Detection of Lipid Domains in Model and Plasma Membranes by Fluorescence Lifetime Imaging Microscopy of Fluorescent Lipid Analogues

Abstract

The presence of lipid domains in cellular membranes and their characteristic features are still an issue of dividing discussion. Several recent studies implicate that lipid domains in plasma membranes of mammalian cells are short lived and in the submicron range. To unravel the lateral heterogeneity of cellular membranes, in particular of mammalian plasma membranes, at this scale various techniques of fluorescence spectroscopy and microscopy have been applied. Measuring fluorescence lifetime of appropriate lipid analogues is a proper approach to detect domains with such properties. Here, the sensitivity of the fluorescence lifetime of 1-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). For GUVs forming microscopically visible lipid domains the fluorescence lifetime in the liquid disordered (ld) and the liquid ordered (lo) phase was clearly distinct being about 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. Application of this approach to mammalian cells revealed that while the fluorescence lifetime is sensitive to the composition of the plasma membrane, distinct lipid domains as found for GUVs were not detected. A broad distribution of the long lifetime was found for C6-NBD-PC inserted in the plasma membrane of these cells centred around 11 ns. However, FLIM studies on lipid domain forming giant vesicles derived from the plasma membrane of HeLa-cells rather support a recent hypothesis that an ensemble of lipid domains being of submicroscopic size exist in the plasma membrane.