GPI-Anchored Proteins do not Reside in Ordered Domains in the Live Cell Plasma Membrane

Abstract

The organization and dynamics of proteins and lipids in the plasma membrane, and their role in membrane functionality, have been subject of a long-lasting debate. Specifically, it is unclear to what extent membrane proteins are affected by their immediate lipid and protein environment and vice versa. Studies on model membranes and plasma membrane spheres indicated characteristic preferences of proteins for ordered or disordered lipid phases, however, whether such phases do indeed exist in live cells is still not known. Here, we used a micropatterning approach combined with super-resolution microscopy to quantify the influence of a glycosylphosphatidylinositol-anchored protein (GPI-AP) - a typical marker of ordered lipid phases - on its molecular environment directly in the live cell plasma membrane. Intriguingly, local enrichment and immobilization of a GPI-anchored mGFP - even at highest densities - did not nucleate the formation of ordered membrane regions. At a molecular scale, immobilized mGFP-GPIs essentially behaved as inert obstacles that reduced the mobility of other membrane constituents according to their molecular size and shape. Overall we conclude that GPI-APs do not significantly influence their membrane environment over distances beyond their actual physical size. Further, our results imply that the outer leaflet of the plasma membrane is in a homogenously mixed single phase state under physiological conditions.