Volume 107, Issue 6, Pages 1364-1374 (September 2014) Ether- versus Ester-Linked Phospholipid Bilayers Containing either Linear or Branched Apolar Chains  Daniel Balleza, Aritz B. Garcia-Arribas, Jesús Sot, Kepa Ruiz-Mirazo, Félix M. Goñi  Biophysical Journal  Volume 107, Issue 6, Pages 1364-1374 (September 2014) DOI: 10.1016/j.bpj.2014.07.036 Copyright © 2014 Biophysical Society Terms and Conditions

Figure 1 DSC thermograms of DPPC, DHPC, DPhoPC, DPhPC, and their binary mixtures. A heating rate of 45°C/h was used. The thermograms were recorded after sample equilibration at the starting temperature. The profiles correspond to heating scans. Biophysical Journal 2014 107, 1364-1374DOI: (10.1016/j.bpj.2014.07.036) Copyright © 2014 Biophysical Society Terms and Conditions

Figure 2 Micrographs of GUVs made of binary mixtures of linear and branched lipids: (A) DPhoPC/DPPC, (B) DPhPC/DPPC, (C and E) DPhoPC/DHPC, and (D and F) DPhPC/DHPC. DPPC and DHPC promote the formation of solid domains and a mixture of solid and reticulated coalescent domains, respectively. All scale bars are 10 μm. To see this figure in color, go online. Biophysical Journal 2014 107, 1364-1374DOI: (10.1016/j.bpj.2014.07.036) Copyright © 2014 Biophysical Society Terms and Conditions

Figure 3 Fluorescence polarization data of bilayers (large unilamellar vesicles) composed of single phospholipids or binary mixtures. (A and C) TMA-DPH anisotropy. (B and D) Laurdan GP. Student’s t-test of significance: ∗∗p < 0.005; ∗∗∗p < 0.001; n.s., nonsignificant. Biophysical Journal 2014 107, 1364-1374DOI: (10.1016/j.bpj.2014.07.036) Copyright © 2014 Biophysical Society Terms and Conditions

Figure 4 Representative AFM-tip indentation curves. Representative force curves from SPBs composed of DPPC (linear, ester) (a), DHPC (linear, ether) (b and b′), DPhPC (branched, ether) (c and c′), DPhoPC, (branched, ester) (d and d′). DHPC, DPhPC, and DPhoPC exhibit two alternative reproducible behaviors. Biophysical Journal 2014 107, 1364-1374DOI: (10.1016/j.bpj.2014.07.036) Copyright © 2014 Biophysical Society Terms and Conditions

Figure 5 Bilayer breakthrough force (Fb) histograms of linear-chain lipids. The force step distribution from AFM-tip indentation curves on DPPC (A) (16.01 ± 2.01 nN, n = 647) and DHPC (B) (13.99 ± 3.13 nN and 21.74 ± 2.43 nN, n = 1716) is shown. Continuous lines represent Gaussian fittings. Each histogram is based on four samples analyzed with four different tips. Biophysical Journal 2014 107, 1364-1374DOI: (10.1016/j.bpj.2014.07.036) Copyright © 2014 Biophysical Society Terms and Conditions

Figure 6 Bilayer breakthrough force (Fb) histograms of branched-chain lipids. The force step distribution from AFM-tip indentation curves from SPBs composed of DPhoPC (A) (8.31 ± 1.11 nN and 10.48 ± 0.84 nN, n = 2279) and DPhPC (B) (0.77 ± 0.26 nN and 3.06 ± 0.76 nN, n = 2767) is shown. Continuous lines represent Gaussian fittings. Each histogram is based on three samples analyzed with three different tips. Biophysical Journal 2014 107, 1364-1374DOI: (10.1016/j.bpj.2014.07.036) Copyright © 2014 Biophysical Society Terms and Conditions

Figure 7 Topographic AFM analysis of DHPC SPBs. (A–E) Contact-mode AFM images of a DHPC supported bilayer, showing changes over time at 23°C: t = 0 (A), t = 15 min (B), and t = 30 min (C), with cross-section details from the blue lines at t = 0 (D) and t = 30 min (F), and DHPC bilayer thickness histogram as summary of the data (E). Black areas represent bare mica support and are used to calculate zero height as reference. Both lipid phases can be easily identified by their significantly different heights (3.82 ± 0.16 nm and 5.71 ± 0.17 nm), which are particularly clear at t = 0 as depicted by the dotted green and red lines (to guide the eye) in the corresponding cross section. Phase segregation disappears over time and becomes nonexistent at t = 30 min and only the thickest phase persists (red dotted line in the corresponding cross section). Thickness measurements were made by cross-section height analysis (n = 100) of AFM images taken from two different samples. Biophysical Journal 2014 107, 1364-1374DOI: (10.1016/j.bpj.2014.07.036) Copyright © 2014 Biophysical Society Terms and Conditions

Figure 8 Selected kinetic profiles of polyalcohol influx through liposomes of various compositions (pure lipids or equimolecular binary mixtures). As a control, the glycine-betaine case of null permeation is included. The time-dependent decrease in absorbance at 450 nm was averaged from at least three experiments, GB, glycine-betaine; G2, ethyleneglycol; G3, glycerol; G4, erythrytol. Measurements were obtained at 20°C unless indicated otherwise. Biophysical Journal 2014 107, 1364-1374DOI: (10.1016/j.bpj.2014.07.036) Copyright © 2014 Biophysical Society Terms and Conditions

Scheme 1 (A and B) Chemical structures of (A) DPPC, DHPC, DPhoPC, and DPhPC (images from Avanti Polar Lipids), and (B) the nonelectrolytes employed in our permeability assays (e-glycol, glycerol, erythritol), and glycine-betaine. Biophysical Journal 2014 107, 1364-1374DOI: (10.1016/j.bpj.2014.07.036) Copyright © 2014 Biophysical Society Terms and Conditions