Research ArticleION TRANSPORT

The channel-kinase TRPM7 regulates antigen gathering and internalization in B cells

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Science Signaling  05 Jun 2018:
Vol. 11, Issue 533, eaah6692
DOI: 10.1126/scisignal.aah6692
  • Fig. 1 TRPM7 controls the actin cytoskeleton in B cells.

    (A to C) Scanning electron microscopy analysis of unstimulated WT and TRPM7-KO DT40 B cells. Images (A) are representative. Data on the numbers of filopodia (B) and filopodia length (C) are means ± SEM of a minimum of 30 cells. (D) Scanning electron microscopy analysis of IgM-stimulated WT and TRPM7-KO DT40 B cells. The dotted line indicates the cell edge. (E to G) Confocal microscopy analysis of actin in WT and TRPM7-KO DT40 B cells stimulated with lipid bilayers containing anti-IgM. Images (E) are representative. Data on the contact area (F) and shape factor (G) are means ± SEM of a minimum of 30 cells. All images and quantified data are representative of three independent experiments. Statistical significance was assessed by Mann-Whitney test. ****P < 0.0001. Scale bars, 5 μm.

  • Fig. 2 B cell spreading and the contraction response are impaired in TRPM7-KO cells.

    (A to E) TIRFM analysis of WT and TRPM7-KO DT40 B cells stimulated with bilayers containing fluorescently labeled anti-IgM. Images (A) are representative, and the contact area (B) and the number of fluorescent antigen clusters over time (C) are means ± SEM of measurements taken from a minimum of 20 cells. Antigen microcluster tracks from individual cells (D) are representative, and the diffusion coefficient of antigen microclusters (E) is means ± SEM of measurements pooled from three independent experiments. DIC, differential interference contrast. (F to I) Confocal microscopy analysis of actin in WT and TRPM7-KO DT40 B cells after stimulation with lipid bilayers containing anti-IgM for 3 min (F and G) or 10 min (H and I). Images (F and H) are representative. Relative fluorescence intensity (FI) plots indicate the distribution of antigen (pink) and actin (green) along the dashed line. Data on total antigen fluorescence intensity at the contact interface (G and I) are means ± SEM of measurements taken from a minimum of 20 cells. AU, arbitrary units. All images and quantified data are representative of three independent experiments, except for the data in (E). Statistical significance for the data in (B) and (C) was assessed by regression analysis (see fig. S1). Statistical significance for the data in (E), (G), and (I) was assessed by Mann-Whitney test. ***P < 0.001, ****P < 0.0001. Scale bars, 5 μm.

  • Fig. 3 TRPM7 kinase activity is important for cell contraction in response to membrane-bound antigen.

    (A) Scanning electron microscopy analysis of unstimulated TRPM7-KD cells. (B to F) TIRFM analysis of TRPM7-KD cells stimulated with bilayers containing fluorescently labeled anti-IgM. Images (A) are representative, and data on the contact area (C) and the number of antigen clusters over time (D) are means ± SEM of measurements taken from at least 20 cells. Antigen microcluster tracks from individual cells (E) are representative, and the diffusion coefficient of antigen microclusters (F) is means ± SEM pooled from three independent experiments. (G to J) Confocal microscopy analysis of TRPM7-KD cells stimulated with lipid bilayers containing anti-IgM for 3 min (G and H) or 10 min (I and J). Relative fluorescence intensity plots indicate the distribution of antigen (pink) and actin (green) along the dashed line. Images (G and I) were quantified. Data on antigen accumulation (H and J) are means ± SEM of measurements taken from a minimum of 20 cells per experiment. (K and L) Western blot analysis of phosphorylated extracellular signal–regulated kinase (pERK) in lysates from WT, TRPM7-KO, and TRPM7-KD cells that were stimulated for the indicated times with anti-IgM antibody. Blots (K) are representative of three independent experiments. Pooled normalized pERK band intensity data (L) are means ± SEM. All images and quantified data are representative of three independent experiments, except for the data in (E) and (L). Statistical significance for the data in (C) and (D) was assessed by regression analysis (see fig. S3). The statistical significance for the data in (F), (H), and (J) was assessed by Mann-Whitney test. One-way analysis of variance (ANOVA) with Tukey’s post hoc test was used to determine statistical significance for the data in (L). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Scale bars, 5 μm.

  • Fig. 4 Antigen gathering is compromised in cells expressing PLC-γ2-T1045A.

    (A to E) TIRFM analysis of PLC-γ2-KO cells expressing PLC-γ2-T1045A and stimulated with lipid bilayers containing fluorescently labeled anti-IgM. Images (A) and antigen microcluster tracks from individual cells (D) are representative of three independent experiments. Data on the contact area (B), the number of antigen clusters over time (C), and the diffusion coefficients of antigen microclusters (E) are means ± SEM of pooled measurements taken from at least 17 cells. Statistical significance was assessed by Mann-Whitney test and regression analysis (see fig. S5). ***P < 0.001, ****P < 0.0001. Scale bars, 5 μm.

  • Fig. 5 Lipid metabolism is altered in TRPM7-KO and TRPM7-KD cells upon activation.

    (A to D) TIRFM analysis of WT, TRPM7-KO, and TRPM7-KD cells expressing a fluorescent biosensor for either PIP2 (A and B) or DAG (C and D) after stimulation with lipid bilayers containing anti-IgM. Images (A and C) are representative of four independent experiments. Fold change data (B and D) are means ± SEM of pooled measurements taken from a minimum of 30 cells per condition. Statistical significance was assessed by regression analysis (see figs. S6 and S7). Scale bars, 5 μm.

  • Fig. 6 Reduced expression of TRPM7 leads to altered antigen accumulation and signaling in primary B cells.

    (A) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of the expression of TRPM7 mRNA in WT and TRPM7+/− primary B cells. Data are means ± SEM of triplicate analysis from four independent experiments. (B to G) Confocal microscopy analysis of WT and TRPM7+/− primary B cells stimulated with lipid bilayers containing anti-Igκ for 1.5 min (B to D) or 10 min (E to G). Images (B and E) are representative of three independent experiments. Antigen accumulation (C and F) and contact area (D and G) are means ± SEM of measurements taken from a minimum of 40 cells per experiment. (H and I) Western blotting analysis of pERK in lysates of WT and TRPM7+/− primary B cells that were stimulated for the indicated times with anti-IgM. Blots (H) are representative of five independent experiments. Pooled normalized pERK band intensity data (I) are means ± SEM. Statistical significance was assessed by Mann-Whitney test. *P < 0.05, **P < 0.01, ****P < 0.0001. Scale bars, 5 μm.

  • Fig. 7 TRPM7 is important for antigen internalization and presentation.

    (A and B) Flow cytometry analysis of surface BCR expression on WT, TRPM7-KO, and TRPM7-KD DT40 B cells after IgM stimulation. Histograms (A) are representative of three independent experiments. Quantified fold change in the mean fluorescence intensity (MFI) (B) is means ± SEM. (C and D) Flow cytometry analysis of surface MHC class II expression on primary murine B cells after anti-κ stimulation with vehicle or NS8593 (channel inhibitor). Histograms (C) are representative of three independent experiments. Quantified MFI (D) data are means ± SEM. (E) A20 B cells were treated with vehicle or NS8593 and incubated with beads coated with HEL. Cells were mixed with HEL-specific 2G7 T cell hybridoma, and antigen presentation was assessed by ELISA measurement of IL-2 secretion 18 hours after stimulation. Fold change IL-2 concentration data representative of three independent experiments are means ± SEM. Statistical significance was assessed by one-way ANOVA with Tukey’s post hoc test or Mann-Whitney (E). *P < 0.05.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/533/eaah6692/DC1

    Fig. S1. Statistical analysis of the spreading and microcluster accumulation in WT and TRPM7-KO DT40 B cells.

    Fig. S2. Expression of TRPM7 in TRPM7-KO cells restores B cell antigen gathering.

    Fig. S3. Statistical analysis of the spreading and microcluster accumulation in WT and TRPM7-KD DT40 B cells.

    Fig. S4. Supplementation with extracellular Mg2+ alters signaling but not the contraction defect in TRPM7-KO and TRPM7-KD cells.

    Fig. S5. Statistical analysis of the spreading and microcluster accumulation in PLC-γ2-T1045A DT40 B cells.

    Fig. S6 Statistical analysis of the PIP2 accumulation in WT, TRPM7-KO, and TRPM7-KD DT40 B cells.

    Fig. S7. Statistical analysis of the DAG accumulation in WT, TRPM7-KO, and TRPM7-KD DT40 B cells.

    Fig. S8. Pharmacological inhibition of TRPM7 increases antigen accumulation in DT40 and primary B cells.

    Fig. S9. Pharmacological inhibition is specific to TRPM7 channels.

    Fig. S10. Antigen internalization is not affected in TRPM7+/− B cells.

    Fig. S11. Supplementation with extracellular Mg2+ is not sufficient to rescue the BCR internalization defect in TRPM7-KO cells.

    Movie S1. B cell spreading and the contraction response are impaired in TRPM7-KO DT40 cells.

    Movie S2. TRPM7 kinase activity is important for cell contraction.

    Movie S3. Antigen gathering is compromised in cells expressing PLC-γ2-T1045A.

    Movie S4. The amount of PIP2 at the cell-bilayer interface is altered in TRPM7-KO and TRPM7-KD cells upon activation.

    Movie S5. The amount of DAG at the cell-bilayer interface is altered in TRPM7-KO and TRPM7-KD cells upon activation.

  • Supplementary Materials for:

    The channel-kinase TRPM7 regulates antigen gathering and internalization in B cells

    Mithunah Krishnamoorthy, Laabiah Wasim, Fathima Hifza Mohamed Buhari, Tiantian Zhao, Trisha Mahtani, Josephine Ho, Sohee Kang, Francina Deason-Towne, Anne-Laure Perraud, Carsten Schmitz, Bebhinn Treanor*

    *Corresponding author. Email: bebhinn.treanor{at}utoronto.ca

    This PDF file includes:

    • Fig. S1. Statistical analysis of the spreading and microcluster accumulation in WT and TRPM7-KO DT40 B cells.
    • Fig. S2. Expression of TRPM7 in TRPM7-KO cells restores B cell antigen gathering.
    • Fig. S3. Statistical analysis of the spreading and microcluster accumulation in WT and TRPM7-KD DT40 B cells.
    • Fig. S4. Supplementation with extracellular Mg2+ alters signaling but not the contraction defect in TRPM7-KO and TRPM7-KD cells.
    • Fig. S5. Statistical analysis of the spreading and microcluster accumulation in PLC-γ2-T1045A DT40 B cells.
    • Fig. S6. Statistical analysis of the PIP2 accumulation in WT, TRPM7-KO, and TRPM7-KD DT40 B cells.
    • Fig. S7. Statistical analysis of the DAG accumulation in WT, TRPM7-KO, and TRPM7-KD DT40 B cells.
    • Fig. S8. Pharmacological inhibition of TRPM7 increases antigen accumulation in DT40 and primary B cells.
    • Fig. S9. Pharmacological inhibition is specific to TRPM7 channels.
    • Fig. S10. Antigen internalization is not affected in TRPM7+/− B cells.
    • Fig. S11. Supplementation with extracellular Mg2+ is not sufficient to rescue the BCR internalization defect in TRPM7-KO cells.
    • Legends for movies S1 to S5

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.avi format). B cell spreading and the contraction response are impaired in TRPM7-KO DT40 cells.
    • Movie S2 (.avi format). TRPM7 kinase activity is important for cell contraction.
    • Movie S3 (.avi format). Antigen gathering is compromised in cells expressing PLC-γ2-T1045A.
    • Movie S4 (.avi format). The amount of PIP2 at the cell-bilayer interface is altered in TRPM7-KO and TRPM7-KDcells upon activation.
    • Movie S5 (.avi format). The amount of DAG at the cell-bilayer interface is altered in TRPM7-KO and TRPM7-KD cells upon activation.

    © 2018 American Association for the Advancement of Science

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