Project summary The unfolded protein response (UPR) is a critically important signaling network that is responsible for maintaining the health of the endoplasmic reticulum (ER). While the typical outcome of UPR activation is cytoprotective, prolonged or excessive UPR activity can drive cells towards apoptotic death. The UPR serves as a potent controller of cell fate, and its dysregulation is known to be implicated in a broad range of human diseases such as diabetes, neurodegeneration, autoimmune disorders, and cancer. The UPR comprises three interconnected branches that exhibit spatiotemporally distinct patterns of activation both in normal development and in disease. A lack of understanding of how the UPR is differentially regulated in different cell types and tissues has so far precluded it from being successfully targeted in human patients. The best-studied branch of the UPR is mediated by the ER membrane-resident bifunctional kinase-RNase IRE1 (inositol-requiring enzyme 1). Recent work demonstrated that IRE1 signaling is closely tied to adhesion, cell migration, and cells’ ability to receive and respond to external cues. Cell signaling is often coupled to mechanical and chemical changes in the local microenvironment, but the involvement of IRE1 and the UPR in this coupling is only beginning to be revealed. Since UPR components are ubiquitously expressed in nearly all cell types, context-dependent regulation offers an attractive potential explanation for the observed large variances in UPR signaling across tissues. However, it remains to be determined what properties of the local environment cause cells to rely on UPR signaling and how this information is communicated. I propose to build on the tools I developed as a postdoc and on the unique combined resources of my two co-mentors to answer these challenging and exciting questions. I will engineer precisely defined growth substrates of varying chemical composition, porosity, stiffness, and 3-dimensional organization. I will then use chemical inhibitors and optogenetic activators of IRE1 to identify which substrate properties render cells reliant on IRE1 signaling and which properties render IRE1 dispensable. Substrate dependence of IRE1 signaling will be functionally separated from general UPR activation and mapped to specific nodes within the UPR. Finally, a targeted approach will identify the specific molecular players responsible for the information flow between ER stress sensors and the extracellular environment. Throughout the mentored phase of the award, I will continue to hone my skills and qualifications as an independent scientist. Working closely with the lab of Dr. Valerie Weaver will provide me with the expertise in cellular mechanobiology and substrate engineering, complementing the deep background of my primary mentor, Dr. Peter Walter, in UPR signaling. Learning how information flows between the ER and the extracellular matrix will reveal exciting new cell biology, result i...