title: Munc18-2 and syntaxin 3 control distinct essential steps in mast cell degranulation.
abstract: Mast cell degranulation requires N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) and mammalian uncoordinated18 (Munc18) fusion accessory proteins for membrane fusion. However, it is still unknown how their interaction supports fusion. In this study, we found that small interfering RNA-mediated silencing of the isoform Munc18-2 in mast cells inhibits cytoplasmic secretory granule (SG) release but not CCL2 chemokine secretion. Silencing of its SNARE-binding partner syntaxin 3 (STX3) also markedly inhibited degranulation, whereas combined knockdown produced an additive inhibitory effect. Strikingly, while Munc18-2 silencing impaired SG translocation, silencing of STX3 inhibited fusion, demonstrating unique roles of each protein. Immunogold studies showed that both Munc18-2 and STX3 are located on the granule surface, but also within the granule matrix and in small nocodazole-sensitive clusters of the cytoskeletal meshwork surrounding SG. After stimulation, clusters containing both effectors were detected at fusion sites. In resting cells, Munc18-2, but not STX3, interacted with tubulin. This interaction was sensitive to nocodazole treatment and decreased after stimulation. Our results indicate that Munc18-2 dynamically couples the membrane fusion machinery to the microtubule cytoskeleton and demonstrate that Munc18-2 and STX3 perform distinct, but complementary, functions to support, respectively, SG translocation and membrane fusion in mast cells.
authors: Inserm UMRS-699, 75018 Paris, France.|Université Paris Diderot, Sorbonne Paris Cite, Laboratoire d'excellence INFLAMEX, 75018 Paris, France.
authors: Cristiana
authors: C
authors: Brochetta
authors: Laboratory of Molecular Immunogenetics, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD, 20892.
authors: Ryo
authors: R
authors: Suzuki
authors: Department of Life Sciences Department of Physiology and Pathology, University of Trieste, Italy.
authors: Francesca
authors: F
authors: Vita
authors: Department of Life Sciences Department of Physiology and Pathology, University of Trieste, Italy.
authors: Maria Rosa
authors: MR
authors: Soranzo
authors: Inserm UMRS-699, 75018 Paris, France.|Université Paris Diderot, Sorbonne Paris Cite, Laboratoire d'excellence INFLAMEX, 75018 Paris, France.
authors: Julien
authors: J
authors: Claver
authors: Inserm UMRS-699, 75018 Paris, France.|Université Paris Diderot, Sorbonne Paris Cite, Laboratoire d'excellence INFLAMEX, 75018 Paris, France.
authors: Lydia Celia
authors: LC
authors: Madjene
authors: Inserm UMRS-699, 75018 Paris, France.|Université Paris Diderot, Sorbonne Paris Cite, Laboratoire d'excellence INFLAMEX, 75018 Paris, France.
authors: Tarik
authors: T
authors: Attout
authors: Inserm UMRS-699, 75018 Paris, France.|Université Paris Diderot, Sorbonne Paris Cite, Laboratoire d'excellence INFLAMEX, 75018 Paris, France.
authors: Joana
authors: J
authors: Vitte
authors: Inserm U978, 93000 Bobigny, France.|Laboratoire d'excellence "Inflamex," Unité de Formation et de Recherche Santé-Médecine-Biologie Humaine, 93000 Bobigny, France.
authors: Nadine
authors: N
authors: Varin-Blank
authors: Department of Life Sciences Department of Physiology and Pathology, University of Trieste, Italy.
authors: Giuliano
authors: G
authors: Zabucchi
authors: Laboratory of Molecular Immunogenetics, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD, 20892.
authors: Juan
authors: J
authors: Rivera
authors: Inserm UMRS-699, 75018 Paris, France.|Université Paris Diderot, Sorbonne Paris Cite, Laboratoire d'excellence INFLAMEX, 75018 Paris, France.
authors: Ulrich
authors: U
authors: Blank
mesh_terms: D000818:Animals; D015550:Cell Degranulation / Q000235:genetics* / Q000276:immunology*; D002460:Cell Line; D003594:Cytoplasmic Granules / Q000378:metabolism; D005786:Gene Expression Regulation; D020868:Gene Silencing; D008407:Mast Cells / Q000276:immunology* / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism; D051938:Munc18 Proteins / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D021381:Protein Transport; D050765:Qa-SNARE Proteins / Q000235:genetics* / Q000378:metabolism; D034622:RNA Interference; D051381:Rats
chemical_list: D051938:Munc18 Proteins; D050765:Qa-SNARE Proteins