Main Text
A key feature of biological membranes that has recently been attracting the attention it deserves is the lipid asymmetry between the two leaflets of the bilayer. The mere fact that nature has developed sophisticated procedures and spends considerable energy to establish and maintain substantially different lipid compositions between the two leaflets of many membranes implies that asymmetry has important biological functions. However, because much of what we know about membrane structure and function has been derived from studies using classical symmetrical lipid membrane models, our knowledge about the structural and functional consequences of lipid asymmetry is still quite modest.
This is about to change. Although protocols to prepare asymmetric planar bilayers have been long available and have had useful applications in investigations of asymmetry and domain formation (1, 2, 3, 4), in the past few years, more and more protocols have been introduced to prepare a wide variety of asymmetric liposomes, which allows a much wider range of studies. Asymmetric liposomes can be formed leaflet by leaflet on water droplets crossing water-oil and oil-water interfaces (5). In another approach, the outer leaflet of large unilamellar (acceptor) lipid vesicles can be partially or virtually fully exchanged with that of an excess population of donor lipid vesicles, catalyzing lipid exchange by cyclodextrins or exchange proteins (6, 7, 8, 9). This method requires the removal of the donor vesicles after exchange, typically by centrifugation, but partial exchange can be achieved by adding the lipid in cyclodextrin-solubilized form, either avoiding (10) or removing (11) excess donor vesicles before the exchange. Local enzymatic conversion of one lipid into another in the outer leaflet has also been achieved (12, 13). Finally, molecular dynamics simulations have been set up for asymmetric membranes (14).
Despite these advances, there is no universal method for preparing asymmetric vesicles. Each method has limitations and advantages. In this issue of Biophysical Journal, Enoki and Feigenson (15) have now added a big advance to the toolbox for preparing asymmetric giant unilamellar vesicles (GUVs). An advantage of GUVs is that single vesicles can be visualized and characterized, which includes the ability to pick individual vesicles with the proper level of exchange and asymmetry from a more or less broad distribution. Furthermore, membrane domains coexisting within one GUV can be seen and characterized separately. Although methods to prepare asymmetric GUVs exist, they have limitations. For oil-water approaches, contaminating oil has been a concern. In lipid-exchange protocols for GUVs, avoiding contaminating donor lipid by removing excess donor lipid before exchange, as noted above, limits experiments to GUVs in which the outer leaflet has undergone only partial exchange (11).
Enoki and Feigenson (15) have solved the issue of donor removal and exchange efficiency by using (a large excess of) donor lipid on a solid support and catalyzing exchange by reversible, calcium-ion-induced hemifusion of the acceptor GUVs with the solid support. To assess asymmetry, Enoki and Feigenson (15) use fluorescent membrane probes of different colors, usually DiI and TFPC, mixed with the outside and the inside lipids, respectively. To show that intensities are changing by the amount predicted for a successful exchange, outside versus inside dye levels were measured after quenching outside dye fluorescence with dithionite. The work provides compelling evidence that exchange takes place for most vesicles in the preparation and to a large degree—up to 98% for some vesicles—but leaves the inner lipid leaflet and the aqueous core of the GUV largely untouched. A few imperfect GUVs produced by this technique are much less of a problem than for bulk methods—data collection can be done on hand-picked, perfectly asymmetric, and stable GUVs.
In addition to efficient exchange, the method 1) allows preparation of vesicles with outer leaflets totally composed of unsaturated lipids that mimic the inner (cytosolic) leaflet lipids, 2) avoids potential issues arising from the use of cyclodextrins, and 3) although only implied, permits preparing asymmetric vesicles with different starting amounts of cholesterol in each leaflet. Cholesterol is known to flip-flop quickly between the leaflets (16), but in a bilayer of otherwise slowly flipping lipids, it cannot freely partition according to its preferences for outside versus inside lipids (17) because the areas of the two leaflets need to match or near-match in terms of total lipid content. When (or if!) cell biologists finally agree on the level of cholesterol asymmetry in cells, cholesterol asymmetry will become an important topic for studies in artificial vesicles.
The key question of whether and how lipid organization in one leaflet “signals” to the other has been addressed by a few prior studies, giving rise to different concepts and views of interleaflet coupling (11, 14, 18, 19, 20, 21, 22, 23). Eukaryotic plasma membranes contain mixtures of high- and low-melting lipids and cholesterol in their outer leaflet, which show a propensity to demix into separate Lo and Ld domains or clusters. But how does this influence and how is it influenced by the inner leaflet lipids, which do not have this propensity?
Enoki and Feigenson demonstrated the power of their model to study coupling between the inner and outer leaflets (15). They could visualize the coupling of Lo and Ld domains in their asymmetric GUVs with a DSPC-DOPC-cholesterol inner leaflet and a DOPC-cholesterol outer leaflet. Similar to what has been reported for asymmetric GUVs with SM in place of DSPC (23), the authors show that the contact with the Lo phase in the DSPC-containing inner leaflet induced ordered domains in the DSPC-lacking outer leaflet (i.e., containing only DOPC and cholesterol). Furthermore, using dye partitioning and generalized polarization to assess lipid packing, they found that the ordered domains in both the order-inducing, DSPC-containing inner leaflet and the responding, DSPC-free outer leaflet were less ordered than the in-register Lo domains in symmetrical DOPC-DSPC-cholesterol vesicles. This greatly strengthens the idea that coupling is a two-way street in which both leaflets influence the formation and final properties of ordered domains. Finally, it was found that Ld domain order also decreased in asymmetric vesicles. This may be due to local depletion of cholesterol that is transferred to the Lo domains induced in the DSPC-free leaflet (23).
As the authors discuss, the effects that govern interleaflet coupling are still not fully understood, and the whole research topic needs more systematic concepts and terminology. The new model established here will undoubtedly help a great deal to accomplish this goal. To summarize, the valuable protocol developed by Enoki and Feigenson (15) will open many new avenues to tackle membrane asymmetry issues of key importance. The asymmetry wave is taking up speed.
Editor: Claudia Steinem.
Contributor Information
Heiko Heerklotz, Email: heiko.heerklotz@pharmazie.uni-freiburg.de.
Erwin London, Email: erwin.london@stonybrook.edu.
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