PROJECT SUMMARY/ABSTRACT Despite recent progresses in mechanobiology research, there is still a fundamental knowledge gap in critical cellular functions and molecular mechanisms, which take place between the molecular and cellular scale. For systematic, quantitative and deterministic studies, there are key challenges to achieve single-molecule precision, heteromolecular control, simultaneous and independent modulation of multiple physicochemical cues. Based on our unique approach combining top-down nanotechnology and bottom-up synthetic biology, we will overcome these challenges and create “smart” cell interfaces which could probe, sense and manipulate cells and the microenvironment, in an unparalleled precise and controllable manner. The overarching goal of my laboratory is to bring molecular insights into cell biology, and advance the knowledge of cell as a machine, whose behaviors and functions can be predicted and directed. Based on our discoveries in geometric underpinning of molecular mechanobiology, we will address three outstanding questions: (i) What underlies the spatial effects in receptor clustering, and how are they force- mediated? (ii) Are there heteromolecular spatial effects in co-clustering and segregation of different receptors, and what are their roles in signaling crosstalk and integration? (iii) How to use the new knowledge in molecular mechanisms and effectively direct cell functions? Considering the interplay and crosstalk between multiple receptors and physicochemical cues, we will reveal the underlying synergistic mechanisms of heteromolecular mechanobiology for cell-extracellular matrix (ECM) signaling (fibroblast as a model) and cell-cell signaling (T cell as a model). Specifically, for cell-ECM signaling, we will investigate the integrin clustering mechanism, its relationship with cellular contractility, and the crosstalk between growth factor receptors and integrins. For cell- cell juxtacrine signaling, we will investigate the synergistic mechanism of mechanosensing and spatial sensing in T cell receptor clustering and triggering, and the heteromolecular co-clustering mechanism between activatory and inhibitory receptors. Finally, the artificial ECM and artificial cell surfaces we developed in this research will be used to direct critical cell functions for in vitro cell engineering and manufacturing, as well as potential in vivo applications. Overall, our technology will break the limit of mechanobiology study with a single type of bioligands on 2D, rigid, static surfaces, provide heteromolecular control over multiple ligands in a 3D, soft, dynamic fashion, and therefore better mimic the complex in vivo environment. This transformative methodology can be customized and adopted in distinct systems to expand our understanding of fundamental molecular and cellular mechanisms, and apply the new knowledge for novel diagnostic and therapeutic methods.