Capping of a phospholipid analog in the plasma membrane of lymphocytes

Abstract

Lipid vesicles were formed by ultrasonication from N-2,4,6-trinitrophenyl,1-acyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole) aminocaproyl phosphatidylethanolamine (Tnp-NBD-PE), a fluorescent- and hapten-conjugated phospholipid analog. The vesicles, which were nonfluorescent due to self-quenching, were further characterized by gel filtration chromatography and electron microscopy. Treatment of EL-4 lymphoma cells with these vesicles at 2°C resulted in immediate dequenching of fluorescence due to dilution of the fluorescent lipid in cell membranes. Control experiments showed Tnp-NBD-PE association with cells was predominantly via a lipid transfer mechanism, with little adsorption of intact vesicles. Fluorescence microscopy of the treated cells revealed bright uniform peripheral ring staining with no apparent internal fluorescence as long as the cells were maintained at 2°C. Fluorescence photobleaching recovery measurements showed that the majority (∼80%) of the Tnp-NBD-PE was free to diffuse in the plasma membrane of the treated cells and had a diffusion coefficient of approximately 8.5 × 10 −9 cm 2/sec. These observations strongly suggest that the exogenously supplied supplied lipid was diluted in the plasma membrane of the EL-4 cells and is initially free to diffuse rapidly in the plane of the membrane consistent with its proper integration in the plasma membrane bilayer. Upon incubation of washed, vesicle-treated cells at 37°C, essentially all the Tnp-NBD-PE became rapidly internalized. However, warming the cells in the presence of bound Tnp antibodies prevented the internalization of the Tnp-NBD-PE and led to the formation of caps. Capping was inhibited by azide and cytochalasin B. These results are discussed in relation to various models for the redistribution of lymphocyte plasma membrane components.