We have developed a cell fusion assay that quantifies SNARE-mediated membrane fusion events by activated expression of β-galactosidase.
RNA interference has been proven very effective to analyze gene function in Drosophila tracheal development. A detailed protocol used by Jiang lab to inject dsRNA into fly embryos to knockdown gene expression is illustrated. This technique has the potential for screening genes required for tissue and organ development in Drosophila.
We describe two methods for conditional trans-complementation of hepatitis C virus (HCV) assembly and the completion of the full viral life cycle, which rely on heterokaryon formation. These techniques are suitable to screen for cell lines that express dominant restriction factors, which preclude production of infectious HCV progeny.
1Department of Biochemistry & Molecular Biology, Wayne State University School of Medicine, 2Cardiovascular Research Institute, Wayne State University School of Medicine, 3Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine
Cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel, has been reported to interact with various proteins and regulate important cellular processes; among them the CFTR PDZ motif-mediated interactions have been well documented. This protocol describes methods we developed to assemble a PDZ-dependent CFTR macromolecular signaling complex in vitro.
1Department of Biomedical Engineering, University of Wisconsin-Madison, 2Department of Biomedical Engineering, Materials Science Program, Laboratory for Optical and Computational Instrumentation, University of Wisconsin-Madison
A method to track cell fusion in living organisms over time is described. The approach utilizes Cre-LoxP recombination to induce luciferase expression upon cell fusion. The luminescent signal generated can be detected in living organisms using biophotonic imaging systems with a sensitivity of detection of ˜1,000 cells in peripheral tissues.
Biophysical and biochemical studies of interactions among membrane-embedded protein domains face many technical challenges, the first of which is obtaining appropriate study material. This article describes a protocol for producing and purifying disulfide-stabilized transmembrane peptide complexes that are suitable for structural analysis by solution nuclear magnetic resonance (NMR) and other analytical applications.
A Faster, High Resolution, mtPA-GFP-based Mitochondrial Fusion Assay Acquiring Kinetic Data of Multiple Cells in Parallel Using Confocal Microscopy
1Department of Neuroscience, Center for Neuroscience Research, Tufts School of Medicine, 2Department of Internal Medicine, Geriatrics & Gerontology, Wake Forest Baptist Medical Center, 3Department of Medicine, Boston University Medical Center
Mitochondrial fusion was measured by tracking the equilibration of photoconverted matrix-targeted GFP across the mitochondrial network over time. Thus far, only one cell could be subjected to an hour long kinetic analysis at a time. We present a method that simultaneously measures multiple cells, thereby speeding up the data collection process.
Cultured muscle cells are an inadequate model to recapitulate innervated muscle in vivo. A functional motor unit can be reproduced in vitro by innervation of differentiated human primary muscle cells using rat embryo spinal cord explants. This article describes how co-cultures of spinal cord explants and muscle cells are established.
A flexible and efficient method for the characterization of cell type-specific protein localization and nucleocytoplasmic shuttling is described. This heterokaryon approach uses fluorescently-labeled fusion proteins to image protein localizations after cell fusion. The protocol is amenable to steady-state localizations or more dynamic determinations based on live cell imaging.
We present an in vitro, two-color fluorescence assay to visualize the fusion of single virus particles with a fluid target bilayer. By labeling viral particles with fluorophores that differentially stain the viral membrane and its interior, we are able to monitor the kinetics of hemifusion and pore formation.
The surgical procedure for delivery of embryonic stem cell-derived endothelial cells to the ischemic hindlimb is demonstrated, with non-invasive tracking by bioluminescence imaging.
A novel and highly efficient two-step affinity chromatography protocol has been developed and is described in detail. The method is based on a small purification tag with two inherent affinities and is applicable to a wide range of target proteins with different properties.
Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy
A method of observing individual DNA molecules in live cells is described. The technique is based on the binding of a fluorescently tagged lac repressor protein to binding sites engineered into the DNA of interest. This method can be adapted to follow many recombinant DNAs in live cells over time.
In this video we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method using electroporation and the subsequent detection of fused cells visualization with fluorescence microscopy.
This video demonstrates the technique of anterior cervical discectomy and fusion in the ovine model.
Protein transduction enables the direct delivery of biologically active proteins into cells. In contrast to conventional methods such as DNA transfection or viral transduction this non-invasive paradigm allows highly efficient cellular manipulation in a titratable manner circumventing cellular toxicity and the risk of oncogenic transformation by permanent genetic modification.
Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System
We have developed a technique to test protein-protein interactions in plant. A yellow fluorescent protein (YFP) is split into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest via Gateway system, enabling expression of fusion proteins. Reconstitution of YFP signal only occurs when the inquest proteins interact.
1Department of Pathology, Case Western Reserve University School of Medicine, 2Cell Biology Program, Case Western Reserve University School of Medicine, 3Case Comprehensive Cancer Center, Case Western Reserve University School of Medicine
To purify zygotes of S. cerevisiae, haploid cells of opposite mating type were engineered to express red or green fluorescent proteins, co-incubated to allow zygote formation, and fractionated using a flow cytometry-based protocol. The highly-enriched fraction enables subsequent "-omic" studies, recovery of initial progeny, and systematic investigation of zygote morphogenesis.
This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.
Photobleaching Assays (FRAP & FLIP) to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells
We describe photobleaching methods including Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) to monitor chromatin protein dynamics in embryonic stem (ES) cells. Chromatin protein dynamics, which is considered to be one of the means to study chromatin plasticity, is enhanced in pluripotent cells.
Elastomeric PGS scaffolds with vascular smooth muscle cells cultured in a pulsatile flow bioreactor may lead to promising small-diameter arterial constructs with native ECM production in a relatively short culture period.
SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again.
With the growing interest in stem cell therapies, molecular imaging techniques are ideal for monitoring stem cell behavior after transplantation. Luciferase reporter genes have enabled non-invasive, repetitive assessment of cell survival, location, and proliferation in vivo. This video will demonstrate how to track hESC proliferation in a living mouse.
Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons
FRAP has been used to quantify the mobility of Green Fluorescence Protein (GFP)-tagged proteins in cultured cells. We examined the mobile/immobile fractions of the GFP by analyzing the fluorescence recovery percentage after photobleaching. In this study, FRAP was performed at spines of hippocampal neurons.
This protocol utilizes a pull down assay to determine the levels of active RhoC GTPase within cells.
The limiting factor in the use of the adult Drosophila eye to study neurodegeneration and cell biology is the difficult imaging of intracellular processes. We describe the dissection of single ommatidia to generate a bona-fide primary neuronal cell culture, which can be subject to drug treatment and advanced imaging.
The method presented here comprises the precise injury of live zebrafish embryos with high-energy laser pulses and the subsequent analysis of these injuries and their recovery with time. We also show how genetically labeled single or groups of skeletal muscle cells can be tracked during and after laser light induced damage.
A method for large-scale purification of the APP intracellular domain (AICD) is described. We also describe methodology to induce in vitro AICD aggregation and visualization by atomic force microscopy. The methods described are useful for biochemical/structural characterization of the AICD and the effects of molecular chaperones on its aggregation.
In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae
Clathrin-mediated endocytosis depends on adaptor proteins that coordinate cargo selection and clathrin coat assembly. Here we describe procedures to study adaptor-clathrin physical interaction and live cell imaging approaches using as a model the yeast endocytic adaptor protein Sla1p.
Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin
We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable.
Microbial biofilms are generally constituted by distinct subpopulations of specialized cells. Single-cell analysis of these subpopulations requires the use of fluorescent reporters. Here we describe a protocol to visualize and monitor several subpopulationswithin B. subtilis biofilms using fluorescence microscopy and flow cytometry.
Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae
Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.
Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays
1Centre for Vaccine Evaluation, Biologics and Genetic Therapies Directorate, HPFB, Health canada, 2National Institute for the Control of Pharmaceutical and Biological Products, The State Food and Drug Administration, Beijing, 3Department of Biochemistry, Microbiology and Immunology, University of Ottawa, 4Microbiology Department, Faculty of Medicine, King Abdulaziz University, 5National Microbiology Laboratory, Public Health Agency of Canada
A simple slot blot method was developed for the quantification of influenza viral hemagglutinin and neuraminidase using universal antibodies targeting their most conserved sequences identified through bioinformatics analyses. This innovative approach may provide a useful alternative to quantitative determination of all viral hemagglutinin and neuraminidase.
Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)
1Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, 2Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 3Department of Physics, Jilin University
We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the λ repressor CI 1.
1Department of Laboratory Medicine & Pathobiology, University of Toronto, 2Division of Urology, Sunnybrook Health Sciences Centre, Toronto, Canada, 3Department of Anatomic Pathology, Sunnybrook Health Sciences Centre, Toronto, Canada, 4Biological Sciences, Sunnybrook Research Institute
Quantitative Real Time polymerase chain reaction (qPCR) is a rapid and sensitive method to investigate the expression levels of various microRNA (miRNA) molecules in tumor samples. Using this method expression of hundreds of different miRNA molecules can be amplified, quantified, and analyzed from the same cDNA template.
Adult cardiac myocytes are primary cells that can be isolated from animal hearts and cultured for several days. Within this culture period adenoviral gene transfer can be used to express genetically encoded biosensors (GEBs) or fluorescent fusion proteins. Both approaches allow cellular investigations by means of confocal microscopy.
Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.
A detailed protocol is described for imaging the real time formation of DNA repair complexes in Bacillus subtilis cells.
Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein
A method to monitor ubiquitin-proteasome activity in living cells is described. A degron-destabilized GFP- (GFP-dgn) and a stable GFP-dgnFS fusion protein are generated and transduced into the cell using a lentiviral expression vector. This technique allows to generate a stable GFP-dgn/GFP-dgnFS expressing cell line in which ubiquitin-proteasome activity can be easily assessed using epifluorescence or flow cytometry.
In situ subcellular fractionation of mammalian cells on microscope coverslips allows the visualisation of protein localisation.
1Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, 2Department of Medicine, Division of Geriatric Medicine and Pittsburgh Institute for Neurodegenerative Diseases, University of Pittsburgh
The ability to produce transgenes for Caenorhabditis elegans using genomic DNA carried by fosmids is particularly attractive as all of the native regulatory elements are retained. Described is a simple and robust procedure for the production of transgenes via recombineering with the galK selectable marker.
The technical aspects of performing LIPS (Luciferase Immunoprecipitation Systems) are described. The overall approach involves expressing chimeric genes encoding antigens fused to Renilla luciferase (Ruc) in mammalian cells. Crude Ruc-antigen extracts are then prepared and, without purification, employed in immunoprecipitation assays to quantify antibodies.
A non-invasive means to evaluate the success of myoblast transplantation is described. The method takes advantage of a unified fusion reporter gene composed of genes whose expression can be imaged with different imaging modalities. Here, we make use of a fluc reporter gene sequence to target cells via bioluminescence imaging.
We describe guidelines to perform a safe and efficient elective fiberoptic intubation in pediatric patients while maintaining spontaneous ventilation.
This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.
This protocol describes how resin embedded brain tissue can be prepared and imaged in the three dimensions in the focussed ion beam, scanning electron microscope.
Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals
1Section on Synaptic Pharmacology/Laboratory for Integrative Neuroscience, National Institutes of Health/National Institute on Alcohol Abuse and Alcoholism, 2Department of Electronics Engineering, Ewha Womans University, 3Section on Transmitter Signaling/Laboratory of Molecular Physiology, National Institutes of Health/National Institute on Alcohol Abuse and Alcoholism
This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.
Stem Cell Transplantation Strategies for the Restoration of Cognitive Dysfunction Caused by Cranial Radiotherapy
Brain tumor patients routinely undergo cranial radiotherapy, and while beneficial, this treatment often results in debilitating cognitive dysfunction. This serious unresolved problem has at present, no clinical recourse, and has driven our efforts to devise stem cell based therapies for the recovery of radiation-induced cognitive decrements.
Preparation of Cell-lines for Conditional Knockdown of Gene Expression and Measurement of the Knockdown Effects on E4orf4-Induced Cell Death
Contribution of the ACF chromatin remodeling factor to E4orf4-induced cell death was measured. The protocol includes selection of cell clones in which doxycycline treatment induces conditional knockdown of the ACF subunits Acf1 and SNF2h, and use of the DAPI assay to measure E4orf4-induced cell death in the inducible cell lines.