Exploring the Canonical and Non-Canonical Functions of Desmosomes

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2023

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Abstract

Desmosomes are cell-cell adhesion complexes that provide tissues with mechanical integrity and their disruption results in severe blistering in the epidermis as well as cardiomyopathies. In this work, we sought to explore the various ways through which desmosomes function in keratinocytes to maintain connections between cells and promote cell sheet cohesion. Our approach examined both their canonical interactions with the intermediate filament cytoskeleton and a novel noncanonical association with RNA-induced silencing complexes.Keratin intermediate filaments form dynamic polymer networks that organize in specific ways dependent on the cell type, the stage of the cell cycle, and the state of the cell. In differentiated cells of the epidermis, they are organized by desmosomes, which provide essential mechanical integrity to this tissue. Despite this, we know little about how keratin organization is controlled and whether desmosomes locally regulate keratin dynamics or merely bind preassembled filaments. Ndel1 is a desmosome-associated protein in the differentiated epidermis that was previously described as being able to promote the assemble of neurofilaments, another class of intermediate filaments, although its function at desmosomes has not been examined. Here, we show that Ndel1 binds directly to keratin subunits through a motif conserved in all intermediate filament proteins. Further, Ndel1 was necessary for robust desmosome-keratin association and was sufficient to reorganize keratins at distinct cellular sites. Lis1, a Ndel1 binding protein, was required for desmosomal localization of Ndel1, but not for its effects on keratin filaments. Finally, we use mouse genetics to demonstrate that loss of Ndel1 results in desmosome defects in the epidermis. Our data thus identify Ndel1 as a desmosome-associated protein that promotes local assembly/reorganization of keratin filaments and is essential for robust desmosome formation. In addition to their canonical adhesive function, desmosomes have many emerging non-canonical functions such as regulating diverse signaling pathways and integrating diverse cytoskeletal networks in keratinocytes. Here we reveal a surprising recruitment of the RNA-induced silencing complex (RISC) to the cell cortex of keratinocytes. Using mutant cell lines, we find that desmosomes, but not adherens junctions, are required for the cortical localization of RISCs. We then identified the RISC-associated mRNAs specifically localized to the cortex using CLEAR-CLIP analysis which showed an enrichment for genes involved in wound healing and cell adhesion. Wound healing assays conducted both in vitro and in vivo revealed that RISCs become depleted from the cell cortex of keratinocytes near the site of injury. Further, perturbation of desmosomal adhesion using antibodies that target the desmosomes, which recapitulates the autoimmune disease pemphigus vulgaris, results in a similar loss of cortical Ago2. Our results define a new function for desmosomes in translational regulation and suggest that they may act as sensors of adhesion integrity which can directly influence the cell’s translatome to quickly restore homeostasis when it is disrupted. Although it is well established that desmosomes are important for the mechanical integrity of some tissues, it is only recently that we have come to understand the diversity of cellular processes they coordinate. Here we propose a mechanism through which desmosomes play a more active role in the organization of intermediate filaments than previously thought. Additionally, we demonstrate that they are involved in translational regulation via recruitment of RISCs and show that this population dynamically responds upon injury. Future investigations can further elucidate the role of the RISCs during wounding by probing the changes to the proteome shortly after wounding to determine the contributions of RISC-bound mRNA to recovery.

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Hlavaty, Daniel Clark (2023). Exploring the Canonical and Non-Canonical Functions of Desmosomes. Dissertation, Duke University. Retrieved from https://hdl.handle.net/10161/29145.

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