Biophysical mechanisms of protein recruitment to raft domains studied using planar model membranes

Ann Carol Kimble-Hill, Purdue University

Abstract

Lipid rafts, which are liquid-ordered domains enriched in sphingolipids and cholesterol, are believed to play a significant role in several cell biological processes, largely due to their ability to segregate membrane proteins on the cell surface. The current study explores the poorly understood topic of biophysical mechanisms of protein recruitment to raft domains in a model raft system based on planar polymer-supported membranes. In particular, we report on the raft recruitment of membrane proteins by association with clustering antibodies, their native binding ligands and glycosyl-phosphatidylinositol (GPI) anchored proteins. While raft recruitment by clustering of antibodies is widely recognized, for the first time this study explores the role of native binding ligands and other raftophilic proteins on the recruitment of individual membrane proteins to lipid rafts. Three different types of proteins have been reconstituted and studied: (i) lipid raft-associated [GPI-anchored proteins Fc gamma-subunit containing receptor (FcγRIII) and urokinase plasminogen activator receptor (uPAR)]; (ii) non-raft-associated [transferrin receptor (TfR)]; and (iii) temporarily raft-associated (integrins αVβ 3 and α5β1). The domain-specific quantification of reconstituted membrane proteins (labeled with dye-conjugated monoclonal antibodies) was achieved with high sensitivity using a combined setup of EPI-fluorescence microscopy (EPI) and confocal fluorescence correlation spectroscopy (FCS). A ligand-induced raft recruitment process was observed for (membrane proteins/binding ligands): FcγRIII/IgA, uPAR/urokinase plasminogen activator (uPA), and αVβ3/vitronectin(VN). In contrast, TfR and α5β1 show recruitment to the more fluid, or liquid-disordered, domains after binding to their ligands, transferring (Tf) and fibronectin (FN). Remarkably, the current study also provides experimental evidence for specific protein-protein interactions that cause raft recruitment of the transmembrane proteins αVβ 3 and α5β1 by the GPI-anchored uPAR. To verify such an intriguing GPI-based recruitment process, additional evidence for the necessary integrin-uPAR complex formation was obtained by tracking uPAR (labeled with dye-conjugated monoclonal antibodies) with and without integrin using wide-field single molecule fluorescence microscopy. The current study is scientifically relevant because it demonstrates that protein-protein interactions may play a more prominent role in protein recruitment to raft domains than proposed by the classical raft hypothesis.

Degree

Ph.D.

Advisors

Naumann, Purdue University.

Subject Area

Biochemistry|Physical chemistry

COinS