Lipid bilayers as model systems for plasma membranes: experimental synergy to tackle growing complexity

Plasma membranes are dynamic and complex structures that play a central role in regulating various cellular processes, including communication, signalling, and transport. They exhibit a highly heterogeneous organization, with cholesterol playing a fundamental role in regulating specific protein localization and dynamics within the lipid bilayer, promoting the formation of functional nanodomains called lipid rafts (LRs). In this study the influence of cholesterol on lipid rafts formation and in the modulation of membrane stiffness and fluidity has been investigated, through a bottom-up approach, i.e. by building multi-component membrane mimics, which represent the outer leaflet of eukaryotic cell membranes. This novel 4-element membrane mimic allowed for adjustments of cholesterol concentration up to physiologically relevant values. It also provided a controlled environment to investigate how cholesterol concentration modulates lipid organization and facilitates LRs assembly, affecting membrane biophysical properties, as stiffness and fluidity, and how these properties modify in different physiological conditions. By means of atomic force microscopy topographic imaging and nanoindentation spectroscopy, confocal laser scanning fluorescence microscopy (CLSM) and fluorescence recovery after photobleaching (FRAP), we have collected quantitative information on membrane properties such as lipid rafts coverage and height, domains stiffness and molecular diffusion within the domains, as a function of cholesterol content. In a second part of the work, we used membranes biophysical characteristics to rationalize the role of membrane fluidity in the interaction with extracellular vesicles, potent nanosized natural vectors which strongly contribute to cell-cell communication. This analysis has relevant implications for the understanding of cellular communication and for eventually testing drugs to regulate EVs uptake.