High Resolution Imaging Methods to Probe FcεRI Membrane Organization & Behavior (139.6)

Abstract

Abstract We have applied a combination of high resolution microscopy approaches, together with lipid mass spec analysis, to study the membrane organization of high affinity IgE receptors (FcεRI), their signaling partners and the local lipid environment. Using immunogold labeling and transmission electron microscopy (TEM) imaging of fixed membrane sheets, we find resting receptors in singlets and small clusters. After crosslinking, clusters increase dramatically in size and recruit specific downstream signaling molecules to form primary signaling domains. Atomic Force Microscopy (AFM) studies provide maps of topographical features of the cytoplasmic face of the membrane and demonstrate the importance of cholesterol to the height of these features. Consistent with this, lipid mass spec analysis of isolated FcεRI membrane patches show that 50% of the lipid surrounding receptors is cholesterol. Recently, we have generated novel quantum dot (QD) based probes to study the dynamics of early events in FcεRI signaling. Monovalent IgE-QD complexes are used to track the lateral motion of individual FcεRI in resting and activated states. These studies document transient co-confinement of resting IgE-bound receptors, as well as the immobilization of receptors after extensive crosslinking. Mathematical models show that clusters form when receptors rapidly exchange between domains or confinement zones, consistent with experimental results. The motion of QD-IgE has also been tracked in live cells transfected with GFP-actin using total internal reflection microscopy (TIRF), providing direct evidence that actin corrals provide one mechanism for defining confinement zones. Supported by NIH RO1 AI051575 (BW), NIH RO1 GM49814 (JM0) and Sandia Labs (LDRD).