# Opening the gate: Complexity and modularity of the nuclear pore scaffold and basket

Elisa Dultz, Valérie Doye

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Elisa Dultz, Valérie Doye. Opening the gate: Complexity and modularity of the nuclear pore scaffold and basket. Current Opinion in Cell Biology, 2025, 92, pp.102461. 10.1016/j.ceb.2024.102461. hal-05358014

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Opening the gate: Complexity and modularity of the nuclear
pore scaffold and basket

Elisa Dultz¹ and Valérie Doye²

¹Institute of Biochemistry, ETH Zürich, 8093 Zürich

² Université Paris Cité, CNRS, Institut Jacques Monod, F-75013 Paris, France.

For correspondence: elisa.dultz@bc.biol.ethz.ch; valerie.doye@ijm.fr
## Abstract

Nuclear pore complexes (NPCs) are giant molecular assemblies that form the gateway between the nucleus and the cytoplasm and accommodate the bidirectional transport of a large variety of cargoes. Recent years have seen tremendous advances in our understanding of their building principles and have in particular called attention to the flexibility and variability of NPC composition and structure. Here, we review these recent advances and discuss how the newest technologies push the boundaries of nuclear pore research forward, with a specific highlight on the NPC scaffold and a prominent pore appendage, the nuclear basket, whose architecture has long been elusive.

## NPC architecture: an entangled puzzle

Nuclear pore complexes (NPCs) are macromolecular assemblies embedded in the nuclear envelope (NE) and act as communication channels between the cytoplasm and the nucleus. The basic organization of NPCs features an eight-fold rotational symmetry, with transmembrane proteins spanning the NE and anchoring a core scaffold formed by an inner ring (IR, also sometimes termed "spoke ring") sandwiched between two outer rings: a cytoplasmic ring (CR) and a nuclear ring (NR). All three rings of the scaffold tether flexible structures: the IR binds to FG-Nups bearing disordered domains featuring repeated Phe-Gly sequences that fill the central channel of the pore and play a key role in the transport process; the CR and NR carry extensions known as the cytoplasmic filaments/mRNA export platform and the nuclear basket, respectively (Figure 1).

The ~30 distinct constituents that build up the NPC (the nucleoporins, Nups), their organization into biochemically stable subcomplexes, and their approximate localization within the NPC was already largely established in several model organisms two decades ago. However, recent technical improvements have allowed tremendous advances in the understanding of the structure of nuclear pores. In particular, improved cryo-electron tomography (cryo-ET) and single-particle cryo-electron microscopy (cryo-EM) reconstructions have provided refined density maps of the NPCs [1-13]. Together with structural determination - including AI-based prediction - of individual nucleoporins and nucleoporin subcomplexes, crosslinking mass-spectrometry analyses, and subcomplex modeling, these integrative studies have yielded a pseudo-atomic view of the NPC scaffold and the structures associated with its cytoplasmic side in multiple species ([1-13], reviewed in [14,15]). These studies, while revealing an overall conservation of NPC structure and composition, also uncovered species-specific features regarding notably, as discussed later in this review, the outer rings and the nuclear basket [16].
Importantly, these detailed maps can provide the basis to understand molecular mechanisms of NPC function and dysfunction. For example, identifying critical interfaces between individual Nups and Nup subcomplexes enables the design of mutations or inhibitors that interfere with these interactions and can shed light on the extensively studied yet still incompletely understood process of NPC assembly and disassembly (reviewed in [20,21]). Along these lines, the use of inhibitory nanobodies raised against conserved Nup interfaces recently revealed a previously overlooked role for NUP98 in NPC assembly [22].

The refined NPC maps also revealed an increasing number of nucleoporins shared between distinct NPC subcomplexes, with possible functional consequences (see Figure 1). For instance, the localization of NUP93 and NUP205 along with the Y-complex to the outer rings is noteworthy as mutations within this very specific subset of Nups cause steroid-resistant nephrotic syndrome, SRNS, a rare and shared hereditary chronic kidney disease [23]. This suggests that affecting the integrity of the NPC outer rings may ultimately cause intrinsic alterations within a very specific kidney cell type.

More generally, the near-atomic resolution of the NPC scaffold provides the basis to understand how specific pathological mutations compromise NPC assembly and function (reviewed in [24]). For example, a recent study localized a disease-linked mutation in NDC1 to the ALADIN-binding interface and consistently revealed a reduced recruitment of ALADIN to the NPC in patient cells [25].

Although the structural model of the NPC is nearly complete, the cryo-ET and modeling approaches failed to position the membrane Nup POM121 within the NPC architecture. An entirely distinct method, Pan Expansion Microscopy (Pan-ExM), very recently enabled the localization of this transmembrane Nup in the vicinity of the nuclear ring in multiple cell types [26]. In addition, while previous studies revealed the presence of NPCs lacking a specific subset of Nups including POM121 in aged iPSC-derived neurons from patients with amyotrophic lateral sclerosis [27], this new analysis found the remaining POM121 to be shifted towards the inner ring of the NPC in patient cells. Pan-ExM thus appears as a complementary tool to visualize changes in NPC architecture in pathological contexts.

## A flexible yet durable scaffold

A major revelation from *in cellulo* cryo-ET studies was that the diameter of the nuclear pore is variable and larger than previously measured on isolated NPCs or nuclear envelopes [28–31] (Table 1). The models show that the individual spokes of the inner ring complex interact with each other only via flexible linker Nups. The spaces between the spokes may provide the passageway
for membrane proteins that need to pass the nuclear pore complex. In addition, the flexible
connectors allow constriction and dilation of the pore in the plane of the nuclear envelope. Such
dilation relative to purified NPCs may be caused by membrane tension and/or active transport
[12,13,26,29-32] and generates a central channel with a width of ~ 60-65 nm that is large enough
to transport huge cargoes (Table 1). Indeed, up to four large ribosomal subunits were detected in
the channel of purified NPCs [33]. Even intact HIV capsids can be transported [34,35]. However,
a recent study based on template matching analyses of the cryo-ET datasets suggests that very
large cargo may sometime cause rupturing of the NPC scaffold [36]. Such rupturing may occur
because, in contrast to the flexible inner ring, the outer rings present a more rigid scaffold that can
dilate only modestly [13,26,30]. These rigid rings are in turn important to template the eight-fold
symmetry of the NPC, as demonstrated by an increased frequency of 7- and 9-fold symmetric
NPCs in cells lacking an outer ring Y-complex component, NUP133 [32]. Thus, the interplay of
rigid outer rings that provide a template during assembly and stability during mechanical tension
with a flexible inner ring that allows the accommodation of a variety of transport substrates
generates a functionally adaptive but stable multiprotein assembly.

The much more accurate information on the pore dimensions and the FG attachment sites from
the current models also provide the basis for efforts aimed at understanding the transport process
through the NPC by *in silico* modelling or with the help of synthetic pores ([37,38] and reviewed in
[39]). Such methods, combined with innovative live imaging studies – including synthetic biology-
based labelling approaches [40] - will be required to characterize the conformation, interactions
and dynamic behavior of the disordered domains harboring FG repeats, which are not accessible
to cryo-ET.

The elusive nuclear pore basket revealed

Although the first high-resolution cryo-ET structures of the nuclear pore complex did not resolve
the nuclear basket, the most recent work has enabled visualization of some features of this
prominent NPC appendage in yeast and mouse [17].

The main structural components of the nuclear basket are large coiled-coil proteins (TPR in
mammals, scMlp1 and scMlp2 in budding yeast) which form filamentous structures extending
into the nucleoplasm ([41,42], Figure 2). Remarkably, while the density of the yeast basket exhibits
a fish-trap structure consistent with the conformation of filaments joined in a distal ring, as was
previously observed in different organisms [43,44], the mouse EM map revealed a nearly straight
extension of the nuclear basket filaments into the nucleoplasm, with no resolved distal ring [17].
However, the current models derived from averaging possibly only reflect one out of a range of
conformations that the nuclear basket can attain. In particular, dimers of purified Mlp1 exhibit
flexible joints [45] that could support flexible basket structures. In the future, template matching approaches [32,36,46] may provide information about the conformation of the nuclear basket filaments at the single NPC level.

The basket FG-Nup NUP153 (scNup60) behaves as a linker Nup that binds to the nuclear membrane via an amphipathic helix [47,48] and interacts with the outer ring as well as the nuclear basket Nups, NUP50 (scNup2) and TPR (scMlp1/2), via short linear motifs (SLiMs) [45,47,49,50] (Figure 2A, B). Interestingly, the filament protein TPR exhibits stable binding to the NPC with a half-life of hours [51, 52], while NUP153 is highly dynamic and required only to recruit but not to maintain TPR at NPCs [52–55].

The current maps still fail to provide sufficient resolution to unambiguously define the stoichiometry and positioning of each individual Nup present in the NPC basket. This is particularly the case for scPml39, initially defined as a nuclear basket interacting protein in yeast [56], but recently recognized to be conserved to metazoans (ZC3HC1, zinc finger C3HC-type containing 1/ NIPA in mammals) [57,58]. ZC3HC1(scPml39) localizes to the distal ring region of the nuclear basket [59] and is responsible for recruiting additional copies of TPR (scMlp1) to the NPC [52,57,59]. How ZC3HC1 and its dependent TPR pool are precisely positioned in the nuclear basket remains to be determined.

## Variations to a theme: nuclear basket diversity

Intriguingly, the analysis of NPCs from different species has revealed significant variability in the stoichiometry of the outer rings (Figure 3). The composition of nuclear pore complexes can also vary between cell types and even within single cells [18,60-65], but how this is regulated remains largely unknown. Recently, some examples of highly regulated modifications that can provide specialized functionality in specific physiological conditions have been characterized in budding yeast. These predominantly comprise changes in the peripheral components, in particular the nuclear basket (Figure 2C). NPCs proximal to the nucleolus are devoid of the basket in S. cerevisiae [60], but it was only recently recognized that Mlp1/2 is also absent from a subset of additional yeast NPCs [61]. Consistent with this, cryoET could resolve a nuclear basket structure only on about a quarter of yeast NPCs [17]. This subclass of NPCs was also characterized by the presence of 16 copies of the Y-complex in the nuclear outer ring – compared to 8 copies in the remaining population. This observation links basket filament assembly to the nuclear outer ring, an interdependence that seems to be conserved in mammalian cells, where defects in NUP133, a Y-complex Nup, lead to the absence of basket filaments on a population of NPCs [32,51,66]. Assembly of a second nuclear outer ring might then be a prerequisite for formation of the nuclear
basket filaments and could underlie the late association of the filaments about 40 minutes after scaffold assembly is complete [67].

In yeast, dynamic regulation of the canonical nuclear basket composition also occurs during different stages of the life cycle: in newly born daughter cells [68,69], in aged yeast cells that reach the end of their replicative life span [63] and during meiosis [53] (Box 1). Remarkably, in all of these cases, the regulation relies on different post-translational modifications of scNup60 (NUP153).

Since basket filaments recruit a number of interactors, the subset of basket-containing NPCs likely takes on additional functions. Basket components have been implicated in promoting and regulating the export of mRNAs [54, 56, 60, 61, 70-73], but also act as a platform for other processes including DNA repair, gene regulation and the generation of a heterochromatin exclusion zone (reviewed in [74,75]).

Interestingly, recent work also links a specific function in regulating DNA replication to NPCs that lack the nuclear basket filaments Mlp1/2 [76], indicating that the two major forms of NPCs in yeast have evolved distinct functional roles. However, in all wild type vertebrate cells analyzed so far, TPR was present on all NPCs and a similar dichotomy of composition and function as in budding yeast has not yet been reported.

## Conclusion

Because of their complexity, nuclear pore complexes are frequently used as blueprints for the characterization of complex macromolecular assemblies. Over the past years the development of new technologies has led to a refined view of their architecture. While *in cellulo* cryoET has enabled considerable progress, this technology was initially limited by the need to average images. The advent of template matching approaches, but also of other more accessible modalities of super-resolution imaging, including in live cells, now provide a better understanding of the diversity of nuclear pores not only between different cell types, but also within a given cell. A broader use of these approaches by the community will make it possible to understand how nuclear pores can adapt to allow and regulate the transport of a wide range of molecules, but also contribute to cell signaling, notably mechanotransduction, or nuclear architecture and function, the deregulation of which impacts on a variety of cell functions and leads to multiple pathologies.

Box 1: NPC variants in budding yeast
* Mlp(+)- and Mlp(-) NPCs assemble distinct protein and RNA/RNP interactomes [61].

* Mlp(-) NPCs may contribute to regulating DNA replication [76].

* The Sir4-associated Nup (Snup) complex composed of several inner and outer ring Nups and Sir4 is localized at the nuclear envelope and acts in telomere silencing [77]. As these complexes lack central channel components and probably do not extend to the cytoplasm, they cannot be considered bona-fide NPC variants.

* During meiosis, budding yeast nuclear basket components are not destroyed together with other Nups but are recycled, which is enabled by their phosphorylation-triggered release from old NPCs and reassociation to new NPCs assembled in nascent spores [53,78].

* In newly born daughter cells, the lysine deacetylase Hos3 localizes at NPCs and deacetylation of Nup60 promotes the release of NPC-associated mRNA export complexes. This results in a delay in S-phase entry in the daughter cells [68,69].

* In replicatively aged cells, extrachromosomal circles accumulate at NPCs and promote their clustering and retention in the mother cell. As a result of activity of the SAGA complex, these clustered NPCs lack components of the cytoplasmic mRNA export platform and the nuclear basket [63].

* During heat stress, Mlp1/2 are released from the NPC and form nuclear foci [62].

## Acknowledgements

We are very grateful to Benoit Palancade (Institut Jacques Monod, Paris), and members of Karsten Weis, ED and VD labs for critical reading of the manuscript and to Sarah Khawaja for proofreading of the manuscript. We apologize to colleagues whose work could not be directly cited due to space limitation. Work in the laboratory of ED is supported by ETH Zurich research grant ETH-33 19-1. Work in the laboratory of VD is supported by the CNRS, the Fondation pour la Recherche Médicale (FRM) under grant No EQU202003010205, "Equipe FRM 2020", the Labex Who Am I? (ANR-11-LABX-0071; Idex ANR-11-IDEX-0005-02) and the Agence Nationale de la Recherche (ANR), under grant number ANR-23-CE12-0035.

## Conflict of interest statement

The authors declare no competing interests.
Figure legends

**Figure 1:** **Schematic overview of the vertebrate NPC and its building blocks.** Nups present in multiple subcomplexes are printed in bold. FG-Nups are indicated. Grey bars connect interacting complexes or Nups. The copy number per NPC indicated for each Nup corresponds to values extrapolated from structural information [1,2,4,6,8,11,12,17], and sometime differ from those determined by quantitative mass spectrometry [18] or imaging data [19]. Question marks indicate uncertain stoichiometry due to lack of or incomplete structural information.

**Figure 2:** **Schematic representation of the interactions organizing the nuclear basket in vertebrates (A) and in budding yeast (B).** (A) NUP153 is anchored in the pore membrane via an amphipathic helix (AH) and recruits the basket components TPR and NUP50. NUP153 also interacts with the Y-complex. Additional copies of TPR are anchored by ZC3HC1. Dimers of coiled-coil regions of TPR are schematically depicted with a helical line. FG repeats are depicted as squiggly lines. SLiMS of NUP153 that mediate interactions within the NPC are represented by rounded rectangle shapes. (B) Analogous to (A) in budding yeast. Nup1 is a paralogue of Nup60, which also is anchored in the pore membrane via an AH and interacts with the NPC scaffold. (C) Variations in budding yeast nuclear basket composition. (i) In meiosis I or upon acute depletion of Nup60, Mlp1/2 and Pml39 as well as Nup1 remain at the NPC [52,53]. (ii) A subset of NPCs in budding yeast lacks Mlp1/2 and Pml39 [56,60,61]. These proteins are also released from the NPC upon heat shock [62]. (iii) During meiosis II all basket Nups are released from the NPC [53]. In replicatively aged yeast cells, a population of NPCs that associates with extrachromosomal circles loses most basket components as well as certain cytoplasmic Nups. Nup1 (not depicted) is retained at the NPC under these conditions [63]. Note that the stoichiometry of the different nuclear basket components is still uncertain and the figure represents one plausible situation.

**Figure 3:** **Structural diversity of the outer rings in different species.** Schematic representation of the diverse outer ring architectures (cytoplasmic rings, CR and nuclear ring, NR) observed in different organisms. The details of the species analyzed and the corresponding references are indicated below. On the cytoplasmic side, the CR complement ranges from largely absent or incomplete Y-complexes in *T. gondii* and *S. pombe* respectively, to a single ring composed of 8 Y-complexes in *S. cerevisiae* and 16 Y-complexes in vertebrates. On the nuclear side, single rings (*S. cerevisiae*, *S. pombe*, *T. gondii*), double rings (vertebrates and a subset of NPCs in *S. cerevisiae*) and triple nuclear rings (in *D. discoideum*) have been observed.

<table><caption>**Table 1:** Average diameters of nuclear pores complexes measured *in cellulo* at resting stage compared to those measured on isolated NPCs or nuclear envelopes, or upon energy depletion in *S. pombe*.</caption><thead><tr><th rowspan="2"></th><th colspan="2">S. cerevisiae [5,29]</th><th colspan="2">S. pombe [31]</th><th colspan="2">human [30]</th></tr><tr><th>in cellulo</th><th>purified NPCs</th><th>in cellulo: resting stage</th><th>in cellulo: energy depletion</th><th>in cellulo</th><th>purified NE</th></tr></thead><tbody><tr><th>membrane to membrane :</th><td>100 nm</td><td>80 nm</td><td>105 nm</td><td>86 nm</td><td>105 nm</td><td>89 nm</td></tr><tr><th>central channel :</th><td>59 nm</td><td>43 nm</td><td>69 nm</td><td>47 nm</td><td>57 nm</td><td>43 nm</td></tr></tbody></table>
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<figure><img src="image_2.png" /><figcaption>Figure 1</figcaption></figure>
<figure><img src="image_3.png" /></figure>

A. Vertebrates

<figure><img src="image_4.png" /></figure>

B. Budding yeast Mlp(+)

<figure><img src="image_5.png" /></figure>

C. Budding yeast variants

i. **meiosis I or depletion of Nup60**

<figure><img src="image_6.png" /></figure>

ii. **Mlp1(−) or heat shock**

<figure><img src="image_7.png" /></figure>

iii. **meiosis II or aged cells**

<figure><img src="image_8.png" /></figure>
<table><caption>Figure 3</caption><thead><tr><th></th><th>Largely absent<br>Y-complexes</th><th>8 incomplete<br>Y-complexes</th><th>8 Y-complexes<br>= single ring</th><th>16 Y-complexes<br>= double ring</th><th>24 Y-complexes<br>= triple ring</th></tr></thead><tbody><tr><th>Complement<br>of the<br>outer rings :</th><td></td><td></td><td></td><td></td><td></td></tr><tr><th>Present on<br>the CR of :</th><td></td><td></td><td></td><td></td><td></td></tr><tr><th>Present on<br>the NR of :</th><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

<figure><img src="image_9.png" /></figure>

vertebrates:Homo sapiens [2,6,12]

Mus musculus [17,32]

Xenopus laevis [4,7,8,11]

<figure><img src="image_10.png" /></figure>

Schizosaccharomyces

pombe

[31]

<figure><img src="image_11.png" /></figure>

Saccharomyces

cerevisiae

[5,17]

<figure><img src="image_12.png" /></figure>

Toxoplasma

gondii

[17]

<figure><img src="image_13.png" /></figure>

Clamydomonas

reinhartii

[3]

<figure><img src="image_14.png" /></figure>

Dictyostelium

discoideum

[13]