Activated FcγRII and signalling molecules revealed in rafts by ultra-structural observations of plasma-membrane sheets

Abstract

To reveal topography of FcgammaRII components of the receptor-signalling complex, large plasma-membrane sheets were obtained by cell cleavage and analysed by immuno-electron microscopy. Non-activated FcgammaRII was dispersed in the plane of the plasma membrane and only rarely was localized in the proximity of Lyn, an Src family tyrosine kinase, and CD55, a glycosylphosphatidylinositol-anchored protein. After FcgammaRII activation by cross-linking with antibodies, clusters of an electron-dense material acquiring about 86% of FcgammaRII and reaching up to 300 nm in diameter were formed within 5 min. These structures also accommodated about 85% of Lyn and 63% of CD55 labels that were located in close vicinity of gold particles attributed to the cross-linked FcgammaRII . The electron-dense structures were also abundant in tyrosine phosphorylated proteins. At their margins PIP2 was preferentially located. Based on a concentration of Lyn, CD55 and activated FcgammaRII , the electron-dense structures seem to reflect coalescent membrane rafts.