Recruitment of a phospholipase C/sphingomyelinase into non-lamellar lipid droplets during hydrolysis of lipid bilayers

When giant unilamellar vesicles (GUVs) composed of sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, and cholesterol are treated with PlcHR2, a phospholipase C/sphingomyelinase from Pseudomonas aeruginosa, the initial stages of lipid hydrolysis do not cause large changes in vesicle morphology ( Ibarguren et al., 2011). However, when hydrolysis progresses confocal fluorescence microscopy reveals the formation of lipid aggregates, whose morphology is not compatible with that of bilayers. Smaller vesicles or droplets can also be seen inside the GUV. Our studies indicate that these aggregates or droplets are enriched in the non-lamellar lipid ceramide, an end-product of PlcHR2 reaction. Moreover, the aggregates/droplets appear enriched in the hydrolytic enzyme PlcHR2. At a final stage GUVs containing the enzyme-enriched droplets disintegrate and vanish from the microscope field. The observed non-lamellar enzyme-rich structures may be related to intermediates in the process of aggregation and fusion although the experimental design prevents vesicle free diffusion in the aqueous medium, thus actual aggregation or fusion cannot be observed.

► Lipid bilayers in the form of GUV have been treated with a phospholipase C/sphingomyelinase.
► Enzyme action leads to formation of nonlamellar domains in the bilayers.
► These domains are enriched in ceramide and diacylglycerol.
► The nonlamellar domains are also enriched in the enzyme.