1. The PIP2 metabolism–derived amplification loop accounts for the enhanced activation of B cells expressing PLAID-associated PLC-γ2 mutants at low temperature.
The PIP2 metabolism–derived amplification loop accounts for the enhanced activation of B cells expressing PLAID-associated PLC-γ2 mutants at low temperature. (A) Quantification of PIP2 mFI at the contact interface between fibronectin-coated coverslips (nonstimulatory) or PLBs tethering anti-chicken IgM surrogate antigens and DT40-WT, DT40–PLC-γ2–KO, and DT40–PLC-γ2–KO B cells with the expression of PLC-γ2-WT, PLC-γ2–Δ19, or PLC-γ2–Δ20-22, respectively. PIP2 molecules were stained with anti-PIP2 antibodies (n = 41 to 52 cells). Bar represents mean ± SEM. (B) PCI between BCR and PIP2 in DT40–PLC-γ2–KO B cells expressing PLC-γ2–Δ19 on PLBs tethering anti-chicken IgM surrogate antigens under either low or physiological temperature (n = 17 cells). Bar represents mean ± SD. (C and D) Quantification of antigen microclusters in DT40–PLC-γ2–KO B cells expressing either PLC-γ2–Δ19 or PLC-γ2–Δ20-22 under low or physiological temperature. (C) Microcluster number. Each dot represents one cell (n = 22 to 31). Bar represents mean ± SD. (D) Integrated FI of each microcluster (n > 800 microclusters). Bar represents mean ± SEM. (E to H) (E) TIRFM images of PIP2 and BCR in human primary B cells expressing PLC-γ2–WT or PLC-γ2–Δ19 under low or physiological temperature. Correlated pixel FI plot of BCR and PIP2 from the magnified areas (3 μm × 3 μm) (right). Scale bars, 2 μm. (F) Quantification of mFI of PIP2 (n = 20 to 24 cells). Bar represents mean ± SEM. (G) Microcluster number. Each dot represents one cell (n = 25 to 38). Bar represents mean ± SD. (H) Integrated FI of each microcluster (n > 166 microclusters). Bar represents mean ± SEM. *P < 0.05, **P < 0.01, ***P < in two-tailed t tests. Data are representative of at least two independent experiments.
Chenguang Xu et al. Sci. Immunol. 2017;2:eaan0787
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