Maitane Ibarguren

Cholesterol displacement by ceramide in sphingomyelin‐containing liquid‐ordered domains, and generation of gel regions in giant lipidic vesicles

Fluorescence confocal microscopy and differential scanning calorimetry are used in combination to study the phase behaviour of bilayers composed of PC:PE:SM:Chol equimolecular mixtures, in the presence or absence of 10 mol% egg ceramide. In the absence of ceramide, separate liquid‐ordered and liquid‐disordered domains are observed in giant unilamellar vesicles. In the presence of ceramide, gel‐like domains appear within the liquid‐ordered regions. The melting properties of these gel‐like domains resemble those of SM:ceramide binary mixtures, suggesting Chol displacement by ceramide from SM:Chol‐rich liquid‐ordered regions. Thus three kinds of domains coexist within a single vesicle in the presence of ceramide: gel, liquid‐ordered, and liquid‐disordered. In contrast, when 10 mol% egg diacylglycerol is added instead of ceramide, homogeneous vesicles, consisting only of liquid‐disordered bilayers, are observed.

Imaging the early stages of phospholipase C/sphingomyelinase activity on vesicles containing coexisting ordered-disordered and gel-fluid domains

The binding and early stages of activity of a phospholipase C/sphingomyelinase from Pseudomonas aeruginosa on giant unilamellar vesicles (GUV) have been monitored using fluorescence confocal microscopy. Both the lipids and the enzyme were labeled with specific fluorescent markers. GUV consisted of a mixture of phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, and cholesterol in equimolar ratios, to which 5–10 mol% of the enzyme end-product ceramide and/or diacylglycerol were occasionally added. Morphological examination of the GUV in the presence of enzyme reveals that, although the enzyme diffuses rapidly throughout the observation chamber, detectable enzyme binding appears to be a slow, random process, with new bound-enzyme-containing vesicles appearing for several minutes. Enzyme binding to the vesicles appears to be a cooperative process. After the initial cluster of bound enzyme is detected, further binding and catalytic activity follow rapidly. After the activity has started, the enzyme is not released by repeated washing, suggesting a “scooting” mechanism for the hydrolytic activity. The enzyme preferentially binds the more disordered domains, and, in most cases, the catalytic activity causes the disordering of the other domains. Simultaneously, peanut- or figure-eight-shaped vesicles containing two separate lipid domains become spherical. At a further stage of lipid hydrolysis, lipid aggregates are formed and vesicles disintegrate.

Multiple phospholipid substrates of phospholipase C/sphingomyelinase HR2 from Pseudomonas aeruginosa

The activity of phospholipase C/sphingomyelinase HR(2) (PlcHR(2)) from Pseudomonas aeruginosa was characterized on a variety of substrates. The enzyme was assayed on liposomes (large unilamellar vesicles) composed of PC:SM:Ch:X (1:1:1:1; mol ratio) where X could be PE, PS, PG, or CL. Activity was measured directly as disappearance of substrate after TLC lipid separation. Previous studies had suggested that PlcHR(2) was active only on PC or SM. However we found that, of the various phospholipids tested, only PS was not a substrate for PlcHR(2). All others were degraded, in an order of preference PC>SM>CL>PE>PG. PlcHR(2) activity was sensitive to the overall lipid composition of the bilayer, including non-substrate lipids.

End-products diacylglycerol and ceramide modulate membrane fusion induced by a phospholipase C/sphingomyelinase from Pseudomonas aeruginosa

A phospholipase C/sphingomyelinase from Pseudomonas aeruginosa has been assayed on vesicles containing phosphatidylcholine, sphingomyelin, phosphatidylethanolamine and cholesterol at equimolar ratios. The enzyme activity modifies the bilayer chemical composition giving rise to diacylglycerol (DAG) and ceramide (Cer). Assays of enzyme activity, enzyme-induced aggregation and fusion have been performed. Ultrastructural evidence of vesicle fusion at various stages of the process is presented, based on cryo-EM observations. The two enzyme lipidic end-products, DAG and Cer, have opposite effects on the bilayer physical properties; the former abolishes lateral phase separation, while the latter generates a new gel phase [Sot et al., FEBS Lett. 582, 3230-3236 (2008)]. Addition of either DAG, or Cer, or both to the liposome mixture causes an increase in enzyme binding to the bilayers and a decrease in lag time of hydrolysis. These two lipids also have different effects on the enzyme activity, DAG enhancing enzyme-induced vesicle aggregation and fusion, Cer inhibiting the hydrolytic activity. These effects are explained in terms of the different physical properties of the two lipids. DAG increases bilayers fluidity and decreases lateral separation of lipids, thus increasing enzyme activity and substrate accessibility to the enzyme. Cer has the opposite effect mainly because of its tendency to sequester sphingomyelin, an enzyme substrate, into rigid domains, presumably less accessible to the enzyme.

Quantitation of cholesterol incorporation into extruded lipid bilayers

Cholesterol incorporation into lipid bilayers, in the form of multilamellar vesicles or extruded large unilamellar vesicles, has been quantitated. To this aim, the cholesterol contents of bilayers prepared from phospholipid:cholesterol mixtures 33-75 mol% cholesterol have been measured and compared with the original mixture before lipid hydration. There is a great diversity of cases, but under most conditions the actual cholesterol proportion present in the extruded bilayers is much lower than predicted. A quantitative analysis of the vesicles is thus required before any experimental study is undertaken.

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 PlcHR(2), 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 PlcHR(2) reaction. Moreover, the aggregates/droplets appear enriched in the hydrolytic enzyme PlcHR(2). 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.

Thermally-induced aggregation and fusion of protein-free lipid vesicles

Membrane fusion is an important phenomenon in cell biology and pathology. This phenomenon can be modeled using vesicles of defined size and lipid composition. Up to now fusion models typically required the use of chemical (polyethyleneglycol, cations) or enzymatic catalysts (phospholipases). We present here a model of lipid vesicle fusion induced by heat. Large unilamellar vesicles consisting of a phospholipid (dioleoylphosphatidylcholine), cholesterol and diacylglycerol in a 43:57:3 mol ratio were employed. In this simple system, fusion was the result of thermal fluctuations, above 60 °C. A similar system containing phospholipid and cholesterol but no diacylglycerol was observed to aggregate at and above 60 °C, in the absence of fusion. Vesicle fusion occurred under our experimental conditions only when (31)P NMR and cryo-transmission electron microscopy of the lipid mixtures used in vesicle preparation showed non-lamellar lipid phase formation (hexagonal and cubic). Non-lamellar structures are probably the result of lipid reassembly of the products of individual fusion events, or of fusion intermediates. A temperature-triggered mechanism of lipid reassembly might have occurred at various stages of protocellular evolution.