Figure 1.

MarvelD3, tricellulin, and occludin are the only members of the tight junction–associated MARVEL protein (TAMP) subfamily. (A) MARVEL domain alignment was ordered after model fitting and tree reconstruction and shaded within each of the four MARVEL protein lineages according to the Blosum62 score. Amino acids are shaded according to physiochemical properties as indicated in the figure. The characteristic acidic residue in the third transmembrane domain (Sanchez-Pulido et al., 2002) is indicated by an asterisk (*). (B) The alignment was used for tree reconstruction, and disparities were calculated according to the fraction of mutated residues. Posterior values are shown as 100-fold original predictions, implying that a bootstrap value of 60 is highly reliable. (C) Gene conservation analysis demonstrates that exons encoding MARVEL domains (∗) show the highest degree of evolutionary conservation within the marvelD3, tricellulin, and occludin genes. Data are presented as a base-by-base conservation measurement, with 0 indicating a neutral region and 1 indicating perfect conservation, from 28 vertebrate species including marsupial, monotreme, and placental mammals, as well as reptile, amphibian, bird, and fish clades. (D) Analysis of transcript expression in the Affymetrix GeneChip Mouse Genome 430 2.0 Array (Santa Clara, CA) reveals a highly correlated (p < 0.001), though nonredundant, pattern of expression for marvelD3, tricellulin, and occludin within epithelial organs. (E) EGFP-MARVEL domain–containing fusion proteins (green) were transfected into Caco-2 human intestinal epithelial cells. Polarized monolayers were grown on Transwell supports, and ZO-1 (red) was detected by immunofluorescent staining. Three-dimensional reconstruction shows that EGFP-marvelD3 splice variants 1 and 2, EGFP-tricellulin, and EGFP-occludin localize to tight junctions and intracellular vesicles. Both EGFP-marvelD3 splice variants are enriched along bicellular tight junctions, similar to occludin, whereas tricellulin is specifically concentrated at tricellular regions. MARVEL proteins from other lineages were found to decorate the apical plasma membrane and intracellular transport vesicles, but not the tight junction. Images are representative of ≥3 experiments, all with similar results. Bar, 10 μm.