Biophysical and structural characterization of H-1-NMR-detectable mobile lipid domains in NIH-3T3 fibroblasts

Nature and subcellular localization of H-1-NMR-detectable mobile lipid doma ins (ML) were investigated by NMR, Nile red fluorescence and electron micro scopy, in NIH-3T3 fibroblasts and their H-ras transformants (3T3(ras)) tran sfected with a high number of oncogene copies, Substantial ML levels (ratio of(CH2)(n)/CH3 peak areas R = 1.56 +/- 0.33) were associated in untransfor med fibroblasts with both (a) intramembrane amorphous lipid vesicles, about 60 nm in diameter, distinct from caveolae ; and (b) cytoplasmic, osmiophil ic lipid bodies surrounded by own membrane, endowed of intramembrane partic les. 2D NMR maps demonstrated that ML comprised both mono- and polyunsatura ted fatty chains. Lower ML signals were detected in 3T3(ras) (R = 0.76 +/- 0.37), under various conditions of cell growth. Very few (if any) lipid bod ies and vesicles were detected in the cytoplasmic or membrane compartments of 3T3(ras) cells with R < 0.4, while only intramembrane lipid vesicles wer e associated with moderate R values. Involvement of phosphatidylcholine hyd rolysis in ML generation was demonstrated by selective inhibition of endoge nous phospholipase C (PC-plc) or by exposure to bacterial PC-pie. This stud y indicates that: (1) both cytoplasmic lipid bodies and membrane vesicles ( possibly in mutual dynamic exchange) may contribute (although to a differen t extent) to ML signals; and (2) high levels of ras-transfection either inh ibit ML formation or facilitate their extrusion from the cell. (C) 1999 Els evier Science B.V. All rights reserved.