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B cell activation involves nanoscale receptor reorganizations and inside-out signaling by Syk.

Kläsener K, Maity PC, Hobeika E, Yang J, Reth M - Elife (2014)

Bottom Line: Binding of antigen to the B cell antigen receptor (BCR) initiates a multitude of events resulting in B cell activation.How the BCR becomes signaling-competent upon antigen binding is still a matter of controversy.We also show that upon binding Syk opens the receptor by an inside-out signaling mechanism that amplifies BCR signaling.

View Article: PubMed Central - PubMed

Affiliation: BIOSS Centre for Biological Signalling Studies, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany Department of Molecular Immunology, Biology III, Faculty of Biology, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany Max Planck Institute for Immunobiology and Epigenetics, Freiburg, Germany.

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Nanoscale IgM-BCR and IgD-BCR dissociation and coreceptor reorganization on B cells expressing only one BCR isotype.(A–C) Representative microscopic images (left) and quantified results (right) of the IgM:IgM (A), the IgM:CD19 (B), and the IgM:CD20 (C) Fab-PLA of resting or activated TKO-M cells stimulated for 5 min with the antigen NIP-BSA, the oxidant pervanadate and the F-actin inhibitor Lat-A. (D–F) Representative microscopic images (left) and quantified results (right) of the IgD:IgD (D), the IgD:CD19 (E), and the IgD:CD20 (F) Fab-PLA of resting or activated TKO-D cells stimulated for 5 min with the antigen NIP-BSA, the oxidant pervanadate and the F-actin inhibitor Lat-A. Scale bar: 5 μm. The quantified data represent the mean and SEM of a minimum of three independent experiments. For each experiment, PLA signals (counts per cell) of each sample were counted from a minimum of 1000 cells and were then normalized to the PLA signal of the resting (A, D, E, F) or NIP-BSA stimulated (B and C) cells.DOI:http://dx.doi.org/10.7554/eLife.02069.012
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fig7s1: Nanoscale IgM-BCR and IgD-BCR dissociation and coreceptor reorganization on B cells expressing only one BCR isotype.(A–C) Representative microscopic images (left) and quantified results (right) of the IgM:IgM (A), the IgM:CD19 (B), and the IgM:CD20 (C) Fab-PLA of resting or activated TKO-M cells stimulated for 5 min with the antigen NIP-BSA, the oxidant pervanadate and the F-actin inhibitor Lat-A. (D–F) Representative microscopic images (left) and quantified results (right) of the IgD:IgD (D), the IgD:CD19 (E), and the IgD:CD20 (F) Fab-PLA of resting or activated TKO-D cells stimulated for 5 min with the antigen NIP-BSA, the oxidant pervanadate and the F-actin inhibitor Lat-A. Scale bar: 5 μm. The quantified data represent the mean and SEM of a minimum of three independent experiments. For each experiment, PLA signals (counts per cell) of each sample were counted from a minimum of 1000 cells and were then normalized to the PLA signal of the resting (A, D, E, F) or NIP-BSA stimulated (B and C) cells.DOI:http://dx.doi.org/10.7554/eLife.02069.012

Mentions: BCR opening, as well as the reorganization of co-receptors, does not require the co-expression of the IgM and IgD on the cell surface. TKO-M and TKO-D cells that only express one class of BCR behave similarly to the TKO-MD cells (Figure 7—figure supplement 1). Resting TKO-D cells display a close proximity between the IgD-BCR and the CD19/CD20 module but lose this interaction upon BCR stimulation, while the IgM-BCR gains contact to the CD19/CD20 module only upon B cell activation. These results suggest that on resting B cells, the CD19/CD20 module resides in a membrane compartment that is also targeted by the IgD-BCR but that is not dependent on IgD expression. To learn more about the lipid composition of this compartment, we labeled the cholera toxin B-subunit (CTB) with one of the PLA oligos. CTB binds with high affinity to GM1 gangliosides, glycolipids that are highly expressed in lipid ordered domains (Kenworthy et al., 2000). The IgD:CTB and IgM:CTB PLA showed that on resting B cells, GM1 gangliosides are found in close proximity to the IgD-BCR, whereas the IgM-BCR gains access to large amounts of these lipids only upon B cell activation (Figure 7H,G). The GPI-linked protein CD52 (Treumann et al., 1995) showed a distribution by Fab-PLA similar to that seen for the GM1 gangliosides (Figure 7—figure supplement 2). Together, these studies suggest that the IgM-BCR and the IgD-BCR are localized on the B cell surface in different membrane areas that are reorganized upon B cell activation.


B cell activation involves nanoscale receptor reorganizations and inside-out signaling by Syk.

Kläsener K, Maity PC, Hobeika E, Yang J, Reth M - Elife (2014)

Nanoscale IgM-BCR and IgD-BCR dissociation and coreceptor reorganization on B cells expressing only one BCR isotype.(A–C) Representative microscopic images (left) and quantified results (right) of the IgM:IgM (A), the IgM:CD19 (B), and the IgM:CD20 (C) Fab-PLA of resting or activated TKO-M cells stimulated for 5 min with the antigen NIP-BSA, the oxidant pervanadate and the F-actin inhibitor Lat-A. (D–F) Representative microscopic images (left) and quantified results (right) of the IgD:IgD (D), the IgD:CD19 (E), and the IgD:CD20 (F) Fab-PLA of resting or activated TKO-D cells stimulated for 5 min with the antigen NIP-BSA, the oxidant pervanadate and the F-actin inhibitor Lat-A. Scale bar: 5 μm. The quantified data represent the mean and SEM of a minimum of three independent experiments. For each experiment, PLA signals (counts per cell) of each sample were counted from a minimum of 1000 cells and were then normalized to the PLA signal of the resting (A, D, E, F) or NIP-BSA stimulated (B and C) cells.DOI:http://dx.doi.org/10.7554/eLife.02069.012
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig7s1: Nanoscale IgM-BCR and IgD-BCR dissociation and coreceptor reorganization on B cells expressing only one BCR isotype.(A–C) Representative microscopic images (left) and quantified results (right) of the IgM:IgM (A), the IgM:CD19 (B), and the IgM:CD20 (C) Fab-PLA of resting or activated TKO-M cells stimulated for 5 min with the antigen NIP-BSA, the oxidant pervanadate and the F-actin inhibitor Lat-A. (D–F) Representative microscopic images (left) and quantified results (right) of the IgD:IgD (D), the IgD:CD19 (E), and the IgD:CD20 (F) Fab-PLA of resting or activated TKO-D cells stimulated for 5 min with the antigen NIP-BSA, the oxidant pervanadate and the F-actin inhibitor Lat-A. Scale bar: 5 μm. The quantified data represent the mean and SEM of a minimum of three independent experiments. For each experiment, PLA signals (counts per cell) of each sample were counted from a minimum of 1000 cells and were then normalized to the PLA signal of the resting (A, D, E, F) or NIP-BSA stimulated (B and C) cells.DOI:http://dx.doi.org/10.7554/eLife.02069.012
Mentions: BCR opening, as well as the reorganization of co-receptors, does not require the co-expression of the IgM and IgD on the cell surface. TKO-M and TKO-D cells that only express one class of BCR behave similarly to the TKO-MD cells (Figure 7—figure supplement 1). Resting TKO-D cells display a close proximity between the IgD-BCR and the CD19/CD20 module but lose this interaction upon BCR stimulation, while the IgM-BCR gains contact to the CD19/CD20 module only upon B cell activation. These results suggest that on resting B cells, the CD19/CD20 module resides in a membrane compartment that is also targeted by the IgD-BCR but that is not dependent on IgD expression. To learn more about the lipid composition of this compartment, we labeled the cholera toxin B-subunit (CTB) with one of the PLA oligos. CTB binds with high affinity to GM1 gangliosides, glycolipids that are highly expressed in lipid ordered domains (Kenworthy et al., 2000). The IgD:CTB and IgM:CTB PLA showed that on resting B cells, GM1 gangliosides are found in close proximity to the IgD-BCR, whereas the IgM-BCR gains access to large amounts of these lipids only upon B cell activation (Figure 7H,G). The GPI-linked protein CD52 (Treumann et al., 1995) showed a distribution by Fab-PLA similar to that seen for the GM1 gangliosides (Figure 7—figure supplement 2). Together, these studies suggest that the IgM-BCR and the IgD-BCR are localized on the B cell surface in different membrane areas that are reorganized upon B cell activation.

Bottom Line: Binding of antigen to the B cell antigen receptor (BCR) initiates a multitude of events resulting in B cell activation.How the BCR becomes signaling-competent upon antigen binding is still a matter of controversy.We also show that upon binding Syk opens the receptor by an inside-out signaling mechanism that amplifies BCR signaling.

View Article: PubMed Central - PubMed

Affiliation: BIOSS Centre for Biological Signalling Studies, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany Department of Molecular Immunology, Biology III, Faculty of Biology, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany Max Planck Institute for Immunobiology and Epigenetics, Freiburg, Germany.

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Related in: MedlinePlus