Transverse lipid organization dictates bending fluctuations in model plasma membranes

Abstract

Membrane undulations play a vital role in many biological processes, including the regulation of membrane protein activity. The asymmetric lipid composition of most biological membranes complicates theoretical description of these bending fluctuations, yet experimental data that would inform any such a theory is scarce. Here, we used neutron spin-echo (NSE) spectroscopy to measure the bending fluctuations of large unilamellar vesicles (LUV) having an asymmetric transbilayer distribution of high- and low-melting lipids. The asymmetric vesicles were prepared using cyclodextrin-mediated lipid exchange, and were composed of an outer leaflet enriched in egg sphingomyelin (ESM) and an inner leaflet enriched in 1-palmitoyl-2-oleoyl-phosphoethanolamine (POPE), which have main transition temperatures of 37 °C and 25 °C, respectively. The overall membrane bending rigidity was measured at three temperatures: 15 °C, where both lipids are in a gel state; 45 °C, where both lipids are in a fluid state; and 30 °C, where there is gel-fluid co-existence. Remarkably, the dynamics for the fluid asymmetric LUVs (aLUVs) at 30 °C and 45 °C do not follow trends predicted by their symmetric counterparts. At 30 °C, compositional asymmetry suppressed the bending fluctuations, with the asymmetric bilayer exhibiting a larger bending modulus than that of symmetric bilayers corresponding to either the outer or inner leaflet. We conclude that the compositional asymmetry and leaflet coupling influence the internal dissipation within the bilayer and result in membrane properties that cannot be directly predicted from corresponding symmetric bilayers.

Fig. 1

(A) ¹H-NMR of aLUV in the presence of the shift reagent Pr³⁺. The red peak represents the protected choline groups on the inner leaflet, the green peak is the outer leaflet choline groups, and the grey peaks arise from other groups on the lipid and residual CD peaks (3.6 and 3.8 ppm). (B) ³¹P-NMR of the aLUV to determine the ratio of ESM (orange) and POPE (blue) present. (C) Cartoon representation of the aLUV generated. The blue presents POPE lipids, and orange represents ESM, highlighting their distribution. (D) SAXS (main) and SANS (inset) data of the aLUV at 30 °C are represented by circles, and the jointly optimized model fits are represented by solid lines. (E) The resultant electron density (ED) profile calculated from the optimized models from D. The black curve is the total ED which is the sum of the terminal methyl groups of the hydrocarbon chains (CH₃), the bulk of the hydrocarbon (CH + CH₂), the headgroup (HG) and the water.

Fig. 2

Top: Accessible length and time scales and corresponding energy and momentum transfer $\left(Q\right)$, for some spectroscopic techniques covering nanoscopic to macroscopic dynamics, covering a number of membrane dynamics. Figure adapted from ref. . Bottom: Normalized intermediate scattering function $I(Q,t)/I(Q,0)$ measured by NSE for the aLUV in D₂O at 30 °C. The inset shows the linear dependence of the relaxation rate $\left({\Gamma }_{\text{ZG}}\right)$ with respect to $Q^3$. Error bars represent one standard deviation here and throughout the manuscript.

Fig. 3

Decay rate ${\Gamma }_{\text{ZG}}$ normalized by $Q^3$ averaged for all $Q^3$ for both aLUVs and LUVs as a function of (A) ESM mole fraction $\left({\chi }_{\text{ESM}}\right)$ at 15 °C (■), 30 °C (●) and 45 °C (▲). aLUV samples are indicated by half-filled points. (B) Bending moduli ($\kappa$) for the fluid aLUVs and symmetric vesicles with compositions corresponding to the inner leaflet, outer leaflet and overall aLUV composition measured with NSE (complete temperature series found in Fig. S4. C) Cartoon visualization of the observed bending rigidity for an asymmetric organization of ESM and POPE (top) and a symmetric mixture of ESM and POPE (bottom) at 30 °C.