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Past Issue in 2018
Volume: 8, Issue: 23
Biochemistry
Wheat Germ Agglutinin (WGA)-SDS-PAGE: A Novel Method for the Detection of O-GlcNAc-modified Proteins by Lectin Affinity Gel Electrophoresis
Diverse cytoplasmic and nuclear proteins dynamically change their molecular functions by O-linked β-N-acetylglucosamine (O-GlcNAc) modification on serine and/or threonine residues. Evaluation of the O-GlcNAcylation level of a specific protein, however, needs multiple and time-consuming steps if using conventional methods (e.g., immune-purification, mass spectrometric analysis). To overcome this drawback, we developed the following easy and rapid method for detection of O-GlcNAcylated proteins of interest. An O-GlcNAc affinity gel layer containing wheat germ agglutinin (WGA), a GlcNAc-specific lectin, selectively induces retardation of the mobility of O-GlcNAcylated proteins during electrophoresis. This WGA-layer thereby separates O-GlcNAcylated and non-modified forms of proteins, allowing the detection and quantification of the O-GlcNAcylation level of these proteins. This new method therefore provides qualitative and quantitative analysis of O-GlcNAcylated proteins in a relatively shorter time compared to conventional methods.
Purification of Globular Actin from Rabbit Muscle and Pyrene Fluorescent Assays to Investigate Actin Dynamics in vitro
Pyrene Fluorescent Assay is established to monitor the dynamic actin nucleation, elongation, capping and disassembly in vitro. This technique provides an easy handle procedure and straightforward visual data analysis. By coupling actin purification and polymerization assays in this protocol, the readers could quickly get the affordable and straightforward assays to study actin dynamics.
In vitro Membrane Interaction and Liposome Fusion Assays Using Recombinant Hepatitis C Virus Envelope Protein E2
In order to study the mechanism underlying the Hepatitis C Virus (HCV) fusion process we have performed assays using phospholipid liposomes and a truncated form of E2 protein, E2661 (amino acids 384-661 of the HCV polyprotein) lacking the transmembrane region. E2661 has been previously generated by using the baculovirus expression system. This form has been used in lipid-protein interaction studies with different model vesicles at different pHs, and monitored using a variety of fluorescent assays. After the analysis of the results, we observed that E2661 is able to insert into lipid bilayers and to induce vesicle aggregation, lipid mixing and liposome leakage, showing higher values of membrane destabilization for negatively charged phospholipids at acidic pH. This is indicative of the role of E2 glycoprotein in the HCV initial infective steps, interacting with the target membranes and producing their destabilization.
Cancer Biology
Cluster Analysis of Endogenous HER2 and HER3 Receptors in SKBR3 Cells
The Human Epidermal Growth Factor Receptor (HER) family of receptor tyrosine kinases consists of four, single pass, transmembrane receptor homologs (HER1-4) that act to regulate many critical processes in normal and tumor cells. HER2 is overexpressed in many tumors, and the deregulated proliferation of cancerous cells is driven by cooperation with its preferred receptor partner, HER3. The assessment of the in-situ organization of tagged HER2 and HER3 using super-resolution microscopy reveals quantitative Single Molecule Localization Microscopy (SMLM) as an ideal bioanalytical tool to characterize receptor clusters. Clustering of receptors is an important regulatory mechanism to prime cells to respond to stimuli so, to understand these processes, it is necessary to measure parameters such as numbers of clusters, cluster radii and the number of localizations per cluster for different perturbations. Previously, Fluorescence Localization Imaging with Photobleaching (FLImP), another nanoscale, single-molecule technique, characterized the oligomerization state of HER1 [or Epidermal Growth Factor Receptors (EGFR)] in cell membranes. To achieve an unprecedented resolution (< 5 nm) for inter-molecular separations in EGFR oligomers using FLImP, very few receptors are tagged, and so this method is unsuitable for measurements of whole receptor populations in cancer cells where receptors are frequently upregulated. Here, in order to detect all receptors involved in cluster formation, we saturate endogenous HER2 and HER3 membrane receptors with ligands at a 1:1 dye to protein ratio, in the presence or absence of therapeutic drugs (lapatinib or bosutinib). This is performed in the commonly used breast cancer cell line model SKBR3 cells, where there are ~1.6 million HER2 receptors/cell and 10,000-40,000 HER3 receptors/cell. The basal state of these receptors is studied using HER2- or HER3-specific Affibodies, and likewise, the active state is probed using the natural HER3 ligand, Neuregulin-beta1 (NRGβ1). Stochastic Optical Reconstruction Microscopy (STORM), one form of SMLM, was used here to image cells, which were chemically fixed to minimize image blurring and provide data (x and y coordinates and standard deviation of the measured localizations) for cluster analysis. Further analysis can also determine proportions of receptor colocalizations. Our findings show that lapatinib-bound HER2, complexed with HER3 via a non-canonical kinase dimer structure, induces higher order oligomers. We hypothesized that nucleation of receptors creates signaling platforms that explain the counterintuitive, increase in cell proliferation upon ligand binding, in the presence of the HER2-inhibitor lapatinib.
Cell Biology
Blinded Visual Scoring of Images Using the Freely-available Software Blinder
In nearly all subfields of biomedical sciences, there are phenotypes that are currently classified by expert visual scoring. In research applications, these classifications require the experimenter to be blinded to the treatment group in order to avoid unintentional bias in scoring. Currently, many labs either use laborious and tedious methods to manually blind the images, require multiple experimenters to gather and score the data blindly or fail to properly blind the data altogether. In this protocol, we present a simple, freely available software that we created that allows the experimenter to blindly score images. In our protocol, the user loads unblinded images and defines a scoring system. The software then shows the user the images in a random order, allowing the user to select a score from their defined scoring system for each image. Furthermore, the software has an optional “quality control” mechanism where the user will be shown some images multiple times to test the robustness of the visual scoring. Finally, the software summarizes the results in an exportable file that includes unblinded summary data for each group and a full list of images with their scores. In this protocol, we briefly present directions for using the software, potential applications, and caveats/limitations to this approach.
Developmental Biology
Notochord Injury Assays that Stimulate Transcriptional Responses in Zebrafish Larvae
Zebrafish have become an increasingly important model organism in the field of wound healing and regenerative medicine, due to their high regenerative capacity coupled with high-resolution imaging in living animals. In a recent study, we described multiple physical and chemical methods to induce notochord injury that led to highly specific transcriptional responses in notochord cellular subpopulations. The notochord is a critical embryonic structure that functions to shape and pattern the vertebrae and spinal column. Here, we describe precision needle injury, tail-notochord amputation, and chemical inhibition of caveolin that trigger a wound-specific wt1b expression response in the notochord sheath cell subpopulation. We propose that these procedures can be used to study distinct cell populations that make up the cellular processes of notochord repair.
Immunology
Flow Cytometry Assay for Recycling of LFA-1 in T-lymphocytes
To enable cells to move forward, cell surface integrins are internalized into an endosomal compartment and subsequently intracellularly transported to be re-exposed at a new site on the cell membrane. Leukocytes are the fastest migrating cell type in the human body, which express the leukocyte-specific integrin LFA-1. Here, we describe a flow cytometry-based assay that allows the quantification of LFA-1 internalization and its re-expression on the cell surface in T lymphocytes. An advantage of using flow cytometry-based assay over biochemical methods is the low number of needed cells. This protocol can be also used to measure recycling of other receptors.
Ultrasound Guided Intra-thymic Injection to Track Recent Thymic Emigrants and Investigate T Cell Development
To track recent thymic emigrants (RTEs) or study T cell development in the thymus, intra-thymic injection of a cellular tag or precursor cells for various T cell lineages is often desired. However, the traditional surgical approach to expose the thymus for intra-thymic injection is time-consuming and can cause a high level of pain and stress to animals, which might disrupt immune homeostasis, potentially confounding the results. Here, we introduce an ultrasound guided intra-thymic injection procedure, which is non-surgical and minimally invasive to animals. This technique is relatively easy to learn and offers an efficient and accurate tool to track RTEs or perform intra-thymic transfer of various cell types to investigate their differentiation.
Integrin Dependent RhoB Activation Assay Using Leukocytes
To be able to migrate, leukocyte needs to re-use its adhesion molecules to move forward. These adhesion molecules are called integrins and are intracellularly transported via endocytosis and exocytosis in order to translocate to a new site on the cell membrane. The intracellular transportation is regulated by different small GTPases including RhoB. Here we describe an activation assay of RhoB in leukocytes migrating on ICAM-1Fc coated dishes using commercially available Rhoteikin coated agarose beads. Although this is a specific protocol for LFA-1 induced RhoB activation, it can also be used for RhoB activation induced by other soluble and insoluble factors.
Microbiology
Quantitative Transformation Efficiency Assay for Bacillus subtilis
Bacillus subtilis (B. subtilis) is a model Gram-positive organism used to study a variety of physiological states and stress responses, one of which is the development of competence. Competence refers to the physiological state of a cell which allows it to be transformed naturally. Through induction of competence, the efficiency of natural transformation can be quantified by plating colony forming units (CFU) and transforming units (TFU). Here we describe a protocol for quantifying relative transformability using B. subtilis.
Field Inoculation and Classification of Maize Ear Rot Caused by Fusarium verticillioides
Maize ear rot is a worldwide fungal disease mainly caused by Fusarium verticillioides and Fusarium graminearum. Maize planted in the field was inoculated with Fusarium verticillioides at the filling stage, 15 days after pollination. Two milliliters of spore suspension with a concentration of 5 x 106/ml was injected into the middle of the top ear using pricking ear method to cause maize ear rot. The thirty days after inoculation was the most suitable time for phenotypic evaluation of Fusarium resistance.
An in vitro Microscopy-based Assay for Microtubule-binding and Microtubule-crosslinking by Budding Yeast Microtubule-associated Protein
In this protocol, we describe a simple microscopy-based method to assess the interaction of a microtubule-associated protein (MAP) with microtubules. The interaction between MAP and microtubules is typically assessed by a co-sedimentation assay, which measures the amount of MAP that co-pellets with microtubules by centrifugation, followed by SDS-PAGE analysis of the supernatant and pellet fractions. However, MAPs that form large oligomers tend to pellet on their own during the centrifugation step, making it difficult to assess co-sedimentation. Here we describe a microscopy-based assay that measures microtubule binding by direct visualization using fluorescently-labeled MAP, solving the limitations of the co-sedimentation assay. Additionally, we recently reported quantification of microtubule bundling by measuring the thickness of individual microtubule structures observed in the microscopy-based assay, making the protocol more advantageous than the traditional microtubule co-pelleting assay.
Plant Science
Genome-wide Estimation of Evolutionary Distance and Phylogenetic Analysis of Homologous Genes
Homologous genes, including paralogs and orthologs, are genes that share sequence homologies within or between different species. Homologous genes originate from a common origin through speciation, genetic duplication or horizontal gene transfer. Estimation of the sequence divergence of homologous genes help us to understand divergence time, which makes it possible to understand the evolutionary patterns of speciation, gene duplication and gene transfer events. This protocol will provide a detailed bioinformatics pipeline on how to identify the homologous genes, compare their sequence divergence and phylogenetic relationships, focusing on homologous genes that show syntenic relationships using soybean (Glycine max) and common bean (Phaseolus vulgaris) as example species.
Using Arabidopsis Mesophyll Protoplasts to Study Unfolded Protein Response Signaling
Various environmental stresses or artificial reagents can trigger unfolded protein accumulation in the endoplasmic reticulum (ER) due to the folding capacity of the ER being exceeded. This is termed ER stress, and triggers the unfolded protein response (UPR). Assays for activation of the UPR in plants include Tunicamycin (Tm)- or dithiothreitol (DTT)-mediated root growth inhibition, analysis of splicing of the UPR-responsive transcription factor bZIP60 (basic Leucine Zipper Domain 60), and upregulation of relevant UPR genes. We provide here a quick and robust method to detect UPR signaling in Arabidopsis thaliana protoplasts. This assay can also be applied to other plant species for which protoplasts can be isolated.