Intrinsic properties of immunoglobulin IgG1 isotype-switched B cell receptors promote microclustering and the initiation of signaling.

Mice, cells, antibodies, plasmids, and transfections Primary B cells were isolated from spleens of Igα-YFP Tg C57BL/6 mice generated and characterized in our lab or normal C57BL/6 mice by negative selection using MACS R sorting as described ( Tolar et al., 2009a ). Mice were treated in accordance with guidelines approved by NIH animal care and use committees. To induce class switching of IgM-BCRs to IgG1-BCRs, we incubated the purified spleen B cells from Igα-YFP Tg mice with 40 μg/ml LPS and 20ng/ml recombinant mouse IL-4 for 72 h following a well established protocol ( Kaisho et al., 1997 ). Purified unconjugated Fab goat anti-mouse Fc IgM (anti-IgM), F(ab′)2 goat anti-mouse IgG F(ab′)2, goat anti-mouse Ig light chain (anti-L), and goat anti-mouse IgG Fc portion were purchased from Jackson ImmunoResearch. Detailed lists of all the antibodies used in this study are provided as supplemental information . Plasmids expressing Igα-YFP were constructed as described ( Sohn et al., 2006 ).

Plasmids expressing γ1-B1-8 fused with a C-terminus

CFP through the linker peptide, GGGAAS (γ1-B1-8-CFP), were constructed as described ( Tolar et al., 2005 ). Using the γ1-B1-8-CFP plasmid as a template, γ1-B1-8-High-CFP (γ1-High) and γ1-B1-8-Low-CFP (γ1-Low) were generated using a QuikChange ™ II XL Site-Directed Mutagenesis Kit (Stratagene). The detailed mutagenesis procedures and acquisition of γ1-High and γ1-Low J558L cells by transfection and cell sorting are provided in supplemental information . Cytoplasmic tail chimeric γ1-High or μ-High mutants To construct the cytoplasmic tail chimera γ1-High and μ-High mutants, γ1-B1-8-High-CFP (γ1-WT) or μ-B1-8-High-CFP (μ-WT) plasmids were respectively used as parent plasmids. Based on γ1-WT, γ1-Cyto N12 γ1, γ1-Cyto μ and γ1-Y384F cytoplasmic tail mutant were acquired using primer A–C respectively ( Figure S6 ) in QuikChange ™ II XL Site-Directed Mutagenesis Kit (Stratagene). Similarly, based on μ-WT, μ-Cyto γ1, μ-Cyto N12 γ1 and μ-TMγ1-Cyto μ are acquired using primer D–G respectively ( Figure S6 ) in mutagenesis polymerase chain reaction (PCR). All the cytoplasmic amino acid sequences and schematic presentations of these WT or mutant IgM or IgG1 heavy chain constructs are given in Figure 6A . We have provided detailed experimental procedures to produce these constructs in supplemental information . Transfections of γ1-WT, γ1-Y384F, γ1-Cyto N12 γ1, or γ1-Cyto μ constructs into purified splenic B cells from C57BL/6 mice were performed using an optimized Amaxa protocol for stimulated C57BL/6 mouse primary spleen B cells with an Amaxa Nucleofector ™ Kit. Briefly, splenic B cells were purified and enriched from the spleen of 10 week-old C57BL/6 mice by negative selection using MACS R sorting. Purified splenic B cells were incubated overnight with 50 μg/ml LPS. The LPS-stimulated B cells were transfected with these DNA constructs respectively using Amaxa mouse B cells Nucleofector ™ solutions and transfection program of Z-001. Transfected B cells were cultured overnight and imaged the next day. Two-color time lapse live cell imaging by TIRFM Cells were placed on planar fluid lipid bilayers containing NIP hapten antigen or biotinylated anti-Ig surrogate antigen prepared as described ( Tolar et al., 2009a ) and provided in detail in supplemental information . TIRF images were acquired at 37 °C on a heated stage by an Olympus IX-81 microscope supported by a TIRF port, CascadeII 512 × 512 electron-multiplying CCD camera (Roper Scientific), Olympus 100 × 1.45 N.A. and Zeiss 100 × 1.4 N.A. objective lens. The acquisition was controlled by Metamorph (Molecular Devices). The exposure time was 100 ms unless specially indicated. Three types of lasers were used: a 442 nm solid state laser; a 488 nm and 514 nm argon gas laser and a 568 nm and 647 nm red krypton and argon gas laser. TIRF images were analyzed by Image Pro Plus (Media Cybernetics), Image J (NIH, US) or Matlab (Mathworks) software as indicated. Before analysis, images were split, aligned, background subtracted and corrected for spectral bleed-through using either Image Pro Plus or Matlab software. Imaging of BCR, antigen and pSyk microclusters by TIRFM were as recently described ( Depoil et al., 2008 ; Tolar et al., 2009a ) and detailed as supplemental information .

Single particle tracking and analysis

J558L cells were incubated with 1nM of Alexa 568-anti-IgG as described ( Tolar et al., 2009a ). The acquisition and analyses of single BCR molecule TIRF images were as described ( Tolar et al., 2009a ). Briefly, a sub-region of roughly 100 × 100 pixels of the available area of the EMCCD chip (512 × 512 pixels) was used to achieve an exposure time of 35 ms per frame. Single BCR molecules were captured on 300 frames in a time course of 10 s in streamline acquisition mode, the time resolution of which was found to be sufficient to reliably track the single molecule BCRs as reported ( Tolar et al., 2009a ). Single molecule tracking was performed using Matlab (Mathworks) code based on available positional fitting and tracking algorithms ( Crocker and Grier, 1996 ; Douglass and Vale, 2008 ). Mean square displacements (MSD) and short-range diffusion coefficients for each BCR molecule trajectories (D 0 , based on time intervals of 35–140 ms) were calculated from positional coordinates as described ( Douglass and Vale, 2008 ). The MSD plot was mathematically fitted into a confined diffusion model by an exponential function to acquire the size of the confinement microdomain. Analysis of fluorescence intensities and sizes of the BCR and antigen microclusters Precise 2D positions and integrated FI of the BCR or antigen microclusters in time lapse TIRF images were obtained by means of least squares fitting of a 2D Gaussian function as below to each of the 2D FI profiles at each time point ( Holtzer et al., 2007 ). f ( x , y ) = z 0 + I 4 ln 2 π σ r 2 e − [ 4 ln 2 ( ( x − x c ) 2 σ r 2 / ε 2 + ( y − y c ) 2 σ r 2 ε 2 ) ] For each microcluster the fit yields ‘personalized’ 2D Gaussian functions to mathematically describe the point spreading of the microcluster in each image at each time point. The ‘personalized’ 2D Gaussian functions give the parameters of local background FI (z 0 ), position (x c , y c ), integrated FI (I), and generalized full width at half maximum peak height (FWHM, σ x σ y ) of the intensity distribution in x and y direction, respectively, as described in detail ( Liu et al., 2010 ). Only microclusters that were successfully tracked for at least 10 steps and only the first 60 steps (120 s) of each track from J558L microclusters were selected for analysis. This selection was necessary in order to avoid tracking and Gaussian fitting errors, both of which arise from spots merging and overlapping at later stages of the observed processes. Arithmetic means and standard errors of the FI or FWHM values of individual microcluster were calculated for all selected spots present in one frame and plotted versus time. Values belonging to the same track were normalized to the first position. The statistical test used to compare the kinetics of microcluster growth is as described ( Baldwin et al., 2007 ; Elso et al., 2003 ; Hammarlund et al., 2003 ) or through online server ( http://bioinf.wehi.edu.au/software/compareCurves/index.html ).

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