A dynamic apical actin cytoskeleton facilitates exocytosis of tear proteins in rabbit lacrimal acinar epithelial cells

Lacrimal acinar cells exocytose mature secretory vesicles at their apical membranes in response to secretagogues. Here I use time-lapse confocal fluorescence microscopy and fluorescence recovery after photobleaching to investigate the changes in apical actin during exocytosis evoked by the muscarinic agonist, carbachol (100 muM). Time-lapse confocal fluorescence microscopy of reconstituted rabbit lacrimal acini transduced with replication-deficient adenovirus (Ad) containing GFP-actin revealed a quiescent apical actin array in resting acini. Carbachol increased apical actin filament turnover and promoted transient actin assembly around apparent fusion intermediates. Fluorescence recovery after photobleaching measurements revealed significant (p ≤ 0.05) increases and decreases, respectively, in mobile fraction (Mf) and turnover times (t1/2) for apical actin filaments in carbachol-stimulated acini relative to untreated acini. 2,3-butanedione monoxime and ML-7 each significantly decreased carbachol-stimulated secretion of bulk protein and the exogenous secretory vesicle marker, syncollin-GFP. This inhibition was accompanied by accumulation of apical actin filaments, suppression of the increased Mf of apical filaments elicited by carbachol, and accumulation of actin-coated structures enriched, in Ad-syncollin-GFP transduced acini, in syncollin-GFP. In contrast, latrunculin B significantly increased carbachol-stimulated secretion of bulk protein and syncollin-GFP.; I also investigated the involvement of the PKCepsilon in apical actin remodeling during exocytosis. Lacrimal acinar PKCepsilon, bound to actin filaments and was co-immunoprecipitated with anti-actin antibody. Confocal fluorescence microscopy and biochemical analysis showed increased association of PKCepsilon with apical actin in stimulated acini. Overexpression of dominant negative (DN) PKCepsilon in lacrimal acini resulted in formation of actin-coated structures at the apical surface and extension of actin-enriched processes from the basolateral surface. Ad-DN-PKCepsilon transduction significantly inhibited carbachol-stimulated secretion of bulk protein and beta-hexosaminidase. Co-transduction of acini with Ad-syncollin-GFP and Ad-DN-PKCepsilon significantly inhibited carbachol-stimulated release of syncollin-GFP; Ad-DN-PKCepsilon transduction also suppressed carbachol-stimulated release of secretory component.; My findings suggest that the increased turnover of apical actin filaments, driven in part by non-muscle myosin II, is an essential step in stimulated exocytosis in lacrimal acinar cells. I also propose that PKCepsilon is essential in the apical actin remodeling in exocytosis.