Localized synthesis of PIP2 by PIP5K is mainly responsible for the PIP2 enrichment outside the BCR microclusters. Localized synthesis of PIP2 by PIP5K.

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Localized synthesis of PIP2 by PIP5K is mainly responsible for the PIP2 enrichment outside the BCR microclusters. Localized synthesis of PIP2 by PIP5K is mainly responsible for the PIP2 enrichment outside the BCR microclusters. (A and B) TIRFM images of PIP2 in DT40-WT, DT40-PIP5Kα-KO, DT40-PIP5Kγ-KO, or DT40-PIP5Kα/γ-DKO B cells on nonstimulating coverslips or after BCR stimulation for 3 min by PLBs containing anti-IgM surrogate antigens. (A) Representative images of the cells on the contact interface. Scale bars, 2 μm. (B) Quantification of PIP2 mFI (n = 28 to 44 cells). Bar represents mean ± SEM. (C to G) Distribution of BCR and PIP2 in the immunological synapse of DT40-PIP5Kα/γ-DKO B cells. (C) TIRFM images; boxed areas (4.5 μm × 4.5 μm) are magnified (lower right). (D) FI profiles of BCR and PIP2 along the white lines in (C). Scale bar, 2 μm. (E) Correlated pixel FI plot of BCR and PIP2 from the boxed areas in (C). (F) FI ratio of PIP2 outside to that inside the microclusters. Each dot represents one microcluster (n = 21 to 25). Bar represents mean ± SD. (G) PCI between BCR and PIP2. Each dot represents one cell (n = 20). 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-PIP5Kα/γ-DKO B cells. Images are pseudo-colored (top row). Boxed areas (4.5 μm × 4.5 μm) in the leftmost TIRFM images are magnified and provided as time-lapse images. BCR microclusters 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 are also given (lower right). Scale bar, 2 μm. (I to L) Distribution of BCR and GFP-PIP5Kα in the immunological synapse of DT40-WT B cells. (I) TIRFM images; boxed areas (4.5 μm × 4.5 μm) are magnified (lower right). Scale bar, 2 μm. (J) FI profiles of BCR and GFP-PIP5Kα along the white lines in (I). (K) Correlated pixel FI plot of BCR and GFP-PIP5Kα from the boxed areas in (I). (L) PCI between BCR and GFP-PIP5Kα or GFP control (n = 21 to 31 cells). Bar represents mean ± SEM. **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