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Cees Dekker - One of the best experts on this subject based on the ideXlab platform.
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DNA origami scaffold for studying intrinsically disordered proteins of the Nuclear Pore Complex
Nature Communications, 2018Co-Authors: Philip Ketterer, Adithya N. Ananth, Diederik S. Laman Trip, Eva Bertosin, Mahipal Ganji, Jaco Van Der Torre, Ankur Mishra, Patrick Onck, Hendrik Dietz, Cees DekkerAbstract:The Nuclear Pore Complex (NPC) is the gatekeeper for Nuclear transport in eukaryotic cells. A key component of the NPC is the central shaft lined with intrinsically disordered proteins (IDPs) known as FG-Nups, which control the selective molecular traffic. Here, we present an approach to realize artificial NPC mimics that allows controlling the type and copy number of FG-Nups. We constructed 34 nm-wide 3D DNA origami rings and attached different numbers of NSP1, a model yeast FG-Nup, or NSP1-S, a hydrophilic mutant. Using (cryo) electron microscopy, we find that NSP1 forms denser cohesive networks inside the ring compared to NSP1-S. Consistent with this, the measured ionic conductance is lower for NSP1 than for NSP1-S. Molecular dynamics simulations reveal spatially varying protein densities and conductances in good agreement with the experiments. Our technique provides an experimental platform for deciphering the collective behavior of IDPs with full control of their type and position.FG-Nups are disordered proteins in the Nuclear Pore Complex (NPC) where they selectively control Nuclear transport. Here authors build NPC-mimics based on DNA origami rings which attach a certain numbers of Nups to analyse those nanoPores by cryoEM and conductance measurements.
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dna origami scaffold for studying intrinsically disordered proteins of the Nuclear Pore Complex
Nature Communications, 2018Co-Authors: Philip Ketterer, Adithya N. Ananth, Eva Bertosin, Mahipal Ganji, Jaco Van Der Torre, Ankur Mishra, Patrick Onck, Hendrik Dietz, Diederik Laman S Trip, Cees DekkerAbstract:The Nuclear Pore Complex (NPC) is the gatekeeper for Nuclear transport in eukaryotic cells. A key component of the NPC is the central shaft lined with intrinsically disordered proteins (IDPs) known as FG-Nups, which control the selective molecular traffic. Here, we present an approach to realize artificial NPC mimics that allows controlling the type and copy number of FG-Nups. We constructed 34 nm-wide 3D DNA origami rings and attached different numbers of NSP1, a model yeast FG-Nup, or NSP1-S, a hydrophilic mutant. Using (cryo) electron microscopy, we find that NSP1 forms denser cohesive networks inside the ring compared to NSP1-S. Consistent with this, the measured ionic conductance is lower for NSP1 than for NSP1-S. Molecular dynamics simulations reveal spatially varying protein densities and conductances in good agreement with the experiments. Our technique provides an experimental platform for deciphering the collective behavior of IDPs with full control of their type and position.
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Single-molecule transport across an individual biomimetic Nuclear Pore Complex
Nature Nanotechnology, 2011Co-Authors: Stefan W. Kowalczyk, Roderick Y. H. Lim, Larisa E. Kapinos, Timothy R. Blosser, Tomás Magalhães, Pauline Van Nies, Cees DekkerAbstract:Nuclear Pore Complexes regulate the selective exchange of RNA and proteins across the Nuclear envelope in eukaryotic cells^ 1 . Biomimetic strategies offer new opportunities to investigate this remarkable transport phenomenon^ 2 . Here, we show selective transport of proteins across individual biomimetic Nuclear Pore Complexes at the single-molecule level. Each biomimetic Complex is constructed by covalently tethering either Nup98 or Nup153 (phenylalanine-glycine (FG) nucleoporins) to a solid-state nanoPore^ 3 . Individual translocation events are monitored using ionic current measurements with sub-millisecond temporal resolution. Transport receptors (Impβ) proceed with a dwell time of ∼2.5 ms for both Nup98- and Nup153-coated Pores, whereas the passage of non-specific proteins is strongly inhibited with different degrees of selectivity. For Pores up to ∼25 nm in diameter, Nups form a dense and low-conducting barrier, whereas they adopt a more open structure in larger Pores. Our biomimetic Nuclear Pore Complex provides a quantitative platform for studying nucleocytoplasmic transport phenomena at the single-molecule level in vitro . Covalently attaching Nuclear Pore proteins to solid-state nanoPores forms a Complex that can selectively transport certain proteins, similar to the Nuclear Pore Complex.
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Single-molecule transport across an individual biomimetic Nuclear Pore Complex.
Nature Nanotechnology, 2011Co-Authors: Stefan W. Kowalczyk, Roderick Y. H. Lim, Larisa E. Kapinos, Timothy R. Blosser, Tomás Magalhães, Pauline Van Nies, Cees DekkerAbstract:Covalently attaching Nuclear Pore proteins to solid-state nanoPores forms a Complex that can selectively transport certain proteins, similar to the Nuclear Pore Complex.
Markus Sauer - One of the best experts on this subject based on the ideXlab platform.
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correlative super resolution fluorescence and electron microscopy of the Nuclear Pore Complex with molecular resolution
Journal of Cell Science, 2014Co-Authors: Anna Löschberger, Christian Franke, Sebastian Van De Linde, Georg Krohne, Markus SauerAbstract:Here, we combine super-resolution fluorescence localization microscopy with scanning electron microscopy to map the position of proteins of Nuclear Pore Complexes in isolated Xenopus laevis oocyte Nuclear envelopes with molecular resolution in both imaging modes. We use the periodic molecular structure of the Nuclear Pore Complex to superimpose direct stochastic optical reconstruction microscopy images with a precision of <20 nm on electron micrographs. The correlative images demonstrate quantitative molecular labeling and localization of Nuclear Pore Complex proteins by standard immunocytochemistry with primary and secondary antibodies and reveal that the Nuclear Pore Complex is composed of eight gp210 (also known as NUP210) protein homodimers. In addition, we find subpopulations of Nuclear Pore Complexes with ninefold symmetry, which are found occasionally among the more typical eightfold symmetrical structures.
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Correlative super-resolution fluorescence and electron microscopy of the Nuclear Pore Complex with molecular resolution
Journal of Cell Science, 2014Co-Authors: Anna Löschberger, Christian Franke, Sebastian Van De Linde, Georg Krohne, Markus SauerAbstract:Here, we combine super-resolution fluorescence localization microscopy with scanning electron microscopy to map the position of proteins of Nuclear Pore Complexes in isolated Xenopus laevis oocyte Nuclear envelopes with molecular resolution in both imaging modes. We use the periodic molecular structure of the Nuclear Pore Complex to superimpose direct stochastic optical reconstruction microscopy images with a precision of
Benjamin Vollmer - One of the best experts on this subject based on the ideXlab platform.
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in situ structural analysis of the human Nuclear Pore Complex
Nature, 2015Co-Authors: Alexander Von Appen, Jan Kosinski, Lenore Sparks, Amanda L Diguilio, Benjamin Vollmer, Marietherese Mackmull, Niccolo Banterle, Luca Parca, Panagiotis L KastritisAbstract:The most comprehensive architectural model to date of the Nuclear Pore Complex reveals previously unknown local interactions, and a role for nucleoporin 358 in Y-Complex oligomerization. The transport of materials between the nucleus and cytoplasm in eukaryotic cells is controlled by the Nuclear Pore Complex. Martin Beck and colleagues have used cryo-electron tomography, mass spectrometry and other analyses to generate the most comprehensive architectural model of the human Nuclear Pore Complex to date. The model reveals previously unknown local interactions, and a role for the transport channel nucleoporin 358 (Nup358) in mediating oligomerization of the Y-Complex within the Nuclear Pore Complex. Nuclear Pore Complexes are fundamental components of all eukaryotic cells that mediate nucleocytoplasmic exchange. Determining their 110-megadalton structure imposes a formidable challenge and requires in situ structural biology approaches. Of approximately 30 nucleoporins (Nups), 15 are structured and form the Y and inner-ring Complexes. These two major scaffolding modules assemble in multiple copies into an eight-fold rotationally symmetric structure that fuses the inner and outer Nuclear membranes to form a central channel of ~60 nm in diameter1. The scaffold is decorated with transport-channel Nups that often contain phenylalanine-repeat sequences and mediate the interaction with cargo Complexes. Although the architectural arrangement of parts of the Y Complex has been elucidated, it is unclear how exactly it oligomerizes in situ. Here we combine cryo-electron tomography with mass spectrometry, biochemical analysis, perturbation experiments and structural modelling to generate, to our knowledge, the most comprehensive architectural model of the human Nuclear Pore Complex to date. Our data suggest previously unknown protein interfaces across Y Complexes and to inner-ring Complex members. We show that the transport-channel Nup358 (also known as Ranbp2) has a previously unanticipated role in Y-Complex oligomerization. Our findings blur the established boundaries between scaffold and transport-channel Nups. We conclude that, similar to coated vesicles, several copies of the same structural building block—although compositionally identical—engage in different local sets of interactions and conformations.
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in situ structural analysis of the human Nuclear Pore Complex
Nature, 2015Co-Authors: Alexander Von Appen, Jan Kosinski, Lenore Sparks, Amanda L Diguilio, Benjamin Vollmer, Marietherese Mackmull, Niccolo Banterle, Alessandro Ori, Luca ParcaAbstract:Nuclear Pore Complexes are fundamental components of all eukaryotic cells that mediate nucleocytoplasmic exchange. Determining their 110-megadalton structure imposes a formidable challenge and requires in situ structural biology approaches. Of approximately 30 nucleoporins (Nups), 15 are structured and form the Y and inner-ring Complexes. These two major scaffolding modules assemble in multiple copies into an eight-fold rotationally symmetric structure that fuses the inner and outer Nuclear membranes to form a central channel of ~60 nm in diameter. The scaffold is decorated with transport-channel Nups that often contain phenylalanine-repeat sequences and mediate the interaction with cargo Complexes. Although the architectural arrangement of parts of the Y Complex has been elucidated, it is unclear how exactly it oligomerizes in situ. Here we combine cryo-electron tomography with mass spectrometry, biochemical analysis, perturbation experiments and structural modelling to generate, to our knowledge, the most comprehensive architectural model of the human Nuclear Pore Complex to date. Our data suggest previously unknown protein interfaces across Y Complexes and to inner-ring Complex members. We show that the transport-channel Nup358 (also known as Ranbp2) has a previously unanticipated role in Y-Complex oligomerization. Our findings blur the established boundaries between scaffold and transport-channel Nups. We conclude that, similar to coated vesicles, several copies of the same structural building block--although compositionally identical--engage in different local sets of interactions and conformations.
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The diverse roles of the Nup93/Nic96 Complex proteins - structural scaffolds of the Nuclear Pore Complex with additional cellular functions.
Biological Chemistry, 2014Co-Authors: Benjamin Vollmer, Wolfram AntoninAbstract:Nuclear Pore Complexes mediate the transport between the cell nucleoplasm and cytoplasm. These 125 MDa structures are among the largest assemblies found in eukaryotes, built from proteins organized in distinct subComplexes that act as building blocks during Nuclear Pore Complex biogenesis. In this review, we focus on one of these subComplexes, the Nup93 Complex in metazoa and its yeast counterpart, the Nic96 Complex. We discuss its essential function in Nuclear Pore Complex assembly as a linker between the Nuclear membrane and the central part of the Pore and its various roles in Nuclear transport processes and beyond.
Michael P. Rout - One of the best experts on this subject based on the ideXlab platform.
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thermodynamic characterization of the multivalent interactions underlying rapid and selective translocation through the Nuclear Pore Complex
Journal of Biological Chemistry, 2018Co-Authors: Ryo Hayama, Michael P. Rout, Samuel Sparks, Lee M Hecht, Kaushik Dutta, Jerome M Karp, Christina M Cabana, David CowburnAbstract:Intrinsically disordered proteins (IDPs) play important roles in many biological systems. Given the vast conformational space that IDPs can explore, the thermodynamics of the interactions with their partners is closely linked to their biological functions. Intrinsically disordered regions of Phe-Gly nucleoporins (FG Nups) that contain multiple phenylalanine-glycine repeats are of particular interest, as their interactions with transport factors (TFs) underlie the paradoxically rapid yet also highly selective transport of macromolecules mediated by the Nuclear Pore Complex. Here, we used NMR and isothermal titration calorimetry to thermodynamically characterize these multivalent interactions. These analyses revealed that a combination of low per-FG motif affinity and the enthalpy-entropy balance prevents high-avidity interaction between FG Nups and TFs, whereas the large number of FG motifs promotes frequent FG-TF contacts, resulting in enhanced selectivity. Our thermodynamic model underlines the importance of functional disorder of FG Nups. It helps explain the rapid and selective translocation of TFs through the Nuclear Pore Complex and further expands our understanding of the mechanisms of "fuzzy" interactions involving IDPs.
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interactome mapping reveals the evolutionary history of the Nuclear Pore Complex
PLOS Biology, 2016Co-Authors: Samson O Obado, Wenzhu Zhang, Brian T Chait, Marc Brillantes, Kunihiro Uryu, Natalia E Ketaren, Mark C Field, Michael P. RoutAbstract:The Nuclear Pore Complex (NPC) is responsible for nucleocytoplasmic transport and constitutes a hub for control of gene expression. The components of NPCs from several eukaryotic lineages have been determined, but only the yeast and vertebrate NPCs have been extensively characterized at the quaternary level. Significantly, recent evidence indicates that compositional similarity does not necessarily correspond to homologous architecture between NPCs from different taxa. To address this, we describe the interactome of the trypanosome NPC, a representative, highly divergent eukaryote. We identify numerous new NPC components and report an exhaustive interactome, allowing assignment of trypanosome nucleoporins to discrete NPC substructures. Remarkably, despite retaining similar protein composition, there are exceptional architectural dissimilarities between opisthokont (yeast and vertebrates) and excavate (trypanosomes) NPCs. Whilst elements of the inner core are conserved, numerous peripheral structures are highly divergent, perhaps reflecting requirements to interface with divergent Nuclear and cytoplasmic functions. Moreover, the trypanosome NPC has almost complete nucleocytoplasmic symmetry, in contrast to the opisthokont NPC; this may reflect divergence in RNA export processes at the NPC cytoplasmic face, as we find evidence supporting Ran-dependent mRNA export in trypanosomes, similar to protein transport. We propose a model of stepwise acquisition of nucleocytoplasmic mechanistic Complexity and demonstrate that detailed dissection of macromolecular Complexes provides fuller understanding of evolutionary processes.
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The Nuclear Pore Complex: bridging Nuclear transport and gene regulation
Nature Reviews Molecular Cell Biology, 2010Co-Authors: Caterina Strambio-de-castillia, Mario Niepel, Michael P. RoutAbstract:The Nuclear Pore Complex is the key regulator of transport between the cytoplasm and nucleus. Emerging evidence suggests it also regulates gene expression by influencing the internal architecture of the nucleus and by coordinating the delivery of genetic information to the cytoplasmic protein synthesis machinery. Although the Nuclear Pore Complex (NPC) is best known for its primary function as the key regulator of molecular traffic between the cytoplasm and the nucleus, a growing body of experimental evidence suggests that this structure participates in a considerably broader range of cellular activities on both sides of the Nuclear envelope. Indeed, the NPC is emerging as an important regulator of gene expression through its influence on the internal architectural organization of the nucleus and its apparently extensive involvement in coordinating the seamless delivery of genetic information to the cytoplasmic protein synthesis machinery.
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Cleave to leave: structural insights into the dynamic organization of the Nuclear Pore Complex.
Molecular Cell, 2002Co-Authors: Svetlana Dokudovskaya, Liesbeth M. Veenhoff, Michael P. RoutAbstract:A detailed understanding of the fine structure of the Nuclear Pore Complex has remained elusive. Now, studies on a small protein domain have shed light on the dynamic organization of this massive assembly.
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the yeast Nuclear Pore Complex composition architecture and transport mechanism
Journal of Cell Biology, 2000Co-Authors: Michael P. Rout, John D Aitchison, Adisetyantari Suprapto, Kelly Hjertaas, Yingming Zhao, Brian T ChaitAbstract:An understanding of how the Nuclear Pore Complex (NPC) mediates nucleocytoplasmic exchange requires a comprehensive inventory of the molecular components of the NPC and a knowledge of how each component contributes to the overall structure of this large molecular translocation machine. Therefore, we have taken a comprehensive approach to classify all components of the yeast NPC (nucleoporins). This involved identifying all the proteins present in a highly enriched NPC fraction, determining which of these proteins were nucleoporins, and localizing each nucleoporin within the NPC. Using these data, we present a map of the molecular architecture of the yeast NPC and provide evidence for a Brownian affinity gating mechanism for nucleocytoplasmic transport.
Vincent Géli - One of the best experts on this subject based on the ideXlab platform.
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The Nuclear Pore Complex prevents sister chromatid recombination during replicative senescence
Nature Communications, 2020Co-Authors: Paula Aguilera, Jenna Whalen, Christopher Minguet, Dmitri Churikov, Catherine Freudenreich, Marie-noëlle Simon, Vincent GéliAbstract:The Nuclear Pore Complex (NPC) has emerged as an important hub for processing various types of DNA damage. Here, we uncover that fusing a DNA binding domain to the NPC basket protein Nup1 reduces telomere relocalization to Nuclear Pores early after telomerase inactivation. This Nup1 modification also impairs the relocalization to the NPC of expanded CAG/CTG triplet repeats. Strikingly, telomerase negative cells bypass senescence when expressing this Nup1 modification by maintaining a minimal telomere length compatible with proliferation through rampant unequal exchanges between sister chromatids. We further report that a Nup1 mutant lacking 36 C-terminal residues recapitulates the phenotypes of the Nup1-LexA fusion indicating a direct role of Nup1 in the relocation of stalled forks to NPCs and restriction of error-prone recombination between repeated sequences. Our results reveal a new mode of telomere maintenance that could shed light on how 20% of cancer cells are maintained without telomerase or ALT. The Nuclear Pore Complex has been linked to DNA damage processing. Here the authors reveal that the Nup1 C-terminus is critical for the relocalization of eroded telomeres to Nuclear Pores and that modification of Nup1 promotes sister chromatid recombination and unleashes a new telomere maintenance mechanism.
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The Nuclear Pore Complex prevents sister chromatid recombination during replicative senescence
Nature Communications, 2020Co-Authors: Paula Aguilera, Jenna Whalen, Christopher Minguet, Dmitri Churikov, Catherine Freudenreich, Marie-noëlle Simon, Vincent GéliAbstract:The Nuclear Pore Complex (NPC) has emerged as an important hub for processing various types of DNA damage. Here, we uncover that fusing a DNA binding domain to the NPC basket protein Nup1 reduces telomere relocalization to Nuclear Pores early after telomerase inactivation. This Nup1 modification also impairs the relocalization to the NPC of expanded CAG/CTG triplet repeats. Strikingly, telomerase negative cells bypass senescence when expressing this Nup1 modification by maintaining a minimal telomere length compatible with proliferation through rampant unequal exchanges between sister chromatids. We further report that a Nup1 mutant lacking 36 C-terminal residues recapitulates the phenotypes of the Nup1-LexA fusion indicating a direct role of Nup1 in the relocation of stalled forks to NPCs and restriction of error-prone recombination between repeated sequences. Our results reveal a new mode of telomere maintenance that could shed light on how 20% of cancer cells are maintained without telomerase or ALT.
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posttranslational marks control architectural and functional plasticity of the Nuclear Pore Complex basket
Journal of Cell Biology, 2016Co-Authors: Carlos A Nino, Vincent Géli, David Guet, Sergine Brutus, Frederic Jourquin, Shweta Mendiratta, Jean Salamero, Catherine DargemontAbstract:The Nuclear Pore Complex (NPC) serves as both the unique gate between the nucleus and the cytoplasm and a major platform that coordinates nucleocytoplasmic exchanges, gene expression, and genome integrity. To understand how the NPC integrates these functional constraints, we dissected here the posttranslational modifications of the Nuclear basket protein Nup60 and analyzed how they intervene to control the plasticity of the NPC. Combined approaches highlight the role of monoubiquitylation in regulating the association dynamics of Nup60 and its partner, Nup2, with the NPC through an interaction with Nup84, a component of the Y Complex. Although major Nuclear transport routes are not regulated by Nup60 modifications, monoubiquitylation of Nup60 is stimulated upon genotoxic stress and regulates the DNA-damage response and telomere repair. Together, these data reveal an original mechanism contributing to the plasticity of the NPC at a molecular-organization and functional level.
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The DNA damage response at eroded telomeres and tethering to the Nuclear Pore Complex.
Nature Cell Biology, 2009Co-Authors: Basheer Khadaroo, Marie-noëlle Simon, M. Teresa Teixeira, Pierre Luciano, Nadine Eckert-boulet, Susanne Manuela Germann, Irene Gallina, Pauline Abdallah, Eric Gilson, Vincent GéliAbstract:Cells with a single short telomere and lacking telomerase mount a damage response consisting of recruitment of DNA damage checkpoint proteins, Cdc13, RPA and Rad52, many generations before senescence and in addition show tethering of the short telomere to the Nuclear Pore Complex.
