The interplay of membrane tension and FtsZ filament condensation on the initiation and progression of cell division in B. subtilis
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eLife Assessment
This fundamental work provides solid evidence that advances our understanding of the physical mechanisms underlying bacterial cell division by examining the role of membrane tension and FtsZ condensation in sequential stages of division. The effect of accDA overexpression on membrane tension was carefully characterized. To further enhance rigor, the authors could consider examining orthogonal perturbations to membrane tension, addressing membrane tension vs. fluidity, and addressing the ability of FtsZ to bend membranes in cells.
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Abstract
Abstract
The first step of cell division is deforming the planar cell membrane inward towards the cytoplasm. As deforming membranes is energetically costly, biology has developed various protein systems to accomplish this task. The mechanisms providing the force to deform bacterial membranes to initiate division remain unknown. In vivo studies have shown the condensation of FtsZ filaments into a sharp ring is required to initiate cell division, an observation mirrored in vitro with FtsZ filaments encapsulated inside liposomes. Similarly, the force for membrane deformation in many eukaryotic deforming systems arises from the local crowding of proteins on the membrane surface. As any membrane deforming system works against the membrane tension, here we modulated the amount of lipid synthesis and thus membrane tension in Bacillus subtilis to examine: 1) if the condensation of FtsZ filaments by FtsZ bundling proteins serves to overcome the cellular membrane tension to deform the membrane inward and 2) how changes to the membrane tension affect the subsequent invagination of the septum. First, we developed methods to simultaneously measure and modulate membrane tension in live cells. Next, we determined how altering the membrane tension affected the cell’s ability to initiate division with reduced levels of FtsZ bundling proteins. While cells depleted of 2 FtsZ bundling proteins were unable to divide, reducing membrane tension to a given threshold restored their ability to initiate division. Likewise, cells with intermediate levels of FtsZ bundling proteins required a lesser decrease in membrane tension to initiate division. We also found that reductions in membrane tension increase the rate of Z ring constriction, with the constriction rate scaling linearly with the membrane tension. Interestingly, while the constriction rate in wild-type B. subtilis is limited by FtsZ treadmilling, the rate of constriction becomes independent of FtsZ’s treadmilling rate when membrane tension is reduced. These experiments give two major insights: First, the filament condensation caused by FtsZ bundling proteins works to overcome membrane tension and deform the membrane inward to initiate division. Second, the rate of septal constriction is limited by membrane tension, suggesting that membrane fluctuations at the tip of the growing septa limit the rate of cell wall synthesis. Finally, our measurements allow the estimation of several physical values of cell division, such as the force required to bend the membrane, but also that the cell membrane provides only 0.1%, a small amount of surface tension relative to the entire cell envelope, indicating 99.9% of the pressure drop occurs across the cell wall. These calculations also indicate that cell division occurs via comparatively very small membrane tension fluctuations relative to the high turgor pressure that exists across the entire cell envelope.
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eLife Assessment
This fundamental work provides solid evidence that advances our understanding of the physical mechanisms underlying bacterial cell division by examining the role of membrane tension and FtsZ condensation in sequential stages of division. The effect of accDA overexpression on membrane tension was carefully characterized. To further enhance rigor, the authors could consider examining orthogonal perturbations to membrane tension, addressing membrane tension vs. fluidity, and addressing the ability of FtsZ to bend membranes in cells.
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Reviewer #1 (Public review):
In this study, Ramirez-Diaz and coworkers address an important and lingering question in the bacterial cell division field, i.e., whether FtsZ polymers bend the cell membrane inwards, using an elegant and innovative approach. The key cell division protein FtsZ is a homolog of tubulin and forms curved polymers in the presence of GTP. It has long been hypothesized that this curvature provides the force to bend the cell membrane inwards, thereby triggering septal synthesis. Several in vitro studies have shown that purified FtsZ, when attached to the membrane, can indeed deform artificial membranes. However, other studies favor the view that only septal peptidoglycan synthesis drives cell division. Ramirez-Diaz has tried to address the membrane deformation theory in vivo by developing a mutant that synthesizes …
Reviewer #1 (Public review):
In this study, Ramirez-Diaz and coworkers address an important and lingering question in the bacterial cell division field, i.e., whether FtsZ polymers bend the cell membrane inwards, using an elegant and innovative approach. The key cell division protein FtsZ is a homolog of tubulin and forms curved polymers in the presence of GTP. It has long been hypothesized that this curvature provides the force to bend the cell membrane inwards, thereby triggering septal synthesis. Several in vitro studies have shown that purified FtsZ, when attached to the membrane, can indeed deform artificial membranes. However, other studies favor the view that only septal peptidoglycan synthesis drives cell division. Ramirez-Diaz has tried to address the membrane deformation theory in vivo by developing a mutant that synthesizes extra lipids. In this way, the membrane tension is lowered, which would facilitate cell division if deformation of the cell membrane by curved FtsZ polymers is a crucial step in cell division. Surprisingly, they showed that this mutant overcomes the cell division block in a sepF ezrA double mutant. In addition, they carefully characterize the membrane characteristics of the mutant and the effect on FtsZ ring formation. With this work, they have set up a very useful model system to study the role of the cell membrane in cell division, and also a new tool to better study the function of the cell division proteins EzrA and SepF. Overall, this is a very important study for the bacterial cell division field with interesting findings and ideas.
Nevertheless, the authors jump to a conclusion that I cannot yet share. The main issue I have is that they focus on membrane tensions, yet what they seem to modulate is membrane fluidity. Both are clearly related but not the same. I think that it is important to extensively address this issue in the manuscript. They (also) use Laurdan generalized polarization as an indication of membrane tension (Figure 1F), but this method is primarily used in the literature to measure membrane fluidity. In addition, they explain the occurrence of strong local fluorescent membrane signals as the occurrence of double membranes (Figure S1D), whereas others have shown that such fluorescent hot spots can, in theory, also be formed by local accumulation of fluid lipids (PMID: 24603761). The reason why it is so important to distinguish fluidity from tension is that for the attachment of FtsZ polymers, the cell makes use of anchor proteins like FtsA that contain an amphipathic alpha helix, which inserts into the inner leaflet of the lipid bilayer. Importantly, this insertion only works when the fatty acids can be "pushed apart", and this is stimulated by unsaturated and short-chain fatty acids that make the membrane more fluid (PMID: 12676941). If a membrane is "more fluid", then it can more easily accommodate an amphipathic helix. Thus, the production of extra membrane material may increase the fluidity of the cell membrane, as the Laurdan GP measurements indicated, which can then facilitate the attachment of FtsA, including the attached FtsZ polymers, to the membrane. In other words, what the authors have observed may not be a stimulation of Z-ring formation due to lowering membrane tension, but rather because of stimulated binding of FtsZ polymers to the cell membrane. It might be that the attachment of late cell division to the Z-ring, which is all transmembrane proteins, is also facilitated in a more fluid lipid environment. The authors have not excluded the latter (by using a mutant depleted for one of the late cell division proteins).
Finally, the authors performed EM studies to measure septa thickness, and surprisingly, they did not seem to observe deformed septa in a sepF-ezrA double mutant, when overexpressing accDA, while it has been shown before that the absence of SepF leads to strongly deformed septa. Since this finding nuances the mode of action of SepF polymers, it should be discussed.
In conclusion, this is an important and interesting study, but it seems crucial for the interpretation of the findings to include a clear discussion on membrane fluidity and its consequences.
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Reviewer #2 (Public review):
Summary:
In this manuscript, Ramirez-Diaz and colleagues set out to examine key physical mechanisms of bacterial cell division, using the Gram-positive model Bacillus subtilis. Specifically, they investigate the hypothesis that condensation of polymers of the master regulator of division FtsZ can deform membranes to initiate division, but that this is limited by membrane tension. They test this by modulating both membrane tension and FtsZ condensation genetically. To modulate membrane tension, they overexpress accDA to increase the rate of phospholipid synthesis and increase the "hidden membrane reservoir", thereby decreasing membrane tension. To modulate FtsZ condensation, they deplete the bundling protein EzrA in a background lacking a second bundling protein, SepF. They confirm the effects of accDA …
Reviewer #2 (Public review):
Summary:
In this manuscript, Ramirez-Diaz and colleagues set out to examine key physical mechanisms of bacterial cell division, using the Gram-positive model Bacillus subtilis. Specifically, they investigate the hypothesis that condensation of polymers of the master regulator of division FtsZ can deform membranes to initiate division, but that this is limited by membrane tension. They test this by modulating both membrane tension and FtsZ condensation genetically. To modulate membrane tension, they overexpress accDA to increase the rate of phospholipid synthesis and increase the "hidden membrane reservoir", thereby decreasing membrane tension. To modulate FtsZ condensation, they deplete the bundling protein EzrA in a background lacking a second bundling protein, SepF. They confirm the effects of accDA overexpression on membrane tension using two different sensors before assessing the relationship between membrane tension, FtsZ condensation, and division. They demonstrate that cells with excess membrane (reduced membrane tension) can divide with reduced bundling protein abundance, suggesting that FtsZ condensation driven by ZBPs normally serves to overcome membrane tension to initiate division. In addition, they find an inverse relationship between membrane tension and FtsZ ring constriction rate, but no effect of membrane tension on FtsZ treadmilling. Estimation of physical parameters leads them to conclude that very small membrane fluctuations are sufficient to initiate division in unperturbed cells and that the membrane contributes only ~0.1% of the total surface tension strength, maintaining cell shape.
Strengths:
The highly quantitative approach of this work is a strength, as is the rigorous assessment of membrane tension with multiple sensors. The model proposed is largely consistent with existing data and provides a mechanism for further study and validation. The study tackles a major outstanding question in bacterial cell biology, and provides a potential mechanism for a key step in replication with broad implications in other organisms.
Weaknesses:
The authors only use one method (overexpression of accDA) to perturb membrane tension, which could influence division in unanticipated ways (e.g., metabolic adaptations and/or activation of signaling pathways). The proposed model for initiation of division posits that FtsZ condensation bends membranes, which is supported by in vitro evidence, but there is no in vivo evidence that FtsZ condensation can bend membranes in cells. It remains possible that the function of FtsZ condensation is to localize sufficient cell wall synthetic activity to build peptidoglycan that rectifies membrane fluctuations.
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