Bilayer Asymmetry Influences Integrin Sequestering in Raft-Mimicking Lipid Mixtures

Abstract

There is growing recognition that lipid heterogeneities in cellular membranes play an important role in the distribution and functionality of membrane proteins. However, the detection and characterization of such heterogeneities at the cellular level remains challenging. Here we report on the poorly understood relationship between lipid bilayer asymmetry and membrane protein sequestering in raft-mimicking model membrane mixtures using a powerful experimental platform comprised of confocal spectroscopy XY-scan and photon-counting histogram analyses. This experimental approach is utilized to probe the domain-specific sequestering and oligomerization state of αvβ3 and α5β1 integrins in bilayers, which contain coexisting liquid-disordered/liquid-ordered (ld/lo) phase regions exclusively in the top leaflet of the bilayer (bottom leaflet contains ld phase). Comparison with previously reported integrin sequestering data in bilayer-spanning lo-ld phase separations demonstrates that bilayer asymmetry has a profound influence on αvβ3 and α5β1 sequestering behavior. For example, both integrins sequester preferentially to the lo phase in asymmetric bilayers, but to the ld phase in their symmetric counterparts. Furthermore, our data show that bilayer asymmetry significantly influences the role of native ligands in integrin sequestering.

Ligand Binding Alters Dimerization and Sequestering of Urokinase Receptors in Raft-Mimicking Lipid Mixtures

“…In the latter case, the confocal spot was kept at a fixed bilayer position and the photon counts through the Alexa 555 channel were collected over 50 s. In these experiments, the correct Biophysical Journal 107(9) 2101-2111 confocal position was identified by maximizing the photon-count rate of probe molecule of interest. In light of the uncertainties about the geometry of the confocal spot (knowledge about confocal shape is required for accurate acquisition of diffusion coefficients via FCS), diffusion coefficients of bilayer-incorporated probe molecules were determined by using a TRITC-DHPE standard, for which the FCS diffusion time could be compared to a diffusion coefficient determined using wide-field single molecule fluorescence microscopy (30). Raw data of photon counts at a fixed bilayer position over time were also used for PCH analysis.…”

“…As reported previously, the microscopy system allows EPI microscopy, confocal fluorescence intensity detection, fluorescence correlation spectroscopy (FCS), and photon counting histogram analysis (PCH) (29,30). EPI analysis was conducted to confirm the integrity of the polymer-tethered lipid bilayer and the existence of coexisting l o and l d lipid phases in ternary raft-mimicking lipid mixtures.…”

“…The reconstitution of uPAR into the planar polymer-tethered lipid bilayer was conducted by adapting the modified Rigaud technique applied to integrins previously (29,30). Specifically, 1.3 Â 10 À11 mol uPAR and 250 mM OG were added together to the preassembled bilayer system and allowed to incubate for 2 h. This surfactant concentration in the presence of the bilayer sample corresponds to~0.002 cmc.…”

“…By applying this powerful experimental platform, we showed that native ligands alter integrin sequestration but not oligomerization in raft-mimicking lipid mixtures (29). In another study, this experimental strategy was pursued to demonstrate that bilayer asymmetry (i.e., presence of l o -l d phase separations exclusively in the top leaflet of the bilayer versus bilayer-spanning l o -l d phase separations) influences integrin sequestration in such lipid mixtures as well (30). Most notably, in the latter work, a v b 3 and a 5 b 1 integrins (in the absence of ligands) were found to have a preference for the l o phase in the asymmetric bilayer, but exhibit an l d phase affinity in their symmetric counterparts.…”

N-cadherin-functionalized polymer-tethered multi-bilayer: a cell surface-mimicking substrate to probe cellular mechanosensitivity

“…As reported before, diffusion coefficients of bilayer-incorporated probe molecules were determined using a TRITC-DHPE standard, for which the FCS diffusion time could be compared to a diffusion coefficient determined using wide-field single molecule fluorescence microscopy. 34 This approach was chosen because uncertainties about the exact geometry of the confocal spot limit the accuracy of lateral diffusion coefficients of probe molecules directly obtained from FCS autocorrelation analysis. Raw data of photon counts at a fixed bilayer position were also analyzed using the PCH method.…”