Resonance energy transfer microscopy: observations of membrane-bound fluorescent probes in model membranes and in living cells.

Abstract

Abstract. A conventional fluorescence microscope was modified to observe the sites of resonance energy transfer (RET) between fluorescent probes in model membranes and in living cells. These modifications, and the parameters necessary to observe RET between membrane-bound fluorochromes, are detailed for a system that uses N-4-nitrobenzo-2-oxa-l,3-diazole (NBD) or fluorescein as the energy donor and sulforhodamine as the energy acceptor. The necessary parameters for RET in this system were first optimized using liposomes. Both quenching of the energy donor and sensitized fluorescence of the energy acceptor could be directly observed in the microscope. RET microscopy was then used in cultured fibroblasts to identify those intracellular organelles labeled by the lipid probe, N-SRh-decylamine (N-SRh-C~). This was done by observing the sites of RET in cells doubly labeled with N-SRh-C~ and an NBD-labeled lipid previously shown to label the endoplasmic reticulum, mitochondria, and nuclear envelope. RET microscopy was also used in cells treated with fluorescein-labeled Lens culinaris agglutinin and a sulforhodamine derivative of phosphatidylcholine to examine the internalization of plasma membrane lipid and protein probes. After internalization, the fluorescent lectin resided in most, but not all of the intracellular compartments labeled by the fluorescent lipid, suggesting sorting of the membrane-bound lectin into a subset of internal compartments. We conclude that RET microscopy can colocalize different membrane-bound components at high resolution, and may be particularly useful in examining temporal and spatial changes in the distribution of fluorescent molecules in membranes of the living cell.ESONANCE energy transfer (RET) ~ is characterized by the transfer of photon energy from one fluorochrome (the donor) to another molecule (the acceptor) that is in close physical proximity. The donor fluorescence is quenched and, if the acceptor is an appropriate fluorescent molecule, it will fluoresce as if excited directly (2). Because RET decreases in proportion to the inverse sixth power of the distance between the two probes, this phenomenon is effective only when the donor and acceptor molecules are within 100 /~ of each other (31). RET therefore provides a convenient method for probing inter-and intramolecular distances in proteins (for a review, see reference 30) and in membranes.1. Abbreviations used in this paper: BHK, baby hamster kidney; (C6-NBD)-PA, 1,2-(oleoyl, NBD-aminocaproyl) phosphatidic acid; (C6-NBD)-PC, 1,2-(acyl, NBD-aminocaproyl) phosphatidylcholine; (C6-SRh)-PC, 1,2-(acyl, SRh-aminocaproyl) phosphatidylcholine; (C6-tBOC)-PC, 1,2- (acyl, tert-butoxycarbonyl-aminocaproyl) phosphatidylcholine; DOPC, dioleoyl phosphatidylcholine; FITC-LCA, fluorescein isothionyl-Lens culinaris agglutinin; HMEM, 18 mM Hepes-buffered Eagle's minimal essential medium without indicator, pH 7.4; NBD, N~-nitrobenzo-2-oxa-1,3-diazole; N-NBD-PE, N-NBD-dioleoyl phosphatidylethanolamine; N-SRh-C~0, N-SRh-decylamine; N-SRh-PE, N-SRh-...

High-Resolution FRET Microscopy of Cholera Toxin B-Subunit and GPI-anchored Proteins in Cell Plasma Membranes

“…CTXB and all of the GPI-anchored proteins studied here have been identified previously as components of lipid rafts defined biochemically (van den Berg et al, 1995;Smart et al, 1996;Strohmeier et al, 1997;Hatanaka et al, 1998). We confirmed this in HeLa cells by incubating labeled cells in PBS containing 1% Triton X-100 for 30 min on ice and then fixing the cells and visualizing the detergent-insoluble membrane fraction by fluorescence microscopy (Mayor and Maxfield, 1995;Kenworthy and Edidin, 1998).…”

“…FRET microscopy detects the proximity of appropriately labeled or intrinsically fluorescent lipids and proteins with a resolution of tens of angstroms (Uster and Pagano, 1986;Herman, 1989;Jovin and Arndt-Jovin, 1989;Tsien et al, 1993;Nagy et al, 1998;Ng et al, 1999;Pollok and Heim, 1999). The efficiency of energy transfer E for a donor and acceptor fluorophore separated by distance r is given by the following: E ϭ 1/{1 ϩ (r/R o ) 6 }, where R o is a characteristic of the donor and acceptor pair and is typically 30 -60 Å.…”

“…One might expect that lipid rafts would concentrate molecules such as GPI-anchored proteins, effectively clustering them. Such domains containing clustered GPI-anchored proteins can sometimes be detected by conventional light or electron microscopy (Matsuura et al 1984;Latker et al 1987;Kobayashi and Robinson, 1991;van den Berg et al, 1995) (reviewed in Anderson [1998]). In other cases clusters of GPI-anchored proteins or other lipid raft components are only apparent when they have been crosslinked with secondary antibodies (Howell et al 1987;Rothberg et al, 1990;Fra et al, 1994;Mayor et al, 1994;Parton et al, 1994;Fujimoto, 1996;Harder et al, 1998).…”

Cutting Edge: Evidence for a Signaling Partnership Between Urokinase Receptors (CD87) and L-Selectin (CD62L) in Human Polymorphonuclear Neutrophils

“…To further demonstrate the specificity of this interaction, no RET was seen when cells were labeled with the combination of a FITC-anti-L-selectin mAb and a TRITC-labeled mAb reactive to another GPI-linked receptor, CD14. Significant RET is indicative of molecular proximity between the fluorochromes within a resolution limit of 7 nm, consistent with a direct "nearest neighbor" interaction between uPAR and L-selectin (18). This type of complex formation is analogous to those described between uPAR and the ␤ 2 integrins CR3 (CD11b/CD18) and CR4 (CD11c/CD18), also demonstrated by RET.…”

Ebola Virus Secretory Glycoprotein (sGP) Diminishes FcγRIIIB-to-CR3 Proximity on Neutrophils

“…Long pass dichroic mirrors of 510 nm and 560 nm were used for FITC and TRITC, respectively. For energy transfer imaging, the 485DF22, 5101p, and 590DF30 filter combination was used (13). In some experiments the RET intensity was quantitated using a photomultiplier tube (Hamamatsu) held in a Products for Research (Danvers, MA) housing.…”