β1-Integrin-Mediated Adhesion Is Lipid-Bilayer Dependent

Abstract

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 (L) domains, thins them, and causes the greatest increase in hydrophobic mismatch between liquid-ordered (L) and L domains among the three amphiphiles, leading to domain separation. In human aortic endothelial cells, BA increased β-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 β-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 L 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 L 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 L lipid domains increased β-integrin-Arg-Gly-Asp-peptide affinity and valency, thus implicating L domains in modulation of integrin adhesion, nascent adhesion formation, and cell migration.

Interplay Between Receptor-Ligand Binding and Lipid Domain Formation Depends on the Mobility of Ligands in Cell-Substrate Adhesion

“…As mentioned in Introduction, there is a discrepancy regarding the interplay between receptor-ligand binding and lipid raft coalescence in adhesion experiments with mobile (Huang et al, 2010;Anderson and Roche, 2015) and immobile ligands (Gaus et al, 2006;Mitchell et al, 2009;Evani and Ramasubramanian, 2016;Son et al, 2017). Our results suggest that the discrepancy may be caused by the mobility of ligands in cell-substrate adhesion.…”

“…Destabilizing raft and protein heterogeneities in vesicles adversely affects the biotin-streptavidin mediated stable adhesion (Zhao et al, 2013). For the adhesion of cells to substrates with immobile ligands mimicking cell-ECM adhesion, experimental studies led to contradictory results regarding the interplay between receptor-ligand binding and coalescence of raft domains (Gaus et al, 2006;Mitchell et al, 2009;Evani and Ramasubramanian, 2016;Son et al, 2017). For example, Son et al (Son et al, 2017) found that integrin-mediated adhesion of human aortic endothelial cells to a substrate facilitates raft domain formation and integrin clustering, which increase the cell-substrate adhesion.…”

Motility of Enzyme-Powered Vesicles

“…Both methods confirm formation of lipid vesicles ( Figure S4 FCS, which was primarily used in this work for observing the changes, is a very sensitive technique for measuring the diffusion changes of molecules. [25][26][27][28] The autocorrelation curves for vesicle tagged and free ATPase show distinct differences in the diffusion time τD, which is directly related to the size of the system according to Stokes-Einstein equation ( Figure S5). To prepare homogeneous and uniform sized ATPase tagged vesicles for FCS measurements, the vesicles were extruded through polycarbonate filters.…”

In situ, fluorescence lifetime-based measurements of cell membrane micromechanics

“…The elastic moduli of membranes are fundamental to the understanding of cellular responses to mechanical stimuli (reviewed in (1)), the sensitivity of ion channels to membrane stretch (reviewed in (2)), responses of cilia to fluid flow (reviewed in (3)) and mechanobiological phenomena responsible for health and disease (reviewed in (4)). Derived from area per lipid (APL), these moduli provide input to models that predict membrane bending fluctuations, by which cells sense their surroundings (5), and hydrophobic matching between lipids and proteins, key constants governing lipid phase separation and protein modulation by lipids (6,7). Thus, APL and associated moduli are fundamental mechanistic links between lipid membrane composition and membrane-related biological functions of cells.…”

“…where kB is Boltzmann constant, T is temperature, and _`a T is the variance of the APL distribution. Assuming a polymer brush model for the lipid bilayer (21), the membrane bending modulus l can be calculated from KA by, l = _ ( nn − % ) T / 24 (7) where DHH is the head-to-head thickness of the bilayer (Table S3) and Do = 1.0 nm is double the distance between the end of the hydrophobic core and center of the…”