M.E. Müller Institute for Structural Biology, Biozentrum, University of Basel, Klingelbergstr. 70, CH-4056 Basel, Switzerland

Running title: NPC becomes alive

* authors contributedequally

corresponding author: [email protected]; phone: +41 61 267 2255; fax: +41 61 267 2109

Abstract

In this review wesummarize the structure and function ofthe nuclearpore complex (NPC). Special emphasis isput on recent findings which reveal theNPC as a dynamic structure inthecontext of cellularevents like nucleocytoplasmic transport, celldivision anddifferentiation, stress response and apoptosis. Evidence for theinvolvement ofnucleoporins in transcription and oncogenesis is discussed, andevolutionarystrategies developed by viruses to cross the nuclear envelope arepresented.  

Keywords: nucleus;nuclear pore complex;nucleocytoplasmic transport; nuclear assembly; apoptosis; rheumatoidarthritis;primary biliary cirrhosis (PBC); systemic lupus erythematosus; cancer

Eukaryotic cells in interphase are characterized by distinct nuclear and cytoplasmic compartments separated by the nuclear envelope (NE), a double membrane that is continuous with the endoplasmic reticulum (ER). Nuclear pore complexes (NPCs) are large supramolecular assemblies embedded in the NE and they provide the sole gateways for molecular trafficking between the cytoplasm and nucleus of interphase eukaryotic cells. They allow passive diffusion of ions and small molecules, and facilitate receptor-mediated transport of signal bearing cargoes, such as proteins, RNAs and ribonucleoprotein (RNP) particles (Görlich and Kutay, 1999; Conti and Izaurralde, 2001; Macara, 2001; Fried and Kutay, 2003). A consensus model of the 3D architecture of the NPC has evolved from extensive electron microscopy (EM) and tomography studies in both yeast and higher eukaryotes (Unwin and Milligan, 1982; Hinshaw et al., 1992; Akey and Radermacher, 1993; Yang et al., 1998; Stoffler et al., 2003; Beck et al., 2004). Accordingly, the NPC consists of an eightfold symmetric central framework. The cytoplasmic ring moiety of the central framework is decorated with eight cytoplasmic filaments, whereas the nuclear ring moiety is topped with eight tenuous filaments that join distally into a massive distal ring and thereby form a distinct nuclear basket (Fig. 1). The overall 3D model of the NPC is conserved from yeast to higher eukaryotes, except for variations in linear dimensions among species (Fahrenkrog et al., 1998; Cohen et al., 2002; Stoffler et al., 2003).

Based on their molecular mass of ~125 MDa (Reichelt et al., 1990) and the high degree of 822 symmetry of their central framework, it is assumed that both vertebrate  and yeast  NPCs are composed of multiple copies of ~30 different proteins, called nucleoporins (Nups) (Panté and Aebi, 1996; Stoffler et al., 1999; Fahrenkrog and Aebi, 2002). Up to date, ~30 yeast (Table 1) and ~26 vertebrate (Table 2) nucleoporins have been identified and characterized (Rout et al., 2000; Cronshaw et al., 2002).

Precise localization ofthenucleoporins in the NPC is essential for understanding their functionsinnucleocytoplasmic transport and in NPC assembly at the molecular level.Immunolocalizationof nucleoporins using immunogold EM has shown that most nucleoporinsaresymmetrically localized at both the cytoplasmic and nuclear peripheryof theNPC and only few of them being located asymmetrically to either thecytoplasmicor nuclear periphery in both yeast and vertebrate NPC (Panté etal., 1994; Fahrenkrog et al.,1998;Rout et al., 2000; Fahrenkrog and Aebi, 2002; Cronshaw et al., 2002). Interestingly, manynucleoporins areorganized into distinct subcomplexes within the NPC structure (Finlay et al., 1991;Panté et al., 1994;Belgareh et al., 2001; Walther et al., 2003). Some of thesesubcomplexes likethe Nup107-160 complex and the Nup62 complex even stay intact duringmitosisand might represent elementar building blocks of the NPC.

Despite the conserved structural features of NPCs among different species as yeast and vertebrates there have been some reports about cell-type specific expression of certain nucleoporins (Wang et al., 1994; Hu and Gerace, 1998; Nothwang et al., 1998; Cai et al., 2002; Coy et al., 2002; Olsson et al., 2004). For example, RanBP2L1, and POMFIL1 represent cell-type specific expressed proteins with homology to the nucleoporins RanBP2/Nup358 and POM121 which are coded by different genes (Wang et al., 1994; Nothwang et al., 1998; Cai et al., 2002; Coy et al., 2002). In contrast, the differentially expressed nucleoporins Nup45 and Nup58, both constituents of the Nup62 NPC subcomplex, seem to be generated by alternative splicing from the same gene (Hu and Gerace, 1998). The most recent report concerns the restricted expression of the transmembrane nucleoporin gp210: the mRNA as well as the expressed protein seem to be specific for cultured embryonic stem cells and certain polarized epithelial cells, whereas no expression could be detected in several cultured cell lines of fibroblastic and epithelial origin (Olsson et al., 2004). This result is of special importance since there seem to be only two transmembrane nucleoporins, gp210 and POM121, to anchor the entire 125 MDa NPC to the nuclear envelope, pointing towards a crucial role for POM121 in the architectural design of this multiprotein assembly.

3. Nuclear pore complex: static versus dynamic structure

3.1. Nucleoporindomain flexibility and nucleoporin turnover at the NPC

Contrary to theassumption that theNPC is a rather static structure, a number of recent studies haveprovidedevidence that some of the nucleoporins are mobile within the NPC. Forexample, ithas been shown that the vertebrate nucleoporin Nup98, originallylocalized to thenuclear basket (Radu et al., 1995), can also be found onthecytoplasmic face of the NPC (Griffiset al., 2003). Nup98 can dynamicallyassociatewith the nuclear pore and shuttlebetween the NPC andintranuclearbodies and additionally between the nucleus and the cytoplasm in atranscription-dependentmanner (Zolotukhin and Felber,1999; Griffis et al.,2002 and 2004). Because Nup98 playsa role in RNAexport (Powers et al., 1997), its mobility proposesthat Nup98might associate with RNA close to its transcription site and thenfurtheraccompany the processed RNA through the NPC into the cytoplasm.

Nup153 plays a role inthe import ofproteins into the nucleus as well as in the export of RNAs and proteinsintothe cytoplasm (Bastos et al., 1996;Shah and Forbes, 1998). Nup153associated with exportcargo shuttles between the nuclear and cytoplasmic faces of the NPC (Nakielny et al., 1999). Initially thevertebrate Nup153had been localized to the distal ring of the nuclear basket (Panté etal., 1994) and close to or at thenuclear ringof the NPC (Walther et al., 2001). Immunogold EM withdomain-specificantibodies against Nup153, in combination with recombinantly expressedepitope-tagged Nup153 in the Xenopusoocytes,has revealed a domain-specific topology within the NPC (Fahrenkrog et al., 2002). Nup153 seems to beanchoredthrough its N-terminal domain to the nuclear ring moiety and itscentralZn-finger domain to the distal ring of the NPC. By contrast, theC-terminaldomain which harbors ~40 FG repeats (about 700 amino acids), appears tobehighly mobile, as it has been localized throughout the nuclear basketand evenat the cytoplasmic periphery of the central pore of the NPC (Fig.2; Fahrenkrog et al. (2002and2004); Fahrenkrogand Aebi (2003)). In their native stateFG-repeatsare unfolded and highly unstructured (Baylisset al., 2000; Allen et al., 2002;Denning et al., 2002 and 2003) sothat theC-terminal domain of Nup153 could extend up to ~200 nmif completely stretched out, meaning that the C-terminal domain canreach allthe way from the distal ring of the nuclear basket through the centralporeinto the cytoplasmic periphery of the NPC. This high mobility andstructuralflexibility of the FG repeats which act as binding/docking sites forcargocomplexes might significantly increase the efficiency of cargotranslocationthrough the NPC.

In addition to this structural flexibility, the association of Nup153 with the NPC exhibits remarkable dynamics: fluorescence recovery after photobleaching (FRAP) experiments have demonstrated that on average Nup153 stays associated with the NPC for no longer than about 40 seconds (Daigle et al., 2001; Griffis et al., 2004; for an overview of the turn over times of individual Nups at the NPC see Rabut et al., 2004a). This is interesting since Nup153 has been documented to be crucial for the formation of structurally intact NPCs. If nuclear envelopes are assembled in vitro from meiotic Xenopus laevis egg extracts depletion of Nup153 from this extract abolishes the incorporation of Tpr, Nup93 and Nup98 into the NPCs (Walther et al., 2001). In an attempt to combine these two findings into a model, two distinct Nup153 pools have been proposed to co-exist at the NPC: a stably associated one which has an architectural function, and a dynamic one exhibiting a high turnover rate (Griffis et al., 2004). Further investigations revealed that similar to Nup98, the mobility of Nup153 is also governed by transcriptional activity, in a way that transcription decreases the dissociation rate from the NPC by a factor of 10 so that  a considerable amount of either protein does not exchange any more, i.e. stably associated with the NPC (Griffis et al., 2004). Dissection of Nup98 and Nup153 demonstrated that RNA polymerase I and RNA polymerase II exert distinct effects on the mobility of these two nucleoporins via distinct domains within the two proteins. As might be expected, these domains are the respective NPC interacting domains that have been described previously (Griffis et al., 2004). However, as yet nothing is known about the mediators of this transcription-dependent mobility nor about the exact molecular modifications of Nup98 and Nup153 that are responsible for their mobile behaviour.

Another aspect of NPC dynamics is revealed by the fact that the NE of higher eukaryotes disintegrates and NPCs disassemble into distinct subcomplexes during mitosis. Although the functional significance of this mitotic disassembly of NPCs is not known, the molecular mechanism leading to this phenomenon appears to be governed by the phosphorylation/dephosphorylation of nucleoporins as has been shown for the transmembrane nucleoporin gp210, as well as for Nup62, Nup153, CAN/Nup214 and RanBP2/Nup358 (Macaulay et al., 1995; Favreau et al., 1996). Examples of nucleoporin subcomplexes that stay intact during mitosis are the Nup107-160 subcomplex, the Nup62 complex, and the CAN/Nup88 subcomplex (Macaulay et al., 1995; Bodoor et al., 1999; Matsuoka et al., 1999; Belgareh et al., 2001). Towards the end of anaphase, NPCs sequentially reassemble around chromatin, and while one would expect this process to be initiated by transmembrane nucleoporins, there are also indications that the association of the Nup107-160 complex with chromatin might be the first step in the reassembly of the NPC after mitosis (Fig. 3; Walther et al. (2003); see also Bodoor et al. (1999)). In the near future the use of defined biochemical fractions or even recombinantly expressed nucleoporins in combination with in vitro assembly reactions and ultrastructural analysis by TEM and SEM will yield further insights into the distinct assembly steps that eventually yield functional NPCs (Finlay et al., 1991; Walther et al., 2001, 2002 and 2003; Liu et al., 2003).

In addition to this reassembly after mitosis, a second mechanism for the de novo formation of NPCs in pre-existing nuclear membranes must take place in lower eukaryotes that go through closed mitosis and therefore never disassemble their NEs. Furthermore, morphometric analysis and FRAP experiments on cells cultured from higher eukaryotes demonstrate that NPCs can also assemble into preformed NEs (Maul et al., 1972; Winey et al., 1997; Daigle et al., 2001). Hence, here we have the interesting situation that two distinct molecular events lead to the formation the same large supramolecular assembly (see also Rabut et al., 2004b). Finally, it should be mentioned that NPCs and nucleoporins might play a more active role in the progression of mitosis than believed earlier. To this end, it has been shown that the giant nucleoporin RanBP2/Nup358 plays an important role in mitotic progression and that the RanGAP1-RanBP2 complex is essential for microtubule-kinetochore interactions during mitosis (Salina et al., 2003; Joseph et al., 2004). Moreover, the Nup107-160 complex localizes in part to kinetochores during mitosis, and depletion of some members of the homologous complexes in yeast and C. elegans leads to defects in cell division (Galy et al., 2003; Baï et al., 2004; Loïodice et al., 2004).

As mentioned above,yeasts and otherlower eukaryotes undergo a closed mitosis so that their NEs and NPCsstayintact during this process. Recently however, it could be shown thateven inthese cells NPCs exhibit cell cycle-dependent dynamics. For example,the yeastnucleoporin Nup53p is associated with Nup170p during interphase butfollowingchanges at the onset of mitosis it becomes associated with anothernucleoporin,Nic96p (Makhnevych et al., 2003). This change presumablyleads to theexposure of a binding site for the transport factor Kap121p, whichafter associationwith Nic96p, prematurely releases its cargo and thereby preventsefficientnuclear import. Hence, this is a good example how physiological changesinnuclear transport activity can be regulated at the level of NPCarchitecture. Stress-inducedstructural reorganization of NPCs in yeast cells and the proteolyticbreakdown ofcertain nucleoporins during apoptosis in higher eukaryotes are furtherexamplesof dynamic NPC modifications (Ferrando-Mayet al., 2001; Shulga and Goldfarb,2003; Kihlmark et al., 2004; Loïodice et al., 2004). In yeast, for example,an increasein NPC permeability is observed under certain stress situations such ashighconcentrations of aliphatic alcohols, the presence of deoxyglucose, orduringchilling of the yeast cultures (Shulgaand Goldfarb, 2003). If yeast cellsdepleted of the nucleoporinNup170p are treated with increasing concentrations of certain alcohols,dissociationof several other nucleoporins, both centrally and peripherally locatedones, isobserved. Whilst no dissociation of any nucleoporin was observed inwild-typecells, the above mentioned results still may be taken as an indicatorforrearrangements occurring in NPC architecture following stress treatmentof the yeastcells (Shulga and Goldfarb,2003).

One hallmark ofapoptotic cells isthe condensation of their chromatin followed by the fragmentation oftheir DNA.Recent studies investigated the question of a possible link betweentheseapoptotic features and changes in the structure and function of NPCs (Ferrando-May et al.,2001; Kihlmark et al.,2004; Loïodice et al., 2004).During apoptosis nucleoporins aresequentially cleaved, amongst the first ones being the transmembranenucleoporin POM121 and RanBP2/Nup358, followed by Nup153, Nup62 and theNE-associatedlamins. Concomitant with these later steps clustering of the NPCs isobserved,while an increased permeability of the nucleus, the redistribution ofnucleartransport factors and the nuclear accumulation of mRNAs are hallmarksofearlier stages of apoptosis (Ferrando-Mayet al., 2001; Kihlmark et al.,2004; Loïodice et al., 2004).To date, the debate is still onwhether these observed changes in NPC architecture and nucleartransport are causallylinked to the later apoptotic program occurring in the nucleus.

Whereas ions and small molecules move in and out of the nucleus by passive diffusion, transport of proteins, RNAs, ribonucleoprotein  particles (RNPs), and ribosomal subunits is an energy-dependent and receptor-mediated process. Because many excellent reviews about nucleocytoplasmic transport have been published in the last few years (c.f. Corbett and Silver, 1997; Mattaj and Englmeier, 1998; Görlich and Kutay, 1999; Kaffman and O'Shea, 1999; Conti and Izaurralde, 2001; Komeili and O'Shea, 2001; Macara, 2001; Weis, 2002; Quimby and Dasso, 2003; Fried and Kutay, 2003; Johnson et al., 2004; Xu and Massagué, 2004), we describe here only the best characterized classical receptor-mediated nucleocytoplasmic transport (Fig. 4a). Accordingly, cargo to be transported in or out of the nucleus harbors a nuclear localization signal (NLS) for nuclear import or a nuclear export signal (NES) for nuclear export. NLS and NES are recognized by soluble transport receptors known as importins and exportins, also called karyopherins. These receptors facilitate transport through the NPC by interaction with FG repeats that many nucleoporins harbor. The key regulator of the directionality of nucleocytoplasmic transport is Ran, a member of the Ras-related GTPase superfamily, by switching between its GDP- and GTP-bound form. The nucleotide exchange factor, RanGEF (or RCCI) is bound to DNA and facilitates RanGTP formation in the nucleus, whereas the GTPase activating protein, RanGAP, that is excluded from the nucleus, assures depletion of the cytoplasm of RanGTP in combination with Ran binding protein RanBP1 and RanBP2. Hence, interaction of Ran with its compartmentalized regulatory proteins RanGEF and RanGAP generates a gradient across the NE with a high RanGTP concentration in the nucleus and a low level in the cytoplasm, and vice versa for RanGDP (Fig. 4b).

5. NPCs and diseases

NPCs via thenucleoporins have beenimplicated in several malignancies such as leukemias and otherscancers, aswell as in certain autoimmune diseases. Furthermore, as gatewaysbetween thecytoplasm and the nucleus they also play a pivotal role in viralinfections.

Several autoimmunediseases arecaused by autoantibodies recognizing nuclear antigens resulting in suchdiversillnesses as systemic lupus erythematosus, rheumatoid arthritis andprimarybiliary cirrhosis (PBC). Anti-NPC autoantibodies identified so farreact withthe nucleoporins Nup62, Nup153, Tpr, Nup358/RanBP2 and thetransmembranenucleoporin gp210 (reviewed in Nesheret al., 2001; Enarson et al., 2004).The epitopes of several of theseantibodies have been mapped but the exact mode of action is unknown.Often,these anti-nucleoporin antibodies co-exist with other autoantibodies sothattheir relevance for disease progression is unclear. Interestingly,anti-gp210autoantibodies are often found in PBC which are negative foranti-mitochondrialantibodies which gives them a potential diagnostic relevance. Inaddition,these antibodies are often associated with more severe forms of thediseasethus also being a possible prognostic marker.

Nucleoporins are involved in several cases of acute myeloid leukemia and a few other hematological malignancies as well as rare cases of other tumors (Table 3) (Cronshaw and Matunis, 2004). For Nup88, its overexpression is associated with malignant tumors (Gould et al., 2000 and 2002; Emterling et al., 2003; Agudo et al., 2004), whereas in most other cases the role of Nups in tumorigenesis stems from chromosomal rearrangements that result in oncogenic fusion proteins. With the exception of the RanBP2/Nup358-ALK fusion protein which localizes to the NPC, in all other known examples the oncogenic fusion protein does not involve the NPC-interacting domain(s) of the nucleoporin and thus is no longer associated with the NPC. The most common oncogenic fusions involve a segment of the gene encoding the FG-repeat domain of the nucleoporin Nup98 which, in turn, becomes linked to genes of the homeobox family of transcription factors. It was found that the Nup98 derived FG-repeat segments of the resulting oncogene interact with the transcriptional coactivators CBP (CREB binding protein) and p300 thereby leading to increased gene transcription (Kasper et al., 1999). Interestingly, FG-repeat segments of two other nucleoporins, Nup153 and CAN/Nup214, could substitute for the Nup98 segment in the oncogenic fusion protein (Kasper et al., 1999). It will be interesting to see if there is a link between this observed transactivation activity and the previously mentioned transcription-dependent mobility of Nup98 within the nucleus and at the NPC. Similarly, leukemias involving CAN/Nup214 chromosomal translocations, the FG-repeat domain of this nucleoporin gets fused to the DNA-binding proteins SET and DEK (von Lindern et al., 1992a and 1992b). In the case of chromosomal rearrangements involving the nucleoporin Tpr, the N-terminal coiled-coil domain of this protein is fused to the protein kinases MET, RAF and TRK and thereby yielding oncogenic dimerization of these kinases (Hays and Watowich, 2003).

Significant progress hasbeenachieved in recent years in the elucidation of the mechanisms thatviruses useto deliver their genetic material to the nucleus for replication (Fig. 5;reviewed in Greberand Fassati (2003) and Gustin (2003)). After fusion at theplasmamembrane or endocytotic uptake the virus particles are in the cytoplasmwherethey encounter different fates. The problem for entry of the geneticmaterialinto the nuclear compartment is the limited diameter of the NPC. Recentexperiments employing protein covered colloidal gold particles showedthat objectsup to 39 nm diameter are able to travers the NPC (Panté andKann, 2002). Thus smaller viruseslike theparvovirus MVM (minute virus of mouse, 25 nm capsid diameter) or HBV(hepatitisB virus, 32-36 nm capsid diameter) are able to cross the NPC withoutthe needfor capsid disassembly. Invivo in thecase of HBV, however, passage through the NPC is facilitated by thephosphorylation of HBV capsids during maturation which makes nuclearlocalization signals accessible for the nuclear transport receptorsimportin-αand -β (Rabe et al., 2003). After passage throughthe centralpore of the NPC virus particles then reach the nuclear basket whereimmaturecapsids are apparently trapped, whereas capsid protein and DNA frommaturedviruses are released into the nucleoplasm (Fig.5a and 6; Rabe et al. (2003)).

In contrast to the situation for HBV, herpes simplex virus (HSV, 125 nm capsid diameter) or adenovirus particles (90 nm diameter) in the cytoplasm are too large to travers the NPC. In a first step these virus particles dock at the cytoplasmic side of the NPC. This docking of HSV particles at the NPC is dependent on the presence of importin-β, whereas adenovirus capsids directly associate with the cytoplasmically localized nucleoporin CAN/Nup214 (Wisnivesky et al., 1999; Ojala et al., 2000; Trotman et al., 2001). HSV then releases its DNA through the NPC into the nucleoplasm, whereas adenovirus particles first trap cytosolic factors like Hsc70, together with histone H1 and transport factors which are necessary for the subsequent capsid disassembly prior to viral DNA-translocation into the nucleus (Fig. 5b and c; Trotman et al. (2001)).

Certain RNA viruses likepicornaviruses (e.g.poliovirus or rhinovirus)and rhabdoviruses replicate in the cytoplasm of host cells and thus donot haveto cross the NE. However also in cells infected with these viruses, NPCstructure and nucleocytplasmic transport are often severely disturbed (Gustin, 2003).It has been shown that theinfection of cells with polioviruses or rhinoviruses results in thecytoplasmicaccumulation of certain nuclear proteins like La, Sam68 or nucleolinwhich theninteract with the viral RNA and might aid in their replication. This isachieved by inhibiting active nucleocytoplasmic transport via theproteolyticdegradation of nucleoporins like Nup62 and CAN/Nup214 (and possiblyothers aswell) (Gustin and Sarnow, 2001and2002). Another strategy isused bythevesicular stomatitis virus (VSV). Its most abundant virion component,the Mprotein, causes cell rounding, inhibits transcription and also blocksnuclearexport of U snRNAs, rRNAs and mRNAs as well as snRNP and classicalnuclearimport. As one cellular protein interacting with M protein thenucleoporinNup98 could be identified which binds transport receptors and has beenshown tobe crucial for nuclear export of mRNAs (von Kobbe et al., 2000).Interestingly during antiviralresponse via interferon signaling Nup98 gets upregulated and thisupregulationreleases the M protein induced mRNA export block (Enninga et al., 2002).

Already by 1985 a“gene-gating”hypothesis was proposed (Blobel,1985) suggesting aninteraction oftheNPC with genes as a means of transcriptional regulation. In recentyearsexperimental evidence for such interactions arised from studies inyeast. Inthis context, the interaction of telomeric chromatin with the NPC,especiallyNup145C, Nup60p, Mlp1p and Mlp2p result in transcriptional silencing (Feuerbach et al., 2002), whereas tethering ofchromosomesto the nucleoporin Nup2p lead to a block in heterochromatin spreading,i.e.Nup2p exhibits chromatin boundary activity, thus leading to geneactivation (Ishii et al., 2002). In the most recentreport genesinteracting with several nucleoporins and transport factors wereidentified (Casolari et al., 2004). This was achieved bychromatinimmunoprecipitation using certain nucleoporins and transport factors asbaitsand analysis of the precipitated gene sequences by microarrayhybridizationanalysis (Casolari et al., 2004). It turned out thatmore frequentlytranscribed genes were clearly associated with certain nucleoporins(amongstothers Mlp1p, Mlp2p, Nup2p and Nup60p) and transport factors, whereastheRanGEF Prp20p was found to interact mainly with infrequentlytranscribed genes(Fig. 7).Future in vivochromosomalpainting andnucleoporin labeling studies together with high resolution lightmicroscopyunder gene inducing conditions will clarify the significance of theseobservations.

In this review we have described the involvement of the NPC in various physiological events and hope to leave the reader with a dynamic impression of that complex and fascinating structure.

Acknowledgements:

This work was supportedby the Human Frontier ScienceProgram (U.A), bythe Swiss National Science Foundation (B.F.), and by the NationalCenter ofCompetence in Research (NCCR) program “NanoscaleScience”(UA). Additionalfunds were provided by the Maurice E. Müller Foundation and bytheKanton BaselStadt.

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