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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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FcɛRI stimulation induces cytoskeletal rearrangement. (A–C) FcɛRI stimulation induces microtubule formation. IgE-sensitized BMMCs were stimulated with either vehicle (A) or DNP-HSA (B) for 5 min and fixed, then double stained with phalloidin-rhodamine (red fluorescence) and antibody to α-tubulin (green fluorescence). Representative images by confocal microscopy are shown. C is a magnified version of the region delineated by the dotted line in B. Arrows indicate the structure of microtubules in BMMCs. Arrowheads indicate the position of MTOC. Bar, 10 μm. (D–I) FcɛRI stimulation induces F-actin ring disassembly. IgE-sensitized BMMCs were stimulated with either vehicle (D–F) or DNP-HSA (G–I) for 5 min. Cells were stained with phalloidin-rhodamine (red fluorescence). E and H show magnified images for D and G, respectively. F and I show intensity of F-actin by pseudo-3D analysis. Bar, 10 μm. (J and K) Microtubule and F-actin do not colocalize after stimulation. IgE-sensitized BMMCs were stimulated with either vehicle (J) and DNP-HSA (K). Cells were stained with phalloidin-rhodamine (red fluorescence) and antibody to α-tubulin (green fluorescence). Arrowheads indicate the position of MTOC. Arrows indicate microtubules at the cortical layer. Bar, 10 μm.
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fig1: FcɛRI stimulation induces cytoskeletal rearrangement. (A–C) FcɛRI stimulation induces microtubule formation. IgE-sensitized BMMCs were stimulated with either vehicle (A) or DNP-HSA (B) for 5 min and fixed, then double stained with phalloidin-rhodamine (red fluorescence) and antibody to α-tubulin (green fluorescence). Representative images by confocal microscopy are shown. C is a magnified version of the region delineated by the dotted line in B. Arrows indicate the structure of microtubules in BMMCs. Arrowheads indicate the position of MTOC. Bar, 10 μm. (D–I) FcɛRI stimulation induces F-actin ring disassembly. IgE-sensitized BMMCs were stimulated with either vehicle (D–F) or DNP-HSA (G–I) for 5 min. Cells were stained with phalloidin-rhodamine (red fluorescence). E and H show magnified images for D and G, respectively. F and I show intensity of F-actin by pseudo-3D analysis. Bar, 10 μm. (J and K) Microtubule and F-actin do not colocalize after stimulation. IgE-sensitized BMMCs were stimulated with either vehicle (J) and DNP-HSA (K). Cells were stained with phalloidin-rhodamine (red fluorescence) and antibody to α-tubulin (green fluorescence). Arrowheads indicate the position of MTOC. Arrows indicate microtubules at the cortical layer. Bar, 10 μm.

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)

FcɛRI stimulation induces cytoskeletal rearrangement. (A–C) FcɛRI stimulation induces microtubule formation. IgE-sensitized BMMCs were stimulated with either vehicle (A) or DNP-HSA (B) for 5 min and fixed, then double stained with phalloidin-rhodamine (red fluorescence) and antibody to α-tubulin (green fluorescence). Representative images by confocal microscopy are shown. C is a magnified version of the region delineated by the dotted line in B. Arrows indicate the structure of microtubules in BMMCs. Arrowheads indicate the position of MTOC. Bar, 10 μm. (D–I) FcɛRI stimulation induces F-actin ring disassembly. IgE-sensitized BMMCs were stimulated with either vehicle (D–F) or DNP-HSA (G–I) for 5 min. Cells were stained with phalloidin-rhodamine (red fluorescence). E and H show magnified images for D and G, respectively. F and I show intensity of F-actin by pseudo-3D analysis. Bar, 10 μm. (J and K) Microtubule and F-actin do not colocalize after stimulation. IgE-sensitized BMMCs were stimulated with either vehicle (J) and DNP-HSA (K). Cells were stained with phalloidin-rhodamine (red fluorescence) and antibody to α-tubulin (green fluorescence). Arrowheads indicate the position of MTOC. Arrows indicate microtubules at the cortical layer. Bar, 10 μm.
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Related In: Results  -  Collection

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fig1: FcɛRI stimulation induces cytoskeletal rearrangement. (A–C) FcɛRI stimulation induces microtubule formation. IgE-sensitized BMMCs were stimulated with either vehicle (A) or DNP-HSA (B) for 5 min and fixed, then double stained with phalloidin-rhodamine (red fluorescence) and antibody to α-tubulin (green fluorescence). Representative images by confocal microscopy are shown. C is a magnified version of the region delineated by the dotted line in B. Arrows indicate the structure of microtubules in BMMCs. Arrowheads indicate the position of MTOC. Bar, 10 μm. (D–I) FcɛRI stimulation induces F-actin ring disassembly. IgE-sensitized BMMCs were stimulated with either vehicle (D–F) or DNP-HSA (G–I) for 5 min. Cells were stained with phalloidin-rhodamine (red fluorescence). E and H show magnified images for D and G, respectively. F and I show intensity of F-actin by pseudo-3D analysis. Bar, 10 μm. (J and K) Microtubule and F-actin do not colocalize after stimulation. IgE-sensitized BMMCs were stimulated with either vehicle (J) and DNP-HSA (K). Cells were stained with phalloidin-rhodamine (red fluorescence) and antibody to α-tubulin (green fluorescence). Arrowheads indicate the position of MTOC. Arrows indicate microtubules at the cortical layer. Bar, 10 μm.
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