Membrane Binding Controls the ATPase Cycle and Localization of MinD in Bacillus subtilis

  1. Helge Feddersen
  2. Charlotte Dyckmanns
  3. Marc Bramkamp

  • Curated by eLife

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    eLife Assessment

    This important study provides convincing 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. The reviewers felt that the revisions, particularly the additional experiments and changes to the text to make the experimental design and conclusions clearer, improve the quality of the manuscript and will increase its impact.

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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 spatial organization of MinD relies on the short-lived temporal residence of MinD dimers at the membrane.

Article activity feed

  1. eLife

    eLife Assessment

    This important study provides convincing 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. The reviewers felt that the revisions, particularly the additional experiments and changes to the text to make the experimental design and conclusions clearer, improve the quality of the manuscript and will increase its impact.

  2. eLife

    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 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 characterization 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 was nicely done and showed that mutations in MinD alter dynamic localization, correlating with PL binding on and off rates in vitro.

    - In the revised manuscript, the authors now demonstrate localization and tracking data for minC and minJ deletion strains, which suggest that MinJ impacts MinD membrane cycling, but MinC does not. Additional in vitro work showed that the PDZ domain of MinJ modifies MinD ATP hydrolysis rates, and the authors propose that MinJ may promote MinD dimer formation.

    Weaknesses of the revised version: No major weaknesses.

  3. eLife

    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.

    Comments on revisions:

    I'm satisfied with the authors response to my private recommendation points. However, I thought that they would also respond to my points mentioned in Public Review part, weaknesses as shown above and update the revised version accordingly.

  4. eLife

    Author response:

    The following is the authors’ response to the original reviews.

    Public Reviews:

    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.

    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.

    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.

    We thank the reviewers for their detailed and constructive comments on our work. We particularly acknowledge that the initial version of our manuscript was difficult to read and might have provoked the impression that the aim was to formulate a new mathematical model of Min dynamics in B. subtilis. However, our work aimed at providing solid (and first) biochemical evidence for the MinD ATPase cycle and the nature of the ATPase stimulation. Furthermore, we aimed at corroborating the in vitro findings with single-molecule microscopy data that provided a detailed in vivo picture of the Min dynamics in living cells. Together, this work combines for the first time in vitro and single-molecule in vivo data. During the revision, we generated a wealth of new data that aimed at unraveling the potential effects of MinC and MinJ on MinD dynamics. A major problem during the revision was the problematic purification of MinJ. The membrane integral MinJ has been shown to be highly susceptible to proteolytic decay during purification attempts. Despite various attempts we did not succeed in the purification of full length MinJ. These efforts also led to the unusual long revision time. We therefore turned to the purification of the soluble part of MinJ, namely the PDZ domain. The revised work now contains in vitro data showing the impact of MinC and MinJ-PDZ on MinD ATPase activity and membrane binding. Furthermore, we now provide single-molecule tracking data of MinD in minC and minJ deletion mutant backgrounds. Importantly, the new data show that MinC has no effect on MinD activities, while the PDZ domain has a mild stimulating effect on MinD´s ATPase activity. In summary, a detailed picture on how MinD dynamics function mechanistically in B. subtills emerges.

    Recommendations for the authors:

    Reviewer #1 (Recommendations for the authors):

    (1) It is important to evaluate MinD ATPase activity, PL binding, and release in the presence of MinC and MinJ. In E. coli, MinD recruits MinC to phospholipids. The presence of MinC could change the on/off rates. It is unknown if MinC or MinJ could alter the ATPase rates or dynamics. Presuming that MinD alone drives the complete dynamic story because stimulation is observed in vitro with phospholipids, it follows that Michaelis Menten kinetics is insufficient. It is acknowledged that MinJ is difficult to purify, but one could test a small cytoplasmic subdomain or MinJ-enriched membranes for MinD recruitment and release.

    Indeed, it is unknown whether MinC or MinJ have an impact on the ATPase rates or protein dynamics of MinD in B. subtilis. To address the potential influence of MinC and MinJ on MinD’s ATPase activity and dynamics, we conducted a series of experiments. MinC was successfully purified, and subsequent BLI and ATPase assays revealed no significant impact on MinD activity in our system, except for a modestly reduced ATPase activity (Figure S 5).

    With regard to MinJ, multiple constructs and purification strategies were attempted. While full-length MinJ could not be purified, we isolated the C-terminal PDZ domain to probe potential interactions. In ATPase assays, the PDZ domain reproducibly increased MinD ATP hydrolysis rates, whereas BLI measurements did not reveal detectable changes in MinD membrane-binding kinetics under these conditions. We agree with the reviewer that membrane-integrated MinJ could exert additional effects on MinD recruitment or release that are not captured by the isolated PDZ domain, and we now discuss this limitation in the revised Discussion.

    Furthermore, we performed single-molecule localization and tracking analyses of MinD in ∆minC and ∆minJ backgrounds. These experiments, found in a newly added Results section and summarized in Fig. S 12, demonstrate that MinJ appears to play a role in maintaining dynamic MinD membrane cycling and preventing excessive confinement or aggregation, whereas MinC has no obvious effect on MinD dynamics.

    (2) It is important to show the reduced ATP hydrolysis by MinD mutant proteins (line 243). Stating that they are catalytically inactive without showing the data is presumptuous, and there may be differences between the mutants. Although I am sure that the authors evaluated activity with phospholipids, it should be shown.

    We have now quantified the ATPase activity for all MinD mutants from the respective EnzChek assay data. These experiments confirm that the G12V, K16A, and D40A mutations effectively abolish catalytic activity, yielding phosphate release rates that are essentially at the background detection limit of the assay. We have included these data in Figure S 7 C and updated the text to reflect these findings.

    (3) The shoulder on MinD-K16A suggests that it is capable of forming a dimer at low equilibrium. The suggestion that it is due to interaction with the inert SEC matrix (line 242) raises more concerns, although this is highly unlikely, given that G12V elutes as a single peak. The possibility of a dimer here also demonstrates the necessity of reporting precise ATPase rates for the mutants.

    Thank you for this comment. Since we shared some of your concerns, we made sure to gather enough evidence before making the respective claims. We conducted both in vivo (single-particle tracking, widefield microscopy) experiments and in vitro experiments with the respective K16A mutant of MinD. Most convincingly, K16A is completely catalytically inactive (see previous answer), while both positive and negative controls behave as expected. Both in vivo and in vitro experiments suggest that the protein still binds membrane despite not being able to form dimers. Similar observations were made in a study conducted by colleagues in parallel (Bohorquez et. al, 2024). Furthermore, K16A exchanges in other Walker motif-containing proteins, including E. coli MinD and RecA, and B. subtilis ParA/Soj, abolish dimer formation completely.

    There are many possible explanations why the observed shoulder during elution could appear, which we did not spell out in the results section. This includes possible conformational heterogeneity, as the protein may adopt multiple stable or semi-stable conformations that slightly differ in hydrodynamic volume. It is also possible, that the shoulder represents small protein aggregates from degradation products or proteolysis, which we indeed observe in the respective SDS-PAGE/Blot (Fig. S6). As written in the text, interactions with the SEC column through e.g. hydrophobic patches sticking out is not uncommon, as the surface charges of the mutant protein is different to the wild type version. On the same note, the buffer may subtly affect the surface properties like charge and hydrophobicity differently to the wild type protein and thus its interaction with the column. In conclusion, we are confident that the orthogonal methods used point towards dimer abolishment in a K16A mutant of MinD, despite displaying a small shoulder during SEC elution.

    (4) BLI data - were the kon and koff rates also determined without ATP, since it is assumed that MinD-K16A does not bind ATP, but has a strong Kd (Table 1). Does ATP modify Kd of wt MinD for PLs?

    Without ATP, MinD did neither properly interact with the sensor-bound liposomes nor follow a regular binding kinetic. Therefore, kinetic constants could not be determined, as the fitting of the curves is not possible. In addition to the respective figure (Fig. S8), we attached the graph of the raw/unfitted data in the supplement (Fig. S 13)- (MinD2 dataset)).

    (5) Local MinJ interactions are proposed to alter the dynamic localization of MinD wt and variants in vivo (line 349-358), which could occur through regulation of ATP hydrolysis, PL binding, or release by MinJ or MinC. Localization dynamics should be measured in minC and minJ mutant strains.

    We thank the reviewer for this important suggestion. In response, we have now directly measured MinD localization dynamics in both ∆minC and ∆minJ backgrounds. We performed single-molecule localization microscopy (SMLM) and single-molecule tracking (SMT) of Halo-MinD expressed from its native locus in these mutant strains, using the same experimental and analytical pipeline applied throughout the study. These new experiments are presented in a newly added Results section and summarized in Figure S12, where we quantitatively compare MinD localization, mobility, diffusion states, and confinement between wild type, ∆minC and ∆minJ cells. The data show that deletion of minJ leads to a pronounced increase in the confined/static MinD fraction and reduced dynamic cycling, whereas deletion of minC causes only subtle changes in MinD dynamics. These findings support a specific role of MinJ in maintaining dynamic MinD membrane cycling in vivo, while MinC has a more modest modulatory effect. We have integrated these results into the Discussion to refine our model of how MinJ and MinC differentially influence MinD dynamics and localization.

    (6) Considering the single molecule population counting and a lack of error presented for the binning of tracks (confined/slow/fast); it is difficult to rationalize why G12V and K16A are defective. The relative proportions of confined/slow/fast between wt, G12V, and K16A seem quite similar (i.e., bubble plot). And the static localization in Fig. 2B does not seem dramatically perturbed. This seems to invoke other cellular regulators as critical for the system's operation in the cell, further pointing to important regulatory roles by MinJ and/or MinC.

    First, regarding the apparent lack of error estimates for the population binning, the uncertainties associated with the SMT-based population fitting are intrinsically very small and fall below the graphical resolution of the plots. This reflects the large number of tracks analyzed and the robustness of the fitting procedure, rather than an omission of error analysis.

    Second, we respectfully disagree that the diffusion-state distributions and static localization patterns of G12V and K16A are similar to those of the wild type. In the context of SMT data, the observed shifts in population sizes are substantial and biologically meaningful. Moreover, the static localization of these mutants is markedly altered: instead of forming a graded enrichment at poles and septa, both mutants display a uniform membrane distribution, similar to e.g. a membrane stain (also see Fig. 2 B). This indicates a loss of regulated recruitment, consistent with impaired interaction with MinJ. Importantly, our biochemical analyses, together with extensive data on conserved Walker-type ATPases carrying analogous G12V and K16A mutations, strongly support the conclusion that these variants are functionally defective despite retaining membrane association.

    Third, we agree about the importance of MinC and MinJ, and have now directly tested the contribution of these interactors by analyzing MinD dynamics in ∆minC and ∆minJ backgrounds. These new data, presented in a newly added Results section and summarized in Fig. S12, support our interpretation by showing that MinJ has a pronounced effect on MinD confinement and dynamic cycling in vivo, whereas MinC has a more modest influence. Together, these findings reinforce the conclusion that the defects of G12V and K16A arise from impaired regulatory cycling through the mutations, but also through impaired interaction with MinJ.

    (7) Interesting that they stored the His-MinD protein at 4C for up to one week and not at -80C as it was in 10% glycerol. Was MinD inactivated by freezing? Did this contribute to the observed aggregation (line 695)?

    We thank the reviewer for raising this point. Prior to this comment, we routinely worked with freshly purified MinD and therefore had not systematically compared storage at 4 °C and -80 °C. In response to the suggestion, we have now directly compared the activity of MinD stored at 4 °C for one week with that of MinD stored at -80 °C for four weeks. We did not observe any significant difference in ATPase activity or overall biochemical behavior between the two storage conditions. These results indicate that freezing does not inactivate MinD and that the aggregation observed in some preparations is unlikely to be caused by storage at 4 °C. We have clarified this point in the materials and methods part of the manuscript and thank the reviewer for prompting this.

    (8) Line 109 - Type. Change "component" to "components".

    (9) Page 4, line 52 change 'machinery' to ‘machine'.

    (10) Page 13, line 248, changed 'manifested' to 'displayed'.

    Thank you for pointing out these typos, which have all been corrected.

    Reviewer #2 (Recommendations for the authors):

    I suggest making changes to sentence Lines 60-62: "In rod-shaped model bacteria like Escherichia coli and Bacillus subtilis, division site selection is governed by two protein systems (15-17): nucleoid occlusion and the Min system." However, it was shown previously that the deletion of both systems in B. subtilis, division site selection wasn't disturbed and other mechanism was suggested to be involved.

    We agree that this information should be part of the introduction. Therefore, we included the following sentence at the indicated position:

    “However, it was previously shown that simultaneous deletion of both systems in B. subtilis did not disturb division site selection, suggesting additional mechanisms to be involved (Rodrigues and Harry, 2012).”

    I suggest changing sentence Lines 85-86: "Dimerized MinD recruits MinC and activates it to prevent FtsZ dynamics (46)". It would be more precise to say: "Dimerized MinD recruits MinC and activates it to inhibit FtsZ oligomerization (46).

    Thank you, we agree and changed the sentence accordingly.

    In Figure S2 mark the two mentioned peaks 31 and 62 kDa to which elution volumes correspond.

    We thank the reviewer for this point. We ran the standards for this column again and fitted them to our peaks (see updated Fig. S2), now demonstrating that the shoulders are indeed not at a size where dimers would elute but rather around ~44.3 kDa. We note that both the Ni-NTA eluate and SEC fractions contain multiple His-tagged degradation products (see revised Fig. S2 and His-MinD blot in Fig. S1). Because the SEC run was performed with excess ADP to suppress ATP-dependent dimerization, we interpret the minor shoulder at ~44.3 kDa as arising from sample heterogeneity due to these degradation products, either by co-elution of fragments or by transient fragment:full-length MinD assemblies, rather than full-length MinD dimerization. This is now also described in the respective Results section.

    Reviewer #3 (Recommendations for the authors):

    The quality of the written manuscript is poor, making it difficult to read and appreciate. Specifically: The introduction is quite long. It takes almost three pages until the primary objective of the paper, identifying determinants of MinD localization in B. subtilis, is clearly stated. The introduction should be shortened to focus specifically on Min system function across species-i.e. prevent aberrant polar septation events. Three or four paragraphs should be sufficient. E.g. 1. Introduction to Min systems generally, 2. A summary of the mechanism underlying MinD oscillation in E. coli, 3. An explanation of similarities and differences between E. coli and B. subtilis, and 4. A paragraph outlining the specific questions to be addressed in this study.

    We have substantially revised the Introduction to address this concern. The revised version is considerably shorter and more focused, and now follows the structure proposed by the reviewer. As a result, the main objective of the manuscript is now stated much earlier, and the overall readability and clarity of the Introduction have been substantially improved.

    The results section is challenging to read, in part due to the inclusion of methods as well as some issues with organization. For example, this section begins with a single sentence describing the need to investigate MinD's ATPase cycle in vitro. This sentence is followed by a header and an entirely new section describing the methods used to purify MinD for biochemical analysis. These details should be in the methods section. Similarly, the first paragraph of the following section, which focuses on the ATPase activity MinD in the presence and absence of liposomes, describes how the commercially available EnzChek phosphate assays works. This is, again, something that belongs in methods, not results.

    We have revised the Results section extensively in response to this comment. In the revised manuscript, we have removed or relocated substantial methodological detail from the Results to the Methods section and streamlined the overall organization. Descriptions of protein purification procedures and standard assay principles, including details of the EnzChek phosphate assay, have been condensed or moved to the Methods where appropriate.

    At the same time, we have retained limited methodological information in the Results where it is essential for understanding the interpretation of non-standard experimental setups or key controls, like SMLM. In these cases, brief methodological context is provided to ensure clarity without requiring frequent cross-referencing to the Methods section.

    Overall, the Results section has been substantially condensed and reorganized to improve readability, while additional experiments added in response to reviewer comments necessarily increase the scope of the section. We believe the revised structure now clearly separates experimental outcomes from methodological detail and improves the flow of the Results.

    The discussion section, at 7 pages, is overly long and includes substantial extraneous information. For example, it begins with a 2.5 page long paragraph that includes a summary of pattern formation during embryogenesis in animals, followed by a brief description of Turing's reaction-diffusion model, and finally, repeating parts of the introduction, a summary of the mechanism underlying MinCDE localization in E. coli. It is only in the middle of this paragraph - near the end of the second page - that the authors turn their attention back to MinD localization in B. subtilis, albeit with a focus on reaction-diffusion-based behaviors of other ParA homologues. A revised discussion section should focus on the primary conclusion of the authors, based on data presented in the results. If the authors would like to make the case that their data fit the Turing reaction-diffusion model, they will need to include mathematically based modeling that demonstrates this point in their results.

    We have substantially revised and condensed the Discussion in response to this comment. In the revised manuscript, we removed the extended introductory material on general pattern formation, embryogenesis, and Turing reaction-diffusion theory, as these topics extended beyond the scope of the present study. We also eliminated redundant summaries of the E. coli MinCDE system that overlapped with the Introduction. The revised Discussion now focuses on the primary conclusions supported by our experimental data, namely the biochemical and in vivo mechanisms governing MinD membrane binding, ATPase activity, and dynamic localization in B. subtilis, as well as the regulatory roles of MinJ and MinC. Importantly, we would like to clarify that we did not intend to claim that the B. subtilis Min system follows a Turing-type reaction-diffusion mechanism. References to general reaction-diffusion concepts were meant to provide contextual background and not to imply a specific mathematical framework for the system studied here. To avoid any possible ambiguity, we have removed these references from the Discussion.

    While the overall length of the Discussion is now comparable to the previous version, this reflects the inclusion of substantial new experimental data added during revision. Importantly, the structure and content of the Discussion have been streamlined to prioritize interpretation of the results rather than general background, resulting in a more focused and cohesive narrative.

    Experimental comments:

    Line 213: Please provide a rationale for the ATPase experiments. What is the expected result for each mutant and why?

    We have clarified the rationale for the ATPase experiments in the revised manuscript by briefly outlining the expected behavior of each MinD mutant. The anticipated ATPase properties of G12V, K16A, and D40A are based on well-established studies of conserved Walker-type ATPases and were implicit in the original experimental design, as they should all be catalytically inactive. To avoid any ambiguity, we now state these expectations explicitly in the manuscript.

    Line 243: ATPase data for the mutant proteins should be included in the supplement.

    We have now quantified the ATPase activity for all MinD mutants from the respective EnzChek assay data. These experiments confirm that the G12V, K16A, and D40A mutations effectively abolish catalytic activity, yielding phosphate release rates that are essentially at the background detection limit of the assay. We have included these data in Figure S 7 C and updated the text to reflect these findings.

    Figure 2B: Please include transverse section fluorescence data for all variants as well as quantitative data on average MinD positioning.

    The quantitative information requested is already provided by our single-molecule localization and tracking (SMLM/SMT) analysis of Halo-MinD and its variants (Fig. 4 A and now S 12 A). This approach represents the averaged spatial distribution of individual MinD localizations collected from dozens of cells per condition and provides substantially higher spatial resolution and quantitative precision than transverse fluorescence profiles obtained by conventional widefield microscopy.

    We therefore believe that the SMLM-based analysis is superior to transverse section fluorescence measurements and more accurately captures average MinD positioning across the cell population. To avoid redundancy, we have retained the SMLM analysis as the quantitative framework for MinD localization.

    Figure 2B: I am not convinced that punctate and membrane-associated are mutually exclusive. Quantitative data on protein localization from transverse fluorescent sections is necessary to make this point.

    Please see the answer above and Fig. 4 A

    Figure 2B: It is impossible to assess the functionality of individual mutants without quantitative data on minicell frequency and cell length.

    We have addressed this point by quantitatively measuring both cell length and minicell frequency for all relevant strains. These analyses were performed on a minimum of n = 430 cells per strain and are now presented in Table S 5. The added data provide a quantitative assessment of mutant functionality and support the phenotypic interpretations shown in Fig. 2B, and is also integrated in the Results section.

    Other comments:

    Line 109: should read "components".

    Thank you, corrected.

    Line 135: Why is this sentence outside the major section of the results?

    It now has been integrated into the major section.

    Line 197: I am not sure I understand this sentence.

    We have revised this sentence to improve clarity and readability.

    Line 218: I do not understand this paragraph.

    We have also rephrased and rewritten this paragraph for clarity and readability.

    Line 223: To make this section focused on the results rather than the method, the authors could simply say "To determine the role of ATP mediated dimerization, we...." (If I am understanding this section correctly).

    We followed this suggestion and revised the text accordingly to focus on the experimental outcome rather than methodological detail.

    Line 273: "depicted" not depictured.

    Thank you, corrected.

    Figure 4: The single-cell data look good in the figure, however, the description of these results and their meaning are nearly impossible to follow in the text.

    We acknowledge that the single-molecule data presented in Fig. 4 are complex. While we have made minor clarifications to improve the flow and wording of the text, we did not substantially reduce the level of detail, as the description of the analytical framework is required for correct interpretation of the results.

    At the same time, we aimed to avoid repeating extensive methodological explanations that are already described in the Materials and Methods section, in line with other reviewer comments. We therefore retained a concise but technically accurate description in the Results to ensure that the biological conclusions drawn from Fig. 4 can be properly understood.

  5. Version published to 10.7554/elife.101517.2 on eLife
  6. Version published to 10.7554/elife.101517 on eLife
  7. Version published to 10.7554/elife.101517.1 on eLife
  8. eLife

    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.

  9. eLife

    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.

  10. eLife

    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.

  11. eLife

    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.

  12. Version published to 10.1101/2024.07.08.602513 on bioRxiv