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Past Issue in 2020
Volume: 10, Issue: 9
Biochemistry
Confocal and Super-resolution Imaging of RNA in Live Bacteria Using a Fluorogenic Silicon Rhodamine-binding Aptamer
Genetically encoded light-up RNA aptamers have been shown to be promising tools for the visualization of RNAs in living cells, helping us to advance our understanding of the broad and complex life of RNA. Although a handful of light-up aptamers spanning the visible wavelength region have been developed, none of them have yet been reported to be compatible with advanced super-resolution techniques, mainly due to poor photophysical properties of their small-molecule fluorogens. Here, we describe a detailed protocol for fluorescence microscopy of mRNA in live bacteria using the recently reported fluorogenic silicon rhodamine binding aptamer (SiRA) featuring excellent photophysical properties. Notably, with SiRA, we demonstrated the first aptamer-based RNA visualization using super-resolution (STED) microscopy. This imaging method can be especially valuable for visualization of RNA in prokaryotes since the size of a bacterium is only a few times greater than the optical resolution of a conventional microscope.
Quantification of Fatty Acids in Mammalian Tissues by Gas Chromatography–Hydrogen Flame Ionization Detection
In mammalian organisms, fatty acids (FAs) exist mostly in esterified forms, as building blocks of phospholipids, triglycerides, and cholesteryl esters, while some exist as non-esterified free FAs. The absolute quantification of FA species in total lipids or in a specific lipid class is critical in lipid-metabolism studies. To quantify FAs in biological samples, gas chromatography–hydrogen flame ionization detection (GC-FID)-based methods have been used as highly robust and reliable techniques. Prior to GC-FID analysis, FAs need to be derivatized to volatile FA methyl esters (FAMEs). The derivatization of unsaturated FAs using classical derivatization methods that rely on high reaction temperature requires skill; consequently, the quantification results are often unreliable. The recently available FA-methylation procedure rapidly and reliably derivatizes a variety of FA species, including poly-unsaturated FAs (PUFAs). To analyze FAs in mammalian tissue samples, lipid extraction and fractionation are also critical for robust analysis. In this report, we describe a whole protocol for the GC-FID-based FA quantification of mammalian tissue samples, including lipid extraction, fractionation, derivatization, and quantification. The protocol is useful when various FAs, especially unsaturated FAs, need to be reliably quantified.
Cancer Biology
Colorimetric RhoB GTPase Activity Assay
The Ras homologous protein (Rho) GTPase subfamily, including RhoA, RhoB, and RhoC are small molecules (~21 kDa) that act as molecular switches in a wide range of signaling pathways to orchestrate biological processes associated with both physiological and tumorigenic cellular states. The Rho GTPases are crucial regulators of actin cytoskeleton rearrangements and FA dynamics and are required for effective cell migration and invasion, as well as cell cycle progression and apoptosis. The Rho GTPases activity is regulated by conformational switching between GTP-bound (active) and GDP-bound (inactive) states. This GTP/GDP cycling is tightly controlled by the guanine nucleotide exchange factors (GEFs), which function as activators by catalyzing the exchange of GDP for GTP and by the GTPase-activating proteins (GAPs), which enable hydrolysis of GTP leading to the Rho GTPase inactivation. Here, we describe a detailed protocol to perform a RhoB G-LISA activation assay to detect the level of GTP-loaded RhoB in vitro. This is the first colorimetric assay designed to specifically measure RhoB activation. This method was developed by adapting the RhoA G-LISA Activation Assay Kit (Cytoskeleton, Inc.) and allow the precise measurement of RhoB activity in less than 3 hours. This rapid methodology can be broadly used to assess the level of GTP-loaded RhoB in any kind of cellular models, to appreciate either the role RhoB activation in physiological processes, diseases, oncogenic transformation or for drug discovery in high throughput screens.
Developmental Biology
Live Cell Imaging of Male Meiosis in Arabidopsis by a Landmark-based System
Live cell imaging has tremendously promoted our understanding of cellular and subcellular processes such as cell division. Here, we present a step-by-step protocol for a robust and easy-to-use live cell imaging approach to study male meiosis in the plant Arabidopsis thaliana as recently established. Our method relies on the concomitant analysis of two reporter genes that highlight chromosome configurations and microtubule dynamics. In combination, these reporter genes allowed the discrimination of five cellular parameters: cell shape, microtubule array, nucleus position, nucleolus position, and chromatin condensation. These parameters can adopt different states, e.g., the nucleus position can be central or lateral. Analyzing how tightly these states are associated gives rise to landmark stages that in turn allow a quantitative and qualitative dissection of meiotic progression. We envision that such an approach can also provide valuable criteria for the analysis of cell differentiation processes outside of meiosis.
Application of Mechanical Forces on Drosophila Embryos by Manipulation of Microinjected Magnetic Particles
Cells generate mechanical forces to shape tissues during morphogenesis. These forces can activate several biochemical pathways and trigger diverse cellular responses by mechano-sensation, such as differentiation, division, migration and apoptosis. Assessing the mechano-responses of cells in living organisms requires tools to apply controlled local forces within biological tissues. For this, we have set up a method to generate controlled forces on a magnetic particle embedded within a chosen tissue of Drosophila embryos. We designed a protocol to inject an individual particle in early embryos and to position it, using a permanent magnet, within the tissue of our choice. Controlled forces in the range of pico to nanonewtons can be applied on the particle with the use of an electromagnet that has been previously calibrated. The bead displacement and the epithelial deformation upon force application can be followed with live imaging and further analyzed using simple analysis tools. This method has been successfully used to identify changes in mechanics in the blastoderm before gastrulation. This protocol provides the details, (i) for injecting a magnetic particle in Drosophila embryos, (ii) for calibrating an electromagnet and (iii) to apply controlled forces in living tissues.
A Modified Semisolid Clonal Culture for Identification of B-1 and B-2 Progenitor Colony Forming Ability of Mouse Embryonic Hemogenic Endothelial Cells
The search for the origin of the first hematopoietic stem cells (HSCs) in the mouse embryo has been a hot topic in the field of developmental hematopoiesis. Detecting lymphoid potential is one of the supportive evidence to show the definitive hematopoietic activity of HSCs. However, the first B-lymphoid potential in the mouse embryos are reported to be biased to innate-like B-1 cell lineage that can develop from hemogenic endothelial cells (HECs) independently of HSCs. On the other hand, conventional adaptive immune B cells (B-2) cells are considered to be exclusively derived from HSCs. Therefore, segregating B-1 and B-2 progenitor potential is important to understand the developmental process of HSCs that are also produced from HECs through intermediate precursors referred to as pre-HSCs. Both HECs and pre-HSCs show endothelial surface phenotype and require stromal support to detect their hematopoietic activity. The method utilizing stromal cell culture followed by modified semisolid clonal culture enables us to detect the number of colony forming units for B-1/B-2 progenitors originally derived from HECs/pre-HSCs, which will reflect the potential of B-1 biased or multi-lineage repopulating HSCs.
Immunology
HIV-CRISPR: A CRISPR/Cas9 Screening Method to Identify Genes Affecting HIV Replication
Screening with CRISPR/Cas9 technology has already led to significant discoveries in the fields of cancer biology, cell biology and virology. Because of the relatively low false discovery rates and the ability to perform high-throughput, pooled approaches, it has rapidly become the assay of choice for screening studies, including whole-genome screens. Here, we describe a CRISPR screening protocol that allows for efficient screening of the entire life cycle of HIV-1 through packaging of the HIV-CRISPR lentiviral genomes by infecting HIV-1 virus in trans.
Evaluation of B Cell Proliferation in vivo by EdU Incorporation Assay
Generation of antibodies is crucial for establishing enduring protection from invading pathogens, as well as for maintaining homeostasis with commensal bacteria at mucosal surfaces. Chronic exposure to microbiota- and dietary- derived antigens results in continuous production of antibody producing cells within the Peyer’s patch germinal center structures. Recently, we have shown that B cells responding to gut-derived antigens colonize the subepithelial dome (SED) in Peyer’s patches and rapidly proliferate independently of their relative BCR affinity. To evaluate B cell proliferation within different niches in Peyer’s patches, we applied in vivo EdU incorporation assay as described in this protocol.
Assessing in vitro and in vivo Trogocytosis By Murine CD4+ T cells
Recognition of antigens by lymphocytes (B, T, and NK) on the surface of an antigen-presenting cell (APC) leads to lymphocyte activation and the formation of an immunological synapse between the lymphocyte and the APC. At the immunological synapse APC membrane and associated membrane proteins can be transferred to the lymphocyte in a process called trogocytosis. The detection of trogocytosed molecules provides insights to the activation state, antigen specificity, and effector functions and differentiation of the lymphocytes. Here we outline our protocol for identifying trogocytosis-positive CD4+ T cells in vitro and in vivo. In vitro, antigen presenting cells are surface biotinylated and pre-loaded with magnetic polystyrene beads before incubating for a short time with in vitro activated CD4+ T cell blasts (90 min) or naïve T cells (3-24 h). After T cell recovery and APC depletion by magnetic separation trogocytosis positive (trog+) cells are identified by streptavidin staining of trogocytosed, biotinylated APC membrane proteins. Their activation phenotype, effector function, and effector differentiation are subsequently analyzed by flow cytometry immediately or after subsequent incubation. Similarly, trogocytosis-positive cells can be identified and similarly analyzed by flow cytometry. Previous studies have described methods for analyzing T cell trogocytosis to identify antigen-specific cells or the antigenic epitopes recognized by the cells. With the current protocol, the effects of trogocytosis on the individual T cell or the ability of trog+ T cells to modulate the activation and function of other immune cells can be assessed over an extended period of time.
Microbiology
A Sensitive Coupled Enzyme Assay for Measuring Kinase and ATPase Kinetics Using ADP-Specific Hexokinase
Kinases and ATPases perform essential biological functions in metabolism and regulation. Activity of these enzymes is commonly measured by coupling ATP consumption to the synthesis of a detectable product. For most assay systems the ATP concentration during the reaction is unknown, compromising the precision of the assay.Using the ADP-specific hexokinase (ADP-HK) from the thermophilic archaeon Thermococcus litoralis the protocol outlined here allows real time coupling of ATP consumption to downstream signal change enabling accurate kinetic measurements. ADP-HK phosphorylates glucose that is then used by glucose-6-phosphate dehydrogenase to reduce NAD+ to NADH which can be measured at 340 nm. We have shown this assay to be sensitive to the detection of micromole quantities of ADP with no detectable background from ATP.
Quantification of Bacteria Residing in Caenorhabditis elegans Intestine
Quantification of intestinal colonization by pathogenic or commensal bacteria constitute a critical part of the analysis to understand host-microbe interactions during different time points of their interplay. Here we detail a method to isolate non-pathogenic and pathogenic bacteria from C. elegans intestines, and classify gut phenotypes induced by bacterial pathogens using fluorescently-tagged bacteria. Furthermore, these methods can be used to isolate and identify new culturable bacterial species from natural microbiomes of wild nematodes.
Viral Double-Stranded RNA Detection by DNase I and Nuclease S1 digestions in Leishmania parasites
Many RNA viruses are found in protozoan parasites. They can be responsible for more serious pathology or treatment failure. For the detection of viral double-stranded RNA (dsRNA), sequence-dependent and -independent methods are available, such as quantitative real-time PCR and immunofluorescence, dot blot, ELISA or sequencing. The technique presented here is sequence-independent and is well detailed in the following protocol, taking the example of Leishmania RNA virus (LRV) in Leishmania guyanensis (Lgy) species. To summarise, the protocol is divided into four major steps: RNA extraction from the parasites, RNA purification, enzymatic digestions with DNase I and Nuclease S1, and visualization by gel electrophoresis. This method can be used to detect other viral dsRNA in other parasites. It provides an additional tool, complementary to other techniques previously cited and it is easy and quite fast to achieve.
Molecular Biology
Split Nano Luciferase-based Assay to Measure Assembly of Japanese Encephalitis Virus
Cells infected with flavivirus release various forms of infectious and non-infectious particles as products and by-products. Comprehensive profiling of the released particles by density gradient centrifugation is informative for understanding viral particle assembly. However, it is difficult to detect low-abundance minor particles in such analyses. We developed a method for viral particle analysis that integrates a high-sensitivity split luciferase system and density gradient centrifugation. This protocol enables high-resolution profiling of particles produced by cells expressing Japanese encephalitis virus factors.
Neuroscience
Rapid Generation of Human Neuronal Cell Models Enabling Inducible Expression of Proteins-of-interest for Functional Studies
CRISPR-Cas9 technology has transformed the ability to edit genomic sequences and control gene expression with unprecedented ease and scale. However, precise genomic insertions of coding sequences using this technology remain time-consuming and inefficient because they require introducing adjacent single-strand cuts through Cas9 nickase action and invoking the host-encoded homology-directed repair program through the concomitant introduction of large repair templates. Here, we present a system for the rapid study of any protein-of-interest in two neuronal cell models following its inducible expression from the human AAVS1 safe harbor locus. With lox-flanked foundation cassettes in the AAVS1 site and a tailor-made plasmid for accepting coding sequences-of-interest in place, the system allows investigators to produce their own neuronal cell models for the inducible expression of any coding sequence in less than a month. Due to the availability of preinserted enhanced green fluorescent protein (EGFP) coding sequences that can be fused to the protein-of-interest, the system facilitates functional investigations that track a protein-of-interest by live-cell microscopy as well as interactome analyses that capitalize on the availability of exquisitely efficient EGFP capture matrices.
Systems Biology
Sequence Alignment Using Machine Learning for Accurate Template-based Protein Structure Prediction
Template-based modeling, the process of predicting the tertiary structure of a protein by using homologous protein structures, is useful when good templates can be available. Indeed, modern homology detection methods can find remote homologs with high sensitivity. However, the accuracy of template-based models generated from the homology-detection-based alignments is often lower than that from ideal alignments. In this study, we propose a new method that generates pairwise sequence alignments for more accurate template-based modeling. Our method trains a machine learning model using the structural alignment of known homologs. When calculating sequence alignments, instead of a fixed substitution matrix, this method dynamically predicts a substitution score from the trained model.
Correction
