Cryo-electron tomography: en route to the molecular anatomy of organisms and tissues DOI Creative Commons
Oda Helene Schiøtz, Sven Klumpe, Jürgen M. Plitzko

et al.

Biochemical Society Transactions, Journal Year: 2024, Volume and Issue: unknown

Published: Dec. 6, 2024

Cryo-electron tomography (cryo-ET) has become a key technique for obtaining structures of macromolecular complexes in their native environment, assessing local organization and describing the molecular sociology cell. While microorganisms adherent mammalian cells are common targets studies, appropriate sample preparation data acquisition strategies larger cellular assemblies such as tissues, organoids or small model organisms have only recently sufficiently practical to allow in-depth structural characterization samples situ. These advances include tailored lift-out approaches using focused ion beam (FIB) milling, improved schemes. Consequently, cryo-ET FIB lamellae from large volume can complement ultrastructural analysis with another level information: anatomy. This review highlights recent developments towards anatomy studies cryo-ET, briefly outlines what be expected near future.

Language: Английский

Detailed characterisation of the trypanosome nuclear pore architecture reveals conserved asymmetrical functional hubs that drive mRNA export DOI Creative Commons
Bernardo Gabiatti,

Johanna Krenzer,

Silke Braune

et al.

PLoS Biology, Journal Year: 2025, Volume and Issue: 23(2), P. e3003024 - e3003024

Published: Feb. 3, 2025

Nuclear export of mRNAs requires loading the mRNP to transporter Mex67/Mtr2 in nucleoplasm, controlled access pore by basket-localised TREX-2 complex and mRNA release at cytoplasmic site DEAD-box RNA helicase Dbp5. Asymmetric localisation nucleoporins (NUPs) transport components as well ATP dependency Dbp5 ensure unidirectionality transport. Trypanosomes possess homologues Mex67/Mtr2, but not or Instead, nuclear is likely fuelled GTP/GDP gradient created Ran GTPase. However, it remains unclear, how directionality achieved since current model trypanosomatid mostly symmetric. We have revisited architecture trypanosome using a novel combination expansion microscopy, proximity labelling streptavidin imaging. could confidently assign NUP76 complex, known Mex67 interaction platform, NUP64/NUP98/NUP75 site. Having defined markers for both sites pore, we set out map all 75 proteins with subregion mass spectrometry data from labelling. This approach several further specific including predicted structural homology components. mapped Ran-based system (RanBPL), (RanGAP, RanBP1) (Ran, MEX67). Lastly, demonstrate, deploying an auxin degron system, that holds essential role consistent possible functional orthology NUP82/88. Altogether, microscopy revealed asymmetric supporting inherent roles directed Our delivered associated inclusive positional information, which can now be interrogated explore trypanosome-specific adaptions basket, control, remodelling.

Language: Английский

Citations

1
Integrative spatiotemporal modeling of biomolecular processes: Application to the assembly of the nuclear pore complex DOI Creative Commons
Andrew P. Latham, Wanlu Zhang, Jeremy O. B. Tempkin

et al.

Proceedings of the National Academy of Sciences, Journal Year: 2025, Volume and Issue: 122(11)

Published: March 14, 2025

Dynamic processes involving biomolecules are essential for the function of cell. Here, we introduce an integrative method computing models these based on multiple heterogeneous sources information, including time-resolved experimental data and physical dynamic processes. First, each time point, a set coarse compositional structural heterogeneity is computed (heterogeneity models). Second, model, static structure (a snapshot model). Finally, selected connected into series trajectories that optimize likelihood both transitions between them trajectory The demonstrated by application to assembly process human nuclear pore complex in context reforming envelope during mitotic cell division, live-cell correlated electron tomography, bulk fluorescence correlation spectroscopy–calibrated quantitative live imaging, model fully assembled complex. Modeling improves precision over modeling alone. applicable wide range time-dependent systems biology available broader scientific community through implementation open source Integrative Platform (IMP) software.

Language: Английский

Citations

1
Expanding insights from in situ cryo-EM DOI
Joshua Hutchings, Elizabeth Villa

Current Opinion in Structural Biology, Journal Year: 2024, Volume and Issue: 88, P. 102885 - 102885

Published: July 11, 2024

Language: Английский

Citations

5
Nuclear basket proteins Mlp1 and Nup2 drive heat shock-induced 3D genome restructuring DOI Creative Commons
Suman Mohajan, Linda S Rubio, David S. Gross

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 2, 2025

ABSTRACT The nuclear pore complex (NPC), a multisubunit located within the envelope, regulates RNA export and import of proteins. Here we address role NPC in driving thermal stress-induced 3D genome repositioning Heat Shock Responsive ( HSR ) genes yeast. We found that two basket proteins, Mlp1 Nup2, although dispensable for integrity, are required into coalesced chromatin clusters, consistent with their strong, heat shock-dependent recruitment to gene regulatory coding regions. clustering occurs predominantly nucleoplasm is independent essential scaffold-associated proteins Nup1 Nup145. Notably, double depletion Nup2 has little effect on formation Factor 1 (Hsf1)-containing transcriptional condensates, Hsf1 Pol II genes, or mRNA abundance. Our results define restructuring extrinsic downstream activation.

Language: Английский

Citations

0
Opening the gate: Complexity and modularity of the nuclear pore scaffold and basket DOI Creative Commons
Elisa Dultz, Valérie Doye

Current Opinion in Cell Biology, Journal Year: 2025, Volume and Issue: 92, P. 102461 - 102461

Published: Jan. 17, 2025

Language: Английский

Citations

0
Frontiers in integrative structural modeling of macromolecular assemblies DOI Creative Commons
Kartik Majila, Shreyas Arvindekar,

M. Jindal

et al.

QRB Discovery, Journal Year: 2025, Volume and Issue: 6

Published: Jan. 1, 2025

Abstract Integrative modeling enables structure determination for large macromolecular assemblies by combining data from multiple experiments with theoretical and computational predictions. Recent advancements in AI-based prediction cryo electron-microscopy have sparked renewed enthusiasm integrative modeling; structures methods can be integrated situ maps to characterize assemblies. This approach previously allowed us others determine the architectures of diverse assemblies, such as nuclear pore complexes, chromatin remodelers, cell–cell junctions. Experimental spanning several scales was used these studies, ranging high-resolution data, X-ray crystallography AlphaFold structure, low-resolution cryo-electron tomography co-immunoprecipitation experiments. Two recurrent challenges emerged across a range studies. First, contained significant fractions disordered regions, necessitating development new regions context ordered regions. Second, needed developed utilize information tomography, timely challenge structural biology is increasingly moving towards characterization. Here, we recapitulate recent developments proteins analysis highlight other opportunities method modeling.

Language: Английский

Citations

0
Strategies for the Viral Exploitation of Nuclear Pore Transport Pathways DOI Creative Commons
Xin Zhang, Keesiang Lim,

Yujia Qiu

et al.

Viruses, Journal Year: 2025, Volume and Issue: 17(2), P. 151 - 151

Published: Jan. 23, 2025

Viruses frequently exploit the host’s nucleocytoplasmic trafficking machinery to facilitate their replication and evade immune defenses. By encoding specialized proteins other components, they strategically target host nuclear transport receptors (NTRs) nucleoporins within spiderweb-like inner channel of pore complex (NPC), enabling efficient access nucleus. This review explores intricate mechanisms governing import export viral with a focus on interplay between factors determinants that are essential for these processes. Given pivotal role shuttling in life cycle, we also examine therapeutic strategies aimed at disrupting pathways. includes evaluating efficacy pharmacological inhibitors impairing assessing potential as antiviral treatments. Furthermore, emphasize need continued research develop targeted therapies leverage vulnerabilities trafficking. Emerging high-resolution techniques, such advanced imaging computational modeling, transforming our understanding dynamic interactions viruses NPC. These cutting-edge tools driving progress identifying novel opportunities uncovering deeper insights into pathogenesis. highlights importance advancements paving way innovative strategies.

Language: Английский

Citations

0
Nuclear Basket Proteins Mlp1 and Nup2 Drive Heat Shock–Induced 3D Genome Restructuring DOI Creative Commons
Suman Mohajan, Linda S Rubio, David S. Gross

et al.

Published: Jan. 31, 2025

The nuclear pore complex (NPC), a multisubunit located within the envelope, regulates RNA export and import of proteins. Here we address role NPC in driving thermal stress-induced 3D genome repositioning _Heat Shock Responsive_ (_HSR_) genes yeast. We found that two basket proteins, Mlp1 Nup2, although dispensable for integrity, are required _HSR_ into coalesced chromatin clusters, consistent with their strong, heat shock-dependent recruitment to gene regulatory coding regions. clustering occurs predominantly nucleoplasm is independent essential scaffold-associated proteins Nup1 Nup145. Notably, double depletion Nup2 has little effect on formation Heat Factor 1 (Hsf1)-containing transcriptional condensates, Hsf1 Pol II genes, or mRNA abundance. Our results define restructuring extrinsic downstream activation.

Language: Английский

Citations

0
Conformational dynamics of the nuclear pore complex central channel DOI Creative Commons

Yu Chen,

Guoli Zhou,

Miao Yu

et al.

Biochemical Society Transactions, Journal Year: 2025, Volume and Issue: 53(01)

Published: Feb. 7, 2025

The nuclear pore complex (NPC) is a vital regulator of molecular transport between the nucleus and cytoplasm in eukaryotic cells. At heart NPC’s function are intrinsically disordered phenylalanineglycine-rich nucleoporins (FG-Nups), which form dynamic permeability barrier within central channel. This nature facilitates efficient nucleocytoplasmic but also poses significant challenges to its characterization, especially nano-confined environment NPC. Recent advances experimental techniques, such as cryo-electron microscopy, atomic force fluorescence magnetic resonance, along with computational modeling, have illuminated conformational flexibility FG-Nups, underpins their functional versatility. review synthesizes these advancements, emphasizing how disruptions FG-Nup behavior—caused by mutations or pathological interactions—contribute diseases neurodegenerative disorders, aging-related decline, viral infections. Despite progress, persist deciphering dynamics crowded cellular environment, under conditions. Addressing gaps critical for advancing therapeutic strategies targeting NPC dysfunction disease progression.

Language: Английский

Citations

0
Correlative In Situ Cryo-ET Reveals Cellular and Viral Remodeling Associated with Selective HIV-1 Core Nuclear Import DOI Creative Commons

Zhen Hou,

Y. Shen, Stanley Fronik

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2025, Volume and Issue: unknown

Published: March 4, 2025

Abstract Lentiviruses like HIV-1 infect non-dividing cells by traversing the nuclear pore, but studying this process has been challenging due to its scarcity and dynamic nature in infected cells. Here, we developed a robust cell-permeabilization system that recapitulates import established an integrated cryo-correlative workflow combining cryo-CLEM, cryo-FIB, cryo-ET for targeted imaging of process. These advancements enabled successful capture 1,899 cores at various stages import. Statistical structural analyses native wild-type mutant revealed depends on both capsid elasticity pore adaptability, as well factors such CPSF6. Brittle fail enter complex (NPC), while CPSF6-binding-deficient stall inside NPC, resulting impaired Intriguingly, pores function selective filters favoring smaller, tube-shaped cores. Our study opens new avenues dissecting biochemistry biology downstream events including core uncoating potentially integration, with unprecedented detail.

Language: Английский

Citations

0