Does Ca²⁺ cause fusion or lysis of unilamellar lipid vesicles?

Abstract

MEMBRANE fusion is the central event in biological processes as diverse as secretion, fertilisation, endocytosis and pathological cell fusion, but as yet its mechanism is poorly understood¹. To elucidate this mechanism, bilayer phospholipid vesicles have been used extensively as models for interacting biomem-branes^2–5. As a result of studies involving differential scanning calorimetry and freeze-fracture electron microscopy, it has been suggested that vesicles made from acidic phospholipids can fuse when exposed to divalent cations^3–6. For phosphatidylserine vesicles, the ‘critical’ Ca²⁺ concentration for membrane fusion seems to be ∼ 1 mM, at which concentration a drastic increase in the permeability of the vesicle membranes has also been reported³. The techniques used to demonstrate ‘fusion’ in this system do not exclude molecular mixing of interacting membranes by mechanisms other than genuine fusion. In the latter case, the membrane-bounded internal compartments of two separate vesicles must become confluent in a process which transfers the contents of the initial vesicles to the resulting vesicle. It is possible, however, that Ca²⁺ may cause rupture of the lipid vesicles, with subsequent reassembly of the membrane fragments to form larger non-vesicular structures. If this is occurring, bilayer vesicles are poor paradigms for biological membrane fusion. One may predict the likelihood of vesicle fusion by calculating their energy of interaction due to electrostatic repulsion and electrodynamic attraction⁷. This approach shows that the addition of 1 mM Ca²⁺ to a suspension of phosphatidylserine vesicles is unlikely to reduce electrostatic repulsion between liposomes sufficiently for any but a fraction of the smallest ones to approach to 0.8 nm separation. This finding suggests that vesicles cannot approach closely enough to fuse and supports the notion that Ca²⁺ may initiate vesicle lysis and reassembly. We report here experimental support for vesicle lysis, based on the redistribution of a marker for the aqueous phase between vesicles and the external compartment on exposure to divalent cations.