Inter-leaflet Organization of Membrane Nanodomains: What Can(not) Be Resolved by FRET?

Plasma membranes as well as their simplified model systems show an inherent nanoscale heterogeneity. As a result of strong interleaflet interactions, these nanoheterogeneities (called here lipid nanodomains) can be found in perfect registration (i.e. nanodomains in the inner leaflet are registered with the nanodomains in the outer leaflet). Alternatively, they might be inter-leaflet independent, anti-registered or located asymmetrically in one bilayer leaflet only. To distinguish these scenarios from each other appears to be an experimental challenge. In this work, we analyzed the potential of Forster resonance energy transfer (FRET) to characterize inter-leaflet organization of nanodomains. We generated in-silico time-resolved fluorescence decays for a large set of virtual as well as real donor/acceptor pairs distributed over the bilayer containing registered, independent, anti-registered or asymmetrically distributed nanodomains. In this way, we were able to identify conditions that gave satisfactory or unsatisfactory resolution. Overall, FRET appears as a robust method that - when using D/A pairs with good characteristics - yields otherwise difficult-to-reach characteristics of membrane lipid nanodomains. STATEMENT OF SIGNIFICANCEThis work first explores the potential of Forster resonance energy transfer (FRET) to characterize inter-leaflet nanodomain coupling and then shows how a FRET experiment can designed to achieve optimal resolution towards nanodomain coupling. Importantly, the analysis identifies as the most critical the following parameters fundamentally affecting the resolution of FRET: the Forster radius and its value related to the inter-layer distance at which donors and acceptors in the opposing membrane leaflets are separated from each other and the donor and acceptor partition coefficients characterizing their distribution between the domain and nondomain region. By setting these parameters correctly, FRET allows for the characterization of inter-leaflet nanodomain organization with unprecedented detail.