Probing Cholesterol-Dependence of Integrin-Urokinase Receptor Complex Formation using Confocal Dual-Color Fluorescence Intensity Analysis

Abstract

The modern view of a plasma membrane is that of a complex supramolecular assembly of notable lipid/protein heterogeneity, in which the local lipid environment may have a profound impact on membrane protein distribution and function. Yet, the underlying mechanisms of lipid-mediated regulation of membrane protein functionality remain elusive, in part due to the limitations of existing methodologies and the often small size and transient nature of lipid/protein heterogeneities in cellular membranes. Exemplary, we still have rather limited knowledge about the significance of lipid composition in hetero-protein complex formation in such membrane systems. To address this fascinating, but poorly understood topic, here we introduce a single molecule-sensitive confocal experimental strategy, comprised of dual-color fluorescence peak analysis and photon counting histogram (PCH) method, which not only allows the detection of hetero-protein complexes in well-defined lipid environments, but also provides accurate insight into the composition of such complexes in the membrane. By applying the described methodology, here we demonstrate the formation of complexes of αvβ3 integrins and GPI-anchored urokinase plasminogen activator receptors (uPAR) in cholesterol-containing model lipid mixtures. PCH analysis of fluorescence intensity signals of complexes confirms furthermore that αvβ3-uPAR complexes are comprised of one integrin and two uPAR proteins. Remarkably, formation of comparable hetero-protein complexes is largely suppressed in a cholesterol-free lipid composition, highlighting the importance of cholesterol in αvβ3-uPAR complex formation. Our results are intriguing in light of the already established significance of cholesterol in integrin function in cellular membranes.