Dataset Information

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 ?v?3 and ?5?1 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 ?v?3 and ?5?1 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.

SUBMITTER: Hussain NF

PROVIDER: S-EPMC3660629 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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Bilayer asymmetry influences integrin sequestering in raft-mimicking lipid mixtures.

Hussain Noor F NF Siegel Amanda P AP Ge Yifan Y Jordan Rainer R Naumann Christoph A CA

Biophysical journal 20130501 10

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 spectr ...[more]

PMID: 23708361

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Project description:Distinct lipid environments, including lipid rafts, are increasingly recognized as a crucial factor affecting membrane protein function in plasma membranes. Unfortunately, an understanding of their role in membrane protein activation and oligomerization has remained elusive due to the challenge of characterizing these often small and transient plasma membrane heterogeneities in live cells. To address this difficulty, we present an experimental model membrane platform based on polymer-supported lipid bilayers containing stable raft-mimicking domains (type I) and homogeneous cholesterol-lipid mixtures (type II) into which transmembrane proteins are incorporated (?(v)?(3) and ?(5)?(1) integrins). These flexible lipid platforms enable the use of confocal fluorescence spectroscopy, including the photon counting histogram method, in tandem with epifluorescence microscopy to quantitatively probe the effect of the binding of native ligands from the extracellular matrix ligands (vitronectin and fibronectin for ?(v)?(3) and ?(5)?(1), respectively) on domain-specific protein sequestration and on protein oligomerization state. We found that both ?(v)?(3) and ?(5)?(1) sequester preferentially to nonraft domains in the absence of extracellular matrix ligands, but upon ligand addition, ?(v)?(3) sequesters strongly into raft-like domains and ?(5)?(1) loses preference for either raft-like or nonraft-like domains. A corresponding photon counting histogram analysis showed that integrins exist predominantly in a monomeric state. No change was detected in oligomerization state upon ligand binding in either type I or type II bilayers, but a moderate increase in oligomerization state was observed for increasing concentrations of cholesterol. The combined findings suggest a mechanism in which changes in integrin sequestering are caused by ligand-induced changes in integrin conformation and/or dynamics that affect integrin-lipid interactions without altering the integrin oligomerization state.

Project description:How lipid asymmetry impacts ordered lipid domain (raft) formation may yield important clues to how ordered domain formation is regulated in vivo. Under some conditions, a sphingomyelin (SM) and cholesterol-rich ordered domain in one leaflet induces ordered domain formation in the corresponding region of an opposite leaflet composed of unsaturated phosphatidylcholine (PC) and cholesterol. In other conditions, the formation of ordered domains in a SM and cholesterol-rich leaflet can be suppressed by an opposite leaflet containing unsaturated PC and cholesterol. To explore how PC unsaturation influences the balance between these behaviors, domain formation was studied in asymmetric and symmetric lipid vesicles composed of egg SM, cholesterol, and either unsaturated dioleoyl PC (DOPC) or 1-palmitoyl 2-oleoyl PC (POPC). The temperature dependence of ordered domain formation was measured using Förster resonance energy transfer, which detects nanodomains as well as large domains. In cholesterol-containing asymmetric SM+PC outside/PC inside vesicles, the PC-containing inner leaflet tended to destabilize ordered domain formation in the SM+PC-containing outer leaflet relative to ordered domain stability in cholesterol-containing symmetric SM/PC vesicles. Residual ordered domain formation was detected in cholesterol-containing asymmetric SM+DOPC outside/DOPC inside vesicles, but ordered domain formation was completely or almost completely suppressed by asymmetry in cholesterol-containing SM+POPC outside/POPC inside vesicles over the entire temperature range measured. Suppression of ordered domain formation in the latter vesicles was confirmed by fluorescence anisotropy measurements. Because mixtures of SM, POPC, and cholesterol form domains in symmetric vesicles, and this lipid composition mimics plasma membranes to a significant degree, it is possible that under some conditions in vivo the loss of lipid asymmetry could trigger ordered domain formation.

Project description:Integrin-mediated adhesion is a central feature of cellular adhesion, locomotion, and endothelial cell mechanobiology. Although integrins are known to be transmembrane proteins, little is known about the role of membrane biophysics and dynamics in integrin adhesion. We treated human aortic endothelial cells with exogenous amphiphiles, shown previously in model membranes, and computationally, to affect bilayer thickness and lipid phase separation, and subsequently measured single-integrin-molecule adhesion kinetics using an optical trap, and diffusion using fluorescence correlation spectroscopy. Benzyl alcohol (BA) partitions to liquid-disordered (Ld) domains, thins them, and causes the greatest increase in hydrophobic mismatch between liquid-ordered (Lo) and Ld domains among the three amphiphiles, leading to domain separation. In human aortic endothelial cells, BA increased ?1-integrin-Arg-Gly-Asp-peptide affinity by 18% with a transition from single to double valency, consistent with a doubling of the molecular brightness of mCherry-tagged ?1-integrins measured using fluorescence correlation spectroscopy. Accordingly, BA caused an increase in the size of focal-adhesion-kinase/paxillin-positive peripheral adhesions and reduced migration speeds as measured using wound-healing assays. Vitamin E, which thickens Lo domains and disperses them by lowering edge energy on domain boundaries, left integrin affinity unchanged but reduced binding probability, leading to smaller focal adhesions and equivalent migration speed relative to untreated cells. Vitamin E reversed the BA-induced decrease in migration speed. Triton X-100 also thickens Lo domains, but partitions to both lipid phases and left unchanged binding kinetics, focal adhesion sizes, and migration speed. These results demonstrate that only the amphiphile that thinned Ld lipid domains increased ?1-integrin-Arg-Gly-Asp-peptide affinity and valency, thus implicating Ld domains in modulation of integrin adhesion, nascent adhesion formation, and cell migration.

Dataset Information

Bilayer asymmetry influences integrin sequestering in raft-mimicking lipid mixtures.

Publications

Bilayer asymmetry influences integrin sequestering in raft-mimicking lipid mixtures.

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