Multi-Color Single Particle Tracking of QD-IgE-FcεRI: Directly Correlating Oligomer Size with Receptor Mobility and Signaling

Abstract

IgE binds to its high affinity receptor, FcεRI, expressed on mast cells and basophils. Crosslinking of IgE-FcεRI complexes leads to intracellular signaling and eventual immune response. Although much is known about this receptor family, the precise mechanism of signal initiation remains elusive. Previous single particle tracking (SPT) experiments have revealed that small, mobile receptors are signaling competent. However, high-speed SPT had typically been limited to two-color tracking, such that aggregates larger than dimers could not be distinguished. In order to better understand the relationship between receptor mobility, aggregate size and signaling, we have developed a novel high-speed hyperspectral line-scanning microscope (HSM) to perform multi-color SPT (mcSPT) of up to 8 colors of quantum dot (QD)- labeled IgE-FcεRI simultaneously on the surface of mast cells. The HSM is unique in its capability to acquire a 4D image (x,y,λ,t) of ∼30 microns⊥2 with 128 spectral channels at ∼30 fps. This combined with the advantageous characteristics of QDs (high quantum yield, single source excitation, narrow emission spectra) provides an exceptional platform for mcSPT. Using the HSM, we can determine receptor aggregate size from the spectral signature, and then correlate aggregate size with mobility. Comparison of aggregate mobility in the presence of the tyrosine kinase inhibitor, PP2, reveals the influence of adaptor protein recruitment on mobility. The HSM also allows for simultaneous measurement of calcium flux (Fluo-4) while performing mcSPT, providing a direct readout of signaling in response to receptor aggregate formation and mobility. These experiments not only address important questions in IgE-FcεRI signal initiation, but also provide new biophysical insight into the long-standing debate over the relationship between receptor oligomer size and mobility in the plasma membrane.