Nanoscale organization of multiple GPI-anchored proteins in living cell membranes

Cholesterol and sphingolipid-enriched "rafts" have long been proposed as platforms for the sorting of specific membrane components including glycosylphosphatidylinositol-anchored proteins (GPI-APs), however, their existence and physical properties have been controversial. Here, we investigate the size of lipid-dependent organization of GPI-APs in live cells, using homo and hetero-FRET-based experiments combined with theoretical modeling. These studies reveal an unexpected organization wherein cell surface GPI-APs are present as monomers and a smaller fraction (20%-40%) as nanoscale (<5 nm) cholesterol-sensitive clusters. These clusters are composed of at most four molecules and accommodate diverse GPI-AP species; crosslinking GPI-APs segregates them from preexisting GPI-AP clusters and prevents endocytosis of the crosslinked species via a GPI-AP-selective pinocytic pathway. In conjunction with an analysis of the statistical distribution of the clusters, these observations suggest a mechanism for functional lipid-dependent clustering of GPI-APs.