Architecture of polymeric claudin meshworks is modulated by tight junction-associated Marvel proteins (TAMPs)

Epithelial tissue barriers are composed of claudin-based polymeric strands. In addition to creating barriers, some claudins assemble into charge-selective channels that accommodate small molecules up to ~5Å diameter, e.g., ions, but not larger molecules, to create the pore pathway. Although tight junction associated Marvel proteins (TAMPs; occludin, tricellulin, and MARVELD3) do not self-assemble into polymers, it is clear that they can regulate the leak pathway that allows macromolecules up to ~125Å diameter to cross tight junctions. In vivo studies have shown that mice with global knockout of either occludin or tricellulin are viable and have only subtle phenotypes. Similarly, only subtle changes are detected in epithelial cells (MDCK or Caco-2) lacking one TAMP. In contrast, marked delays in barrier development and increased leak pathway permeability are apparent when any two or all three TAMPs are eliminated. We sought to understand the impact of TAMPs on claudin meshwork architecture and function. HYPOTHESIS: We hypothesized that each TAMP would have distinct effects on claudin polymerization and tight junction strand architecture. METHODS: Claudin proteins were expressed individually or in combination with a TAMP. After fixation, claudins or TAMPs were immmunolabelled with STAR RED or STAR ORANGE, respectively. Claudin strand structures and localization of each protein within meshworks were visualized using super-resolution STED microscopy. RESULTS: Consistent with prior studies, meshworks formed by individual claudins differed when expressed in U2OS osteosarcoma cells. For example, claudin-1 was concentrated in punctae along strands within a dense meshwork. In contrast, claudin-3 formed continuous strands that defined a meshwork with relatively small spaces. When co-expressed with individual claudins, each TAMP had distinct effects on meshwork architectures. Tricellulin increased mesh density, i.e., reduced the area of enclosed spaces. Consistent with this, the density of nodes, i.e., sites at which two or more strands intersect, were more frequent in the presence of tricellulin and tended to form right angles. In contrast, occludin induced intracellular claudin aggregates, interfered with strand assembly, and elongated both internode distances and the spaces defined by the meshwork. CONCLUSION: These data show that TAMPs modify polymeric claudin meshwork architecture. Although TAMPs had distinct effects on meshwork architecture of individual claudins, common themes characterized the effect of each TAMP across multiple claudins. In molecular terms, these data are difficult to understand in the absence of defined sites of TAMP-claudin interactions. However, our previous work has shown that, at least for claudins 1 and 2, ZO-1 can mediate indirect interactions with occludin. Such scaffolding functions of ZO-1 or ZO-2 or interactions with other proteins may also underlie the distinct enrichment of tricellulin at tricellular tight junctions, versus occludin and MARVELD3 localization within both bicellular and tricellular tight junction regions, as well as the unique anchoring and exchange properties of each TAMP. Thus, future studies will focus on defining the potential contributions of ZO-1/ZO-2 and F-actin TAMP effects on claudin architecture. NIDDK grants R01DK061931, R01DK068271, R21GM144801, and P30DK034854 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.