The spatial relationship between FcRL4 and the BCR during formation of the immune synapse. (A-D) FcRL4⁻ B cells and FcRL4⁺ B cells stably expressing FcRL4-YFP were stained with Fab goat Abs specific for human IgM (Fab anti-IgM) conjugated with DyLight649 to label the BCR. The cells were placed on planar lipid bilayers containing ICAM and Fab anti-IgM at 37°C and 2-color time-lapse live cell TIRF images were recorded at 2-second intervals for 10 minutes using an Olympus IX-81 microscope equipped with a TIR illumination port, IX2 ZDC autofocus laser system, and 100 × 1.45 numerical aperture objectives lens. Images were processed as described in “Preparation of PLBs and live cell imaging” to quantify the data. (A) The distribution of the surface BCR in the contact area of FcRL4⁻ and FcRL4⁺ B cells with the bilayer 5 minutes after cells contacted the bilayers. For FcRL4⁺ cells, images of the FcRL4 alone and merged images with BCR are given. Scale bars represent 10 μm. (B) Normalized mean fluorescence intensity (NMFI) of the surface BCR in the contact area between the cells and the bilayers with time calculated from time-lapse live-cell TIRF images are given. The mean ± SD was calculated and compared between FcRL4⁻ (n = 18) and FcRL4⁺ (n = 20) B cells. (C) Dynamics of the co-localization between FcRL4 and the BCR in the contact area of FcRL4⁺ B cells and the planar lipid bilayer over time. Shown are FcRL4, BCR, and merged images at the indicated time points. (D) The colocalization coefficient (Rr) is given for FcRL4 and the BCR versus time calculated as mean ± SD by intensity correlation analysis between the BCR and FcRL4 in FcRL4⁺ cells (n = 10 from 3 different experiments). (E-G) Cells expressing either wild-type FcRL4 or all 3 tyrosines in its cytoplasmic tail-mutated FcRL4 (FFF) were labeled with both Cy3-Fab anti-IgM and Alexa Fluor 647–conjugated FcRL4-specific mAb on ice and placed on the planar lipid bilayer at 37°C. After 10 minutes of incubation on the bilayer, cells were fixed with 4% paraformaldehyde and imaged as in panel A. (E) Distribution in the contact area of the BCR (red) and either WT FcRL4 or FcRL4 (FFF; green) and merged images are shown. (F) Given are the NMFIs of the BCR in the contact area between the cells and the bilayers calculated from TIRF images for cells expressing wild-type FcRL4 (n = 50) and FcRL4 (FFF; n = 57) from the representative of 3 independent experiments. (G). Rr calculated as mean ± SD as described in panel D is given for the BCR and either wild-type FcRL4 (n = 20) or FcRL4 (FFF; n = 30).