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First published online 27 November 2002
doi: 10.1242/jcs.00216

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Evidence of a role for Munc18-2 and microtubules in mast cell granule exocytosis

¹ Unité d'Immuno-Allergie, Institut Pasteur, Paris, France
² Unité INSERM 363, ICGM, Hôpital Cochin, Paris, France
³ Molecular Inflammation Section, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892, USA

View larger version (29K): [in a new window]   Fig. 1. Characterisation of Munc18 isoforms in mast cells. (A) Some Munc18 isoforms are expressed in RBL cells. Total brain (1 µg) and RBL cell lysates (2x 105 cell equivalents) were resolved on SDS-PAGE, transferred to nitrocellulose and probed with antibodies to Munc18-1 (1/1000), Munc18-2 or Munc18-3 (all at 1 µg/ml). (B) Munc18-2 and Munc18-3 are expressed in mast cell lines or primary mast cells. Lysates of RBL, C57.1, BMMC and MCP-5/L cells were resolved on SDS-PAGE, transferred to nitrocellulose and probed with anti-Munc18-2 or Munc18-3.

View larger version (42K): [in a new window]   Fig. 2. Interaction of Munc18 isoforms with syntaxins. (A) Proteins from RBL cell lysates were immunoprecipitated with antibodies (5 µg) to syntaxin 2, 3 or 4. Immunoprecipitates were resolved on SDS-PAGE and immunoblotted with antibodies to Munc18-2 or Munc18-3 (upper panel) or with antibodies to syntaxin 2, 3 and 4 (lower panel) (all at 1 µg/ml). (B) Proteins from RBL cell lysates were immunoprecipitated with antibodies (5 µg) to Munc18-2 or GST control, resolved on SDS-PAGE and immunoblotted with antibodies to syntaxin 2, 3 and 4 (upper panel) and Munc18-2 (lower panel).

View larger version (87K): [in a new window]   Fig. 3. Localisation of Munc18 and syntaxins isoforms. (A) Co-localisation with PM marker. RBL cells were seeded overnight on glass coverslips. After permeabilisation, cells were incubated with antibody to FcRI ß chain (1 µg/ml) as a PM marker and either antibodies to Munc18-2, Munc18-3, syntaxin 3 or syntaxin 4 (all at 2 µg/ml). Cells were visualised by confocal microscopy as described in Materials and Methods. Single optical sections through individual cells as well as the merge (green and red fluorescence) from several cells are presented. In the inset of the upper panel the green and red fluorescence are superimposed to the DIC image. Arrows highlight area of red fluorescence in cellular protrusions. (B) Co-localisation with a SG marker. RBL cells were treated as above before incubation with antibody to RMCP II (1/500) as a SG marker, and antibodies to either Munc18-2 or syntaxin 3 (all at 2 µg/ml). For BMMCs, cells were allowed to adhere to L-polylysine-coated coverslips for 1 hour before staining with antibody to serotonin (1/50) as a SG marker, and antibodies to either Munc18-2 or syntaxin 3. Cells were visualised by confocal microscopy as described in Materials and Methods. Single optical sections through individual cells are presented. For BMMCs only the merge (green and red fluorescence) is shown. Bars, 5 µm. Images are representative of at least three experiments.

View larger version (78K): [in a new window]   Fig. 4. Munc18-2 localisation after FcRI-dependent stimulation and relation to cytoskeletal elements. (A) Analysis of the composition of a large Munc18-2-positive compartment. IgE-sensitised RBL cells seeded on coverslips were stimulated for 30 minutes with DNP-HSA and probed with antibody to Munc18-2 (2 µg/ml) for confocal analysis as described in Materials and Methods. Raw confocal acquisitions of a single cell were subjected to mathematical analysis using Huygens software to improve resolution. A DIC image of the cell as well as an expanded region containing a Munc18-2-positive compartment (red fluorescence), probably comprised of at least seven discernible SGs, is shown. Bar, 2 µm. (B) RBL cells were seeded on glass coverslips, sensitised with IgE overnight and either not stimulated (-Ag) or stimulated with DNP-HSA for 10 minutes (+Ag). Cells were processed for staining with antibodies to Munc18-2 (in red) and either phalloidin-FITC (1/40) or antibody to -tubulin (1 µg/ml) (both in green). Cells were visualised by confocal microscopy as described in Materials and Methods. Comparison of a single section, a 3D and a XZ projection are shown for stimulated cells. Single sections of unstimulated cells are shown in the inset. A DIC image of the cell stained for Munc18-2 and -tubulin is also shown as an inset. All size bars represent 5 µm.

View larger version (69K): [in a new window]   Fig. 5. Role of the cytoskeleton in Munc18-2 polarisation. RBL cells seeded on coverslips were treated with cytochalasin D (10 µM) or vehicles for 30 minutes (A) and nocodazole (33 µM) or vehicle for 1 hour (B) prior to incubation with antibodies to Munc18-2 (red fluorescence) and phalloidin-FITC (green fluorescence) or with antibodies to Munc18-2 (red) and -tubulin and RMCP II (both in green) as indicated. Cells were visualised by confocal microscopy as described in Materials and Methods. The merged images (green and red fluorescence) are shown, except for RMCP II staining, which is superimposed to the DIC image. Bars, 5 µm. Images are representative of at least three experiments. Note that the less focalised appearance of Munc18-2 staining in A is probably due to the solvent used for cell treatment. The nuclear appearance of Munc18-2 in nocodazole-treated cells in B (right panel) is caused by a low signal to noise ratio following redistribution of Munc18-2 to a diffuse staining pattern.

View larger version (29K): [in a new window]   Fig. 6. Effect of cytoskeleton-perturbing agents on secretion (A) and Munc18-2 relocation (B). (A) RBL cells were seeded in 96-well plates and sensitised overnight with IgE. Cells were treated with the indicated concentrations of either cytochalasin D for 30 minutes or nocodazole for 1 hour, or corresponding vehicles prior to stimulation with DNP-HSA. Released SG content was determined by ß-hexosminidase release. (B) RBL cells seeded on coverslips were incubated with either cytochalasin D (10 µM) for 1 hour, nocodazole (33 µM) for 1 hour or EGTA (5 mM) for 15 minutes before stimulation with DNP-HSA for 10 minutes. Cells were processed for confocal analysis and probed with antibodies to Munc18-2 (red) and either phalloidin-FITC (green) or antibody to -tubulin (green) as before. Cells were visualised by confocal microscopy as described in Materials and Methods. The merged images (green and red fluorescence) of several, as well as single cells are shown. As after treatment with cytochalasin D phalloidin staining is decreased, presentation of an overlay of the DIC image with Munc18-2 staining is preferred. Note also that after nocodazole-treatment MT staining either disappears (single cell panel) or is severely restricted to the MTOC. Bars, 5 µm. Images are representative of at least three experiments.