Distribution and Dynamics of RBL IgE Receptors (FcɛRI) Quantitatively Observed on Planar Ligand-Presenting Surfaces

Abstract

There is considerable interest in the signaling mechanisms of immunoreceptors, especially when triggered with membrane-bound ligands. When T cells, B cells, or mast cells bind to monovalent ligands on fluid supported lipid bilayers, receptor clustering, signaling, and receptor redistribution into immunological synapses follow. We have quantitatively studied the kinetics of redistribution of IgE receptors (FcɛRI) on RBL-2H3 mast cell surfaces. To separate the kinetics of receptor redistribution from cell spreading, the initial cell-substrate contact time was precisely defined (±50 ms) by micropipette cell manipulation. Using total internal reflection fluorescence, the distribution and dynamics of receptor clusters were imaged. We find strong quantitative evidence that initial receptor aggregation occurs at cellular protrusions, which are visible bright spots/regions on cells contacting ligand-free surfaces. The initial size of these regions is independent of the substrate and the presence or absence of ligand, and they were found to be randomly distributed over the interfacial contact area. Using a finite-element diffusion model, we found that the initial rate of accumulation of receptors at the protrusions is consistent with diffusion-limited trapping with D ∼10−1 μm2/s. At longer timescales, individual clusters on ligand-bearing membranes were observed to move with both a diffusive and a directed component of motion; clusters eventually coalesced near the center of the contact region (∼ 1 minute). The dynamics of the early cluster motion is similar to the dynamics of membrane fluctuations of cells on ligand-free fluid membranes. Thus, the same cellular machinery may be responsible for both processes.Sandia is a multi-program laboratory operated by Sandia Corporation, a Lockheed Martin Company, for the United States Department of Energy under Contract DE-ACO4-94AL85000.