Measurements of diffusion and clustering of integrin receptors in the plasma membrane of live cells using fluorescence-based techniques

Cell membranes display a complex, dynamic organization of lipids, proteins and other small molecules. This organization may arise from varied protein-protein interactions including interactions between receptors, effectors and ligand molecules or from formation of specialized domains within the plasma membrane such as lipid rafts. The involvement of integrin cell surface receptors in membrane complexes is intensively known. Integrins\u27 interactions with other components in these complexes can alter many signal transduction cascades, thus modulating integrins\u27 own functions and affecting various cellular processes. Integrins are heterodimers formed by the non-covalent association of an α and a β subunit. Each subunit consists of a single transmembrane domain, a large extracellular domain and a short cytoplasmic domain. While ligand binding to integrin extracellular domains allows transmission of signals into the cell (outside-in signaling), binding of cytoskeletal proteins to integrin cytoplasmic domains permits inside-out signaling. In this way, integrins mediate bidirectional signaling across the plasma membrane and control a variety of cellular processes including cell adhesion, mobility, growth, survival, proliferation, and differentiation. The work described in this dissertation aims to achieve a better understanding of membrane organization by identifying the factors that affect integrin dynamics. Using molecular biology and fluorescence microscopy techniques, we have measured integrin clustering and diffusion properties under altered environments such as reduced membrane cholesterol levels, reduced cytoplasmic protein concentrations and reduced membrane protein concentrations. Additionally, we have also studied the effects of post-translational modifications on integrin dynamics. The fluorescence techniques used in this work include fluorescence resonance energy transfer (FRET) to study integrin clustering, fluorescence recovery after photobleaching (FRAP) and single particle tracking (SPT) to study integrin diffusion. A number of cytoplasmic and membrane proteins were identified that alter integrin diffusion and clustering. Reducing the levels of cholesterol from the cell membrane resulted in more mobile integrins and affected diffusion of integrins in confined domains. Removing a potential palmitoylation site in αPS2CβPS integrins resulted in more mobile integrins. The role of other proteins, cholesterol and palmitoylation in altering integrin diffusion and clustering may be the result of partitioning of integrins into lipid nanodomains, which are heterogeneous regions in the cell membrane containing higher concentration of lipids and proteins as compared to the bulk membrane and play a very important role in cell signaling