Patterned Ligand Surfaces For The Study Of Egf And Ige Receptor-Mediated Signaling

Bio-patterned surfaces have enabled the exploration of a wide range of cellular activities. The surfaces described in this work have contributed to the understanding of epidermal growth factor receptor (EGFR) and IgE receptor (Fc!RI) regulated signaling processes as well as mechanisms underlying microglia and macrophage mediated amyloid-" degradation. Through standard photolithographic techniques and a polymer lift-off method, receptor ligands, including growth factor, IgE hapten, or fibrillar "-amyloid, may be patterned on selected substrates. Cells settle on these surfaces, and fluorescence imaging (confocal or TIRF) can be used to visualize specific cellular components concentrating at regions of patterned ligand. A novel radial fluorescence analysis method quantifies the intensity of labeled cellular components at patterned features to evaluate distributions of recruitment across different cell areas (e.g., the cell middle vs. cell periphery). In NIH-3T3 cells stably expressing EGFR, we find EGFR recruitment to patterned EGF is accompanied by stimulated tyrosine phosphorylation while F-actin and endogenous "1containing integrins also concentrate, but with differential spatial localizations across the cell compared to EGFR. F-actin accumulation depends on phosphatidylinositol 4,5bisphosphate (PI(4,5)P2): pharmacological inhibitors of PI(4,5)P2 synthesis and acute recruitment of phosphoinositide 5'-phosphatases to the plasma membrane reduce F-actin concentration with EGF patterns. Prompted by our earlier studies with the actin cytoskeleton and focal adhesion proteins, we now demonstrate that !1- and !3-containing integrins co-cluster with Fc"RI in RBL mast cells at patterned antigen surfaces. Additionally, we find important IgE receptor downstream signaling molecules, including Syk, PLC#1, and LAT, recruited to these same sites in a spatial arrangement determined by the form of antigen presentation, as part of either mobile lipid bilayers or immobilized protein. F-actin polymerization further regulates the extent of clustering for these signaling molecules at different patterned antigen surfaces. Stimulated intracellular trafficking of integrins and H-Ras with mast cell recycling endosomes is also demonstrated. Taken together, our work with patterned ligand surfaces has allowed for systematic study of spatially regulated EGF and IgE receptor signal transduction with new insight on the role of the actin cytoskeleton and integrins within these systems