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

The ability to specifically monitor the behavior of the inner monolayer lip ids of membranous vesicles during the membrane fusion process is useful tec hnically and experimentally. In this study, we have identified N-NBD-phosph atidylserine as a reducible probe particularly suitable for inner monolayer fusion assays because of its low rate of membrane translocation after redu ction of the outer monolayer probes by dithionite. Data are presented on tr anslocation as a function of temperature, vesicle size, membrane compositio n, and serum protein concentration. Translocation as a result of the fusion event itself was also characterized. We further show here that a second me mbrane-localized probe, a long wavelength carbocyanine dye referred to a di I(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 un labeled 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 lab eling and aqueous contents mixing assays for cation-dependent fusion of lip osomes composed of phosphatidylserine and phosphatidylethanolamine. The res ults showed that the inner monolayer fusion assay eliminates certain artifa cts and reflects fairly closely the rate of non-leaky mixing of aqueous con tents due to fusion, while outer monolayer mixing always precedes mixing of aqueous contents. In fact, vesicle aggregation and outer monolayer lipid m ixing were found to occur over very long periods of time without inner mono layer mixing at low cation concentrations. Externally added lysophosphatidy lcholine inhibited vesicle aggregation, outer monolayer mixing and any subs equent fusion. The state of vesicle aggregation and outer monolayer exchang e that occurs below the fusion threshold may represent a metastable interme diate state that may be useful for further studies of the mechanism of memb rane fusion. (C) 2000 Elsevier Science B.V. All rights reserved.