Lipid Behavior in Integrin α2 Clustering

Abstract

It is becoming increasingly more evident that transmembrane proteins can influence lipid organization in biological membranes. Our previous studies in model membranes have shown that peptides affect the partitioning of lipids in the bilayer. We now want to further investigate these protein-lipid interactions in a more biological context, by looking at integrin clustering in giant plasma membrane vesicles (GMPVs). Integrins are cell surface proteins that traverse the plasma membrane and mediate, for example, cell signaling and virus entry. Binding of Echovirus 1 to integrin α2 induces integrin clustering and the subsequent internalization of the virus-integrin complex. Clustering is crucial for virus entry, but can also be achieved through the addition of integrin specific antibodies to cells. We are interested in the possible reorganization of the membrane upon clustering as well as the influence of plasma membrane lipids on clustering. For this purpose we have prepared GMPVs from SAOS cells expressing α2 integrin, and induced antibody-mediated clustering in them. We are using lipid probes to visualize the liquid ordered and liquid disordered phase, and fluorescent secondary antibodies to detect α2 integrin in the GPMVs with confocal microscopy. We will look at lipid organization in cells expressing integrin mutants, which are unable to cluster and/or internalize. And we will treat the cells and/or GPMVs in ways that alter their lipid content, such as deplete membrane cholesterol with cyclodextrin, and monitor the resulting changes in integrin and lipid behavior in GMPVs. With these experiments we hope to gain further insight into the interplay of transmembrane proteins and lipids in biological membranes. Also, understanding the lipid behavior in integrin clustering will aid in understanding what governs clustering and how it affects membrane architecture. This information can in turn be beneficial for developing ways of preventing virus entry.