title: Rab27b regulates exocytosis of secretory vesicles in acinar epithelial cells from the lacrimal gland.
abstract: Tear proteins are supplied by the regulated fusion of secretory vesicles at the apical surface of lacrimal gland acinar cells, utilizing trafficking mechanisms largely yet uncharacterized. We investigated the role of Rab27b in the terminal release of these secretory vesicles. Confocal fluorescence microscopy analysis of primary cultured rabbit lacrimal gland acinar cells revealed that Rab27b was enriched on the membrane of large subapical vesicles that were significantly colocalized with Rab3D and Myosin 5C. Stimulation of cultured acinar cells with the secretagogue carbachol resulted in apical fusion of these secretory vesicles with the plasma membrane. Evaluation of morphological changes by transmission electron microscopy of lacrimal glands from Rab27b(-/-) and Rab27(ash/ash)/Rab27b(-/-) mice, but not ashen mice deficient in Rab27a, showed changes in abundance and organization of secretory vesicles, further confirming a role for this protein in secretory vesicle exocytosis. Glands lacking Rab27b also showed increased lysosomes, damaged mitochondria, and autophagosome-like organelles. In vitro, expression of constitutively active Rab27b increased the average size but retained the subapical distribution of Rab27b-enriched secretory vesicles, whereas dominant-negative Rab27b redistributed this protein from membrane to the cytoplasm. Functional studies measuring release of a cotransduced secretory protein, syncollin-GFP, showed that constitutively active Rab27b enhanced, whereas dominant-negative Rab27b suppressed, stimulated release. Disruption of actin filaments inhibited vesicle fusion to the apical membrane but did not disrupt homotypic fusion. These data show that Rab27b participates in aspects of lacrimal gland acinar cell secretory vesicle formation and release.
authors.affiliation: School of Pharmacy, University of Southern California, Los Angeles, CA 90033, USA.
authors.forename: Lilian
authors.initials: L
authors.lastname: Chiang
authors.forename: Julie
authors.initials: J
authors.lastname: Ngo
authors.forename: Joel E
authors.initials: JE
authors.lastname: Schechter
authors.forename: Serhan
authors.initials: S
authors.lastname: Karvar
authors.forename: Tanya
authors.initials: T
authors.lastname: Tolmachova
authors.forename: Miguel C
authors.initials: MC
authors.lastname: Seabra
authors.forename: Alistair N
authors.initials: AN
authors.lastname: Hume
authors.forename: Sarah F
authors.initials: SF
authors.lastname: Hamm-Alvarez
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism; D000704:Analysis of Variance; D000818:Animals; D002217:Carbachol / Q000494:pharmacology; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D005089:Exocytosis / Q000187:drug effects; D007765:Lacrimal Apparatus / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D008297:Male; D008561:Membrane Fusion; D051379:Mice; D018345:Mice, Knockout; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D018715:Microscopy, Video; D009218:Myosins / Q000378:metabolism; D021381:Protein Transport; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D022142:Secretory Vesicles / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D013997:Time Factors; D014162:Transfection; D020691:rab GTP-Binding Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D020692:rab3 GTP-Binding Proteins / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D002217:Carbachol; C512958:Rab27b protein, mouse; D009218:Myosins; D020691:rab GTP-Binding Proteins; D020692:rab3 GTP-Binding Proteins