Mechanistic Insights into MinD Regulation and Pattern Formation in Bacillus subtilis
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eLife assessment
This useful study provides data suggesting that subcellular localization of the spatial regulator of cell division, MinD, is an intrinsic feature of the protein's ability to associate with the membrane as both a dimer and a monomer. These findings distinguish the behavior of MinD in B. subtilis from its counterpart in E. coli and suggest that there is not a need to invoke additional localization factors. However, all three reviewers agreed that the study is incomplete: experimentally, quantitation and assessment of MinD behavior in the presence of proteins previously implicated in its localization are missing, among other assays, and the molecular modeling necessary to support the authors' conclusion that their data support a reaction-diffusion model is completely absent. Finally, the manuscript itself is difficult to read with an overly long discussion and disorganized introduction and results sections, and it will require significant revision.
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Abstract
Bacteria precisely regulate the place and timing of their cell division. One of the best-understood systems for division site selection is the Min system in Escherichia coli . In E. coli , the Min system displays remarkable pole-to-pole oscillation, creating a time-averaged minimum at the cell’s geometric center, which marks the future division site. Interestingly, the Gram-positive model species Bacillus subtilis also encodes homologous proteins: the cell division inhibitor MinC and the Walker-ATPase MinD. However, B. subtilis lacks the activating protein MinE, which is essential for Min dynamics in E. coli . We have shown before that the B. subtilis Min system is highly dynamic and quickly relocalizes to active sites of division. This raised questions about how Min protein dynamics are regulated on a molecular level in B. subtilis . Here, we show with a combination of in vitro experiments and in vivo single-molecule imaging that the ATPase activity of B. subtilis MinD is activated solely by membrane binding. Additionally, both monomeric and dimeric MinD bind to the membrane, and binding of ATP to MinD is a prerequisite for fast membrane detachment. Single-molecule localization microscopy data confirm membrane binding of monomeric MinD variants. However, only wild type MinD enriches at cell poles and sites of ongoing division, likely due to interaction with MinJ. Monomeric MinD variants and locked dimers remain distributed along the membrane and lack the characteristic pattern formation. Single-molecule tracking data further support that MinD has a freely diffusive population, which is increased in the monomeric variants and a membrane binding defective mutant. Thus, MinD dynamics in B. subtilis do not require any unknown protein component and can be fully explained by MinD’s binding and unbinding kinetics with the membrane. The generation of MinD patterns relies on the short-lived temporal residence of MinD dimers at the membrane.
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eLife assessment
This useful study provides data suggesting that subcellular localization of the spatial regulator of cell division, MinD, is an intrinsic feature of the protein's ability to associate with the membrane as both a dimer and a monomer. These findings distinguish the behavior of MinD in B. subtilis from its counterpart in E. coli and suggest that there is not a need to invoke additional localization factors. However, all three reviewers agreed that the study is incomplete: experimentally, quantitation and assessment of MinD behavior in the presence of proteins previously implicated in its localization are missing, among other assays, and the molecular modeling necessary to support the authors' conclusion that their data support a reaction-diffusion model is completely absent. Finally, the manuscript itself is difficult to …
eLife assessment
This useful study provides data suggesting that subcellular localization of the spatial regulator of cell division, MinD, is an intrinsic feature of the protein's ability to associate with the membrane as both a dimer and a monomer. These findings distinguish the behavior of MinD in B. subtilis from its counterpart in E. coli and suggest that there is not a need to invoke additional localization factors. However, all three reviewers agreed that the study is incomplete: experimentally, quantitation and assessment of MinD behavior in the presence of proteins previously implicated in its localization are missing, among other assays, and the molecular modeling necessary to support the authors' conclusion that their data support a reaction-diffusion model is completely absent. Finally, the manuscript itself is difficult to read with an overly long discussion and disorganized introduction and results sections, and it will require significant revision.
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Reviewer #1 (Public review):
Summary:
In this work, the authors investigate the molecular dynamics of MinD, a component of the Bacillus subtilis Min system, in vitro and in vivo. In Escherichia coli the Min system is highly dynamic and displays rapid pole-to-pole oscillation whereby a time average minimum of the Min proteins at mid-cell is established. However, in B. subtilis, this is not the case, and there is no MinE present. MinD in B. subtilis dynamically relocalizes from the poles to division sites and binds to MinC and MinJ, which mediates its interaction with DivIVA. This paper reports the biochemical characterization of B. subtilis MinD in vitro and dynamics of MinD variants in vivo, providing mechanistic insight into the mechanism of dynamic localization.
Strengths:
In the current study, the authors perform a detailed …
Reviewer #1 (Public review):
Summary:
In this work, the authors investigate the molecular dynamics of MinD, a component of the Bacillus subtilis Min system, in vitro and in vivo. In Escherichia coli the Min system is highly dynamic and displays rapid pole-to-pole oscillation whereby a time average minimum of the Min proteins at mid-cell is established. However, in B. subtilis, this is not the case, and there is no MinE present. MinD in B. subtilis dynamically relocalizes from the poles to division sites and binds to MinC and MinJ, which mediates its interaction with DivIVA. This paper reports the biochemical characterization of B. subtilis MinD in vitro and dynamics of MinD variants in vivo, providing mechanistic insight into the mechanism of dynamic localization.
Strengths:
In the current study, the authors perform a detailed biochemical characterizion of the in vitro ATPase activity of MinD and demonstrate that rapid hydrolysis is elicited by adding phospholipids. They further show using a collection of substitution mutants of MinD that both monomers and dimers bind to the membrane, and ATP occupancy changes the on and off rates. Identification, quantification, and tracking of discrete Halo-MinD populations were nicely done and showed that mutations in MinD alter dynamic localization, correlating with PL binding on and off rates in vitro.
Weaknesses:
While the study shows that MinD in B. subtilis utilizes a different (MinE-independent) activation mechanism, it remains to be determined the extent to which MinJ and/or MinC play a role.
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Reviewer #2 (Public review):
Summary:
Feddersen & Bramkamp determined important characteristics of how MinD protein binds/dissociates to/from the membrane, and dimerizes in relation to its ATPase activity. The presented data clearly shows the differences in function of MinD homologs from B. subtilis and E. coli.
Strengths:
The work presents well-executed experiments that lead to interesting conclusions and a new model of how Min system works during B. subtilis mid-cell division. Importantly, this model is supported by in vitro characterization of well-chosen mutants in the functional domains of MinD. Outstandingly, most of the in vitro data are confirmed by single-molecule localization microscopy.
Weaknesses:
The authors immobilized liposomes, for which they used E. coli total lipids, to measure ATPase activity and liposome association …
Reviewer #2 (Public review):
Summary:
Feddersen & Bramkamp determined important characteristics of how MinD protein binds/dissociates to/from the membrane, and dimerizes in relation to its ATPase activity. The presented data clearly shows the differences in function of MinD homologs from B. subtilis and E. coli.
Strengths:
The work presents well-executed experiments that lead to interesting conclusions and a new model of how Min system works during B. subtilis mid-cell division. Importantly, this model is supported by in vitro characterization of well-chosen mutants in the functional domains of MinD. Outstandingly, most of the in vitro data are confirmed by single-molecule localization microscopy.
Weaknesses:
The authors immobilized liposomes, for which they used E. coli total lipids, to measure ATPase activity and liposome association and dissociation of B. subtilis MinD. For these experiments would be more suitable to use B. subtilis total lipids as more biologically relevant data could be gained.
Although the work is in detail and nicely compares the function of B. subtilis Min system with E. coli Min system, it lacks the comparison of the Min system function in other rod-shaped Gram-positive bacteria. I would suggest including in the Discussion the complexity of other Min systems. Especially, this complexity is seen in other rod-shaped and spore formers such as Clostridial species in which one of these Min systems or both are present, an oscillating E. coli Min system type and more static as in B. subtilis.
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Reviewer #3 (Public review):
Experimentally, this study provides sufficient data to support the authors' conclusion that MinD dimerization but not ATPase activity is both necessary and sufficient for concentrating it and its binding partner, the division inhibitor MinC, at cell poles. Biochemical data appears to be rigorously acquired and includes proper controls. Although cytological data are consistent with the authors' model, quantitative information on MinD localization in a statistically relevant set of cells is missing (e.g. Figure 2B).
The study's other major conclusion, as outlined in their discussion, that a reaction-diffusion model explains MinD localization in wild-type cells, is unsubstantiated. If they would like to make this a major conclusion of the final manuscript, they will need to include modeling that takes into …
Reviewer #3 (Public review):
Experimentally, this study provides sufficient data to support the authors' conclusion that MinD dimerization but not ATPase activity is both necessary and sufficient for concentrating it and its binding partner, the division inhibitor MinC, at cell poles. Biochemical data appears to be rigorously acquired and includes proper controls. Although cytological data are consistent with the authors' model, quantitative information on MinD localization in a statistically relevant set of cells is missing (e.g. Figure 2B).
The study's other major conclusion, as outlined in their discussion, that a reaction-diffusion model explains MinD localization in wild-type cells, is unsubstantiated. If they would like to make this a major conclusion of the final manuscript, they will need to include modeling that takes into account biochemical and cytological data.
From a presentation perspective, the manuscript is challenging to read and will require substantial rewriting and revision prior to publication.
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