Membrane heterogeneities in the formation of B cell receptor-Lyn kinase microclusters and the immune synapse

Abstract

Antigen binding to the B cell receptors (BCRs) induces BCR clustering, phosphorylation of BCRs by the Src family kinase Lyn, initiation of signaling, and formation of an immune synapse. We investigated B cells as they first encountered antigen on a membrane using live cell high resolution total internal reflection fluorescence microscopy in conjunction with fluorescence resonance energy transfer. Newly formed BCR microclusters perturb the local membrane microenvironment, leading to association with a lipid raft probe. This early event is BCR intrinsic and independent of BCR signaling. Association of BCR microclusters with membrane-tethered Lyn depends on Lyn activity and persists as microclusters accumulate and form an immune synapse. Membrane perturbation and BCR-Lyn association correlate both temporally and spatially with the transition of microclustered BCRs from a "closed" to an "open" active signaling conformation. Visualization and analysis of the earliest events in BCR signaling highlight the importance of the membrane microenvironment for formation of BCR-Lyn complexes and the B cell immune synapse.

Figure 1.

Time-lapse FRET imaging using TIRFM reveals the spatial and temporal dynamics of the change in the lipid environment of the BCR during antigen contact. (A) Time-lapse TIRFM images of CH27 B cells expressing Igα-YFP and Lyn16-CFP contacting a planar lipid bilayer containing only ICAM-1 or ICAM-1 and the antigen PC₁₀-BSA. Three-channel TIRFM images were acquired using an electron-multiplier CCD camera. The CFP and YFP images are shown. FRET was calculated by sensitized acceptor emission as described in Materials and methods. Corrected net FRET (Fc = F − β × D − γ × A) and FRET efficiency from representative cells are shown as color-coded scaled images. A merged image of CFP (red), YFP (blue), and Ea (green) is also shown, and the relative FIs across the cells indicated by red lines (scale, 20 μm) are given. (B) FIs of Igα-YFP, Lyn16-CFP, and FRET efficiencies with time for the cells imaged in A. Note that a 10:1 ratio of CFP to YFP FIs is equivalent to a 1:1 molar ratio of CFP to YFP under our experimental setting as described in Materials and methods. (C) Mean + SEM (error bars) of the FRET efficiencies for multiple cells expressing Igα-YFP and either Lyn16-CFP (12 cells) or Ger-CFP (six cells). The data are from one of three independent experiments.

Figure 2.

The interactions of BCR clusters and the raft lipid probe are not dependent on the initiation of BCR signaling or on the actin cytoskeleton. (A) CFP, YFP, and Ea time-lapse images (acquired as in Fig. 1) of CH27 B cells expressing Igα-YFP and the raft lipid probe, Lyn16-CFP, at 4, 60, and 600 s after an encounter with an ICAM-1– and antigen-containing bilayer. A merged image of YFP and Ea and the relative FIs of YFP (red) and Ea (green) across the contact area of the merged image indicated by red lines (scale, 20 μm) are given. CH27 B cells were either untreated or pretreated with the Src family kinase inhibitor PP₂ (50 μm for 1 h at 37°C) or latrunculin B (10 μm for 30 min at 37°C). (B) Three-channel images (CFP, YFP, and FRET) of individual cells as performed in A were collected at 2-s intervals, and the FRET efficiency (Ea) was calculated as in Fig. 1 and described in Materials and methods. The mean Ea + SEM from six untreated cells, five PP2-treated cells, and six latrunculin B–treated cells is shown. (C) CFP, YFP, Ea, and merged images of J558L B cells expressing either the wild-type NIP-specific BCR (Igα_YY/Igβ_YY)-YFP or the mutant signaling-deficient BCR (Igα_YY→_FF/Igβ_YY→_FF)-YFP and Lyn16-CFP 1 min after the B cells encountered ICAM-1– and antigen-containing bilayers. Bars, 10 μm. The tyrosine to phenylalanine mutations in the ITAM of Igα/Igβ are indicated as asterisks in the BCR diagram (GFPs and BCR are not drawn to scale). (D and E) The number of BCR microclusters (D) and the FRET efficiency (Ea; E) were determined for single J558L cells expressing either the wild-type BCR or ITAM mutant BCR during time-lapse TIRF microscope imaging for 10 min after the cells contacted ICAM-1– and antigen-containing bilayers. The number of BCR clusters and FRET efficiencies were determined as described in Materials and methods at the time of peak FRET. Frequency plots of representative cells taken from the data of four independent experiments are shown. Horizontal lines are the median values of all individual points. D, 15–20 cells; E, 10–15 cells. P-values determined by t test are also shown.

Figure 3.

Conformational change in the BCR cytoplasmic domains correlates temporally and spatially with the BCR's association with the lipid raft probe. (A) J558L cells expressing B1-8γ-CFP and Igα-YFP were allowed to spread on bilayers containing NIP₁₄-BSA. TIRFM images were acquired, and FRET efficiency was calculated from tracking of individual clusters of the BCR as described in Materials and methods. Data represent mean ± SEM (error bars) from 14 clusters from multiple cells in four independent experiments. The corrected average FI of the BCR in clusters is also shown. Corresponding phases of the cluster lifetime are shown at the top. (B) Images of J558L cells expressing B1-8γ-CFP and Igα-YFP spreading on a NIP₁₄-BSA–containing bilayer. IgG-CFP (left) and YFP/CFP fluorescence ratio images at 442-nm laser illumination at the indicated times from the initial contact are shown.

Figure 4.

The antigen-induced association of Lyn kinase with BCR. (A) CFP, YFP, Ea, and merged time-lapse images of CH27 B cells expressing LynFL-CFP and Igα-YFP 0–600 s after the encounter of either ICAM-1 alone or ICAM-1– and antigen-containing bilayers. CFP and YFP FIs and Ea across the contact area indicated by red lines (scale, 20 μm) are given. (B) Quantification of the Igα-YFP and LynFL-CFP FIs and Ea plotted against time for cells imaged in A.

Figure 5.

FRET between antigen-clustered BCR and Lyn kinase is prolonged and sensitive to PP2 inhibition. (A) Comparison of FRET efficiencies between Igα-YFP and Lyn16-CFP and between Igα-YFP and LynFL-CFP with time. (B) A comparison of the Ea between Igα-YFP and LynFL-CFP in the presence or absence of PP2. The calculation of FRET efficiency was performed as described in Materials and methods. Mean + SEM (error bars) of calculated Ea are shown with time for 12 cells expressing Lyn16-CFP, six cells expressing LynFL-CFP, and three cells expressing LynFL-CFP treated with PP2.

Figure 6.

BCR clustering precedes by several seconds the association of clustered BCR with raft lipids and Lyn kinase. (A) The merged TIRFM images of CFP (red), YFP (blue), and Ea (green; top) and quantification of the fold increases of CFP and YFP FIs and Ea for the BCR microclusters (bottom). The images were taken at 8-s intervals of CH27 B cells expressing either Igα-YFP and the lipid raft probe, Lyn16-CFP (top row), or Igα-YFP and LynFL kinase, LynFL-CFP (bottom row), as the cell first encounters an ICAM-1– and antigen-containing bilayer. The clusters marked by white arrows were quantified as the fold intensities relative to the intensities of the first frame in each channel showing a signal over background over a 4-μm² region of interest surrounding the clusters. Bars, 2 μm. (B) TIRFM time-lapse images were taken of CH27 B cells expressing either Igα-YFP and Lyn16-CFP or Igα-YFP and LynFL-CFP as they contacted PC₁₀-BSA– and ICAM-1–containing bilayers. Individual BCR clusters that formed in the periphery of the contact area and moved to the central synapse were tracked, and the pattern of movement of single BCR clusters was analyzed by time-lapse Ea images as described in Materials and methods. The time delay before a cluster that formed in the periphery moved to the center was determined. A total of ∼200 clusters from cells expressing either both Igα-YFP and Lyn16-CFP (four cells) or both Igα-YFP and LynFL-CFP (three cells) were included in the analyses. Error bars represent SEM.