Asymmetric phase transitions in lipid bilayers: coupling or bending?

Biomembranes show asymmetric lipid composition of their two leaflets. The phenomenon that ordered domains in one leaflet may affect the order of the other has been referred to as interleaflet coupling and discussed in terms of transmembrane signaling. Many coupling mechanisms have been proposed; one potential mechanism should arise from the fact that the isolated melting of an ordered, e.g., gel phase gives rise to a significant expansion of this leaflet, resulting in a mismatch between the intrinsic areas of the leaflets. This asymmetry stress can be accommodated in a number of ways. One is interleaflet coupling - individually higher- and lower-melting leaflets melt together at intermediate melting temperature. Alternatively, the membrane may bend towards the larger-intrinsic-area leaflet, bud and release very small daughter vesicles (DVs). Here, we prepared lipid-asymmetric large unilamellar vesicles (aLUVs) with low-melting stearyl-oleyl-phosphatidylcholine (SOPC) in the inner and SOPC with ∼20 mol% of high-melting dipalmitoyl phosphatidylglycerol (DPPG) in the outer leaflet. Phase transitions in aLUVs versus LUVs were recorded with pressure perturbation calorimetry; vesicle budding was monitored by asymmetric flow field-flow fractionation (AF4) and visualized by cryo-transmission electron microscopy. An HPLC protocol was established to quantify the total DPPG content; zeta potential was used to detect outer-leaflet DPPG. It turned out to be possible to prepare aLUVs at 5 and 15 °C, with the outer leaflet partially in the gel phase. The properties of the final aLUVs depended on the preparation temperature. aLUVs prepared at 5 and 15 °C caused the budding of exovesicles upon heating and only weak coupling of the phase transitions of the leaflets. aLUVs prepared at 30 °C with both leaflets in the fluid state showed stronger coupling upon asymmetric freezing/melting at lower temperature. This is in line with the hypotheses that (i) the exchange of lipid between close-to lipid-saturated cyclodextrin and acceptor vesicles at a given temperature results in largely stress-free bilayers and (ii) that outside budding and coupling are, to some extent, alternative responses of the bilayer to asymmetric expansion. These hypotheses help explaining our and some literature data; the overall understanding and prediction of coupling for any given aLUV system remains an urgent, open question.