Structure, dynamics and function ofnuclear pore complexesMaximiliano A. D’Angelo and Martin W. Hetzer

Molecular and Cell Biology Laboratory, Salk Institute for Biological Studies, La Jolla, CA 92037, USA

Review

Nuclear pore complexes are large aqueous channels thatpenetrate the nuclear envelope, thereby connecting thenuclear interior with the cytoplasm. Until recently, thesemacromolecular complexes were viewed as static struc-tures, the only function of which was to control themolecular trafficking between the two compartments.It has now become evident that this simplistic scenario isinaccurate and that nuclear pore complexes are highlydynamic multiprotein assemblies involved in diversecellular processes ranging from the organization of thecytoskeleton to gene expression. In this review, wediscuss the most recent developments in the nuclear-pore-complex field, focusing on the assembly, disassem-bly, maintenance and function of this macromolecularstructure.

IntroductionA hallmark of eukaryotic cells is the compartmentalizationof the genetic material inside the nucleus. By restrictingthe accessibility of cytoplasmic proteins to DNA with thephysical barrier of the nuclear envelope (NE), eukaryoticcells have achieved a complexity in transcriptional regu-lation not found in prokaryotes. Furthermore, the NEprovides additional levels of regulation of gene expression,such as the selective export of newly synthesized mRNAinto the ribosome-containing cytoplasm and the establish-ment of higher-order levels of organization of the nucleargenome.

The NE comprises two concentric lipid bilayers, theouter and inner nuclear membranes (ONM and INM,respectively) [1]. The ONM is continuous with the endo-plasmic reticulum (ER) and studded with ribosomes,whereas the INM is characterized by a set of integralmembrane proteins [1]. Large multiprotein structuresknown as nuclear pore complexes (NPCs) penetrate theNE at sites at which the INM and the ONM are fused [1].

NPCs function as gatekeepers of the nucleus, perform-ing the essential cellular role of mediating the exchange ofmolecules between the nucleoplasm and the cytoplasm [2].Ions and small metabolites can diffuse through NPCs;however, molecules possessing a mass >40 kDa need tobe actively transported. Nucleocytoplasmic transport is acomplicated process that is carried out by a large family oftransport receptors known as karyopherins in yeastand importins and exportins in mammals, the latternamed depending on their direction of transport [3]. Themolecular mechanism of nuclear transport has recentlybeen the subject of several major reviews and, thus, is not

Corresponding author: Hetzer, M.W. (hetzer@salk.edu).

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discussed [2,4]. Here, we focus on the latest advances in ourunderstanding of nuclear pore assembly, disassembly,maintenance and function.

NPC structureOwing to their function as exclusive nucleocytoplasmictransport channels and unique structural features, NPCshave been actively studied since their discovery in the1950s. Although the initial descriptions of the overallNPC structure were performed >40 years ago [5], it wasnot until recently that new techniques, such as cryo-elec-tron tomography, field-emission in-lens scanning electronmicroscopy (FEISEM), 4pi and atomic force microscopyand improved cell-fixation protocols could provide adetailed picture of its three-dimensional organization[6–11]. Overall, the NPC is an eightfold-symmetrical struc-ture comprising a NE-embedded scaffold that surrounds acentral transport channel and two rings – the cytoplasmicand nuclear rings – to which eight filaments are attached(Figure 1a). Whereas the cytoplasmic filaments have looseends, the nuclear filaments are joined in a distal ring,forming a structure known as the nuclear basket. Eventhough the size of the NPC varies between species, itsoverall structure is evolutionarily conserved from yeasts tomammals [12,13].

Despite their high molecular mass of �60–125 MDa inmammals and �40–60 MDa in yeasts, proteomic analyseshave revealed that NPCs contain only �30 differentproteins, known as nucleoporins or nups [13–16](Table 1). Except for three transmembrane proteins thatare believed to anchor the NPC to the NE [17,18], all othernucleoporins are soluble. Owing to the eightfold symmetryof pores, each nucleoporin is present in copies of eight ormultiples of eight, resulting in �500–1000 nups per pore.Remarkably, nucleoporins have a very limited set ofdomains, restricted to b-propellers, a-solenoids, phenyl-alanine-glycine (FG) repeats, coiled–coiled and transmem-brane domains [19,20]. Most of these proteins associate inbiochemically stable subcomplexes that are believed tofunction as the ‘building blocks’ of the NPC (Figure 1b).

Recently, a detailed 3D model for the position andabundance of each nup in the Saccharomyces cerevisiaeNPC structure was proposed based on experimental dataobtained frommolecular, biochemical and structural infor-mation of the NPCs and their components [20,21]. In themodeled structure, the scaffold of theNPC is formed by twomain protein subcomplexes that, through linker proteins,anchor a set of FG-containing nucleoporins [20]. TheFG-rich nucleoporins, which can contain 4–48 GLFG,

Ltd. All rights reserved. doi:10.1016/j.tcb.2008.07.009 Available online 9 September 2008

Figure 1. Nuclear pore complex (NPC) structure and composition. (a) Schematic illustration of the NPC structure. (b) Predicted localization of subcomplexes and

nucleoporins within the NPC. The members of the Nup214 complex (Nup214, Nup88), Nup98 complex (Nup98, Rae1), Nup107–160 complex (Nup160, Nup133, Nup107,

Nup96, Nup75, Nup43, Nup37, Sec13, Seh1), Nup62 complex (Nup62, Nup58, Nup54, Nup45) and Nup93–205 complex (Nup205, Nup188, Nup155, Nup93, Nup35) are

enclosed in the same box. Green lines indicate the location of the three transmembrane nucleoporins, red lines indicate the location of peripheral components and blue

lines indicate the location of scaffold subcomplexes.

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FxFG, PxFG or SxFG amino acid repeats, represent a thirdof the total pore proteins and fill the central channel of theNPC, extending into the cytoplasmic and nucleoplasmicsides. The FG domains have an unfolded structure and areresponsible for interaction with transport receptors [22–

26]. Notably, it has been demonstrated recently that, athigh concentrations, the FG repeats of the S. cerevisiaenucleoporin Nsp1p form a 3D hydrogel that reproduces thepermeability properties of the NPC [27,28]. These resultsindicate that the FG nucleoporins form a sieve-likemeshwork through weak hydrophobic interactions that

functions as a barrier, restricting the passage of moleculesthrough the NPC, and also support the ‘selective phase’model for the pore permeability barrier [29,30] (Box 1). Tocarry cargoes through the NPC, the transport receptorsovercome this barrier by interacting with the FG repeats,locally dissolving the FG–FG meshwork.

Although the existence of a 3D hydrogel has yet to beproven in vivo, the formation of the FG meshwork issupported by a study showing that the FG nucleoporinslocated at the NPC central channel interact through theirFG motifs in vitro and in vivo [31]. Nevertheless, the FG

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Table 1. Mammalian, S. cerevisiae and C. elegans nucleoporinshomologsa,b

Mammalian S. cerevisiae C. elegans

Nup35 Nup53p Npp-19

Nup37 – –

Nup43 – C09G9.2

Nup50 Nup2p Npp-16

Nup54 Nup57p Npp-1

Nup58/45 Nup49p Npp-4

Nup62 Nsp1p Npp-11

Nup75 Nup85p Npp-2

Nup88 Nup82p –

Nup93 Nic96p Npp-13

Nup96 Nup145Cp Npp-10

Nup98 Nup145Np Nup100p Nup116p Npp-10

Nup107 Nup84p Npp-5

Nup133 Nup133p Npp-15

Nup153 Nup1p Nup2p Nup60p Npp-7

Nup155 Nup157p Nup170p Npp-8

Nup160 Nup120p Npp-6

Nup188 Nup188p –

Nup205 Nup192p Npp-3

Nup214 Nup159p Npp-14

Nup358 (RanBP2) – Npp-9

Sec13R Sec13p Npp-20

Seh1 Sehp Npp-18

Pom121 – –

Gp210 – Npp-12

Ndc1 Ndc1p Npp-22

Tpr Mlp1p Mlp2p Npp-21

RAE1 Gle2p Npp-17

ALADIN – –

NLP1(hCG1) Nup42p –

– Nup59p –

– Nup116p –

– Nup100p –

– Pom134p –

– Pom152p –aNote that some nucleoporins, such as Nup98, Nup153, Nup155, Tpr and Nup2p,

share homology with more than one nucleoporin.bA dash (–) indicates that no homologs have yet been described in the organism.

Box 1. Models of NPC selectivity

NPCs can perform 1000 translocations events per second, shuttling

a mass of �100 MDa second�1 [29]. However, these structures

efficiently restrict the passages of large molecules that lack

nuclear-transport signals. Although it has long been known that

the major diffusion barrier of the NPC resides in its central channel,

how nuclear pores achieve their transport selectivity is still a matter

of debate. Several models have been proposed to explain the NPC

selectivity, which take into account that active transport through the

pore occurs by facilitated diffusion and requires the interaction of

transport receptors with the FG nucleoporins.

The ‘virtual gate model’ [33] predicts that the highly dense FG

nucleoporins of the central channel generate an entropic barrier that

prevents the passage of inert molecules. For a molecule that is freely

diffusing in the cytoplasm, entering the overcrowded central

channel of the pore implies an immediate restriction of its liberty

of movement and a drop of its entropy. The entropic price that a

molecule has to pay to move through the pore increases with its

size, thereby lowering its probability of crossing the channel. The

interaction of a molecule with the FG nucleoporins increases the

probability of it entering and moving through the pore. This model

proposes the existence of an energetic barrier and not a physical

barrier controlling NPC permeability.

The ‘spaghetti oil model’ [25] proposes that extended FG repeats

fill and obstruct the pore. The idea is that the FG repeats are

constantly moving at physiological temperature and can be pushed

to one side by translocation complexes. In this model, transport

receptors move through the FG spaghetti by a binding and release

mechanism. Because the FG-transport receptor interactions are

weak, the carriers would bind the FG repeats only transiently and,

once released, they would be free to diffuse through the pore until

they find another nucleoporin to bind. The continuous binding–

release–diffusion cycle would enable the transporter to move

through the pore by a facilitated diffusion.

In contrast to the previous models, the ‘selective phase model’

[29] indicates that the FG nucleoporins from the central channel

form a sieve-like meshwork through weak hydrophobic interactions.

This gel-like network prevents the passage of inert molecules larger

than its pore size. In this model, the transport receptors would

selectively partition into the semi-liquid-pore phase and move

through the central channel by competing with the FG–FG interac-

tions locally dissolving the meshwork. Thus, by interacting with the

FG nucleoporins, the carriers would help molecules carrying

nuclear-transport signals cross the pore by increasing their

solubility in the central channel. This model proposes the existence

of a physical barrier that determines NPC selectivity, whereby the

exclusion size of the pore is defined by the pore size of the sieve-like

meshwork.

Finally, the ‘reduction of dimensionality model’ [26] proposes that

transport receptors function as ferries that can slide on the surface

of an FG surface. In this model, a continuous FG surface extends

from the cytoplasmic filaments to the nucleoplasmic basket and

throughout the walls of the central channel. Transport receptors

bind to FG surface in the cytoplasmic or nucleoplasmic side of the

NPC and move through the pore by a two-dimensional walk. The

model predicts the existence of a selectivity filter in the central

channel, which is also generated by the FG repeats, with an

unobstructed narrow tube in the center that would enable the

diffusion of small molecules. The transport-receptor-cargo com-

plexes would overcome the filter because they can bind and enter

the FG surface through its extremes. However, inert molecules that

are too large to diffuse through the central tube and cannot bind the

FG repeats would not be able to enter the filter and move across

the pore.

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repeats of the nucleoporins that constitute the cytoplasmicand nuclear filaments have not been found to interact withthe FG domains of the nucleoporins from the centralchannel, thereby indicating that the peripheral FG nucleo-porins could function as ‘entropic bristles’ preventing theentry of molecules to the pore. Functioning as ‘entropicbristles’ means that, by movement, the filaments reducethe available space to access the pore channel, thus, gen-erating in this way an entropic barrier that decreases theprobability of the molecules entering the pore withoutphysically repelling them. To access this limited spaceand enter the pore, a molecule would have to reduce itsentropy, which is energetically unfavorable. In support ofthis idea, it has recently been reported that the FGdomains of Nup153, a main component of the nuclearbasket, form a brush-like structure in vitro with entro-pic-repulsion properties [32]. According to this, the fila-ments would help to maintain the permeability barrier, inagreement with the ‘virtual gate’ model [33] (Box 1), whichproposes that the probability of a molecule to overcome theentropic cost of entering a limited space packed with non-interacting nucleoporins decreases with increasing size(Box 1). The interaction of transport receptors with theFG repeats enables largemolecules to overcome the energyloss and move through the entropic barrier.

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It is worth mentioning that, in the selective-phasemodel, the formation of the sieve-like meshwork wouldalso generate an entropic barrier for those molecules thatcan enter the pores of the FG network. This barrier wouldreduce the probability of the molecules entering the pores

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but would not define the permeability limit of the NPC,which would be determined by the meshwork pore size.

Although these findings point towards the FG networkas the main mechanism for pore selectivity, they are notyet sufficient to completely discard other proposed modelsfor NPC permeability [25,26,29,33] (Box 1). It is possiblethat a combination of these models would be required toexplain all the reported NPC properties.

Dynamic organization of NPCs

A remarkable, yet largely uncharacterized, feature of theNPCs is their dynamic molecular organization. Using asystematic approach, in which 19 GFP-tagged nucleopor-ins were studied by fluorescence recovery after photo-bleaching (FRAP), it was shown that the residence timesof different nups at the NPC varied from a few seconds to>70 h depending on their location or function [34].Although the proteins that form the NPC scaffold, suchas members of the Nup107–160 complex, are stablyembedded in the NE during interphase (having residencetimes longer than the average cell cycle), the more periph-eral components, such as Nup153 and Nup50, are highlydynamic, having residence times of seconds to minutes. Athird class of nucleoporins, which are believed to work aslinkers between the scaffold and the peripheral nucleopor-ins, have intermediate residence times.

The functional importance of ‘dynamic nucleoporins’ isstill unclear. It has been suggested that mobile nucleopor-ins could help to deliver cargo to the NPC. In support ofthis, the mobility of two RNA-binding nucleoporins,Nup153 and Nup98, is transcription dependent, indicatingthat they could assist newly transcribed RNAs to reach thepore and be exported to the cytoplasm [35]. Although atransport-dependent function of dynamic nups seemsplausible, it is also possible that these proteins havepore-independent functions (see later). A dynamic organ-ization of NPCs could also be indicative of changes inprotein composition in response to altered transportrequirements. The presence of tissue and developmen-tal-specific nucleoporins has been reported [36–39]; how-ever, there is no evidence that NPCs of differentcomposition coexist in the same cell.

The dynamic properties of NPCs can also be observed atthe structural level. Early cryo-electron microscopy (EM)studies found that theNPCswere present in either an openor closed conformation, the latter characterized by thepresence of a central plug or transporter [40]. Historically,the central plug was considered to be a structural com-ponent of the NPC, but recent data have indicated that itrepresents translocating cargo. Transmission electronmicroscopy (TEM) and FEISEM have identified differentconformations of theNPCnuclear basket in response to theexport of large ribonucleoprotein particles [41]. Cryo-elec-tron tomography has shown two different structural statesof the cytoplasmic filaments and variability in the positionof the narrowest constriction of the central channel [7].Furthermore, the NPC has been shown to alter itsconformation in response to Ca2+, glucocorticoids andATP [42–47].

The molecular basis of the structural changes in porearchitecture and its physiological role is still a mystery.

One possibility is that the location of nucleoporins isactively reorganized for functional purposes within theNPC structure. Consistent with this, two nucleoporins,Nup153 and Nup214, have been found in different regionsof the NPC in a transport-dependent manner [48,49].Moreover, the yeast NPC has been shown to undergocell-cycle-specific rearrangements [50]. It has beenobserved that, by changing interacting partners, somenucleoporinsmodify their location at the yeast NPC, affect-ing specific nuclear-transport pathways [50]. Anotherpossibility is that structural changes of the NPC are aconsequence of alterations in nucleoporin conformationand not in their location. Recently, the crystal structureof a-helical regions of Nup58/45, a nucleoporin from thepore central channel, was solved. It was found that the a-helical domains formed tetramers through the interactionof two antiparallel dimers [51]. The presence of differenttypes of tetramer conformations that were laterally dis-placed along the axis of the dimer–dimer interface wasfound. Thus, it has been proposed that eight Nup58/45tetramers would be built around the central channel andwould regulate the aperture or diameter of the NPC bysliding along the dimer interface. Also supporting a nucleo-porin conformational change, Nup153 has been reported tocollapse reversibly into a compact conformation when itsFG repeats bind to the transport receptor karyopherin b

(the yeast homolog of importin b) [52].

NPC life cycleAssembly

The biogenesis of nuclear pores is essential for cell survivaland proliferation. There are two phases during themetazoan cell cycle in which NPCs need to be assembled:first, at the end ofmitosis, when theNE reforms around thesegregated chromosomes and second, during interphase inwhich the cells double their number of pores in preparationfor the next round of division. Even though both processeslead to the same final structure, they occur under verydifferent conditions. Mitotic-NPC assembly takes placeconcomitantly with reformation of the nuclear membranearound segregated chromosomes. At this time, NPCs arerebuilt from disassembled subcomplexes that were dis-persed into the cytoplasm during breakdown of the NE(Figure 2). By contrast, NPC assembly during interphaseoccurs in an intactNEusing newly synthesized nups and ina cellular environment in which the nucleus and thecytoplasm are physically separated (Figure 2). Interphaseassembly is the only existing mechanism in organisms,such as yeasts, that undergo closed mitosis (i.e. their NEdoes not break down during cell division). Althoughaccumulating evidence indicates that both types of poreassembly might occur through similar processes, owingto their very different environments it is likely that differ-ences will be uncovered.

Mitotic assembly

As mentioned, NPC assembly during mitosis occurs whenthe NEs form around the chromatin of the two nascentdaughters [53,54]. Data obtained from mammalian cellsand an in vitro nuclear-assembly system based on Xenopusegg extracts have shown that mitotic-NPC assembly is a

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Figure 2. NPC assembly during mitosis and interphase. Mitotic-NPC assembly occurs concomitantly with the formation of new nuclear envelopes (NEs) around chromatin.

During this time, NPCs assemble by recycling subcomplexes that were dispersed into the cytoplasm during NPC and NE breakdown. Note that, during mitosis, the

cytoplasmic and nuclear contents are mixed together. Mitotic assembly is a step-wise process that begins with the recruitment of structural nups to chromatin during early

anaphase. By contrast, during interphase, NPCs assemble into an intact NE when the nucleus and cytoplasm are physically separated. During this process, NPCs use newly

synthesized nucleoporins present on both sides of the nuclear envelope.

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highly organized step-wise process. Several reports haveshown that nucleoporins are recruited to chromatin in asequential manner starting in early anaphase [55–59].Recently, in an elegant systematic analysis of nucleoporinrecruitment in vivo, the kinetics of assembly of eightdifferent NPC subcomplexes in living cells has been ana-lyzed [60]. Taken together, these results indicate thatmitotic-NPC assembly begins on chromatin with therecruitment of the scaffold Nup107–160 complex, knownto be essential for NPC assembly [57], followed by therecruitment of a small fraction of the total Nup153 andNup50 proteins. It is commonly assumed, although not

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firmly demonstrated, that the early recruited nucleoporins(Elys/Mel-28, Nup107–160 complex, Nup153, Nup50 and,possibly, other nucleoporins that have not yet been ana-lyzed) form a chromatin-bound intermediate, sometimesreferred to as a ‘prepore’, that functions as a bindingplatform for the recruitment of transmembrane nucleopor-ins and, later, the more peripheral proteins. Notably, therecruitment of several nucleoporins occurs after NPCshave started to transport, supporting the idea thatnot all nups are essential for pore function [60,61] orthat different NPC functions or transport pathways areactivated at different times of assembly.

Figure 3. Importin b and RanGTPase regulation of NPC assembly. (a) During mitosis, importin b binds and sequesters the Elys/Mel-28 nucleoporin (Mel-28), preventing its

interaction with chromatin. When the importin-b–Mel-28 complexes are in the proximity of DNA, where there is a high concentration of RanGTP owing to the chromatin

association of the Ran GDP–GTP exchange factor [regulator of chromosome condensation (RCC1)], RanGTP binds to the transport receptor, thereby releasing Mel-28 and

enabling it to bind to chromatin. Following the same mechanism, the importin-b-bound Nup107–160 complex is released by RanGTP in the proximity of DNA and recruited

to chromatin through Mel-28. The chromatin-bound Nup107–160 complex can then recruit other nucleoporins in a step-wise manner. (b) NPC assembly during interphase

requires the RanGTP-dependent release of the Nup107–160 complex from importin b on the cytoplasmic and nuclear side of the NE. How the released complexes

coordinate the formation of a functional NPC from both sides of the nuclear envelope is still unclear.

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The step-wise mechanism of NPC assembly and theexistence of intermediate structures on chromatin is sup-ported by EM studies [62–65]. Evidence from severalrecent reports has indicated that the early recruitmentof the Nup107–160 complex to chromatin is mediated byElys/Mel-28, which was initially discovered as an AT-hook-containing transcription factor [66–69]. The chromatinassociations of nups and the interactions between manyof them are regulated by the ratio of importin b to thesmall GTPase Ran [58,70]. During mitosis, importin b

associates with and sequesters a subset of nups, includingElys/Mel-28 and the Nup107–160 complex, preventingtheir association with chromatin and interaction with

other nups [58,68] (Figure 3a). The RanGTP-dependentrelease of nucleoporins from the import receptor is anearly step in NPC assembly and, because RanGTP isconcentrated around chromosomes, occurs in the proximityof the chromosomal DNA. In this way, importin b andRanGTPase coordinate the spatial positioning of theassembly of NPCs during mitosis.

Interphase assembly

During interphase, the number of pores doubles to preparethe cells for re-entering mitosis. Previously, it was believedthat NPC doubling was restricted to S-phase [71]; however,recent studies have indicated that NPCs are assembled

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continuously from G1 to G2 phases [72,73]. Little is knownabout the mechanism of interphase NPC assembly.Genetic studies in S. cerevisiae have identified nucleopor-ins essential for the formation of NPCs [74–76] and arequirement for karyopherin b and RanGTP [77,78]. How-ever, the cellular mechanism used to assemble NPCs intoan intact NE remains unclear. In metazoans, the assemblyof NPCs into the NE also requires importin b and RanGTP[79]. Moreover, RanGTP and the essential Nup107–160complex are required from both sides of the NE, indicatingthat interphase NPC assembly requires the coordination ofnuclear and cytoplasmic events (Figure 3b).

EM studies inDrosophila embryos have shown that newNPCs can form in an intact nuclear envelope that lackspre-existing pores [80]. Consistent with this finding,assembly of NPCs in regions devoid of pores has beenobserved inHeLa nuclei by live imaging [79]. Furthermore,experiments using in vitro-assembled nuclei have indi-cated that NPCs can form de novo, without using subunitsfrom pre-existing pores [79].

Taken together, these results have led to the currentview of interphase NPC assembly, which indicates thatnuclear pore complexes form independently of pre-existingpores and from both sides of the nuclear envelope, follow-ing a mechanism that is at least partially conserved withmitotic-NPC assembly. This raises the question of whetherthe chromatin-bound intermediates observed during mito-sis indeed represent that part of the pore that is insertedinto theNE from the nucleoplasmic side during interphase.If so, the mitotic- and interphase-assembly mechanismswould involve a coordinated interaction between chroma-tin-bound subcomplexes, cytoplasmic subcomplexes andtransmembrane NPC components to assemble the multi-protein NPC into the double membrane of the NE.

Disassembly

NPC disassembly has only been described during mitosisand there is no evidence of pores being dismantled duringinterphase. Similar to the assembly process, NPC disas-sembly takes place through an ordered process. Partiallydisassembled pores have been observed in Drosophilaembryos [80] and in in vitro NE-disassembly studies[81]. In Drosophila embryos, only one kind of disassemblyintermediate was found during prophase, indicative of ahighly synchronized process, the existence of other extre-mely unstable intermediates or the existence of only oneintermediate in this organism [80]. By contrast, a mixedpopulation of different partially disassembled NPCs wasobserved in the same disassembling nuclei using the invitro Xenopus system, indicating the existence of an asyn-chronous mechanism [81].

The step-wise disassembly of NPCs has been followedduringNE breakdown of starfish oocytes. In these cells, theprocess starts with the release of the basket nucleoporinsNup98 and Nup153 and is followed by the release ofNup214 from the cytoplasmic ring. The dissociation ofperipheral nucleoporins into the cytoplasm leads to agradual loss of selective nuclear permeability.

Using a kinetic assay in mammalian cells [60], it hasbeen shown that NPC disassembly occurs synchronously,being faster than assembly and, intriguingly, not following

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its exact reverse order. For example, nucleoporins that arerecruited to chromatin early during assembly, such asmembers of the Nup107–160 complex, are released beforenucleoporins that are recruited later, such as Nup58.

In support of these results, it has been observed that inDrosophila the scaffold Nup107 protein is also releasedfrom NPCs after the basket components Nup153 and Mtor(the homolog of themammalian Tpr protein) but before thecentral channel nucleoporin Nup58 [82].

In the yeasts S. cerevisiae and Schizosaccharomycespombe, which divide without NE breakdown, no disassem-bly of pores has been reported; however, in other fungiundergoing closed mitosis, such as Aspergillus nidulans, apartial disassembly of pores also occurs [83]. In these cells,the cytoplasmic dispersal of several FG nucleoporins start-ing in prophase leaves partially disassembled pores at theNE, which have an increase in permeability and enablemitotic regulators to access the nucleus.

Although the molecular mechanisms of pore disassem-bly have not been investigated in detail, all the currentevidence points towards mitotic phosphorylation as thetrigger of the disassembly process. Several groups havereported mitotic-specific hyperphosphorylation of nucleo-porins both in vivo and in vitro [61,84–87]. The Ser/Thrkinase CDC2 (Cdk1) seems to be a universal mitotic kinaseinvolved in nucleoporin phosphorylation [83,84,88,89];however, the mammalian protein kinase A (PKA) andprotein kinase C (PKC), the S. cerevisiae casein kinaseand the Aspergillus nidulands Ser/Thr kinase NIMA havealso been shown to phosphorylate NPC components[39,83,88,90]. How mitotic phosphorylation initiatesNPC disassembly is still unclear, but it is possible thatit induces the dissociation of subcomplexes and preventstheir association, because nucleoporins have to be depho-sphorylated to reassemble the pore [58].

Maintenance

The mitotic disassembly–reassembly cycle of NPCs inmetazoans ensures that every nuclear pore gets renewedduring each cell-division cycle. Yet, how NPCs are main-tained during interphase is unknown. As mentioned, sev-eral nucleoporins are dynamic and can come on and off thepore, indicating that they are exchanged or recycled fromNPCs during interphase. By contrast, the NPC scaffoldsare very stable and only exchange once per cell cycle, whenthe NE breaks down. This raises the question of how NPCscaffold proteins are maintained when the cells stop divid-ing (e.g. after differentiation). Are old pores disassembledand replaced by newNPCs? This would imply the existenceof a post-mitotic disassembly mechanism. As mentioned,no evidence of such amechanism has been described so far.Or, do the NPC scaffolds remain embedded in the NE forthe entire life span of the cell? If this is the case, then insome long-lived cells such as neurons, the core of the NPCswould remain in the NE for extensive periods of time.Because of this, the pores could be subject to age-depend-ent damage that could affect their function and possiblycompromise nuclear integrity. In favor of this idea, altera-tions in the nuclear structure and mislocalization ofnuclear proteins have been linked recently to the agingphenomena [91,92].

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Transport-independent functions of nupsAs described, the main function of NPCs is to control thepassage of molecules between the nucleus and the cyto-plasm. A tight regulation of nucleocytoplasmic transport isessential for cell homeostasis. Consequently, alterations inNPC members or the nuclear-transport process have astrong impact on cell growth and survival and, not surpris-ingly, have been associated with several diseases such ascancer [93,94] and the rare autosomal recessive disordertriple A syndrome [95].

Besides functioning as gatekeepers of the nucleus, it hasbecome evident that NPCs and nucleoporins are impli-cated in many other biological functions [1]. NPCs havebeen shown to anchor and modulate the activity of sumoy-lating and desumoylating enzymes [96]. Interestingly,Nup358, the main component of the cytoplasmic filaments,is an active E3 ligase in the sumoylation reaction [97,98].Thus, through the SUMO (small ubiquitin-related modi-fiers) pathway, NPCs are indirectly involved in the regu-lation of numerous cellular processes such as genetranscription, DNA replication, DNA damage and repair,chromosome segregation, genome stability, cell death andsenescence.

Three nucleoporins –Nup153, Nup358 and gp210 – havebeen associated with nuclear-envelope breakdown[1,99,100]. Although Nup153 and Nup358 contribute tothis process by recruiting the coatomer coat protein I(COPI) complex to the NE, the role of gp210 in NE break-down is still unclear. Conversely, several other nucleopor-ins have been reported to have a role in NE assembly.These include the transmembrane nucleoporins Pom121and NDC1, and the soluble nucleoporins Elys/Mel-28,Nup35 and Nup155, the latter two being part of the samesubcomplex [17,99,101–103].

In the past few years, many components of the NPChave been described as having important roles duringmitosis. Nup170p, the yeast homolog of mammalianNup155, was the first nucleoporin linked to kinetochorefunction and chromosome segregation [104]. Subsequently,Nup358 was found anchored to kinetochores and spindlesthrough the export receptor CRM1 (chromosome regionmaintenance 1). Depletion of Nup358 in cells has revealedits essential role in kinetochore assembly and interactionwith microtubules [100,105]. More recently, the Nup107–

160 complex and Elys/Mel-28 were found at kinetochoresduring mitosis [106–108]. The recruitment of the Nup107–

160 complex to these structures was shown to be depend-ent on the NDC80 complex and CENP-F (centromereprotein F) and to occur upstream of Nup358. Cells depletedof several members of the Nup107–160 complex together,or the Seh1 component alone, showed a mitotic delay andfailed to attach microtubules to kinetochores properly,resulting in abnormal chromosome congression. Althoughthese cells showed activation of the spindle checkpoint andlonger spindles in metaphase, no defects in spindle assem-bly were observed [107]. In a different study, the Nup107–

160 complex was shown to be required for assembly of thebipolar spindle in vitro, although no activation of thespindle checkpoint was observed in extracts depleted ofthe complex [109]. The differences observed could beattributable to the fact that the Nup107–160 complex

strongly localizes to spindles assembled in vitro but onlytransiently associates with cellular spindles during pro-metaphase [109]. A role for the Nup107–160 complexes inspindle assembly and function is supported by the findingsthat fission yeasts lacking Nup120p, a homolog of thehuman Nup160 protein, show abnormal spindles [110].Rae1 is a shuttling nucleoporin that has also been associ-ated with spindle formation [14,15,111,112]. It was foundthat this protein is part of a large ribonucleoprotein com-plex that controls microtubule dynamics and has an essen-tial role in spindle assembly [112]. Finally, RNAiexperiments in Caenorhabditis elegans have revealed arole for several nucleoporins in the regulation of spindleorientation [113].

Very recently, a small fraction of the Nup358 nucleo-porin, the main constituent of the NPC cytoplasmic fila-ments, was found in the cytoplasm of cells co-localizingwith microtubules during interphase. In this study,Nup358 was found to interact with and regulate thedynamics of interphasemicrotubules directly, thus, havinga role in organization of the cytoskeleton [114].

Lastly, several reports have associated NPCs with chro-matin organization and the regulation of gene expression(for reviews, see Refs [115,116]), especially in lower eukar-yotes. The nuclear periphery has historically been associ-ated with gene silencing and repression (for a review, seeRef. [117]). The presence of heterochromatin patches, gene-poor chromosomal regions and silent genomic domainsadjacent to the NE support this hypothesis. In agreementwith this idea, NPCs were initially associated with silentchromatin regions [118,119]. In budding yeast, Nup145p isresponsible for tethering silent telomeric chromatin to theNPCs through the myosin-like protein (MLP)1 and MLP2proteins [118], although the role of the MLP proteins intelomere silencing and anchoring has been recently chal-lenged [120]. Opposing the classical view of the nuclearperiphery, there is now increasing evidence indicating thatthis region also functions as a gene-activating domain. In ascreening for proteins with chromatin boundary activity,the Nup2p nucleoporin was found to block the propagationof heterochromatin in specific domains, thus, preventingtheir silencing, and it is thought to do so by recruitingchromatin to pores [121]. These studies indicate that chro-matin activity can be modulated by relocation to the NPC.Chromatin immunoprecipitations (CHIP) studies withantibodies against NPC components have shown thatmany nucleoporins preferentially associate with transcrip-tionally active genes [122]. The results of this technique donot directly connect active genes to the NPC structure andit remains a possibility that the DNA binding of thenucleoporins occurs entirely in the nuclear interior. How-ever, the fact that the NPC-associated protein Sus1 hasbeen described to be a member of the Spt-Ada-Gcn5-acet-yltransferase (SAGA) histone acetylase complex involvedin transcriptional initiation [123] and required for theconfinement of GAL genes to NPCs during transcriptionalactivation in S. cerevisiae [124] directly links nuclear poresto active transcription. The relocation of genes to poresduring activation has been reported for several othergenes, including the HXK1 (encoding hexokinase-1),INO1 (encoding an inositol 1-phosphate synthase) and

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several mating-response genes, although there are somecontroversial findings in the nucleoporin requirements forgene tethering (for reviews, see Refs [115,125]). Also sup-porting a role of NPCs in transcriptional activation, exper-iments in which the Nup2p was fused to the microccocalnuclease have revealed that the nuclease cleavage occursmainly in promoter regions, indicating the association ofNup2p with actively transcribed genes [126].

How genes are activated at the NPC is not well under-stood, but the Rap1 transcription factor seems to have animportant role. CHIP analyses have indicated that mostnups preferentially associate with genes containing Rap1-binding sites [122]. Furthermore, the Rap1-dependentactivation in yeast was shown to occur through the Nup84pcomplex (the yeast Nup107–160 complex homolog) at theNPC [127]. This result led to the idea of a ‘reverse recruit-mentmechanism’, indicating that, during activation, genesmove to a platform tethered to the NPC containing thepreassembled transcriptional machinery [127].

In higher eukaryotes, little is known about the connec-tion between the NPC and regulation of gene expression,but the fact that the SAGA-dependent anchorage of genesto the NPC has been reported in Drosophila [128] and thattwo nucleoporins are linked to the transcriptional regula-tion of dosage compensation in this organism [129] indicatethat the mechanism might be conserved. Interestingly, arecent report has shown the association of Nup93, a stablecomponent of the NPC scaffold, with several regions of thehuman genome [130]. But, in this case, the chromosomeregions associatedwithNup93were enriched inmarkers ofheterochromatin, circling back to first findings in buddingyeast indicating the association of NPCs with silent chro-matin. Notably, the chromosome-association of Nup93varied depending on the global histone acetylation stateof chromatin.

The association of chromatin to NPCs, together withthe fact that in higher eukaryotesNPCs are immobile [55],indicates that pores could function as positional markersat the NE for the organization and maintenance of thegenome architecture. The relative location of severalgenes or chromosome regions within the cell nucleushas been shown to vary in response to metabolic changesand during cell differentiation. The findings that theDNA–NPC interactions are dynamic and change depend-ing on the chromatin state indicate that NPCs could beinvolved, at least in part, in regulating these genomicrearrangements. Supporting a role of NPCs in celldifferentiation, Nup133, a member of the scaffoldNup107–160 complex, has recently been described to beimportant for neural differentiation during embryogen-esis, which potentially highlights a new aspect of NPC andnucleoporin function [131].

Concluding remarksOwing to its interesting properties and increasing reper-toire of functions, the biology of NPCs has become an areaof focus for many fields. Despite the substantial progressthat has been made in the understanding of NPC bio-genesis and function in the past decade, there are stilllarge gaps to fill. The actual location of every nucleoporininside the NPC structure, the detailed molecular mechan-

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isms of assembly, disassembly and translocation throughthe pore, and how NPCs perform their transport-indepen-dent functions are just some of the mysteries that might beclarified in the coming years.

AcknowledgementsWe apologize to all colleagues whose work could not be cited directlyowing to space limitation. We thank members of the Hetzer laboratoryand Marcela Raices for helpful suggestions and critical reading of themanuscript.

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