Lipid Structure and Composition Control Consequences of Interleaflet Coupling in Asymmetric Vesicles

Abstract

Using Förster resonance energy transfer, raft/liquid-ordered-domain formation was assessed in asymmetric vesicles containing outer leaflets composed of high-Tm (melting temperature) saturated phosphatidylcholines (diCPC, diCPC, diCPC, or diCPC), low-Tm unsaturated dioleoylphosphatidylcholine (DOPC) and cholesterol, and inner leaflets composed of lipids that by themselves would not form ordered domains (DOPC and cholesterol). Ordered-domain formation in the outer leaflet was compared to that in symmetric vesicles with the same lipid composition as the asymmetric vesicle outer leaflets. The difference between ordered-domain thermal stability in asymmetric and symmetric vesicles was highly dependent on high-Tm PC acyl-chain length. At one extreme, in diCPC-containing asymmetric vesicles, the outer leaflet did not segregate to form ordered domains over the entire experimental temperature range even though ordered domains formed in the symmetric vesicles, indicating the inner leaflet dominated outer-leaflet physical behavior in the asymmetric vesicles. At the other extreme, in diCPC-containing asymmetric vesicles, ordered domains formed over the entire temperature range at which they were present in symmetric vesicles, indicating the inner leaflet did not dominate outer-leaflet physical behavior. DiCPC- and diCPC-containing vesicles exhibited intermediate behaviors. A different set of vesicles was prepared with high-Tm lipid sphingomyelin (SM) in place of saturated phosphatidylcholine, and the % SM was varied. The thermal stability of outer-leaflet ordered domains in asymmetric vesicles was found to decrease more than in symmetric vesicles as SM levels decreased, indicating that the inner leaflet increasingly dominated outer leaflet physical state as SM levels decreased. Overall, inhibition of outer-leaflet ordered-domain formation in asymmetric vesicles by inner-leaflet lipids decreased as the ability of outer-leaflet lipids to form an ordered state by themselves increased, i.e., when outer-leaflet high-Tm lipid content or acyl-chain length increased. This has implications for how ordered-domain formation may be controlled in vivo.

Asymmetric Bilayers by Hemifusion: Method and Leaflet Behaviors

“…In a sense, different researchers describe leaflet coupling for a particular system without an agreed definition of what ''coupling'' means. Beyond phase-separated domains in the two leaflets being in register, coupling can mean that one phase influences the melting temperature (4,41) or the diffusion coefficient of the apposed leaflet (42), the order parameter in the apposed leaflet is changed (41,42), or that the extent of interdigitation has changed (42). Nonetheless, theoretical/phenomenological models have been proposed to estimate coupling strengths (23)(24)(25).…”

The Beauty of Asymmetric Membranes: Reconstitution of the Outer Membrane of Gram-Negative Bacteria

“…WANG et al observed in asymmetric vesicles that the inhibition of outer-leaflet ordered-domain formation by inner-leaflet lipids decreased. He explains this by an increased ability of outer-leaflet lipids to form an ordered state by themselves (Wang and London, 2018). From coarse-grained molecular dynamics simulations Fowler et al (2016) proposed a two-step model in which membranes undergo a shift from an anti-registered phase to registered symmetric phase after bilayer equilibration.…”

Phospholipid exchange shows insulin receptor activity is supported by both the propensity to form wide bilayers and ordered domains (rafts)

“…If there are no domains, one would predict they should have had similar levels of activation. Second, studies in plasma membrane vesicles formed after lipid exchange and in artificial asymmetric lipid vesicles show that lipid compositions increasing the propensity to form Lo domains do result in increased formation of ordered lipid domains under physiologic or near physiologic conditions [42], [48]. Third, the domain model is favored by the close relationship between decreased sensitivity of IR activity to phosphatases in lipid substitutions that favor Lo domain formation.…”

“…This correlation can be seen clearly in Figure 4 in which IR autophosphorylation activity is graphed against the gel-to-Ld phase transition temperatures (Tm) [Supplementary table 2] of the lipids in vesicles containing a single lipid species. The gel state is highly ordered, and in artifical asymmetric membranes high Tm is associated with a greater tendency to form Lo domains when cholesterol is present [42].…”