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

Sohn HW, Tolar P, Pierce SK - J. Cell Biol. (2008)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852, USA.

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.

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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.
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fig3: 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.

Mentions: Previous studies using FRET confocal microscopy provided evidence that antigen binding from solution resulted in a conformational change in the BCR cytoplasmic domains from a “closed” to an “open” form and simultaneous phosphorylation of the BCR (Tolar et al., 2005). We observed that when clustered by antigen, BCRs containing a membrane Ig–CFP and Igα-YFP showed an initial increase in FRET, reflecting the close molecular proximity of the cytoplasmic domains of the clustered BCRs, and then a drop in FRET, indicating that the cytoplasmic domains within the BCR clusters moved apart or opened. To determine whether the observed FRET between Igα-YFP and Lyn16-CFP correlated either temporally or spatially with the antigen-induced transition in the BCR to an open form, we measured FRET in a J558L cell line expressing an NIP-specific BCR containing membrane Ig–CFP and Igα-YFP as the B cells encountered a bilayer containing NIP and ICAM-1 (Fig. 3). Tracking the initial BCR microclusters individually from their formation to the generation of the synapse showed that within the first few seconds of formation, FRET in the microclusters sharply increased, indicating the induced close molecular proximity of the cytoplasmic domains of the BCRs within the microclusters (Fig. 3 A). Despite the continued accumulation of BCRs in the clusters, the FRET level reached a peak and then dropped, which is consistent with a synchronized opening of the cytoplasmic domains of the BCR clusters as previously described (Tolar et al., 2005).


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

Sohn HW, Tolar P, Pierce SK - J. Cell Biol. (2008)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2483512&req=5

fig3: 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.
Mentions: Previous studies using FRET confocal microscopy provided evidence that antigen binding from solution resulted in a conformational change in the BCR cytoplasmic domains from a “closed” to an “open” form and simultaneous phosphorylation of the BCR (Tolar et al., 2005). We observed that when clustered by antigen, BCRs containing a membrane Ig–CFP and Igα-YFP showed an initial increase in FRET, reflecting the close molecular proximity of the cytoplasmic domains of the clustered BCRs, and then a drop in FRET, indicating that the cytoplasmic domains within the BCR clusters moved apart or opened. To determine whether the observed FRET between Igα-YFP and Lyn16-CFP correlated either temporally or spatially with the antigen-induced transition in the BCR to an open form, we measured FRET in a J558L cell line expressing an NIP-specific BCR containing membrane Ig–CFP and Igα-YFP as the B cells encountered a bilayer containing NIP and ICAM-1 (Fig. 3). Tracking the initial BCR microclusters individually from their formation to the generation of the synapse showed that within the first few seconds of formation, FRET in the microclusters sharply increased, indicating the induced close molecular proximity of the cytoplasmic domains of the BCRs within the microclusters (Fig. 3 A). Despite the continued accumulation of BCRs in the clusters, the FRET level reached a peak and then dropped, which is consistent with a synchronized opening of the cytoplasmic domains of the BCR clusters as previously described (Tolar et al., 2005).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852, USA.

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.

Show MeSH
Related in: MedlinePlus