Small, mobile FcepsilonRI receptor aggregates are signaling competent

Abstract

Crosslinking of IgE-bound FcepsilonRI triggers mast cell degranulation. Previous fluorescence recovery after photobleaching (FRAP) and phosphorescent anisotropy studies suggested that FcepsilonRI must immobilize to signal. Here, single quantum dot (QD) tracking and hyperspectral microscopy methods were used for defining the relationship between receptor mobility and signaling. QD-IgE-FcepsilonRI aggregates of at least three receptors remained highly mobile over extended times at low concentrations of antigen that induced Syk kinase activation and near-maximal secretion. Multivalent antigen, presented as DNP-QD, also remained mobile at low doses that supported secretion. FcepsilonRI immobilization was marked at intermediate and high antigen concentrations, correlating with increases in cluster size and rates of receptor internalization. The kinase inhibitor PP2 blocked secretion without affecting immobilization or internalization. We propose that immobility is a feature of highly crosslinked immunoreceptor aggregates and a trigger for receptor internalization, but is not required for tyrosine kinase activation leading to secretion.

Figure 1. Antigen Induced Immobilization and Degranulation are Dose-Dependent

(A) The relative change in diffusion coefficient as a function of time. Cells were treated with DNP₂₅-BSA at the indicated doses (in μg/ml) 10 s into each image series. Each trace is an average from at least nine individual cells. (B) Example trajectories of QD-IgE-FcεRI after treatment with 0.001 μg/ml (left column) or 1 μg/ml (right column) DNP₂₅-BSA. (C) Cumulative Probability Analysis (CPA) plot shows the distribution of diffusion coefficients for 1 μg/ml DNP_n-BSA on RBL-2H3 cells. (D–G) Degranulation assay results showing percentage of total β-hexosaminidase released from cells stimulated with the indicated doses of antigen for (D) DNP₂₅-BSA, (E) DNP₁₂-BSA, (F) DNP₄-BSA, and (G) DNP₂-BSA. Error bars represent standard deviation. (H) CPA plot of diffusion coefficients for QD-IgE-FcεRI tracked on BMMC. (I) Degranulation assay results on BMMC.

Figure 2. Antigen-Induced Aggregates of at Least Three QD-IgE-FcεRI Remain Mobile

Cells were labeled with five colors of QD-IgE and then stimulated with 0.1 μg/ml DNP-BSA. Selected images of diffusing QD-IgE-FcεRI are shown and the spectra of selected aggregates (red rectangles) are displayed to the right of each image. (A) An aggregate composed of QD565-, QD585-, and QD655-IgE-FcεRI diffusing together for over two minutes. (B) An aggregate composed of QD565-, QD625-, and QD655-IgE-FcεRI diffusing together for 20 s. Variation in the peak intensities over time is attributed to QD “blinking.” Pseudo-colored RGB images were generated by displaying 635–655 nm as red, 575–630 nm as green, and 540–570 nm as blue. Scale bars represent 2 μm.

Figure 3. DNP-QD Remains Mobile at Activating Doses

(A) Degranulation assay plot showing percentage of total β-hexosaminidase released from cells stimulated with the indicated doses of DNP-QD655 (white bars) or DNP-QD585 (black bars). Error bars represent standard deviation. (B) Cells were primed with IgE_anti-DNP and then labeled with 1 pM DNP-QD655. The cumulative probability of the diffusion rate of DNP-QD655 was then calculated before (thick line) and two min after (thin line) addition of 500 pM DNP-QD585.

Figure 4. Phosphorylation Kinetics for Syk and the FcεRI β and γ Subunits

(A) Tyrosine phosphorylation of the 72 kD band (Syk) in RBL-2H3 cell lysates after stimulation with increasing doses of DNP-BSA. (B) Quantification of blot shown in A. (C) Tyrosine phosphorylation of anti-FcεRIβ immunoprecipitates showing the kinetics of β and γ subunit tyrosine phosphorylation in RBL-2H3 cells treated with increasing doses of DNP-BSA. (D,E) Quantification of blots shown in C for FcεRIβ (D) and FcεRIγ (E). Closed symbols in B, D, and E designate antigen doses which induced marked and rapid immobilization.

Figure 5. Immobilization is insensitive to PP2 treatment and correlates with internalization

(A) Western blot showing phosphotyrosine signal from RBL-2H3 cells stimulated with 0.1 μg/ml DNP-BSA in the absence or presence of PP2. (B) Quantification of the 72 kD (Syk) and 55 kD (Lyn) bands from the blot shown in A. Intensity is normalized to the peak signal for each protein. (C) CPA plot showing diffusion of QD-IgE-FcεRI before (solid lines) or 1 min after (dashed lines) addition of 1 μg/ml DNP-BSA in the absence (red lines) or presence (blue lines) of PP2. (D) Percent internalization of IgE-FcεRI as a function of time and antigen dose in the presence (open symbols) or absence (filled symbols) of PP2. All antigen doses are in μg/ml.

Figure 6. FcεRI cluster size increases as a function of antigen dose

(A–C) Electron micrographs from membrane sheets with 5 nm gold particles marking the positions of the β-subunit of FcεRI. Membrane sheet preparations were from resting cells (A), or cells treated with 0.001 (B) or 1 (C) μg/ml DNP-BSA for 1 min. (D) Quantification of receptor clustering after 1 min of stimulation at the indicated doses of DNP-BSA, based upon 10 micrographs per condition. Arrowhead in B indicates a clathrin-coated pit. Scale bars in A–C represent 0.1 μm.

Figure 7. Direct Crosslinking is Required for Immobilization

(A) Cells were primed with Alexa647-IgE_anti-dansyl and Alexa488-IgE_anti-DNP and then stimulated for 1 min with DNP-BSA, dansyl-BSA, or both (see Supplemental Experimental Procedures). Scale bars represent 5 μm. (B) The cumulative probability plot of the diffusion coefficient of QD-IgE_anti-DNP for four conditions is shown. Cells were labeled with QD-IgE_anti-DNP and then primed with either IgE_anti-DNP or IgE_anti-dansyl. The diffusion rate of QD-IgE_anti-DNP was then measured in IgE_anti-DNP primed cells before (red, solid line) and after (red, dashed line) addition of 1 μg/ml DNP-BSA. The diffusion rate of QD-IgE_anti-DNP was also measured in IgE_anti-dansyl primed cells before (blue, solid line) and after (blue, dashed line) addition of 1 μg/ml dansyl-BSA.