Quantitative nanoscale analysis of IgE-FcεRI clustering and coupling to early signaling proteins

Abstract

Antigen-mediated cross-linking of IgE bound to its receptor, FcεRI, initiates a transmembrane signaling cascade that results in mast cell activation in the allergic response. Using immunogold labeling of intact RBL mast cells and scanning electron microscopy (SEM), we visualize molecular reorganization of IgE-FcεRI and early signaling proteins on both leaflets of the plasma membrane, without the need for ripped off membrane sheets. As quantified by pair correlation analysis, we observe dramatic changes in the nanoscale distribution of IgE-FcεRI after binding of multivalent antigen to stimulate transmembrane signaling, and this is accompanied by similar clustering of Lyn and Syk tyrosine kinases, and adaptor protein LAT. We find that Lyn co-redistributes with IgE-FcεRI into clusters that cross-correlate throughout 20 min of stimulation. Inhibition of tyrosine kinase activity reduces the numbers of both IgE-FcεRI and Lyn in stimulated clusters. Coupling of these proteins is also decreased when membrane cholesterol is reduced either before or after antigen addition. These results provide evidence for involvement of FcεRI phosphorylation and cholesterol-dependent membrane structure in the interactions that accompany IgE-mediated activation of RBL mast cells. More generally, this SEM view of intact cell surfaces provides new insights into the nanoscale organization of receptor-mediated signaling complexes in the plasma membrane.

Figure 1. Cell surface topology and immuno-gold distribution is visualized with SEM

A) Composite SED image of the dorsal surface on intact RBL cells. Immuno-gold particles (10nm) labeling IgE-FcεRI are superimposed in yellow; particle centers are determined using BSD in conjunction with automated image processing. Images are acquired at 15K magnification, and scale bar is 5 µm. B–D) Higher magnification images from (A) in areas indicated; scale bars are 500 nm. E–G) SEM images acquired at higher magnification (35K) for increased lateral resolution and for automated identification of gold particle location; scale bars are 500 nm. E) Raw BSD image. F) Gold particle centers identified from BSD micrographs with automated image processing algorithms (Supplemental Figure S1). Apparent topology in (E–G) arises from carbon coating and is uncorrelated with gold image processing. G) Reconstructed particle centers overlaid on SED distributions.

Figure 2. Gold particles labeling proteins are self-clustered in resting cells

A) Clustering is evident in representative reconstructed SEM (BSD) images of gold particles that label IgE bound to the receptor FcεRI. B) Pair auto-correlation functions of gold particle distributions from many images quantify immuno-gold labeled IgE-FcεRI in the plasma membrane. (C–D) Double label experiments indicate that self-clustering of golds labeling IgE-FcεRI complexes is dominated by multiple gold particles binding to single target proteins. C) Images of A488- or FITC-conjugated IgE-FcεRI that are distinctively immuno-labeled with 5nm (red) and 10nm (blue) gold particles, respectively. D) Auto-correlation functions of 5nm particles (red points) indicate self-clustering. In contrast, the cross-correlation function for the 5nm vs. 10nm particles is 1 within error bounds at all r (black points). The presence of significant self-clustering (red points) in the absence of co-clustering (black points) indicates that over-counting of multiple gold binding to individual target proteins dominates observations of self-clustering in this single label experiment. E) Correlation functions, g(r) vs. r, are evaluated and averaged over many images of specified target proteins (Table 1) and indicate that gold particles are significantly self-clustered at short distances in all cases. In subsequent figures, we only show correlation functions for r>50nm to avoid any self-clustering artifacts associated with multiple labels bound to single target proteins . The correlation curves in (E) are fit for r >10 nm by Gaussian functions centered at r=0.

Figure 3. IgE-FcεRI, Lyn, Syk and LAT rapidly redistribute into large clusters after addition of antigen to crosslink IgE-FcεRI, and clusters decrease in size over 20 min

A) Representative reconstructed BSD images of plasma membrane with immuno-gold labeling of specified target proteins (10 nm gold particles). Left panels: SEM samples prepared from cells incubated at 37°C in the absence of stimulus; right panels: SEM samples prepared from cells stimulated with antigen for 1 min at 37°C. Scale bars are 500 nm. B) Gold particle distributions are quantified for multiple images (N>100; Table 1) using pair auto-correlation functions. Correlation curves are well fit by a single exponential for r>50nm. Extracted fit parameters are plotted in Figure 4, and those for IgE-FcεRI and Lyn are tabulated in Supplemental Table S1A.

Figure 4. Physical properties of clustered target proteins as a function of stimulation time are measured or extracted from correlation functions

Auto-correlation functions determined from many images of specified target proteins are well fit to a single exponential as shown in Figure 3B, and parameters are determined as described in the text: correlation length, ξ (A); average number of proteins per cluster, N_cl (B); average surface density, ρ (C); increased density of proteins within clusters, ρ_cl/ρ (D). These parameters for IgE-FcεRI and Lyn are tabulated in Supplemental Table S1A.

Figure 5. Double label experiments reveal that Lyn, but not Thy-1, co-redistributes with IgE-FcεRI after cells are stimulated with antigen

A) Representative reconstructed BSD images from cells that were stimulated (or not) for times indicated and specified target proteins were immuno-gold labeled. IgE-FcεRI (5 nm gold particles) and Lyn (10 nm gold particles) were double labeled, or IgE-FcεRI (10 nm gold particles) and Thy-1 (5 nm gold particles) were double labeled. Scale bars are 200nm. B) Gold particle distributions for many images from double label experiments represented in (A) are quantified using cross-correlation functions, and curves are well fit by an exponential. Extracted fit parameters for IgE-FcεRI and Lyn are tabulated in Supplemental Table S1B.

Figure 6. Co-redistribution of Lyn with cross-linked IgE-FcεRI is sensitive to Src family kinase activity and plasma membrane cholesterol levels

A) Representative reconstructed BSD images of immuno-gold labeled IgE-FcεRI and Lyn in antigen stimulated cells and parallel samples treated with PP1 prior to stimulation or with MβCD either before or after stimulation. Scale bar is 500 nm. B) Auto-correlation functions quantify particle distributions from many images in experiments represented in (A), and curves are fit to exponentials for r>50nm as in Figure 3 (solid lines). C) Parameters ξ, N_cl, ρ_cl/ρ are extracted from fits shown in (B); values are tabulated in Supplemental Table S1C.

Figure 7. Lyn clustering is qualitatively reproduced using a minimal model that incorporates composition fluctuations and a Lyn activation state that depends on the local membrane environment

(A) Snapshots from simulations incorporating fluctuations of liquid-ordered (orange pixels) and liquid-disordered (white pixels) membrane regions, IgE-FcεRI complexes (small red circles), and Lyn in an active (yellow circles) or inactive (green circles) state. IgE-FcεRI distributions are fixed throughout the simulation and are taken from the images shown in Figure 6. Orange, ordered domains localize around clustered IgE-FcεRI receptors because crosslinked receptors prefer to partition into ordered regions. Lyn is allowed to move and also partitions into ordered regions. In addition, Lyn can be in two distinct states depending on local lipid environment. Lyn is in an active state when surrounded by liquid-ordered components, and experiences a strong binding affinity for IgE-FcεRI. Otherwise Lyn is inactive and interacts weakly with IgE-FcεRI. A detailed description of the model and its implementation is provided in Materials and Methods. Lyn is nearly randomly distributed in unstimulated cells (−Ag). In simulations of antigen (+Ag) treated cells, most Lyn is activated and associated with receptors. In simulations of PP1 treated cells, Lyn is restricted to be in an inactive state (independent of its local environment) and fewer Lyn proteins partition into IgE-FcεRI rich clusters due to the absence of strong direct binding between components. The fraction of liquid-ordered membrane is reduced in simulations of MβCD treated cells. Few Lyn proteins are activated or bound to receptor because Lyn rarely is surrounded by liquid-ordered components. As a result, Lyn is only weakly localized within receptor rich domains. (B) Pair correlation functions for simulated Lyn proteins are calculated from 50 snapshots like those shown in A. Lyn is most strongly clustered in simulations with clustered receptors, and clustering is reduced in simulations mimicking cells treated with PP1 or MβCD.