Biophysical and structural characterization of 1H-NMR-detectable mobile lipid domains in NIH-3T3 fibroblasts.
Nature and subcellular localization of 1H-NMR-detectable mobile lipid domains (ML) were investigated by NMR, Nile red fluorescence and electron microscopy, in NIH-3T3 fibroblasts and their H-ras transformants (3T3ras) transfected 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 untransformed fibroblasts with both (a) intramembrane amorphous lipid vesicles, about 60 nm in diameter, distinct from caveolae; and (b) cytoplasmic, osmiophilic lipid bodies surrounded by own membrane, endowed of intramembrane particles. 2D NMR maps demonstrated that ML comprised both mono- and polyunsaturated fatty chains. Lower ML signals were detected in 3T3ras (R=0.76+/-0.37), under various conditions of cell growth. Very few (if any) lipid bodies and vesicles were detected in the cytoplasmic or membrane compartments of 3T3ras cells with R<0.4, while only intramembrane lipid vesicles were associated with moderate R values. Involvement of phosphatidylcholine hydrolysis in ML generation was demonstrated by selective inhibition of endogenous phospholipase C (PC-plc) or by exposure to bacterial PC-plc. This study indicates that: (1) both cytoplasmic lipid bodies and membrane vesicles (possibly in mutual dynamic exchange) may contribute (although to a different extent) to ML signals; and (2) high levels of ras-transfection either inhibit ML formation or facilitate their extrusion from the cell.
Ferretti
A
A
Laboratory of Cell Biology, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161, Rome, Italy.
Knijn
A
A
Iorio
E
E
Pulciani
S
S
Giambenedetti
M
M
Molinari
A
A
Meschini
S
S
Stringaro
A
A
Calcabrini
A
A
Freitas
I
I
Strom
R
R
Arancia
G
G
Podo
F
F
D016475:3T3 Cells; D000818:Animals; D002461:Cell Line, Transformed; D002849:Chromatography, Gas; D005347:Fibroblasts / Q000737:chemistry* / Q000648:ultrastructure; D005434:Flow Cytometry; D005614:Freeze Fracturing; D008055:Lipids / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D010078:Oxazines
D008055:Lipids; D010078:Oxazines; C044808:nile red