Introduction of antigenic phospholipids into the plasma membrane of mammalian cells: organization and antibody-induced lipid redistribution

Phosphatidylethanolamine bearing the 2,4,6-trinitrophenyl hapten was introduced into the surface membrane of mammalian fibrobalsts by incubating the cells with small unilamellar vesicles containing this hapten-conjugated lipid. Consistent with integration of the antigen into the plasma membrane lipid bilayer, the exogenously supplied lipid was observed by immunofluorescence to diffuse rapidly (D≥0.6x10^-8 cm²/sec) over the surface of polykaryons formed between vesicle- and non-vesicle-treated cells. Association of the exogenous lipids with cells via adsorption of vesicles to the plasma membrane was rigorously excluded by a combination of ultrastructural and immunofluorescence studies. The distribution of the integrated antigenic lipid in the plasma membranes of vesicle-treated cells was followed by immunofluorescence microscopy. The exogenously supplied hapten-conjugated phospholipid was observed to be uniformly distributed and remained so for up to 1 hr at 37°C. However, upon the addition of bivalent, but not monovalent, antihapten antibodies, the phospholipid underwent a rapid temperature-dependent redistribution, forming small patches that eventually coalesced into one or more large aggregates. This unexpected finding is discussed in terms of the mode of insertion of the lipid into the cell surface and the possible mechanisms by which bivalent ligands might alter the mobility and distribution of cell surface phospholipids.