PROJECT SUMMARY The goal of this proposal is to provide a mechanistic understanding of how cell division in bacteria is controlled at a molecular level. Understanding these mechanisms, which are broadly conserved among the bacteria, is important because it can reveal potential targets for new antibacterial agents that inhibit cell division and stop cell propagation. The division process in bacteria involves two stages. In the first distinct step, FtsZ protofilament assembly, the Z-ring, forms. In Escherichia coli, the model organism for this study, the first step occurs early in the cell cycle. Only after a significant delay, which can last half of the cell cycle, does the second stage of cell division start. In this later step, septal peptidoglycan synthesis begins, and the cell constricts. In this stage, more than two dozen proteins are involved, most of them in a complex referred to as the divisome. It is not yet understood what determines the onset of either the first or the second stage of the division, both of which are critical for the cell's survival. This significant gap in our knowledge exists even though many proteins involved in cell division are known and their pairwise binding interactions mapped out. The difficulty in understanding processes controlling cell division arises from the presence of a large number of different interactions within the divisome and of many pathways that are partially redundant. Moreover, the protein assemblies, which in most studies are viewed as static, are highly dynamic, turning over in a matter of seconds in an energy-dependent process such as treadmilling. The complexity of the problem requires not only further experiments but the integration of the existing experimental results into a comprehensive modeling framework. Accordingly, we combine state-of-the-art experimental methods with stochastic cell cycle simulations and 3D modeling of assembly reactions of proteins involved in cell division. On the latter front, we leverage our previous work and ongoing collaborations. In the experimental work, we use molecular biology and genetic methods alongside high throughput and super-resolution microscopy, and we develop novel microfluidic devices for this research. These techniques have already generated large amounts of information-rich data that, among other findings, have shed new light on processes leading to the assembly of Z-ring from individual FtsZ protofilaments and determining the role of DNA replication over the control of the progression of the second stage of the division. The proposed work aims to consolidate these past findings into a single mechanistic framework. The knowledge gained will enhance our understanding of fundamental cellular processes in bacteria and provide a basis for designing effective antibacterial therapies that target bacterial cell division.