Project Summary Homeostasis is a fundamental feature of physiological control in all cells. In the past five years, a new homeostatic principle co-discovered by us has been changing our view of cell-size control. This principle, known as the “adder”, states that cells add fixed size between birth and division irrespective of the cell size at birth. The adder principle is therefore distinct from many biological controls due to its passive nature, as the adder-like cells do not employ any apparent size sensing or feedback mechanisms to trigger division when they reach a fixed critical size. The proposed program is an extension of our success in cell-size control research to understand a broader class of physiological controls. First, we will study the mechanisms responsible for the precision and robustness of physiological processes. We will focus on replication initiation in bacteria as it can serve as a tractable model to solve these long- standing problems. Another important aspect of physiological control is how cells allocate their resources to growth. The current paradigm based on E. coli is that cells balance supply and demand of amino acids to maximize growth rate under all growth conditions. These models are important because they have been able to explain the tradeoff between production of cellular energy vs. production of proteins that is pertinent to cancer. However, we have obtained experimental results in B. subtilis that directly challenged this E. coli centric view of growth control. We will thus seek more general principles of cellular resource allocation that encompass both E. coli and B. subtilis, and ideally beyond bacteria. Finally, we will extend our previous work on cell-size control to investigate how cells ensure physiological equilibrium when proteins and organelles partition asymmetrically, which is important in the context of inheritance and cellular aging. These questions require multidisciplinary approaches from physiology to development of novel technologies. To this end, we will work with collaborators who are leaders in their research fields. Furthermore, over a decade we have been making major efforts to democratize technologies to the research community. We expect the knowledge and technology that will be generated from our proposed research to open exciting new research avenues and facilitate other important discoveries in physiology and cell biology.