Unraveling complex nanoscale lipid dynamics in simple model biomembranes: Insights from fluorescence correlation spectroscopy in super-resolution stimulated emission depletion mode

•Quantification of nanoscale dynamical heterogeneity in model membranes using STED – FCS.•Emergence of nanoscale domains due to interaction of pore – forming toxin (PFT).•High cholesterol content is sufficient to induce nanoscale heterogeneity in a single component membrane.•Length scales of domains formed in a two-component system reduces upon addition of cholesterol. Dynamic heterogeneity (DH) at nanoscale due to lipid-lipid and/or lipid-protein interactions in cell membranes plays a crucial role in determining a broad range of important cell functions. In cell membranes, the dimensions of these nanodomains have been postulated to be in the order of 10’s of nm and transient in nature. While the structural features of membranes have been studied in detail, little is known about their dynamical characteristics due to paucity of techniques which can probe nanoscale phenomena with simultaneous high temporal resolution. A combination of super-resolution stimulated emission depletion (STED) and fluorescence correlation spectroscopy (FCS) technique can overcome this limitation and provide information about the nanoscale dynamic heterogeneity in cell membranes. Using STED-FCS and FCS diffusion law, we provide an understanding of how nanoscale dynamically organizing lipid platforms can emerge in minimal system of model biomembranes. To illustrate the utility of the technique we have chosen cholesterol containing supported lipid bilayers and demonstrated the role of cholesterol concentration and/or added pore-forming protein, Listeriolysin O (LLO) in determining onset of lipid DH. In addition we have also looked at multi-component lipid bilayers with and without cholesterol to infer about the role of phospholipid and cholesterol composition on lipid dynamics. These results on simple biomimetic systems provide insights into fundamental pathways for the emergence of complex nanodomain substructures with implications for a wide variety of membrane mediated cellular events and depict the significant contribution that STED-FCS can make in resolving several outstanding issues in membrane biology.