Imaging the Nanoscale Structure of Endocytosis with Correlative Super-Resolution Light and Electron Microscopy

Abstract

Platinum replicas of unroofed cells can be imaged with an electron microscope to produce high resolution images of the structure of the cytoplasmic side of plasma membrane. It has been challenging, however, to identify proteins within these samples. Super-resolution localization microscopy can image fluorescently-labeled molecules with better than 20 nm precision. We have developed a correlative super-resolution light and platinum replica EM method (CLEM) that couples these two complementary methods to produce images where identified proteins are mapped within the dense structural context of the cell. This correlative method is uniquely suited to investigate the nanometer-scale molecular organization of the plasma membrane and associated organelles such as endocytic and exocytic vesicles. Using this CLEM method, we studied 19 key proteins involved in clathrin-mediated endocytosis. Our data provide a comprehensive molecular architecture of endocytic structures at the nanoscale. We discover that endocytic proteins distribute into three distinct spatial zones; inside, outside, and at the edge of the clathrin coat. The presence and amount of many factors within these zones change during organelle maturation. We propose that the formation and curvature of single clathrin-coated pits is regulated by the recruitment, re-organization, and loss of proteins within these three nanoscale zones during endocytosis.