Experimental Estimation of Membrane Tension Induced by Osmotic Pressure

Abstract

Osmotic pressure (Π) induces the stretching of plasma membranes of cells or lipid membranes of vesicles, which plays various roles in physiological functions. However, there have been no experimental estimations of the membrane tension of vesicles upon exposure to Π. In this report, we estimated experimentally the lateral tension of the membranes of giant unilamellar vesicles (GUVs) when they were transferred into a hypotonic solution. First, we investigated the effect of Π on the rate constant, k_p, of constant-tension (σ_ex)-induced rupture of dioleoylphosphatidylcholine (DOPC)-GUVs using the method developed by us recently. We obtained the σ_ex dependence of k_p in GUVs under Π and by comparing this result with that in the absence of Π, we estimated the tension of the membrane due to Π at the swelling equilibrium, σ_osm^eq. Next, we measured the volume change of DOPC-GUVs under small Π. The experimentally obtained values of σ_osm^eq and the volume change agreed with their theoretical values within the limits of the experimental errors. Finally, we investigated the characteristics of the Π-induced pore formation in GUVs. The σ_osm^eq corresponding to the threshold Π at which pore formation is induced is similar to the threshold tension of the σ_ex-induced rupture. The time course of the radius change of GUVs in the Π-induced pore formation depends on the total membrane tension, σ_t; for small σ_t, the radius increased with time to an equilibrium one, which remained constant for a long time until pore formation, but for large σ_t, the radius increased with time and pore formation occurred before the swelling equilibrium was reached. Based on these results, we discussed the σ_osm^eq and the Π-induced pore formation in lipid membranes.

Figure 1

Effect of Π on constant-tension-induced rupture of DOPC-GUVs. (A) A scheme of the measurement for ΔC⁰ = C_in⁰ − C_out = 2.8 mM, where C_in⁰ is the initial sucrose concentration inside a GUV and C_out is the glucose concentration on the outside of the GUV. (B) Time course of the fraction of intact DOPC-GUVs without rupture among all of the examined GUVs, P_intact(t), in the presence of Π due to ΔC⁰ = 2.8 mM and tension due to the micropipette aspiration: σ_ex = 3.3 mN/m (open triangles), 3.8 mN/m (open squares), and 4.3 mN/m (open circles). The number of single GUVs examined was 18−22 in each experiment. The solid lines represent the best-fit curves of Eq. 8. (C) Dependence of k_p on external tension for ΔC⁰ = 1.9 mM (solid triangle), ΔC⁰ = 2.8 mM (solid rhombus), and ΔC⁰ = 0 mM (open squares) (i.e., no osmotic pressure). Error bars show standard errors. The solid lines show the best fits in accordance with the theoretical curves corresponding to Eq. 9 using Γ = 10.5 pN and D_r = 165 nm²/s. The data for ΔC⁰ = 0.0 mM (open squares) and its fitting curve are reprinted from (27) with permission from the American Chemical Society. The dashed line and the dotted line correspond to the theoretical Eq. 9 using σ_t = σ_ex + 2.6 mN/m and σ_t = σ_ex + 3.8 mN/m, respectively.

Figure 2

Increase in volume of DOPC-GUV induced by Π. (A1) A DIC image of a GUV held at the tip of a micropipette using a small aspiration pressure in chamber A, which contained an isotonic solution. (A2) A DIC image of a GUV held at the tip of a micropipette after the GUV was transferred into another chamber, chamber B, containing a hypotonic solution. d_p is the internal diameter of the micropipette and ΔL is the change of the projection length. The bar corresponds to 10 μm. (B) Time course of volume change of a GUV after it was transferred into chamber B, which contained 96.0 mM (i.e., ΔC⁰ = 2.0 mM; solid circles), 95.0 mM (i.e., ΔC⁰ = 3.0 mM; open squares), and 98.0 mM (i.e., ΔC⁰ = 0.0 mM, open circles) glucose solutions.

Figure 3

Large Π-induced pore formation in DOPC-GUVs. (A) Phase-contrast microscopic images of a GUV after it was transferred into chamber B, which contained 90.0 mM glucose solution (i.e., ΔC⁰ = 8.0 mM). The numbers below each image show the time in seconds after the transfer of the GUV into chamber B. Scale bar, 25 μm. The arrows show a rapid, transient leakage of sucrose from the DOPC-GUV. (B) The fraction of GUV where a transient leakage occurred during the first 6 min, P_Leak (360 s), as a function of ΔC⁰. Mean values and standard errors of P_Leak (360 s) for each ΔC⁰ were determined among three independent experiments using 10−15 GUVs for each experiment. Error bars show standard errors. (C) P_Leak (360 s) as a function of membrane tension due to Π at equilibrium, ${\sigma }_{\text{osm}}^{\text{eq}}={\sigma }_{\text{t}}$ (open squares). ΔC⁰ values in (B) were converted into ${\sigma }_{\text{osm}}^{\text{eq}}$. For comparison, the data in Fig. 1 were added (solid circles); the values of the fraction of ruptured GUVs during the first 6 min among all the examined GUVs, P_pore (360 s), were obtained for the theoretical values of ${\sigma }_{\text{t}}={\sigma }_{\text{ex}}+{\sigma }_{\text{osm}}^{\text{eq}}$ for each condition of the experiments of Fig. 1.

Figure 4

The time courses of the fractional change of radius of a GUV, Δr/r₀, under Π and σ_ex. The curves represent the Δr/r₀ results for several GUVs for (A) ΔC⁰ = 3.0 mM and σ_ex = 4.0 mN/m and (B) ΔC⁰ = 4.0 mM and σ_ex = 5.0 mN/m.

Figure 5

Dependence of the free-energy profile of a prepore in a DOPC-GUV on its radius for various tensions due to Π, ${\sigma }_{\text{osm}}^{\text{eq}}$: (A) 9.0, (B) 7.0, (C) 5.0, and (D) 3.0 mN/m. U(r) is calculated according to Eq. 10 using Γ = 10.5 pN.