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.