PROJECT ABSTRACT Our proposal will examine a central mechanism of cell death pathways—the formation of large membrane pores during the final stages of cell death. Recent studies have demonstrated that this process involves Ninjurin1 (Ninj1), a membrane-spanning protein that multimerizes to form large membrane pores that lead to membrane rupture. These membrane ruptures are essential for advanced signs of cell death and for the release of larger inflammatory molecules required to mount an effective response to pathogens. The multimerization of Ninj1 is critical for membrane rupture, but little is known about the specific mechanisms underlying Ninj1 multimer formation or the direct implications of this oligomerization. This proposal will determine how Ninj1 is activated by upstream steps of the cell death pathway, including caspase-8 and caspase-1-driven signaling, and how gasdermin family members regulate this process. We will also examine the contribution of Ninj1 activation in cell death pathways in the innate immune response to pathogens using mouse models of Yersinia infection. Our preliminary experiments demonstrated that Ninj1 expressed in myeloid cells is required for the host response to Yersinia bacteria. We also showed that Ninj1 controls myeloid-cell driven defenses to Yersinia infection in mice, underscoring the importance of Ninj1 in the innate immune response to bacterial infections. Furthermore, our data suggest that GSDMD has a regulatory role in Ninj1 oligomerization initiated via both caspase-8 and caspase-1. We hypothesize that GSDM proteins are modulating Ninj1 oligomerization and host responses to caspase-8 and caspase-1 stimulations, and that Ninj1 oligomerization is a critical component in effector-mediated immunity to Yersinia infections. Using Yersinia infections, cytokines, inhibitors, expression systems and other tools, we will define the pathway controlling Ninj1 oligomerization during innate immune signaling to cell death and inflammation to elucidate how this central step in multiple cell death pathways is regulated.