Imaging FtsZ Rings In Vitro by Negative-Stain EM

Abstract

In prokaryotes, the major cytoskeleton protein involved in cell division is FtsZ, a homolog of tubulin. In vivo, light microscopy studies have shown that FtsZ forms a ring (Z ring) at midcell during cell division. However, the substructure of the Z ring remains a mystery. In vitro, FtsZ assembles into short, single-stranded protofilaments (pfs), but it is unknown how these 120 nm long pfs come together to form a ring that encompasses the ∼3000 nm circumference of the cell. One possibility is that they associate by lateral contacts to make ribbons of protofilaments. Another is that the pfs are not in direct contact but communicate via distortions of the membrane. We have developed a lipid tubule system that has enabled us to image the Z-ring substructure by negative-stain EM. Here we took advantage of the self-assembling lipid, DC 8,9, PC, which spontaneously forms 500 nm diameter tubules in solution. We added membrane-targeted FtsZ (mts-FtsZ), which has been shown to form Z rings inside or outside of tubular liposomes. The mts-FtsZ assembled characteristic Z rings imaged by fluorescence light microscopy. Negative-stain EM showed that these Z rings consist of ribbons of pfs, which were one-subunit thick. Fourier transform analysis of these images confirmed the close packing (∼5 nm) of individual pfs in the Z ring. Overall, the data suggest that lateral contacts are important for Z ring assembly. In addition, these results were similar for FtsZ from E. coli and M. tuberculosis, suggesting that lateral contacts are an intrinsic property of Z rings.