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  1. Author Response:

    Joint Public Review:

    Davis et al. parameterize a published, coarse-grained classical density functional theory (DFT) model to describe the free energy landscape of the FG-NTR system. They leverage their previously published experimental data (Zahn et al. eLife, 2016) to develop the model of inter-molecular cohesion calculations, which were tuned to reproduce their previous experimental results. The authors investigate NTR binding behavior to the planar film of FG-nups, first for single NTRs and then by combinations of NTRs. They confirm that the higher concentration of NTRs in the FG-nup films decreases their affinity to the film, which provides one rationale to explain the "transport paradox" of NTRs, which bind specifically to FG-nups but transit the NPC extremely rapidly and at high density. The second result is that increasing the concentration of one of the transport receptors in the film (by increasing its bulk concentration) reduces the adsorbed amount of the other transport receptor (whose concentrations is fixed). Last, the authors thus suggest that within some NTR concentration regimes there emerges a phase separation of the two NTRs such that NTF2 (small NTRs) locate near the surface while importin beta (large NTRs) go to the film/solution interface, implying the existence of separate transport pathways inside the NPC, which has been reported previously in experimental findings.

    There was broad enthusiasm for the model, which was found to be interesting, relevant, and to have successfully delivered testable insights. In general, the conclusions were found to be supported by the model outcomes. The segregation of small and large NTRs to different regions of the film was found to be an interesting result. Some results were found to be less exciting, for example the effect of competition between NTRs as they possess only repulsive interactions in the model.

    While there was some disagreement about the quality of the writing, there was a consensus that the explanation of the motivation, methodology, and impact of the conclusions was not sufficient. In particular, the reviewers felt there was a lack of sufficient context related to prior work in the field in the introduction and discussion and the need to better articulate the impact of the findings in the study. Thus, although the work was found by some to be a meaningful contribution addressing two important questions in the NPC field: how different NTRs are organized within the permeability barrier and if NTR organization and dynamics contribute to the efficient rates of nucleocytoplasmic transport through the crowded environment of the NPC, this point needs to be made clearer. Moreover, more attention is needed to previous theoretical works related to protein adsorption in polymer brushes.

    There was a consensus that the authors could have increased the impact of the work by broadening the study to investigate (or at a minimum discuss) 1) how the combination of NTRs with inert molecules behave (i.e. does the addition of NTRs influence the exclusion of inert cargo?); and 2) how cargo bound to the NTRs (particularly NTF2, which has a single cargo - Ran) influences the results (e.g. would the importin-beta effect be exacerbated by its coupling to an "inert" cargo?). A related theme was concern over the potential impact that the geometry of the NPC in vivo would have on the model outcomes, which speaks to the biological relevance. While the authors mention this issue in the Discussion, more directly addressing whether they can speculate on how their results will change for a cylindrical geometry and how the calculations would compare in a system with opposing surfaces (i.e., two surfaces modified by polymer brushes) was warranted. The latter system was felt to be a good proxy to understand how the effects of nanoconfinement in a cylindrical geometry may affect the results.

    We thank the reviewers for the thorough and comprehensive scientific evaluation of our manuscript and for the constructive feedback as articulated in their comments.

    Summary of Major Changes:

    We have added 28 individual data plots, in the form of four additional supplemental figures, in response to the comments of the reviewers. Importantly, we have produced an additional main figure containing a qualitative phase diagram that concisely summarizes the essential physical picture resulting from our work. In response to criticism about the explanation of our work, we have made substantial changes to the introduction, methods, results, and discussion sections in line with the feedback from the reviewers. Overall, we believe that the manuscript is much stronger than before in terms of the science, the relation to the existing literature, and the clarity in conveying our major assumptions.

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  2. Evaluation Summary:

    This theoretical study describes the interaction of a planar brush or film of the resident unstructured components of the nuclear pore complex (NPC) called nucleoporins (FG-nups) and different nuclear transport receptors (NTRs). The authors describe impacts of competitive binding that give rise to enrichment of the NTRs, NTF2 and importin-beta, at different depths of the FG-nup film, which could relate to experimental observations in other studies, as well as evidence that crowding could promote the rate of nuclear transport by modulating FG-NTR binding/unbinding. The conclusions were found to be generally supported by the data, relevant to the field of nuclear transport, and able to make specific predictions that can be experimentally tested in the future, although previous studies in the field and the novelty could be better described.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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  3. Joint Public Review:

    Davis et al. parameterize a published, coarse-grained classical density functional theory (DFT) model to describe the free energy landscape of the FG-NTR system. They leverage their previously published experimental data (Zahn et al. eLife, 2016) to develop the model of inter-molecular cohesion calculations, which were tuned to reproduce their previous experimental results. The authors investigate NTR binding behavior to the planar film of FG-nups, first for single NTRs and then by combinations of NTRs. They confirm that the higher concentration of NTRs in the FG-nup films decreases their affinity to the film, which provides one rationale to explain the "transport paradox" of NTRs, which bind specifically to FG-nups but transit the NPC extremely rapidly and at high density. The second result is that increasing the concentration of one of the transport receptors in the film (by increasing its bulk concentration) reduces the adsorbed amount of the other transport receptor (whose concentrations is fixed). Last, the authors thus suggest that within some NTR concentration regimes there emerges a phase separation of the two NTRs such that NTF2 (small NTRs) locate near the surface while importin beta (large NTRs) go to the film/solution interface, implying the existence of separate transport pathways inside the NPC, which has been reported previously in experimental findings.

    There was broad enthusiasm for the model, which was found to be interesting, relevant, and to have successfully delivered testable insights. In general, the conclusions were found to be supported by the model outcomes. The segregation of small and large NTRs to different regions of the film was found to be an interesting result. Some results were found to be less exciting, for example the effect of competition between NTRs as they possess only repulsive interactions in the model.

    While there was some disagreement about the quality of the writing, there was a consensus that the explanation of the motivation, methodology, and impact of the conclusions was not sufficient. In particular, the reviewers felt there was a lack of sufficient context related to prior work in the field in the introduction and discussion and the need to better articulate the impact of the findings in the study. Thus, although the work was found by some to be a meaningful contribution addressing two important questions in the NPC field: how different NTRs are organized within the permeability barrier and if NTR organization and dynamics contribute to the efficient rates of nucleocytoplasmic transport through the crowded environment of the NPC, this point needs to be made clearer. Moreover, more attention is needed to previous theoretical works related to protein adsorption in polymer brushes.

    There was a consensus that the authors could have increased the impact of the work by broadening the study to investigate (or at a minimum discuss) 1) how the combination of NTRs with inert molecules behave (i.e. does the addition of NTRs influence the exclusion of inert cargo?); and 2) how cargo bound to the NTRs (particularly NTF2, which has a single cargo - Ran) influences the results (e.g. would the importin-beta effect be exacerbated by its coupling to an "inert" cargo?). A related theme was concern over the potential impact that the geometry of the NPC in vivo would have on the model outcomes, which speaks to the biological relevance. While the authors mention this issue in the Discussion, more directly addressing whether they can speculate on how their results will change for a cylindrical geometry and how the calculations would compare in a system with opposing surfaces (i.e., two surfaces modified by polymer brushes) was warranted. The latter system was felt to be a good proxy to understand how the effects of nanoconfinement in a cylindrical geometry may affect the results.

    Was this evaluation helpful?