Project Summary Intercellular junctions and their cytoskeletal connections are essential for maintaining tissue stability and function. This is particularly true of the multi-layered epidermis as it is exposed to high levels of mechanical stress while maintaining an essential physical and immune barrier. Critical contributors to the epidermis' ability to maintain the epidermal barrier while simultaneously adjusting to stress are attachments between keratin intermediate filaments (IF) and intercellular junctions called desmosomes. Within desmosomes, a cytoskeletal linker protein called desmoplakin (DP) is responsible for anchoring the IF to the junctions. DP is ubiquitously expressed in all desmosome-forming cells and is regulated by post-translational modification (PTM) of its C-terminal tail domain to control DP-IF affinity. Consistent with the importance of this phospho-regulatory region of DP for desmosome function, genetic deletions of the C-terminal region cause multiple diseases associated with severe cardio- cutaneous and lethal epidermal barrier defects. Functionally, when the C-terminal motif is in its hypo- phosphorylated state, DP exhibits its highest affinity towards IF. In experimental models, the increased DP-IF association had protective effects in mature cell sheets by generating stronger, more stable desmosomes. Conversely, during desmosome assembly hypo-phosphorylated DP accumulates on the IF network, disrupting it's trafficking to desmosome junctions, and interfering with the formation and maturation of new cell-cell desmosome junctions. Given the strong molecular phenotypes associated with DP's hypo-phosphorylated form in vitro, a coordinated process must exist to regulate DP phosphorylation for proper desmosome function. We previously identified GSK3 as the kinase responsible for phosphorylating DP; however, the protein phosphatase responsible for negatively regulating DP phosphorylation was previously unknown. My preliminary data identified PP2A-B55 as capable of binding to and dephosphorylating DP's C-terminus. This proposal will test the hypothesis that PP2A-B55 regulates DP phosphorylation during the dynamic process of desmosome assembly and in response to mechanical stress to allow cells to respond to the changing properties and specialized functions of the stratified epidermis. Aim 1 will employ cutting-edge microscopy and proteomics- based approaches to identify the mechanism by which PP2A regulates DP during desmosome assembly. Aim 2 will establish how PP2A phospho-regulation of DP impacts 1) desmosome-dependent cell adhesion and 2) the desmosomal response to mechanical stress using novel mechano-biology techniques. The proposed work will provide important insight into how phosphatase signaling controls desmosome assembly and function to maintain the human epidermal barrier and, therefore, how dysregulated phosphatase signaling could contribute to barrier-related diseases. Together, the proposed research and career development plan will provide a springboard for my development as an independent investigator and planned K99/R00 application.

Project Narrative The machinery responsible for holding skin cells together is critical for the maintenance of tissue integrity and barrier function of the stratified epidermis. This proposal will leverage cutting-edge microscopy and proximity- labeling proteomics strategies to address how protein modifications of this critical machinery impacts the skin's form and function. Together, the scientific insights into the mechanisms regulating this machinery, coupled with the training opportunities proposed in this application will form the necessary foundation for my long-term scientific and career goals.