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Fc{epsilon}RI-mediated mast cell degranulation requires calcium-independent microtubule-dependent translocation of granules to the plasma membrane.

Nishida K, Yamasaki S, Ito Y, Kabu K, Hattori K, Tezuka T, Nishizumi H, Kitamura D, Goitsuka R, Geha RS, Yamamoto T, Yagi T, Hirano T - J. Cell Biol. (2005)

Bottom Line: Drugs affecting microtubule dynamics effectively suppressed the FcepsilonRI-mediated translocation of granules to the plasma membrane and degranulation.Thus, the degranulation process can be dissected into two events: the calcium-independent microtubule-dependent translocation of granules to the plasma membrane and calcium-dependent membrane fusion and exocytosis.Finally, we show that the Fyn/Gab2/RhoA (but not Lyn/SLP-76) signaling pathway plays a critical role in the calcium-independent microtubule-dependent pathway.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Cytokine Signaling, RIKEN Research Center for Allergy and Immunology, Kanagawa 230-0045, Japan.

ABSTRACT
The aggregation of high affinity IgE receptors (Fcepsilon receptor I [FcepsilonRI]) on mast cells is potent stimulus for the release of inflammatory and allergic mediators from cytoplasmic granules. However, the molecular mechanism of degranulation has not yet been established. It is still unclear how FcepsilonRI-mediated signal transduction ultimately regulates the reorganization of the cytoskeleton and how these events lead to degranulation. Here, we show that FcepsilonRI stimulation triggers the formation of microtubules in a manner independent of calcium. Drugs affecting microtubule dynamics effectively suppressed the FcepsilonRI-mediated translocation of granules to the plasma membrane and degranulation. Furthermore, the translocation of granules to the plasma membrane occurred in a calcium-independent manner, but the release of mediators and granule-plasma membrane fusion were completely dependent on calcium. Thus, the degranulation process can be dissected into two events: the calcium-independent microtubule-dependent translocation of granules to the plasma membrane and calcium-dependent membrane fusion and exocytosis. Finally, we show that the Fyn/Gab2/RhoA (but not Lyn/SLP-76) signaling pathway plays a critical role in the calcium-independent microtubule-dependent pathway.

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The Fyn/Gab2-mediated pathway controls microtubule formation. (A) Fyn and Gab2 are required for mast cell degranulation. Either wild-type, Gab2-, Fyn-, Lyn-, or SLP-76–deficient BMMCs were sensitized with IgE and then stimulated with various concentrations of DNP-HSA (Antigen) as indicated for 30 min. β-Hexosaminidase release was measured for indication of mast cell degranulation. The values indicate means ± SD of three separate experiments. Statistical analysis was performed using the t test. Single asterisk indicates P < 0.05 vs. wild-type BMMCs. Double asterisk indicates P < 0.01 vs. antigen-induced wild-type BMMCs. (B) Fyn-dependent Gab2 phosphorylation. Either wild-type or Fyn-deficient BMMCs were sensitized with IgE and then stimulated with 100 ng/ml DNP-HSA. Immunoprecipitates of anti-Gab2 antibody were probed with anti-phosphotyrosine (top) or anti-Gab2 antibody (bottom). (C) FcɛRI stimulation increases polymerization of tubulin. Either wild-type, Gab2-, or Fyn-deficient BMMCs were sensitized with IgE and stimulated with 100 ng/ml DNP-HSA for the times indicated. Stimulated cells were lysed in 0.1% Triton X-100. Triton X-100–insoluble fraction was subjected to Western blot analysis using anti-α-tubulin antibody. Lysate from each of the same samples were stained with CBB for loading control. One representative of three experiments is shown for each panel. Ratio is indicated as relative intensity (versus intensity in unstimulated cells). Poly-MT, polymeric tubulin. (D–M) Fyn and Gab2 are required for FcɛRI-induced microtubule formation. Either wild-type, Gab2-, Fyn-, Lyn-, or SLP-76–deficient BMMCs were sensitized with IgE and then stimulated with vehicle (D–H) or DNP-HSA (I–M) for 5 min. Stimulated cells were processed for double staining with phalloidin-rhodamine (red fluorescence) and antibody to α-tubulin (green fluorescence). Representative images are shown. Bar, 10 μm.
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fig5: The Fyn/Gab2-mediated pathway controls microtubule formation. (A) Fyn and Gab2 are required for mast cell degranulation. Either wild-type, Gab2-, Fyn-, Lyn-, or SLP-76–deficient BMMCs were sensitized with IgE and then stimulated with various concentrations of DNP-HSA (Antigen) as indicated for 30 min. β-Hexosaminidase release was measured for indication of mast cell degranulation. The values indicate means ± SD of three separate experiments. Statistical analysis was performed using the t test. Single asterisk indicates P < 0.05 vs. wild-type BMMCs. Double asterisk indicates P < 0.01 vs. antigen-induced wild-type BMMCs. (B) Fyn-dependent Gab2 phosphorylation. Either wild-type or Fyn-deficient BMMCs were sensitized with IgE and then stimulated with 100 ng/ml DNP-HSA. Immunoprecipitates of anti-Gab2 antibody were probed with anti-phosphotyrosine (top) or anti-Gab2 antibody (bottom). (C) FcɛRI stimulation increases polymerization of tubulin. Either wild-type, Gab2-, or Fyn-deficient BMMCs were sensitized with IgE and stimulated with 100 ng/ml DNP-HSA for the times indicated. Stimulated cells were lysed in 0.1% Triton X-100. Triton X-100–insoluble fraction was subjected to Western blot analysis using anti-α-tubulin antibody. Lysate from each of the same samples were stained with CBB for loading control. One representative of three experiments is shown for each panel. Ratio is indicated as relative intensity (versus intensity in unstimulated cells). Poly-MT, polymeric tubulin. (D–M) Fyn and Gab2 are required for FcɛRI-induced microtubule formation. Either wild-type, Gab2-, Fyn-, Lyn-, or SLP-76–deficient BMMCs were sensitized with IgE and then stimulated with vehicle (D–H) or DNP-HSA (I–M) for 5 min. Stimulated cells were processed for double staining with phalloidin-rhodamine (red fluorescence) and antibody to α-tubulin (green fluorescence). Representative images are shown. Bar, 10 μm.

Mentions: Gab2- or Fyn-deficient BMMCs showed a decreased β-hexosaminidase release (Fig. 5 A). We further confirmed that the FcɛRI-induced phosphorylation of Gab2 was decreased in Fyn-deficient BMMCs (Fig. 5 B). More importantly, the FcɛRI-induced formation of polymeric tubulin was decreased in Fyn- and Gab2-deficient BMMCs, as shown in Fig. 5 C. Consistent with this, the microtubule formation was impaired in Fyn- or Gab2-deficient BMMCs, but was intact in Lyn- and SLP-76–deficient BMMCs (Fig. 5, D–M).


Fc{epsilon}RI-mediated mast cell degranulation requires calcium-independent microtubule-dependent translocation of granules to the plasma membrane.

Nishida K, Yamasaki S, Ito Y, Kabu K, Hattori K, Tezuka T, Nishizumi H, Kitamura D, Goitsuka R, Geha RS, Yamamoto T, Yagi T, Hirano T - J. Cell Biol. (2005)

The Fyn/Gab2-mediated pathway controls microtubule formation. (A) Fyn and Gab2 are required for mast cell degranulation. Either wild-type, Gab2-, Fyn-, Lyn-, or SLP-76–deficient BMMCs were sensitized with IgE and then stimulated with various concentrations of DNP-HSA (Antigen) as indicated for 30 min. β-Hexosaminidase release was measured for indication of mast cell degranulation. The values indicate means ± SD of three separate experiments. Statistical analysis was performed using the t test. Single asterisk indicates P < 0.05 vs. wild-type BMMCs. Double asterisk indicates P < 0.01 vs. antigen-induced wild-type BMMCs. (B) Fyn-dependent Gab2 phosphorylation. Either wild-type or Fyn-deficient BMMCs were sensitized with IgE and then stimulated with 100 ng/ml DNP-HSA. Immunoprecipitates of anti-Gab2 antibody were probed with anti-phosphotyrosine (top) or anti-Gab2 antibody (bottom). (C) FcɛRI stimulation increases polymerization of tubulin. Either wild-type, Gab2-, or Fyn-deficient BMMCs were sensitized with IgE and stimulated with 100 ng/ml DNP-HSA for the times indicated. Stimulated cells were lysed in 0.1% Triton X-100. Triton X-100–insoluble fraction was subjected to Western blot analysis using anti-α-tubulin antibody. Lysate from each of the same samples were stained with CBB for loading control. One representative of three experiments is shown for each panel. Ratio is indicated as relative intensity (versus intensity in unstimulated cells). Poly-MT, polymeric tubulin. (D–M) Fyn and Gab2 are required for FcɛRI-induced microtubule formation. Either wild-type, Gab2-, Fyn-, Lyn-, or SLP-76–deficient BMMCs were sensitized with IgE and then stimulated with vehicle (D–H) or DNP-HSA (I–M) for 5 min. Stimulated cells were processed for double staining with phalloidin-rhodamine (red fluorescence) and antibody to α-tubulin (green fluorescence). Representative images are shown. Bar, 10 μm.
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Related In: Results  -  Collection

Show All Figures
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fig5: The Fyn/Gab2-mediated pathway controls microtubule formation. (A) Fyn and Gab2 are required for mast cell degranulation. Either wild-type, Gab2-, Fyn-, Lyn-, or SLP-76–deficient BMMCs were sensitized with IgE and then stimulated with various concentrations of DNP-HSA (Antigen) as indicated for 30 min. β-Hexosaminidase release was measured for indication of mast cell degranulation. The values indicate means ± SD of three separate experiments. Statistical analysis was performed using the t test. Single asterisk indicates P < 0.05 vs. wild-type BMMCs. Double asterisk indicates P < 0.01 vs. antigen-induced wild-type BMMCs. (B) Fyn-dependent Gab2 phosphorylation. Either wild-type or Fyn-deficient BMMCs were sensitized with IgE and then stimulated with 100 ng/ml DNP-HSA. Immunoprecipitates of anti-Gab2 antibody were probed with anti-phosphotyrosine (top) or anti-Gab2 antibody (bottom). (C) FcɛRI stimulation increases polymerization of tubulin. Either wild-type, Gab2-, or Fyn-deficient BMMCs were sensitized with IgE and stimulated with 100 ng/ml DNP-HSA for the times indicated. Stimulated cells were lysed in 0.1% Triton X-100. Triton X-100–insoluble fraction was subjected to Western blot analysis using anti-α-tubulin antibody. Lysate from each of the same samples were stained with CBB for loading control. One representative of three experiments is shown for each panel. Ratio is indicated as relative intensity (versus intensity in unstimulated cells). Poly-MT, polymeric tubulin. (D–M) Fyn and Gab2 are required for FcɛRI-induced microtubule formation. Either wild-type, Gab2-, Fyn-, Lyn-, or SLP-76–deficient BMMCs were sensitized with IgE and then stimulated with vehicle (D–H) or DNP-HSA (I–M) for 5 min. Stimulated cells were processed for double staining with phalloidin-rhodamine (red fluorescence) and antibody to α-tubulin (green fluorescence). Representative images are shown. Bar, 10 μm.
Mentions: Gab2- or Fyn-deficient BMMCs showed a decreased β-hexosaminidase release (Fig. 5 A). We further confirmed that the FcɛRI-induced phosphorylation of Gab2 was decreased in Fyn-deficient BMMCs (Fig. 5 B). More importantly, the FcɛRI-induced formation of polymeric tubulin was decreased in Fyn- and Gab2-deficient BMMCs, as shown in Fig. 5 C. Consistent with this, the microtubule formation was impaired in Fyn- or Gab2-deficient BMMCs, but was intact in Lyn- and SLP-76–deficient BMMCs (Fig. 5, D–M).

Bottom Line: Drugs affecting microtubule dynamics effectively suppressed the FcepsilonRI-mediated translocation of granules to the plasma membrane and degranulation.Thus, the degranulation process can be dissected into two events: the calcium-independent microtubule-dependent translocation of granules to the plasma membrane and calcium-dependent membrane fusion and exocytosis.Finally, we show that the Fyn/Gab2/RhoA (but not Lyn/SLP-76) signaling pathway plays a critical role in the calcium-independent microtubule-dependent pathway.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Cytokine Signaling, RIKEN Research Center for Allergy and Immunology, Kanagawa 230-0045, Japan.

ABSTRACT
The aggregation of high affinity IgE receptors (Fcepsilon receptor I [FcepsilonRI]) on mast cells is potent stimulus for the release of inflammatory and allergic mediators from cytoplasmic granules. However, the molecular mechanism of degranulation has not yet been established. It is still unclear how FcepsilonRI-mediated signal transduction ultimately regulates the reorganization of the cytoskeleton and how these events lead to degranulation. Here, we show that FcepsilonRI stimulation triggers the formation of microtubules in a manner independent of calcium. Drugs affecting microtubule dynamics effectively suppressed the FcepsilonRI-mediated translocation of granules to the plasma membrane and degranulation. Furthermore, the translocation of granules to the plasma membrane occurred in a calcium-independent manner, but the release of mediators and granule-plasma membrane fusion were completely dependent on calcium. Thus, the degranulation process can be dissected into two events: the calcium-independent microtubule-dependent translocation of granules to the plasma membrane and calcium-dependent membrane fusion and exocytosis. Finally, we show that the Fyn/Gab2/RhoA (but not Lyn/SLP-76) signaling pathway plays a critical role in the calcium-independent microtubule-dependent pathway.

Show MeSH
Related in: MedlinePlus