title: Investigating mast cell secretory granules; from biosynthesis to exocytosis.
abstract: Mast Cells (MC) are secretory cells of the immune system that accomplish their physiological and pathological functions by releasing pre-formed and newly synthesized allergic, inflammatory and immunoregulatory mediators. MCs' mediators affect multiple tissues and organs culminating in allergic and immune responses. The synthesis, storage and release of the MC mediators are highly regulated. The pre-formed mediators are packed in cytoplasmic secretory granules (SG) that fuse with the plasma membrane and release their content by regulated exocytosis. We present a protocol, based on the co-expression of a gene of interest with a reporter gene that is targeted to the SGs and is released in a regulated fashion alongside the endogenous SG mediators. The protocol enables high resolution four dimensional confocal analyses of the MC SGs and monitoring their timeline from biogenesis to triggered exocytosis. Thus, using this protocol for screening genes of interest for their phenotypic and functional impact allows deciphering the molecular mechanisms that govern the biogenesis and exocytosis of the MC SGs and identifying the regulators involved. Thereby, further insights into the cellular mechanisms that account for MCs function in health and disease should be provided.
authors: Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel Aviv University; Division of Allergy and Immunology, Department of Pediatrics, Cincinnati Children's Hospital Medical Center, University of Cincinnati College of Medicine.
authors: Nurit P
authors: NP
authors: Azouz
authors: Laboratory of Membrane Trafficking Mechanisms, Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University.
authors: Mitsunori
authors: M
authors: Fukuda
authors: Division of Allergy and Immunology, Department of Pediatrics, Cincinnati Children's Hospital Medical Center, University of Cincinnati College of Medicine.
authors: Marc E
authors: ME
authors: Rothenberg
authors: Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel Aviv University; histol3@post.tau.ac.il.
authors: Ronit
authors: R
authors: Sagi-Eisenberg
mesh_terms: D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D003594:Cytoplasmic Granules / Q000737:chemistry / Q000276:immunology / Q000378:metabolism / Q000502:physiology*; D005089:Exocytosis / Q000502:physiology; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D008247:Lysosomes / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D008407:Mast Cells / Q000737:chemistry / Q000276:immunology / Q000378:metabolism / Q000502:physiology*; D051381:Rats; D022142:Secretory Vesicles / Q000276:immunology / Q000378:metabolism / Q000502:physiology*; D014162:Transfection / Q000379:methods
chemical_list: D049452:Green Fluorescent Proteins
references: Int Rev Cell Mol Biol. 2009;275:89-131
references: 19491054
references: Trends Immunol. 2007 May;28(5):234-41
references: 17400512
references: IUBMB Life. 2011 Oct;63(10):873-80
references: 21905201
references: Int Arch Allergy Immunol. 2010;151(1):1-7
references: 19672091
references: Adv Exp Med Biol. 2011;716:212-34
references: 21713659
references: J Allergy Clin Immunol. 2006 Jun;117(6):1214-25; quiz 1226
references: 16750977
references: Physiol Rev. 1997 Oct;77(4):1033-79
references: 9354811
references: Adv Exp Med Biol. 2011;716:186-211
references: 21713658
references: Blood. 1999 Oct 1;94(7):2333-42
references: 10498605
references: EMBO J. 1994 Mar 15;13(6):1287-96
references: 8137813
references: Annu Rev Pharmacol Toxicol. 1983;23 :331-51
references: 6191653
references: Eur J Immunol. 2010 Jul;40(7):1843-51
references: 20583030
references: J Immunol. 2006 Apr 1;176(7):4141-6
references: 16547250
references: Immunol Today. 1990 Dec;11(12):458-64
references: 2073318
references: Immunol Rev. 2007 Jun;217:65-78
references: 17498052
references: Front Immunol. 2012 Jun 04;3:134
references: 22675325
references: Adv Exp Med Biol. 2011;716:270-88
references: 21713661
references: J Immunol. 2014 May 1;192(9):4043-53
references: 24696234
references: J Innate Immun. 2011;3(5):495-507
references: 21654154
references: Adv Exp Med Biol. 2011;716:2-12
references: 21713648
references: Cell Mol Life Sci. 2011 Mar;68(6):965-75
references: 21069421
references: J Allergy Clin Immunol. 2010 Jul;126(1):140-9
references: 20538331
references: Curr Opin Immunol. 2010 Oct;22(5):643-8
references: 20884193
references: J Immunol. 2012 Sep 1;189(5):2169-80
references: 22826321