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Nuclear pore complex

Toby Mathieson, Holger Franken, Jan Kosinski, Nils Kurzawa, Nico Zinn, Gavain Sweetman, Daniel Poeckel, Vikram S Ratnu, Maike Schramm, Isabelle Becher, Michael Steidel, Kyung-Min Noh, Giovanna Bergamini, Martin Beck, Marcus Bantscheff, Mikhail M Savitski
A better understanding of proteostasis in health and disease requires robust methods to determine protein half-lives. Here we improve the precision and accuracy of peptide ion intensity-based quantification, enabling more accurate protein turnover determination in non-dividing cells by dynamic SILAC-based proteomics. This approach allows exact determination of protein half-lives ranging from 10 to >1000 h. We identified 4000-6000 proteins in several non-dividing cell types, corresponding to 9699 unique protein identifications over the entire data set...
February 15, 2018: Nature Communications
Liping Yang, Rong Wang, Shixing Yang, Zexu Ma, Shaoli Lin, Yuchen Nan, Qisheng Li, Qiyi Tang, Yan-Jin Zhang
Movement of macromolecules between the cytoplasm and the nucleus occurs through the nuclear pore complex (NPC). Karyopherins comprise a family of soluble transport factors facilitating nucleocytoplasmic translocation of proteins through the NPC. In this study, we discovered that karyopherin alpha6 (KPNA6, also known as importin alpha7) was required for the optimal replication of porcine reproductive and respiratory syndrome virus (PRRSV) and Zika virus (ZIKV), which are positive-sense, single-stranded RNA viruses replicating in the cytoplasm...
February 14, 2018: Journal of Virology
Adithya N Ananth, Ankur Mishra, Steffen Frey, Arvind Dwarkasing, Roderick Versloot, Erik van der Giessen, Dirk Görlich, Patrick Onck, Cees Dekker
Nuclear pore complexes (NPCs) lined with intrinsically disordered FG-domains act as selective gatekeepers for molecular transport between the nucleus and the cytoplasm in eukaryotic cells. The underlying physical mechanism of the intriguing selectivity is still under debate. Here, we probe the transport of ions and transport receptors through biomimetic NPCs consisting of Nsp1 domains attached to the inner surface of solid-state nanopores. We examine both wildtype FG-domains and hydrophilic SG-mutants. FG-nanopores showed a clear selectivity as transport receptors can translocate across the pore whereas other proteins cannot...
February 14, 2018: ELife
Michael P Hughes, Michael R Sawaya, David R Boyer, Lukasz Goldschmidt, Jose A Rodriguez, Duilio Cascio, Lisa Chong, Tamir Gonen, David S Eisenberg
Subcellular membraneless assemblies are a reinvigorated area of study in biology, with spirited scientific discussions on the forces between the low-complexity protein domains within these assemblies. To illuminate these forces, we determined the atomic structures of five segments from protein low-complexity domains associated with membraneless assemblies. Their common structural feature is the stacking of segments into kinked β sheets that pair into protofilaments. Unlike steric zippers of amyloid fibrils, the kinked sheets interact weakly through polar atoms and aromatic side chains...
February 9, 2018: Science
Wanlu Zhang, Annett Neuner, Diana Rüthnick, Timo Sachsenheimer, Christian Lüchtenborg, Britta Brügger, Elmar Schiebel
The paralogous Brr6 and Brl1 are conserved integral membrane proteins of the nuclear envelope (NE) with an unclear role in nuclear pore complex (NPC) biogenesis. Here, we analyzed double-degron mutants of Brr6/Brl1 to understand this function. Depletion of Brr6 and Brl1 caused defects in NPC biogenesis, whereas the already assembled NPCs remained unaffected. This NPC biogenesis defect was not accompanied by a change in lipid composition. However, Brl1 interacted with Ndc1 and Nup188 by immunoprecipitation, and with transmembrane and outer and inner ring NPC components by split yellow fluorescent protein analysis, indicating a direct role in NPC biogenesis...
February 9, 2018: Journal of Cell Biology
Raquel Sales Gil, Ines J de Castro, Jerusalem Berihun, Paola Vagnarelli
The nuclear envelope (NE) is a unique topological structure formed by lipid membranes (Inner and Outer Membrane: IM and OM) interrupted by open channels (Nuclear Pore complexes). Besides its well-established structural role in providing a physical separation between the genome and the cytoplasm and regulating the exchanges between the two cellular compartments, it has become quite evident in recent years that the NE also represents a hub for localized signal transduction. Mechanical, stress, or mitogen signals reach the nucleus and trigger the activation of several pathways, many effectors of which are processed at the NE...
February 6, 2018: Biochemical Society Transactions
M Soheilypour, M Mofrad
Despite extensive research on how mRNAs are quality controlled prior to export into the cytoplasm, the exact underlying mechanisms are still under debate. Specifically, it is unclear how quality control proteins at the entry of the nuclear pore complex (NPC) distinguish normal and aberrant mRNAs. While some of the involved components are suggested to act as switches and recruit different factors to normal versus aberrant mRNAs, some experimental and computational evidence suggests that the combined effect of the regulated stochastic interactions between the involved components could potentially achieve an efficient quality control of mRNAs...
February 12, 2018: Nucleus
Samuel Sparks, Deniz B Temel, Michael P Rout, David Cowburn
The largely intrinsically disordered phenylalanine-glycine-rich nucleoporins (FG Nups) underline a selectivity mechanism that enables the rapid translocation of transport factors (TFs) through the nuclear pore complexes (NPCs). Conflicting models of NPC transport have assumed that FG Nups undergo different conformational transitions upon interacting with TFs. To selectively characterize conformational changes in FG Nups induced by TFs we performed small-angle neutron scattering (SANS) with contrast matching...
January 29, 2018: Structure
Haruhiko Asakawa, Yasushi Hiraoka, Tokuko Haraguchi
Cellular structures and biomolecular complexes are not simply assemblies of proteins, but are organized with defined numbers of protein molecules in precise locations. Thus, evaluating the spatial localization and numbers of protein molecules is of fundamental importance in understanding cellular structures and functions. The amounts of proteins of interest have conventionally been determined by biochemical methods. However, biochemical measurements based on the population average have limitations: it is sometimes difficult to determine the amounts of insoluble proteins or low expression proteins localized in small portions of the cell...
2018: Methods in Molecular Biology
Jean-Pierre Korb
The nuclear magnetic relaxation dispersion (NMRD) technique consists of measurement of the magnetic-field dependence of the longitudinal nuclear-spin-lattice relaxation rate 1/T1. Usually, the acquisition of the NMRD profiles is made using a fast field cycling (FFC) NMR technique that varies the magnetic field and explores a very large range of Larmor frequencies (10 kHz < ω0/(2π) <40 MHz). This allows extensive explorations of the fluctuations to which nuclear spin relaxation is sensitive...
February 2018: Progress in Nuclear Magnetic Resonance Spectroscopy
Asumi Ochiai, Junpei Imoto, Mizuki Suetake, Tatsuki Komiya, Genki Furuki, Ryohei Ikehara, Shinya Yamasaki, Gareth T W Law, Toshihiko Ohnuki, Bernd Grambow, Rodney C Ewing, Satoshi Utsunomiya
Trace U was released from the Fukushima Daiichi Nuclear Power Plant (FDNPP) during the meltdowns, but the speciation of the released components of the nuclear fuel remains unknown. We report, for the first time, the atomic-scale characteristics of nano-fragments of the nuclear fuels that were released from the FDNPP into the environment. Nano-fragments of an intrinsic U-phase were discovered to be closely associated with radioactive cesium-rich microparticles (CsMPs) in paddy soils collected ~4 km from the FDNPP...
January 29, 2018: Environmental Science & Technology
Xiuying Li, Peiyuan Kang, Zhuo Chen, Sneha Lal, Li Zhang, Jeremiah J Gassensmith, Zhenpeng Qin
Efficient delivery to the cell nucleus remains a significant challenge for many biomolecules, including anticancer drugs, proteins and DNAs. Despite numerous attempts to improve nuclear import including the use of nuclear localization signal (NLS) peptides and nanoparticle carriers, they are limited by the nanoparticle size, conjugation method, dependence on the functional nuclear import and intracellular trafficking mechanisms. To overcome these limitations, here we report that the nanomechanical force from plasmonic nanobubbles increases nuclear membrane permeability and promotes universal uptake of macromolecules into the nucleus, including macromolecules that are larger than the nuclear pore complex and would otherwise not enter the nucleus...
January 29, 2018: Chemical Communications: Chem Comm
Tatjana Schneckenburger, Jens Riefstahl, Klaus Fischer
Background: Aliphatic (poly)hydroxy carboxylic acids [(P)HCA] occur in natural, e.g. soils, and in technical (waste disposal sites, nuclear waste repositories) compartments . Their distribution, mobility and chemical reactivity, e.g. complex formation with metal ions and radionuclides, depend, among others, on their adsorption onto mineral surfaces. Aluminium hydroxides, e.g. gibbsite [α-Al(OH)3], are common constituents of related solid materials and mimic the molecular surface properties of clay minerals...
2018: Environmental Sciences Europe
Ryo Hayama, Samuel Sparks, Lee M Hecht, Kaushik Dutta, Christina M Cabana, Jerome M Karp, Michael P Rout, David Cowburn
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 (NPC)...
January 26, 2018: Journal of Biological Chemistry
Stéphane Isabettini, Mirjam E Baumgartner, Peter Fischer, Erich J Windhab, Marianne Liebi, Simon Kuster
Bicelles are tunable disk-like polymolecular assemblies formed from a large variety of lipid mixtures. Applications range from membrane protein structural studies by nuclear magnetic resonance (NMR) to nanotechnological developments including the formation of optically active and magnetically switchable gels. Such technologies require high control of the assembly size, magnetic response and thermal resistance. Mixtures of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and its lanthanide ion (Ln3+) chelating phospholipid conjugate, 1,2-dimyristoyl-sn-glycero-3-phospho-ethanolamine-diethylene triaminepentaacetate (DMPE-DTPA), assemble into highly magnetically responsive assemblies such as DMPC/DMPE-DTPA/Ln3+ (molar ratio 4:1:1) bicelles...
January 3, 2018: Journal of Visualized Experiments: JoVE
Reinoud de Groot, Joel Lüthi, Helen Lindsay, René Holtackers, Lucas Pelkmans
High-content imaging using automated microscopy and computer vision allows multivariate profiling of single-cell phenotypes. Here, we present methods for the application of the CISPR-Cas9 system in large-scale, image-based, gene perturbation experiments. We show that CRISPR-Cas9-mediated gene perturbation can be achieved in human tissue culture cells in a timeframe that is compatible with image-based phenotyping. We developed a pipeline to construct a large-scale arrayed library of 2,281 sequence-verified CRISPR-Cas9 targeting plasmids and profiled this library for genes affecting cellular morphology and the subcellular localization of components of the nuclear pore complex (NPC)...
January 23, 2018: Molecular Systems Biology
Ramona Jühlen, Mirko Peitzsch, Sebastian Gärtner, Dana Landgraf, Graeme Eisenhofer, Angela Huebner, Katrin Koehler
Mutations in the AAAS gene coding for the nuclear pore complex protein ALADIN lead to the autosomal recessive disorder triple A syndrome. Triple A patients present with a characteristic phenotype including alacrima, achalasia and adrenal insufficiency. Patient fibroblasts show increased levels of oxidative stress, and several in vitro studies have demonstrated that the nucleoporin ALADIN is involved in both the cellular oxidative stress response and adrenal steroidogenesis. It is known that ALADIN knock-out mice lack a phenotype resembling human triple A syndrome...
January 23, 2018: Biology Open
Patrick D Ellis Fisher, Qi Shen, Bernice Akpinar, Luke K Davis, Kenny Kwok Hin Chung, David Baddeley, Andela Saric, Thomas J Melia, Bart W Hoogenboom, Chenxiang Lin, C Patrick Lusk
Nuclear pore complexes (NPCs) form gateways that control molecular exchange between the nucleus and the cytoplasm. They impose a diffusion barrier to macromolecules and enable the selective transport of nuclear transport receptors with bound cargo. The underlying mechanisms that establish these permeability properties remain to be fully elucidated, but require unstructured nuclear pore proteins rich in Phe-Gly (FG)-repeat domains of different types, such as FxFG and GLFG. While physical modeling and in vitro approaches have provided a framework for explaining how the FG network contributes to the barrier and transport properties of the NPC, it remains unknown whether the number and/or the spatial positioning of different FG-domains along a cylindrical, ~40 nm diameter transport channel contributes to their collective properties and function...
January 19, 2018: ACS Nano
Shotaro Otsuka, Anna M Steyer, Martin Schorb, Jean-Karim Hériché, M Julius Hossain, Suruchi Sethi, Moritz Kueblbeck, Yannick Schwab, Martin Beck, Jan Ellenberg
The nuclear envelope has to be reformed after mitosis to create viable daughter cells with closed nuclei. How membrane sealing of DNA and assembly of nuclear pore complexes (NPCs) are achieved and coordinated is poorly understood. Here, we reconstructed nuclear membrane topology and the structures of assembling NPCs in a correlative 3D EM time course of dividing human cells. Our quantitative ultrastructural analysis shows that nuclear membranes form from highly fenestrated ER sheets whose holes progressively shrink...
January 2018: Nature Structural & Molecular Biology
Pan Chen, Daniel L Levy
Xenopus egg extract represents a powerful cell-free biochemical tool for studying organelle assembly and function. Large quantities of cytoplasm can be isolated, and biochemical manipulation of extract composition and cell cycle state is relatively straightforward. In this protocol, we describe the reconstitution of nuclear assembly by adding a chromatin source to interphasic X. laevis egg extract. Intact nuclei assemble within 30-45 min of initiating the reaction, followed by nuclear growth. We also describe methods for imaging and quantifying nuclear import kinetics...
January 10, 2018: Cold Spring Harbor Protocols
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