In vitro tubulation of liposomes
The reconstitution of contractile Z rings inside tubular liposomes was an important step towards reconstruction of the cell division machinery in vitro (Osawa et al, 2008). As we were investigating the interaction of FtsZ with liposomes, we discovered that FtsZ-YFP-mts could produce concave depressions and tubulate liposomes when added to the outside.
A variety of proteins can tubulate lipid vesicles. Dynamin forms small (∼50 nm diameter) membrane tubules by assembling a distinctive helical array that wraps around the outside of the membrane tubules (Sweitzer and Hinshaw, 1998). Proteins containing a BAR domain can also tubulate liposomes, and like dynamin they form helical filaments that wrap around the outside of the tubules. The BAR domains are banana-shaped helical bundles that curve the membrane by binding with their concave surface to the outer surface of the membrane (Takei et al, 1998; Farsad and De Camilli, 2003; Shimada et al, 2007). A subclass of BAR domains, called I-BAR, also tubulates liposomes, but bends the membrane in the opposite direction. Thus, the I-BAR domains bind the membrane on their convex surface and form an array on the inside of the tubules (Saarikangas et al, 2009). BAR domains bind the membrane by a group of positively charged aa's on the concave surface; for I-BAR domains the positive aa's are on the convex surface.
The most relevant tubulation system for our consideration is MinD itself, since we used the amphipathic helix from MinD as the membrane tether for FtsZ. Hu et al (2002) found that MinD plus ATP tubulated liposomes by forming polymers that wrapped circumferentially around the tubules. In contrast to dynamin, BAR domains and MinD, where the polymers wrap circumferentially around the membrane tubules, the FtsZ-mts protofilaments seem aligned parallel to the axis of the tubules. Recently Dajkovic et al (2008) demonstrated a similar axial arrangement of filaments when liposomes were tubulated by an equimolar (6 μM) mixture of FtsZ, MinD and MinC. In this mixed system, the lipid concentration was increased to a point where 6 μM MinD alone would not tubulate (Dajkovic et al, 2008). We suggest that the tubulation in this mixed system is driven by FtsZ assembly, with the MinC–MinD providing just the tether to the membrane. This would account for the filaments running parallel to the tubule axis, the same as in our system with directly tethered FtsZ-mts.
Direction of the force on the membrane
Z rings on the inside of tubular liposomes are bound to the concave membrane surface and constrict it to a more concave curvature (smaller diameter). How is this related to the forces on membranes generated by FtsZ-mts on the outside? A key observation is that the initial distortion of the membrane is the formation of concave depressions. These concave depressions on the outside would require a bending force on the membrane in the same direction as the Z ring on the inside (Figure 6B and C). A simple mechanism for bending the membrane would be for the FtsZ protofilaments themselves to have a preferred bent conformation, which generates a bending force on the attached membrane (Erickson et al, 1996; Erickson, 1997; Allard and Cytrynbaum, 2009). Our observations now favour a bending mechanism over alternative models based on lateral bonds and sliding protofilaments (discussed in Erickson, 2009).
Figure 6. (A) A molecular model of FtsZ showing the attachment points of tethers at the C- and N-terminal positions. The model is from Pseudomonas FtsZ (pdb: 10 fu; Cordell et al, 2003) visualized with PyMol (DeLano, WL The PyMOL Molecular Graphics System (2002) on World Wide Web, http://www.pymol.org). The top left model shows the FtsZ from the ‘front’ view, as tubulin appears from the outside of a microtubule. The lower left shows the FtsZ from the left side. The normal C-terminal tether emerges from aa G316 on the front surface. The artificial N-terminal tether that we created is attached to the N-terminal methionine, continues through a short linker and is anchored to D10, the first aa visible in the crystal structure. These two attachment points are on the front and back faces, approximately 180 degrees apart. The right-hand model shows FtsZ subunits connected into a curved protofilament, with a 5-degree bend (5 degrees was arbitrarily chosen to illustrate the direction of curvature) at each interface, viewed from the left. The C-terminal attachment G316 is on the convex surface labelled ‘front’ and the N-terminal attachment is on the concave surface. A coloured version of this figure is available at The EMBO Journal online. (B) A model of membrane deformations generated by bending force of FtsZ filaments. When the mts is attached at the C- or N-terminus, the bent protofilaments form a concave depression (left panel) or convex bulge (right panel), respectively. The direction of bending to make a concave depression is the same as that of Z-ring constriction. (C) A model of Z-ring constriction by FtsZ filaments that have a preferred curvature. The scheme to the left shows FtsZ filaments scattered on the cylindrical membrane; because of their curvature they will align circumferentially. The scheme in the middle shows the filaments approaching each other and coalescing to make a Z ring. The scheme to the right shows the ring constricting.
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The idea that the concave depressions are generated by a preferred curvature of the bound protofilaments, is supported by our discovery that switching the mts from the C to the N-terminus switched the membrane distortion from concave depressions to convex protrusions. The geometry of mts attachment is shown in Figure 6A. The tether attaching the C-terminal mts to the globular FtsZ domain is anchored to the front of the globular FtsZ domain, corresponding to the outside of a microtubule. When the tether is switched to the N-terminus it is anchored to the back, approximately 180 degrees away. To explain the concave and convex bending of the membrane, we propose that the FtsZ protofilament has a preferential bend, with the C-terminal tether attachment on the convex side (Figure 6A). If the mts extends from the convex side of the protofilament, it will generate a concave depression in the membrane, and if it extends from the concave side of the protofilament it will generate a convex protrusion (Figure 6B). An implicit assumption is that the tether–mts binds the membrane on the face to which it is anchored, rather than looping around the protofilament.
There is now evidence for at least two curved conformations of FtsZ protofilaments, discussed in more detail by Erickson (2009). A highly curved conformation with a 22-degree bend between subunits produces mini-rings on cationic lipid monolayers and helical tubes in DEAE dextran, both measuring ∼24 nm in outside diameter. An intermediate curved conformation with a 2.5-degree bend produces a circular form of ∼200 nm in diameter. The concave depressions and convex protrusions of the membranes are on the order of 1000 nm in diameter. This is similar to the diameter of an undivided bacterium, and much less curved than either curved protofilament conformation. We therefore suggest that, in the liposome system, the protofilaments are only able to bend the membranes a fraction of the way towards their preferred curvature. (We also note that the relation of these curved conformations to GTP hydrolysis is more complex than the earlier proposal, and is not yet understood; Erickson, 2009.)
One problem raised by the curved protofilament model in Figure 6 is that its direction of curvature is opposite of what we expect by analogy to tubulin rings. Tubulin rings are curved perpendicular to the wall of the microtubule, leaving the outside, kinesin-binding face of the microtubule on the inside of the ring (Wang and Nogales, 2005; Moores and Milligan, 2008; Tan et al, 2008). In order for FtsZ-mts to make concave depressions on the membrane, we had to put the corresponding face on the outside of the ring (Figure 6A). One possibility is that the membrane bending is not produced by the highly curved, mini-ring conformation, but by the intermediate curved conformation. We have no information about the direction of this curvature, and it could be in the opposite direction to the highly curved.
Another consideration is the structure of the supposedly flexible tether linking the globular domains to the membrane. A tether of 50 aa's would be 17 nm long if fully extended. It would seem that a flexible tether this long would easily permit the protofilament to roll over on its side and bend in the plane of the membrane, without generating any bending force on the membrane. However, a chain of unstructured aa's behaves as a worm-like chain and tends to collapse on itself. The average end-to-end length of a 50-aa peptide is ∼4 nm (Ohashi et al, 2007). The worm-like chain will behave as an entropic spring that will generate a force if its ends are extended. Bustamante et al (1994) provide a formula that calculates a force of 10 pN to be needed to stretch the end-to-end distance to one-half of its contour length (a persistence length 0.5 nm was used for this calculation). Lan et al (2007) estimated that a force of ∼8 pN would be needed to divide cells. The force of this entropic spring is similar in magnitude and may be sufficient to keep the front face of the protofilament facing the membrane.
The concave depressions and the convex bulges are incomplete arcs of membrane, which provides another argument that the force is generated by bending protofilaments. Mechanisms where contraction is produced by sliding of protofilaments (Horger et al, 2008; Lan et al, 2009) would not be able to generate concavities, since the filaments are only tethered to the fluid bilayer and would be free to slide without generating force.
The concave depressions are the first membrane distortions formed, and in most conditions they are followed by tubules extruding from the vertices of the concavities. The extrusion of tubules is probably related to the bending that forms concave depressions, but the mechanism is not yet clear. Our preliminary assumption is that the vertices of the concavities are surrounded by FtsZ protofilaments or sheets aligned parallel to the axis of the initial tubule, all trying to bend the membrane outward. However, because they are balanced on all sides, the net result may be to pull membrane from the liposome into the growing tubule. We should note also that FtsZ with the mts on the N-terminus also produced tubules, but much fewer than with the C-terminal mts (data not shown). It is not clear how tubulation could be generated by bending forces in these opposite directions.
We now have two in vitro reconstitution systems for investigating how FtsZ interacts with the membrane and generates force on it. The Z rings inside tubular liposomes appear to be an excellent mimic of the Z rings in bacterial cells, but so far they are only found in multi-lamellar tubular liposomes, which are not frequent. The concave depressions and tubulation reaction are much easier to achieve, and the majority of liposomes on the slide show the same response. This system has already provided new evidence that protofilament bending is the basis of the constriction force. It will be a new tool to explore the mechanisms of Z-ring assembly and force generation.