Novel inner monolayer fusion assays reveal differential monolayer mixing associated with cation-dependent membrane fusion

Abstract

The ability to specifically monitor the behavior of the inner monolayer lipids of membranous vesicles during the membrane fusion process is useful technically and experimentally. In this study, we have identified N-NBD-phosphatidylserine as a reducible probe particularly suitable for inner monolayer fusion assays because of its low rate of membrane translocation after reduction of the outer monolayer probes by dithionite. Data are presented on translocation as a function of temperature, vesicle size, membrane composition, and serum protein concentration. Translocation as a result of the fusion event itself was also characterized. We further show here that a second membrane-localized probe, a long wavelength carbocyanine dye referred to a diI(5)C18ds, appears to form a membrane-bound resonance energy transfer pair with N-NBD-PS, and its outer monolayer fluorescence can also be eliminated by dithionite treatment. Lipid dilution of these probes upon fusion with unlabeled membranes leads to an increase in NBD donor fluorescence, and hence is a new type of inner monolayer fusion assay. These inner monolayer probe mixing assays were compared to random lipid labeling and aqueous contents mixing assays for cation-dependent fusion of liposomes composed of phosphatidylserine and phosphatidylethanolamine. The results showed that the inner monolayer fusion assay eliminates certain artifacts and reflects fairly closely the rate of non-leaky mixing of aqueous contents due to fusion, while outer monolayer mixing always precedes mixing of aqueous contents. In fact, vesicle aggregation and outer monolayer lipid mixing were found to occur over very long periods of time without inner monolayer mixing at low cation concentrations. Externally added lysophosphatidylcholine inhibited vesicle aggregation, outer monolayer mixing and any subsequent fusion. The state of vesicle aggregation and outer monolayer exchange that occurs below the fusion threshold may represent a metastable intermediate state that may be useful for further studies of the mechanism of membrane fusion.

Negatively Charged Lipids as a Potential Target for New Amphiphilic Aminoglycoside Antibiotics

“…Second, hemifusion suggested by confocal microscopy on GUVs implies the fusion of the outer layers of each vesicle where the stable bilayer at the interface of the independent compartments is composed of the two inner layers (52). To confirm this process, we monitored membrane fusion and content mixing by using the self-quenching properties of R 18 and the quenching of ANTS by DPX (46,47), respectively.…”

Phosphoinositides and SNARE chaperones synergistically assemble and remodel SNARE complexes for membrane fusion

“…To test for lysis, we used dithionite (S 2 O 4 2Ϫ ), a membrane-impermeable reducing agent that destroys the fluorescence of NBD-PE (43), as described (14,44). The fluorescence of exposed NBD-PE was rapidly diminished by S 2 O 4 2Ϫ added either 32 min or 6 min before the addition of Sec17p/Sec18p/HOPS (Fig.…”

IM30 triggers membrane fusion in cyanobacteria and chloroplasts

“…To determine whether IM30-induced liposome fusion depends on the Mg 2 þ concentration, fusion assays were performed and evaluated essentially as described in Meers et al 55 . 0.8 mM LissRhodPE (FRET-donor) and 0.8 mM NBD-PE (FRET-acceptor) were added to 0.1 mM lipids (60% MGDG, 40% DOPG, w/w) and liposomes were prepared as described.…”

“…NBD-PE fluorescence emission at 535 nm was followed at 25°C after excitation at 460 nm, with both the excitation and emission slit-width set at 5 nm. To determine the FRET value for 100% fusion, 0.1 mM mock-fused liposomes were prepared, containing 0.08 mM of both, LissRhodPE and NBD-PE 55 .…”