Micro-Patterned Ligand Surfaces Provide New Insights Into Spatial Regulation Of Egf Receptor Signaling And Ige Receptor Endocytosis

Fc[epsilon]RI, the high affinity receptor for immunoglobulin E (IgE), is primarily found on the surface of mast cells and basophils. Fc[epsilon]RI signaling is triggered by the crosslinking of receptor-bound IgE by a multivalent antigen (Ag). This initiates a series of intracellular signaling events that eventually lead to secretion of mediators such as histamines and cytokines, which are responsible for allergic reactions. In addition, Ag-crosslinked IgE-Fc[epsilon]RI complexes are internalized. Although the sequelae of signaling events leading to release of preformed mediators has been extensively studied, much less is known about the Fc[epsilon]RI internalization pathway. Recent investigations of our laboratory have focused on the roles that the plasma membrane and the actin cytoskeleton play in Fc[epsilon]RI-mediated signaling leading to secretory events. However, the tools available to study spatial regulation of cellular responses were limited. Towards overcoming this limitation, we previously developed micro/nanofabricated surfaces with patterned arrays of ligand to spatially control the stimuli provided to cells. This enabled us to visualize the formation of signaling complexes at the plasma membrane of the cells. My research has now extended the application of these micro-patterned surfaces to study the assembly of Fc[epsilon]RI endocytic machinery, in conjunction with fluorimetry, flow cytometry, and confocal microscopy. I found that inhibitors of F-actin cycling cause i crosslinked IgE-Fc[epsilon]RI complexes too localize in membrane invaginations that are not cleaved from the plasma membrane. I provided evidence that Fc[epsilon]RI internalization is sensitive to cholesterol, phosphoinositide synthesis at the plasma membrane, and the tyrosine kinase Syk. My experiments with micro-patterned ligand surfaces showed that recruitment of F-actin to the Fc[epsilon]RI signaling complexes is diminished in the presence of inhibitors of phosphoinositide synthesis. I have further extended the application of micro-patterned ligand surfaces to study the spatial regulation of epidermal growth factor (EGF) receptor (EGFR) signaling by developing a scheme to covalently immobilize micron-sized patches of functionally active EGF. I utilized NIH-3T3 cells stably over-expressing EGFR to detect the formation of EGFR signaling complexes via fluorescence confocal microscopy. I detected actin cytoskeleton-dependent recruitment of EGFR signaling partners H-Ras, MEK, and phosphorylated Erk to the EGFR signaling complexes on the plasma membrane of cells. In addition, I observed recruitment of F-actin and the adaptor protein paxillin, including phosphorylated paxillin, to regions where EGFR is clustered at the EGF patches. I also found that recruitment of F-actin and Erk to EGFR signaling complexes is diminished in the presence of inhibitors of phosphoinositide synthesis. Integrin [alpha]-5, the most abundant integrin in NIH-3T3 cells, is excluded from these EGFR signaling complexes. These results demonstrate that micro-patterned EGF surfaces are valuable for investigation of spatially orchestrated interactions of signaling proteins with EGFR at the plasma membrane. i