Actin and non-muscle myosin II facilitate apical exocytosis of tear proteins in rabbit lacrimal acinar epithelial cells

Abstract

The acinar epithelial cells of the lacrimal gland exocytose the contents of mature secretory vesicles containing tear proteins at their apical membranes in response to secretagogues. Here we use time-lapse confocal fluorescence microscopy and fluorescence recovery after photobleaching to investigate the changes in actin filaments located beneath the apical membrane during exocytosis evoked by the muscarinic agonist, carbachol (100 μM). Time-lapse confocal fluorescence microscopy of apical actin filaments in reconstituted rabbit lacrimal acini transduced with replication-deficient adenovirus containing GFP-actin revealed a relatively quiescent apical actin array in resting acini. Carbachol markedly increased apical actin filament turnover and also 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 (t½) for apical actin filaments in carbachol-stimulated acini relative to untreated acini. The myosin inhibitors, 2,3-butanedione monoxime (BDM, 10 mM, 15 minutes) and ML-7 (40 μM, 15 minutes), significantly decreased carbachol-stimulated secretion of bulk protein and the exogenous secretory vesicle marker, syncollin-GFP; these agents also promoted accumulation of actin-coated structures which were enriched, in transduced acini, in syncollin-GFP, confirming their identity as fusion intermediates. Actin-coated fusion intermediates were sized consistent with incorporation of multiple rather than single secretory vesicles; moreover, BDM and ML-7 caused a shift towards formation of multiple secretory vesicle aggregates while significantly increasing the diameter of actin-coated fusion intermediates. Our findings suggest that the increased turnover of apical actin filaments and the interaction of actin with non-muscle myosin II assembled around aggregates of secretory vesicles facilitate exocytosis in lacrimal acinar epithelial cells.

Rab27b regulates exocytosis of secretory vesicles in acinar epithelial cells from the lacrimal gland

“…Acinar cells isolated from LG from New Zealand White rabbits (1.8 -2.2 kg) (Irish Farms, Norco, CA) were sequentially incubated in Mg 2ϩ -and Ca 2ϩ -free HBSS supplemented with EDTA and Ham's medium supplemented with collagenase, hyaluronidase, and DNAse, and cultured for 2 days in Peter's serum-free culture medium (18). These cultured cells reform acinuslike structures displaying distinct apical and basolateral domains (9,17,24). For imaging of fixed cells, LG acinar cells were seeded on coverslips at 2 ϫ 10 6 cells per well in 12-well plates coated with Matrigel.…”

“…Myosin motors and actin filaments are believed to facilitate terminal aspects of SV fusion with APM and content extrusion; studies in LG acinar cells have collectively suggested that stimulation with the muscarinic agonist, carbachol (CCH), results in transient disassembly of the actin barrier beneath the APM concurrent with the assembly of actin "coats," which aid in the compound fusion of SV homotypically and with the APM (24, 46, 52). Both the unconventional myosin motor, myosin 5C, and non-muscle myosin II are thought to participate in aspects of terminal SV fusion (24,29). Microtubules and cytoplasmic dynein have also been shown to play a general role in the maturation and maintenance of SV in the LG (50).…”

“…Mature LG acinar cell SV localize beneath an actin-rich network underlying the APM; on stimulation, SV appear to undergo compound fusion followed by exocytosis of their contents at the APM (25). Myosin motors and actin filaments are believed to facilitate terminal aspects of SV fusion with APM and content extrusion; studies in LG acinar cells have collectively suggested that stimulation with the muscarinic agonist, carbachol (CCH), results in transient disassembly of the actin barrier beneath the APM concurrent with the assembly of actin "coats," which aid in the compound fusion of SV homotypically and with the APM (24,46,52). Both the unconventional myosin motor, myosin 5C, and non-muscle myosin II are thought to participate in aspects of terminal SV fusion (24,29).…”

Powering membrane traffic in endocytosis and recycling

“…For example, the fusion of lysosomes with the plasma membrane during wound repair depends on myosin IIb 122 . Also, in chromaffin cells, a mutant regulatory myosin light chain decreases docking and exocytosis 123 , and inhibitors of myosin light chain kinase or the myosin ATPase reduce the exocytosis of tear secretory vesicles in lacrimal acinar epithelial cells 124 . In neurons, the interaction of neural cell-adhesion molecule-180 (NCAM180) with the myosin light chain kinase is required to replenish the pool of synaptic vesicles at the active zone after high levels of exocytosis 125 , perhaps by regulating presynaptic myosin IIb 126 .…”

Serous Cells in the Parotid Glands of Two Species of Tamarins: Polarized Secretory Granules

“…An additional possibility is that actin filaments, which are involved in the translocation of salivary serous granules to a secretory surface (Segawa and Riva, 1996;Jerdeva et al, 2005), are attached only to the spherulecontaining pole and pull this pole toward the cell surface so that it perforce becomes the forepart of the granule about to be exocytosed. The absence of obvious actin filaments in our preparations might be due to the wellknown propensity of osmium tetroxide to cause the depolymerization of this molecule (Boyles et al, 1985).…”