The Experts below are selected from a list of 51 Experts worldwide ranked by ideXlab platform
Xingtian Xuan - One of the best experts on this subject based on the ideXlab platform.
-
The levels of Tyrosine and Brutons Tyrosine Kinase (Btk) phosphorylation in B cell receptor (BCR) clusters in response to soluble antigen (sAg) are reduced in Rictor knockout (KO) B cells.
2017Co-Authors: Lu Huang, Yongjie Zhang, Linlin Niu, Jinzhi Wang, Xiaoyu Sun, Xiaoming Bai, Xingtian XuanAbstract:Splenic B cells were incubated with Alexa Fluor (AF) 546–monobiotinylated (mB)-Fab′–anti- immunoglobulin (Ig) without (−) or with streptavidin (sAg) at 4°C, washed, and warmed to 37°C for varying lengths of time. After fixation and permeabilization, the cells were stained for phosphoTyrosine (pY) and phosphorylated Brutons Tyrosine Kinase (pBtk) and analyzed using confocal microscopy (CFm) or flow cytometry (A, B, F and G). The mean fluorescence intensity (MFI) of pY and pBtk were generated using NIS-Elements AR 3.2 software (C and D). The Pearson’s correlation coefficients between BCR and pY and pBtk staining in sAg-stimulated cells were determined using NIS-Elements AR 3.2 software (E). Flow cytometry analysis of the MFI of pY, pBtk, and phosphorylated extracellular regulated protein Kinases (pErk) after stimulation with sAgs (F, G and I). Ca2+ flux analysis of splenic B cells activated with soluble monobiotinylated (mB)-Fab′–anti- immunoglobulin (Ig) plus streptavidin using flow cytometry (H). Western blot analysis of the pBtk, pAkt (Serine 473 [Ser473]), pErk, and pY (80 kDa) after stimulation with sAgs (J); actin was used as a loading control. Shown are representative images at indicated times and the average values (±SD) of about 50 cells from 3 independent experiments. Scale bars, 2.5 μm. Mann–Whitney U test was used to do the statistics, *p < 0.01. The numerical data (for C, D, E, F, G, and I) can be found in S1 Data.
-
Rictor deficiency deregulates the dephosphorylation of ezrin and actin inhibition can rescue the defects of B cell receptor (BCR) signaling.
2017Co-Authors: Lu Huang, Yongjie Zhang, Linlin Niu, Jinzhi Wang, Xiaoyu Sun, Xiaoming Bai, Xingtian XuanAbstract:Splenic B cells were pretreated with or without 0.05 μM Latrunculin B for 30 min and then incubated with Alexa Fluor (AF) 546–monobiotinylated (mB)-Fab′–anti-immunoglobulin (Ig) without (−) or with streptavidin (sAg) at 4°C, washed, and warmed to 37°C for varying lengths of time in the presence of Latrunculin B. (F) Flow cytometry analysis of BCR internalization by quantifying the percentage of biotin-F(ab′)2–anti-immunoglobulin (Ig)–labeled BCR remaining on the cell surface after the 37°C chase in the presence of Latrunculin B after treatment. Shown are the average percentages (±SD) from 3 independent experiments. *p < 0.01 compared to control B cells. After fixation and permeabilization, the cells were stained for Alexa Fluor 488 (AF488)-phallodin, phosphorylated Ezrin (pEzrin) (A-C), phosphorylated Brutons Tyrosine Kinase (pBtk), and phosphoTyrosine (pY) (G-I) and analyzed using confocal microscopy (CFm) and flow cytometry (D-F and J-K). The Pearson’s correlation coefficients between BCR and phosphorylated SH2-containing inositol phosphatase (pSHIP) staining in soluble antigen (sAg)-stimulated cells were determined using NIS-Elements AR 3.2 software (L). Shown are representative images at indicated times and the average values (±SD) of about 50 cells from 3 independent experiments. Scale bars, 2.5 μm. One-way ANOVA with the Tukey test was used to do multiple group comparisons, *p < 0.01. The numerical data (for E, F, J, K, and L) can be found in S1 Data.
-
Rictor positively regulates B cell receptor signaling by modulating actin reorganization via ezrin.
Public Library of Science (PLoS), 2017Co-Authors: Lu Huang, Yongjie Zhang, Linlin Niu, Jinzhi Wang, Xiaoyu Sun, Xiaoming Bai, Xingtian XuanAbstract:As the central hub of the metabolism machinery, the mammalian target of rapamycin complex 2 (mTORC2) has been well studied in lymphocytes. As an obligatory component of mTORC2, the role of Rictor in T cells is well established. However, the role of Rictor in B cells still remains elusive. Rictor is involved in B cell development, especially the peripheral development. However, the role of Rictor on B cell receptor (BCR) signaling as well as the underlying cellular and molecular mechanism is still unknown. This study used B cell-specfic Rictor knockout (KO) mice to investigate how Rictor regulates BCR signaling. We found that the key positive and negative BCR signaling molecules, phosphorylated Brutons Tyrosine Kinase (pBtk) and phosphorylated SH2-containing inositol phosphatase (pSHIP), are reduced and enhanced, respectively, in Rictor KO B cells. This suggests that Rictor positively regulates the early events of BCR signaling. We found that the cellular filamentous actin (F-actin) is drastically increased in Rictor KO B cells after BCR stimulation through dysregulating the dephosphorylation of ezrin. The high actin-ezrin intensity area restricts the lateral movement of BCRs upon stimulation, consequently reducing BCR clustering and BCR signaling. The reduction in the initiation of BCR signaling caused by actin alteration is associated with a decreased humoral immune response in Rictor KO mice. The inhibition of actin polymerization with latrunculin in Rictor KO B cells rescues the defects of BCR signaling and B cell differentiation. Overall, our study provides a new pathway linking cell metablism to BCR activation, in which Rictor regulates BCR signaling via actin reorganization
-
B cell receptor (BCR) cluster formation, B cell spreading, and BCR signalsome are reduced and actin polymerization is enhanced in Rictor knockout (KO) B cells.
2017Co-Authors: Lu Huang, Yongjie Zhang, Linlin Niu, Jinzhi Wang, Xiaoyu Sun, Xiaoming Bai, Xingtian XuanAbstract:Splenic B cells were incubated with Alexa Fluor (AF) 546– monobiotinylated (mB)-Fab′–anti- immunoglobulin (Ig) without (−) or with streptavidin (sAg) at 4°C, washed, and warmed to 37°C for varying lengths of time. After fixation and permeabilization, cells were stained for Alexa Fluor 488 (AF488)-phallodin. Cells were analyzed using confocal microscopy (CFm) and flow cytometry. Shown are representative images at indicated times (A and B) and the average values (±SD) of mean fluorescence intensity (MFI) of phallodin in the cytoplasm and on the plasma membrane at 10 min (C). About 50 cells from 3 independent experiments using NIS-Elements AR 3.2 software and the MFI of phallodin using flow cytometry (D). Splenic B cells from wild-type (WT) and Rictor KO mice were incubated with AF546-mB-Fab′–anti-Ig tethered to lipid bilayers at 37°C for indicated times. Cells were fixed, permeabilized, and stained. Shown are representative images (E and F) and the average values (± SD) of the MFI of filamentous actin (F-actin) (G), the MFI of the BCR (H), B cell contact area (I), and the MFI of phosphorylated Brutons Tyrosine Kinase pBtk (J) in the contact zone. Total internal reflection fluorescent microscopy (TIRFm) analysis of the spatial relationship of BCR with phosphoTyrosine (pY) and pBtk in the contact zone of splenic B cells incubated with membrane-tethered Fab′–anti-immunoglobulin (Ig). The colocalization coefficients between BCR and pY and pBtk staining were determined using NIS-Elements AR 3.2 software (K). The data were generated using 20–90 cells from 3 independent experiments. Scale bars, 2.5 μm. Mann–Whitney U test was used to do the statistics, *p < 0.01. The numerical data (for C, D, G, H, I, J, and K) can be found in S1 Data.
Lu Huang - One of the best experts on this subject based on the ideXlab platform.
-
The levels of Tyrosine and Brutons Tyrosine Kinase (Btk) phosphorylation in B cell receptor (BCR) clusters in response to soluble antigen (sAg) are reduced in Rictor knockout (KO) B cells.
2017Co-Authors: Lu Huang, Yongjie Zhang, Linlin Niu, Jinzhi Wang, Xiaoyu Sun, Xiaoming Bai, Xingtian XuanAbstract:Splenic B cells were incubated with Alexa Fluor (AF) 546–monobiotinylated (mB)-Fab′–anti- immunoglobulin (Ig) without (−) or with streptavidin (sAg) at 4°C, washed, and warmed to 37°C for varying lengths of time. After fixation and permeabilization, the cells were stained for phosphoTyrosine (pY) and phosphorylated Brutons Tyrosine Kinase (pBtk) and analyzed using confocal microscopy (CFm) or flow cytometry (A, B, F and G). The mean fluorescence intensity (MFI) of pY and pBtk were generated using NIS-Elements AR 3.2 software (C and D). The Pearson’s correlation coefficients between BCR and pY and pBtk staining in sAg-stimulated cells were determined using NIS-Elements AR 3.2 software (E). Flow cytometry analysis of the MFI of pY, pBtk, and phosphorylated extracellular regulated protein Kinases (pErk) after stimulation with sAgs (F, G and I). Ca2+ flux analysis of splenic B cells activated with soluble monobiotinylated (mB)-Fab′–anti- immunoglobulin (Ig) plus streptavidin using flow cytometry (H). Western blot analysis of the pBtk, pAkt (Serine 473 [Ser473]), pErk, and pY (80 kDa) after stimulation with sAgs (J); actin was used as a loading control. Shown are representative images at indicated times and the average values (±SD) of about 50 cells from 3 independent experiments. Scale bars, 2.5 μm. Mann–Whitney U test was used to do the statistics, *p < 0.01. The numerical data (for C, D, E, F, G, and I) can be found in S1 Data.
-
Rictor deficiency deregulates the dephosphorylation of ezrin and actin inhibition can rescue the defects of B cell receptor (BCR) signaling.
2017Co-Authors: Lu Huang, Yongjie Zhang, Linlin Niu, Jinzhi Wang, Xiaoyu Sun, Xiaoming Bai, Xingtian XuanAbstract:Splenic B cells were pretreated with or without 0.05 μM Latrunculin B for 30 min and then incubated with Alexa Fluor (AF) 546–monobiotinylated (mB)-Fab′–anti-immunoglobulin (Ig) without (−) or with streptavidin (sAg) at 4°C, washed, and warmed to 37°C for varying lengths of time in the presence of Latrunculin B. (F) Flow cytometry analysis of BCR internalization by quantifying the percentage of biotin-F(ab′)2–anti-immunoglobulin (Ig)–labeled BCR remaining on the cell surface after the 37°C chase in the presence of Latrunculin B after treatment. Shown are the average percentages (±SD) from 3 independent experiments. *p < 0.01 compared to control B cells. After fixation and permeabilization, the cells were stained for Alexa Fluor 488 (AF488)-phallodin, phosphorylated Ezrin (pEzrin) (A-C), phosphorylated Brutons Tyrosine Kinase (pBtk), and phosphoTyrosine (pY) (G-I) and analyzed using confocal microscopy (CFm) and flow cytometry (D-F and J-K). The Pearson’s correlation coefficients between BCR and phosphorylated SH2-containing inositol phosphatase (pSHIP) staining in soluble antigen (sAg)-stimulated cells were determined using NIS-Elements AR 3.2 software (L). Shown are representative images at indicated times and the average values (±SD) of about 50 cells from 3 independent experiments. Scale bars, 2.5 μm. One-way ANOVA with the Tukey test was used to do multiple group comparisons, *p < 0.01. The numerical data (for E, F, J, K, and L) can be found in S1 Data.
-
Rictor positively regulates B cell receptor signaling by modulating actin reorganization via ezrin.
Public Library of Science (PLoS), 2017Co-Authors: Lu Huang, Yongjie Zhang, Linlin Niu, Jinzhi Wang, Xiaoyu Sun, Xiaoming Bai, Xingtian XuanAbstract:As the central hub of the metabolism machinery, the mammalian target of rapamycin complex 2 (mTORC2) has been well studied in lymphocytes. As an obligatory component of mTORC2, the role of Rictor in T cells is well established. However, the role of Rictor in B cells still remains elusive. Rictor is involved in B cell development, especially the peripheral development. However, the role of Rictor on B cell receptor (BCR) signaling as well as the underlying cellular and molecular mechanism is still unknown. This study used B cell-specfic Rictor knockout (KO) mice to investigate how Rictor regulates BCR signaling. We found that the key positive and negative BCR signaling molecules, phosphorylated Brutons Tyrosine Kinase (pBtk) and phosphorylated SH2-containing inositol phosphatase (pSHIP), are reduced and enhanced, respectively, in Rictor KO B cells. This suggests that Rictor positively regulates the early events of BCR signaling. We found that the cellular filamentous actin (F-actin) is drastically increased in Rictor KO B cells after BCR stimulation through dysregulating the dephosphorylation of ezrin. The high actin-ezrin intensity area restricts the lateral movement of BCRs upon stimulation, consequently reducing BCR clustering and BCR signaling. The reduction in the initiation of BCR signaling caused by actin alteration is associated with a decreased humoral immune response in Rictor KO mice. The inhibition of actin polymerization with latrunculin in Rictor KO B cells rescues the defects of BCR signaling and B cell differentiation. Overall, our study provides a new pathway linking cell metablism to BCR activation, in which Rictor regulates BCR signaling via actin reorganization
-
B cell receptor (BCR) cluster formation, B cell spreading, and BCR signalsome are reduced and actin polymerization is enhanced in Rictor knockout (KO) B cells.
2017Co-Authors: Lu Huang, Yongjie Zhang, Linlin Niu, Jinzhi Wang, Xiaoyu Sun, Xiaoming Bai, Xingtian XuanAbstract:Splenic B cells were incubated with Alexa Fluor (AF) 546– monobiotinylated (mB)-Fab′–anti- immunoglobulin (Ig) without (−) or with streptavidin (sAg) at 4°C, washed, and warmed to 37°C for varying lengths of time. After fixation and permeabilization, cells were stained for Alexa Fluor 488 (AF488)-phallodin. Cells were analyzed using confocal microscopy (CFm) and flow cytometry. Shown are representative images at indicated times (A and B) and the average values (±SD) of mean fluorescence intensity (MFI) of phallodin in the cytoplasm and on the plasma membrane at 10 min (C). About 50 cells from 3 independent experiments using NIS-Elements AR 3.2 software and the MFI of phallodin using flow cytometry (D). Splenic B cells from wild-type (WT) and Rictor KO mice were incubated with AF546-mB-Fab′–anti-Ig tethered to lipid bilayers at 37°C for indicated times. Cells were fixed, permeabilized, and stained. Shown are representative images (E and F) and the average values (± SD) of the MFI of filamentous actin (F-actin) (G), the MFI of the BCR (H), B cell contact area (I), and the MFI of phosphorylated Brutons Tyrosine Kinase pBtk (J) in the contact zone. Total internal reflection fluorescent microscopy (TIRFm) analysis of the spatial relationship of BCR with phosphoTyrosine (pY) and pBtk in the contact zone of splenic B cells incubated with membrane-tethered Fab′–anti-immunoglobulin (Ig). The colocalization coefficients between BCR and pY and pBtk staining were determined using NIS-Elements AR 3.2 software (K). The data were generated using 20–90 cells from 3 independent experiments. Scale bars, 2.5 μm. Mann–Whitney U test was used to do the statistics, *p < 0.01. The numerical data (for C, D, G, H, I, J, and K) can be found in S1 Data.
Jumaa Hassan - One of the best experts on this subject based on the ideXlab platform.
-
Scientific Reports / Secreted IgM deficiency leads to increased BCR signaling that results in abnormal splenic B cell development
Nature, 2017Co-Authors: Tsiantoulas Dimitrios, Binder, Christoph J., Kiss Mate, Bartolini-gritti Barbara, Bergthaler Andreas, Mallat Ziad, Jumaa HassanAbstract:Mice lacking secreted IgM (sIgM /) antibodies display abnormal splenic B cell development, which results in increased marginal zone and decreased follicular B cell numbers. However, the mechanism by which sIgM exhibit this effect is unknown. Here, we demonstrate that B cells in sIgM / mice display increased B cell receptor (BCR) signaling as judged by increased levels of phosphorylated Brutons Tyrosine Kinase (pBtk), phosphorylated Spleen Tyrosine Kinase (pSyk), and nuclear receptor Nur77. Low dosage treatment with the pBtk inhibitor Ibrutinib reversed the altered B cell development in the spleen of sIgM / mice, suggesting that sIgM regulate splenic B cell differentiation by decreasing BCR signaling. Mechanistically, we show that B cells, which express BCRs specific to hen egg lysozyme (HEL) display diminished responsiveness to HEL stimulation in presence of soluble anti-HEL IgM antibodies. Our data identify sIgM as negative regulators of BCR signaling and suggest that they can act as decoy receptors for self-antigens that are recognized by membrane bound BCRs.(VLID)460668
-
Mimicry of a constitutively active pre-B cell receptor in acute lymphoblastic leukemia cells.
eScholarship University of California, 2005Co-Authors: Feldhahn Niklas, Klein Florian, Mooster Jana, Hadweh Paul, Sprangers Mieke, Wartenberg Maria, Bekhite Mohamed, Hofmann Wolf-karsten, Herzog Sebastian, Jumaa HassanAbstract:Pre-B cells undergo apoptosis unless they are rescued by pre-B cell receptor-dependent survival signals. We previously showed that the BCR-ABL1 Kinase that is expressed in pre-B lymphoblastic leukemia bypasses selection for pre-B cell receptor-dependent survival signals. Investigating possible interference of BCR-ABL1 with pre-B cell receptor signaling, we found that neither SYK nor SLP65 can be phosphorylated in response to pre-B cell receptor engagement. Instead, Brutons Tyrosine Kinase (BTK) is constitutively phosphorylated by BCR-ABL1. Activated BTK is essential for survival signals that otherwise would arise from the pre-B cell receptor, including activation of PLCgamma1, autonomous Ca2+ signaling, STAT5-phosphorylation, and up-regulation of BCLX(L). Inhibition of BTK activity specifically induces apoptosis in BCR-ABL1+ leukemia cells to a similar extent as inhibition of BCR-ABL1 Kinase activity itself. However, BCR-ABL1 cannot directly bind to full-length BTK. Instead, BCR-ABL1 induces the expression of a truncated splice variant of BTK that acts as a linker between the two Kinases. As opposed to full-length BTK, truncated BTK lacks Kinase activity yet can bind to BCR-ABL1 through its SRC-homology domain 3. Acting as a linker, truncated BTK enables BCR-ABL1-dependent activation of full-length BTK, which initiates downstream survival signals and mimics a constitutively active pre-B cell receptor
Linlin Niu - One of the best experts on this subject based on the ideXlab platform.
-
The levels of Tyrosine and Brutons Tyrosine Kinase (Btk) phosphorylation in B cell receptor (BCR) clusters in response to soluble antigen (sAg) are reduced in Rictor knockout (KO) B cells.
2017Co-Authors: Lu Huang, Yongjie Zhang, Linlin Niu, Jinzhi Wang, Xiaoyu Sun, Xiaoming Bai, Xingtian XuanAbstract:Splenic B cells were incubated with Alexa Fluor (AF) 546–monobiotinylated (mB)-Fab′–anti- immunoglobulin (Ig) without (−) or with streptavidin (sAg) at 4°C, washed, and warmed to 37°C for varying lengths of time. After fixation and permeabilization, the cells were stained for phosphoTyrosine (pY) and phosphorylated Brutons Tyrosine Kinase (pBtk) and analyzed using confocal microscopy (CFm) or flow cytometry (A, B, F and G). The mean fluorescence intensity (MFI) of pY and pBtk were generated using NIS-Elements AR 3.2 software (C and D). The Pearson’s correlation coefficients between BCR and pY and pBtk staining in sAg-stimulated cells were determined using NIS-Elements AR 3.2 software (E). Flow cytometry analysis of the MFI of pY, pBtk, and phosphorylated extracellular regulated protein Kinases (pErk) after stimulation with sAgs (F, G and I). Ca2+ flux analysis of splenic B cells activated with soluble monobiotinylated (mB)-Fab′–anti- immunoglobulin (Ig) plus streptavidin using flow cytometry (H). Western blot analysis of the pBtk, pAkt (Serine 473 [Ser473]), pErk, and pY (80 kDa) after stimulation with sAgs (J); actin was used as a loading control. Shown are representative images at indicated times and the average values (±SD) of about 50 cells from 3 independent experiments. Scale bars, 2.5 μm. Mann–Whitney U test was used to do the statistics, *p < 0.01. The numerical data (for C, D, E, F, G, and I) can be found in S1 Data.
-
Rictor deficiency deregulates the dephosphorylation of ezrin and actin inhibition can rescue the defects of B cell receptor (BCR) signaling.
2017Co-Authors: Lu Huang, Yongjie Zhang, Linlin Niu, Jinzhi Wang, Xiaoyu Sun, Xiaoming Bai, Xingtian XuanAbstract:Splenic B cells were pretreated with or without 0.05 μM Latrunculin B for 30 min and then incubated with Alexa Fluor (AF) 546–monobiotinylated (mB)-Fab′–anti-immunoglobulin (Ig) without (−) or with streptavidin (sAg) at 4°C, washed, and warmed to 37°C for varying lengths of time in the presence of Latrunculin B. (F) Flow cytometry analysis of BCR internalization by quantifying the percentage of biotin-F(ab′)2–anti-immunoglobulin (Ig)–labeled BCR remaining on the cell surface after the 37°C chase in the presence of Latrunculin B after treatment. Shown are the average percentages (±SD) from 3 independent experiments. *p < 0.01 compared to control B cells. After fixation and permeabilization, the cells were stained for Alexa Fluor 488 (AF488)-phallodin, phosphorylated Ezrin (pEzrin) (A-C), phosphorylated Brutons Tyrosine Kinase (pBtk), and phosphoTyrosine (pY) (G-I) and analyzed using confocal microscopy (CFm) and flow cytometry (D-F and J-K). The Pearson’s correlation coefficients between BCR and phosphorylated SH2-containing inositol phosphatase (pSHIP) staining in soluble antigen (sAg)-stimulated cells were determined using NIS-Elements AR 3.2 software (L). Shown are representative images at indicated times and the average values (±SD) of about 50 cells from 3 independent experiments. Scale bars, 2.5 μm. One-way ANOVA with the Tukey test was used to do multiple group comparisons, *p < 0.01. The numerical data (for E, F, J, K, and L) can be found in S1 Data.
-
Rictor positively regulates B cell receptor signaling by modulating actin reorganization via ezrin.
Public Library of Science (PLoS), 2017Co-Authors: Lu Huang, Yongjie Zhang, Linlin Niu, Jinzhi Wang, Xiaoyu Sun, Xiaoming Bai, Xingtian XuanAbstract:As the central hub of the metabolism machinery, the mammalian target of rapamycin complex 2 (mTORC2) has been well studied in lymphocytes. As an obligatory component of mTORC2, the role of Rictor in T cells is well established. However, the role of Rictor in B cells still remains elusive. Rictor is involved in B cell development, especially the peripheral development. However, the role of Rictor on B cell receptor (BCR) signaling as well as the underlying cellular and molecular mechanism is still unknown. This study used B cell-specfic Rictor knockout (KO) mice to investigate how Rictor regulates BCR signaling. We found that the key positive and negative BCR signaling molecules, phosphorylated Brutons Tyrosine Kinase (pBtk) and phosphorylated SH2-containing inositol phosphatase (pSHIP), are reduced and enhanced, respectively, in Rictor KO B cells. This suggests that Rictor positively regulates the early events of BCR signaling. We found that the cellular filamentous actin (F-actin) is drastically increased in Rictor KO B cells after BCR stimulation through dysregulating the dephosphorylation of ezrin. The high actin-ezrin intensity area restricts the lateral movement of BCRs upon stimulation, consequently reducing BCR clustering and BCR signaling. The reduction in the initiation of BCR signaling caused by actin alteration is associated with a decreased humoral immune response in Rictor KO mice. The inhibition of actin polymerization with latrunculin in Rictor KO B cells rescues the defects of BCR signaling and B cell differentiation. Overall, our study provides a new pathway linking cell metablism to BCR activation, in which Rictor regulates BCR signaling via actin reorganization
-
B cell receptor (BCR) cluster formation, B cell spreading, and BCR signalsome are reduced and actin polymerization is enhanced in Rictor knockout (KO) B cells.
2017Co-Authors: Lu Huang, Yongjie Zhang, Linlin Niu, Jinzhi Wang, Xiaoyu Sun, Xiaoming Bai, Xingtian XuanAbstract:Splenic B cells were incubated with Alexa Fluor (AF) 546– monobiotinylated (mB)-Fab′–anti- immunoglobulin (Ig) without (−) or with streptavidin (sAg) at 4°C, washed, and warmed to 37°C for varying lengths of time. After fixation and permeabilization, cells were stained for Alexa Fluor 488 (AF488)-phallodin. Cells were analyzed using confocal microscopy (CFm) and flow cytometry. Shown are representative images at indicated times (A and B) and the average values (±SD) of mean fluorescence intensity (MFI) of phallodin in the cytoplasm and on the plasma membrane at 10 min (C). About 50 cells from 3 independent experiments using NIS-Elements AR 3.2 software and the MFI of phallodin using flow cytometry (D). Splenic B cells from wild-type (WT) and Rictor KO mice were incubated with AF546-mB-Fab′–anti-Ig tethered to lipid bilayers at 37°C for indicated times. Cells were fixed, permeabilized, and stained. Shown are representative images (E and F) and the average values (± SD) of the MFI of filamentous actin (F-actin) (G), the MFI of the BCR (H), B cell contact area (I), and the MFI of phosphorylated Brutons Tyrosine Kinase pBtk (J) in the contact zone. Total internal reflection fluorescent microscopy (TIRFm) analysis of the spatial relationship of BCR with phosphoTyrosine (pY) and pBtk in the contact zone of splenic B cells incubated with membrane-tethered Fab′–anti-immunoglobulin (Ig). The colocalization coefficients between BCR and pY and pBtk staining were determined using NIS-Elements AR 3.2 software (K). The data were generated using 20–90 cells from 3 independent experiments. Scale bars, 2.5 μm. Mann–Whitney U test was used to do the statistics, *p < 0.01. The numerical data (for C, D, G, H, I, J, and K) can be found in S1 Data.
Jinzhi Wang - One of the best experts on this subject based on the ideXlab platform.
-
The levels of Tyrosine and Brutons Tyrosine Kinase (Btk) phosphorylation in B cell receptor (BCR) clusters in response to soluble antigen (sAg) are reduced in Rictor knockout (KO) B cells.
2017Co-Authors: Lu Huang, Yongjie Zhang, Linlin Niu, Jinzhi Wang, Xiaoyu Sun, Xiaoming Bai, Xingtian XuanAbstract:Splenic B cells were incubated with Alexa Fluor (AF) 546–monobiotinylated (mB)-Fab′–anti- immunoglobulin (Ig) without (−) or with streptavidin (sAg) at 4°C, washed, and warmed to 37°C for varying lengths of time. After fixation and permeabilization, the cells were stained for phosphoTyrosine (pY) and phosphorylated Brutons Tyrosine Kinase (pBtk) and analyzed using confocal microscopy (CFm) or flow cytometry (A, B, F and G). The mean fluorescence intensity (MFI) of pY and pBtk were generated using NIS-Elements AR 3.2 software (C and D). The Pearson’s correlation coefficients between BCR and pY and pBtk staining in sAg-stimulated cells were determined using NIS-Elements AR 3.2 software (E). Flow cytometry analysis of the MFI of pY, pBtk, and phosphorylated extracellular regulated protein Kinases (pErk) after stimulation with sAgs (F, G and I). Ca2+ flux analysis of splenic B cells activated with soluble monobiotinylated (mB)-Fab′–anti- immunoglobulin (Ig) plus streptavidin using flow cytometry (H). Western blot analysis of the pBtk, pAkt (Serine 473 [Ser473]), pErk, and pY (80 kDa) after stimulation with sAgs (J); actin was used as a loading control. Shown are representative images at indicated times and the average values (±SD) of about 50 cells from 3 independent experiments. Scale bars, 2.5 μm. Mann–Whitney U test was used to do the statistics, *p < 0.01. The numerical data (for C, D, E, F, G, and I) can be found in S1 Data.
-
Rictor deficiency deregulates the dephosphorylation of ezrin and actin inhibition can rescue the defects of B cell receptor (BCR) signaling.
2017Co-Authors: Lu Huang, Yongjie Zhang, Linlin Niu, Jinzhi Wang, Xiaoyu Sun, Xiaoming Bai, Xingtian XuanAbstract:Splenic B cells were pretreated with or without 0.05 μM Latrunculin B for 30 min and then incubated with Alexa Fluor (AF) 546–monobiotinylated (mB)-Fab′–anti-immunoglobulin (Ig) without (−) or with streptavidin (sAg) at 4°C, washed, and warmed to 37°C for varying lengths of time in the presence of Latrunculin B. (F) Flow cytometry analysis of BCR internalization by quantifying the percentage of biotin-F(ab′)2–anti-immunoglobulin (Ig)–labeled BCR remaining on the cell surface after the 37°C chase in the presence of Latrunculin B after treatment. Shown are the average percentages (±SD) from 3 independent experiments. *p < 0.01 compared to control B cells. After fixation and permeabilization, the cells were stained for Alexa Fluor 488 (AF488)-phallodin, phosphorylated Ezrin (pEzrin) (A-C), phosphorylated Brutons Tyrosine Kinase (pBtk), and phosphoTyrosine (pY) (G-I) and analyzed using confocal microscopy (CFm) and flow cytometry (D-F and J-K). The Pearson’s correlation coefficients between BCR and phosphorylated SH2-containing inositol phosphatase (pSHIP) staining in soluble antigen (sAg)-stimulated cells were determined using NIS-Elements AR 3.2 software (L). Shown are representative images at indicated times and the average values (±SD) of about 50 cells from 3 independent experiments. Scale bars, 2.5 μm. One-way ANOVA with the Tukey test was used to do multiple group comparisons, *p < 0.01. The numerical data (for E, F, J, K, and L) can be found in S1 Data.
-
Rictor positively regulates B cell receptor signaling by modulating actin reorganization via ezrin.
Public Library of Science (PLoS), 2017Co-Authors: Lu Huang, Yongjie Zhang, Linlin Niu, Jinzhi Wang, Xiaoyu Sun, Xiaoming Bai, Xingtian XuanAbstract:As the central hub of the metabolism machinery, the mammalian target of rapamycin complex 2 (mTORC2) has been well studied in lymphocytes. As an obligatory component of mTORC2, the role of Rictor in T cells is well established. However, the role of Rictor in B cells still remains elusive. Rictor is involved in B cell development, especially the peripheral development. However, the role of Rictor on B cell receptor (BCR) signaling as well as the underlying cellular and molecular mechanism is still unknown. This study used B cell-specfic Rictor knockout (KO) mice to investigate how Rictor regulates BCR signaling. We found that the key positive and negative BCR signaling molecules, phosphorylated Brutons Tyrosine Kinase (pBtk) and phosphorylated SH2-containing inositol phosphatase (pSHIP), are reduced and enhanced, respectively, in Rictor KO B cells. This suggests that Rictor positively regulates the early events of BCR signaling. We found that the cellular filamentous actin (F-actin) is drastically increased in Rictor KO B cells after BCR stimulation through dysregulating the dephosphorylation of ezrin. The high actin-ezrin intensity area restricts the lateral movement of BCRs upon stimulation, consequently reducing BCR clustering and BCR signaling. The reduction in the initiation of BCR signaling caused by actin alteration is associated with a decreased humoral immune response in Rictor KO mice. The inhibition of actin polymerization with latrunculin in Rictor KO B cells rescues the defects of BCR signaling and B cell differentiation. Overall, our study provides a new pathway linking cell metablism to BCR activation, in which Rictor regulates BCR signaling via actin reorganization
-
B cell receptor (BCR) cluster formation, B cell spreading, and BCR signalsome are reduced and actin polymerization is enhanced in Rictor knockout (KO) B cells.
2017Co-Authors: Lu Huang, Yongjie Zhang, Linlin Niu, Jinzhi Wang, Xiaoyu Sun, Xiaoming Bai, Xingtian XuanAbstract:Splenic B cells were incubated with Alexa Fluor (AF) 546– monobiotinylated (mB)-Fab′–anti- immunoglobulin (Ig) without (−) or with streptavidin (sAg) at 4°C, washed, and warmed to 37°C for varying lengths of time. After fixation and permeabilization, cells were stained for Alexa Fluor 488 (AF488)-phallodin. Cells were analyzed using confocal microscopy (CFm) and flow cytometry. Shown are representative images at indicated times (A and B) and the average values (±SD) of mean fluorescence intensity (MFI) of phallodin in the cytoplasm and on the plasma membrane at 10 min (C). About 50 cells from 3 independent experiments using NIS-Elements AR 3.2 software and the MFI of phallodin using flow cytometry (D). Splenic B cells from wild-type (WT) and Rictor KO mice were incubated with AF546-mB-Fab′–anti-Ig tethered to lipid bilayers at 37°C for indicated times. Cells were fixed, permeabilized, and stained. Shown are representative images (E and F) and the average values (± SD) of the MFI of filamentous actin (F-actin) (G), the MFI of the BCR (H), B cell contact area (I), and the MFI of phosphorylated Brutons Tyrosine Kinase pBtk (J) in the contact zone. Total internal reflection fluorescent microscopy (TIRFm) analysis of the spatial relationship of BCR with phosphoTyrosine (pY) and pBtk in the contact zone of splenic B cells incubated with membrane-tethered Fab′–anti-immunoglobulin (Ig). The colocalization coefficients between BCR and pY and pBtk staining were determined using NIS-Elements AR 3.2 software (K). The data were generated using 20–90 cells from 3 independent experiments. Scale bars, 2.5 μm. Mann–Whitney U test was used to do the statistics, *p < 0.01. The numerical data (for C, D, G, H, I, J, and K) can be found in S1 Data.
