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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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The antigen-induced association of Lyn kinase with BCR. (A) CFP, YFP, Ea, and merged time-lapse images of CH27 B cells expressing LynFL-CFP and Igα-YFP 0–600 s after the encounter of either ICAM-1 alone or ICAM-1– and antigen-containing bilayers. CFP and YFP FIs and Ea across the contact area indicated by red lines (scale, 20 μm) are given. (B) Quantification of the Igα-YFP and LynFL-CFP FIs and Ea plotted against time for cells imaged in A.
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fig4: The antigen-induced association of Lyn kinase with BCR. (A) CFP, YFP, Ea, and merged time-lapse images of CH27 B cells expressing LynFL-CFP and Igα-YFP 0–600 s after the encounter of either ICAM-1 alone or ICAM-1– and antigen-containing bilayers. CFP and YFP FIs and Ea across the contact area indicated by red lines (scale, 20 μm) are given. (B) Quantification of the Igα-YFP and LynFL-CFP FIs and Ea plotted against time for cells imaged in A.

Mentions: Association of the antigen-induced BCR microclusters with the lipid raft probe has been suggested to play a role in facilitating the association of clustered BCR with the Lyn kinase. To directly characterize the interaction of the BCR with Lyn, CH27 B cells were analyzed that expressed Igα-YFP and full-length Lyn (LynFL) linked by six amino acids at the C terminus to CFP (LynFL-CFP). Images of B cells engaging a bilayer containing only ICAM-1 showed extensive colocalization of Igα-YFP with LynFL-CFP but no FRET (Fig. 4 A). In contrast, images of B cells engaging an ICAM-1– and antigen-containing bilayer showed significant FRET at the first points of contact where LynFL-CFP colocalized with Igα-YFP (Fig. 4 A). FRET between LynFL-CFP and Igα-YFP persisted as the BCR microclusters formed a central synapse. Quantification of the FIs and FRET over the contact area with time showed that although the relative FIs of Igα-YFP and LynFL-CFP were similar with time in the presence or absence of antigen, FRET was only detected when antigen was present (Fig. 4 B). Thus, the FRET cannot be attributed to a change in the ratios of YFP and CFP. The concentration of FRET between the BCR and LynFL in the center synapse was in contrast to the observation for the lipid raft probe and BCR, in which case FRET was highest in the cell's periphery. A time-lapse video illustrated this difference, showing that FRET between the BCR and raft lipid probe was more restricted to the cells' periphery in contrast to the FRET between Lyn and the BCR that was concentrated in the synapse (Fig. S1 and Videos 1–8, available at http://www.jcb.org/cgi/content/full/jcb.200802007/DC1).


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)

The antigen-induced association of Lyn kinase with BCR. (A) CFP, YFP, Ea, and merged time-lapse images of CH27 B cells expressing LynFL-CFP and Igα-YFP 0–600 s after the encounter of either ICAM-1 alone or ICAM-1– and antigen-containing bilayers. CFP and YFP FIs and Ea across the contact area indicated by red lines (scale, 20 μm) are given. (B) Quantification of the Igα-YFP and LynFL-CFP FIs and Ea plotted against time for cells imaged in A.
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Related In: Results  -  Collection

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fig4: The antigen-induced association of Lyn kinase with BCR. (A) CFP, YFP, Ea, and merged time-lapse images of CH27 B cells expressing LynFL-CFP and Igα-YFP 0–600 s after the encounter of either ICAM-1 alone or ICAM-1– and antigen-containing bilayers. CFP and YFP FIs and Ea across the contact area indicated by red lines (scale, 20 μm) are given. (B) Quantification of the Igα-YFP and LynFL-CFP FIs and Ea plotted against time for cells imaged in A.
Mentions: Association of the antigen-induced BCR microclusters with the lipid raft probe has been suggested to play a role in facilitating the association of clustered BCR with the Lyn kinase. To directly characterize the interaction of the BCR with Lyn, CH27 B cells were analyzed that expressed Igα-YFP and full-length Lyn (LynFL) linked by six amino acids at the C terminus to CFP (LynFL-CFP). Images of B cells engaging a bilayer containing only ICAM-1 showed extensive colocalization of Igα-YFP with LynFL-CFP but no FRET (Fig. 4 A). In contrast, images of B cells engaging an ICAM-1– and antigen-containing bilayer showed significant FRET at the first points of contact where LynFL-CFP colocalized with Igα-YFP (Fig. 4 A). FRET between LynFL-CFP and Igα-YFP persisted as the BCR microclusters formed a central synapse. Quantification of the FIs and FRET over the contact area with time showed that although the relative FIs of Igα-YFP and LynFL-CFP were similar with time in the presence or absence of antigen, FRET was only detected when antigen was present (Fig. 4 B). Thus, the FRET cannot be attributed to a change in the ratios of YFP and CFP. The concentration of FRET between the BCR and LynFL in the center synapse was in contrast to the observation for the lipid raft probe and BCR, in which case FRET was highest in the cell's periphery. A time-lapse video illustrated this difference, showing that FRET between the BCR and raft lipid probe was more restricted to the cells' periphery in contrast to the FRET between Lyn and the BCR that was concentrated in the synapse (Fig. S1 and Videos 1–8, available at http://www.jcb.org/cgi/content/full/jcb.200802007/DC1).

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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