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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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FRET between antigen-clustered BCR and Lyn kinase is prolonged and sensitive to PP2 inhibition. (A) Comparison of FRET efficiencies between Igα-YFP and Lyn16-CFP and between Igα-YFP and LynFL-CFP with time. (B) A comparison of the Ea between Igα-YFP and LynFL-CFP in the presence or absence of PP2. The calculation of FRET efficiency was performed as described in Materials and methods. Mean + SEM (error bars) of calculated Ea are shown with time for 12 cells expressing Lyn16-CFP, six cells expressing LynFL-CFP, and three cells expressing LynFL-CFP treated with PP2.
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fig5: FRET between antigen-clustered BCR and Lyn kinase is prolonged and sensitive to PP2 inhibition. (A) Comparison of FRET efficiencies between Igα-YFP and Lyn16-CFP and between Igα-YFP and LynFL-CFP with time. (B) A comparison of the Ea between Igα-YFP and LynFL-CFP in the presence or absence of PP2. The calculation of FRET efficiency was performed as described in Materials and methods. Mean + SEM (error bars) of calculated Ea are shown with time for 12 cells expressing Lyn16-CFP, six cells expressing LynFL-CFP, and three cells expressing LynFL-CFP treated with PP2.

Mentions: FRET between Igα-YFP and either Lyn16-CFP or LynFL-CFP was quantified with time over the entire contact area of the B cells interacting with ICAM-1– and antigen-containing bilayers and was compared (Fig. 5 A). FRET between Lyn16-CFP and Igα-YFP increased rapidly upon initial B cell contact with the antigen-containing bilayer during the initial contact phase and then decreased as B cells spread and BCR clusters moved to the center of the synapse. FRET between LynFL-CFP and Igα-YFP showed an initial small peak that corresponded in time to the FRET between Igα-YFP and Lyn16-CFP during the initial contact phase (Fig. 5 A). The initial FRET peak between Igα-YFP and LynFL-CFP was followed by a peak in FRET that was significantly greater in magnitude and persisted for longer as the BCR clusters moved to the center of the synapse. Importantly, in cells treated with PP2 to block Lyn's activity, FRET between Igα-YFP and LynFL-CFP was limited to only the first peak in the initial contact phase (Fig. 5 B). Thus, Lyn appears to interact with the BCR clusters first in a PP2-resistant fashion, presumably mediated by lipid–protein interactions between Lyn and the BCR, and then in a PP2-sensitive phase, presumably by protein–protein interactions between the BCR and a kinase-active Lyn. Collectively, these observations indicate that over the contact area, BCR interactions with the lipid raft probe preceded those of the BCR and Lyn and that these lipid–protein interactions are more transient or unstable as compared with the protein–protein interactions between Lyn and the BCR.


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)

FRET between antigen-clustered BCR and Lyn kinase is prolonged and sensitive to PP2 inhibition. (A) Comparison of FRET efficiencies between Igα-YFP and Lyn16-CFP and between Igα-YFP and LynFL-CFP with time. (B) A comparison of the Ea between Igα-YFP and LynFL-CFP in the presence or absence of PP2. The calculation of FRET efficiency was performed as described in Materials and methods. Mean + SEM (error bars) of calculated Ea are shown with time for 12 cells expressing Lyn16-CFP, six cells expressing LynFL-CFP, and three cells expressing LynFL-CFP treated with PP2.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2483512&req=5

fig5: FRET between antigen-clustered BCR and Lyn kinase is prolonged and sensitive to PP2 inhibition. (A) Comparison of FRET efficiencies between Igα-YFP and Lyn16-CFP and between Igα-YFP and LynFL-CFP with time. (B) A comparison of the Ea between Igα-YFP and LynFL-CFP in the presence or absence of PP2. The calculation of FRET efficiency was performed as described in Materials and methods. Mean + SEM (error bars) of calculated Ea are shown with time for 12 cells expressing Lyn16-CFP, six cells expressing LynFL-CFP, and three cells expressing LynFL-CFP treated with PP2.
Mentions: FRET between Igα-YFP and either Lyn16-CFP or LynFL-CFP was quantified with time over the entire contact area of the B cells interacting with ICAM-1– and antigen-containing bilayers and was compared (Fig. 5 A). FRET between Lyn16-CFP and Igα-YFP increased rapidly upon initial B cell contact with the antigen-containing bilayer during the initial contact phase and then decreased as B cells spread and BCR clusters moved to the center of the synapse. FRET between LynFL-CFP and Igα-YFP showed an initial small peak that corresponded in time to the FRET between Igα-YFP and Lyn16-CFP during the initial contact phase (Fig. 5 A). The initial FRET peak between Igα-YFP and LynFL-CFP was followed by a peak in FRET that was significantly greater in magnitude and persisted for longer as the BCR clusters moved to the center of the synapse. Importantly, in cells treated with PP2 to block Lyn's activity, FRET between Igα-YFP and LynFL-CFP was limited to only the first peak in the initial contact phase (Fig. 5 B). Thus, Lyn appears to interact with the BCR clusters first in a PP2-resistant fashion, presumably mediated by lipid–protein interactions between Lyn and the BCR, and then in a PP2-sensitive phase, presumably by protein–protein interactions between the BCR and a kinase-active Lyn. Collectively, these observations indicate that over the contact area, BCR interactions with the lipid raft probe preceded those of the BCR and Lyn and that these lipid–protein interactions are more transient or unstable as compared with the protein–protein interactions between Lyn and the BCR.

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