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Key Points Structural, biochemical and cell biology investigations of the nuclear pore complex (NPC) over the past 60 years have contributed to explaining its canonical features. Recently, in vitro and in situ investigations have converged to produce the first pseudo-atomic model of the NPC central core. It seems that nucleoporins, the building blocks of the NPC, are conformationally labile and locally adapt to serve multiple structural roles. A network of short linear motifs is essential for holding the scaffold of the NPC together and offers a novel and attractive scientific concept of how scaffolding might be regulated by post-translational modifications. Prospectively, these structural insights might trigger novel types of investigations that will shed light on the mechanism behind nucleocytoplasmic transport (as well as other functions of the NPC) and the disease-relevance of nucleoporin dysfunction. Nuclear pore complexes (NPCs) are large protein assemblies that form channels in the nuclear envelope and constitute major routes for nucleocytoplasmic communication. Insights into the complex structure of NPCs provide the basis for understanding their functions and reveal how the dysfunction of their structural components, nucleoporins, contributes to human disease. Nuclear pore complexes (NPCs) fuse the inner and outer nuclear membranes to form channels across the nuclear envelope. They are large macromolecular assemblies with a complex composition and diverse functions. Apart from facilitating nucleocytoplasmic transport, NPCs are involved in chromatin organization, the regulation of gene expression and DNA repair. Understanding the molecular mechanisms underlying these functions has been hampered by a lack of structural knowledge about the NPC. The recent convergence of crystallographic and biochemical in vitro analysis of nucleoporins (NUPs), the components of the NPC, with cryo-electron microscopic imaging of the entire NPC in situ has provided first pseudo-atomic view of its central core and revealed that an unexpected network of short linear motifs is an important spatial organization principle. These breakthroughs have transformed the way we understand NPC structure, and they provide an important base for functional investigations, including the elucidation of the molecular mechanisms underlying clinically manifested mutations of the nucleocytoplasmic transport system.

Necroptosis and pyroptosis are two forms of programmed cell death with a common feature of plasma membrane rupture. Here we studied the morphology and mechanism of pyroptosis in comparison with necroptosis. Different from necroptosis, pyroptosis undergoes membrane blebbing and produces apoptotic body-like cell protrusions （termed pyroptotic bodies） prior to plasma membrane rupture. The rupture in necroptosis is explosion-like, whereas in pyroptosis it leads to flattening of cells. It is known that the execution of necroptosis is mediated by mixed lineage kinase domain-like （MLKL） oligomers in the plasma membrane, whereas gasdermin-D （GSDMD） mediates pyroptosis after its cleavage by caspase-1 or caspase-11. We show that N-terminal fragment of GSDMD （GSDMD-N） generated by caspase cleavage also forms oligomer and migrates to the plasma membrane to kill cells. Both MLKL and GSDMD-N are lipophilic and the N-terminal sequences of both proteins are important for their oligomerization and plasma membrane translocation. Unlike MLKL which forms channels on the plasma membrane that induces influx of selected ions which osmotically swell the cells to burst, GSDMD-N forms non-selective pores and does not rely on increased osmolarity to disrupt cells. Our study reveals the pore-forming activity of GSDMD and channel-forming activity of MLKL determine different ways of plasma membrane rupture in pyroptosis and necroptosis.

A better understanding of proteostasis in health and disease requires robust methods to determine protein half-lives. Here we improve the precision and accuracy of peptide ion intensity-based quantification, enabling more accurate protein turnover determination in non-dividing cells by dynamic SILAC-based proteomics. This approach allows exact determination of protein half-lives ranging from 10 to >1000 h. We identified 4000–6000 proteins in several non-dividing cell types, corresponding to 9699 unique protein identifications over the entire data set. We observed similar protein half-lives in B-cells, natural killer cells and monocytes, whereas hepatocytes and mouse embryonic neurons show substantial differences. Our data set extends and statistically validates the previous observation that subunits of protein complexes tend to have coherent turnover. Moreover, analysis of different proteasome and nuclear pore complex assemblies suggests that their turnover rate is architecture dependent. These results illustrate that our approach allows investigating protein turnover and its implications in various cell types. The proteome-wide characterization of proteostasis depends on robust approaches to determine protein half-lives. Here, the authors improve the accuracy and precision of mass spectrometry-based quantification, enabling reliable protein half-life determination in several non-dividing cell types.

Mechanical force controls fundamental cellular processes in health and disease, and increasing evidence shows that the nucleus both experiences and senses applied forces. Such forces can lead to the nuclear translocation of proteins, but whether force controls nucleocytoplasmic transport, and how, remains unknown. Here we show that nuclear forces differentially control passive and facilitated nucleocytoplasmic transport, setting the rules for the mechanosensitivity of shuttling proteins. We demonstrate that nuclear force increases permeability across nuclear pore complexes, with a dependence on molecular weight that is stronger for passive than for facilitated diffusion. Owing to this differential effect, force leads to the translocation of cargoes into or out of the nucleus within a given range of molecular weight and affinity for nuclear transport receptors. Further, we show that the mechanosensitivity of several transcriptional regulators can be both explained by this mechanism and engineered exogenously by introducing appropriate nuclear localization signals. Our work unveils a mechanism of mechanically induced signalling, probably operating in parallel with others, with potential applicability across signalling pathways. Andreu et al. show that force regulates nucleocytoplasmic transport by weakening the permeability barrier of nuclear pore complexes, affecting passive and facilitated diffusion in different ways.

Nuclear pore complexes play central roles as gatekeepers of RNA and protein transport between the cytoplasm and nucleoplasm. However, their large size and dynamic nature have impeded a full structural and functional elucidation. Here we determined the structure of the entire 552-protein nuclear pore complex of the yeast Saccharomyces cerevisiae at sub-nanometre precision by satisfying a wide range of data relating to the molecular arrangement of its constituents. The nuclear pore complex incorporates sturdy diagonal columns and connector cables attached to these columns, imbuing the structure with strength and flexibility. These cables also tie together all other elements of the nuclear pore complex, including membrane-interacting regions, outer rings and RNA-processing platforms. Inwardly directed anchors create a high density of transport factor-docking Phe-Gly repeats in the central channel, organized into distinct functional units. This integrative structure enables us to rationalize the architecture, transport mechanism and evolutionary origins of the nuclear pore complex. The structure of the yeast nuclear pore complex, determined at sub-nanometre precision using an integrative approach that combines a wide range of data, reveals details of its architecture, transport mechanism and evolutionary origins. Structure of yeast nuclear pore complex Nuclear pore complexes (NPCs) are the gateways for the transport of RNA and proteins into and out of the nucleus. Although previous work has provided structural insights into parts of NPCs, getting a full picture of these complexes has proved difficult owing to their large size and dynamic nature. A multidisciplinary team of researchers now report the structure of the entire 552-protein NPC of the yeast at a sub-nanometre level using an integrative approach including mass spectrometry, cryo-electron tomography and chemical crosslinking. The structure provides a detailed view of the functional elements of the NPC and clarifies its architecture, mechanism of action and evolutionary origins.

The nuclear envelope (NE) is a spherical double membrane with elastic properties. How NE shape and elasticity are regulated by lipid chemistry is unknown. Here we discover lipid acyl chain unsaturation as essential for NE and nuclear pore complex (NPC) architecture and function. Increased lipid saturation rigidifies the NE and the endoplasmic reticulum into planar, polygonal membranes, which are fracture prone. These membranes exhibit a micron-scale segregation of lipids into ordered and disordered phases, excluding NPCs from the ordered phase. Balanced lipid saturation is required for NPC integrity, pore membrane curvature and nucleocytoplasmic transport. Oxygen deprivation amplifies the impact of saturated lipids, causing NE rigidification and rupture. Conversely, lipid droplets buffer saturated lipids to preserve NE architecture. Our study uncovers a fundamental link between lipid acyl chain structure and the integrity of the cell nucleus with implications for nuclear membrane malfunction in ischaemic tissues. Romanauska and Köhler manipulate the levels of endogenously produced saturated acyl chains in yeast and show that nuclear envelope and nuclear pore complex function are uniquely sensitive to lipid acyl chain unsaturation.

Understanding how the nuclear pore complex (NPC) is assembled is of fundamental importance to grasp the mechanisms behind its essential function and understand its role during the evolution of eukaryotes 1 – 4 . There are at least two NPC assembly pathways—one during the exit from mitosis and one during nuclear growth in interphase—but we currently lack a quantitative map of these events. Here we use fluorescence correlation spectroscopy calibrated live imaging of endogenously fluorescently tagged nucleoporins to map the changes in the composition and stoichiometry of seven major modules of the human NPC during its assembly in single dividing cells. This systematic quantitative map reveals that the two assembly pathways have distinct molecular mechanisms, in which the order of addition of two large structural components, the central ring complex and nuclear filaments are inverted. The dynamic stoichiometry data was integrated to create a spatiotemporal model of the NPC assembly pathway and predict the structures of postmitotic NPC assembly intermediates. Single-molecule calibrated live microscopy and computational modelling have revealed that human nuclear pore complex assembly takes different pathways during the exit from mitosis and during nuclear growth in interphase.

The cytoplasmic mislocalization and aggregation of TAR DNA-binding protein-43 (TDP-43) is a common histopathological hallmark of the amyotrophic lateral sclerosis and frontotemporal dementia disease spectrum (ALS/FTD). However, the composition of aggregates and their contribution to the disease process remain unknown. Here we used proximity-dependent biotin identification (BioID) to interrogate the interactome of detergent-insoluble TDP-43 aggregates and found them enriched for components of the nuclear pore complex and nucleocytoplasmic transport machinery. Aggregated and disease-linked mutant TDP-43 triggered the sequestration and/or mislocalization of nucleoporins and transport factors, and interfered with nuclear protein import and RNA export in mouse primary cortical neurons, human fibroblasts and induced pluripotent stem cell–derived neurons. Nuclear pore pathology is present in brain tissue in cases of sporadic ALS and those involving genetic mutations in TARDBP and C9orf72. Our data strongly implicate TDP-43-mediated nucleocytoplasmic transport defects as a common disease mechanism in ALS/FTD.

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.

Nuclear pore complexes (NPCs) fuse the inner and outer membranes of the nuclear envelope. They comprise hundreds of nucleoporins (Nups) that assemble into multiple subcomplexes and form large central channels for nucleocytoplasmic exchange 1 , 2 . How this architecture facilitates messenger RNA export, NPC biogenesis and turnover remains poorly understood. Here we combine in situ structural biology and integrative modelling with correlative light and electron microscopy and molecular perturbation to structurally analyse NPCs in intact Saccharomyces cerevisiae cells within the context of nuclear envelope remodelling. We find an in situ conformation and configuration of the Nup subcomplexes that was unexpected from the results of previous in vitro analyses. The configuration of the Nup159 complex appears critical to spatially accommodate its function as an mRNA export platform, and as a mediator of NPC turnover. The omega-shaped nuclear envelope herniae that accumulate in nup116Δ cells 3 conceal partially assembled NPCs lacking multiple subcomplexes, including the Nup159 complex. Under conditions of starvation, herniae of a second type are formed that cytoplasmically expose NPCs. These results point to a model of NPC turnover in which NPC-containing vesicles bud off from the nuclear envelope before degradation by the autophagy machinery. Our study emphasizes the importance of investigating the structure–function relationship of macromolecular complexes in their cellular context. In-cell structural studies in Saccharomyces cerevisiae reveal that the configuration of the Nup159 complex is a key determinant of the mRNA export function of the nuclear pore complex, and suggest a model in which nuclear pore complexes are degraded via the autophagy machinery.