Figure 3. In vitro reconstitution of bacterial cell membrane constriction by the FtsZ ring from purified components.

(A) Thermotoga maritima FtsA (TmFtsA) and Thermotoga maritima FtsZ (TmFtsZ) form spirals on a flat lipid monolayer, as indicated by a white dotted line. The filaments tend to appear as double strands (doublets). Negative-stain electron microscopy. (B) Transmission electron cryomicroscopy allows resolution of the inner and outer leaflet of undisturbed liposomes (top panel). When TmFtsA is added to the outside, an additional layer of density corresponding to FtsA becomes apparent (middle panel). Recruitment of TmFtsZ by TmFtsA leads to the formation of two layers (bottom panel). Taken together, we conclude that FtsA is sandwiched between the membrane and FtsZ filaments (bottom panel). See also Figure 3—figure supplement 1 and Figure 3—figure supplement 2. (C–G) Constriction sites are efficiently formed when TmFtsA and TmFtsZ are encapsulated in liposomes that have sizes comparable to bacterial cells. Five representative liposomes are shown using transmission electron cryomicroscopy (hence are 2D projections of 3D objects). Importantly, constriction sites are only formed where a ring made of the two proteins is present (black arrowheads) and not at other sites where filaments are located. The TmFtsA and TmFtsZ layers are clearly visible (inset H, same as boxed area ‘1’ in C; inset J, same as boxed area ‘2’ in C and inset I, which is from Figure 4 electron cryotomography data) and the protein's organisation mirrors that present in E. coli cells (compare with Figure 2C). The distance of 12 nm between TmFtsZ and the membrane (inset J) resembles that found in over-expressing cells (see Figure 2G and also Figure 5C). (E) Intriguingly, liposomes are being constricted (partially) in the absence of added nucleotide. Scale bars: 50 nm in (A–C), 25 nm for insets.

DOI: http://dx.doi.org/10.7554/eLife.04601.013

Figure 3—figure supplement 1. TmFtsZ and TmFtsA on the outside of liposomes and in the presence of GMPCPP deform liposomes.

(A) Low-magnification (upper panel). More detailed snapshots (lower panel) show that the filaments are on the outside; however, they do not form rings but curved structures that are positioned in areas of negative membrane curvature that they probably induce. (B) Schematic representation of the curvature produced by co-polymerisation of FtsA and FtsZ, which have differing repeat distances of 5 and 4 nm, respectively. Since FtsA binds to the membrane, this arrangement will lead to negative curvature. Hence, the intrinsic, negative curvature of the FtsA:FtsZ filaments fits the curvature of the membrane on the inside. However, on the outside, the membrane curvature is positive, as is also shown in Figure 4—figure supplement 1.

DOI: http://dx.doi.org/10.7554/eLife.04601.014

Figure 3—figure supplement 2. Control experiments showing that both TmFtsA and TmFtsZ form straight filaments when polymerised separately. And liposomes deform mostly after dilution.

(A) When mixed, FtsA and FtsZ form curved filaments (right panel). (B) TmFtsZ does not bind to liposomes on its own. Random electron cryomicroscopy images taken immediately after detergent dilution were analysed for liposome deformations. The plot in (C) shows the number of liposomes, out of 63, that are perfectly round (as per solidity quantity, defined in (ImageJ)). Clearly, liposomes become more deformed over a 30-min period after dilution. (D) Shows a spherical liposome without proteins added and (E) at time point 0 min, right after dilution. Scale bars 50 nm.

DOI: http://dx.doi.org/10.7554/eLife.04601.015