Measuring Conformational Changes in Proteins at Single Sites of Endocytosis with FRET-based Correlative Imaging

Abstract

Clathrin-mediated endocytosis is the primary internalization mechanism of mammalian cells. Conformational changes in proteins regulate this process. To investigate conformational changes during endocytosis, we develop a new correlative lifetime-based fluorescence resonance energy transfer (FRET) and platinum replica transmission electron microscopy method named FRET-CLEM. Here, FRET-based atomic distances can be mapped directly to individual clathrin-coated structures visualized by electron microscopy at the plasma membrane. Using this method, we reveal novel conformational changes in clathrin light chain, which regulate curvature and endocytosis in living mammalian cells. FRET-CLEM will help develop a complete mechanistic model of endocytosis.