Preferential binding and re-organization of nanoscale domains on model lipid membranes by pore-forming toxins: insight from STED-FCS

Potential nanodomains in cellular membranes are widely believed to be targeted by proteins and other biomolecules to enable execution of critical cellular functions. However, characterization of these nanodomains remains elusive, primarily due to the diffraction limit of a conventional optical microscope. Herein using super-resolution STED microscopy coupled with fluorescence correlation spectroscopy (STED-FCS), we provide experimental evidence of lipid nanodomains present in model membranes comprised of phosphocholine-cholesterol binary mixture, and its reorganization induced by pore-forming toxins (PFTs). In this study, we used two different types of PFTs, namely α-PFT (cytolysin A) and β-PFT (listeriolysin O), that preferentially associate as well as reorganize cholesterol-containing saturated and unsaturated phosphocholine membranes. The emergence of nanodomains due to the lipid–lipid and lipid-PFT interactions was quantified at a length scales of ∼50–150 nm using FCS diffusion law enabled by variation of spot sizes in the super-resolution STED microscopy. Our results shed light on the usefulness of super-resolution microscopy to quantify the underlying nanoscale domains in model membranes with implications for a wide variety of membrane-mediated cellular events observed in real cell membranes. (paper)