Non-Invasive Imaging of Membrane Dynamics Accompanied with Endocytosis in Living Cells by Atomic Force Microscopy

Abstract

Macromolecular dynamics around the plasma membrane (membrane proteins, cytoskeleton, lipid bilayer, etc.) play a crucial role in cell signaling, cell motility and cell adhesion. Nano-scale imaging of plasma membrane dynamics has been a difficult task due to a lack of fluorescent labeling method for lipid bilayers in a living cell. Here, we utilized a high-speed atomic force microscope (HS-AFM) to directly visualize the dynamics of the plasma membrane with sub-second time resolution.Imaging of a living COS-7 cell in a serum-free medium exhibited a number of membrane invaginations with a diameter ranging from 90 to 300 nm, which corresponds well to the size of clathrin-mediated endocytosis. Using this method, we identified various unique properties of endocytosis: (i)The duration time of the invagination was 50∼300 sec, and by treating the cell with dynasore (inhibitor for dynamin), the duration time was prolonged to over 1,000 sec, demonstrating that the closing step of the invagination is dependent on dynamin. (ii)Formation of the pit was accompanied with dynamic morphological changes of the invagination; adjacent pits were fused into a larger pit or the pit was split into several smaller pits. (iii)In the final stage of pit closure, different from the canonical model in which dynamin pinches off the membrane vesicle from the flat plasma membrane, the invaginations were capped by a brief swelling of a peripheral region of the membrane.Furthermore, a hybrid HS-AFM with a confocal fluorescence microscope enabled simultaneous imaging of the morphological dynamics of the plasma membrane and the localization of various membrane-associated proteins essential for maturation of endocytic vesicle such as FCHo, Dynamin, etc. This technique will allow for elucidation of the detailed processes of endocytosis with the dynamics of membrane proteins and lipid bilayers.