Fluorescence method for measuring the kinetics of fusion between biological membranes

Abstract

An assay is presented that allows continuous and sensitive monitoring of membrane fusion in both artificial and biological membrane systems. The method relies upon the relief of fluorescence self-quenching of octadecyl Rhodamine B chloride. When the probe is incorporated into a lipid bilayer at concentrations up to 9 mol% with respect to total lipid, the efficiency of self-quenching is proportional to its surface density. Upon fusion between membranes labeled with the probe and nonlabeled membranes, the decrease in surface density of the fluorophore results in a concomitant, proportional increase in fluorescence intensity, allowing kinetic and quantitative measurements of the fusion process. The kinetics of fusion between phospholipid vesicles monitored with this assay were found to be the same as those determined with a fusion assay based on resonance energy transfer [Struck, D. K., Hoekstra, D., & Pagano, R. E. (1981) Biochemistry 20, 4093-4099]. Octadecyl Rhodamine B chloride can be readily inserted into native biological membranes by addition of an ethanolic solution of the probe. Evidence is presented showing that the dilution of the fluorophore, occurring when octadecyl Rhodamine containing influenza virus is mixed with phospholipid vesicles at pH 5.0, but not pH 7.4, resulted from virus-vesicle fusion and was not related to processes other than fusion. Furthermore, by use of this method, the kinetics of fusion between Sendai virus and erythrocyte ghosts and virus-induced fusion of ghosts were readily revealed. Dilution of the probe was not observed upon prior treatment of fluorescently labeled Sendai virus with trypsin.(ABSTRACT TRUNCATED AT 250 WORDS)

Dynamics of ionic lipophilic probes in micelles: picosecond fluorescence depolarization measurements

“…The mechanism for the cytotoxic behavior of MC540, however, is controversial and remains an active area of research.28-30 ODRB (Figure 1) is a cationic lipophilic fluorescent probe which has been used in studies of electronic excitation transport in micelles3I and vesicles,3z in distance determinations in biological complexes by resonance energy transfer,33 and in the monitoring of cell fusion. 34 MC540 is anchored to a micelle by the two tetramethylenic tails, with the benzoxazole end functionalized by the anionic sulfonate group situated at the surface of the micelle. For this orientation, MC540's transition moments are preferentially aligned along the normal to the surface of the micelle.…”

Observation of single influenza virus-cell fusion and measurement by fluorescence video microscopy.

“…Considerable insight has been gained recently by studying the fusion of viruses with cell plasma membranes by spectrofluorometric methods (1)(2)(3)(4)(5)(6). Virus particles are labeled with the lipid analogue octadecylrhodamine B (R18), which intercalates in the viral coat at concentrations that cause selfquenching ofthe fluorochrome.…”

Gene transfection by quantitatively reconstituted Sendai envelope proteins into liposomes

“…It has been well documented that the F glycoprotein of Sendai virus promotes the virus -mediated membrane fusion even though the precise mechanism for the process has not been clarified yet. 21,22 Also, it has been considered that the Sendai HN glycoprotein serves as an anchoring protein, specifically attaching to sialoglycoproteins and sialoglycolipids in cell membranes. In addition to the anchoring function, some scientists proposed that the HN glycoprotein also plays an active role in the fusion process.…”