bims-nucpor 2022-04-17 papers

bims-nucpor

Biomed News

on Nuclear pore complex and nucleoporins in stress, aging and disease

Issue of 2022–04–17
thirty-two papers selected by
Sara Mingu, Johannes Gutenberg University

Analysis of Nuclear Pore Complex Permeability in Mammalian Cells and Isolated Nuclei Using Fluorescent Dextrans.
Localizing Total mRNA in Plant Cells.
Using Single Molecule RNA FISH to Determine Nuclear Export and Transcription Phenotypes in Drosophila Tissues.
Analysis of Nuclear Pore Complexes in Caenorhabditis elegans by Live Imaging and Functional Genomics.
Validation of Nuclear Pore Complex Protein-Protein Interactions by Transient Expression in Plants.
Multivalent Interactions with Intrinsically Disordered Proteins Probed by Surface Plasmon Resonance.
Visualizing Nuclear Pore Complexes in Xenopus Egg Extracts.
Nuclear Pore Complex Assembly Using Xenopus Egg Extract.
Analysis of Ubiquitylation and SUMOylation of Yeast Nuclear Pore Complex Proteins.
Speed Microscopy: High-Speed Single Molecule Tracking and Mapping of Nucleocytoplasmic Transport.
Subcellular Fractionation Suitable for Studies of RNA and Protein Trafficking.
Atomic Force Microscopy for Structural and Biophysical Investigations on Nuclear Pore Complexes.
Live CLEM Imaging of Tetrahymena to Analyze the Dynamic Behavior of the Nuclear Pore Complex.
Imaging Fluorescent Nuclear Pore Complex Proteins in C. elegans.
Protein Retargeting in Aspergillus nidulans to Study the Function of Nuclear Pore Complex Proteins.
Analysis of Nucleoporin Function Using Inducible Degron Techniques.
Phosphorylation but Not Oligomerization Drives the Accumulation of Tau with Nucleoporin Nup98.
Transformation of Chaetomium thermophilum and Affinity Purification of Native Thermostable Protein Complexes.
Scanning Electron Microscopy (SEM) and Immuno-SEM of Nuclear Pore Complexes from Amphibian Oocytes, Mammalian Cell Cultures, Yeast, and Plants.
Affinity Isolation of Endogenous Saccharomyces Cerevisiae Nuclear Pore Complexes.
Visualizing Nuclear Pore Complex Assembly In Situ in Human Cells at Nanometer Resolution by Correlating Live Imaging with Electron Microscopy.
High-Resolution Imaging and Analysis of Individual Nuclear Pore Complexes.
Purification of Cdk-CyclinB-Kinase-Targeted Phosphopeptides from Nuclear Envelope.
Monitoring of Chromatin Organization at the Nuclear Pore Complex, Inner Nuclear Membrane, and Nuclear Interior in Live Cells by Fluorescence Ratiometric Imaging of Chromatin (FRIC).
NPC Structure in Model Organisms: Transmission Electron Microscopy and Immunogold Labeling Using High-Pressure Freezing/Freeze Substitution of Yeast, Worms, and Plants.
Binding Affinity Measurement of Nuclear Export Signal Peptides to Their Exporter CRM1.
Crystallization of Nuclear Export Signals or Small-Molecule Inhibitors Bound to Nuclear Exporter CRM1.
Roles of Nucleoporin RanBP2/Nup358 in Acute Necrotizing Encephalopathy Type 1 (ANE1) and Viral Infection.
Bimolecular Fluorescence Complementation: Quantitative Analysis of In Cell Interaction of Nuclear Transporter Importin α with Cargo Proteins.
Hormone-Inducible Transport Reporter Assay to Study Nuclear Import Defects in Neurodegenerative Diseases.
Molecular Basis of Functional Effects of Phosphorylation of the C-Terminal Domain of the Rabies Virus P Protein.
Bioinformatics analysis of the expression and clinical significance of the NUP210 Gene in acute myeloid leukaemia.

Methods Mol Biol. 2022 ;2502 69-80

Analysis of Nuclear Pore Complex Permeability in Mammalian Cells and Isolated Nuclei Using Fluorescent Dextrans.

Marcela Raices, Maximiliano A D'Angelo.

  In eukaryotic cells the nuclear envelope encloses the genome separating it from the rest of the cell. Nuclear pore complexes are large multi protein channels that perforate the nuclear envelope, connecting the nucleus and the cytoplasm. Besides controlling nucleocytoplasmic molecule exchange, nuclear pore complexes create a permeability barrier that defines the maximum size of molecules that can freely diffuse into the nucleus. Accumulating evidence indicate that the permeability barrier of the nucleus can vary in different cellular conditions, during aging and in disease. Here we provide a simple protocol to analyze changes in nuclear permeability in plasma membrane-permeabilized cells and isolated nuclei using fluorescent dextrans of different sizes and confocal microscopy. The methods described herein represent a valuable resource to researchers studying the function of nuclear pore complexes and the dynamics of nuclear permeability in different cell types and processes.

Keywords:  Dextran; Diffusion; Nuclear permeability; Nuclear pore complex; Permeability barrier

DOI:  https://doi.org/10.1007/978-1-0716-2337-4_4
Methods Mol Biol. 2022 ;2502 105-111

Localizing Total mRNA in Plant Cells.

Geraint Parry.

  Visualizing the location of the total cellular mRNA pool can be important to understand how different genes affect cellular physiology. Over the past decade researchers investigating RNA processing, nuclear transport and the function of the nuclear pore complex have used in situ hybridization protocol to visualize and quantify the accumulation of the total mRNA pool within the plant cell nucleus.

Keywords:  In situ hybridization; Nuclear pore complex; Nuclear transport; Nucleus; mRNA

DOI:  https://doi.org/10.1007/978-1-0716-2337-4_7
Methods Mol Biol. 2022 ;2502 113-125

Using Single Molecule RNA FISH to Determine Nuclear Export and Transcription Phenotypes in Drosophila Tissues.

Jennifer R Aleman, Shawn C Little, Maya Capelson.

  Single molecule RNA fluorescence in situ hybridization (smRNA FISH) is a widely used method for examining cellular localization of RNA and assessing gene expression outputs. The Nuclear Pore Complex (NPC) is a nuclear macro-complex known to both mediate nucleocytoplasmic transport and influence transcription via interactions with chromatin. Consequently, depletion of NPC proteins can result in defects in either transcription or nuclear export of mRNA. To distinguish between these two different functions of NPC components, it is preferable to analyze transcription and mRNA export simultaneously or in the same cell. Here, we present a smRNA FISH protocol with downstream custom MATLAB image analysis for application in Drosophila larval salivary gland tissues. This method can detect both nuclear export and transcriptional phenotypes in the same cell and as a single assay, and can be adapted to many other cell types and organisms.

Keywords:  Confocal microscopy; Drosophila; Larval salivary gland; Nuclear pore; Nuclear transport; RNA FISH; Transcription; mRNA export

DOI:  https://doi.org/10.1007/978-1-0716-2337-4_8
Methods Mol Biol. 2022 ;2502 161-182

Analysis of Nuclear Pore Complexes in Caenorhabditis elegans by Live Imaging and Functional Genomics.

Patricia de la Cruz Ruiz, Raquel Romero-Bueno, Peter Askjaer.

  Nuclear pore complexes (NPCs) are essential to communication of macromolecules between the cell nucleus and the surrounding cytoplasm. RNA synthesized in the nucleus is exported through NPCs to function in the cytoplasm, whereas transcription factors and other proteins are selectively and actively imported. In addition, many NPC constituents, known as nuclear pore proteins (nucleoporins or nups), also play critical roles in other processes, such as genome organization, gene expression, and kinetochore function. Thanks to its genetic amenability and transparent body, the nematode Caenorhabditis elegans is an attractive model to study NPC dynamics. We provide here an overview of available genome engineered strains and FLP/Frt-based tools to study tissue-specific functions of individual nucleoporins. We also present protocols for live imaging of fluorescently tagged nucleoporins in intact tissues of embryos, larvae, and adult and for analysis of interactions between nucleoporins and chromatin by DamID.

Keywords:  CRISPR-Cas9; Caenorhabditis elegans; DamID; FLP; Live imaging; NPC; Nuclear pore complex; Nucleocytoplasmic transport; Nucleoporin; npp

DOI:  https://doi.org/10.1007/978-1-0716-2337-4_11
Methods Mol Biol. 2022 ;2502 235-243

Validation of Nuclear Pore Complex Protein-Protein Interactions by Transient Expression in Plants.

Fumika Ikeda, Kentaro Tamura.

  The nuclear pore complex (NPC) is the largest protein complex, consisting of multiple copies of over 30 different nucleoporins. The interactions between the nucleoporins are critical elements for the NPC functions of the nuclear envelope in plant cells. In recent years, transient expression-based validations of protein-protein interactions have been widely used in plants. Bimolecular fluorescence complementation assay and coimmunoprecipitation assays are powerful tools to identify the molecules that interact with specific proteins. Here, as an example, we describe these techniques using nucleoporin protein interactions in plants.

Keywords:  Agroinfiltration; Arabidopsis thaliana; Bimolecular fluorescence complementation; Coimmunoprecipitation; Nicotiana benthamiana; Nicotiana tabacum; Nuclear pore complex

DOI:  https://doi.org/10.1007/978-1-0716-2337-4_15
Methods Mol Biol. 2022 ;2502 311-328

Multivalent Interactions with Intrinsically Disordered Proteins Probed by Surface Plasmon Resonance.

Larisa E Kapinos, Roderick Y H Lim.

  Multivalent interactions underpin associations between intrinsically disordered proteins (IDPs) and their binding partners. This is a subject of considerable interest and governs how nuclear transport receptors (NTRs) orchestrate the nucleocytoplasmic transport (NCT) of signal-specific cargoes through nuclear pore complexes (NPCs) in eukaryotic cells. Specifically, IDPs termed phenylalanine-glycine nucleoporins (FG Nups) exert multivalent interactions with NTRs to facilitate their transport selectivity and speed through the NPC. Here, we document the use of surface plasmon resonance (SPR) to quantify the affinity and kinetics of NTR-FG Nup binding as a function of FG Nup surface density. Moreover, we describe an in situ method that measures conformational height changes that occur in a FG Nup layer following NTR-binding. Protocols by which the as-obtained SPR results are treated with respect to mass transport limitations are further described. Overall, the SPR methodology described here can be applied to studying multivalent interactions and the role of avidity in diverse biological and biointerfacial systems.

Keywords:  Biological interface; Exportin; FG Nucleoporins; Importin; Intrinsically disordered proteins; Karyopherin; Multivalent interactions; Nuclear pore complex; Steady state; Surface plasmon resonance

DOI:  https://doi.org/10.1007/978-1-0716-2337-4_21
Methods Mol Biol. 2022 ;2502 395-405

Visualizing Nuclear Pore Complexes in Xenopus Egg Extracts.

Sampada Mishra, Daniel L Levy.

  The nuclear pore complex (NPC) is the conduit in the nuclear envelope through which proteins and RNA are transported between the cytoplasm and nucleus. Xenopus egg extracts that support de novo assembly of nuclei have provided a robust system to study NPC structure and function because the biochemical composition of the extract can be easily manipulated. Here we describe how to assemble nuclei in Xenopus egg extract, how to visualize and analyze NPCs in both live and fixed samples, and different approaches to altering nucleocytoplasmic transport in extract.

Keywords:  Immunofluorescence; NPC; Nuclear import; Nuclear pore complexes; Spindown; Xenopus egg extracts; mAb414

DOI:  https://doi.org/10.1007/978-1-0716-2337-4_25
Methods Mol Biol. 2022 ;2502 51-66

Nuclear Pore Complex Assembly Using Xenopus Egg Extract.

Guillaume Holzer, Wolfram Antonin.

  Xenopus egg extract is a powerful tool for the in vitro investigation of complex cellular mechanisms. Here we describe how to obtain and employ interphase Xenopus egg extract to study nuclear pore complex assembly and how to analyze the process using Western blot or immunofluorescence based assays. The function of proteins can be conveniently assayed by high-efficient antibody mediated depletion.

Keywords:  Annulate lamellae; Immunofluorescence; Interphase egg extract; Nuclear assembly; Nuclear pore complex; Western blot; Xenopus laevis

DOI:  https://doi.org/10.1007/978-1-0716-2337-4_3
Methods Mol Biol. 2022 ;2502 259-269

Analysis of Ubiquitylation and SUMOylation of Yeast Nuclear Pore Complex Proteins.

Catherine Dargemont.

  Posttranslational modifications and in particular ubiquitylation and SUMOylation of the nuclear pore complex (NPC), have been shown to regulate some of its functions, particularly in response to diverse stress signals.Although proteomic approaches are extremely powerful to identify substrates and modification sites, dissecting specific mechanisms and regulation functions of ubiquitylation and SUMOylation of the diverse NPC proteins, in different genetic backgrounds or cell environmental conditions, requires specific biochemical assays based on purification and precise analysis of 6His-tagged ubiquitylated or SUMOylated protein of interest. Here we describe an approach that can be easily employed without specific equipment. It allowed to successfully analyze yeast NPC proteins but can easily be adapted to the study of the mammalian NPC.

Keywords:  6His-Tag purification; Nuclear pore complex; SUMO; Ubiquitin; Yeast

DOI:  https://doi.org/10.1007/978-1-0716-2337-4_17
Methods Mol Biol. 2022 ;2502 353-371

Speed Microscopy: High-Speed Single Molecule Tracking and Mapping of Nucleocytoplasmic Transport.

Steven J Schnell, Mark Tingey, Weidong Yang.

  The nuclear pore complex (NPC) functions as a gateway through which molecules translocate into and out of the nucleus. Understanding the transport dynamics of these transiting molecules and how they interact with the NPC has great potentials in the discovery of clinical targets. Single-molecule microscopy techniques are powerful tools to provide sub-diffraction limit information about the dynamic and structural details of nucleocytoplasmic transport. Here we detail single-point edge-excitation subdiffraction (SPEED) microscopy, a high-speed superresolution microscopy technique designed to track and map proteins and RNAs as they cross native NPCs.

Keywords:  Live cell imaging; Nucleocytoplasmic transport; Single-molecule microscopy; Single-point edge-excitation subdiffraction microscopy; Sub–diffraction limit imaging; Superresolution light microscopy

DOI:  https://doi.org/10.1007/978-1-0716-2337-4_23
Methods Mol Biol. 2022 ;2502 91-104

Subcellular Fractionation Suitable for Studies of RNA and Protein Trafficking.

Biljana Culjkovic-Kraljacic, Katherine L B Borden.

  The nuclear pore complex is the major conduit for trafficking between the nucleus and cytoplasm. Nuclear import and export of both proteins and RNAs represent important functional steps for many biological processes. One of the major means to study NPC activity and the nuclear and cytoplasmic distribution of proteins and RNAs is through biochemical fractionation. Here, we describe detailed methods to generate high quality nuclear and cytoplasmic fractions simultaneously capturing RNA and proteins which can be used subsequently for a wide array of biochemical characterizations including proteomics and next generation sequencings.

Keywords:  Localization; Nuclear- Cytoplasmic Fractionation; Protein; RNA

DOI:  https://doi.org/10.1007/978-1-0716-2337-4_6
Methods Mol Biol. 2022 ;2502 299-310

Atomic Force Microscopy for Structural and Biophysical Investigations on Nuclear Pore Complexes.

Ivan Liashkovich, Gonzalo Rosso, Victor Shahin.

  Atomic force microscopy (AFM) enables simultaneous generation of topographical and biophysical maps of surfaces of biological samples at nanoresolution in physiologically relevant environments. Here, we describe the application of AFM to study nuclear pore complexes from structural and biophysical aspects.

Keywords:  Atomic force microscopy; Nanoimaging; Nanomechanics; Nuclear envelope; Nuclear pore complexes

DOI:  https://doi.org/10.1007/978-1-0716-2337-4_20
Methods Mol Biol. 2022 ;2502 473-492

Live CLEM Imaging of Tetrahymena to Analyze the Dynamic Behavior of the Nuclear Pore Complex.

Tokuko Haraguchi, Hiroko Osakada, Masaaki Iwamoto.

  Tetrahymena is a fascinating organism for studying the nuclear pore complex because it has two structurally and functionally distinct nuclei (macronucleus and micronucleus) within a cell, and there are two compositionally distinct nuclear pore complexes (NPCs) with different functions in each nucleus. Therefore, it is possible to link the function of a specific constituent protein with the nuclear function of the macronucleus and micronucleus. Additionally, these NPCs undergo dynamic changes in their structures and compositions during nuclear differentiation. Live CLEM imaging, a method of correlative light and electron microscopy (CLEM) combined with live cell imaging, is a powerful tool for visualizing these dynamic changes of specific molecules/structures of interest at high resolution. Here, we describe Live CLEM that can be applied to the study of the dynamic behavior of NPCs in Tetrahymena cells undergoing nuclear differentiation.

Keywords:  CLEM; Ciliate; Correlative light and electron microscopy; Electron microscopy; Fluorescence microscopy; Live cell imaging; Nuclear envelope; Nuclear pore complex; Tetrahymena

DOI:  https://doi.org/10.1007/978-1-0716-2337-4_30
Methods Mol Biol. 2022 ;2502 373-393

Imaging Fluorescent Nuclear Pore Complex Proteins in C. elegans.

Courtney Lancaster, Giulia Zavagno, James Groombridge, Adelaide Raimundo, David Weinkove, Tim Hawkins, Joanne Robson, Martin W Goldberg.

  C. elegans is a well-characterized and relatively simple model organism, making it attractive for studying nuclear pore complex proteins in cell and developmental biology. C. elegans is transparent and highly amendable to genetic manipulation. Therefore, it is possible to generate fluorescently tagged proteins and combine this with various light microscopy techniques to study protein behavior in space and time. Here, we provide protocols to prepare both fixed and live C. elegans for confocal and light sheet microscopy. This enables the analysis of nuclear pore complex proteins from embryonic stages to the aging adult.

Keywords:  3D-SIM; C. elegans; Confocal; Fluorescence microscopy; Light sheet; Nuclear pore complex; Nucleus; Superresolution; fluorescent protein

DOI:  https://doi.org/10.1007/978-1-0716-2337-4_24
Methods Mol Biol. 2022 ;2502 183-201

Protein Retargeting in Aspergillus nidulans to Study the Function of Nuclear Pore Complex Proteins.

Subbulakshmi Suresh, Stephen A Osmani.

  Targeting a protein of interest to a subcellular location by linking it to another protein is a commonly used approach to help determine function in many model systems. Such targeting strategies rely on the creation of functional protein-protein fusions followed by microscopic examination if one or both proteins have fluorescent tags. In this paper, using the model filamentous fungus Aspergillus nidulans, we describe methods to link GFP-tagged proteins to other proteins in the cell by fusing the latter with a GFP-Binding Protein (GBP) that has a high affinity for GFP. This method enables rapid generation of strains with linked proteins in filamentous fungi by sexual crossing or transformations. Additionally, if these two linked proteins stably associate with subcellular structures, it is possible to link the structures using this approach. For example, we used this method to link Nuclear Pore Complexes (NPCs) with mitotic chromatin in A. nidulans. This was done to show that the NPC protein Nup2, that uniquely transitions from NPC onto mitotic chromatin, couples NPC segregation with chromatin segregation by bridging these two structures. In the absence of Nup2, we used the described approach to show that an artificial NPC-chromatin bridge was sufficient for faithful NPC segregation.

Keywords:  Aspergillus; Filamentous fungi; GFP-binding protein; Green fluorescent protein; Nuclear pore complex; Protein retargeting

DOI:  https://doi.org/10.1007/978-1-0716-2337-4_12
Methods Mol Biol. 2022 ;2502 129-150

Analysis of Nucleoporin Function Using Inducible Degron Techniques.

Vasilisa Aksenova, Alexei Arnaoutov, Mary Dasso.

  Over the last decade, the use of auxin-inducible degrons (AID) to control the stability of target proteins has revolutionized the field of cell biology. AID-mediated degradation helps to overcome multiple hurdles that have been encountered in studying multisubunit protein complexes, like the nuclear pore complex (NPC), using classical biochemical and genetic methods. We have used the AID system for acute depletion of individual members of the NPC, called nucleoporins, in order to distinguish their roles both within established NPCs and during NPC assembly.Here, we describe a protocol for CRISPR/Cas9-mediated gene targeting of genes with the AID tag. As an example, we describe a step-by-step protocol for targeting of the NUP153 gene. We also provide recommendations for screening strategies and integration of the sequence encoding the Transport Inhibitor Response 1 (TIR1) protein, a E3-Ubiquitin ligase subunit necessary for AID-dependent protein degradation. In addition, we discuss applications of the NUP-AID system and functional assays for analysis of NUP-AID tagged cell lines.

Keywords:  Auxin-Inducible Degradation; CRISPR/Cas9; Nuclear Pore Complex

DOI:  https://doi.org/10.1007/978-1-0716-2337-4_9
Int J Mol Sci. 2022 Mar 23. pii: 3495. [Epub ahead of print]23(7):

Phosphorylation but Not Oligomerization Drives the Accumulation of Tau with Nucleoporin Nup98.

Lisa Diez, Larisa E Kapinos, Janine Hochmair, Sabrina Huebschmann, Alvaro Dominguez-Baquero, Amelie Vogt, Marija Rankovic, Markus Zweckstetter, Roderick Y H Lim, Susanne Wegmann.

  Tau is a neuronal protein that stabilizes axonal microtubules (MTs) in the central nervous system. In Alzheimer's disease (AD) and other tauopathies, phosphorylated Tau accumulates in intracellular aggregates, a pathological hallmark of these diseases. However, the chronological order of pathological changes in Tau prior to its cytosolic aggregation remains unresolved. These include its phosphorylation and detachment from MTs, mislocalization into the somatodendritic compartment, and oligomerization in the cytosol. Recently, we showed that Tau can interact with phenylalanine-glycine (FG)-rich nucleoporins (Nups), including Nup98, that form a diffusion barrier inside nuclear pore complexes (NPCs), leading to defects in nucleocytoplasmic transport. Here, we used surface plasmon resonance (SPR) and bio-layer interferometry (BLI) to investigate the molecular details of Tau:Nup98 interactions and determined how Tau phosphorylation and oligomerization impact the interactions. Importantly, phosphorylation, but not acetylation, strongly facilitates the accumulation of Tau with Nup98. Oligomerization, however, seems to inhibit Tau:Nup98 interactions, suggesting that Tau-FG Nup interactions occur prior to oligomerization. Overall, these results provide fundamental insights into the molecular mechanisms of Tau-FG Nup interactions within NPCs, which might explain how stress-and disease-associated posttranslational modifications (PTMs) may lead to Tau-induced nucleocytoplasmic transport (NCT) failure. Intervention strategies that could rescue Tau-induced NCT failure in AD and tauopathies will be further discussed.

Keywords:  FG-Nups; MAPT; nuclear pore complex; posttranslational modifications

DOI:  https://doi.org/10.3390/ijms23073495
Methods Mol Biol. 2022 ;2502 35-50

Transformation of Chaetomium thermophilum and Affinity Purification of Native Thermostable Protein Complexes.

Nikola Kellner, Ed Hurt.

  Chaetomium thermophilum, a eukaryotic thermophile, is an aspiring organism holding great potential for various biochemical and biotechnological applications. Prerequisite for genetic manipulation is a reliable transformation system for target genes combined with selection markers operating at the high growth temperatures of the fungus. Here, we present a detailed protocol for Chaetomium thermophilum protoplast transformation to allow stable chromosomal integration of constructs into its genome, rendering this eukaryotic thermophile a valuable resource for affinity purification of native thermostable protein complexes, like nuclear pore subcomplexes.

Keywords:  Affinity purification; Chaetomium thermophilum; Genetic transformation; Nuclear pore complex (NPC); Thermophile

DOI:  https://doi.org/10.1007/978-1-0716-2337-4_2
Methods Mol Biol. 2022 ;2502 417-437

Scanning Electron Microscopy (SEM) and Immuno-SEM of Nuclear Pore Complexes from Amphibian Oocytes, Mammalian Cell Cultures, Yeast, and Plants.

Martin W Goldberg, Jindřiška Fišerová.

  Scanning electron microscopy (SEM) can be used to image nuclear pore complex (NPC) surface structure of from a number of organisms and model systems. With a field emission SEM , this is a medium resolution technique where details of the organization of various components can be directly imaged. Some components, such as the NPC baskets and cytoplasmic filaments, are difficult to visualize in any other way. Protein components can be identified by immunogold labeling. Any surface that can be exposed can potentially be studied by SEM . Several overlapping protocols for SEM sample preparation and immunogold labeling of NPCs are given here. Various parameters for sample preparation, fixation, immunogold labeling, drying, metal coating, and imaging are detailed which have been optimized for different types of specimens and desired endpoints.

Keywords:  Cell culture; Envelope; Immunogold; Labeling; Mammalian; Nuclear; Plant; Scanning electron microscopy; Xenopus; Yeast

DOI:  https://doi.org/10.1007/978-1-0716-2337-4_27
Methods Mol Biol. 2022 ;2502 3-34

Affinity Isolation of Endogenous Saccharomyces Cerevisiae Nuclear Pore Complexes.

Ilona Nudelman, Javier Fernandez-Martinez, Michael P Rout.

  Studying protein complexes in vitro requires the production of a relatively pure sample that maintains the full complement, native organization, and function of that complex. This can be particularly challenging to achieve for large, multi-component, membrane embedded complexes using the traditional recombinant expression and reconstitution methodologies. However, using affinity capture from native cells, suitable whole endogenous protein complexes can be isolated. Here we present a protocol for the affinity isolation of baker's yeast (S. cerevisiae) nuclear pore complexes, which are ~50 MDa assemblies made up of 552 distinct proteins and embedded in a double-membraned nuclear envelope. Producing this sample allowed us for the first time to perform analyses to characterize the mass, stoichiometry, morphology, and connectivity of this complex and to obtain its integrative structure with ~9 Å precision. We believe this methodology can be applied to other challenging protein complexes to produce similar results.

Keywords:  Affinity capture; Baker’s yeast; Electron microscopy; Endogenous macromolecular assembly; Native Isolation; Nuclear pore complex; S. cerevisiae; Structural and functional analyses

DOI:  https://doi.org/10.1007/978-1-0716-2337-4_1
Methods Mol Biol. 2022 ;2502 493-512

Visualizing Nuclear Pore Complex Assembly In Situ in Human Cells at Nanometer Resolution by Correlating Live Imaging with Electron Microscopy.

Helena Bragulat-Teixidor, M Julius Hossain, Shotaro Otsuka.

  In eukaryotic cells that undergo open mitosis, nuclear pore complex assembly proceeds via two distinct pathways: postmitotic and interphase assembly. Studying both assembly processes is challenging because postmitotic assembly is fast, interphase assembly is rare and sporadic, and assembly intermediates in both pathways are very small with a diameter below 100 nm. Here, we present a protocol for studying nuclear pore complex biogenesis in situ in cultured human cells in a spatiotemporally resolved and quantitative manner by combining live imaging with three-dimensional electron microscopy. The method described here can also be applied for studying other cell cycle-associated events with high spatiotemporal resolution.

Keywords:  Cell cycle; Correlative light-electron microscopy; Electron tomography; High-pressure freezing; Live-cell imaging; Mitosis; Nuclear envelope

DOI:  https://doi.org/10.1007/978-1-0716-2337-4_31
Methods Mol Biol. 2022 ;2502 461-471

High-Resolution Imaging and Analysis of Individual Nuclear Pore Complexes.

Boris Fichtman, Saroj G Regmi, Mary Dasso, Amnon Harel.

  Field emission scanning electron microscopy (FESEM) is a well-established technique for acquiring three-dimensional surface images of nuclear pore complexes (NPCs). We present an optimized protocol for the exposure of mammalian cell nuclei and direct surface imaging of nuclear envelopes by FESEM, allowing for a detailed morphological comparison of individual NPCs, without the need for averaging techniques. This provides a unique high resolution tool for studying the effects of cellular stress, specific genetic manipulations and inherited diseases on the ultrastructure of human NPCs.

Keywords:  Auxin-inducible degron; Central channel particles; Mammalian cell culture; Nuclear envelope; Nuclear pore complex; Scanning electron microscopy

DOI:  https://doi.org/10.1007/978-1-0716-2337-4_29
Methods Mol Biol. 2022 ;2502 271-282

Purification of Cdk-CyclinB-Kinase-Targeted Phosphopeptides from Nuclear Envelope.

Justin D Blethrow, Amanda L DiGuilio, Joseph S Glavy.

  We describe a method for rapid identification of protein kinase substrates within the nuclear envelope. Open mitosis in higher eukaryotes is characterized by nuclear envelope breakdown (NEBD) concerted with disassembly of the nuclear lamina and dissociation of nuclear pore complexes (NPCs) into individual subcomplexes. Evidence indicates that reversible phosphorylation events largely drive this mitotic NEBD. These posttranslational modifications likely disrupt structurally significant interactions among nucleoporins (Nups), lamina and membrane proteins of the nuclear envelope (NE). It is therefore critical to determine when and where these substrates are phosphorylated. One likely regulator is the mitotic kinase: Cdk1-Cyclin B. We employed an "analog-sensitive" Cdk1 to bio-orthogonally and uniquely label its substrates in the NE with a phosphate analog tag. Subsequently, peptides covalently modified with the phosphate analogs are rapidly purified by a tag-specific covalent capture and release methodology. In this manner, we were able to confirm the identity of known Cdk1 targets in the NE and discover additional candidates for regulation by mitotic phosphorylation.

Keywords:  ATP analog N6-(benzyl)ATP-γ-phosphorothioate; Analog-sensitive kinase (as-kinase); Cdk1-Cyclin B kinase (cdk1); Mass Spectrometry (MS, MS/MS); Nuclear Envelope (NE); Nuclear Envelope Breakdown (NEBD); Nuclear Lamina; Nuclear Pore Complex (NPC); nucleoporins (Nups)

DOI:  https://doi.org/10.1007/978-1-0716-2337-4_18
Methods Mol Biol. 2022 ;2502 151-160

Monitoring of Chromatin Organization at the Nuclear Pore Complex, Inner Nuclear Membrane, and Nuclear Interior in Live Cells by Fluorescence Ratiometric Imaging of Chromatin (FRIC).

Frida Niss, Cecilia Bergqvist, Anna-Lena Ström, Einar Hallberg.

  The image analysis tool FRIC (Fluorescence Ratiometric Imaging of Chromatin) quantitatively monitors dynamic spatiotemporal distribution of euchromatin and total chromatin in live cells. A vector (pTandemH) assures stoichiometrically constant expression of the histone variants Histone 3.3 and Histone 2B, fused to EGFP and mCherry, respectively. Quantitative ratiometric (H3.3/H2B) imaging displayed a concentrated distribution of heterochromatin in the periphery of U2OS cell nuclei. As a proof of concept, peripheral heterochromatin responded to experimental manipulation of histone acetylation as well as expression of the mutant lamin A protein "progerin," which causes Hutchinson-Gilford Progeria Syndrome. In summary FRIC is versatile, unbiased, robust, requires a minimum of experimental steps and is suitable for screening purposes.

Keywords:  Chromatin; Fluorescence ratiometric; Live imaging; Nuclear membrane; Nuclear pore complex

DOI:  https://doi.org/10.1007/978-1-0716-2337-4_10
Methods Mol Biol. 2022 ;2502 439-459

NPC Structure in Model Organisms: Transmission Electron Microscopy and Immunogold Labeling Using High-Pressure Freezing/Freeze Substitution of Yeast, Worms, and Plants.

A Christine Richardson, Jindřiška Fišerová, Martin W Goldberg.

  The nuclear pore complex (NPC) is a large elaborate structure embedded within the nuclear envelope, and intimately linked to the cytoskeleton, nucleoskeleton, and chromatin. Many different cargoes pass through its central channel and along the membrane at its periphery. The NPC is dismantled and reassembly, fully or partially, every cell cycle. In post-mitotic cells it consists of a combination of hyper-stable and highly dynamic proteins. Because of its size, dynamics, heterogeneity and integration, it is not possible to understand its structure and molecular function by any one, or even several, methods. For decades, and to this day, thin section transmission electron microscopy (TEM) has been a central tool for understanding the NPC, its associations, dynamics and role in transport as it can uniquely answer questions concerning fine structural detail within a cellular context. Using immunogold labeling specific components can also be identified within the ultrastructural context. Model organisms such as Saccharomyces cerevisiae are also central to NPC studies but have not been used extensively in structural work. This is because the cell wall presents difficulties with structural preservation and processing for TEM. In recent years, high-pressure freezing and freeze substitution have overcome these problems, as well as opened up methods to combine immunogold labeling with detailed structural analysis. Other model organisms such as the worm Caenorhabditis elegans and the plant Arabidopsis thaliana have been underused for similar reasons, but with similar solutions, which we present here. There are also many advantages to using these methods, adapted for use in mammalian systems, due to the instant nature of the initial fixation, to capture rapid processes such as nuclear transport, and preservation of dynamic membranes.

Keywords:  C. elegans; Freeze substitution; High-pressure freezing; Immunogold; Plant; Thin section transmission electron microscopy; Yeast

DOI:  https://doi.org/10.1007/978-1-0716-2337-4_28
Methods Mol Biol. 2022 ;2502 245-256

Binding Affinity Measurement of Nuclear Export Signal Peptides to Their Exporter CRM1.

Ho Yee Joyce Fung, Yuh Min Chook.

  CRM1 recognizes hundreds to thousands of protein cargoes by binding to the eight to fifteen residue-long nuclear export signals (NESs) within their polypeptide chains. Various assays to measure the binding affinity of NESs for CRM1 have been developed. CRM1 binds to NESs with a wide range of binding affinities, with dissociation constants that span from low nanomolar to tens of micromolar. An optimized binding affinity assay with improved throughput was recently developed to measure binding affinities of NES peptides for CRM1 in the presence of excess RanGTP. The assay can measure affinities, with multiple replicates, for up to seven different NES peptides per screening plate. Here, we present a protocol for the purification of the necessary proteins and for measuring CRM1-NES binding affinities.

Keywords:  Binding affinity; CRM1; Fluorescence polarization; NES; Nuclear export signals; XPO1

DOI:  https://doi.org/10.1007/978-1-0716-2337-4_16
Methods Mol Biol. 2022 ;2502 285-297

Crystallization of Nuclear Export Signals or Small-Molecule Inhibitors Bound to Nuclear Exporter CRM1.

Ho Yee Joyce Fung, Yuh Min Chook.

  The Karyopherin protein CRM1 or XPO1 is the major nuclear export receptor that regulates nuclear exit of thousands of macromolecules in the cell. CRM1 recognizes protein cargoes by binding to their 8-15 residue-long nuclear export signals (NESs). A ternary CRM1-Ran-RanBP1 complex engineered to be suitable for crystallization has enabled structure determination by X-ray crystallography of CRM1 bound to many NES peptides and small-molecule inhibitors. Here, we present a protocol for the purification of the individual proteins, formation of the ternary CRM1-Ran-RanBP1 complex and crystallization of this complex for X-ray crystallography.

Keywords:  CRM1; KPT; LMB; Leptomycin B; NES; Nuclear export signals; SINE; X-ray crystallography; XPO1

DOI:  https://doi.org/10.1007/978-1-0716-2337-4_19
Int J Mol Sci. 2022 Mar 24. pii: 3548. [Epub ahead of print]23(7):

Roles of Nucleoporin RanBP2/Nup358 in Acute Necrotizing Encephalopathy Type 1 (ANE1) and Viral Infection.

Jing Jiang, Yifan E Wang, Alexander F Palazzo, Qingtang Shen.

  Ran Binding Protein 2 (RanBP2 or Nucleoporin358) is one of the main components of the cytoplasmic filaments of the nuclear pore complex. Mutations in the RANBP2 gene are associated with acute necrotizing encephalopathy type 1 (ANE1), a rare condition where patients experience a sharp rise in cytokine production in response to viral infection and undergo hyperinflammation, seizures, coma, and a high rate of mortality. Despite this, it remains unclear howRanBP2 and its ANE1-associated mutations contribute to pathology. Mounting evidence has shown that RanBP2 interacts with distinct viruses to regulate viral infection. In addition, RanBP2 may regulate innate immune response pathways. This review summarizes recent advances in our understanding of how mutations in RANBP2 contribute to ANE1 and discusses how RanBP2 interacts with distinct viruses and affects viral infection. Recent findings indicate that RanBP2 might be an important therapeutic target, not only in the suppression of ANE1-driven cytokine storms, but also to combat hyperinflammation in response to viral infections.

Keywords:  RanBP2; acute necrotizing encephalopathy type 1 (ANE1); cytokines; viruses

DOI:  https://doi.org/10.3390/ijms23073548
Methods Mol Biol. 2022 ;2502 215-233

Bimolecular Fluorescence Complementation: Quantitative Analysis of In Cell Interaction of Nuclear Transporter Importin α with Cargo Proteins.

Alexander Lee, Marie A Bogoyevitch, David A Jans.

  Bimolecular fluorescence complementation utilizes the ability of two complementary nonfluorescent fragments to reconstitute and emit fluorescence when brought together through specific interaction of attached protein fragments of interest. It has been used in several different contexts to study protein-protein interaction. Here we apply the method for the first time to study interaction of the nuclear transporter importin α and its cargoes in a cellular context. By using image analysis to quantify the extent of nuclear complexation, it is possible to gain insight into the strength of interaction in cells.

Keywords:  Bimolecular fluorescence complementation; CLSM; Importin α; Nuclear transport

DOI:  https://doi.org/10.1007/978-1-0716-2337-4_14
Methods Mol Biol. 2022 ;2502 81-90

Hormone-Inducible Transport Reporter Assay to Study Nuclear Import Defects in Neurodegenerative Diseases.

Saskia Hutten, Dorothee Dormann.

  In the recent years, defective nuclear import has emerged as an important pathomechanism of neurodegenerative diseases, particularly in amyotrophic lateral sclerosis (ALS). Here, specific nuclear RNA binding proteins (RBPs) mislocalize and aggregate in the cytoplasm of neurons and glial cells in degenerating brain regions. Bona fide transport assays that measure nuclear import in a quantitative manner allow one to distinguish whether disease-linked RBP mutations that cause cytosolic RBP mislocalization directly result in reduced nuclear import or cause increased cytoplasmic localization of the RBP through other mechanisms. Here we describe the quantitative analysis of nuclear import rates of RBPs using a hormone-inducible system by live cell imaging.

Keywords:  Hormone-inducible; Live cell imaging; Neurodegeneration; Nuclear import; Nuclear localization sequence; Quantitative transport assay; RNA binding protein

DOI:  https://doi.org/10.1007/978-1-0716-2337-4_5
J Virol. 2022 Apr 11. e0011122

Molecular Basis of Functional Effects of Phosphorylation of the C-Terminal Domain of the Rabies Virus P Protein.

Jingyu Zhan, Ericka Watts, Aaron M Brice, Riley D Metcalfe, Ashley M Rozario, Ashish Sethi, Fei Yan, Toby D M Bell, Michael D W Griffin, Gregory W Moseley, Paul R Gooley.

  The rabies virus (RABV) phosphoprotein (P protein) is expressed as several isoforms, which differ in nucleocytoplasmic localization and microtubule (MT) association, mediated by several sequences, including nuclear localization (NLS) and export (NES) sequences. This appears to underpin a functional diversity enabling multiple functions in viral replication and modulation of host biology. Mechanisms regulating trafficking are poorly defined, but phosphorylation by protein kinase C (PKC) in the P protein C-terminal domain (PCTD) regulates nuclear trafficking, mediated by PCTD-localized NLS/NES sequences, indicating that phosphorylation contributes to functional diversity. The molecular mechanism underlying the effects of PKC, and potential roles in regulating other host-cell interactions are unresolved. Here, we assess effects of phosphorylation on the P3 isoform, which differs from longer isoforms through an ability to localize to the nucleus and associate with MTs, which are associated with antagonism of interferon (IFN) signaling. We find that phosphomimetic mutation of the PKC site S210 inhibits nuclear accumulation and MT association/bundling. Structural analysis indicated that phosphomimetic mutation induces no significant structural change to the NLS/NES but results in the side chain of N226 switching its interactions from E228, within the NES, to E210. Intriguingly, N226 is the sole substituted residue between the PCTD of the pathogenic IFN-resistant RABV strain Nishigahara and a derivative attenuated IFN-sensitive strain Ni-CE, inhibiting P3 nuclear localization and MT association. Thus, S210 phosphorylation appears to impact on N226/E228 to regulate P protein localization, with N226 mutation in Ni-CE mimicking a constitutively phosphorylated state resulting in IFN sensitivity and attenuation. IMPORTANCE Rabies virus P protein is a multifunctional protein with critical roles in replication and manipulation of host-cell processes, including subversion of immunity. This functional diversity involves interactions of several P protein isoforms with the cell nucleus and microtubules. Previous studies showed that phosphorylation of the P protein C-terminal domain (PCTD) at S210, near nuclear trafficking sequences, regulates nucleocytoplasmic localization, indicating key roles in functional diversity. The molecular mechanisms of this regulation have remained unknown. Here, we show that phosphomimetic mutation of S210 regulates nuclear localization and MT association. This regulation does not appear to result from disrupted PCTD structure, but rather from a switch of specific side chain interactions of N226. Intriguingly, N226 was previously implicated in P protein nuclear localization/MT association, immune evasion, and RABV pathogenesis, through undefined mechanisms. Our data indicate that the S210-N226 interface is a key regulator of virus-host interactions, which is significant for pathogenesis.

Keywords:  P protein; lyssavirus; microtubule association; nuclear export; nuclear import; protein phosphorylation; protein structure-function; rabies

DOI:  https://doi.org/10.1128/jvi.00111-22
Hematology. 2022 Dec;27(1): 456-462

Bioinformatics analysis of the expression and clinical significance of the NUP210 Gene in acute myeloid leukaemia.

Mengqi Li, He Zhao.

  : Nucleoporin 210 (NUP210) is a membrane-spanning nuclear protein known to be involved in the development of solid tumours; however, its role in haematological cancers has not been investigated. This study aimed to assess the expression and prognostic potential of NUP210 gene expression in patients with acute myeloid leukaemia (AML).
: In this study, we assessed the expression and prognostic potential of NUP210 gene expression in patients with AML through bioinformatics analysis of The Cancer Genome Atlas and Genotype-Tissue Expression databases.
:The expression of NUP210 mRNA in bone marrow was significantly increased in patients with AML compared to that in healthy individuals and was correlated with AML subtypes according to French-American-British classification as well as with bone marrow blast counts and patient sex (P < 0.05). The high NUP210 expression level was an independent biomarker of poor prognosis in the total AML population (P < 0.05) and separately in female but not male patients.
: Our results of NUP210 mRNA analyses revealed, for the first time, that NUP210 transcription was upregulated in patients with AML and positively associated with unfavourable AML prognosis, suggesting that NUP210 expression can be used as guidance in patient stratification for targeted therapy.

Keywords:  Acute myeloid leukaemia; gene expression; nucleoporin 210; overall survival; prognostic biomarker

DOI:  https://doi.org/10.1080/16078454.2022.2061107