Synthesizing Fluorescently Labeled dsRNAs and sRNAs to Visualize Fungal RNA Uptake.
Abstract Fungal pathogens are responsible for severe crop losses worldwide. Defending crops against fungal disease is critical for global food security; however, most current disease management approaches rely on chemical fungicides that can leave dangerous residues in the environment. RNA interference (RNAi) is an important process through which RNA molecules target and silence complementary genes, regulating gene expression during both transcription and translation. Recently, it has been discovered that some species of fungi can efficiently take up RNAs originating from their host plant and the environment. If t...
Source: Mol Biol Cell - July 28, 2020 Category: Molecular Biology Authors: Hamby R, Wang M, Qiao L, Jin H Tags: Methods Mol Biol Source Type: research

A Single-Molecule RNA Mobility Assay to Identify Proteins that Link RNAs to Molecular Motors.
Abstract mRNA transport and localization is a key aspect of posttranscriptional gene regulation. While the transport of many mRNAs is thought to occur through the recruitment of molecular motors, it has been a challenge to identify RNA-binding proteins (RBPs) that directly interact with motors by conventional assays. In order to identify RBPs and their specific domains that are responsible for recruiting a motor to transport granules, we have developed a single-molecule RNA mobility assay that enables quantifying the effect of a tethered RBP on the movement of an RNA. We demonstrate that tethering of RNAs to myosi...
Source: Mol Biol Cell - July 28, 2020 Category: Molecular Biology Authors: Bhaskar V, Jia M, Chao JA Tags: Methods Mol Biol Source Type: research

Live-Cell CRISPR Imaging in Plant Cells with a Telomere-Specific Guide RNA.
Abstract Chromatin organization is highly dynamic in living cells. Therefore, it might have a regulatory role over biological mechanisms like transcription, replication, and DNA repair. To elucidate how these mechanisms are regulated, it is required to establish imaging methods to visualize the chromatin dynamic in living cells. Here, we provide a protocol for a live plant cell imaging technique based on application of two orthologs of the bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) from Streptococcus pyogenes and Staphylococcus aureus. This technique us...
Source: Mol Biol Cell - July 28, 2020 Category: Molecular Biology Authors: Khosravi S, Dreissig S, Schindele P, Wolter F, Rutten T, Puchta H, Houben A Tags: Methods Mol Biol Source Type: research

Live-Cell Imaging of Genomic Loci Using CRISPR/Molecular Beacon Hybrid Systems.
Abstract The ability to monitor the behavior of specific genomic loci in living cells can offer tremendous opportunities for deciphering the molecular basis driving cellular physiology and disease evolution. Toward this goal, clustered regularly interspersed short palindromic repeat (CRISPR)-based imaging systems have been developed, with tagging of either the nuclease-deactivated mutant of the CRISPR-associated protein 9 (dCas9) or the CRISPR single-guide RNA (sgRNA) with fluorescent protein (FP) molecules currently the major strategies for labeling. Recently, we have demonstrated the feasibility of tagging the s...
Source: Mol Biol Cell - July 28, 2020 Category: Molecular Biology Authors: Wu X, Ying Y, Mao S, Krueger CJ, Chen AK Tags: Methods Mol Biol Source Type: research

Using RNA Tags for Multicolor Live Imaging of Chromatin Loci and Transcription in Drosophila Embryos.
Abstract Elucidating the biological implications of higher order chromatin architectures in animal development requires simultaneous, quantitative measurements of chromatin dynamics and transcriptional activity in living specimen. Here we describe a multicolor labeling and live imaging approach in embryos of the fruit fly Drosophila melanogaster. The method allows simultaneous measurement of movements of specific loci and their transcriptional activity for developmental genes, enabling new approaches to probe the interaction between 3D chromatin architecture and regulation of gene expression in development. P...
Source: Mol Biol Cell - July 28, 2020 Category: Molecular Biology Authors: Chen H, Gregor T Tags: Methods Mol Biol Source Type: research

TRAP-SEQ of Eukaryotic Translatomes Applied to the Detection of Polysome-Associated Long Noncoding RNAs.
Abstract Translating ribosome affinity purification (TRAP) technology allows the isolation of polysomal complexes and the RNAs associated with at least one 80S ribosome. TRAP consists of the stabilization and affinity purification of polysomes containing a tagged version of a ribosomal protein. Quantitative assessment of the TRAP RNA is achieved by direct sequencing (TRAP-SEQ), which provides accurate quantitation of ribosome-associated RNAs, including long noncoding RNAs (lncRNAs). Here we present an updated procedure for TRAP-SEQ, as well as a primary analysis guide for identification of ribosome-associated lncR...
Source: Mol Biol Cell - July 28, 2020 Category: Molecular Biology Authors: Traubenik S, Blanco F, Zanetti ME, Reynoso MA Tags: Methods Mol Biol Source Type: research

In vitro models for ASD-patient-derived iPSCs and cerebral organoids.
Abstract Autism spectrum disorder (ASD) is a set of pervasive neurodevelopmental disorders. The causation is multigenic in most cases, which makes it difficult to model the condition in vitro. Advances in pluripotent stem cell technology has made it possible to generate in vitro models of human brain development. Induced pluripotent stem cells (iPSCs) can be generated from somatic cells and have the ability to differentiate to all of the body's cells. This chapter aims to give an overview of the iPSC technology for generating neural cells and cerebral organoids as models for neurodevelopment and how these models a...
Source: Mol Biol Cell - July 28, 2020 Category: Molecular Biology Authors: Hohmann SS, Ilieva M, Michel TM Tags: Prog Mol Biol Transl Sci Source Type: research

Bench to Bedside and Back: The Evolving Story of Alpha-1 Antitrypsin Deficiency.
PMID: 32716630 [PubMed - as supplied by publisher] (Source: Mol Biol Cell)
Source: Mol Biol Cell - July 27, 2020 Category: Molecular Biology Authors: Chapman KR Tags: Am J Respir Cell Mol Biol Source Type: research

Lost in Translation: ER-mitochondria Crosstalk in Idiopathic Pulmonary Fibrosis.
PMID: 32716642 [PubMed - as supplied by publisher] (Source: Mol Biol Cell)
Source: Mol Biol Cell - July 27, 2020 Category: Molecular Biology Authors: Bueno M, Rojas M Tags: Am J Respir Cell Mol Biol Source Type: research

Cytoskeleton-a crucial key in host cell for coronavirus infection.
Abstract The emerging coronavirus pandemic is threatening the public health all over the world. Cytoskeleton is an intricate network involved in controlling cell shape, cargo transport, signal transduction, and cell division. Infection biology studies have illuminated essential roles for cytoskeleton in mediating the outcome of host‒virus interactions. In this review, we discuss the dynamic interactions between actin filaments, microtubules, intermediate filaments, and coronaviruses. In one round of viral life cycle, coronaviruses surf along filopodia on the host membrane to the entry sites, utilize specific int...
Source: Mol Biol Cell - July 27, 2020 Category: Molecular Biology Authors: Wen Z, Zhang Y, Lin Z, Shi K, Jiu Y Tags: J Mol Cell Biol Source Type: research

Gene dysregulation analysis builds a mechanistic signature for prognosis and therapeutic benefit in colorectal cancer.
Abstract The implementation of cancer precision medicine requires biomarkers or signatures for predicting prognosis and therapeutic benefits. Most of current efforts in this field are paying much more attention to predictive accuracy than to molecular mechanistic interpretability. Mechanism-driven strategy has recently emerged, aiming to build signatures with both predictive power and explanatory power. Driven by this strategy, we developed a robust gene dysregulation analysis framework with machine learning algorithms, which is capable of exploring gene dysregulations underlying carcinogenesis from high-dimension...
Source: Mol Biol Cell - July 27, 2020 Category: Molecular Biology Authors: Li Q, Dai W, Liu J, Li YX, Li YY Tags: J Mol Cell Biol Source Type: research

Rice OVATE family protein 6 regulates leaf angle by modulating secondary cell wall biosynthesis.
Abstract Secondary cell wall not only provides rigidity and mechanical resistance to plants, but also has a large impact on plant growth and adaptation to environments. Biosynthesis of secondary cell wall is regulated by a complicated signaling transduction network; however, it is still unclear how the transcriptional regulation of secondary cell wall biosynthesis works at the molecular level. Here, we report in rice that OVATE family proteins 6 (OsOFP6) is a positive regulator in modulating expression of the genes related to biosynthesis of the secondary cell wall. Transgenic plants with knock-down of OsOFP6 by R...
Source: Mol Biol Cell - July 26, 2020 Category: Molecular Biology Authors: Sun X, Ma Y, Yang C, Li J Tags: Plant Mol Biol Source Type: research

Germline mutagenesis of Nasonia vitripennis through ovarian delivery of CRISPR-Cas9 ribonucleoprotein.
This article is protected by copyright. All rights reserved. PMID: 32715554 [PubMed - as supplied by publisher] (Source: Mol Biol Cell)
Source: Mol Biol Cell - July 26, 2020 Category: Molecular Biology Authors: Chaverra-Rodriguez D, Dalla Benetta E, Heu CC, Rasgon JL, Ferree PM, Akbari OS Tags: Insect Mol Biol Source Type: research

Disorder for Dummies: Functional Mutagenesis of Transient Helical Segments in Disordered Proteins.
Abstract Most cytosolic eukaryotic proteins contain a mixture of ordered and disordered regions. Disordered regions facilitate cell signaling by concentrating sites for posttranslational modifications and protein-protein interactions into arrays of short linear motifs that can be reorganized by RNA splicing. The evolution of disordered regions looks different from their ordered counterparts. In some cases, selection is focused on maintaining protein binding interfaces and PTM sites, but sequence heterogeneity is common. In other cases, simple properties like charge, length, or end-to-end distance are maintained. M...
Source: Mol Biol Cell - July 24, 2020 Category: Molecular Biology Authors: Daughdrill GW Tags: Methods Mol Biol Source Type: research

How to Annotate and Submit a Short Linear Motif to the Eukaryotic Linear Motif Resource.
on TJ Abstract Over the past few years, it has become apparent that approximately 35% of the human proteome consists of intrinsically disordered regions. Many of these disordered regions are rich in short linear motifs (SLiMs) which mediate protein-protein interactions. Although these motifs are short and often partially conserved, they are involved in many important aspects of protein function, including cleavage, targeting, degradation, docking, phosphorylation, and other posttranslational modifications. The Eukaryotic Linear Motif resource (ELM) was established over 15 years ago as a repository to store an...
Source: Mol Biol Cell - July 24, 2020 Category: Molecular Biology Authors: Gouw M, Alvarado-Valverde J, Čalyševa J, Diella F, Kumar M, Michael S, Van Roey K, Dinkel H, Gibson TJ Tags: Methods Mol Biol Source Type: research

Cell-Free Protein Synthesis of Small Intrinsically Disordered Proteins for NMR Spectroscopy.
Abstract Cell-free protein synthesis (CFPS) is an established method to produce recombinant proteins and has been used in a wide variety of applications. The use of CFPS has almost from the onset been favorably linked to the production of isotopically labelled proteins for NMR spectroscopy as the resulting labelling of the produced protein is defined by the chosen amino acids during reaction setup. Here we describe how to set up production and isotopic labelling of small intrinsically disordered proteins (IDPs) for NMR spectroscopy applications using an E. coli-based CFPS system in batch mode. PMID: 32696360 ...
Source: Mol Biol Cell - July 24, 2020 Category: Molecular Biology Authors: Isaksson L, Pedersen A Tags: Methods Mol Biol Source Type: research

Protocol for Investigating the Interactions Between Intrinsically Disordered Proteins and Membranes by Neutron Reflectometry.
Abstract Several intrinsically disordered proteins (IDPs) exhibit high affinity for lipid membranes. Among the different biophysical methods to probe protein-lipid interaction, neutron reflectometry (NR) can provide direct and structural detailed information on the location of the IDP with respect to the membrane. Supported lipid bilayers are commonly used as cell membrane models in such experiments. NR measurements can be collected on the supported lipid bilayer before and after the interaction with the IDP to characterize whether the protein molecules are mainly located on the membrane surface (interaction with ...
Source: Mol Biol Cell - July 24, 2020 Category: Molecular Biology Authors: Luchini A, Arleth L Tags: Methods Mol Biol Source Type: research

Determining Binding Kinetics of Intrinsically Disordered Proteins by NMR Spectroscopy.
Abstract The unique structural flexibility of intrinsically disordered proteins (IDPs) is central to their diverse functions in cellular processes. Protein-protein interactions involving IDPs are frequently transient and dynamic in nature. Nuclear magnetic resonance (NMR) spectroscopy is an especially powerful tool for characterizing the structural propensities, dynamics, and interactions of IDPs. Here we describe applications of the Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiment in combination with NMR titrations to characterize the kinetics and mechanisms of interactions between intrinsically ...
Source: Mol Biol Cell - July 24, 2020 Category: Molecular Biology Authors: Yang K, Arai M, Wright PE Tags: Methods Mol Biol Source Type: research

In Vitro Transition Temperature Measurement of Phase-Separating Proteins by Microscopy.
We describe a method to measure the in vitro transition temperature of a sub-10 μL sample of a phase-separating solution using transmitted light microscopy. Through monitoring changes in transition temperature with solution conditions, this approach allows the impact of additional biomolecules and additives to be quantitatively assessed and permits the construction of phase diagrams. PMID: 32696385 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - July 24, 2020 Category: Molecular Biology Authors: Holland J, Crabtree MD, Nott TJ Tags: Methods Mol Biol Source Type: research

Multiple Site-Specific Phosphorylation of IDPs Monitored by NMR.
Abstract In line with their high accessibility, disordered proteins are exquisite targets of kinases. Eukaryotic organisms use the so-called intrinsically disordered proteins (IDPs) or intrinsically disordered regions of proteins (IDRs) as molecular switches carrying intracellular information tuned by reversible phosphorylation schemes. Solvent-exposed serines and threonines are abundant in IDPs, and, consistently, kinases often modify disordered regions of proteins at multiple sites. In this context, nuclear magnetic resonance (NMR) spectroscopy provides quantitative, residue-specific information that permits map...
Source: Mol Biol Cell - July 24, 2020 Category: Molecular Biology Authors: Julien M, Bouguechtouli C, Alik A, Ghouil R, Zinn-Justin S, Theillet FX Tags: Methods Mol Biol Source Type: research

Detection of Multisite Phosphorylation of Intrinsically Disordered Proteins Using Quantitative Mass-Spectrometry.
Abstract Intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) within proteins have attracted considerable attention in recent years. Several important biological signaling mechanisms including protein-protein interactions and post-translational modifications can be easily mediated by IDPs and IDRs due to their flexible structure. These regions can encode linear sequences that are indispensable in cell-signaling networks and circuits. For example, the linear multisite phosphorylation networks encoded in disordered protein sequences play a key role in cell-cycle regulation where the p...
Source: Mol Biol Cell - July 24, 2020 Category: Molecular Biology Authors: Valk E, Maljavin A, Loog M Tags: Methods Mol Biol Source Type: research

Recording In-Cell NMR-Spectra in Living Mammalian Cells.
Abstract At the foundation of many cellular processes as well as a large number of diseases is the (mis)folding of important intrinsically disordered proteins (IDPs). Despite tremendous scientific efforts, the factors driving their structural changes within the cellular context remain poorly understood. In-cell NMR spectroscopy enables investigation of IDPs directly in the living eukaryotic cell enabling investigation of its intermolecular interactions and ensuing modifications at an unprecedented atomic resolution. In the following protocol, we describe how to prepare in-cell NMR samples of IDPs within eukaryotic...
Source: Mol Biol Cell - July 24, 2020 Category: Molecular Biology Authors: Matečko-Burmann I, Burmann BM Tags: Methods Mol Biol Source Type: research

In-Cell NMR of Intrinsically Disordered Proteins in Mammalian Cells.
Abstract In-cell NMR enables structural insights at atomic resolution of proteins in their natural environment. To date, very few methods have been developed to study proteins by in-cell NMR in mammalian systems. Here we describe a detailed protocol to conduct in-cell NMR on the intrinsically disordered protein of alpha-Synuclein (αSyn) in mammalian cells. This chapter includes a simplified expression and purification protocol of recombinant αSyn and its delivery into mammalian cells. The chapter also describes how to assess the cell leakage that might occur to the cells, the setup of the instrument, a...
Source: Mol Biol Cell - July 24, 2020 Category: Molecular Biology Authors: Gerez JA, Prymaczok NC, Riek R Tags: Methods Mol Biol Source Type: research

WAVE1 and WAVE2 have distinct and overlapping roles in controlling actin assembly at the leading edge.
Abstract SCAR/WAVE proteins and Arp2/3 complex assemble branched actin networks at the leading edge. Two isoforms of SCAR/WAVE, WAVE1 and WAVE2, reside at the leading edge, yet it has remained unclear whether they perform similar or distinct roles. Further, there have been conflicting reports about the Arp2/3-independent biochemical activities of WAVE1 on actin filament elongation. To investigate this in vivo, we knocked out WAVE1 and WAVE2 genes, individually and together, in B16-F1 melanoma cells. We demonstrate that WAVE1 and WAVE2 are redundant for lamellipodia formation and motility. However, there is a signi...
Source: Mol Biol Cell - July 22, 2020 Category: Molecular Biology Authors: Tang Q, Schaks M, Koundinya N, Yang C, Pollard LW, Svitkina TM, Rottner K, Goode BL Tags: Mol Biol Cell Source Type: research

The Borealin Dimerization Domain Interacts with Sgo1 to Drive Aurora B-mediated Spindle Assembly.
Abstract The Chromosomal Passenger Complex (CPC), which includes the kinase Aurora B, is a master regulator of meiotic and mitotic processes that ensure the equal segregation of chromosomes. Sgo1 is thought to play a major role in the recruitment of the CPC to chromosomes, but the molecular mechanism and contribution of Sgo1-dependent CPC recruitment is currently unclear. Using Xenopus egg extracts and biochemical reconstitution, we found that Sgo1 directly interacts with the dimerization domain of the CPC subunit Borealin. Borealin and the PP2A phosphatase complex can simultaneously bind to the coiled coil domain...
Source: Mol Biol Cell - July 22, 2020 Category: Molecular Biology Authors: Bonner MK, Haase J, Saunders H, Gupta H, Li BI, Kelly AE Tags: Mol Biol Cell Source Type: research

PCH-2TRIP13 regulates spindle checkpoint strength.
lla N Abstract Spindle checkpoint strength is dictated by the number of unattached kinetochores, cell volume and cell fate. We show that the conserved AAA-ATPase, PCH-2/TRIP13, which remodels the checkpoint effector Mad2 from an active conformation to an inactive one, controls checkpoint strength in C. elegans. Having previously established that this function is required for spindle checkpoint activation, we demonstrate that in cells genetically manipulated to decrease in cell volume, PCH-2 is no longer required for the spindle checkpoint or recruitment of Mad2 at unattached kinetochores. This role is not limited ...
Source: Mol Biol Cell - July 22, 2020 Category: Molecular Biology Authors: Défachelles L, Russo AE, Nelson CR, Bhalla N Tags: Mol Biol Cell Source Type: research

Scribble, Lgl1, and myosin II form a complex in vivo to promote directed cell migration.
In this study, we show that Scrib, through its LRR domain, forms a complex in vivo with Lgl1. Scrib also forms a complex with myosin II, and Scrib, Lgl1, and myosin II co-localize at the leading edge of migrating cells. The cellular localization and the cytoskeletal association of Scrib and Lgl1 are interdependent, as depletion of either protein affects its counterpart. In addition, depletion of either Scrib or Lgl1 disrupts the cellular localization of myosin II. We show that depletion of either Scrib or Lgl1 affects cell adhesion through the inhibition of focal adhesion disassembly. Finally, we show that Scrib and Lgl1 a...
Source: Mol Biol Cell - July 22, 2020 Category: Molecular Biology Authors: Abedrabbo M, Ravid S Tags: Mol Biol Cell Source Type: research

AnnotatorJ: an ImageJ plugin to ease hand-annotation of cellular compartments.
Horváth P Abstract AnnotatorJ combines single-cell identification with deep learning and manual annotation. Cellular analysis quality depends on accurate and reliable detection and segmentation of cells so that the subsequent steps of analyses e.g. expression measurements may be carried out precisely and without bias. Deep learning has recently become a popular way of segmenting cells, performing unimaginably better than conventional methods. However, such deep learning applications may be trained on a large amount of annotated data to be able to match the highest expectations. High-quality annotations are...
Source: Mol Biol Cell - July 22, 2020 Category: Molecular Biology Authors: Hollandi R, Diósdi Á, Hollandi G, Moshkov N, Horváth P Tags: Mol Biol Cell Source Type: research

Endothelial Permeability Assays In Vitro.
Abstract The endothelium is a thin layer of squamous cells that acts as a semipermeable barrier regulating vascular permeability to let molecules pass through it thereby maintaining tissue fluid homeostasis. Physiological increase in endothelial or vascular permeability is transient, transpired by post-tissue injury during the initial phases of healing, whereas pathological permeability is persistent commonly witnessed in conditions such as atherosclerosis, chronic inflammation, tumor growth, and diabetic retinopathy. The in vivo or in situ use of animal models in the assessment of permeability not only raises ine...
Source: Mol Biol Cell - July 21, 2020 Category: Molecular Biology Authors: Adil MS, Somanath PR Tags: Methods Mol Biol Source Type: research

Editorial introduction.
Abstract Welcome to this Fourth Special Issue of Molecular Biology of the Cell on Forces on and within Cells. As with our other Special Issues, the journal's goal here is to focus attention on a major new direction in cell biology. In this case, it is the field of mechanobiology, which endeavours, broadly, to understand how mechanical forces are harnessed to drive cellular function and how force can also be a mode of biological information that regulates cell behavior. The collection of papers that we have in this issue reflects many current efforts to address these questions. While each of these papers is a disti...
Source: Mol Biol Cell - July 21, 2020 Category: Molecular Biology Authors: Yap AS Tags: Mol Biol Cell Source Type: research

Role of Hypoxia-Inducible Factors in Regulating Right Ventricular Function and Remodeling during Chronic Hypoxia-Induced Pulmonary Hypertension.
Abstract Pulmonary hypertension (PH) and right ventricular (RV) hypertrophy frequently develop in patients with hypoxic lung disease. Chronic alveolar hypoxia (CH) promotes sustained pulmonary vasoconstriction and pulmonary artery (PA) remodeling by acting on lung cells, resulting in the development of PH. RV hypertrophy develops in response to PH, but arterial hypoxemia in CH may influence that response by activating Hypoxia-Inducible Factor-1α (HIF-1α) and/or HIF-2α in cardiomyocytes. Indeed, other studies show that attenuation of PH in CH fails to prevent RV remodeling, suggesting that PH-inde...
Source: Mol Biol Cell - July 21, 2020 Category: Molecular Biology Authors: Smith KA, Waypa GB, Dudley VJ, Budinger GRS, Abdala-Valencia H, Bartom E, Schumacker PT Tags: Am J Respir Cell Mol Biol Source Type: research

Type I Collagen Signaling Regulates Opposing Fibrotic Pathways through Alpha2Beta1 Integrin.
Abstract Fibrosis is characterized by fibroblast activation, leading to matrix remodeling culminating in a stiff, type I collagen rich fibrotic matrix. Alveolar epithelial cell (AEC) apoptosis is also a major feature of fibrogenesis and AEC apoptosis is sufficient to initiate a robust lung fibrotic response. TGFβ is a major driver of fibrosis and can induced both AEC apoptosis and fibroblast activation. We and others have previously shown that changes in extracellular matrix stiffness and composition can regulate the cellular response to TGFβ. In the present study we find that type I collagen signaling p...
Source: Mol Biol Cell - July 21, 2020 Category: Molecular Biology Authors: Agarwal M, Goheen M, Jia S, Ling S, White ES, Kim KK Tags: Am J Respir Cell Mol Biol Source Type: research

Functional Genomics of ATP-Binding Cassette Transporter A3 (ABCA3) Variants.
Abstract Rare or private, biallelic, variants in the ATP-binding cassette transporter A3 (ABCA3) gene are the most common monogenic causes of lethal neonatal respiratory failure and childhood interstitial lung disease. Functional characterization of fewer than 10% of over 200 disease-associated ABCA3 variants (majority missense) suggests either disruption of ABCA3 protein trafficking (type I) or of ATPase-mediated phospholipid transport (type II). Therapies remain limited and non-specific. A scalable platform is required for functional characterization of ABCA3 variants and discovery of pharmacologic correctors. T...
Source: Mol Biol Cell - July 21, 2020 Category: Molecular Biology Authors: Wambach JA, Yang P, Wegner DJ, Heins HB, Luke C, Li F, White FV, Cole FS Tags: Am J Respir Cell Mol Biol Source Type: research

Live-Cell Imaging and Analysis of Nuclear Body Mobility.
Abstract The cell nucleus contains different domains and nuclear bodies, whose position relative to each other inside the nucleus can vary depending on the physiological state of the cell. Changes in the three-dimensional organization are associated with the mobility of individual components of the nucleus. In this chapter, we present a protocol for live-cell imaging and analysis of nuclear body mobility. Unlike other similar protocols, our image analysis pipeline includes non-rigid compensation for global motion of the nucleus before particle tracking and trajectory analysis, leading to precise detection of intra...
Source: Mol Biol Cell - July 20, 2020 Category: Molecular Biology Authors: Sorokin DV, Arifulin EA, Vassetzky YS, Sheval EV Tags: Methods Mol Biol Source Type: research

Laser Targeted Oligo Ligation (LTOL) to Identify DNA Sequences in the Vicinity of a Single Subnuclear Structure in a Single Cell.
Abstract Gene loci are organized around nuclear substructures, forming gene hubs which provide a level of transcriptional control. To date, most techniques used to investigate the genes in these hubs have been based on using material from bulk cells. This makes identifying specific gene associations difficult. Here we describe the Laser Targeted Oligo Ligation (LTOL) technique that was developed to identify DNA sequences around a single subnuclear structure on a single-cell basis by targeting these regions with two-photon irradiation. PMID: 32681480 [PubMed - as supplied by publisher] (Source: Mol Biol Cell)
Source: Mol Biol Cell - July 20, 2020 Category: Molecular Biology Authors: Anchel D, Ching RW Tags: Methods Mol Biol Source Type: research

Studying Proton Gradients Across the Nuclear Envelope.
Sennoune SR Abstract The existence of nuclear pore complexes in the nuclear envelope has led to the assumption that ions move freely from the cytosol into the nucleus, and that the molecular mechanisms at the plasma membrane that regulate cytosolic pH also regulate nuclear pH. Furthermore, studies to measure pH in the nucleus have produced contradictory results, since it has been found that the nuclear pH is either similar to the cytosol or more alkaline than the cytosol. However, most studies of nuclear pH have lacked the rigor needed to understand pH regulation in the nucleus. A major problem has been the lack o...
Source: Mol Biol Cell - July 20, 2020 Category: Molecular Biology Authors: Martínez-Zaguilán R, Sennoune SR Tags: Methods Mol Biol Source Type: research

COMBinatorial Oligonucleotide FISH (COMBO-FISH) with Uniquely Binding Repetitive DNA Probes.
Abstract During the last decade, genome sequence databases of many species have been more and more completed so that it has become possible to further develop a recently established technique of FISH (Fluorescence In Situ Hybridization) called COMBO-FISH (COMBinatorial Oligo FISH). In contrast to standard FISH techniques, COMBO-FISH makes use of a bioinformatic search in sequence databases for probe design, so that it can be done for any species so far sequenced. In the original approach, oligonucleotide stretches of typical lengths of 15-30 nucleotides were selected in such a way that they only co-localize at the...
Source: Mol Biol Cell - July 20, 2020 Category: Molecular Biology Authors: Hausmann M, Lee JH, Sievers A, Krufczik M, Hildenbrand G Tags: Methods Mol Biol Source Type: research

Genome-Wide Mapping of UV-Induced DNA Damage with CPD-Seq.
Abstract Exposure to ultraviolet (UV) radiation is the major risk factor for skin cancers. UV induces helix-distorting DNA damage such as cyclobutane pyrimidine dimers (CPDs). If not repaired, CPDs can strongly block DNA and RNA polymerases and cause mutagenesis or cell death. Nucleotide excision repair (NER) is critical for the removal of UV-induced photolesions including CPDs in the cell. Investigating CPD formation and repair across the genome is important for understanding the mechanisms by which these lesions promote somatic mutations in skin cancers. Here we describe a high-throughput, single nucleotide-reso...
Source: Mol Biol Cell - July 20, 2020 Category: Molecular Biology Authors: Mao P, Wyrick JJ Tags: Methods Mol Biol Source Type: research

Macromolecular Crowding Measurements with Genetically Encoded Probes Based on F örster Resonance Energy Transfer in Living Cells.
Macromolecular Crowding Measurements with Genetically Encoded Probes Based on Förster Resonance Energy Transfer in Living Cells. Methods Mol Biol. 2020;2175:169-180 Authors: Mouton SN, Veenhoff LM, Boersma AJ Abstract Genetically encoded Förster resonance energy transfer (FRET)-based probes allow a sensitive readout for different or specific parameters in the living cell. We previously demonstrated how FRET-based probes could quantify macromolecular crowding with high spatio-temporal resolution and under various conditions. Here, we present a protocol developed for the use of FRET-based crow...
Source: Mol Biol Cell - July 20, 2020 Category: Molecular Biology Authors: Mouton SN, Veenhoff LM, Boersma AJ Tags: Methods Mol Biol Source Type: research

Analysis of a Nuclear Intrinsically Disordered Proteome.
Abstract Intrinsically disordered proteins (IDPs) play crucial roles in cell functioning, although they do not possess defined three-dimensional architecture. They are highly abundant in the cell nucleus, and the vast majority of transcription factors (TFs) contain extended regions of intrinsic disorder. IDPs do not respond to denaturing conditions in a standard manner, and this can be used for their separation from structured proteins. Here we describe a protocol for the isolation and characterization of nuclear IDPs in which heat treatment is used for enrichment of IDPs in samples. The whole workflow comprises t...
Source: Mol Biol Cell - July 20, 2020 Category: Molecular Biology Authors: Skupien-Rabian B, Jankowska U, Kedracka-Krok S Tags: Methods Mol Biol Source Type: research

Timing of Cytosine Methylation on Newly Synthesized RNA by Electron Microscopy.
We describe here a methodology to dissect the timing of modification of cytosine to 5-methylcytosine (5mC or m5C) in relation to RNA elongation and processing. To do this we use chlorouridine and iodouridine, two synthetically modified nucleotide bases which can be recognized by RNA polymerase II and incorporated into nascent RNA. These modified bases are added to a cell culture for defined intervals of time, and then immunocytochemical staining using antibodies against the modified nucleotides is carried out. This procedure allows us to identify the range of time in which 5mC is produced in nascent mRNA. This method provi...
Source: Mol Biol Cell - July 20, 2020 Category: Molecular Biology Authors: Zannino L, Siciliani S, Biggiogera M Tags: Methods Mol Biol Source Type: research

The Nucleus of Intestinal Cells of the Bacterivore Nematode Caenorhabditis elegans as a Sensitive Sensor of Environmental Pollutants.
Abstract Prevalent environmental challenges are climate change, the biodiversity crisis, and the global scale of environmental pollution. We identified the cell nucleus as a sensitive sensor for bio-effects of pollutants such as mercury and nanoparticles. As a major route of pollutant uptake into organisms is ingestion, we have developed a test system that uses single intestinal cells of the nematode roundworm Caenorhabditis elegans. Microscopic observation of the cell nucleus in reporter worms for the methyltransferase fibrillarin (FIB-1::GFP) revealed nuclear staining patterns that are specific for pollutants su...
Source: Mol Biol Cell - July 20, 2020 Category: Molecular Biology Authors: Piechulek A, Berwanger L, Hemmerich P, von Mikecz A Tags: Methods Mol Biol Source Type: research

Tuning the Expression of Long Noncoding RNA Loci with CRISPR Interference.
Abstract Long noncoding RNAs (lncRNAs) have emerged as important regulators of gene expression networks. Over 50,000 lncRNA loci have been annotated in the human genome, but only a subset has been involved in regulation of key cellular processes, organismal development, and diseases. Hence, the functional role for the majority of the lncRNA genes remains unknown. With the recent developments of different CRISPR/Cas9 technologies, the function of lncRNAs can now be examined. CRISPR interference (CRISPRi) is one of these methods that can be used to inhibit the expression of any genomic locus including lncRNAs. This ...
Source: Mol Biol Cell - July 20, 2020 Category: Molecular Biology Authors: Stojic L Tags: Methods Mol Biol Source Type: research

Identification of Chromatin Binding Sites for Long Noncoding RNAs by Chromatin Oligo-Affinity Precipitation (ChOP).
Abstract Revealing the interactions of long noncoding RNAs (LncRNAs) with specific genomic regions is of basic importance to explore the mechanisms by which they regulate gene expression. Chromatin oligo-affinity precipitation (ChOP) technique was the first method developed to analyze the association of LncRNAs with genomic regions in the chromatin context. The first step of the procedure is cell cross-linking, aimed at stabilizing the RNA-protein-DNA complexes. Next, after chromatin fragmentation, the RNA complexes are pulled down through hybridization with antisense oligonucleotides tagged with biotin and purifi...
Source: Mol Biol Cell - July 20, 2020 Category: Molecular Biology Authors: Rossi MN, Maione R Tags: Methods Mol Biol Source Type: research

Knockdown of Nuclear lncRNAs by Locked Nucleic Acid (LNA) Gapmers in Nephron Progenitor Cells.
Abstract Despite recent advance in our understanding on the role of long noncoding RNAs (lncRNAs), the function of the vast majority of lncRNAs remains poorly understood. To characterize the function of lncRNAs, knockdown studies are essential. However, the conventional silencing methods for mRNA, such as RNA interference (RNAi), may not be as efficient against lncRNAs, partly due to the mismatch of the localization of lncRNAs and RNAi machinery. To circumvent such limitation, a new technique has recently been developed, i.e., locked nucleic acid (LNA) gapmers. This system utilizes RNase H that distributes evenly ...
Source: Mol Biol Cell - July 20, 2020 Category: Molecular Biology Authors: Nishikawa M, Yanagawa N Tags: Methods Mol Biol Source Type: research

Targeting Polyadenylation for Retention of RNA at Chromatin.
om UA Abstract The various steps of RNA polymerase II transcription, including transcription initiation, splicing, and termination, are interlinked and tightly coordinated. Efficient 3'end processing is defined by sequence motifs emerging in the nascent-transcribed RNA strand and the cotranscriptional binding of regulatory proteins. The processing of a mature 3'end consists of cleavage and polyadenylation and is coupled with RNA polymerase II transcription termination and the dissociation of the nascent RNA transcript from the chromatin-associated transcriptional template. The subcellular and subnuclear topologica...
Source: Mol Biol Cell - July 20, 2020 Category: Molecular Biology Authors: Ntini E, Vang Ørom UA Tags: Methods Mol Biol Source Type: research

In Vivo Crosslinking of Histone and RNA-Binding Proteins.
Abstract Protein-protein interactions are essential in various cellular processes including regulation of gene expression, formation of protein complexes, and cellular signaling transduction. In particular, several proteins in the nucleus interact to regulate transcription and RNA splicing. These protein-protein interactions are short and weak and occur through transient processes, making it difficult to identify these interactions. In addition, detection of interacting partners in vitro using cell lysates cannot provide complete information due to the loss of spatial organization and changes in protein modificati...
Source: Mol Biol Cell - July 20, 2020 Category: Molecular Biology Authors: Kim YE, Kim KE, Kim KK Tags: Methods Mol Biol Source Type: research

Mapping Transcriptome-Wide and Genome-Wide RNA-DNA Contacts with Chromatin-Associated RNA Sequencing (ChAR-seq).
Abstract RNAs play key roles in the cell as molecular intermediates for protein synthesis and as regulators of nuclear processes such as splicing, posttranscriptional regulation, or chromatin remodeling. Various classes of non-coding RNAs, including long non-coding RNAs (lncRNAs), can bind chromatin either directly or via interaction with chromatin binding proteins. It has been proposed that lncRNAs regulate cell-state-specific genes by coordinating the locus-dependent activity of chromatin-modifying complexes. Yet, the vast majority of lncRNAs have unknown functions, and we know little about the specific loci the...
Source: Mol Biol Cell - July 20, 2020 Category: Molecular Biology Authors: Limouse C, Jukam D, Smith OK, Fryer KA, Straight AF Tags: Methods Mol Biol Source Type: research

Proximity RNA-seq: A Sequencing Method to Identify Co-localization of RNA.
Abstract RNA localization is an important regulatory layer of gene expression and cell functioning. The protocol guides through the Proximity RNA-seq method, in which RNA molecules are sequenced in their spatial, cellular context to derive RNA co-localization and transcriptome organization. Transcripts in individual subcellular particles from chemically crosslinked cells are tagged with the same, unique DNA barcode in water-in-oil emulsion droplets. First, single DNA barcodes are PCR amplified and immobilized on single, small magnetic beads in droplets. Subsequently, 3' ends of bead-bound barcode copies are tailed...
Source: Mol Biol Cell - July 20, 2020 Category: Molecular Biology Authors: Morf J, Wingett SW Tags: Methods Mol Biol Source Type: research

Analysis  of RNA-DNA Triplex Structures In Vitro and In Vivo.
Analysis of RNA-DNA Triplex Structures In Vitro and In Vivo. Methods Mol Biol. 2020;2161:229-246 Authors: Postepska-Igielska A, Blank-Giwojna A, Grummt I Abstract RNA can bind within the major groove of purine-rich DNA via Hoogsteen base pairing and form a triple helical RNA-DNA structure that anchors the RNA to specific DNA sequences, thereby targeting RNA-associated regulatory proteins to distinct genomic sites. Here we present methods to analyze the potential of a given RNA to form triplexes in vitro and to validate these structures in vivo. PMID: 32681516 [PubMed - as supplied by publish...
Source: Mol Biol Cell - July 20, 2020 Category: Molecular Biology Authors: Postepska-Igielska A, Blank-Giwojna A, Grummt I Tags: Methods Mol Biol Source Type: research