Saturday, September 7, 2024
9/7/2024
PROJECT SUMMARY Intercellular adhesion is essential for tissue development and maintenance. One such adhesive structure is the zonula adherens junction (ZA) where the tension sensitive protein -catenin (cat) couples transmembrane cadherins to the actin cytoskeleton. While the essential function of ZA components is well-established, we know comparatively less about the dynamic regulation of this structure. During tissue morphogenesis, cells must accommodate neighbor exchanges while maintaining crucial barrier function. Here we address the overarching hypothesis that cat integrates actomyosin based mechanical signals with post-translational modification (i.e., phosphorylation) to control epithelial barrier structure and function. Our team has identified an evolutionarily-conserved phosphorylation scheme within a flexible region of cat linking the F-actin binding domain to its mechanosensitive middle-domain. Phosphorylation at these sites is required for strong cell-cell adhesion in widely used dog kidney epithelial cell line, as well as development in flies. But mechanistic underpinnings and critical contribution to mammalian development remain incompletely understood. We find that phospho-cat appears enriched at the zonula adherens junction and is reduced by actomyosin inhibition. We also find that a phospho-mimic form of cat phenocopies this ZA enrichment and adopts a more opened conformation in vitro. These data suggest actomyosin contractility promotes cat phosphorylation, leading to a conformational change that favors recruitment of a key binding partner required for cat junctional enrichment, ZA structure and barrier function. To address this model, Aim 1 will address whether Afadin, a multi-domain scaffold protein implicated in ZA structure, is a key effector of phospho-cat, making use of a newly mapped binding site in cat. Aim 2 will establish the in vivo relevance of this mechanism through characterization of a novel cat phospho-null mouse with postnatal developmental delay. Together, these aims will fill a fundamental gap in the understanding of how a central cell-cell adhesive component is regulated by mechanical and chemical modification to control epithelial barrier function, with implications for diseases caused by cat mutation, such as butterfly-shaped patterned eye dystrophy and vitreoretinopathy. I will carry out these research aims under the mentorship of my sponsor, Dr. Cara Gottardi, with her expertise in epithelial cell biology. Our strategy will be complemented by a training plan that enhances skill in quantitative image microscopy, methods to interrogate epithelial barrier function, and tissue-level phenotypic analysis in a mouse model. As these training goals are applicable to a range of research questions, they will be foundational to my career development as a physician scientist, with current interest in ophthalmology, given that cat missense mutations causally contribute to eye disease, and the mouse developed in Aim 2 may provide an avenue for future independent research.
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