The class V myosin motor, myosin 5c, localizes to mature secretory vesicles and facilitates exocytosis in lacrimal acini

Abstract

Marchelletta RR, Jacobs DT, Schechter JE, Cheney RE, Hamm-Alvarez SF. The class V myosin motor, myosin 5c, localizes to mature secretory vesicles and facilitates exocytosis in lacrimal acini. Am J Physiol Cell Physiol 295: C13-C28, 2008. First published April 23, 2008 doi:10.1152/ajpcell.00330.2007.-We investigated the role of the actin-based myosin motor, myosin 5c (Myo5c) in vesicle transport in exocrine secretion. Lacrimal gland acinar cells (LGAC) are the major source for the regulated secretion of proteins from the lacrimal gland into the tear film. Confocal fluorescence and immunogold electron microscopy revealed that Myo5c was associated with secretory vesicles in primary rabbit LGAC. Upon stimulation of secretion with the muscarinic agonist, carbachol, Myo5c was also detected in association with actin-coated fusion intermediates. Adenovirus-mediated expression of green fluorescent protein (GFP) fused to the tail domain of Myo5c (Ad-GFP-Myo5c-tail) showed that this protein was localized to secretory vesicles. Furthermore, its expression induced a significant (P Յ 0.05) decrease in carbachol-stimulated release of two secretory vesicle content markers, secretory component and syncollin-GFP. Adenovirus-mediated expression of GFP appended to the full-length Myo5c (Ad-GFP-Myo5c-full) was used in parallel with adenovirus-mediated expression of GFP-Myo5c-tail in LGAC to compare various parameters of secretory vesicles labeled with either GFP-labeled protein in resting and stimulated LGAC. These studies revealed that the carbachol-stimulated increase in secretory vesicle diameter associated with compound fusion of secretory vesicles that was also exhibited by vesicles labeled with GFP-Myo5c-full was impaired in vesicles labeled with GFP-Myo5c-tail. A significant decrease in GFP labeling of actin-coated fusion intermediates was also seen in carbachol-stimulated LGAC transduced with GFPMyo5c-tail relative to LGAC transduced with GFP-Myo5c-full. These results suggest that Myo5c participates in apical exocytosis of secretory vesicles.actin; lacrimal gland; tear film THE LACRIMAL GLAND (LG) is the principal source of proteins released into the tear film. These proteins play critical roles in protection of the ocular surface from pathogens and also provide nutrients and growth factors essential for maintenance of the cornea (3, 50). The lacrimal gland acinar cells (LGAC) constitute ϳ85% of the LG and are largely responsible for this regulated secretion of tear proteins including secretory immunoglobulin A (sIgA), secretory component (SC), lysosomal hydrolases, and growth factors, among others (50). The development of decreased LG output occurs in individuals with syndromes ranging in severity from mild dry eye to the autoimmune disorder,

Directing exocrine secretory vesicles to the apical membrane by actin cables generated by the formin mDia1

“…We favor such a mechanism, because myosin V family motors have been shown to mediate secretory vesicle transport both in Saccharomyces cerevisiae, in which actin cables generated by the formin Bni1-related1 (Bnr1), located at the bud neck, serve as tracks for vesicle transport to the growing bud (22), and in our previous study of secretion via Dia-generated cables in Drosophila tubular organs (11). Furthermore, the velocity of vesicle movement over actin bundles that we report is similar to the measured velocity of vesicle transport via myosin Vc, the predominant myosin V isoform in the exocrine pancreas (23,24).…”

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

“…In previous studies, we showed that only 20 -30% of total cellular syncollin-GFP is released during a typical stimulation period with CCH (14,25). This plotting format thus enables detection of differences between treatment groups and enables clear comparison to previous work in which the role of other effectors in exocytosis of syncollin-GFP (Myo5C, Rab3D, actin filaments) has been probed (14,25,29). In data plots, values were expressed as a percentage increase above the total release from stimulated acini of cells expressing WT in the presence of CCH at the maximal time of release (30 min), normalized to 1.…”

“…As a measure of regulated secretion, syncollin release was measured in LG acinar cell cotransduced with Ad-syncollin-GFP and the Xp-WT, -CA, or -DN constructs as described previously (20,24,29). The bathing media, which is continuous with the apical lumina of the cultured cells, was collected at time points after CCH addition.…”

“…3C). Recent work in our group has shown that myosin 5C is enriched on LG acinar cell SV and that inhibition of myosin 5C impairs CCH-stimulated SV exocytosis (29). Colocalization coefficients were quantified for comparison purposes, and values demonstrated greater colocalization in the resting state than when following CCH stimulation of exocytosis for both Rab3D and myosin 5C (Table 1).…”

“…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).…”