High affinity IgE receptor diffusional dynamics measured by single quantum dot tracking in resting and activated cells

The high affinity IgE receptor (FcεRI) of mast cells and basophils binds circulating IgE with sub‐nanomolar affinity. Crosslinking of IgE‐FcεRI complexes by multivalent allergen initiates a complex signaling cascade that leads to the release of inflammatory mediators. In order to study the dynamics of FcεRI signaling, we have generated two novel quantum dot (QD) based probes: monovalent QD‐IgE (binds FcεRI without crosslinking) and polyvalent 2,4‐dinitrophenol (DNP)‐QD (crosslinks DNP‐specific IgE). Through single QD tracking on the apical membrane of RBL‐2H3 cells, we observe a two‐fold decrease in the diffusion coefficient of IgE‐FcεRI complexes in the presence of saturating (dark) IgE‐FcεRI, suggesting homotypic interactions. By simultaneously tracking QD‐IgE and DNP‐QD, we observe that, at low allergen levels, DNP‐QD crosslinked receptors remain mobile, suggesting that receptor immobilization is not critical for signal initiation. Mathematical models incorporating these data show that slower diffusion rates of nearby receptors initiate clustering consistent with the distribution of immunolabeled receptors seen in electron micrographs. Taken together, these data indicate a mechanism through which FcεRI receptors maintain proximity and enhance the probability of signaling complex formation.