PROJECT SUMMARY 1R01GM149073 PARENT GRANT Cells perform diverse processes, such as cell division, growth, motility, formation of adhesions, and tissue morphogenesis, under a wide range of mechanical environments. Central to these processes are mechanical forces, which may come from outside the cell or be generated internally and which are integrated with signaling pathways to guide the cellular process. The cell’s macromolecular cytoskeletal machinery, including the actin- based myosin II motors and actin crosslinking proteins, assemble, function and then disassemble in response to these forces and signaling pathways. This dynamic force-responsive assembly provides self-tuning of the machinery, leading to natural positive and negative feedback and further allows mechanical inputs to be converted into signaling outputs. Using Dictyostelium cells, we discovered that many of these components are pre-assembled in the cytoplasm in the form of mechanoresponsive Contractility Kits (CKs), which allow for highly efficient responses to force inputs. The CKs include myosin II, cortexillin I, IQGAP1, IQGAP2, plus several other proteins that we know of. For this application, substantial published and unpublished data motivate the questions to be answered, and our work extends from Dictyostelium to human proteins and model systems. We begin by leveraging our suite of experimental and modeling platforms, including a new modeling framework called SpringSaLaD, which allows for molecularly motivated, particle-based, stochastic simulations of biochemical processes. Using SpringSaLaD, we are modeling the formation of CKs by drawing upon measured in vivo concentrations, diffusion constants, and in vivo “apparent” KDs. From this model, we have made an initial list of predictions about the features of the CKs, which we will test in Dictyostelium. We will also explore the kinetics of assembly and disassembly of the CKs with and without mechanical force. For assembly, we will determine the molecular basis for force-dependent assembly of the CKs and nonmuscle myosin II bipolar thick filament (BTF), using interference scattering mass spectrometry. For disassembly, we will use magnetic tweezers to measure the compliance within the BTF and then determine how this compliance restricts the activity of the myosin heavy chain kinase (MHCKC for Dictyostelium and PKCzeta for NMIIB). We have also found that the setpoint of mechanosensitive accumulation (mechanoaccumulation) by Dictyostelium myosin II and human NMIIB has an optimum of 20% assembly fraction. Further, NMIIB’s setpoint is cell type- and cell cycle stage-specific. We will use the framework we have established to determine the consequences of setpoint positioning on cell behavior, including NMIIB dynamics, cell division, and gene expression. We will incorporate this information into our computational models for myosin II mechanoaccumulation, expanding the models to include the components of the CKs. In sum, this research...