The role of lateral tension in calcium-induced DPPS vesicle rupture

Abstract

We assess the role of lateral tension in rupturing anionic dipalmitoylphosphatidyserine (DPPS), neutral dipalmitoylphosphatidylcholine (DPPC), and mixed DPPS-DPPC vesicles. Binding of Ca(2+) is known to have a significant impact on the effective size of DPPS lipids and little effect on the size of DPPC lipids in bilayer structures. In the present work we utilized laser transmission spectroscopy (LTS) to assess the effect of Ca(2+)-induced stress on the stability of the DPPS and DPPC vesicles. The high sensitivity and resolution of LTS has permitted the determination of the size and shape of liposomes in solution. The results indicate a critical size after which DPPS single shell vesicles are no longer stable. Our measurements indicate Ca(2+) promotes bilayer fusion up to a maximum diameter of ca. 320 nm. These observations are consistent with a straightforward free-energy-based model of vesicle rupture involving lateral tension between lipids regulated by the binding of Ca(2+). Our results support a critical role of lateral interactions within lipid bilayers for controlling such processes as the formation of supported bilayer membranes and pore formation in vesicle fusion. Using this free energy model we are able to infer a lower bound for the area dilation modulus for DPPS (252 pN/nm) and demonstrate a substantial free energy increase associated with vesicle rupture.

Figure 1

Laser Transmission Spectroscopy results are shown for DPPS (A), a 3:1 DPPS-DPPC mixture (B), and DPPC (C). Upon the addition of 20 mM Ca²⁺ (pink curves), the observed particle size for DPPS is observed to increase dramatically, while the mixed and DPPC vesicles shows little effect. The systems were treated with EDTA (dotted line) to assess for vesicle fusion. The pure DPPS and mixed vesicle initial curves has been scaled down as indicated for clarity.

Figure 2

The serial addition of 3 μL of 20 mM Ca²⁺ is plotted (A–E). Upon the addition of 6 μL of 20 mM Ca²⁺, mass aggregation is observed. Addition of additional increments of Ca²⁺ results in rupture of the large vesicles and kinetically slower aggregation of the remaining small vesicles.

Figure 3

LTS size distributions are plotted for (A) DPPS, (B) 3:1 mixture DPPS-DPPC, and (C) DPPC vesicles that result from the addition of the indicated amounts of 20 mM Ca²⁺. In the plots for DPPS (A) and in the 3:1 mixture (B), the intensity of the initial distribution has been scaled as indicated for clarity.

Figure 4

The total surface area detected in the sample is plotted as a function of the amount of Ca²⁺ added. The dots are the experimental data points for pure DPPS (red), a 3:1 DPPS-DPPC mixed vesicle (blue), and pure DPPC (green). The solid red line is a single exponential fit to the DPPS data. Neither the mixed system nor pure DPPC fit to an exponential and a linear fit is provided as a guide to the eye.

Figure 5

The AFM image of a mica surface in a solution of DPPS vesicles is shown before (A) and after (B) the addition of Ca²⁺. In both images the height profile is shown across the line (1) in inset. In the presence of Ca²⁺, a terrace with a 5 nm step is observed, consistent with a supported bilayer membrane.

Figure 6

The calculated free energy associated with increased vesicle diameter according to equation 9 is shown for DPPS and DPPC vesicles. The dashed line represents the maximum size vesicle that was observed in the LTS measurements utilizing DPPS. The plot clearly shows the tension induced by Ca²⁺ condensation incurs a substantial energy penalty.