Three‐dimensional imaging of the mucus secretory process in the cryofixed frog respiratory epithelium

Abstract

Using the frog palate as a representative model of human mucociliary epithelium, we analyzed, after quick freezing fixation, the three-dimensional (3-D) respiratory mucus secretory release with high voltage (200-300 kV) transmission electron microscopy (TEM). The 3-D vision of the mucus release from the secretory cells was obtained as stereo-pairs and "bas-relief" images after analysis of stereo-pairs using an image analyzer. After standard glutaraldehyde fixation, the secretory cells showed a typical goblet shape with secretory granules heterogeneous in size and electron-density which often fuse together. On the other hand, quick-frozen secretory cells exhibited a columnar shape and their membrane-bound secretory granules contained a homogeneously dark matrix. The expanded gel mucus layer was preserved and its depth never exceeded 2 microns. When the epithelium was immersed in culture medium in presence of cholinergic agonist, a marked discharge of mucus was observed and the granules swelled at the apex of the secretory cell before being discharged in the lumen. In native cryofixed epithelium, the secretory granules exhibited a marked deformability during the process of their extrusion from the secretory cell. Clusters of secretory granules surrounded by cytoplasmic material were observed in the extracellular lumen, suggesting an apocrine-type secretion. These observations indicate that rapid cryofixation and 3-D stereoscopic imaging enable a unique opportunity to analyze, without artifact, the mucous secretory process. We speculate that, apart from the classical merocrine-type secretion mechanism, the respiratory mucus may be released, at least partly by an apocrine-type secretion.

Mucin Granule Intraluminal Organization in Living Mucous/Goblet Cells

“…7) clearly suggested the presence of partially discharged granules with limited SHGFP-MUC5AC/CK and likely no intragranular matrix. Hence, these results support the notion that transient simple exocytosis, rather than massive compound exocytosis (63) or apocrine secretion (64), is prevalent in HT29-SGFP-MUC5AC/CK cells under the conditions assayed. Such a mechanism would suggest that the decondensation and expansion of the mucin matrix take place extracellularly, either as the matrix traverses the fusion pore or shortly thereafter.…”

MUC16 is produced in tracheal surface epithelium and submucosal glands and is present in secretions from normal human airway and cultured bronchial epithelial cells

“…MUC1, where the N-terminal domain is shed from the cell surface leaving the C-terminal in the membrane (Thathiah, Blobel, & Carson, 2003). Cryofixation studies have indicated that secretion from goblet cells may, in part, occur via an apocrine mechanism involving discharge of whole secretory granules (Puchelle, Beorcia, Ménager, Zahm, & Ploton, 1991).…”

Regulation of depth and composition of airway surface liquid

“…This system of "mucociliary clearance" serves to remove particles trapped in the mucous gel. The concept of two layers of airway surface liquid (ASL) has been supported by a variety of microscopy studies [2][3][4][5] (fig. 1).…”

“…Frozen tissues, cryosectioned and then freeze-dried and substituted have provided excellent differentiation between the sol and the gel [2,5]. However, freeze substitution is associated with tissue shrinkage of 11% in the linear dimension [44], and portions of the ASL may be fractured away during cryosectioning [45].…”