Blurred molecular epidemiological lines between the two dominant methicillin-resistant Staphylococcus aureus clones

Article activity feed

1. 

   Review Commons

   Oct 10, 2022

   Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Reply to the reviewers

   The authors do not wish to provide a response at this time. We aim to provide a revised version of the manuscript within two month.

   Read the original source
2. 

   Review Commons

   Oct 10, 2022

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #3

   Evidence, reproducibility and clarity

   In recent years, significant progress has been made in defining the molecular details of many structural features of the nuclear pore complex (NPC). However, one area that remains ill-defined is the interface between the core structures of the NPC and the pore membrane domain. This is an especially intriguing area when one considers that the NPC contains several integral proteins and numerous peripheral membrane proteins contain amphipathic helices whose functions and interactions with the membrane, as well as with one another, remain largely undefined.

   In this manuscript by Amm et al., the authors have examined the functional role …

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #3

   Evidence, reproducibility and clarity

   In recent years, significant progress has been made in defining the molecular details of many structural features of the nuclear pore complex (NPC). However, one area that remains ill-defined is the interface between the core structures of the NPC and the pore membrane domain. This is an especially intriguing area when one considers that the NPC contains several integral proteins and numerous peripheral membrane proteins contain amphipathic helices whose functions and interactions with the membrane, as well as with one another, remain largely undefined.

   In this manuscript by Amm et al., the authors have examined the functional role of the integral membrane Nup Ndc1 and its interactions with various peripheral membrane Nups, including members of the Nup84 complex (termed the Y-complex) and the linker Nups Nup53 and Nup59. The authors show that Ndc1 interacts with specific members of the Nup84 complex, namely Nup120 and Nup133, supporting the idea that Ndc1 functions, in part, to anchor this NPC substructure to the pore membrane. In addition, they identified an amphipathic helix (AH) within the C-terminal half of Ndc1, and they showed that it can directly bind to membranes. Importantly, they have used genetic assays to show that the Ndc1-AH functionally interacts with AHs present at the C-terminus of Nup53 and Nup59. Strikingly, they show that the lethal phenotype detected in strains lacking Ndc1 can be suppressed by the deletion of NUP53, but not NUP59, and, more specifically, only the loss of the C-terminal AH Nup53 was required to suppress the lethal phenotype of the ndc1 null mutation. Further ultrastructural analysis of these mutants revealed that, while these mutants were viable, they exhibited extensive NE expansion phenotypes.

   Overall, the data presented in this manuscript are of high quality, and the experiments are well controlled. My specific comments are relatively minor and listed below.

   Minor points

   1. The authors state "Serial ultrathin sections of fixed yeast cells overexpressing ProtA-CtNdc1 revealed that these unusual extranuclear membrane proliferations exhibited pore-like structures with diameters similar to the diameter of NPCs within the nuclear membrane (Fig. 2C)." This is not entirely clear from the data. I suggest the authors provide direct measurements that support their statement.

   2. The authors examined the total cellular lipid content following overexpression of Ndc1-AH-containing constructs, as well as ProtA-ScHmg1. There is little discussion of the significance of these results, which would provide a clear justification for including these data in the manuscript.

   3. There are numerous typographical and grammatical errors throughout the manuscript that need to be addressed.

   Significance

   The results presented in this manuscript provide further insight into the molecular interactions between Nups and the pore membrane. They suggest that AHs present in a subset of Nups perform linked functions and contribute, in part, to nuclear membrane biogenesis. As such, these results are an important advance in our knowledge of NPC structure and function. They will be of general interest to those studying the function of NPCs and, more generally, NE and organelle biogenesis.

   Reviewer expertise: NPC structure and function, NE biogenesis, yeast model system.

   Read the original source
3. 

   Review Commons

   Oct 10, 2022

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #2

   Evidence, reproducibility and clarity

   Amm et al report on the role of new motifs and interactions between the essential and conserved integral nuclear pore membrane protein Ndc1 and other key components of the yeast nuclear pore complex. They show that members of the Y-subcomplex that coats the pore membrane bind directly to Ndc1 and identify an amphipathic helix at the C-terminus of Ndc1 that displays genetic interactions with other nucleoporins carrying analogous amphipathic helices. The authors find that cells can survive without Ndc1 when these related amphipathic helices from other nups are coincidentally deleted.

   Despite significant recent advances in our …

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #2

   Evidence, reproducibility and clarity

   Amm et al report on the role of new motifs and interactions between the essential and conserved integral nuclear pore membrane protein Ndc1 and other key components of the yeast nuclear pore complex. They show that members of the Y-subcomplex that coats the pore membrane bind directly to Ndc1 and identify an amphipathic helix at the C-terminus of Ndc1 that displays genetic interactions with other nucleoporins carrying analogous amphipathic helices. The authors find that cells can survive without Ndc1 when these related amphipathic helices from other nups are coincidentally deleted.

   Despite significant recent advances in our structural understanding of the nuclear pore complex, how the NPC associates with the curved nuclear membrane remains poorly understood. Previous studies in yeast have uncovered significant redundancy in this association but again the basis for this remains unclear. Therefore, I find this study on the amphipathic helix of Ndc1 and its interaction with other membrane binding components of the NPC an important and timely contribution to the field. Technically, the paper is solid and I find that most of the authors' conclusions are well supported by the evidence they provide (but see below for few experimental issues). Overall, the paper is well written, and despite the use of several mutants and methodologies, it is easy to read. I think the paper's significance would improve if the authors could present some "larger picture" view on how the Ndc1 helix and/or domains they describe interact with the Nup84 complex and the pore membrane or other elements of the NPC. For example, the authors make the remarkable finding that removal of Nup53 makes ndc1 nulls able to survive. Would it be possible to use existing models of the yeast NPC and provide some structural explanation of why that is? However, I would like to emphasize that this is not required to support the main claims of the paper and should only be considered if the authors wish to provide a more "molecular" view of their findings.

   Specific experimental issues and clarifications:

   • A major part of the manuscript describes a detailed structure-function analysis of Ndc1. The link between the two domains of ScNdc1 studied and their effects on membrane proliferation could be better defined: specifically, can the authors exclude that the N-domain of Ndc1 that includes its transmembrane domain, is not also involved in the membrane proliferation phenotype shown in Fig2A and C? It also seems as if GAL-ProtA-ScNdc1 (1-260) also causes growth inhibition (Fig. 2F). How do cells with GAL-ProtA-ScNdc1 (1-260) look like? Finally, although the authors convincingly show that overexpression of 261-655 inhibits growth, from the EM it seems as its effects on membrane proliferation is not the same as that of the overexpression of full-length Ndc1 (compare Fig. 3D vs Fig. 2D).

   • Figure 1A: Do the CtNups shown under "Input" represent 100% of what used in the binding reaction? If so, please indicate at the figure.

   • CtNup120 and CtPom133 would migrate close to CtPom152, which could make visualization by Coomassie stain a bit tricky - if the authors could provide SDS PAGE gels with lower %, that would be helpful. Along similar lines, how do the authors know that CtNup120beta does not bind the CtNdc1 if these two appear to migrate at the same size (Fig. 1D)?

   • Figure 1B, GUVs: Why do the authors use CtNup85 for the GUV experiment instead of CtNup84 that was used in Fig. 1A?

   • Moreover, CtNup120 and CtNup133 ...BC08/SCL1 (Fig. 1C)" Don't see this in Fig. 1C

   • The imaging of ProtA-AHNdc1-eGFP (Fig. 3C) is not great and the localization of the AH does not look very clear - can the authors provide better micrographs? Perhaps co-expression of a red ER reporter or similar reporter would also help.

   • The ndc1 nup53 double mutant appears to display a striking cold-sensitive growth defect (Supplemental Figure 6A, compare 23 vs 30C). Can the authors comment on this?

   Significance

   Despite significant recent advances in our structural understanding of the nuclear pore complex, how the NPC associates with the curved nuclear membrane remains poorly understood. Previous studies in yeast have uncovered significant redundancy in this association but again the basis for this remains unclear. Therefore, I find this study on the amphipathic helix of Ndc1 and its interaction with other membrane binding components of the NPC an important and timely contribution to the field. Technically, the paper is solid and I find that most of the authors' conclusions are well supported by the evidence they provide (but see below for few experimental issues). Overall, the paper is well written, and despite the use of several mutants and methodologies, it is easy to read. I think the paper's significance would improve if the authors could present some "larger picture" view on how the Ndc1 helix and/or domains they describe interact with the Nup84 complex and the pore membrane or other elements of the NPC. For example, the authors make the remarkable finding that removal of Nup53 makes ndc1 nulls able to survive. Would it be possible to use existing models of the yeast NPC and provide some structural explanation of why that is? However, I would like to emphasize that this is not required to support the main claims of the paper and should only be considered if the authors wish to provide a more "molecular" view of their findings.

   Audience: Mostly the following - Nuclear pore complex, nuclear envelope, and possibly some membrane biologists.

   My field of expertise: Cell biology, Nuclear envelope.

   Read the original source
4. 

   Review Commons

   Oct 10, 2022

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #1

   Evidence, reproducibility and clarity

   Ndc1 is a transmembrane nucleoporin, essential for insertion of the nuclear pore complex (NPC) and spindle pole body (SPB) into the nuclear envelope (NE). How NE-associated proteins contribute to the bending and fusion of membranes during NPC insertion has not been fully elucidated. Here, the authors report a number of loosely connected, interesting observations related to Ndc1 function. Their main findings are the following: (i) The N-terminal transmembrane domain of Ndc1 mediates the membrane recruitment of two Y-complex nucleoporins. Therefore, these interactions are likely to contribute to NPC biogenesis. (ii) Over-expression of …

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #1

   Evidence, reproducibility and clarity

   Ndc1 is a transmembrane nucleoporin, essential for insertion of the nuclear pore complex (NPC) and spindle pole body (SPB) into the nuclear envelope (NE). How NE-associated proteins contribute to the bending and fusion of membranes during NPC insertion has not been fully elucidated. Here, the authors report a number of loosely connected, interesting observations related to Ndc1 function. Their main findings are the following: (i) The N-terminal transmembrane domain of Ndc1 mediates the membrane recruitment of two Y-complex nucleoporins. Therefore, these interactions are likely to contribute to NPC biogenesis. (ii) Over-expression of a novel amphipathic helix (AH) in the non-essential C-terminus of Ndc1, and of a similar AH in the non-essential nucleoporin Nup53, alters the lipid composition and nuclear morphology of yeast cells, although the underlying mechanisms remain unknown. (iii) The essential function of Ndc1 can be suppressed by deleting the amphipathic helix from Nup53, or by deleting the transmembrane nucleoporin POM34. Surviving strains have altered nuclear morphology (NE expansions), and are sensitive to membrane-fluidizing drugs, suggesting that NPC assembly is somehow linked to lipid homeostasis.

   Overall, the experiments are of high technical quality, are presented in a clear way, and the conclusions are well-supported by the data. I have some minor suggestions for clarifications, which can be addressed by textual changes or by additional experiments.

   1. When overexpressed in budding yeast, the C-terminal domain of Ndc1 is toxic and induces membrane expansion with NPC-like openings, which the authors describe as enlarged ER membranes (Figure 2). Could these be NE expansions instead? ER and NE membranes are continuous but perhaps this issue could be addressed by examining the distribution of fluorescent markers specific for each compartment.

   2. The essential function of Ndc1 can be suppressed by deleting the amphipathic helix from Nup53 or by deleting POM34. These experiments are done using a plasmid shuffle strategy, in which Ndc1 is temporarily expressed from a low copy plasmid. I wonder if surviving strains are stable, or whether they survive for a limited time only due to stabilisation of the Ndc1 protein in the absence of Nup53 or Pom34. Could the authors discard this possibility, for example by checking whether viable double mutants are recovered after backcrossing of the survivor strains?

   3. Cells over-expressing Ndc1, and surviving ndc1-delta strains display ER and/or NE expansions. It would be interesting to discuss these observations in the context of nuclear morphology studies by the Cohen-Fix and Liakopoulos labs, among others, showing NE expansion is partially dependent on the coordination between lipid synthesis, cell growth rate, and cell cycle progression (doi: 10.1091/mbc.E18-04-0204, 10.1091/mbc.e05-09-0839, 10.1016/j.cub.2012.04.022).

   4. Related to the previous point: nuclear membrane expansions caused by metaphase arrest usually overlap with the nucleolus, and appear DAPI-negative. Did the authors examine nucleolar distribution relative to NE expansion in cells shown in figure 4C? Along the same lines, what is the cell cycle distribution of cells with ER/NE expansion? If they are delayed in mitosis, nuclear morphology defects may be a secondary consequence of cell cycle progression defects, themselves due to NPC and/or SPB insertion problems.

   5. I suggest to rephrase the last sentence of the abstract: "nuclear membrane biogenesis dependent on a balanced ratio between amphipathic motifs in diverse nucleoporins is essential for interphase NPC biogenesis". This study does not directly assess NPC biogenesis and therefore, the interesting link between lipids and NPC biogenesis remains correlative.

   6. It would be useful to include some information on the number of cells observed in the EM figures.

   7. Results, first page: "Moreover, CtNup120 and CtNup133 did not associate with GUVs containing the unrelated inner nuclear membrane protein BC08/SCL1 (Fig. 1C)" should be Figure S1C.

   8. P. 19: "Prompted by the finding that Ndc1 and Nup53/Nup59 amphipathic motifs may (modify?) the nuclear ... "

   I am an expert in yeast genetics and cell cycle progression.

   Significance

   Significance: This report describes novel functional motifs in the Ndc1 protein that may be important for NPC assembly, and intriguing genetic interactions between NPC assembly and lipid homeostasis pathways. Although the mechanisms linking Ndc1 motifs with NE expansion and lipid composition remain unclear, these observations will be interesting for researchers working on NPC biogenesis and nuclear morphology.

   Reviewer Expertise: yeast genetics, cell cycle progression and NPCs.

   Read the original source
5. 

   Version published to 10.1101/501833v3 on bioRxiv

   Sep 18, 2019
6. Version published to 10.1093/ofid/ofz302

   Jun 27, 2019
7. 

   Version published to 10.1101/501833v2 on bioRxiv

   Mar 21, 2019
8. 

   Version published to 10.1101/501833v1 on bioRxiv

   Dec 21, 2018