Bim-FCS Analysis of Membrane Protein Diffusion Reveals Dynamics of Membrane Cytoskeleton and Lipid Domains in Intact Cells

Abstract

Signaling transmembrane proteins are experiencing a heterogeneous structure due to membrane cytoskeleton meshwork and selective association with cholesterol stabilized lipid domains throughout the cell membrane. The limited optical resolution due to diffraction limits direct observation of both cytoskeleton structure and cholesterol stabilized micro-domains.Fluorescence correlation spectroscopy (FCS) allows to measure membrane protein diffusion, but does not resolve the heterogeneity. We analyze the diffusion of GFP-tagged membrane proteins in multiple areas of increasing size simultaneous using TIRF illumination and camera based FCS. The binned-imaging FCS (bim-FCS) allows distinguishing free Brownian diffusing proteins from proteins interacting with the membrane cytoskeleton and from proteins transiently entering sub-microscopic domains of reduced mobility. Using bimFCS it is possible to study how subtle changes modulate the structural nanodomains or the functional interaction with them, and how these changes propagate with time.We investigate effect of membrane meshwork on diffusion of transmembrane proteins and on GPI anchored proteins which associate with cholesterol stabilized micro-domains in intact cells. We show in real-time how the protein-membrane ultrastructure interactions are modulated by protein dimerization or lipid cross-linking. We perform Monte Carlo simulations of bimFCS experiments with square pixels to analyze the effect of cytoskeleton barriers and cholesterol domains on the diffusion. We are able to quantitatively model the changes induced by protein or lipid cross-linking.