Structure of cytoplasmic ring of nuclear pore complex by integrative cryo-EM and AlphaFold

Science, Journal Year: 2022, Volume and Issue: 376(6598)

Published: June 9, 2022

INTRODUCTION The nuclear pore complex (NPC) is the molecular conduit in membrane of eukaryotic cells that regulates import and export biomolecules between nucleus cytosol, with vertebrate NPCs ~110 to 125 MDa mass ~120 nm diameter. are organized into four main rings: cytoplasmic ring (CR) at cytosolic side, inner luminal on plane membrane, facing nucleus. Each possesses an approximate eightfold symmetry composed multiple copies different nucleoporins. have been implicated numerous biological processes, their dysfunctions associated a growing number serious human diseases. However, despite pioneering studies from many groups over past two decades, we still lack full understanding NPCs' organization, dynamics, complexity. RATIONALE We used Xenopus laevis oocyte as model system for structural characterization because each large NPC particles can be visualized native membranes without aid detergent extraction. single-particle cryo-electron microscopy (cryo-EM) analysis data collected stage tilt angles three-dimensional reconstruction structure prediction AlphaFold building. RESULTS reconstructed CR map X. 6.9 6.7 Å resolutions protomer core region, respectively, predicted structures individual nucleoporins using no high-resolution models Nups were available. For any ambiguous subunit interactions, also structures, which further guided fitting protomer. placed nucleoporin or density obtain almost atomic model, outer Y-complexes, Nup205, Nup214-Nup88-Nup62 complex, one Nup155, five Nup358. In particular, largest protein NPC, Nup358, having S-shaped globular domain, coiled-coil largely disordered C-terminal region containing phenylalanine-glycine (FG) repeats previously shown form gel-like condensate phase selective cargo passage. Four Nup358 clamp around Y-complexes stabilize CR, fifth situates center cluster clamps. homo-oligomeric, likely specifically pentameric, may provide avidity recruitment lowering threshold condensation biogenesis. CONCLUSION Our offer example integrative cryo-EM general approach attaining more precise megadalton complexes medium-resolution maps. accurate complete presented here expands our interactions represents substantial step forward toward architecture implications function, biogenesis, regulation. [Figure: see text].

Language: Английский

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Bringing Structure to Cell Biology with Cryo-Electron Tomography

Annual Review of Biophysics, Journal Year: 2023, Volume and Issue: 52(1), P. 573 - 595

Published: May 9, 2023

Recent advances in cryo-electron microscopy have marked only the beginning of potential this technique. To bring structure into cell biology, modality tomography has fast developed a bona fide situ structural biology technique where structures are determined their native environment, cell. Nearly every step cryo-focused ion beam-assisted electron (cryo-FIB-ET) workflow been improved upon past decade, since first windows were carved cells, unveiling macromolecular networks near-native conditions. By bridging and cryo-FIB-ET is advancing our understanding structure–function relationships environment becoming tool for discovering new biology.

Language: Английский

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Visualizing the disordered nuclear transport machinery in situ

Nature, Journal Year: 2023, Volume and Issue: 617(7959), P. 162 - 169

Published: April 26, 2023

Abstract The approximately 120 MDa mammalian nuclear pore complex (NPC) acts as a gatekeeper for the transport between nucleus and cytosol 1 . central channel of NPC is filled with hundreds intrinsically disordered proteins (IDPs) called FG-nucleoporins (FG-NUPs) 2,3 Although structure scaffold has been resolved in remarkable detail, actual machinery built up by FG-NUPs—about 50 MDa—is depicted an 60-nm hole even highly tomograms and/or structures computed artificial intelligence 4–11 Here we directly probed conformations vital FG-NUP98 inside NPCs live cells permeabilized intact using synthetic biology-enabled site-specific small-molecule labelling approach paired time-resolved fluorescence microscopy. Single cell measurements distance distribution segments combined coarse-grained molecular simulations allowed us to map uncharted environment nanosized channel. We determined that provides—in terminology Flory polymer theory 12 —a ‘good solvent’ environment. This enables FG domain adopt expanded thus control cytoplasm. With more than 30% proteome being formed from IDPs, our study opens window into resolving disorder–function relationships IDPs situ, which are important various processes, such cellular signalling, phase separation, ageing viral entry.

Language: Английский

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Nuclear transport proteins: structure, function, and disease relevance

Signal Transduction and Targeted Therapy, Journal Year: 2023, Volume and Issue: 8(1)

Published: Nov. 10, 2023

Abstract Proper subcellular localization is crucial for the functioning of biomacromolecules, including proteins and RNAs. Nuclear transport a fundamental cellular process that regulates many macromolecules within nuclear or cytoplasmic compartments. In humans, approximately 60 are involved in transport, nucleoporins form membrane-embedded pore complexes, karyopherins cargoes through these Ran system ensure directed rapid transport. Many play additional essential roles mitosis, biomolecular condensation, gene transcription. Dysregulation linked to major human diseases such as cancer, neurodegenerative diseases, viral infections. Selinexor (KPT-330), an inhibitor targeting export factor XPO1 (also known CRM1), was approved 2019 treat two types blood cancers, dozens clinical trials ongoing. This review summarizes three decades research data this field but focuses on structure function individual from recent studies, providing cutting-edge holistic view role health disease. In-depth knowledge rapidly evolving has potential bring new insights into biology, pathogenic mechanisms, therapeutic approaches.

Language: Английский

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The molecular architecture of the nuclear basket

Cell, Journal Year: 2024, Volume and Issue: 187(19), P. 5267 - 5281.e13

Published: Aug. 9, 2024

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

Language: Английский

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Protein import into peroxisomes occurs through a nuclear pore–like phase

Science, Journal Year: 2022, Volume and Issue: 378(6625)

Published: Dec. 15, 2022

Peroxisomes are ubiquitous organelles whose dysfunction causes fatal human diseases. Most peroxisomal proteins imported from the cytosol in a folded state by soluble receptor PEX5. How cargo crosses membrane is unknown. Here, we show that import similar to nuclear transport. The protein PEX13 contains conserved tyrosine (Y)- and glycine (G)-rich YG domain, which forms selective phase resembling formed phenylalanine-glycine (FG) repeats within pores. resides two orientations oligomerize suspend meshwork lipid bilayer. Purified domains form hydrogels into PEX5 selectively partitions, using aromatic amino acid motifs, bringing along. thus an aqueous conduit through delivers peroxisomes.

Language: Английский

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The Nuclear Pore Complex: Birth, Life, and Death of a Cellular Behemoth

Cells, Journal Year: 2022, Volume and Issue: 11(9), P. 1456 - 1456

Published: April 25, 2022

Nuclear pore complexes (NPCs) are the only transport channels that cross nuclear envelope. Constructed from ~500–1000 nucleoporin proteins each, they among largest macromolecular assemblies in eukaryotic cells. Thanks to advances structural analysis approaches, construction principles and architecture of NPC have recently been revealed at submolecular resolution. Although overall structure inventory nucleoporins conserved, NPCs exhibit significant compositional functional plasticity even within single cells surprising variability their assembly pathways. Once assembled, remain seemingly unexchangeable post-mitotic There a number as yet unresolved questions about how versatility composition is established, monitor state or could be renewed. Here, we review current progress our understanding key aspects lifecycle.

Language: Английский

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Structure and Function of the Nuclear Pore Complex

Cold Spring Harbor Perspectives in Biology, Journal Year: 2022, Volume and Issue: unknown, P. a041264 - a041264

Published: Sept. 12, 2022

The nucleus, a genome-containing organelle eponymous of eukaryotes, is enclosed by double membrane continuous with the endoplasmic reticulum. nuclear pore complex (NPC) an ∼110-MDa, ∼1000-protein channel that selectively transports macromolecules across envelope and thus plays central role in regulated flow genetic information from transcription to translation. Its size, complexity, flexibility have hindered determination atomistic structures intact NPCs. Recent studies overcome these hurdles combining biochemical reconstitution docking high-resolution NPC subcomplexes into cryo-electron tomographic reconstructions physiological validation. Here, we provide overview near-atomic composite structure human NPC, milestone toward unlocking molecular understanding mRNA export, NPC-associated diseases, viral host-pathogen interactions, serving as paradigm for studying similarly large complexes.

Language: Английский

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Lipid saturation controls nuclear envelope function

Nature Cell Biology, Journal Year: 2023, Volume and Issue: 25(9), P. 1290 - 1302

Published: Aug. 17, 2023

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

Language: Английский

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Phase separation of intrinsically disordered FG-Nups is driven by highly dynamic FG motifs

Proceedings of the National Academy of Sciences, Journal Year: 2023, Volume and Issue: 120(25)

Published: June 12, 2023

The intrinsically disordered FG-Nups in the central channel of nuclear pore complex (NPC) form a selective permeability barrier, allowing small molecules to traverse by passive diffusion, while large can only translocate with help transport receptors. exact phase state barrier remains elusive. In vitro experiments have shown that some undergo separation into condensates display NPC-like properties. Here, we use molecular dynamics simulations at amino acid resolution study characteristics each yeast NPC. We find GLFG-Nups and reveal FG motifs act as highly dynamic hydrophobic stickers are essential for formation FG-Nup featuring droplet-spanning percolated networks. Additionally, an mixture resembles NPC stoichiometry observe condensate is formed containing multiple GLFG-Nups. this also driven FG-FG interactions, similar homotypic condensates. Based on observed behavior, different be divided two classes: (mostly GLFG-type) located network many short-lived peripheral FxFG-type) entry exit likely entropic brush.

Language: Английский

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