Title: Protein assemblies as genetically encoded mechanical actuators for intracellular mechanobiology research Project Summary Cells are continuously subjected to mechanical cues that regulate diverse biochemical and biophysical processes. In the rapidly growing field of mechanobiology, various methods, including but not limited to substrate engineering, optical/magnetic tweezers, atomic force microscopy, pipette aspiration, and microfluidics, have been developed and exploited for mechanical manipulation of live cells. Though with grand success, these paradigms primarily apply mechanical forces at the cellular surface, while direct intracellular perturbation remains underexplored. The limited capability of intracellular force exertion impedes in-depth investigation of critical fundamental questions such as how forces are translated inside the cells and regulate the output functions. Therefore, we plan to fill the technological gap by developing a toolbox of genetically encoded peptides/proteins as intracellular mechanical actuators. We will rationally design and engineer peptides/proteins that can spontaneously form in-cellulo nanoscopic or microscopic assemblies with various sizes, shapes, surface chemistries, and mechanical properties to mimic intracellular mechanical milieu. The corresponding biological responses of cells will be probed by molecular sensors and optical microscopy. With the assistance of numerical simulation, the mechanical interactions between protein assemblies and subcellular structures will be recapitulated and correlated to the change of biological processes. The tools, once developed, will be exploited to study mechanoresponses of membrane receptors and cell nuclei. The genetically encoded mechanical actuators can afford several distinct advantages: 1) It allows direct, chronic, and precise exertion of force intracellularly; 2) A large number of cells can be transformed and characterized simultaneously, enabling high-throughput probing and analysis; 3) The genetic delivery of the tool and the contact-free perturbation makes it readily applicable to more complex and physiologically relevant biological systems, such as organoids, ex-vivo tissues, and even the live animal models. We believe the new genetically encodable tools can shift the paradigm for intracellular mechanobiology research and help advance our understanding of organelle mechanosensing,