Fusion and phase separation monitored by lifetime changes of a fluorescent phospholipid probe

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Abstract
The sensitivity of the fluorescence lifetime of 1-palmitoyl-2[[2-[4-(6-phenyl-trans-1,3,5-hexatrienyl)phenyl]ethyl]carbonyl]-3-sn-phosphatidylcholine (DPHpPC) to its local concentration in lipid bilayers was used to monitor both lipid mixing and phase separation occurring during membrane vesicle fusion. Vesicles containing 2 mol % DPHpPC were mixed with a 10-fold excess of vesicles devoid of probe. Upon addition of a fusogen, mixing of bilayer lipids associated with fusion was followed as an increase in the fluorescence lifetime of DPHpPC. Ca2+-induced fusion of phosphatidylserine vesicles served to test the method and was shown to have an exponential half-time of 7 s. Phase separation (between the phosphatidylserine head groups of bulk lipid and the phosphatidylcholine head groups of the probe) was monitored by DPHpPC under the same conditions used to follow lipid mixing due to fusion. Phase separation was not significant until 10 min after Ca2+ addition and was completely reversible by disodium ethylenediaminetetraacetate addition. Vesicle aggregation induced by Ca2+ addition to mixed phosphatidylserine/phosphatidylcholine vesicles did not alter the DPHpPC lifetime indicating that close association of vesicles did not promote intervesicular exchange of the probe. In addition, we have investigated the effects of Ca2+ on the fluorescence properties of this probe and of the head-group-labeled flourescent probes N-(4-nitro-2,1,3-benzoxadiazolyl)phosphatidylethanolamine and N-(lissamine Rhodamine B sulfonyl)dioleoylphosphatidylethanolamine, which are used in the fluorescence energy transfer assay of Struck et al. [Struck, D.K., Hoekstra, D., and Pagano, R.E. (1981) Biochemistry 20, 4093-4099]. Ca2+ was shown to quench the fluorescence intensity of the head-group-labeled probes, while the hydrophobic environment surrounding the fluorescent moiety of DPHpPC appeared to protect it from the direct influence of such water-soluble fusogens. Overall, monitoring of DPHpPC fluorescence lifetime offered some significant advantages over current methods for detecting phospholipid reorganizations accompanying membrane fusion.