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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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Granule translocation does not require calcium, but does microtubule formation, Fyn, and Gab2. (A) FcɛRI stimulation induces the translocation of granules to the plasma membrane. BMMCs expressing CD63-GFP were sensitized for 6 h with IgE and stimulated with either vehicle (left), DNP-HSA in normal medium (middle), or DNP-HSA in calcium-free medium (right) for 10 min. Cells were fixed with 4% PFA for 30 min, and then attached to glass slides by using cytospin. CD63-GFP was visualized by confocal microscopy. Representative images are shown. Bar, 10 μm. Arrowheads show the structure of granules in the BMMC. (B) The fusion of CD63-containing granules to the plasma membrane is calcium dependent. IgE-sensitized BMMCs were stimulated with either vehicle (left), DNP-HSA in normal medium (middle), or DNP-HSA in calcium-free medium (right) for 10 min. Cell surface expression of CD63 was detected by FACS using anti-CD63. The number in the figures indicates the percentage of CD63-positive cells. (C) Calcium is not required for FcɛRI-induced granule translocation. BMMCs expressing CD63-GFP were sensitized for 6 h with IgE and stimulated with DNP-HSA (Ag) in various conditions as indicated for 10 min. Cells were fixed with 4% PFA for 30 min, and attached to glass slides by using cytospin. Cells were stained with phalloidin-rhodamine (red fluorescence) to detect F-actin. Both F-actin and CD63-GFP were visualized by confocal microscopy. We calculated the frequency of cells showing granule translocation to the plasma membrane according to the following criteria. The first criterion was the increase of yellow-color fluorescence around the plasma membrane, which was a result of the merge of phalloidin-rhodamine and CD63-GFP signal. The second was the obvious decrease of the cytoplasmic area containing CD63-GFP as compared with that of nonstimulated cells. The cells that satisfied both criteria were considered to be positive for granule translocation. Representative images obtained in various conditions were shown in the bottom panel. We counted at least 90 independent GFP-positive cells for each experiment. Statistical analysis was performed using the t test. Double asterisk indicates P < 0.01 vs. antigen-induced BMMCs in normal condition. Bar, 10 μm. (D) Fyn and Gab2 are required for FcɛRI-induced granule translocation. Either wild-type, Gab2-, Fyn-, Lyn-, or SLP-76–deficient BMMCs introduced with CD63-GFP were sensitized for 6 h with IgE and stimulated with DNP-HSA for 10 min. The frequency of cells showing granule translocation to the plasma membrane was determined as described in the figure legend for panel C. The values are shown as means ± SD of three separate experiments. Statistical analysis was performed using the t test. Double asterisk indicates P < 0.01 vs. wild-type mast cells. (E–J) Partial colocalization of CD63-containing granule with microtubules. IgE-sensitized BMMCs expressing CD63-GFP were stimulated with either vehicle (E–G) or DNA-HSA (H–J) for 5 min. Cells were stained with antibody to α-tubulin (red fluorescence). Bar, 1 μm.
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fig4: Granule translocation does not require calcium, but does microtubule formation, Fyn, and Gab2. (A) FcɛRI stimulation induces the translocation of granules to the plasma membrane. BMMCs expressing CD63-GFP were sensitized for 6 h with IgE and stimulated with either vehicle (left), DNP-HSA in normal medium (middle), or DNP-HSA in calcium-free medium (right) for 10 min. Cells were fixed with 4% PFA for 30 min, and then attached to glass slides by using cytospin. CD63-GFP was visualized by confocal microscopy. Representative images are shown. Bar, 10 μm. Arrowheads show the structure of granules in the BMMC. (B) The fusion of CD63-containing granules to the plasma membrane is calcium dependent. IgE-sensitized BMMCs were stimulated with either vehicle (left), DNP-HSA in normal medium (middle), or DNP-HSA in calcium-free medium (right) for 10 min. Cell surface expression of CD63 was detected by FACS using anti-CD63. The number in the figures indicates the percentage of CD63-positive cells. (C) Calcium is not required for FcɛRI-induced granule translocation. BMMCs expressing CD63-GFP were sensitized for 6 h with IgE and stimulated with DNP-HSA (Ag) in various conditions as indicated for 10 min. Cells were fixed with 4% PFA for 30 min, and attached to glass slides by using cytospin. Cells were stained with phalloidin-rhodamine (red fluorescence) to detect F-actin. Both F-actin and CD63-GFP were visualized by confocal microscopy. We calculated the frequency of cells showing granule translocation to the plasma membrane according to the following criteria. The first criterion was the increase of yellow-color fluorescence around the plasma membrane, which was a result of the merge of phalloidin-rhodamine and CD63-GFP signal. The second was the obvious decrease of the cytoplasmic area containing CD63-GFP as compared with that of nonstimulated cells. The cells that satisfied both criteria were considered to be positive for granule translocation. Representative images obtained in various conditions were shown in the bottom panel. We counted at least 90 independent GFP-positive cells for each experiment. Statistical analysis was performed using the t test. Double asterisk indicates P < 0.01 vs. antigen-induced BMMCs in normal condition. Bar, 10 μm. (D) Fyn and Gab2 are required for FcɛRI-induced granule translocation. Either wild-type, Gab2-, Fyn-, Lyn-, or SLP-76–deficient BMMCs introduced with CD63-GFP were sensitized for 6 h with IgE and stimulated with DNP-HSA for 10 min. The frequency of cells showing granule translocation to the plasma membrane was determined as described in the figure legend for panel C. The values are shown as means ± SD of three separate experiments. Statistical analysis was performed using the t test. Double asterisk indicates P < 0.01 vs. wild-type mast cells. (E–J) Partial colocalization of CD63-containing granule with microtubules. IgE-sensitized BMMCs expressing CD63-GFP were stimulated with either vehicle (E–G) or DNA-HSA (H–J) for 5 min. Cells were stained with antibody to α-tubulin (red fluorescence). Bar, 1 μm.

Mentions: To visualize the granules, we introduced a CD63-GFP fusion protein into BMMCs. Before FcɛRI stimulation, we observed that CD63-containing granule structures were retained in cytoplasm (Fig. 4 A, left) as reported by Nishikata et al. (1992). After FcɛRI stimulation, the CD63-containing granules were translocated to the plasma membrane (Fig. 4 A, middle; Fig. S1 A, available at http://www.jcb.org/cgi/content/full/jcb.200501111/DC1). Consistent with this, FACS analysis showed the increase of CD63 cell surface expression, indicating that granule–plasma membrane fusion occurred (Fig. 4 B, middle).


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)

Granule translocation does not require calcium, but does microtubule formation, Fyn, and Gab2. (A) FcɛRI stimulation induces the translocation of granules to the plasma membrane. BMMCs expressing CD63-GFP were sensitized for 6 h with IgE and stimulated with either vehicle (left), DNP-HSA in normal medium (middle), or DNP-HSA in calcium-free medium (right) for 10 min. Cells were fixed with 4% PFA for 30 min, and then attached to glass slides by using cytospin. CD63-GFP was visualized by confocal microscopy. Representative images are shown. Bar, 10 μm. Arrowheads show the structure of granules in the BMMC. (B) The fusion of CD63-containing granules to the plasma membrane is calcium dependent. IgE-sensitized BMMCs were stimulated with either vehicle (left), DNP-HSA in normal medium (middle), or DNP-HSA in calcium-free medium (right) for 10 min. Cell surface expression of CD63 was detected by FACS using anti-CD63. The number in the figures indicates the percentage of CD63-positive cells. (C) Calcium is not required for FcɛRI-induced granule translocation. BMMCs expressing CD63-GFP were sensitized for 6 h with IgE and stimulated with DNP-HSA (Ag) in various conditions as indicated for 10 min. Cells were fixed with 4% PFA for 30 min, and attached to glass slides by using cytospin. Cells were stained with phalloidin-rhodamine (red fluorescence) to detect F-actin. Both F-actin and CD63-GFP were visualized by confocal microscopy. We calculated the frequency of cells showing granule translocation to the plasma membrane according to the following criteria. The first criterion was the increase of yellow-color fluorescence around the plasma membrane, which was a result of the merge of phalloidin-rhodamine and CD63-GFP signal. The second was the obvious decrease of the cytoplasmic area containing CD63-GFP as compared with that of nonstimulated cells. The cells that satisfied both criteria were considered to be positive for granule translocation. Representative images obtained in various conditions were shown in the bottom panel. We counted at least 90 independent GFP-positive cells for each experiment. Statistical analysis was performed using the t test. Double asterisk indicates P < 0.01 vs. antigen-induced BMMCs in normal condition. Bar, 10 μm. (D) Fyn and Gab2 are required for FcɛRI-induced granule translocation. Either wild-type, Gab2-, Fyn-, Lyn-, or SLP-76–deficient BMMCs introduced with CD63-GFP were sensitized for 6 h with IgE and stimulated with DNP-HSA for 10 min. The frequency of cells showing granule translocation to the plasma membrane was determined as described in the figure legend for panel C. The values are shown as means ± SD of three separate experiments. Statistical analysis was performed using the t test. Double asterisk indicates P < 0.01 vs. wild-type mast cells. (E–J) Partial colocalization of CD63-containing granule with microtubules. IgE-sensitized BMMCs expressing CD63-GFP were stimulated with either vehicle (E–G) or DNA-HSA (H–J) for 5 min. Cells were stained with antibody to α-tubulin (red fluorescence). Bar, 1 μm.
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fig4: Granule translocation does not require calcium, but does microtubule formation, Fyn, and Gab2. (A) FcɛRI stimulation induces the translocation of granules to the plasma membrane. BMMCs expressing CD63-GFP were sensitized for 6 h with IgE and stimulated with either vehicle (left), DNP-HSA in normal medium (middle), or DNP-HSA in calcium-free medium (right) for 10 min. Cells were fixed with 4% PFA for 30 min, and then attached to glass slides by using cytospin. CD63-GFP was visualized by confocal microscopy. Representative images are shown. Bar, 10 μm. Arrowheads show the structure of granules in the BMMC. (B) The fusion of CD63-containing granules to the plasma membrane is calcium dependent. IgE-sensitized BMMCs were stimulated with either vehicle (left), DNP-HSA in normal medium (middle), or DNP-HSA in calcium-free medium (right) for 10 min. Cell surface expression of CD63 was detected by FACS using anti-CD63. The number in the figures indicates the percentage of CD63-positive cells. (C) Calcium is not required for FcɛRI-induced granule translocation. BMMCs expressing CD63-GFP were sensitized for 6 h with IgE and stimulated with DNP-HSA (Ag) in various conditions as indicated for 10 min. Cells were fixed with 4% PFA for 30 min, and attached to glass slides by using cytospin. Cells were stained with phalloidin-rhodamine (red fluorescence) to detect F-actin. Both F-actin and CD63-GFP were visualized by confocal microscopy. We calculated the frequency of cells showing granule translocation to the plasma membrane according to the following criteria. The first criterion was the increase of yellow-color fluorescence around the plasma membrane, which was a result of the merge of phalloidin-rhodamine and CD63-GFP signal. The second was the obvious decrease of the cytoplasmic area containing CD63-GFP as compared with that of nonstimulated cells. The cells that satisfied both criteria were considered to be positive for granule translocation. Representative images obtained in various conditions were shown in the bottom panel. We counted at least 90 independent GFP-positive cells for each experiment. Statistical analysis was performed using the t test. Double asterisk indicates P < 0.01 vs. antigen-induced BMMCs in normal condition. Bar, 10 μm. (D) Fyn and Gab2 are required for FcɛRI-induced granule translocation. Either wild-type, Gab2-, Fyn-, Lyn-, or SLP-76–deficient BMMCs introduced with CD63-GFP were sensitized for 6 h with IgE and stimulated with DNP-HSA for 10 min. The frequency of cells showing granule translocation to the plasma membrane was determined as described in the figure legend for panel C. The values are shown as means ± SD of three separate experiments. Statistical analysis was performed using the t test. Double asterisk indicates P < 0.01 vs. wild-type mast cells. (E–J) Partial colocalization of CD63-containing granule with microtubules. IgE-sensitized BMMCs expressing CD63-GFP were stimulated with either vehicle (E–G) or DNA-HSA (H–J) for 5 min. Cells were stained with antibody to α-tubulin (red fluorescence). Bar, 1 μm.
Mentions: To visualize the granules, we introduced a CD63-GFP fusion protein into BMMCs. Before FcɛRI stimulation, we observed that CD63-containing granule structures were retained in cytoplasm (Fig. 4 A, left) as reported by Nishikata et al. (1992). After FcɛRI stimulation, the CD63-containing granules were translocated to the plasma membrane (Fig. 4 A, middle; Fig. S1 A, available at http://www.jcb.org/cgi/content/full/jcb.200501111/DC1). Consistent with this, FACS analysis showed the increase of CD63 cell surface expression, indicating that granule–plasma membrane fusion occurred (Fig. 4 B, middle).

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