Coordinating nucleoporin condensation and nuclear pore complex assembly

The nuclear pore complex (NPC) is among the most elaborate protein complexes in eukaryotes. While ribosomes and proteasomes are known to require dedicated assembly machinery, our understanding of NPC assembly is at a relatively early stage. Defects in NPC assembly or homeostasis are tied to movement disorders, including dystonia and amyotrophic lateral sclerosis (ALS), as well as aging, requiring a better understanding of these processes to enable therapeutic intervention. Here, we discuss recent progress in the understanding of NPC assembly and highlight how related defects in human disorders can shed light on NPC biogenesis. We propose that the condensation of phenylalanine‐glycine repeat nucleoporins needs to be carefully controlled during NPC assembly to prevent aberrant condensation, aggregation, or amyloid formation.

The nuclear pore complex (NPC) is among the most elaborate protein complexes in eukaryotes.While ribosomes and proteasomes are known to require dedicated assembly machinery, our understanding of NPC assembly is at a relatively early stage.Defects in NPC assembly or homeostasis are tied to movement disorders, including dystonia and amyotrophic lateral sclerosis (ALS), as well as aging, requiring a better understanding of these processes to enable therapeutic intervention.Here, we discuss recent progress in the understanding of NPC assembly and highlight how related defects in human disorders can shed light on NPC biogenesis.We propose that the condensation of phenylalanine-glycine repeat nucleoporins needs to be carefully controlled during NPC assembly to prevent aberrant condensation, aggregation, or amyloid formation.Keywords: condensation; fusogen; membrane fusion; molecular chaperones; nuclear envelope; nuclear pore complex; phase separation Nuclear pore complexes (NPCs) are the gateways between the nucleus and the cytoplasm of the eukaryotic cell and enable transport between them.NPCs are composed of over 30 different proteins termed nucleoporins (Nups).Nups are each present in different stoichiometries, adding up to ~1000 individual proteins and a total mass of approximately 110 MDa per NPC.In general, NPCs share a similar overall organization across eukaryotic species.Recent studies reveal the structure of the NPC in unprecedented detail and show how distinct subcomplexes are orchestrated to yield a complex, yet modular structure with an eightfold rotational symmetry [1][2][3][4][5][6][7][8][9].NPCs are embedded in the nuclear envelope via transmembrane Nups and consist of an inner ring flanked by two outer rings on the cytoplasmic and nuclear facing sides consisting predominantly of the Nup107-160 complex (also referred to as the Y-complex).Specialized Nups are attached to each of the outer rings; the cytoplasmic filaments facilitating an mRNA export platform on the cytoplasmic ring; and the nuclear basket on the nuclear facing ring.A subset of Nups designated FG-Nups feature intrinsically disordered regions (IDRs) consisting of phenylalanine-glycine repeat regions that form the selective permeable barrier within the NPC [10,11].
The FG-Nups comprise about a third of the Nups within the NPC.When taken out of the context of the NPC and purified in vitro, FG-Nups have the ability to phase separate and form gel-like condensates [12,13].The FG motifs are considered to be the driving force behind the interaction between the FG domains and the formation of a meshwork within the NPC that ensures Abbreviations AAA+ ATPases, ATPases associated with a variety of cellular activities; ALS, amyotrophic lateral sclerosis; Cryo-EM/Cryo-ET, Cryo-electron microscopy/cryo-electron tomography; FG-Nups, Phenylalanine-glycine repeat nucleoporins; IDR, intrinsically disordered region; INM, inner nuclear membrane; NLS, nuclear localization signal; NPC, nuclear pore complex; NTR, nuclear transport receptor; ONM, outer nuclear membrane; PNS, perinuclear space.a selective barrier with the ability to exclude random passage of cargo [12][13][14].Interestingly, specific amino acids in the spacers between the FG motifs also contribute significantly to the interactions between IDRs and provide the different FG-Nups with distinct properties contributing to their function and localization within the NPC [15][16][17][18][19][20][21][22].Small molecules can passively diffuse through NPCs but selective nucleocytoplasmic transport of molecules larger than ~40 kDa requires nuclear transport receptors (NTRs) that can ferry cargo through the permeability barrier [23].NTRs contain multiple specific binding sites for the FG motifs on the FG-Nups through which they can rapidly associate and dissociate [24][25][26].NTRs are not only required for transport across the nuclear envelope but are crucial for the function of NPCs [27][28][29][30][31]. Removal of NTRs from NPCs results in a loss of barrier function and, as a result, less selective transport [27,32,33].Conversely, excess NTRs result in a strengthening of the barrier [33].How the selective permeability barrier is formed and how this facilitates the fast nucleocytoplasmic transport mechanism continue to be an active area of investigation [15,21,22,[34][35][36][37][38][39].
While X-ray crystallography combined with electron microscopy (cryo-EM/cryo-ET) and artificial intelligence-based modeling systems have materially advanced our structural understanding of the NPC, the intricate assembly process of this megadalton multi-subunit complex has remained largely elusive.Compared with other multiprotein complexes in eukaryotes, NPC assembly is additionally complicated by the need to traverse two membranes designated the inner and outer nuclear membrane (INM/ONM) that enclose the perinuclear space (PNS).Despite recent progress in delineating proteins and motifs responsible for membrane interactions [40], a detailed structural view of how the NPC interacts with the nuclear envelope and how membrane insertion and assembly are coordinated remains to be developed, defining an important area for further investigation.Interestingly, fundamentally distinct strategies have evolved over the course of evolution to solve the problem of NPC assembly in relationship to the nuclear envelope, thus accounting for different constraints imposed by a dynamic and functionally diversified nuclear envelope in a variety of physiological settings and organisms.In the following, we review recent progress in this area and suggest that nuclear envelope membrane dynamics, FG-Nup condensation, and NPC assembly are spatially and temporally controlled to enable orderly NPC assembly, requiring dedicated assembly machinery which largely remains to be identified, especially in higher eukaryotes.

Nuclear pore complex biogenesis
In metazoans, three modes of NPC biogenesis have been observed: (a) postmitotic insertion during nuclear envelope reassembly in human cells after mitotic nuclear envelope breakdown [41] (Fig. 1A), (b) 'insideout' biogenesis in human cells when new NPCs are inserted via membrane remodeling initiated at the INM [42] (Fig. 1B), and (c) during Drosophila melanogaster oogenesis, where Nups condense into cytosolic precursors that progress into NPCs [43,44] (Fig. 1C).Each of these NPC assembly events exhibits a different genetic requirement for Nups in the early stages of assembly [43,[45][46][47][48][49][50], upon which other Nups-or Nup subcomplexes-are built.As NPC assembly modes have been reviewed in detail recently [41,51,52], we will focus our discussion on 'inside-out' biogenesis, which is the major mode of NPC assembly in higher eukaryotes after the nuclear envelope has reformed following open mitosis.It is estimated that this assembly mode accounts for 50% of NPCs in dividing human cells, and it is thought to be the major route to assemble NPCs in non-dividing cells [53].Thus, inside-out biogenesis is critical for neurons and other cell types with low mitotic indices.In lower eukaryotes undergoing closed mitosis, including the yeast Saccharomyces cerevisiae, this modality is probably the sole pathway for NPC assembly.
Since the nuclear envelope is already established prior to inside-out biogenesis, newly synthesized Nups need to be transported from the site of synthesis at cytosolic ribosomes to the nuclear compartment via pre-existing NPCs (Fig. 2, steps 1-3).Thus, mechanisms must presumably be in place to prevent the premature assembly of larger elements of the NPC structure, which would be an impediment to nuclear import given the size constraints for nuclear transport [54][55][56][57].Equally important, the finding that purified FG-Nups form condensates that can mature into gels, aggregates, and in some cases amyloids (depending on amino acid composition) [12,13,28,58], suggests that these aggregates or amyloids need to be counteracted by molecular chaperones or other suitable machinery to keep FG-Nups in a transport-competent state.Upon arrival in the nuclear compartment, however, such factors need to dissociate to enable staged NPC assembly in juxtaposition to the INM (Fig. 2, step 4).
In human cells, the Nups Pom121 and Nup153 are recruited first to biogenesis sites [47][48][49]59].Nup153 can bind to lamins, which line the nuclear envelope, and anchor the NPC at the nuclear envelope [60], possibly determining NPC insertion sites.How exactly the initial membrane curvature during inside-out NPC biogenesis is generated is presently unclear, but it is evident from EM analysis that a budding event brings the INM to within a fusogenic distance to the ONM during the process [42] (Fig. 2, step 5).Nups containing amphipathic helices, including Nup53, Nup153, and Nup133, can sense, and are proposed to even induce, membrane curvature [40,47,50,61,62].Although membrane bending by Nup153 is not necessary for its function in interphase NPC biogenesis [47], in yeast Nup1 and Nup60 (the Nup153-homologs) and Nup53 do play a crucial role in remodeling the membrane at NPC assembly sites [63,64].While the fusogen responsible for fusing INM and ONM in higher eukaryotes has so far escaped detection, significant progress was made in yeast where Brr6 and Brl1, together with Apq12, have been functionally tied to this process, revealing a critical role for an amphipathic helix in Brl1 and Apq12 [65][66][67][68].Mutations in either specific components of the NPC or the fusogenic machinery cause nuclear envelope herniations, which are also known as nuclear envelope blebs [66,[69][70][71][72].These are presumably related to arrested intermediate stages of this process, for example due to a failure in INM/ONM fusion.Interestingly, overexpression of a Brl1 mutant results in multilayered, onion-like nuclear envelope herniations [65].These feature a uniform intermembrane distance that is not observed in torsin-deficient human cells [73].
The formation of herniations supports the notion that the assembly of NPCs and the accompanying membrane dynamics are closely coordinated to ensure that the permeability barrier is established prior to membrane fusion [40,71,74,75].Once fusion has occurred, cytosolic elements of the NPC, including Nup358, are added from the cytosolic side to complete the structure [51,59] (Fig. 2, step 6).Consistent with this temporal order of events, yeast deficient in Brl1 feature herniations that contain inner and outer ring components but lack elements of the cytosolic export platform [65].Thus, the overall succession of membrane dynamics during inside-out NPC biogenesis (Fig. 2, steps 1-6) is probably highly similar, if not identical across species [59,76].However, it is noteworthy that several key players in the pathway await identification, particularly in higher eukaryotes that lack, for example, clear orthologs of Brl1 and Brr6.In addition, factors that stabilize FG-Nups during assembly are just beginning to emerge.

Compromised interphase NPC assembly in a neurological disorder
A plethora of experimental and pathological conditions are characterized by an accumulation of nuclear envelope herniations [71], raising the question of if and how these structures relate to defects in NPC biogenesis.Mutations in torsin ATPases, for example, robustly provoke the formation of nuclear envelope herniations across species ranging from nematodes to fruit flies, mouse neurons, and human cell lines [73,[77][78][79][80][81].Torsins are unusual members of the AAA+ ATPase protein superfamily, which is composed of ATPases associated with diverse cellular activities [78].Notably, torsins represent the only members of the AAA+ superfamily to reside within the ER/nuclear envelope membrane system [82].Despite belonging to the AAA+ superfamily, torsins are not inherently active ATPases as they lack a catalytic arginine required for ATP hydrolysis within their active site [83,84].In order to hydrolyze ATP, torsinA must interact with a protein cofactor that complements the active site by donating the missing arginine [84][85][86][87].Two such cofactors have been identified as type II ER/nuclear envelope transmembrane proteins laminaassociated polypeptide 1 (LAP1) and luminal domain like LAP1 (LULL1) [84,85,88].
An autosomal dominant mutation in the gene encoding torsinA is the cause for the most common congenital form of the movement disorder childhoodonset dystonia (DYT1 dystonia) that is characterized by involuntary muscle contractions and highly debilitating posturing.The mutation results in an in-frame deletion of glutamine residue 302 or 303 and is referred to as torsinA DE302 [89,90].This mutation perturbs the interaction with LAP1 or LULL1 [84,87], highlighting the importance of the torsin-cofactor complex in neurological development.In mouse models, deletion of torsinA during a developmental window causes cells of the central nervous system to develop nuclear envelope herniations and the onset of dystonic symptoms [80,91].Importantly, a burst of NPC biogenesis was observed in developing mouse neurons [81,92], making neurons an attractive model system to study inside-out biogenesis in a mammalian context as neurons are completely reliant on this assembly mode due to their quiescent nature.This is presumably one major reason why torsinA mutations affect primarily neurons, apart from torsinA being the dominantly expressed torsin paralog in early neuronal development [80].
In torsin-deficient cell lines, nuclear envelope herniations can be induced abundantly and can be studied via EM tomography [73,93].Remarkably, these herniations are quite analogous to those observed in yeast upon mutation of Brl1 [65], in that they contain several Nups at an electron-dense neck that presumably resembles an immature NPC but lack the cytosolic subunit Nup358 that would only be added after the INM/ONM fusion has occurred [42,73,94].Similar observations were made in mouse models [81,95].Furthermore, torsin-deficient cells can be grown in large quantities, enabling a compositional analysis of nuclear envelope herniations by combining classical subcellar fractionation with comparative proteomics.This enabled the identification of machinery that associates with defective or immature NPCs, including the poorly understood protein MLF2, as well as Hsp70 and various DNAJ proteins that are known cofactors of Hsp70s [94,96].Further derivatization of MLF2 with GFP and APEX provided the first live-cell imaging and spatial proteomic tools to study the formation and detailed composition of nuclear envelope herniations, respectively [94,97].Interestingly, nuclear envelope herniations form in a highly synchronous manner coinciding with a burst of NPC biogenesis observed previously [42,51,98].Furthermore, herniation formation depends on the transmembrane Nup Pom121 [94] and Nup153 [93], which were previously shown to be essential for this mode of NPC biogenesis [47,49,50].The precise function of torsins during NPC biogenesis is still unclear, and while a role in membrane fusion is a possibility, other, more indirect ties are equally feasible, defining a clear need for future investigation.Regardless, these data clearly support a functional link of torsins to inside-out NPC biogenesis.Using impairment of torsins as a tool provides a unique opportunity to identify candidates for NPC assembly machinery that would otherwise be difficult to capture due to their transient association with rapidly forming NPCs.
Many mechanisms have been proposed to explain how torsinA mutation causes DYT1 dystonia [99].However, most models have insufficiently accounted for the exclusively childhood onset and reduced penetrance traits of the disease.Due to the transient nature of the hallmark nuclear envelope herniations, persistent yet mild nuclear transport defects [77,95,100] resulting from perturbed assembly are unlikely to account for the pathogenesis entirely.One recently developed model can address these two unique features: nuclear envelope herniations arise in torsindeficient cells due to defective interphase NPC biogenesis and impose a window of vulnerability.During this window, a metastable proteome is generated both through the aberrant stabilization of ubiquitylated proteins that would normally be destined for degradation and the pronounced sequestration of chaperones within nuclear envelope herniations (Fig. 2).This model hypothesizes that in ~30% of disease allele carriers, the metastable proteome is pushed to a proteotoxic level following some secondary insult such as a fever or exposure to environmental stress [97].Future studies are needed to test this model and probe proteome stability in DYT1 dystonia models.Conceptually however, these and other data suggest that NPC assembly defects may have consequences that go well beyond nuclear transport defects, and that detrimental, gain-of-function properties should be considered in other pathological settings in the future.

Emerging mechanisms safeguarding FG-Nup assembly
Many large complexes in cells, including the proteasome, ribosomes, and mitochondrial complexes, require assistance from assembly factors [101][102][103] or rely on co-translational assembly during their biogenesis.While co-translational assembly of NPC subcomplexes has been observed recently [104,105], it has remained largely unclear how FG-Nups are protected from making inappropriate interactions during de novo NPC biogenesis and how their timely release after nuclear import is coordinated.NPC biogenesis starts in some cases with an abundant amount of condensation-prone Nups, as seen for Nup358 in D. melanogaster [43], NSP1 in yeast [106,107].Condensates of these Nups can be precursors for NPC biogenesis, but are also observed independent of NPC biogenesis in D. melanogaster [108].In yeast, Nup condensates comprise newly synthesized Nups that are inherited by the daughter cell [106].Moreover, for some Nups a substantial fraction is not incorporated into NPCs, either en route to biogenesis or due to dynamic association and dissociation from the mature NPC, both in yeast and in human cells [59,76,109].Maintenance of these Nups is crucial as they are at risk of undergoing phase transition into aggregates, but also to prevent them from creating premature or ectopic NPC assembly events.
One class of proteins that are known to bind and release Nups are the NTRs, not only within the NPC but also during mitosis when NPCs disassemble and reassemble at the nuclear envelope upon completion of mitosis [59,110].Interestingly, importins can prevent aggregation of RNA-binding proteins with prion-like domains [111][112][113], arginine-rich dipeptide repeat proteins [114], and proteins with basic domains [115].The intrinsic properties of both RNA-binding proteins and FG-Nups to transition into an aggregated form might have similar underlying mechanisms.Thus, one possibility is that FG-Nups are captured by NTRs cotranslationally-as was recently shown [104,116]-to keep them in an import-competent state, and that they are released on the nuclear side via the canonical Ran GTPase cycle [117].Another attractive mechanism was identified in yeast, where localized translation was observed for specific Nups directly at the nuclear envelope [105].
But what about FG-Nups that escape cotranslational 'detection' by NTRs, or those not bearing nuclear localization signals (NLSs)?And which mechanisms are at work to ensure that the selective phase forms adequately within the central transport channel rather than ectopically in the cytoplasm or nucleoplasm after FG-Nups are released from NTRs? Here, proteins identified in nuclear envelope herniations of torsin-deficient cells deserve further scrutiny, in particular DNAJB6, MLF2, and Hsp70, which form a ternary complex [94,97].Indeed, proteins that maintain FG-Nup condensates in a functional state and counteract their progression into an aggregated state have gained more attention as possible assembly factors of the NPC.Molecular chaperones are known to prevent the transition into aggregates of various protein substrates and have recently been shown to maintain FG-Nups in a physiological state [93,97].The Hsp70 co-chaperone DNAJB6 can prevent aggregation of FG-Nups and loss of DNAJB6 results in the formation of stacks of NPCs in the cytoplasm [93].In addition, the ternary complex of MLF2, DNAJB6, and Hsp70 counteracts the amyloid formation of FG-Nups [97].The loss of DNAJB6 also affects the uniform distribution of NPCs in the nuclear envelope [93].
Interestingly, both the mislocalization of NPC components to the cytoplasm and the disturbed spatial distribution are observed upon loss of torsins in mouse neurons as well [81].Given that the chromatin-and torsin-associated INM protein LAP1 was tied to nuclear envelope dynamics recently [118,119], it will be important to scrutinize the spatial organization of inside-out NPC biogenesis.It is tempting to speculate that NPC biogenesis events at the nuclear envelope are enriched in specialized nuclear envelope segments of distinct lipid composition, possibly representing newly synthesized lipid territories [120].Similarly, MLF2, as an emerging diagnostic marker of the earliest NPC assembly events, is distributed non-randomly in clusters with a diameter of approximately 1 lm in diameter in torsin-deficient cells [94].These contain small MLF2-positive areas juxtaposed against the INM where the membrane curvature starts to form [94]. Given that the MLF2/DNAJB6/Hsp70 complex tunes the properties of FG-Nup condensates in vitro, we speculate that the condensation of FG-Nups could be a driver for the initial membrane curvature, presumably in conjunction with transmembrane Nups that connect this condensation process to the INM.Since a 'velcro function' of the FG-Nup Nup116 in yeast has been reported recently [69], such INM-confined condensates may act as seed regions to recruit additional NPC subcomplexes allowing further maturation of the NPC progenitor.

NPC maintenance
Proper NPC maturation is crucial in embryonic development in zebrafish where NPC maturation leads to more efficient transport [121].In contrast to embryogenesis, in aging yeast, NPCs lose stoichiometry over time, and this is linked to aging phenotypes including a decrease in shuttling of transcription factors, an increase in nucleocytoplasmic compartmentalization, and an increase in nuclear envelope herniations [122].Altered NPC stoichiometry and compromised compartmentalization were also observed in Caenorhabditis elegans and mammalian cells [123].One of the Nups that loses abundance during aging is Nsp1, which is an FG-Nup that resides in both the NPC and in the cytoplasm in condensates [106].Surprisingly, Nsp1 is involved in the maintenance of the phase state of other FG-Nup and the prevention of progression into an aggregated state [106], similar to the chaperone DNAJB6 [93].The loss of Nsp1 leads to a reduced cytoplasmic and condensated amount of Nsp1, which subsequently leads to NPC assembly defects [106].Recently, loss of NPC integrity and nucleocytoplasmic shuttling have emerged as processes that are disturbed in in several aging-related neurodegenerative diseases, including Alzheimer's disease and ALS [124].Mutations in Nups themselves can lead to diseases, including Dystonia [125,126], and nucleocytoplasmic transport can become disturbed by mutations in aggregation-prone proteins that sequester importins, Nups, or components of the Ran gradient needed for efficient transport [122][123][124][127][128][129][130].These findings highlight the need for protein quality control at the nuclear envelope to ensure maintenance of the NPC and its efficient transport capabilities [131].Likely more factors will be identified that are required to establish and maintain cellular compartmentalization and transport capacity.

Conclusion and perspectives
Our understanding of the fundamental modes of assembly (Fig. 1) and accompanying nuclear envelope membrane dynamics (Figs 1 and 2) has significantly increased over the past decade.This has allowed for a better understanding of the temporal order of assembling NPC subunits, which provide important diagnostic markers for advanced imaging modalities.However, several key questions that remain unanswered define important areas for future investigation.How are the intrinsic properties of FG-Nups controlled in space and time to prevent ectopic condensation?While NTRs and chaperone complexes are emerging as important players, the molecular basis for the exquisite temporal synchrony of inside-out NPC biogenesis and the related membrane expansion leading to herniations is still enigmatic.Furthermore, with cytosolic NUP condensation being observed independent of NPC biogenesis [108], it is tempting to speculate that such condensates might fulfill additional roles that are unrelated to nuclear transport, which remain to be determined.Lastly, the fusogenic machinery acting within the nuclear envelope of higher eukaryotes remains to be identified.Since defects in biogenesis and maintenance of NPCs are increasingly connected to human pathology, it is evident that knowing the identity of specific machinery tied to these processes will provide excellent molecular markers that can be harnessed diagnostically or as readouts for the development of pharmacological strategies.

Fig. 1 .
Fig. 1.Postmitotic, 'inside-out', and condensate-based nuclear pore complex (NPC) biogenesis.(A) During open mitosis in mammalian cells, the nuclear envelope (NE) and NPCs are taken apart and reassembled after mitosis.For correct timely and spatial reassembly, the complexes containing ELYS and Nup107-160 are released from importin b by a RanGTP 'cloud' that surrounds the condensed chromatin (1).ELYS binds the chromatin with an AT-hook (2) and this complex recruits vesicles with the transmembrane Nup Pom121 (3).Pom121 facilitates membrane fusion and other Nups recruitment to the assembling NPC (4), giving rise to mature NPCs in a closed nuclear envelope (5).(B) The details of 'inside-out' NPC biogenesis in mammalian cells are still not fully described, but it is thought that Nup153 and Pom121 are the first Nups at biogenesis sites.To initiate NPC biogenesis from the nuclear side, Nup153 is imported into the nucleus (1) where it recruits the Nup107-160 complex (2).At the NPC biogenesis initiation sites, an NPC precursor is assembled (3) and the inner nuclear membrane protrudes toward the outer nuclear membrane prior to fusion of the membranes (4).When nuclear envelope fusion is complete, addition of the cytoplasmic ring Nups including NUP358 (5) leads to a mature NPC(6).(C) Condensate-based NPC biogenesis has been shown in D. melanogaster oocytes and starts with the condensation of Nup358 in nurse cells, where also Nup358 mRNA is present (1).Nup358 is maintained as a condensate due to high levels of RanGDP.In the ooplasm, high levels of RanGTP leads to de-condensation of Nup358 (2) and allows other Nups to interact and form NPCs (3).The NPCs are first assembled into cytoplasmic membrane stacks called annulate lamellae (4) as a storge for later incorporation into the nuclear envelope where they mature into full NPCs.