Research ArticleANTIGEN RECEPTOR SIGNALING

A PIP2-derived amplification loop fuels the sustained initiation of B cell activation

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Science Immunology  17 Nov 2017:
Vol. 2, Issue 17, eaan0787
DOI: 10.1126/sciimmunol.aan0787
  • Fig. 1 PIP2 is depleted inside but enriched outside the BCR microclusters.

    (A) Schematic of a PIP2 biosensor. (B to F) Distribution of BCR and PIP2 in the immunological synapse of DT40-WT B cells. (B) TIRFM images; the images in the boxed areas (4.5 μm × 4.5 μm) are magnified (right). Scale bars, 2 μm. (C) FI profiles of BCR and GFP–PLC-δ–PH (or GFP control) along the white lines in (B). (D) Pixel FI of BCR and GFP–PLC-δ–PH (or GFP control) from the boxed areas in (B). (E) FI ratio of GFP–PLC-δ–PH (or GFP control) outside to that inside the microclusters. Each dot represents one microcluster (n = 29). (F) Pearson’s correlation index (PCI) between BCR and GFP–PLC-δ–PH (or GFP control). Each dot represents one cell (n = 20 to 25). Bar represents mean ± SD. (G and H) Merged binary mask of BCR and the PALM image of PIP2 (mEos3.2–PLC-δ–PH, left) or the control mEos3.2–PLC-δ–PH–Mut (right) in the immunological synapse of DT40-WT B cells (3 μm × 3 μm regions) (G). Scale bars, 500 nm. Enrichment of PIP2 within the BCR microclusters (H). Each dot represents a 3 μm × 3 μm region (n = 26 to 29). Bar represents mean ± SD. (I) Two-color time-lapse TIRFM images of BCR and PIP2 [red fluorescent protein (RFP)–PLC-δ–PH] in the immunological synapse of DT40-WT 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 are also given (lower right). Scale bar, 2 μm. (J to M) (J) Representative time sequence images (first two rows) of BCR and PIP2 (RFP–PLC-δ–PH) in primary B1-8 primary B cells before and after photoactivation of the caged-NP antigen. The images in the boxed areas (3 μm × 3 μm) are magnified and merged, as shown in the third row (BCR, red; PIP2, green). The correlated pixel FI of BCR and RFP–PLC-δ–PH in the magnified regions are also shown (bottom row). Normalized mFI of BCR (K) and RFP–PLC-δ–PH (L) along time (n = 13 to 14). Scale bar, 2 μm. (M) PCI between BCR and PIP2 along time (n = 11). Bar represents mean ± SEM. a.u., arbitrary units. ns, not significant; ***P < 0.001 in two-tailed t tests. Data are representative of two or three independent experiments.

  • Fig. 2 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.

  • Fig. 3 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.

  • Fig. 4 DAG-derived PA mediates the recruitment and localization of PIP5Kα to the B cell immunological synapse.

    (A) Schematic of the potential PIP2 synthesis–feedback loop from the product of PIP2 hydrolysis DAG; DAG is converted into PA catalyzed by DGK and then PA activates PIP5K to regenerate PIP2. (B to D) Quantification of DGKζ in either DT40-WT or DT40–PLC-γ2–KO B cells. (B) Representative confocal images of equatorial and bottom planes of the DT40-WT and DT40–PLC-γ2–KO cells expressing GFP-DGKζ on fibronectin-coated coverslips (0 min) and PLBs tethering anti-IgM surrogate antigens. Scale bars, 2 μm. (C) Ratio of the mFI of GFP-DGKζ (plasma membrane localization to cytosol localization) in DT40-WT and DT40–PLC-γ2–KO B cells, or in DT40–PLC-γ2–KO B cells with the exogenous expression of PLC-γ2–WT, PLC-γ2–LD, or PLC-γ2–R564A (n = 28 to 52 cells). PM, plasma membrane. (D) Ratio of the mFI of GFP-DGKζ (bottom localization to equatorial localization; n = 28 to 52 cells). Bar represents mean ± SEM. (E to H) (E) TIRFM images of GFP-PIP5Kα and PA (RFP-Spo20) in DT40-WT B cells on PLBs tethering anti-IgM surrogate antigens. Boxed areas (4.5 μm × 4.5 μm) are magnified (right). Scale bar, 2 μm. (F) Correlated pixel FI plot of GFP-PIP5Kα and PA (top) and BCR and PA (bottom) from the boxed areas in (E). (G) FI profiles of GFP-PIP5Kα, PA, and BCR along the white lines in (E). (H) PCI between BCR and PA, or between GFP-PIP5Kα and PA (n = 28 to 40 cells). Bar represents mean ± SEM. (I and J) Quantification of immunological synapse recruitment of PIP5Kα in DT40-WT or DT40-DGKζ-KO B cells. (I) Representative images of the equatorial and bottom planes of the DT40-WT or DT40-DGKζ-KO B cells expressing GFP-PIP5Kα on fibronectin-coated coverslips (0 min) and PLBs tethering anti-IgM surrogate antigens. Scale bars, 2 μm. (J) Ratio of mFI of GFP-PIP5Kα (bottom localization to equatorial localization; n = 18 to 26 cells). Bar represents mean ± SEM. (K to N) (K) TIRFM images of BCR and GFP-PIP5Kα in DT40-DGKζ-KO B cells on PLBs tethering anti-IgM surrogate antigens. Scale bar, 2 μm. (L) FI profiles of BCR and GFP-PIP5Kα along the white line in (K). Correlated pixel FI plot (M) of BCR and GFP-PIP5Kα from the magnified areas in (K). (N) PCI between BCR and PIP5Kα at indicated time points (n = 16 to 27 cells). Bar represents mean ± SEM. (O and P) (O) TIRFM images of PIP2 stained with anti-PIP2 antibodies within the immunological synapse of DT40-WT or DT40-DGKζ-KO B cells. Scale bars, 2 μm. (P) Quantification of mFI of PIP2 within the immunological synapse (n = 30 to 46 cells). Bar represents mean ± SEM. (Q to T) DT40-DGKζ-KO B cells expressing GFP–PLC-δ–PH on PLBs tethering anti-IgM surrogate antigens at 3 min. (Q) Representative TIRFM images. Images in the boxed areas (4.5 μm × 4.5 μm) are magnified. Scale bar, 2 μm. (R) FI profiles of BCR and PIP2 along the white line in (Q). The r value between two signals is given. (S) Correlated pixel FI plot of BCR and PIP2 in the boxed area in (Q). (T) PCI between BCR and PIP2. Each dot represents one cell (n = 15 cells). Bar represents mean ± SD. **P < 0.01, ***P < 0.001 in two-tailed t tests. Data are representative of two or three independent experiments.

  • Fig. 5 The fast Brownian motility of DAG in B cell immunological synapse.

    (A and B) Representative TIRFM images of DT40-WT B cells expressing PKCθ-C1–GFP (A) or GFP-DGKζ (B) on PLBs containing anti-IgM surrogate antigens (left). FI profiles of BCR and DAG or GFP-DGKζ (middle) along the white line in the left images. Correlated pixel FI plot of BCR and DAG or GFP-DGKζ (right) in the boxed area in the left images. Scale bars, 2 μm. (C to H) Single-molecule tracking of DAG (PKCθ-C1–mEos3.2) or PIP2 (mEos3.2–PLC-δ–PH) in DT40-WT B cells on PLBs tethering anti-IgM surrogate antigens. (C) Trajectories were pseudo-colored according to the value of the diffusion coefficients (n is given above the figures). (D) Mean square displacement (MSD) plots. Bar represents mean ± SEM. (E) Diffusion coefficients with the median indicated by black bars. (F) Cumulative probability of diffusion coefficients of DAG and PIP2. (G) The probability distribution of diffusion coefficients and fitting to two populations showing slow (red line) and fast (green line) diffusion. The mean diffusion coefficients of fast and slow population are given above the plots. (H) The percentage of the slow population in DAG or PIP2. (I to K) (I) DAG or PIP2 trajectories are randomly colored and are plotted on the background of BCR microclusters. The right panels are the magnified regions from the boxed areas (left) with time series. Scale bars, 2 μm. (J) The percentage of trajectories crossing the BCR microcluster borders. (K) The percentage of the trajectories showing the direction from inside to outside the BCR microclusters. n = 6 cells. Bar represents mean ± SEM. **P < 0.01, ***P < 0.001 in two-tailed t tests. Data are representative of three independent experiments.

  • Fig. 6 Both PIP2 depletion inside and synthesis outside the BCR microclusters are essential for the initiation of B cell activation.

    (A) Schematic presentation for the targeted PIP2 manipulation system inside the BCR microclusters. PLC-γ2–LD fused to Ins54p (or PIP5Km) is recruited to early BCR microclusters after antigen recognition. (B to D) Quantification of antigen microclusters in DT40-WT B cells and DT40-WT B cells with the expression of the indicated type of plasmids, as shown in (C). (B) TIRFM images of antigen microclusters in DT40-WT B cells with the expression of PLC-γ2–LD–Ins54p or PLC-γ2–LD–PIPKm. Scale bars, 2 μm. (C) Number of microcluster in DT40-WT B cells with the expression of the indicated type of plasmids (n = 29 to 39 cells). Bar represents mean ± SD. (D) Integrated FI of the antigen microclusters in cells as in (C) (n > 500 microclusters). Bar represents mean ± SEM. (E and F) (E) Number of antigen microclusters in DT40–PLC-γ2–KO B cells with the exogenous expression of PLC-γ2–LD–Ins54p or PLC-γ2–LD–Ins54p-D281A with or without the addition of PA micelles (n = 32 to 50 cells). Bar represents mean ± SEM. (F) Integrated FI of microclusters in cells as in (E) (n > 500 microclusters). Bar represents mean ± SEM. (G) Schematic representation of the targeted PIP2 manipulation system outside the BCR microclusters. Before rapamycin addition, the FKBP fused to Ins54p (or PIP5Km) will localize to the cytosol. Instead, the FKBP fused to Ins54p (or PIP5Km) will be recruited to the CD45-(ET)-FRB after the addition of rapamycin. (H to J) (H) Representative TIRFM images of DT40-WT B cells on PLBs tethering anti-IgM surrogate antigens with the expression of the indicated type of plasmids in the CD45-targeted PIP2 manipulation system. Scale bars, 2 μm. (I) Number of BCR microclusters in the B cells expressing the indicated type of plasmids in the CD45-targeted PIP2 manipulation system. Each dot represents one cell (n = 19 to 35 cells). Bar represents mean ± SD. (J) Integrated FI of the BCR microclusters in the cells as in I (n > 216 microclusters). Bar represents mean ± SEM. LD, PLC-γ2–LD; Rap, rapamycin. *P < 0.05, **P < 0.01, ***P < 0.001 in two-tailed t tests. Data are representative of at least two independent experiments.

  • Fig. 7 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 < 0.001 in two-tailed t tests. Data are representative of at least two independent experiments.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/2/17/eaan0787/DC1

    Materials and Methods

    Fig. S1. PIP2 is enriched in the immunological synapse upon B cell activation.

    Fig. S2. Expression of PIP5Kα, PIP5Kβ, and PIP5Kγ in DT40 B cells.

    Fig. S3. DGKζ is essential for converting DAG into PA after B cell activation.

    Fig. S4. BCR signaling–induced PA generation is converted from the PLC-γ2–derived DAG by DGKζ.

    Fig. S5. The validation of the PIP2 density manipulation system.

    Fig. S6. Cold treatment induces the regeneration of excessive amount of PIP2 in B cells expressing PLAID-associated PLC-γ2 mutants.

    Fig. S7. Model depicting the PIP2-derived amplification loop for the robust initiation of B cell activation.

    Table S1. Sequence of primers used in reverse transcription polymerase chain reaction for detecting the expression of PIP5K and DGK isoenzymes in DT40 B cells.

    Movie S1. The depletion of PIP2 inside the BCR microclusters and the enrichment of PIP2 at the region outside the BCR microclusters in DT40-WT B cells.

    Movie S2. PIP2 is depleted inside the BCR microclusters and enriched at the region outside the BCR microclusters in B1-8 primary B cells on a caged-NP antigen system.

    Movie S3. The depletion of PIP2 inside the BCR microclusters and the enrichment of PIP2 at the region outside the BCR microclusters are impaired in DT40-PLC-γ2-KO B cells.

    Movie S4. Enrichment of PIP2 at the region outside the BCR microclusters in a PIP5K-dependent manner.

    Movie S5. DAG is more often than PIP2 to cross the BCR microcluster border.

    Raw data Excel file

    References (4248)

  • Supplementary Materials

    Supplementary Material for:

    A PIP2-derived amplification loop fuels the sustained initiation of B cell activation

    Chenguang Xu, Hengyi Xie, Xingdong Guo, Haipeng Gong, Lei Liu, Hai Qi, Chenqi Xu, Wanli Liu*

    *Corresponding author. Email: liulab{at}tsinghua.edu.cn

    Published 17 November 2017, Sci. Immunol. 2, eaan0787 (2017)
    DOI: 10.1126/sciimmunol.aan0787

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. PIP2 is enriched in the immunological synapse upon B cell activation.
    • Fig. S2. Expression of PIP5Kα, PIP5Kβ, and PIP5Kγ in DT40 B cells.
    • Fig. S3. DGKζ is essential for converting DAG into PA after B cell activation.
    • Fig. S4. BCR signaling–induced PA generation is converted from the PLC-γ2– derived DAG by DGKζ.
    • Fig. S5. The validation of the PIP2 density manipulation system.
    • Fig. S6. Cold treatment induces the regeneration of excessive amount of PIP2 in B cells expressing PLAID-associated PLC-γ2 mutants.
    • Fig. S7. Model depicting the PIP2-derived amplification loop for the robust initiation of B cell activation.
    • Table S1. Sequence of primers used in reverse transcription polymerase chain reaction for detecting the expression of PIP5K and DGK isoenzymes in DT40 B cells.
    • Legends for movies S1 to S5
    • References (42–48).

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.avi format). The depletion of PIP2 inside the BCR microclusters and the enrichment of PIP2 at the region outside the BCR microclusters in DT40-WT B cells.
    • Movie S2 (.avi format). PIP2 is depleted inside the BCR microclusters and enriched at the region outside the BCR microclusters in B1-8 primary B cells on a caged-NP antigen system.
    • Movie S3 (.avi format). The depletion of PIP2 inside the BCR microclusters and the enrichment of PIP2 at the region outside the BCR microclusters are impaired in DT40-PLC-γ2-KO B cells.
    • Movie S4 (.avi format). Enrichment of PIP2 at the region outside the BCR microclusters in a PIP5K-dependent manner.
    • Movie S5 (.avi format). DAG is more often than PIP2 to cross the BCR microcluster border.
    • Raw data Excel file

    Download Movies S1–S5

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