Focal adhesion proteins connect IgE receptors to the cytoskeleton as revealed by micropatterned ligand arrays

Patterned surfaces that present specific ligands in spatially defined arrays are used to examine structural linkages between clustered IgE receptors (IgE-Fc epsilon RI) and the cytoskeleton in rat basophilic leukemia (RBL) mast cells. We showed with fluorescence microscopy that cytoskeletal F-actin concentrates in the same regions as cell surface IgE-Fc epsilon RI that bind to the micrometer-size patterned ligands. However, the proteins mediating these cytoskeletal connections and their functional relevance were not known. We now show that whereas the adaptor proteins ezrin and moesin do not detectably concentrate with the array of clustered IgE-Fc epsilon RI, focal adhesion proteins vinculin, paxillin, and talin, which are known to link F-actin with integrins, accumulate in these regions on the same time scale as F-actin. Moreover, colocalization of these focal adhesion proteins with clustered IgE-Fc epsilon RI is enhanced after addition of fibronectin-RGD peptides. Significantly, the most prominent rat basophilic leukemia cell integrin (alpha 5) avoids the patterned regions occupied by the ligands and associates preferentially with exposed regions of the silicon substrate. Thus, spatial separation provided by the patterned surface reveals that particular focal adhesion proteins, which connect to the actin cytoskeleton, associate with ligand-cross-linked IgE-Fc epsilon RI, independently of integrins. We investigated the functional role of one of these proteins, paxillin, in IgE-Fc epsilon RI-mediated signaling by using small interfering RNA. From these results, we determine that paxillin reduces stimulated phosphorylation of the Fc epsilon RI beta subunit but enhances stimulated Ca2+ release from intracellular stores. The results suggest that paxillin associated with clustered IgE-Fc epsilon RI has a net positive effect on Fc epsilon RI signaling.