Fig 1.
Illustration for applying the electric field in GUVs.
(A) Schematic diagram of experimental set-up for the constant electric tension-induced rupture of GUVs under osmotic pressure. (B) An IRE signal of pulse width 200 μs.
Fig 2.
The influence of osmotic pressure on the constant electric tension-induced stochastic rupture of DOPG/DOPC/chol (46/39/15)-GUVs in a buffer containing physiological ions.
(A) Phase contrast images of rupture of (i) first (ii) second and (iii) third ‘single DOPG/DOPC/chol (46/39/15)-GUV’ at tension, σc = 6.5 mN/m and the osmolarity difference between the inside and outside GUVs, ΔC0 = 15 mOsm/L. The field direction is shown with an arrow in the left side. The numbers above in each image indicate the time in seconds after applying of σc due to electric field. The white bar corresponds to a length of 15 μm. The time of stochastic rupture/intact in several single DOPG/DOPC/chol (46/39/15)-GUVs under ΔC0 = 15 mOsm/L at (B) σc = 6.5 mN/m (C) σc = 6.0 mN/m and (D) σc = 5.0 mN/m. The number of measured GUVs in B, C and D was 18. The bars in B, C, D represent ruptured and intact GUVs. The cross mark (×) above the bars in C, D indicates the intact GUVs until time 60 s. (E) The σc dependent Ppore (60 s) value for DOPG/DOPC/chol (46/39/15)-GUVs under ΔC0 = 0 (green open square), ΔC0 = 15 mOsm/L (cyan open triangle) and ΔC0 = 19 mOsm/L (pink open circle). Average values and standard deviations of Ppore (60 s) at σc were determined for 2–3 independent experiments, each with 15–24 GUVs, for each value of σc.
Fig 3.
Constant electric tension-dependent rupture of DOPG/DOPC/chol (46/39/15)-GUVs under different osmotic pressures.
(A) The time course of the fraction of intact DOPG/DOPC/chol (46/39/15)-GUVs at tensions σc = 6.5, 6.0 and 5.0 mN/m. The solid lines represent the best-fitted single exponential decay function of Eq (5). (B) The σc dependent kp value for DOPG/DOPC/chol (46/39/15)-GUVs under the osmolarity difference between the inside and outside of GUVs, ΔC0 = 0 (green open square), ΔC0 = 15 mOsm/L (cyan open triangle) and ΔC0 = 19 mOsm/L (pink open circle). Average values with standard deviations of kp at σc were determined for 2–3 independent experiments, each with 15–24 GUVs, for each value of σc. The solid (green, cyan and pink) lines were the best fit theoretical curves corresponding to Eq (3) using line tension Γ = 12.9 pN, B = 2.14 mN/m and AF = 8.4×105 m2s−1 J−1. The cyan line and the pink line correspond to the theoretical Eq (3) using σt = σc + 2.3 mN/m and σt = σc + 4.0 mN/m, respectively. (C) The ΔC0 dependent membrane tension (σoseq) (experimental and theoretical). Experimental values were determined by analyzing the constant electric tension-induced rupture of GUVs. Their mean values with standard deviations are shown.
Table 1.
An estimation of membrane tension of DOPG/DOPC/chol (46/39/15)-GUVs for ΔC0 = 15 mOsm/L and Cout = 373 mOsm/L.
Table 2.
An estimation of membrane tension of DOPG/DOPC/chol (46/39/15)-GUVs for ΔC0 = 19 mOsm/L and Cout = 369 mOsm/L.
Fig 4.
The time of stochastic rupture of several ‘single DOPG/DOPC (40/60)-GUVs’.
(A) The osmolarity difference between the inside and outside of GUVs, ΔC0 = 13 mOsm/L (B) ΔC0 = 17 mOsm/L at σc = 3.0 mN/m. The number of measured GUVs in A and B was 19. The bars in A, B represent ruptured and intact GUVs. The cross mark (×) above the bars in A indicates the intact GUVs until time 60 s. (C) The σc dependent Ppore (60 s) value for DOPG/DOPC (40/60)-GUVs under ΔC0 = 0 (black open square), ΔC0 = 13 mOsm/L (blue open triangle) and ΔC0 = 17 mOsm/L (red open circle). (D) The σc dependent kp value for DOPG/DOPC (40/60)-GUVs under ΔC0 = 0 (black open square), ΔC0 = 13 mOsm/L (blue open triangle) and ΔC0 = 17 mOsm/L (red open circle). Average values with standard deviations of kp at σc were determined for 2–3 independent experiments, each with 15–24 GUVs, for each value of σc. The solid (black, blue and red) lines were the best-fitted theoretical curves corresponding to Eq (3) using line tension Γ = 12.1 pN, B = 1.76 mN/m and AF = 8.8×106 m2s−1 J−1. The blue line and the red line correspond to the theoretical Eq (3) using σt = σc + 2.1 mN/m and σt = σc + 3.5 mN/m, respectively. (E) The ΔC0 dependent membrane tensions (σoseq) (experimental and theoretical) at swelling equilibrium. Experimental values are determined by analyzing the constant electric tension-induced rupture of GUVs. Their mean values with standard deviations are shown. (F) Comparison of experimentally determined membrane tension for DOPG/DOPC (40/60) and DOPG/DOPC/chol (46/39/15)-GUVs under different ΔC0.
Table 3.
An estimation of membrane tension of DOPG/DOPC (40/60)-GUVs for ΔC0 = 13 mOsm/L and Cout = 375 mOsm/L.
Table 4.
An estimation of membrane tension of DOPG/DOPC (40/60)-GUVs for ΔC0 = 17 mOsm/L and Cout = 371 mOsm/L.
Fig 5.
Comparison of the experimentally estimated membrane tension for DOPG/DOPC (40/60)-GUVs using the IRE technique and the micropipette technique.
The data on the membrane tension using the micropipette technique was taken from [11].
Fig 6.
Phase contrast microscopic image of DOPG/DOPC/chol (46/39/15)-GUVs under the influence of the osmolarity difference between the inside and outside of GUVs, ΔC0 = 29 mOsm/L.
Large osmotic pressure-induced leakage of sucrose due to the pore formation in the membranes of GUVs. The GUVs labeled by 1, 2 and 3 leaked out sucrose from their inside. The scale bar corresponds to a length of 50 μm.
Fig 7.
The prepore radius dependent free-energy profile of a prepore of DOPG/DOPC/chol (46/39/15)-GUVs under tension.
(A) 9.0 mN/m (B) 8.0 mN/m (C) 6.0 mN/m and (D) 5.0 mN/m. U(r, σt) was calculated according to Eq (6) using Γ = 12.9 pN.