Membrane elasticity modulated by cholesterol in model of porcine eye lens-lipid membrane

Abstract

Experimental evidence shows that the eye lens loses its elasticity dramatically with age. It has also been reported that the cholesterol (Chol) content in the eye lens fiber cell plasma membrane increases significantly with age. High Chol content leads to the formation of cholesterol bilayer domains (CBDs) in the lens membrane. The role of high Chol associated with lens elasticity is unclear. The purpose of this research is to investigate the membrane elasticity of the model of porcine lens-lipid (MPLL) membrane with increasing Chol content to elucidate the role of high Chol in lens membrane elasticity. In this study, we used atomic force microscopy (AFM) to study the mechanical properties (breakthrough force and area compressibility modulus (K_A)) of the MPLL membrane with increasing Chol content where K_A is the measure of membrane elasticity. We varied Chol concentration in Chol/MPLL membrane from 0 to ∼71 mol%. Supported Chol/MPLL membranes were prepared by fusion of small unilamellar vesicles (SUVs) on top of a flat mica surface. SUVs of the Chol/MPLL lipid mixture were prepared with the rapid solvent exchange method followed by probe-tip sonication. For the Chol/MPLL mixing ratio of 0, AFM image showed the formation of two distinct phases of the membrane, i.e., liquid-disordered phase (l_d) and solid-ordered phase (s_o) membrane. However, with Chol/MPLL mixing ratio of 0.5 and above, only liquid-ordered phase (l_o) membrane was formed. Also, two distinct breakthrough forces corresponding to l_d and s_o were observed for Chol/MPLL mixing ratio of 0, whereas only one breakthrough force was observed for membranes with Chol/MPLL mixing ratio of 0.5 and above. No significant difference in the membrane surface roughness was measured with increasing Chol content for these membranes; however, breakthrough force and K_A for l_o membrane increased when Chol/MPLL mixing ratio was increased from 0.5 to 1. Interestingly above the Chol/MPLL mixing ratio of 1, both breakthrough force and K_A decreased, indicating the formation of CBDs. Furthermore, these results showed that membrane elasticity increases at high Chol content, suggesting that high Chol content in lens membrane might be responsible for maintaining lens membrane elasticity.

Keywords: Atomic force microscopy; Cholesterol; Lens elasticity; Lens membrane elasticity; Mechanical properties; Model lens-lipid membrane; Supported lipid membrane.

Fig. 1:

Representative topographical images of the membranes with different Chol/MPLL mixing ratios. The membrane consists of Chol/MPLL mixing ratio 0 (A), 0.5 (B), 1.0 (C), 1.5 (D), 2.0 (E) and 2.5 (F). The height profile of the membranes across purple lines in the images is shown below the images. Solid ordered phase (s_o) and liquid disordered phases (l_d) are obtained in the membrane without Chol, i.e., Chol/MPLL mixing ratio of 0 (A), but only a single liquid order phase (l_o) is obtained for all other membranes containing Chol. Single-phase in the membranes is due to the presence of Chol. The height difference between the l_d and s_o in (A) is almost 1 nm. Image scale = 1 μm

Fig. 2:

Collection of the force curves in Chol/MPLL membranes obtained from one representative set of experiments. The y-axis represents the force, whereas the x-axis represents the separation distance of the AFM tip from the mica substrate. The Chol/MPLL membrane with a mixing ratio of 0 consists of two phases, and hence force curves in those phases also display different rupture events. The green force curves are for the s_o phase (gel phase), while the red curves represent the l_d phase (fluid phase). The Chol/MPLL mixing ratio of 0.5 and above consists of only l_o phase (fluid phase) membrane. As the Chol content increases from Chol/MPLL mixing ratio 0.5 to 2.5, the BT force initially increases for l_o membrane, i.e., when Chol/MPLL mixing ratio increases from 0.5 to 1.0, whereas BT force for the l_o membrane starts to decrease above Chol/MPLL mixing ratio of 1.0.

Fig. 3:

Representative force curves obtained for the MPLL membranes (A) without Chol, (B) MPLL containing different Chol content, and (C) average breakthrough (BT) forces for different Chol/MPLL mixing ratios. In (A), the force curves for l_d and s_o phase are represented by red and green, respectively. (B) represents the force curves for Chol/MPLL membrane with mixing ratios of 0.5, 1.0, 1.5, 2.0, and 2.5 in brown, golden, light green, blue, and purple, respectively. (C) Shows the average BT forces for the Chol/MPLL membranes where green open circle and red open triangle represents BT force for s_o and l_d membranes, respectively. The filled purple squares are the average breakthrough force for l_o membranes at different Chol content in the MPLL membrane. The average BT force for l_o membranes increases initially (from mixing ratio 0.5 to 1.0), and as Chol/MPLL mixing ratio increases further, the BT force decreases, indicating the presence of CBDs.

Fig. 4:

Representative probability distribution of the BT forces for the force curves obtained from MPLL membranes with different Chol content. The green bars represent the BT forces for the fluid phase membrane across the different Chol/MPLL mixing ratios, while the red bar represents the breakthrough force for the gel phase MPLL membrane. The solid black lines are the Gaussian fit for the distribution.

Fig. 5:

Representative fit of equation 1 with the force curve and average membrane’s area compressibility modulus (K_A) of the Chol/MPLL membranes with different mixing ratios. The representative force curve in (A) is taken from Chol/MPLL mixing ratio of 2.0. In (B), the open green circle is the average K_A for s_o phase membrane, while the open red triangle is for l_d phase of Chol/MPLL mixing ratio of 0. The purple filled squares are the average K_A values for the membranes containing different amounts of Chol, which are l_o phase membranes. At Chol/MPLL mixing ratio of 0, K_A of gel phase is greater than the fluid phase membrane. For Chol/MPLL mixing ratio of 0.5 to 2.5, we clearly observe the increase in K_A with the addition of Chol (from mixing ratio 0.5 to 1.0) initially, and above the mixing ratio of 1.0, the average K_A decreases with further addition of Chol, indicating that the elasticity of the membrane increases with the Chol content in membranes above mixing ratio of 1.0.

Fig. 6:

Height images of the MPLL membrane patches containing different Chol content. Membrane patches with Chol/MPLL mixing ratio 0 (A), 0.5 (B), 1.0 (C), 1.5 (D), 2.0 (E), and 2.5 (F) are shown in the figure. Membrane patches were prepared by briefly incubating the diluted SUV solution, and images were taken without flushing. Islands of s_o domains within l_d can be seen in the patches of MPLL membrane without Chol, whereas no such domains are seen in other membrane patches containing Chol. The membrane thickness was measured using the rotating box function of the nanoscope analysis software at the patches boundary in the image and averaged from three independent experiments. The height difference between the two phases in Chol/MPLL mixing ratio of 0 is ~ 1 nm, which is similar to the one obtained from the complete membrane (see Fig. 1A). The average thickness for the l_o membrane increases initially (Chol/MPLL mixing ratio of 0.5 to 1.0) and decreases with more Chol in the membrane. Image scale for (A-F) is 1 μm.

Fig. 7:

Membrane thickness and rupture depth of the Chol/MPLL membrane with different Chol content. The green open circle in Fig. A is the average thickness for s_o membrane, while the red open triangle is the average thickness for the l_d membrane in Chol/MPLL membrane without Chol. The purple filled squares are the average thickness of l_o membranes at different Chol/MPLL mixing ratios. The thickness for the l_o membrane initially increases and decreases after it peaks at Chol/MPLL mixing ratio of 1. In Fig. B, green bar is the rupture depth of the s_o membrane and red bar is for l_d membrane in MPLL membrane without Chol. The purple bars are the average rupture depth for l_o membranes across different Chol/MPLL mixing ratios. The average rupture depth for the s_o membrane is smaller than that of fluid phase membranes.