Functional nanoscale coupling of Lyn kinase with IgE-FcεRI is restricted by the actin cytoskeleton in early antigen-stimulated signaling

Abstract

The allergic response is initiated on the plasma membrane of mast cells by phosphorylation of the receptor for immunoglobulin E (IgE), FcεRI, by Lyn kinase after IgE-FcεRI complexes are cross-linked by multivalent antigen. Signal transduction requires reorganization of receptors and membrane signaling proteins, but this spatial regulation is not well defined. We used fluorescence localization microscopy (FLM) and pair-correlation analysis to measure the codistribution of IgE-FcεRI and Lyn on the plasma membrane of fixed cells with 20- to 25-nm resolution. We directly visualized Lyn recruitment to IgE-FcεRI within 1 min of antigen stimulation. Parallel FLM experiments captured stimulation-induced FcεRI phosphorylation and colocalization of a saturated lipid-anchor probe derived from Lyn's membrane anchorage. We used cytochalasin and latrunculin to investigate participation of the actin cytoskeleton in regulating functional interactions of FcεRI. Inhibition of actin polymerization by these agents enhanced colocalization of IgE-FcεRI with Lyn and its saturated lipid anchor at early stimulation times, accompanied by augmented phosphorylation within FcεRI clusters. Ising model simulations provide a simplified model consistent with our results. These findings extend previous evidence that IgE-FcεRI signaling is initiated by colocalization with Lyn in ordered lipid regions and that the actin cytoskeleton regulates this functional interaction by influencing the organization of membrane lipids.

FIGURE 1:

Pair-correlation analysis of two-color FLM images measures stimulation time-dependent nanoscale colocalization of IgE-FcεRI and Lyn. Cells expressing Lyn-mEos3.2 and sensitized with Dy654 IgE are stimulated with 500 ng/ml DNP-BSA for 0, 1, 3, 6, or 12 min, fixed, and imaged as described in Materials and Methods. (A) Representative two-color FLM images of cells for each stimulation time point. Regions indicated by white boxes are magnified in the insets. The map of single-probe localizations obtained through FLM imaging is convolved with 2D Gaussian functions with radii of 50 nm in whole images and 20 nm in insets for display. (B) Individual and average pair cross-correlation functions for cells fixed at each stimulation time point. Individual Lyn/IgE-FcεRI cross-correlation functions are fitted to Eq. 1 as described in Materials and Methods. Fits of these individual cross-correlation functions to Eq. 1 are used to extract the fit parameters A (amplitude) and ξ (correlation length, in nanometers) for each correlation function. Measured individual cross-correlations with fits are shown in light colors; averages of the individual cross-correlation functions measured at a given time point are shown in black (eight or nine cells per time point). (C) Fit parameters A and ξ generated by the fit of individual Lyn/IgE-FcεRI cross-correlation functions averaged and plotted as a function of stimulation time. Error bars represent SEM.

FIGURE 2:

Phosphotyrosine/IgE-FcεRI cross-correlation amplitude increases with stimulation time. (A) Two-color FLM images of A488 anti-phosphotyrosine and Dy654 IgE in cells fixed after stimulation for 0 (left) or 6 min (right). Regions indicated by white boxes are magnified in the insets. (B) Individual and average pair cross-correlation functions for cells imaged for each stimulation time point. Individual cross-correlation functions are fitted to Eq. 1 as in Figure 1. Measured individual cross-correlations with fits are shown in light colors; average cross-correlation functions are shown in black (six cells per time point). (C) Cross-correlation fit parameters A and ξ as a function of stimulation time for A488 phosphotyrosine/Dy654 IgE-labeled cells stimulated for 0, 1, 3, or 6 min and averaged over multiple cells for each time point.

FIGURE 3:

Inhibition of actin polymerization enhances Lyn/IgE-FcεRI cross-correlation amplitude. (A) Representative Lyn-mEos3.2/Dy654 IgE FLM images of cells stimulated for 6 min without (top) or with treatment with cytochalasin (middle) or latrunculin (bottom). (B) Lyn/IgE-FcεRI cross-correlation fit parameters A and ξ as a function of stimulation time for RBL-2H3 cells expressing Lyn-mEos3.2 and sensitized with Dy654 IgE that were stimulated for 0, 1, 3, 6, or 12 min with and without treatment with actin-disrupting drugs. Fit parameters are averaged over multiple cells for each time point (eight or nine cells per time point for untreated cells, nine cells per time point for cytochalasin-treated cells, and seven cells per time point for latrunculin-treated cells). Lyn/IgE-FcεRI cross-correlation function fit parameters for untreated cells are reproduced from Figure 1. Error bars denote SEM. (C) Average cross-correlation functions for the three treatment conditions for unstimulated cells (top), cells stimulated for 1 min (middle), and the average integral of the cross-correlation function minus 1 from 0 to 300 nm for 0- and 1-min stimulated time points (bottom).

FIGURE 4:

IgE-FcεRI-correlated phosphotyrosine is enhanced due to inhibition of actin polymerization. (A) Phosphotyrosine/IgE-FcεRI cross-correlation fit parameters A and ξ as a function of stimulation time for RBL-2H3 cells sensitized with Dy654 IgE and stimulated for 0, 1, 3, or 6 min with and without treatment with actin-disrupting drugs, followed by immunolabeling with A488 4G10 anti-phosphotyrosine (A488 pY). Fit parameters are averaged over multiple cells for each time point (six cells per time point for untreated cells, seven cells per time point for cytochalasin and latrunculin-treated cells). pY/IgE-FcεRI cross-correlation function fit parameters are also plotted and are reproduced from Figure 2. Inset, values for A at the unstimulated time point on an expanded scale. Equivalent data for Syk-negative cells shown in B are plotted in A (filled symbols) for direct comparison to wild-type RBL-2H3 cells. (B) Average pY/IgE-FcεRI cross-correlation fit parameters for A488 pY/Dy654 IgE two-color images of Syk-negative cells that were stimulated for 6 min with and without treatment with actin-disrupting drugs. Fit parameters are averaged over multiple cells for each treatment condition (four cells for all treatment conditions). All error bars denote SEM.

FIGURE 5:

Lipid anchorage of mEos3.2 constructs determines antigen-induced recruitment to IgE/FcεRI and the effects of cytoskeletal perturbation. (A) Representative two-color images of cells expressing PM-mEos3.2 (top) or mEos3.2-GG (bottom) sensitized with Dy654 IgE and stimulated for 6 min. (B) Average PM/IgE-FcεRI and GG/IgE-FcεRI cross-correlation fit parameters A and ξ as a function of stimulation time in RBL-2H3 cells. Cells were stimulated for 1, 3, 6, or 12 min with and without treatment with 1 μM latrunculin. Fit parameters are averaged over multiple cells for each time point (seven cells per time point for cells expressing both constructs and for both treatment conditions). Error bars denote SEM.

FIGURE 6:

Actin disruption enhances FcεRI receptor-PM interactions in simulations of the 2D Ising model. (A) Simulation snapshots (left) and time-averaged positions (right) of receptors (FcεRI) and PM (lipid anchor of Lyn) in simulations of the 2D Ising model coupled to a static actin network and performed as described in Materials and Methods. Receptors (red), PM (green), and actin (not shown) all prefer to be in contact with the same type of components, referred to as ordered lipids (L_o, dark gray pixels in simulation snapshots). Receptors are clustered within actin corrals by a Gaussian-shaped potential that does not directly affect the localization of other components in clustered simulations. Simulations were conducted over a range of corral sizes. Representative simulations with small (top) and large (bottom) average corral sizes are shown. The sizes indicated represent the square root of the median corral area. (B) Pair cross-correlation function, c(r), tabulated between receptors and PM in the absence (top) and presence (bottom) of receptor clustering. The curves on the right (blurred c(r)) are convolved with a Gaussian-shaped filter with σ = 24 nm in each channel to facilitate comparison with experimental results (Supplemental Figure S7). (C) Simulation snapshots (left) and time-averaged positions (right) of receptors and PM, equivalent to A for the case in which the disordered (L_d) components (light gray pixels) preferentially associate with the actin meshwork, whereas receptors and PM still associate with L_o components (dark gray pixels). (D) Summary of amplitude values, c(r = 0), from blurred receptor-PM cross-correlation functions over the range of median corral sizes and for the three types of membrane-actin interactions: order preferring (blue), disorder preferring (red), and locally randomized preference (green). Example simulation images, snapshots, and correlation functions from simulations of the latter two cases are shown in Supplemental Figure S9. Data from simulations run in the absence of actin are plotted as a single point (black diamond). The horizontal dashed line is set by the y-axis value of this point. The vertical dashed line indicates the diameter of receptor clusters (100 nm).