Project Summary Our abilities to engineer synthetic cell systems that can communicate with living cells remain limited. The long-term goal is to engineer cell-like systems with increasingly complex biomimetic functions that can serve as cell replacement or augment functions of natural cells. The objective of this proposal is to develop a mechanosensitive synthetic cell that can respond to an increase in shear stress, which is most prevalent in the cardiovascular system, and secrete bioactive molecules to effect living cells. Cells in our bodies constantly sense and respond to microenvironmental stimuli, including passive and active physical stimuli, such as extracellular matrix rigidity, adhesive ligand density, tension, compression, and fluid shear flow. The rationale underlying this proposal is that completion will result in a novel biomimetic cell-like system as a novel shear stress-responsive ‘material’ that can interface with natural living cells. The majority of engineered biomaterials respond to differences in the biochemical environment (e.g. differences in redox, pH, and enzyme composition) between normal and diseased tissues. By comparison, there has been relatively less effort in exploiting forces for stimulus-responsive behaviors. The synthetic cell idea is inspired by natural platelets’ ability to bind and respond to elevated shear stress and secrete granule contents when bound to a surface. The proposed work consists of three specific aims: 1) Characterize shear stress response of mechanosensing vesicles, 2) Couple mechanosensing with exocytosis in synthetic cells, 3) Test intercellular communication of shear stress-activated synthetic cells with endothelial cells in vitro. We will pursue these aims using an innovative approach of repurposing mechanosensitive channel for shear stress sensing and using peptide-based membrane fusion. Our lab was the first group to demonstrate mechanosensing synthetic cells and we have significant expertise in bottom-up synthetic biology. The proposed research is significant, because it will be the first synthetic cell system developed to communicate with mammalian cells using calcium-triggered secretion. The work will develop fundamental strategies for coupling mechanosensing to a biochemical response in synthetic cells. This will open new avenue for other researchers interested in developing more complex cell-like systems. The results will have an important positive impact immediately because it will support the idea that mechanosensitive channels can sense lateral membrane tension due to shear stress and long-term because they lay the groundwork of engineering synthetic cells with other sensing abilities.