Biophysical Society Conference | Estes Park 2023

Membrane Budding and Fusion

Wednesday Speaker Abstracts

MEASURING THE NANOSCALE STRUCTURE OF ENDOCYTOSIS WITH LIGHT AND ELECTRON MICROSCOPY Justin W. Taraska 1 ; 1 NIH, NHLBI, Bethesda, MD, USA Clathrin-mediated endocytosis is the key mechanism human cells use to internalize receptors, nutrients, hormones, and other cargo. It is fundamental to the physiology of cells and tissues. We use super-resolution light, EM, correlative light and EM (CLEM), and cryo-electron tomography to image protein complexes within the dense native environment of the cell. First, using CLEM, we studied over a dozen key proteins involved in clathrin-mediated endocytosis. Our data provides a comprehensive molecular architecture of endocytic vesicles in human cells. We discovered that key regulatory and cargo proteins distribute into distinct nanoscale spatial zones; inside, outside, and at the edge of the clathrin coat in human cells. The presence and amount of many factors within these zones change during vesicle curvature. From these data we present a model that the formation and curvature of single endocytic vesicles is driven by the recruitment, re-organization, and loss of proteins within these nanoscale zones. Next, we used large-scale transmission electron microscopy to study clathrin-mediated endocytosis across many types of mammalian cells. We created and analyzed ultra-high precision platinum replica TEM images of over 33,000 endocytic structures from nearly two hundred cells and eight different cell types. We also developed a new cryo-EM tomography method that allows us to image lattice organization within single clathrin structures and other proteins in the native environment of a plasma membrane in the frozen state. This lets us directly study proteins at molecular resolutions inside cells. Our primary finding is that flat clathrin lattices are loosely assembled and spontaneously curve without energy-dependent clathrin exchange or recruited proteins. This is a new model of how clathrin-mediated endocytosis works in eukaryotic cells to regulate signaling and traffic across the plasma membrane.

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