Visualizing lipid raft dynamics and early signaling events during antigen receptor-mediated B-lymphocyte activation

Abstract

Recent biochemical evidence indicates that an early event in signal transduction by the B-cell antigen receptor (BCR) is its translocation to specialized membrane subdomains known as lipid rafts. We have taken a microscopic approach to image lipid rafts and early events associated with BCR signal transduction. Lipid rafts were visualized on primary splenic B lymphocytes from wild-type or anti-hen egg lysozyme BCR transgenic mice, and on a mature mouse B-cell line Bal 17 by using fluorescent conjugates of cholera toxin B subunit or a Lyn-based chimeric protein, which targets green fluorescent protein to the lipid raft compartment. Time-lapse imaging of B cells stimulated via the BCR with the antigen hen egg lysozyme, or surrogate for antigen anti-IgM, demonstrated that lipid rafts are highly dynamic entities, which move laterally on the surface of these cells and coalesce into large regions. These regions of aggregated lipid rafts colocalized with the BCR and tyrosine-phosphorylated proteins. Microscopic imaging of live B cells also revealed an inducible colocalization of lipid rafts with the tyrosine kinase Syk and the receptor tyrosine phosphatase CD45. These two proteins play indispensable roles in BCR-mediated signaling but are not detectable in biochemically purified lipid raft fractions. Strikingly, BCR stimulation also induced the formation of long, thread-like filopodial projections, similar to previously described structures called cytonemes. These B-cell cytonemes are rich in lipid rafts and actin filaments, suggesting that they might play a role in long-range communication and/or transportation of signaling molecules during an immune response. These results provide a window into the morphological and molecular organization of the B-cell membrane during the early phase of BCR signaling.

Figure 1

Correlation between lipid raft staining with cholera toxin B and Lyn24GFP. (A) A marker specific to lipid rafts was constructed by fusing the N-terminal 24 amino acids of the Src family tyrosine kinase Lyn, which contain N-terminal myristylation (myr) and palmitoylation (palm) sites, to the N terminus of green fluorescent protein (see MATERIALS AND METHODS). The resulting chimera was termed Lyn24GFP. (B) Bal 17 cells were transiently transfected with the Lyn24GFP construct and after overnight expression stained with 25 μg/ml CTB-TRITC. Images were acquired of surface planar sections simultaneously using the fluorescein and rhodamine filters, and single-color and overlay images are shown as indicated.

Figure 2

Lipid rafts coalesce and polarize upon BCR ligation. Bal 17 cells (A) or splenic B cells from C57BL/6 mice (B) were left unstimulated (left) or stimulated with 25 μg/ml anti-mouse IgM F(ab′)₂ for 10 min (right), fixed with 4% PFA, and stained with CTB-TRITC. The cell suspension was placed on a glass slide, covered with a coverslip, and images were acquired, using the rhodamine-phycoerythrin-Texas Red filter, of a surface planar section for splenic B cells (B), or a Z-stack of 50 adjacent focal planes for Bal 17 cells followed by three-dimensional reconstruction by using the Softworx program (A). The areas of the cell that are enriched for lipid rafts are indicated by white arrows.

Figure 3

BCR colocalizes with lipid rafts upon ligation. Bal 17 cells were stained with CTB-FITC as described in text, followed by treatment with 25 μg/ml rhodamine conjugates of anti-IgM Fab fragment or anti-IgM F(ab′)₂ fragment at room temperature. MD4 B cells were stimulated with 10 mg/ml plate-bound HEL. Images were collected after 10 min, by using the fluorescein and rhodamine filters of the Deltavision microscope. Lipid raft staining is shown in green, BCR in red and the colocalization regions in yellow as indicated by the white arrows.

Figure 4

CD45 colocalizes with lipid rafts after BCR cross-linking. (A) Bal 17 cells were stimulated (+) or not (−) with 25 μg/ml anti-IgM F(ab′)₂ for 10 min at 37°C. MD4 B cells were stimulated with plate-bound HEL for 10 min at 37°C. The stimulation was stopped by fixing the cells with 4% PFA followed by staining lipid rafts with CTB-TRITC. The cells were washed and preblocked with the Fc blocking antibody 2.4G2 for 10 min followed by washing and staining with anti-CD45-FITC for 30 min at 4°C. After washing the cells were visualized using the fluorescein and rhodamine filters of the Deltavision microscope. The panels showing CD45 and CTB staining in unstimulated cells are representative of 90% of the population in which no colocalization was observed (refer to Table 1 for quantitation data). In the other 10% of cells, some colocalization was seen. (B) Bal 17 cells were adhered onto a poly-l-lysine–coated glass dish for 1 h and after blocking Fc receptor binding by treating with 2.4G2 for 10 min, the cells were stained with FITC-conjugated anti-CD45 for 30 min at 4°C. The cells were washed and subsequently stained with CTB-TRITC. The dish was fixed on a stage adapter, the microscope focused on the cells and images acquired using the fluorescein and rhodamine filters in the unstimulated state (top). Anti-IgM F(ab′)₂ (50 μg/ml) was added to the dish and image acquisition was started simultaneously. Images were collected over a period of 10 min with a time lapse of 15 s. Surface planar views of the cells are shown, with CD45 in green, lipid rafts in red and regions of colocalization in yellow indicated by white arrows. The time course of CD45 colocalization, after BCR stimulation, can be viewed in movie format (Movie 1) in the supplementary information section.

Figure 5

Transferrin receptor (CD71) does not colocalize with lipid rafts either before or after BCR ligation. Bal 17 cells were either left unstimulated (left) or stimulated (right) with 25 μg/ml anti-IgM F(ab′)₂ fragment for 10 min at 37°C. The reaction was stopped by fixing the cells with 4% PFA for 20 min at room temperature. The cells were washed and stained with CTB-TRITC as described in text, followed by blocking with 2.4G2 antibody for 10 min. After washing, the cells were stained with FITC-conjugated anti-CD71 (anti-transferrin receptor) antibody for 30 min at 4°C. Images were collected using the fluorescein and rhodamine filters of the Deltavision microscope. Lipid raft staining is shown in red, CD71 in green, and the colocalization regions in yellow.

Figure 6

Syk translocates to lipid rafts upon BCR cross-linking with a concomitant association of tyrosine phosphorylated proteins with aggregated lipid rafts. (A) Bal 17 cells stably expressing Syk-GFP were stimulated with 25 μg/ml anti-mouse IgM at room temperature. Next, 12 μl of the cell suspension was placed on a slide and covered with a coverslip. After focusing on cells, image acquisition was started and continued every 10 s for a period of 10 min. All 60 images were saved as a time-lapse movie (supplementary data, Movie 2). Shown are still images at seven time points as indicated. The pattern of patching and capping seen in the upper cell was more commonly observed than the pattern seen for the lower cell, which has patches of Syk concentration at 10 min but has not yet started capping. (B) Bal 17 cells were transfected with 20 μg of Syk-GFP fusion construct. After 24 h, cells were adhered onto a poly-l-lysine–coated glass dish for 1 h and stained with CTB-TRITC. The dish was fixed on a stage adapter, and images were acquired using the fluorescein and rhodamine filters in the unstimulated state (top row). Anti-IgM F(ab′)₂ (50 μg/ml) was added to the dish and images were acquired again after 10 min of stimulation (bottom row). Surface views of the cells are shown, with Syk-GFP in green, lipid rafts in red, and regions of colocalization in yellow. (C) Bal 17 cells were preincubated (bottom row) or not (top and middle rows) with 10 μM PP1 and either left unstimulated (top row), or stimulated with 25 μg/ml anti-IgM F(ab′)₂ (middle and bottom rows) for 10 min at 37°C, and the reaction stopped by fixing the cells in 4% PFA. The cells were then stained with CTB-TRITC as described in text, washed twice, permeabilized with 0.5% saponin, stained with anti-phosphotyrosine-FITC, and washed twice. Images were acquired using the fluorescein and rhodamine filters. Phosphotyrosine staining is shown in green, lipid rafts in red, and regions of colocalization in yellow.

Figure 7

Induction of cytoneme-like extensions of the B-cell membrane upon BCR engagement. (A–C) Bal 17 cells or (D) primary splenic B cells were stained with CTB-TRITC as described in text and images of unstimulated cells (left) were acquired. (A) Cells were stimulated with 25 μg/ml anti-IgM F(ab′)₂ at room temperature and images were acquired at 30 min. The white arrowhead and arrow indicate the concentrated lipid raft staining at a branch point and the tip of the B-cell cytoneme, respectively. (B) This experiment was similar to A except that the cells were plated sparsely and the image shown is at 40 min after anti-IgM cross-linking. The length of the cytonemes was measured using the Softworx tools and the thickness was measured at the limit of resolution of light microscopy to be approx. 0.2–0.4. (C) In this experiment, images were acquired of a Z-stack of 50 adjacent focal planes of a cell that had been stained with CTB-TRITC and stimulated with anti-IgM for 20 min. (D) Splenic B cells were similarly treated with anti-IgM at room temperature and images acquired at 30 min. The left panel shows a representative unstimulated cell and the middle and right panels show two representative cells with induced cytonemes. In the middle panel, the body of cell 1 is out of focus but the cytoneme stained with CTB-TRITC can be seen clearly. Cell 2 in the right panel shows two protruding cytonemes.

Figure 8

Punctate staining pattern for lipid rafts and actin along the length of B cell-cytonemes. (A) Bal 17 cells were stained with CTB-TRITC and cells were stimulated with 25 μg/ml anti-IgM F(ab′)₂ at room temperature and images were acquired at 15 min. (B) Bal 17 cells were transfected with an expression plasmid encoding an actin-GFP fusion protein and after 24 h the transfectants were adhered to glass Delta T dishes. The cells were stimulated with 50 μg/ml anti-IgM F(ab′)₂ at room temperature for 15 min and images were acquired using a GFP filter set on the Deltavision microscope. The white arrows indicate the punctate staining pattern for lipid rafts (A) and actin-GFP (B) of the B cell cytonemes of stimulated cells.