Nuclear pores dilate and constrict in cellulo

Science, Journal Year: 2021, Volume and Issue: 374(6573)

Published: Dec. 9, 2021

In eukaryotic cells, nuclear pore complexes (NPCs) fuse the inner and outer membranes mediate nucleocytoplasmic exchange. They are made of 30 different nucleoporins form a cylindrical architecture around an aqueous central channel. This is highly dynamic in space time. Variations NPC diameter have been reported, but physiological circumstances molecular details remain unknown. Here, we combined cryo–electron tomography with integrative structural modeling to capture movie respective large-scale conformational changes cellulo. Although NPCs exponentially growing cells adopted dilated conformation, they reversibly constricted upon cellular energy depletion or conditions hypertonic osmotic stress. Our data point model where envelope membrane tension linked conformation NPC.

Language: Английский

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AI-based structure prediction empowers integrative structural analysis of human nuclear pores

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

Published: June 9, 2022

INTRODUCTION The eukaryotic nucleus pro-tects the genome and is enclosed by two membranes of nuclear envelope. Nuclear pore complexes (NPCs) perforate envelope to facilitate nucleocytoplasmic transport. With a molecular weight ∼120 MDa, human NPC one larg-est protein complexes. Its ~1000 proteins are taken in multiple copies from set about 30 distinct nucleoporins (NUPs). They can be roughly categorized into classes. Scaf-fold NUPs contain folded domains form cylindrical scaffold architecture around central channel. Intrinsically disordered line extend channel, where they interact with cargo highly dynamic. It responds changes tension conforma-tional breathing that manifests dilation constriction movements. Elucidating architecture, ultimately at atomic resolution, will important for gaining more precise understanding function dynamics but imposes substantial chal-lenge structural biologists. RATIONALE Considerable progress has been made toward this goal joint effort field. A synergistic combination complementary approaches turned out critical. In situ biology techniques were used reveal overall layout defines spatial reference modeling. High-resolution structures many determined vitro. Proteomic analysis extensive biochemical work unraveled interaction network NUPs. Integra-tive modeling combine different types data, resulting rough outline scaffold. Previous struc-tural models NPC, however, patchy limited accuracy owing several challenges: (i) Many high-resolution individual have solved distantly related species and, consequently, do not comprehensively cover their counterparts. (ii) scaf-fold interconnected intrinsically linker straight-forwardly accessible common techniques. (iii) intimately embraces fused inner outer distinctive topol-ogy cannot studied isolation. (iv) conformational limits resolution achievable structure determination. RESULTS study, we artificial intelligence (AI)-based prediction generate an exten-sive repertoire subcomplexes. various interfaces so far remained structurally uncharac-terized. Benchmarking against previous unpublished x-ray cryo-electron micros-copy revealed unprecedented accu-racy. We obtained well-resolved tomographic maps both constricted dilated states hu-man NPC. Using integrative modeling, fit-ted microscopy maps. explicitly included traced trajectory through scaf-fold. elucidated great detail how mem-brane-associated transmembrane distributed across fusion topology membranes. architectural model increases coverage twofold. extensively validated our earlier new experimental data. completeness enabled microsecond-long coarse-grained simulations within explicit membrane en-vironment solvent. These prevents otherwise stable double-membrane small diameters absence tension. CONCLUSION Our 70-MDa atomically re-solved covers >90% captures occur during constriction. also reveals anchoring sites NUPs, identification which prerequisite complete dy-namic study exempli-fies AI-based may accelerate elucidation subcellular ar-chitecture resolution. [Figure: see text].

Language: Английский

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The cellular environment shapes the nuclear pore complex architecture

Nature, Journal Year: 2021, Volume and Issue: 598(7882), P. 667 - 671

Published: Oct. 13, 2021

Abstract Nuclear pore complexes (NPCs) create large conduits for cargo transport between the nucleus and cytoplasm across nuclear envelope (NE) 1–3 . These multi-megadalton structures are composed of about thirty different nucleoporins that distributed in three main substructures (the inner, cytoplasmic nucleoplasmic rings) around central channel 4–6 Here we use cryo-electron tomography on DLD-1 cells were prepared using cryo-focused-ion-beam milling to generate a structural model human NPC its native environment. We show that—compared with previous models obtained from purified NEs—the inner ring our is substantially wider; volume increased by 75% rings reorganized. Moreover, membrane exhibits asymmetry inner-ring complex. Using targeted degradation Nup96, scaffold nucleoporin rings, observe interdependence each modulating maintaining asymmetry. Our findings highlight inherent flexibility suggest cellular environment has considerable influence dimensions architecture.

Language: Английский

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Architecture of the cytoplasmic face of the nuclear pore

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

Published: June 9, 2022

INTRODUCTION The subcellular compartmentalization of eukaryotic cells requires selective transport folded proteins and protein-nucleic acid complexes. Embedded in nuclear envelope pores, which are generated by the circumscribed fusion inner outer membranes, pore complexes (NPCs) sole bidirectional gateways for nucleocytoplasmic transport. ~110-MDa human NPC is an ~1000-protein assembly that comprises multiple copies ~34 different proteins, collectively termed nucleoporins. symmetric core composed ring encircling central channel rings formed Y‑shaped coat nucleoporin (CNCs) anchored atop both sides envelope. decorated with compartment‑specific asymmetric basket cytoplasmic filament nucleoporins, establish directionality provide docking sites factors small guanosine triphosphatase Ran. nucleoporins also play essential role irreversible remodeling messenger ribonucleoprotein particles (mRNPs) as they exit channel. Unsurprisingly, NPC's face represents a hotspot disease‑associated mutations commonly targeted viral virulence factors. RATIONALE Previous studies established near-atomic composite structure combining (i) biochemical reconstitution to elucidate interaction network between (ii) crystal single-particle cryo-electron microscopy determination reveal their three-dimensional shape molecular details interactions, (iii) quantitative tomography (cryo-ET) maps intact uncover stoichiometry positioning, (iv) cell‑based assays validate physiological relevance structural findings. In this work, we extended our approach architecture NPC. RESULTS Using reconstitution, elucidated protein-protein protein-RNA networks Chaetomium thermophilum establishing evolutionarily conserved heterohexameric complex (CFNC) held together heterotrimeric coiled‑coil hub tethers two separate mRNP‑remodeling Further analysis series structures revealed metazoan‑specific NUP358 16 distinct domains, including N‑terminal S‑shaped α‑helical solenoid followed oligomerization element, numerous Ran‑interacting E3 ligase domain, C‑terminal prolyl‑isomerase domain. Physiologically validated into cryo-ET pentameric bundles, conjoined through domains stalk regions CNC, projecting flexibly attached far ~600 Å cytoplasm. assays, demonstrated dispensable architectural integrity assembled interphase RNA export but required efficient translation. After assignment, remaining 4-shaped cryo‑ET density matched dimensions CFNC hub, close proximity outer-ring NUP93. Whereas N-terminal NUP93 sensor motif anchors properly related heterotrimer ring, confirmed reused anchoring By contrast, C. CFNCs divergent mechanism involves sensors located unstructured portions CNC unassigned occupies binding on face, component ELYS equivalent position unoccupied, suggesting mechanisms other than steric competition promote distribution CONCLUSION We have substantially advanced characterization nucleoporins' attachment at faces Our near‑atomic provides framework elucidating basis mRNP remodeling, factor interference function, underlying diseases [Figure: see text].

Language: Английский

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HIV-1 uncoating by release of viral cDNA from capsid-like structures in the nucleus of infected cells

eLife, Journal Year: 2021, Volume and Issue: 10

Published: April 27, 2021

HIV-1 replication commences inside the cone-shaped viral capsid, but timing, localization, and mechanism of uncoating are under debate. We adapted a strategy to visualize individual reverse-transcribed cDNA molecules their association with cellular proteins using fluorescence correlative-light-and-electron-microscopy (CLEM). specifically detected nuclei, not in cytoplasm. Nuclear initially co-localized fluorescent integrase fusion (IN-FP) CA (capsid) protein, cDNA-punctae separated from IN-FP/CA over time. This phenotype was conserved primary target cells, nuclear complexes exhibiting strong CA-signals all cell types. CLEM revealed capsid-like structures apparently broken capsid-remnants at position IN-FP signals elongated chromatin-like punctae lacking IN-FP. Our data argue for by physical disruption rather than cooperative disassembly CA-lattice, followed separation pre-integration complex.

Language: Английский

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Architecture of the linker-scaffold in the nuclear pore

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

Published: June 9, 2022

INTRODUCTION In eukaryotic cells, the selective bidirectional transport of macromolecules between nucleus and cytoplasm occurs through nuclear pore complex (NPC). Embedded in envelope pores, ~110-MDa human NPC is an ~1200-Å-wide ~750-Å-tall assembly ~1000 proteins, collectively termed nucleoporins. Because NPC's eightfold rotational symmetry along nucleocytoplasmic axis, each ~34 different nucleoporins multiples eight. Architecturally, symmetric core composed inner ring encircling central channel two outer rings anchored on both sides envelope. its role flow genetic information from DNA to RNA protein, commonly targeted viral infections nucleoporin constituents are associated with a plethora diseases. RATIONALE Although arrangement most scaffold was determined by quantitative docking crystal structures into cryo-electron tomographic (cryo-ET) maps intact NPCs, topology molecular details their cohesion multivalent linker have remained elusive. Recently, situ cryo-ET reconstructions NPCs various species indicated that capable reversible constriction dilation response variations membrane tension, thereby modulating diameter ~200 Å. We combined biochemical reconstitution, high-resolution single-particle microscopy (cryo-EM) structure determination, maps, physiological validation elucidate architecture linker-scaffold interaction network not only essential for integrity but also confers plasticity robustness necessary allow withstand such large-scale conformational changes. RESULTS By biochemically mapping scaffold-binding regions all fungal determining cryo-EM complexes, we completed characterization tractable established evolutionary conservation, despite considerable sequence divergence. series Nup188 Nup192 hubs bound Nic96, Nup145N, Nup53 binding regions, revealing proteins form distinct question mark-shaped keystones evolutionarily conserved hetero‑octameric complexes. Linkers bind surface pockets short defined motifs, flanking forming additional disperse interactions reinforce binding. Using structure‑guided functional analysis

Language: Английский

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In-Cell Structural Biology by NMR: The Benefits of the Atomic Scale

Chemical Reviews, Journal Year: 2022, Volume and Issue: 122(10), P. 9497 - 9570

Published: March 31, 2022

In-cell structural biology aims at extracting information about proteins or nucleic acids in their native, cellular environment. This emerging field holds great promise and is already providing new facts outlooks of interest both fundamental applied levels. NMR spectroscopy has important contributions on this stage: It brings a broad variety nuclei the atomic scale, which ensures its versatility uniqueness. Here, we detail methods, knowledge, applications biomedical engineering related to in-cell by NMR. We finally propose brief overview main other techniques (EPR, smFRET, cryo-ET, etc.) draw some advisable developments for In era large-scale screenings deep learning, accurate qualitative experimental evidence are as essential ever understand interior life cells. can generate such it does so scale. review meant deliver comprehensive but accessible information, with advanced technical details reflections nature results, future field.

Language: Английский

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The HIV capsid mimics karyopherin engagement of FG-nucleoporins

Nature, Journal Year: 2024, Volume and Issue: unknown

Published: Jan. 24, 2024

Abstract HIV can infect non-dividing cells because the viral capsid overcome selective barrier of nuclear pore complex and deliver genome directly into nucleus 1,2 . Remarkably, intact is more than 1,000 times larger size limit prescribed by diffusion 3 This in central channel composed intrinsically disordered nucleoporin domains enriched phenylalanine–glycine (FG) dipeptides. Through multivalent FG interactions, cellular karyopherins their bound cargoes solubilize this phase to drive nucleocytoplasmic transport 4 By performing an vitro dissection complex, we show that a pocket on surface similarly interacts with motifs from multiple nucleoporins interaction licences capsids penetrate FG-nucleoporin condensates. karyopherin mimicry model addresses key conceptual challenge for role entry offers explanation as how exogenous entity much any known cargo may be able non-destructively breach envelope.

Language: Английский

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HIV-1 capsids enter the FG phase of nuclear pores like a transport receptor

Nature, Journal Year: 2024, Volume and Issue: 626(8000), P. 843 - 851

Published: Jan. 24, 2024

Abstract HIV-1 infection requires nuclear entry of the viral genome. Previous evidence suggests that this proceeds through pore complexes (NPCs), with 120 × 60 nm capsid squeezing an approximately 60-nm-wide central channel 1 and crossing permeability barrier NPC. This can be described as FG phase 2 is assembled from cohesively interacting phenylalanine–glycine (FG) repeats 3 selectively permeable to cargo captured by transport receptors (NTRs). Here we show assemblies target NPCs efficiently in NTR-independent manner bind directly several types repeats, including barrier-forming cohesive repeats. Like NTRs, readily partitions into vitro serve NPC mimic excludes much smaller inert probes such mCherry. Indeed, protein greatly enhanced assembly, which also allows encapsulated clients enter. Thus, our data indicate behaves like NTR, its interior serving a container. Because capsid-coating trans -acting NTRs would increase diameter 10 or more, suggest ‘self-translocating’ undermines size restrictions imposed scaffold, thereby bypassing otherwise effective infection.

Language: Английский

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High-confidence 3D template matching for cryo-electron tomography

Nature Communications, Journal Year: 2024, Volume and Issue: 15(1)

Published: May 11, 2024

Abstract Visual proteomics attempts to build atlases of the molecular content cells but automated annotation cryo electron tomograms remains challenging. Template matching (TM) and methods based on machine learning detect structural signatures macromolecules. However, their applicability limited in terms both abundance size targets. Here we show that performance TM is greatly improved by using template-specific search parameter optimization including higher-resolution information. We establish a pipeline with systematically tuned parameters for automated, objective comprehensive identification structures confidence 10 100-fold above noise level. demonstrate high-fidelity high-confidence localizations nuclear pore complexes, vaults, ribosomes, proteasomes, fatty acid synthases, lipid membranes microtubules, individual subunits inside crowded eukaryotic cells. provide software tools generic implementation our method broadly applicable towards realizing visual proteomics.

Language: Английский

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