Cytoskeletal Participation in Stimulated Secretion and Compensatory Apical Plasma Membrane Retrieval in Lacrimal Gland Acinar Cells

Costa, Silvia R. da; Andersson, Sofia V.; Yarber, Francie A.; Okamoto, Curtis T.

doi:10.1007/978-1-4615-0717-8_26

Cited by 8 publications

(3 citation statements)

References 19 publications

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“…Isolated LGAC were cultured for 2-3 days in 100-mm round culture dishes at a density of 3.0 ϫ 10 7 cells or on glass coverslips in 12-well dishes coated with Matrigel (Invitrogen) at a density of 2.0 ϫ 10 7 cells. We have established that under these conditions, the cultured cells reestablish distinct apical and basolateral domains, form mSVs, and position these vesicles beneath lumena formed between adjacent APM of adjoining cells (7,14,15,47).…”

Section: Methodsmentioning

confidence: 97%

“…Immunoblots of LG lysate were developed using SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL), and films were scanned and imaged using Adobe Photoshop. LGAC lysate was prepared by 10 passes of 3.0 ϫ 10 7 LGAC through a 23-gauge needle three times on ice in 150 l of RIPA buffer containing protease inhibitor cocktail.…”

Section: Sds-page and Western Blot Analysismentioning

confidence: 99%

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

Marchelletta

Jacobs²,

Schechter

et al. 2008

American Journal of Physiology-Cell Physiology

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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,

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Section: Methodsmentioning

confidence: 97%

Section: Sds-page and Western Blot Analysismentioning

confidence: 99%

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

Marchelletta

Jacobs²,

Schechter

et al. 2008

American Journal of Physiology-Cell Physiology

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“…Cultured acinar cells were stimulated to secrete with CCH (100 µM, 15 m). For nocodazole treatments, cultured cells were incubated on ice for 5 m to induce depolymerization of microtubules prior to the addition of 33 µM nocodazole for 60 m at 37°C as previously described [68]. For subapical SV recovery studies, acinar cells were stimulated with CCH and then washed 4 times with warm culture media before incubation in fresh media re-perfused with nocodazole.…”

Section: Methodsmentioning

confidence: 99%

Direct Imaging of RAB27B-Enriched Secretory Vesicle Biogenesis in Lacrimal Acinar Cells Reveals Origins on a Nascent Vesicle Budding Site

Chiang

Karvar

2012

PLoS ONE

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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.

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Efron

2012

Contact Lens Complications

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Cytoskeletal Participation in Stimulated Secretion and Compensatory Apical Plasma Membrane Retrieval in Lacrimal Gland Acinar Cells

Cited by 8 publications

References 19 publications

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

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

Direct Imaging of RAB27B-Enriched Secretory Vesicle Biogenesis in Lacrimal Acinar Cells Reveals Origins on a Nascent Vesicle Budding Site

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