Changes in Cholesterol Level Alter Integrin Sequestration in Raft-Mimicking Lipid Mixtures  Yifan Ge, Jiayun Gao, Rainer Jordan, Christoph A. Naumann  Biophysical Journal  Volume 114, Issue 1, Pages 158-167 (January 2018) DOI: 10.1016/j.bpj.2017.11.005 Copyright © 2017 Biophysical Society Terms and Conditions

Figure 1 Representative EPI micrographs of NBD-DHPE distribution in a polymer-tethered lipid bilayer that contains equimolar concentrations of DOPC and DPPC and varying CHOL molar concentration of 25, 28, 33, 35, 37, and 40 mol%. Biophysical Journal 2018 114, 158-167DOI: (10.1016/j.bpj.2017.11.005) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 2 Analysis of domain-specific TRITC-DHPE diffusivity (A) and Eraft data of NBD-DHPE distribution (B) suggest that variation in membrane CHOL between 25 and 37 mol% CHOL alters lipid packing differences between lo and ld domains of a polymer-tethered lipid bilayer containing DOPC/DPPC/CHOL mixtures (1:1 DOPC/DPPC molar ratio); p < 0.005. Error bars represent standard deviation of data sets from 15 different bilayer regions (out of three samples). Biophysical Journal 2018 114, 158-167DOI: (10.1016/j.bpj.2017.11.005) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 3 Representative CS-XY scans (raw data) of NBD-DHPE (A–C and G–I) and Alexa 555 MAb-tagged αvβ3-integrin distributions (D–F and J–L) in DOPC/DPPC/CHOL mixtures containing equimolar amounts of DOPC and DPPC and varying CHOL molar concentrations of 25, 33, and 37 mol%, as obtained through the NBD and Alexa 555 channels of the confocal detection system (p < 0.01). Separate data sets are presented for αvβ3 (A–F) and αvβ3 + VN (G–L). Quantification of the CS-XY data in terms of the parameter Eraft (see Eq. 1) demonstrates the profound influence of CHOL on the sequestration of αvβ3 before and after VN addition (M); p < 0.01 (box size; CS-XY scan: 12 × 12 μm2, pixel size: 0.5 μm). Error bars in (M) represent SD of Eraft acquisitions from 15 different bilayer regions (out of three samples). To see this figure in color, go online. Biophysical Journal 2018 114, 158-167DOI: (10.1016/j.bpj.2017.11.005) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 4 Representative time scans of confocal fluorescence intensity (A) and corresponding PCH curves (B) of Alexa 555 MAb-labeled αvβ3 in lo and ld domains in a polymer-tethered lipid bilayer comprised of an equimolar ratio of DOPC and DPPC and 33 mol% CHOL. Parameter Xdimer from PCH analysis demonstrates that changes in CHOL content do not alter the predominantly monomeric nature of integrins in the bilayer (C) (p < 0.01). Data in (B) and (C) are based on 50 independent data sets for each lipid composition. Errors bars represent standard deviation. Biophysical Journal 2018 114, 158-167DOI: (10.1016/j.bpj.2017.11.005) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 5 Populations of αvβ3 and uPAR, Xmigrate (defined by Eq. 2), in a polymer-tethered lipid bilayer, which alter their phase preference in a ternary DOPC/DPPC/CHOL mixture in response to a change in membrane CHOL from 33 to 37 mol%; p < 0.005. Data are based on 15 independent data sets of Eraft at 33 and 37 mol% CHOL. Errors bars represent corresponding standard deviation. Biophysical Journal 2018 114, 158-167DOI: (10.1016/j.bpj.2017.11.005) Copyright © 2017 Biophysical Society Terms and Conditions