Boxcar Imaging FCS Reveals Membrane Raft Stabilization Kinetics in Antigen-Stimulated Mast Cells

Antigen (Ag) crosslinking of immunoglobulin E-receptor (IgE-FcεRI) complexes in mast cells and consequent coupling with Lyn tyrosine kinase in the plasma membrane inner leaflet stimulates transmembrane signaling to initiate allergic and inflammatory responses. As established previously, this coupling requires formation of liquid-ordered (Lo)-like regions (aka "rafts") around the nano-clustered receptors to facilitate lipid-based partitioning of Lyn via its membrane anchor, followed by receptor phosphorylation mediated by protein-protein interactions. Imaging fluorescence correlation spectroscopy (ImFCS) was previously used to measure diffusion of Lyn-EGFP and its lipid anchor PM-EGFP (both Lo-preferring) as well as EGFP-GG (inner leaflet lipid probe, liquid-disordered (Ld)-preferring) and showed that the membrane reorganized within 15 minutes after Ag addition. To quantify the transition kinetics between the resting and Ag-stimulated steady-states, we have now developed Boxcar ImFCS for time-resolved diffusion measurements on sub-minute scale. We found that Ag stimulation causes gradual diffusion decreases for Lyn-EGFP and PM-EGFP with distinctive half-times ( $t_{1/2}$ ) of 6.9 min and 12 min, respectively, showing that Lyn's protein-based interactions accelerate its diffusional transition. Simultaneously, EGFP-GG gradually changes to faster diffusion with $t_{1/2}=9.4\text{min}$ . In comparison, $t_{1/2}=5.0\text{min}$ for recruitment of cytoplasmic Syk by phosphorylated FcεRI, consistent with initiation of transmembrane signaling before global membrane reorganization and raft condensation is completed by large, stabilized Ag-IgE-FcεRI clusters. Boxcar ImFCS extends the analytical power of ImFCS to reveal dynamic membrane processes that may accompany stimuli-receptor interactions and their sequalae.