Differential organization of tonic and chronic B cell antigen receptors in the plasma membrane

Abstract

Stimulation of the B cell antigen receptor (BCR) triggers signaling pathways that promote the differentiation of B cells into plasma cells. Despite the pivotal function of BCR in B cell activation, the organization of the BCR on the surface of resting and antigen-activated B cells remains unclear. Here we show, using STED super-resolution microscopy, that IgM-containing BCRs exist predominantly as monomers and dimers in the plasma membrane of resting B cells, but form higher oligomeric clusters upon stimulation. By contrast, a chronic lymphocytic leukemia-derived BCR forms dimers and oligomers in the absence of a stimulus, but a single amino acid exchange reverts its organization to monomers in unstimulated B cells. Our super-resolution microscopy approach for quantitatively analyzing cell surface proteins may thus help reveal the nanoscale organization of immunoreceptors in various cell types.

Fig. 1

Arrangement of mIgM-containing BCRs in the plasma membrane of human B cells. a Schematic depiction of the staining procedure and the generation of plasma membrane sheets. b, e, h Images of membrane sheets of unstimulated Ramos B cells (b), unstimulated primary B cells (e), and Ramos cells stimulated with anti-IgM F(ab’)₂ fragments for 30 min on ice (h). Membrane sheets were stained with the membrane marker R18 (confocal images pseudo-colored in green) and anti-human IgM affibody-Star635P (STED images displayed in red). White squares on central images indicate the zoomed regions that are displayed in the images to their right. Scale bars represent 5 µm and 1 µm for the low and high zoom images respectively. c, f, i Example histograms showing the fluorescence intensity distributions of single Star635P-conjugated affibodies on coverslips (black curves, control) and mIgM-spots present in membrane sheets (red curves, mIgM-BCRs). d, g, j Percentages of different mIgM-BCR arrangements in plasma membrane sheets were calculated after pooling the spot intensities of dozens of membrane sheets together and then fitting the intensity distribution to a sum of Gaussian curves (Supplementary Fig. 2). Data were obtained from four and three independent experiments including a total of 30 membranes from unstimulated and 15 membranes from F(ab’)₂-stimulated Ramos B cells, respectively. The experiments with primary human B cells were performed three independent times from two different donors and 30 membrane sheets were analyzed in total. Error bars represent the 68% confidence interval, corresponding to 1 standard deviation. Histogram’s source data are provided as a Source Data file

Fig. 2

mIgM-BCRs do not arrange in large protein islands at the plasma membrane. a Schematic depiction of the TIRF/dSTORM setup imaging depth. b Representative TIRF/dSTORM image of an intact Ramos B cell stained with CF^TM647-conjugated anti-IgM affibodies. The dotted white trace delineates the area of the plasma membrane close to the glass coverslip imaged under TIRF/dSTORM mode. The white square indicates the zoomed area shown in the image to the right. Scale bars represent 1 µm and 0.5 µm for the low and high zoom, respectively. c Scatter plot displaying the spot size (FWHM) distribution unstimulated Ramos cells and as resolution reference the scatter plot of single CF^TM647- affibodies spread on a glass coverslip

Fig. 3

Cell surface arrangement of a CLL-derived mIgM-BCR. a, d, g Plasma membrane sheets of DG75 B cells (a), an mIgM^neg variant expressing a CLL-derived mIgM-BCR (CLL-BCR, d) or the R38A-mutant variant thereof (CLL-R38A-BCR, g) were analyzed as in Fig. 1. White squares indicate the zoomed regions. Scale bars represent 5 µm for the low zoom images and 1 µm for the high zoom STED images. b, e, h Example histograms of the spot fluorescence intensities for each cell type. c, f, i Percentages of different mIgM-BCR arrangements were derived as in Fig. 1. Data were obtained from three independent experiments including 16 membranes for DG75, 17 membranes for the CLL-BCR, and 12 membranes for the CLL-R38A-BCR (raw data in Supplementary Fig. 5). Error bars represent the 68% confidence interval, corresponding to 1 standard deviation. Histogram’s source data are provided as a Source Data file