Bacterial cytokinesis is accomplished by the essential ‘divisome’ machinery. The most widely conserved divisome component, FtsZ, is a tubulin homolog that polymerizes into the ‘FtsZ-ring’ (‘Z-ring’). Previous in vitro studies suggest that Z-ring contraction serves as a major constrictive force generator to limit the progression of cytokinesis. Here, we applied quantitative superresolution imaging to examine whether and how Z-ring contraction limits the rate of septum closure during cytokinesis in Escherichia coli cells. Surprisingly, septum closure rate was robust to substantial changes in all Z-ring properties proposed to be coupled to force generation: FtsZ’s GTPase activity, Z-ring density, and the timing of Z-ring assembly and disassembly. Instead, the rate was limited by the activity of an essential cell wall synthesis enzyme and further modulated by a physical divisome–chromosome coupling. These results challenge a Z-ring–centric view of bacterial cytokinesis and identify cell wall synthesis and chromosome segregation as limiting processes of cytokinesis.

The FtsZ protein, a tubulin-like GTPase, plays a pivotal role in prokaryotic cell division. In vivo it localizes to the midcell and assembles into a ring-like structure-the Z-ring. The Z-ring serves as an essential scaffold to recruit all other division proteins and generates contractile force for cytokinesis, but its supramolecular structure remains unknown. Electron microscopy (EM) has been unsuccessful in detecting the Z-ring due to the dense cytoplasm of bacterial cells, and conventional fluorescence light microscopy (FLM) has only provided images with limited spatial resolution (200–300 nm) due to the diffraction of light. Hence, given the small sizes of bacteria cells, identifying the in vivo structure of the Z-ring presents a substantial challenge. Here, we used photoactivated localization microscopy (PALM), a single molecule-based super-resolution imaging technique, to characterize the in vivo structure of the Z-ring in E. coli. We achieved a spatial resolution of ∼35 nm and discovered that in addition to the expected ring-like conformation, the Z-ring of E. coli adopts a novel compressed helical conformation with variable helical length and pitch. We measured the thickness of the Z-ring to be ∼110 nm and the packing density of FtsZ molecules inside the Z-ring to be greater than what is expected for a single-layered flat ribbon configuration. Our results strongly suggest that the Z-ring is composed of a loose bundle of FtsZ protofilaments that randomly overlap with each other in both longitudinal and radial directions of the cell. Our results provide significant insight into the spatial organization of the Z-ring and open the door for further investigations of structure-function relationships and cell cycle-dependent regulation of the Z-ring.

Summary In most bacterial cells, cell division is dependent on the polymerization of the FtsZ protein to form a ring-like structure (Z-ring) at the midcell. Despite its essential role, the molecular architecture of the Z-ring remains elusive. In this work we examine the roles of two FtsZ-associated proteins, ZapA and ZapB, in the assembly dynamics and structure of the Z-ring in E. coli cells. In cells deleted of zapA or zapB, we observed abnormal septa and highly dynamic FtsZ structures. While details of these FtsZ structures are difficult to discern under conventional fluorescence microscopy, single-molecule based superresolution imaging method Photoactivated Localization Microscopy (PALM) reveals that these FtsZ structures arise from disordered arrangements of FtsZ clusters. Quantitative analysis finds these clusters are larger and comprise more molecules than a single FtsZ protofilament, and likely represent a distinct polymeric species that is inherent to the assembly pathway of the Z-ring. Furthermore, we find these clusters are not due to the loss of ZapB-MatP interaction in ΔzapA and ΔzapB cells. Our results suggest that the main function of ZapA and ZapB in vivo may not be to promote the association of individual protofilaments but to align FtsZ clusters that consist of multiple FtsZ protofilaments.