Editor's evaluation: Crowding-induced phase separation of nuclear transport receptors in FG nucleoporin assemblies
Year: 2021
Type: peer-review
Abstract: Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Methods Data availability References Decision letter Author response Article and author information Metrics Abstract The rapid (<1 ms) transport of biological material to and from the cell nucleus is regulated by the nuclear pore complex (NPC). At the core of the NPC is a permeability barrier consisting of intrinsically disordered phenylalanine-glycine nucleoporins (FG Nups). Various types of nuclear transport receptors (NTRs) facilitate transport by partitioning in the FG Nup assembly, overcoming the barrier by their affinity to the FG Nups, and comprise a significant fraction of proteins in the NPC barrier. In previous work (Zahn et al., 2016), we revealed a universal physical behaviour in the experimentally observed binding of two well-characterised NTRs, Nuclear Transport Factor 2 (NTF2) and the larger Importin-β (Imp-β), to different planar assemblies of FG Nups, with the binding behaviour defined by negative cooperativity. This was further validated by a minimal physical model that treated the FG Nups as flexible homopolymers and the NTRs as uniformly cohesive spheres. Here, we build upon our original study by first parametrising our model to experimental data, and next predicting the effects of crowding by different types of NTRs. We show how varying the amounts of one type of NTR modulates how the other NTR penetrates the FG Nup assembly. Notably, at similar and physiologically relevant NTR concentrations, our model predicts demixed phases of NTF2 and Imp-β within the FG Nup assembly. The functional implication of NTR phase separation is that NPCs may sustain separate transport pathways that are determined by inter-NTR competition. Editor's evaluation 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-β, 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. https://doi.org/10.7554/eLife.72627.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Embedded in the nuclear envelope are nuclear pore complexes (NPCs), large hour-glass shaped channels (inner diameter ∼40 nm) that regulate biomolecular transport between the cytoplasm and nucleoplasm (Alberts, 1994; Wente, 2000). The NPC presents an exclusion barrier to inert molecules, with the degree of exclusion increasing with molecular size. This physical barrier arises from a dense (mass density 100–300 mg/ml) assembly of moderately cohesive intrinsically disordered phenylalanine-glycine nucleoproteins (FG Nups; Hoogenboom et al., 2021). In addition, the barrier contains relatively high numbers (∼20–100) of nuclear transport receptors (NTRs), globular proteins that facilitate the translocation of cargo by transiently binding to the FG Nups (Lowe et al., 2015; Kim et al., 2018; Hayama et al., 2018). The known roles of NTRs in nucleocytoplasmic transport include ferrying specific cargo in and/or out of the nucleoplasm, returning RanGTP to the cytoplasm, and enhancing the exclusion of inert molecules (Jovanovic-Talisman et al., 2009; Aitchison and Rout, 2012; Lowe et al., 2015; Jovanovic-Talisman and Zilman, 2017; Kapinos et al., 2017). However, it remains to be fully elucidated how different NTRs organise themselves within the permeability barrier and how this organisation affects transport (Stanley et al., 2017; Jovanovic-Talisman and Zilman, 2017; Hoogenboom et al., 2021). Qualitatively, NTRs are required to facilitate cargo transport, yet their presence in high numbers poses a significant risk of jamming the transport channel due to crowding effects (Hoogenboom et al., 2021). These seemingly contradictory phenomena have inspired various propositions about more subtle roles of NTRs in the NPC, such as their being essential to maintaining the barrier to non-specific transport; and the existence of spatially segregated, separate transport pathways through the NPC, where different NTRs and/or cargoes may take different trajectories through the barrier (Shah and Forbes, 1998; Yang and Musser, 2006; Naim et al., 2007; Fiserova et al., 2010; Yamada et al., 2010; Ma et al., 2012; Kapinos et al., 2014; Lowe et al., 2015; Lim et al., 2015; Ma et al., 2016; Kapinos et al., 2017; Perez Sirkin et al., 2021). Furthermore, NTRs of varying size and affinity to the FG Nups may play different roles in maintaining efficient transport, e.g., some smaller and more cohesive NTRs may play the role of a cross-linker, modulating the distribution of FG Nup mass in the pore, thereby influencing the trajectories of larger NTRs or cargoes (Perez Sirkin et al., 2021). An alternative mechanism, involving the switching between import and export transport states, has also been proposed (Kapon et al., 2008). Another observation to keep in mind is that the apparent binding affinities of NTRs to FG Nups in vitro appear too tight to enable the fast transport as seen in native NPCs; this can be strongly modulated, however, by the presence of other cellular proteins that compete – non-specifically – with the NTRs close to the FG Nup mass (Tetenbaum-Novatt et al., 2012; Lennon et al., 2021). It is difficult to test different hypotheses regarding how NTR crowding affects the NPC barrier in an in vivo setting, due to the complexity of probing multitudes of diverse proteins in a dense nanoscale channel, as is the case in the NPC. To circumvent this complexity, various studies have reverted to much simpler in vitro FG Nup and NTR assemblies that resemble the physical environment of the NPC, e.g., considering the behaviour of FG Nups in solutions or assemblies with a similar mass density of FG Nups as found in the NPC (∼200 mg/ml) (Ghavami et al., 2016; Davis et al., 2020). Particularly well-controlled model systems are polymer film assemblies, where copies of an FG Nup are anchored to a planar surface and where NTRs (typically of one type) are introduced in the bulk volume above the surface (Eisele et al., 2010; Schoch et al., 2012; Eisele et al., 2012; Kapinos et al., 2014; Zahn et al., 2016; Vovk et al., 2016). It merits emphasising that FG Nup polymer film assemblies are but a simplified model for the NPC in that, typically, only one type of FG Nup and one type of NTR are probed, whereas in the NPC there are multiple different types of FG Nups and NTRs in the inner channel; and in that – in polymer film assemblies – the geometry of the nanoconfinement of the FG Nups and NTRs arises from a single hard planar wall, as compared with the more complex cylindrical confinement in the NPC. Nonetheless, they provide a most suitable platform to discover general principles of FG Nup and NTR behaviour that may be obscured in experiments on in vivo NPCs; and provide a foundation for a step-by-step increase in the level of complexity towards that of real NPCs. For the behaviour of planar films containing one type of FG Nup and one type of NTR, the main findings thus far have been: (1) that NTRs of one type (such as NTF2 and Importin-β [Imp-β]) bind to FG Nups in a rather generic way, suggesting universal physical principles – based on negative cooperative binding – might govern their behaviour (Vovk et al., 2016; Zahn et al., 2016); (2) NTRs readily penetrate the FG Nup films, with only limited effects on the collective morphology of the FG Nups (little swelling or compaction) (Eisele et al., 2010; Kapinos et al., 2014; Wagner et al., 2015; Vovk et al., 2016; Zahn et al., 2016); (3) that such systems can replicate the basic selective mechanism in the NPC, i.e., inert proteins tend not to penetrate the collective FG Nup phases but NTRs do, consistent with in vivo NPC functionality and with experiments on bulk solutions of FG Nups and NTRs (Schmidt and Görlich, 2015; Schmidt and Görlich, 2016); (4) the number of transport proteins in the FG Nup films can vary by orders of magnitude as a function of NTR numbers in solution above the film, in a highly non-Langmuir manner, where complex many-body interactions preclude the use of simple one-to-one binding models (Eisele et al., 2010; Schoch et al., 2012; Kapinos et al., 2014; Wagner et al., 2015; Vovk et al., 2016; Zahn et al., 2016). With the caveat that only a subset of NTRs have been probed, investigations of planar assemblies of FG Nups and NTRs highlight the fine-tuned balance of FG Nup attachment density, FG Nup cohesion, FG Nup-NTR affinities, the amount of non-specific proteins in the system, and NTR concentrations, where minor changes in this balance can lead to qualitatively different binding scenarios (Vovk et al., 2016; Zahn et al., 2016; Stanley et al., 2017; Lennon et al., 2021). Taking a next step towards the complexity of the in vivo NPC and considering the large number (∼20–100) of NTRs of different sizes and affinities in the NPC inner channel (Lowe et al., 2015; Peters, 2009a; Peters, 2009b; Lim et al., 2015; Kim et al., 2018; Hoogenboom et al., 2021), one may next ask how the binding affinity of a specific NTR to a planar assembly of FG Nups depends on the binding behaviour of other NTRs. Physiologically, the answers to this question may explain how binding and thereby transport of specific NTRs, and their associated cargoes, can be modulated (if at all) by the presence of other types of NTRs, since this would directly impact on the transport function of the NPC. More generally, such answers will aid our understanding of how the NPC maintains fast and efficient transport whilst hosting a dense milieu of FG Nup motifs, NTRs, and cargoes. Finally, a systematically probing of crowding effects provides a means to assess various conceptual models of NPC transport such as the 'Kap-centric' barrier model (Wagner et al., 2015) and reduction-of-dimensionality models (Peters, 2005). Here, we aim to understand how the crowding by different NTRs may affect the organisation and thereby transport functionality of FG Nup assemblies. Focusing on planar FG Nup assemblies as a well-controlled model system (Zahn et al., 2016), we use physical modelling to probe how the binding of one type of NTR could be affected by other types of NTRs, in a way that can be tested by currently available experimental setups. To explore the effects of mixed NTR crowding, we model a ternary mixture containing two different NTRs and one type of FG Nup in a polymer film assembly. When modelling FG Nups and NTRs, one can take various coarse-grained approaches, for instance one can take an all-atom approach (Miao and Schulten, 2009; Gamini et al., 2014; Raveh et al., 2016), or account only for the amino acids (Ghavami et al., 2013, Ghavami et al., 2014; Ghavami et al., 2018), or work only with the generic patterning of FG/hydrophobic/hydrophilic/charged 'patches' (Tagliazucchi and Szleifer, 2015; Matsuda and Mofrad, 2021; Davis et al., 2021), or completely neglect sequence details altogether in a 'homopolymer' approach (Moussavi-Baygi et al., 2011; Osmanovic et al., 2012; Osmanović et al., 2013b; Vovk et al., 2016; Zahn et al., 2016; Timney et al., 2016; Davis et al., 2020). Each approach has its strengths and weaknesses. For instance higher-resolution modelling can account for greater molecular complexity, but with the difficulty in probing large time and length scales. In contrast, homopolymer modelling provides access to larger time and length scales with more robust parameterisation and simplicity of execution, at the expense of (sub)molecular detail. In this work, we build on our previous minimal modelling framework based on treating FG Nups as sticky and flexible homopolymers and NTRs as uniformly cohesive spheres (Osmanović et al., 2013b; Zahn et al., 2016; Davis et al., 2020). Results Minimal physical modelling facilitates the understanding of many aspects of NPC functionality in terms of general principles, but it requires quantitatively accurate parameter settings to make meaningful predictions (Osmanović et al., 2013a; Jovanovic-Talisman and Zilman, 2017; Hoogenboom et al., 2021). In this work, the minimal modelling framework we employ is that of coarse-grained classical density functional theory (DFT), that has been previously used to model aspects of the NPC permeability barrier (Osmanović et al., 2013b; Zahn et al., 2016; Davis et al., 2020). To ensure that the setting of the parameters in our DFT model – outlined in the Computational methods section (below) – is commensurate with the behaviour of FG Nups (Nsp1) and NTRs (NTF2 or Imp-β) as probed in experiments, we make use of experimental data on FG Nup-NTR polymer film assemblies where the macroscopic binding between one type of FG Nup (Nsp1) and one type of NTR (NTF2 or Imp-β) was quantitatively probed (see Figure 1) Zahn et al., 2016. The experiments focused on a polymer film consisting of Nsp1 domains, attached to a flat surface at physiologically relevant densities (≈3.3 polymers/100 nm2), interacting with either NTF2 or Imp-β over a physiologically relevant range of concentrations (0.01–10 µM). Figure 1 with 2 supplements see all Download asset Open asset Setting the polymer-particle cohesion strengths {ϵFG-NTF2,ϵFG-Impβ} through comparison of density functional theory (DFT) results with experimental binding isotherms for the cases of NTF2 (left) and Importin-β (Imp-β) (right) binding to an Nsp1 film (Zahn et al., 2016) (top). Concomitant film thicknesses as found in DFT and experiment (bottom). The experimental Nsp1 surface attachment densities were 4.9 pmol/cm2 and 5.1 pmol/cm2 for NTF2 and Imp-β, respectively. The parametrised cohesion strengths ϵFG-NTF2=2.4±0.1 kBT and ϵFG-Impβ=2.3±0.1 kBT correspond to the modelled NTF2 and Imp-β particles, respectively. Filled bands (in all four panels) denote a tolerance of ±0.1 kBT in the polymer-particle cohesion strengths. The thicknesses of the filled bands for the bottom two panels are similar to the thickness of the line connecting DFT data points (blue and orange). Figure 1—source code 1 Simulation parameters for the classical density functional theory code. https://cdn.elifesciences.org/articles/72627/elife-72627-fig1-code1-v2.zip Download elife-72627-fig1-code1-v2.zip For consistency with the available experimental data we focus on the FG Nup Nsp1, which we treat as a homogeneous, flexible, and cohesive polymer consisting of M=300 beads of diameter d(3)=0.76 nm (two amino acids per bead), resulting in the approximately correct persistence length for FG Nups (Lim et al., 2006; Zahn et al., 2016; Hayama et al., 2019; Davis et al., 2020). The intermolecular and intramolecular cohesive properties of FG Nups arise from a combination of hydrophobic motifs, e.g., FG, FxFG, and GLFG, and charged residues along the sequence which, in our model, are subsumed into one single cohesion parameter ϵFG-FG (equivalent to ϵ(33) in the Computational methods section). In addition to the FG Nups, we also include the presence of the NTRs, NTF2 and Imp-β, which we model as uniformly cohesive spheres of diameters dNTF2=4 nm (same as d(1) in the Computational methods section) and dImp-β=6 nm (same as d(2) in the Computational methods section), respectively (Zahn et al., 2016). The cohesive properties of the NTRs strictly refer to the attraction between the NTRs and FG Nups, arising at least in part from the hydrophobic grooves and charged regions on the former and the hydrophobic motifs and charged regions on the latter (Kumeta et al., 2012; Kim et al., 2013; Hayama et al., 2018; Frey et al., 2018). We do not consider any attractive interactions between NTRs themselves as there is no empirical evidence that suggests there are cohesive interactions between NTRs, a choice that is consistent with previous work and shown to well replicate experimental data of NTR binding to FG Nup assemblies (Osmanović et al., 2013b; Zahn et al., 2016; Vovk et al., 2016). As with the FG Nup intermolecular and intramolecular cohesive interactions, we subsume all contributions to the respective cohesive interactions FG Nup – NTF2 and FG Nup – Imp-β through two more cohesion parameters ϵFG-NTF2 and ϵFG-Impβ (equivalent to the cohesion variables ϵ(12) and ϵ(13), respectively). We begin the parametrisation of our model with the setting of ϵFG-FG so as to accurately reproduce the experimental thickness of Nsp1 planar film assemblies, at similar anchoring densities, as was done previously (Zahn et al., 2016; Fisher et al., 2018; Davis et al., 2020) (see Figure 1—figure supplement 1). With the here chosen interaction potential, the resulting cohesion strength is ϵFG-FG=0.275±0.025 kBT (with experiments conducted at ≈23 ∘C), that yields a film thickness τ=26±2 nm, in our model defined as the height above the surface below which 95% of the total polymer density is included. We note that the value of ϵFG-FG found here is different to that of our previous work (Zahn et al., 2016), mainly due to the different choice of interaction potential. Nonetheless, both the model here and the model in Zahn et al., 2016 are parametrised using the same experimental data and produce the same film thicknesses. To further validate this value of ϵFG-FG, we checked whether the polymer film would exclude inert molecules, a basic property that has been observed for Nsp1 assemblies (Schmidt and Görlich, 2015; Schmidt and Görlich, 2016). The inert molecules are modelled as non-cohesive spheres of diameter d(i), with i labelling the particle type, and their inclusion/exclusion in the film is quantified through the potential of mean force (PMF) W(i)(z) given as (1) W(i)(z)=c(i)(z)+Vext(i)(z)+∫ρ(3)(z)u(i)(z−z′)dz′−μ(i),≈−kBTln(ρ(i)(z)ρbulk(i)), where c(i)(z) is the one-body direct correlation function (see equation 19), Vext(i)(z) is the external potential (see equation 11), ρ(3)(z) is the polymer number density, u(i)(z) is the i.e., over the polymer-particle cohesive potential (see equation and is the potential to et al., and For the inert molecules, the polymer-particle attraction in equation is As non-cohesive with increasing diameters and nm) a greater potential barrier upon into the polymer film (see Figure 1—figure supplement that our simple model of an Nsp1 film one of the of the permeability barrier as seen in the the degree of exclusion of inert molecules increasing with molecular size et al., 2009; et al., 2015; Ghavami et al., 2016). We note that the presence of a close to the anchoring in the density for inert particle diameters and 2 nm is due to the in the polymer density to the surface with a potential well close to the see Figure 1—figure supplement the of the in the density depends upon the anchoring density of the FG Nups shown We that of diameters nm of In experiments, the size exclusion of the NPC was determined as nm, that this is rather and and Peters, et al., et al., In our model, this empirical size to at least one of magnitude greater the 1 1—figure supplement This of magnitude of barrier is of a physiologically relevant and is consistent with – – modelling work that into account the amino sequence of the FG Nups in a pore geometry (Ghavami et al., 2016), as well as with a more study that the of of varying size and and Mofrad, 2021). shown that the parametrised polymer model for Nsp1 films the experimentally observed film thickness and the size of the NPC, we our focus to setting the parameters for the NTRs, NTF2 and The cohesion strengths ϵFG-NTF2 (same as in the Computational methods section) and ϵFG-Impβ (same as in the Computational methods section) are by isotherms as in DFT to in experiment (Zahn et al., 2016) (see Figure 1). we the total density of NTRs in the film as the total NTR within the film thickness by the to of (2) NTR density in where is the number density of the NTRs. With only one parameter for NTR the Nup interaction the DFT binding isotherms are found to be in with experiment over orders of magnitude in bulk NTR 1 as was previously by a similar DFT model were attached to the of a in Zahn et al., 2016. For the here chosen interaction potential, the resulting parametrised cohesion strengths are ϵFG-NTF2=2.4±0.1 kBT and ϵFG-Impβ=2.3±0.1 kBT for NTF2 and Imp-β, respectively. It out that for the two NTRs, the Imp-β particle an larger volume diameter as compared with the NTF2 However, given the in and a in the respective of intermolecular interactions (see equation we directly the cohesive properties of the two NTRs based on the cohesion strengths ϵFG-NTF2 and ϵFG-Impβ the film thicknesses from DFT – as a function of NTR – are also in with experiment 1 At this all the essential interaction parameters and ϵFG-Impβ have been by between DFT and we the effects of crowding of one type of NTR on the We molecular crowding through two the fraction where is the number density of a NTR by and the W(i)(z) of a NTR, in the presence of other NTRs and the FG Nups (see Figure 2 and equation 1). For high crowding, one the fraction of a NTR to increase in magnitude and for the to more (with to the same system but with which is as a greater potential barrier a potential We that both NTF2 and Imp-β of and density within the Nsp1 film upon increasing their respective bulk concentrations and 2 The density arise from effects mainly by a hard planar wall, where to to a flat the of next to a planar is a et al., et al., As is in both the observed fraction was close to the surface µM). For the here chosen it is that the fraction and will be by the interactions with the and the crowding of other NTRs, with significant effects arising from the of the surface We note that the density for the Imp-β particle show greater as compared with the NTF2 particle the same above the which is since the Imp-β is larger in size and thus more effects et al., Figure 2 with 1 supplement see all Download asset Open asset nuclear transport bulk and of the potential well within the Nsp1 film, for systems containing one type of NTR density functional theory where is the number density and is the particle as a function of the height above the flat surface for NTF2 (left) and Importin-β at various concentrations (top). of mean force for the same systems as on the For both NTF2 and Imp-β, the in magnitude negative within the bulk of the upon an increase in bulk NTR 2 increasing the in solution from to results in an in the value of the The implication of this is that, at of in the film, it is relatively for NTRs to from the polymer film, for NTRs to the polymer film from the solution above This may be to the of i.e., molecular crowding, of the NTRs between the Nsp1 The results of Figure 2 are relevant to the NPC where fast transport transport per in with – selective – there are various of the transport (Hoogenboom et al., 2021), results show how NTR crowding may facilitate the of NTRs from the NPC, that a of kBT in well would a rate for we 2 the presence of NTRs in the NPC inner channel (Lowe et al., 2015; Peters, 2009a; Peters, 2009b; Lim et al., 2015; Kim et al., 2018; Hoogenboom et al., 2021), we how varying the available bulk of one type of NTR – in this case would affect the barrier for inert as seen in our modelled FG Nup film (see Figure 1—figure supplement As we increase the bulk of NTF2 from to we that the amount of inert particles, with diameters in the range nm, within the FG Nup film, as compared to inert in a film with FG Nups only (see Figure supplement The amount of inert in the film is also quantified by an increase in the with the increase in being larger for of greater size (see Figure supplement This is in with experimental observations of an of the NPC barrier to large inert through an presence of NTRs et al., and consistent with the that NTRs to the of the NPC transport barrier (Peters, 2009a; Peters, 2009b; Lim et al., As a next we explore how the between NTRs may affect the and distribution of NTRs in FG Nup assemblies. we model the mixed crowding effects in a system containing the NTRs, NTF2 and Imp-β, in an Nsp1 polymer film (see Figures and and their respective Figure As in the case with one type of NTR, we probe the binding and polymer film but this time the amount of one NTR at a physiologically relevant (Zahn et al., 2016) whilst varying the of the other NTR increasing the bulk of NTF2 from to the bulk (in of Imp-β at 1 the amount of Imp-β and the Imp-β is towards the surface of the Nsp1 polymer film and Figure supplement 1). the density of Imp-β within the film, which are at of out upon increasing the amount of NTF2 to This that the presence of NTF2 directly modulates the distribution of Imp-β within the upon increasing NTF2 from the bulk of Imp-β we NTR phase an phase within the FG Nup film and an phase at the film We can this phase separation to the crowding of the NTRs, since by the affinity between NTF2 and the FG Nups, thereby the competitive of NTF2 over Imp-β, we an of Imp-β in the FG Nup polymer film (see Figure supplement Figure with 2 supplements see all Download asset Open asset separation in a ternary phenylalanine-glycine nucleoporins (FG transport polymer film assembly. functional theory and of mean force for Nsp1 polymer films in the presence of NTF2 and Importin-β
Source: eLife
Author: Megan C King
Institution: Yale University
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $2,500
OpenAlex ID: W4214505263