FEBS Letters 582 (2008) 2004–2016

Minireview Nuclear pore complex assembly through the : Regulation and membrane organization

Wolfram Antonina,*, Jan Ellenbergb,*, Elisa Dultzb a Friedrich Miescher Laboratory of the Max-Planck-Society, Spemannstraße 39, 72076 Tu¨bingen, Germany b European Molecular Biology Laboratory, Meyerhofstraße 1, 69117 Heidelberg, Germany

Received 6 February 2008; accepted 28 February 2008

Available online 6 March 2008

Edited by Ulrike Kutay

generate the channel through the double membrane of the nu- Abstract In , all macromolecules traffic between the nucleus and the cytoplasm through nuclear pore complexes clear envelope in which the NPC is embedded and we will dis- (NPCs), which are among the largest supramolecular assemblies cuss here how this could be achieved. NPC assembly is in cells. Although their composition in yeast and metazoa is well regulated in space and time and some of the principles of this characterized, understanding how NPCs are assembled and form regulation have become evident in the last years. However, in the pore through the double membrane of the addition to being a target of cell cycle regulation, it has and how both processes are controlled still remains a challenge. emerged that the NPC itself controls several aspects of cell cy- Here, we summarize what is known about the biogenesis of cle progression and we will therefore review what is known NPCs throughout the cell cycle with special focus on the mem- about the crosstalk between the NPC and cell cycle regulation. brane reorganization and the regulation that go along with NPC assembly. 1.1. Overall structure Ó 2008 Federation of European Biochemical Societies. Pub- lished by Elsevier B.V. All rights reserved. NPCs are very large macromolecular assemblies of an esti- mated mass of 66 Mega (MDa) in yeast and Keywords: Nuclear pore complex; Nuclear assembly; Cell 125 MDa in vertebrates. Despite this mass difference, the fun- cycle; damental architecture of NPCs is conserved between yeast and metazoa as shown by three-dimensional models of the NPC derived from electron microscopy. Recently, state of the art cryo-electron tomography of NPCs in Dictyostelium discoideum has achieved a resolution of below 1. Introduction to the nuclear pore complex 6nm [2,3]. These studies confirm and extend previous struc- tural observations (Fig. 1A): the complex has an eight-fold The nuclear envelope in eukaryotic cells provides a physical rotational symmetry and can be divided in three main parts: barrier between the , the site of DNA replication a central core in the plane of the nuclear envelope, a nuclear and , and the cytoplasm, the site of trans- basket, and cytoplasmic filaments. The central core is formed lation. This separation allows and requires regulated transport by three distinct rings structures all of which contact the mem- of macromolecules such as , RNA and ribonucleopro- branes of the nuclear envelope: Centrally, the spoke ring is tein particles across the nuclear envelope. The sole sites of this composed of eight clamp-shaped structures that are attached transport are the nuclear pore complexes (NPCs). The traffic to the membrane at two sites. This spoke ring is sandwiched through the NPC is highly specific and regulated, nevertheless between a cytoplasmic and a nucleoplasmic ring. The central incredibly efficient with several hundred translocation events core is decorated by eight extended filaments on the cytoplas- per second and NPC. How NPCs mechanistically mediate this mic side and by a basket-like structure connected with a distal enormous flux of macromolecules is still controversially dis- ring on the nuclear side. The improved resolution of Beck et al. cussed [1]. allowed the visualization of a luminal connector element that Although being indispensable to understand the mechanism spans the space between outer and inner nuclear membrane of transport, a clear picture of the molecular structure of the and is attached to opposite sides of the membrane, where the NPC is still lacking and many questions about how it is assem- cytoplasmic and nuclear rings make contact. bled and maintained remain open. The assembly of the NPC As informative as this cryo-electron tomography studies are, must be accompanied with changes in membrane structure to they do not provide sufficient resolution to study molecular de- tails of the individual proteins of the NPC, the . Obtaining atomic resolution data of entire NPCs by X-ray *Corresponding authors. Fax: +49 7071 601801 (W. Antonin); fax: +49 crystallography seems an impossible undertaking for the near 6221 38798328 (J. Ellenberg). future. However, significant progress has been made in under- E-mail addresses: [email protected] (W. Antonin), standing the structures of single nucleoporins. Several high res- [email protected] (J. Ellenberg), [email protected] (E. Dultz). olution structures of nucleoporins or fragments of Abbreviations: NPC, nuclear pore complex; ER, endoplasmic reticu- nucleoporins are available [4–7, and references therein]. With lum; MDa, Mega Dalton; ALPS, ArfGAP1 packing sensor more and more high resolution structures of individual

0014-5793/$34.00 Ó 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2008.02.067 W. Antonin et al. / FEBS Letters 582 (2008) 2004–2016 2005

Fig. 1. Structure of the NPC. (A) Reconstruction of the Dictiostylium NPC from cryo-electron tomography (adapted by permission from Macmillan Publishers Ltd.: Nature, Beck et al., copyright 2007). Cytoplasmic filaments are not visible and a mass density in the centre of the NPC which is at least partially ascribed to transported substrate has been omitted. Membranes are shown in grey. (B) Diagram of the modular structure of the metazoan NPC composed of different biochemically characterized subcomplexes. The exact position of many nucleoporins in the NPC is unknown and therefore drawn schematically. Nucleoporins and complexes recruited during the first, second, third and forth step of NPC assembly as described in the text are colored in green, yellow, red and blue, respectively. For nucleoporins colored in grey, the assembly time points have not been analyzed so far. nucleoporins and eventually NPC subcomplexes of the NPC or stretches of them devoid of FG repeats are thought to be being solved and further improvements in electron microscopy structured and form the backbone of the overall architecture techniques it will hopefully become feasible to fit atomic reso- of the NPC. Bioinformatic analysis predicts that the structured lution structures into the overall low resolution structure of the parts of nucleoporins consists of only two different fold types, NPC in the not too distant future. repeating alpha helixes (in an alpha-solenoid fold) and zigzag- Recently, Alber et al. took an alternative approach to ging beta sheets (in a beta-propeller fold) [10]. Many of the bridge the gap between the overall structural and biochemical solved structures of nucleoporins support this prediction, but information [8,9]: integrating data about the stoichiometry, recent data shows that the structural repertoire of the NPC localization, shape of and protein–protein interactions be- is more complex. Although yeast Nic96p and Nup145 do con- tween each of the nucleoporins they modeled the best fit to tain the predicted alpha-helices, those are not arranged in a place each nucleoporin in the frame of the known size and typical fold [5,6,11]. symmetry of the yeast NPC. This model predicts that the cen- The NPC is firmly anchored in and has extensive contact tral core is composed of two inner and two outer rings. The with the nuclear membrane, however, only three nucleoporins, outer rings could relate to the nucleoplasmic and cytoplasmic the pore membrane proteins (Poms) contain transmembrane rings described previously by electron microscopy. It remains domains and only one of them, Ndc1, is conserved [12]. to be seen to which extent the inner rings would correspond Pom152p in yeast is speculated to have its equivalent in to the one spoke ring seen before. The authors also propose gp210 in metazoa as both contain a single transmembrane re- that each of the eight symmetrical spokes is split into two gion and expose the largest part of the protein to the inter- very similar columns formed by duplicated or homologous membrane space of the nuclear envelope. The third, Pom34p nucleoporins thus suggesting that the entire complex may is only found in yeast and has no structural similarity to the have a 16-fold rotational symmetry. However, this interesting vertebrate Pom121. predicted feature awaits confirmation by direct structural The modular organization of the NPC is not only evident in investigation. its overall eight-fold symmetry with central spokes, nucleoplas- mic basket and cytoplasmic filaments. The NPC can also be 1.2. Composition dissected biochemically into distinct subcomplexes both in Given their enormous size, NPCs are built by a surprisingly yeast and higher eukaryotes ([8], see [7] for review), which small number of approximately 30 nucleoporins. Nucleoporins are thought to act as building blocks of the NPC (Fig. 1B). can be quite large with up to 360 kDa and due to the eight-fold Three major complexes make up the major mass of the symmetry of the NPC are thought to come in multiples of NPC, in vertebrates these are the Nup107–160, Nup93 and eight. Nucleoporins can be roughly categorized into ‘‘unstruc- Nup62 complexes. These are stably bound within the NPC, tured’’ and ‘‘structured’’ based on the presence or absence of whereas several more peripheral nucleoporins exchange rap- hydrophobic – (FG) repeats. FG repeat idly with a free pool [13]. Although the individual nucleoporins containing stretches of nucleoporins presumably line the chan- often show only limited sequence conservation between yeast nel with unstructured filaments that are assumed to constitute and vertebrates, the overall structure and composition of these the permeability barrier of the NPC. In contrast, nucleoporins subcomplexes appear to be conserved during evolution. 2006 W. Antonin et al. / FEBS Letters 582 (2008) 2004–2016

1.3. NPC membrane interaction the curvature of membranes is difficult to evaluate, but cer- NPCs are positioned at the sites where inner and outer nu- tainly will be an area of further research. Homo- and heterool- clear membranes join. Viewed from the membrane perspective igomerization of membrane proteins could amplify this effect. these are places of unusual membrane bending: The nuclear Interestingly, the metazoan transmembrane nucleoporin gp210 pore membrane has a convex (positive) curvature along the oligomerizes [19] as does Pom152p in yeast, which also inter- surface of the membrane connection between the outer and acts with Pom34p [8]. the inner nuclear membrane and a concave (negative) curva- Third, scaffolding of the membrane by peripheral membrane ture in the central plane of the pore membrane [14] proteins is an important way to change membrane curvature. (Fig. 2A). Currently, it is largely unclear how this bent nuclear Vesicle coat proteins such as COPI, COPII and clathrin influ- pore membrane could be formed and stably maintained. How- ence membrane bending by polymerizing into curved struc- ever, several mechanisms can be envisioned in analogy to other tures. It has been proposed that proteins of the Nup84 cell biological processes [15,16]. Although most of the exam- complex in yeast (Nup107–160 in vertebrates) and vesicle coat- ples are known from dynamic processes like endocytosis and ing complexes have a similar overall structure composed of al- vesicle budding and fusion, mechanisms that induce dynamic pha-solenoids and beta-propeller folds and a common changes in membrane curvature could also stabilize a bent evolutionary origin [20]. Similar to vesicle coats the Nup84 membrane. complex might shape the curved region of the nuclear pore First, the lipid components of the pore membrane could pro- membrane. Using a computational approach to calculate the mote or generate membrane curvature. In vitro, high propor- composition and arrangement of nucleoporins in the NPC, it tions of specific are capable of changing the has been suggested that the Nup84 complex indeed localizes morphology and local curvature of liposomes [17]. The chem- close to the membrane [8]. Consistent with this, the Sec13- ical properties of different lipid acyl chains or headgroups can Nup145C proteins of the Nup84 complex, crystallize as a het- support positive or negative membrane curvatures when asym- erooctamer that forms a slightly curved unit. metrically distributed between the two lipid leaflets of a mem- However, even COPII and clathrin lattices do not share an brane and both mechanisms have been suggested to be identical construction principle [21] and also the Sec13- involved in endocytosis. As lipids are able to diffuse and mix Nup145C structure shows a different arrangement from the rapidly in the plane of cell membranes, a mechanism to restrict Sec13-Sec31 in the COPII complex [5]. Further studies of the lipid movement would be required at the NPC. Theoretically, COPI cage and the Nup107–160 complex should reveal transmembrane regions of transmembrane nucleoporins as whether common principles indeed exist and will shed light well as integral membrane proteins in close proximity to the on their evolutionary relationship. In addition it will be inter- pore could constitute a diffusion barrier within the pore mem- esting to examine whether and how potential scaffolding fac- brane. These transmembrane regions could provide a binding tors would be adopted to the special requirement of surface for specific lipids and thus maintain their steady state stabilizing both a positive and a negative curvature of the pore enrichment at the site of high membrane curvature. Peripheral membrane. membrane lipid binding proteins associated with the NPC Scaffolding from the lumenal side could also be envisioned, could have similar functions. although no candidate factors have been described so far. Second, transmembrane proteins with a conical shape can The protein mass detected as the lumenal ring or connector have an effect on local curvature as has recently been proposed by electron microscopy might be attributed to the large lume- for the , which are required to maintain proper tubu- nal domains of the transmembrane nucleoporins Pom152p in lar (ER) structure [18]. Given that the yeast or gp210 in metazoa, but peripheral membrane associ- structures of only so few integral membrane proteins are ated proteins of the intermembrane space might as well con- known, the global contribution of transmembrane regions to tribute to that. As both Pom152p in yeast and gp210 in

Fig. 2. NPC formation requires membrane deformation. (A) Topology of the pore membrane: the nuclear pore membrane has a positive (i.e. convex) curvature along the surface connection between the outer and the inner membrane and a negative (i.e. concave) curvature in the plane of the pore. (B) The phospholipid bilayer of membranes can be deformed by (a) changes in composition of lipids that possess a different diameter of the head and tail regions resulting in a cone shape; (b) influence of membrane proteins that have a conical shape and/or oligomerize; (c) scaffolding of the bilayer from either side; (d) amphipathic helix insertion in one leaflet of the bilayer. W. Antonin et al. / FEBS Letters 582 (2008) 2004–2016 2007 metazoa are not essential for NPC formation and NPCs exist lus nidulans [31], and other variations between open and closed without the respective proteins [22–24], an electron micro- are likely to exist. In the following, we will therefore scopic analysis of NPCs lacking these proteins would shed mainly focus on mechanisms and regulations of NPC assembly light on this possibility. in metazoa. Fourth, insertion of amphipathic alpha-helices in a parallel orientation with respect to the plane of the membrane bilayer can increase the curvature of the membrane. Formation and incorporation of the amphipathic helix of epsin 1 has been 3. Postmitotic NPC assembly shown to directly affect membrane curvature during endocyto- sis [25]. Interestingly, some nucleoporins have a predicted Arf- 3.1. Coordinated assembly of NPCs and nuclear membranes GAP1 lipid packing sensor (ALPS) motif, which can insert Nuclear assembly after mitosis has been studied extensively bulky hydrophobic residues between loosely packed lipids in the past, both in intact cells and in cell free assays, the most and form an amphipathic helix on highly curved membranes prominent using Xenopus egg extracts and sperm . A [26]. Human Nup133 contains an ALPS motif that has been central task during the formation of the new nucleus is to coor- experimentally verified to fold in an amphipathic helix and dinate the assembly of NPCs, nuclear membranes and nuclear to be targeted to curved membranes [27]. It is tempting to spec- lamina into a seamless functional nuclear envelope. Two mod- ulate that such a mechanism could help formation and/or sta- els for the coordination of NPC formation and nuclear mem- bilization of the pore membrane curvature. Yeast Nup53p is brane attachment to chromosomes have been discussed. Under predicted to form an amphipathic alpha-helix and binds to certain conditions in in vitro assays, enclosure of the chroma- membranes. Nup53p overexpression leads to nuclear mem- tin by pore-free nuclear membranes can be observed [32–34].It brane proliferations interrupted by pore-like structures lacking has therefore been suggested that postmitotic NPC assembly fully assembled NPCs [28]. In addition to Nup53p, these struc- starts by a fusion of the outer and inner nuclear membrane tures contained two transmembrane nucleoporins, suggesting to allow for membrane hole formation into which NPCs could that Nup53p may have a role in pore formation. Finally, assemble. As will be discussed in detail below, a number of re- two additional vertebrate and four yeast nucleoporins also cent studies strongly suggest an alternative mechanism, in have predicted ALPS motifs, however, these regions are not which postmitotic NPC assembly is initiated by chromatin evolutionarily conserved and it is unclear whether they repre- binding of several nucleoporins independent of nuclear mem- sent functional domains able to insert into membranes. branes. This would lead to the formation of NPC-like prepores The four principle mechanisms of NPC membrane interac- on chromatin, structures that have long been described by elec- tion discussed above are not mutually exclusive. Quite the con- tron microscopy [35], followed by a coordinated assembly of trary, it is likely that membrane curvature is achieved by a NPCs and formation of a closed nuclear envelope. combination of several mechanisms as in other cellular pro- It has been a debate whether the nuclear envelope is formed cesses such as clathrin mediated endocytosis where clathrin, by outgrowth of the ER as suggested at least in mammalian epsin and several other factors act synergistically to deform tissue culture cells [36–38] or from nuclear envelope precursor the membrane. vesicles [39,40]. However, it is very likely that these vesicles arise through fragmentation of larger membrane structures of the mitotic ER during extract preparation. Indeed, also 2. NPC assembly in vitro data show that nuclear envelope formation can pro- ceed via an outgrowth of the ER network [41]: tubular tips The organization of multiple copies of about 30 different grow out from the ER, establish initial contacts to the chroma- nucleoporins, i.e. several hundred polypeptides, into an up to tin, flatten and form an ER-like network on the chromatin sur- 125 MDa large structure which is embedded in the double face [41,42] (Fig. 3). Initial contacts of the tubules on membrane of the nuclear envelope is a fascinating example chromatin are likely mediated by inner nuclear membrane pro- of self-assembly and a formidable challenge to understand teins that are dispersed in the mitotic ER and show an intrinsic mechanistically. In multicellular eukaryotes that have an open DNA binding property [43]. To form a closed nuclear double mitosis, NPC assembly occurs during two different phases of membrane from a tubular network would then require closure the cell cycle. First, since the nuclear envelope breaks down of the holes and gaps. It is attractive to imagine that mem- at the beginning of mitosis to give the cytoplasmic microtu- branes would simply expand across the chromatin surface with bules access to chromosomes, both nuclear membranes and the last remaining hole stabilized by the newly formed NPC at NPCs need to reassemble around the segregated chromosomes the sites of the chromatin bound prepores [41] (Fig. 3). Pre- at the end of mitosis. Second, new NPCs assemble into the in- sumably, this involves interactions of the prepore with trans- tact nuclear envelope during cell growth and in response to membrane nucleoporins that reside in the ER in mitosis. changes in metabolic activity. This mechanism obviates the need for a final closing step of This clear separation of mitotic and NPC assem- the membrane network and the need for membrane fusions bly cannot be made in many fungi: in to allow NPC integration. that has a closed mitosis and assembles the mitotic spindle in- side the nucleus the nuclear envelope including the NPCs stays 3.2. NPC assembly is regulated in space and time intact during mitosis, although NPC transport properties ap- NPC assembly has to be coordinated with chromosome seg- pear to be modulated in a cell cycle dependent manner [29]. regation and nuclear membrane formation and therefore must Other fungi perform a complete disassembly of the nuclear be precisely regulated in time. In addition, spatial regulation is envelope and NPCs such as Ustilago maydis [30] or a partial required to restrict NPC assembly to chromatin and prevent disassembly of peripheral NPC components such as Aspergil- ectopic NPC formation in the cytoplasm. 2008 W. Antonin et al. / FEBS Letters 582 (2008) 2004–2016

The trigger of chromosome segregation is the activation of the anaphase promoting complex (APC) by the mitotic spindle checkpoint, which leads to the cleavage of by separase and thereby to the initiation of anaphase. The second major action of the APC at the transition from metaphase to ana- phase is the inactivation of the mitotic kinase Cdk1 by degra- dation of its activating cyclin. With declining mitotic kinase activity, phosphatases eliminate many mitosis specific protein phosphorylations. Several nucleoporins including members of the early assembling Nup107–160 complex are specifically phosphorylated in mitosis [49–52] and their dephosphorylation is thought to temporally control their ability to bind to chro- matin and to each other upon anaphase onset, although clear data is lacking. A candidate for anaphase dephosphorylation of nucleopo- rins is the type 1 protein phosphatase (PP1) (reviewed in [53]). PP1 is expressed in several isoforms in human cells and involved in many different cellular pathways. Its localization and substrate specificity is regulated by a large number of tar- geting subunits. Several examples for the involvement of PP1 in nuclear assembly have been established. For example, PP1 is recruited to chromatin by the targeting subunit Repo-Man after the onset of anaphase and inactivates a so far unknown chromosome compaction mechanism [54]. A different targeting subunit, PP1 nuclear targeting subunit (PNUTS), also affects PP1-dependent chromatin condensation [55]. Furthermore, recruitment of PP1 to the NE by AKAP149 is required for B dephosphorylation in to allow lamina assembly [56–58]. It will be very interesting to test whether PP1 is also responsible for nucleoporin dephosphorylation and if so which targeting factors are involved. The above examples of PP1 function illustrate that the local activation of phosphatases by targeting subunits allow spatial in addition to temporal regulation. Known spatial regulators for the deposition of nucleoporins on chromatin are the small GTPase and its counterplayer, the transport receptor b. In its GTP bound form, Ran can displace importin b from its binding partners and thereby regulates nucleocytoplasmic transport in interphase cells, as well as spindle formation and nuclear assembly in Fig. 3. Model for postmitotic nuclear envelope assembly. After mitosis. Several nucleoporins including Nup153, Nup358 and prepore (green) formation on chromatin (blue), ER tubules attach the Nup107–160 complex bind to importin b during mitosis, and form a tubular network, flatten to form membrane sheets that finally merge around the pores. an interaction which prevents their assembly into NPCs [59,60]. Since RanGTP is produced on chromatin by its GTP exchange factor RCC1, importin b inhibited nucleoporins The first step of NPC assembly is the binding of several are released there and can assemble into NPCs. While this re- nucleoporins directly to chromatin, where they may form prep- lease could in principle also happen during earlier stages of ores. The recently identified nucleoporin ELYS/Mel28 could mitosis, the phosphorylation status of the nucleoporins may constitute the anchor on chromatin via its AT hook, a small prevent their association with chromatin before anaphase. In DNA binding motif [44–47]. Its binding is required for the addition, the Ran cycle itself appears to be regulated by phos- association of the Nup107–160 complex, a central building phorylation of RCC1 during mitosis which could provide addi- block of the NPC. Both ELYS/Mel28 and the Nup107–160 tional temporal control to this spatial regulator [61–63]. complex are essential for pore formation, and pore-free nuclei Finally, ubiquitinylation is another regulatory principle in- form in their absence in Xenopus egg extracts. Two additional volved in mitotic exit. APC mediated ubiquitinylation does nucleoporins start to associate with chromatin already prior to not only lead to the degradation of mitotic effector proteins membrane association, Nup153 and Nup50 [48]. However, the but modification by ubiquitin can also serve as regulatory major fraction of both binds to the nuclear envelope only later mechanism independent of degradation. Ubiquitinylation has when nuclear import is reinitiated and they may not be mech- not yet been implicated directly in NPC assembly, but in other anistically involved in prepore formation. processes of nuclear reformation at the end of mitosis. An How could these early steps of NPC assembly be regulated example is the p97 mediated extraction of ubiquitinylated Aur- in time? The association of nucleoporins with chromatin be- ora B kinase from chromatin during anaphase, a prerequisite gins when chromosomes start to segregate in anaphase [48]. for chromatin decondensation and nuclear envelope formation W. Antonin et al. / FEBS Letters 582 (2008) 2004–2016 2009

[64]. It will be interesting to investigate whether ubiquitinyla- matin binding and the coordination with nuclear membrane tion is also directly involved in regulating NPC assembly. assembly may be the key points of regulation. Once fully assembled, the NPC is an extremely stable complex and its 3.3. NPC reassembly occurs in a stepwise manner structural core lasts throughout interphase [13]. After formation of a prepore on chromatin, the second step in NPC reassembly is the association of membranes with chro- 3.4. Postmitotic NPC assembly and exit from mitosis matin and the accumulation of nuclear envelope specific mem- Nuclear envelope assembly in telophase reestablishes the brane proteins including the membrane nucleoporin Pom121. separation of the nuclear and cytoplasmic compartments. This Pom121 concentrates at sites on chromatin where the reformation of the compartment barrier can actively contrib- Nup107–160 complex is already bound [48]. Together, ute to the process of mitotic exit (Fig. 4). For example, several Pom121 and the Nup107–160 complex may have a checkpoint proteins involved in mitotic spindle function are controlled in function in coordinating the assembly of NPCs and the nuclear their activity by interacting with importin b in a Ran depen- membranes [23]. In assembly reactions in Xenopus egg extract dent manner. This allows activating them in proximity to chro- depleted of Pom121, vesicles binding to chromatin fail to fuse matin during mitosis, but once the NPC is assembled into a to form a closed nuclear envelope. Co-depletion of the transport competent state it promotes their import into the Nup107–160 complex releases this block in fusion and leads forming nucleus. Together with APC mediated degradation to the formation of a closed (although pore-free) nuclear enve- this facilitates the rapid clearance of spindle factors from the lope. How such a checkpoint that coordinates NPC assembly during mitotic exit [75] and the reestablishment of with nuclear membrane closure around chromatin could work the interphase microtubule cytoskeleton. mechanistically is still unknown. In addition, recent work suggests that assembly of the NPC In the third step of NPC assembly, the Nup93 and then the is coupled to the regulation of DNA replication [76].InXeno- Nup62 complex associate with the reforming nuclear envelope pus egg extract, DNA replication licensing is inhibited already [65,66]. Recent analyses in living NRK cells have shown that in telophase with the nuclear import of the licensing inhibitor import activity of the NPC starts concomitant with the associ- geminin. Therefore, licensing has to be completed prior to ation of both complexes [48]. The Nup93 complex has been assembly of the NPC. Indeed it was found that chromatin implicated in several structural aspects of the NPC. Nup53, bound licensing complexes promote chromatin association of one member of the complex, interacts with the membrane the nucleoporin ELYS/Mel28 and formation of the nuclear nucleoporin Ndc1, establishing a link between the pore mem- envelope. When the loading of licensing complexes was inhib- brane and the soluble nucleoporins [52,67]. Nup155, another ited with excess geminin, nuclear envelope formation was de- member of the Nup93 complex, is required for NPC and nucle- layed. Such a mechanism would ensure that, at least during ar envelope formation in Xenopus egg extract and Caenorhab- the rapid early divisions of Xenopus embryos, replication ditis elegans [68]. In addition, the Nup93 complex contributes licensing occurs prior to NPC assembly. significantly to the exclusion limit of the NPC [69,70]. Thus, These two examples highlight that NPC assembly and rees- the Nup93 complex may play a major structural role in the tablishment of nucleocytoplasmic compartmentalization are assembly and the formation of NPCs from prepores. not only subject to cell cycle mediated regulation, but they The Nup93 complex had very similar association kinetics to themselves contribute to regulate the process of mitotic exit Nup98 and both associated briefly before the Nup62 complex, and later steps of the cell cycle. consistent with a function in recruiting the Nup62 complex. In- deed, defects in Nup62 association have been observed in fibroblasts from Nup98 knock-out mice [71]. The Nup62 com- 4. Nuclear envelope breakdown plex contains nucleoporins with many FG repeats that are in- volved in various transport activities. Together, the addition of Nucleocytoplasmic compartmentalization is removed at the these two large subcomplexes appears to lead to the structural end of prophase when the nuclear envelope breaks down. and functional completion of an NPC assembly intermediate Although much less is known about the regulation of nuclear that can already import protein substrates. envelope breakdown compared to nuclear assembly, recent The last step of NPC assembly is the addition of peripheral work has lead to some progress in understanding the steps of nucleoporins including Nup214, Tpr and the major pools of nuclear envelope disassembly and the role of the NPC therein. Nup153 and Nup50 as well as the membrane nucleoporin gp210 [48,65,66,72]. Their late assembly is consistent with the 4.1. NPC disassembly is an early event in nuclear envelope fact that several of them are dispensable for NPC assembly breakdown in Xenopus egg extract and suggests that they are not required The earliest event of nuclear envelope disassembly observed for nuclear import, which has indeed been demonstrated for so far is an increase in the permeability of the nuclear envelope Nup214 and gp210 [73,74]. [77,78]. This continuous increase in nuclear envelope perme- In summary, NPC assembly can be divided into four major ability coincidences with the dissociation of nucleoporins from steps: prepore formation on chromatin, membrane association, the nuclear envelope, suggesting that disassembly of the NPC completion of the NPC core and addition of peripheral nucle- causes the initial permeabilization. As observed by electron oporins. While dephosphorylation and Ran/importin b pro- microscopy in starfish oocytes and Drosophila embryos, NPC vide global temporal and spatial regulation of NPC disassembly appears to occur via structural intermediates that assembly, the order of nucleoporin association may also de- in Drosophila have similar morphologies to assembly interme- pend on the availability of binding sites at the forming NPC diates [79]. However, one intermediate dominated all prophase in a self-assembly process. Therefore, the early steps of chro- nuclei, indicating that other intermediates may be very tran- 2010 W. Antonin et al. / FEBS Letters 582 (2008) 2004–2016

Fig. 4. Crosstalk between the NPC and cell cycle regulation. Schematic representation of the regulatory mechanisms connecting cell cycle progression and the NPC. Not that the table is not exhaustive. Details and references are given in the text. sient. This fits well with observations in living mammalian ylation in NPC disassembly, it will be necessary to map the cells, where the whole process of NPC disassembly is com- cell cycle dependent phosphorylations on all nucleoporins, as- pleted within a few minutes and most nucleoporins dissociate sign the responsible kinases and analyze the molecular func- from the NE rather synchronously [48]. The temporal trigger tion of the individual phosphorylations. for NPC disassembly could be the destabilization of central After their dissociation from the NPC, the mitotically stable protein–protein contacts between core subcomplexes by rapid nucleoporin subcomplexes were initially thought to simply dif- mitotic phosphorylation of NPC components. Spatial regula- fuse in the cytoplasm until the end of mitosis. However, evi- tion of disassembly may not be required, since all NPCs disas- dence is accumulating that many nucleoporins have specific semble at the same time including annulate lamellae (NPC-like functions during mitosis (Fig. 4). Several nucleoporins localize structures in the ER) present in the cytoplasm of embryonic to , where they have roles in spindle assembly and and cancer cells. chromosome segregation. In fact, also the mitotic checkpoint An important phosphorylation substrate to initiate NPC proteins Mad1 and Mad2 localize to NPCs during interphase disassembly could be the transmembrane nucleoporin and relocalize to kinetochores during mitosis, pointing to a gp210. Its depletion in C. elegans compromises nuclear enve- strong connection between kinetochores and NPCs [83].In lope breakdown during the early embryonic divisions [73,80]. addition, Nup98 and its binding partner Rae1 inhibit the ana- In Xenopus egg extract treated with Fab fragments against phase promoting complex, APC-Cdh1, to prevent premature gp210, nuclei do not disassemble: all analyzed nucleoporins activation of separase and sister chromatid segregation in pro- remained bound at the nuclear envelope and the nuclei did metaphase [84,85]. The very early dissociation of Nup98 from not become permeable for 70 kDa dextrans [73]. gp210 is spe- the NPC during the initial permeabilization process [48,77] cifically phosphorylated in mitosis [49] which appears to may be important for this function. destabilize its anyway transient interaction with the NPC [81]. This phosphorylation may be carried out by Cdk1, which has been shown to be important for the regulation 4.2. Nuclear envelope breakdown requires membrane of the early permeabilization and fenestration of the nuclear redistribution to the ER and lamina depolymerization envelope in vitro [78]. Mammalian NIMA kinase homologues The next step in nuclear envelope breakdown is the forma- (Neks) also participate in mitotic regulation and might play a tion of holes and the complete opening of the nucleus. In star- role in the disassembly of the mammalian NPC (for review fish oocytes, holes have been observed to form by enlargement see [82]). During the semi-closed mitosis of the fungus A. of the membrane pores at former NPC sites [77]. In contrast to nidulans, both Cdk1 and NIMA kinase are required for the this model, pore closure after NPC removal has recently been release of a subset of nucleoporins from the nuclear envelope suggested, when pore-free nuclear envelopes were observed by [31]. To further understand the role of nucleoporin phosphor- electron microscopy during the disassembly of nuclei in W. Antonin et al. / FEBS Letters 582 (2008) 2004–2016 2011

Xenopus egg extract [86]. However, such a model would not 5. NPC assembly in interphase explain the continuous increase in nuclear envelope permeabil- ity during prophase in both starfish oocytes and mammalian Cycling mammalian cells double the number of NPCs in cells [77,87]. In mammalian cells, the holes are subsequently interphase to prepare for a new cell division [98,99], and enlarged by microtubule mediated rupturing of the nuclear NPC assembly therefore has to occur at a significant rate after lamina [88,89], a process that appears to be controlled by the mitosis. We currently know very little about the mechanism spatial regulators Ran and importin b [78]. The collapse of and regulation of NPC assembly in interphase. Since the order nucleocytoplasmic compartimentalization upon complete nu- of nucleoporin addition to the forming pore may be ruled by clear envelope permeablisation leads to an immediated in- mutual interactions between them, a straightforward model crease in microtubule nucleation [90] possibly by the release is to assume that interphase assembly follows a similar mech- of mitotic microtubule regulators from the nucleus. anism to postmitotic assembly and also uses comparable regu- The third step of nuclear envelope breakdown is the redis- latory pathways. However, the cellular environment could tribution of nuclear membranes into the ER. While some in- hardly be more dissimilar. Postmitotic NPC assembly in ana- ner nuclear membrane proteins like lamin B receptor phase relies on an abundant free pool of nucleoporins from redistribute throughout the ER already several minutes be- the mother cell, initiates on exposed chromatin and is coordi- fore NPC disassembly is completed, others including the nated with nuclear membrane assembly. By contrast, inter- membrane nucleoporin Pom121 remain bound in the nuclear phase assembly starts when most free nucleoporins have membrane longer [88]. Two models of nuclear membrane been consumed by postmitotic assembly and has to insert redistribution have been proposed. One view states that nu- NPCs into the intact nuclear envelope that is tightly inter- clear membranes feed directly into the ER [36,38,88], whereas linked with the and peripheral chromatin. another suggests that nuclear membranes vesiculate and dis- Conceptually, several distinct mechanisms can be envisioned perse [39,40]. Recently, when inducing nuclear envelope for interphase NPC assembly [100]. Existing NPCs could breakdown of in vitro assembled nuclei, ER-like tubules were duplicate by a growth and splitting mechanism. Such a mech- observed to form from the nuclear surface as well as vesicles anism would require a fusion of the pore membrane regions to [86] providing support for both models. However, all nuclear generate two holes out of one [101]. NPCs with a more than membrane proteins investigated to date in intact cells reside eight-fold symmetry that could be interpreted as intermediates in the tubular-cisternal membrane network of the ER in of NPC growth and splitting have been observed [102] but are mitosis [36–38,88,91–94], strongly supporting that although so rare that it seems unlikely that they account for the dou- vesicles may exist, the final mitotic reservoir of nuclear mem- bling of NPC numbers during the cell cycle. Based on recent branes is the ER. Interestingly depletion of proteins involved data, it appears more likely, that NPCs form in interphase in peripheral ER structure blocks nuclear envelope break- without reusing previously assembled NPCs. Newly formed down in C. elegans [80]. Although a direct mechanistic link NPCs were found not to contain major amounts of nucleopo- was not yet established this suggests that dispersal of the nu- rins from preexisting NPCs and the interphase assembly events clear membranes needs a connection to and/or presence of documented by live cell imaging so far occurred in previously the ER. pore-free areas of the nuclear envelope [103]. Such de novo Depolymerization of the nuclear lamina occurs only after assembly could start either by formation of a prepore on chro- complete opening of the nuclear membranes as the last step matin analogous to postmitotic assembly or follow a distinct of nuclear disassembly [77,88]. Remaining lamina containing route in which creation of a new hole in the double membrane nuclear envelope fragments are removed from chromatin along could be the first step. microtubules and migrate towards the centrosomes until the Regardless of how de novo NPC assembly is initiated, the are completely dissolved latest in metaphase. The disas- complex ultimately has to be embedded into the double mem- sembly of the lamina depends on PKCbII mediated phosphor- brane of the nucleus. Mechanistically, two main scenarios for ylation [95]. PKC activation and its translocation to the this key event can be envisioned. In the first, the nuclear enve- nucleus in prophase may depend on the increase of diacylglyc- lope could locally disassemble to allow embedding of a prepore erol levels at the nucleus [96,97], indicating a potential role of followed by regrowth of the ER cisternae, similar to postmitot- lipid signaling in mitotic entry. ic NPC assembly. It has been argued that local nuclear enve- In summary, NPC disassembly is an early event of and lope breakdown would be a devastating event for the cell probably the trigger for nuclear envelope breakdown. NPC because it would destroy the separation of nucleus and cyto- disassembly is likely to be regulated by cell cycle specific plasm. However, as long as such a nuclear membrane discon- phosphorylation although the full repertoire of kinases and tinuity could be locally and temporally restricted the enormous additional factors is currently unknown. Disassembly of the transport capacity of the NPCs would likely compensate the NPC is followed by the formation of holes that completely local breakdown of the diffusion barrier. Although in principle open the nucleus to cytoplasmic structures. In the next step, possible, so far no convincing electron microscopic evidence nuclear membranes redistribute into the ER and finally the for local nuclear membrane discontinuities in the interphase lamina depolymerizes. In mammalian cells, formation of nucleus have been documented that could account for NPC large nuclear envelope holes and removal of lamina remnants assembly. from chromosomes is additionally facilitated by microtubules In the second, arguably more likely scenario, a convergence that attach to the nucleus already at the beginning of pro- and fusion of the outer and inner nuclear membrane would phase. Some examples illustrate the function and feedback form a hole into which the NPC would immediately assemble of NPC disassembly in cell cycle progression and regulation without compromising the diffusion barrier. The factors that and with progression of the field more are likely to be char- could mediate the approximation of the two membranes across acterized. the 20–40 nm perinuclear space or their fusion are currently 2012 W. Antonin et al. / FEBS Letters 582 (2008) 2004–2016

Fig. 5. NPC formation in interphase. (A) Pore formation in the intact double membrane of the nuclear envelope requires membrane approximation and fusion. (B) Mechanisms of membrane approximation and/or fusion: (a) transmembrane proteins could mediate membrane approximation and/or fusion similar to SNARE proteins e.g. mediating synaptic vesicle fusion; (b) lumenal proteins could mediate membrane deformation to the intermembrane space as do clathrin, COPI and COPII vesicle components during vesicle budding; (c) membrane deformation to the intermembrane space could be also mediated by factors on the nuclear and/or cytoplasmic site similar to membrane invaginations during multi-vesicular body formation or viral budding at the plasma membrane.

unknown. As membrane pore formation and NPC assembly but some lineages lost either gp210 or Ndc1 from their gen- would be temporally and spatially tightly coordinated, it is omes [12]. Neither gp210 nor Ndc1 are essential in C. elegans tempting to speculate that the same factors are involved in [69,104] and in A. nidulans, double mutants lacking both both events. Pom152p and Ndc1p surprisingly are viable (Hui-Lin Liu, Ste- What would be the protein machinery that forms a hole into phen Osmani, personal communication). In summary, it re- the double membrane of the nucleus? Mechanistically, mem- mains to be seen whether the two modes of NPC assembly brane approximation and/or fusion can be envisioned in anal- in mitosis and in interphase have distinct requirements of ogy to other cellular processes involving membrane transmembrane nucleoporins and if and how these proteins deformation and/or fusion (Fig. 5). with very different topology and domain structures could sub- Similar to vesicle or viral fusion with the target membrane, stitute for each other. transmembrane proteins residing on either one or both mem- Initial membrane contacts between the inner and/or outer brane sides of the nuclear envelope could be involved. There- nuclear membrane could be established by bulging of one or fore, transmembrane nucleoporins have long been considered both membranes into the intermembrane space. In analogy as prime candidates to fulfill this function. However, in yeast, to vesicle budding, this deformation could be mediated by depletion of Pom152p and Pom34p alone or in combination luminal proteins which could bend the membrane similar to has no effect on NPC number and assembly [22]. Depletion vesicle coat complexes. However, up to now no such lumenal of the third transmembrane nucleoporins Ndc1p lead to a de- candidates have been identified nor have bud-like structures fect in NPC structure and function, an effect that was increased been observed at the nuclear envelope by electron microscopy when Pom152p was co-depleted indicating some redundancy except for virus infected cells. Furthermore, it is unclear how [22]. Nevertheless, it is not clear whether this defect can be di- such coated deformed membrane surfaces would then allow rectly assigned to pore formation. for bilayer fusion. Alternatively, membrane deformation and In metazoa, the role of the three transmembrane nucleopo- approximation could be initiated by proteins from the cyto- rins in interphase NPC assembly has not been addressed di- plasmic and/or nucleoplasmic side. Candidates for such an ac- rectly. Ndc1 and Pom121 are required for postmitotic NPC tion might be non-transmembrane structural nucleoporins, like assembly in vertebrates and a function in interphase pore the Nup107–160 complex, which has been shown to be re- assembly cannot be excluded. While gp210 is not required quired for interphase NPC assembly on both sides of the nu- for postmitotic NPC assembly, its knockdown in cultured cells clear envelope [103]. However, it remains to be seen whether leads to a reduction in NPC staining, consistent with a role in this requirement is connected to the membrane binding activity interphase NPC assembly or maintenance. Surprisingly, none of the complex that was proposed based on structural similar- of the three metazoan transmembrane nucleoporins are found ity to coat complex proteins [20]. Along similar lines, Nup53 in all species. Pom121 has so far only been identified in verte- with its predicted amphipathic helix is another candidate for brates, gp210 and Ndc1 show a more widespread distribution, this process. W. Antonin et al. / FEBS Letters 582 (2008) 2004–2016 2013

In addition to membrane fusion, the insertion of NPCs into cently been made, a large number of important questions await the intact nuclear envelope likely also requires a local disrup- to be addressed. Which for example are the phosphorylations tion of the lamina. Such local disruptions could be generated and other posttranslational modifications that regulate the dis- in a similar way to the ones induced by herpes viruses during and reassembly of NPCs during mitosis and what is their effect the egress of their capsids from the nucleus. These capsids on protein–protein and protein–membrane interactions? How are assembled in the nucleus but are too large to exit the nu- is the association of membranes with the NPC regulated in cleus via NPCs. Instead, the capsids bud through the nuclear postmitotic assembly and how the fusion of the outer and in- membranes after local solubilization of the lamina via PKC in- ner nuclear membranes during assembly in interphase? How, duced phosphorylation of lamin B [105]. PKC mediated phos- once the NPC has assembled, is the unusual topology of the phorylation also regulates lamina disassembly in apoptosis and pore membrane stabilized? How are all these processes re- mitosis and could thus also occur during interphase NPC versed when the NPC is disassembled in mitosis? And finally, assembly. what functions other than are performed Very little is known about how NPC assembly in interphase by nucleoporins at other locations in the cell or in other cellu- is regulated. Since NPCs are immobile in the nuclear envelope lar processes in mitosis as well as in interphase? Given its cen- in mammalian cells [92], their positioning has to be controlled tral position between the two major cellular compartments, the already upon assembly when the membranes are induced to nucleus and cytoplasm, it would not be surprising if the NPC fuse and the lamina is disrupted. The spatial regulator RanGTP was involved in many more cellular processes than have been has been shown to be essential for the insertion of NPCs into discovered so far. intact nuclei in Xenopus egg extract on the cytoplasmic as well as the nuclear side [103], but additional regulators are probably Acknowledgements: We thank Ohad Medalia for providing Fig. 1a and involved in locally defining the sites of NPC assembly. Possible Martin Vo¨tsch, Verena Dreesen and Petra Riedinger for help with pre- factors could be lamins and lamina associated proteins. For paring the figures. example, the loss of the lamin A interacting protein Sun1, which colocalizes with NPCs, as well as of lamin A itself have been found to alter the distribution of NPCs on the nuclear sur- References face [106–108]. In yeast, the deletion of many nucleoporins and other proteins leads to a prominent clustering of NPCs in one [1] Weis, K. 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