Reconstitution of contractile FtsZ rings in liposomes

Abstract

FtsZ is a tubulin homolog and the major cytoskeletal protein in bacterial cell division. It assembles into the Z ring, which contains FtsZ and a dozen other division proteins, and constricts to divide the cell. We have constructed a membrane-targeted FtsZ (FtsZ-mts) by splicing an amphipathic helix to its C terminus. When mixed with lipid vesicles, FtsZ-mts was incorporated into the interior of some tubular vesicles. There it formed multiple Z rings that could move laterally in both directions along the length of the liposome and coalesce into brighter Z rings. Brighter Z rings produced visible constrictions in the liposome, suggesting that FtsZ itself can assemble the Z ring and generate a force. No other proteins were needed for assembly and force generation.

Fig. 1

(A) Model of the Z ring. The Z ring is constructed from overlapping short protofilaments and averages 3 to 9 protofilaments in thickness, depending on the bacterial strain. (B) FtsZ is normally tethered to the membrane by FtsA. The Cterminal peptide (orange) of FtsZ binds FtsA, and FtsA binds the membrane by its amphipathic helix (purple). (C) In FtsZ-mts, the FtsA-binding peptide is replaced with yellow fluorescent protein (YFP) and an amphipathic helix.

Fig. 2

FtsZ-mts was expressed in E. coli depleted of wild-type FtsZ, which forms long filaments because division is blocked. FtsZ-mts formed numerous Z rings and tight-pitch helices.

Fig. 3

FtsZ-mts and GTP were mixed with liposomes. Although the FtsZ-mts was initially outside the liposomes, some tubular, multilamellar liposomes were formed that enclosed FtsZ-mts and GTP. The FtsZ assembled into Z rings in these tubular liposomes. (A) A liposome with three bright Z rings, each centered on a constriction. The fluorescent FtsZ is shown in yellow, superimposed on the differential interference contrast image of the liposome. Arrows indicate Z rings. (B) The bright Z ring near the middle (indicated by the arrow) is forming a visible constriction and on the right side appears to have detached some inner layers of the multilamellar wall. (C) A liposome is shown here with a visible constriction at the Z ring when first observed, 5 to 10 min after making the specimen. Six minutes later the constriction has narrowed markedly. See movie S1 for a 10-min series showing Z rings coalescing into brighter ones, which generate constrictions.

Fig. 4

A Z ring and its constriction abruptly relaxes. For this preparation, the GTP concentration was reduced from 400 to 100 µM and should have been exhausted in ~20 min. The first image was obtained ~10 min after mixing liposomes with FtsZ-mts and GTP, and images were taken every 10 s for another 60 min. Elapsed times after the first image are indicated on the frames. See movie S2 for the complete time series.