Fluorometric methods for assaying membrane fusion exploit processes, such as nonradiative energy transfer, fluorescence quenching and pyrene excimer formation, that are dependent on probe concentration. Assays of membrane fusion report either the mixing of membrane lipids (described here) or the mixing of the aqueous contents of the fused entities (Assays of Volume Change, Membrane Fusion and Membrane Permeability—Note 14.3). Probes for Lipids and Membranes—Chapter 13 describes additional methods for detecting membrane fusion based on image analysis.
NBD–Rhodamine Energy Transfer
Principle: Struck, Hoekstra and Pagano introduced lipid-mixing assays based on NBD–rhodamine energy transfer. In this method (Figure 1), membranes labeled with a combination of fluorescence energy transfer donor and acceptor lipid probes—typically NBD-PE and N-Rh-PE (N360, L1392; Fatty Acid Analogs and Phospholipids—Section 13.2), respectively—are mixed with unlabeled membranes. Fluorescence resonance energy transfer (FRET), detected as rhodamine emission at ~585 nm resulting from NBD excitation at ~470 nm, decreases when the average spatial separation of the probes is increased upon fusion of labeled membranes with unlabeled membranes. The reverse detection scheme, in which FRET increases upon fusion of membranes that have been separately labeled with donor and acceptor probes, has also proven to be a useful lipid-mixing assay.
Applications of the NBD–rhodamine assay are described in footnoted references.
Figure 1. Pictorial representation of a lipid-mixing assay based on fluorescence resonance energy transfer (FRET). The average spatial separation of the donor (D) and acceptor (A) lipid probes increases upon fusion of labeled membranes with unlabeled membranes, resulting in decreased efficiency of proximity-dependent FRET (represented by yellow arrows). Decreased FRET efficiency is registered by increased donor fluorescence intensity and decreased acceptor fluorescence intensity.
Octadecyl Rhodamine B Self-Quenching
Principle: Lipid-mixing assays based on self-quenching of octadecyl rhodamine B (R18, O246; Other Nonpolar and Amphiphilic Probes—Section 13.5) were originally described by Hoekstra and co-workers. Octadecyl rhodamine B self-quenching occurs when the probe is incorporated into membrane lipids at concentrations of 1–10 mole percent. Unlike phospholipid analogs, octadecyl rhodamine B can readily be introduced into existing membranes in large amounts. Fusion with unlabeled membranes results in dilution of the probe, which is accompanied by increasing fluorescence (excitation/emission maxima 560/590 nm) (Figure 2). The assay may be compromised by effects such as spontaneous transfer of the probe to unlabeled membranes, quenching of fluorescence by proteins and probe-related inactivation of viruses; the prevalence of these effects is currently debated.
Figure 2. Pictorial representation of a lipid-mixing assay based on fluorescence self-quenching. Fluorescence of octadecyl rhodamine B (O246), incorporated at >1:100 with respect to host membrane lipids, is quenched due to dye–dye interactions. Fusion with unlabeled membranes causes dispersion of the probe, resulting in a fluorescence increase that is represented here by a color change from black to green.
Pyrene Excimer Formation
Principle: Pyrene-labeled fatty acids (e.g., P31, P96, P243; Fatty Acid Analogs and Phospholipids—Section 13.2) can be biosynthetically incorporated into viruses and cells in sufficient quantities to produce the degree of labeling required for long-wavelength pyrene excimer fluorescence (Figure 3). This excimer fluorescence is diminished upon fusion of labeled membranes with unlabeled membranes (Figure 4). Fusion can be monitored by following the increase in the ratio of monomer (~400 nm) to excimer (~470 nm) emission, with excitation at about 340 nm. This method appears to circumvent some of the potential artifacts of the octadecyl rhodamine B self-quenching technique and, therefore, provides a useful alternative for virus–cell fusion applications.
Figure 3. Excimer formation by pyrene in ethanol. Spectra are normalized to the 371.5 nm peak of the monomer. All spectra are essentially identical below 400 nm after normalization. Spectra are as follows: 1) 2 mM pyrene, purged with argon to remove oxygen; 2) 2 mM pyrene, air-equilibrated; 3) 0.5 mM pyrene (argon-purged); and 4) 2 µM pyrene (argon-purged). The monomer-to-excimer ratio (371.5 nm/470 nm) is dependent on both pyrene concentration and the excited-state lifetime, which is variable because of quenching by oxygen.
Figure 4. Pictorial representation of a lipid-mixing assay based on pyrene excimer formation (Figure 3). Locally concentrated pyrene-labeled lipid probes emit red-shifted fluorescence due to formation of excimers (excited-state dimers). Probe dilution by unlabeled lipids as a result of membrane fusion is registered by the replacement of excimer emission by blue-shifted monomer fluorescence.
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