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J. Biol. Chem., Vol. 283, Issue 45, 30828-30837, November 7, 2008

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Detection of Lipid Domains in Model and Cell Membranes by Fluorescence Lifetime Imaging Microscopy of Fluorescent Lipid Analogues^*

From the Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, Institut für Biologie/Biophysik, Invalidenstrasse 42, Berlin D-10115, Germany

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 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.

Received for publication, February 21, 2008 , and in revised form, July 24, 2008.

^* This work was supported by the Deutsche Forschungsgemeinschaft (Grants FG 475 and SFB 740) and the European Union (Grant MRTN-CT-2004-005330) (to A. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ To whom correspondence should be addressed: Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, Institut für Biologie/Biophysik, Invalidenstrasse 43, Berlin D-10115, Germany. Tel.: 49-30-2093-8830; Fax: 49-30-2093-8585; E-mail: andreas.herrmann{at}rz.hu-berlin.de.

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