scholarly journals 3D super-resolution fluorescence microscopy maps the variable molecular architecture of the Nuclear Pore Complex

2020 ◽  
Author(s):  
Vilma Jimenez Sabinina ◽  
M. Julius Hossain ◽  
Jean-Karim Hériché ◽  
Philipp Hoess ◽  
Bianca Nijmeijer ◽  
...  
Keyword(s):  
3D Structure ◽  
Super Resolution ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Molecular Architecture ◽  
Nuclear Ring ◽  
Molecular Specificity ◽  
Pore Complexes ◽  
Super Resolution Microscopy ◽  
Exciting Possibility

AbstractNuclear pore complexes (NPCs) are large macromolecular machines that mediate the traffic between the nucleus and the cytoplasm. In vertebrates, each NPC consists of ∼1000 proteins, termed nucleoporins, and has a mass of over 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 3D super-resolution 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 over ten thousand 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.

scholarly journals 3D super-resolution fluorescence microscopy maps the variable molecular architecture of the Nuclear Pore Complex

2021 ◽  
pp. mbc.E20-11-0728
Author(s):  
Vilma Jimenez Sabinina ◽  
M. Julius Hossain ◽  
Jean-Karim Hériché ◽  
Philipp Hoess ◽  
Bianca Nijmeijer ◽  
...  
Keyword(s):  
3D Structure ◽  
Super Resolution ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Molecular Architecture ◽  
Nuclear Ring ◽  
Molecular Specificity ◽  
Pore Complexes ◽  
Super Resolution Microscopy ◽  
Exciting Possibility

Nuclear pore complexes (NPCs) are large macromolecular machines that mediate the traffic between the nucleus and the cytoplasm. In vertebrates, each NPC consists of ∼1000 proteins, termed nucleoporins, and has a mass of over 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 3D super-resolution 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 over ten thousand 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.

Nuclear Pore Transport: Insight in Situ

1998 ◽  
Vol 4 (S2) ◽  
pp. 956-957
Author(s):  
U. Aebi ◽  
D. Stoffler ◽  
B. Feja ◽  
K. Goldie
Keyword(s):  
Nuclear Periphery ◽  
3D Structure ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Molecular Architecture ◽  
Energy Filtering ◽  
3D Architecture ◽  
Bidirectional Transport ◽  
Pore Complexes

The nuclear envelope (NE) physically separates the genetic machinery residing in the nucleus from protein synthesis taking place in the cytoplasm. Bidirectional transport of proteins, RNAs and RNP particles between these two compartments is mediated by the nuclear pore complexes (NPCs), large (-120 MDa) supramolecular assemblies embedded in the NE and being built of -100 different polypeptides, called nucleoporins. Extensive structural studies have revealed the 3D architecture of NPCs, and epitopes of several nucleoporins have been localized within their 3D structure.In an attempt to go beyond the current consensus model of the NPC (Fig. lc, inset), we have embarked on a more systematic structural analysis of the molecular architecture of native NPCs. This is achieved by field-emission (FETEM; Fig. la) or energy-filtering (EFTEM; Fig. lb) TEM of completely unfixed and unstained Xenopus oocyte NEs embedded in thick (i.e. -200 nm) amorphous ice so that the 3D organization of the cytoplasmic and nuclear periphery of the NPCs (i.e. the cytoplasmic fibrils and nuclear baskets) is fully preserved (Fig. lb).

scholarly journals O-GlcNAc modification of nuclear pore complexes accelerates bi-directional transport

2020 ◽  
Author(s):  
Tae Yeon Yoo ◽  
Timothy J Mitchison
Keyword(s):  
Nuclear Import ◽  
Nuclear Transport ◽  
Super Resolution ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Permeability Barrier ◽  
Nuclear Pore Complexes ◽  
Facilitated Diffusion ◽  
Import And Export ◽  
Pore Complexes

AbstractMacromolecular transport across the nuclear envelope depends on facilitated diffusion through nuclear pore complexes (NPCs). The interior of NPCs contains a permeability barrier made of phenylalanine-glycine (FG) repeat domains that selectively facilitates the permeation of cargoes bound to nuclear transport receptors (NTRs). FG repeats in NPC are a major site of O-linked N-acetylglucosamine (O-GlcNAc) modification, but the functional role of this modification in nucleocytoplasmic transport is unclear. We developed high-throughput assays based on optogenetic probes to quantify the kinetics of nuclear import and export in living human cells. We found that increasing O-GlcNAc modification of the NPC accelerated NTR-facilitated nucleocytoplasmic transport of proteins in both directions, and decreasing modification slowed transport. Super-resolution imaging revealed strong enrichment of O-GlcNAc at the FG-repeat barrier. O-GlcNAc modification also accelerated passive permeation of a small, inert protein through NPCs. We conclude that O-GlcNAc modification accelerates nucleocytoplasmic transport by enhancing the non-specific permeability the FG-repeat barrier, perhaps by steric inhibition of interactions between FG repeats.SummaryNuclear pore complexes mediate nuclear transport and are highly modified with O-linked N-acetylglucosamine (O-GlcNAc) on FG repeat domains. Using a new quantitative live-cell imaging assay, Yoo and Mitchison demonstrate acceleration of nuclear import and export by O-GlcNAc modification.

scholarly journals Correction: Components of Coated Vesicles and Nuclear Pore Complexes Share a Common Molecular Architecture

PLoS Biology ◽  
2005 ◽  
Vol 3 (2) ◽  
pp. e80 ◽  
Author(s):  
Damien Devos ◽  
Svetlana Dokudovskaya ◽  
Frank Alber ◽  
Rosemary Williams ◽  
Brian T Chait ◽  
...  
Keyword(s):  
Coated Vesicles ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Molecular Architecture ◽  
Pore Complexes

scholarly journals Actin assembly ruptures the nuclear envelope by prying the lamina away from nuclear pores and nuclear membranes in starfish oocytes

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Natalia Wesolowska ◽  
Ivan Avilov ◽  
Pedro Machado ◽  
Celina Geiss ◽  
Hiroshi Kondo ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Super Resolution ◽  
Germinal Vesicle ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Actin Assembly ◽  
Nuclear Membranes ◽  
Pore Complexes ◽  
Distinct Membrane ◽  
Nuclear Rupture

The nucleus of oocytes (germinal vesicle) is unusually large and its nuclear envelope (NE) is densely packed with nuclear pore complexes (NPCs) that are stockpiled for embryonic development. We showed that breakdown of this specialized NE is mediated by an Arp2/3-nucleated F-actin ‘shell’ in starfish oocytes, in contrast to microtubule-driven tearing in mammalian fibroblasts. Here, we address the mechanism of F-actin-driven NE rupture by correlated live-cell, super-resolution and electron microscopy. We show that actin is nucleated within the lamina, sprouting filopodia-like spikes towards the nuclear membranes. These F-actin spikes protrude pore-free nuclear membranes, whereas the adjoining stretches of membrane accumulate NPCs that are associated with the still-intact lamina. Packed NPCs sort into a distinct membrane network, while breaks appear in ER-like, pore-free regions. We reveal a new function for actin-mediated membrane shaping in nuclear rupture that is likely to have implications in other contexts, such as nuclear rupture observed in cancer cells.

scholarly journals Ubiquitylation of the nuclear pore complex controls nuclear migration during mitosis in S. cerevisiae

2012 ◽  
Vol 196 (1) ◽  
pp. 19-27 ◽  
Author(s):  
Akira Hayakawa ◽  
Anna Babour ◽  
Lucie Sengmanivong ◽  
Catherine Dargemont
Keyword(s):  
Posttranslational Modifications ◽  
Systematic Approach ◽  
Protein Transport ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Molecular Architecture ◽  
Dynein Light Chain ◽  
Large Protein ◽  
Pore Complexes ◽  
Cytoplasmic Side

Nuclear pore complexes (NPCs) correspond to large protein transport complexes responsible for selective nucleocytoplasmic exchange. Although research has revealed much about the molecular architecture and roles of the NPC subcomplexes, little is known about the regulation of NPC functions by posttranslational modifications. We used a systematic approach to show that more than half of NPC proteins were conjugated to ubiquitin. In particular, Nup159, a nucleoporin exclusively located on the cytoplasmic side of the NPC, was monoubiquitylated by the Cdc34/SCF (Skp1–Cdc53–F-box E3 ligase) enzymes. Preventing this modification had no consequences on nuclear transport or NPC organization but strongly affected the ability of Nup159 to target the dynein light chain to the NPC. This led to defects in nuclear segregation at the onset of mitosis. Thus, defining ubiquitylation of the yeast NPC highlights yet-unexplored functions of this essential organelle in cell division.

scholarly journals Determining the architecture of nuclear ring of Xenopus laevis nuclear pore complex using integrated approaches

2021 ◽  
Author(s):  
He Ren ◽  
Linhua Tai ◽  
Yun Zhu ◽  
Chun Chan ◽  
Qun Zhao ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Nucleocytoplasmic Transport ◽  
Complex Model ◽  
Nuclear Pore ◽  
Xenopus Laevis Oocytes ◽  
Nuclear Pore Complexes ◽  
Cell Experiment ◽  
Nuclear Ring ◽  
Integrated Approaches ◽  
Pore Complexes

The nuclear pore complexes (NPCs) are large protein assemblies as a physical gate to regulate nucleocytoplasmic transport. Here, using integrated approaches including cryo-electron microscopy, hybrid homology modeling and cell experiment, we determined the architecture of the nuclear ring (NR) from Xenopus laevis oocytes NPC at subnanometer resolution. In addition to the improvement of the Y complex model, eight copies of Nup205 and ELYS were assigned in NR. Nup205 connects the inner and outer Y complexes and contributes to the assembly and stability of the NR. By interacting with both the inner Nup160 and the nuclear envelope (NE), the N-terminal β-propeller and α-solenoid domains of ELYS were found to be essential for accurate assembly of the NPC on the NE.

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