Journal: The Journal of Cell Biology

Article Title: PI(4,5)P2 determines the threshold of mechanical force–induced B cell activation

doi: 10.1083/jcb.201711055

Figure Lengend Snippet: Activation of IgA-, IgD-, and IgM-BCR exhibit distinct mechanical force thresholds compared with IgE- or IgG-BCR. (A) Schematic representation of the NP-specific BCR-expressing B cells and NP-TGTs with the predefined tension forces of 12 pN, 43 pN, and 56 pN, respectively, which are mainly used in this study. (B–F) Representative TIRFM images and quantification of the total FI of BCR accumulation into immunological synapse in contact with 12-pN, 43-pN, and 56-pN NP-TGTs in Ramos B cells expressing NP-specific IgM-BCR (B), IgD-BCR (C), IgA-BCR (D), IgG-BCR (E), and IgE-BCR (F), respectively. (G–I) Representative fluorescent images (G) and quantifications of the percentage of pERK-positive J558L cells expressing IgM-BCR (H) or IgG-BCR (I) in response to NP-TGTs. Cells were stained with an antibody to phosphorylated ERK (red) and a nucleus dye Hoechst (white). About 70–110 cells were scored for each data point. (J) Sequence of WT, Y/F, and SSVV/AAAA cytoplasmic tails. (K and L) Representative TIRFM images and quantification of the total FI of BCR recruitment into immunological synapses on contact surfaces of 12-pN, 43-pN, and 56-pN NP-TGTs in J558L cells expressing NP-specific IgG-BCR with cytoplasmic mutations of Y/F to block the ITT signaling motif (K) or SSVV/AAAA to block the PDZ-binding motif (L). Error bars represent means ± SEM. Two-tailed t tests were performed for the statistical comparisons. Data are from at least 25 cells and represent at least two independent experiments. Bars: (B–F, K, and L) 3 µm; (G) 9 µm.

Article Snippet: Alexa Fluor 647 or Cy3 AffiniPure Fab fragment goat anti–mouse IgG1 Fc fragment specific and Cy5-Fab fragment goat anti–mouse IgM µ chain specific (Jackson ImmunoResearch Laboratories, Inc.) were used for mouse IgG- and IgM-BCR cell surface staining, respectively.

Techniques: Activation Assay, Expressing, Staining, Sequencing, Blocking Assay, Binding Assay, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: PI(4,5)P2 determines the threshold of mechanical force–induced B cell activation

doi: 10.1083/jcb.201711055

Figure Lengend Snippet: The PM-tethered status of the IgG-tail is required for the low threshold of mechanical force–induced activation. (A) Schematic illustration showing the FRET system that was used to detect the interaction between mTFP (FRET donor) fused to the C terminus of the cytoplasmic tail (WT, Mut1, and Mut2) and R18 dye (FRET acceptor) stained on the PM. (B) Sequence of the cytoplasmic tail of WT, Mut1 (the flexible 25 aa), and Mut2 (inserting a 25-aa flexible linker between the transmembrane domain of the IgG-BCR and N terminus of the IgG-tail). (C and D) Dequenching FRET to measure the FRET efficiency between mTFP and R18 in WT, Mut1–, and Mut2–IgG-BCR–expressing DT40 B cells. FRET efficiency was calculated as detailed in Materials and methods. DQ, dequenched; Q, quenched. (C) Representative confocal images of DT40 B cells expressing WT and Mut2–IgG-BCR are shown. Bars, 1.1 µm. (D) FRET efficiency was measured and plotted. Error bars represent means ± SD. (E and F) Statistical quantification of the synaptic recruitment of IgG-BCR and pSyk in J558L cells expressing Mut1– (E) or Mut2–IgG-BCR (F). Error bars represent means ± SEM. Two-tailed t tests were performed for the statistical comparisons. Data are from at least 30 cells over three independent experiments.

Article Snippet: Alexa Fluor 647 or Cy3 AffiniPure Fab fragment goat anti–mouse IgG1 Fc fragment specific and Cy5-Fab fragment goat anti–mouse IgM µ chain specific (Jackson ImmunoResearch Laboratories, Inc.) were used for mouse IgG- and IgM-BCR cell surface staining, respectively.

Techniques: Activation Assay, Staining, Sequencing, Expressing, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: PI(4,5)P2 determines the threshold of mechanical force–induced B cell activation

doi: 10.1083/jcb.201711055

Figure Lengend Snippet: Both positively charged and hydrophobic features of IgG-tail are important for its PM association orientation. (A) Left: Schematic illustration showing the FRET system to detect the interaction between mTFP (FRET donor) fused to the C terminus of the cytoplasmic tail (WT or Mut (3–6)) and R18 dye (FRET acceptor) stained on the PM. Right: Sequence and biochemical characteristic analysis of the cytoplasmic tail of WT, Mut3/4 (reducing both hydrophobicity and positively charged proportion of IgG-tail), Mut5 (keeping hydrophobic level intact but reducing the pI value), and Mut6 (keeping pI value intact but decreasing hydrophobicity). Acidic, basic, and hydrophobic residues are colored in blue, red, and green, respectively. (B–D) Dequenching FRET to measure the FRET efficiency between mTFP and R18 in WT, Mut3–, Mut4–, Mut5–, and Mut6–IgG-BCR–expressing DT40 B cells. (B) FRET efficiency was calculated as detailed in Materials and methods. Representative confocal images of WT and Mut5-IgG-BCR are shown. Bars, 1.1 µm. DQ, dequenched; Q, quenched. (C and D) FRET efficiency was measured and plotted. Error bars represent means ± SD. (E–H) Statistical quantification of the synaptic recruitment of IgG-BCR and pSyk in J558L cells expressing Mut3– (E), Mut4– (F), Mut5– (G), and Mut6–IgG-BCR (H). Error bars represent means ± SEM. Two-tailed t tests were performed for the statistical comparisons. Data are from at least 30 cells over three independent experiments.

Article Snippet: Alexa Fluor 647 or Cy3 AffiniPure Fab fragment goat anti–mouse IgG1 Fc fragment specific and Cy5-Fab fragment goat anti–mouse IgM µ chain specific (Jackson ImmunoResearch Laboratories, Inc.) were used for mouse IgG- and IgM-BCR cell surface staining, respectively.

Techniques: Staining, Sequencing, Expressing, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: PI(4,5)P2 determines the threshold of mechanical force–induced B cell activation

doi: 10.1083/jcb.201711055

Figure Lengend Snippet: PM-tethered WT IgG-BCR moves faster at resting stage and slower after encountering 12-pN NP-TGTs in comparison with PM-untethered mutant IgG-BCR, Mut5, and Mut6. (A–C) A series of mathematical comparisons of the diffusion of WT IgG-BCR, Mut5–IgG-BCR, or Mut6–IgG-BCR molecules in J558L cells at resting stage (A), stimulated with 56 pN NP-TGT (B), or with 12 pN NP-TGT (C) in cumulative probability distribution plots (left), mean square displacement (MSD) plots (middle), or scatter plots (right). Bars represent median values. Data are from at least 30 cells over two independent experiments.

Article Snippet: Alexa Fluor 647 or Cy3 AffiniPure Fab fragment goat anti–mouse IgG1 Fc fragment specific and Cy5-Fab fragment goat anti–mouse IgM µ chain specific (Jackson ImmunoResearch Laboratories, Inc.) were used for mouse IgG- and IgM-BCR cell surface staining, respectively.

Techniques: Mutagenesis, Diffusion-based Assay

Journal: The Journal of Cell Biology

Article Title: PI(4,5)P2 determines the threshold of mechanical force–induced B cell activation

doi: 10.1083/jcb.201711055

Figure Lengend Snippet: Positively charged residues in the PM-tethered IgG-tail are responsible for the low–mechanical force threshold of IgG-BCR activation. (A) Left: Schematic illustration showing the FRET system to detect the interaction between mTFP (FRET donor) fused to the C terminus of the cytoplasmic tail (WT and Mut7/8/9/10) and R18 dye (FRET acceptor) stained on the PM. Right: Sequence and biochemical characteristic analysis of the cytoplasmic tail of WT, Mut7 (artificially designed cytoplasmic tail with high pI by high proportion of positively charged residues), Mut8 (artificially designed cytoplasmic tail with low pI as control for Mut7), Mut9, and Mut10 (artificially designed cytoplasmic tail with high hydrophobic level but almost electroneutral). Acidic, basic, and hydrophobic residues are colored in blue, red, and green, respectively. (B–D) Dequenching FRET to measure the FRET efficiency between mTFP and R18 in WT, Mut7–, Mut8–, and Mut9–IgG-BCR–expressing DT40 B cells. FRET efficiency was calculated as detailed in Materials and methods. (B) Representative confocal images of DT40 B cells expressing WT, Mut7–, Mut8–, and Mut9–IgG-BCR are shown. Bars, 1.1 µm. DQ, dequenched; Q, quenched. (C and D) FRET efficiency was measured and plotted. Error bars represent means ± SD. (E–H) Statistical quantification for the total FI of accumulated BCRs or pSyk in the immunological synapse of Mut7– (E), Mut8– (F), Mut9– (G), and Mut10–IgG-BCR (H)–expressing J558L cells. Error bars represent means ± SEM. Two-tailed t tests were performed for the statistical comparisons. Data are from at least 28 cells over three independent experiments.

Article Snippet: Alexa Fluor 647 or Cy3 AffiniPure Fab fragment goat anti–mouse IgG1 Fc fragment specific and Cy5-Fab fragment goat anti–mouse IgM µ chain specific (Jackson ImmunoResearch Laboratories, Inc.) were used for mouse IgG- and IgM-BCR cell surface staining, respectively.

Techniques: Activation Assay, Staining, Sequencing, Expressing, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: PI(4,5)P2 determines the threshold of mechanical force–induced B cell activation

doi: 10.1083/jcb.201711055

Figure Lengend Snippet: PI(4,5)P2 is significantly enriched in WT IgG-BCR membrane microdomains in quiescent B cells. (A) Representative conventional TIRF images of IgG-BCRs and superresolution images of mEos3.2-based lipid biosensors of PI(4,5)P2, PS, PI(3,4,5)P3, PA, and DAG. (B) Statistical quantification of enrichment of mEos3.2-based lipid biosensors of PI(4,5)P2, PS, PI(3,4,5)P3, PA, and DAG within WT IgG-BCRs. (C) Representative conventional TIRF images of IgG-BCRs and superresolution images of the mEos3.2-based PI(4,5)P2 biosensor within IgG-BCRs of WT, Mut5, and Mut6 in Ramos B cells (left). Statistical quantification of enrichment of mEos3.2-based PI(4,5)P2 biosensor within IgG-BCRs (right). (D) Two-color dSTORM and PALM images of BCR labeled with Alexa Fluor 647 goat Fab anti–mouse IgG1 Fc fragment and PI(4,5)P2 labeled with PH-PLCδ-mEos3.2 in Ramos B cells expressing IgG-BCRs with cytoplasmic tail of WT, Mut5, and Mut6. The relative fluorescence intensity distributions of BCR (red) and PI(4,5)P2 (green) along the white dashed lines are shown on the right. (E–G) L-function (E), Pearson correlation index analyses (F), and percentages of overlapped clusters (G) were used to quantify the spatial distribution of BCR with PI(4,5)P2 in Ramos B cells expressing IgG-BCRs with cytoplasmic tail of WT, Mut5, and Mut6 in quiescent B cells. (H) Representative conventional TIRF images of IgG-BCRs and superresolution images of mEos3.2-based lipid biosensors of PI(4,5)P2 in Ramos B cells expressing IgG-BCRs with cytoplasmic tails of WT or Mut7. (I and J) Pearson’s correlation index analyses (I) and enrichment analyses (J) were used to quantify the spatial distribution of BCR with PI(4,5)P2 in Ramos B cells expressing IgG-BCRs with cytoplasmic tails of WT and Mut7 in quiescent states. (K) Representative conventional TIRF images of IgG-BCRs and superresolution images of mEos3.2-based lipid biosensors of PI(4,5)P2 in 293T cells expressing IgG-BCRs with cytoplasmic tails of WT or Mut9. Enlarged images are marked by white squares in main images. Bars: (A, main images) 4 µm; (A, enlarged images) 320 nm; (C, D, H, and K, main images) 2 µm; (C, D, H, and K, enlarged images) 200 nm. (L and M) Pearson’s correlation index analyses (L) and enrichment analyses (M) were used to quantify the spatial distribution of BCR with PI(4,5)P2 in 293T cells expressing IgG-BCRs with cytoplasmic tails of WT and Mut9 in quiescent B cells. Error bars represent means ± SEM. Two-tailed t tests were performed for statistical comparisons. Data are from at least 25 cells over two independent experiments.

Article Snippet: Alexa Fluor 647 or Cy3 AffiniPure Fab fragment goat anti–mouse IgG1 Fc fragment specific and Cy5-Fab fragment goat anti–mouse IgM µ chain specific (Jackson ImmunoResearch Laboratories, Inc.) were used for mouse IgG- and IgM-BCR cell surface staining, respectively.

Techniques: Labeling, Expressing, Fluorescence, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: PI(4,5)P2 determines the threshold of mechanical force–induced B cell activation

doi: 10.1083/jcb.201711055

Figure Lengend Snippet: Depletion of PI(4,5)P2 within IgG-BCR membrane microdomains increases the mechanical force threshold in IgG-BCR activation. (A) A schematic diagram depicting the PI(4,5)P2 depletion through membrane-anchored Ins54p in B cells expressing WT IgG-BCRs. Phosphatase-dead Ins54p (named Lyn16-Ins54p phosphatase-dead control) was used as control for WT Ins54p (named Lyn16-Ins54p-WT). (B) Representative confocal images of PI(4,5)P2 lipid biosensor (GFP-PH) and Lyn-Ins54p-mCherry on DT40 B cell membrane are shown. (C) Statistical quantification of the ratio of MFI of GFP-PH on the PM versus MFI of GFP-PH in cytosol of DT40 B cells expressing Lyn-Ins54p phosphatase-dead control or Lyn16-Ins54p-WT. Error bars represent means ± SD. (D) Statistical quantification of the synaptic accumulation of WT IgG-BCR of J558L cells expressing Lyn16-Ins54p phosphatase-dead control or Lyn16-Ins54p-WT encountering 12-pN or 56-pN NP-TGT sensors. (E) A schematic diagram depicting the PI(4,5)P2 depletion within membrane microdomains of WT IgG-BCR through a rapamycin-based inducible system of WT IgG-BCR–FRB and FKBP-Ins54p-WT. Phosphatase-dead Ins54p (named FKBP-Ins54p phosphatase-dead control) was used as control for FKBP-Ins54p-WT. (F) Representative conventional TIRF images of FKBP-Ins54p-BFP, WT IgG-BCRs, and superresolution images of mEos3.2-based lipid biosensors of PI(4,5)P2 in 293T cells expressing FKBP-Ins54p-WT or FKBP-Ins54p phosphatase-dead controls after preincubation with rapamycin for 5 min. (G) Statistical quantification of enrichment of mEos3.2-based PI(4,5)P2 biosensor within WT IgG-BCR membrane microdomains. (H) Representative TIRFM images of FKBP-Ins54p-mCherry or BCRs in J558L cells expressing either FKBP-Ins54p-WT or FKBP-Ins54p phosphatase-dead control when encountering 12-pN or 56-pN NP-TGTs. Enlarged images in F are marked by white squares in main images. Bars: (B, F [main images], and H) 2 µm; (F, enlarged images) 200 nm. (I) Quantification of the total FI of BCR accumulation into immunological synapses in contact with the surfaces of 12-pN or 56-pN NP-TGTs. Error bars represent means ± SEM. Two-tailed t tests were performed for the statistical comparisons. Data are from at least 20 cells over two independent experiments, except as specifically described.

Article Snippet: Alexa Fluor 647 or Cy3 AffiniPure Fab fragment goat anti–mouse IgG1 Fc fragment specific and Cy5-Fab fragment goat anti–mouse IgM µ chain specific (Jackson ImmunoResearch Laboratories, Inc.) were used for mouse IgG- and IgM-BCR cell surface staining, respectively.

Techniques: Activation Assay, Expressing, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: PI(4,5)P2 determines the threshold of mechanical force–induced B cell activation

doi: 10.1083/jcb.201711055

Figure Lengend Snippet: Enrichment of PI(4,5)P2 within IgG-BCR membrane microdomains lowers the mechanical force threshold in IgG-BCR activation. (A) Schematic representation showing the amount of PI(4,5)P2 increasing through membrane-anchored PIP5K in B cells expressing Mut6–IgG-BCRs. Kinase-dead PIP5K (named Lyn16-PIP5K kinase-dead control) was used as control for WT PIP5K (named Lyn16-PIP5K-WT). (B) Representative confocal images of PI(4,5)P2 sensor (GFP-PH) and Lyn-PIP5K-mCherry on DT40 B cell membrane are shown. (C) Statistical quantification of ratio of MFI of GFP-PH on the PM versus the MFI of GFP-PH in the cytosol of DT40 B cells expressing Lyn-PIP5K kinase-dead control or Lyn16-PIP5K-WT. Error bars represent means ± SD. (D and E) Statistical quantifications of the synaptic accumulation of Mut6–IgG-BCR (D) or WT IgM-BCR (E) of J558L cells expressing Lyn16-PIP5K kinase-dead control or Lyn16-PIP5K-WT encountering 12-pN or 56-pN NP-TGT sensors. (F) A schematic diagram depicting the PI(4,5)P2-increasing system within membrane microdomains of Mut6–IgG-BCR through a rapamycin-based inducible system of Mut6–IgG-BCR–FRB and FKBP-PIP5K-WT. Kinase-dead PIP5K (named FKBP-PIP5K kinase-dead control) was used as control for FKBP-PIP5K-WT. (G) Representative conventional TIRF images of FKBP-PIP5K-BFP and Mut6–IgG-BCRs and superresolution images of mEos3.2-based lipid biosensors of PI(4,5)P2 in 293T cells expressing FKBP-PIP5K-WT or FKBP-PIP5K kinase-dead control after preincubation with rapamycin for 5 min. (H) Statistical quantification of enrichment of mEos3.2-based PI(4,5)P2 biosensor within Mut6–IgG-BCRs. (I) Representative TIRFM images of FKBP-PIP5K-mCherry or Mut6–IgG-BCRs in J558L cells expressing either FKBP-PIP5K-WT or FKBP-PIPK5 kinase-dead control when encountering 12-pN or 56-pN NP-TGTs. In G, enlarged areas are marked by white squares in main images. Bars: (B, G [main images], and I) 2 µm; (G, enlarged images) 200 nm. (J) Quantification of the total FI of BCR accumulation into immunological synapses in contact with surfaces of 12-pN or 56-pN NP-TGTs. Error bars represent means ± SEM. Two-tailed t tests were performed for the statistical comparisons. Data are from at least 20 cells over two independent experiments except specific descriptions.

Article Snippet: Alexa Fluor 647 or Cy3 AffiniPure Fab fragment goat anti–mouse IgG1 Fc fragment specific and Cy5-Fab fragment goat anti–mouse IgM µ chain specific (Jackson ImmunoResearch Laboratories, Inc.) were used for mouse IgG- and IgM-BCR cell surface staining, respectively.

Techniques: Activation Assay, Expressing, Two Tailed Test