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While the mechanisms by which chemical signals control cell fate have been well studied, the impact of mechanical inputs on cell fate decisions is not well understood. Here, using the well-defined system of keratinocyte differentiation in the skin, we examine whether and how direct force transmission to the nucleus regulates epidermal cell fate. Using a molecular biosensor, we find that tension on the nucleus through linker of nucleoskeleton and cytoskeleton (LINC) complexes requires integrin engagement in undifferentiated epidermal stem cells and is released during differentiation concomitant with decreased tension on A-type lamins. LINC complex ablation in mice reveals that LINC complexes are required to repress epidermal differentiation in vivo and in vitro and influence accessibility of epidermal differentiation genes, suggesting that force transduction from engaged integrins to the nucleus plays a role in maintaining keratinocyte progenitors. This work reveals a direct mechanotransduction pathway capable of relaying adhesion-specific signals to regulate cell fate.

Desmosomes are adhesive complexes found at sites of intercellular contact that are essential for mediating cell-cell adhesion. These junctions undergo regulated assembly and reorganization during processes such as embryogenesis and wound healing. Plakophilins (PKPs) are armadillo family members related to the classic cadherin-associated protein p120ctn. PKPs localize to the cytoplasmic plaque of intercellular junctions and participate in linking the intermediate filament (IF)-binding protein desmoplakin (DP) to desmosomal cadherins. During desmosome assembly PKP2 associates with DP in plaque precursors that form in the cytoplasm in response to cell-cell contact, which translocate to nascent desmosomes. Here we provide evidence that PKP2 governs DP assembly dynamics by scaffolding a DP-PKP2-PKCá complex, which is disrupted by PKP2 knockdown. The behavior of a phosphorylation-deficient DP mutant that associates more tightly with IF is mimicked by PKP2- and PKCα knockdown and PKC pharmacological inhibition, all of which impair junction assembly. siRNA-mediated PKP2 knockdown is accompanied by increased phosphorylation of PKC substrates, raising the possibility that global alterations in PKC signaling may contribute to pathogenesis of congenital defects caused by PKP2 deficiency. An intact actin cytoskeleton is required for the efficient assembly of these plaque precursors, which closely associate with both actin and IF during assembly. However, the molecular mechanism underlying actin involvement in the assembly of desmosomes is not well understood. Here we demonstrate that PKP2 directs myosin-dependent actin contractility and actin reorganization. Cells deficient in PKP2 exhibit constitutive myosin activation accompanied by blunted Rho GTPase membrane localization and activation, and the inability to properly reorganize the actin cytoskeleton. These studies demonstrate that Rho activity is required early during junction assembly but must be downregulated later in order for junction maturation to occur. Taken together, our results suggest that PKP2 regulates actin contractility signaling in a temporal and spatially restricted manner that tailors these activities to desmosome assembly-specific events. The two major desmosome compartments (membrane and plaque) undergo distinct assembly pathways. How PKPs are involved in the coordination of these two pathways is not clear. Here, I show a dramatic defect in desmosomal cadherin assembly (but not adherens junction assembly) in cells deficient for PKP2. Further, siRNA-mediated PKP2 knockdown led to an enhancement in the colocalization of desmosomal plaque component, DP, with desmosomal cadherins and other arm proteins. Taken together these data suggest that PKP2 is required for efficient desmosome assembly. From this work, I conclude that PKP2 plays an important role in the coordination of desmosomal cadherin and plaque components and in the regulation of signaling pathways important for temporal and spatial control of actin reorganization and DP-IF interactions during desmosome assembly.

Abstract While the mechanisms by which chemical signals control cell fate have been well studied, how mechanical inputs impact cell fate decisions are not well understood. Here, using the well-defined system of keratinocyte differentiation in the skin, we examine whether and how direct force transmission to the nucleus regulates epidermal cell fate. Using a molecular biosensor, we find that tension on the nucleus through Linker of Nucleoskeleton and Cytoskeleton (LINC) complexes requires integrin engagement in undifferentiated epidermal stem cells, and is released during differentiation concomitant with decreased tension on A-type lamins. LINC complex ablation in mice reveals that LINC complexes are required to repress epidermal differentiation in vivo and in vitro and influence accessibility of epidermal differentiation genes, suggesting that force transduction from engaged integrins to the nucleus plays a role in maintaining keratinocyte progenitors. This work reveals a direct mechanotransduction pathway capable of relaying adhesion-specific signals to regulate cell fate. Competing Interest Statement The authors have declared no competing interest. Footnotes * This revised manuscript includes three major additions: 1) further characterization of the N2G-JM-TSMod LINC complex tension sensor (Fig. 1); 2) ATAC-seq analysis demonstrating precocious accessibility of epidermal differentiation genes in the absence of LINC complexes (Fig. 5) and 3) use of mouse keratinocytes lacking beta-1 integrin to probe the requirement for cell-ECM engagement to both drive high tension on the LINC complex (Fig. 1) and to repress epidermal differentiation (Fig. 4). Supplementary files for bioinformatic analysis and the links to the raw sequencing data are also provided. * https://www.ncbi.nlm.nih.gov/bioproject/PRJNA636991