Dual RNA-Seq of Chlamydia and Host Cells.
Abstract During the infection of a host cell by a bacterial pathogen, a cascading series of gene expression changes occurs as each organism manipulates or responds to the other via defense or survival strategies. Unraveling this complex interplay is key for our understanding of bacterial virulence and host response pathways for the development of novel therapeutics. Dual RNA sequencing (dual RNA-Seq) has recently been developed to simultaneously capture host and bacterial transcriptomes from an infected cell. Leveraging the sensitivity and resolution allowed by RNA-seq, dual RNA-Seq can be applied to any bacteria-...
Source: Mol Biol Cell - August 8, 2019 Category: Molecular Biology Authors: Marsh JW, Hayward RJ, Shetty A, Mahurkar A, Humphrys MS, Myers GSA Tags: Methods Mol Biol Source Type: research

Isolation and Propagation of Single Inclusion-Derived Chlamydia Using Laser Microdissection.
Abstract Other than its routine application for capturing pure cell populations from fixed tissue sections for diverse downstream molecular assays, laser microdissection enables isolation of single live cells. Here we describe a method for the isolation of single Chlamydia trachomatis-infected cells using a laser microdissection system, in which the dissected samples are captured via gravity. Cells infected by C. trachomatis at low multiplicity of infection are marked with the fluorescent Golgi-specific probe BODIPY® FL C5-ceramide, to facilitate identification of the cells with chlamydial inclusions under the...
Source: Mol Biol Cell - August 8, 2019 Category: Molecular Biology Authors: Podgorny OV, Polina NF, Lazarev VN Tags: Methods Mol Biol Source Type: research

A Coinfection Model to Evaluate Chlamydia Inc Protein Interactions.
te; I Abstract Chlamydia trachomatis resides and replicates within a membranous vacuole, termed the inclusion. A group of Type III secreted effector proteins, the inclusion membrane proteins (Inc), are embedded within the inclusion membrane and facilitate the interaction of the inclusion with host cell organelles. These interactions are vital for bacterial replication and allow for the acquisition of essential nutrients from the host cell. However, it is not known if Inc proteins function independently or require interactions with other Inc proteins to function. This chapter describes a system to test the homotypi...
Source: Mol Biol Cell - August 8, 2019 Category: Molecular Biology Authors: Ende R, Derré I Tags: Methods Mol Biol Source Type: research

High-Throughput Screening for Novel Inhibitors of Intracellular Pathogens, Including Chlamydia trachomatis.
Abstract High-throughput drug screening (HTS) is a powerful tool that can be used rapidly to identify new potential bacterial inhibitors and/or compounds which enhance host cell control of pathogens, which can then go on to be developed as novel therapeutics. Typically screening is commonly done in artificial culture medium; however, obligate intracellular pathogens, such as Chlamydia trachomatis, cannot be tested this way. Intracellular screening methods allow for such pathogens to undergo HTS, while still giving reliable and consistent data. Plus, as well as identifying new potential bacterial inhibitors, it is ...
Source: Mol Biol Cell - August 8, 2019 Category: Molecular Biology Authors: Brown AC, Kushner NL Tags: Methods Mol Biol Source Type: research

Electron Microscopy to Study the Fine Structure of the Pneumococcal Cell.
Abstract Electron microscopy allows for studying bacterial ultrastructure at high resolutions. Two types of electron microscopes are used for this purpose. The transmission electron microscope allows for access to inner bacterial ultrastructure when imaging ultrathin sections as well as cell wall-attached structures by negative staining, whereas scanning electron microscopy allows for the detection of structures on the bacterial cell surface alone or to study the interplay between pneumococci and their host cells. This chapter deals with recommendations for well-adapted methodologies to examine pneumococcal ultras...
Source: Mol Biol Cell - August 7, 2019 Category: Molecular Biology Authors: Hammerschmidt S, Rohde M Tags: Methods Mol Biol Source Type: research

Immunofluorescent Staining and High-Resolution Microscopy to Study the Pneumococcal Cell.
Abstract Immunofluorescent staining using antibodies to detect specific proteins allows for visualization of proteins of interest in a biological sample. In recent years, there have been important advances in the microscopy equipment used for imaging, and we can now perform so-called high-resolution microscopy. Through high-resolution microscopy we can not only study biological processes but also visualize them. PMID: 30929203 [PubMed - indexed for MEDLINE] (Source: Mol Biol Cell)
Source: Mol Biol Cell - August 7, 2019 Category: Molecular Biology Authors: Iovino F, Henriques-Normark B Tags: Methods Mol Biol Source Type: research

Construction of Fluorescent Pneumococci for In Vivo Imaging and Labeling of the Chromosome.
Abstract Advances in fluorescence imaging techniques and development and optimization of fluorescent proteins recent years have made major impacts on different fields of pneumococcal research. This chapter provides methodology for construction of fluorescent pneumococcal strains using fusions to DNA-binding proteins. By expressing fluorescent proteins fused to HlpA, a pneumococcal nucleoid binding protein, brightly fluorescent pneumococci are generated. HlpA fusions may be used both for in vivo imaging of pneumococci as well as for marking the nucleoid in cell biology studies. Furthermore, it also explains how to ...
Source: Mol Biol Cell - August 7, 2019 Category: Molecular Biology Authors: Kjos M Tags: Methods Mol Biol Source Type: research

High-Resolution and Super-Resolution Immunofluorescent Microscopy Ex Vivo to Study Pneumococcal Interactions with the Host.
Abstract In vivo imaging, meaning imaging tissues in living animals, is still a developing technique. However, microscopy imaging ex vivo remains a very important tool that allows for visualization of biological and pathological processes occurring in vivo. As described in Chap. 5, imaging of animal and human tissue postmortem can be performed at high resolution. Recently, imaging of human tissues infected with pneumococci using an even higher resolution, the so-called super-resolution with STED, has been reported. PMID: 30929205 [PubMed - indexed for MEDLINE] (Source: Mol Biol Cell)
Source: Mol Biol Cell - August 7, 2019 Category: Molecular Biology Authors: Iovino F, Henriques-Normark B Tags: Methods Mol Biol Source Type: research

Mass Spectrometry to Study the Bacterial Proteome from a Single Colony.
Abstract Mass spectrometry (MS) has been widely used in recent years for bacterial identification and typing. Single bacterial colonies are regarded as pure cultures of bacteria grown from single cells. In this chapter, we describe a method for identifying bacteria at the species level with 100% accuracy using the proteomes of bacterial cultures from single colonies. In this chapter, six reference strains of gram-negative and gram-positive bacteria are analyzed, producing results of high reproducibility, as examples of bacterial identification through the application of liquid chromatography-tandem mass spectromet...
Source: Mol Biol Cell - August 7, 2019 Category: Molecular Biology Authors: Zhou J, Zhang L, Chuan H, Sloan A, Tsang R, Cheng K Tags: Methods Mol Biol Source Type: research

Bead-Based Flow-Cytometric Cell Counting of Live and Dead Bacteria.
Abstract Flow cytometry (FCM) is based on the detection of scattered light and fluorescence to identify cells with characteristics of interest. Many flow cytometers cannot precisely control the flow through its interrogation point and hence the volume and concentration of the sample cannot be immediately obtained. Here we describe the optimization and evaluation of a bead-based method for absolute cell counting applicable to basic flow cytometers without specialized counting features. Prior to the application of this method to an unknown concentration of a species of bacteria, a calibration experiment should ...
Source: Mol Biol Cell - August 7, 2019 Category: Molecular Biology Authors: Ou F, McGoverin C, White J, Swift S, Vanholsbeeck F Tags: Methods Mol Biol Source Type: research

In Vitro Adhesion, Invasion, and Transcytosis of Streptococcus pneumoniae with Host Cells.
Abstract Physical interactions of bacteria with host cells are often a principal aspect of bacterial pathogenesis. In the case of Streptococcus pneumoniae (Spn), which does not produce a secreted toxin, adhesion to and/or invasion of host cells is necessary for colonization of the nasopharynx and subsequently to cause opportunistic disease in its human host. Knowledge of how pneumococci interact with host cells thereby helps to explain its biology and may identify potential targets for intervention. One of the simplest, yet powerful, assays that can be leveraged to dissect the molecular basis of this vital host-pa...
Source: Mol Biol Cell - August 7, 2019 Category: Molecular Biology Authors: Brissac T, Orihuela CJ Tags: Methods Mol Biol Source Type: research

In Vivo Mouse Models to Study Pneumococcal Host Interaction and Invasive Pneumococcal Disease.
Abstract Animal models are fundamental tools to study the biology of physiological processes and disease pathogenesis. To study invasive pneumococcal disease (IPD), many models using mice in particular have been established and developed during recent years. Thanks to the advances of the research in the pneumococcal field, nowadays, there is the possibility to use defined mouse models to study each disease caused by the pneumococcus. In this chapter mouse models for pneumonia, bacteremia, and meningitis are described. Since pneumococci are commensal pathogens found to a high extent in healthy individuals. Hen...
Source: Mol Biol Cell - August 7, 2019 Category: Molecular Biology Authors: Iovino F, Sender V, Henriques-Normark B Tags: Methods Mol Biol Source Type: research

Two-Photon Intravital Imaging of Leukocytes in the Trachea During Pneumococcal Infection.
Abstract Two-photon intravital imaging (2P-IVM) of the murine trachea is a powerful technique for real-time imaging of immune cell recruitment and trafficking during airborne pathogen infections. Neutrophils are an important component of the innate immune response that are able to rapidly infiltrate the airway mucosa in response to Streptococcus pneumoniae infection. Here we describe a protocol to visualize in vivo neutrophil extravasation and cell dynamics in the tracheal tissue of a S. pneumoniae-infected mouse using 2P-IVM. To perform this protocol, we infected and imaged the trachea of a lysozyme M green fluor...
Source: Mol Biol Cell - August 7, 2019 Category: Molecular Biology Authors: Palomino-Segura M, Gonzalez SF Tags: Methods Mol Biol Source Type: research

IVIS Spectrum CT to Image the Progression of Pneumococcal Infections In Vivo.
Abstract Imaging through the IVIS Spectrum CT system does not provide the resolution at cellular level like the high-resolution or super-resolution microscopy. Rather, it detects bacterial infections in specific anatomical compartments/organs of the animals. The IVIS Spectrum imaging system is a unique imaging technology that allows for real-time monitoring of disease progression in living animals through the use of either bioluminescent or fluorescent probes. PMID: 30929216 [PubMed - indexed for MEDLINE] (Source: Mol Biol Cell)
Source: Mol Biol Cell - August 7, 2019 Category: Molecular Biology Authors: Sierakowiak A, Henriques-Normark B, Iovino F Tags: Methods Mol Biol Source Type: research

Biotinylation Assay to Determine LFA-1 Recycling in Motile T-Lymphocytes.
Abstract The cycles of internalization of the cell surface β2 integrin receptor lymphocyte function-associated antigen 1 (LFA-1) and its re-exposure on the plasma membrane are important for T-cell trafficking. Biotinylation of cells enables to measure surface expression of receptors, and after reducing surface biotin with reducing buffer, enables to measure the internalization of receptors. Here, by using biotin in combination with reducing buffer and recombinant intercellular adhesion molecule-1 (rICAM-1)-coated dishes and subsequent Western immunoblot analysis, we describe how to measure internalization of ...
Source: Mol Biol Cell - August 4, 2019 Category: Molecular Biology Authors: Samuelsson M, Svensson LM Tags: Methods Mol Biol Source Type: research

A Protocol to Study T-Cell Signaling in an Immune Synapse by Microscopy.
Abstract The immune synapse is a complex cellular structure that enables cell-cell communications between immune cells, mainly at the interface of an effector T-cell and an antigen-presenting cell (APC) that expresses the appropriate peptide-MHC complexes. With progressive technological advances, there has been increasing interest in understanding molecular events that take place in motile T-lymphocytes at the immune synapse. Here, we provide an optimized method to induce the formation of an immune synapse between a T-cell and an APC in vitro. The experimental protocol described herein would be useful in character...
Source: Mol Biol Cell - August 4, 2019 Category: Molecular Biology Authors: Fazil MHUT, Kumar P, Verma NK Tags: Methods Mol Biol Source Type: research

Enzyme-Linked Immunosorbent Assay for T-Cell Dependent Immunogenicity Assessment of Therapeutic Peptides.
Abstract Immunogenicity assessment of therapeutic peptides, proteins, oligonucleotides, and hybrid molecules, such as nucleopeptides, is a major aspect in understanding their safety and efficacy. Both T-cell independent and dependent immune reactions contribute to an immunogenic response against antigen, including secretion of cytokines and production of an antigen-specific antibody. Various assays exist for detecting and quantifying such immunogenic responses by human T-cells ex vivo or in mouse serum, which primarily include enzyme-linked immunosorbent assay (ELISA, direct and indirect), flow-cytometry and surfa...
Source: Mol Biol Cell - August 4, 2019 Category: Molecular Biology Authors: Chalasani MLS, Lakshminarayanan R, Verma NK Tags: Methods Mol Biol Source Type: research

Modified Intravital Microscopy to Assess Vascular Health and T-Cell Motility.
Abstract The ability to study the microcirculation in real time is key to elucidating how the immune system and the associated microvasculature interact and influence one another within the lymph node (LN). Here, we present a method for near in-situ imaging of the inguinal LN. In particular, this method is ideal for the assessment of overall vascular health that influences immune functions and for the evaluation of T-cell motility. We focus on imaging of the microvasculature of the LN, paying particular attention to methods that ensure the study of healthy vessels, the ability to maintain imaging of viable vessels...
Source: Mol Biol Cell - August 4, 2019 Category: Molecular Biology Authors: Payne GW, Mitchell K, Sellers SL Tags: Methods Mol Biol Source Type: research

Computational Analysis of Protein-Protein Interactions in Motile T-Cells.
Abstract Analysis of protein-protein interactions is important for better understanding of molecular mechanisms involved in immune regulation and has potential for elaborating avenues for drug discovery targeting T-cell motility. Currently, only a small fraction of protein-protein interactions have been characterized in T-lymphocytes although there are several detection methods available. In this regard, computational approaches garner importance, with the continued explosion of genomic and proteomic data, for handling protein modeling and protein-protein interactions in large scale. Here, we describe a computatio...
Source: Mol Biol Cell - August 4, 2019 Category: Molecular Biology Authors: Kumar S, Fazil MHUT, Ahmad K, Tripathy M, Rajapakse JC, Verma NK Tags: Methods Mol Biol Source Type: research

Identification of Long Noncoding RNAs in the Developing Endosperm of Maize.
Abstract Maize endosperm consists of three distinct types of tissues, including the starchy endosperm (SE), the basal endosperm transfer cell layer (BETL), and the aleurone cell layer (AL). Compartmentalization of these tissues during endosperm differentiation makes the endosperm development an excellent model to study changes in gene expression during development. By utilizing cryo-dissection of developing endosperm, morphologically distinct samples can be obtained for transcriptome and epigenome analysis. Here, we describe methods for the isolation of tissues from developing maize endosperm and for the transcrip...
Source: Mol Biol Cell - August 4, 2019 Category: Molecular Biology Authors: Kim E, Xiong Y, Kang BH, Sung S Tags: Methods Mol Biol Source Type: research

Medium-Throughput RNA In Situ Hybridization of Serial Sections from Paraffin-Embedded Tissue Microarrays.
Abstract (m)RNA spatiotemporal pattern of distribution is of key importance to decipher gene function. In this post-genomic era, numerous transcriptomic studies are made publicly available, sometimes reaching a tissular resolution and even more rarely the cellular level. This "one tissue-numerous genes" information can be completed by the reverse "one gene-numerous tissues" picture through traditional RNA in situ hybridization (ISH). Here, we present a method including (1) principles of transcriptomic data mining to be performed prior and following ISH and (2) a detailed step-by-step medium-thr...
Source: Mol Biol Cell - August 4, 2019 Category: Molecular Biology Authors: Francoz E, Ranocha P, Dunand C, Burlat V Tags: Methods Mol Biol Source Type: research

Purification and Functional Analysis of Plant Long Noncoding RNAs (lncRNA).
Abstract More than 70% of eukaryotic genomes are transcribed into RNA transcripts, the majority of these transcripts are noncoding protein, and their biological functions are largely unknown. Over the last decade, the application of high-throughput sequencing technologies has led to the description of almost all cellular coding and noncoding RNA transcripts except perhaps for those transcripts that are lowly abundant or those present only in specific cells that are underrepresented in sampled tissue(s). An often underrepresented class of noncoding are long noncoding RNAs (lncRNAs), and these often play key regulat...
Source: Mol Biol Cell - August 4, 2019 Category: Molecular Biology Authors: Do T, Qu Z, Searle I Tags: Methods Mol Biol Source Type: research

An Easy-to-Follow Pipeline for Long Noncoding RNA Identification: A Case Study in Diploid Strawberry Fragaria vesca.
Abstract Long noncoding RNAs (lncRNAs), defined as transcripts longer than 200 nucleotides without coding potential, are a new class of regulatory molecules with roles in diverse biological processes. New lncRNAs can readily be identified by mining RNA-seq data from a wide range of plant species. However, challenges remain as to how one can distinguish functional lncRNAs from mRNAs coding for small peptides or products of pseudogenes without any function. In this chapter, stepwise instruction is provided using RNA-seq datasets of developing wild strawberry fruit to illustrate each step. The workflow can be divided...
Source: Mol Biol Cell - August 4, 2019 Category: Molecular Biology Authors: Kang C, Liu Z Tags: Methods Mol Biol Source Type: research

Identification of Long Noncoding RNA-Protein Interactions Through In Vitro RNA Pull-Down Assay with Plant Nuclear Extracts.
Abstract Recent advances in next-generation sequencing have revealed that majority of the plant genome is transcribed into long noncoding RNA (lncRNA). Many lncRNAs function by interacting with proteins and forming regulatory complexes. RNA-protein interactions are vital in controlling core cellular processes like transcription and translation. Therefore, identifying proteins that interact with lncRNAs is the first step to deciphering lncRNA functions. Here, we describe an RNA-protein pull-down assay, which enables the identification of proteins that interact with an RNA under study. As an example, we describe pul...
Source: Mol Biol Cell - August 4, 2019 Category: Molecular Biology Authors: Seo JS, Chua NH Tags: Methods Mol Biol Source Type: research

In Vivo Genome-Wide RNA Structure Probing with Structure-seq.
Abstract In vivo genome-wide RNA structure probing provides a global view of RNA structure as it occurs in the cell and can assist in elucidating important functional aspects of RNA structure. Structure-seq2 provides high-quality data on transcriptome-wide RNA structure in vivo but contains numerous steps that require technical precision. In this chapter we present the steps needed to produce high-quality structural data with a focus on controls and troubleshooting. Structure-seq2 can be applied to numerous organisms including plants, humans, and bacteria and is amenable to a wide variety of RNA-modifying chemical...
Source: Mol Biol Cell - August 4, 2019 Category: Molecular Biology Authors: Ritchey LE, Su Z, Assmann SM, Bevilacqua PC Tags: Methods Mol Biol Source Type: research

Using Protein Interaction Profile Sequencing (PIP-seq) to Identify RNA Secondary Structure and RNA-Protein Interaction Sites of Long Noncoding RNAs in Plants.
Abstract From the moment of transcription, RNA molecules are continuously bound by RNA-binding proteins (RBPs). While the majority of research has focused on how these RBPs regulate posttranscriptional gene regulation of messenger RNAs (mRNAs), the majority of cellular RNAs do not code for proteins, such as ribosomal RNAs, transfer RNAs, and microRNAs. Since these RNAs do not code for protein, their function is mainly determined by their interactions with RBPs as well as their intramolecular base pairing, or RNA secondary structure. One class of noncoding RNAs termed long noncoding RNAs (lncRNAs) have recently bec...
Source: Mol Biol Cell - August 4, 2019 Category: Molecular Biology Authors: Kramer MC, Gregory BD Tags: Methods Mol Biol Source Type: research

In Situ Hi-C for Plants: An Improved Method to Detect Long-Range Chromatin Interactions.
Abstract Recently, long noncoding RNAs (lncRNAs) are shown to be implicating nuclear domain organization and gene regulation by mediating long-range chromatin interactions. Chromosome conformation capture (3C) is a method used to study such long-range interaction between two different loci in the 3D nuclear space. Through successive improvement in resolution and throughput, 3C, chromosome conformation capture on chip (4C), and chromosome conformation capture carbon copy (5C) to Hi-C methods were developed to study interactions between loci from one versus one scale to an unprecedented genome-wide resolution. In si...
Source: Mol Biol Cell - August 4, 2019 Category: Molecular Biology Authors: Padmarasu S, Himmelbach A, Mascher M, Stein N Tags: Methods Mol Biol Source Type: research

Molecular Dynamic Simulations to Probe Water Permeation Pathways of GPCRs.
Abstract Rhodopsin is a light-driven G protein-coupled receptor mediating signal transduction in eyes. The molecular dynamics (MD) simulations are powerful computational tools to investigate molecular behavior of proteins and internal water molecules which are related to the function of proteins; however, the MD simulations of the rhodopsin require several technical setups for accurate calculations. This chapter discusses practical methods for setting up the MD simulations of the rhodopsin [preparation of initial systems, condition files for MD simulation package GROMACS, and data analysis]. The data analysis incl...
Source: Mol Biol Cell - August 4, 2019 Category: Molecular Biology Authors: Tomobe K, Yamamoto E, Yasuoka K Tags: Methods Mol Biol Source Type: research

A Combined Cell-Free Protein Synthesis and Fluorescence-Based Approach to Investigate GPCR Binding Properties.
Abstract Fluorescent labeling of de novo synthesized proteins is in particular a valuable tool for functional and structural studies of membrane proteins. In this context, we present two methods for the site-specific fluorescent labeling of difficult-to-express membrane proteins in combination with cell-free protein synthesis. The cell-free protein synthesis system is based on Chinese Hamster Ovary Cells (CHO) since this system contains endogenous membrane structures derived from the endoplasmic reticulum. These so-called microsomes enable a direct integration of membrane proteins into a biological membrane. In th...
Source: Mol Biol Cell - August 4, 2019 Category: Molecular Biology Authors: Zemella A, Richter T, Thoring L, Kubick S Tags: Methods Mol Biol Source Type: research

Furan Cross-Linking Technology for Investigating GPCR-Ligand Interactions.
Abstract Interactions between G protein-coupled receptors and their ligands hold extensive potential for drug discovery. Studying these interactions poses technical problems due to their transient nature and the inherent difficulties when working with G protein-coupled receptors (GPCR) that are only functional in a membrane setting. Here, we describe the use of a furan-based chemical cross-linking methodology to achieve selective covalent coupling between a furan-modified peptide ligand and its native GPCR present on the surface of living cells under normal cell culture conditions. This methodology relies on the o...
Source: Mol Biol Cell - August 4, 2019 Category: Molecular Biology Authors: Van Troys M, Vannecke W, Ampe C, Madder A Tags: Methods Mol Biol Source Type: research

Chemoselective Acylation of Hydrazinopeptides to Access Fluorescent Probes for Time-Resolved FRET Assays on GPCRs.
net D Abstract Fluorescence techniques represent a powerful tool to investigate dynamic and functional architecture of GPCRs. Thus, fluorescent GPCR ligands have found various applications in cellular imaging, in the development of binding assays as replacements for radioligands in the study of ligand-receptor but also in receptor-receptor interactions at the cell surface or in native tissues. To extend the applicability of these techniques, the design and the synthesis of fluorescent probes are critical steps. As there are numerous peptide receptors in the GPCR family, fluorescent peptide-based probes are of impo...
Source: Mol Biol Cell - August 4, 2019 Category: Molecular Biology Authors: Ramanoudjame SM, Esteoulle L, Riché S, Margathe JF, Durroux T, Karpenko IA, Bonnet D Tags: Methods Mol Biol Source Type: research

Time-Resolved FRET-Based Assays to Characterize G Protein-Coupled Receptor Hetero-oligomer Pharmacology.
Abstract Although G protein-coupled receptor (GPCR) oligomerization is a matter of debate, it has been shown that the nature of the GPCR partners within the oligomers can influence the pharmacological properties of the receptors. Therefore, finding specific ligands for homo- or hetero-oligomers opens new perspectives for drug discovery. However, no efficient experimental strategy to screen for such ligands existed yet. Indeed, conventional binding strategies do not discriminate ligand binding on GPCR monomers, homo- or hetero-oligomers. To address this issue, we recently developed a new assay based on a time-resol...
Source: Mol Biol Cell - August 4, 2019 Category: Molecular Biology Authors: Heuninck J, Hounsou C, Dupuis E, Trinquet E, Mouillac B, Pin JP, Bonnet D, Durroux T Tags: Methods Mol Biol Source Type: research

Combining Conformational Profiling of GPCRs with CRISPR/Cas9 Gene Editing Approaches.
rt TE Abstract Ligand-biased signaling could have a significant impact on drug discovery programs. As such, many approaches to screening now target a larger section of the signaling responses downstream of an individual G protein-coupled receptor (GPCR). Biosensor-based platforms have been developed to capture signaling signatures. Despite the ability to use such signaling signatures, they may still be particular to an individual cell type and thus such platforms may not be portable from cell to cell, necessitating further cell-specific biosensor development. We have developed a complementary strategy based on cap...
Source: Mol Biol Cell - August 4, 2019 Category: Molecular Biology Authors: Bourque K, Devost D, Inoue A, Hébert TE Tags: Methods Mol Biol Source Type: research

Measuring GPCR Stoichiometry Using Types-1, -2, and -3 Bioluminescence Resonance Energy Transfer-Based Assays.
Abstract How G protein-coupled receptors are assembled is a matter of considerable interest owing in large part to their remarkable pharmacological importance. For determining receptor stoichiometry, resonance energy transfer-based methods offer considerable advantages insofar as they provide the necessary spatial resolution, and because measurements can be made in situ, relatively easily. This chapter describes three complementary stoichiometric assays that rely on measurements of bioluminescence resonance energy transfer. These quantitative approaches make it possible to identify true protein-protein interaction...
Source: Mol Biol Cell - August 4, 2019 Category: Molecular Biology Authors: Felce JH, James JR, Davis SJ Tags: Methods Mol Biol Source Type: research

Quantification and Comparison of Signals Generated by Different FRET-Based cAMP Reporters.
Abstract A variety of FRET-based biosensors are currently in use for real-time monitoring of dynamic changes of intracellular cAMP. Due to differences in sensor properties, unique features of the cell type under examination and diverse specifications of the imaging setups in different laboratories, data generated using these sensors may not be immediately comparable within the same study or across studies. To facilitate comparison, often FRET data are normalized and expressed as fractional change of the maximal FRET response at sensor saturation. However, this approach may lead to misinterpretation of the underlyi...
Source: Mol Biol Cell - August 4, 2019 Category: Molecular Biology Authors: Koschinski A, Zaccolo M Tags: Methods Mol Biol Source Type: research

Measurement of β-Arrestin Recruitment at GPCRs Using the Tango Assay.
Measurement of β-Arrestin Recruitment at GPCRs Using the Tango Assay. Methods Mol Biol. 2019;1947:257-267 Authors: Laroche G, Giguère PM Abstract Intracellular signal transduced by G protein-coupled receptors (GPCRs) is tightly controlled by a guanine nucleotide-binding complex made of G protein Gα, Gβ, and Gγ subunits, as well as a growing array of regulatory and accessory proteins such as arrestins. G protein-independent β-arrestin recruitment at GPCRs is universally accepted as the canonical interactor system and it has been found to be a powerful tracker of most G...
Source: Mol Biol Cell - August 4, 2019 Category: Molecular Biology Authors: Laroche G, Giguère PM Tags: Methods Mol Biol Source Type: research

Super-Resolution Imaging of G Protein-Coupled Receptors Using Ground State Depletion Microscopy.
Abstract G protein-coupled receptors (GPCRs) comprise the largest family of integral membrane proteins, which are coupled to heterotrimeric G proteins to influence cell signaling. Subsequent to G protein activation, agonist-stimulated G protein-coupled receptor kinase (GRK) phosphorylation results in the recruitment of β-arrestin proteins, which form both stable and unstable complexes with GPCRs. β-Arrestins when bound to GPCRs not only contribute to the uncoupling of G protein signaling but also to the redistribution of GPCRs to clathrin-coated pits via their association with both clathrin and &bet...
Source: Mol Biol Cell - August 4, 2019 Category: Molecular Biology Authors: Caetano Crowley FA, Heit B, Ferguson SSG Tags: Methods Mol Biol Source Type: research

Analysis of Spatial Assembly of GPCRs Using Photoactivatable Dyes and Localization Microscopy.
Abstract Super-resolution imaging has provided unprecedented insight in the molecular complexities of fundamental cell biological questions. For G protein-coupled receptors (GPCRs), its application to the study of receptor homomers and heteromers have unveiled the diversity of complexes these GPCRs can form at the plasma membrane at a structural and functional level. Here, we describe our methodological approach of photoactivated localization microscopy with photoactivatable dyes (PD-PALM) to visualize and quantify the spatial assembly of GPCR heteromers at the plasma membrane. PMID: 30969426 [PubMed - indexe...
Source: Mol Biol Cell - August 4, 2019 Category: Molecular Biology Authors: Jonas KC, Hanyaloglu AC Tags: Methods Mol Biol Source Type: research

Imaging of Tissue-Specific and Temporal Activation of GPCR Signaling Using DREADD Knock-In Mice.
Abstract Engineered G protein-coupled receptors (DREADDs, designer receptors exclusively activated by designer drugs) are convenient tools for specific activation of GPCR signaling in many cell types. DREADDs have been utilized as research tools to study numerous cellular and physiologic processes, including regulation of neuronal activity, behavior, and metabolism. Mice with random insertion transgenes and adeno-associated viruses have been widely used to express DREADDs in individual cell types. We recently created and characterized ROSA26-GsDREADD knock-in mice to allow Cre recombinase-dependent expression of a...
Source: Mol Biol Cell - August 4, 2019 Category: Molecular Biology Authors: Akhmedov D, Kirkby NS, Mitchell JA, Berdeaux R Tags: Methods Mol Biol Source Type: research

Long non-coding RNA Malat1 activated autophagy, hence promoting cell proliferation and inhibiting apoptosis by sponging miR-101 in colorectal cancer.
Conclusion: Long non-coding RNA Malat1 activated autophagy and promoted cell proliferation, yet inhibited apoptosis by sponging miR-101 in colorectal cancer cells. PMID: 31372165 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - August 4, 2019 Category: Molecular Biology Authors: Si Y, Yang Z, Ge Q, Yu L, Yao M, Sun X, Ren Z, Ding C Tags: Cell Mol Biol Lett Source Type: research

The Contribution of Differential Scanning Calorimetry for the Study of Peptide/Lipid Interactions.
Abstract Membrane-active peptides include a variety of molecules such as antimicrobial (AMP), cell-penetrating (CPP), viral, and amyloid peptides that are implicated in several pathologies. They constitute important targets because they are either at the basis of novel therapies (drug delivery for CPPs or antimicrobial activity for AMPs) or they are the agents causing these pathologies (viral and amyloid peptides). They all share the common property of interacting with the cellular lipid membrane in their mode of action. Therefore, a better understanding of the peptide/lipid (P/L) interaction is essential to help ...
Source: Mol Biol Cell - August 2, 2019 Category: Molecular Biology Authors: Jobin ML, Alves ID Tags: Methods Mol Biol Source Type: research

Intrinsic Thermodynamics of Protein-Ligand Binding by Isothermal Titration Calorimetry as Aid to Drug Design.
Abstract Isothermal titration calorimetry (ITC) is one of the main techniques to determine specific interactions between molecules dissolved in aqueous solution. This technique is commonly used in drug development programs when low-molecular-weight molecules are sought that bind tightly and specifically to a protein (disease target) molecule. The method allows a complete thermodynamic characterization of an interaction, i.e., ITC enables direct determination of the model-independent observed interaction change in enthalpy (ΔH) and a model-dependent observed interaction affinity (change in Gibbs free energy, ...
Source: Mol Biol Cell - August 2, 2019 Category: Molecular Biology Authors: Paketurytė V, Zubrienė A, Ladbury JE, Matulis D Tags: Methods Mol Biol Source Type: research

Thermodynamics of Molecular Machines Using Incremental ITC.
Abstract Molecular biomachines, such as DNA and RNA polymerases or the ribosome, are fascinating biological assemblies able to swiftly perform repeated and highly regulated tasks, with a remarkable accuracy. Significant advances in structural studies during the past 20 years provided a wealth of information regarding their architecture and considerably contributed to a better understanding of their mechanism of action. However, the three-dimensional structure of a biological nanomachine alone does not provide access to its detailed mechanism of action, even when obtained at atomic resolution. When combined wi...
Source: Mol Biol Cell - August 2, 2019 Category: Molecular Biology Authors: Meyer B, da Veiga C, Dumas P, Ennifar E Tags: Methods Mol Biol Source Type: research

Characterization of Microtubule-Associated Proteins (MAPs) and Tubulin Interactions by Isothermal Titration Calorimetry (ITC).
Abstract Microtubules are highly dynamic structures which play a central role in many cellular processes such as cell division, intracellular transport, and migration. Their dynamics is tightly regulated by stabilizing and destabilizing microtubule-associated proteins (MAPs), such as tau and stathmin. Many approaches have been developed to study interactions between tubulin and MAPs. However, isothermal titration calorimetry (ITC) is the only direct thermodynamic method that enables a full thermodynamic characterization of the interaction after a single titration experiment. We provide here the protocols to apply ...
Source: Mol Biol Cell - August 2, 2019 Category: Molecular Biology Authors: Tsvetkov PO, La Rocca R, Malesinski S, Devred F Tags: Methods Mol Biol Source Type: research

Tinkering with Binding Polynomials in Isothermal Titration Calorimetry.
Abstract Isothermal titration calorimetry (ITC) has become the preferred experimental technique for characterizing intermolecular interactions between biological molecules. Among the several advantages, the use of natural non-labeled molecules and the determination of the complete thermodynamic profile for the interaction in solution remain as the primary features that have promoted ITC to the forefront of experimental biophysics. The experimental design in ITC may range from studying a simple direct binary macromolecule-ligand interaction to studying the homotropic or heterotropic cooperative effect between ligan...
Source: Mol Biol Cell - August 2, 2019 Category: Molecular Biology Authors: Claveria-Gimeno R, Vega S, Abian O, Velazquez-Campoy A Tags: Methods Mol Biol Source Type: research

Introduction to the  Immune System.
Introduction to the Immune System. Methods Mol Biol. 2019;2024:1-24 Authors: McComb S, Thiriot A, Akache B, Krishnan L, Stark F Abstract The immune system in a broad sense is a mechanism that allows a living organism to discriminate between "self" and "nonself." Examples of immune systems occur in multicellular organisms as simple and ancient as sea sponges. In fact, complex multicellular life would be impossible without the ability to exclude external life from the internal environment. This introduction to the immune system will explore the cell types and soluble factors inv...
Source: Mol Biol Cell - August 2, 2019 Category: Molecular Biology Authors: McComb S, Thiriot A, Akache B, Krishnan L, Stark F Tags: Methods Mol Biol Source Type: research

Antigen Identification for Cell-Binding Antibodies Using Ligand-Directed Crosslinking and Biotin Transfer.
We describe the synthesis of the ASB crosslinker, labelling of the ligand with ASB, and cell binding of the labelled ligands. Next, biotin affinity purification and trypsin digestion of cell surface proteins that have been crosslinked by ASB are described. Lastly, several hints and tips to improve the proteomic analysis for these types of samples are provided. PMID: 31364049 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - August 2, 2019 Category: Molecular Biology Authors: Tremblay TL, Hill JJ Tags: Methods Mol Biol Source Type: research

Whole-Genome Phage Display Libraries: A Powerful Tool for Antigen Discovery.
Abstract In the last two decades, phage display technology has been used for investigating complex biological processes and isolating molecules of practical value in several applications. Bacteriophage lambda, representing a classical cloning and expression system, has also been exploited for generating display libraries of small peptides and protein domains. More recently, large cDNA and whole-genome lambda display libraries of human pathogens have been generated for the discovery of new antigens for biomedical applications. Here, we describe the construction of a whole-genome library of a common pathogen-Strepto...
Source: Mol Biol Cell - August 2, 2019 Category: Molecular Biology Authors: Beghetto E, Gargano N Tags: Methods Mol Biol Source Type: research

Construction and Screening of an  Antigen-Derived Peptide Library Displayed on Yeast Cell Surface for CD4+ T Cell Epitope Identification.
Construction and Screening of an Antigen-Derived Peptide Library Displayed on Yeast Cell Surface for CD4+ T Cell Epitope Identification. Methods Mol Biol. 2019;2024:213-234 Authors: Wen F, Smith MR, Zhao H Abstract Antigenic peptides (termed T cell epitopes) are assembled with major histocompatibility complex (MHC) molecules and presented on the surface of antigen-presenting cells (APCs) for T cell recognition. T cells engage these peptide-MHCs using T cell receptors (TCRs). Because T cell epitopes determine the specificity of a T cell immune response, their prediction and identification are impo...
Source: Mol Biol Cell - August 2, 2019 Category: Molecular Biology Authors: Wen F, Smith MR, Zhao H Tags: Methods Mol Biol Source Type: research

Enrichment of Phosphorylated MHC Peptides with Immobilized Metal Affinity Chromatography and Titanium Dioxide Particles.
Abstract Phosphorylation is one of the most important forms of posttranslational modification. Dysregulation of phosphorylation is implicated in tumorigenesis, with cancerous signaling pathways activated by kinases. For immunotherapy with neoantigen-based peptides, phosphopeptides derived from aberrantly phosphorylated proteins presented by major histocompatibility complex (MHC) are promising candidates due to their specificity to elicit cytotoxic T-cell responses. Unlike other MHC peptides, phosphorylated MHC peptides cannot be predicted from DNA sequences, and their identification relies on the direct detection ...
Source: Mol Biol Cell - August 2, 2019 Category: Molecular Biology Authors: Chen R, Li J Tags: Methods Mol Biol Source Type: research