Imaging non-classical mechanical responses of lipid membranes using molecular rotors

Abstract

Lipid packing in cellular membranes has a direct effect on membrane tension and microviscosity, and plays a central role in cellular adaptation, homeostasis and disease. According to conventional mechanical descriptions, viscosity and tension are directly interconnected, with increased tension leading to decreased membrane microviscosity. However, the intricate molecular interactions that combine to build the structure and function of a cell membrane suggest a more complex relationship between these parameters. In this work, a viscosity-sensitive fluorophore ('molecular rotor') is used to map changes in microviscosity in model membranes under conditions of osmotic stress. Our results suggest that the relationship between membrane tension and microviscosity is strongly influenced by the bilayer's lipid composition. In particular, we show that the effects of increasing tension are minimised for membranes that exhibit liquid disordered (L_d) - liquid ordered (L_o) phase coexistence; while, surprisingly, membranes in pure gel and L_o phases exhibit a negative compressibility behaviour, i.e. they soften upon compression.

Fig. 1. Molecular structure of the thiophene dye 1 used in this work (a) and its time-resolved decay traces in toluene/castor oil mixtures of variable viscosity (b) and in lipid bilayers (DOPC and DPPC LUVs), (c). Dashed line in (c) represents DPPC gel to liquid transition temperature.

Fig. 2. The effect of DOPC : DPPC : cholesterol ternary LUV composition on lifetime variations of 1 expressed as: Δτ_w = (τ_w/τ_w,σ=0) − 1 compared at 23 °C to iso-osmotic conditions for (a) membrane tension (ΔC = 0.36 M, σ = 0.098 mN m⁻¹); (b) membrane compression (ΔC = −1.08 M, σ = −0.063 mN m⁻¹) and (c) difference between hypo- and hyper-osmotic lifetime divided by lifetime under iso-osmotic conditions. Inset shows the expected change in lipid packing for a membrane following classical mechanics. Bars show mean ± S.D (n = 3).

Fig. 3. Calculated strains from SAXS/WAXS traces for DOPC and DPPC bilayers under pressure. (a) Strain normal to the membrane plane, defined as ε_z = Δd_HH/d_HH,0, (b) Area strain of the membrane plane, defined as ε_A = ΔA/A₀. See ESI for detailed information.

Fig. 4. Effect of cholesterol content and temperature on buffering of membrane stress in ternary DOPC : DPPC : cholesterol LUVs. Clear bars represent data at T = 23 °C; dashed bars represent data from a single phase at T = 45 °C. Bars show mean ± S.D. (n = 3).

Fig. 5. Example FLIM images of DOPC GUVs under (a) hypo-osmotic (ΔC = 0.18 M) (b) iso-osmotic (ΔC = 0 M) and (c) hyper-osmotic (ΔC = −0.36 M) conditions. (d) Average GUV τ_wn ≥ 10. Scale bar: 30 μm.

Fig. 6. Example FLIM images of 40 : 40 : 20 DOPC : DPPC : cholesterol GUVs under (a) hypo-osmotic (ΔC = 0.18 M) (b) iso-osmotic (ΔC = 0 M) and (c) hyper-osmotic (ΔC = −0.36 M) conditions. L_o/L_d coexistence is evident, τ_w for L_d and L_o phases are shown in (d). n ≥ 10. Scale bar: 30 μm.

Fig. 7. Hypothetical mechanisms for tension (left) and compression (right) buffering. When tension is applied, the L_d phase (red) expands and tension is ultimately transmitted to the L_o domains (green). Enough tension would decrease lipid packing in the L_o phase, but this is unfavourable and instead the L_o phase solubilizes into the L_d matrix. In contrast, increased compression would initially increase packing of DOPC molecules in the L_d phase. This would be followed by buckling out of the L_o domains and, eventually, ejection of excess lipid to relieve membrane stress (the last step is more likely to happen in larger GUVs).