Small, mobile FcepsilonRI receptor aggregates are signaling competent.

Reagents Mouse monoclonal

IgE anti-DNP was prepared as described in ( Liu et al., 1980 ). Mouse monoclonal IgE anti-dansyl was from BD Biosciences (San Jose, CA). DNP n -BSA was from Invitrogen (Carlsbad, CA) or Biosearch Technologies (Novato, CA). For unit conversion, 5 nM IgE and 14 nM DNP-BSA are ~1 μg/ml. PP2 was from Calbiochem (La Jolla, CA), anti FcεRIβ antibody was a gift of J. Rivera (NIH, Bethesda, MD) and anti-phosphotyrosine antibodies (PY20/PY99) were from Santa Cruz Biotechnology (Santa Cruz, CA). HRP-conjugated secondary antibodies and SuperSignal® West Pico Chemiluminescent Substrate kits were from Pierce Protein Research Products (Rockford, IL).

Reagent Synthesis QD-IgE

Detailed methods are described in ( Andrews et al., 2008 ). DNP-QD A 1 mM biotin-DNP stock solution was prepared by dissolving 1 mg DNP-X-biocytin-X succinimidyl ester (Molecular Probes) in 100 μl dimethylsulfoxide (Sigma) followed by reaction with ammonium bicarbonate (Sigma) in a 1:10 stoichiometric ratio in water for 30 minutes to quench the succinimidyl ester group. DNP-QD was prepared by reacting biotin-DNP with the indicated Qdot® Streptavidin conjugate (Invitrogen) in a 50:1 ratio in PBS + 1% BSA for four hours at 4°C; the manufacturer estimates each QD is conjugated to 3–5 strepavidins. DNP-QD was then purified by dialysis against PBS and the final concentration determined by absorption at 532 nm. Stock solutions of QD-IgE and DNP-QDs were stored at 4°C and used within four weeks. Alexa-IgE Fluorescent IgE was prepared using Alexa Fluor® 488 or Alexa Fluor® 633 Microscale Protein Labeling Kits (Invitrogen, Carlsbad, CA). Dansyl-BSA This reagent was prepared by modification of the protocol provided with Dansyl-X, SE (Invitrogen, Carlsbad, CA) to yield a dansyl:BSA ratio of 69.3 as measured by absorbance at 335 and 280 nm.

Cell Culture Rat basophilic leukemia

(RBL-2H3) cells were grown as adherent monolayers in minimum essential medium with 10% fetal bovine serum (MEM/FBS) (Invitrogen, Carlsbad, CA) as described in ( Wilson et al., 2000 ). BMMC were differentiated from mast cell progenitors in murine bone marrow by culturing in IL-3-containing medium for 4–6 week as in ( Kashiwakura et al., 2008 ). Degranulation, Western Blotting and Internalization Assays RBL cell monolayers were grown in 24-well tissue culture plates for 24 hours and primed with 5 nM IgEanti-DNP or IgEanti-dansyl; BMMC were primed in suspension overnight, then harvested, washed and allowed to settle onto 8-well chambers for 2 hrs in phenol red-free RPMI without IL-3. Release of β-hexosaminidase was measured as described in ( Ortega et al., 1988 ). Immunoprecipitations and immunoblotting protocols were performed as in ( Wilson et al., 1995 ) and ( Hernandez-Hansen et al., 2004 ). Measurements of fluorescently-labeled IgE internalization were performed by flow cytometry, as previously described ( Barker et al., 1995 ); where indicated in legend, cells were pretreated with 10 μM PP2 and maintained in the inhibitor throughout the assay.

Single QD tracking

SPT was performed using an Olympus IX71 inverted microscope equipped with a 60× 1.3 N.A. water objective and an electron multiplying CCD camera (Andor iXon 887) as in ( Andrews et al., 2008 ). Samples were maintained at 34–36 °C by an objective heater (Bioscience Tools, San Diego, CA). For tracking QD-IgE-FcεRI complexes, RBL-2H3 cells were labeled with 500 pM QD655-IgE anti-DNP in HBSS for 10 min at 37°C prior to imaging. Where indicated, cells were incubated with 50 nM IgE anti-DNP or IgE anti-dansyl for 30 min at 37 °C in MEM/FBS, washed, and stimulated with the indicated doses of DNP n -BSA +/− 10 μM PP2 or dansyl-BSA while imaging at 20–33 frames/s at 35 °C. For tracking DNP-QDs, cells were primed by 30 min incubation with 50 nM “dark” IgEanti-DNP at 37 °C, followed by addition of DNP-QD655 or DNP-QD585 at the microscope, as specified in legends. The resulting image series were analyzed and diffusion coefficients obtained using previously described single particle tracking algorithms ( Andrews et al., 2008 ).

Kinetics of immobilization assay

RBL-2H3 cells were labeled with a 1:1 mixture of 500 pM QD625- and QD705-IgE anti-DNP for 10 min at 37 °C in HBSS, washed, and incubated at 37 °C for 30 min with 140 nM IgE anti-DNP . Cells were washed and imaged at 20 frames/s at 35 °C in 200 μl HBSS; after ~10s, 100 μl of 3X DNP n -BSA was added at indicated concentrations. Instantaneous diffusion coefficients were calculated as described ( Andrews et al., 2008 ) and the traces from multiple cells (resting, n=51; 10 μg/ml, n=11; 1 μg/ml, n=9; 0.1 μg/ml, n=10; 0.01 μg/ml, n=10; 0.001 μg/ml, n=16) averaged to generate the final plot. BMMC experiments Cells were labeled with 5 nM IgE anti-DNP in suspension overnight, washed and incubated with 1 nM QD655-IgE anti-DNP in 1 ml phenol-red free RPMI for 30 min at 37°C. Cells were resuspended in Hanks-BSA and permitted to settle onto 8-well chambers prior to imaging.

Analysis

Image processing was performed using Matlab (The MathWorks, Inc., Natick, MA) in conjunction with the image processing library DIPImage (Delft University of Technology). Descriptions of specific analysis routines has been reported previously ( Andrews et al., 2008 ). D 1–3 , values are reported as medians and interquartile range is provided as a measure of statistical dispersion ( Table S1 ).

Hyperspectral Microscopy

Cells on 15 mm round coverslips were incubated for 15 min at RT with a mixture of QD655-, QD625-, QD585-, QD565-, and QD525-IgE at 400 pM each in HBSS, washed, and coverslips mounted onto 25×75 mm glass slides using a 2 mm rubber spacer. Samples were treated +/− 0.1 μg/ml DNP 25 -BSA and imaged by hyperspectral microscopy as described in ( Sinclair et al., 2006 ). Confocal images were acquired at 0.25 frames/s for 60 frames. The resulting image series were subtracted for dark current and despiked ( Jones et al., 2008 ), then displayed using a custom image analysis program ( Haaland et al., 2009 ). Regions of interest could then be manually selected and the spectra from these regions obtained.

Electron Microscopy

Cells on glass coverslips were primed overnight with 5 nM IgE anti-DNP . Washed cells were stimulated with indicated doses of DNP-BSA for 1 min, followed by fixation (7 min, 0.5% paraformaldehyde) and membrane sheets prepared as previously described ( Wilson et al., 2000 ). Digital images were acquired using a Hitachi H600 transmission electron microscope, followed by image processing and statistical analyses as previously described ( Zhang et al., 2006 ). For plotting cluster size, we employed a cut-off distance of 50 pixels (43 nm), such that receptors within this distance of each other were considered part of the same cluster.

Supplementary Material Supplementary Figures/Text Video1 Video2 Video3 Video4