Morphological changes induced by phospholipase C and by sphingomyelinase on large unilamellar vesicles: a cryo-transmission electron microscopy study of liposome fusion

Cryo-transmission electron microscopy has been applied to the study of the changes induced by phospholipase C on large unilamellar vesicles containing phosphatidylcholine, as well as to the action of sphingomyelinase on vesicles containing sphingomyelin. In both cases vesicle aggregation occurs as the earliest detectable phenomenon; later, each system behaves differently. Phospholipase C induces vesicle fusion through an intermediate consisting of aggregated and closely packed vesicles (the "honeycomb structure") that finally transforms into large spherical vesicles. The same honeycomb structure is also observed in the absence of enzyme when diacylglycerols are mixed with the other lipids in organic solution, before hydration. In this case the sample then evolves toward a cubic phase. The fact that the same honeycomb intermediate can lead to vesicle fusion (with enzyme-generated diacylglycerol) or to a cubic phase (when diacylglycerol is premixed with the lipids) is taken in support of the hypothesis according to which a highly curved lipid structure ("stalk") would act as a structural intermediate in membrane fusion. Sphingomyelinase produces complete leakage of vesicle aqueous contents and an increase in size of about one-third of the vesicles. A mechanism of vesicle opening and reassembling is proposed in this case.