Paired Recording to Study Electrical Coupling Between Photoreceptors in Mouse Retina.
Abstract Gap junction-mediated electrical coupling between retinal photoreceptors is an important determinant of photoreceptor function. Yet, quantitative measurements of the junctional conductance between coupled photoreceptors are required to fully assess the effects of coupling on visual performance. Such measurements have been obtained in salamander and other lower vertebrate retinas but are difficult to acquire in mammalian retinas, in part because of the much smaller size of photoreceptors in mammals. Here, we describe in detail a dual whole-cell patch-clamp technique we recently developed to measure the jun...
Source: Mol Biol Cell - December 3, 2019 Category: Molecular Biology Authors: Jin N, Zhang Z, Mankiewicz KA, Ribelayga CP Tags: Methods Mol Biol Source Type: research

T Cell Reprogramming Against Cancer.
Abstract Advances in academic and clinical studies during the last several years have resulted in practical outcomes in adoptive immune therapy of cancer. Immune cells can be programmed with molecular modules that increase their therapeutic potency and specificity. It has become obvious that successful immunotherapy must take into account the full complexity of the immune system and, when possible, include the use of multifactor cell reprogramming that allows fast adjustment during the treatment. Today, practically all immune cells can be stably or transiently reprogrammed against cancer. Here, we review works rel...
Source: Mol Biol Cell - December 1, 2019 Category: Molecular Biology Authors: Katz SG, Rabinovich PM Tags: Methods Mol Biol Source Type: research

Identification of Cell Surface Targets for CAR T Cell Immunotherapy.
Abstract Immunotherapy has become a prominent approach for the treatment of cancer. Targeted killing of malignant cells by adoptive transfer of chimeric antigen receptor (CAR) T cells is a promising immunotherapy technique in oncology. However, the identification of cell surface antigens unique to tumor cells against which CAR T cells can be engineered has historically been challenging and not well documented in solid tumors. Here, we describe a generalized method to construct a cell subtype-specific surface antigen profile (i.e., surfaceome) from cell lines and identify high-confidence antigens as effective targe...
Source: Mol Biol Cell - December 1, 2019 Category: Molecular Biology Authors: DeLucia DC, Lee JK Tags: Methods Mol Biol Source Type: research

Microfluidic Approach for Highly Efficient Viral Transduction.
Abstract Lentiviral vectors enable gene transfer into target cells, but manufacturing is complex, scale-limited, and costly. Here, we describe the use of microfluidic devices for efficient ex vivo gene transfer. Up to four- to fivefold reductions in viral vector usage and two- to fourfold reductions in transduction times can be obtained by using this method. PMID: 31776918 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - December 1, 2019 Category: Molecular Biology Authors: Tran R, Lam WA Tags: Methods Mol Biol Source Type: research

Single-Cell Cytokine Analysis to Characterize CAR-T Cell Activation.
Abstract With the increase in the implementation of adoptive transfer anti-CD19 chimeric antigen receptor (CAR) T cell therapy, we introduce a novel platform to study their heterogeneous cytokine response at the single-cell level following activation. Here, we describe an activation platform which incubates single CAR-T cells with single-target CD19 antigen-presenting cells (APCs) and measures the resulting cytokine secretions. This can be compared to the more traditional bulk activation modalities. Both pipelines of activation are measured using our high-throughput, multiplexed single-cell secretomic microchip co...
Source: Mol Biol Cell - December 1, 2019 Category: Molecular Biology Authors: Finck A, Fan R Tags: Methods Mol Biol Source Type: research

Testing the Specificity of Compounds Designed to Inhibit CPT1A in T Cells.
Abstract In response to antigen and costimulation, T cells undergo a series of metabolic transitions that fulfill the biosynthetic demands of clonal expansion, differentiation, and effector function. Following antigen clearance, the oxidation of long-chain fatty acids (LCFAO) has been implicated in the transition from effector to central memory T cells. However, studies demonstrating a role for LCFAO in memory T-cell development have largely relied on the use of etomoxir (ETO), a small molecule inhibitor of the long-chain fatty acid transporter CPT1A. Understanding how the depletion of nutrients including LCFA tha...
Source: Mol Biol Cell - December 1, 2019 Category: Molecular Biology Authors: O'Connor RS, Milone MC Tags: Methods Mol Biol Source Type: research

Engineering of Natural Killer Cells for Clinical Application.
Abstract The clinical success of chimeric antigen receptor-directed T cells in leukemia and lymphoma has boosted the interest in cellular therapy of cancer. It has been known for nearly half a century that a subset of lymphocytes called natural killer (NK) cells can recognize and kill cancer cells, but their clinical potential as therapeutics has not yet been fully explored. Progress in methods to expand and genetically modify human NK cells has resulted in technologies that allow the production of large numbers of highly potent cells with specific anticancer activity. Here, we describe clinically applicable proto...
Source: Mol Biol Cell - December 1, 2019 Category: Molecular Biology Authors: Shimasaki N, Campana D Tags: Methods Mol Biol Source Type: research

In Vivo Assessment of NK Cell-Mediated Cytotoxicity by Adoptively Transferred Splenocyte Rejection.
Abstract NK cells are innate lymphocytes that are vital to clearance of virally infected or malignantly transformed cells. Assessment of the cytotoxic response is an important component of NK cell research and investigation of human disease. Standard assays of NK cell-mediated cytotoxicity of CD107a degranulation or 51Cr release assay utilize cultured or freshly isolated NK cell populations in vitro. In addition to requirements to maintain multiple target cell lines and radioactivity precautions in the case of 51Cr, these are in vitro evaluations of a complex in vivo function. Here, we describe the in vivo assessm...
Source: Mol Biol Cell - December 1, 2019 Category: Molecular Biology Authors: Schloemer NJ, Abel AM, Thakar MS, Malarkannan S Tags: Methods Mol Biol Source Type: research

Immunomodulation of NK Cell Activity.
Abstract Natural killer (NK) cells can kill virus-infected cells and tumor cells without prior sensitization and secrete numerous cytokines and chemokines that modulate the activity of different cells of the immune system. The recognition of target cells is mediated by germ line-encoded receptors, and the activity of NK cells can be further regulated by soluble factors such as cytokines and Toll-like receptor ligands. Thus, NK cells display an exciting potential as a powerful immunotherapeutic tool against malignant diseases, and different strategies are being tested aiming to overcome tumor-induced NK cell suppre...
Source: Mol Biol Cell - December 1, 2019 Category: Molecular Biology Authors: Domaica CI, Sierra JM, Zwirner NW, Fuertes MB Tags: Methods Mol Biol Source Type: research

An Overview of Advances in Cell-Based Cancer Immunotherapies Based on the Multiple Immune-Cancer Cell Interactions.
SG Abstract Tumors have a complex ecosystem in which behavior and fate are determined by the interaction of diverse cancerous and noncancerous cells at local and systemic levels. A number of studies indicate that various immune cells participate in tumor development (Fig. 1). In this review, we will discuss interactions among T lymphocytes (T cells), B cells, natural killer (NK) cells, dendritic cells (DCs), tumor-associated macrophages (TAMs), neutrophils, and myeloid-derived suppressor cells (MDSCs). In addition, we will touch upon attempts to either use or block subsets of immune cells to target cancer. ...
Source: Mol Biol Cell - December 1, 2019 Category: Molecular Biology Authors: Zhang J, Späth SS, Weissman SM, Katz SG Tags: Methods Mol Biol Source Type: research

Rapid Production of Physiologic Dendritic Cells (phDC) for Immunotherapy.
Abstract Generation of large numbers of dendritic cells (DC) for research or immunotherapeutic purposes typically involves in vitro conversion of murine bone marrow precursors or human blood monocytes to DC via cultivation with supraphysiologic concentrations of cytokines such as GM-CSF and IL-4 for up to 7 days. Alternatively, our group has recently established a new approach, based on the underlying mechanism of action of a widely used cancer immunotherapy termed Extracorporeal Photochemotherapy (ECP). Our method of rapid and cytokine-free production of therapeutically relevant DC populations, lev...
Source: Mol Biol Cell - December 1, 2019 Category: Molecular Biology Authors: Hanlon D, Sobolev O, Han P, Ventura A, Vassall A, Kibbi N, Yurter A, Robinson E, Filler R, Tatsuno K, Edelson RL Tags: Methods Mol Biol Source Type: research

Reprogramming Exosomes for Immunotherapy.
Abstract Exosomes are nanosized vesicles secreted by nearly all types of cells and play important roles in intercellular communication. Given their unique and important pharmacological properties, exosomes have been emerging as a new class of cell-free therapeutics. Herein, we describe exosomes developed against epidermal growth factor receptor (EGFR), a key factor in epithelial malignancies, involved in enhanced tumor growth, invasion, and metastasis. The exosomes are genetically modified for displaying two distinct types of monoclonal antibodies on the exosome surface, resulting in novel synthetic multivalent an...
Source: Mol Biol Cell - December 1, 2019 Category: Molecular Biology Authors: Cheng Q, Shi X, Zhang Y Tags: Methods Mol Biol Source Type: research

Nanoparticles for Immune Cell Reprogramming and Reengineering of Tumor Microenvironment.
Abstract Nanoparticles in cancer therapy have garnered significant attention in the past few decades. Cancer immunotherapy, which is aptly called "the new-generation cancer therapy," is slowly making remarkable strides in the improvement of patient outcome and longevity. Taken together, nanoparticles in immune therapy have the potential to offer advantages of both nanoparticles and immune therapy on a single platform. PMID: 31776928 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - December 1, 2019 Category: Molecular Biology Authors: Bhise K, Sau S, Alzhrani R, Iyer AK Tags: Methods Mol Biol Source Type: research

Genome-Wide CRISPRi/a Screening in an In Vitro Coculture Assay of Human Immune Cells with Tumor Cells.
SG Abstract Cell-based immunotherapy has achieved preclinical success in certain types of cancer patients, with a few approved cell-based products for clinical use. These achievements revitalized the field of cell engineering/ immunotherapy and brought attention to the opportunities that cell-based immunotherapeutics can offer to patients. On the other hand, obvious indications emphasize the need for a better understanding of the biological mechanisms involved in the immune response. This knowledge may not only ameliorate safety and efficacy, but also determine the possibilities and limitations in use of immune c...
Source: Mol Biol Cell - December 1, 2019 Category: Molecular Biology Authors: Zhang J, Späth SS, Katz SG Tags: Methods Mol Biol Source Type: research

The Propagation and Quantification of Two Emerging Oncolytic Viruses: Vesicular Stomatitis (VSV) and Zika (ZIKV).
Abstract Developments in genetic engineering have allowed researchers and clinicians to begin harnessing viruses to target and kill cancer cells, either through direct lysis or through recruitment of antiviral immune responses. Two powerful viruses in the fight against cancer are the single-stranded RNA viruses vesicular stomatitis virus and Zika virus. Here, we describe methods to propagate and titer these two viruses. We also describe a simple cell-killing assay to begin testing modified viruses for increased potential killing of glioblastoma cells. PMID: 31776931 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - December 1, 2019 Category: Molecular Biology Authors: Means RE, Roy SG, Katz SG Tags: Methods Mol Biol Source Type: research

Radiolabeling and Imaging of Adoptively Transferred Immune Cells by Positron Emission Tomography.
This article describes a two-step radiometal labeling procedure utilizing the bifunctional siderophore p-isothiocyanatobenzyl-desferrioxamine (DFO-Bz-NCS) that chelates 89Zr with high affinity and binds covalently to primary amines of cell-surface proteins via its isothiocyanate moiety. Cells labeled with 89Zr-DFO-Bz-NCS remain viable and retain the radiolabel, enabling repetitive PET imaging of adoptively transferred immune cells with high sensitivity and specificity for up to 2 weeks. PMID: 31776932 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - December 1, 2019 Category: Molecular Biology Authors: Najjar AM Tags: Methods Mol Biol Source Type: research

Functional Analysis of Human Hematopoietic Stem Cells In Vivo in Humanized Mice.
Abstract Ex vivo generation and expansion of functional hematopoietic stem cells represents the holy grail of reprogramming and would constitute a major advance in stem cell therapies and generation of blood cellular products. In vivo testing is critical to assure proper cell intrinsic function in an organismal context. Here we describe methods for the generation of human hematopoiesis chimeric mice and evaluation of hematopoietic stem cell function. The choice of mouse model, stem cell source, and transplantation route can be adjusted to suit the desired application. PMID: 31776933 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - December 1, 2019 Category: Molecular Biology Authors: Song Y, Gbyli R, Fu X, Halene S Tags: Methods Mol Biol Source Type: research

Monitoring Allogeneic CAR-T Cells Using Flow Cytometry.
Abstract Chimeric antigen receptor T cell (CAR-T) therapies have now entered mainstream clinical practice with two approved autologous CAR-T products targeting CD19 and numerous other products in early and late phase clinical trials. This has led to a demand for highly sensitive, specific, and easily reproducible methods to monitor CAR-T cells in patients. Here we describe a flow cytometry based protocol for detection of allogeneic CAR-T cells and for monitoring their phenotype and numbers in blood and bone marrow of patients following CAR-T treatment. PMID: 31776934 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - December 1, 2019 Category: Molecular Biology Authors: Jozwik A, Dunlop A, Sanchez K, Benjamin R Tags: Methods Mol Biol Source Type: research

Key Factors in Clinical Protocols for Adoptive Cell Therapy in Melanoma.
Abstract Adoptive cell therapy (ACT) with autologous tumor infiltrating lymphocytes (TIL) has been studied for patients with advanced metastatic cancers (primarily melanoma) for decades and has changed significantly during that period. Treatment with TIL includes ex vivo cell activation and expansion followed by re-infusion of these cells into the patient. After cell infusion, patients receive Interleukin-2 (IL-2). Objective response rates up to 52% have been seen in patients with metastatic melanoma. Efforts to improve TIL therapy include better selection and expansion of tumor-reactive lymphocytes, optimization ...
Source: Mol Biol Cell - December 1, 2019 Category: Molecular Biology Authors: Considine B, Hurwitz ME Tags: Methods Mol Biol Source Type: research

Place of Academic GMP Facilities in Modern Cell Therapy.
Abstract Academic medicine, in general, serves a dual role in advancing the scientific field as well as providing the highest quality clinical care. In the last decade we have observed significant development of commercial cell and gene therapy products with rapid growth of the industry. Currently, hospital-based Good Manufacturing Practice (GMP) facilities, which are used to support primarily academic investigator-initiated clinical trials, are increasingly involved in interactions with industry.The purpose of this piece is to review the role of academic GMP facilities in development of cellular therapies. For th...
Source: Mol Biol Cell - December 1, 2019 Category: Molecular Biology Authors: Bersenev A, Fesnak A Tags: Methods Mol Biol Source Type: research

Detection of Inositol Phosphates by Split PH Domains.
Abstract The pleckstrin homology (PH) domain is a family of structurally conserved proteins which can bind inositol phosphate derivatives. Some proteins involved in cellular signaling and cytoskeletal organization possess split PH domains that assemble into a structure which can bind specific inositol phosphates. Here we describe the design of split PH domain from a structurally well-characterized PH domain of phospholipase C (PLC) δ1 and Bruton's tyrosine kinase (Btk), which selectively bind Ins(1,4,5)P3 and Ins(1,3,4,5)P4, respectively. The PH domains fold into a functional structure when the split halves ...
Source: Mol Biol Cell - November 29, 2019 Category: Molecular Biology Authors: Sakaguchi R, Tajima S, Mori Y, Morii T Tags: Methods Mol Biol Source Type: research

Dictyostelium discoideum as a Model to Study Inositol Polyphosphates and Inorganic Polyphosphate.
rdi A Abstract The yeast Saccharomyces cerevisiae has given us much information on the metabolism and function of inositol polyphosphates and inorganic polyphosphate. To expand our knowledge of the metabolic as well as functional connections between inositol polyphosphates and inorganic polyphosphate, we have refined and developed techniques to extract and analyze these molecules in a second eukaryotic experimental model, the amoeba Dictyostelium discoideum. This amoeba, possessing a well-defined developmental program, is ideal to study physiological changes in the levels of inositol polyphosphates and inorganic p...
Source: Mol Biol Cell - November 29, 2019 Category: Molecular Biology Authors: Desfougères Y, Saiardi A Tags: Methods Mol Biol Source Type: research

Characterizing Enzymes of the Diphosphoinositol Polyphosphate Phosphohydrolase (DIPP) Family.
Abstract The diphosphoinositol polyphosphate phosphohydrolases are a subset of the Nudix hydrolase family of enzymes. As such, they metabolize a wide range of substrates, including diphosphoinositol polyphosphates (inositol diphosphates, inositol pyrophosphates), dinucleotide phosphates, nucleosides as well as 5-phosphoribosyl 1-pyrophosphate and inorganic polyphosphate. Here, we describe protocols to optimize these enzymes, with consideration to buffer composition and sample preparation and how to analyze the metabolism of these substrates using high-performance liquid chromatography, giving advice where pitfalls...
Source: Mol Biol Cell - November 29, 2019 Category: Molecular Biology Authors: Winward L, Kilari RS, Safrany ST Tags: Methods Mol Biol Source Type: research

Metabolic Labeling of Inositol Phosphates and Phosphatidylinositols in Yeast and Mammalian Cells.
Abstract Inositol phosphate (IP) and phosphatidylinositol (PI) signaling are critical signal transduction pathways responsible for generating numerous receptor-mediated cellular responses. Biochemical and genetic studies have revealed diverse roles of IP and PI signaling in eukaryotic signaling, but detailed characterization of unique IP and PI signaling profiles in response to different agonists and among cell types remains largely unexplored. Here, we outline steady-state inositol metabolic-labeling techniques that can be leveraged to assess the IP and PI signaling state in eukaryotic cells. This flexible techni...
Source: Mol Biol Cell - November 29, 2019 Category: Molecular Biology Authors: Hale AT, Clarke BP, York JD Tags: Methods Mol Biol Source Type: research

Back-Pyrophosphorylation Assay to Detect In Vivo InsP7-Dependent Protein Pyrophosphorylation in Mammalian Cells.
Abstract Protein pyrophosphorylation involves the transfer of a high-energy β-phosphate from inositol pyrophosphates, such as diphosphoinositol pentakisphosphate (InsP7) to phosphorylated serine residues. Over a decade of research has established several proteins, involved in diverse physiological processes, as substrates of InsP7-mediated pyrophosphorylation. However, the need for detection of this posttranslational modification on endogenous proteins is paramount. "Back-pyrophosphorylation" is a simple technique to test whether a native protein undergoes InsP7-mediated pyrophosphorylation inside c...
Source: Mol Biol Cell - November 29, 2019 Category: Molecular Biology Authors: Chanduri M, Bhandari R Tags: Methods Mol Biol Source Type: research

Investigating the InsP3 Receptor in Living Cells by Caged InsP3.
Abstract The inositol 1,4,5-trisphosphate receptor (InsP3R) operates as an intracellular ligand-gated Ca2+ channel and plays a pivotal role in cellular Ca2+ homeostasis across all living cells. It is activated following membrane receptor-ligand interactions and stimulation of subsequent signaling cascades involving the enzymatic breakdown of the membrane lipid phosphatidyl-4,5-bisphosphate (PIP2) into the membrane-delimited second messenger diacylglycerol (DAG) and the diffusible second messenger inositol-1,4,5-trisphophate (InsP3). Modulation of InsP3R's activity is thus involved in a plethora of physiological an...
Source: Mol Biol Cell - November 29, 2019 Category: Molecular Biology Authors: Hui X, Lipp P Tags: Methods Mol Biol Source Type: research

High-Throughput Differential Scanning Fluorimetry of GFP-Tagged Proteins.
Abstract Differential scanning fluorimetry is useful for a wide variety of applications including characterization of protein function, structure-activity relationships, drug screening, and optimization of buffer conditions for protein purification, enzyme activity, and crystallization. A limitation of classic differential scanning fluorimetry is its reliance on highly purified protein samples. This limitation is overcome through differential scanning fluorimetry of GFP-tagged proteins (DSF-GTP). DSF-GTP specifically measures the unfolding and aggregation of a target protein fused to GFP through its proximal pertu...
Source: Mol Biol Cell - November 29, 2019 Category: Molecular Biology Authors: Sorenson AE, Schaeffer PM Tags: Methods Mol Biol Source Type: research

Measuring Small Molecule Binding to Escherichia coli AcrB by Surface Plasmon Resonance.
Abstract Antimicrobial resistance (AMR) is rapidly becoming one of the great healthcare challenges. A common mechanism employed by pathogenic bacteria to avoid the action of certain antibiotics is to overexpress efflux pumps that can extrude these drugs from the cell rendering them ineffective. Small molecule inhibitors that target bacterial efflux pumps provide a route toward reversing AMR. Here, we describe the application of surface plasmon resonance (SPR) technology to characterize protein:small molecule interactions between the inner membrane protein AcrB subunit of the Escherichia coli AcrA-AcrB-TolC efflux ...
Source: Mol Biol Cell - November 29, 2019 Category: Molecular Biology Authors: Polyak SW, Mowla R, Venter H Tags: Methods Mol Biol Source Type: research

Electrophoretic Mobility Shift Assays with GFP-Tagged Proteins (GFP-EMSA).
Abstract The electrophoretic mobility shift assay (EMSA) is commonly used for the study of nucleic acid-binding proteins. The technique can be used to demonstrate that a protein is binding to RNA or DNA through visualization of a shift in electrophoretic mobility of the nucleic acid band. A major disadvantage of the EMSA is that it does not always provide an absolute certitude that the band shift is due to the protein under scrutiny, as contaminants in the sample could also cause the band shift. Here we describe a variation of the standard EMSA allowing to visualize with added certitude, the co-localized band shif...
Source: Mol Biol Cell - November 29, 2019 Category: Molecular Biology Authors: Sorenson AE, Schaeffer PM Tags: Methods Mol Biol Source Type: research

High-Throughput Assessment of Metabolism-Induced Toxicity of Compounds on a 384-Pillar Plate.
Abstract A variety of oxidative and conjugative enzymes are involved in the metabolism of compounds including drugs, which can be converted into toxic metabolites by Phase I drug-metabolizing enzymes (DMEs), such as the cytochromes P450 (CYP450s), and/or detoxified by Phase II DMEs, such as UDP-glucuronosyltransferases (UGTs), sulfotransferases (SULTs), and glutathione S-transferases (GSTs). Traditionally, primary hepatocytes containing a complete set of DMEs have been widely used as a gold standard to assess metabolism-induced compound toxicity. However, primary hepatocytes are expensive, have high donor variabil...
Source: Mol Biol Cell - November 29, 2019 Category: Molecular Biology Authors: Kang SY, Yu KN, Joshi P, Lee MY Tags: Methods Mol Biol Source Type: research

Lipoxygenases as Targets for Drug Development.
Abstract Lipoxygenases are key enzymes that catalyze the polyunsaturated fatty acids such as arachidic acid, linoleic acid (LA), and others unsaturated fatty acids. They are involved in important functions such as cell structure, metabolism, and signal transduction mechanisms, finally mediating cell death process, especially ferroptosis, a novel type of cell death modality. Our present protocol described a colorimetric assay for measuring lipoxygenase activity as well as a high-performance liquid chromatography/electrospray ionization tandem mass spectrometry method for the quantification of arachidonic acid metab...
Source: Mol Biol Cell - November 29, 2019 Category: Molecular Biology Authors: Mao XY Tags: Methods Mol Biol Source Type: research

Enzymatic Analysis of Reconstituted Archaeal Exosomes.
Abstract The archaeal exosome is a protein complex with phosphorolytic activity. It is built of a catalytically active hexameric ring containing the archaeal Rrp41 and Rrp42 proteins, and a heteromeric RNA-binding platform. The platform contains a heterotrimer of the archaeal Rrp4 and Csl4 proteins (which harbor S1 and KH or Zn-ribbon RNA binding domains), and comprises additional archaea-specific subunits. The latter are represented by the archaeal DnaG protein, which harbors a novel RNA-binding domain and tightly interacts with the majority of the exosome isoforms, and Nop5, known as a part of an rRNA methylatin...
Source: Mol Biol Cell - November 27, 2019 Category: Molecular Biology Authors: Evguenieva-Hackenberg E, Gauernack AS, Hou L, Klug G Tags: Methods Mol Biol Source Type: research

Mapping Exosome-Substrate Interactions In Vivo by UV Cross-Linking.
Abstract The RNA exosome complex functions in both the accurate processing and rapid degradation of many classes of RNA in eukaryotes and Archaea. Functional and structural analyses indicate that RNA can either be threaded through the central channel of the exosome or more directly access the active sites of the ribonucleases Rrp44 and Rrp6, but in most cases, it remains unclear how many substrates follow each pathway in vivo. Here we describe the method for using an UV cross-linking technique termed CRAC to generate stringent, transcriptome-wide mapping of exosome-substrate interaction sites in vivo and at base-p...
Source: Mol Biol Cell - November 27, 2019 Category: Molecular Biology Authors: Delan-Forino C, Tollervey D Tags: Methods Mol Biol Source Type: research

Thiouridine-to-Cytidine Conversion Sequencing (TUC-Seq) to Measure mRNA Transcription and Degradation Rates.
Abstract The study of RNA dynamics, specifically RNA transcription and decay rates, has gained increasing attention in recent years because various mechanisms have been discovered that affect mRNA half-life, thereby ultimately controlling protein output. Therefore, there is a need for methods enabling minimally invasive, simple and high-throughput determination of RNA stability that can be applied to determine RNA transcription and decay rates in cells and organisms. We have recently developed a protocol which we named TUC-seq to directly distinguish newly synthesized transcripts from the preexisting pool of trans...
Source: Mol Biol Cell - November 27, 2019 Category: Molecular Biology Authors: Lusser A, Gasser C, Trixl L, Piatti P, Delazer I, Rieder D, Bashin J, Riml C, Amort T, Micura R Tags: Methods Mol Biol Source Type: research

Comparative Poly(A)+ RNA Interactome Capture of RNA Surveillance Mutants.
Abstract RNA exosome complexes degrade many different RNA substrates. Substrate selection and targeting to the exosome complex rely on cofactors, which bind to the substrate RNA, recruit the exosome complex, and help to remodel the associated ribonucleoprotein particle to facilitate RNA degradation. These cofactors are RNA-binding proteins, but their interaction with RNA may be very transient because the RNAs they are bound to are rapidly turned over by the exosome complex. Hence, the cofactors involved in the degradation of many exosome substrates are unknown. Here, we describe comparative poly(A)+ RNA interactom...
Source: Mol Biol Cell - November 27, 2019 Category: Molecular Biology Authors: Kilchert C, Hester S, Castello A, Mohammed S, Vasiljeva L Tags: Methods Mol Biol Source Type: research

In Vitro Characterization of the Activity of the Mammalian RNA Exosome on mRNAs in Ribosomal Translation Complexes.
Abstract The RNA exosome is a multisubunit protein complex that exhibits a 3' to 5' exoribonuclease activity, endoribonuclease activity, and participates in a variety of RNA processing and degradation pathways in both the nucleus and the cytoplasm. Exosomes interact with various cofactors which target them to specific RNA substrates and processes. Investigation of the mechanisms by which mammalian RNA exosomes are targeted to specific RNA substrates requires the development of in vitro approaches for purification of exosomes and their co-factors, assembly of substrates and monitoring of the exosomal activity. Here...
Source: Mol Biol Cell - November 27, 2019 Category: Molecular Biology Authors: Zinoviev A, Hellen CUT, Pestova TV Tags: Methods Mol Biol Source Type: research

Chemical Cross-Linking and Mass Spectrometric Analysis of the Endogenous Yeast Exosome Complexes.
Abstract Chemical cross-linking and mass spectrometric readout (CX-MS) has become a useful toolkit for structural analysis of protein complexes. CX-MS enables rapid detection of a larger number of cross-link peptides from the chemically cross-linked protein assembly, providing invaluable cross-link spatial restraints to understand the architecture of the complex. Since CX-MS is complementary with other structural and computational modeling tools, it can be used for integrative structural determination of large native protein assemblies. However, due to technical limitations, current CX-MS applications have still b...
Source: Mol Biol Cell - November 27, 2019 Category: Molecular Biology Authors: Xiang Y, Shen Z, Shi Y Tags: Methods Mol Biol Source Type: research

Cryo-Electron Microscopy of Endogenous Yeast Exosomes.
Abstract The RNA exosome is a multisubunit complex typically composed of a catalytically inactive core and the Rrp44 protein, which contains 3'-to-5' exo- and endo-RNase activities. With assistance from nuclear or cytoplasmic cofactors, functional studies implicated the exosome as a critical player in the turnover of almost all RNA species, including mRNAs, rRNA, tRNAs, and other noncoding RNAs. Here, we describe the purification of the yeast 10-subunit exosome and 11-subunit Exosome-Ski7, as well as subsequent sample screening by negative staining EM and structural analysis by cryo-EM. PMID: 31768987 [PubMed...
Source: Mol Biol Cell - November 27, 2019 Category: Molecular Biology Authors: Liu JJ, Wang HW Tags: Methods Mol Biol Source Type: research

Strategies for Generating RNA Exosome Complexes from Recombinant Expression Hosts.
Abstract The eukaryotic RNA exosome is a conserved and ubiquitous multiprotein complex that possesses multiple RNase activities and is involved in a diverse array of RNA degradation and processing events. While much of our current understanding of RNA exosome function has been elucidated using genetics and cell biology based studies of protein functions, in particular in S. cerevisiae, many important contributions in the field have been enabled through use of in vitro reconstituted complexes. Here, we present an overview of our approach to purify exosome components from recombinant sources and reconstitute them in...
Source: Mol Biol Cell - November 27, 2019 Category: Molecular Biology Authors: Weick EM, Zinder JC, Lima CD Tags: Methods Mol Biol Source Type: research

Purification and Reconstitution of the S. cerevisiae TRAMP and Ski Complexes for Biochemical and Structural Studies.
Abstract The RNA exosome is a macromolecular machine that degrades a large variety of RNAs from their 3'-end. It comprises the major 3'-to-5' exonuclease in the cell, completely degrades erroneous and overly abundant RNAs, and is also involved in the precise processing of RNAs. To degrade transcripts both specifically and efficiently the exosome functions together with compartment-specific cofactors. In the yeast S. cerevisiae, the exosome associates with the Ski complex in the cytoplasm and with Mtr4 alone or with Mtr4 as part of the TRAMP complex in the nucleus. Here we describe how to produce, purify, and assem...
Source: Mol Biol Cell - November 27, 2019 Category: Molecular Biology Authors: Keidel A, Conti E, Falk S Tags: Methods Mol Biol Source Type: research

What a Difference 30  Years Makes! A Perspective on Changes in Research Methodologies Used to Study Toxoplasma gondii.
What a Difference 30 Years Makes! A Perspective on Changes in Research Methodologies Used to Study Toxoplasma gondii. Methods Mol Biol. 2020;2071:1-25 Authors: Boothroyd JC Abstract Toxoplasma gondii is a remarkable species with a rich cell, developmental, and population biology. It is also sometimes responsible for serious disease in animals and humans and the stages responsible for such disease are relatively easy to study in vitro or in laboratory animal models. As a result of all this, Toxoplasma has become the subject of intense investigation over the last several decades, becoming a model o...
Source: Mol Biol Cell - November 24, 2019 Category: Molecular Biology Authors: Boothroyd JC Tags: Methods Mol Biol Source Type: research

High-Throughput Measurement of Microneme Secretion in Toxoplasma gondii.
Abstract Micronemes are specialized secretory organelles present in all motile forms of apicomplexan parasites. Microneme vesicles hold adhesins and other proteins that are secreted to facilitate parasite attachment, invasion of host cells, and egress following replication-all processes indispensable for cell-to-cell transmission of these obligate intracellular parasites. Defining the signaling pathways that lead to microneme secretion is an important part of understanding the infectious cycle of apicomplexan parasites. However, the classical method of measuring microneme secretion by immunoblotting for microneme ...
Source: Mol Biol Cell - November 24, 2019 Category: Molecular Biology Authors: Brown KM, Sibley LD, Lourido S Tags: Methods Mol Biol Source Type: research

Plate-Based Quantification of Stimulated Toxoplasma Egress.
Abstract Apicomplexans are obligate parasites that replicate inside host cells, within a subcellular compartment called the parasitophorous vacuole. Egress is the process by which apicomplexan parasites like Toxoplasma gondii exit from host cells, rupturing the parasitophorous vacuole and host-cell plasma membranes in the process. T. gondii retains the ability to egress throughout most of its intracellular replicative cycle, and this process has been associated with parasite signaling pathways that include the modulation of intracellular calcium, cyclic nucleotides, phosphatidic acid, and pH, which can be manipula...
Source: Mol Biol Cell - November 24, 2019 Category: Molecular Biology Authors: Shortt E, Lourido S Tags: Methods Mol Biol Source Type: research

Genetic Indicators for Calcium Signaling Studies in Toxoplasma gondii.
Abstract Fluctuations of the cytosolic calcium ion (Ca2+) concentration regulate a variety of cellular functions in all eukaryotes. Cells express a sophisticated set of mechanisms to balance the cytosolic Ca2+ levels and the signals that elevate Ca2+ in the cytosol are compensated by mechanisms that reduce it. Alterations in Ca2+-dependent homeostatic mechanisms are the cause of many prominent diseases in humans, such as heart failure or neuronal death.The genetic tractability of Toxoplasma gondii and the availability of genetic tools enabled the use of Genetically Encoded Calcium Indicators (GECIs) expressed in t...
Source: Mol Biol Cell - November 24, 2019 Category: Molecular Biology Authors: Vella SA, Calixto A, Asady B, Li ZH, Moreno SNJ Tags: Methods Mol Biol Source Type: research

Phenotyping Toxoplasma Invasive Skills by Fast Live Cell Imaging.
Abstract Host cell invasion by Toxoplasma gondii/T. gondii tachyzoites is an obligate but complex multistep process occurring in second-scale. To capture the dynamic nature of the whole entry process requires fast and high-resolution live cell imaging. Recent advances in T. gondii/host cell genome editing and in quantitative live cell imaging-image acquisition and processing included-provide a systematic way to accurately phenotype T. gondii tachyzoite invasive behaviour and to highlight any variation or default from a standard scenario. Therefore, applying these combined strategies allows gaining deeper insights ...
Source: Mol Biol Cell - November 24, 2019 Category: Molecular Biology Authors: Pavlou G, Tardieux I Tags: Methods Mol Biol Source Type: research

Experimental Approaches for Examining Apicoplast Biology.
Abstract Genetic manipulation is a powerful tool to study gene function but identifying the direct and primary functional outcomes of any gene depletion is crucial for this strategy to be productive. This is a major challenge for the study of apicoplast biology, because, in the absence of an efficient isolation method, apicoplast functions must be assayed in the parasite. These assays should be performed dynamically from the time of gene depletion, and include standards and controls that separate direct from indirect phenotypes. Here, we describe a pipeline for apicoplast functional analysis and highlight relevant...
Source: Mol Biol Cell - November 24, 2019 Category: Molecular Biology Authors: Biddau M, Ovciarikova J, Sheiner L Tags: Methods Mol Biol Source Type: research

Toxoplasma gondii: Bradyzoite Differentiation In Vitro and In Vivo.
Abstract Toxoplasma gondii, a member of the Apicomplexa, is known for its ability to infect an impressive range of host species. It is a common human infection that causes significant morbidity in congenitally infected children and immunocompromised patients. This parasite can be transmitted by bradyzoites, a slowly replicating life stage found within intracellular tissue cysts, and oocysts, the sexual life cycle stage that develops in domestic cats and other Felidae. T. gondii bradyzoites retain the capacity to revert back to the quickly replicating tachyzoite life stage, and when the host is immune compromised u...
Source: Mol Biol Cell - November 24, 2019 Category: Molecular Biology Authors: Mayoral J, Di Cristina M, Carruthers VB, Weiss LM Tags: Methods Mol Biol Source Type: research

Three-Dimensional Reconstruction of Toxoplasma-Neuron Interactions In Situ.
Abstract How tissue and cellular architecture affects host cell-microbe interactions in vivo remains poorly defined because imaging these interactions in complex tissue is difficult and standard in vitro cultures do not mimic whole organ architecture. Here we describe a method that combines new tissue clearing techniques, high-resolution imaging, and three-dimensional reconstruction to overcome these barriers and allow in situ imaging of host cell-microbe interactions in complex tissue. We use the interactions between neurons and Toxoplasma gondii, a ubiquitous, protozoan parasite that establish a lifelong central...
Source: Mol Biol Cell - November 24, 2019 Category: Molecular Biology Authors: Cabral CM, Merritt EF, Koshy AA Tags: Methods Mol Biol Source Type: research

Model Systems for Studying Mechanisms of Ocular Toxoplasmosis.
We present methods for in vitro and in vivo studies of ocular toxoplasmosis, including dissection of the human eye, and culture and infection of differentiated cell populations from the retina, as well as induction of mouse ocular toxoplasmosis by intraocular, or sequential systemic and intraocular, inoculations, and imaging of toxoplasmic retinal lesions. PMID: 31758460 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - November 24, 2019 Category: Molecular Biology Authors: Smith JR, Ashander LM, Ma Y, Rochet E, Furtado JM Tags: Methods Mol Biol Source Type: research

Assays to Evaluate Toxoplasma-Macrophage Interactions.
We describe multiplex approaches for measuring arginase activity, indoleamine 2,3 dioxygenase activity, cell death, and parasite growth during Toxoplasma-macrophage interactions. These assays can be used to compare how different Toxoplasma strains differ in their interaction with macrophages, and we describe how to properly assess Toxoplasma strain differences in Toxoplasma-macrophage interactions. PMID: 31758462 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - November 24, 2019 Category: Molecular Biology Authors: Mukhopadhyay D, Saeij JPJ Tags: Methods Mol Biol Source Type: research