Multi-Color Quantum Dot Tracking to Correlate FcɛR1 Aggregate Size with Mobility

Abstract

The high affinity IgE receptor, FcɛRI, is the primary immune receptor expressed on mast cells and basophils. These receptors bind circulating IgE with high affinity and are activated when multivalent allergen crosslinks IgE-bound receptors, initiating a complex signaling cascade that leads to the release of inflammatory mediators by degranulation. It is also well documented that FcɛRI crosslinking leads to receptor immobilization. However, the exact mechanism of crosslink-induced activation is not completely understood.We have used single quantum dot (QD) tracking to characterize the mobility of QD-IgE-FcɛRI in the resting and activated state. We have previously shown that crosslink-induced immobilization is depended on the actin cytoskeleton as well as the antigen dose. Furthermore, we have determined that small, mobile clusters are signaling competent and that immobilization is the trigger for internalization rather than signal initiation. In these experiments, a combination of QD-IgE and dark IgE were crosslinked with multivalent antigen; therefore, the cluster size could not be directly correlated with mobility. We have now developed an experimental strategy and algorithmic approach to differentiate between monomers, dimers, trimers, and tetramers. Using a four-color beam splitter, we can simultaneously track FcɛRI complexes labeled with four spectrally distinct QDs (525, 585, 655, and 705) at rates up to 30 frames/s. From these data, we use QD localization and channel overlay accuracies to identify monomers, dimers, trimers, and tetramers and determine the diffusion coefficient for each type of aggregate. We demonstrate this approach with both simulated and experimental data.