Project Summary The nuclear envelope compartmentalizes genomic and cytoplasmic activities. It also originates a group of lipid- derived paracrine signals collectively called eicosanoids. Eicosanoids, such as leukotrienes and prostaglandins, are essential lipid mediators of leukocyte recruitment to infection and tissue damage. Too little eicosanoid sig- naling causes infection susceptibility, too much can inflict inflammatory bystander injury onto already damaged organs, and lead to vicious auto-inflammatory circles, for example, during asthma, Crohn’s disease, ischemia, and cystic fibrosis. Thus, it is of utmost medical importance to understand and therapeutically control this path- way. To initiate eicosanoid synthesis, cytosolic phospholipase A2 (cPLA2) attaches to the inner or outer nuclear membrane where it cleaves arachidonic acid, the common fatty acid precursor of all eicosanoids. For long, cPLA2 was thought to be exclusively regulated by chemical signals, such as calcium ions, and protein phosphorylation, and the reasons for cPLA2’s nuclear localization remained enigmatic. However, our recent research suggests an unexpected role for nuclear membrane stretch as novel, key regulator of cPLA2 and the eicosanoid cascade in zebrafish, and human cells. I hypothesize that nuclear biomechanics control innate immune responses through stretch-sensitive nuclear membrane interactions of inflammatory enzymes, such as cPLA2, 5-lipoxygenase, and likely others. We will test this hypothesis with an integrated, multilevel approach that ranges from in vitro recon- stitution to whole animal experiments. First, we will dissect the mechanism of stretch sensing by cPLA2 and related enzymes by combining microscopic, biophysical, and biochemical assays on artificial bilayers, isolated nuclei, and intact cells. Second, we will investigate the cytoskeletal regulation of nuclear membrane mecha- notransduction in various cell culture models of biomechanical stress, as well as by intravital imaging in a zebrafish infection model. Our work sets out to establish a novel paradigm of nuclear function and cellular mech- anosensing that can be targeted for therapeutic benefit in the future.