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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 p150Glued, 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.
Highlights
Apically-secreting epithelial cells of the lacrimal gland are organized around lumina continuous with tear ducts which drain contents on to the ocular surface
Classical studies of transport vesicle budding in professional secretory cells suggest that secretory vesicles (SV) budding and fission occur in the basolaterally-organized Golgi stacks and trans-Golgi network (TGN) [15,16,17], but much of this data is based on static techniques such as electron microscopy
Clusters of three or more of these cells reform functional reconstituted acini in vitro, with apical domains organized around luminal regions which are sites of SV exocytosis and which remain open to the culture media
Summary
Apically-secreting epithelial cells of the lacrimal gland are organized around lumina continuous with tear ducts which drain contents on to the ocular surface. Rab27b interacts with effectors which mediate interaction with the actin cytoskeleton during transport to the plasma membrane for fusion (MyRIP/Exophilin8/Slac2-c) and with the SNARE protein affiliated granule docking (Granuphilin/Exophilin2/Slp4) which suggest its involvement in the final stages of exocytosis [39,43]. These studies suggest that Rab27b is at least associated with mature SV in the subapical region, if not a wider SV population. Rab27b can be be utilized to investigate SV formation and maturation, in addition to events involving terminal exocytosis
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