Formation of a mast cell synapse: Fc epsilon RI membrane dynamics upon binding mobile or immobilized ligands on surfaces

Abstract

Fc epsilonRI on mast cells form a synapse when presented with mobile, bilayer-incorporated Ag. In this study, we show that receptor reorganization within the contacting mast cell membrane is markedly different upon binding of mobile and immobilized ligands. Rat basophilic leukemia mast cells primed with fluorescent anti-DNP IgE were engaged by surfaces presenting either bilayer-incorporated, monovalent DNP-lipid (mobile ligand), or chemically cross-linked, multivalent DNP (immobilized ligand). Total internal reflection fluorescence imaging and electron microscopy methods were used to visualize receptor reorganization at the contact site. The spatial relationships of Fc epsilonRI to other cellular components at the synapse, such as actin, cholesterol, and linker for activation of T cells, were also analyzed. Stimulation of mast cells with immobilized polyvalent ligand resulted in typical levels of degranulation. Remarkably, degranulation also followed interaction of mast cells, with bilayers presenting mobile, monovalent ligand. Receptors engaged with mobile ligand coalesce into large, cholesterol-rich clusters that occupy the central portion of the contacting membrane. These data indicate that Fc epsilonRI cross-linking is not an obligatory step in triggering mast cell signaling and suggest that dense populations of mobile receptors are capable of initiating low-level degranulation upon ligand recognition.

FIGURE 1

Illustration and characterization of surfaces. A. Representation of IgE-primed cells contacting different types of antigen presenting surfaces. B. Tracking of fluorescent IgE bound to 5 mol%, 25 mol% DNP-lipid/POPC lipid bilayers and crosslinked DNP-BSA surfaces. Arrows indicate starting point and initial direction of each track. Diffusion coefficients were calculated from tracking of IgE^AF488 bound to each tested surface (Table at right). C. Sequential images of FRAP performed on a 25 mol% bilayer showing pre-bleach, bleach spot, and recovery. Calculated diffusion coefficients for each surface are indicated in the associated table. *Statistically significant with P-value ≤ 0.01; n=5.

FIGURE 2

Receptor behavior and distribution is dependent upon the type of contact surface. A. TIRF imaging of IgE^AF488-primed cells engaged with mobile (left column) or immobile (middle and right columns) ligand presenting surfaces over a 12 minute period. Images are contrast enhanced. All scale bars represent 5 μm. Disappearance of signal in the central region of the cell settled onto the EGS-DNP-BSA surface is due to photobleaching indicated by another cell imaged under similar conditions without extensive laser exposure (inset image). B. TEM images of membrane sheets from cells settled onto mobile (DNP-lipid in bilayer), immobilized (EGS-DNP-BSA) ligand, or non-activating surface. FcεRI β is labeled with 12 nm or 6 nm gold. Line in top panel delineates area of receptor coalescence within the membrane. Hopkins tests are located below each image for which they were performed. Images are contrast enhanced. Scale bars are 100 nm. C. Percent β-hexosaminadase release from cells settled onto glass, mobile ligand, immobilized ligand, or stimulated by soluble antigen. Asterisks indicate results that are significantly higher than spontaneous release (bare glass). D. Fluorescence recovery of a bleached portion of a synaptic region formed on a 25 mol% DNP-lipid bilayer. The region outlined in a dashed box indicates region bleached. Bleach and post-bleach images taken at 6 and 8 minute time points are in the right column. Grayscale range indicator shows amount of fluorescent recovery in each post-bleach image. All images were corrected for within-scan photobleaching by scaling to a non-bleached region of the synapse.

FIGURE 3

Actin reorganization at the adherent surface is dependent upon ligand mobility. A. TIRF imaging of GFP-actin RBL-2H3 cells primed with IgE^Dy520 settled onto glass, mobile (DNP-lipid) ligand, or immobilized (EGS-DNP₂₄-BSA) ligand. Scale bars are 5 μm. B. Confocal imaging of IgE^AF488-primed RBL-2H3 cells fixed with paraformaldehyde and labeled with rhodamine phalloidin. Top row is a cell settled onto a 25 mol% DNP-lipid bilayer and bottom row is a cell settled onto a DNP₂₄-BSA surface. Scale bars are 5 μm. C. Low magnification TEM of membrane sheets from cells bound to an EGS-DNP₂₄-BSA EM grid (top) or a bilayer coated EM grid (bottom). Boxed regions are magnified views of cytoskeletal structures for each sheet. Scale bars are 100 nm. All images are contrast enhanced.

FIGURE 4

Fluorescent cholesterol derivatives reveal strong association with mobile but not immobile FcεRI. A. Fluorescent cholesterol analog used in membrane repletion with a short chain PEG linker between the FITC molecule and cholesterol. B. TIRF imaging of RBL-2H3 cells repleted with FITC cholesterol and primed with IgE^Dy520 after contact with glass (top row), mobile ligand (middle row), or immobilized ligand (bottom row). Pearson’s coefficient calculation from each series of fluorescent images is in brackets. Scale bars are 5 μm. C. TEM membrane sheets from cells settled onto EM grids coated with mobile or immobilized ligand. Regions of receptor (12 nm gold) and cholesterol (6 nm gold) colocalization on the mobile surface are outlined. On the immobile surface, regions of cholesterol are outlined in a thin line while receptors are circled with a thick line. Ripley’s tests for coincidence of label for FcεRI β and FITC (cholesterol) are shown as insets. Scale bars are 100 nm. All images are contrast enhanced.

FIGURE 5

LAT clusters do not colocalize with FcεRI at the synapse. A. Membrane sheets from IgE primed cells settled onto mobile (top) or immobilized (bottom) ligand for six minutes and labeled for FcεRI β (12 nm gold) or LAT (6 nm gold). LAT clusters are circled with thin lines on each sheet to indicate general cluster size and location relative to receptors (circled with thick lines). Scale bars are 100 nm. Ripley’s tests for coincidence of LAT and FcεRI β label are inset in each figure. B. Paraformaldehyde fixed IgE^DNP-primed RBL-2H3 cells settled onto 25 mol% DNP-lipid (top row) or DNP₂₄-BSA (bottom row) surfaces for 6 minutes. After permeabilization, cells were dually labeled for phosphorylated LAT (AF555) and FcεRI β (Cy5; psuedocolored). Images are contrast enhanced.