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Structural dynamics of the nuclear pore complex
Accepted Manuscript Title: Structural Dynamics of the Nuclear Pore Complex Authors: Yusuke Sakiyama, Radhakrishnan Panatala, Roderick Y.H. Lim PII: DOI: Reference:
S1084-9521(16)30351-2 http://dx.doi.org/doi:10.1016/j.semcdb.2017.05.021 YSCDB 2227
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Seminars in Cell & Developmental Biology
Received date: Accepted date:
22-5-2017 29-5-2017
Please cite this article as: Sakiyama Yusuke, Panatala Radhakrishnan, Lim Roderick Y.H.Structural Dynamics of the Nuclear Pore Complex.Seminars in Cell and Developmental Biology http://dx.doi.org/10.1016/j.semcdb.2017.05.021 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Structural Dynamics of the Nuclear Pore Complex Yusuke Sakiyama1,†, Radhakrishnan Panatala1,†, Roderick Y.H. Lim1,* 1
Biozentrum and the Swiss Nanoscience Institute, University of Basel, Klingelbergstrasse 70,
4056 Basel, Switzerland *
Corresponding Author:
Roderick Y. H. Lim Biozentrum and the Swiss Nanoscience Institute University of Basel, Klingelbergstrasse 70 4056 Basel, Switzerland Phone: +41 61 267 2083 Fax: +41 61 267 2109 E-mail: [email protected] †These
authors contributed equally.
Abstract Nuclear pore complexes (NPCs) are the sole conduits that facilitate macromolecular exchange between the nucleus and cytosol. Recent advancements have led to a more highly resolved NPC structure. However, our understanding of the NPC modus operandi that facilitates transport selectivity, and speed, of diverse cargoes remains incomplete. For the most part, assorted cargo-complexes of different sizes traverse the NPC central channel in milliseconds, yet little is known about the nanoscopic movements of its barrier-forming Phe-Gly nucleoporins (FG Nups) and related sub-structures at transport-relevant time and length scales. Here, we discuss how such dynamic FG Nups may confer NPCs an effective permeability barrier according to the functional needs of the cell. Moreover, we postulate that such structural flexibility might resonate throughout the NPC framework from the cytoplasmic filaments to the nuclear basket. Keywords: Nuclear pore complex; intrinsically disordered proteins; structural dynamics; central plug; nuclear basket; high-speed atomic force microscopy
Table of Contents 1
Introduction
1.1
Nucleocytoplasmic Transport
1.2
NPC Components
1.3
NPC Barrier Models
1.3.1
The Selective Phase
1.3.2
Virtual Gating/Polymer Brush
1.3.3
Kap-Centric Control
2
NPC Structure Determination
2.1
Static Measurements
2.2
Time-Resolved Measurements
2.3
NPC Dynamics Resolved by High-Speed Atomic Force Microscopy
3
Outlook
1
Introduction
1.1
Nucleocytoplasmic Transport Nucleocytoplasmic transport (NCT) is exceptionally selective, efficient and robust
within the complex cellular milieu [1–3]. This underpins how specific proteins and mRNA are continuously exchanged across the nuclear envelope (NE) being perforated by nuclear pore complexes (NPCs) [4–6] that link the nuclear genome to the site of protein synthesis viz. the endoplasmic reticulum (ER) in the cytosol [7]. Though NPCs form the only gateways between nucleus and the cytosol, they are not simple pores. Each NPC is a multiprotein complex that amounts to an overall mass of ∼60 MDa [8] and ∼120 MDa [9] in yeast and metazoans, respectively. NPCs are assembled from approximately 30 different proteins known as nucleoporins (Nups) [10], which are present in multiples of eight conferring a characteristic octagonal structure that spans speciesdependent diameters of 50 to 100 nm [11–13]. The NPC central channel is adorned with several intrinsically disordered nucleoporins termed FG Nups being rich in phenylalanineglycine (FG) repeats [14]. Although the NPC is permeable to small molecules below ~40 kDa, the FG Nups form a selective barrier that generally impedes large non-specific macromolecules [15,16]. Rapid and exclusive access through the NPC is reserved for soluble nuclear transport receptors [17] (i.e., karyopherins or Kaps) that bind the FG Nups. In this manner, the classical 97 kDa import receptor karyopherinβ1 (Kapβ1 or importinβ [18] or Kap95 in yeast) navigates the pore in milliseconds [19]. Hence, NPCs regulate cargo translocation via biochemical selectivity and not size exclusion per se. During import, Kapβ1 identifies, binds and shuttles specific cargoes that contain nuclear localization signals (i.e., NLS) using Kap/importin- as an adaptor [20]. Kap-cargo
translocation is itself a diffusional process [21], whereas cargo directionality is regulated by the GTPase Ran, which exists in GTP- and GDP-bound forms localized to the nucleus and cytosol, respectively [22]. In the nucleus, RanGTP binds Kapβ1 to concomitantly release imported cargoes from Kapβ1. Following its return to the cytosol, RanGTP is hydrolyzed by RanGAP (Ran GTPase-activating protein) to RanGDP, which releases Kapβ1 for another import cycle [23]. RanGDP is then recycled to the nucleus by its specific carrier, NTF2 (nuclear transport factor 2) [24]. The Ran loop is finally closed by RCC1 (RanGEF, Ran Guanine nucleotide Exchange Factor), which catalyzes the recharging of RanGDP to RanGTP in the nucleus [23]. Presently, the NPC modus operandi that facilitates the selectivity and speed of signalspecific cargoes remains unclear. To be precise, nucleocytoplasmic transport in vivo proceeds rapidly through NPCs in a matter of milliseconds. In contrast, our understanding of FG Nup behavior has derived predominantly from static (i.e., time-independent) in vitro experimentation. Although significant strides in cryo-electron microscopy have led to a highly resolved ~20 Å structure of the NPC [25,26], the FG Nups themselves remain unresolved. The purpose of this review is to contemplate NPC function as a dynamic transport machine. This is exemplified by high-speed atomic force microscopy (HS-AFM) movie sequences that reveal nanoscopic dynamic movements within the FG Nups, cytoplasmic filaments and the nuclear basket of native NPCs for the first time [27]. 1.2
NPC Components Nups can be divided into four groups based on their amino acid sequence motifs,
structural characteristics and position within the NPC (Figure 1a). These are the transmembrane (TM) Nups, scaffold Nups, barrier-forming FG Nups, and, at its extremities the cytoplasmic filaments and nuclear basket Nups. TM Nups, which comprise of Gp210,
Pom121 and Ndc1 in vertebrates, anchor NPCs to the NE [28] and play a key role in NPC biogenesis and assembly [29]. Scaffold Nups link the FG Nups to the TM Nups, and consist of three sub-complexes – the outer ring Nups, inner ring Nups and linker Nups [28]. The outer ring Nups form a Y-shaped complex that stabilizes the highly-curved pore membrane [30] and may exhibit a hinge-like motion during NCT [31]. The FG Nups are intrinsically disordered proteins that harbor FG-binding sites for Kaps in order to facilitate selective transport [14] whilst simultaneously impeding the entry of non-specific cargoes. The FG Nups consist of nearly one-third of all nucleoporins and line the NPC from the cytoplasmic filaments to the central channel and all the way to the distal ring. We refer the reader to Refs. [32,33] for expert reviews on the Nups. 1.3
NPC Barrier Models
1.3.1 The Selective Phase Görlich et al. proposed that cohesive hydrophobic interactions among FG repeats cross-link the FG Nups into a sieve-like hydrogel or selective phase, and that its mesh size sets the cutoff limit for non-specific cargoes (Figure 1b). In this regard, Kapβ1 complexes translocated through a wtNsp1 hydrogel up to 20’000 times faster than inert macromolecules of a similar size [34]. In contrast, a mutant-Nsp1 where Phe residues were substituted with Ser did not form gels [35]. Subsequently, Nup98-depleted nuclei were incapable of cargo accumulation whereas adding back highly cohesive Nup98 helped to restore active nuclear transport and the permeability barrier [36]. Later, translocation experiments conducted with a mix of different FG Nups viz. Nup62, Nup54, Nup58 indicated that the selectivity barrier performed more effectively in a composite gel state than in an individual gel state [37]. Additionally, TEM analysis revealed that the FG Nups formed highly ordered amyloid fibrils
that aggregated into a hydrogel harboring sub-100 nm-diameter pores under molecular crowding conditions [38]. 1.3.2 Virtual Gating/Polymer Brush Rout et al. proposed that the NPC acted as a ‘virtual gate’ where the bristling motions of the FG Nups would restrict non-specific macromolecules from crossing the central channel [39] (Figure 1b). They then postulated that FG Nup-binding would enhance the probability of Kap-cargo complexes to translocate across the central channel. This idea was supported by Lim et al. who found that surface-tethered Nup153 exhibited polymer brush-like behavior by extending beyond the hydrodynamic radius of the molecule [40]. Moreover, the binding of low nM concentrations of Kapβ1 led to a compaction of the Nup153 brushes, which was corroborated in situ in Xenopus oocyte NPCs by immunogold electron microscopy [41]. 1.3.3 Kap-Centric Control Kapβ1 consists of approximately 10 hydrophobic grooves that can all potentially bind FG-repeats [42]. Although individual FG-repeats exert multiple fast low affinity interactions with a Kap [43,44], how this facilitates fast selective transport remains unclear. Our own data indicates that the affinity and kinetics of Kap1-FG Nup binding is modulated by Kap1 concentration due to multivalent binding with the FG Nups [45,46]. Using surface plasmon resonance, we uncovered the emergence of fast exchange kinetics where Kaps bind weakly to the FG Nup layer at physiological Kap concentrations (∼10 µM) due to limited penetration into the preoccupied FG Nups. This also impacted on other transport factors, such as, the Ran importer NTF2 [47]. Interestingly, such an effect was used to regulate the two-dimensional diffusion of colloidal beads functionalized with Kap1 on a surface of FG Nups [48] thereby recapitulating reduction of dimensionality as proposed by Peters [49]. Importantly,
experiments now show that Kapβ1 exhibits at least two kinetically distinct pools in digitoninpermeabilized cells [50]. On this basis, the model of Kap-centric control (Figure 1b) proposes that Kaps constitute bona fide constituents of the NPC to regulate transport selectivity and speed [51]. 2
NPC Structure Determination
2.1
Static Measurements The structure of the NPC has been historically and extensively studied by electron
microscopy (EM) [52]. Using energy-filtering transmission electron microscopy (EFTEM), Stoffler et al. obtained the first tomographic 3-D reconstruction of native X. laevis NPCs [13] (Figure 2a). Following the development of cryo-electron tomography (cryo-ET) [5,53], the human NPC structure has now been resolved to 23 Å using a direct electron detector [25] (Figure 2b). Then, by docking nucleoporin crystal structures onto the reconstructed human NPC, Lin et al. showed that flexible linker sequences mediated the assembly of the inner ring complex [54]. Subsequently, the authors proposed that the FG-repeats of FG Nups would emanate circumferentially toward the adjacent spoke rather than pointing radially to the center of the channel. Further cryo-ET structural analysis by Eibauer et al. resolved the X. laevis NPC up to 20 Å at different states of transport. They inferred that the FG Nups assemble to form two molecular gates consisting of a selective phase at the central channel ring and a polymer brush-like entropic barrier at the nucleoplasmic ring [26] (Figure 2c). Despite achieving significant gains in spatial resolution, EM methods require different treatments such as sample fixation/freezing and structural averaging procedures that preclude resolving the dynamic conformations of the FG Nups and other sub-structures. The same holds true for super resolution (SR) microscopy approaches, which was used to
structurally assess the NPC scaffold [55] and to visualize the octameric ring and FG repeats within the central channel [56] (Figure 2d). Nevertheless, static snapshots of conformational changes in the FG Nups have been acquired using immunogold labels under different transport conditions [57,58]. Fluorescence methods have also been used to study the NPC permeability barrier under different transport conditions. For instance, single-point edgeexcitation diffraction (SPEED) microscopy was used to obtain the three-dimensional transport routes of passive diffusion and facilitated translocation through native NPCs [59]. Using fluorescently labeled Kaps or FG domains, Ma et al could identify sub-regions of FG repeats within the NPC central channel and the related Kap-FG interaction zones [60]. An advantage of using atomic force microscopy (AFM) is that it allows for imaging and stiffness measurements [61] of native NPCs. Nevertheless, conventional AFM is limited to slow imaging speeds (~1 min per image) as well as spatial resolution. Still, this was sufficient to resolve the opening and closing of the nuclear basket with respect to changing Ca +2 conditions [62] (Figure 2e). In other AFM studies, Kramer et al. investigated the NPC channel in the absence and presence of Kapβ1 [63,64], and determined that the NPC undergoes nanoscopic changes with the addition of cytochrome C to initiate apoptosis [65] (Figure 2f). 2.2
Time-Resolved Measurements NCT in vivo proceeds rapidly through NPCs. Kaps import in ~5 ms [19] while 3 MDa
mRNPs translocate quickly in less than 20 ms but spend an overall time of ~200 ms between docking and release at the nuclear basket and cytoplasmic [66]. Hence, it is the dynamic spatiotemporal behavior of the FG Nups and not their static properties (i.e., timeindependent) that regulates NPC transport. Towards this goal, Cardarelli et al. managed to resolve FG Nup dynamics using fluorescence correlation spectroscopy to track in real-time
the rapid motion of Nup153 within the NPC [67]. Interestingly, this coincided with Kapβ1 movement during nuclear import suggesting that FG Nup-mediated molecular motion might represent an intrinsic feature of selective gating in intact NPCs [58]. Nevertheless, it should be noted that the FG Nups themselves remained structurally invisible in such fluorescencebased studies. 2.3
NPC Dynamics Resolved by High-Speed Atomic Force Microscopy We recently employed HS-AFM to resolve the dynamic FG Nup behavior within
individual NPCs obtained from X. laevis NPCs [27]. A key advantage of HS-AFM lies in being able to capture molecules-at-work in their native environments at a spatial-temporal resolution of approximately 1 nm and 100 ms, respectively [68]. In marked contrast, HS-AFM records molecular movies consisting of several hundred frames per minute in comparison to a conventional AFM that is limited to capturing an individual image in the same amount of time. Moreover, disturbances to the FG Nups are absolutely minimized by the application of low piconewton forces and microsecond tip-sample contact times [69]. A gallery of various HS-AFM movie sequences obtained under physiological conditions is shown in Figure 3. Starting in Fig. 3a, several NPCs can be seen from the cytoplasmic face in a typical zoomed out image of the NE. Surrounding the central channel of each NPC are eight protruding cytoplasmic filaments that exhibit nanoscopic “rocking” movements as shown in Fig. 3b. Closer inspection of different NPCs further reveals the presence of occlusions at their central channels, which is common for approximately 50% of all NPCs observed. Zoom-ins of two different NPCs in Fig. 3c reveal the presence of macromolecular particles being flanked by the rapid dynamic movements of nanoscopic filaments being the FG Nups. Previously [27], we had shown that the FG Nups fluctuate dynamically giving the
transient appearance of extended, retracted, entangled and radial conformational states in vacant NPCs (Fig. 3d). Here, because the particles exhibit smaller dynamic movements than the FG Nups, they are likely to be cargoes caught-in-transit or so-called “central plugs”. Still, it cannot be ruled out if larger, less mobile FG Nup condensates are forming in the pores [70]. We had also used HS-AFM to resolve the eight nuclear filaments that assemble into a distal ring in gluteraldehyde-fixed nuclear baskets [27]. In the absence of fixation, distal rings can exhibit either opened or closed states (Fig. 4), which suggests that specific signals such as Kap-binding, or Ca2+ [62] might shift the equilibrium to either conformation. Outlook It remains challenging to resolve the motion of functional structures in the NPC at the millisecond timescales that govern cargo translocation. Using HS-AFM, we have shown that dynamic fluctuations dominate FG Nup behavior, although they can also transiently intermingle [27]. Whether these short-lived interactions are facilitated by physicochemical forces or result from physical entanglements remains unknown. Moreover, it is unresolved if similar behavior is exhibited by the FG Nups located deeper in the central channel. Regardless, this dynamic behavior manifests characteristics of the selective phase [34,35] and the polymer brush/virtual gating [6,40,41] and in doing so reconciles static interpretations of the FG Nup barrier. Accordingly, we hypothesize that the NPC permeability barrier depends effectively on the observational frame of reference of incoming cargoes in relation to the rapid conformational changes in the FG Nups [27]. This argues that the appearance of the FG Nups is likely different to different cargoes depending on the timescales of their encounter and the size of the cargo complex. Hence, a small molecule that diffuses quickly has a high probability
to enter the NPC, as it would encounter less FG Nup fluctuations within a narrow time window (i.e., a lower barrier). In contrast, large non-specific cargoes that diffuse more slowly would experience a more pronounced barrier given the collective cloud-like motion of the FG Nups within a longer time window. Clearly, a combination of all-atom molecular dynamics and coarse-grained simulations [70–75] (reviewed in [76]) should provide further insight into this process. Likewise, theoretical and computational approaches [77,78] may shed light on the molecular details underlying the mechanism of Kap-centric control. As shown above, other sub-structural components of the NPC show to exhibit dynamic behavior. This is consistent with computational [79] and AFM [80] studies that predict structural plasticity in the NPC being important for accommodating a wide variety of transport cargoes of differing sizes. Could the central channel dilate in some signal specific manner, such as via allosteric coupling between Nup58 and Nup54 in the mid-plane ring [81,82]? Or, are such movements prohibited within a fully assembled Nup62•58•54 complex [83]? Regardless, this raises intriguing questions as to how FG Nups facilitate large cargo translocation in the central channel [84], and on the other hand whether structural movements and NPC plasticity might serve a functional role. As a case in point, the distal ring of the nuclear basket lies directly below the central channel on the inner nuclear membrane. How do large virus capsids [85] bypass this structure to enter the nucleoplasm? Or, how do large mRNP export complexes [66] avoid the nuclear basket to enter the NPC from the nucleoplasm? Can specific signals trigger the opening and closing of the distal ring to alleviate such spatial constraints, such as by the RanGTPdependent turnover of Kap1 from Nup153 [50], or in a calcium-specific manner [62]? Taken together, the NPC is much more than a gated pore. It is physically coupled to the NE by TM Nups and the transduction of mechanical forces by linker of the nucleoskeleton and
cytoskeleton (LINC) complexes [86] might induce nanoscopic deformations within the NPC that would impact NCT. Other thought-provoking possibilities include the transient uncoupling of neighboring Nups to allow membrane-tethered proteins a translocate path that cuts across the NPC [87]. By surpassing static ‘averaged-out’ behavior, ultrafast imaging techniques such as HS-AFM provide the opportunity to watch native NPCs at work in space and time.
Acknowledgments Y.S. is funded by a PhD Fellowship from the Swiss Nanoscience Institute at the University of Basel. R.K. is funded by the Swiss National Science Foundation as part of the NCCR in Molecular Systems Engineering. R.Y.H.L. acknowledges support from the Swiss National Science Foundation grant 31003A_170041.
References [1] Fahrenkrog B, Aebi U. The nuclear pore complex: nucleocytoplasmic transport and beyond. Nat Rev Mol Cell Biol 2003;4:757–66. doi:10.1038/nrm1230. [2]
Nigg EA. Nucleocytoplasmic transport: signals, mechanisms and regulation. Nature 1997;386:779–87. doi:10.1038/386779a0.
[3]
Terry LJ, Shows EB, Wente SR. Crossing the nuclear envelope: hierarchical regulation of nucleocytoplasmic transport. Science 2007;318:1412–6. doi:10.1126/science.1142204.
[4]
Alber F, Dokudovskaya S, Veenhoff LM, Zhang W, Kipper J, Devos D, et al. The molecular architecture of the nuclear pore complex. Nature 2007;450:695–701. doi:10.1038/nature06405.
[5]
Beck M, Förster F, Ecke M, Plitzko JM, Melchior F, Gerisch G, et al. Nuclear pore complex structure and dynamics revealed by cryoelectron tomography. Science 2004;306:1387–90. doi:10.1126/science.1104808.
[6]
Rout MP, Aitchison JD, Suprapto A, Hjertaas K, Zhao Y, Chait BT. The yeast nuclear pore complex: composition, architecture, and transport mechanism. J Cell Biol 2000;148:635–51.
[7]
Strambio-De-Castillia C, Niepel M, Rout MP. The nuclear pore complex: bridging nuclear transport and gene regulation. Nat Rev Mol Cell Biol 2010;11:490–501. doi:10.1038/nrm2928.
[8]
Rout MP, Blobel G. Isolation of the yeast nuclear pore complex. J Cell Biol 1993;123:771–83.
[9]
Reichelt R, Holzenburg A, Buhle EL, Jarnik M, Engel A, Aebi U. Correlation between structure and mass distribution of the nuclear pore complex and of distinct pore complex components. J Cell Biol 1990;110:883–94.
[10] Cronshaw JM, Krutchinsky AN, Zhang W, Chait BT, Matunis MJ. Proteomic analysis of the mammalian nuclear pore complex. J Cell Biol 2002;158:915–27. doi:10.1083/jcb.200206106. [11] Elad N, Maimon T, Frenkiel-Krispin D, Lim RYH, Medalia O. Structural analysis of the
nuclear pore complex by integrated approaches. Curr Opin Struct Biol 2009;19:226– 32. doi:10.1016/j.sbi.2009.02.009. [12] Frenkiel-Krispin D, Maco B, Aebi U, Medalia O. Structural analysis of a metazoan nuclear pore complex reveals a fused concentric ring architecture. J Mol Biol 2010;395:578–86. doi:10.1016/j.jmb.2009.11.010. [13] Stoffler D, Feja B, Fahrenkrog B, Walz J, Typke D, Aebi U. Cryo-electron tomography provides novel insights into nuclear pore architecture: implications for nucleocytoplasmic transport. J Mol Biol 2003;328:119–30. doi:10.1016/S00222836(03)00266-3. [14] Yamada J, Phillips JL, Patel S, Goldfien G, Calestagne-Morelli A, Huang H, et al. A bimodal distribution of two distinct categories of intrinsically disordered structures with separate functions in FG nucleoporins. Mol Cell Proteomics 2010;9:2205–24. doi:10.1074/mcp.M000035-MCP201. [15] Popken P, Ghavami A, Onck PR, Poolman B, Veenhoff LM. Size-dependent leak of soluble and membrane proteins through the yeast nuclear pore complex. Mol Biol Cell 2015;26:1386–94. doi:10.1091/mbc.E14-07-1175. [16] Timney BL, Raveh B, Mironska R, Trivedi JM, Kim SJ, Russel D, et al. Simple rules for passive diffusion through the nuclear pore complex. J Cell Biol 2016;215:57–76. doi:10.1083/jcb.201601004. [17] Chook YM, Süel KE. Nuclear import by karyopherin-βs: recognition and inhibition. Biochim Biophys Acta 2011;1813:1593–606. doi:10.1016/j.bbamcr.2010.10.014. [18] Bayliss R, Littlewood T, Stewart M. Structural basis for the interaction between FxFG nucleoporin repeats and importin-beta in nuclear trafficking. Cell 2000;102:99–108. [19] Dange T, Grünwald D, Grünwald A, Peters R, Kubitscheck U. Autonomy and robustness of translocation through the nuclear pore complex: a single-molecule study. J Cell Biol 2008;183:77–86. doi:10.1083/jcb.200806173. [20] Lange A, Mills RE, Lange CJ, Stewart M, Devine SE, Corbett AH. Classical nuclear localization signals: definition, function, and interaction with importin alpha. J Biol Chem 2007;282:5101–5. doi:10.1074/jbc.R600026200.
[21] Yang W, Gelles J, Musser SM. Imaging of single-molecule translocation through nuclear pore complexes. Proc Natl Acad Sci U S A 2004;101:12887–92. doi:10.1073/pnas.0403675101. [22] Görlich D, Panté N, Kutay U, Aebi U, Bischoff FR. Identification of different roles for RanGDP and RanGTP in nuclear protein import. EMBO J 1996;15:5584–94. [23] Stewart M. Molecular mechanism of the nuclear protein import cycle. Nat Rev Mol Cell Biol 2007;8:195–208. doi:10.1038/nrm2114. [24] Ribbeck K, Lipowsky G, Kent HM, Stewart M, Görlich D. NTF2 mediates nuclear import of Ran. EMBO J 1998;17:6587–98. doi:10.1093/emboj/17.22.6587. [25] Appen A von, Kosinski J, Sparks L, Ori A, DiGuilio AL, Vollmer B, et al. In situ structural analysis of the human nuclear pore complex. Nature 2015;526:140–3. doi:10.1038/nature15381. [26] Eibauer M, Pellanda M, Turgay Y, Dubrovsky A, Wild A, Medalia O. Structure and gating of the nuclear pore complex. Nat Commun 2015;6:7532. doi:10.1038/ncomms8532. [27] Sakiyama Y, Mazur A, Kapinos LE, Lim RYH. Spatiotemporal dynamics of the nuclear pore complex transport barrier resolved by high-speed atomic force microscopy. Nat Nanotechnol 2016;11:719–23. doi:10.1038/nnano.2016.62. [28] Grossman E, Medalia O, Zwerger M. Functional architecture of the nuclear pore complex. Annu Rev Biophys 2012;41:557–84. doi:10.1146/annurev-biophys-050511102328. [29] Rothballer A, Kutay U. Poring over pores: nuclear pore complex insertion into the nuclear envelope. Trends Biochem Sci 2013;38:292–301. doi:10.1016/j.tibs.2013.04.001. [30] Kampmann M, Blobel G. Three-dimensional structure and flexibility of a membranecoating module of the nuclear pore complex. Nat Struct Mol Biol 2009;16:782–8. doi:10.1038/nsmb.1618. [31] Debler EW, Ma Y, Seo H-S, Hsia K-C, Noriega TR, Blobel G, et al. A fence-like coat for the nuclear pore membrane. Mol Cell 2008;32:815–26.
doi:10.1016/j.molcel.2008.12.001. [32] Wente SR, Rout MP. The nuclear pore complex and nuclear transport. Cold Spring Harb Perspect Biol 2010;2:a000562. doi:10.1101/cshperspect.a000562. [33] Knockenhauer KE, Schwartz TU. The nuclear pore complex as a flexible and dynamic gate. Cell 2016;164:1162–71. doi:10.1016/j.cell.2016.01.034. [34] Frey S, Görlich D. FG/FxFG as well as GLFG repeats form a selective permeability barrier with self-healing properties. EMBO J 2009;28:2554–67. doi:10.1038/emboj.2009.199. [35] Frey S, Görlich D. A saturated FG-repeat hydrogel can reproduce the permeability properties of nuclear pore complexes. Cell 2007;130:512–23. doi:10.1016/j.cell.2007.06.024. [36] Hülsmann BB, Labokha AA, Görlich D. The permeability of reconstituted nuclear pores provides direct evidence for the selective phase model. Cell 2012;150:738–51. doi:10.1016/j.cell.2012.07.019. [37] Labokha AA, Gradmann S, Frey S, Hülsmann BB, Urlaub H, Baldus M, et al. Systematic analysis of barrier-forming FG hydrogels from Xenopus nuclear pore complexes. EMBO J 2013;32:204–18. doi:10.1038/emboj.2012.302. [38] Milles S, Huy Bui K, Koehler C, Eltsov M, Beck M, Lemke EA. Facilitated aggregation of FG nucleoporins under molecular crowding conditions. EMBO Rep 2013;14:178–83. doi:10.1038/embor.2012.204. [39] Rout MP, Aitchison JD, Magnasco MO, Chait BT. Virtual gating and nuclear transport: the hole picture. Trends Cell Biol 2003;13:622–8. doi:10.1016/j.tcb.2003.10.007. [40] Lim RYH, Huang N-P, Köser J, Deng J, Lau KHA, Schwarz-Herion K, et al. Flexible phenylalanine-glycine nucleoporins as entropic barriers to nucleocytoplasmic transport. Proc Natl Acad Sci U S A 2006;103:9512–7. doi:10.1073/pnas.0603521103. [41] Lim RYH, Fahrenkrog B, Köser J, Schwarz-Herion K, Deng J, Aebi U. Nanomechanical basis of selective gating by the nuclear pore complex. Science 2007;318:640–3. doi:10.1126/science.1145980.
[42] Isgro TA, Schulten K. Binding dynamics of isolated nucleoporin repeat regions to importin-beta. Structure 2005;13:1869–79. doi:10.1016/j.str.2005.09.007. [43] Milles S, Mercadante D, Aramburu IV, Jensen MR, Banterle N, Koehler C, et al. Plasticity of an ultrafast interaction between nucleoporins and nuclear transport receptors. Cell 2015;163:734–45. doi:10.1016/j.cell.2015.09.047. [44] Hough LE, Dutta K, Sparks S, Temel DB, Kamal A, Tetenbaum-Novatt J, et al. The molecular mechanism of nuclear transport revealed by atomic-scale measurements. Elife 2015;4. doi:10.7554/eLife.10027. [45] Schoch RL, Kapinos LE, Lim RYH. Nuclear transport receptor binding avidity triggers a self-healing collapse transition in FG-nucleoporin molecular brushes. Proc Natl Acad Sci U S A 2012;109:16911–6. doi:10.1073/pnas.1208440109. [46] Kapinos LE, Schoch RL, Wagner RS, Schleicher KD, Lim RYH. Karyopherin-centric control of nuclear pores based on molecular occupancy and kinetic analysis of multivalent binding with FG nucleoporins. Biophys J 2014;106:1751–62. doi:10.1016/j.bpj.2014.02.021. [47] Wagner RS, Kapinos LE, Marshall NJ, Stewart M, Lim RYH. Promiscuous binding of Karyopherinβ1 modulates FG nucleoporin barrier function and expedites NTF2 transport kinetics. Biophys J 2015;108:918–27. doi:10.1016/j.bpj.2014.12.041. [48] Schleicher KD, Dettmer SL, Kapinos LE, Pagliara S, Keyser UF, Jeney S, et al. Selective transport control on molecular velcro made from intrinsically disordered proteins. Nat Nanotechnol 2014;9:525–30. doi:10.1038/nnano.2014.103. [49] Peters R. Translocation through the nuclear pore complex: selectivity and speed by reduction-of-dimensionality. Traffic 2005;6:421–7. doi:10.1111/j.16000854.2005.00287.x. [50] Lowe AR, Tang JH, Yassif J, Graf M, Huang WYC, Groves JT, et al. Importin-β modulates the permeability of the nuclear pore complex in a Ran-dependent manner. Elife 2015;4. doi:10.7554/eLife.04052. [51] Lim RYH, Huang B, Kapinos LE. How to operate a nuclear pore complex by Kap-centric control. Nucleus 2015;6:366–72. doi:10.1080/19491034.2015.1090061.
[52] Akey CW, Goldfarb DS. Protein import through the nuclear pore complex is a multistep process. J Cell Biol 1989;109:971–82. [53] Beck M, Lucić V, Förster F, Baumeister W, Medalia O. Snapshots of nuclear pore complexes in action captured by cryo-electron tomography. Nature 2007;449:611–5. doi:10.1038/nature06170. [54] Lin DH, Stuwe T, Schilbach S, Rundlet EJ, Perriches T, Mobbs G, et al. Architecture of the symmetric core of the nuclear pore. Science 2016;352:aaf1015. doi:10.1126/science.aaf1015. [55] Szymborska A, de Marco A, Daigle N, Cordes VC, Briggs JAG, Ellenberg J. Nuclear pore scaffold structure analyzed by super-resolution microscopy and particle averaging. Science 2013;341:655–8. doi:10.1126/science.1240672. [56] Göttfert F, Wurm CA, Mueller V, Berning S, Cordes VC, Honigmann A, et al. Coaligned dual-channel STED nanoscopy and molecular diffusion analysis at 20 nm resolution. Biophys J 2013;105:L01–3. doi:10.1016/j.bpj.2013.05.029. [57] Paulillo SM, Powers MA, Ullman KS, Fahrenkrog B. Changes in nucleoporin domain topology in response to chemical effectors. J Mol Biol 2006;363:39–50. doi:10.1016/j.jmb.2006.08.021. [58] Fahrenkrog B, Maco B, Fager AM, Köser J, Sauder U, Ullman KS, et al. Domain-specific antibodies reveal multiple-site topology of Nup153 within the nuclear pore complex. J Struct Biol 2002;140:254–67. [59] Ma J, Goryaynov A, Sarma A, Yang W. Self-regulated viscous channel in the nuclear pore complex. Proc Natl Acad Sci U S A 2012;109:7326–31. doi:10.1073/pnas.1201724109. [60] Ma J, Goryaynov A, Yang W. Super-resolution 3D tomography of interactions and competition in the nuclear pore complex. Nat Struct Mol Biol 2016;23:239–47. doi:10.1038/nsmb.3174. [61] Bestembayeva A, Kramer A, Labokha AA, Osmanović D, Liashkovich I, Orlova EV, et al. Nanoscale stiffness topography reveals structure and mechanics of the transport barrier in intact nuclear pore complexes. Nat Nanotechnol 2015;10:60–4. doi:10.1038/nnano.2014.262.
[62] Stoffler D, Goldie KN, Feja B, Aebi U. Calcium-mediated structural changes of native nuclear pore complexes monitored by time-lapse atomic force microscopy. J Mol Biol 1999;287:741–52. doi:10.1006/jmbi.1999.2637. [63] Kramer A, Liashkovich I, Ludwig Y, Shahin V. Atomic force microscopy visualises a hydrophobic meshwork in the central channel of the nuclear pore. Pflugers Arch 2008;456:155–62. doi:10.1007/s00424-007-0396-y. [64] Kramer A, Ludwig Y, Shahin V, Oberleithner H. A pathway separate from the central channel through the nuclear pore complex for inorganic ions and small macromolecules. J Biol Chem 2007;282:31437–43. doi:10.1074/jbc.M703720200. [65] Kramer A, Liashkovich I, Oberleithner H, Ludwig S, Mazur I, Shahin V. Apoptosis leads to a degradation of vital components of active nuclear transport and a dissociation of the nuclear lamina. Proc Natl Acad Sci U S A 2008;105:11236–41. doi:10.1073/pnas.0801967105. [66] Grünwald D, Singer RH. In vivo imaging of labelled endogenous β-actin mRNA during nucleocytoplasmic transport. Nature 2010;467:604–7. doi:10.1038/nature09438. [67] Cardarelli F, Lanzano L, Gratton E. Capturing directed molecular motion in the nuclear pore complex of live cells. Proc Natl Acad Sci U S A 2012;109:9863–8. doi:10.1073/pnas.1200486109. [68] Ando T, Uchihashi T, Fukuma T. High-speed atomic force microscopy for nanovisualization of dynamic biomolecular processes. Prog Surf Sci 2008;83:337–437. doi:10.1016/j.progsurf.2008.09.001. [69] Uchihashi T, Kodera N, Ando T. Guide to video recording of structure dynamics and dynamic processes of proteins by high-speed atomic force microscopy. Nat Protoc 2012;7:1193–206. doi:10.1038/nprot.2012.047. [70] Osmanovic D, Bailey J, Harker AH, Fassati A, Hoogenboom BW, Ford IJ. Bistable collective behavior of polymers tethered in a nanopore. Phys Rev E Stat Nonlin Soft Matter Phys 2012;85:061917. doi:10.1103/PhysRevE.85.061917. [71] Moussavi-Baygi R, Mofrad MRK. Rapid Brownian Motion Primes Ultrafast Reconstruction of Intrinsically Disordered Phe-Gly Repeats Inside the Nuclear Pore
Complex. Sci Rep 2016;6:29991. doi:10.1038/srep29991. [72] Ando D, Gopinathan A. Cooperative Interactions between Different Classes of Disordered Proteins Play a Functional Role in the Nuclear Pore Complex of Baker’s Yeast. PLoS ONE 2017;12:e0169455. doi:10.1371/journal.pone.0169455. [73] Mincer JS, Simon SM. Simulations of nuclear pore transport yield mechanistic insights and quantitative predictions. Proc Natl Acad Sci U S A 2011;108:E351–8. doi:10.1073/pnas.1104521108. [74] Gamini R, Han W, Stone JE, Schulten K. Assembly of Nsp1 nucleoporins provides insight into nuclear pore complex gating. PLoS Comput Biol 2014;10:e1003488. doi:10.1371/journal.pcbi.1003488. [75] Tagliazucchi M, Peleg O, Kröger M, Rabin Y, Szleifer I. Effect of charge, hydrophobicity, and sequence of nucleoporins on the translocation of model particles through the nuclear pore complex. Proc Natl Acad Sci U S A 2013;110:3363–8. doi:10.1073/pnas.1212909110. [76] Osmanović D, Fassati A, Ford IJ, Hoogenboom BW. Physical modelling of the nuclear pore complex. Soft Matter 2013;9:10442. doi:10.1039/c3sm50722j. [77] Vovk A, Gu C, Opferman MG, Kapinos LE, Lim RY, Coalson RD, et al. Simple biophysics underpins collective conformations of the intrinsically disordered proteins of the Nuclear Pore Complex. Elife 2016;5. doi:10.7554/eLife.10785. [78] Zahn R, Osmanović D, Ehret S, Araya Callis C, Frey S, Stewart M, et al. A physical model describing the interaction of nuclear transport receptors with FG nucleoporin domain assemblies. Elife 2016;5. doi:10.7554/eLife.14119. [79] Wolf C, Mofrad MRK. On the octagonal structure of the nuclear pore complex: insights from coarse-grained models. Biophys J 2008;95:2073–85. doi:10.1529/biophysj.108.130336. [80] Liashkovich I, Meyring A, Kramer A, Shahin V. Exceptional structural and mechanical flexibility of the nuclear pore complex. J Cell Physiol 2011;226:675–82. doi:10.1002/jcp.22382. [81] Koh J, Blobel G. Allosteric regulation in gating the central channel of the nuclear pore
complex. Cell 2015;161:1361–73. doi:10.1016/j.cell.2015.05.013. [82] Solmaz SR, Chauhan R, Blobel G, Melčák I. Molecular architecture of the transport channel of the nuclear pore complex. Cell 2011;147:590–602. doi:10.1016/j.cell.2011.09.034. [83] Chug H, Trakhanov S, Hülsmann BB, Pleiner T, Görlich D. Crystal structure of the metazoan Nup62•Nup58•Nup54 nucleoporin complex. Science 2015;350:106–10. doi:10.1126/science.aac7420. [84] Tu L-C, Fu G, Zilman A, Musser SM. Large cargo transport by nuclear pores: implications for the spatial organization of FG-nucleoporins. EMBO J 2013;32:3220–30. doi:10.1038/emboj.2013.239. [85] Rabe B, Vlachou A, Panté N, Helenius A, Kann M. Nuclear import of hepatitis B virus capsids and release of the viral genome. Proc Natl Acad Sci U S A 2003;100:9849–54. doi:10.1073/pnas.1730940100. [86] Ungricht R, Kutay U. Mechanisms and functions of nuclear envelope remodelling. Nat Rev Mol Cell Biol 2017;18:229–45. doi:10.1038/nrm.2016.153. [87] Meinema AC, Laba JK, Hapsari RA, Otten R, Mulder FAA, Kralt A, et al. Long unfolded linkers facilitate membrane protein import through the nuclear pore complex. Science 2011;333:90–3. doi:10.1126/science.1205741.
Figure Captions Figure 1: (a) Schematic representation of nucleoporin sub-structures within a vertebrate NPC. Darker colors denote dynamic components including the barrier-forming FG Nups. (b) NPC barrier models. In all cases, small molecules (red watermarked) diffuse freely through the barrier whereas large non-specific molecules (red) are withheld. Only Kaps (dark green) can traverse the barrier due to binding with the FG-repeats. (Left) The Selective Phase consists of FG Nups that form an intertwined molecular sieve. (Center) Virtual Gating proposes that the FG Nups behave as polymer brush-like barrier. (Right) Kap-centric control argues that Kaps are integral constituents of the FG Nup barrier. Strongly bound Kaps (dark green) diffuse slowly (grey double-headed arrow) whereas weakly bound Kaps (light green) exhibit rapid translocation (black double-headed arrow). Figure 2: Static views of NPC structure. (a) 3D-reconstruction by zero-loss filtered electron tomography. CF = cytoplasmic filaments; CP = central plug; HA = “handling” points that anchor the NPC to the NE. Reprinted from Journal of Molecular Biology 328:1, Stoffler et.al., Cryoelectron Tomography Provides Novel Insights into Nuclear Pore Architecture: Implications for Nucleocytoplasmic Transport, 741-752 (2003) with permission from Elsevier. (b) Orthoslice through the nucleocytoplasmic axis of a human NPC obtained by CET. Reproduced with permission from Nature 526: 140-143 copyright (2015) Macmillan Magazines Ltd. (c) Tomogram of a native X. laevis NPC suggesting possible transport routes across the NPC (solid and dashed lines). Reproduced with permission from Nature Communications 6:7532 copyright (2015) Macmillan Magazines Ltd. (d) STED reveals octagonal subunits of a TM Nup and the central channel. Scale, 500 nm. Reprinted from Biophysical Journal 105:1 Goettfert et al., Coaligned Dual-Channel STED Nanoscopy and Molecular Diffusion Analysis at 20 nm
Resolution, L01-L03 (2013) with permission from Elsevier. (e) Calcium-mediated opening and closing of the distal ring by time-lapse AFM. Reprinted from Journal of Molecular Biology 287:4 , Stoffler et.al., Calcium-mediated structural changes of native nuclear pore complexes monitored by time-lapse atomic force microscopy, 741-752 (1999) with permission from Elsevier. (f) Nanoscopic changes to NPCs upon exposure to Cytochrome C images by AFM. Reprinted from Proc. Natl. Acad Sci USA 105:32, 11236-11241 Kramer et al., (2008) with permission from National Academy of Sciences. Figure 3: Dynamic views of X. laevis NPC structure obtained by HS-AFM (a) Zoomed out image reveals several NPCs that decorate the cytoplasmic-facing outer nuclear membrane. Scale, 100 nm. (Imaging parameters: 100 x 100 pixels, 400 x 400 nm. 780 ms/frame.) (b) Single NPC showing eight distinct cytoplasmic filaments. A “rocking” back-and-forth motion (denoted by arrows) was observed for an individual filament across consecutive image frames. Scale, 50 nm. (Imaging parameters: 200 x 200 pixels, 200 x 200 nm. 1990 ms/frame.) (c) (First row) Close inspection of the same NPC as (b) reveals occlusions within the central channel. (Imaging parameters: 200 x 200 pixels, 200 x 200 nm. 1990 ms/frame.) Scale, 50 nm. Zooming into the central channel further shows dynamic interactions between the FG Nups (white arrows) and the occluding particles (bright spots). Scale, 10 nm. (Imaging parameters: 80 x 80 pixels, 80 x 80 nm. 280 ms/frame.) (Second row) Particles seen occluding within a different NPC. (Imaging parameters: 200 x 200 pixels, 200 x 200 nm. 1990 ms/frame.) Zoom-ins show similar FG Nup dynamics as above. (Imaging parameters: 80 x 80 pixels, 70 x 70 nm 310 ms/frame.) Scale, same as first row (d) Behavior of a vacant NPC. Scale, 50 nm. (Imaging parameters: 100 x 100 pixels, 150 x 150 nm. 1360 ms/frame.) Zoom-ins show various FG Nup dynamic conformational states. From left to right: “radial”, “retracted”, “extended” and “entangled”. (Imaging parameters: 80x80 pixels, 70x70 nm, 180 ms/frame.)
Figure 4: HS-AFM reveals the nuclear basket of X. laevis NPCs. The nuclear filaments are seen to adopt either a (a) “closed” or (b) “open” distal ring conformation. Scale, 50nm. Imaging parameters: (a) 100x100 pixels, 180 nm, 1990 ms/frame. (b) 100x100 pixels, 200x200 nm. 1000 ms/frame. [Note: All samples were imaged in buffered solution without chemical treatments.]
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S1084-9521(16)30351-2 http://dx.doi.org/doi:10.1016/j.semcdb.2017.05.021 YSCDB 2227
To appear in:
Seminars in Cell & Developmental Biology
Received date: Accepted date:
22-5-2017 29-5-2017
Please cite this article as: Sakiyama Yusuke, Panatala Radhakrishnan, Lim Roderick Y.H.Structural Dynamics of the Nuclear Pore Complex.Seminars in Cell and Developmental Biology http://dx.doi.org/10.1016/j.semcdb.2017.05.021 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Structural Dynamics of the Nuclear Pore Complex Yusuke Sakiyama1,†, Radhakrishnan Panatala1,†, Roderick Y.H. Lim1,* 1
Biozentrum and the Swiss Nanoscience Institute, University of Basel, Klingelbergstrasse 70,
4056 Basel, Switzerland *
Corresponding Author:
Roderick Y. H. Lim Biozentrum and the Swiss Nanoscience Institute University of Basel, Klingelbergstrasse 70 4056 Basel, Switzerland Phone: +41 61 267 2083 Fax: +41 61 267 2109 E-mail: [email protected] †These
authors contributed equally.
Abstract Nuclear pore complexes (NPCs) are the sole conduits that facilitate macromolecular exchange between the nucleus and cytosol. Recent advancements have led to a more highly resolved NPC structure. However, our understanding of the NPC modus operandi that facilitates transport selectivity, and speed, of diverse cargoes remains incomplete. For the most part, assorted cargo-complexes of different sizes traverse the NPC central channel in milliseconds, yet little is known about the nanoscopic movements of its barrier-forming Phe-Gly nucleoporins (FG Nups) and related sub-structures at transport-relevant time and length scales. Here, we discuss how such dynamic FG Nups may confer NPCs an effective permeability barrier according to the functional needs of the cell. Moreover, we postulate that such structural flexibility might resonate throughout the NPC framework from the cytoplasmic filaments to the nuclear basket. Keywords: Nuclear pore complex; intrinsically disordered proteins; structural dynamics; central plug; nuclear basket; high-speed atomic force microscopy
Table of Contents 1
Introduction
1.1
Nucleocytoplasmic Transport
1.2
NPC Components
1.3
NPC Barrier Models
1.3.1
The Selective Phase
1.3.2
Virtual Gating/Polymer Brush
1.3.3
Kap-Centric Control
2
NPC Structure Determination
2.1
Static Measurements
2.2
Time-Resolved Measurements
2.3
NPC Dynamics Resolved by High-Speed Atomic Force Microscopy
3
Outlook
1
Introduction
1.1
Nucleocytoplasmic Transport Nucleocytoplasmic transport (NCT) is exceptionally selective, efficient and robust
within the complex cellular milieu [1–3]. This underpins how specific proteins and mRNA are continuously exchanged across the nuclear envelope (NE) being perforated by nuclear pore complexes (NPCs) [4–6] that link the nuclear genome to the site of protein synthesis viz. the endoplasmic reticulum (ER) in the cytosol [7]. Though NPCs form the only gateways between nucleus and the cytosol, they are not simple pores. Each NPC is a multiprotein complex that amounts to an overall mass of ∼60 MDa [8] and ∼120 MDa [9] in yeast and metazoans, respectively. NPCs are assembled from approximately 30 different proteins known as nucleoporins (Nups) [10], which are present in multiples of eight conferring a characteristic octagonal structure that spans speciesdependent diameters of 50 to 100 nm [11–13]. The NPC central channel is adorned with several intrinsically disordered nucleoporins termed FG Nups being rich in phenylalanineglycine (FG) repeats [14]. Although the NPC is permeable to small molecules below ~40 kDa, the FG Nups form a selective barrier that generally impedes large non-specific macromolecules [15,16]. Rapid and exclusive access through the NPC is reserved for soluble nuclear transport receptors [17] (i.e., karyopherins or Kaps) that bind the FG Nups. In this manner, the classical 97 kDa import receptor karyopherinβ1 (Kapβ1 or importinβ [18] or Kap95 in yeast) navigates the pore in milliseconds [19]. Hence, NPCs regulate cargo translocation via biochemical selectivity and not size exclusion per se. During import, Kapβ1 identifies, binds and shuttles specific cargoes that contain nuclear localization signals (i.e., NLS) using Kap/importin- as an adaptor [20]. Kap-cargo
translocation is itself a diffusional process [21], whereas cargo directionality is regulated by the GTPase Ran, which exists in GTP- and GDP-bound forms localized to the nucleus and cytosol, respectively [22]. In the nucleus, RanGTP binds Kapβ1 to concomitantly release imported cargoes from Kapβ1. Following its return to the cytosol, RanGTP is hydrolyzed by RanGAP (Ran GTPase-activating protein) to RanGDP, which releases Kapβ1 for another import cycle [23]. RanGDP is then recycled to the nucleus by its specific carrier, NTF2 (nuclear transport factor 2) [24]. The Ran loop is finally closed by RCC1 (RanGEF, Ran Guanine nucleotide Exchange Factor), which catalyzes the recharging of RanGDP to RanGTP in the nucleus [23]. Presently, the NPC modus operandi that facilitates the selectivity and speed of signalspecific cargoes remains unclear. To be precise, nucleocytoplasmic transport in vivo proceeds rapidly through NPCs in a matter of milliseconds. In contrast, our understanding of FG Nup behavior has derived predominantly from static (i.e., time-independent) in vitro experimentation. Although significant strides in cryo-electron microscopy have led to a highly resolved ~20 Å structure of the NPC [25,26], the FG Nups themselves remain unresolved. The purpose of this review is to contemplate NPC function as a dynamic transport machine. This is exemplified by high-speed atomic force microscopy (HS-AFM) movie sequences that reveal nanoscopic dynamic movements within the FG Nups, cytoplasmic filaments and the nuclear basket of native NPCs for the first time [27]. 1.2
NPC Components Nups can be divided into four groups based on their amino acid sequence motifs,
structural characteristics and position within the NPC (Figure 1a). These are the transmembrane (TM) Nups, scaffold Nups, barrier-forming FG Nups, and, at its extremities the cytoplasmic filaments and nuclear basket Nups. TM Nups, which comprise of Gp210,
Pom121 and Ndc1 in vertebrates, anchor NPCs to the NE [28] and play a key role in NPC biogenesis and assembly [29]. Scaffold Nups link the FG Nups to the TM Nups, and consist of three sub-complexes – the outer ring Nups, inner ring Nups and linker Nups [28]. The outer ring Nups form a Y-shaped complex that stabilizes the highly-curved pore membrane [30] and may exhibit a hinge-like motion during NCT [31]. The FG Nups are intrinsically disordered proteins that harbor FG-binding sites for Kaps in order to facilitate selective transport [14] whilst simultaneously impeding the entry of non-specific cargoes. The FG Nups consist of nearly one-third of all nucleoporins and line the NPC from the cytoplasmic filaments to the central channel and all the way to the distal ring. We refer the reader to Refs. [32,33] for expert reviews on the Nups. 1.3
NPC Barrier Models
1.3.1 The Selective Phase Görlich et al. proposed that cohesive hydrophobic interactions among FG repeats cross-link the FG Nups into a sieve-like hydrogel or selective phase, and that its mesh size sets the cutoff limit for non-specific cargoes (Figure 1b). In this regard, Kapβ1 complexes translocated through a wtNsp1 hydrogel up to 20’000 times faster than inert macromolecules of a similar size [34]. In contrast, a mutant-Nsp1 where Phe residues were substituted with Ser did not form gels [35]. Subsequently, Nup98-depleted nuclei were incapable of cargo accumulation whereas adding back highly cohesive Nup98 helped to restore active nuclear transport and the permeability barrier [36]. Later, translocation experiments conducted with a mix of different FG Nups viz. Nup62, Nup54, Nup58 indicated that the selectivity barrier performed more effectively in a composite gel state than in an individual gel state [37]. Additionally, TEM analysis revealed that the FG Nups formed highly ordered amyloid fibrils
that aggregated into a hydrogel harboring sub-100 nm-diameter pores under molecular crowding conditions [38]. 1.3.2 Virtual Gating/Polymer Brush Rout et al. proposed that the NPC acted as a ‘virtual gate’ where the bristling motions of the FG Nups would restrict non-specific macromolecules from crossing the central channel [39] (Figure 1b). They then postulated that FG Nup-binding would enhance the probability of Kap-cargo complexes to translocate across the central channel. This idea was supported by Lim et al. who found that surface-tethered Nup153 exhibited polymer brush-like behavior by extending beyond the hydrodynamic radius of the molecule [40]. Moreover, the binding of low nM concentrations of Kapβ1 led to a compaction of the Nup153 brushes, which was corroborated in situ in Xenopus oocyte NPCs by immunogold electron microscopy [41]. 1.3.3 Kap-Centric Control Kapβ1 consists of approximately 10 hydrophobic grooves that can all potentially bind FG-repeats [42]. Although individual FG-repeats exert multiple fast low affinity interactions with a Kap [43,44], how this facilitates fast selective transport remains unclear. Our own data indicates that the affinity and kinetics of Kap1-FG Nup binding is modulated by Kap1 concentration due to multivalent binding with the FG Nups [45,46]. Using surface plasmon resonance, we uncovered the emergence of fast exchange kinetics where Kaps bind weakly to the FG Nup layer at physiological Kap concentrations (∼10 µM) due to limited penetration into the preoccupied FG Nups. This also impacted on other transport factors, such as, the Ran importer NTF2 [47]. Interestingly, such an effect was used to regulate the two-dimensional diffusion of colloidal beads functionalized with Kap1 on a surface of FG Nups [48] thereby recapitulating reduction of dimensionality as proposed by Peters [49]. Importantly,
experiments now show that Kapβ1 exhibits at least two kinetically distinct pools in digitoninpermeabilized cells [50]. On this basis, the model of Kap-centric control (Figure 1b) proposes that Kaps constitute bona fide constituents of the NPC to regulate transport selectivity and speed [51]. 2
NPC Structure Determination
2.1
Static Measurements The structure of the NPC has been historically and extensively studied by electron
microscopy (EM) [52]. Using energy-filtering transmission electron microscopy (EFTEM), Stoffler et al. obtained the first tomographic 3-D reconstruction of native X. laevis NPCs [13] (Figure 2a). Following the development of cryo-electron tomography (cryo-ET) [5,53], the human NPC structure has now been resolved to 23 Å using a direct electron detector [25] (Figure 2b). Then, by docking nucleoporin crystal structures onto the reconstructed human NPC, Lin et al. showed that flexible linker sequences mediated the assembly of the inner ring complex [54]. Subsequently, the authors proposed that the FG-repeats of FG Nups would emanate circumferentially toward the adjacent spoke rather than pointing radially to the center of the channel. Further cryo-ET structural analysis by Eibauer et al. resolved the X. laevis NPC up to 20 Å at different states of transport. They inferred that the FG Nups assemble to form two molecular gates consisting of a selective phase at the central channel ring and a polymer brush-like entropic barrier at the nucleoplasmic ring [26] (Figure 2c). Despite achieving significant gains in spatial resolution, EM methods require different treatments such as sample fixation/freezing and structural averaging procedures that preclude resolving the dynamic conformations of the FG Nups and other sub-structures. The same holds true for super resolution (SR) microscopy approaches, which was used to
structurally assess the NPC scaffold [55] and to visualize the octameric ring and FG repeats within the central channel [56] (Figure 2d). Nevertheless, static snapshots of conformational changes in the FG Nups have been acquired using immunogold labels under different transport conditions [57,58]. Fluorescence methods have also been used to study the NPC permeability barrier under different transport conditions. For instance, single-point edgeexcitation diffraction (SPEED) microscopy was used to obtain the three-dimensional transport routes of passive diffusion and facilitated translocation through native NPCs [59]. Using fluorescently labeled Kaps or FG domains, Ma et al could identify sub-regions of FG repeats within the NPC central channel and the related Kap-FG interaction zones [60]. An advantage of using atomic force microscopy (AFM) is that it allows for imaging and stiffness measurements [61] of native NPCs. Nevertheless, conventional AFM is limited to slow imaging speeds (~1 min per image) as well as spatial resolution. Still, this was sufficient to resolve the opening and closing of the nuclear basket with respect to changing Ca +2 conditions [62] (Figure 2e). In other AFM studies, Kramer et al. investigated the NPC channel in the absence and presence of Kapβ1 [63,64], and determined that the NPC undergoes nanoscopic changes with the addition of cytochrome C to initiate apoptosis [65] (Figure 2f). 2.2
Time-Resolved Measurements NCT in vivo proceeds rapidly through NPCs. Kaps import in ~5 ms [19] while 3 MDa
mRNPs translocate quickly in less than 20 ms but spend an overall time of ~200 ms between docking and release at the nuclear basket and cytoplasmic [66]. Hence, it is the dynamic spatiotemporal behavior of the FG Nups and not their static properties (i.e., timeindependent) that regulates NPC transport. Towards this goal, Cardarelli et al. managed to resolve FG Nup dynamics using fluorescence correlation spectroscopy to track in real-time
the rapid motion of Nup153 within the NPC [67]. Interestingly, this coincided with Kapβ1 movement during nuclear import suggesting that FG Nup-mediated molecular motion might represent an intrinsic feature of selective gating in intact NPCs [58]. Nevertheless, it should be noted that the FG Nups themselves remained structurally invisible in such fluorescencebased studies. 2.3
NPC Dynamics Resolved by High-Speed Atomic Force Microscopy We recently employed HS-AFM to resolve the dynamic FG Nup behavior within
individual NPCs obtained from X. laevis NPCs [27]. A key advantage of HS-AFM lies in being able to capture molecules-at-work in their native environments at a spatial-temporal resolution of approximately 1 nm and 100 ms, respectively [68]. In marked contrast, HS-AFM records molecular movies consisting of several hundred frames per minute in comparison to a conventional AFM that is limited to capturing an individual image in the same amount of time. Moreover, disturbances to the FG Nups are absolutely minimized by the application of low piconewton forces and microsecond tip-sample contact times [69]. A gallery of various HS-AFM movie sequences obtained under physiological conditions is shown in Figure 3. Starting in Fig. 3a, several NPCs can be seen from the cytoplasmic face in a typical zoomed out image of the NE. Surrounding the central channel of each NPC are eight protruding cytoplasmic filaments that exhibit nanoscopic “rocking” movements as shown in Fig. 3b. Closer inspection of different NPCs further reveals the presence of occlusions at their central channels, which is common for approximately 50% of all NPCs observed. Zoom-ins of two different NPCs in Fig. 3c reveal the presence of macromolecular particles being flanked by the rapid dynamic movements of nanoscopic filaments being the FG Nups. Previously [27], we had shown that the FG Nups fluctuate dynamically giving the
transient appearance of extended, retracted, entangled and radial conformational states in vacant NPCs (Fig. 3d). Here, because the particles exhibit smaller dynamic movements than the FG Nups, they are likely to be cargoes caught-in-transit or so-called “central plugs”. Still, it cannot be ruled out if larger, less mobile FG Nup condensates are forming in the pores [70]. We had also used HS-AFM to resolve the eight nuclear filaments that assemble into a distal ring in gluteraldehyde-fixed nuclear baskets [27]. In the absence of fixation, distal rings can exhibit either opened or closed states (Fig. 4), which suggests that specific signals such as Kap-binding, or Ca2+ [62] might shift the equilibrium to either conformation. Outlook It remains challenging to resolve the motion of functional structures in the NPC at the millisecond timescales that govern cargo translocation. Using HS-AFM, we have shown that dynamic fluctuations dominate FG Nup behavior, although they can also transiently intermingle [27]. Whether these short-lived interactions are facilitated by physicochemical forces or result from physical entanglements remains unknown. Moreover, it is unresolved if similar behavior is exhibited by the FG Nups located deeper in the central channel. Regardless, this dynamic behavior manifests characteristics of the selective phase [34,35] and the polymer brush/virtual gating [6,40,41] and in doing so reconciles static interpretations of the FG Nup barrier. Accordingly, we hypothesize that the NPC permeability barrier depends effectively on the observational frame of reference of incoming cargoes in relation to the rapid conformational changes in the FG Nups [27]. This argues that the appearance of the FG Nups is likely different to different cargoes depending on the timescales of their encounter and the size of the cargo complex. Hence, a small molecule that diffuses quickly has a high probability
to enter the NPC, as it would encounter less FG Nup fluctuations within a narrow time window (i.e., a lower barrier). In contrast, large non-specific cargoes that diffuse more slowly would experience a more pronounced barrier given the collective cloud-like motion of the FG Nups within a longer time window. Clearly, a combination of all-atom molecular dynamics and coarse-grained simulations [70–75] (reviewed in [76]) should provide further insight into this process. Likewise, theoretical and computational approaches [77,78] may shed light on the molecular details underlying the mechanism of Kap-centric control. As shown above, other sub-structural components of the NPC show to exhibit dynamic behavior. This is consistent with computational [79] and AFM [80] studies that predict structural plasticity in the NPC being important for accommodating a wide variety of transport cargoes of differing sizes. Could the central channel dilate in some signal specific manner, such as via allosteric coupling between Nup58 and Nup54 in the mid-plane ring [81,82]? Or, are such movements prohibited within a fully assembled Nup62•58•54 complex [83]? Regardless, this raises intriguing questions as to how FG Nups facilitate large cargo translocation in the central channel [84], and on the other hand whether structural movements and NPC plasticity might serve a functional role. As a case in point, the distal ring of the nuclear basket lies directly below the central channel on the inner nuclear membrane. How do large virus capsids [85] bypass this structure to enter the nucleoplasm? Or, how do large mRNP export complexes [66] avoid the nuclear basket to enter the NPC from the nucleoplasm? Can specific signals trigger the opening and closing of the distal ring to alleviate such spatial constraints, such as by the RanGTPdependent turnover of Kap1 from Nup153 [50], or in a calcium-specific manner [62]? Taken together, the NPC is much more than a gated pore. It is physically coupled to the NE by TM Nups and the transduction of mechanical forces by linker of the nucleoskeleton and
cytoskeleton (LINC) complexes [86] might induce nanoscopic deformations within the NPC that would impact NCT. Other thought-provoking possibilities include the transient uncoupling of neighboring Nups to allow membrane-tethered proteins a translocate path that cuts across the NPC [87]. By surpassing static ‘averaged-out’ behavior, ultrafast imaging techniques such as HS-AFM provide the opportunity to watch native NPCs at work in space and time.
Acknowledgments Y.S. is funded by a PhD Fellowship from the Swiss Nanoscience Institute at the University of Basel. R.K. is funded by the Swiss National Science Foundation as part of the NCCR in Molecular Systems Engineering. R.Y.H.L. acknowledges support from the Swiss National Science Foundation grant 31003A_170041.
References [1] Fahrenkrog B, Aebi U. The nuclear pore complex: nucleocytoplasmic transport and beyond. Nat Rev Mol Cell Biol 2003;4:757–66. doi:10.1038/nrm1230. [2]
Nigg EA. Nucleocytoplasmic transport: signals, mechanisms and regulation. Nature 1997;386:779–87. doi:10.1038/386779a0.
[3]
Terry LJ, Shows EB, Wente SR. Crossing the nuclear envelope: hierarchical regulation of nucleocytoplasmic transport. Science 2007;318:1412–6. doi:10.1126/science.1142204.
[4]
Alber F, Dokudovskaya S, Veenhoff LM, Zhang W, Kipper J, Devos D, et al. The molecular architecture of the nuclear pore complex. Nature 2007;450:695–701. doi:10.1038/nature06405.
[5]
Beck M, Förster F, Ecke M, Plitzko JM, Melchior F, Gerisch G, et al. Nuclear pore complex structure and dynamics revealed by cryoelectron tomography. Science 2004;306:1387–90. doi:10.1126/science.1104808.
[6]
Rout MP, Aitchison JD, Suprapto A, Hjertaas K, Zhao Y, Chait BT. The yeast nuclear pore complex: composition, architecture, and transport mechanism. J Cell Biol 2000;148:635–51.
[7]
Strambio-De-Castillia C, Niepel M, Rout MP. The nuclear pore complex: bridging nuclear transport and gene regulation. Nat Rev Mol Cell Biol 2010;11:490–501. doi:10.1038/nrm2928.
[8]
Rout MP, Blobel G. Isolation of the yeast nuclear pore complex. J Cell Biol 1993;123:771–83.
[9]
Reichelt R, Holzenburg A, Buhle EL, Jarnik M, Engel A, Aebi U. Correlation between structure and mass distribution of the nuclear pore complex and of distinct pore complex components. J Cell Biol 1990;110:883–94.
[10] Cronshaw JM, Krutchinsky AN, Zhang W, Chait BT, Matunis MJ. Proteomic analysis of the mammalian nuclear pore complex. J Cell Biol 2002;158:915–27. doi:10.1083/jcb.200206106. [11] Elad N, Maimon T, Frenkiel-Krispin D, Lim RYH, Medalia O. Structural analysis of the
nuclear pore complex by integrated approaches. Curr Opin Struct Biol 2009;19:226– 32. doi:10.1016/j.sbi.2009.02.009. [12] Frenkiel-Krispin D, Maco B, Aebi U, Medalia O. Structural analysis of a metazoan nuclear pore complex reveals a fused concentric ring architecture. J Mol Biol 2010;395:578–86. doi:10.1016/j.jmb.2009.11.010. [13] Stoffler D, Feja B, Fahrenkrog B, Walz J, Typke D, Aebi U. Cryo-electron tomography provides novel insights into nuclear pore architecture: implications for nucleocytoplasmic transport. J Mol Biol 2003;328:119–30. doi:10.1016/S00222836(03)00266-3. [14] Yamada J, Phillips JL, Patel S, Goldfien G, Calestagne-Morelli A, Huang H, et al. A bimodal distribution of two distinct categories of intrinsically disordered structures with separate functions in FG nucleoporins. Mol Cell Proteomics 2010;9:2205–24. doi:10.1074/mcp.M000035-MCP201. [15] Popken P, Ghavami A, Onck PR, Poolman B, Veenhoff LM. Size-dependent leak of soluble and membrane proteins through the yeast nuclear pore complex. Mol Biol Cell 2015;26:1386–94. doi:10.1091/mbc.E14-07-1175. [16] Timney BL, Raveh B, Mironska R, Trivedi JM, Kim SJ, Russel D, et al. Simple rules for passive diffusion through the nuclear pore complex. J Cell Biol 2016;215:57–76. doi:10.1083/jcb.201601004. [17] Chook YM, Süel KE. Nuclear import by karyopherin-βs: recognition and inhibition. Biochim Biophys Acta 2011;1813:1593–606. doi:10.1016/j.bbamcr.2010.10.014. [18] Bayliss R, Littlewood T, Stewart M. Structural basis for the interaction between FxFG nucleoporin repeats and importin-beta in nuclear trafficking. Cell 2000;102:99–108. [19] Dange T, Grünwald D, Grünwald A, Peters R, Kubitscheck U. Autonomy and robustness of translocation through the nuclear pore complex: a single-molecule study. J Cell Biol 2008;183:77–86. doi:10.1083/jcb.200806173. [20] Lange A, Mills RE, Lange CJ, Stewart M, Devine SE, Corbett AH. Classical nuclear localization signals: definition, function, and interaction with importin alpha. J Biol Chem 2007;282:5101–5. doi:10.1074/jbc.R600026200.
[21] Yang W, Gelles J, Musser SM. Imaging of single-molecule translocation through nuclear pore complexes. Proc Natl Acad Sci U S A 2004;101:12887–92. doi:10.1073/pnas.0403675101. [22] Görlich D, Panté N, Kutay U, Aebi U, Bischoff FR. Identification of different roles for RanGDP and RanGTP in nuclear protein import. EMBO J 1996;15:5584–94. [23] Stewart M. Molecular mechanism of the nuclear protein import cycle. Nat Rev Mol Cell Biol 2007;8:195–208. doi:10.1038/nrm2114. [24] Ribbeck K, Lipowsky G, Kent HM, Stewart M, Görlich D. NTF2 mediates nuclear import of Ran. EMBO J 1998;17:6587–98. doi:10.1093/emboj/17.22.6587. [25] Appen A von, Kosinski J, Sparks L, Ori A, DiGuilio AL, Vollmer B, et al. In situ structural analysis of the human nuclear pore complex. Nature 2015;526:140–3. doi:10.1038/nature15381. [26] Eibauer M, Pellanda M, Turgay Y, Dubrovsky A, Wild A, Medalia O. Structure and gating of the nuclear pore complex. Nat Commun 2015;6:7532. doi:10.1038/ncomms8532. [27] Sakiyama Y, Mazur A, Kapinos LE, Lim RYH. Spatiotemporal dynamics of the nuclear pore complex transport barrier resolved by high-speed atomic force microscopy. Nat Nanotechnol 2016;11:719–23. doi:10.1038/nnano.2016.62. [28] Grossman E, Medalia O, Zwerger M. Functional architecture of the nuclear pore complex. Annu Rev Biophys 2012;41:557–84. doi:10.1146/annurev-biophys-050511102328. [29] Rothballer A, Kutay U. Poring over pores: nuclear pore complex insertion into the nuclear envelope. Trends Biochem Sci 2013;38:292–301. doi:10.1016/j.tibs.2013.04.001. [30] Kampmann M, Blobel G. Three-dimensional structure and flexibility of a membranecoating module of the nuclear pore complex. Nat Struct Mol Biol 2009;16:782–8. doi:10.1038/nsmb.1618. [31] Debler EW, Ma Y, Seo H-S, Hsia K-C, Noriega TR, Blobel G, et al. A fence-like coat for the nuclear pore membrane. Mol Cell 2008;32:815–26.
doi:10.1016/j.molcel.2008.12.001. [32] Wente SR, Rout MP. The nuclear pore complex and nuclear transport. Cold Spring Harb Perspect Biol 2010;2:a000562. doi:10.1101/cshperspect.a000562. [33] Knockenhauer KE, Schwartz TU. The nuclear pore complex as a flexible and dynamic gate. Cell 2016;164:1162–71. doi:10.1016/j.cell.2016.01.034. [34] Frey S, Görlich D. FG/FxFG as well as GLFG repeats form a selective permeability barrier with self-healing properties. EMBO J 2009;28:2554–67. doi:10.1038/emboj.2009.199. [35] Frey S, Görlich D. A saturated FG-repeat hydrogel can reproduce the permeability properties of nuclear pore complexes. Cell 2007;130:512–23. doi:10.1016/j.cell.2007.06.024. [36] Hülsmann BB, Labokha AA, Görlich D. The permeability of reconstituted nuclear pores provides direct evidence for the selective phase model. Cell 2012;150:738–51. doi:10.1016/j.cell.2012.07.019. [37] Labokha AA, Gradmann S, Frey S, Hülsmann BB, Urlaub H, Baldus M, et al. Systematic analysis of barrier-forming FG hydrogels from Xenopus nuclear pore complexes. EMBO J 2013;32:204–18. doi:10.1038/emboj.2012.302. [38] Milles S, Huy Bui K, Koehler C, Eltsov M, Beck M, Lemke EA. Facilitated aggregation of FG nucleoporins under molecular crowding conditions. EMBO Rep 2013;14:178–83. doi:10.1038/embor.2012.204. [39] Rout MP, Aitchison JD, Magnasco MO, Chait BT. Virtual gating and nuclear transport: the hole picture. Trends Cell Biol 2003;13:622–8. doi:10.1016/j.tcb.2003.10.007. [40] Lim RYH, Huang N-P, Köser J, Deng J, Lau KHA, Schwarz-Herion K, et al. Flexible phenylalanine-glycine nucleoporins as entropic barriers to nucleocytoplasmic transport. Proc Natl Acad Sci U S A 2006;103:9512–7. doi:10.1073/pnas.0603521103. [41] Lim RYH, Fahrenkrog B, Köser J, Schwarz-Herion K, Deng J, Aebi U. Nanomechanical basis of selective gating by the nuclear pore complex. Science 2007;318:640–3. doi:10.1126/science.1145980.
[42] Isgro TA, Schulten K. Binding dynamics of isolated nucleoporin repeat regions to importin-beta. Structure 2005;13:1869–79. doi:10.1016/j.str.2005.09.007. [43] Milles S, Mercadante D, Aramburu IV, Jensen MR, Banterle N, Koehler C, et al. Plasticity of an ultrafast interaction between nucleoporins and nuclear transport receptors. Cell 2015;163:734–45. doi:10.1016/j.cell.2015.09.047. [44] Hough LE, Dutta K, Sparks S, Temel DB, Kamal A, Tetenbaum-Novatt J, et al. The molecular mechanism of nuclear transport revealed by atomic-scale measurements. Elife 2015;4. doi:10.7554/eLife.10027. [45] Schoch RL, Kapinos LE, Lim RYH. Nuclear transport receptor binding avidity triggers a self-healing collapse transition in FG-nucleoporin molecular brushes. Proc Natl Acad Sci U S A 2012;109:16911–6. doi:10.1073/pnas.1208440109. [46] Kapinos LE, Schoch RL, Wagner RS, Schleicher KD, Lim RYH. Karyopherin-centric control of nuclear pores based on molecular occupancy and kinetic analysis of multivalent binding with FG nucleoporins. Biophys J 2014;106:1751–62. doi:10.1016/j.bpj.2014.02.021. [47] Wagner RS, Kapinos LE, Marshall NJ, Stewart M, Lim RYH. Promiscuous binding of Karyopherinβ1 modulates FG nucleoporin barrier function and expedites NTF2 transport kinetics. Biophys J 2015;108:918–27. doi:10.1016/j.bpj.2014.12.041. [48] Schleicher KD, Dettmer SL, Kapinos LE, Pagliara S, Keyser UF, Jeney S, et al. Selective transport control on molecular velcro made from intrinsically disordered proteins. Nat Nanotechnol 2014;9:525–30. doi:10.1038/nnano.2014.103. [49] Peters R. Translocation through the nuclear pore complex: selectivity and speed by reduction-of-dimensionality. Traffic 2005;6:421–7. doi:10.1111/j.16000854.2005.00287.x. [50] Lowe AR, Tang JH, Yassif J, Graf M, Huang WYC, Groves JT, et al. Importin-β modulates the permeability of the nuclear pore complex in a Ran-dependent manner. Elife 2015;4. doi:10.7554/eLife.04052. [51] Lim RYH, Huang B, Kapinos LE. How to operate a nuclear pore complex by Kap-centric control. Nucleus 2015;6:366–72. doi:10.1080/19491034.2015.1090061.
[52] Akey CW, Goldfarb DS. Protein import through the nuclear pore complex is a multistep process. J Cell Biol 1989;109:971–82. [53] Beck M, Lucić V, Förster F, Baumeister W, Medalia O. Snapshots of nuclear pore complexes in action captured by cryo-electron tomography. Nature 2007;449:611–5. doi:10.1038/nature06170. [54] Lin DH, Stuwe T, Schilbach S, Rundlet EJ, Perriches T, Mobbs G, et al. Architecture of the symmetric core of the nuclear pore. Science 2016;352:aaf1015. doi:10.1126/science.aaf1015. [55] Szymborska A, de Marco A, Daigle N, Cordes VC, Briggs JAG, Ellenberg J. Nuclear pore scaffold structure analyzed by super-resolution microscopy and particle averaging. Science 2013;341:655–8. doi:10.1126/science.1240672. [56] Göttfert F, Wurm CA, Mueller V, Berning S, Cordes VC, Honigmann A, et al. Coaligned dual-channel STED nanoscopy and molecular diffusion analysis at 20 nm resolution. Biophys J 2013;105:L01–3. doi:10.1016/j.bpj.2013.05.029. [57] Paulillo SM, Powers MA, Ullman KS, Fahrenkrog B. Changes in nucleoporin domain topology in response to chemical effectors. J Mol Biol 2006;363:39–50. doi:10.1016/j.jmb.2006.08.021. [58] Fahrenkrog B, Maco B, Fager AM, Köser J, Sauder U, Ullman KS, et al. Domain-specific antibodies reveal multiple-site topology of Nup153 within the nuclear pore complex. J Struct Biol 2002;140:254–67. [59] Ma J, Goryaynov A, Sarma A, Yang W. Self-regulated viscous channel in the nuclear pore complex. Proc Natl Acad Sci U S A 2012;109:7326–31. doi:10.1073/pnas.1201724109. [60] Ma J, Goryaynov A, Yang W. Super-resolution 3D tomography of interactions and competition in the nuclear pore complex. Nat Struct Mol Biol 2016;23:239–47. doi:10.1038/nsmb.3174. [61] Bestembayeva A, Kramer A, Labokha AA, Osmanović D, Liashkovich I, Orlova EV, et al. Nanoscale stiffness topography reveals structure and mechanics of the transport barrier in intact nuclear pore complexes. Nat Nanotechnol 2015;10:60–4. doi:10.1038/nnano.2014.262.
[62] Stoffler D, Goldie KN, Feja B, Aebi U. Calcium-mediated structural changes of native nuclear pore complexes monitored by time-lapse atomic force microscopy. J Mol Biol 1999;287:741–52. doi:10.1006/jmbi.1999.2637. [63] Kramer A, Liashkovich I, Ludwig Y, Shahin V. Atomic force microscopy visualises a hydrophobic meshwork in the central channel of the nuclear pore. Pflugers Arch 2008;456:155–62. doi:10.1007/s00424-007-0396-y. [64] Kramer A, Ludwig Y, Shahin V, Oberleithner H. A pathway separate from the central channel through the nuclear pore complex for inorganic ions and small macromolecules. J Biol Chem 2007;282:31437–43. doi:10.1074/jbc.M703720200. [65] Kramer A, Liashkovich I, Oberleithner H, Ludwig S, Mazur I, Shahin V. Apoptosis leads to a degradation of vital components of active nuclear transport and a dissociation of the nuclear lamina. Proc Natl Acad Sci U S A 2008;105:11236–41. doi:10.1073/pnas.0801967105. [66] Grünwald D, Singer RH. In vivo imaging of labelled endogenous β-actin mRNA during nucleocytoplasmic transport. Nature 2010;467:604–7. doi:10.1038/nature09438. [67] Cardarelli F, Lanzano L, Gratton E. Capturing directed molecular motion in the nuclear pore complex of live cells. Proc Natl Acad Sci U S A 2012;109:9863–8. doi:10.1073/pnas.1200486109. [68] Ando T, Uchihashi T, Fukuma T. High-speed atomic force microscopy for nanovisualization of dynamic biomolecular processes. Prog Surf Sci 2008;83:337–437. doi:10.1016/j.progsurf.2008.09.001. [69] Uchihashi T, Kodera N, Ando T. Guide to video recording of structure dynamics and dynamic processes of proteins by high-speed atomic force microscopy. Nat Protoc 2012;7:1193–206. doi:10.1038/nprot.2012.047. [70] Osmanovic D, Bailey J, Harker AH, Fassati A, Hoogenboom BW, Ford IJ. Bistable collective behavior of polymers tethered in a nanopore. Phys Rev E Stat Nonlin Soft Matter Phys 2012;85:061917. doi:10.1103/PhysRevE.85.061917. [71] Moussavi-Baygi R, Mofrad MRK. Rapid Brownian Motion Primes Ultrafast Reconstruction of Intrinsically Disordered Phe-Gly Repeats Inside the Nuclear Pore
Complex. Sci Rep 2016;6:29991. doi:10.1038/srep29991. [72] Ando D, Gopinathan A. Cooperative Interactions between Different Classes of Disordered Proteins Play a Functional Role in the Nuclear Pore Complex of Baker’s Yeast. PLoS ONE 2017;12:e0169455. doi:10.1371/journal.pone.0169455. [73] Mincer JS, Simon SM. Simulations of nuclear pore transport yield mechanistic insights and quantitative predictions. Proc Natl Acad Sci U S A 2011;108:E351–8. doi:10.1073/pnas.1104521108. [74] Gamini R, Han W, Stone JE, Schulten K. Assembly of Nsp1 nucleoporins provides insight into nuclear pore complex gating. PLoS Comput Biol 2014;10:e1003488. doi:10.1371/journal.pcbi.1003488. [75] Tagliazucchi M, Peleg O, Kröger M, Rabin Y, Szleifer I. Effect of charge, hydrophobicity, and sequence of nucleoporins on the translocation of model particles through the nuclear pore complex. Proc Natl Acad Sci U S A 2013;110:3363–8. doi:10.1073/pnas.1212909110. [76] Osmanović D, Fassati A, Ford IJ, Hoogenboom BW. Physical modelling of the nuclear pore complex. Soft Matter 2013;9:10442. doi:10.1039/c3sm50722j. [77] Vovk A, Gu C, Opferman MG, Kapinos LE, Lim RY, Coalson RD, et al. Simple biophysics underpins collective conformations of the intrinsically disordered proteins of the Nuclear Pore Complex. Elife 2016;5. doi:10.7554/eLife.10785. [78] Zahn R, Osmanović D, Ehret S, Araya Callis C, Frey S, Stewart M, et al. A physical model describing the interaction of nuclear transport receptors with FG nucleoporin domain assemblies. Elife 2016;5. doi:10.7554/eLife.14119. [79] Wolf C, Mofrad MRK. On the octagonal structure of the nuclear pore complex: insights from coarse-grained models. Biophys J 2008;95:2073–85. doi:10.1529/biophysj.108.130336. [80] Liashkovich I, Meyring A, Kramer A, Shahin V. Exceptional structural and mechanical flexibility of the nuclear pore complex. J Cell Physiol 2011;226:675–82. doi:10.1002/jcp.22382. [81] Koh J, Blobel G. Allosteric regulation in gating the central channel of the nuclear pore
complex. Cell 2015;161:1361–73. doi:10.1016/j.cell.2015.05.013. [82] Solmaz SR, Chauhan R, Blobel G, Melčák I. Molecular architecture of the transport channel of the nuclear pore complex. Cell 2011;147:590–602. doi:10.1016/j.cell.2011.09.034. [83] Chug H, Trakhanov S, Hülsmann BB, Pleiner T, Görlich D. Crystal structure of the metazoan Nup62•Nup58•Nup54 nucleoporin complex. Science 2015;350:106–10. doi:10.1126/science.aac7420. [84] Tu L-C, Fu G, Zilman A, Musser SM. Large cargo transport by nuclear pores: implications for the spatial organization of FG-nucleoporins. EMBO J 2013;32:3220–30. doi:10.1038/emboj.2013.239. [85] Rabe B, Vlachou A, Panté N, Helenius A, Kann M. Nuclear import of hepatitis B virus capsids and release of the viral genome. Proc Natl Acad Sci U S A 2003;100:9849–54. doi:10.1073/pnas.1730940100. [86] Ungricht R, Kutay U. Mechanisms and functions of nuclear envelope remodelling. Nat Rev Mol Cell Biol 2017;18:229–45. doi:10.1038/nrm.2016.153. [87] Meinema AC, Laba JK, Hapsari RA, Otten R, Mulder FAA, Kralt A, et al. Long unfolded linkers facilitate membrane protein import through the nuclear pore complex. Science 2011;333:90–3. doi:10.1126/science.1205741.
Figure Captions Figure 1: (a) Schematic representation of nucleoporin sub-structures within a vertebrate NPC. Darker colors denote dynamic components including the barrier-forming FG Nups. (b) NPC barrier models. In all cases, small molecules (red watermarked) diffuse freely through the barrier whereas large non-specific molecules (red) are withheld. Only Kaps (dark green) can traverse the barrier due to binding with the FG-repeats. (Left) The Selective Phase consists of FG Nups that form an intertwined molecular sieve. (Center) Virtual Gating proposes that the FG Nups behave as polymer brush-like barrier. (Right) Kap-centric control argues that Kaps are integral constituents of the FG Nup barrier. Strongly bound Kaps (dark green) diffuse slowly (grey double-headed arrow) whereas weakly bound Kaps (light green) exhibit rapid translocation (black double-headed arrow). Figure 2: Static views of NPC structure. (a) 3D-reconstruction by zero-loss filtered electron tomography. CF = cytoplasmic filaments; CP = central plug; HA = “handling” points that anchor the NPC to the NE. Reprinted from Journal of Molecular Biology 328:1, Stoffler et.al., Cryoelectron Tomography Provides Novel Insights into Nuclear Pore Architecture: Implications for Nucleocytoplasmic Transport, 741-752 (2003) with permission from Elsevier. (b) Orthoslice through the nucleocytoplasmic axis of a human NPC obtained by CET. Reproduced with permission from Nature 526: 140-143 copyright (2015) Macmillan Magazines Ltd. (c) Tomogram of a native X. laevis NPC suggesting possible transport routes across the NPC (solid and dashed lines). Reproduced with permission from Nature Communications 6:7532 copyright (2015) Macmillan Magazines Ltd. (d) STED reveals octagonal subunits of a TM Nup and the central channel. Scale, 500 nm. Reprinted from Biophysical Journal 105:1 Goettfert et al., Coaligned Dual-Channel STED Nanoscopy and Molecular Diffusion Analysis at 20 nm
Resolution, L01-L03 (2013) with permission from Elsevier. (e) Calcium-mediated opening and closing of the distal ring by time-lapse AFM. Reprinted from Journal of Molecular Biology 287:4 , Stoffler et.al., Calcium-mediated structural changes of native nuclear pore complexes monitored by time-lapse atomic force microscopy, 741-752 (1999) with permission from Elsevier. (f) Nanoscopic changes to NPCs upon exposure to Cytochrome C images by AFM. Reprinted from Proc. Natl. Acad Sci USA 105:32, 11236-11241 Kramer et al., (2008) with permission from National Academy of Sciences. Figure 3: Dynamic views of X. laevis NPC structure obtained by HS-AFM (a) Zoomed out image reveals several NPCs that decorate the cytoplasmic-facing outer nuclear membrane. Scale, 100 nm. (Imaging parameters: 100 x 100 pixels, 400 x 400 nm. 780 ms/frame.) (b) Single NPC showing eight distinct cytoplasmic filaments. A “rocking” back-and-forth motion (denoted by arrows) was observed for an individual filament across consecutive image frames. Scale, 50 nm. (Imaging parameters: 200 x 200 pixels, 200 x 200 nm. 1990 ms/frame.) (c) (First row) Close inspection of the same NPC as (b) reveals occlusions within the central channel. (Imaging parameters: 200 x 200 pixels, 200 x 200 nm. 1990 ms/frame.) Scale, 50 nm. Zooming into the central channel further shows dynamic interactions between the FG Nups (white arrows) and the occluding particles (bright spots). Scale, 10 nm. (Imaging parameters: 80 x 80 pixels, 80 x 80 nm. 280 ms/frame.) (Second row) Particles seen occluding within a different NPC. (Imaging parameters: 200 x 200 pixels, 200 x 200 nm. 1990 ms/frame.) Zoom-ins show similar FG Nup dynamics as above. (Imaging parameters: 80 x 80 pixels, 70 x 70 nm 310 ms/frame.) Scale, same as first row (d) Behavior of a vacant NPC. Scale, 50 nm. (Imaging parameters: 100 x 100 pixels, 150 x 150 nm. 1360 ms/frame.) Zoom-ins show various FG Nup dynamic conformational states. From left to right: “radial”, “retracted”, “extended” and “entangled”. (Imaging parameters: 80x80 pixels, 70x70 nm, 180 ms/frame.)
Figure 4: HS-AFM reveals the nuclear basket of X. laevis NPCs. The nuclear filaments are seen to adopt either a (a) “closed” or (b) “open” distal ring conformation. Scale, 50nm. Imaging parameters: (a) 100x100 pixels, 180 nm, 1990 ms/frame. (b) 100x100 pixels, 200x200 nm. 1000 ms/frame. [Note: All samples were imaged in buffered solution without chemical treatments.]
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