LIPID DOMAIN-STRUCTURE OF THE PLASMA-MEMBRANE REVEALED BY PATCHING OFMEMBRANE-COMPONENTS

Lateral assemblies of glycolipids and cholesterol, ''rafts,'' have bee n implicated to play a role in cellular processes like membrane sortin g, signal transduction, and cell adhesion. We studied the structure of raft domains in the plasma membrane of non-polarized cells. Overexpre ssed plasma membrane markers were evenly distributed in the plasma mem brane. We compared the patching behavior of pairs of raft markers (def ined by insolubility in Triton X-100) with pairs of raft/non-raft mark ers. For this purpose we cross-linked glycosyl-phosphatidylinositol (G PI)-anchored proteins placental alkaline phosphatase (PLAP), Thy-1, in fluenza virus hemagglutinin (HA), and the raft lipid ganglioside GM1 u sing antibodies and/or cholera toxin. The patches of these raft marker s overlapped extensively in BHK cells as well as in Jurkat T-lymphoma cells. Importantly, patches of GPI-anchored FLAP accumulated src-like protein tyrosine kinase fyn, which is thought to be anchored in the cy toplasmic leaflet of raft domains. In contrast patched raft components and patches of transferrin receptor as a non-raft marker were sharply separated. Taken together, our data strongly suggest that coalescence of cross-linked raft elements is mediated by their common lipid envir onments, whereas separation of raft and non-raft patches is caused by the immiscibility of different lipid phases. This view is supported by the finding that cholesterol depletion abrogated segregation. Our res ults are consistent with the view that raft domains in the plasma memb rane of non-polarized cells are normally small and highly dispersed bu t that raft size can be modulated by oligomerization of raft component s.