Liposome division by a simple bacterial division machinery

Abstract

We previously reconstituted Z rings in tubular multilamellar liposomes with FtsZ-YFP-mts, where mts is a membrane-targeting amphiphilic helix. These reconstituted Z rings generated a constriction force but did not divide the thick-walled liposomes. Here we developed a unique system to observe Z rings in unilamellar liposomes. FtsZ-YFP-mts incorporated inside large, unilamellar liposomes formed patches that produced concave distortions when viewed at the equator of the liposome. When viewed en face at the top of the liposome, many of the patches were seen to be small Z rings, which still maintained the concave depressions. We also succeeded in reconstituting the more natural, two-protein system, with FtsA and FtsZ-YFP (having the FtsA-binding peptide instead of the mts). Unilamellar liposomes incorporating FtsA and FtsZ-YFP showed a variety of distributions, including foci and linear arrays. A small fraction of liposomes had obvious Z rings. These Z rings could constrict the liposomes and in some cases appeared to complete the division, leaving a clear septum between the two daughter liposomes. Because complete liposome divisions were not seen with FtsZ-mts, FtsA may be critical for the final membrane scission event. We demonstrate that reconstituted cell division machinery apparently divides the liposome in vitro.

Keywords: bacteria; cytoskeleton; tubulin.

Fig. 1.

Z rings and related structures formed by FtsZ-YFP-mts in unilamellar liposomes, without FtsA*, immobilized in a soft agarose gel. (A, A′, and A″) The focus is on a plane through the middle of the liposome. The DIC image (Right) shows many membrane protrusions, which are concave depressions as seen from inside the liposome. The fluorescence signal shows small patches of FtsZ-YFP-mts at these sites (arrows). A′ and A″ show vesicles with more sparse patches to more easily identify them with membrane protrusions. (B) The focus is on the upper surface of the same liposome as in A. Small Z rings are seen on the inner surface of liposome (arrows). Patches (arrowheads) were also observed. (C) Another liposome viewed en face, showing Z rings and patches. (D) A Z ring on the inner surface of a small liposome appeared as two bright dots connected by a faint line. After 80 s, the Z ring had decreased in diameter. The bright dots (Z ring seen edge on) were also visible in the DIC images (arrows). (E) A Z ring formed in a cylindrical protrusion. (F) The Z ring shown in E constricted the protrusion 10 min later. The arrows indicate Z rings and constriction sites. (G) FtsZ foci in this thin tubular extension are exactly localized at the membrane kissing points. These Z rings are smaller than the 250-nm resolution of the light microscope. In all experiments, liposomes contained 7 µM FtsZ-YFP-mts with 1 mM GTP. (Scale bar, 10 µm.)

Fig. 2.

Localization of FtsZ-YFP assembled with FtsA* in unilamellar liposomes immobilized in a soft agarose gel. (A) FtsZ-YFP associated with the membrane at small puncta and more diffusely. Much of the diffuse fluorescence in the center of the liposome may be soluble FtsZ-YFP. (B) FtsZ-YFP filament bundles growing from foci (arrows). The image is focused on a plane through the middle of the liposome, and the bundles grow mostly into the liposome center. (C) This is the same liposome in B but the focus is on the upper surface. Some bundles appear to grow along the liposome membrane. (D and E) The FtsZ-YFP bundles grow and connect, forming a network on the membrane. The focus is on a plane through the middle of the liposome for the upper panel and on the upper surface of the same liposome for the lower panel. In one set of quantitative experiments, the diffuse membrane binding distribution shown in A was seen in 2.5% (5/200) of liposomes. The bundle formation shown in B–E was seen in 14% of liposomes (28/200). Almost all liposomes had foci on the membrane and/or inside liposomes. Liposomes contained 7 µM FtsZ-YFP, 7 µM FtsA*, 2 mM GTP, and 0.5 mM ATP. (Scale bars, 10 µm.)

Fig. 3.

Time lapse recording of liposome division by FtsZ-YFP plus FtsA*. The recording started with a Z ring (arrow) at a moderate constriction at time 0. The constriction deepened over time. There is a clear gap at the constriction site at 603 s, but at 777 s, the gap is replaced with a complete septum. The liposome contained 7 µM FtsZ-YFP, 7 µM FtsA*, 2 mM GTP, and 0.5 mM ATP. (Scale bar, 10 µm.)