Author ORCID Identifier
0000-0002-2386-4800
Date of Graduation
5-2024
Document Type
Thesis (MS)
Program Affiliation
Biomedical Sciences
Degree Name
Masters of Science (MS)
Advisor/Committee Chair
Burton Dickey, M.D.
Committee Member
Michael Tuvim, Ph.D.
Committee Member
Roberto Adachi, M.D.
Committee Member
Harry Karmouty-Quintana, Ph.D.
Committee Member
Jichao Chen, Ph.D.
Abstract
There are two rates at which airway epithelial cells secrete mucin. The low baseline rate accounts for ciliary clearance, and the high stimulated rate obstructs airways in protection from helminths or in obstructive pulmonary diseases. The exocytosis of mucins occurs via the four-helix SNARE complex. The SNARE proteins contributing helices are the vesicle-SNARE VAMP, and the target-SNAREs SNAP and Syntaxin. Coiling of the complex fuses the granule and plasma membranes to release mucins into the extracellular space. The VAMP and SNAP isoforms mediating baseline and stimulated secretion are known, but the Syntaxin (Stx) isoform remains unknown.
Three candidate exocytic Stx, 3, 4, and 11, were localized using immunofluorescence microscopy. To determine function, cell-specific deletion in club cells was accomplished by crossing the Stx mutant/floxed mice to Scgb1a1tm1(icre)fjd or tamoxifen-inducible Scgb1a1tm1(cre/ERT)blh/J mice. To measure mucin secretion defects, intracellular mucin was stained with Periodic Acid Fluorescence Schiff’s reagent (PAFS) and quantified with image analysis. In uninflamed mice, the baseline rates of secretion and production are similar so minimal intracellular mucin is quantified in wild-type mice, and a defect in mutant mice can be identified as spontaneous mucin accumulation. To identify a stimulated secretion defect, mucin production was increased with IL-13 and then mucin secretion was stimulated with ATP. A defect in mutant mice can be identified as the failure to release intracellular mucin in response to ATP.
In immunofluorescent assays of airway epithelium Stx3 localized apically, Stx4 localized to the basolateral membrane, and Stx11 was undetected. Stx4 airway specific deletant mice did not have viable airways. Stx11 conditional knockout mice showed no defect in baseline or agonist-stimulated mucin secretion. Stx3 homozygous airway epithelial deletion was embryonically lethal, so Stx3 tamoxifen-inducible Cre mice were used for experiments. These mice showed a three-fold increase in baseline intracellular mucin content, and stimulated secretion experiments revealed a severe defect. At a subcellular level, Stx3 localized prominently to mucin granules, suggesting that stimulated mucin secretion involves compound exocytosis.
Our findings demonstrate that Stx3 mediates baseline and stimulated airway mucin secretion while Stx 4 and 11 do not play roles in the regulated exocytic machinery. Complete knowledge of the composition and function of the mucin exocytic machinery may allow its therapeutic manipulation in airway obstructive diseases such as asthma and COPD, as suggested by the recent identification of a lead compound that selectively blocks stimulated mucin secretion.
Keywords
mucins, exocytosis, SNARE complex, Muc5ac, Muc5b, asthma, COPD