PROJECT SUMMARY A my scale splitting middle) and use studies, cytoskeletal protein variants coli conformation conformational genetics, protein result, interact phase to interactions, subtilis cell oligomeric the and these provide city-cell's cell is like a city, with an organized yet dynamic infrastructure grouped into specialties. For nearly 30 years, lab has investigated how the simplest cells — bacteria — organize themselves at the cellular and molecular to divide and proliferate. We mainly f ocus on how bacteria such as E. coli achieve the daunting task of themselves in two at the right time (once their genetic material is duplicated) and place (exactly in the every 20 minutes. The keys to this s uccess are ancient versions of protein polymers of actin (FtsA) tubulin (FtsZ), which our lab visualized for the first time in living bacteria over 25 years ago. Today, we state of the art super-resolution imaging along with molecular genetics, protein biochemistry, interaction and in vitro reconstitution, to gain more detailed insights into the structure and regulation of these polymers and their associated proteins, which comprise the dynamic membrane-associated nanomachine (the divisome) that divides bacterial cells. Our early characterization of hypermorphic of FtsA and FtsZ paved the way for a model, now strongly supported by numerous studies, that the E. divisome initially assembles in an inactive form, then becomes activated mainly by changes in protein and oligomeric state. Nonetheless, many f t he mechanisms that trigger and respond to these and oligomeric changes remain unclear. In the last grant period, we used our expertise in cellular imaging, and in vivo crosslinking to discover novel and physiologically significant protein- interactions among essential divisome proteins and develop new tools to study their function. As a we overturned several long-established assumptions about how FtsA, FtsZ and t heir binding partners and function. In addition, in collaborative studies we explored the ability of FtsZ to form l iquid-liquid condensates, which have the potential to transform how we think about the divisome and its response stress. In this proposal, we plan to continue our investigation of the cellular implications of molecular-scale using the E. coli divisome as the model but also expanding our studies of the divisome of Bacillus , the predominant Gram-positive model. We will address the most pressing questions about bacterial division, which include how protein-protein interactions and switches in protein conformation and tate sequentially trigger setup and assembly of the divisome; (2) how a third stage, synthesis of division eptum, is licensed and activated; (3) how the divisome interfaces with chromosome segregation cell wall synthesis. As we have done extensively in the past and in the last grant period, we will leverage approaches with collaborations. Our ongoing investigation of how the simplest ells divide continues to greater...