Membrane order and molecular dynamics associated with IgE receptor cross-linking in mast cells

Abstract

Cholesterol-rich microdomains (or "lipid rafts") within the plasma membrane have been hypothesized to exist in a liquid-ordered phase and play functionally important roles in cell signaling; however, these microdomains defy detection using conventional imaging. To visualize domains and relate their nanostructure and dynamics to mast cell signaling, we use two-photon (760 nm and 960 nm) fluorescence lifetime imaging microscopy and fluorescence polarization anisotropy imaging, with comparative one-photon anisotropy imaging and single-point lifetime and anisotropy decay measurements. The inherent sensitivity of ultrafast excited-state dynamics and rotational diffusion to the immediate surroundings of a fluorophore allows for real-time monitoring of membrane structure and organization. When the high affinity receptor for IgE (FcepsilonRI) is extensively cross-linked with anti-IgE, molecules associated with cholesterol-rich microdomains (e.g., saturated lipids (the lipid analog diI-C(18) or glycosphingolipids)) and lipid-anchored proteins coredistribute with cross-linked IgE-FcepsilonRI. We find an enhancement in fluorescence lifetime and anisotropy of diI-C(18) and Alexa 488-labeled IgE-FcepsilonRI in the domains where these molecules colocalize. Our results suggest that fluorescence lifetime and, particularly, anisotropy permit us to correlate the recruitment of lipid molecules into more ordered domains that serve as platforms for IgE-mediated signaling.

FIGURE 1

Confocal fluorescence imaging and steady-state fluorescence anisotropy imaging of RBL-2H3 cells. diI-C₁₈ (λ_ex = 543 nm) in the plasma membrane (B and E) colocalizes with A488-IgE (λ_ex = 488 nm) on the cell surface (A and D) upon extensive cross-linking by α-IgE. Steady-state anisotropy images of diI-C₁₈ (C and F) are shown in the absence or presence of α-IgE, respectively. Higher anisotropy, or membrane order, increases toward the red end of the color scale. Bar, 5 μm.

FIGURE 2

2P-FLIM of RBL-2H3 cells. Representative 2P-FLIM images of diI-C₁₈ (D and H) and A488-IgE (B and F) in the plasma membrane of cells that have been cross-linked (F and H) with α-IgE or not (B and D) (λ_ex = 760 nm). The average lifetime (〈τ_fl〉, indicated by the color scale) was determined by fitting the lifetime decays at the pixels (binning factor = 1) highlighted by arrows, yielding A488-IgE (− α-IgE) = 1.14 ± 0.09 ns; A488-IgE (+ α-IgE) = 1.34 ± 0.03 ns; diI-C₁₈ (− α-IgE) = 0.57 ± 0.04 ns; and diI-C₁₈ (+ α-IgE) = 0.71 ± 0.03 ns. Corresponding DIC images (A, C, E, and G) show cell viability after 2P-FLIM. Histograms demonstrate an increase in the average lifetime distributions of diI-C₁₈ (J) and A488-IgE (I) that occur for cells upon cross-linking (curves 1 and 3) versus uncross-linked cells (curves 2 and 4). The free fluorescent markers in solution decay as a single exponential with average lifetimes of 0.43 ± 0.04 ns (n = 3) for 10 μM diI-C₁₈ in DMSO and 3.1 ± 0.2 ns (n = 3) for 10 μM A488 in water (images not shown). Bar, 5 μm.

FIGURE 3

1P time-resolved fluorescence lifetime of diI-C₁₈ and A488-IgE in living mast cells, under different conditions of IgE cross-linking. Representative single-point fluorescence lifetime decays for diI-C₁₈ (A) in the plasma membrane of mast cells in the presence (curve 1) or absence (curve 2) of α-IgE; and A488-IgE (B) on the surface of mast cells with (curve 3) or without (curve 4) cross-linking (λ_ex = 480 nm for both diI-C₁₈ and A488-IgE). Triexponential diI-C₁₈ decays were measured at magic angle polarization and biexponential A488-IgE decays were calculated from the measured parallel and perpendicularly polarized fluorescence decays (following the denominator of Eq. 2). In the calculated magic angle fluorescence decays, the fitting was started beyond the FWHM of the system response function. All decays were fit following Eq. 1, and the fit parameters of time-resolved fluorescence decays are summarized in Table 1. Comparison between both lifetime methods (with and without deconvolution) is included in the text where appropriate. With samples having long excited state lifetimes (i.e., Alexa 488), there are no significant differences in the fitting parameters with each method.

FIGURE 4

1P time-resolved fluorescence anisotropy of diI-C₁₈ and A488-IgE in living mast cells, under different conditions of IgE cross-linking. Representative single-point time-resolved anisotropy decays of diI-C₁₈ (A) in the plasma membrane of mast cells with (curve 1) or without (curve 2) IgE cross-linking and A488-IgE (B) on the surface of cells in the presence (curve 3) or absence (curve 4) of α-IgE (λ_ex = 480 nm for both diI-C₁₈ and A488-IgE). Curves were fit, following Eq. 2, as a single (diI-C₁₈ labeled cells) or biexponential (A488-IgE labeled cells), both with an additional residual anisotropy component. Points of the decays at times well beyond the excited state lifetime of the probe where noise levels were high (3 ns for diI-C₁₈) were not included in the fit. The fit parameters of time-resolved fluorescence anisotropy decays are summarized in Table 2. The observed associated-anisotropy feature (47) in diI-C₁₈ without cross-linking was not reproducible in all our measurements and may be attributed to the low signal/noise at times much longer than the excited-state lifetime (see Fig. 3).

FIGURE 5

Model of A488-IgE and diI-C₁₈ orientation and location within the plasma membrane in the absence (A) and presence (B) of IgE receptor cross-linking. Ordered regions of the membrane are indicated by high concentrations of cholesterol, sphingomyelin, glycosphingolipids, and glycerophospholipids. Upon receptor cross-linking, these domains cluster to nucleate a larger, functional domain.