title: Direct imaging of RAB27B-enriched secretory vesicle biogenesis in lacrimal acinar cells reveals origins on a nascent vesicle budding site.
abstract: This study uses YFP-tagged Rab27b expression in rabbit lacrimal gland acinar cells, which are polarized secretory epithelial cells, to characterize early stages of secretory vesicle trafficking. Here we demonstrate the utility of YFP-Rab27b to delineate new perspectives on the mechanisms of early vesicle biogenesis in lacrimal gland acinar cells, where information is significantly limited. Protocols were developed to deplete the mature YFP-Rab27b-enriched secretory vesicle pool in the subapical region of the cell, and confocal fluorescence microscopy was used to track vesicle replenishment. This analysis revealed a basally-localized organelle, which we termed the "nascent vesicle site," from which nascent vesicles appeared to emerge. Subapical vesicular YFP-Rab27b was co-localized with p150(Glued), a component of the dynactin cofactor of cytoplasmic dynein. Treatment with the microtubule-targeted agent, nocodazole, did not affect release of mature secretory vesicles, although during vesicle repletion it significantly altered nascent YFP-Rab27b-enriched secretory vesicle localization. Instead of moving to the subapical region, these vesicles were trapped at the nascent vesicle site which was adjacent to, if not a sub-compartment of, the trans-Golgi network. Finally, YFP-Rab27b-enriched secretory vesicles which reached the subapical cytoplasm appeared to acquire the actin-based motor protein, Myosin 5C. Our findings show that Rab27b enrichment occurs early in secretory vesicle formation, that secretory vesicles bud from a visually discernable nascent vesicle site, and that transport from the nascent vesicle site to the subapical region requires intact microtubules.
authors.affiliation: Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, California, United State of America.
authors.forename: Lilian
authors.initials: L
authors.lastname: Chiang
authors.forename: Serhan
authors.initials: S
authors.lastname: Karvar
authors.forename: Sarah F
authors.initials: SF
authors.lastname: Hamm-Alvarez
mesh_terms: D061354:Acinar Cells / Q000166:cytology / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D002451:Cell Compartmentation; D005260:Female; D006056:Golgi Apparatus / Q000378:metabolism; D021621:Imaging, Three-Dimensional / Q000379:methods*; D007765:Lacrimal Apparatus / Q000166:cytology*; D008870:Microtubules / Q000378:metabolism; D008954:Models, Biological; D009218:Myosins / Q000378:metabolism; D011485:Protein Binding; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D022142:Secretory Vesicles / Q000378:metabolism*; D020691:rab GTP-Binding Proteins / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D009218:Myosins; D020691:rab GTP-Binding Proteins