Research ArticleDEGRANULATION

Tomosyn functions as a PKCδ-regulated fusion clamp in mast cell degranulation

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Science Signaling  03 Jul 2018:
Vol. 11, Issue 537, eaan4350
DOI: 10.1126/scisignal.aan4350
  • Fig. 1 Mast cells express tomosyn, which inhibits degranulation.

    (A) RT-PCR analysis of STXBP5 mRNA splice variant expression in RBL-2H3 and BMMCs. The relative size of the big (b), medium (m), and short (s) isoforms are indicated. Images are representative of two independent experiments. (B) Western blot (IB) analysis for tomosyn in lysates from BMMCs and RBL-2H3 cells. Blots are representative of three independent experiments. (C) Confocal microscopy analysis of tomosyn distribution in BMMCs before and after PMA/ionomycin (ion) stimulation. Single optical section images are representative of three independent experiments. (D) Western blot analysis for tomosyn in lysates of RBL-2H3 cells transfected with control (siCtrl) and tomosyn siRNAs (si1Tomosyn, si2Tomosyn, and si3Tomosyn). Blots (top) are representative of at least three independent experiments. Tomosyn band intensities normalized to actin (bottom) are means ± SEM pooled from all experiments. (E) β-Hexosaminidase release after IgE/dinitrophenyl–human serum albumin (DNP-HSA) and PMA/ionomycin stimulation in RBL-2H3 cells transfected with control and tomosyn siRNA. Graphed data are means ± SEM from eight independent experiments per group. (F) Western blot analysis for tomosyn in lysates of BMMCs transfected with control and tomosyn siRNA. Blots (left) are representative of at least three independent experiments. Tomosyn band intensities normalized to actin (right) are means ± SEM pooled from all experiments. (G) Flow cytometry analysis of CD63 cell surface exposure on resting and IgE/DNP-HSA–stimulated BMMCs transfected with control and tomosyn siRNA. Contour plots are representative of four independent experiments. Graphed data are the mean percentage of CD63+ cells ± SEM pooled from all experiments. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 by one-way analysis of variance (ANOVA) followed by Dunnett’s test (D and F) and two way-ANOVA followed by Bonferroni’s correction (E and G). Scale bars, 5 μm.

  • Fig. 2 BMMC stimulation induces a switch in tomosyn interaction with STX4 or STX3.

    (A and C) Confocal analysis of tomosyn proximity to STX4 (A) or STX3 (C) determined by PLA in BMMC at the indicated times of BMMCs after PMA/ionomycin stimulation. Three-dimensional (3D) reconstitution images (left) of the proximity of the indicated targets (red) and cellular nuclei (blue) are representative of three independent experiments. Quantified data (right) are means ± SEM of at least 45 cells per group pooled from all experiments. (B and D) Co-IP of tomosyn with STX4 (B) or STX3 (D) from lysates of BMMC stimulated for the indicated times with IgE/DNP-HSA. Blots are representative of at least three independent experiments (top). Tomosyn band intensities normalized to STX (bottom) are means ± SEM pooled from all experiments. **P ≤ 0.01 and ***P ≤ 0.001 by one-way ANOVA followed by Dunnett’s test. Scale bars, 5 μm.

  • Fig. 3 PKA and PKC control the interaction of tomosyn with STX4 and STX3.

    (A and B) Co-IP of tomosyn with STX4 (A) or STX3 (B) from lysates of BMMC treated either with vehicle (Veh), PKA inhibitor (PKAi), or PKC inhibitor (PKCi) for 15 min before stimulation for the indicated times with IgE/DNP-HSA. Blots (left) are representative of five independent experiments. Tomosyn band intensities normalized to resting cells (right) are means ± SEM pooled from all experiments. *P ≤ 0.05 and **P ≤ 0.01 by one-way ANOVA followed by Dunnett’s test.

  • Fig. 4 BMMC activation stimulates tomosyn Thr phosphorylation dependent on PKC activity.

    (A) Mass spectroscopy analysis of tomosyn immunoprecipitates from BMMC stimulated for the indicated times with IgE/DNP-HSA. Possible phosphorylation sites were localized and compiled using the average normalized abundance of three independent experiments. (B) Western blot analysis for p-Thr on tomosyn immunoprecipitates from lysates of BMMC stimulated for the indicated times with IgE/DNP-HSA. Blots are representative of three independent experiments. p-Thr tomosyn band intensities normalized to tomosyn (right) are means ± SEM pooled from all experiments. (C) Western blot analysis for p-Thr on tomosyn immunoprecipitates from lysates of BMMC treated either with control, PKA, or PKC inhibitors for 15 min before IgE/DNP-HSA stimulation for the indicated times. Blots (left) are representative of three independent experiments. p-Thr tomosyn band intensities normalized to resting cells (right) are means ± SEM pooled from all experiments. *P < 0.05 and ***P < 0.001 by one-way ANOVA followed by Dunnett’s test (B and C).

  • Fig. 5 PKCδ-mediated tomosyn Thr phosphorylation controls its interaction with STX partners.

    (A) Western blot analysis for PKCδ in lysates from BMMCs transfected with control (siCtrl) and PKCδ siRNAs (si1PKCδ and si2PKCδ). Blots (left) are representative of three independent experiments. PKCδ band intensities normalized to actin (right) are means ± SEM pooled from all experiments. (B) Flow cytometry analysis of CD63 cell surface exposure on resting and IgE/Ag-stimulated BMMCs transfected with control and PKCδ. Contour plots (right) are representative of four independent experiments. Graphed data are the mean percentage of CD63+ cells ± SEM pooled from all experiments. (C) Western blot analysis of p-Thr on tomosyn immunoprecipitates from lysates of BMMCs transfected with control or PKCδ siRNA stimulated with IgE/DNP-HSA for the indicated times. Blots are representative of six independent experiments. p-Thr tomosyn band intensities normalized to resting cells (right) are means ± SEM pooled from all experiments. (D and E) Confocal analysis of tomosyn proximity to STX4 (D) or STX3 (E) determined by PLA in control and PKCδ siRNA knockdown BMMCs at the indicated times after PMA/ionomycin stimulation. 3D reconstitution images (top) of the proximity of the indicated targets (red) and cellular nuclei (blue) are representative of three independent experiments. Quantified data (bottom) are means ± SEM of at least 15 cells per condition in each experiment. *P < 0.05, **P < 0.01, and ***P < 0.001 by Student’s t test (A) and two-way ANOVA followed by Bonferroni’s correction (B to D). Scale bars, 5 μm. n.s., not significant.

  • Fig. 6 Stimulation of human basophils oppositely regulates the interaction of tomosyn with STX partners and effect of total IgE concentrations on tomosyn amount.

    (A and B) Confocal analysis of tomosyn proximity to STX4 (A) or STX3 (B) determined by PLA in resting and anti-IgE–stimulated human basophils. 3D reconstitution images (left) of the proximity of the indicated targets (red), cellular nuclei (blue), and FcεRI (green) are representative of three independent experiments. Quantified data (right) are means ± SEM of at least 42 cells per group pooled from all experiments. (C) Flow cytometry analysis of tomosyn abundance in human basophils from allergic and nonallergic subjects. Data are the individual geometric mean fluorescence intensity (gMFI) of tomosyn normalized to isotype controls from 10 independent experiments. The median + interquartile range is indicated for healthy nonallergic, allergic, and allergic patients classified by serum IgE concentration. (D) Correlation of tomosyn abundance data in (C) with serum total IgE concentration in nonallergic and allergic subjects. The best-fit line and Spearman’s correlation analysis are indicated. **P < 0.01 by Mann-Whitney test. Scale bars, 2 μm.

  • Fig. 7 IgE increases tomosyn protein in mast cells, and transfection of tomosyn decreases mast cell degranulation.

    (A) Flow cytometry analysis of tomosyn abundance in mast cells after culture for 7 days with indicated concentrations of IgE. Data are means ± SEM from at least three independent experiments. (B) Flow cytometry analysis of CD63 cell surface exposure on green fluorescent protein–positive (GFP+) resting and IgE/DNP-HSA–stimulated BMMCs transfected with GFP-control and GFP-tomosyn. Contour plots (left) are representative of four independent experiments. Graphed data (right) are the mean percentage of CD63+ cells ± SEM pooled from all experiments. *P < 0.05 by one way-ANOVA followed by Dunnett’s test (A) or two-way ANOVA followed by Bonferroni’s correction (B).

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/537/eaan4350/DC1

    Fig. S1. Tomosyn silencing in RBL-2H3 cells does not modify expression of SNARE proteins.

    Fig. S2. Tomosyn silencing in BMMCs does not modulate IL-6, TNF-α, and CCL2 secretion.

    Fig. S3. Plasma membrane and cytosolic localization of tomosyn complexes with STX4, STX3, and SNAP-23.

    Fig. S4. Kinetic of β-hexosaminidase release in BMMCs.

    Fig. S5. Tomosyn/STX4 dissociation and tomosyn/STX3 association do not depend on calcium mobilization.

    Fig. S6. PKCβ is required for mast cell degranulation but does not phosphorylate tomosyn.

    Fig. S7. Basophil gating strategy by flow cytometry and basophil activation by immunofluorescence.

    Fig. S8. Proposed model for tomosyn action in mast cells.

    Table S1. Patient characteristics.

    Table S2. List of antibodies used in the study.

    Table S3. List of siRNAs used in the study.

    • This PDF file includes:
    • Fig. S1. Tomosyn silencing in RBL-2H3 cells does not modify expression of SNARE proteins.
    • Fig. S2. Tomosyn silencing in BMMCs does not modulate IL-6, TNF-α, and CCL2 secretion.
    • Fig. S3. Plasma membrane and cytosolic localization of tomosyn complexes with STX4, STX3, and SNAP-23.
    • Fig. S4. Kinetic of β-hexosaminidase release in BMMCs.
    • Fig. S5. Tomosyn/STX4 dissociation and tomosyn/STX3 association do not depend on calcium mobilization.
    • Fig. S6. PKCβ is required for mast cell degranulation but does not phosphorylate tomosyn.
    • Fig. S7. Basophil gating strategy by flow cytometry and basophil activation by immunofluorescence.
    • Fig. S8. Proposed model for tomosyn action in mast cells.
    • Table S1. Patient characteristics.
    • Table S2. List of antibodies used in the study.
    • Table S3. List of siRNAs used in the study.

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