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 sphingomye linase on vesicles containing sphingomyelin. In both cases vesicle agg regation occurs as the earliest detectable phenomenon; later, each sys tem behaves differently. Phospholipase C induces vesicle fusion throug h an intermediate consisting of aggregated and closely packed vesicles (the ''honeycomb structure'') that finally transforms into large sphe rical vesicles. The same honeycomb structure is also observed in the a bsence of enzyme when diacylglycerols are mixed with the other lipids in organic solution, before hydration. In this case the sample then ev olves toward a cubic phase. The fact that the same honeycomb intermedi ate 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 curv ed lipid structure (''stalk'') would act as a structural intermediate in membrane fusion, Sphingomyelinase produces complete leakage of vesi cle 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.