Project Summary Cell wall constriction, the synthesis of new cell wall and the splitting of the old cell wall, is an additional step during cell division that occurs in walled bacteria. The synthesis and degradation of the cell wall, made of peptidoglycan (PG), must be highly coordinated because mis-regulation can result in fatal lysis of the cell. The divisome, a macromolecular complex of over 30 proteins, is responsible for this extremely coordinated process. FtsZ, a tubulin homolog and GTPase, is an essential divisome protein that begins cell division by polymerizing into a ring-like structure (Z-ring) at the midcell. This Z-ring acts as a scaffold (known as the Z-track) for other divisome proteins including the septal PG (sPG) synthase complex, FtsWI, and regulators FtsN and FtsQLB. Divisome proteins can exit the Z-track onto a second track (sPG-track) where sPG synthesis occurs. This application proposes to study the role of the FtsQLB complex in regulating the activity of FtsWI in with high spatiotemporal resolution during cell wall constriction in E. coli. Aim 1 uses single molecule tracking (SMT) and three-dimensional (3D) superresolution imaging to investigate how FtsQLB is coupled to the Z-track. I will examine how FtsQLB responds to altered FtsZ dynamics. Aim 2 focuses on the modulation of the sPG synthesis activity of FtsWI. Using the same SMT and imaging approaches I will determine if FtsQLB is coupled with FtsWI on the sPG track. Finally, Aim 3 uses superfission and dominant negative mutants to dissect the roles of key protein-protein interactions of FtsQLB, FtsN, and FtsWI. The use of single-molecule live cell imaging combined with perturbational analysis will enable me to identify molecular determinants responsible for cell wall constriction, providing insight into the spatial and temporal regulation of bacterial cell division. Due to the highly conserved nature of the bacterial cell wall, mechanistic insights learned from this study can be applicable to a wide range of bacterial species to facilitate the development of antimicrobial drugs.