Comprehensive structure and functional adaptations of the yeast nuclear pore complex.

Nuclear pore complexes (NPCs) mediate the nucleocytoplasmic transport of macromolecules. Here we provide a structure of the isolated yeast NPC in which the inner ring is resolved by cryo-EM at sub-nanometer resolution to show how flexible connectors tie together different structural and functional layers. These connectors may be targets for phosphorylation and regulated disassembly in cells with an open mitosis. Moreover, some nucleoporin pairs and transport factors have similar interaction motifs, which suggests an evolutionary and mechanistic link between assembly and transport. We provide evidence for three major NPC variants that may foreshadow functional specializations at the nuclear periphery. Cryo-electron tomography extended these studies, providing a model of the in situ NPC with a radially expanded inner ring. Our comprehensive model reveals features of the nuclear basket and central transporter, suggests a role for the lumenal Pom152 ring in restricting dilation, and highlights structural plasticity that may be required for transport.

Structure and Function of the Nuclear Pore Complex Cytoplasmic mRNA Export Platform.

The last steps in mRNA export and remodeling are performed by the Nup82 complex, a large conserved assembly at the cytoplasmic face of the nuclear pore complex (NPC). By integrating diverse structural data, we have determined the molecular architecture of the native Nup82 complex at subnanometer precision. The complex consists of two compositionally identical multiprotein subunits that adopt different configurations. The Nup82 complex fits into the NPC through the outer ring Nup84 complex. Our map shows that this entire 14-MDa Nup82-Nup84 complex assembly positions the cytoplasmic mRNA export factor docking sites and messenger ribonucleoprotein (mRNP) remodeling machinery right over the NPC's central channel rather than on distal cytoplasmic filaments, as previously supposed. We suggest that this configuration efficiently captures and remodels exporting mRNP particles immediately upon reaching the cytoplasmic side of the NPC.

Implications of a multiscale structure of the yeast nuclear pore complex.

Nuclear pore complexes (NPCs) direct the nucleocytoplasmic transport of macromolecules. Here, we provide a composite multiscale structure of the yeast NPC, based on improved 3D density maps from cryogenic electron microscopy and AlphaFold2 models. Key features of the inner and outer rings were integrated into a comprehensive model. We resolved flexible connectors that tie together the core scaffold, along with equatorial transmembrane complexes and a lumenal ring that anchor this channel within the pore membrane. The organization of the nuclear double outer ring reveals an architecture that may be shared with ancestral NPCs. Additional connections between the core scaffold and the central transporter suggest that under certain conditions, a degree of local organization is present at the periphery of the transport machinery. These connectors may couple conformational changes in the scaffold to the central transporter to modulate transport. Collectively, this analysis provides insights into assembly, transport, and NPC evolution.

Dissecting the Structural Dynamics of the Nuclear Pore Complex.

Cellular processes are largely carried out by macromolecular assemblies, most of which are dynamic, having components that are in constant flux. One such assembly is the nuclear pore complex (NPC), an ∼50 MDa assembly comprised of ∼30 different proteins called Nups that mediates selective macromolecular transport between the nucleus and cytoplasm. We developed a proteomics method to provide a comprehensive picture of the yeast NPC component dynamics. We discovered that, although all Nups display uniformly slow turnover, their exchange rates vary considerably. Surprisingly, this exchange rate was relatively unrelated to each Nup's position, accessibility, or role in transport but correlated with its structural role; scaffold-forming Nups exchange slowly, whereas flexible connector Nups threading throughout the NPC architecture exchange more rapidly. Targeted perturbations in the NPC structure revealed a dynamic resilience to damage. Our approach opens a new window into macromolecular assembly dynamics.

One Ring to Rule them All? Structural and Functional Diversity in the Nuclear Pore Complex.

The nuclear pore complex (NPC) is the massive protein assembly that regulates the transport of macromolecules between the nucleus and the cytoplasm. Recent breakthroughs have provided major insights into the structure of the NPC in different eukaryotes, revealing a previously unsuspected diversity of NPC architectures. In parallel, the NPC has been shown to be a key player in regulating essential nuclear processes such as chromatin organization, gene expression, and DNA repair. However, our knowledge of the NPC structure has not been able to address the molecular mechanisms underlying its regulatory roles. We discuss potential explanations, including the coexistence of alternative NPC architectures with specific functional roles.

Integrative structure and functional anatomy of a nuclear pore complex.

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.

Spatial coherence from Nd3+ quantum emitters mediated by a plasmonic chain.

Controlling the coherence properties of rare earth emitters in solid-state platforms in the absence of an optical cavity is highly desirable for quantum light-matter interfaces and photonic networks. Here, we demonstrate the possibility of generating directional and spatially coherent light from Nd3+ ions coupled to the longitudinal plasmonic mode of a chain of interacting Ag nanoparticles. The effect of the plasmonic chain on the Nd3+ emission is analyzed by Fourier microscopy. The results reveal the presence of an interference pattern in which the Nd3+ emission is enhanced at specific directions, as a distinctive signature of spatial coherence. Numerical simulations corroborate the need of near-field coherent coupling of the emitting ions with the plasmonic chain mode. The work provides fundamental insights for controlling the coherence properties of quantum emitters at room temperature and opens new avenues towards rare earth based nanoscale hybrid devices for quantum information or optical communication in nanocircuits.

A strategy for dissecting the architectures of native macromolecular assemblies.

It remains particularly problematic to define the structures of native macromolecular assemblies, which are often of low abundance. Here we present a strategy for isolating complexes at endogenous levels from GFP-tagged transgenic cell lines. Using cross-linking mass spectrometry, we extracted distance restraints that allowed us to model the complexes' molecular architectures.

A Robust Workflow for Native Mass Spectrometric Analysis of Affinity-Isolated Endogenous Protein Assemblies.

The central players in most cellular events are assemblies of macromolecules. Structural and functional characterization of these assemblies requires knowledge of their subunit stoichiometry and intersubunit connectivity. One of the most direct means for acquiring such information is so-called "native mass spectrometry (MS)", wherein the masses of the intact assemblies and parts thereof are accurately determined. It is of particular interest to apply native MS to the study of endogenous protein assemblies-i.e., those wherein the component proteins are expressed at endogenous levels in their natural functional states, rather than the overexpressed (sometimes partial) constructs commonly employed in classical structural studies, whose assembly can introduce stoichiometry artifacts and other unwanted effects. To date, the application of native MS to the elucidation of endogenous protein complexes has been limited by the difficulty in obtaining pristine cell-derived assemblies at sufficiently high concentrations for effective analysis. Here, to address this challenge, we present a robust workflow that couples rapid and efficient affinity isolation of endogenous protein complexes with a sensitive native MS readout. The resulting workflow has the potential to provide a wealth of data on the stoichiometry and intersubunit connectivity of endogenous protein assemblies-information that is key to successful integrative structural elucidation of biological systems.

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.

Molecular architecture and function of the SEA complex, a modulator of the TORC1 pathway.

The TORC1 signaling pathway plays a major role in the control of cell growth and response to stress. Here we demonstrate that the SEA complex physically interacts with TORC1 and is an important regulator of its activity. During nitrogen starvation, deletions of SEA complex components lead to Tor1 kinase delocalization, defects in autophagy, and vacuolar fragmentation. TORC1 inactivation, via nitrogen deprivation or rapamycin treatment, changes cellular levels of SEA complex members. We used affinity purification and chemical cross-linking to generate the data for an integrative structure modeling approach, which produced a well-defined molecular architecture of the SEA complex and showed that the SEA complex comprises two regions that are structurally and functionally distinct. The SEA complex emerges as a platform that can coordinate both structural and enzymatic activities necessary for the effective functioning of the TORC1 pathway.

Integrative structure-function mapping of the nucleoporin Nup133 suggests a conserved mechanism for membrane anchoring of the nuclear pore complex.

The nuclear pore complex (NPC) is the sole passageway for the transport of macromolecules across the nuclear envelope. Nup133, a major component in the essential Y-shaped Nup84 complex, is a large scaffold protein of the NPC's outer ring structure. Here, we describe an integrative modeling approach that produces atomic models for multiple states of Saccharomyces cerevisiae (Sc) Nup133, based on the crystal structures of the sequence segments and their homologs, including the related Vanderwaltozyma polyspora (Vp) Nup133 residues 55 to 502 (VpNup133(55-502)) determined in this study, small angle X-ray scattering profiles for 18 constructs of ScNup133 and one construct of VpNup133, and 23 negative-stain electron microscopy class averages of ScNup133(2-1157). Using our integrative approach, we then computed a multi-state structural model of the full-length ScNup133 and validated it with mutational studies and 45 chemical cross-links determined via mass spectrometry. Finally, the model of ScNup133 allowed us to annotate a potential ArfGAP1 lipid packing sensor (ALPS) motif in Sc and VpNup133 and discuss its potential significance in the context of the whole NPC; we suggest that ALPS motifs are scattered throughout the NPC's scaffold in all eukaryotes and play a major role in the assembly and membrane anchoring of the NPC in the nuclear envelope. Our results are consistent with a common evolutionary origin of Nup133 with membrane coating complexes (the protocoatomer hypothesis); the presence of the ALPS motifs in coatomer-like nucleoporins suggests an ancestral mechanism for membrane recognition present in early membrane coating complexes.

The Molecular Architecture of the Nuclear Basket.

The nuclear pore complex (NPC) is the sole mediator of nucleocytoplasmic transport. Despite great advances in understanding its conserved core architecture, the peripheral regions can exhibit considerable variation within and between species. One such structure is the cage-like nuclear basket. Despite its crucial roles in mRNA surveillance and chromatin organization, an architectural understanding has remained elusive. Using in-cell cryo-electron tomography and subtomogram analysis, we explored the NPC's structural variations and the nuclear basket across fungi (yeast; S. cerevisiae), mammals (mouse; M. musculus), and protozoa (T. gondii). Using integrative structural modeling, we computed a model of the basket in yeast and mammals that revealed how a hub of Nups in the nuclear ring binds to basket-forming Mlp/Tpr proteins: the coiled-coil domains of Mlp/Tpr form the struts of the basket, while their unstructured termini constitute the basket distal densities, which potentially serve as a docking site for mRNA preprocessing before nucleocytoplasmic transport.

Dynamic molecular mechanism of the nuclear pore complex permeability barrier.

Nuclear pore complexes (NPCs) mediate nucleocytoplasmic transport of specific macromolecules while impeding the exchange of unsolicited material. However, key aspects of this gating mechanism remain controversial. To address this issue, we determined the nanoscopic behavior of the permeability barrier directly within yeast S. cerevisiae NPCs at transport-relevant timescales. We show that the large intrinsically disordered domains of phenylalanine-glycine repeat nucleoporins (FG Nups) exhibit highly dynamic fluctuations to create transient voids in the permeability barrier that continuously shape-shift and reseal, resembling a radial polymer brush. Together with cargo-carrying transport factors the FG domains form a feature called the central plug, which is also highly dynamic. Remarkably, NPC mutants with longer FG domains show interweaving meshwork-like behavior that attenuates nucleocytoplasmic transport in vivo. Importantly, the bona fide nanoscale NPC behaviors and morphologies are not recapitulated by in vitro FG domain hydrogels. NPCs also exclude self-assembling FG domain condensates in vivo, thereby indicating that the permeability barrier is not generated by a self-assembling phase condensate, but rather is largely a polymer brush, organized by the NPC scaffold, whose dynamic gating selectivity is strongly enhanced by the presence of transport factors.

Atomic structure of the nuclear pore complex targeting domain of a Nup116 homologue from the yeast, Candida glabrata.

The nuclear pore complex (NPC), embedded in the nuclear envelope, is a large, dynamic molecular assembly that facilitates exchange of macromolecules between the nucleus and the cytoplasm. The yeast NPC is an eightfold symmetric annular structure composed of ~456 polypeptide chains contributed by ~30 distinct proteins termed nucleoporins. Nup116, identified only in fungi, plays a central role in both protein import and mRNA export through the NPC. Nup116 is a modular protein with N-terminal "FG" repeats containing a Gle2p-binding sequence motif and a NPC targeting domain at its C-terminus. We report the crystal structure of the NPC targeting domain of Candida glabrata Nup116, consisting of residues 882-1034 [CgNup116(882-1034)], at 1.94 Å resolution. The X-ray structure of CgNup116(882-1034) is consistent with the molecular envelope determined in solution by small-angle X-ray scattering. Structural similarities of CgNup116(882-1034) with homologous domains from Saccharomyces cerevisiae Nup116, S. cerevisiae Nup145N, and human Nup98 are discussed.

Affinity Isolation of Endogenous Saccharomyces Cerevisiae Nuclear Pore Complexes.

Studying protein complexes in vitro requires the production of a relatively pure sample that maintains the full complement, native organization, and function of that complex. This can be particularly challenging to achieve for large, multi-component, membrane embedded complexes using the traditional recombinant expression and reconstitution methodologies. However, using affinity capture from native cells, suitable whole endogenous protein complexes can be isolated. Here we present a protocol for the affinity isolation of baker's yeast (S. cerevisiae) nuclear pore complexes, which are ~50 MDa assemblies made up of 552 distinct proteins and embedded in a double-membraned nuclear envelope. Producing this sample allowed us for the first time to perform analyses to characterize the mass, stoichiometry, morphology, and connectivity of this complex and to obtain its integrative structure with ~9 Å precision. We believe this methodology can be applied to other challenging protein complexes to produce similar results.

Silver Nanoparticle Chains for Ultra-Long-Range Plasmonic Waveguides for Nd3+ Fluorescence.

Plasmonic waveguides have been shown to be a promising approach to confine and transport electromagnetic energy beyond the diffraction limit. However, ohmic losses generally prevent their integration at micrometric or millimetric scales. Here, we present a gain-compensated plasmonic waveguide based on the integration of linear chains of Ag nanoparticles on an optically active Nd3+-doped solid-state gain medium. By means of dual confocal fluorescence microscopy, we demonstrate long-range optical energy propagation due to the near-field coupling between the plasmonic nanostructures and the Nd3+ ions. The subwavelength fluorescence guiding is monitored at distances of around 100 µm from the excitation source for two different emission ranges centered at around 900 nm and 1080 nm. In both cases, the guided fluorescence exhibits a strong polarization dependence, consistent with the polarization behavior of the plasmon resonance supported by the chain. The experimental results are interpreted through numerical simulations in quasi-infinite long chains, which corroborate the propagation features of the Ag nanoparticle chains at both excitation (λexc = 590 nm) and emission wavelengths. The obtained results exceed by an order of magnitude that of previous reports on electromagnetic energy transport using linear plasmonic chains. The work points out the potential of combining Ag nanoparticle chains with a small interparticle distance (~2 nm) with rare-earth-based optical gain media as ultra-long-range waveguides with extreme light confinement. The results offer new perspectives for the design of integrated hybrid plasmonic-photonic circuits based on rare-earth-activated solid-state platforms.

Measuring in vivo protein turnover and exchange in yeast macromolecular assemblies.

We present a comprehensive and robust protocol to track the dynamics of all proteins in a complex in yeast cells. A single member of the protein assembly is tagged and conditionally expressed, minimizing the perturbations to the protein complex. Then, SILAC labeling and affinity purification are used for the assessment of the whole protein complex dynamics. This method can determine and distinguish both subunit turnover and exchange specifically in an assembly to provide a comprehensive picture of assembly dynamics. For complete details on the use and execution of this protocol, please refer to Hakhverdyan et al. (2021).

Prevalence of advanced heart failure and use of palliative care in admitted patients: Findings from the EPICTER study.

INTRODUCTION AND AIM: Palliative care in patients with advanced heart failure is strongly recommended by Clinical Practice Guidelines. We aimed to calculate the prevalence of advanced heart failure in admitted patients, to describe their management, and to analyse the factors that influence their referral to specialised palliative care. PATIENTS AND METHODS: Cross-sectional, multicentre study that consecutively included patients admitted for heart failure in 74 Spanish hospitals. If they met criteria for advanced heart failure, their treatment, complications and procedures were recorded. RESULTS: A total of 3153 patients were included. Of them, 739 (23%) met criteria for advanced heart failure. They were more likely to be women, older and to have a history of anaemia, chronic kidney disease and cognitive impairment. For their management, furosemide infusions (30%) and vasodilators (21%) were used. Refractory symptoms were treated with opioids (47%) and benzodiazepines (44%). Palliative care was only provided in the last hours of life in 48% of them. A multidisciplinary approach, involving palliative care specialists was sought in 15% of these patients. Treatment with furosemide infusions, an advanced New York Heart Association functional class, to meet advanced HF criteria and the presence of cancer were associated with the referral to specialised palliative care. CONCLUSIONS: Almost one in four patients admitted with HF met criteria of advanced disease. They were older and had more comorbidities. Specialist palliative care services were involved in only a minority of patients, mainly those who were highly symptomatic or had cancer.