Summary There is an increasing appreciation that mechanical signals play key roles in tissue homeostasis. This project seeks to define the cellular and molecular mechanisms by which force transmission to the nucleus through Linker of Nucleoskeleton and Cytoskeleton (LINC) complexes regulates differentiation using skin as a model system, yielding insights into fundamental mechanisms in cell biology and mechanobiology. Central to this proposal is the need for an imaging technology that is capable of 1) resolving specifically labeled nuclear features (e.g. a genomic locus, specific histone modification, or proteins of interest), 2) in the context of the surrounding nuclear morphology, 3) at a resolution approaching the size of nucleosomes (~10 nanometers). However, as no readily accessible microscopy method currently meets all of these requirements, there is an urgent need for new imaging techniques that allow researchers to visualize specific structures of interest in the unbiased nanoscale context of the surrounding nuclear organization. We recently developed pan-Expansion Microcopy (pan-ExM), which provides optical contrast equivalent to EM heavy-metal stains using standard confocal microscopy but remains fully compatible with molecular techniques such as immunolabeling. In this project, we will 1), validate structural preservation of the expanded nucleus in the epidermis when using the pan-ExM protocol by investigating the integrity of the DNA, folding of the genome and nucleosome structure; and 2), apply pan-ExM to investigate how LINC complex ablation impacts nuclear remodeling during epidermal differentiation. For the latter, we will test how the folding and compartmentalization of the “epidermal differentiation complex” (EDC), a specialized region of the genome housing over 60 epidermal differentiation genes, are altered in the absence of LINC complexes, and explore how key chromatin factors in epidermal differentiation are recruited to (or removed from) the EDC during epidermal differentiation. Our project will have high impact in two aspects: it will provide answers to the fundamental question of how mechanical cues can be translated to alter gene expression in the epidermis, thereby laying the groundwork for a larger research program. Second, pan-ExM represents a novel tool that will accelerate research, particularly by dissemination of protocols validated in the epidermis to any interested research lab, thereby providing a much-needed research tool that will allow researchers to systematically investigate the connection between chromatin structure and function both in normal epidermal differentiation and also in disease contexts.