtomogram averaging to these latticed coats present in curved and flat morphologies in HeLa cells. We determine a high-resolution curved structure with density consistent with in vitro studies showing the AP2 ß2 appendage bound to clathrin coats. We compare averages of differing curved states and highlight structural variations such as alternative orientations (or splaying) of clathrin heavy chain ankle and N-terminal domains under lattice vertices. Additionally, low-curvature coats exhibit more irregularity within the latticed triskelia network. These results could inform new models of clathrin-clathrin and clathrin-adaptor interactions during endocytic transition state.