Abstract
The presence of lipid domains in cellular membranes and their characteristic features are still an issue of dividing discussion. Several recent studies implicate lipid domains in plasma membranes of mammalian cells as short lived and in the submicron range. Measuring the fluorescence lifetime of appropriate lipid analogues is a proper approach to detect domains with such properties. Here, the sensitivity of the fluorescence lifetime of1-palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]-hexanoyl]-sn-glycero-3-phospholipid (C6-NBD-phospholipid) analogues has been employed to characterize lipid domains in giant unilamellar vesicles (GUVs) and the plasma membrane of mammalian cells by fluorescence lifetime imaging (FLIM). Fluorescence decay of C6-NBD-phosphatidylcholine is characterized by a short and long lifetime. For GUVs forming microscopically visible lipid domains the longer lifetime in the liquid disordered (ld) and the liquid ordered (lo) phase was clearly distinct, being approximately 7 ns and 11 ns, respectively. Lifetimes were not sensitive to variation of cholesterol concentration of domain-forming GUVs indicating that the lipid composition and physical properties of those lipid domains are well defined entities. Even the existence of submicroscopic domains can be detected by FLIM as demonstrated for GUVs of palmitoyloleoyl phosphatidylcholine/N-palmitoyl-d-sphingomyelin/cholesterol mixtures. A broad distribution of the long lifetime was found for C6-NBD-phosphatidylcholine inserted in the plasma membrane of HepG2 and HeLa cells centered around 11 ns. FLIM studies on lipid domains forming giant vesicles derived from the plasma membrane of HeLa cells may suggest that a variety of submicroscopic lipid domains exists in the plasma membrane of intact cells.
Highlights
fluorescence lifetime imaging microscopy (FLIM) Is Suitable to Detect Transient Small Lipid Domains— To study domains of submicroscopic dimension methods are required of which the resolution does not interfere with the size and the dynamics of those domains
To resolve two distinct lipid environments by FLIM, lipid analogues must not interchange between the environments during emission, otherwise the two different lifetimes would average out
Domains should be stable at least for about three times the fluorescence lifetime of the analogue, e.g. for ϳ30 ns in the case of a C6-NBD-lipid analogue
Summary
Materials—Phospholipids and NBD-labeled lipids were obtained from Avanti Polar Lipids (Birmingham, AL), and cholesterol from Sigma-Aldrich. Preparation of Cells and Giant Plasma Membrane Vesicles— HeLa and HepG2 cells were grown in Dulbecco’s modified Eagle’s medium containing 4.5 g/liter glucose, supplemented with 10% heat-inactivated fetal bovine serum, and penicillin/ streptomycin and routinely passaged in 25-cm plastic culture flasks (in the case of HepG2 cells coated with collagen A), medium was changed every 3– 4 days. After washing with cold DPBS, cell labeling with C6-NBD-PC was performed for 20 min on ice HepG2 cells were prepared according to the same protocol with minor changes: cells were seeded on poly-D-lysine-coated dishes, grown for at least 3– 4 days, the final concentration of labeled lipids was 0.5–1 M, and all washing steps were done with HBSSϩ. Appropriate fitting of NBD fluorescence decay in domain-forming GUVs showed three lifetime components. Pseudocolor-coded according to the average lifetime (av) of the pixels
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