Cryo-electron Microscopy Reveals the Structure of the Nuclear Pore Complex

The nuclear pore complex (NPC) is a giant protein assembly that penetrates the double layers of the nuclear membrane. The overall structure of the NPC has approximately eightfold symmetry and is formed by approximately 30 nucleoporins. The great size and complexity of the NPC have hindered the study of its structure for many years until recent breakthroughs were achieved by integrating the latest high-resolution cryo-electron microscopy (cryo-EM), the emerging artificial intelligence-based modeling and all other available structural information from crystallography and mass spectrometry. Here, we review our latest knowledge of the NPC architecture and the history of its structural study from in vitro to in situ with progressively improved resolutions by cryo-EM, with a particular focus on the latest subnanometer-resolution structural studies. The future directions for structural studies of NPCs are also discussed.     

Toward a more complete 3-D structure of the nuclear pore complex

The nuclear pore complex (NPC) is a large supramolecular assembly embedded in the double-membraned nuclear envelope (NE) that plays a pivotal role in the exchange of macromolecules and particles between the nucleus and the cytoplasm. Applying various methods of sample preparation to Xenopus laevis whole nuclei and isolated NEs in combination with conventional transmission electron microscopy and digital image processing, we have characterized several distinct components of the NPC, including massive cytoplasmic and more tenuous nuclear rings, NPCs devoid of their cytoplasmic or both rings, and prominent "knobs" that protrude from the periphery of the NPC proper into the lumen of the NE. Moreover, by quick freezing/freeze drying/rotary metal shadowing isolated NEs, we have visualized two distinct types of NPC-associated filaments: (1) eight short, highly twisted filaments that project from the cytoplasmic ring and sometimes collapse into short cylinders; and (2) eight long, thin filaments that protrude from the nuclear ring and whose ends join to form a distal ring centered above the NPC such that the assembly resembles a "fishtrap." These nuclear fishtraps are sensitive to divalent cations: removal unfolds them and addition reforms them. The significance of these various structural components in terms of current NPC models is discussed, and the emerging asymmetry of the NPC relative to its nuclear and cytoplasmic face is stressed.     

Interactions between Mad1p and the nuclear transport machinery in the yeast Saccharomyces cerevisiae

In addition to its role in nucleocytoplasmic transport, the nuclear pore complex (NPC) acts as a docking site for proteins whose apparent primary cellular functions are unrelated to nuclear transport, including Mad1p and Mad2p, two proteins of the spindle assembly checkpoint (SAC) machinery. To understand this relationship, we have mapped domains of yeast Saccharomyces cerevisiae Mad1p that interact with the nuclear transport machinery, including further defining its interactions with the NPC. We showed that a Kap95p/Kap60p-dependent nuclear localization signal, positioned in the C-terminal third of Mad1p, is required for its efficient targeting to the NPC. At the NPC, Mad1p interacts with Nup53p and a presumed Nup60p/Mlp1p/Mlp2p complex through two coiled coil regions within its N terminus. When the SAC is activated, a portion of Mad1p is recruited to kinetochores through an interaction that is mediated by the C-terminal region of Mad1p and requires energy. We showed using photobleaching analysis that in nocodazole-arrested cells Mad1p rapidly cycles between the Mlp proteins and kinetochores. Our further analysis also showed that only the C terminus of Mad1p is required for SAC function and that the NPC, through Nup53p, may act to regulate the duration of the SAC response.     

Structure of the nuclear pore complex goes atomic

The nuclear pore complex (NPC) is a supra-molecular assembly that mediates substance and information flow across the nuclear envelope (NE). Due to its extraordinary size and complexity, the NPC remains one of the most challenging tasks in structural elucidation at atomic resolution. Recent breakthroughs in cryo-electron microscopy (cryo-EM) reconstruction, Machine Learning empowered structure prediction and biochemical reconstitution have combined to yield molecular models of the NPC at unprecedented accuracy. Furthermore, in cellulo cryo-electron tomography (cryo-ET) structures reveal substantial structural dynamics of the NPC. These advances shed light on the organizational principles and functions of the NPC.     

Systematic deletion and mitotic localization of the nuclear pore complex proteins of Aspergillus nidulans

To define the extent of the modification of the nuclear pore complex (NPC) during Aspergillus nidulans closed mitosis, a systematic analysis of nuclear transport genes has been completed. Thirty genes have been deleted defining 12 nonessential and 18 essential genes. Several of the nonessential deletions caused conditional phenotypes and self-sterility, whereas deletion of some essential genes caused defects in nuclear structure. Live cell imaging of endogenously tagged NPC proteins (Nups) revealed that during mitosis 14 predicted peripheral Nups, including all FG repeat Nups, disperse throughout the cell. A core mitotic NPC structure consisting of membrane Nups, all components of the An-Nup84 subcomplex, An-Nup170, and surprisingly, An-Gle1 remained throughout mitosis. We propose this minimal mitotic NPC core provides a conduit across the nuclear envelope and acts as a scaffold to which dispersed Nups return during mitotic exit. Further, unlike other dispersed Nups, An-Nup2 locates exclusively to mitotic chromatin, suggesting it may have a novel mitotic role in addition to its nuclear transport functions. Importantly, its deletion causes lethality and defects in DNA segregation. This work defines the dramatic changes in NPC composition during A. nidulans mitosis and provides insight into how NPC disassembly may be integrated with mitosis.     

Nucleoporin domain topology is linked to the transport status of the nuclear pore complex

Nuclear pore complexes (NPCs) facilitate macromolecular exchange between the nucleus and cytoplasm of eukaryotic cells. The vertebrate NPC is composed of approximately 30 different proteins (nucleoporins), of which around one third contain phenylalanine-glycine (FG)-repeat domains that are thought to mediate the main interaction between the NPC and soluble transport receptors. We have recently shown that the FG-repeat domain of Nup153 is flexible within the NPC, although this nucleoporin is anchored to the nuclear side of the NPC. By using domain-specific antibodies, we have now mapped the domain topology of Nup214 in Xenopus oocytes and in human somatic cells by immuno-EM. We have found that whereas Nup214 is anchored to the cytoplasmic side of the NPC via its N-terminal and central domain, its FG-repeat domain appears flexible, residing on both sides of the NPC. Moreover, the spatial distribution of the FG-repeat domains of both Nup153 and Nup214 shifts in a transport-dependent manner, suggesting that the location of FG-repeat domains within the NPC correlates with cargo/receptor interactions and that they concomitantly move with cargo through the central pore of the NPC.     

Cell cycle regulated transport controlled by alterations in the nuclear pore complex

Eukaryotic cells have developed mechanisms for regulating the nuclear transport of macromolecules that control various cellular events including movement through defined stages of the cell cycle. In yeast cells, where the nuclear envelope remains intact throughout the cell cycle, these transport regulatory mechanisms must also function during mitosis. We have uncovered a mechanism for regulating transport that is controlled by M phase specific molecular rearrangements in the nuclear pore complex (NPC). These changes allow a transport inhibitory nucleoporin, Nup53p, to bind the karyopherin Kap121p specifically during mitosis, slowing its movement through the NPC and inducing cargo release. Yeast strains that possess defects in the function of Kap121p or the fidelity of the inhibitory pathway are delayed in mitosis. We propose that fluctuations in Kap121p transport mediated by the NPC contribute to controlling the subcellular distribution of molecules that direct progression through mitosis.     

Nup180, a novel nuclear pore complex protein localizing to the cytoplasmic ring and associated fibrils

Using an autoimmune serum from a patient with overlap connective tissue disease we have identified by biochemical and immunocytochemical approaches an evolutionarily conserved nuclear pore complex (NPC) protein with an estimated molecular mass of 180 kD and an isoelectric point of approximately 6.2 which we have designated as nup180. Extraction of isolated nuclear envelopes with 2 M urea and chromatography of the solubilized proteins on WGA-Sepharose demonstrated that nup180 is a peripheral membrane protein and does not react with WGA. Affinity-purified antibodies yielded a punctate immunofluorescent pattern of the nuclear surface of mammalian cells and stained brightly the nuclear envelope of cryosectioned Xenopus oocytes. Nuclei reconstituted in vitro in Xenopus egg extract were also stained in the characteristic punctate fashion. Immunogold EM localized nup180 exclusively to the cytoplasmic ring of NPCs and short fibers emanating therefrom into the cytoplasm. Antibodies to nup180 did not inhibit nuclear protein transport in vivo nor in vitro. Despite the apparent lack of involvement in NPC assembly or nucleocytoplasmic transport processes, the conservation of nup180 across species and its exclusive association with the NPC cytoplasmic ring suggests an important, though currently undefined function for this novel NPC protein.     

Localization of Pom121 to the inner nuclear membrane is required for an early step of interphase nuclear pore complex assembly

The nuclear pore complex (NPC) is a large protein assembly that mediates molecular trafficking between the cytoplasm and the nucleus. NPCs assemble twice during the cell cycle in metazoans: postmitosis and during interphase. In this study, using small interfering RNA (siRNA) in conjunction with a cell fusion-based NPC assembly assay, we demonstrated that pore membrane protein (Pom)121, a vertebrate-specific integral membrane nucleoporin, is indispensable for an early step in interphase NPC assembly. Functional domain analysis of Pom121 showed that its nuclear localization signals, which bind to importin β via importin α and likely function with RanGTP, play an essential role in targeting Pom121 to the interphase NPC. Furthermore, a region of Pom121 that interacts with the inner nuclear membrane (INM) and lamin B receptor was found to be crucial for its NPC targeting. Based on these findings and on evidence that Pom121 localizes at the INM in the absence of a complete NPC structure, we propose that the nuclear migration of Pom121 and its subsequent interaction with INM proteins are required to initiate interphase NPC assembly. Our data also suggest, for the first time, the importance of the INM as a seeding site for "prepores" during interphase NPC assembly.     

Nuclear side conformational changes in the nuclear pore complex following calcium release from the nuclear membrane

Changes in nuclear pore complex (NPC) structure are studied following treatments modifying the cisternal calcium levels located between the two lipid bilayers that together form the nuclear envelope. Since the NPC forms the only known passageway across the nuclear envelope, it plays a central role in nucleocytoplasmic transport. Understanding the origin of conformational changes that may affect this trafficking or modify cargo interactions with the NPC is, therefore, necessary to completely understand the function of these complex molecules. In previous studies on the cytoplasmic side of the nuclear envelope, a central mass was observed in the pore of the NPC and its location was shown to be sensitive to the cisternal calcium levels. Here we report atomic force microscopy (AFM) measurements on the nuclear side of the envelope, which also reveal a cisternal calcium dependence in the conformational state of the NPC. These measurements, made at the single nuclear pore level, reveal a displacement of the central mass towards the nuclear side of the membrane following treatments with adenophostin A, a specific agonist of calcium channels (inositol 1,4,5-trisphosphate (IP(3)) receptors) located in the nuclear envelope. We further demonstrate that these conformational changes are observed in nuclear pores lacking the basket structure while samples prepared in the presence of protease inhibitors retain baskets and block AFM measurements of the channel. While these measurements are unable to distinguish whether the central mass is cargo or an integral component of the NPC, its dose-dependent displacement with cisternal calcium levels does suggest links to transport or to changes in cargo interactions with the NPC. Taken together with previous measurements done on the cytoplasmic side of the nuclear envelope, these studies argue against a piston-like displacement of the central mass and instead suggest a more complicated mechanism. One possibility involves a concerted collapse of the NPC rings towards one another following cisternal calcium release, thus leading to the apparent emergence of the central mass from each side of the NPC.     

Rapid translocation of NTF2 through the nuclear pore of isolated nuclei and nuclear envelopes

The mechanism by which macromolecules are translocated through the nuclear pore complex (NPC) is little understood. However, recent measurements of nuclear transport in permeabilized cells showed that molecules binding to phenylalanine-glycine-rich repeats (FG repeats) in NPC proteins were translocated much faster through the NPC than molecules not interacting with FG repeats. We have studied that substrate preference of the NPC in isolated oocyte nuclei and purified nuclear envelopes by optical single transporter recording. NTF2, the transport receptor of RanGDP, was exported ~30 times faster than green fluorescent protein, an inert molecule of approximately the same size. The data confirm that restricted diffusion of inert molecules and facilitated transport of FG-repeat binding proteins are basic types of translocation through the NPC, demonstrating that the functional integrity of the NPC can be conserved in isolated nuclei and nuclear envelopes and thus opening new avenues to the analysis of nucleocytoplasmic transport.     

Posttranslational marks control architectural and functional plasticity of the nuclear pore complex basket

The nuclear pore complex (NPC) serves as both the unique gate between the nucleus and the cytoplasm and a major platform that coordinates nucleocytoplasmic exchanges, gene expression, and genome integrity. To understand how the NPC integrates these functional constraints, we dissected here the posttranslational modifications of the nuclear basket protein Nup60 and analyzed how they intervene to control the plasticity of the NPC. Combined approaches highlight the role of monoubiquitylation in regulating the association dynamics of Nup60 and its partner, Nup2, with the NPC through an interaction with Nup84, a component of the Y complex. Although major nuclear transport routes are not regulated by Nup60 modifications, monoubiquitylation of Nup60 is stimulated upon genotoxic stress and regulates the DNA-damage response and telomere repair. Together, these data reveal an original mechanism contributing to the plasticity of the NPC at a molecular-organization and functional level.     

Interactions and three-dimensional localization of a group of nuclear pore complex proteins

We have used antibodies directed against a number of nuclear pore complex (NPC) proteins to determine their mutual interactions and location within the three-dimensional structure of the NPC. A monoclonal antibody, termed QE5, recognized three NPC polypeptides, p250, NUP153, and p62 on Western blots, and labeled the nuclear envelope of several cultured cell lines by immunofluorescence microscopy. These three polypeptides contained O-linked N- acetylglucosamine residues and were released from the NPC by detergent/high-salt treatment as discrete high molecular weight complexes. p250 was found in association with a novel 75 kD protein, NUP153 was released as a homo-oligomer of about 1 megadalton, and p62 was associated with polypeptides of 58 and 54 kD (previously reported by Finlay, D. R., E. Meier, P. Bradley, J. Horecka, and D. J. Forbes. 1991. J. Cell Biol. 114:169-183). p75, p58, and p54 were not galactosylated in vitro. Xenopus oocyte NEs were labeled with gold- conjugated QE5 and prepared for electron microscopy by quick freezing/freeze drying/rotary metal shadowing. This EM preparation method enabled us to more precisely localize the epitopes of this antibody to the cytoplasmic filaments and the nuclear basket of the NPC. Since QE5 recognizes three O-linked NPC glycoproteins, its labeling was compared with that of the lectin wheat germ agglutinin which recognizes O-linked N-acetylglucosamine moieties. The two probes were found to yield similar, although not identical, distributions of label. To identify the individual proteins with particular NPC components, we have used an anti-peptide antibody against NUP153 and a monospecific anti-p250 polyclonal antibody. Labeling with these two antibodies has documented that NUP153 is a constituent of the nuclear basket with at least one of its epitopes residing in its terminal ring, whereas p250 is a constituent of the cytoplasmic filaments.     

Phosphomimetic mutation of the mitotically phosphorylated serine 1880 compromises the interaction of the transmembrane nucleoporin gp210 with the nuclear pore complex

The nuclear pore complexes (NPCs) reversibly disassemble and reassemble during mitosis. Disassembly of the NPC is accompanied by phosphorylation of many nucleoporins although the function of this is not clear. It was previously shown that in the transmembrane nucleoporin gp210 a single serine residue at position 1880 is specifically phosphorylated during mitosis. Using amino acid substitution combined with live cell imaging, time-lapse microscopy and FRAP, we investigated the role of serine 1880 in binding of gp210 to the NPC in vivo. An alanine substitution mutant (S1880A) was significantly more dynamic at the NPC compared to the wild-type protein, suggesting that serine 1880 is important for binding of gp210 to the NPC. Moreover a glutamate substitution (S1880E) closely mimicking phosphorylated serine specifically interfered with incorporation of gp210 into the NPC and compromised its post-mitotic recruitment to the nuclear envelope of daughter nuclei. Our findings are consistent with the idea that mitotic phosphorylation acts to dissociate gp210 from the structural elements of the NPC.     

Domain topology of nucleoporin Nup98 within the nuclear pore complex

Nuclear pore complexes (NPCs) facilitate selective transport of macromolecules across the nuclear envelope in interphase eukaryotic cells. NPCs are composed of roughly 30 different proteins (nucleoporins) of which about one third are characterized by the presence of phenylalanine-glycine (FG) repeat domains that allow the association of soluble nuclear transport receptors with the NPC. Two types of FG (FG/FxFG and FG/GLFG) domains are found in nucleoporins and Nup98 is the sole vertebrate nucleoporin harboring the GLFG-type repeats. By immuno-electron microscopy using isolated nuclei from Xenopus oocytes we show here the localization of distinct domains of Nup98. We examined the localization of the C- and N-terminal domain of Nup98 by immunogold-labeling using domain-specific antibodies against Nup98 and by expressing epitope tagged versions of Nup98. Our studies revealed that anchorage of Nup98 to NPCs through its C-terminal autoproteolytic domain occurs in the center of the NPC, whereas its N-terminal GLFG domain is more flexible and is detected at multiple locations within the NPC. Additionally, we have confirmed the central localization of Nup98 within the NPC using super resolution structured illumination fluorescence microscopy (SIM) to position Nup98 domains relative to markers of cytoplasmic filaments and the nuclear basket. Our data support the notion that Nup98 is a major determinant of the permeability barrier of NPCs.     

Fluorescence Anisotropy Reveals Order and Disorder of Protein Domains in the Nuclear Pore Complex

We present a new approach for studying individual protein domains within the nuclear pore complex (NPC) using fluorescence polarization microscopy. The NPC is a large macromolecular complex, the size and complexity of which presents experimental challenges. Using fluorescence anisotropy and exploiting the symmetry of the NPC and its organization in the nuclear envelope, we have resolved order and disorder of individual protein domains. Fluorescently tagging specific domains of individual nucleoporins revealed both rigid and flexible domains: the tips of the FG domains are disordered, whereas the NPC-anchored domains are ordered. Our technique allows the collection of structural information in vivo, providing the ability to probe the organization of protein domains within the NPC. This has particular relevance for the FG domain nucleoporins, which are crucial for nucleocytoplasmic transport.     

The human homologue of yeast CRM1 is in a dynamic subcomplex with CAN/Nup214 and a novel nuclear pore component Nup88.

The oncogenic nucleoporin CAN/Nup214 is essential in vertebrate cells. Its depletion results in defective nuclear protein import, inhibition of messenger RNA export and cell cycle arrest. We recently found that CAN associates with proteins of 88 and 112 kDa, which we have now cloned and characterized. The 88 kDa protein is a novel nuclear pore complex (NPC) component, which we have named Nup88. Depletion of CAN from the NPC results in concomitant loss of Nup88, indicating that the localization of Nup88 to the NPC is dependent on CAN binding. The 112 kDa protein is the human homologue of yeast CRM1, a protein known to be required for maintenance of correct chromosome structure. This human CRM1 (hCRM1) localized to the NPC as well as to the nucleoplasm. Nuclear overexpression of the FG-repeat region of CAN, containing its hCRM1-interaction domain, resulted in depletion of hCRM1 from the NPC. In CAN-/- mouse embryos lacking CAN, hCRM1 remained in the nuclear envelope, suggesting that this protein can also bind to other repeat-containing nucleoporins. Lastly, hCRM1 shares a domain of significant homology with importin-beta, a cytoplasmic transport factor that interacts with nucleoporin repeat regions. We propose that hCRM1 is a soluble nuclear transport factor that interacts with the NPC.     

Nup153 affects entry of messenger and ribosomal ribonucleoproteins into the nuclear basket during export

A specific messenger ribonucleoprotein (RNP) particle, Balbiani ring (BR) granules in the dipteran Chironomus tentans, can be visualized during passage through the nuclear pore complex (NPC). We have now examined the transport through the nuclear basket preceding the actual translocation through the NPC. The basket consists of eight fibrils anchored to the NPC core by nucleoprotein Nup153. On nuclear injection of anti-Nup153, the transport of BR granules is blocked. Many granules are retained on top of the nuclear basket, whereas no granules are seen in transit through NPC. Interestingly, the effect of Nup153 seems distant from the antibody-binding site at the base of the basket. We conclude that the entry into the basket is a two-step process: an mRMP first binds to the tip of the basket fibrils and only then is it transferred into the basket by a Nup153-dependent process. It is indicated that ribosomal subunits follow a similar pathway.