Single molecule NSOM microscopy resolves distinct nano-scale compartments on the cell membrane

Recruitment of proteins to lipid domains known as rafts has been proposed as important mechanism to regulate their activity on a variety of cells. However, the association of transmembrane receptors with lipid rafts remains largely debated, and a direct visualization of these interactions has been challenging. We have exploited single molecule high-resolution near-field scanning optical microscopy (NSOM) in aqueous conditions to resolve for the first time individual lipids and receptor domains with an accuracy of 3-7 nm. We show that the glycosphyngolipid GM1, a major lipid raft component, organizes in nano-domains of ∼10-150nm in diameter on monocytes and dendritic cells. We further demonstrate that the putative raft-associated transmembrane integrin LFA-1 pre-organizes in nano-clusters spatially distinctive but significantly proximal to those of GM-1, with 20-25% of LFA-1 being within < 50nm proximity to GM1. In contrast, we show that the non-raft associated transferrin receptor is excluded from GM1 nanodomains. These remarkable results suggest that proximal but distinct compartmentalization occurs at the nm scale having direct consequences on the current view of rafts as stable protein-recruiting platforms. Different current models to explain our data as well as recent experiments on the dynamics of individual LFA-1 clusters and their potential interaction with lipid rafts will be presented.