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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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Related in: MedlinePlus

RhoA is required for FcɛRI-induced microtubule formation and degranulation. (A) FcɛRI stimulation induces RhoA and Rac activation. RhoA and Rac activity were determined by Rhotekin RBD and PAK CRIB pull-down assay, respectively. Either wild-type or Gab2-deficient BMMCs were sensitized with IgE and then stimulated with DNP-HSA (Ag) for the times indicated. Cell lysates were incubated with GST-Rhotekin RBD (left) or GST-PAK CRIB (right) fusion protein. RhoA-GTP and Rac-GTP forms were detected by anti-RhoA and anti-Rac antibodies, respectively. One representative of three experiments is shown for each panel. (B) RhoA is involved in mast cell degranulation. RBL-2H3 cells were stably transfected with Flag-RhoA N19 (DN-RhoA) and pcDNA3 (mock). DN-RhoA or mock-introduced cells were sensitized with 0.5 μg/ml IgE for 12 h, and 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. Double asterisk indicates P < 0.01 vs. mock-introduced RBL-2H3 cells. Expression of Flag-tagged DN-RhoA in RBL-2H3 stable transfectants were visualized immunoblotting with anti-Flag antibodies (right). (C) RhoA is required for FcɛRI-induced increase of cell surface expression of CD63. BMMCs transfected with retroviral vector encoding IRES-GFP (Mock) or IRES-GFP Flag-RhoA N19 (DN-RhoA) were sensitized for 6 h with IgE. Cells were stimulated with either vehicle (gray histogram) or DNP-HSA (green line) for 10 min. Antigen-induced surface expression of CD63 was detected with anti-CD63 antibody, followed by FACS analysis. The number in the figures indicates the percentage of CD63-positive cells. (D) RhoA controls FcɛRI-mediated microtubule formation. BMMCs transfected with retroviral vector encoding IRES-GFP (Mock) or IRES-GFP Flag-RhoA N19 (DN-RhoA) were sensitized with IgE for 6 h. Cells were stimulated with DNP-HSA for 5 min. Cells were stained with antibody to α-tubulin (red fluorescence). The region delineated by the dotted line indicates mock-transfected cells (left) and RhoA N19–transfected cells (right), respectively. Representative images are shown. Bar, 10 μm. (E) Quantification of effect of DN-RhoA on microtubule formation BMMCs transfected with retroviral vector encoding IRES-GFP (mock) or IRES-GFP Flag-RhoA N19 (DN-RhoA) were sensitized with IgE for 6 h. Cells were stimulated with DNP-HSA for 5 min. Cells were stained with antibody to α-tubulin (red fluorescence). Mock or DN-RhoA transfected BMMCs were selected and mean fluorescence intensity of them was measured by Leica confocal software version 2.5. Statistical analysis was performed using the t test. Double asterisk indicates P < 0.01 vs. mock-infected BMMCs.
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fig6: RhoA is required for FcɛRI-induced microtubule formation and degranulation. (A) FcɛRI stimulation induces RhoA and Rac activation. RhoA and Rac activity were determined by Rhotekin RBD and PAK CRIB pull-down assay, respectively. Either wild-type or Gab2-deficient BMMCs were sensitized with IgE and then stimulated with DNP-HSA (Ag) for the times indicated. Cell lysates were incubated with GST-Rhotekin RBD (left) or GST-PAK CRIB (right) fusion protein. RhoA-GTP and Rac-GTP forms were detected by anti-RhoA and anti-Rac antibodies, respectively. One representative of three experiments is shown for each panel. (B) RhoA is involved in mast cell degranulation. RBL-2H3 cells were stably transfected with Flag-RhoA N19 (DN-RhoA) and pcDNA3 (mock). DN-RhoA or mock-introduced cells were sensitized with 0.5 μg/ml IgE for 12 h, and 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. Double asterisk indicates P < 0.01 vs. mock-introduced RBL-2H3 cells. Expression of Flag-tagged DN-RhoA in RBL-2H3 stable transfectants were visualized immunoblotting with anti-Flag antibodies (right). (C) RhoA is required for FcɛRI-induced increase of cell surface expression of CD63. BMMCs transfected with retroviral vector encoding IRES-GFP (Mock) or IRES-GFP Flag-RhoA N19 (DN-RhoA) were sensitized for 6 h with IgE. Cells were stimulated with either vehicle (gray histogram) or DNP-HSA (green line) for 10 min. Antigen-induced surface expression of CD63 was detected with anti-CD63 antibody, followed by FACS analysis. The number in the figures indicates the percentage of CD63-positive cells. (D) RhoA controls FcɛRI-mediated microtubule formation. BMMCs transfected with retroviral vector encoding IRES-GFP (Mock) or IRES-GFP Flag-RhoA N19 (DN-RhoA) were sensitized with IgE for 6 h. Cells were stimulated with DNP-HSA for 5 min. Cells were stained with antibody to α-tubulin (red fluorescence). The region delineated by the dotted line indicates mock-transfected cells (left) and RhoA N19–transfected cells (right), respectively. Representative images are shown. Bar, 10 μm. (E) Quantification of effect of DN-RhoA on microtubule formation BMMCs transfected with retroviral vector encoding IRES-GFP (mock) or IRES-GFP Flag-RhoA N19 (DN-RhoA) were sensitized with IgE for 6 h. Cells were stimulated with DNP-HSA for 5 min. Cells were stained with antibody to α-tubulin (red fluorescence). Mock or DN-RhoA transfected BMMCs were selected and mean fluorescence intensity of them was measured by Leica confocal software version 2.5. Statistical analysis was performed using the t test. Double asterisk indicates P < 0.01 vs. mock-infected BMMCs.

Mentions: Cytoskeletal organization is regulated by small G proteins such as Rho, Rac, and Cdc42 (Etienne-Manneville and Hall, 2002). We examined RhoA and Rac activation in wild-type and Gab2-deficient BMMCs, by measuring the GTP-bound (active) RhoA and Rac after FcɛRI stimulation. The amount of GST-Rhotekin–bound RhoA was significantly decreased in Gab2-deficient BMMCs compared with wild-type cells at 5 min after stimulation (Fig. 6 A). We did not, however, observe any difference in the time course of Rac activation between Gab2-deficient and wild-type BMMCs (Fig. 6 A).


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)

RhoA is required for FcɛRI-induced microtubule formation and degranulation. (A) FcɛRI stimulation induces RhoA and Rac activation. RhoA and Rac activity were determined by Rhotekin RBD and PAK CRIB pull-down assay, respectively. Either wild-type or Gab2-deficient BMMCs were sensitized with IgE and then stimulated with DNP-HSA (Ag) for the times indicated. Cell lysates were incubated with GST-Rhotekin RBD (left) or GST-PAK CRIB (right) fusion protein. RhoA-GTP and Rac-GTP forms were detected by anti-RhoA and anti-Rac antibodies, respectively. One representative of three experiments is shown for each panel. (B) RhoA is involved in mast cell degranulation. RBL-2H3 cells were stably transfected with Flag-RhoA N19 (DN-RhoA) and pcDNA3 (mock). DN-RhoA or mock-introduced cells were sensitized with 0.5 μg/ml IgE for 12 h, and 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. Double asterisk indicates P < 0.01 vs. mock-introduced RBL-2H3 cells. Expression of Flag-tagged DN-RhoA in RBL-2H3 stable transfectants were visualized immunoblotting with anti-Flag antibodies (right). (C) RhoA is required for FcɛRI-induced increase of cell surface expression of CD63. BMMCs transfected with retroviral vector encoding IRES-GFP (Mock) or IRES-GFP Flag-RhoA N19 (DN-RhoA) were sensitized for 6 h with IgE. Cells were stimulated with either vehicle (gray histogram) or DNP-HSA (green line) for 10 min. Antigen-induced surface expression of CD63 was detected with anti-CD63 antibody, followed by FACS analysis. The number in the figures indicates the percentage of CD63-positive cells. (D) RhoA controls FcɛRI-mediated microtubule formation. BMMCs transfected with retroviral vector encoding IRES-GFP (Mock) or IRES-GFP Flag-RhoA N19 (DN-RhoA) were sensitized with IgE for 6 h. Cells were stimulated with DNP-HSA for 5 min. Cells were stained with antibody to α-tubulin (red fluorescence). The region delineated by the dotted line indicates mock-transfected cells (left) and RhoA N19–transfected cells (right), respectively. Representative images are shown. Bar, 10 μm. (E) Quantification of effect of DN-RhoA on microtubule formation BMMCs transfected with retroviral vector encoding IRES-GFP (mock) or IRES-GFP Flag-RhoA N19 (DN-RhoA) were sensitized with IgE for 6 h. Cells were stimulated with DNP-HSA for 5 min. Cells were stained with antibody to α-tubulin (red fluorescence). Mock or DN-RhoA transfected BMMCs were selected and mean fluorescence intensity of them was measured by Leica confocal software version 2.5. Statistical analysis was performed using the t test. Double asterisk indicates P < 0.01 vs. mock-infected BMMCs.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171390&req=5

fig6: RhoA is required for FcɛRI-induced microtubule formation and degranulation. (A) FcɛRI stimulation induces RhoA and Rac activation. RhoA and Rac activity were determined by Rhotekin RBD and PAK CRIB pull-down assay, respectively. Either wild-type or Gab2-deficient BMMCs were sensitized with IgE and then stimulated with DNP-HSA (Ag) for the times indicated. Cell lysates were incubated with GST-Rhotekin RBD (left) or GST-PAK CRIB (right) fusion protein. RhoA-GTP and Rac-GTP forms were detected by anti-RhoA and anti-Rac antibodies, respectively. One representative of three experiments is shown for each panel. (B) RhoA is involved in mast cell degranulation. RBL-2H3 cells were stably transfected with Flag-RhoA N19 (DN-RhoA) and pcDNA3 (mock). DN-RhoA or mock-introduced cells were sensitized with 0.5 μg/ml IgE for 12 h, and 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. Double asterisk indicates P < 0.01 vs. mock-introduced RBL-2H3 cells. Expression of Flag-tagged DN-RhoA in RBL-2H3 stable transfectants were visualized immunoblotting with anti-Flag antibodies (right). (C) RhoA is required for FcɛRI-induced increase of cell surface expression of CD63. BMMCs transfected with retroviral vector encoding IRES-GFP (Mock) or IRES-GFP Flag-RhoA N19 (DN-RhoA) were sensitized for 6 h with IgE. Cells were stimulated with either vehicle (gray histogram) or DNP-HSA (green line) for 10 min. Antigen-induced surface expression of CD63 was detected with anti-CD63 antibody, followed by FACS analysis. The number in the figures indicates the percentage of CD63-positive cells. (D) RhoA controls FcɛRI-mediated microtubule formation. BMMCs transfected with retroviral vector encoding IRES-GFP (Mock) or IRES-GFP Flag-RhoA N19 (DN-RhoA) were sensitized with IgE for 6 h. Cells were stimulated with DNP-HSA for 5 min. Cells were stained with antibody to α-tubulin (red fluorescence). The region delineated by the dotted line indicates mock-transfected cells (left) and RhoA N19–transfected cells (right), respectively. Representative images are shown. Bar, 10 μm. (E) Quantification of effect of DN-RhoA on microtubule formation BMMCs transfected with retroviral vector encoding IRES-GFP (mock) or IRES-GFP Flag-RhoA N19 (DN-RhoA) were sensitized with IgE for 6 h. Cells were stimulated with DNP-HSA for 5 min. Cells were stained with antibody to α-tubulin (red fluorescence). Mock or DN-RhoA transfected BMMCs were selected and mean fluorescence intensity of them was measured by Leica confocal software version 2.5. Statistical analysis was performed using the t test. Double asterisk indicates P < 0.01 vs. mock-infected BMMCs.
Mentions: Cytoskeletal organization is regulated by small G proteins such as Rho, Rac, and Cdc42 (Etienne-Manneville and Hall, 2002). We examined RhoA and Rac activation in wild-type and Gab2-deficient BMMCs, by measuring the GTP-bound (active) RhoA and Rac after FcɛRI stimulation. The amount of GST-Rhotekin–bound RhoA was significantly decreased in Gab2-deficient BMMCs compared with wild-type cells at 5 min after stimulation (Fig. 6 A). We did not, however, observe any difference in the time course of Rac activation between Gab2-deficient and wild-type BMMCs (Fig. 6 A).

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