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Originally published as Biophys J. BioFAST on February 26, 2007.
doi:10.1529/biophysj.106.094730
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Biophysical Journal 92:3564-3574 (2007)
© 2007 The Biophysical Society

Fluorescence Resonance Energy Transfer between Lipid Probes Detects Nanoscopic Heterogeneity in the Plasma Membrane of Live Cells

Prabuddha Sengupta, David Holowka and Barbara Baird

Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853-1301

Correspondence: Address reprint requests to Barbara Baird, Dept. of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853-1301. Tel.: 607-255-4095; Fax: 607-255-4137; E-mail: bab13{at}cornell.edu.

Fluorescence resonance energy transfer (FRET) between matched carbocyanine lipid analogs in the plasma membrane outer leaflet of RBL mast cells was used to investigate lateral distributions of lipids and to develop a general method for quantitative measurements of lipid heterogeneity in live cell membranes. FRET measured as fluorescence quenching of long-chain donor probes such as DiO-C18 is greater with long-chain, saturated acceptor probes such as DiI-C16 than with unsaturated or shorter-chain acceptors with the same chromophoric headgroup compared at identical concentrations. FRET measurements between these lipid probes in model membranes support the conclusion that differential donor quenching is not caused by nonideal mixing or spectroscopic differences. Sucrose gradient analysis of plasma membrane-labeled, Triton X-100-lysed cells shows that proximity measured by FRET correlates with the extent of lipid probe partitioning into detergent-resistant membranes. FRET between DiO-C16 and DiI-C16 is sensitive to cholesterol depletion and disruption of liquid order (Lo) by short-chain ceramides, and it is enhanced by cross linking of Lo-associated proteins. Consistent results are obtained when homo-FRET is measured by decreased fluorescence anisotropy of DiI-C16. These results support the existence of nanometer-scale Lo/liquid disorder heterogeneity of lipids in the outer leaflet of the plasma membrane in live cells.




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