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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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Calcium is dispensable for microtubule formation, but essential for F-actin disassembly. (A–F) Microtubule formation is calcium independent. IgE-sensitized BMMCs were stimulated with either vehicle (A), DNP-HSA in normal medium (B), DNP-HSA in calcium-free medium (C), DNP-HSA in calcium-free medium containing EGTA (D), DNP-HSA in normal medium containing xestospongin C (XeC, 10 μM) (E), or DNP-HSA in calcium-free medium containing EGTA and XeC (F). 5 min after stimulation, cells were fixed and processed for double staining with phalloidin-rhodamine (red fluorescence) and antibody to α-tubulin (green fluorescence). Representative images by confocal microscopy are shown. Bar, 10 μm. (G) Measurement of intracellular calcium IgE-sensitized BMMCs were stimulated with either DNP-HSA (red line), DNP-HSA in calcium-free medium containing EGTA (blue line), or DNP-HSA in calcium-free medium containing EGTA and XeC (green line) for indicated periods. Intracellular calcium was measured as described in Materials and methods. (H) F-actin disassembly is dependent on calcium. IgE-sensitized BMMCs were incubated with either vehicle (1), DNP-HSA in normal medium (2), DNP-HSA in calcium-free medium (3), or DNP-HSA in calcium-free medium containing EGTA (4) for 5 min. The F-actin ring was detected by phalloidin-rhodamine. The number of cells showing F-actin disassembly was estimated using a quantitative intensity analysis of pseudo-3D image. The values indicate means ± SD of three separate experiments. Statistical analysis was performed using the t test. Double asterisk indicates P < 0.01 vs. antigen (Ag)-stimulated BMMCs in normal medium. (I) Intracellular calcium is essential for mast cell degranulation. IgE-sensitized BMMCs were stimulated with either vehicle (1), DNP-HSA in normal medium (2), DNP-HSA in calcium-free medium (3), or DNP-HSA in calcium-free medium containing EGTA (4) 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. Ag-stimulated BMMCs in normal medium.
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fig2: Calcium is dispensable for microtubule formation, but essential for F-actin disassembly. (A–F) Microtubule formation is calcium independent. IgE-sensitized BMMCs were stimulated with either vehicle (A), DNP-HSA in normal medium (B), DNP-HSA in calcium-free medium (C), DNP-HSA in calcium-free medium containing EGTA (D), DNP-HSA in normal medium containing xestospongin C (XeC, 10 μM) (E), or DNP-HSA in calcium-free medium containing EGTA and XeC (F). 5 min after stimulation, cells were fixed and processed for double staining with phalloidin-rhodamine (red fluorescence) and antibody to α-tubulin (green fluorescence). Representative images by confocal microscopy are shown. Bar, 10 μm. (G) Measurement of intracellular calcium IgE-sensitized BMMCs were stimulated with either DNP-HSA (red line), DNP-HSA in calcium-free medium containing EGTA (blue line), or DNP-HSA in calcium-free medium containing EGTA and XeC (green line) for indicated periods. Intracellular calcium was measured as described in Materials and methods. (H) F-actin disassembly is dependent on calcium. IgE-sensitized BMMCs were incubated with either vehicle (1), DNP-HSA in normal medium (2), DNP-HSA in calcium-free medium (3), or DNP-HSA in calcium-free medium containing EGTA (4) for 5 min. The F-actin ring was detected by phalloidin-rhodamine. The number of cells showing F-actin disassembly was estimated using a quantitative intensity analysis of pseudo-3D image. The values indicate means ± SD of three separate experiments. Statistical analysis was performed using the t test. Double asterisk indicates P < 0.01 vs. antigen (Ag)-stimulated BMMCs in normal medium. (I) Intracellular calcium is essential for mast cell degranulation. IgE-sensitized BMMCs were stimulated with either vehicle (1), DNP-HSA in normal medium (2), DNP-HSA in calcium-free medium (3), or DNP-HSA in calcium-free medium containing EGTA (4) 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. Ag-stimulated BMMCs in normal medium.

Mentions: Microtubule and actin filament networks function cooperatively in the process of vesicle and organelle transport (Goode et al., 2000). We examined whether FcɛRI stimulation generates changes in cytoskeletal proteins such as tubulin or actin in bone marrow–derived mast cells (BMMCs). After FcɛRI stimulation, we observed an enhancement of the intensity of tubulin staining (Fig. 1, A and B). We also observed the microtubule structures between the cortical layer and cytoplasmic region of the cells (Fig. 1 C). At the same time, we noted disassembly of the cortical F-actin fluorescent ring (Fig. 1, D–I). FcɛRI stimulation increased the number of cells displaying fragmentation of the cortical F-actin rings (from 4.5 ± 4.4% in unstimulated cells to 55.2 ± 1.7% in stimulated cells, n = 300 cells; Fig. 2 H). Microtubule and F-actin did not appear to colocalize after FcɛRI stimulation (Fig. 1, J and K). Microtubule was localized to the area into which the F-actin ring had collapsed (Fig. 1 K).


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)

Calcium is dispensable for microtubule formation, but essential for F-actin disassembly. (A–F) Microtubule formation is calcium independent. IgE-sensitized BMMCs were stimulated with either vehicle (A), DNP-HSA in normal medium (B), DNP-HSA in calcium-free medium (C), DNP-HSA in calcium-free medium containing EGTA (D), DNP-HSA in normal medium containing xestospongin C (XeC, 10 μM) (E), or DNP-HSA in calcium-free medium containing EGTA and XeC (F). 5 min after stimulation, cells were fixed and processed for double staining with phalloidin-rhodamine (red fluorescence) and antibody to α-tubulin (green fluorescence). Representative images by confocal microscopy are shown. Bar, 10 μm. (G) Measurement of intracellular calcium IgE-sensitized BMMCs were stimulated with either DNP-HSA (red line), DNP-HSA in calcium-free medium containing EGTA (blue line), or DNP-HSA in calcium-free medium containing EGTA and XeC (green line) for indicated periods. Intracellular calcium was measured as described in Materials and methods. (H) F-actin disassembly is dependent on calcium. IgE-sensitized BMMCs were incubated with either vehicle (1), DNP-HSA in normal medium (2), DNP-HSA in calcium-free medium (3), or DNP-HSA in calcium-free medium containing EGTA (4) for 5 min. The F-actin ring was detected by phalloidin-rhodamine. The number of cells showing F-actin disassembly was estimated using a quantitative intensity analysis of pseudo-3D image. The values indicate means ± SD of three separate experiments. Statistical analysis was performed using the t test. Double asterisk indicates P < 0.01 vs. antigen (Ag)-stimulated BMMCs in normal medium. (I) Intracellular calcium is essential for mast cell degranulation. IgE-sensitized BMMCs were stimulated with either vehicle (1), DNP-HSA in normal medium (2), DNP-HSA in calcium-free medium (3), or DNP-HSA in calcium-free medium containing EGTA (4) 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. Ag-stimulated BMMCs in normal medium.
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Related In: Results  -  Collection

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fig2: Calcium is dispensable for microtubule formation, but essential for F-actin disassembly. (A–F) Microtubule formation is calcium independent. IgE-sensitized BMMCs were stimulated with either vehicle (A), DNP-HSA in normal medium (B), DNP-HSA in calcium-free medium (C), DNP-HSA in calcium-free medium containing EGTA (D), DNP-HSA in normal medium containing xestospongin C (XeC, 10 μM) (E), or DNP-HSA in calcium-free medium containing EGTA and XeC (F). 5 min after stimulation, cells were fixed and processed for double staining with phalloidin-rhodamine (red fluorescence) and antibody to α-tubulin (green fluorescence). Representative images by confocal microscopy are shown. Bar, 10 μm. (G) Measurement of intracellular calcium IgE-sensitized BMMCs were stimulated with either DNP-HSA (red line), DNP-HSA in calcium-free medium containing EGTA (blue line), or DNP-HSA in calcium-free medium containing EGTA and XeC (green line) for indicated periods. Intracellular calcium was measured as described in Materials and methods. (H) F-actin disassembly is dependent on calcium. IgE-sensitized BMMCs were incubated with either vehicle (1), DNP-HSA in normal medium (2), DNP-HSA in calcium-free medium (3), or DNP-HSA in calcium-free medium containing EGTA (4) for 5 min. The F-actin ring was detected by phalloidin-rhodamine. The number of cells showing F-actin disassembly was estimated using a quantitative intensity analysis of pseudo-3D image. The values indicate means ± SD of three separate experiments. Statistical analysis was performed using the t test. Double asterisk indicates P < 0.01 vs. antigen (Ag)-stimulated BMMCs in normal medium. (I) Intracellular calcium is essential for mast cell degranulation. IgE-sensitized BMMCs were stimulated with either vehicle (1), DNP-HSA in normal medium (2), DNP-HSA in calcium-free medium (3), or DNP-HSA in calcium-free medium containing EGTA (4) 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. Ag-stimulated BMMCs in normal medium.
Mentions: Microtubule and actin filament networks function cooperatively in the process of vesicle and organelle transport (Goode et al., 2000). We examined whether FcɛRI stimulation generates changes in cytoskeletal proteins such as tubulin or actin in bone marrow–derived mast cells (BMMCs). After FcɛRI stimulation, we observed an enhancement of the intensity of tubulin staining (Fig. 1, A and B). We also observed the microtubule structures between the cortical layer and cytoplasmic region of the cells (Fig. 1 C). At the same time, we noted disassembly of the cortical F-actin fluorescent ring (Fig. 1, D–I). FcɛRI stimulation increased the number of cells displaying fragmentation of the cortical F-actin rings (from 4.5 ± 4.4% in unstimulated cells to 55.2 ± 1.7% in stimulated cells, n = 300 cells; Fig. 2 H). Microtubule and F-actin did not appear to colocalize after FcɛRI stimulation (Fig. 1, J and K). Microtubule was localized to the area into which the F-actin ring had collapsed (Fig. 1 K).

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