Limits...
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.

Show MeSH

Related in: MedlinePlus

Microtubule dynamics are required for mast cell degranulation. (A–J) The effect of cytoskeletal inhibitors on microtubule formation and F-actin disassembly. IgE-sensitized BMMCs were pretreated with either vehicle (A and F), latrunculin B (B and G), jasplakinolide (C and H), nocodazole (D and I), or Taxol (E and J) as described in Materials and methods. Then, cells were stimulated with either vehicle (A–E) or DNP-HSA (F–J) in the presence of each inhibitor. Cells were visualized by confocal microscopy. Bar, 10 μm. (K–N) The effect of cytoskeletal inhibitors on mast cell degranulation. IgE-sensitized BMMCs were pretreated with either latrunculin B (K), jasplakinolide (L), nocodazole (M), or Taxol (N) as described in Materials and methods. Then, cells were stimulated with various concentrations of DNP-HAS as indicated for 30 min in the presence of each inhibitor. 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. without inhibitor. Double asterisk indicates P < 0.01 vs. without inhibitor.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171390&req=5

fig3: Microtubule dynamics are required for mast cell degranulation. (A–J) The effect of cytoskeletal inhibitors on microtubule formation and F-actin disassembly. IgE-sensitized BMMCs were pretreated with either vehicle (A and F), latrunculin B (B and G), jasplakinolide (C and H), nocodazole (D and I), or Taxol (E and J) as described in Materials and methods. Then, cells were stimulated with either vehicle (A–E) or DNP-HSA (F–J) in the presence of each inhibitor. Cells were visualized by confocal microscopy. Bar, 10 μm. (K–N) The effect of cytoskeletal inhibitors on mast cell degranulation. IgE-sensitized BMMCs were pretreated with either latrunculin B (K), jasplakinolide (L), nocodazole (M), or Taxol (N) as described in Materials and methods. Then, cells were stimulated with various concentrations of DNP-HAS as indicated for 30 min in the presence of each inhibitor. 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. without inhibitor. Double asterisk indicates P < 0.01 vs. without inhibitor.

Mentions: We next demonstrated the need for both microtubule formation and F-actin ring disassembly in FcɛRI-mediated degranulation using various cytoskeletal inhibitors. First, we tested drugs affecting F-actin, latrunculin B (which disrupts F-actin), and jasplakinolide (which stabilizes F-actin). Latrunculin B treatment induced the disassembly of the F-actin rings (Fig. 3, B and G) and significantly enhanced β-hexosaminidase release (Fig. 3 K). In contrast, jasplakinolide suppressed F-actin ring disassembly (Fig. 3, C and H) and slightly reduced β-hexosaminidase release (Fig. 3 L). These results are consistent with the idea that the disassembly of the F-actin ring is required, at least in part, for mast cell degranulation in a calcium-dependent manner.


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)

Microtubule dynamics are required for mast cell degranulation. (A–J) The effect of cytoskeletal inhibitors on microtubule formation and F-actin disassembly. IgE-sensitized BMMCs were pretreated with either vehicle (A and F), latrunculin B (B and G), jasplakinolide (C and H), nocodazole (D and I), or Taxol (E and J) as described in Materials and methods. Then, cells were stimulated with either vehicle (A–E) or DNP-HSA (F–J) in the presence of each inhibitor. Cells were visualized by confocal microscopy. Bar, 10 μm. (K–N) The effect of cytoskeletal inhibitors on mast cell degranulation. IgE-sensitized BMMCs were pretreated with either latrunculin B (K), jasplakinolide (L), nocodazole (M), or Taxol (N) as described in Materials and methods. Then, cells were stimulated with various concentrations of DNP-HAS as indicated for 30 min in the presence of each inhibitor. 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. without inhibitor. Double asterisk indicates P < 0.01 vs. without inhibitor.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Microtubule dynamics are required for mast cell degranulation. (A–J) The effect of cytoskeletal inhibitors on microtubule formation and F-actin disassembly. IgE-sensitized BMMCs were pretreated with either vehicle (A and F), latrunculin B (B and G), jasplakinolide (C and H), nocodazole (D and I), or Taxol (E and J) as described in Materials and methods. Then, cells were stimulated with either vehicle (A–E) or DNP-HSA (F–J) in the presence of each inhibitor. Cells were visualized by confocal microscopy. Bar, 10 μm. (K–N) The effect of cytoskeletal inhibitors on mast cell degranulation. IgE-sensitized BMMCs were pretreated with either latrunculin B (K), jasplakinolide (L), nocodazole (M), or Taxol (N) as described in Materials and methods. Then, cells were stimulated with various concentrations of DNP-HAS as indicated for 30 min in the presence of each inhibitor. 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. without inhibitor. Double asterisk indicates P < 0.01 vs. without inhibitor.
Mentions: We next demonstrated the need for both microtubule formation and F-actin ring disassembly in FcɛRI-mediated degranulation using various cytoskeletal inhibitors. First, we tested drugs affecting F-actin, latrunculin B (which disrupts F-actin), and jasplakinolide (which stabilizes F-actin). Latrunculin B treatment induced the disassembly of the F-actin rings (Fig. 3, B and G) and significantly enhanced β-hexosaminidase release (Fig. 3 K). In contrast, jasplakinolide suppressed F-actin ring disassembly (Fig. 3, C and H) and slightly reduced β-hexosaminidase release (Fig. 3 L). These results are consistent with the idea that the disassembly of the F-actin ring is required, at least in part, for mast cell degranulation in a calcium-dependent manner.

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