By tethering the intermediate filament (IF) cytoskeleton to the plasma membrane, the desmosomal plaque component desmoplakin (DP) strengthens adhesion mediated by the transmembrane desmosomal cadherins. DP mutations result in potentially lethal disorders of the skin and heart. The loss of mechanical tissue integrity caused by desmosome dysfunction is commonly thought to underlie disease pathogenesis. Duing this MERIT award period, however, we showed that DP is not simply a physical connector. It is also a signaling scaffold that tunes its own dynamic association with IF as well as the cortical actin cytoskeleton to modulate epithelial cell mechanics. We identified previously unrecognized binding partners of an intrinsically disordered region (IDR) in the DP C-terminus that makes the DP-IF complex tunable. How this DP scaffold changes as keratinocytes transit from the basal proliferating layer to the superficial layers to meet the needs of the developing tissue is still poorly understood. Our overarching hypothesis is that DP and associated plakophilins localize and integrate the activities of signaling mediators and actomyosin modulators when and where they are needed for junction assembly, adhesion strengthening, and epidermal morphogenesis. Here, we will use a combination of 2- and 3-dimensional in vitro human models and DP-deficient human disease and animal models to: 1. Define the mechanism of desmoplakin-intermediate filament complex formation and cooperation with desmosomal cadherins to modulate adhesion and promote early epidermal morphogenesis. We hypothesize that DP forms cytoplasmic precursors comprising plakophilins, actin regulators, RNAs, and RNA binding proteins necessary for junction-associated actin polymerization, altered cell mechanics and cell fate. To test this, we will define a) the machinery responsible for DP precursor formation, b) the role of the DP IDR as a stress sensor, c) how DP cooperates with desmosomal cadherins to promote actin remodeling, and d) the role of DP in stratification; 2. Determine the contribution of the desmoplakin-intermediate filament complex to late epidermal morphogenesis and the tight junction barrier. We hypothesize that DP recruits Rho Guanine nucleotide exchange factors (GEFs) and other maturation factors including phosphatases to locally activate RhoA and strengthen DP-IF interactions, which in turn promotes high cortical tension and tight junction assembly in superficial layers of the epidermis. To test this we will use genetically or pharmacologically targeted epidermal cultures, mouse and human disease models of DP dysfunction to: a) evaluate Rho localization/activity, cell mechanics and TJ structure/function in RhoGEF-deficient or DP mutant conditions with or without local restoration of tension via optogenetic activation of RhoA, and b) evaluate the role of phosphatases (PP)1/2A and associated DP post-translational modifications on cell mechanics and TJs. The proposed work will shed light on how alterations in DP-IF protein networks tailor the cell cortex in each layer to support early and late events in epidermal differentiation, and how interfering with this network contributes to cardiocutaneous disorders.
|Effective start/end date||6/1/21 → 5/31/26|
- National Institute of Arthritis and Musculoskeletal and Skin Diseases (2R01AR043380-26)