PROJECT SUMMARY Cytokinesis, the division of a cell into two daughter cells, serves as an elegant cell behavior that highlights the biomechanical systems required for many cell shape change processes. Over the life of this grant, we have demonstrated how an interplay of active force production, cortical tension, surface curvature, and viscoelasticity drive cytokinesis furrow ingression. We identified key molecular pathways that control these properties and found that the circuitry is wired like a control system complete with feedback loops that allows mechanical and chemical signals to tune the accumulation of the contractile machinery. Finally, we have applied these concepts to other systems, such as myoblast fusion, entosis, hepatocyte mechanics, pancreatic cancer, and lung biology, demonstrating the power of using a model organism (Dictyostelium) and a model process (cytokinesis) as a concept generator for more complex systems. In this proposal, we continue to build upon our understanding of cytokinesis and the mechanosensitive contractile network by developing a biochemically grounded map of the protein interactions that constitute this network (Aim 1) and discern the functional roles of five unusual suspects in cell shape control (Aim 2). In Aim 1, we will map the biochemical interactions revealed through a combination of proteomics and genetic analyses using several state-of-the-art methodologies. We will use fluorescence cross-correlation spectroscopy to measure cellular concentrations, complex sizes (reflected in diffusion coefficients), and the strengths of the biochemical interactions (`in vivo Kd'). Using Single Molecule Pulldown, we will measure the stoichiometry of each component. Finally, using a combination of Structured Illumination Microscopy, Lattice Light Sheet Microscopy, and confocal imaging, we will develop a more complete picture of the sub-cellular distribution and architecture of the system. These studies will then give us a physical biochemical interaction map of the contractile network. In Aim 2, we will pursue functional studies of five unexpected proteins implicated in the mechanosensory contractile system that have been revealed through two or more proteomics and/or genetics strategies. These proteins include adenine nucleotide translocase (AncA), methylmalonate semialdehyde dehydrogenase (Mmsdh), two ribonucleotide proteins (RNP1A and RNP1B), and discoidin complex. Each protein offers a unique entry point into deciphering new mechanisms of cell shape control. AncA provides an in-road into the interface between cell mechanics and metabolism. Mmsdh suggests a possible mode of regulation of contractility through propionylation. The RNP1s are predicted to be intrinsically disordered, but interact genetically and biochemically with one of the main nodes of the contractility network. Finally, the discoidin complex is a lectin, which may provide part of the anchoring complex that links the contractile network to the plasma...