Crosslinking by ZapD drives the assembly of short FtsZ filaments into toroidal structures in solution

Reviewer #1 (Public Review):

Summary:

The major result in the manuscript is the observation of the higher order structures in a cryoET reconstruction that could be used for understanding the assembly of toroid structures. The cross-linking ability of ZapD dimers result in bending of FtsZ filaments to a constant curvature. Many such short filaments are stitched together to form a toroid like structure. The geometry of assembly of filaments - whether they form straight bundles or toroid like structures - depends on the relative concentrations of FtsZ and ZapD.

Strengths:

In addition to a clear picture of the FtsZ assembly into ring-like structures, the authors have carried out basic biochemistry and biophysical techniques to assay the GTPase activity, the kinetics of assembly, and the ZapD to FtsZ ratio.

Weaknesses:

The discussion does not provide an overall perspective that correlates the cryoET structural organisation of filaments with the biophysical data.

The crosslinking nature of ZapD is already established in the field. The work carried out is important to understand the ring assembly of FtsZ. However, the availability of the cryoET observations can be further analysed in detail to derive many measurements that will help validate the model, and obtain new insights.

Reviewer #2 (Public Review):

Summary:

In this paper, the authors set out to better understand the mechanism by which the FtsZ-associated protein ZapD crosslinks FtsZ filaments to assemble a large scale cytoskeletal assembly. For this aim, they use purified proteins in solution and a combination of biochemical, biophysical experiments and cryo-EM. The most significant finding of this study is the observation of FtsZ toroids that form at equimolar concentrations of the two proteins.

Strengths:

Many experiments in this paper confirm previous knowledge about ZapD. For example, it shows that ZapD promotes the assembly of FtsZ polymers, that ZapD bundles FtsZ filaments, that ZapD forms dimers and that it reduces FtsZ's GTPase activity.

The most novel discovery is the observation of different assemblies as a function of ZapD:FtsZ ratio. In addition, using CryoEM to describe the structure of toroids and bundles, the papers provides some information about the orientation of ZapD in relation to FtsZ filaments. For example, they found that the organization of ZapD in relation to FtsZ filaments is "intrinsic heterogeneous" and that FtsZ filaments were crosslinked by ZapD dimers pointing in all directions. The authors conclude that it is this plasticity that allows for the formation of toroids and its stabilization. Unfortunately, a high-resolution structure of the protein organization was not possible.

Weaknesses:

While the data is convincing, their interpretation has some substantial weaknesses that the authors should address for the final version of this paper.

For example, as the authors are the first to describe FtsZ-ZapD toroids, a discussion why this has not been observed in previous studies would be very interesting, i.e. is it due to buffer conditions, sample preparation?

At parts of the manuscript, the authors try a bit too hard to argue for the physiological significance of these toroids. This, however, is at least very questionable, because:
The typical diameter is in the range of 0.25-1.0 μm, which requires some flexibility of the filaments to be able to accommodate this. It's difficult to see how a FtsZ-ZapD toroid, which appears to be quite rigid with a narrow size distribution of 502 nm {plus minus} 55 nm could support cell division rather than stalling it at that cell diameter. which the authors say is similar to the E. coli cell.

For cell division, FtsZ filaments are recruited to the membrane surface via an interaction of FtsA or ZipA the C-terminal peptide of FtsZ. As ZapD also binds to this peptide, the question arises who wins this competition or where is ZapD when FtsZ is recruited to the membrane surface? Can such a toroidal structure of FtsZ filaments form on the membrane surface? Additional experiments would be helpful, but a more detailed discussion on how the authors think ZapD could act on membrane-bound filaments would be essential.

The authors conclude that the FtsZ filaments are dynamic, which is essential for cell division. But the evidence for dynamic FtsZ filaments within these toroids seems rather weak, as it is solely the partial reassembly after addition of GTP. As ZapD significantly slows down GTP hydrolysis, I am not sure it's obvious to make this conclusion.

On a similar note, on page 5 the authors claim that ZapD would transiently interact with FtsZ filaments. What is the evidence for this? They also say that this transient interaction could have a "mechanistic role in the functionality of FtsZ macrostructures." Could they elaborate?

The author should also improve in putting their findings into the context of existing knowledge. For example:

The authors observe a straightening of filament bundles with increasing ZapD concentration. This seems consistent with what was found for ZapA, but this is not explicitly discussed (Caldas et al 2019)

A paragraph summarizing what is known about the properties of ZapD in vivo would be essential: i.e. what has been found regarding its intracellular copy number, location and dynamics?

In the introduction, the authors write that "GTP binding and hydrolysis induce a conformational change in each monomer that modifies its binding potential, enabling them to follow a treadmilling behavior". This seems inaccurate, as shown by Wagstaff et al. 2022, the conformational change of FtsZ is not associated with the nucleotide state. In addition, they write that FtsZ polymerization depends on the GTPase activity. It would be more accurate to write that polymerization depends on GTP, and disassembly on GTPase activity.

On page 2 they also write that "the mechanism underlying bundling of FtsZ filaments is unknown". I would disagree, the underlying mechanism is very well known (see for example Schumacher, MA JBC 2017), but how this relates to the large-scale organization of FtsZ filaments was not clear.

The authors describe the toroid as a dense 3D mesh, how would this be compatible with the Z-ring and its role for cell division? I don't think this corresponds to the current model of the Z-ring (McQuillen & Xiao, 2020). Apart from the fact it's a ring, I don't think the organization of FtsZ obviously similar to the current of the Z-ring in the bacterial cell, in particular because it's not obvious how FtsZ filaments can bind ZapD and membrane anchors simultaneously.

The authors write that "most of these modulators" interact with FtsZ's CTP, but then later that ZapD is the only Zap protein that binds CTP. This seems to be inconsistent. Why not write that membrane anchors usually bind the CTP, most Zaps do not, but ZapD is the exception?

I also have some comments regarding the experiments and their analysis:

Regarding cryoET: the filaments appear like flat bands, even in the absence of ZapD, which further elongates these bands. Is this due to an anisotropic resolution? This distortion makes the conclusion that ZapD forms bi-spherical dimers unconvincing.

The authors say that the cryoET visualization provides crucial information on the length of the filaments within this toroid. How long are they? Could the authors measure it?

Regarding the dimerization mutant of ZapD: there is actually no direct confirmation that mZapD is monomeric. Did the authors try SEC MALS or AUC? Accordingly, the statement that dimerization is "essential" seems exaggerated (although likely true).

What do the authors mean that toroid formation is compatible with robust persistence length? I.e. What does robust mean? It was recently shown that FtsZ filaments are actually surprisingly flexible, which matches well the fact that the diameter of the Z-ring must continuously decrease during cell division (Dunajova et al Nature Physics 2023).

the authors claim that their observations suggest „that crosslinkers ... allows filament sliding in an organized fashion". As far as I know there is no evidence of filament sliding, as FtsZ monomers in living cells and in vitro are static.

What is the „proto-ring FtsA protein"?

The authors refer to „increasing evidence" for „alternative network remodling mechanisms that do not rely on chemical energy consumption as those in which entropic forces act through diffusible crosslinkers, similar to ZapD and FtsZ polymers." A reference should be given, I assume the authors refer to the study by Lansky et al 2015 of PRC on microtubules. However, I am not sure how the authors made the conclusion that this applies to FtsZ and ZapD, on which evidence is this assumption based?

Some inconsistencies in supplementary figure 3: The normalized absorbances in panel a do not seem to agree with the absolute absorbance shown in panel e, i.e. compare maximum intensity for ZapD = 20 µM and 5 µM in both panels.

It's not obvious to me why the structure formed by ZapD and FtsZ disassembles after some time even before GTP is exhausted, can the authors explain? As the structures disassemble, how is the "steady-state turbidity" defined? Do the structures also disassemble when they use a non-hydrolyzable analog of GTP?

Conclusion:

Despite some weaknesses in the interpretation of their findings, I think this paper will likely motivate other structural studies on large scale assemblies of FtsZ filaments and its associated proteins. A systematic comparison of the effects of ZapA, ZapC and ZapD and how their different modes of filament crosslinking can result in different filament networks will be very useful to understand their individual roles and possible synergistic behavior.

Reviewer #3 (Public Review):

Summary:

The authors provide the first image analysis by cryoET of toroids assembled by FtsZ crosslinked by ZapD. Previously toroids of FtsZ alone have been imaged only in projection by negative stain EM. The authors attempt to distinguish ZapD crosslinks from the underlying FtsZ filaments. I did not find this distinction convincing, especially because it seems inconsistent with the 1:1 stoichiometry demonstrated by pelleting. I was intrigued by one image showing straight filament pairs, which may suggest a new model for how ZapD crosslinks FtsZ filaments.

Strengths:

(1) The first image analysis of FtsZ toroids by cryoET.
(2) The images are accompanied by pelleting assays that convincingly establish a 1:1 stoichiometry of FtsZ:ZapD subunits.
(3) Fig. 5 shows an image of a pair of FtsZ filaments crosslinked by ZapD. This seems to have higher resolution than the toroids. Importantly, it suggests a new model for the structure of FtsZ-ZapD that resolves previously unrecognized conflicts. (This is discussed below under weaknesses, because it is so far only supported by a single image.)

Weaknesses:

This paper reports a study by cryoEM of polymers and bundles assembled from FtsZ plus ZapD. Although previous studies by other labs have focused on straight bundles of filaments, the present study found toroids mixed with these straight bundles, and they focused most of their study on the toroids. In the toroids they attempt to delineate FtsZ filaments and ZapD crosslinks. A major problem here is with the stoichiometry. Their pelleting assays convincingly established a stoichiometry of 1:1, while the mass densities identified as ZapD are sparse and apparently well below the number of FtsZ (FtsZ subunits are not resolved in the reconstructions, but the continuous sheets or belts seem to have a lot more mass than the identified crosslinks.) Apart from the stoichiometry I don't find the identification of crosslinks to be convincing. It is missing an important control - cryoET of toroids assembled from pure FtsZ, without ZapD.

However, if I ignore these and jump to Fig. 5, I think there is an important discovery that resolves controversies in the present study as well as previous ones, controversies that were not even recognized. The controversy is illustrated by the Schumacher 2017 model (their Fig. 7), which is repeated in a simplified version in Fig. 1a of the present mss. That model has a two FtsZ filaments in a plane facing ZapD dimers which bridge them. In this planar model the C-terminal linker, and the ctd of FtsZ that binds ZapD facing each other and the ZapD in the middle, with. The contradiction arises because the C-terminus needs to face the membrane in order to attach and generate a bending force. The two FtsZ filaments in the planar model are facing 90{degree sign} away from the membrane. A related contradiction is that Houseman et al 2016 showed that curved FtsZ filaments have the C terminus on the outside of the curve. In a toroid the C termini should all be facing the outside. If the paired filaments had the C termini facing each other, they could not form a toroid because the two FtsZ filaments would be bending in opposite directions.

Fig. 5 of the present mss seems to resolve this by showing that the two FtsZ filaments and ZapD are not planar, but stacked. The two FtsZ filaments have their C termini facing the same direction, let's say up, toward the membrane, and ZapD binds on top, bridging the two. The spacing of the ctd binding sites on the Zap D dimer is 6.5 nm, which would fit the ~8 nm width of the paired filament complex observed in the present cryoEM (Fig S13). In the Schumacher model the width would be about 20 nm. Importantly, the stack model has the ctd of each filament facing the same direction, so the paired filaments could attach to the membrane and bend together (using ctd's not bound by ZapD). Finally, the new arrangement would also provide an easy way for the complex to extend from a pair of filaments to a sheet of three or four or more.

A problem with this new model from Fig. 5 is that it is supported by only a single example of the paired FtsZ-ZapD complex. If this is to be the basis of the interpretation, more examples should be shown. Maybe examples could be found with three or four FtsZ filaments in a sheet.

What then should be done with the toroids? I am not convinced by the identification of ZapD as "connectors." I think it is likely that the ZapD is part of the belts that I discuss below, although the relative location of ZapD in the belts is not resolved. It is likely that the resolution in the toroid reconstructions of Fig. 4, S8,9 is less than that of the isolated pf pair in Fig. 5c.

Importantly, If the authors want to pursue the location of ZapD in toroids, I suggest they need to compare their ZapD-containing toroids with toroids lacking ZapD. Popp et al 2009 have determined a variety of solution conditions that favor the assembly of toroids by FtsZ with no added protein crosslinker. It would be very interesting to investigate the structure of these toroids by the present cryoEM methods, and compare them to the FtsZ-ZapD toroids. I suspect that the belts seen in the ZapD toroids will not be found in the pure FtsZ toroids, confirming that their structure is generated by ZapD.

Author Response

We acknowledge the editors and reviewers for their careful and thoughtful review of the preprint. Their comments and suggestions will be very useful in improving the manuscript's revised version, which we plan to submit in the coming weeks.