title: Rac1 and Rac2 control distinct events during antigen-stimulated mast cell exocytosis.
abstract: The release of preformed mediators from immune cells is through a process described as exocytosis. In mast cells, exocytosis is regulated by several coordinated intracellular signaling pathways. Here, we investigated the role of the hematopoietic-specific Rho GTPase, Rac2, and the ubiquitously expressed Rac1, in controlling mast cell exocytosis. These two isoforms showed equivalent levels of expression in mouse BMMCs. Although Rac1 and Rac2 share 92% sequence identity, they were not functionally redundant, as Rac2-/-BMMCs were defective in exocytosis, even though Rac1 levels were unaffected. Antigen-stimulated WT mast cells underwent a series of morphological transitions: initial flattening, followed by actin-mediated peripheral membrane ruffling and calcium influx, which preceded exocytosis. Whereas membrane ruffling was unaffected in Rac2-/-BMMCs, calcium influx was decreased significantly. Calcium influx was studied further by examining SOCE. In Rac2-/-BMMCs, the activation of PLCγ1 and calcium release from intracellular stores occurred normally; however, activation of plasma membrane calcium channels was defective, shown by the lack of extracellular calcium influx and a reduction of YFP-STIM1 puncta at the plasma membrane. Additionally, we used the small molecule Rac inhibitor, EHT 1864, to target Rac signaling acutely in WT BMMCs. EHT 1864 blocked exocytosis and membrane ruffling completely in conjunction with exocytosis. Our findings suggest that antigen-stimulated membrane ruffling in mast cells is a Rac1-mediated process, as this persisted in the absence of Rac2. Therefore, we define distinct modes of Rac-regulated mast cell exocytosis: Rac2-mediated calcium influx and Rac1-mediated membrane ruffling.
authors.affiliation: Department of Cell Biology and.
authors.forename: Alicia
authors.initials: A
authors.lastname: Baier
authors.affiliation: Department of Cell Biology and.|Department of Medicine, University of Örebro, Örebro, Sweden.
authors.forename: Vivian N E
authors.initials: VN
authors.lastname: Ndoh
authors.affiliation: The Pulmonary Research Group, Department of Medicine, University of Alberta, Edmonton, Alberta, Canada; and.
authors.forename: Paige
authors.initials: P
authors.lastname: Lacy
authors.affiliation: Department of Cell Biology and gary.eitzen@ualberta.ca.
authors.forename: Gary
authors.initials: G
authors.lastname: Eitzen
mesh_terms: D000818:Animals; D000941:Antigens / Q000276:immunology* / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects / Q000235:genetics / Q000276:immunology*; D002462:Cell Membrane / Q000235:genetics / Q000276:immunology; D005089:Exocytosis / Q000187:drug effects / Q000235:genetics / Q000276:immunology*; D008407:Mast Cells / Q000276:immunology*; D051379:Mice; D018345:Mice, Knockout; D009479:Neuropeptides / Q000037:antagonists & inhibitors / Q000235:genetics / Q000276:immunology*; D011753:Pyrones / Q000494:pharmacology; D011804:Quinolines / Q000494:pharmacology; D020744:rac GTP-Binding Proteins / Q000235:genetics / Q000276:immunology*; D020830:rac1 GTP-Binding Protein / Q000037:antagonists & inhibitors / Q000235:genetics / Q000276:immunology*; D000097766:RAC2 GTP-Binding Protein
chemical_list: D000941:Antigens; D009479:Neuropeptides; D011753:Pyrones; D011804:Quinolines; D020744:rac GTP-Binding Proteins; D020830:rac1 GTP-Binding Protein; D000097766:RAC2 GTP-Binding Protein; C506907:EHT 1864; C484602:Rac1 protein, mouse
keywords: Rho GTPase; actin dynamics; calcium flux; degranulation