Measurement of the membrane curvature preference of phospholipids reveals only weak coupling between lipid shape and leaflet curvature

Abstract

In biological processes, such as fission, fusion and trafficking, it has been shown that lipids of different shapes are sorted into regions with different membrane curvatures. This lipid sorting has been hypothesized to be due to the coupling between the membrane curvature and the lipid's spontaneous curvature, which is related to the lipid's molecular shape. On the other hand, theoretical predictions and simulations suggest that the curvature preference of lipids, due to shape alone, is weaker than that observed in biological processes. To distinguish between these different views, we have directly measured the curvature preferences of several lipids by using a fluorescence-based method. We prepared small unilamellar vesicles of different sizes with a mixture of egg-PC and a small mole fraction of N-nitrobenzoxadiazole (NBD)-labeled phospholipids or lysophospholipids of different chain lengths and saturation, and measured the NBD equilibrium distribution across the bilayer. We observed that the transverse lipid distributions depended linearly on membrane curvature, allowing us to measure the curvature coupling coefficient. Our measurements are in quantitative agreement with predictions based on earlier measurements of the spontaneous curvatures of the corresponding nonfluorescent lipids using X-ray diffraction. We show that, though some lipids have high spontaneous curvatures, they nevertheless showed weak curvature preferences because of the low values of the lipid molecular areas. The weak curvature preference implies that the asymmetric lipid distributions found in biological membranes are not likely to be driven by the spontaneous curvature of the lipids, nor are lipids discriminating sensors of membrane curvature.

Fig. 1.

The schematic diagram of the POPC, NBD-lyso-PPE, NBD-DPPE, NBD-lyso-OPE and NBD-DOPE lipids used in this study. POPC is the major component of egg-PC. The structures of NBD-lyso-MPE, NBD-DMPE, NBD-lyso-OPS and NBD-DOPS lipids are shown in Fig. S1 (see SI Appendix).

Fig. 2.

Sizes of the SUVs. (A) The size distribution of SUVs extruded through polycarbonate membrane filters with the indicated pore sizes measured by dynamic light scattering (DLS). Mean diameters and standard deviations measured by fitting with the log-normal distribution function are shown in Table 1. (B) Cryo-TEM images of egg-PC + NBD-lyso-PPE SUVs of different size distributions. The images were taken about 24 hours after preparation. SUVs are of mean diameter (i) 31 ± 13 nm, (ii) 50 ± 18 nm, (iii) 70 ± 21 nm and (iv) 83 ± 25 nm (Table 1). Unilamellar, bilamellar, and trilamellar vesicles are marked with single, double, and triple arrowheads, respectively.

Fig. 3.

Fluorescent-quenching method for estimating the fractional outer leaflet NBD-labeled lipid distribution in the SUVs. (A) SUVs of 83 nm mean diameter were prepared from egg-PC with a small mole fraction (0.01%) of NBD-DPPE. Addition of 15 mM SDT caused a fast decrease in total fluorescence due to NBD quenching. The following slow decrease is mainly due to outward translocation of inner leaflet NBD-DPPE lipids. Addition of 20 mM detergent, Triton-X100, dissolves the membrane, exposing the rest of the NBD-DPPE lipids in the inner leaflet to SDT, causing the total fluorescence to drop to zero. Data were normalized after background subtraction. (B) Fluorescence quenching of the SUV outer leaflet NBD-lyso-PPE (Left) and NBD-DPPE (Right) lipids by SDT. The fast drop in fluorescence, I _o, represents the fractional amount of NBD-lyso-PPE or NBD-DPPE present in the outer leaflet of the SUVs. In case of NBD-lyso-PPE, I _o increases with decreasing SUV diameter. The opposite effect is observed for NBD-DPPE lipids.

Fig. 4.

The transverse distribution, n _o, of the NBD-labeled lipids as a function of SUV bilayer curvature, H. The dotted lines correspond to symmetric distribution, n _s (Eq. 4). (A) The NBD-lyso-PPE (▾) lipids show preference for the positive curvature outer leaflet, whereas the NBD-DPPE (▪) lipids show preference for the inner leaflet. (B) The unsaturated NBD-lyso-OPE (▾) lipids do not show any significant preference for any of the leaflets as it overlaps with n _s. The unsaturated NBD-DOPE (▪) lipids prefer the negative curvature inner leaflet. By fitting the data with Eq. 5 (the red and blue lines for the single- and double-chain lipids, respectively) we estimated the curvature coupling coefficient, Γ, and spontaneous curvature, c ₀, of the lipids (Table 2).