Architecture of the ring formed by the tubulin homologue FtsZ in bacterial cell division

Thank you for sending your work entitled “Architecture of the FtsZ ring in vivo and in vitro” for consideration at eLife. Your article has been favorably evaluated by Richard Losick (Senior editor), Werner Kühlbrandt (Reviewing editor), and 3 reviewers, one of whom, Ethan Garner, has agreed to share his identity.

The reviewers found that this paper gives some of the best images of FtsZ rings attained thus far, both in vivo and in vitro. There are also number of findings of importance in this work that could shift the current perception of how bacteria divide.

A number of the findings described in this paper have recently been published by others, in particular the reconstitution of constriction of FtsA and FtsZ inside vesicles, and spirals of FtsZ / FtsA on lipid bilayers. While the preliminary publications of these results is unfortunate, and could be interpreted as stealing some of the authors’ “thunder”, the reviewers do not feel the authors should be penalized for this, as their manuscript puts all these observations into a more coherent interpretation than the previous studies, as well as showing high-resolution images.

The reviewers therefore find that with some modifications, in particular to the discussion, this paper should be suitable for eLife. The Reviewing editor has assembled the following comments to help you prepare a revised submission.

1) The text states that “the filaments are ... uninterrupted everywhere the missing wedge allows it”, but one reviewer noted filaments apparently terminating inside the visible region (as assessed by the extent of the adjacent membranes) in Figures 1C (bottom), 1F (right bottom), 1H (bottom), Figure 1–figure supplement 2 panels B (right bottom) and D (left bottom), Figure 1–figure supplement 3 panels B (bottom) and E (left and right top), Figure 1–figure supplement 4 panels B (bottom). That is actually about half the filaments shown in the figures. This claim should therefore be re-examined and clarified. The text further claims that the lack of complete rings in several cells could be explained by their orientation with respect to the tilt axis. A single sentence should be added simply listing the angular orientations of the cells with and without apparently complete rings so readers can see how consistent the trend is. Representative tomographic volumes should be deposited in a suitable database upon publication.

2) Previous electron cryotomography from the Jensen lab and high-resolution fluorescence imaging of GFP-FtsZ from at least 3 labs support an often-discontinuous Z ring. The contrast between the authors result and the current literature view is hardly addressed and should be discussed more fully.

3) The results for the in vitro reconstitution of FtsZ and FtsA in lipid vesicles are quite beautiful and the quality of the images is impressive. The question is what they mean; in other words does this reconstitution of FtsA and FtsZ filaments in vitro have any significance to cell division in vivo? One of the reviewers doubts it, because there may not be enough FtsA in the cell to make a continuous filament. Please address this issue.

4) The authors' data supports an idea that “filament sliding” could be powering division. This is a historic model in the field. The model presented by the authors is slightly different from previous sliding models, and they should explain these differences clearly.

5) The authors need to explain how their filament-sliding model agrees with the dynamics that have been observed in vivo and in vitro. The authors state that the filaments should slide against each other, and also acknowledge that the filaments turn over.

6) The authors propose that membrane constriction is driven by “repeated filament shortening through nucleotide turnover”, and on first pass, this would appear to fit with the rapid recoveries observed in vitro. But, on closer inspection, this becomes hard to reconcile with other observations:

First, if there are very few long filaments in the cell, and filaments exchange at the ends, how can the filaments constantly turn over at the rapid rate that has been observed in vitro and in vivo?

Second, if the filaments are both long, and turn over at the ends, why do Z-rings recover from all positions equally after photobleaching? If the filaments are long, going around the cell, and recovering from polymerization/depolymerisation at the ends, the rings should recover from the sides, not all over the bleached length.

7) How do the authors reconcile “sliding” with both the in vitro observations of Loose (see “The bacterial cell division proteins FtsA and FtsZ self-organize into dynamic cytoskeletal patterns”, Martin Loose and Timothy J. Mitchison) and the single-molecule tracking of FtsZ (see “Investigating Intracellular Dynamics of FtsZ Cytoskeleton with Photoactivation Single-Molecule Tracking”, Lili Niu Ji Yu)? The in vitro loose work shows that the filaments slide, but the monomers within filaments are immobile, i.e. the filaments are treadmilling, not “sliding”. Similarly, photoactivated FtsZ has shown monomers to be immobile in vivo, also in line with turnover by treadmilling, with static monomers in the filament. The authors need to address these points.

8) Why do the authors assume the sliding must occur between protfilaments? Could it not also occur between filament pairs?

9) The authors should discuss how simple protein crowding of non-filamentous proteins alone can induce bending or tubulation (see “Steric confinement of proteins on lipid membranes can drive curvature and tubulation”, Stachowiak JC1, Hayden CC, Sasaki DY; “Membrane bending by protein-protein crowding.” Stachowiak JC1, Schmid EM, Ryan CJ, Ann HS, Sasaki DY, Sherman MB, Geissler PL, Fletcher DA, Hayden CC). This “bending” by protein crowding could give rise to constrictive forces similar to (or in addition to) constriction resulting from FtsA-induced curvature. This “crowding effect”, driven by depletion interactions would push the filaments together. These depletion interactions could be very strong in vivo, due to the crowded nature of the cell.

1) The text states that “the filaments are ... uninterrupted everywhere the missing wedge allows it”, but one reviewer noted filaments apparently terminating inside the visible region (as assessed by the extent of the adjacent membranes) in Figures 1C (bottom), 1F (right bottom), 1H (bottom), Figure 1–figure supplement 2 panels B (right bottom) and D (left bottom), Figure 1–figure supplement 3 panels B (bottom) and E (left and right top), Figure 1–figure supplement 4 panels B (bottom). That is actually about half the filaments shown in the figures. This claim should therefore be re-examined and clarified. The text further claims that the lack of complete rings in several cells could be explained by their orientation with respect to the tilt axis. A single sentence should be added simply listing the angular orientations of the cells with and without apparently complete rings so readers can see how consistent the trend is. Representative tomographic volumes should be deposited in a suitable database upon publication.

For the Caulobacter cells, the quality of tomograms recorded and their careful analysis in terms of cell orientation with respect to the tilt axis made it possible to derive that the FtsZ ring is most likely continuous (it is the simplest explanation for the data).

For E. coli the reviewers question if it is demonstrated, with the figure panels listed, that it also contains a continuous ring. We agree that the E. coli data is weaker (because the cells are much thicker). However we strongly believe that E. coli also contains a continuous ring, as the architecture of the Z-ring between two species of gram-negative bacteria sharing the same cell division proteins would be expected to be retained. We would argue that the rings sometimes appear to be non-continuous in WT E. coli tomograms simply because the tomograms and images are not as good as for Caulobacter (much more noise, as is obvious from the images) and this was in fact the reason why we employed Caulobacter in the first place.

Please also note in this context that when over-expressed, the Z-ring is definitely continuous and these septa lead to normal divisions as far as we can tell.

To soften our conclusions somewhat, we have added the following text: “Based on these observations we concluded that E. coli Z-rings were, like in C. crescentus, probably continuous and consisted of single-layered bands that are 5-10 filaments wide.”

Figure 1—figure supplement 1 Panel C shows the effect of different orientations of cells with respect to the tilt axis and the reader is referred to this section in the main text.

As for database submission, we have deposited the following two most important datasets from our work, EMDB numbers now also included in the manuscript: EMD-2814 – Caulobacter tomogram, Figure 1A; EMD-2815 – FtsAZ liposome reconstitution tomogram, Figure 4A, 5A, Video 10.

2) Previous electron cryotomography from the Jensen lab and high-resolution fluorescence imaging of GFP-FtsZ from at least 3 labs support an often-discontinuous Z ring. The contrast between the authors result and the current literature view is hardly addressed and should be discussed more fully.

A paragraph addressing these issues has been added: “By imaging completely unmodified cells, utilising recent advances in cryo-EM and acquiring tomograms of cells parallel to the tilt axis we conclude that the FtsZ ring is most likely continuous, made of shorter overlapping filaments. Previous analysis of C. crescentus cells by cryo-ET also showed that the FtsZ ring consists of overlapping filaments inside the inner membrane, although not all cells showed continuous rings {Li et al., 2007, EMBO J, 26, 4694-708}. Equally, results obtained with super resolution fluorescence microscopy techniques (Holden et al., 2014) showed punctuated fluorescence, possibly indicating non-continuous rings. We think it is important to point out that fluorescence microscopy only images the labelled species and intensity fluctuations within the ring may have arisen from using non-functional GFP fusions and/or their over-expression. Or fluctuations coming from overlapping filaments may have been over-emphasised during image analysis because of very low signal-to-noise.”

3) The results for the in vitro reconstitution of FtsZ and FtsA in lipid vesicles are quite beautiful and the quality of the images is impressive. The question is what they mean; in other words does this reconstitution of FtsA and FtsZ filaments in vitro have any significance to cell division in vivo? One of the reviewers doubts it, because there may not be enough FtsA in the cell to make a continuous filament. Please address this issue.

We do think that the in vitro architecture is similar and relevant to what happens in vivo. The fact that there is not enough FtsA to make a continuous filament (at least at the onset of the constriction) is part of our model where a continuous FtsAZ co-filament is possible only at later stages where the membrane curvature allows it (Figure 4–figure supplement 2). FtsZ and FtsA will co-polymerise, until the repeat mismatch makes that energetically unfavourable at a given bending angle (curvature). This 'desire' to continue to polymerise leads to further bending and we would propose that there will first be short stretches of FtsA that then elongate when fully constricted (panel 1F might show a bit of FtsA filaments, actually, since there are additional filaments at 8 nm distance from the membrane).

4) The authors' data supports an idea that “filament sliding” could be powering division. This is a historic model in the field. The model presented by the authors is slightly different from previous sliding models, and they should explain these differences clearly.

We are not sure which paper the reviewers refer to. The sliding model is at length discussed in (Erickson, 2009) and this is/was cited so we do not have to discuss all the various historical models at length.

5) The authors need to explain how their filament-sliding model agrees with the dynamics that have been observed in vivo and in vitro. The authors state that the filaments should slide against each other, and also acknowledge that the filaments turn over.

Yes, we propose that the FtsZ ring made of short overlapping filaments stays in accordance with the dynamics that have been observed by others, as this will enable the filaments to grow and shrink. Already discussed in the manuscript text. Filaments are free to depolymerise/polymerise as long as the ring remains continuous (because otherwise no force can be applied to the membrane). This is a self-selecting principle, of course, and one of the attractive features of our model. A whole paragraph in the Discussion section is devoted to this problem and has been slightly modified to increase clarity.

6) The authors propose that membrane constriction is driven by “repeated filament shortening through nucleotide turnover”, and on first pass, this would appear to fit with the rapid recoveries observed in vitro. But, on closer inspection, this becomes hard to reconcile with other observations:

First, if there are very few long filaments in the cell, and filaments exchange at the ends, how can the filaments constantly turn over at the rapid rate that has been observed in vitro and in vivo?

Second, if the filaments are both long, and turn over at the ends, why do Z-rings recover from all positions equally after photobleaching? If the filaments are long, going around the cell, and recovering from polymerization/depolymerisation at the ends, the rings should recover from the sides, not all over the bleached length.

We do not envisage that there are long filaments going round cells. Unfortunately, the cellular tomograms do not have enough resolution to elucidate this with confidence; when looking along the long cell axes, several filaments are viewed in projection so starts and ends cannot be determined. And in the perpendicular orientation, signal is definitely too low to identify individual filaments.

We propose that there might be many short overlapping filaments forming a ring, producing many ends, enough for the dynamics to appear continuous. Recovery of ring-sub fragments after bleaching may also happen because of sliding and many ends of short filaments enable recovery everywhere.

7) How do the authors reconcile “sliding” with both the in vitro observations of Loose (see “The bacterial cell division proteins FtsA and FtsZ self-organize into dynamic cytoskeletal patterns”, Martin Loose and Timothy J. Mitchison) and the single-molecule tracking of FtsZ (see “Investigating Intracellular Dynamics of FtsZ Cytoskeleton with Photoactivation Single-Molecule Tracking”, Lili Niu Ji Yu)? The in vitro loose work shows that the filaments slide, but the monomers within filaments are immobile, i.e. the filaments are treadmilling, not “sliding”. Similarly, photoactivated FtsZ has shown monomers to be immobile in vivo, also in line with turnover by treadmilling, with static monomers in the filament. The authors need to address these points.

We think it is correct to state that filament treadmilling cannot lead to constriction (because subunits do not move), unless end-tracking is involved, which no known component in the system performs. So we think it is telling that in the mentioned work no constriction was observed, in contrast to the work presented here.

The in vivo tracking is a difficult experiment and, again, fluorescently labelled FtsZ was used. It may well be that that protein was non-functional (and this is what was tracked) but we agree that similar experiments need to be investigated in the future. We plan to do this with our liposomes, where small chemical dyes may be employed, mitigating problems with large fusion proteins.

8) Why do the authors assume the sliding must occur between protfilaments? Could it not also occur between filament pairs?

Yes, this is possible and in vivo we often observed filaments that were associated into doublets and the sliding could of course occur between such filament pairs. This has not been discussed further because it does not change the model and requires more knowledge about FtsZ/FtsA filament doublet formation.

9) The authors should discuss how simple protein crowding of non-filamentous proteins alone can induce bending or tubulation (see “Steric confinement of proteins on lipid membranes can drive curvature and tubulation”, Stachowiak JC1, Hayden CC, Sasaki DY; “Membrane bending by protein-protein crowding.” Stachowiak JC1, Schmid EM, Ryan CJ, Ann HS, Sasaki DY, Sherman MB, Geissler PL, Fletcher DA, Hayden CC). This “bending” by protein crowding could give rise to constrictive forces similar to (or in addition to) constriction resulting from FtsA-induced curvature. This “crowding effect”, driven by depletion interactions would push the filaments together. These depletion interactions could be very strong in vivo, due to the crowded nature of the cell.

This is a very attractive idea. However, it is not clear if this is still valid for proteins acting on negatively curved membranes (e.g., inside cells/liposomes). Also, if this was the case then FtsA (which is the membrane anchor here) itself would trigger significant membrane deformations from the outside and, at least in our hands, one really needs FtsZ to observe the tubulation. No action taken since there are many theoretical studies published with many different ideas, which makes this more suitable for a future review, we would suggest.