RESUMO
Visual proteomics attempts to build atlases of the molecular content of cells but the automated annotation of cryo electron tomograms remains challenging. Template matching (TM) and methods based on machine learning detect structural signatures of macromolecules. However, their applicability remains limited in terms of both the abundance and size of the molecular targets. Here we show that the performance of TM is greatly improved by using template-specific search parameter optimization and by including higher-resolution information. We establish a TM pipeline with systematically tuned parameters for the automated, objective and comprehensive identification of structures with confidence 10 to 100-fold above the noise level. We demonstrate high-fidelity and high-confidence localizations of nuclear pore complexes, vaults, ribosomes, proteasomes, fatty acid synthases, lipid membranes and microtubules, and individual subunits inside crowded eukaryotic cells. We provide software tools for the generic implementation of our method that is broadly applicable towards realizing visual proteomics.
Assuntos
Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Complexo de Endopeptidases do Proteassoma , Proteômica , Ribossomos , Software , Tomografia com Microscopia Eletrônica/métodos , Microscopia Crioeletrônica/métodos , Ribossomos/ultraestrutura , Ribossomos/metabolismo , Complexo de Endopeptidases do Proteassoma/ultraestrutura , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/química , Humanos , Proteômica/métodos , Poro Nuclear/ultraestrutura , Poro Nuclear/metabolismo , Microtúbulos/ultraestrutura , Microtúbulos/metabolismo , Ácido Graxo Sintases/metabolismo , Aprendizado de Máquina , Imageamento Tridimensional/métodos , Algoritmos , Processamento de Imagem Assistida por Computador/métodosRESUMO
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.
Assuntos
Microscopia Crioeletrônica , Complexo de Proteínas Formadoras de Poros Nucleares , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/ultraestrutura , Humanos , Mutação , Poro Nuclear/metabolismo , Poro Nuclear/ultraestrutura , Poro Nuclear/química , Glicina/química , Glicina/metabolismo , Fenilalanina/química , Fenilalanina/metabolismo , Sequências Repetitivas de Aminoácidos , Ligação Proteica , Modelos Moleculares , Interações Hidrofóbicas e HidrofílicasRESUMO
The nuclear pore complex (NPC) is the bidirectional gate that mediates the exchange of macromolecules or their assemblies between nucleus and cytoplasm1-3. 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 factors4,5. 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.
Assuntos
Transporte Ativo do Núcleo Celular , Poro Nuclear , Saccharomyces cerevisiae , Microscopia Crioeletrônica , Poro Nuclear/química , Poro Nuclear/metabolismo , Poro Nuclear/ultraestrutura , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/ultraestrutura , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Fenilalanina , Glicina , Simulação por Computador , SolventesRESUMO
Volume electron microscopy is an important imaging modality in contemporary cell biology. Identification of intracellular structures is a laborious process limiting the effective use of this potentially powerful tool. We resolved this bottleneck with automated segmentation of intracellular substructures in electron microscopy (ASEM), a new pipeline to train a convolutional neural network to detect structures of a wide range in size and complexity. We obtained dedicated models for each structure based on a small number of sparsely annotated ground truth images from only one or two cells. Model generalization was improved with a rapid, computationally effective strategy to refine a trained model by including a few additional annotations. We identified mitochondria, Golgi apparatus, endoplasmic reticulum, nuclear pore complexes, caveolae, clathrin-coated pits, and vesicles imaged by focused ion beam scanning electron microscopy. We uncovered a wide range of membrane-nuclear pore diameters within a single cell and derived morphological metrics from clathrin-coated pits and vesicles, consistent with the classical constant-growth assembly model.
Assuntos
Processamento de Imagem Assistida por Computador , Microscopia Eletrônica , Redes Neurais de Computação , Clatrina , Retículo Endoplasmático/ultraestrutura , Complexo de Golgi/ultraestrutura , Microscopia Eletrônica/métodos , Mitocôndrias/ultraestrutura , Poro Nuclear/ultraestrutura , Cavéolas/ultraestrutura , Biologia CelularRESUMO
The nuclear pore complex (NPC), one of the largest protein complexes in eukaryotes, serves as a physical gate to regulate nucleocytoplasmic transport. Here, we determined the 8 Å resolution cryo-electron microscopic (cryo-EM) structure of the outer rings containing nuclear ring (NR) and cytoplasmic ring (CR) from the Xenopus laevis NPC, with local resolutions reaching 4.9 Å. With the aid of AlphaFold2, we managed to build a pseudoatomic model of the outer rings, including the Y complexes and flanking components. In this most comprehensive and accurate model of outer rings to date, the almost complete Y complex structure exhibits much tighter interaction in the hub region. In addition to two copies of Y complexes, each asymmetric subunit in CR contains five copies of Nup358, two copies of the Nup214 complex, two copies of Nup205 and one copy of newly identified Nup93, while that in NR contains one copy of Nup205, one copy of ELYS and one copy of Nup93. These in-depth structural features represent a great advance in understanding the assembly of NPCs.
Assuntos
Poro Nuclear , Oócitos , Animais , Inteligência Artificial , Microscopia Crioeletrônica , Poro Nuclear/química , Poro Nuclear/metabolismo , Poro Nuclear/ultraestrutura , Oócitos/metabolismo , Xenopus laevisRESUMO
In eukaryotic cells, nuclear pore complexes (NPCs) fuse the inner and outer nuclear membranes and mediate nucleocytoplasmic exchange. They are made of 30 different nucleoporins and form a cylindrical architecture around an aqueous central channel. This architecture is highly dynamic in space and time. Variations in NPC diameter have been reported, but the physiological circumstances and the molecular details remain unknown. Here, we combined cryoelectron tomography with integrative structural modeling to capture a molecular movie of the respective large-scale conformational changes in cellulo. Although NPCs of exponentially growing cells adopted a dilated conformation, they reversibly constricted upon cellular energy depletion or conditions of hypertonic osmotic stress. Our data point to a model where the nuclear envelope membrane tension is linked to the conformation of the NPC.
Assuntos
Membrana Nuclear/fisiologia , Poro Nuclear/fisiologia , Poro Nuclear/ultraestrutura , Transporte Ativo do Núcleo Celular , Fenômenos Biomecânicos , Microscopia Crioeletrônica , Citoplasma/metabolismo , Metabolismo Energético , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional , Modelos Biológicos , Membrana Nuclear/ultraestrutura , Complexo de Proteínas Formadoras de Poros Nucleares/química , Pressão Osmótica , Schizosaccharomyces/crescimento & desenvolvimento , Schizosaccharomyces/ultraestrutura , Proteínas de Schizosaccharomyces pombe/química , Estresse FisiológicoRESUMO
The nuclear basket (NB) scaffold, a fibrillar structure anchored to the nuclear pore complex (NPC), is regarded as constructed of polypeptides of the coiled-coil dominated protein TPR to which other proteins can bind without contributing to the NB's structural integrity. Here we report vertebrate protein ZC3HC1 as a novel inherent constituent of the NB, common at the nuclear envelopes (NE) of proliferating and non-dividing, terminally differentiated cells of different morphogenetic origin. Formerly described as a protein of other functions, we instead present the NB component ZC3HC1 as a protein required for enabling distinct amounts of TPR to occur NB-appended, with such ZC3HC1-dependency applying to about half the total amount of TPR at the NEs of different somatic cell types. Furthermore, pointing to an NB structure more complex than previously anticipated, we discuss how ZC3HC1 and the ZC3HC1-dependent TPR polypeptides could enlarge the NB's functional repertoire.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Neoplasias/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Poro Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Oócitos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Xenopus/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas de Ciclo Celular/genética , Feminino , Células HCT116 , Células HeLa , Humanos , Macaca mulatta , Neoplasias/genética , Neoplasias/ultraestrutura , Poro Nuclear/genética , Poro Nuclear/ultraestrutura , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas Nucleares/genética , Oócitos/ultraestrutura , Ligação Proteica , Proteínas Proto-Oncogênicas/genética , Transdução de Sinais , Proteínas de Xenopus/genética , Xenopus laevisRESUMO
Nuclear pore complexes (NPCs) are large macromolecular machines that mediate the traffic between the nucleus and the cytoplasm. In vertebrates, each NPC consists of â¼1000 proteins, termed nucleoporins, and has a mass of more than 100 MDa. While a pseudo-atomic static model of the central scaffold of the NPC has recently been assembled by integrating data from isolated proteins and complexes, many structural components still remain elusive due to the enormous size and flexibility of the NPC. Here, we explored the power of three-dimensional (3D) superresolution microscopy combined with computational classification and averaging to explore the 3D structure of the NPC in single human cells. We show that this approach can build the first integrated 3D structural map containing both central as well as peripheral NPC subunits with molecular specificity and nanoscale resolution. Our unbiased classification of more than 10,000 individual NPCs indicates that the nuclear ring and the nuclear basket can adopt different conformations. Our approach opens up the exciting possibility to relate different structural states of the NPC to function in situ.
Assuntos
Microscopia de Fluorescência/métodos , Complexo de Proteínas Formadoras de Poros Nucleares/ultraestrutura , Poro Nuclear/ultraestrutura , Animais , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Humanos , Poro Nuclear/metabolismo , Poro Nuclear/fisiologia , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismoRESUMO
Single molecule localization microscopy offers in principle resolution down to the molecular level, but in practice this is limited primarily by incomplete fluorescent labeling of the structure. This missing information can be completed by merging information from many structurally identical particles. In this work, we present an approach for 3D single particle analysis in localization microscopy which hugely increases signal-to-noise ratio and resolution and enables determining the symmetry groups of macromolecular complexes. Our method does not require a structural template, and handles anisotropic localization uncertainties. We demonstrate 3D reconstructions of DNA-origami tetrahedrons, Nup96 and Nup107 subcomplexes of the nuclear pore complex acquired using multiple single molecule localization microscopy techniques, with their structural symmetry deducted from the data.
Assuntos
Substâncias Macromoleculares/química , Substâncias Macromoleculares/ultraestrutura , Imagem Individual de Molécula/métodos , Algoritmos , Linhagem Celular , Simulação por Computador , DNA/química , DNA/ultraestrutura , Humanos , Imageamento Tridimensional , Conformação Molecular , Poro Nuclear/química , Poro Nuclear/ultraestrutura , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/ultraestrutura , Razão Sinal-Ruído , Imagem Individual de Molécula/estatística & dados numéricosRESUMO
Nuclear lamin isoforms form fibrous meshworks associated with nuclear pore complexes (NPCs). Using datasets prepared from subpixel and segmentation analyses of 3D-structured illumination microscopy images of WT and lamin isoform knockout mouse embryo fibroblasts, we determined with high precision the spatial association of NPCs with specific lamin isoform fibers. These relationships are retained in the enlarged lamin meshworks of Lmna-/- and Lmnb1-/- fibroblast nuclei. Cryo-ET observations reveal that the lamin filaments composing the fibers contact the nucleoplasmic ring of NPCs. Knockdown of the ring-associated nucleoporin ELYS induces NPC clusters that exclude lamin A/C fibers but include LB1 and LB2 fibers. Knockdown of the nucleoporin TPR or NUP153 alters the arrangement of lamin fibers and NPCs. Evidence that the number of NPCs is regulated by specific lamin isoforms is presented. Overall the results demonstrate that lamin isoforms and nucleoporins act together to maintain the normal organization of lamin meshworks and NPCs within the nuclear envelope.
Assuntos
Simulação por Computador , Embrião de Mamíferos/metabolismo , Fibroblastos/metabolismo , Lamina Tipo A/metabolismo , Lamina Tipo B/metabolismo , Poro Nuclear/metabolismo , Animais , Linhagem Celular , Embrião de Mamíferos/ultraestrutura , Fibroblastos/ultraestrutura , Lamina Tipo A/genética , Lamina Tipo B/genética , Camundongos , Camundongos Knockout , Poro Nuclear/genética , Poro Nuclear/ultraestrutura , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismoRESUMO
Human immunodeficiency virus (HIV-1) remains a major health threat. Viral capsid uncoating and nuclear import of the viral genome are critical for productive infection. The size of the HIV-1 capsid is generally believed to exceed the diameter of the nuclear pore complex (NPC), indicating that capsid uncoating has to occur prior to nuclear import. Here, we combined correlative light and electron microscopy with subtomogram averaging to capture the structural status of reverse transcription-competent HIV-1 complexes in infected T cells. We demonstrated that the diameter of the NPC in cellulo is sufficient for the import of apparently intact, cone-shaped capsids. Subsequent to nuclear import, we detected disrupted and empty capsid fragments, indicating that uncoating of the replication complex occurs by breaking the capsid open, and not by disassembly into individual subunits. Our data directly visualize a key step in HIV-1 replication and enhance our mechanistic understanding of the viral life cycle.
Assuntos
Capsídeo/metabolismo , HIV-1/metabolismo , Poro Nuclear/metabolismo , Transporte Ativo do Núcleo Celular , Capsídeo/ultraestrutura , Microscopia Crioeletrônica , Células HEK293 , Infecções por HIV/virologia , HIV-1/ultraestrutura , Humanos , Modelos Biológicos , Poro Nuclear/ultraestrutura , Poro Nuclear/virologia , Transcrição Reversa , Vírion/metabolismo , Internalização do Vírus , Fatores de Poliadenilação e Clivagem de mRNA/metabolismoRESUMO
The nuclear pore complex (NPC) is a massive nuclear envelope-embedded protein complex, the canonical function of which is to mediate selective nucleocytoplasmic transport. In addition to its transport function, the NPC has been shown to interact with the underlying chromatin and to influence both activating and repressive gene regulatory processes, contributing to the establishment and the epigenetic maintenance of cell identity. In this review, we discuss diverse gene regulatory functions of NPC components and emerging mechanisms underlying these functions, including roles in genome architecture, transcription complex assembly, chromatin remodeling, and coordination of transcription and mRNA export. These functional roles highlight the importance of the NPC as a nuclear scaffold directing genome organization and function.
Assuntos
Transporte Ativo do Núcleo Celular/genética , Cromatina/ultraestrutura , Genoma/genética , Poro Nuclear/ultraestrutura , Cromatina/genética , Regulação da Expressão Gênica/genética , Humanos , Membrana Nuclear/genética , Membrana Nuclear/ultraestrutura , Poro Nuclear/genéticaRESUMO
It is recognized that HIV-1 capsid cores are disassembled in the cytoplasm, releasing their genomes into the nucleus through nuclear pores, but there is also evidence showing the capsid (CA) exists in the nucleus. Whether HIV-1 enters the nucleus and how it enters the nucleus through the undersized nuclear pore remains mysterious. Based on multicolor labeling and real-time imaging of the viral and cellular components, our observations via light and electron microscopy suggest that HIV-1 selectively gathered at the microtubule organization center (MTOC), leading the nearby nuclear envelope (NE) to undergo deformation, invagination and restoration to form a nuclear vesicle in which the viral particles were wrapped; then, the inner membrane of the nuclear vesicle ruptured to release HIV-1 into the nucleus. This unexpected discovery expands our understanding of the complexity of HIV-1 nuclear entry, which may provide new insights to HIV-1 virology.
Assuntos
Proteínas do Capsídeo/metabolismo , Núcleo Celular/metabolismo , Endocitose , HIV-1/metabolismo , Poro Nuclear/metabolismo , Vírion/metabolismo , Transporte Ativo do Núcleo Celular , Linhagem Celular Tumoral , Núcleo Celular/ultraestrutura , Núcleo Celular/virologia , Células HEK293 , Infecções por HIV/virologia , HIV-1/genética , HIV-1/fisiologia , Humanos , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Centro Organizador dos Microtúbulos/metabolismo , Centro Organizador dos Microtúbulos/virologia , Membrana Nuclear/metabolismo , Membrana Nuclear/ultraestrutura , Membrana Nuclear/virologia , Poro Nuclear/ultraestrutura , Poro Nuclear/virologia , Imagem com Lapso de Tempo/métodos , Vírion/ultraestruturaRESUMO
Nuclear pore complexes (NPCs) are the main conduits for molecular exchange across the nuclear envelope. The NPC is a modular assembly of ~500 individual proteins, called nucleoporins or nups. Most scaffolding nups are organized in two multimeric subcomplexes, the Nup84 or Y complex and the Nic96 or inner ring complex. Working in S. cerevisiae, and to study the assembly of these two essential subcomplexes, we here develop a set of twelve nanobodies that recognize seven constituent nucleoporins of the Y and Nic96 complexes. These nanobodies all bind specifically and with high affinity. We present structures of several nup-nanobody complexes, revealing their binding sites. Additionally, constitutive expression of the nanobody suite in S. cerevisiae detect accessible and obstructed surfaces of the Y complex and Nic96 within the NPC. Overall, this suite of nanobodies provides a unique and versatile toolkit for the study of the NPC.
Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares/química , Poro Nuclear/ultraestrutura , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/ultraestrutura , Anticorpos de Domínio Único/química , Sequência de Aminoácidos , Animais , Afinidade de Anticorpos , Especificidade de Anticorpos , Sítios de Ligação , Camelídeos Americanos , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Cinética , Modelos Moleculares , Poro Nuclear/química , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Biblioteca de Peptídeos , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/isolamento & purificação , Anticorpos de Domínio Único/metabolismoRESUMO
Single-molecule localization microscopy (SMLM) is a powerful tool for studying intracellular structure and macromolecular organization at the nanoscale. The increasingly massive pointillistic data sets generated by SMLM require the development of new and highly efficient quantification tools. Here we present FOCAL3D, an accurate, flexible and exceedingly fast (scaling linearly with the number of localizations) density-based algorithm for quantifying spatial clustering in large 3D SMLM data sets. Unlike DBSCAN, which is perhaps the most commonly employed density-based clustering algorithm, an optimum set of parameters for FOCAL3D may be objectively determined. We initially validate the performance of FOCAL3D on simulated datasets at varying noise levels and for a range of cluster sizes. These simulated datasets are used to illustrate the parametric insensitivity of the algorithm, in contrast to DBSCAN, and clustering metrics such as the F1 and Silhouette score indicate that FOCAL3D is highly accurate, even in the presence of significant background noise and mixed populations of variable sized clusters, once optimized. We then apply FOCAL3D to 3D astigmatic dSTORM images of the nuclear pore complex (NPC) in human osteosaracoma cells, illustrating both the validity of the parameter optimization and the ability of the algorithm to accurately cluster complex, heterogeneous 3D clusters in a biological dataset. FOCAL3D is provided as an open source software package written in Python.
Assuntos
Imageamento Tridimensional/métodos , Imagem Individual de Molécula/métodos , Algoritmos , Análise por Conglomerados , Conjuntos de Dados como Assunto , Humanos , Poro Nuclear/ultraestrutura , Osteossarcoma/ultraestrutura , Linguagens de Programação , Software , Células Tumorais CultivadasRESUMO
While the static structure of the nuclear pore complex (NPC) continues to be refined with cryo-EM and x-ray crystallography, in vivo conformational changes of the NPC remain under-explored. We developed sensors that report on the orientation of NPC components by rigidly conjugating mEGFP to different NPC proteins. Our studies show conformational changes to select domains of nucleoporins (Nups) within the inner ring (Nup54, Nup58, Nup62) when transport through the NPC is perturbed and no conformational changes to Nups elsewhere in the NPC. Our results suggest that select components of the NPC are flexible and undergo conformational changes upon engaging with cargo.
Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Poro Nuclear/química , Poro Nuclear/ultraestrutura , Linhagem Celular , Humanos , Conformação Molecular , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/ultraestrutura , Conformação ProteicaRESUMO
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 exchange1,2. 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 cells3 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.
Assuntos
Microscopia Crioeletrônica , Poro Nuclear/metabolismo , Poro Nuclear/ultraestrutura , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/ultraestrutura , Autofagia , Modelos Moleculares , Poro Nuclear/química , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , TomografiaRESUMO
The mechanisms underlying turnover of the nuclear pore complex (NPC) and the component nucleoporins (Nups) are still poorly understood. In this study, we found that the budding yeast Saccharomyces cerevisiae triggers NPC degradation by autophagy upon the inactivation of Tor kinase complex 1. This degradation largely depends on the selective autophagy-specific factor Atg11 and the autophagy receptor-binding ability of Atg8, suggesting that the NPC is degraded via receptor-dependent selective autophagy. Immunoelectron microscopy revealed that NPCs embedded in nuclear envelope-derived double-membrane vesicles are sequestered within autophagosomes. At least two pathways are involved in NPC degradation: Atg39-dependent nucleophagy (selective autophagy of the nucleus) and a pathway involving an unknown receptor. In addition, we found the interaction between Nup159 and Atg8 via the Atg8-family interacting motif is important for degradation of this nucleoporin not assembled into the NPC. Thus, this study provides the first evidence for autophagic degradation of the NPC and Nups, which we term "NPC-phagy" and "nucleoporinophagy."
Assuntos
Família da Proteína 8 Relacionada à Autofagia/genética , Proteínas Relacionadas à Autofagia/genética , Autofagia/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Poro Nuclear/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Proteínas de Transporte Vesicular/genética , Autofagossomos/efeitos dos fármacos , Autofagossomos/metabolismo , Autofagia/efeitos dos fármacos , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Regulação Fúngica da Expressão Gênica , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Microscopia Imunoeletrônica , Poro Nuclear/efeitos dos fármacos , Poro Nuclear/ultraestrutura , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Ligação Proteica , Inibidores de Proteínas Quinases/farmacologia , Proteólise/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Sirolimo/farmacologia , Proteínas de Transporte Vesicular/metabolismoRESUMO
Dynamic tracking of the spatiotemporal coordination among various organelles to in-depth understanding of the mechanism of autophagy have attracted considerable attention. However, the monitor of nucleoli participation in autophagy was somehow neglected. Herein, we report a RNA-targeted bioprobe (ADAP) with high selective permeability into nuclear pore complexes, which induced a two-photon (TP) fluorescence "off-on" response by groove combination with RNA, dynamically monitoring the autophagy process among multiorganelles (nucleoli, mitochondria, and mitochondria-containing lysosomes). This work provides a simple and convenient way to observe the dynamic behavior of multiorganelles during the autophagy process, which benefits the understanding of cellular metabolism.