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Int J Mol Sci
2020 Jan 23;213:. doi: 10.3390/ijms21030761.
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Subcellular Localization of the TFF Peptides xP1 and xP4 in the Xenopus laevis Gastric/Esophageal Mucosa: Different Secretion Modes Reflecting Diverse Protective Functions.
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The TFF peptides xP1 and xP4 from Xenopus laevis are orthologs of TFF1 and TFF2, respectively. xP1 is secreted as a monomer from gastric surface mucous cells and is generally not associated with mucins, whereas xP4 is a typical secretory peptide from esophageal goblet cells, and gastric mucous neck and antral gland cells tightly associated as a lectin with the ortholog of mucin MUC6. Both TFF peptides have diverse protective functions, xP1 as a scavenger for reactive oxygen species preventing oxidative damage and xP4 as a constituent of the water-insoluble adherent inner mucus barrier. Here, we present localization studies using immunofluorescence and immunoelectron microscopy. xP1 is concentrated in dense cores of secretory granules of surface mucous cells, whereas xP4 mixes with MUC6 in esophageal goblet cells. Of note, we observe two different types of goblet cells, which differ in their xP4 synthesis, and this is even visible morphologically at the electron microscopic level. xP4-negative granules are recognized by their halo, which is probably the result of shrinkage during the processing of samples for electron microscopy. Probably, the tight lectin binding of xP4 and MUC6 creates a crosslinked mucous network forming a stabile granule matrix, which prevents shrinkage.
Figure 1. Labeled ultrathin methacrylate (Lowicryl K11M) sections of X. laevis stomachfundus. Fluorescence microscopy of sequential serial sections labeled for: (A) xP1 with antiserum anti-xP1-1 (Cy3, yellow); (B) xP4 with antiserum anti-xP4-1 (Cy3, yellow); (C) mucin with GSA-II (FITC, green); (D) mucin with antiserum HIK1083 (FITC, green). DNA of the cell nuclei was stained with DAPI (blue). Scale bar: 20 µm. (E) Electron micrograph of gold labeling with the anti-xP1-1 antiserum (detected with protein A-15 nm gold particles) and mucin staining with GSA-II (12 nm gold conjugates); scale bar: 2 µm.
Figure 2. Labeled ultrathin methacrylate (Lowicryl K11M) sections of X. laevis esophagus. (A–D) Fluorescence microscopy of a single section labeled for xP4 with antiserum anti-xP4-1 (Cy3, yellow), mucin with GSA-II (FITC, green), and DNA of cell nuclei with DAPI (blue): xP4 (A), GSA-II (B), xP4/GSA-II double exposure (C), and xP4/GSA-II/DAPI triple exposure (D); scale bar: 10 µm. (E, F) Electron micrographs of gold labeling with the anti-xP4-1 antiserum (detected with protein A-15 nm gold particles) and mucin staining with GSA-II (12 nm gold conjugates) at two orthogonal orientations of the sectioning planes. The three types of secretory granules are marked in (F); scale bars: 2 µm.
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