Mucin granule intraluminal organization

Abstract

Mucus secretions have played a central role in the evolution of multicellular organisms, enabling adaptation to widely differing environments. In vertebrates, mucus covers and protects the epithelial cells in the respiratory, gastrointestinal, urogenital, visual, and auditory systems, amphibian's epidermis, and the gills in fishes. Deregulation of mucus production and/or composition has important consequences for human health. For example, mucus obstruction of small airways is observed in chronic airway diseases, including chronic obstructive pulmonary disease, asthma, and cystic fibrosis. The major protein component in the mucus is a family of large, disulfide-bonded glycoproteins known as gel-forming mucins. These proteins are accumulated in large, regulated secretory granules (the mucin granules) that occupy most of the apical cytoplasm of specialized cells known as mucous/goblet cells. Since mucin oligomers have contour dimensions larger than the mucin granule average diameter, the question arises how these highly hydrophilic macromolecules are organized within these organelles. I review here the intraluminal organization of the mucin granule in view of our knowledge on the structure, biosynthesis, and biophysical properties of gel-forming mucins, and novel imaging studies in living mucous/goblet cells. The emerging concept is that the mucin granule lumen comprises a partially condensed matrix meshwork embedded in a fluid phase where proteins slowly diffuse.

Figure 1.

Structure and biosynthesis of gel-forming mucins. (A) Schematic representation of the structure of MUC5AC (> 5,000 amino acid residues), a typical gel-forming mucin, showing the major protein domains found in its structure, as deduced from cDNA and genomic sequencing (e.g., Refs. 58, 59). P, signal peptide; U, Ser/Thr-rich unique sequences; M, tandemly repeated Ser/Thr-rich sequences-containing domains; D, D-domains; CS, Cys subdomains; C, C-domains; CK, CK (cystine-knot)-like domain. The U and M domains are heavily O-glycosylated. D-, CS-, C-, and CK-like domains are under-glycosylated and Cys-rich protein domains. (B) Current model for the biosynthesis and covalent assembly of gel-forming mucins. Note that the covalent assembly of mucins involves the formation of inter-chain disulfide bonds between the C-terminal CK-domain in two monomers while in the endoplasmic reticulum, and inter-chain disulfide bonds connecting the N-terminal D-domains in dimers to form oligomers of variable size.

Figure 2.

The matrix core model. This model suggests a zipper-like mucin matrix condensation in which Ca²⁺ ions bind negatively charged mucin oligomers (black lines), filling, together with a hydration shell, most of the space between them. Secretory proteins (P) are entrapped among the condensed matrix. The condensed core is encircled by a fluid aqueous phase in which free Ca²⁺ and other ions are in equilibrium with their immobile counterpart in the periphery of the matrix core.

Figure 3.

FRAP studies in living mucous/goblet cells. (A) XY confocal images of HT29–18N2 mucous/goblet cells expressing mucin-GFP showing a cluster of cells (a) or a single cell (b). Note the difference in fluorescence intensities in the granular mass and the rest of the cell, which virtually cannot be seen unless the granule signal is oversaturated. Scale bars = 8 μm and 1 μm in a and b, respectively. (B) Expected normalized FRAP curve profiles for a mucin granule in which the lumen has a condensed matrix core where proteins are unable to diffuse (a) or a penetrable matrix meshwork (panel b) following the main premise of the matrix core paradigm or the matrix meshwork paradigm, respectively. The matrix core model predicts the absence of fluorescence recovering in the bleached area. Conversely, the matrix meshwork model predicts recovery of the signal assuming that the size and tertiary structure of the protein probe allow for its diffusion through the pores of the meshwork. P, pre-bleach granule; B, granule immediately after bleaching; PB, granule once fluorescence recovery reaches a plateau. (C) Representative FRAP analysis involving bleaching of a circular spot of a mucin granule in a living mucous/goblet cell, showing XY confocal images before, immediately after bleaching, and 5 s after bleaching, respectively, at two different magnifications (a). Scale bars = 0.5 μm. Also shown is the actual normalized FRAP bleaching/recovery curve (b). P, pre-bleach image; B, granule immediately after bleaching image; PB, image of the granule once fluorescence recovery reaches a plateau. Scale bars = 1 μm.

Figure 4.

The matrix meshwork model. Our studies suggest the formation of a meshwork in a fluid phase in which proteins (P) and ions (Ca²⁺) can diffuse through its pores and interact with the matrix components, including protein- and O-glycan-rich regions, respectively. Ca²⁺ -dependent condensation might be in effect but would not involve the total collapse of the matrix.