Detergent-resistant membrane fractions contribute to the total 1H NMR-visible lipid signal in cells.
Leukocytes and other cells show an enhanced intensity of mobile lipid in their 1H NMR spectra under a variety of conditions. Such conditions include stimulation, which has recently been shown to involve detergent-resistant, plasma membrane domains (DRMs) often called lipid rafts. As there is much speculation surrounding the origin of cellular NMR-visible lipid, we analysed subcellular fractions, including DRMs, by NMR spectroscopy. We demonstrated that DRMs isolated by density gradient centrifugation from lymphoid (CEM-T4, stimulated Jurkat cells), and monocytoid (THP-1) cells produced NMR-visible, lipid signals. Large scale subfractionation of THP-1 cells determined that while cytoplasmic lipid droplets constituted much of the total NMR-visible lipid, the contribution of DRMs was significant. Qualitative and quantitative lipid analyses revealed that DRMs and lipid droplets differed in their lipid composition. DRMs were enriched in cholesterol and ganglioside GM1, and contained relatively unsaturated fatty acids compared with the lipid droplets. Both lipid droplets and DRMs contained neutral lipids (triacylgycerols, cholesterol ester, fatty acids in THP-1 cells) that could, in addition to phospholipids, contribute to the NMR-visible lipid. The lipid droplets also exhibited different protein profiles and contained 500-fold less protein than DRMs, confirming that DRMs and droplets were fractionated as separate entities. The NMR-visible lipid in DRMs is therefore unlikely to be a contaminant from lipid droplets. We propose a micropartitioning of the NMR-visible mobile lipid of whole cells between intracellular lipid droplets, where most of this lipid resides, and detergent-resistant plasma membrane domains.
Wright
Lesley C
LC
Centre for Infectious Diseases and Microbiology, Institute of Clinical Pathology & Medical Research, Westmead Hospital, Australia. lesleyw@icpmr.wsahs.nsw.gov.au
Djordjevic
Julianne T
JT
Schibeci
Stephen D
SD
Himmelreich
Uwe
U
Muljadi
Nick
N
Williamson
Peter
P
Lynch
Garry W
GW
D002458:Cell Fractionation; D002460:Cell Line; D002784:Cholesterol / Q000378:metabolism; D003902:Detergents / Q000737:chemistry*; D005227:Fatty Acids / Q000378:metabolism; D006801:Humans; D008055:Lipids / Q000737:chemistry*; D008214:Lymphocytes / Q000737:chemistry / Q000166:cytology; D009682:Magnetic Resonance Spectroscopy; D021962:Membrane Microdomains / Q000737:chemistry*; D014280:Triglycerides / Q000378:metabolism
D003902:Detergents; D005227:Fatty Acids; D008055:Lipids; D014280:Triglycerides; D002784:Cholesterol