SUMMARY The nuclear lamina is a compositionally complex structure that serves functions in chromatin organization, transcriptional regulation, genome protection and mechanotransduction. In cells and tissues, the nuclear lamina is mechanically integrated into the actin, microtubule, and intermediate filament cytoskeletons via nuclear envelope-spanning Linker of Nucleoskeleton and Cytoskeleton (LINC) complexes. Through these cytoskeletal connections, forces exerted on plasma membrane adhesions from either the extracellular matrix or from adjacent cells can be transmitted to the nuclear interior. In cells in which LINC complex function has been altered, investigators have observed correlative changes in gene regulation. However, to date it has been extremely challenging to decipher whether mechanical forces transmitted by the LINC complex, potentially through interactions at the nuclear lamina, directly influence specific genetic programs. Our published work and preliminary studies demonstrate that the LINC complex enables a critical crosstalk between cellular adhesions, the cytoskeleton, and components of the nuclear periphery. Most important for this proposal, we used a mouse model to reveal that A-type lamins and the LINC complex drive opposite effects on pro-fibrotic signaling, which is classically driven by SMAD-dependent signaling downstream of TGFβ. Taking these insights together with our global transcriptome studies, we suggest that tension exerted on the nuclear lamina by the LINC complex influences nuclear events necessary for SMAD gene targets to be properly regulated by TGFβ inputs (despite normal cytoplasmic events necessary to drive this signaling pathway). Building on this, here we propose three complementary Aims that will address both molecular mechanisms as well as physiological contexts in which these mechanisms play critical roles. First, we will take an unbiased approach to define how the LINC complex, in combination with substrate inputs from the extracellular matrix, influences the nuclear lamina interactome in situ, ultimately employing a cross-linking mass spectrometry approach. Second, we will investigate the mechanisms by which LINC complex ablation influences SMAD function in the nucleus, including the analysis of SMAD target binding, nuclear position of SMAD target genes, and the influence of integral inner nuclear membrane proteins on SMAD-dependent gene output. Lastly, we will test if (and how) LINC complex function intersects with that of A-type lamins in this TGFβ–SMAD-fibrotic axis using both in vitro and in vivo approaches, including mouse models of interstitial fibrosis of the myocardium and lung injury models of pulmonary fibrosis. Taken together, the Aims of this proposal will reveal both molecular mechanisms of mechanotransduction through the LINC complex while also placing this detailed understanding into its physiological and disease contexts.