RESUMEN
Ribosomes translate the genetic code into proteins. Recent technical advances have facilitated in situ structural analyses of ribosome functional states inside eukaryotic cells and the minimal bacterium Mycoplasma. However, such analyses of Gram-negative bacteria are lacking, despite their ribosomes being major antimicrobial drug targets. Here we compare two E. coli strains, a lab E. coli K-12 and human gut isolate E. coli ED1a, for which tetracycline exhibits bacteriostatic and bactericidal action, respectively. Using our approach for close-to-native E. coli sample preparation, we assess the two strains by cryo-ET and visualize their ribosomes at high resolution in situ. Upon tetracycline treatment, these exhibit virtually identical drug binding sites, yet the conformation distribution of ribosomal complexes differs. While K-12 retains ribosomes in a translation-competent state, tRNAs are lost in the vast majority of ED1a ribosomes. These structural findings together with the proteome-wide abundance and thermal stability assessments indicate that antibiotic responses are complex in cells and can differ between different strains of a single species, thus arguing that all relevant bacterial strains should be analyzed in situ when addressing antibiotic mode of action.
Asunto(s)
Antibacterianos , Escherichia coli , Ribosomas , Tetraciclina , Ribosomas/metabolismo , Ribosomas/efectos de los fármacos , Antibacterianos/farmacología , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Tetraciclina/farmacología , Microscopía por Crioelectrón , ARN de Transferencia/metabolismo , ARN de Transferencia/genética , Humanos , Sitios de Unión , Biosíntesis de Proteínas/efectos de los fármacos , Escherichia coli K12/efectos de los fármacos , Escherichia coli K12/genética , Escherichia coli K12/metabolismoRESUMEN
The thermophilic fungus Chaetomium thermophilum has been used extensively for biochemical and high-resolution structural studies of protein complexes. However, subsequent functional analyses of these assemblies have been hindered owing to the lack of genetic tools compatible with this thermophile, which are typically suited to other mesophilic eukaryotic model organisms, in particular the yeast Saccharomyces cerevisiae. Hence, we aimed to find genes from C. thermophilum that are expressed under the control of different sugars and examine their associated 5' untranslated regions as promoters responsible for sugar-regulated gene expression. To identify sugar-regulated promoters in C. thermophilum, we performed comparative xylose- versus glucose-dependent gene expression studies, which uncovered a number of enzymes with induced expression in the presence of xylose but repressed expression in glucose-supplemented media. Subsequently, we cloned the promoters of the two most stringently regulated genes, the xylosidase-like gene (XYL) and xylitol dehydrogenase (XDH), obtained from this genome-wide analysis in front of a thermostable yellow fluorescent protein (YFP) reporter. With this, we demonstrated xylose-dependent YFP expression by both Western blotting and live-cell imaging fluorescence microscopy. Prompted by these results, we expressed the C. thermophilum orthologue of a well-characterized dominant-negative ribosome assembly factor mutant, under the control of the XDH promoter, which allowed us to induce a nuclear export defect on the pre-60S subunit when C. thermophilum cells were grown in xylose- but not glucose-containing medium. Altogether, our study identified xylose-regulatable promoters in C. thermophilum, which might facilitate functional studies of genes of interest in this thermophilic eukaryotic model organism.
Asunto(s)
Chaetomium , Azúcares , Azúcares/metabolismo , Xilosa/metabolismo , Chaetomium/genética , Chaetomium/metabolismo , Saccharomyces cerevisiae/genética , Glucosa/metabolismoRESUMEN
Cryo-electron tomograms capture a wealth of structural information on the molecular constituents of cells and tissues. We present DeePiCt (deep picker in context), an open-source deep-learning framework for supervised segmentation and macromolecular complex localization in cryo-electron tomography. To train and benchmark DeePiCt on experimental data, we comprehensively annotated 20 tomograms of Schizosaccharomyces pombe for ribosomes, fatty acid synthases, membranes, nuclear pore complexes, organelles, and cytosol. By comparing DeePiCt to state-of-the-art approaches on this dataset, we show its unique ability to identify low-abundance and low-density complexes. We use DeePiCt to study compositionally distinct subpopulations of cellular ribosomes, with emphasis on their contextual association with mitochondria and the endoplasmic reticulum. Finally, applying pre-trained networks to a HeLa cell tomogram demonstrates that DeePiCt achieves high-quality predictions in unseen datasets from different biological species in a matter of minutes. The comprehensively annotated experimental data and pre-trained networks are provided for immediate use by the community.
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Mitocondrias , Ribosomas , Humanos , Células HeLa , Tomografía con Microscopio Electrónico/métodos , Retículo Endoplásmico , Procesamiento de Imagen Asistido por Computador/métodosRESUMEN
Muscle size is controlled by the PI3K-PKB/Akt-mTORC1-FoxO pathway, which integrates signals from growth factors, energy and amino acids to activate protein synthesis and inhibit protein breakdown. While mTORC1 activity is necessary for PKB/Akt-induced muscle hypertrophy, its constant activation alone induces muscle atrophy. Here we show that this paradox is based on mTORC1 activity promoting protein breakdown through the ubiquitin-proteasome system (UPS) by simultaneously inducing ubiquitin E3 ligase expression via feedback inhibition of PKB/Akt and proteasome biogenesis via Nuclear Factor Erythroid 2-Like 1 (Nrf1). Muscle growth was restored by reactivation of PKB/Akt, but not by Nrf1 knockdown, implicating ubiquitination as the limiting step. However, both PKB/Akt activation and proteasome depletion by Nrf1 knockdown led to an immediate disruption of proteome integrity with rapid accumulation of damaged material. These data highlight the physiological importance of mTORC1-mediated PKB/Akt inhibition and point to juxtaposed roles of the UPS in atrophy and proteome integrity.
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Complejo de la Endopetidasa Proteasomal , Ubiquitina , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteostasis , Proteoma/metabolismo , Músculo Esquelético/metabolismoRESUMEN
INTRODUCTION The eukaryotic nucleus pro-tects the genome and is enclosed by the two membranes of the nuclear envelope. Nuclear pore complexes (NPCs) perforate the nuclear envelope to facilitate nucleocytoplasmic transport. With a molecular weight of â¼120 MDa, the human NPC is one of the larg-est protein complexes. Its ~1000 proteins are taken in multiple copies from a set of about 30 distinct nucleoporins (NUPs). They can be roughly categorized into two classes. Scaf-fold NUPs contain folded domains and form a cylindrical scaffold architecture around a central channel. Intrinsically disordered NUPs line the scaffold and extend into the central channel, where they interact with cargo complexes. The NPC architecture is highly dynamic. It responds to changes in nuclear envelope tension with conforma-tional breathing that manifests in dilation and constriction movements. Elucidating the scaffold architecture, ultimately at atomic resolution, will be important for gaining a more precise understanding of NPC function and dynamics but imposes a substantial chal-lenge for structural biologists. RATIONALE Considerable progress has been made toward this goal by a joint effort in the field. A synergistic combination of complementary approaches has turned out to be critical. In situ structural biology techniques were used to reveal the overall layout of the NPC scaffold that defines the spatial reference for molecular modeling. High-resolution structures of many NUPs were determined in vitro. Proteomic analysis and extensive biochemical work unraveled the interaction network of NUPs. Integra-tive modeling has been used to combine the different types of data, resulting in a rough outline of the NPC scaffold. Previous struc-tural models of the human NPC, however, were patchy and limited in accuracy owing to several challenges: (i) Many of the high-resolution structures of individual NUPs have been solved from distantly related species and, consequently, do not comprehensively cover their human counterparts. (ii) The scaf-fold is interconnected by a set of intrinsically disordered linker NUPs that are not straight-forwardly accessible to common structural biology techniques. (iii) The NPC scaffold intimately embraces the fused inner and outer nuclear membranes in a distinctive topol-ogy and cannot be studied in isolation. (iv) The conformational dynamics of scaffold NUPs limits the resolution achievable in structure determination. RESULTS In this study, we used artificial intelligence (AI)-based prediction to generate an exten-sive repertoire of structural models of human NUPs and their subcomplexes. The resulting models cover various domains and interfaces that so far remained structurally uncharac-terized. Benchmarking against previous and unpublished x-ray and cryo-electron micros-copy structures revealed unprecedented accu-racy. We obtained well-resolved cryo-electron tomographic maps of both the constricted and dilated conformational states of the hu-man NPC. Using integrative modeling, we fit-ted the structural models of individual NUPs into the cryo-electron microscopy maps. We explicitly included several linker NUPs and traced their trajectory through the NPC scaf-fold. We elucidated in great detail how mem-brane-associated and transmembrane NUPs are distributed across the fusion topology of both nuclear membranes. The resulting architectural model increases the structural coverage of the human NPC scaffold by about twofold. We extensively validated our model against both earlier and new experimental data. The completeness of our model has enabled microsecond-long coarse-grained molecular dynamics simulations of the NPC scaffold within an explicit membrane en-vironment and solvent. These simulations reveal that the NPC scaffold prevents the constriction of the otherwise stable double-membrane fusion pore to small diameters in the absence of membrane tension. CONCLUSION Our 70-MDa atomically re-solved model covers >90% of the human NPC scaffold. It captures conforma-tional changes that occur during dilation and constriction. It also reveals the precise anchoring sites for intrinsically disordered NUPs, the identification of which is a prerequisite for a complete and dy-namic model of the NPC. Our study exempli-fies how AI-based structure prediction may accelerate the elucidation of subcellular ar-chitecture at atomic resolution. [Figure: see text].
Asunto(s)
Inteligencia Artificial , Proteínas de Complejo Poro Nuclear , Poro Nuclear , Transporte Activo de Núcleo Celular , Microscopía por Crioelectrón , Humanos , Simulación de Dinámica Molecular , Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/química , ProteómicaRESUMEN
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.
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Membrana Nuclear/fisiología , Poro Nuclear/fisiología , Poro Nuclear/ultraestructura , Transporte Activo de Núcleo Celular , Fenómenos Biomecánicos , Microscopía por Crioelectrón , Citoplasma/metabolismo , Metabolismo Energético , Procesamiento de Imagen Asistido por Computador , Imagenología Tridimensional , Modelos Biológicos , Membrana Nuclear/ultraestructura , Proteínas de Complejo Poro Nuclear/química , Presión Osmótica , Schizosaccharomyces/crecimiento & desarrollo , Schizosaccharomyces/ultraestructura , Proteínas de Schizosaccharomyces pombe/química , Estrés FisiológicoRESUMEN
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.
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Cápside/metabolismo , VIH-1/metabolismo , Poro Nuclear/metabolismo , Transporte Activo de Núcleo Celular , Cápside/ultraestructura , Microscopía por Crioelectrón , Células HEK293 , Infecciones por VIH/virología , VIH-1/ultraestructura , Humanos , Modelos Biológicos , Poro Nuclear/ultraestructura , Poro Nuclear/virología , Transcripción Reversa , Virión/metabolismo , Internalización del Virus , Factores de Escisión y Poliadenilación de ARNm/metabolismoRESUMEN
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.
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Microscopía por Crioelectrón , Poro Nuclear/metabolismo , Poro Nuclear/ultraestructura , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/ultraestructura , Autofagia , Modelos Moleculares , Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , TomografíaRESUMEN
Cryo electron tomography with subsequent subtomogram averaging is a powerful technique to structurally analyze macromolecular complexes in their native context. Although close to atomic resolution in principle can be obtained, it is not clear how individual experimental parameters contribute to the attainable resolution. Here, we have used immature HIV-1 lattice as a benchmarking sample to optimize the attainable resolution for subtomogram averaging. We systematically tested various experimental parameters such as the order of projections, different angular increments and the use of the Volta phase plate. We find that although any of the prominently used acquisition schemes is sufficient to obtain subnanometer resolution, dose-symmetric acquisition provides considerably better outcome. We discuss our findings in order to provide guidance for data acquisition. Our data is publicly available and might be used to further develop processing routines.
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Microscopía por Crioelectrón/métodos , Tomografía con Microscopio Electrónico/métodos , Benchmarking , Microscopía por Crioelectrón/normas , Bases de Datos Factuales , Tomografía con Microscopio Electrónico/normas , VIH-1/química , VIH-1/ultraestructura , Sustancias Macromoleculares/química , Sustancias Macromoleculares/ultraestructura , Modelos Moleculares , Biología Molecular/métodos , Biología Molecular/normas , Virión/química , Virión/ultraestructuraRESUMEN
The ability to comprehensively characterize biological samples, including tissues and body fluids, opens up new possibilities to diagnose and treat diseases and to better understand fundamental biological processes. For this purpose, suitable experimental workflows need to be designed. In this context, materials with particular chemoselective properties are used for the enrichment of certain classes of (bio)molecules. Metal oxides such as titanium dioxide have become the materials of choice for the large-scale study of protein phosphorylation in phosphoproteomics. Despite their widespread use, the main factors influencing their performance (for example, affinity and specificity) are not completely understood. This understanding is, however, crucial to develop improved materials and methods. Here, we used the nanocasting method to prepare microspheres of seven metal oxides with comparable textural properties, allowing an objective comparison of the materials and their binding properties. We evaluated these materials with samples of different complexity, ranging from synthetic peptides to whole cell lysates, using liquid chromatography-tandem mass spectrometry as a readout. A set of more than 7000 identified phosphopeptides allowed us to study differences between the metal oxide sorbents in detail. Importantly, the performance of the affinity materials was found to be mainly correlated with the oxides' isoelectric points (IEPs), with the materials that enriched the highest number of phosphopeptides having an IEP of around 6. This included the widely used TiO2 and ZrO2, but also In2O3 that was not previously known to possess affinity to phosphates. This finding supports the conclusion that the IEP has a stronger influence than the particular type of metal oxide and contrasts earlier reports that compared a limited number of materials with often unknown textural properties. Taken together, we introduce new metal oxides suitable for phosphopeptide enrichment, provide deeper insight into the properties that affect their performance, and offer a strategy to optimize enrichment protocols and materials.