The Quest for the Blueprint of the Nuclear Pore Complex

Abstract

During my postdoc interview in June of 1998, I asked Günter why he was moving more towards the nucleus in his latest studies. He said, “Well Joe, that’s where everything starts.” By the end of the interview, I accepted the postdoc. He had a way of making everything sound so cool. Günter’s progression was natural, since the endoplasmic reticulum and the nucleus are the only organelles that share the same membrane. The nuclear envelope extends into a double membrane system with Nuclear Pore Complexes embedded in the pore membrane openings. Even while writing this review, I remember Günter stressing; it is the Nuclear Pore Complex. Just saying Nuclear Pore doesn’t encompass the full magnitude of its significance. The Nuclear Pore Complex is one of the largest collection of proteins that fit together for an overall function: transport. This review will cover the Blobel lab contributions in the quest for the blueprint of the Nuclear Pore Complex from isolation of the nuclear envelope and nuclear lamin to the ring structures.

Introduction

The nucleus and endoplasmic reticulum are the only organelles to share the same related membrane. The Nuclear Envelope (NE) is defined as a specialized endoplasmic reticulum membrane with a double bilayer containing inner and outer membrane systems [1]. While this separation protects the genome, it also is needed to organize nuclear entry and exit for basic cellular processes. The NE separates into three domains: the outer nuclear membrane (ONM), the inner nuclear membrane (INM) and the pore membrane (POM) [2,3]. The POM is the region of the NE where INM and ONM merge. The POM curvatures are coated with specific membrane proteins believed to anchor Nuclear Pore Complexes (NPCs) like the prongs of a diamond ring. NPCs, the gateways for macromolecular traffic into and out of the nucleus, are set in circular openings [4,2,5,6,3]. There are extremely regulated pathways that control nuclear entry and exit of molecules such as transcription factors, RNAs, kinases and viral particles. Overall, proteins with transport signal sequences to be imported or exported from the nucleus 1) bind to transport receptors, 2) are transported through an NPC and 3) translocate from the NPC to intranuclear or cytoplasmic target sites [7,4,8,9,5,3](Fig. 1a, see legend). Additional functions include recognition of properly spliced mRNA or ribosomal subunits for export (Fig. 1b–c). In addition, viruses exploit the NPC to passage into the nucleus from the cytoplasm [10,11]. Nuclear localization signal (NLS) can be collected from host proteins or contained within viral expressed proteins allowing for viral component translocation across NPCs (Fig. 1d–f, bottom panel)[12,13].

Fig. 1. Functions of the Nuclear Pore Complex (NPC).

Molecules smaller than 40 kDa can passively diffuse across the NPC but higher molecular weight proteins, a coordinated receptor-mediated transport to facilitate crossing into the nucleus [4,9,91,143–145]. Binding molecules, known as karyopherins (kaps), fasten to cargo in the cytosol and carry it into the nucleus (shown in panel a); in contrast, they bind cargo in the nucleus and deliver it to the cytoplasm. Kaps recognize their cargo by binding short amino acid sequence segments of the protein [146]. Cargos have nuclear localization signal (NLS) that are classically rich in basic residues for import (shaded area of protein in panel a) [4,9,147,146]. This form of transport is coupled to a cycle of GTP-binding and hydrolysis conducted through the protein Ran (not shown)[8]. The concentration of RanGTP is much higher in the nucleus. Therefore, once the complex has entered the nucleus, the high concentration allows for the disassembly and release of the components [9]. Another function of the NPC is to export properly spliced mRNA through recognition of spliced areas by recognition protein like MEX and TAP (panel b) and the export of ribosomal subunits from the nucleus (panel c). In bottom panels (d-f), viral transport through the NPC using microtubule network in combination with motor proteins like kinesin (panel d) lead to the docking of viral components to the NPC (panel e). Then translocation of viral genetic material and integration complexes into the nucleus (panel F) and disengaged viral coating traveling back up the microtubular network (panel f)[12,13](Pencil sketches courtesy of Grace Glavy).

Foundation for the Blueprint of the NPC

University of Wisconsin, Ph.D.: Van Potter’s Lab

My labmates and I were discussing how one of Günter’s interns was taking a transatlantic cruise back to Germany and how wild that was; Günter interjected, “That’s how I arrived in New York.” Except he also brought his Volkswagen beetle on the ship and then drove it to Madison, Wisconsin for his medical research internship. Günter later decided to stay and pursue his Ph.D. in Van Potter’s laboratory at the University of Wisconsin. In 1966, Blobel and Van Potter described a protocol to generate a highly quantitative separation of nuclei from a homogenate of rat liver [14]. The rat liver offered a uniform population of cells containing nuclei. This protocol used concentrated sucrose solutions rather than detergents or citric acid which permitted better fractionation of cytoplasmic components while using certain parts of the Chauveau method [15–17] (Fig. 2a)[14]. To reduce contaminants seen with the Chauveau method, sucrose concentrations were raised to 2.3M. In the end, an estimated 91 percent of the nuclear DNA was recovered and nuclei contain 4.3 percent of the total RNA. Remnants of the ER with ribosomal attachment sites pointed out (Fig. 2a) were removed through low concentration treatment with Triton X-100 or deoxycholate and Tween 40 in Fig. 2b with little disruption to DNA content [14]. This work helped set the stage to isolate the NE and the nuclear matrix [18–31].

Fig. 2. Isolated Rat Liver Nuclei.

2a. Transmission electron microscopy (TEM) of isolated rat liver Nuclei, arrows identify residual ER ribosomal attachment sites. 2b. TEM of rat liver Nuclei after treatment with 5% Triton X-100 to eliminate ER and ribosomal attachment sites (Adapted from Blobel, G., Potter, VR. 1966 [14]). Freeze-cleaved preparation of rat liver nuclei, shown is the outer leaflet (2c)(Labeled as A&D within image) then the inner leaflet (2d) (Labeled as B&C within image). Bottom panel (2e-2h) arrows direct to corresponding NPC freeze fracture impressions. (Adapted from Monneron, A., Blobel, G., Palade, G.E. 1972 [26].

Rockefeller University, Post-Doc: George Palade’s Lab

In classic work in George Palade’s lab, freeze-cleaved preparations of nuclear fractions showed the two membrane configuration of the NE indicated in Fig. 2c [26]. In Fig. 2c, the face of the inner leaflet is marked as A (Fig. 2c), the complementary outer leaflet is marked as B (Fig. 2d) [26]. Higher magnification craters embedded in the double membrane [26]. These noted polygonal impressions in the NE are individual NPCs (bottom panel 2e-2h)[26,32,33,6,34]. The pores were referred to as circumvallate papillae like structures with long whiskers and found to have a mostly octagonal conformation [26,32,33]. Fractionation of nuclear component was enhanced by selective exposure to charged molecules [26]. Different concentrations of MgCl₂ could uncover the different layers of the NE by competing with ionic interactions [26]. It was thought that with increasing concentrations and combinations of divalent cations, one could tear down the structure leading to ghost pores [35].

Principal Investigator: Rockefeller University

One of the first papers from the Blobel lab dealt with the attachment of the NPC. It was found that envelope-denuded nuclei (partially stripped of membrane) still retained their shape and NPCs maintained their position (Fig. 3a–b (NPCs), p1). After 2% Triton X-100 treatment, the eight-fold symmetry is clearly visible with distinct features (Fig. 3c–d, p2)[36,27,28,37]. The isolated NPCs from several different species maintain this octagonal configuration [38–43]. This arrangement may be necessary to ensure transport clearance for large protein complexes such as ribosomal subunits. Triton X-100 treatments greatly reduced phospholipids in the final pellets. DNA content and histone concentrations were not significantly affected. Early studies of the NE and NPC were ineffective in eliminating DNA and lamina. In later studies the addition of DNAse I digestion steps [44,29] reduced DNA content and revealed lamina connections to NPCs. Triton X-100 treatment followed by salt treatment led to the extraction of lamina-NPC fractions with little DNA/RNA and/or histone content [29,27]. NPCs remained intact and attached to lamina as seen by EM [29]. An early schematic diagram of the nuclear periphery lists the basic components as nuclear envelope, NE; NPC, pc; outer nuclear membrane, om; perinuclear space, ps; inner nuclear membrane, im; peripheral lamina, pl; heterochromatin, hc and ribosomes, r (Fig. 3e)[29]. The NE could be mapped as three distinct structures: the double membrane system, NPCs, and peripheral lamina [29,27], all separate from the nuclear matrix, which is the network of fibres found inside the cell nucleus. Further refinement of the NE was initially achieved through multiple DNAse digestion at pH 8.5 then pH 7.5 (further removal of histones observed) followed by Triton X-100 treatment and salt treatments [36]. Mild sonication led to laminar fragmentation with NPCs remaining intact [36]. Development of anti-sera against the lamina fractions allowed for accuracy while characterizing NE preparations [45]. Through reduction and alkylation, three distinct proteins between 60 and 70 kilodaltons were separated and used to produce an antiserum against each [45]. Later identified as lamin A/C and lamin B, these antisera produced nuclear staining with slight cytoplasmic background in some cases [45–49]. Lamin A/C was found depolymerized during mitosis while lamin B retained attachment to membrane or membrane fragments [46,50]. Intermediate filaments were found to attach to the lamins through studies focused on vimentin and desmin attachments [51,52]. Binding studies with radioactive lamin B labeled its receptor and attachment point to the NE [49,53]. In 1985, Günter proposed the “Gene Gating” hypothesis in PNAS (Fig. 3f) which postulates the idea that transcribed genes are positioned in close proximity to NPCs thereby stream-lining mRNA export [54]. From this early electron microscopy (EM), the position of euchromatin “active/open” and heterochromatin “inactive/closed” could be observed (Fig. 3f). Due to this relationship, NPCs potentially play a role in the adjustment of genomic structure corresponding to different stages in development, differentiation, and the cell cycle. NPCs are proposed as gene gating organelles capable of interacting specifically with expressing genes [54]. All transcripts of a specific gene are projected to exit the nucleus via the NPC to which the gene is gated [54]. The orderly distribution of NPC may reflect underlying genomic organization. Additionally, the nuclear lamin is proposed to participate in organization of compacted regions of the genome [54].

Fig. 3. Eight-fold Symmetry of NPCs.

The periphery of isolated rat liver nuclei after treatment with 2% Triton X-100 (a & b). Panels c & d demonstrate eight-fold symmetry of NPCs. (e) Schematic diagram of the nuclear periphery, lists the basic components as nuclear envelope, NE; the nuclear pore complex, pc; outer nuclear membrane, om; perinuclear space, ps; inner nuclear membrane, im; peripheral lamina, pl; heterochromatin, hc and ribosomes, r. (Adapted from Aaronson, RP., Blobel, G. 1974 & Aaronson, RP., Blobel, G. 1975 [28,29]). (f) Schematic diagram of the position of euchromatin “active/open” and heterochromatin “inactive/closed” relative to NPCs. NPCs are proposed as gene gating organelles capable of interacting specifically with expressing genes [54](Gift from Günter Blobel).

Nucleoporins: Proteins of the NPC.

The proteins composing the NPC were termed nucleoporins (Nups) in 1986 and initially discovered by immune approaches similar to the nuclear lamina. Each NPC contains eight subunits or spokes (Fig. 3 c & d)[37,28,29]. By injection of the Triton X-100 treated rat liver nuclei into a BALB/c mouse, followed by spleen excision and fusion to hybridoma [55,56], a panel of monoclonal antibodies was produced against the nuclear components. Screens of this panel revealed a number of NPC reactive antibodies, none better than clone number 414 (Mab414)[55,56]. Initial studies revealed a specific recognition of a protein at 62 kilodaltons. Initially called p62 but later referenced as Nup62 for its membership as nuclear pore protein and molecular weight (Fig. 4A)[55,56]. This is the major theme of the nomenclature: Nup classification followed by kilodalton weight (Fig. 4A, MAb Lab Art). Of the reactive antibody clones, MAb414 gave a very clear NE or nuclear rim staining by immunofluorescence (Fig. 4B). This landmark study produced the gold standard antibody for decades. While the initial discovery was Nup62, MAb414 recognized more Nups. Nups: 358, 214, 153 and 62 all reacted (Fig. 4A). This was the reason for such pronounced nuclear rim staining, not one Nup but four. Our MAb Lab Art (western blot from my lab’s refrigerator door) answers the question before it’s asked, as MAb recognizes FG domains of these Nups (Fig. 4A). FG domains of Nup62 are used to further purify the antibody from ascites. Purified MAb414 immunoprecipitation in 2% Triton X-100 pulls down all four proteins plus Nup88, CRM1 and Karyopherin beta 1 as identified by mass spectrometry (Glavy, unpublished). A number of Nups have O-linked GlcNAc glycosylation including Nups: 358, 214, 153 and 62 (Fig. 4A). Through a combination of molecular genetics and immunoselection using the panel of monoclonal antibodies against Triton X-100 treated rat liver nuclei, several Nups were localized and isolated to begin characterization [57,58]. Immunoselection was achieved both by antibodies and glycoprotein recognition. Further work in the Blobel lab led to the identification of more Nups as well as pore membrane proteins (POM) both in yeast and human cells [57–64]. A procedure using wheat germ agglutinin (WGA) as a ligand binds a particular class of glycoprotein (N-Acetyl glucosamine groups or sialic acid residues) from the NPC-lamina fraction. Wheat germ agglutinins recognize sugar groups attached to amino acids of NE proteins, POM proteins, and Nups [59–64].

Fig. 4. MAb414 Lab Art-Western Analysis of MAb414.

4a. HeLa nuclear envelope preparation compared to cell extract. Lane 1: Heparin Treated HeLa Nuclear Envelope, labeled NE, Lane 2: HeLa cell extract (labeled Cell Ex). Proteins were separated on 4–20% SDS/PAGE and transferred to nitrocellulose for western analysis probed with MAb414. Enriched NE clearly reacts with Nups 62, 153, 214, 358. Immunofluorescence Microscopy Analysis of MAb414. 4b-4d. HeLa cells were analyzed by immunofluorescence microscopy. Labeled with MAb414 yielding a pronounced nuclear rim staining (red, 4b), Nucleus stained with DAPI (blue, 4c) and merged images (4d).

Although, officially, the Blobel lab only published one collaborative paper with the Brian Chait group at the Rockefeller on the cycle dependent phosphorylation of Y-Nups [65] this had a profound influence on the field because it led to many collaborations with former Blobel post docs with the Chait lab [66–69]. Through two separate studies, the composition of both the yeast and rat NPCs were inventoried [66–69]. In the case of rat liver nuclei preps, the isolated nuclei were subjected to a series of refined treatments [67,69]. The first step was the classic DNase/RNase digestion to eliminate nucleic acids [67,69]. This crude NE was then treated with low molecular weight heparin to further reduce DNA/histone content [67,69]. Next, enriched NE was subjected to Triton-X100 treatment allowing a NPC-lamina fraction to be isolated. This fraction was then solubilized with Emigen BB detergent (Zwitterionic, Amphoteric surfactant) [67,69]. The Chait lab used mass spectrometry (MS) to characterize the purified nucleoporin fraction [66–69]. C4-separated Emigen BB treated fractions were analyzed by SDS-PAGE and stained bands were excised for tryptic digestion followed by mass spectrometric analysis. Mammalian NPCs contained at least seven novel Nups including ALADIN, Nup358, POM210, POM121, NDC1, Nup43, and Nup37 from yeast (Fig. 5)[67]. However results for both yeast and rat revealed approximately thirty Nups exist in both NPCs. These Nups are present in multiple copies, which added to the puzzle of this large transporter. Cloning of several Nups laid the foundation for structural studies leading to the investigations into the blueprint of the NPC.

Fig. 5. Nucleoporin (Nups) Classification, Y-complex And Double Ring Structure.

Shown is the grouping of Nups for both yeast and human Nups (left panel)[3,1]. Structural model of individual Y-complex (middle panel, yellow). Human Double Ring structure formed by collections Y-complex dimers (red/blue and yellow/green)(right panel). [40].

The Quest

The NPC is a collection of a thousand proteins that form subunits leading to the overall structure (Fig. 5)[5,1,3]. The human NPC is approximately 110 MDa with a diameter of ≈60 nm and approximately 33 Nups [1,3,70]. As shown in Fig. 5, the Nups are sorted into 7 groups: Y-complex, Adaptor, Channel, Cytoplasmic, Basket, Mobile and POM. To help give a clearer beginning to the blueprint of the NPC, the stoichiometry was assessed by a mass spectrometry approach to calculate the copy number of individual Nups [71].

Y-complex:

Nup160, Nup133, Nup107, Nup96, Nup75, Nup43, Nup37, Seh1, and Sec13

Y-complex is the most abundant with nine members on the cytoplasmic side (human NPC: Nup160, Nup133, Nup107, Nup96, Nup75, Nup43, Nup37, Seh1, and Sec 13) and ten on the nuclear side with the addition of ELYS (Fig. 5)[72,73,5,3,65,74–77]. These Y-Nups can be pulled down both at G1 and mitosis as an intact unit [65]. Early negative-stained electron microscopy revealed a simple Y shape to this complex, which was later confirmed by Cryo-electron tomography (Cryo-ET) studies, hence the nomenclature [78,40,79]. The tenth member, ELYS, can be found under select conditions but is guaranteed to be crosslinked with the nuclear Y-complex [40,72,73]. ELYS is the only Y-complex protein found with a copy number of 16; the rest have 32. [71]. The complex is sometimes referred to as the coat nucleoporin complex (CNC)[76] as the Y-Nups form an imbricated ring structure of the NPC as shown in Fig. 5 [40]. Y-complexes within the ring are arranged in a head-to-tail fashion [43,40]. Sec13 is classified as a Y-complex member but has multiple locations and binding partners such Sec31 and Sec16 of the COPII vesicles in the ER and members of mTOR regulating SEA/GATOR2 complex [3,80,81]. The first crystal structure of the Y-complex from the Blobel lab was the N-terminus of Nup133 which possessed a seven bladed propeller [82]. Following structures from the Blobel lab (Nup107–133 [83], Nup107-Nup85-Nup96 [84], Nup85-Seh1 [85], and Nup96-Sec13 [86]) helped to begin to resolve the Y-complex configuration.

Adaptor Nups:

Nup93, Nup205, Nup188, Nup155, and Nup35

The Adaptor complex includes five members in humans: Nup93, Nup205, Nup188, Nup155, and Nup35 [87,41]. The X-ray crystal structure of Nup93 reveals an elongated, alpha-helical structure [88]. This form is evolutionarily conserved and therefore functionally maintained [88,89]. Members of the Adaptor complex contain mainly alpha-helical domains [88]. Nup93 and Nup155 are most abundant with 48 copies within the NPC [71]. The adaptor complex aids inner ring (IR) stabilization [88] and is needed for correct NPC assembly and homeostasis [88,89]. This has been confirmed by structure studies that Nup93 is a key component of the IR. Furthermore, Nup62 (Channel Nup) has been shown to interact with Nup93, illustrating interdependence between IR Nups and the Channel Nups [90]. Using an integrated approach (Fig. 6), we found in our studies that 32 copies of both Nup188 and Nup205 (yellow), Nup93 (red), Nup155 (green) and the Channel Nups (Next section: Nup:62, 58–45, 54) fit into the IR with additional Nup155 protein reaching up connecting to the outer ring (green). As shown in Fig. 6, the IRs comprise units similar to the Y-complex, suggesting their evolutionary connection [41], and resolving the ssubdomain of Nup205 and Nup188 and the asymmetric structures of Nup93 have been confirmed by structure studies to be a key component of the IR [41].

Fig. 6. Cross-section of the NPC.

NPC viewed from the cytosol side (top) to the nuclear side (bottom), structure is based on cryo-electron tomography (23Å)[41,40,43]. The curvature of the NE is shown: outer nuclear membrane (ONM), luminal curve (LC) and inner nuclear membrane (INM). The NPC is labeled: Inner Ring (IR) Cytoplasmic Rings (CRs) and Nuclear Ring (NRs)[41].

Channel Nups:

Nup62, Nup58-Nup45, and Nup54

Channel Nups have stretches of FG (Phe-Gly) repetitive residues, which are separated, by polar spacer regions of variable lengths [4,91–93]. FG repeat domains form unstructured regions that form weak interactions with transporting proteins called karyopherins (kaps)(Fig. 1)[4]. Channel Nups are located in the inner pore region or central core inner ring [39,38]. The complex includes Nup62, Nup58-Nup45, and Nup54 (Fig. 6)[94–96]. At one point this complex was referred to as the central plug region of the NPC. While these transport Nups line the inner NPC, it is unlikely that they form a plug against transport, but rather a dynamic transport area of the complex. The FG repeat domains form tentacle-like structures that emanate from and line the channel of the pore to form an FG barrier, which is highly disordered and dynamic [97,98]. In Fig. 6, they are shown to line the IR region. The crystal structure of Nup58-Nup45 produced by the Blobel lab displayed lateral displacements along the alpha-helical axes of their dimeric building blocks [95]. This gave rise to the hypothesis that this section of ring could slide and hence change the overall diameter [95]. Structural analysis of a larger Nup62–58-54 complex denied that sliding could occur while maintaining the trimeric structure [96]. Other channel Nups contain similar alpha-helical regions and may also slide [5], so questions still remain.

Nuclear Basket Nups:

Nup153, Nup50, and Tpr

Nup153 and Nup50 along with Tpr make up the nuclear basket and provide the surface to utilize binding areas for transport. Tpr (Translocated promoter region protein) is an unusual Nup (Nucleoporin Tpr) with more filament protein properties and is required for trafficking across the NE. Tpr coiled-coiled domains help give reliable support to form and maintain the nuclear basket [99]. A recent investigation implies that Tpr may be connected to the regulation of the total number of NPCs [100]. A mechanism for this regulation through extracellular signal– regulated kinases (ERK) pathways needs further investigation [100]. Tpr acts as a framework component in the nuclear segment and functions to tether chromatin. In addition, Tpr is anchored by Nup153 [101]. Tpr plays a role in mitotic spindle checkpoint signaling [102,103]. Furthermore, in concert with Nup153, it participates in both nuclear import and export pathways for proteins with or without nuclear export signal (NES) as well as the export of mRNA [102–104]. Within the nuclear basket, Nup153 is associated with Tpr and contains four zinc fingers. These zinc fingers increase the local Ran concentration to assist nuclear transport (167). Along with Nup50, Nup153 helps to terminate karyopherin mediated transport [4]. Nup153 provides the scaffold for Nup50 localization in the NPC [105].

Cytoplasmic Filament Nups:

Nup358, Nup214, Nup88, Nup2, Aladin, Rae1, and Gle1

As in the case of Channel Nups, Cytoplasmic Filament Nups possess FG regions. Their floppy tentacle nature is incompatible with crystal formation. Thus far, only non-FG regions of these Nups have been reported. The X-ray crystal structure of the non-FG repeat N-terminus of Nup214 reveals a seven bladed β-propeller with a segment of its C-terminus bound to the propeller [106]. Selective knockdown of Nup214 followed by Cryo-ET demonstrates that this subcomplex protrudes into the cytoplasmic ring region [40]. This position secures FG repeats onto the framework of the rings to facilitate nuclear transport [40]. Nup214, Nup88, and Gle1 interact with several Nups within the NPC. Nup358 is the largest Nup with multi-domain regions. It contains multiple Ran-binding domains including zinc finger domains and SUMO E3 ligase binding domains with many disordered areas mixed in [3,28]. The N-terminal alpha-helical domains serve as the anchor at the NPC while the C-terminus extends into the cytosol. Further application of gene-silencing/Cryo-ET uncovered a role for Nup358 to stabilize the cytoplasmic imbricated double ring structure [43]. These findings alter the once clear boundary concept between Y-complexes and Cytoplasmic filaments [43].

Mobile Nup:

Nup98

Nup98 is found both at NPC and within the nucleus [107–110,3]. It has multiple functions and binding partners [110,3,107]. Nup98 has roles in transport, mitotic progression, gene expression, epigenetic changes and viral infection [111–113,110,114,115]. It is not classified as a complex Nup with multiple locations along both sides of the NPC [107] and estimated to have a copy number of 48 within the human NPC [71]. Nup98 arises from a Nup98-Nup96 precursor form that splits by a self-cleavage domain similar to those found in Drosophila Hedgehog and Flavobacterium glycosylasparaginase [116,107]. This precursor gives rise to a mobile Nup (Nup98) and Y-complex Nup (Nup96). Yet to be explained is the fact that Nup96 is present in a different copy number of 32 [71].

The N-terminus of Nup98 contains FG repeats used as a docking site for kaps, but it not classified as an FG Nup but rather as a mobile Nup [107,108]. Nup98 ferries substrates between internuclear regions to NPCs, while regulating transcription of select genes implicated in development and growth [117–120,114,121]. Nup98 facilitates mRNA export from the nucleus [107,122]. Mitotic Phospho-Nup98 is a determining factor in NPC disassembly [119,120]. In NE breakdown (NEBD), the combination of phosphorylation and microtubule-based tearing are believed to lead to the resulting NEBD [123]. Several kinases lead to the hyperphosphorylation of Nup98 and Nup35 that may be contributing factors to NPC disassembly [119,124–128]. One of Nup98’s interacting partners is Rae1; together they act as temporal regulators of the anaphase-promoting complex [129]. Vesicular stomatitis virus (VSV) matrix (M) protein binds Nup98 and inhibits nuclear export of mRNA [122,130]. Studies suggest that Nup98 primes virus-stimulated genes to assist coordinating antiviral response [131].

Inhibition of Nup98 expression has been linked to impaired full expression of p21 and may regulate p53 [132]. Nup98 gene translocations have been linked to several forms of leukemia [114]. Chromosomal translocations involving Nup98 also alter expression of Nup96 [114,107]. Disrupted expression of Nup96 may serve as an additional contributing factor to disease phenotypes that arise from Nup98 translocations with transcription factors, as Nup96 plays a role in the regulation of export of mRNAs that are associated with immunity and cell cycle regulation [114,111,133]. Chromosome translocations of Nup98 are most frequently observed in acute myeloid leukemia (AML), chronic myeloid leukemia in blast crisis (CML-bc), and myelodysplastic syndrome (MDS) [134].

POM Nups:

gp210, Pom121, NDC1, and Pom33

The pore membrane (POM) curvature region of the NE is lined with select membrane proteins believed to anchor NPCs like the settings or prongs of a diamond ring [2,3]. POM Nup proteins are involved in the initiation of pore complex formation, stabilization, release and reformation of the NPC. To date, four POM Nups: gp210, Pom121, NDC1, and Pom33 have been classified through proteomic and genetic screenings. The largest POM protein, gp210 (Pom210), contains a single transmembrane domain as do the rest of the POM proteins. Gp210 was found to be essential for viability in HeLa cells [135]. Knockdowns of gp210 can trigger NE aberrant structures and NPC clusters [135].

NDC1 is found both in the POM region and the spindle pole body (SPB) during mitosis. It functions at the SPB, in yeast [136], helping to anchor the SPB at the NE, but in human cells its purpose is still unknown. RNAi experiments suggest a functional link between NE membrane NDC1 and members of the adaptor complex: Nup93 and Nup205 [137]. These results suggest an anchor point function for the Nup93 subcomplex [90]. The elimination of Pom121 results in the failure to assemble NPCs and to form continuous nuclear membranes [138]. XL-MS shows a direct linkage between Nup133 and Pom121 at the POM interface [40]. POM depletion studies point to the need for additional components to restore overall POM function[139]. NDC1 was shown to form a linkage between the NE and soluble Nups [140,137,139], and depletion of NDC1 shows markedly reduced Channel Nup staining compared to NDC1/control siRNA treatment [137,139].

The POM protein, Pom33 (TMEM33) is required for proper NPC distribution [141,142] as well as assembly. But a large percentage of Pom33 resides in the ER [142,141]. While an element of the tubular endoplasmic reticulum (ER) network, Pom33 also acts as a POM protein at NPCs [142,141]. The C-terminus of Pom33 contains several amphipathic regions that both bind to Kap123 and serve as attachment points that aid in positioning the POM region while N-terminal transmembrane regions anchor the POM [142].

EM and Cryo-ET Studies

Advances in a field are only possible with a solid foundation. Günter Blobel’s early work to isolate the NE helped lead to the characterization of the NPC. Even based on the first sets of EM images the eight-fold symmetry of the NPC was revealed (Fig.3) [28,29,38–43]. This eight-fold rotational symmetric structure is conserved across species [3,70]. The preserved arrangement may be related or proportional to conserved components of the cell such as preribosomal particle transport or perhaps the length and transport of mRNA or even related to the number of particles that can be transported at one time through the NPC. Although the symmetry remains, species differ in the layer construction of the entire NPC [3]. The NPC is a huge assemblage of proteins (110 MDa) making whole structure analyses a challenge [5,1,3]. Yet progress has been made through innovative techniques combining solid biochemistry/cell biology, proteomics, Cryo-ET and structural modelling.

Cytoplasmic Rings (CRs) and Nuclear Rings (NRs)

The Y-complex is a robust assembly [65,40]. For structural studies, the human Y-complex could be expressed from individual proteins to form a complex or the intact complex could be isolated [65,40,75,78]. It was reported in the Blobel lab that the Y-complex could be isolated as nine-member complex with minimal detergent application both at interphase and mitosis [65]. These properties were very advantageous for further analysis by crosslinking mass spectrometry (XL-MS) and structure analysis by Cryo-ET [40]. As shown in Fig. 5 (middle panel, yellow), the single Y-complex has two arms: long and short, leading to a stem region. The human Y-complexes form a basic element dimer of two shifted vertices (Fig. 5, dimers of Y-complexes, right panel, cytoplasmic side: red/blue dimer and yellow/green dimer) [40]. These elements, arranged in a head-to-tail conformation, assemble into two imbricated rings (Fig. 5, right panel), one each at the cytoplasmic side and the nuclear side of the NPC (Fig. 6. CR and NR)[40]. Nup358 RNAi perturbation experiments followed by structural modeling, found that Nup358 fastens the CRs to the NPC [43,40]. Reduction of Nup358 specifically affected CRs quantities [43] creating a hollow area in the rings. Only the Y-complex CRs, not the Y-complex NRs, were affected [43] both proving sidedness and the double ring structure. In the model organism, green alga Chlamydomonas reinhardtii, the CRs contains only 8 copies of the Y-shape due to the lack of Nup358 [148]. This type of local interaction and conformations are most likely key to building an operational NPC.

Complexity of the Inner Ring (IR)

Based on our findings summarized in Fig. 6, the packing of the IR is by the assembly of channel and adaptor Nups. Within the IR, Nups 205,188, 93 and 155 integrate with the channel Nups [41]. Additionally Nup155 is set along the outer portions of the IR [41]. The IR comprises four core modules, each containing one copy of these channel and adaptor Nup combinations [41]. In the end, they come together through inter-complex interactions and oligomerize to form four horizontally stacked rings (Fig. 6.)[41]. The architectural principles are similar to the Y-complex of the outer rings of the CRs and NRs [41]. One of the fundamental hallmarks of this region is a membrane-binding coat of the NPC formed by the membrane engagement of the CR, NR and Nup155 [41]. Adaptor Nups 205 and 188 (Fig. 6, yellow) bind both to IR and Y-complex rings giving an interconnected stack of rings. Fig. 6 gives the current blueprint of the rings from top to bottom of the NPC. A key point of the structure is the combination of Nups in each ring and how they interlink between rings to form stacked layers. Not resolved or shown are the cytoplasmic Nups and the nuclear basket.

Summary

The combination of crystal structures (see recent review [3]) and cryo-ET reconstructions have made great advances in this quest for the blueprint of the NPC. Further studies will yield insights into the entire NPC structure. One vital area will be to elucidate the entire structure of the POM region and its role in the placement of the NPC. Next is to solve the structures of the components in the nuclear basket and cytoplasmic Nups. A method that will shed light on the regions of NPC is a combination of cryo-ET with selected knockdowns or knockouts of specific Nups [40,43]. Thus far, these advances were possible because of past experimentation. Günter Blobel’s work extended beyond the ER into the nucleus where everything starts. The separation of the nuclei to the point of intact NE set the stage to dive into structural studies. Günter’s endeavors led the way to study the NPC by individual crystal structures leading to further innovation and insights into the blueprint of the NPC.