Negative-stain electron microscopy of inside-out FtsZ rings reconstituted on artificial membrane tubules show ribbons of protofilaments

Abstract

FtsZ, the primary cytoskeletal element of the Z ring, which constricts to divide bacteria, assembles into short, one-stranded filaments in vitro. These must be further assembled to make the Z ring in bacteria. Conventional electron microscopy (EM) has failed to image the Z ring or resolve its substructure. Here we describe a procedure that enabled us to image reconstructed, inside-out FtsZ rings by negative-stain EM, revealing the arrangement of filaments. We took advantage of a unique lipid that spontaneously forms 500 nm diameter tubules in solution. We optimized conditions for Z-ring assembly with fluorescence light microscopy and then prepared specimens for negative-stain EM. Reconstituted FtsZ rings, encircling the tubules, were clearly resolved. The rings appeared as ribbons of filaments packed side by side with virtually no space between neighboring filaments. The rings were separated by variable expanses of empty tubule as seen by light microscopy or EM. The width varied considerably from one ring to another, but each ring maintained a constant width around its circumference. The inside-out FtsZ rings moved back and forth along the tubules and exchanged subunits with solution, similarly to Z rings reconstituted outside or inside tubular liposomes. FtsZ from Escherichia coli and Mycobacterium tuberculosis assembled rings of similar structure, suggesting a universal structure across bacterial species. Previous models for the Z ring in bacteria have favored a structure of widely scattered filaments that are not in contact. The ribbon structure that we discovered here for reconstituted inside-out FtsZ rings provides what to our knowledge is new evidence that the Z ring in bacteria may involve lateral association of protofilaments.

Figure 1

Representative light microscopy images of FtsZ rings on tubules. Tubules were composed of 60% DC8,9PC, 20% DOPG, and 20% PC. Tubules were mixed with protein and GTP, and then viewed with the light microscope. (A) Tubules alone in DIC. (B and C) FtsZ-YFP-mts Z rings formed inside tubules. (D–F) Mts-FtsZ-YFP Z rings formed around the outside of tubules.

Figure 2

Mts-FtsZ-YFP Z rings were dynamic. (A) A time-lapse series of fluorescence images of the disappearance of FtsZ rings associated with the exhaustion of GTP. (B) Time-lapse series of fluorescence images of FtsZ ring movement. (C) Time-lapse series of fluorescence images of the FRAP assay. Plotted below is the intensity of photobleached region over time. Recovery half-time: ∼45 s. All times listed in seconds.

Figure 3

Negative-stain EM images of FtsZ rings on tubules. (A) Tubules alone. Scale bar: 500 nm. (B–F) Mts-FtsZ-YFP Z rings around the outside of lipid tubules. Scale bar: 500 nm. (G and H) FtsZ-YFP-mts Z rings inside lipid tubules. Scale bar: 200 nm. (I–L) Mts-FtsZ-YFP Z rings outside lipid tubules. Scale bar: 200 nm. Representative Fourier transform of Z-ring images (M).

Figure 4

Negative-stain EM of mixed mts/wt FtsZ rings on tubules: (A and B) 1:1 mts/wt, (C–F) 1:3 mts/wt, and (G and H) 1:5 mts/wt. Scale bar: 500 nm (A, C, E, and G) and 200 nm (B, D, F, and H).

Figure 5

Representative negative-stain EM images of minimal FtsZ rings formed on tubules. Protein concentration: 1.75 μM. Scale bar: 500 nm.

Figure 6

M. tuberculosis mts-FtsZ-YFP Z rings on tubules detected via light microscopy (A and B) or negative-stain EM (C–G). Closer view of the FtsZ ring by negative-stain EM (H) and its Fourier transform (I). Scale bar: 500 nm (C and D), 200 nm (E–G), and 100 nm (H).