Single-particle tracking of immunoglobulin E receptors (FcεRI) in micron-sized clusters and receptor patches

Abstract

When mast cells contact a monovalent antigen-bearing fluid lipid bilayer, IgE-loaded FcεRI receptors aggregate at contact points and trigger degranulation and the release of immune activators. We used two-color total internal reflection fluorescence microscopy and single-particle tracking to show that most fluorescently labeled receptor complexes diffuse freely within these micron-size clusters, with a diffusion coefficient comparable to free receptors in resting cells. At later times, when the small clusters coalesce to form larger patches, receptors diffuse even more rapidly. In all cases, Monte Carlo diffusion simulations ensured that the tracking results were free of bias, and distinguished biological from statistical variation. These results show the diversity in receptor mobility in mast cells, demonstrating at least three distinct states of receptor diffusivity.

Figure 1

(color web and print): Fluorescent IgE-FcεRI receptor (IgE-FcεRI) complexes undergo confined diffusion in clusters and central patches. (A,D) Total internal reflection fluorescence microscope image of gravity settling RBL cell on fluid lipid bilayer containing 12 mol% DNP-CAP PE lipid after ~30 s (A) and ~4 min (D) of initial contact. The Red box highlights a cluster or central patch in which a single fluorescent IgE-FcεRI complex was tracked at 20 frames/s. Bar = 5 µm. (B) Demonstrates that IgE-FcεRI complex trajectory (red) is restricted to the area occupied by the cluster (green). The cluster image (green) was obtained from an intensity sum over a 4.6 s time series. The cluster moved approximately 140 nm in this time period. (The tracked receptor remained within the patch at all times.) Bar = 200 nm. (C) Mean-squared displacement (MSD) plots of IgE-FcεRI complex trajectory shown in (B) (circles) and cluster trajectory (squares) suggest that IgE-FcεRI diffusivity is an order of magnitude larger than receptor diffusivity. The MSD plot of the tracked IgE-FcεRI complex (circles) indicates confined diffusion by its downward curvature and asymptotic approach to a finite MSD value. From the asymptotic MSD value the estimated circular domain radius is $\sqrt{0.027}$ µm or 160 nm. The MSD plot for the cluster (squares) was linear characterizing simple diffusion. The solid line represents a fit to confined diffusion (circles) or free diffusion (squares). (E) Demonstrates that IgE-FcεRI complex trajectory (red) is restricted to the area occupied by the central patch (green). The patch image (green) was obtained from an intensity sum over a 3.3 s time series. Bar = 200 nm. (F) MSD plot of IgE-FcεRI complex trajectory shown in (E). The IgE-FcεRI complex diffusion was restricted with an estimated circular domain radius of $\sqrt{0.21}$ µm or 460 nm. Moreover, for IgE-FcεRI complexes (circles), the initial slope of the MSD plot in (E) is much stepper than the slope in (C) suggesting that receptor diffuse faster in the central patch than in the cluster.

Figure 2

Experimental (solid black lines) and Monte Carlo simulated (solid grey lines) average (logarithmic mean) diffusivity of IgE-FcεRI receptor complexes confined to central patches (circles) and clusters (squares) as well as average diffusivity of freely diffusing clusters (triangles). For each population, the dashed line above and below the average represents one standard deviation in the lognormal distribution. The average diffusion coefficient 〈D〉 and corral radius 〈R〉 obtained from experiments were used as input parameters for the simulation. Here, the averaging brackets denote the logarithmic mean. 〈D₀〉 is the average diffusivity obtained from 50,000 simulations which incorporated dynamic and static localization uncertainty estimated from experimental data. The data presented here suggests that the variation in diffusivity observed on cells (dashed black lines) cannot be explained on the basis of statistics (dashed grey lines). Thus, there is additional variation of biological origin.

Figure 3

(color web and print): Fluorescent IgE-FcεRI receptor (IgE-FcεRI) trajectory obtained from single-particle tracking at 20 frames/s. Total internal reflection fluorescence (TIRF) microscope images were collected ~30 s after initial cell-substrate contact. The color coding indicates the relative brightness of the IgE-FcεRI, with red being dimmest and blue brightest. The IgE-FcεRI is initially confined in the bottom left cluster (grey) and then transits to the top cluster. During transit the receptor appears to be no longer in close proximity (dimmer in TIRF) to the supported lipid bilayer. Moreover, the IgE-FcεRI appears to diffuse (as determined from linear MSD plot) more rapidly (larger hops) when in transit between clusters indicating release of diffusional constraints over the cell surface. Scale bar = 500 nm.

Figure 4

Histogram in polar coordinates of IgE-FcεRI receptor complex (IgE-FcεRI) hop direction, relative to receptor cluster hop direction, which is represented by the arrow. Here, receptors were no more mobile than the clusters themselves and a total of 509 cluster-receptor vector pairs obtained from individual hops (every 50 ms) were analyzed (7 IgE-FcεRI and cluster trajectories). 251 IgE-FcεRI displacement vectors had a component in the direction of cluster motion (dark grey) and an within statistical variation equal amount of 258 had a downward component (light grey). Thus, slowly diffusing receptors are not influenced by cluster motion and are mobile within the cluster.