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The nucleus contains the cell's genetic material, which directs cellular activity via gene regulation. The physical barrier of the nuclear envelope needs to be permeable to a variety of macromolecules and signals. The most prominent gateways for the transport of macromolecules are the nuclear pore complexes (NPCs). The NPC is the largest multiprotein complex in the cell, and is composed of multiple copies of ∼30 different proteins called nucleoporins. Although much progress has been made in dissecting the NPC structure in vertebrates and yeast, the molecular architecture and physiological function of nucleoporins in plants remain poorly understood. In this review, we summarize the current knowledge regarding the plant NPC proteome and address structural and functional aspects of plant nucleoporins, which support the fundamental cellular machinery.

HIV-1 infection requires nuclear entry of the viral genome. Previous evidence suggests that this entry proceeds through nuclear pore complexes (NPCs), with the 120 × 60 nm capsid squeezing through an approximately 60-nm-wide central channel 1 and crossing the permeability barrier of the NPC. This barrier can be described as an FG phase 2 that is assembled from cohesively interacting phenylalanine–glycine (FG) repeats 3 and is selectively permeable to cargo captured by nuclear transport receptors (NTRs). Here we show that HIV-1 capsid assemblies can target NPCs efficiently in an NTR-independent manner and bind directly to several types of FG repeats, including barrier-forming cohesive repeats. Like NTRs, the capsid readily partitions into an in vitro assembled cohesive FG phase that can serve as an NPC mimic and excludes much smaller inert probes such as mCherry. Indeed, entry of the capsid protein into such an FG phase is greatly enhanced by capsid assembly, which also allows the encapsulated clients to enter. Thus, our data indicate that the HIV-1 capsid behaves like an NTR, with its interior serving as a cargo container. Because capsid-coating with trans -acting NTRs would increase the diameter by 10 nm or more, we suggest that such a ‘self-translocating’ capsid undermines the size restrictions imposed by the NPC scaffold, thereby bypassing an otherwise effective barrier to viral infection. The HIV-1 capsid behaves like a nuclear transport receptor entering and traversing an FG phase, with its interior serving as a cargo container, bypassing an otherwise effective barrier to viral infection.

Nuclear pore complexes (NPCs) mediate nucleocytoplasmic exchange, which is essential for eukaryotes. Mutations in the central scaffolding components of NPCs are associated with genetic diseases, but how they manifest only in specific tissues remains unclear. This is exemplified in Nup133-deficient mouse embryonic stem cells, which grow normally during pluripotency, but differentiate poorly into neurons. Here, using an innovative in situ structural biology approach, we show that Nup133 −/− mouse embryonic stem cells have heterogeneous NPCs with non-canonical symmetries and missing subunits. During neuronal differentiation, Nup133-deficient NPCs frequently disintegrate, resulting in abnormally large nuclear envelope openings. We propose that the elasticity of the NPC scaffold has a protective function for the nuclear envelope and that its perturbation becomes critical under conditions that impose an increased mechanical load onto nuclei. Taniguchi et al. structurally analyse nuclear pore complex architecture in situ during differentiation, which is associated with mechanical constraints on the nuclear envelope. They link nuclear pore complex elasticity to nuclear envelope integrity in differentiation.

The most comprehensive architectural model to date of the nuclear pore complex reveals previously unknown local interactions, and a role for nucleoporin 358 in Y-complex oligomerization. A detailed model of the human nuclear pore complex The transport of materials between the nucleus and cytoplasm in eukaryotic cells is controlled by the nuclear pore complex. Martin Beck and colleagues have used cryo-electron tomography, mass spectrometry and other analyses to generate the most comprehensive architectural model of the human nuclear pore complex to date. The model reveals previously unknown local interactions, and a role for the transport channel nucleoporin 358 (Nup358) in mediating oligomerization of the Y-complex within the nuclear pore complex. Nuclear pore complexes are fundamental components of all eukaryotic cells that mediate nucleocytoplasmic exchange. Determining their 110-megadalton structure imposes a formidable challenge and requires in situ structural biology approaches. Of approximately 30 nucleoporins (Nups), 15 are structured and form the Y and inner-ring complexes. These two major scaffolding modules assemble in multiple copies into an eight-fold rotationally symmetric structure that fuses the inner and outer nuclear membranes to form a central channel of ~60 nm in diameter 1 . The scaffold is decorated with transport-channel Nups that often contain phenylalanine-repeat sequences and mediate the interaction with cargo complexes. Although the architectural arrangement of parts of the Y complex has been elucidated, it is unclear how exactly it oligomerizes in situ . Here we combine cryo-electron tomography with mass spectrometry, biochemical analysis, perturbation experiments and structural modelling to generate, to our knowledge, the most comprehensive architectural model of the human nuclear pore complex to date. Our data suggest previously unknown protein interfaces across Y complexes and to inner-ring complex members. We show that the transport-channel Nup358 (also known as Ranbp2) has a previously unanticipated role in Y-complex oligomerization. Our findings blur the established boundaries between scaffold and transport-channel Nups. We conclude that, similar to coated vesicles, several copies of the same structural building block—although compositionally identical—engage in different local sets of interactions and conformations.

The nuclear pore complex (NPC) is the bidirectional gate that mediates the exchange of macromolecules or their assemblies between nucleus and cytoplasm 1 – 3 . The assembly intermediates of the ribosomal subunits, pre-60S and pre-40S particles, are among the largest cargoes of the NPC and the export of these gigantic ribonucleoproteins requires numerous export factors 4 , 5 . Here we report the cryo-electron microscopy structure of native pre-60S particles trapped in the channel of yeast NPCs. In addition to known assembly factors, multiple factors with export functions are also included in the structure. These factors in general bind to either the flexible regions or subunit interface of the pre-60S particle, and virtually form many anchor sites for NPC binding. Through interactions with phenylalanine-glycine (FG) repeats from various nucleoporins of NPC, these factors collectively facilitate the passage of the pre-60S particle through the central FG repeat network of the NPC. Moreover, in silico analysis of the axial and radial distribution of pre-60S particles within the NPC shows that a single NPC can take up to four pre-60S particles simultaneously, and pre-60S particles are enriched in the inner ring regions close to the wall of the NPC with the solvent-exposed surface facing the centre of the nuclear pore. Our data suggest a translocation model for the export of pre-60S particles through the NPC. We report the cryo-electron microscopy structure of native pre-60S particles trapped in the channel of the yeast nuclear pore complex, suggesting a translocation model for the export of pre-60S particles through the complex.

HIV can infect non-dividing cells because the viral capsid can overcome the selective barrier of the nuclear pore complex and deliver the genome directly into the nucleus 1 , 2 . Remarkably, the intact HIV capsid is more than 1,000 times larger than the size limit prescribed by the diffusion barrier of the nuclear pore 3 . This barrier in the central channel of the nuclear pore is composed of intrinsically disordered nucleoporin domains enriched in phenylalanine–glycine (FG) dipeptides. Through multivalent FG interactions, cellular karyopherins and their bound cargoes solubilize in this phase to drive nucleocytoplasmic transport 4 . By performing an in vitro dissection of the nuclear pore complex, we show that a pocket on the surface of the HIV capsid similarly interacts with FG motifs from multiple nucleoporins and that this interaction licences capsids to penetrate FG-nucleoporin condensates. This karyopherin mimicry model addresses a key conceptual challenge for the role of the HIV capsid in nuclear entry and offers an explanation as to how an exogenous entity much larger than any known cellular cargo may be able to non-destructively breach the nuclear envelope. Dissection of the nuclear pore complex provides a model in which the HIV capsid enters the nucleus through karyopherin mimicry, a mechanism likely to be conserved across other viruses.

Nuclear pore complexes (NPCs) consist of around 1000 protein subunits, are embedded in the membrane that surrounds the nucleus, and regulate transport between the nucleus and the cytoplasm. Although the overall shape of NPCs is known, the details of this macromolecular complex have been obscure. Now, Lin et al. have reconstituted the pore components, determined the interactions between them, and fitted them into a tomographic reconstruction. Kosinski et al. have provided an architectural map of the inner ring of the pore. Science , this issue pp. 10.1126/science.aaf1015 and 363 Reconstitution, spectroscopy, and crystallography allow the construction of a model of the human nuclear pore. The nuclear pore complex (NPC) controls the transport of macromolecules between the nucleus and cytoplasm, but its molecular architecture has thus far remained poorly defined. We biochemically reconstituted NPC core protomers and elucidated the underlying protein-protein interaction network. Flexible linker sequences, rather than interactions between the structured core scaffold nucleoporins, mediate the assembly of the inner ring complex and its attachment to the NPC coat. X-ray crystallographic analysis of these scaffold nucleoporins revealed the molecular details of their interactions with the flexible linker sequences and enabled construction of full-length atomic structures. By docking these structures into the cryoelectron tomographic reconstruction of the intact human NPC and validating their placement with our nucleoporin interactome, we built a composite structure of the NPC symmetric core that contains ~320,000 residues and accounts for ~56 megadaltons of the NPC’s structured mass. Our approach provides a paradigm for the structure determination of similarly complex macromolecular assemblies.

Nucleoporins rich in phenylalanine/glycine (FG) residues form the permeability barrier within the nuclear pore complex and are implicated in several pathological cellular processes, including oncogenic fusion condensates. The self-association of FG-repeat proteins and interactions between FG-repeats play a critical role in these activities by forming hydrogel-like structures. Here we show that mutation of specific FG repeats of Nup98 can strongly decrease the protein’s self-association capabilities. We further present a cryo-electron microscopy structure of a Nup98 peptide fibril with higher stability per residue compared with previous Nup98 fibril structures. The high-resolution structure reveals zipper-like hydrophobic patches which contain a GLFG motif and are less compatible for binding to nuclear transport receptors. The identified distinct molecular properties of different regions of the nucleoporin may contribute to spatial variations in the self-association of FG-repeats, potentially influencing transport processes through the nuclear pore. Here, the authors demonstrate that mutations in the FG repeats of Nup98 significantly reduce its self-association capabilities and present a cryoEM structure exhibiting higher stability per residue then previously observed, suggesting spatial variations in self-association.

The nuclear pore complex (NPC) mediates nucleocytoplasmic transport through the nuclear envelope. How the NPC assembles into this double membrane boundary has remained enigmatic. Here, we captured temporally staged assembly intermediates by correlating live cell imaging with high-resolution electron tomography and super-resolution microscopy. Intermediates were dome-shaped evaginations of the inner nuclear membrane (INM), that grew in diameter and depth until they fused with the flat outer nuclear membrane. Live and super-resolved fluorescence microscopy revealed the molecular maturation of the intermediates, which initially contained the nuclear and cytoplasmic ring component Nup107, and only later the cytoplasmic filament component Nup358. EM particle averaging showed that the evagination base was surrounded by an 8-fold rotationally symmetric ring structure from the beginning and that a growing mushroom-shaped density was continuously associated with the deforming membrane. Quantitative structural analysis revealed that interphase NPC assembly proceeds by an asymmetric inside-out extrusion of the INM. The nucleus is the compartment within our cells that contains most of our genetic material. It is separated from the rest of the cell by a boundary called the nuclear envelope, which consists of two layers of membrane. All transport in and out of the nucleus has to pass through channels in the envelope, formed by large protein assemblies called the nuclear pore complexes. Each nuclear pore complex is composed of multiple copies of over 30 different proteins termed nucleoporins and there are several hundred proteins per pore. Before a cell divides in two, the nucleus has to grow and new nuclear pore complexes must be assembled into the double membrane barrier of the nuclear envelope. The assembly process would require the two nuclear membranes to fuse. However, exactly how nuclear pore complexes are assembled has been controversially debated for over 15 years, because no one has directly observed any of the intermediate stages during the assembly process. Now, Otsuka et al. have captured images of the different steps involved in assembling a nuclear pore complex in a human cell. This was achieved by observing living human cells in which the nucleus was growing and then studying them using advanced techniques such as high-resolution three-dimensional electron tomography and super-resolution microscopy. Otsuka et al. saw dome-shaped bumps or protrusions in the inner nuclear membrane that grew wider and deeper until they fused with the flat outer nuclear membrane. A ring of proteins surrounded the base of these protrusions from the beginning, and the membrane was deformed by a mushroom-shaped collection of proteins. Analysis of the molecules involved in these stages showed that assembly intermediates initially contained nucleoporins that face into the nucleus, and only later were nucleoporins that face into the rest of the cell added to the complex. The discovery that nuclear pore complexes assemble via an inside-out mechanism in human cells provides a new conceptual framework to interpret existing genetic and biochemical data. The findings also provide a new approach to explore the assembly process in much more detail and ask how nuclear pores first evolved.

Nuclear pore complexes (NPCs) are proteinaceous assemblies of approximately 50 MDa that selectively transport cargoes across the nuclear envelope. To determine the molecular architecture of the yeast NPC, we collected a diverse set of biophysical and proteomic data, and developed a method for using these data to localize the NPC’s 456 constituent proteins (see the accompanying paper). Our structure reveals that half of the NPC is made up of a core scaffold, which is structurally analogous to vesicle-coating complexes. This scaffold forms an interlaced network that coats the entire curved surface of the nuclear envelope membrane within which the NPC is embedded. The selective barrier for transport is formed by large numbers of proteins with disordered regions that line the inner face of the scaffold. The NPC consists of only a few structural modules that resemble each other in terms of the configuration of their homologous constituents, the most striking of these being a 16-fold repetition of ‘columns’. These findings provide clues to the evolutionary origins of the NPC. Gatekeeper for the Nucleus The nuclear pore complex plays a crucial role in the cell, as gatekeeper for traffic between the cytoplasm and the interior of the nucleus. It is a large supramolecular complex made up of multiple copies of about 30 different proteins — 456 protein molecules in all. Cell biologists would love to know how each of the pore molecules are placed, but so far this has eluded conventional structural studies. Now, a new proteomics-based technique has provided a detailed view of the architecture of the yeast nuclear pore complex. Half of the complex is made of a core scaffold forming a network coating the surface of the nuclear envelope membrane within which the complex is embedded. The selective transport barrier is formed by the many proteins lining the inner face of the scaffold. Despite its size, there are only a few structural modules in the complex; this underlying simplicity provides possible pointers to an evolutionary origin from a 'primordial' nuclear pore complex. In the cover graphic, the 100-nm diameter pores are shown in the silver-grey nuclear envelope.