(432d) pH-Dependent Lipid Phase-Separation Forming Defective Interfaces: Effects On Lipid Bilayer Fusion

Lipid membrane fusion involves the mixing of lipids from separate bilayers. Fusion has been widely studied using model membranes since it is an essential process for cell survival. Fusion takes place in diverse cellular activities such as exocytosis, vesicle transport among cell organelles, release of neurotransmitters at synapses, fertilization, viral infection, and intracellular membrane trafficking. In therapeutic applications using lipid bilayer vesicles as the delivery carrier, in order to deliver the therapeutic agents intracellularly, fusion between the cellular membrane and the lipid vehicle is critical. Fusion can be triggered by different events: one being formation of defects on the approaching bilayers. In this work, we present a mechanism of introducing lipid packing defects on bilayers resulting in complete fusion. Our findings on vesicle aggregation, total lipid mixing assays, and inner leaflet mixing studies support this statement. Fusion is studied using lipid bilayers in the form of unilamellar vesicles. We are studying phospholipid membranes composed of lipid pairs with phosphatidylcholine and phosphatidic acid headgroups that form phase separated domains with lowering pH. We have demonstrated that lipid pairs in the gel state with non-matching acyl-chain lengths form pH-dependent phase separated domains that, in addition, have leaky interfacial boundaries. Our current studies indicate correlation between extensive vesicle fusogenicity and the presence of leaky interfacial boundaries attributed to lipid packing discontinuities within the interfaces of phase separated lipid domains. We hypothesize that in these systems intervesicular fusion is driven by the tendency of the system (vesicle suspension) to minimize the total length of defective interfaces of the phase-separated lipid domains that are characterized by high line tensions.

Increasing aggregation in vesicle suspensions with decrease in pH is observed, and aggregation rates are strongly dependent on the lipid vesicle concentration. Our findings demonstrate complete vesicle fusion with increasing formation of defective interfaces when bilayers are composed of lipid pairs with non-matching acyl-tail lengths. Fusion rates are strongly dependent on the introduced pH-gradient and the lipid vesicle concentration. For bilayers containing lipid pairs with matching acyl-tail lengths, inner monolayer lipid mixing studies demonstrate hemifusion, and suggest an important role for the overall stiffness of the bilayer membrane. These observations support the hypothesis that pronounced hydrophobic mismatch within the interfaces of phase-separated lipid domains is strongly correlated to the cause that drives fusion in these lipid vesicles.