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Nuclear pore complexes in the maintenance of genome integrity
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Nuclear pore complexes in the maintenance of genome integrity Lucas Bukata, Stephanie L Parker and Maximiliano A D’Angelo Maintaining genome integrity is crucial for successful organismal propagation and for cell and tissue homeostasis. Several processes contribute to safeguarding the genomic information of cells. These include accurate replication of genetic information, detection and repair of DNA damage, efficient segregation of chromosomes, protection of chromosome ends, and proper organization of genome architecture. Interestingly, recent evidence shows that nuclear pore complexes, the channels connecting the nucleus with the cytoplasm, play important roles in these processes suggesting that these multiprotein platforms are key regulators of genome integrity. Addresses Cardiovascular Research Institute, Biochemistry and Biophysics Department, University of California San Francisco, San Francisco, CA 94158, United States Corresponding author: D’Angelo, Maximiliano A ([email protected])
Current Opinion in Cell Biology 2013, 25:378–386 This review comes from a themed issue on Cell nucleus Edited by Edith Heard and Danesh Moazed For a complete overview see the Issue and the Editorial Available online 6th April 2013 0955-0674/$ – see front matter, # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ceb.2013.03.002
Introduction Nuclear pore complexes (NPCs) are aqueous channels that penetrate the nuclear envelope and connect the nucleus with the cytoplasm [1]. These highly conserved structures are built by the repetition of 30 different proteins called nucleoporins, most of which associate in stable subcomplexes (Figure 1) [2,3,4]. Although the control of nucleocytoplasmic molecule exchange is considered the main function of NPCs, these structures and their components have important transport-independent functions. In this review we will focus on the roles of NPCs and nucleoporins in DNA replication, DNA damage, telomere maintenance, and chromosome segregation. These newly discovered functions for NPCs imply that these structures might be central players in the maintenance of genome integrity.
NPCs in DNA repair and replication The ability to sense and repair DNA damage is probably the most important factor in genomic stability. Alterations in the mechanisms that regulate these processes generally lead to an increased rate in DNA mutations and result in Current Opinion in Cell Biology 2013, 25:378–386
genomic instability. Because more mutations augment the probability of acquiring a selective advantage, most cancer cells show alterations in the DNA damage response (DDR), and genomic instability is a hallmark of transformed cells. The first link between NPCs and the DDR machinery was revealed when a genome-wide screen implicated five nucleoporin genes in the repair of ionizing radiation damage in yeast cells [5]. Notably, the identified nucleoporins (Nup84, Nup120, Nup133, Nup170 and Nup188) were all members of the NPC scaffold structure (Figure 1) [4]. Mutants of Nup84, Nup120, Nup133, from the Nup84 complex, were also sensitive to other DNA damaging agents including UVlight, DNA methylating agents, DNA strand breaks inductors, replication and topoisomerase I inhibitors [5], and show defects in double strand break (DSB) repair and targeted recombination [5]. The increased sensitivity to DNA damaging agents in nucleoporin mutants was also identified in several independent studies [6–10,11]. Further supporting the role of nucleoporins in DDR, mutations in the Nup84 complex have a synthetic lethality interaction with RAD27 and RAD52, which play critical roles in DNA replication and repair [12]. Accumulation of RAD52 foci, a marker for DSB accumulation, was observed with deletion of these nucleoporins, in double mutants of the nuclear basket proteins Mlp1 and Mlp2, and in deletion mutants for Nup60, which anchors these nucleoporins to the NPC. No foci were detected in nucleoporin mutants involved in mRNA export, protein import or NPC distribution [13], suggesting that the DNA damage is not a secondary effect of nucleocytoplasmic transport alterations. The Nup84 complex and Nup60 are required for the recruitment of the SUMO protease Ulp1 to NPCs [14] (Figure 2a). Interestingly, Ulp1 mutants that cannot localize to NPCs show DNA damage accumulation phenotypes similar to nucleoporin mutants [13]. Furthermore, Ulp1 overexpression can partially rescue the DNA damage defects of these strains as well as their colethality phenotypes with RAD27 and RAD52 mutants [13]. These findings indicate that the Nup84 complex and Nup60 might regulate DNA damage repair by recruiting Ulp1 to the nuclear envelope (Figure 2a). Supporting this idea, depletion of these nucleoporins results in the loss of Ulp1 from NPCs and affects cellular sumoylation patterns [13,14,15]. Among the affected proteins in nucleoporin depletion mutants is yKu70, which shows decreased sumoylation levels that are apparently responsible for the loss of its function [13]. As yKu70 plays a key role in the non-homologous end-joining (NHEJ) repair of DSBs [16], the loss of its activity could provide a molecular explanation to the DDR phenotypes observed in www.sciencedirect.com
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Figure 1
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Molecular organization of the nuclear pore complex. Schematic representation of the structure and composition of NPCs in yeast and vertebrates. Nucleoporins that are part of the same subcomplexes are enclosed in boxes. Homologous nucleoporins/subcomplexes between yeast and vertebrates share the same colors.
the nucleoporin mutants. Yet, given that many proteins involved in DDR can be sumoylated [17], it is possible that Ulp1 plays a role in the modification of many of these substrates. Another link between the NPC, the SUMO pathway and DNA damage repair was recently described in a study showing that yeast replication forks containing double DSBs relocate to NPCs for repair in a SUMO dependent manner (Figure 2b) [18]. This process requires interaction between the Nup84 complex and the STUbL complex [18], a SUMO-targeted ubiquitin ligase complex composed of Slx5 and Slx8 that preferentially ubiquitinates polysumoylated targets [19]. While localization of the STUbL complex to NPCs is independent of DNA damage and this complex is not required for the recruitment of DSB, its NPC association is critical for the repair of collapsed replication forks and persistent DSB. In the proposed model, DNA damaged sites marked by the accumulation of a sumoylated protein are tethered to the NPC through the STUbL complex. The relocalization of the damaged DNA to the nuclear periphery requires Mec1 and Tel1, the yeast homologs of kinases ATM and ATR [20,21], and is critical for the repair process. Considering the ubiquitin ligase activity of the STUbL complex, it is likely that the degradation of the sumoylated proteins at the site of damage is required for repair to begin. Interestingly, decreasing the Ulp1 levels www.sciencedirect.com
has no effect in the process [18], suggesting the existence of distinct parallel mechanisms for DNA repair through the NPC/SUMO pathway. However, it is still possible that the sumo protease activity of Ulp1 is required to release the recruited DNA from Slx5 after or during damage repair (Figure 2b). The findings that NPCs would act as DNA repair centers regulated by the SUMO pathway is consistent with the emerging idea of compartmentalized DDR repair sites in yeast and with the proposed role of sumoylation in DNA repair and the maintenance of genome integrity (reviewed in [17,22]). Little is known about the role of NPCs in the DDR of mammals. Similar to Drosophila and plants, the presence of NPC-associated SUMO-regulating proteins has been described in mammalian cells [23–25], but their involvement in DDR has not been studied. But the recent findings that downregulation of Nup153 leads to delayed DNA repair and decreases cell survival suggests this NPC function is conserved [26]. Although Nup153 seems to regulate DNA damage repair mostly by controlling the nuclear import of 53BP1 [26,27], a key regulator of DDR [28], some experimental data suggest a 53BP-independent role for Nup153 in regulating the choice between homologous recombination and NHEJ [26]. A link between NPCs and DNA replication is also emerging. Besides the genetic interactions between different Current Opinion in Cell Biology 2013, 25:378–386
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NPCs DNA repair and telomere maintenance. (a) The SUMO protease Ulp1 associates with the NPC through the Nup84 subcomplex and Nup60. At the NPC, Ulp1 regulates the sumoylation status of proteins that play key roles in DNA damage repair. (b) When a DSB (yellow star) is generated during DNA replication, the replication fork collapses and relocates to the NPC in a Mec/Tel1 dependent manner. NPC association requires the STUbL complex (Slx5/Slx8), which potentially tethers DNA through its interaction with sumoylated proteins that accumulate at the site of damage. The STUbL complex might regulate DNA repair by ubiquitinating specific targets and inducing their degradation by the proteasome. As the STUbL complex and Ulp1 are anchored to the NPC by the Nup84 subcomplex it is possible to speculate that desumoylation by Ulp1 might be implicated in the release of DNA from the NPC during or after the repair process. (c) The Mlp1 and Mlp2 nuclear basket nucleoporins regulate telomere length through a yet unknown mechanism. (d) Critically short telomeres are recognized as DNA damage and relocalize to NPCs for repair, potentially through the STUbL complex pathway. (e) Nucleoporins of the Nup84 complex are essential for telomere tethering to the nuclear periphery and for the efficient repair of subtelomeric double strand breaks (DSBs).
nucleoporins and RAD27 described above, the nucleoporin Elys was found to bind the replication licensing proteins Mcm2–7 directly in Xenopus extracts [29], suggesting a potential role in DNA replication. Supporting this idea, Elys Zebrafish mutants show reduced levels Current Opinion in Cell Biology 2013, 25:378–386
of Mcm2 on chromatin, activated DDR, and increased sensitivity to replication stress [30]. Activated DDR in response to Elys depletion was also observed in mouse epithelial cells [31], indicating a conserved function in mammals. Besides the potential role of NPC components www.sciencedirect.com
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in coordinating replication licensing, a recent report shows that during S-phase the release of genes from NPCs is required for their replication [32]. Considering this, it is possible to speculate that by controlling the tethering of specific genes and chromosomal regions to the nuclear periphery NPCs might play a role in regulating their replication timing.
NPCs in telomere maintenance Telomeres are nucleoprotein complexes that protect the ends of linear chromosomes [33]. These structures are required for the efficient replication of chromosome ends and to prevent their recognition as DSBs [34]. Telomere dysfunction is considered a major contributor to genome instability and cancer [33]. In budding yeast, telomeres localize in clusters at the nuclear periphery [35]. Three NPC components, Nup145p, Mlp1 and Mlp2, were initially proposed to be responsible for the peripheral localization of chromosome ends [8,36]. However, their role in telomere tethering was later questioned [11]. More recently, several components of the Nup84 complex were described to be essential for the peripheral localization of chromosome XI telomere [37]. However, another report showed that telomere positioning was not affected in NPC clustering mutants, suggesting that NPCs are not responsible for telomere tethering [38]. These contentious findings might be explained if different tethering mechanisms exist for different chromosome ends, telomeres states, or phases of the cell cycle. Despite the controversial role of NPCs in telomere anchoring, several lines of evidence support their function in telomere maintenance and repair. First, Mlp1 and Mlp2 are important for the control of telomere length [11,15] (Figure 2c). Moreover, in telomerase negative yeast cells, critically short telomeres recruit proteins of the homologous recombination repair machinery as well as checkpoint proteins and relocalize to NPCs [39] (Figure 2d). This suggests that eroded telomeres are recognized as DNA damage and move to NPC to start the repair process, potentially through the Slx5/Slx8-dependent mechanisms described above. NPC tethering of chromosome ends may also be important for the repair of subtelomeric DSBs (Figure 2e). In the same study showing that the Nup84 complex is responsible for anchoring telomere XI-L, mutants for these nucleoporins showed a decreased ability to repair subtelomeric DSBs [37]. Given that NPCs seem to act as centers for DNA repair, the localization of telomeres to these structures might explain why DSB repair is 20fold more efficient in subtelomeric regions than in other chromosomal domains [40]. In mammals, telomeres have recently been shown to localize to the nuclear periphery during postmitotic nuclear assembly [41] and a role for Lamin A proteins in telomere maintenance has been suggested [42]. Yet, www.sciencedirect.com
whether NPCs play a role in telomere tethering, maintenance or repair in mammalian cells has not yet been studied.
Nucleoporins in chromosome segregation and cytokinesis The precise segregation of chromosomes during cell division is critical to ensure the faithful transmission of the genetic information. Alterations in this process might result in genomic instability and often lead to chromosomal rearrangements or the gain or loss of entire chromosomes (aneuploidy) [43,44]. During mitosis, replicated chromosomes condense, microtubules reorganize in the mitotic spindle, chromosomes attach to the spindle through their kinetochores and segregate toward opposing centrosomes. In recent years, it has become clear that nucleoporins play critical roles in mitosis by regulating specific steps of these processes [45–47] (Figure 3). Nup170, the yeast homolog of mammalian Nup155, was the first nucleoporin described to have mitotic functions. Nup170 mutants affect kinetochore integrity (Figure 3a) and show increased chromosome loss and nondisjunction indicating abnormal chromosome segregation [48]. Nup358/RanBP2 is a component of the NPC cytoplasmic filaments (Figure 1). This nucleoporin is an E3 SUMO ligase [49,50] that forms a complex with two other proteins, RanGAP and the SUMO E2 conjugating protein Ubc9 [23,51]. During mitosis this complex binds to kinetochores and spindle poles and plays a critical role in kinetochore assembly and microtubule attachment [52,53] (Figure 3a). Nup358/RanBP2 also sumoylates and recruits toposisomerase II to centromeres, a critical step in sister chromatid separation [54] (Figure 3a). In a similar way, Nup358/RanBP2 associates and sumoylates a component of the Chromosome Passenger Complex (CPC), a mitotic regulator with key functions in spindle and kinetochore assembly, potentially modulating the activity of this complex [55]. Owing to its various functions, Nup358/RanBP2 alterations result in multiple mitotic defects, including mislocalization of several kinetochore-associated proteins, multiple mitotic spindles, chromosome misalignment, anaphase bridges, and aneuploidy [52–54]. Unsurprisingly, mutant mice that are heterozygous for this nucleoporin have a higher tendency of developing spontaneous tumors [54]. Rae1, a nucleoporin sharing homology with the mitotic checkpoint protein Bub3 [56], is essential for spindle assembly and chromosome segregation [56,57,58–61] (Figure 3a). Rae1 interacts with multiple partners and seems to regulate spindle formation and function through several mechanisms (Figure 3a). Rae1 forms part of a large ribonucleoprotein (RNP) complex that binds microtubules directly and controls microtubule dynamics and spindle formation [57]. Additionally, Rae1 interacts Current Opinion in Cell Biology 2013, 25:378–386
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Figure 3
(a) Kinetochore Assembly Nup358-RanGAP-Ubc9 Nup107 -160 Complex Nup170* Kinetochore
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Mitotic functions of nucleoporins. (a) The Nup358 regulates kinetochore assembly and microtubule attachment and also assists in spindle formation and sister chromatid separation through its SUMO ligase properties. In most cases, Nup358 works in a subcomplex with sumoylated RanGAP and the E2 sumo-conjugating enzyme Ubc9. The Nup107–160 complex is essential for kinetochore assembly and function. It promotes microtubule– kinetochore attachment and the formation of k-fibers, which facilitate spindle assembly. The Nup107–160 complex has also been proposed to directly regulate spindle assembly, although there is contradicting evidence for this role. Nup170 (*the yeast homologue of vertebrate Nup155) functions in chromosome segregation by maintaining kinetochore integrity. Rae1 regulates spindle assembly and microtubule bundling through its interaction with multiple proteins in including the cohesion subunit SMC1, the nuclear mitotic apparatus protein NuMA and the nucleoporin Nup98. Rae1 also forms part of a RNP complex that regulates microtubule dynamics and spindle formation. (b) Nup133 mediates the tethering of centrosomes to the nuclear envelope in early prophase. Centrosome association to the nuclear envelope is essential for the early stages of spindle formation. (c) Changes in the expression of several nucleoporins, including, Nup153, Elys, and SEH1, have been linked to defects in cytokinesis. Specifically, depletion of Seh1 and Elys, result in both cytokinesis defects and (d) multinucleated cells. The multinucleated phenotype is also found with overexpression of Nup153 or Tpr, as well as aberrant regulation of Nup88 (overexpression or downregulation).
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with the nuclear mitotic apparatus (NuMA) protein, which functions in the organization and attachment of microtubules to spindle poles [58]. This interaction potentially increases the ability of NuMA to crosslink and bundle microtubules, a requirement for correct spindle formation [59]. Rae1 also interacts with the cohesin subunit SMC1, and the Rae1–SMC1 complex is essential for proper spindle formation [62]. Moreover, a Rae1– Nup98 complex was found to be important for spindle assembly and to inhibit the degradation of the mitotic checkpoint protein Securin by the anaphase-promoting complex (APC) [60,61], possibly controlling the time of chromosome segregation. Consistent with this idea, haploinsufficiency of both proteins results in premature segregation of sister chromatids and aneuploidy [60]. Mitotic functions have also been attributed to the mammalian Nup107–160 complex, the homolog of yeast Nup84 complex. Most components of this complex have been shown to partially localize to kinetochores during mitosis [63,64,65]. The association of the Nup107–160 complex with kinetochores is required for anchoring Nup358/RanBp2 [64]. Interestingly, the depletion of Seh1, which efficiently reduces the levels of these nucleoporins at kinetochores without affecting NPC function, resulted in misaligned chromosomes, kinetochore-spindle attachment defects, spindle checkpoint activation, and mitotic delays [64,66] (Figure 3a). Similar phenotypes in chromosome congression and checkpoint activation were observed with the depletion of Seh1 and Nup107 in a later study, although normal kinetochore– microtubule attachments were observed [66]. The latter attributed the function of the Nup107–160 complex in chromosome alignment and segregation to the recruitment Aurora B and the CPC proteins to centromeres [66]. These studies described alterations in mitotic spindle length and activated spindle checkpoints, but showed no defects in spindle formation [64,66]. However, the findings that several components of the Nup107–160 complex localize throughout the entire mitotic spindle and spindle poles [67], that this complex is required for the in vitro assembly of bipolar spindles [67], and that mutations in a member of the Schizosaccharomyces pombe homologous complex show abnormal mitotic spindles [68] suggest a role in spindle formation (Figure 3a). Consistent with this idea, recent findings show the Nup107–160 complex recruits and works cooperatively with the microtubule nucleation complex g-TuRC to promote the formation of k-fibers and facilitate spindle assembly [69] (Figure 3a). Additionally, the Nup107–160 complex member Nup133 was recently described to mediate the tethering of centrosomes to the nuclear envelope in prophase [70] (Figure 3b). Given that the loss of centrosome attachment to the nuclear envelope transiently results in aberrant spindles, Nup133 might be important for the early stages of mitotic spindle formation [70]. Interestingly, the association of www.sciencedirect.com
centrosomes with the nuclear envelope is required for the release of scaffold nucleoporins in Caenorhabditis elegans and regulates the timing of mitotic onset [71]. Depletion of two Nup107–160 complex components, Seh1 and Elys, results in multinucleated cells indicating cytokinesis defects [66] (Figure 3c,d). Abnormal cytokinesis is also observed with depletion of Nup153, a component of the NPC nuclear basket (Figures 1 and 3c). Downregulating Nup153 leads to mislocalization of Aurora B [72], delayed dissociation of the spindle checkpoint protein Mad1 from kinetochores [73], and accumulation of unresolved midbodies [72–74]. These phenotypes are partially mimicked by downregulation of Nup50, which also becomes mislocalized with Nup153 depletion [72]. Conversely, Nup153 overexpression results in multipolar spindles and multinucleated cells [73] (Figure 3d). Nup153 regulates the localization and phosphorylation of Mad1 during metaphase/anaphase transition and is required for spindle checkpoint activity and mitotic exit, providing a potential molecular mechanism for the observed phenotype [73]. Tpr, a mammalian homolog of Mlp1/Mlp2 that interacts with Nup153, binds Mad1 and Mad2 proteins as well as the molecular motors important for spindle function, dynein and dynactin [75,76]. Tpr depletion affects levels and localization of spindle checkpoint proteins and results in a chromosome lagging phenotype [75,76]. Similar to Nup153, overexpression of Tpr results in multinucleated cells [75] (Figure 3d), a phenotype also observed with depletion or overexpression of Nup88, a nucleoporin misregulated in many cancer cells [77] (Figure 3d).
Conclusions Nuclear pore complexes are emerging as key regulators of numerous cellular functions. In addition to the described role of NPCs in DNA repair, replication, protection and segregation, these structures have been shown to play a role in the organization of genome architecture, another pillar in genome stability (recently reviewed in [1,78,79]). As NPCs play a role in most, if not all, processes that regulate genome integrity, it is not surprising that an increasing number of alterations in these structures are linked to cancer. The detailed molecular mechanisms through which NPCs regulate and coordinate these processes are not clear, but it is foreseeable that soon we will begin to understand these processes and establish how these structures contribute to the faithful transmission of genetic information.
Acknowledgements We apologize to all colleagues whose work could not be cited directly owing to space limitation. We thank Marcela Raices for designing and generating all figures and for the critical reading of the manuscript. M.A.D. is a Pew Scholar in the Biomedical Sciences, supported by the Pew Charitable Trust. This work was also supported by the award 10SDG2610290 from the American Heart Association. Current Opinion in Cell Biology 2013, 25:378–386
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31. Gao N, Davuluri G, Gong W, Seiler C, Lorent K, Furth EE, Kaestner KH, Pack M: The nuclear pore complex protein elys is required for genome stability in mouse intestinal epithelial progenitor cells. Gastroenterology 2011, 140:1547-1555 e1510. 32. Bermejo R, Capra T, Jossen R, Colosio A, Frattini C, Carotenuto W, Cocito A, Doksani Y, Klein H, Gomez-Gonzalez B, Aguilera A et al.: The replication checkpoint protects fork stability by releasing transcribed genes from nuclear pores. Cell 2011, 146:233-246. This work shows that nuclear pore complex associated genes need to be released from the structure to allow replication. 33. De Lange T: Telomere-related genome instability in cancer. Cold Spring Harb Symp Quant Biol 2005, 70:197-204. 34. Verdun RE, Karlseder J: Replication and protection of telomeres. Nature 2007, 447:924-931. 35. Taddei A, Gasser SM: Structure and function in the budding yeast nucleus. Genetics 2012, 192:107-129. 36. Feuerbach F, Galy V, Trelles-Sticken E, Fromont-Racine M, Jacquier A, Gilson E, Olivo-Marin JC, Scherthan H, Nehrbass U: Nuclear architecture and spatial positioning help establish transcriptional states of telomeres in yeast. Nat Cell Biol 2002, 4:214-221. 37. Therizols P, Fairhead C, Cabal GG, Genovesio A, Olivo-Marin JC, Dujon B, Fabre E: Telomere tethering at the nuclear periphery is essential for efficient DNA double strand break repair in subtelomeric region. J Cell Biol 2006, 172:189-199. This study demonstrates that yeast telomeres interact with NPCs and extends the previous evidence for Nup84 complex in DNA repair. 38. Schober H, Ferreira H, Kalck V, Gehlen LR, Gasser SM: Yeast telomerase and the sun domain protein mps3 anchor telomeres and repress subtelomeric recombination. Genes Dev 2009, 23:928-938. 39. Khadaroo B, Teixeira MT, Luciano P, Eckert-Boulet N, Germann SM, Simon MN, Gallina I, Abdallah P, Gilson E, Geli V, Lisby M: The DNA damage response at eroded telomeres and tethering to the nuclear pore complex. Nat Cell Biol 2009, 11:980-987. This work links the DNA damage response at eroded telomeres to changes in NPC tethering. 40. Ricchetti M, Dujon B, Fairhead C: Distance from the chromosome end determines the efficiency of double strand break repair in subtelomeres of haploid yeast. J Mol Biol 2003, 328:847-862. Demonstrates that double strand break DNA repair is more efficient in subtelomeric regions, which allows genome plasticity at chromosomal ends and, thus, cell survival. 41. Crabbe L, Cesare AJ, Kasuboski JM, Fitzpatrick JA, Karlseder J: Human telomeres are tethered to the nuclear envelope during postmitotic nuclear assembly. Cell Rep 2012, 2:1521-1529. This study shows that telomeres are tethered to the nuclear envelope during nuclear reassembly. The authors propose that the inner nuclear membrane protein Sun1 mediates telomere association with the nuclear envelope. 42. Gonzalez-Suarez I, Redwood AB, Perkins SM, Vermolen B, Lichtensztejin D, Grotsky DA, Morgado-Palacin L, Gapud EJ, Sleckman BP, Sullivan T, Sage J et al.: Novel roles for a-type lamins in telomere biology and the DNA damage response pathway. EMBO J 2009, 28:2414-2427. 43. Thompson SL, Bakhoum SF, Compton DA: Mechanisms of chromosomal instability. Curr Biol 2010, 20:R285-R295. 44. Ito D, Matsumoto T: Molecular mechanisms and function of the spindle checkpoint, a guardian of the chromosome stability. Adv Exp Med Biol 2010, 676:15-26. 45. Chatel G, Fahrenkrog B: Nucleoporins: leaving the nuclear pore complex for a successful mitosis. Cell Signal 2011, 23:15551562. 46. Nakano H, Wang W, Hashizume C, Funasaka T, Sato H, Wong RW: Unexpected role of nucleoporins in coordination of cell cycle progression. Cell Cycle 2011, 10:425-433. 47. Wozniak R, Burke B, Doye V: Nuclear transport and the mitotic apparatus: an evolving relationship. Cell Mol Life Sci 2010, 67:2215-2230. www.sciencedirect.com
48. Kerscher O, Hieter P, Winey M, Basrai MA: Novel role for a Saccharomyces cerevisiae nucleoporin, nup170p, in chromosome segregation. Genetics 2001, 157:1543-1553. 49. Pichler A, Knipscheer P, Saitoh H, Sixma TK, Melchior F: The ranbp2 sumo e3 ligase is neither hect- nor ring-type. Nat Struct Mol Biol 2004, 11:984-991. 50. Pichler A, Gast A, Seeler JS, Dejean A, Melchior F: The nucleoporin ranbp2 has sumo1 e3 ligase activity. Cell 2002, 108:109-120. 51. Saitoh H, Pu R, Cavenagh M, Dasso M: Ranbp2 associates with ubc9p and a modified form of rangap1. Proc Natl Acad Sci U S A 1997, 94:3736-3741. 52. Salina D, Enarson P, Rattner JB, Burke B: Nup358 integrates nuclear envelope breakdown with kinetochore assembly. J Cell Biol 2003, 162:991-1001. 53. Joseph J, Liu ST, Jablonski SA, Yen TJ, Dasso M: The rangap1– ranbp2 complex is essential for microtubule–kinetochore interactions in vivo. Curr Biol 2004, 14:611-617. 54. Dawlaty MM, Malureanu L, Jeganathan KB, Kao E, Sustmann C, Tahk S, Shuai K, Grosschedl R, van Deursen JM: Resolution of sister centromeres requires ranbp2-mediated sumoylation of topoisomerase II alpha. Cell 2008, 133:103-115. 55. Klein UR, Haindl M, Nigg EA, Muller S: Ranbp2 and senp3 function in a mitotic sumo2/3 conjugation–deconjugation cycle on borealin. Mol Biol Cell 2009, 20:410-418. 56. Babu JR, Jeganathan KB, Baker DJ, Wu X, Kang-Decker N, van Deursen JM: Rae1 is an essential mitotic checkpoint regulator that cooperates with bub3 to prevent chromosome missegregation. J Cell Biol 2003, 160:341-353. 57. Blower MD, Nachury M, Heald R, Weis K: A rae1-containing ribonucleoprotein complex is required for mitotic spindle assembly. Cell 2005, 121:223-234. A Xenopus egg-based spindle assembly assay is used to identify the mRNA export protein Rae1 as a spindle assembly factor and reveals an unexpected function for RNA in spindle formation. 58. Radulescu AE, Cleveland DW: Numa after 30 years: the matrix revisited. Trends Cell Biol 2010, 20:214-222. 59. Wong RW, Blobel G, Coutavas E: Rae1 interaction with numa is required for bipolar spindle formation. Proc Natl Acad Sci U S A 2006, 103:19783-19787. 60. Jeganathan KB, Malureanu L, van Deursen JM: The rae1–nup98 complex prevents aneuploidy by inhibiting securin degradation. Nature 2005, 438:1036-1039. 61. Jeganathan KB, Baker DJ, van Deursen JM: Securin associates with apccdh1 in prometaphase but its destruction is delayed by rae1 and nup98 until the metaphase/anaphase transition. Cell Cycle 2006, 5:366-370. 62. Wong RW: Interaction between rae1 and cohesin subunit smc1 is required for proper spindle formation. Cell Cycle 2010, 9:198200. 63. Loiodice I, Alves A, Rabut G, Van Overbeek M, Ellenberg J, Sibarita JB, Doye V: The entire nup107–160 complex, including three new members, is targeted as one entity to kinetochores in mitosis. Mol Biol Cell 2004, 15:3333-3344. This study identifies three new members of the Nup107–160 complex, Nup37 Nup43 and Seh1, and shows that this entire complex targets to kinetochores from prophase to anaphase. 64. Zuccolo M, Alves A, Galy V, Bolhy S, Formstecher E, Racine V, Sibarita JB, Fukagawa T, Shiekhattar R, Yen T, Doye V: The human nup107–160 nuclear pore subcomplex contributes to proper kinetochore functions. EMBO J 2007, 26:1853-1864. This work reveals the molecular functions underlying the anchoring of the Nup107–160 complex to kinetochores. 65. Galy V, Askjaer P, Franz C, Lopez-Iglesias C, Mattaj IW: Mel-28, a novel nuclear-envelope and kinetochore protein essential for zygotic nuclear-envelope assembly in C. elegans. Curr Biol 2006, 16:1748-1756. 66. Platani M, Santarella-Mellwig R, Posch M, Walczak R, Swedlow JR, Mattaj IW: The nup107–160 nucleoporin complex Current Opinion in Cell Biology 2013, 25:378–386
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promotes mitotic events via control of the localization state of the chromosome passenger complex. Mol Biol Cell 2009, 20:5260-5275. 67. Orjalo AV, Arnaoutov A, Shen Z, Boyarchuk Y, Zeitlin SG, Fontoura B, Briggs S, Dasso M, Forbes DJ: The nup107–160 nucleoporin complex is required for correct bipolar spindle assembly. Mol Biol Cell 2006, 17:3806-3818. 68. Bai SW, Rouquette J, Umeda M, Faigle W, Loew D, Sazer S, Doye V: The fission yeast nup107–120 complex functionally interacts with the small gtpase ran/spi1 and is required for mrna export, nuclear pore distribution, and proper cell division. Mol Cell Biol 2004, 24:6379-6392. 69. Mishra RK, Chakraborty P, Arnaoutov A, Fontoura BM, Dasso M: The nup107–160 complex and gamma-turc regulate microtubule polymerization at kinetochores. Nat Cell Biol 2010, 12:164-169. Describes that kinetochore associated Nup107–160 works in concert with the g-TuRC complex to nucleate microtubules and promote the assembly of k-fibers, which help to connect kinetochores to spindle poles. 70. Bolhy S, Bouhlel I, Dultz E, Nayak T, Zuccolo M, Gatti X, Vallee R, Ellenberg J, Doye V: A nup133-dependent npc-anchored network tethers centrosomes to the nuclear envelope in prophase. J Cell Biol 2011, 192:855-871. Exposes a role for the nucleoporin Nup133 in mediating the attachment of centrosomes to the nuclear envelope during prophase, a key step in the early events of mitotic spindle assembly. 71. Hachet V, Busso C, Toya M, Sugimoto A, Askjaer P, Gonczy P: The nucleoporin nup205/npp-3 is lost near centrosomes at mitotic onset and can modulate the timing of this process in caenorhabditis elegans embryos. Mol Biol Cell 2012, 23: 3111-3121.
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Nuclear pore complexes in the maintenance of genome integrity Lucas Bukata, Stephanie L Parker and Maximiliano A D’Angelo Maintaining genome integrity is crucial for successful organismal propagation and for cell and tissue homeostasis. Several processes contribute to safeguarding the genomic information of cells. These include accurate replication of genetic information, detection and repair of DNA damage, efficient segregation of chromosomes, protection of chromosome ends, and proper organization of genome architecture. Interestingly, recent evidence shows that nuclear pore complexes, the channels connecting the nucleus with the cytoplasm, play important roles in these processes suggesting that these multiprotein platforms are key regulators of genome integrity. Addresses Cardiovascular Research Institute, Biochemistry and Biophysics Department, University of California San Francisco, San Francisco, CA 94158, United States Corresponding author: D’Angelo, Maximiliano A ([email protected])
Current Opinion in Cell Biology 2013, 25:378–386 This review comes from a themed issue on Cell nucleus Edited by Edith Heard and Danesh Moazed For a complete overview see the Issue and the Editorial Available online 6th April 2013 0955-0674/$ – see front matter, # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ceb.2013.03.002
Introduction Nuclear pore complexes (NPCs) are aqueous channels that penetrate the nuclear envelope and connect the nucleus with the cytoplasm [1]. These highly conserved structures are built by the repetition of 30 different proteins called nucleoporins, most of which associate in stable subcomplexes (Figure 1) [2,3,4]. Although the control of nucleocytoplasmic molecule exchange is considered the main function of NPCs, these structures and their components have important transport-independent functions. In this review we will focus on the roles of NPCs and nucleoporins in DNA replication, DNA damage, telomere maintenance, and chromosome segregation. These newly discovered functions for NPCs imply that these structures might be central players in the maintenance of genome integrity.
NPCs in DNA repair and replication The ability to sense and repair DNA damage is probably the most important factor in genomic stability. Alterations in the mechanisms that regulate these processes generally lead to an increased rate in DNA mutations and result in Current Opinion in Cell Biology 2013, 25:378–386
genomic instability. Because more mutations augment the probability of acquiring a selective advantage, most cancer cells show alterations in the DNA damage response (DDR), and genomic instability is a hallmark of transformed cells. The first link between NPCs and the DDR machinery was revealed when a genome-wide screen implicated five nucleoporin genes in the repair of ionizing radiation damage in yeast cells [5]. Notably, the identified nucleoporins (Nup84, Nup120, Nup133, Nup170 and Nup188) were all members of the NPC scaffold structure (Figure 1) [4]. Mutants of Nup84, Nup120, Nup133, from the Nup84 complex, were also sensitive to other DNA damaging agents including UVlight, DNA methylating agents, DNA strand breaks inductors, replication and topoisomerase I inhibitors [5], and show defects in double strand break (DSB) repair and targeted recombination [5]. The increased sensitivity to DNA damaging agents in nucleoporin mutants was also identified in several independent studies [6–10,11]. Further supporting the role of nucleoporins in DDR, mutations in the Nup84 complex have a synthetic lethality interaction with RAD27 and RAD52, which play critical roles in DNA replication and repair [12]. Accumulation of RAD52 foci, a marker for DSB accumulation, was observed with deletion of these nucleoporins, in double mutants of the nuclear basket proteins Mlp1 and Mlp2, and in deletion mutants for Nup60, which anchors these nucleoporins to the NPC. No foci were detected in nucleoporin mutants involved in mRNA export, protein import or NPC distribution [13], suggesting that the DNA damage is not a secondary effect of nucleocytoplasmic transport alterations. The Nup84 complex and Nup60 are required for the recruitment of the SUMO protease Ulp1 to NPCs [14] (Figure 2a). Interestingly, Ulp1 mutants that cannot localize to NPCs show DNA damage accumulation phenotypes similar to nucleoporin mutants [13]. Furthermore, Ulp1 overexpression can partially rescue the DNA damage defects of these strains as well as their colethality phenotypes with RAD27 and RAD52 mutants [13]. These findings indicate that the Nup84 complex and Nup60 might regulate DNA damage repair by recruiting Ulp1 to the nuclear envelope (Figure 2a). Supporting this idea, depletion of these nucleoporins results in the loss of Ulp1 from NPCs and affects cellular sumoylation patterns [13,14,15]. Among the affected proteins in nucleoporin depletion mutants is yKu70, which shows decreased sumoylation levels that are apparently responsible for the loss of its function [13]. As yKu70 plays a key role in the non-homologous end-joining (NHEJ) repair of DSBs [16], the loss of its activity could provide a molecular explanation to the DDR phenotypes observed in www.sciencedirect.com
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Figure 1
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Molecular organization of the nuclear pore complex. Schematic representation of the structure and composition of NPCs in yeast and vertebrates. Nucleoporins that are part of the same subcomplexes are enclosed in boxes. Homologous nucleoporins/subcomplexes between yeast and vertebrates share the same colors.
the nucleoporin mutants. Yet, given that many proteins involved in DDR can be sumoylated [17], it is possible that Ulp1 plays a role in the modification of many of these substrates. Another link between the NPC, the SUMO pathway and DNA damage repair was recently described in a study showing that yeast replication forks containing double DSBs relocate to NPCs for repair in a SUMO dependent manner (Figure 2b) [18]. This process requires interaction between the Nup84 complex and the STUbL complex [18], a SUMO-targeted ubiquitin ligase complex composed of Slx5 and Slx8 that preferentially ubiquitinates polysumoylated targets [19]. While localization of the STUbL complex to NPCs is independent of DNA damage and this complex is not required for the recruitment of DSB, its NPC association is critical for the repair of collapsed replication forks and persistent DSB. In the proposed model, DNA damaged sites marked by the accumulation of a sumoylated protein are tethered to the NPC through the STUbL complex. The relocalization of the damaged DNA to the nuclear periphery requires Mec1 and Tel1, the yeast homologs of kinases ATM and ATR [20,21], and is critical for the repair process. Considering the ubiquitin ligase activity of the STUbL complex, it is likely that the degradation of the sumoylated proteins at the site of damage is required for repair to begin. Interestingly, decreasing the Ulp1 levels www.sciencedirect.com
has no effect in the process [18], suggesting the existence of distinct parallel mechanisms for DNA repair through the NPC/SUMO pathway. However, it is still possible that the sumo protease activity of Ulp1 is required to release the recruited DNA from Slx5 after or during damage repair (Figure 2b). The findings that NPCs would act as DNA repair centers regulated by the SUMO pathway is consistent with the emerging idea of compartmentalized DDR repair sites in yeast and with the proposed role of sumoylation in DNA repair and the maintenance of genome integrity (reviewed in [17,22]). Little is known about the role of NPCs in the DDR of mammals. Similar to Drosophila and plants, the presence of NPC-associated SUMO-regulating proteins has been described in mammalian cells [23–25], but their involvement in DDR has not been studied. But the recent findings that downregulation of Nup153 leads to delayed DNA repair and decreases cell survival suggests this NPC function is conserved [26]. Although Nup153 seems to regulate DNA damage repair mostly by controlling the nuclear import of 53BP1 [26,27], a key regulator of DDR [28], some experimental data suggest a 53BP-independent role for Nup153 in regulating the choice between homologous recombination and NHEJ [26]. A link between NPCs and DNA replication is also emerging. Besides the genetic interactions between different Current Opinion in Cell Biology 2013, 25:378–386
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NPCs DNA repair and telomere maintenance. (a) The SUMO protease Ulp1 associates with the NPC through the Nup84 subcomplex and Nup60. At the NPC, Ulp1 regulates the sumoylation status of proteins that play key roles in DNA damage repair. (b) When a DSB (yellow star) is generated during DNA replication, the replication fork collapses and relocates to the NPC in a Mec/Tel1 dependent manner. NPC association requires the STUbL complex (Slx5/Slx8), which potentially tethers DNA through its interaction with sumoylated proteins that accumulate at the site of damage. The STUbL complex might regulate DNA repair by ubiquitinating specific targets and inducing their degradation by the proteasome. As the STUbL complex and Ulp1 are anchored to the NPC by the Nup84 subcomplex it is possible to speculate that desumoylation by Ulp1 might be implicated in the release of DNA from the NPC during or after the repair process. (c) The Mlp1 and Mlp2 nuclear basket nucleoporins regulate telomere length through a yet unknown mechanism. (d) Critically short telomeres are recognized as DNA damage and relocalize to NPCs for repair, potentially through the STUbL complex pathway. (e) Nucleoporins of the Nup84 complex are essential for telomere tethering to the nuclear periphery and for the efficient repair of subtelomeric double strand breaks (DSBs).
nucleoporins and RAD27 described above, the nucleoporin Elys was found to bind the replication licensing proteins Mcm2–7 directly in Xenopus extracts [29], suggesting a potential role in DNA replication. Supporting this idea, Elys Zebrafish mutants show reduced levels Current Opinion in Cell Biology 2013, 25:378–386
of Mcm2 on chromatin, activated DDR, and increased sensitivity to replication stress [30]. Activated DDR in response to Elys depletion was also observed in mouse epithelial cells [31], indicating a conserved function in mammals. Besides the potential role of NPC components www.sciencedirect.com
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in coordinating replication licensing, a recent report shows that during S-phase the release of genes from NPCs is required for their replication [32]. Considering this, it is possible to speculate that by controlling the tethering of specific genes and chromosomal regions to the nuclear periphery NPCs might play a role in regulating their replication timing.
NPCs in telomere maintenance Telomeres are nucleoprotein complexes that protect the ends of linear chromosomes [33]. These structures are required for the efficient replication of chromosome ends and to prevent their recognition as DSBs [34]. Telomere dysfunction is considered a major contributor to genome instability and cancer [33]. In budding yeast, telomeres localize in clusters at the nuclear periphery [35]. Three NPC components, Nup145p, Mlp1 and Mlp2, were initially proposed to be responsible for the peripheral localization of chromosome ends [8,36]. However, their role in telomere tethering was later questioned [11]. More recently, several components of the Nup84 complex were described to be essential for the peripheral localization of chromosome XI telomere [37]. However, another report showed that telomere positioning was not affected in NPC clustering mutants, suggesting that NPCs are not responsible for telomere tethering [38]. These contentious findings might be explained if different tethering mechanisms exist for different chromosome ends, telomeres states, or phases of the cell cycle. Despite the controversial role of NPCs in telomere anchoring, several lines of evidence support their function in telomere maintenance and repair. First, Mlp1 and Mlp2 are important for the control of telomere length [11,15] (Figure 2c). Moreover, in telomerase negative yeast cells, critically short telomeres recruit proteins of the homologous recombination repair machinery as well as checkpoint proteins and relocalize to NPCs [39] (Figure 2d). This suggests that eroded telomeres are recognized as DNA damage and move to NPC to start the repair process, potentially through the Slx5/Slx8-dependent mechanisms described above. NPC tethering of chromosome ends may also be important for the repair of subtelomeric DSBs (Figure 2e). In the same study showing that the Nup84 complex is responsible for anchoring telomere XI-L, mutants for these nucleoporins showed a decreased ability to repair subtelomeric DSBs [37]. Given that NPCs seem to act as centers for DNA repair, the localization of telomeres to these structures might explain why DSB repair is 20fold more efficient in subtelomeric regions than in other chromosomal domains [40]. In mammals, telomeres have recently been shown to localize to the nuclear periphery during postmitotic nuclear assembly [41] and a role for Lamin A proteins in telomere maintenance has been suggested [42]. Yet, www.sciencedirect.com
whether NPCs play a role in telomere tethering, maintenance or repair in mammalian cells has not yet been studied.
Nucleoporins in chromosome segregation and cytokinesis The precise segregation of chromosomes during cell division is critical to ensure the faithful transmission of the genetic information. Alterations in this process might result in genomic instability and often lead to chromosomal rearrangements or the gain or loss of entire chromosomes (aneuploidy) [43,44]. During mitosis, replicated chromosomes condense, microtubules reorganize in the mitotic spindle, chromosomes attach to the spindle through their kinetochores and segregate toward opposing centrosomes. In recent years, it has become clear that nucleoporins play critical roles in mitosis by regulating specific steps of these processes [45–47] (Figure 3). Nup170, the yeast homolog of mammalian Nup155, was the first nucleoporin described to have mitotic functions. Nup170 mutants affect kinetochore integrity (Figure 3a) and show increased chromosome loss and nondisjunction indicating abnormal chromosome segregation [48]. Nup358/RanBP2 is a component of the NPC cytoplasmic filaments (Figure 1). This nucleoporin is an E3 SUMO ligase [49,50] that forms a complex with two other proteins, RanGAP and the SUMO E2 conjugating protein Ubc9 [23,51]. During mitosis this complex binds to kinetochores and spindle poles and plays a critical role in kinetochore assembly and microtubule attachment [52,53] (Figure 3a). Nup358/RanBP2 also sumoylates and recruits toposisomerase II to centromeres, a critical step in sister chromatid separation [54] (Figure 3a). In a similar way, Nup358/RanBP2 associates and sumoylates a component of the Chromosome Passenger Complex (CPC), a mitotic regulator with key functions in spindle and kinetochore assembly, potentially modulating the activity of this complex [55]. Owing to its various functions, Nup358/RanBP2 alterations result in multiple mitotic defects, including mislocalization of several kinetochore-associated proteins, multiple mitotic spindles, chromosome misalignment, anaphase bridges, and aneuploidy [52–54]. Unsurprisingly, mutant mice that are heterozygous for this nucleoporin have a higher tendency of developing spontaneous tumors [54]. Rae1, a nucleoporin sharing homology with the mitotic checkpoint protein Bub3 [56], is essential for spindle assembly and chromosome segregation [56,57,58–61] (Figure 3a). Rae1 interacts with multiple partners and seems to regulate spindle formation and function through several mechanisms (Figure 3a). Rae1 forms part of a large ribonucleoprotein (RNP) complex that binds microtubules directly and controls microtubule dynamics and spindle formation [57]. Additionally, Rae1 interacts Current Opinion in Cell Biology 2013, 25:378–386
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Figure 3
(a) Kinetochore Assembly Nup358-RanGAP-Ubc9 Nup107 -160 Complex Nup170* Kinetochore
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Mitotic functions of nucleoporins. (a) The Nup358 regulates kinetochore assembly and microtubule attachment and also assists in spindle formation and sister chromatid separation through its SUMO ligase properties. In most cases, Nup358 works in a subcomplex with sumoylated RanGAP and the E2 sumo-conjugating enzyme Ubc9. The Nup107–160 complex is essential for kinetochore assembly and function. It promotes microtubule– kinetochore attachment and the formation of k-fibers, which facilitate spindle assembly. The Nup107–160 complex has also been proposed to directly regulate spindle assembly, although there is contradicting evidence for this role. Nup170 (*the yeast homologue of vertebrate Nup155) functions in chromosome segregation by maintaining kinetochore integrity. Rae1 regulates spindle assembly and microtubule bundling through its interaction with multiple proteins in including the cohesion subunit SMC1, the nuclear mitotic apparatus protein NuMA and the nucleoporin Nup98. Rae1 also forms part of a RNP complex that regulates microtubule dynamics and spindle formation. (b) Nup133 mediates the tethering of centrosomes to the nuclear envelope in early prophase. Centrosome association to the nuclear envelope is essential for the early stages of spindle formation. (c) Changes in the expression of several nucleoporins, including, Nup153, Elys, and SEH1, have been linked to defects in cytokinesis. Specifically, depletion of Seh1 and Elys, result in both cytokinesis defects and (d) multinucleated cells. The multinucleated phenotype is also found with overexpression of Nup153 or Tpr, as well as aberrant regulation of Nup88 (overexpression or downregulation).
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with the nuclear mitotic apparatus (NuMA) protein, which functions in the organization and attachment of microtubules to spindle poles [58]. This interaction potentially increases the ability of NuMA to crosslink and bundle microtubules, a requirement for correct spindle formation [59]. Rae1 also interacts with the cohesin subunit SMC1, and the Rae1–SMC1 complex is essential for proper spindle formation [62]. Moreover, a Rae1– Nup98 complex was found to be important for spindle assembly and to inhibit the degradation of the mitotic checkpoint protein Securin by the anaphase-promoting complex (APC) [60,61], possibly controlling the time of chromosome segregation. Consistent with this idea, haploinsufficiency of both proteins results in premature segregation of sister chromatids and aneuploidy [60]. Mitotic functions have also been attributed to the mammalian Nup107–160 complex, the homolog of yeast Nup84 complex. Most components of this complex have been shown to partially localize to kinetochores during mitosis [63,64,65]. The association of the Nup107–160 complex with kinetochores is required for anchoring Nup358/RanBp2 [64]. Interestingly, the depletion of Seh1, which efficiently reduces the levels of these nucleoporins at kinetochores without affecting NPC function, resulted in misaligned chromosomes, kinetochore-spindle attachment defects, spindle checkpoint activation, and mitotic delays [64,66] (Figure 3a). Similar phenotypes in chromosome congression and checkpoint activation were observed with the depletion of Seh1 and Nup107 in a later study, although normal kinetochore– microtubule attachments were observed [66]. The latter attributed the function of the Nup107–160 complex in chromosome alignment and segregation to the recruitment Aurora B and the CPC proteins to centromeres [66]. These studies described alterations in mitotic spindle length and activated spindle checkpoints, but showed no defects in spindle formation [64,66]. However, the findings that several components of the Nup107–160 complex localize throughout the entire mitotic spindle and spindle poles [67], that this complex is required for the in vitro assembly of bipolar spindles [67], and that mutations in a member of the Schizosaccharomyces pombe homologous complex show abnormal mitotic spindles [68] suggest a role in spindle formation (Figure 3a). Consistent with this idea, recent findings show the Nup107–160 complex recruits and works cooperatively with the microtubule nucleation complex g-TuRC to promote the formation of k-fibers and facilitate spindle assembly [69] (Figure 3a). Additionally, the Nup107–160 complex member Nup133 was recently described to mediate the tethering of centrosomes to the nuclear envelope in prophase [70] (Figure 3b). Given that the loss of centrosome attachment to the nuclear envelope transiently results in aberrant spindles, Nup133 might be important for the early stages of mitotic spindle formation [70]. Interestingly, the association of www.sciencedirect.com
centrosomes with the nuclear envelope is required for the release of scaffold nucleoporins in Caenorhabditis elegans and regulates the timing of mitotic onset [71]. Depletion of two Nup107–160 complex components, Seh1 and Elys, results in multinucleated cells indicating cytokinesis defects [66] (Figure 3c,d). Abnormal cytokinesis is also observed with depletion of Nup153, a component of the NPC nuclear basket (Figures 1 and 3c). Downregulating Nup153 leads to mislocalization of Aurora B [72], delayed dissociation of the spindle checkpoint protein Mad1 from kinetochores [73], and accumulation of unresolved midbodies [72–74]. These phenotypes are partially mimicked by downregulation of Nup50, which also becomes mislocalized with Nup153 depletion [72]. Conversely, Nup153 overexpression results in multipolar spindles and multinucleated cells [73] (Figure 3d). Nup153 regulates the localization and phosphorylation of Mad1 during metaphase/anaphase transition and is required for spindle checkpoint activity and mitotic exit, providing a potential molecular mechanism for the observed phenotype [73]. Tpr, a mammalian homolog of Mlp1/Mlp2 that interacts with Nup153, binds Mad1 and Mad2 proteins as well as the molecular motors important for spindle function, dynein and dynactin [75,76]. Tpr depletion affects levels and localization of spindle checkpoint proteins and results in a chromosome lagging phenotype [75,76]. Similar to Nup153, overexpression of Tpr results in multinucleated cells [75] (Figure 3d), a phenotype also observed with depletion or overexpression of Nup88, a nucleoporin misregulated in many cancer cells [77] (Figure 3d).
Conclusions Nuclear pore complexes are emerging as key regulators of numerous cellular functions. In addition to the described role of NPCs in DNA repair, replication, protection and segregation, these structures have been shown to play a role in the organization of genome architecture, another pillar in genome stability (recently reviewed in [1,78,79]). As NPCs play a role in most, if not all, processes that regulate genome integrity, it is not surprising that an increasing number of alterations in these structures are linked to cancer. The detailed molecular mechanisms through which NPCs regulate and coordinate these processes are not clear, but it is foreseeable that soon we will begin to understand these processes and establish how these structures contribute to the faithful transmission of genetic information.
Acknowledgements We apologize to all colleagues whose work could not be cited directly owing to space limitation. We thank Marcela Raices for designing and generating all figures and for the critical reading of the manuscript. M.A.D. is a Pew Scholar in the Biomedical Sciences, supported by the Pew Charitable Trust. This work was also supported by the award 10SDG2610290 from the American Heart Association. Current Opinion in Cell Biology 2013, 25:378–386
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