Asymmetric Bilayers by Hemifusion: Method and Leaflet Behaviors

Abstract

We describe a new method to prepare asymmetric giant unilamellar vesicles (aGUVs) via hemifusion. Hemifusion of giant unilamellar vesicles and a supported lipid bilayer, triggered by calcium, promotes the lipid exchange of the fused outer leaflets mediated by lipid diffusion. We used different fluorescent dyes to monitor the inner and the outer leaflets of the unsupported aGUVs. We confirmed that almost all newly exchanged lipids in the aGUVs are found in the outer leaflet of these asymmetric vesicles. In addition, we test the stability of the aGUVs formed by hemifusion in preserving their contents during the procedure. For aGUVs prepared from the hemifusion of giant unilamellar vesicles composed of 1,2-distearoyl-sn-glycero-3phosphocholine/1,2-dioleoyl-sn-glycero-3-phosphocholine/cholesterol ¼ 0.39/0.39/0.22 and a supported lipid bilayer of 1,2-dioleoyl-sn-glycero-3-phosphocholine/cholesterol ¼ 0.8/0.2, we observed the exchanged lipids to alter the bilayer properties. To access the physical and chemical properties of the asymmetric bilayer, we monitored the dye partition coefficients of individual leaflets and the generalized polarization of the fluorescence probe 6-dodecanoyl-2-[ N-methyl-N-(carboxymethyl)amino] naphthalene, a sensor for the lipid packing/order of its surroundings. For a high percentage of lipid exchange (>70%), the dye partition indicates induced-disordered and induced-ordered domains. The induced domains have distinct lipid packing/order compared to the symmetric liquid-disordered and liquid-ordered domains.

Plasma membranes are asymmetric in lipid unsaturation, packing and protein shape

“…The atomistic simulations predict that inner leaflet lipids would have different physical properties from outer leaflet, consistent with extensive experimental observations of symmetric membranes. However, the biophysical consequences of lipid asymmetry in either model or biological membranes remain poorly understood because methodologies for constructing asymmetric model membranes have only recently become widely accessible 7 – 9 , 11 , 12 . We probed the biophysical asymmetry of live cell PMs using a fluorescent reporter of membrane packing (Di-4-ANEPPDHQ, Di4).…”

“…Finally, membrane asymmetries could be used as organellar identifiers for selective sorting of lipids and proteins between organelles. Finally, it has been shown that membrane asymmetry can affect the properties of membrane domains 9 , 11 , 43 and possibly the partitioning of proteins between ordered and disordered lipid environments 49 , 50 , making the magnitude and mechanisms of physical coupling between disparate leaflets in asymmetric membranes an essential open question 42 . Ultimately, deciphering the purpose of membrane asymmetry is central to understanding the functions of cell membranes.…”

“…While the connections between membrane composition and physical properties have been extensively addressed in symmetric bilayers, little is known about how these insights translate to compositionally asymmetric membranes. Recently, robust protocols for producing asymmetric synthetic membranes have been developed 7 – 9 and used to characterize physical coupling between leaflets 10 , 11 , effects of lipid asymmetry on protein conformation 7 , 12 , and the influence of proteins on lipid distribution between leaflets 13 . However, the extent to which membrane properties are coupled across the bilayer in living cells remains unknown.…”

Contributions and Limitations of Biophysical Approaches to Study of the Interactions between Amphiphilic Molecules and the Plant Plasma Membrane

“…Asymmetrical membrane models have been developed in the last fifteen years, notably driven by the London [55,56] and Heerklotz [57,58] groups. They can be prepared by several methods, such as by using cyclodextrins as lipid carrier molecules [59], using enzymes [57], by different microfluidic technologies [60], or by hemifusion between models with different lipid compositions [61]. The development of these asymmetrical models has been mainly focused on mammalian lipid membranes.…”

Kiss and Run Asymmetric Vesicles to Investigate Coupling

“…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.…”