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N-wasp is essential for the negative regulation of B cell receptor signaling.

Liu C, Bai X, Wu J, Sharma S, Upadhyaya A, Dahlberg CI, Westerberg LS, Snapper SB, Zhao X, Song W - PLoS Biol. (2013)

Bottom Line: The increased signaling in N-WASP knockout B cells is concurrent with increased accumulation of F-actin at the B-cell surface, enhanced B-cell spreading on the antigen-presenting membrane, delayed B-cell contraction, inhibition in the merger of signaling active BCR microclusters into signaling inactive central clusters, and a blockage of BCR internalization.The activation of N-WASP is suppressed by Bruton's tyrosine kinase-induced WASP activation, and is restored by the activation of SH2 domain-containing inositol 5-phosphatase that inhibits WASP activation.Our results reveal a new mechanism for the negative regulation of BCR signaling and broadly suggest an actin-mediated mechanism for signaling down-regulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology & Molecular Genetics, University of Maryland, College Park, Maryland, United States of America.

ABSTRACT
Negative regulation of receptor signaling is essential for controlling cell activation and differentiation. In B-lymphocytes, the down-regulation of B-cell antigen receptor (BCR) signaling is critical for suppressing the activation of self-reactive B cells; however, the mechanism underlying the negative regulation of signaling remains elusive. Using genetically manipulated mouse models and total internal reflection fluorescence microscopy, we demonstrate that neuronal Wiskott-Aldrich syndrome protein (N-WASP), which is coexpressed with WASP in all immune cells, is a critical negative regulator of B-cell signaling. B-cell-specific N-WASP gene deletion causes enhanced and prolonged BCR signaling and elevated levels of autoantibodies in the mouse serum. The increased signaling in N-WASP knockout B cells is concurrent with increased accumulation of F-actin at the B-cell surface, enhanced B-cell spreading on the antigen-presenting membrane, delayed B-cell contraction, inhibition in the merger of signaling active BCR microclusters into signaling inactive central clusters, and a blockage of BCR internalization. Upon BCR activation, WASP is activated first, followed by N-WASP in mouse and human primary B cells. The activation of N-WASP is suppressed by Bruton's tyrosine kinase-induced WASP activation, and is restored by the activation of SH2 domain-containing inositol 5-phosphatase that inhibits WASP activation. Our results reveal a new mechanism for the negative regulation of BCR signaling and broadly suggest an actin-mediated mechanism for signaling down-regulation.

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Differential effects of WASP and/or N-WASP KO on BCR signaling.(A–E) TIRFM and IRM analysis of pY staining in the contact zone of mouse splenic B cells incubated with membrane-tethered Fab′–anti-Ig. Shown are representative images (A–D) and the MFI (±SD) of pY in the B-cell contact zone (E) from three independent experiments. Bars, 2.5 µm. (F and G) The FIRs of pY to the BCR were plotted versus the TFI of the BCR in individual BCR clusters. Each open symbol represents a BCR cluster. The simulated values (solid symbol) were generated by LOSSE nonlinear regression using the Stat software. (H and I) TIRFM analysis of phosphorylated Btk (pBtk) and SHIP-1 (pSHIP) in the contact zone of mouse splenic B cells stimulated with membrane-tethered Fab′–anti-Ig. Shown are the average MFI (±SD) of pBtk and pSHIP in the B-cell contact zone from three independent experiments. *p<0.01, compared to littermate control B cells. (J) Ca2+ flux analysis of splenic B cells activated with soluble mB-Fab′–anti-Ig plus streptavidin using flow cytometry. Shown are representative results from three independent experiments.
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pbio-1001704-g003: Differential effects of WASP and/or N-WASP KO on BCR signaling.(A–E) TIRFM and IRM analysis of pY staining in the contact zone of mouse splenic B cells incubated with membrane-tethered Fab′–anti-Ig. Shown are representative images (A–D) and the MFI (±SD) of pY in the B-cell contact zone (E) from three independent experiments. Bars, 2.5 µm. (F and G) The FIRs of pY to the BCR were plotted versus the TFI of the BCR in individual BCR clusters. Each open symbol represents a BCR cluster. The simulated values (solid symbol) were generated by LOSSE nonlinear regression using the Stat software. (H and I) TIRFM analysis of phosphorylated Btk (pBtk) and SHIP-1 (pSHIP) in the contact zone of mouse splenic B cells stimulated with membrane-tethered Fab′–anti-Ig. Shown are the average MFI (±SD) of pBtk and pSHIP in the B-cell contact zone from three independent experiments. *p<0.01, compared to littermate control B cells. (J) Ca2+ flux analysis of splenic B cells activated with soluble mB-Fab′–anti-Ig plus streptavidin using flow cytometry. Shown are representative results from three independent experiments.

Mentions: The effects of WASP and/or N-WASP KO on BCR clustering and B-cell morphology suggest their involvement in BCR signaling. To test this hypothesis, we analyzed the impact of WASP and/or N-WASP KO on tyrosine phosphorylation (pY) at the cell surface in response to membrane-tethered Fab′–anti-Ig using TIRFM. Similar to what we have shown previously [21], pY was first detected at BCR microclusters at early times during the interaction of littermate control B cells with membrane-tethered Fab′–anti-Ig (∼3 min) and then at the outer edge of the BCR central cluster at later times (∼7 min) (Figure 3A). The MFI of pY staining rapidly increased upon BCR binding, peaked at 3 min, and then decreased (Figure 3E). The distribution and levels of pY in the contact zone of WKO B cells followed a qualitatively similar pattern as in control B cells, but the increasing magnitude of pY MFI in the contact zone of WKO B cells was significantly smaller than that of control B cells (Figure 3B,E). Double KO of WASP and N-WASP caused a further reduction in the levels of pY in the B-cell contact zone (Figure 3D,E). However, the pY staining in the contact zone of cNKO B cells remained punctate and colocalized with BCR clusters at 7 min (Figure 3C). The peak level of pY in the contact zone of cNKO B cells was similar to that of control B cells, but its attenuation was significantly delayed (Figure 3E). These results suggest that N-WASP is involved in both stimulation and attenuation of BCR signaling.


N-wasp is essential for the negative regulation of B cell receptor signaling.

Liu C, Bai X, Wu J, Sharma S, Upadhyaya A, Dahlberg CI, Westerberg LS, Snapper SB, Zhao X, Song W - PLoS Biol. (2013)

Differential effects of WASP and/or N-WASP KO on BCR signaling.(A–E) TIRFM and IRM analysis of pY staining in the contact zone of mouse splenic B cells incubated with membrane-tethered Fab′–anti-Ig. Shown are representative images (A–D) and the MFI (±SD) of pY in the B-cell contact zone (E) from three independent experiments. Bars, 2.5 µm. (F and G) The FIRs of pY to the BCR were plotted versus the TFI of the BCR in individual BCR clusters. Each open symbol represents a BCR cluster. The simulated values (solid symbol) were generated by LOSSE nonlinear regression using the Stat software. (H and I) TIRFM analysis of phosphorylated Btk (pBtk) and SHIP-1 (pSHIP) in the contact zone of mouse splenic B cells stimulated with membrane-tethered Fab′–anti-Ig. Shown are the average MFI (±SD) of pBtk and pSHIP in the B-cell contact zone from three independent experiments. *p<0.01, compared to littermate control B cells. (J) Ca2+ flux analysis of splenic B cells activated with soluble mB-Fab′–anti-Ig plus streptavidin using flow cytometry. Shown are representative results from three independent experiments.
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Related In: Results  -  Collection

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pbio-1001704-g003: Differential effects of WASP and/or N-WASP KO on BCR signaling.(A–E) TIRFM and IRM analysis of pY staining in the contact zone of mouse splenic B cells incubated with membrane-tethered Fab′–anti-Ig. Shown are representative images (A–D) and the MFI (±SD) of pY in the B-cell contact zone (E) from three independent experiments. Bars, 2.5 µm. (F and G) The FIRs of pY to the BCR were plotted versus the TFI of the BCR in individual BCR clusters. Each open symbol represents a BCR cluster. The simulated values (solid symbol) were generated by LOSSE nonlinear regression using the Stat software. (H and I) TIRFM analysis of phosphorylated Btk (pBtk) and SHIP-1 (pSHIP) in the contact zone of mouse splenic B cells stimulated with membrane-tethered Fab′–anti-Ig. Shown are the average MFI (±SD) of pBtk and pSHIP in the B-cell contact zone from three independent experiments. *p<0.01, compared to littermate control B cells. (J) Ca2+ flux analysis of splenic B cells activated with soluble mB-Fab′–anti-Ig plus streptavidin using flow cytometry. Shown are representative results from three independent experiments.
Mentions: The effects of WASP and/or N-WASP KO on BCR clustering and B-cell morphology suggest their involvement in BCR signaling. To test this hypothesis, we analyzed the impact of WASP and/or N-WASP KO on tyrosine phosphorylation (pY) at the cell surface in response to membrane-tethered Fab′–anti-Ig using TIRFM. Similar to what we have shown previously [21], pY was first detected at BCR microclusters at early times during the interaction of littermate control B cells with membrane-tethered Fab′–anti-Ig (∼3 min) and then at the outer edge of the BCR central cluster at later times (∼7 min) (Figure 3A). The MFI of pY staining rapidly increased upon BCR binding, peaked at 3 min, and then decreased (Figure 3E). The distribution and levels of pY in the contact zone of WKO B cells followed a qualitatively similar pattern as in control B cells, but the increasing magnitude of pY MFI in the contact zone of WKO B cells was significantly smaller than that of control B cells (Figure 3B,E). Double KO of WASP and N-WASP caused a further reduction in the levels of pY in the B-cell contact zone (Figure 3D,E). However, the pY staining in the contact zone of cNKO B cells remained punctate and colocalized with BCR clusters at 7 min (Figure 3C). The peak level of pY in the contact zone of cNKO B cells was similar to that of control B cells, but its attenuation was significantly delayed (Figure 3E). These results suggest that N-WASP is involved in both stimulation and attenuation of BCR signaling.

Bottom Line: The increased signaling in N-WASP knockout B cells is concurrent with increased accumulation of F-actin at the B-cell surface, enhanced B-cell spreading on the antigen-presenting membrane, delayed B-cell contraction, inhibition in the merger of signaling active BCR microclusters into signaling inactive central clusters, and a blockage of BCR internalization.The activation of N-WASP is suppressed by Bruton's tyrosine kinase-induced WASP activation, and is restored by the activation of SH2 domain-containing inositol 5-phosphatase that inhibits WASP activation.Our results reveal a new mechanism for the negative regulation of BCR signaling and broadly suggest an actin-mediated mechanism for signaling down-regulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology & Molecular Genetics, University of Maryland, College Park, Maryland, United States of America.

ABSTRACT
Negative regulation of receptor signaling is essential for controlling cell activation and differentiation. In B-lymphocytes, the down-regulation of B-cell antigen receptor (BCR) signaling is critical for suppressing the activation of self-reactive B cells; however, the mechanism underlying the negative regulation of signaling remains elusive. Using genetically manipulated mouse models and total internal reflection fluorescence microscopy, we demonstrate that neuronal Wiskott-Aldrich syndrome protein (N-WASP), which is coexpressed with WASP in all immune cells, is a critical negative regulator of B-cell signaling. B-cell-specific N-WASP gene deletion causes enhanced and prolonged BCR signaling and elevated levels of autoantibodies in the mouse serum. The increased signaling in N-WASP knockout B cells is concurrent with increased accumulation of F-actin at the B-cell surface, enhanced B-cell spreading on the antigen-presenting membrane, delayed B-cell contraction, inhibition in the merger of signaling active BCR microclusters into signaling inactive central clusters, and a blockage of BCR internalization. Upon BCR activation, WASP is activated first, followed by N-WASP in mouse and human primary B cells. The activation of N-WASP is suppressed by Bruton's tyrosine kinase-induced WASP activation, and is restored by the activation of SH2 domain-containing inositol 5-phosphatase that inhibits WASP activation. Our results reveal a new mechanism for the negative regulation of BCR signaling and broadly suggest an actin-mediated mechanism for signaling down-regulation.

Show MeSH
Related in: MedlinePlus