In Vivo Structure of the E. coli FtsZ-ring Revealed by Photoactivated Localization Microscopy (PALM)

Abstract

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.

In the Beginning, Escherichia coli Assembled the Proto-ring: An Initial Phase of Division

“…FtsZ ring images have been obtained from C. crescentus by electron cryotomography and by two-and three-dimensional super-resolution imaging and from E. coli using photoactivated localization microscopy. In both cases, they are compatible with a scattered arrangement of short filaments that accumulate within a limited space, forming a loose ring assembly (37)(38)(39).…”

Evidence That Bacteriophage λ Kil Peptide Inhibits Bacterial Cell Division by Disrupting FtsZ Protofilaments and Sequestering Protein Subunits

“…Although the average number of FtsZ subunits per protofilament varies with buffer composition (9), protofilaments are always too short to encompass the entire 3-4-m circumference of a rod-shaped bacterium. Hence, these basic structural units must further assemble into higher order structures to form the observed Z-ring in vivo (10,11). Indeed, protofilaments can be arranged in vitro into several higher order structures involving lateral interactions in the presence of crowding agents (7,12), calcium ions (13), or certain buffer conditions (14).…”

Extreme C Terminus of Bacterial Cytoskeletal Protein FtsZ Plays Fundamental Role in Assembly Independent of Modulatory Proteins

“…Cryo-EM tomography indicates that the FtsZ ring is made up of short filaments (ϳ100 nm) concentrated at the division site (9). Photoactivated localization microscopy suggests that FtsZ protofilaments are arranged around midcell in a randomly oriented loose bundle (10). Lateral interactions are mediated in part through the actions of FtsZ modulatory proteins, including the widely conserved ZapA protein, as well as ZipA and SepF, which are limited to the ␥ group of Gram-negative bacteria and the Firmicutes, respectively (11).…”