Diameter Dependence of Transport through Nuclear Pore Complex Mimics Studied Using Optical Nanopores

Reviewer #1 (Public Review):

The contribution of Klughammer et al reports on the fabrication and functionalization of zero-mode waveguides of different diameters as a mimic system for nuclear pore complexes. Moreover, the researchers performed molecular transport measurements on these mimic systems (together with molecular dynamic simulations) to assess the contribution of pore diameter and Nsp functionalization on the translocation rates of BSA, the nuclear transport protein Kap95 and finally the impact of different Kap95 concentrations on BSA translocation and overall selectivity of the mimicked pores as a function of their diameter. In order to assess the effect of the Nsp1 on the coated pores to the translocation rates and molecular selectivity they also conducted separated experiments on bare nano-pores, i.e., without coating, and of different diameters. One of the most novel aspects of this contribution is the detection scheme used to assess the translocation rates & selectivity, i.e., the use of an optical scheme based on single molecule fluorescence detection as compared to previous works that have mostly relied on conductance measurements. The results are in general convincing, the experiments carefully performed and the procedures explained in detail. Some weaknesses are identified on the FDTD simulations and interpretation of the single molecule data since they might be affected by quenching of the dye in close proximity to the pore. These weaknesses should be clarified and discussed properly.

Importantly, this study provides new insights on the mechanisms of nuclear transport contributing to further our understanding on how real nuclear-pore complexes (i.e., in living cell) can regulate molecular transport. The recent findings that the nuclear pore complexes are sensitive to mechanical stimulation by modulating their effective diameters, adds an additional level of interest to the work reported here, since the authors thoroughly explored different nano-pore diameters and quantified their impact on translocation and selectivity. There are multiple avenues for future research based on the system developed here, including higher throughout detection, extending to truly multicolor schemes or expanding the range of FG-Nups, nuclear transport proteins or cargos that need to be efficiently l transported to the nucleus through the nuclear pore complexes. As a whole, this is an important contribution to the field.

Reviewer #2 (Public Review):

• The central component of the Nuclear Pore Complex (NPC) that controls nucleocytoplasmic transport is the assembly of the intrinsically disordered proteins (IDPs) that line its passageway. Nanopore based mimics functionalized with these IDPs have been an important tool in understanding the mechanisms of protein transport through the NPC. This paper develops a new type of nanopore NPC mimic that acts as Zero Mode Waveguide enabling optical detection of protein translocations on the single molecule level in pores of different diameters. This is a significant improvement over previous mimics, where optical detection was used only for measurement of bulk fluxes, while single molecule detection relied on electrochemical methods that potentially introduce substantial artifacts. Studying the dependence of transport on the pore diameter is interesting because of its important connections to mechanosensitivity of protein partitioning in cells, which can be difficult to directly control and study in live cells.

• The authors study the transport of individual transport proteins in the dilute regime, and compare the transport of the transport proteins that naturally carry cargoes through the NPC with the transport of BSA that serves as a neutral control. The paper confirms the insights of previous work by the same and other authors - IDP functionalized nanopores are selective in a sense that they conduct the transport proteins well while blocking the passage of BSA. As reported in the paper, the selectivity disappears at large pore diameters which become similar to empty pores because the IDPs don't stretch far enough to cover the pore cross-section.

• The authors use one-bead-per-amino acid coarse grained modeling of the IDPs that they developed and validated previously, to model the distribution of the IDPs in the pores. Combining the simulations with the recently developed "void" model of transport through IDP network and phenomenological transport models, they provide an explanation for the observed reduction in the flux of the neutral control proteins compared to that of transport proteins. The translocation of transport proteins is not modeled directly.

• Together, the experimental and the computational results constitute convincing evidence that points toward the correctness of our current understanding of the physical mechanisms of NPC transport.

• The authors study interference between the transport proteins and the neutral control proteins at high concentrations of the latter, where the pore is occupied by multiple transport proteins. The results appear to be different from previous observations (but more study is needed). I think more discussion of how the results seem with the previous work and what are the potential implication for NPC transport would be welcome.

• The authors use simulations and phenomenological models of transport to analyze the crowded regime. It appears there are some inconsistencies in the application of these models in the dilute and crowded regimes, that should be clarified.

• Some details of the experimental system and the appropriateness of the transport models should be explained more - such as the role of the hydrodynamic pressure gradient.

Reviewer #3 (Public Review):

In this study, a minimalistic setup was used to investigate the selectivity of the nuclear pore complex as a function of its diameter. For this an array of solid-state pores was designed in a free-standing palladium membrane and attached to a PDMS-based fluidic cell, which could be mounted on a confocal microscope. In this way, the frequency of translocation events could be measured in an unbiased manner, i.e., no voltage was applied in this setup to facilitate them as it was done previously (Kowalczyk et al., 2011; Ananth et al, 2018; Fragasso et al., 2021, 2022), and therefore they can be considered as spontaneous. Moreover, the pores exhibited the key properties of the nuclear pore complex: (i) the size of the pore, (ii) disordered FG Nups specifically attached in the central channel; (ii) transport receptors that can shuttle through the central channel by binding to the FG Nups. Additionally, the properties of such minimalistic system could be well controlled. This gave the authors an advantage to monitor the translocation of multiple fluorescently labeled molecules (e.g. Kap95 and BSA) simultaneously, in real time and under well controlled conditions.

Strength:
By being able to adjust each system parameter independently, the authors were able to monitor a reciprocal influence of active transporters, such as Kap95, and passive diffusion (using BSA as passive cargo) at different pore sizes and protein concentrations. It was discovered that up to a certain pore size (ca. 50-60 nm, which is close to the diameter of the physiological nuclear pore complex) and the Nsp1 density, Kap95 binding in the pore significantly increases selectivity as it was previously predicted by 'Kap-centric control' model (Kapinos, et al, 2014, Wagner et al, 2015). However, in pores larger than 60 nm, this effect was fading and becoming negligible in very large pores (> 60 nm), showing that the pores could become leaky and less selective due to stretching, as has been previously suggested (Andreu et al., 2022). It was also shown that passive molecules, such as BSA, had no effect on the Kap95 translocation frequency through the pore.

The experimental data were also supported by coarse-grained modelling of Nsp1-coated pores, and the theoretical prediction correlates qualitatively with the experimentally obtained data. These simulations show that there is a relationship between pore diameter and Nsp1 conformation. Based on these simulations, the authors suggest that in small pores (<60 nm) Kap95 increases selectivity by interacting with the Nsp1-FG domains across the pore, whereas this is less likely for larger pore diameters and Kap95 may collapse the Nsp1-FG domains along the pore walls, making them more permeable.

Weaknesses:
However, the simulations did not consider an effect of Kap95 on the conformation of the Nsp1 layer within the pore, which weakens the conclusion of Kap95-induced collapse, even though it seems very plausible.
In addition, there is a discrepancy in the frequency of translocation events in different experimental setups reported in different studies. The authors suggest that this may be due to differences in the sensitivity of detection methods.

Strength of evidence:
However, this does not detract from the results obtained in this work, as the conclusions are based on the relative changes compared to the numerous controls within the same experimental setup and a careful evaluation of all possible sources of error.