Localized hydrolysis of PIP2 by PLC-γ2 is mainly responsible for the PIP2 depletion inside the BCR microclusters. Localized hydrolysis of PIP2 by PLC-γ2.

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Localized hydrolysis of PIP2 by PLC-γ2 is mainly responsible for the PIP2 depletion inside the BCR microclusters. Localized hydrolysis of PIP2 by PLC-γ2 is mainly responsible for the PIP2 depletion inside the BCR microclusters. (A and B) Representative TIRFM images of DiO, PIP2, and BCR on the contact interface in DT40-WT B cells on nonstimulating (A) or antigen-coated (B) coverslips. Cells were stained with DiO and anti-PIP2 antibodies. Correlated pixel FI plot of BCR and DiO or PIP2 (right) in the boxed area (4.5 μm × 4.5 μm) in the left images. Scale bars, 2 μm. (C) Representative TIRFM images of BCR and Lact-C2-GFP on the nonstimulating or antigen-coated contact interfaces in DT40-WT B cells. Correlated pixel FI plot of BCR and PS (right) in the boxed area (4.5 μm × 4.5 μm) in the left images. Scale bars, 2 μm. (D to F) Distribution of BCR and PIP2 in the immunological synapse of DT40–PLC-γ2–KO B cells. (D) TIRFM images; the images in the boxed areas (4.5 μm × 4.5 μm) are magnified (lower right). Scale bar, 2 μm. (E) FI profiles of BCR and GFP–PLC-δ–PH along the white lines in (D). (F) Pixel FI of BCR and GFP–PLC-δ–PH from the boxed areas in (D). (G) Enrichment of PIP2 (mEos3.2–PLC-δ–PH, from PALM imaging) within the BCR microclusters. Each dot represents a 3 μm × 3 μm region (n = 32 to 34). Bar represents mean ± SD. (H) Two-color time-lapse TIRFM images of BCR and PIP2 (RFP–PLC-δ–PH) in the immunological synapse of DT40–PLC-γ2–KO B cells. Images are pseudo-colored (left). Boxed areas (3 μm × 3 μm) are magnified in time sequence. BCR microcluster regions are denoted by white circles in BCR images and by the corresponding black circles in PIP2 images. FI profiles of BCR and PIP2 on the white line in the TIRFM images are given (upper right). Correlated pixel FI plots of BCR and PIP2 (lower right) are also given. Scale bar, 2 μm. (I and J) (I) TIRFM images of PIP2 stained with anti-PIP2 antibodies within the immunological synapse of either DT40-WT or DT40–PLC-γ2–KO B cells. Scale bars, 2 μm. (J) Quantification of mFI of PIP2 within the B cell immunological synapse (n = 40 to 60 cells). Bar represents mean ± SEM. r, correlation. **P < 0.01, ***P < 0.001 in two-tailed t tests. Data are representative of two or three independent experiments. Chenguang Xu et al. Sci. Immunol. 2017;2:eaan0787 Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works