RESUMO
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 nucleoporins (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.
RESUMO
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.
RESUMO
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.
Assuntos
Poro Nuclear , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Proteínas de Membrana TransportadorasRESUMO
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.
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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.
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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.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Membrana Nuclear/metabolismo , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
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.
Assuntos
Adaptação Fisiológica , Poro Nuclear/metabolismo , Saccharomyces cerevisiae/fisiologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Fluorescência , Simulação de Acoplamento Molecular , Membrana Nuclear/metabolismo , Poro Nuclear/química , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Domínios Proteicos , Reprodutibilidade dos Testes , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
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.
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Molecular virology tools are critical for basic studies of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and for developing new therapeutics. Experimental systems that do not rely on viruses capable of spread are needed for potential use in lower-containment settings. In this work, we use a yeast-based reverse genetics system to develop spike-deleted SARS-CoV-2 self-replicating RNAs. These noninfectious self-replicating RNAs, or replicons, can be trans-complemented with viral glycoproteins to generate replicon delivery particles for single-cycle delivery into a range of cell types. This SARS-CoV-2 replicon system represents a convenient and versatile platform for antiviral drug screening, neutralization assays, host factor validation, and viral variant characterization.
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RNA Viral/genética , Replicon/fisiologia , SARS-CoV-2/genética , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Antivirais/farmacologia , Linhagem Celular , Humanos , Interferons/farmacologia , Testes de Sensibilidade Microbiana , Mutação , Plasmídeos , RNA Viral/metabolismo , Replicon/genética , Genética Reversa , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/fisiologia , Saccharomyces cerevisiae/genética , Glicoproteína da Espícula de Coronavírus/genética , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Pseudotipagem Viral , Vírion/genética , Vírion/fisiologia , Replicação ViralRESUMO
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).
Assuntos
Cromatografia de Afinidade/métodos , Substâncias Macromoleculares , Proteômica/métodos , Proteínas de Saccharomyces cerevisiae , Marcação por Isótopo , Substâncias Macromoleculares/análise , Substâncias Macromoleculares/química , Substâncias Macromoleculares/metabolismo , Proteólise , Proteínas de Saccharomyces cerevisiae/análise , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
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.
Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares , Poro Nuclear , Núcleo Celular , CitoplasmaRESUMO
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 â¼50 MDa assembly comprised of â¼30 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.
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Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Poro Nuclear/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Poro Nuclear/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
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.
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Insuficiência Cardíaca , Cuidados Paliativos , Estudos Transversais , Feminino , Insuficiência Cardíaca/diagnóstico , Insuficiência Cardíaca/epidemiologia , Insuficiência Cardíaca/terapia , Hospitalização , Humanos , PrevalênciaRESUMO
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.
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Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Poro Nuclear/química , Poro Nuclear/metabolismo , Saccharomyces cerevisiae/química , Reagentes de Ligações Cruzadas/química , Espectrometria de Massas , Modelos Moleculares , Estabilidade Proteica , Transporte Proteico , Transporte de RNARESUMO
The study of the nuclear pore complex (NPC) is a fascinating endeavor, as it not only implies uncovering the 'engineering marvel' of its architecture and function, but also provides a key window into a significant evolutionary event: the origin of the eukaryotic cell. The combined efforts of many groups in the field, with the help of novel methodologies and new model organisms, are facilitating a much deeper understanding of this complex assembly. Here we cover recent advances on the characterization of the structure of the NPC scaffold and of the biophysical mechanisms that define the permeability barrier. We identify common architectural and functional principles between those two NPC compartments, expanding the previous protocoatomer hypothesis to suggest possible evolutionary origins for the FG nucleoporins and the NPC permeability barrier.
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Células Eucarióticas/citologia , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , Células Eucarióticas/metabolismo , Evolução Molecular , Poro Nuclear/química , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/químicaRESUMO
The membrane ring that equatorially circumscribes the nuclear pore complex (NPC) in the perinuclear lumen of the nuclear envelope is composed largely of Pom152 in yeast and its ortholog Nup210 (or Gp210) in vertebrates. Here, we have used a combination of negative-stain electron microscopy, nuclear magnetic resonance, and small-angle X-ray scattering methods to determine an integrative structure of the â¼120 kDa luminal domain of Pom152. Our structural analysis reveals that the luminal domain is formed by a flexible string-of-pearls arrangement of nine repetitive cadherin-like Ig-like domains, indicating an evolutionary connection between NPCs and the cell adhesion machinery. The 16 copies of Pom152 known to be present in the yeast NPC are long enough to form the observed membrane ring, suggesting how interactions between Pom152 molecules help establish and maintain the NPC architecture.
Assuntos
Glicoproteínas de Membrana/química , Poro Nuclear/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Adesão Celular , Glicoproteínas de Membrana/metabolismo , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Domínios Proteicos , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
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.
Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares/química , Poro Nuclear/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Leveduras/metabolismo , Transporte Ativo do Núcleo Celular , Proteínas Fúngicas , Complexo de Proteínas Formadoras de Poros Nucleares/ultraestrutura , RNA Mensageiro , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/ultraestruturaRESUMO
Here, we describe an affinity isolation protocol. It uses cryomilled yeast cell powder for producing cell extracts and antibody-conjugated paramagnetic beads for affinity capture. Guidelines for determining the optimal extraction solvent composition are provided. Captured proteins are eluted in a denaturing solvent (sodium dodecyl sulfate polyacrylamide gel electrophoresis sample buffer) for gel-based proteomic analyses. Although the procedures can be modified to use other sources of cell extract and other forms of affinity media, to date we have consistently obtained the best results with the method presented.
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Cromatografia de Afinidade/métodos , Substâncias Macromoleculares/isolamento & purificação , Complexos Multienzimáticos/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Saccharomyces cerevisiae/química , Marcadores de Afinidade/metabolismo , Eletroforese , Proteoma/análiseRESUMO
This protocol describes the isolation of native protein complexes by density gradient ultracentrifugation. The outcome of an affinity capture and native elution experiment is generally a mixture of (1) the complex(es) associated with the protein of interest under the specific conditions of capture, (2) fragments of the complex generated by degradation or disassembly during the purification procedure, and (3) the protease or reagent used to natively elute the sample. To separate these components and isolate a homogeneous complex, an additional step of purification is required. Rate-zonal density gradient ultracentrifugation is a reliable and powerful technique for separating particles based on their hydrodynamic volume. The density gradient is generated by mixing low- and high-density solutions of a suitable low-molecular-weight inert solute (e.g., sucrose or glycerol). The gradient is formed in a solvent that could be any of the solvents used for the affinity capture and native elution and should help to preserve the structure and activity of the assembly.