Characterization of the lens fiber cell tricellular junctional complex and its dependency on delta-catenin

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Abstract: Lens fiber cells are organized into a regular hexagonal array which promotes the transparency and refractive power of the lens. Junctional adhesion complexes are thought to facilitate this cellular organization but most efforts have focused on proteins and their associated complexes localized within bicellular membranes, where two cell membranes meet. The repeating hexagonal cellular pattern also naturally creates a different type of junction at the confluence of three cells called the tricellular junction which, in other epithelia, is inhabited by a distinct complement of proteins. However, the majority of what is known about these distinctive proteins are part of tricellular tight-junctions (tTJs) while little knowledge exists on the protein complexes that make up tricellular adherens junctions (tAJs) in vertebrates. The tAJ of lens fiber cells are large due to their extreme length and makes them an ideal model for elucidating the biology of this structure. We have recently demonstrated that the cadherin-associated protein δ-catenin is among proteins that specifically localize to the tricellular, but not the bicellular junctions, of lens fiber cells in mice. Furthermore, preliminary proteomic data suggests δ- catenin associates with a distinct set of proteins that include members of desmosomal junctions and GTPase signaling regulators. Importantly, human mutations within the encoding gene of δ-catenin, CTNND2, are associated with both cortical cataracts and high myopia, supporting the notion that both δ-catenin and tricellular junctions are important for lens physiology. However, what functional role δ-catenin plays in the lens or within the tricellular junctional complex of any tissue has not been assessed. In this exploratory proposal, the hypothesis that δ-catenin is required for the normal organization of the tricellular adherens junctional complex of lens fiber cells which preserves lens refraction and transparency will be tested with two aims. In the first aim, the requirement of δ-catenin for lens function will be tested. Additionally, we will perform a series of in vivo and ex vivo interaction assays to test for the presence of hypothesized candidate protein interactions within the tAJ. This analysis will be performed in lens fiber cell lysates and tissue of control or mutant mice lacking functional δ- catenin and followed up with super-resolution fluorescent microscopy to determine tAJ localization. In the second aim, we will test whether the δ-catenin associated protein complex is dependent or independent of the tAJ residing protein afadin and/or mechanical force. Preliminary data indicates that afadin localizes exclusively to tAJs and not bicellular membranes of lens fiber cells and is required for lens fiber cell organization and transparency. The complement of tAJ proteins that depend on afadin to associate with δ-catenin will be determined from proteomic analysis and high-resolution microscopy. Together these experiments will be the first to characterize the function of δ-catenin in the lens and identify the constituent proteins of the tricellular adherens junctional complex of lens fiber cells.