Imaging SNAREs at work in 'unroofed' cells - approaches that may be of general interest for functional studies on membrane proteins

When cultured cells are subjected to a brief ultrasound pulse, their upper parts burst, but the basal plasma membranes with their embedded membrane-protein complexes remain intact. Such two-dimensional, paraformaldehyde-fixed plasma membrane sheets have been used in the past to visualize the morphology of the inner plasmalemmal leaflet by electron or light microscopy. More recently, fluorescence microscopy of unfixed native membranes has been applied to study SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) function. For instance, biochemical reactions of the plasmalemmal SNARES with soluble fluorescent SNARES, patching of SNARE and raft domains, and online monitoring of SNARE-mediated membrane fusion has been performed. The results obtained with the membrane sheet system have added some novel aspects to our understanding of the regulation of neuronal exocytosis. Surprisingly, SNARES are concentrated in cholesterol-dependent microdomains that are different from membrane rafts. SNARES in such domains are highly reactive, and define sites for vesicle exocytosis. Secretory granules that fuse on the membrane sheets are retrieved intact in a dynamin-dependent process, suggesting that the 'kiss-and-run' mechanism is not a reversed SNARE reaction, but is driven by a biochemically different mechanism. So far, studies of this type have focused on neuronal exocytosis; however, the method might be widely applicable. Data obtained with this system are derived from a 100% pure plasma membrane preparation that is only several seconds old, and membrane proteins are studied in their natural microenvironment that is defined by local lipid composition and putative bound proteins. Hence this approach yields results that most probably reflect the situation in a live cell.