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.     

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.     

Structural Analysis of a Metazoan Nuclear Pore Complex Reveals a Fused Concentric Ring Architecture

The sole gateway for molecular exchange between the cytoplasm and the nucleus is the nuclear pore complex (NPC). This large supramolecular assembly mediates transport of cargo into and out of the nucleus and fuse the inner and outer nuclear membranes to form an aqueous translocation channel. The NPC is composed of eight proteinaceous asymmetric units forming a pseudo-8-fold symmetric passage. Due to its shear size, complexity, and plastic nature, dissecting the high-resolution three-dimensional structure of the NPC in its hydrated state is a formidable challenge. Toward this goal, we applied cryo-electron tomography to spread nuclear envelopes from Xenopus oocytes. To compensate for perturbations of the 8-fold symmetry of individual NPCs, we performed symmetry-independent asymmetric unit averaging of three-dimensional tomographic NPC volumes to eventually yield a refined model at 6.4 nm resolution. This approach revealed novel structural features, particularly in the spoke-ring complex and luminal domains. Fused concentric ring architecture of the spoke-ring complex was found along the translocation channel. Additionally, a comparison of the refined Xenopus model to that of its Dictyostelium homologue yielded similar pore diameters at the level of the three canonical rings, although the Xenopus NPC was found to be 30% taller than the Dictyostelium pore. This discrepancy is attributed primarily to the relatively low homology and different organization of some nucleoporins in the Dictyostelium genome as compared to that of vertebrates. Nevertheless, the experimental conditions impose a preferred axial orientation of the NPCs within spread Xenopus oocyte nuclear envelopes. This may at least in part explain the increased height of the reconstructed vertebrate NPCs compared to those obtained from tomographic reconstruction of intact Dictyostelium nuclei.     

The Nuclear Pore Complex and Nuclear Transport

Internal membrane bound structures sequester all genetic material in eukaryotic cells. The most prominent of these structures is the nucleus, which is bounded by a double membrane termed the nuclear envelope (NE). Though this NE separates the nucleoplasm and genetic material within the nucleus from the surrounding cytoplasm, it is studded throughout with portals called nuclear pore complexes (NPCs). The NPC is a highly selective, bidirectional transporter for a tremendous range of protein and ribonucleoprotein cargoes. All the while the NPC must prevent the passage of nonspecific macromolecules, yet allow the free diffusion of water, sugars, and ions. These many types of nuclear transport are regulated at multiple stages, and the NPC carries binding sites for many of the proteins that modulate and modify the cargoes as they pass across the NE. Assembly, maintenance, and repair of the NPC must somehow occur while maintaining the integrity of the NE. Finally, the NPC appears to be an anchor for localization of many nuclear processes, including gene activation and cell cycle regulation. All these requirements demonstrate the complex design of the NPC and the integral role it plays in key cellular processes.Taxonomically speaking, all life on earth falls into one of two fundamental groups, the prokaryotes and the eukaryotes. The prokaryotes, the first group to evolve, are single cell organisms bounded by a single membrane. About 1.5 billion years later, a series of evolutionary innovations led to the emergence of eukaryotes. Eukaryotes have multiple inner membrane structures that allow for compartmentalization within the cell, and therefore differentiation of the cell and regulation within it. Ultimately, the greater cellular complexity of eukaryotes allowed them to adopt a multicellular lifestyle, as seen in the plants, fungi and animals of today (reviewed in Field and Dacks 2009).Internal membrane bound structures sequester all genetic material in eukaryotic cells. The most prominent of these structures, which gives the eukaryotes their Greek-rooted name, is the nucleus—the central “kernel” (gr. “karyo-”) of the cell. The nucleus is bounded by a double membrane termed the nuclear envelope (NE), which separates the nucleoplasm and genetic material from the surrounding cytoplasm. However the genetic material in the nucleus is not totally isolated from the rest of the cell. Studded throughout the NE are portals called nuclear pore complexes (NPCs). The NPC is a highly selective, bidirectional transporter for a tremendous range of cargoes. Going into the nucleus, these cargoes include inner nuclear membrane proteins and all the proteins in the nucleoplasm. Going out are RNA-associated proteins that are assembled into ribosomal subunits or messenger ribonucleoproteins (mRNPs). Once transported, the NPC must ensure these cargos are retained in their respective nuclear and cytoplasmic compartments. All the while the NPC must prevent the passage of nonspecific macromolecules, yet allow the free diffusion of water, sugars, and ions. These many types of nuclear transport are regulated at multiple stages, providing a powerful extra level of cellular control that is not necessary in prokaryotes. Assembly, maintenance, and repair of the NPC must somehow occur while maintaining the integrity of the NE. Finally, the NPC appears to be an anchor for localization of many nuclear processes, including gene activation and cell cycle regulation (reviewed in Ahmed and Brickner 2007; Hetzer and Wente 2009). All these requirements demonstrate the complex design of the NPC and the integral role it plays in key cellular processes.     

Mapping the Polarity Interactome

Interactions between proteins are an essential part of biology, and the desire to identify these interactions has led to the development of numerous technologies to systematically map protein–protein interactions at a large scale. As in most cellular processes, protein interactions are central to the control of cell polarity, and a full understanding of polarity will require comprehensive knowledge of the protein interactions involved. At its core, cell polarity is established through carefully regulated mutually inhibitory interactions between several groups of cortical proteins. While several interactions have been identified, the dynamics and molecular mechanisms that control these interactions are not well understood. Cell polarity also needs to be integrated with cellular processes including junction formation, cytoskeletal organization, organelle positioning, protein trafficking, and functional specialization of membrane domains. Moreover, polarized cells need to respond to external cues that coordinate polarity at the tissue level. Identifying the protein–protein interactions responsible for integrating polarity with all of these processes remains a major challenge, in part because the mechanisms of polarity control vary in different contexts and with developmental times. Because of their unbiased nature, systematic large-scale protein–protein interaction mapping approaches can be particularly helpful to identify such mechanisms. Here, we discuss methods commonly used to generate proteome-wide interactome maps, with an emphasis on advances in our understanding of cell polarity that have been achieved through application of such methods.     

核孔复合体的动态组装

核孔是介导所有大分子入核出核的唯一通道。在整个生命活动中,核孔复合体的组成蛋白总是处于动态变化中。核孔复合体的动态组装改变了核质转运状态,并最终改变了细胞的功能。     

Structural Characterization by Cross-linking Reveals the Detailed Architecture of a Coatomer-related Heptameric Module from the Nuclear Pore Complex

Most cellular processes are orchestrated by macromolecular complexes. However, structural elucidation of these endogenous complexes can be challenging because they frequently contain large numbers of proteins, are compositionally and morphologically heterogeneous, can be dynamic, and are often of low abundance in the cell. Here, we present a strategy for the structural characterization of such complexes that has at its center chemical cross-linking with mass spectrometric readout. In this strategy, we isolate the endogenous complexes using a highly optimized sample preparation protocol and generate a comprehensive, high-quality cross-linking dataset using two complementary cross-linking reagents. We then determine the structure of the complex using a refined integrative method that combines the cross-linking data with information generated from other sources, including electron microscopy, X-ray crystallography, and comparative protein structure modeling. We applied this integrative strategy to determine the structure of the native Nup84 complex, a stable hetero-heptameric assembly (∼600 kDa), 16 copies of which form the outer rings of the 50-MDa nuclear pore complex (NPC) in budding yeast. The unprecedented detail of the Nup84 complex structure reveals previously unseen features in its pentameric structural hub and provides information on the conformational flexibility of the assembly. These additional details further support and augment the protocoatomer hypothesis, which proposes an evolutionary relationship between vesicle coating complexes and the NPC, and indicates a conserved mechanism by which the NPC is anchored in the nuclear envelope.Macromolecular complexes are the building blocks that drive virtually all cellular and biological processes. In each eukaryotic cell, there exist many hundreds of these protein complexes (1–3), the majority of which are still poorly understood in terms of their structures, dynamics, and functions. The classical structure determination approaches of nuclear magnetic resonance, X-ray crystallography, and electron microscopy (EM)¹ remain challenged in attempts to determine the high-resolution structures of large, dynamic, and flexible complexes in a living cell (4). Thus, additional robust and rapid methods are needed, ideally working in concert with these classical approaches, to allow the greatest structural and functional detail in characterizations of macromolecular assemblies.Integrative modeling approaches help address this need, providing powerful tools for determining the structures of endogenous protein complexes (5, 6) by relying on the collection of an extensive experimental dataset, preferably coming from diverse sources (both classical and new) and different levels of resolution. These data are translated into spatial restraints that are used to calculate an ensemble of structures by satisfying the restraints, which in turn can be analyzed and assessed to determine precision and estimate accuracy (5, 7). A major advantage of this approach is that it readily integrates structural data from different methods and a wide range of resolutions, spanning from a few angstroms to dozens of nanometers. This strategy has been successfully applied to a number of protein complexes (8–16). However, it has proven difficult and time-consuming to generate a sufficient number of accurate spatial restraints to enable high-resolution structural characterization; thus, the determination of spatial restraints currently presents a major bottleneck for widespread application of this integrative approach. An important step forward is therefore the development of technologies for collecting high-resolution and information-rich spatial restraints in a rapid and efficient manner, ideally from endogenous complexes isolated directly from living cells.Chemical cross-linking with mass spectrometric readout (CX-MS) (17, 18) has recently emerged as an enabling approach for obtaining residue-specific restraints on the structures of proteins and protein complexes (19–25). In a CX-MS experiment, the purified protein complex is chemically conjugated by a functional group-specific cross-linker, and this is followed by proteolytic digestion and analysis of the resulting peptide mixture by mass spectrometry (MS). However, because of the complexity of the peptide mixtures and low abundance of most of the informative cross-linked species, comprehensive detection of these cross-linked peptides has proven challenging. This challenge increases substantially in studies of endogenous complexes of modest to low abundance, which encompass the great majority of assemblies in any cell (26, 27). In addition, because most cross-linkers used for CX-MS target primary amines, comprehensive detection of cross-links is further limited by the occurrence of lysine, which constitutes only ∼6% of protein sequences, although these lysine residues are generally present on protein surfaces. The use of cross-linkers with different chemistries and reactive groups, especially toward abundant residues, would increase the cross-linking coverage and could be of great help for downstream structural analysis (28).The nuclear pore complex (NPC) is one of the largest protein assemblies in the cell and is the sole mediator of macromolecular transport between the nucleus and the cytoplasm. The NPC is formed by multiple copies of ∼30 different proteins termed nucleoporins (Nups) that are assembled into discrete subcomplexes (8, 29). These building blocks are arranged into eight symmetrical units called spokes that are radially connected to form several concentric rings. The outer rings of the NPC are mainly formed by the Nup84 complex (a conserved complex, termed the Nup107–Nup160 complex in vertebrates). In budding yeast, the Nup84 complex is an essential, Y-shaped assembly of ∼600 kDa that is formed by seven nucleoporins (Nup133, Nup120, Nup145c, Nup85, Nup84, Seh1, and Sec13 in Saccharomyces cerevisiae) (30). The Nup84 complex has been shown to have a common evolutionary origin with vesicle coating complexes (VCCs), such as COPII, COPI, and clathrin (31, 32), but the evolutionary relationships between these VCCs have not been fully delineated. The Nup84 complex has been extensively characterized; several of its components have been analyzed via X-ray crystallography (33, 34), its overall shape has been defined by means of negative-stain electron microscopy (14, 30, 35, 36), and recently efforts were made to define the protein contacts in the Nup84 complex via CX-MS in humans (35) and a thermophilic fungus (37). Finally, we recently used an integrative modeling approach combining domain mapping, negative-stain electron microscopy (38), and publicly available crystal structures to generate a medium-resolution map of the native Nup84 complex (14). However, despite all these efforts, the fine features of the complex, and in particular the intricate domain orientations and contacts within the complex''s hub, remain poorly described.To address these issues, we present here an optimized CX-MS strategy for robust and in-depth structural characterization of endogenous protein complexes. To test the strategy, we generated a comprehensive high-quality CX-MS dataset on the endogenous Nup84 complex using two complementary cross-linkers, disuccinimidyl suberate (DSS) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). Using the resulting cross-linking restraints together with other sources of information (including electron microscopy, X-ray crystallography, and comparative modeling), we computed a detailed structure of the endogenous Nup84 complex. In addition to providing the overall architecture of the yeast Nup84 complex, the resulting structure reveals the previously unknown architecture of its pentameric structural hub. Our results demonstrate that the present approach provides a robust framework for the standardized generation and use of CX-MS spatial restraints toward the structural characterization of endogenous protein complexes.     

Energetics of Transport through the Nuclear Pore Complex

Molecular transport across the nuclear envelope in eukaryotic cells is solely controlled by the nuclear pore complex (NPC). The NPC provides two types of nucleocytoplasmic transport: passive diffusion of small molecules and active chaperon-mediated translocation of large molecules. It has been shown that the interaction between intrinsically disordered proteins that line the central channel of the NPC and the transporting cargoes is the determining factor, but the exact mechanism of transport is yet unknown. Here, we use coarse-grained molecular dynamics simulations to quantify the energy barrier that has to be overcome for molecules to pass through the NPC. We focus on two aspects of transport. First, the passive transport of model cargo molecules with different sizes is studied and the size selectivity feature of the NPC is investigated. Our results show that the transport probability of cargoes is significantly reduced when they are larger than ∼5 nm in diameter. Secondly, we show that incorporating hydrophobic binding spots on the surface of the cargo effectively decreases the energy barrier of the pore. Finally, a simple transport model is proposed which characterizes the energy barrier of the NPC as a function of diameter and hydrophobicity of the transporting particles.     

Nuclear Pore Complex Structure in Birds

The nuclear envelope consists of two parallel membranes enclosing an aqueous lumen. In places there are pores in both membranes at which the two membranes are joined. Within these pores reside the nuclear pore complexes. The current structural models of the nuclear pore complex have been derived from a number of studies using different electron microscopical techniques. Recently, using surface imaging techniques such as field emission in-lens scanning electron microscopy, novel structures have been identified, particularly at the periphery of the structure, most notably the nucleoplasmic basket. One limitation of the current models is that they are based almost entirely on nuclear envelopes isolated from amphibian oocytes and a pressing question is whether this structure is the same in other organisms and tissues. Here we have studied the structure of nuclear envelopes isolated from bird oocytes. We show that the overall structure is remarkably conserved. In particular, recently discovered peripheral structures appear very similar. We see variations in basket conformation but believe that this is related to the functional states of individual pore complexes.     

Adaptation to High Ethanol Reveals Complex Evolutionary Pathways

Tolerance to high levels of ethanol is an ecologically and industrially relevant phenotype of microbes, but the molecular mechanisms underlying this complex trait remain largely unknown. Here, we use long-term experimental evolution of isogenic yeast populations of different initial ploidy to study adaptation to increasing levels of ethanol. Whole-genome sequencing of more than 30 evolved populations and over 100 adapted clones isolated throughout this two-year evolution experiment revealed how a complex interplay of de novo single nucleotide mutations, copy number variation, ploidy changes, mutator phenotypes, and clonal interference led to a significant increase in ethanol tolerance. Although the specific mutations differ between different evolved lineages, application of a novel computational pipeline, PheNetic, revealed that many mutations target functional modules involved in stress response, cell cycle regulation, DNA repair and respiration. Measuring the fitness effects of selected mutations introduced in non-evolved ethanol-sensitive cells revealed several adaptive mutations that had previously not been implicated in ethanol tolerance, including mutations in PRT1, VPS70 and MEX67. Interestingly, variation in VPS70 was recently identified as a QTL for ethanol tolerance in an industrial bio-ethanol strain. Taken together, our results show how, in contrast to adaptation to some other stresses, adaptation to a continuous complex and severe stress involves interplay of different evolutionary mechanisms. In addition, our study reveals functional modules involved in ethanol resistance and identifies several mutations that could help to improve the ethanol tolerance of industrial yeasts.     

Reconstructing the Complex Evolutionary History of Hepatitis B Virus

A detailed analysis of the evolutionary history of hepatitis B virus (HBV) was undertaken using 39 mammalian hepadnaviruses for which complete genome sequences were available, including representatives of all six human genotypes, as well as a large sample of small S gene sequences. Phylogenetic trees of these data were ambiguous, supporting no single place of origin for HBV, and depended heavily on the underlying model of DNA substitution. In some instances genotype F, predominant in the Americas, was the first to diverge, suggesting that the virus arose in the New World. In other trees, however, sequences from genotype B, prevalent in East Asia, were the most divergent. An attempt was also made to determine the rate of nucleotide substitution in the C open reading frame and then to date the origin of HBV. However, no relationship between time and number of substitutions was found in two independent data sets, indicating that a reliable molecular clock does not exist for these data. Both the pattern and the rate of nucleotide substitution are therefore complex phenomena in HBV and hinder any attempt to reconstruct the past spread of this virus. Received: 5 December 1998 / Accepted: 23 February 1999     

Conserved Spatial Organization of FG Domains in the Nuclear Pore Complex

Selective transport through the nuclear pore complex (NPC) requires nucleoporins containing natively unfolded phenylalanine-glycine (FG) domains. Several differing models for their dynamics within the pore have been proposed. We characterize the behavior of the FG nucleoporins in vivo using polarized fluorescence microscopy. Using nucleoporins tagged with green fluorescent protein along their FG domains, we show that some of these proteins are ordered, indicating an overall orientational organization within the NPC. This orientational ordering of the FG domains depends on their specific context within the NPC, but is independent of active transport and cargo load. For most nups, behavior does not depend on the FG motifs. These data support a model whereby local geometry constrains the orientational organization of the FG nups. Intriguingly, homologous yeast and mammalian proteins show conserved behavior, suggesting functional relevance. Our findings have implications for mechanistic models of NPC transport.     

Quantitative Analysis of Membrane Protein Transport Across the Nuclear Pore Complex

Nuclear transport of the Saccharomyces cerevisiae membrane proteins Src1/Heh1 and Heh2 across the NPC is facilitated by a long intrinsically disordered linker between the nuclear localization signal (NLS) and the transmembrane domain. The import of reporter proteins derived from Heh2 is dependent on the FG‐Nups in the central channel, and the linker can position the transport factor‐bound NLS in the vicinity of the FG‐Nups in the central channel, while the transmembrane segment resides in the pore membrane. Here, we present a quantitative analysis of karyopherin‐mediated import and passive efflux of reporter proteins derived from Heh2, including data on the mobility of the reporter proteins in different membrane compartments. We show that membrane proteins with extralumenal domains up to 174 kDa, terminal to the linker and NLS, passively leak out of the nucleus via the NPC, albeit at a slow rate. We propose that also during passive efflux, the unfolded linker facilitates the passage of extralumenal domains through the central channel of the NPC .