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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 interactions of BCR clusters and the raft lipid probe are not dependent on the initiation of BCR signaling or on the actin cytoskeleton. (A) CFP, YFP, and Ea time-lapse images (acquired as in Fig. 1) of CH27 B cells expressing Igα-YFP and the raft lipid probe, Lyn16-CFP, at 4, 60, and 600 s after an encounter with an ICAM-1– and antigen-containing bilayer. A merged image of YFP and Ea and the relative FIs of YFP (red) and Ea (green) across the contact area of the merged image indicated by red lines (scale, 20 μm) are given. CH27 B cells were either untreated or pretreated with the Src family kinase inhibitor PP2 (50 μm for 1 h at 37°C) or latrunculin B (10 μm for 30 min at 37°C). (B) Three-channel images (CFP, YFP, and FRET) of individual cells as performed in A were collected at 2-s intervals, and the FRET efficiency (Ea) was calculated as in Fig. 1 and described in Materials and methods. The mean Ea + SEM from six untreated cells, five PP2-treated cells, and six latrunculin B–treated cells is shown. (C) CFP, YFP, Ea, and merged images of J558L B cells expressing either the wild-type NIP-specific BCR (IgαYY/IgβYY)-YFP or the mutant signaling-deficient BCR (IgαYY→FF/IgβYY→FF)-YFP and Lyn16-CFP 1 min after the B cells encountered ICAM-1– and antigen-containing bilayers. Bars, 10 μm. The tyrosine to phenylalanine mutations in the ITAM of Igα/Igβ are indicated as asterisks in the BCR diagram (GFPs and BCR are not drawn to scale). (D and E) The number of BCR microclusters (D) and the FRET efficiency (Ea; E) were determined for single J558L cells expressing either the wild-type BCR or ITAM mutant BCR during time-lapse TIRF microscope imaging for 10 min after the cells contacted ICAM-1– and antigen-containing bilayers. The number of BCR clusters and FRET efficiencies were determined as described in Materials and methods at the time of peak FRET. Frequency plots of representative cells taken from the data of four independent experiments are shown. Horizontal lines are the median values of all individual points. D, 15–20 cells; E, 10–15 cells. P-values determined by t test are also shown.
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fig2: The interactions of BCR clusters and the raft lipid probe are not dependent on the initiation of BCR signaling or on the actin cytoskeleton. (A) CFP, YFP, and Ea time-lapse images (acquired as in Fig. 1) of CH27 B cells expressing Igα-YFP and the raft lipid probe, Lyn16-CFP, at 4, 60, and 600 s after an encounter with an ICAM-1– and antigen-containing bilayer. A merged image of YFP and Ea and the relative FIs of YFP (red) and Ea (green) across the contact area of the merged image indicated by red lines (scale, 20 μm) are given. CH27 B cells were either untreated or pretreated with the Src family kinase inhibitor PP2 (50 μm for 1 h at 37°C) or latrunculin B (10 μm for 30 min at 37°C). (B) Three-channel images (CFP, YFP, and FRET) of individual cells as performed in A were collected at 2-s intervals, and the FRET efficiency (Ea) was calculated as in Fig. 1 and described in Materials and methods. The mean Ea + SEM from six untreated cells, five PP2-treated cells, and six latrunculin B–treated cells is shown. (C) CFP, YFP, Ea, and merged images of J558L B cells expressing either the wild-type NIP-specific BCR (IgαYY/IgβYY)-YFP or the mutant signaling-deficient BCR (IgαYY→FF/IgβYY→FF)-YFP and Lyn16-CFP 1 min after the B cells encountered ICAM-1– and antigen-containing bilayers. Bars, 10 μm. The tyrosine to phenylalanine mutations in the ITAM of Igα/Igβ are indicated as asterisks in the BCR diagram (GFPs and BCR are not drawn to scale). (D and E) The number of BCR microclusters (D) and the FRET efficiency (Ea; E) were determined for single J558L cells expressing either the wild-type BCR or ITAM mutant BCR during time-lapse TIRF microscope imaging for 10 min after the cells contacted ICAM-1– and antigen-containing bilayers. The number of BCR clusters and FRET efficiencies were determined as described in Materials and methods at the time of peak FRET. Frequency plots of representative cells taken from the data of four independent experiments are shown. Horizontal lines are the median values of all individual points. D, 15–20 cells; E, 10–15 cells. P-values determined by t test are also shown.

Mentions: To determine whether association of the clustered BCRs with the raft lipid probe was dependent on either signaling or the function of the actin cytoskeleton, B cells were treated with either PP2 to inhibit Src family kinases (Hanke et al., 1996) or with latrunculin B to disassemble the actin cytoskeleton (Brown and Song, 2001) before exposure to ICAM-1– and antigen-containing bilayers. Our previous study showed that PP2 blocked FRET between Igα-CFP and Lyn16-YFP in cells encountering antigen in solution (Sohn et al., 2006). Here, we found that although PP2 affected the ability of B cells to both spread on the bilayer and organize the BCR in a synapse, it did not block FRET between Igα-YFP and Lyn16-CFP upon BCR antigen binding (Fig. 2 A). Thus, association of the BCR with raft lipids showed different requirements for Src family kinase activity when the B cell engaged antigen in solution versus on a membrane. The requirement of Src family kinase activity when antigen is bound from solution may reflect a role of these kinases in maintaining some feature of the cell membrane or local membrane topology, requirements that contact between the B cell membrane and the antigen-containing bilayer overcomes. In addition, although PP2 does not block FRET between Igα-YFP and Lyn16-CFP, both the FRET spatial and kinetic patterns were altered by treatment with PP2 (Fig. 2, A and B), presumably reflecting a requirement for Src family kinase activity in these downstream processes. Similar results were obtained in cells treated with latrunculin B. FRET between Igα-YFP and Lyn16-CFP was observed after antigen-induced BCR clustering, but both the kinetics and spatial pattern of FRET were affected as compared with untreated cells. Collectively, these observations indicate that association of the BCRs with raft lipids that occurs within the first several seconds of BCR-antigen engagement is independent of signaling and association with the actin cytoskeleton. However, importantly, failure to either signal or associate with the actin cytoskeleton significantly affected both the spatial distribution and kinetics of the BCR–lipid raft probe interactions by mechanisms that remain to be elucidated.


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 interactions of BCR clusters and the raft lipid probe are not dependent on the initiation of BCR signaling or on the actin cytoskeleton. (A) CFP, YFP, and Ea time-lapse images (acquired as in Fig. 1) of CH27 B cells expressing Igα-YFP and the raft lipid probe, Lyn16-CFP, at 4, 60, and 600 s after an encounter with an ICAM-1– and antigen-containing bilayer. A merged image of YFP and Ea and the relative FIs of YFP (red) and Ea (green) across the contact area of the merged image indicated by red lines (scale, 20 μm) are given. CH27 B cells were either untreated or pretreated with the Src family kinase inhibitor PP2 (50 μm for 1 h at 37°C) or latrunculin B (10 μm for 30 min at 37°C). (B) Three-channel images (CFP, YFP, and FRET) of individual cells as performed in A were collected at 2-s intervals, and the FRET efficiency (Ea) was calculated as in Fig. 1 and described in Materials and methods. The mean Ea + SEM from six untreated cells, five PP2-treated cells, and six latrunculin B–treated cells is shown. (C) CFP, YFP, Ea, and merged images of J558L B cells expressing either the wild-type NIP-specific BCR (IgαYY/IgβYY)-YFP or the mutant signaling-deficient BCR (IgαYY→FF/IgβYY→FF)-YFP and Lyn16-CFP 1 min after the B cells encountered ICAM-1– and antigen-containing bilayers. Bars, 10 μm. The tyrosine to phenylalanine mutations in the ITAM of Igα/Igβ are indicated as asterisks in the BCR diagram (GFPs and BCR are not drawn to scale). (D and E) The number of BCR microclusters (D) and the FRET efficiency (Ea; E) were determined for single J558L cells expressing either the wild-type BCR or ITAM mutant BCR during time-lapse TIRF microscope imaging for 10 min after the cells contacted ICAM-1– and antigen-containing bilayers. The number of BCR clusters and FRET efficiencies were determined as described in Materials and methods at the time of peak FRET. Frequency plots of representative cells taken from the data of four independent experiments are shown. Horizontal lines are the median values of all individual points. D, 15–20 cells; E, 10–15 cells. P-values determined by t test are also shown.
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fig2: The interactions of BCR clusters and the raft lipid probe are not dependent on the initiation of BCR signaling or on the actin cytoskeleton. (A) CFP, YFP, and Ea time-lapse images (acquired as in Fig. 1) of CH27 B cells expressing Igα-YFP and the raft lipid probe, Lyn16-CFP, at 4, 60, and 600 s after an encounter with an ICAM-1– and antigen-containing bilayer. A merged image of YFP and Ea and the relative FIs of YFP (red) and Ea (green) across the contact area of the merged image indicated by red lines (scale, 20 μm) are given. CH27 B cells were either untreated or pretreated with the Src family kinase inhibitor PP2 (50 μm for 1 h at 37°C) or latrunculin B (10 μm for 30 min at 37°C). (B) Three-channel images (CFP, YFP, and FRET) of individual cells as performed in A were collected at 2-s intervals, and the FRET efficiency (Ea) was calculated as in Fig. 1 and described in Materials and methods. The mean Ea + SEM from six untreated cells, five PP2-treated cells, and six latrunculin B–treated cells is shown. (C) CFP, YFP, Ea, and merged images of J558L B cells expressing either the wild-type NIP-specific BCR (IgαYY/IgβYY)-YFP or the mutant signaling-deficient BCR (IgαYY→FF/IgβYY→FF)-YFP and Lyn16-CFP 1 min after the B cells encountered ICAM-1– and antigen-containing bilayers. Bars, 10 μm. The tyrosine to phenylalanine mutations in the ITAM of Igα/Igβ are indicated as asterisks in the BCR diagram (GFPs and BCR are not drawn to scale). (D and E) The number of BCR microclusters (D) and the FRET efficiency (Ea; E) were determined for single J558L cells expressing either the wild-type BCR or ITAM mutant BCR during time-lapse TIRF microscope imaging for 10 min after the cells contacted ICAM-1– and antigen-containing bilayers. The number of BCR clusters and FRET efficiencies were determined as described in Materials and methods at the time of peak FRET. Frequency plots of representative cells taken from the data of four independent experiments are shown. Horizontal lines are the median values of all individual points. D, 15–20 cells; E, 10–15 cells. P-values determined by t test are also shown.
Mentions: To determine whether association of the clustered BCRs with the raft lipid probe was dependent on either signaling or the function of the actin cytoskeleton, B cells were treated with either PP2 to inhibit Src family kinases (Hanke et al., 1996) or with latrunculin B to disassemble the actin cytoskeleton (Brown and Song, 2001) before exposure to ICAM-1– and antigen-containing bilayers. Our previous study showed that PP2 blocked FRET between Igα-CFP and Lyn16-YFP in cells encountering antigen in solution (Sohn et al., 2006). Here, we found that although PP2 affected the ability of B cells to both spread on the bilayer and organize the BCR in a synapse, it did not block FRET between Igα-YFP and Lyn16-CFP upon BCR antigen binding (Fig. 2 A). Thus, association of the BCR with raft lipids showed different requirements for Src family kinase activity when the B cell engaged antigen in solution versus on a membrane. The requirement of Src family kinase activity when antigen is bound from solution may reflect a role of these kinases in maintaining some feature of the cell membrane or local membrane topology, requirements that contact between the B cell membrane and the antigen-containing bilayer overcomes. In addition, although PP2 does not block FRET between Igα-YFP and Lyn16-CFP, both the FRET spatial and kinetic patterns were altered by treatment with PP2 (Fig. 2, A and B), presumably reflecting a requirement for Src family kinase activity in these downstream processes. Similar results were obtained in cells treated with latrunculin B. FRET between Igα-YFP and Lyn16-CFP was observed after antigen-induced BCR clustering, but both the kinetics and spatial pattern of FRET were affected as compared with untreated cells. Collectively, these observations indicate that association of the BCRs with raft lipids that occurs within the first several seconds of BCR-antigen engagement is independent of signaling and association with the actin cytoskeleton. However, importantly, failure to either signal or associate with the actin cytoskeleton significantly affected both the spatial distribution and kinetics of the BCR–lipid raft probe interactions by mechanisms that remain to be elucidated.

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