Ebola viruses cause severe hemorrhagic fevers in humans and non-human primates, with case fatality rates as high as 90%. There are no approved vaccines or specific treatments for the disease caused by these viruses, and work with infectious Ebola viruses is restricted to biosafety level 4 laboratories, significantly limiting the research on these viruses. Lifecycle modeling systems model the virus lifecycle under biosafety level 2 conditions; however, until recently such systems have been limited to either individual aspects of the virus lifecycle, or a single infectious cycle. Tetracistronic minigenomes, which consist of Ebola virus non-coding regions, a reporter gene, and three Ebola virus genes involved in morphogenesis, budding, and entry (VP40, GP1,2, and VP24), can be used to produce replication and transcription-competent virus-like particles (trVLPs) containing these minigenomes. These trVLPs can continuously infect cells expressing the Ebola virus proteins responsible for genome replication and transcription, allowing us to safely model multiple infectious cycles under biosafety level 2 conditions. Importantly, the viral components of this systems are solely derived from Ebola virus and not from other viruses (as is, for example, the case in systems using pseudotyped viruses), and VP40, GP1,2 and VP24 are not overexpressed in this system, making it ideally suited for studying morphogenesis, budding and entry, although other aspects of the virus lifecycle such as genome replication and transcription can also be modeled with this system. Therefore, the tetracistronic trVLP assay represents the most comprehensive lifecycle modeling system available for Ebola viruses, and has tremendous potential for use in investigating the biology of Ebola viruses in future. Here, we provide detailed information on the use of this system, as well as on expected results.
28 Related JoVE Articles!
In Vivo Modeling of the Morbid Human Genome using Danio rerio
Institutions: Duke University Medical Center, Duke University, Duke University Medical Center.
Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo
complementation in zebrafish. Zebrafish (Danio rerio
) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo,
and can be genetically manipulated.1
These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.
Molecular Biology, Issue 78, Genetics, Biomedical Engineering, Medicine, Developmental Biology, Biochemistry, Anatomy, Physiology, Bioengineering, Genomics, Medical, zebrafish, in vivo, morpholino, human disease modeling, transcription, PCR, mRNA, DNA, Danio rerio, animal model
Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules
Institutions: Princeton University.
The aim of de novo
protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific properties, such as binding affinity, agonist or antagonist behavior, or stability, relative to the native sequence. Protein design lies at the center of current advances drug design and discovery. Not only does protein design provide predictions for potentially useful drug targets, but it also enhances our understanding of the protein folding process and protein-protein interactions. Experimental methods such as directed evolution have shown success in protein design. However, such methods are restricted by the limited sequence space that can be searched tractably. In contrast, computational design strategies allow for the screening of a much larger set of sequences covering a wide variety of properties and functionality. We have developed a range of computational de novo
protein design methods capable of tackling several important areas of protein design. These include the design of monomeric proteins for increased stability and complexes for increased binding affinity.
To disseminate these methods for broader use we present Protein WISDOM (http://www.proteinwisdom.org), a tool that provides automated methods for a variety of protein design problems. Structural templates are submitted to initialize the design process. The first stage of design is an optimization sequence selection stage that aims at improving stability through minimization of potential energy in the sequence space. Selected sequences are then run through a fold specificity stage and a binding affinity stage. A rank-ordered list of the sequences for each step of the process, along with relevant designed structures, provides the user with a comprehensive quantitative assessment of the design. Here we provide the details of each design method, as well as several notable experimental successes attained through the use of the methods.
Genetics, Issue 77, Molecular Biology, Bioengineering, Biochemistry, Biomedical Engineering, Chemical Engineering, Computational Biology, Genomics, Proteomics, Protein, Protein Binding, Computational Biology, Drug Design, optimization (mathematics), Amino Acids, Peptides, and Proteins, De novo protein and peptide design, Drug design, In silico sequence selection, Optimization, Fold specificity, Binding affinity, sequencing
Test Samples for Optimizing STORM Super-Resolution Microscopy
Institutions: National Physical Laboratory.
STORM is a recently developed super-resolution microscopy technique with up to 10 times better resolution than standard fluorescence microscopy techniques. However, as the image is acquired in a very different way than normal, by building up an image molecule-by-molecule, there are some significant challenges for users in trying to optimize their image acquisition. In order to aid this process and gain more insight into how STORM works we present the preparation of 3 test samples and the methodology of acquiring and processing STORM super-resolution images with typical resolutions of between 30-50 nm. By combining the test samples with the use of the freely available rainSTORM processing software it is possible to obtain a great deal of information about image quality and resolution. Using these metrics it is then possible to optimize the imaging procedure from the optics, to sample preparation, dye choice, buffer conditions, and image acquisition settings. We also show examples of some common problems that result in poor image quality, such as lateral drift, where the sample moves during image acquisition and density related problems resulting in the 'mislocalization' phenomenon.
Molecular Biology, Issue 79, Genetics, Bioengineering, Biomedical Engineering, Biophysics, Basic Protocols, HeLa Cells, Actin Cytoskeleton, Coated Vesicles, Receptor, Epidermal Growth Factor, Actins, Fluorescence, Endocytosis, Microscopy, STORM, super-resolution microscopy, nanoscopy, cell biology, fluorescence microscopy, test samples, resolution, actin filaments, fiducial markers, epidermal growth factor, cell, imaging
Robust 3D DNA FISH Using Directly Labeled Probes
Institutions: The Babraham Institute, The Babraham Institute, University of Cambridge .
3D DNA FISH has become a major tool for analyzing three-dimensional organization of the nucleus, and several variations of the technique have been published. In this article we describe a protocol which has been optimized for robustness, reproducibility, and ease of use. Brightly fluorescent directly labeled probes are generated by nick-translation with amino-allyldUTP followed by chemical coupling of the dye. 3D DNA FISH is performed using a freeze-thaw step for cell permeabilization and a heating step for simultaneous denaturation of probe and nuclear DNA. The protocol is applicable to a range of cell types and a variety of probes (BACs, plasmids, fosmids, or Whole Chromosome Paints) and allows for high-throughput automated imaging. With this method we routinely investigate nuclear localization of up to three chromosomal regions.
Genetics, Issue 78, Molecular Biology, Biochemistry, Cellular Biology, Genomics, Epigenetics, Cell Nucleus, Fluorescence, In Situ Hybridization, FISH, 3D DNA FISH, fluorescence in situ hybridization, nuclear structure, fluorescently labeled probes, visualization, imaging, DNA, chromosomes, sequencing, probes, assay
Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy
Institutions: University of Maine.
Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.
Basic Protocol, Issue 82, Microscopy, Super-resolution imaging, Multicolor, single molecule, FPALM, Localization microscopy, fluorescent proteins
Helical Organization of Blood Coagulation Factor VIII on Lipid Nanotubes
Institutions: University of Texas Medical Branch, University of Texas Medical Branch, University of Texas Medical Branch.
Cryo-electron microscopy (Cryo-EM)1
is a powerful approach to investigate the functional structure of proteins and complexes in a hydrated state and membrane environment2
Coagulation Factor VIII (FVIII)3
is a multi-domain blood plasma glycoprotein. Defect or deficiency of FVIII is the cause for Hemophilia type A - a severe bleeding disorder. Upon proteolytic activation, FVIII binds to the serine protease Factor IXa on the negatively charged platelet membrane, which is critical for normal blood clotting4
. Despite the pivotal role FVIII plays in coagulation, structural information for its membrane-bound state is incomplete5
. Recombinant FVIII concentrate is the most effective drug against Hemophilia type A and commercially available FVIII can be expressed as human or porcine, both forming functional complexes with human Factor IXa6,7
In this study we present a combination of Cryo-electron microscopy (Cryo-EM), lipid nanotechnology and structure analysis applied to resolve the membrane-bound structure of two highly homologous FVIII forms: human and porcine. The methodology developed in our laboratory to helically organize the two functional recombinant FVIII forms on negatively charged lipid nanotubes (LNT) is described. The representative results demonstrate that our approach is sufficiently sensitive to define the differences in the helical organization between the two highly homologous in sequence (86% sequence identity) proteins. Detailed protocols for the helical organization, Cryo-EM and electron tomography (ET) data acquisition are given. The two-dimensional (2D) and three-dimensional (3D) structure analysis applied to obtain the 3D reconstructions of human and porcine FVIII-LNT is discussed. The presented human and porcine FVIII-LNT structures show the potential of the proposed methodology to calculate the functional, membrane-bound organization of blood coagulation Factor VIII at high resolution.
Bioengineering, Issue 88, Cryo-electron microscopy, Lipid nanotubes, Helical assembly, Membrane-bound organization, Coagulation factor VIII
In Situ SIMS and IR Spectroscopy of Well-defined Surfaces Prepared by Soft Landing of Mass-selected Ions
Institutions: Pacific Northwest National Laboratory.
Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of materials that are inaccessible using conventional synthesis techniques. Coupling soft landing with in situ
characterization using secondary ion mass spectrometry (SIMS) and infrared reflection absorption spectroscopy (IRRAS) enables analysis of well-defined surfaces under clean vacuum conditions. The capabilities of three soft-landing instruments constructed in our laboratory are illustrated for the representative system of surface-bound organometallics prepared by soft landing of mass-selected ruthenium tris(bipyridine) dications, [Ru(bpy)3
(bpy = bipyridine), onto carboxylic acid terminated self-assembled monolayer surfaces on gold (COOH-SAMs). In situ
time-of-flight (TOF)-SIMS provides insight into the reactivity of the soft-landed ions. In addition, the kinetics of charge reduction, neutralization and desorption occurring on the COOH-SAM both during and after ion soft landing are studied using in situ
Fourier transform ion cyclotron resonance (FT-ICR)-SIMS measurements. In situ
IRRAS experiments provide insight into how the structure of organic ligands surrounding metal centers is perturbed through immobilization of organometallic ions on COOH-SAM surfaces by soft landing. Collectively, the three instruments provide complementary information about the chemical composition, reactivity and structure of well-defined species supported on surfaces.
Chemistry, Issue 88, soft landing, mass selected ions, electrospray, secondary ion mass spectrometry, infrared spectroscopy, organometallic, catalysis
Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy (f3D-SIM)
Institutions: University of Technology, Sydney.
Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the diffraction of light allowing super-resolution of live samples. There are currently three main types of super-resolution techniques – stimulated emission depletion (STED), single-molecule localization microscopy (including techniques such as PALM, STORM, and GDSIM), and structured illumination microscopy (SIM). While STED and single-molecule localization techniques show the largest increases in resolution, they have been slower to offer increased speeds of image acquisition. Three-dimensional SIM (3D-SIM) is a wide-field fluorescence microscopy technique that offers a number of advantages over both single-molecule localization and STED. Resolution is improved, with typical lateral and axial resolutions of 110 and 280 nm, respectively and depth of sampling of up to 30 µm from the coverslip, allowing for imaging of whole cells. Recent advancements (fast 3D-SIM) in the technology increasing the capture rate of raw images allows for fast capture of biological processes occurring in seconds, while significantly reducing photo-toxicity and photobleaching. Here we describe the use of one such method to image bacterial cells harboring the fluorescently-labelled cytokinetic FtsZ protein to show how cells are analyzed and the type of unique information that this technique can provide.
Molecular Biology, Issue 91, super-resolution microscopy, fluorescence microscopy, OMX, 3D-SIM, Blaze, cell division, bacteria, Bacillus subtilis, Staphylococcus aureus, FtsZ, Z ring constriction
Magnetic Tweezers for the Measurement of Twist and Torque
Institutions: Delft University of Technology.
Single-molecule techniques make it possible to investigate the behavior of individual biological molecules in solution in real time. These techniques include so-called force spectroscopy approaches such as atomic force microscopy, optical tweezers, flow stretching, and magnetic tweezers. Amongst these approaches, magnetic tweezers have distinguished themselves by their ability to apply torque while maintaining a constant stretching force. Here, it is illustrated how such a “conventional” magnetic tweezers experimental configuration can, through a straightforward modification of its field configuration to minimize the magnitude of the transverse field, be adapted to measure the degree of twist in a biological molecule. The resulting configuration is termed the freely-orbiting magnetic tweezers. Additionally, it is shown how further modification of the field configuration can yield a transverse field with a magnitude intermediate between that of the “conventional” magnetic tweezers and the freely-orbiting magnetic tweezers, which makes it possible to directly measure the torque stored in a biological molecule. This configuration is termed the magnetic torque tweezers. The accompanying video explains in detail how the conversion of conventional magnetic tweezers into freely-orbiting magnetic tweezers and magnetic torque tweezers can be accomplished, and demonstrates the use of these techniques. These adaptations maintain all the strengths of conventional magnetic tweezers while greatly expanding the versatility of this powerful instrument.
Bioengineering, Issue 87, magnetic tweezers, magnetic torque tweezers, freely-orbiting magnetic tweezers, twist, torque, DNA, single-molecule techniques
Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization
Institutions: University of Notre Dame.
The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model congenital abnormalities. During the first day of life, the zebrafish embryo progresses through many developmental stages including fertilization, cleavage, gastrulation, segmentation, and the organogenesis of structures such as the kidney, heart, and central nervous system. The anatomy of a young zebrafish embryo presents several challenges for the visualization and analysis of the tissues involved in many of these events because the embryo develops in association with a round yolk mass. Thus, for accurate analysis and imaging of experimental phenotypes in fixed embryonic specimens between the tailbud and 20 somite stage (10 and 19 hours post fertilization (hpf), respectively), such as those stained using whole mount in situ
hybridization (WISH), it is often desirable to remove the embryo from the yolk ball and to position it flat on a glass slide. However, performing a flat mount procedure can be tedious. Therefore, successful and efficient flat mount preparation is greatly facilitated through the visual demonstration of the dissection technique, and also helped by using reagents that assist in optimal tissue handling. Here, we provide our WISH protocol for one or two-color detection of gene expression in the zebrafish embryo, and demonstrate how the flat mounting procedure can be performed on this example of a stained fixed specimen. This flat mounting protocol is broadly applicable to the study of many embryonic structures that emerge during early zebrafish development, and can be implemented in conjunction with other staining methods performed on fixed embryo samples.
Developmental Biology, Issue 89, animals, vertebrates, fishes, zebrafish, growth and development, morphogenesis, embryonic and fetal development, organogenesis, natural science disciplines, embryo, whole mount in situ hybridization, flat mount, deyolking, imaging
From Voxels to Knowledge: A Practical Guide to the Segmentation of Complex Electron Microscopy 3D-Data
Institutions: Lawrence Berkeley National Laboratory, Lawrence Berkeley National Laboratory, Lawrence Berkeley National Laboratory.
Modern 3D electron microscopy approaches have recently allowed unprecedented insight into the 3D ultrastructural organization of cells and tissues, enabling the visualization of large macromolecular machines, such as adhesion complexes, as well as higher-order structures, such as the cytoskeleton and cellular organelles in their respective cell and tissue context. Given the inherent complexity of cellular volumes, it is essential to first extract the features of interest in order to allow visualization, quantification, and therefore comprehension of their 3D organization. Each data set is defined by distinct characteristics, e.g.
, signal-to-noise ratio, crispness (sharpness) of the data, heterogeneity of its features, crowdedness of features, presence or absence of characteristic shapes that allow for easy identification, and the percentage of the entire volume that a specific region of interest occupies. All these characteristics need to be considered when deciding on which approach to take for segmentation.
The six different 3D ultrastructural data sets presented were obtained by three different imaging approaches: resin embedded stained electron tomography, focused ion beam- and serial block face- scanning electron microscopy (FIB-SEM, SBF-SEM) of mildly stained and heavily stained samples, respectively. For these data sets, four different segmentation approaches have been applied: (1) fully manual model building followed solely by visualization of the model, (2) manual tracing segmentation of the data followed by surface rendering, (3) semi-automated approaches followed by surface rendering, or (4) automated custom-designed segmentation algorithms followed by surface rendering and quantitative analysis. Depending on the combination of data set characteristics, it was found that typically one of these four categorical approaches outperforms the others, but depending on the exact sequence of criteria, more than one approach may be successful. Based on these data, we propose a triage scheme that categorizes both objective data set characteristics and subjective personal criteria for the analysis of the different data sets.
Bioengineering, Issue 90, 3D electron microscopy, feature extraction, segmentation, image analysis, reconstruction, manual tracing, thresholding
Zinc-finger Nuclease Enhanced Gene Targeting in Human Embryonic Stem Cells
Institutions: Monash University.
One major limitation with current human embryonic stem cell (ESC) differentiation protocols is the generation of heterogeneous cell populations. These cultures contain the cells of interest, but are also contaminated with undifferentiated ESCs, non-neural derivatives and other neuronal subtypes. This limits their use in in vitro
and in vivo
applications, such as in vitro
modeling for drug discovery or cell replacement therapy. To help overcome this, reporter cell lines, which offer a means to visualize, track and isolate cells of interest, can be engineered. However, to achieve this in human embryonic stem cells via conventional homologous recombination is extremely inefficient. This protocol describes targeting of the Pituitary homeobox 3
) locus in human embryonic stem cells using custom designed zinc-finger nucleases, which introduce site-specific double-strand DNA breaks, together with a PITX3
-specific DNA donor vector. Following the generation of the PITX3
reporter cell line, it can then be differentiated using published protocols for use in studies such as in vitro
Parkinson’s disease modeling or cell replacement therapy.
Molecular Biology, Issue 90, Electroporation, human embryonic stem cell, genome editing, reporter cell line, midbrain dopaminergic neurons
Designing a Bio-responsive Robot from DNA Origami
Institutions: Bar-Ilan University.
Nucleic acids are astonishingly versatile. In addition to their natural role as storage medium for biological information1
, they can be utilized in parallel computing2,3
, recognize and bind molecular or cellular targets4,5
, catalyze chemical reactions6,7
, and generate calculated responses in a biological system8,9
. Importantly, nucleic acids can be programmed to self-assemble into 2D and 3D structures10-12
, enabling the integration of all these remarkable features in a single robot linking the sensing of biological cues to a preset response in order to exert a desired effect.
Creating shapes from nucleic acids was first proposed by Seeman13
, and several variations on this theme have since been realized using various techniques11,12,14,15
. However, the most significant is perhaps the one proposed by Rothemund, termed scaffolded DNA origami16
. In this technique, the folding of a long (>7,000 bases) single-stranded DNA 'scaffold'
is directed to a desired shape by hundreds of short complementary strands termed 'staples'
. Folding is carried out by temperature annealing ramp. This technique was successfully demonstrated in the creation of a diverse array of 2D shapes with remarkable precision and robustness. DNA origami was later extended to 3D as well17,18
The current paper will focus on the caDNAno 2.0 software19
developed by Douglas and colleagues. caDNAno is a robust, user-friendly CAD tool enabling the design of 2D and 3D DNA origami shapes with versatile features. The design process relies on a systematic and accurate abstraction scheme for DNA structures, making it relatively straightforward and efficient.
In this paper we demonstrate the design of a DNA origami nanorobot that has been recently described20
. This robot is 'robotic' in the sense that it links sensing to actuation, in order to perform a task. We explain how various sensing schemes can be integrated into the structure, and how this can be relayed to a desired effect. Finally we use Cando21
to simulate the mechanical properties of the designed shape. The concept we discuss can be adapted to multiple tasks and settings.
Bioengineering, Issue 77, Genetics, Biomedical Engineering, Molecular Biology, Medicine, Genomics, Nanotechnology, Nanomedicine, DNA origami, nanorobot, caDNAno, DNA, DNA Origami, nucleic acids, DNA structures, CAD, sequencing
Live Cell Cycle Analysis of Drosophila Tissues using the Attune Acoustic Focusing Cytometer and Vybrant DyeCycle Violet DNA Stain
Institutions: University of Michigan .
Flow cytometry has been widely used to obtain information about DNA content in a population of cells, to infer relative percentages in different cell cycle phases. This technique has been successfully extended to the mitotic tissues of the model organism Drosophila melanogaster
for genetic studies of cell cycle regulation in vivo
. When coupled with cell-type specific fluorescent protein expression and genetic manipulations, one can obtain detailed information about effects on cell number, cell size and cell cycle phasing in vivo
. However this live-cell method has relied on the use of the cell permeable Hoechst 33342 DNA-intercalating dye, limiting users to flow cytometers equipped with a UV laser. We have modified this protocol to use a newer live-cell DNA dye, Vybrant DyeCycle Violet, compatible with the more common violet 405nm laser. The protocol presented here allows for efficient cell cycle analysis coupled with cell type, relative cell size and cell number information, in a variety of Drosophila
tissues. This protocol extends the useful cell cycle analysis technique for live Drosophila
tissues to a small benchtop analyzer, the Attune Acoustic Focusing Cytometer, which can be run and maintained on a single-lab scale.
Molecular Biology, Issue 75, Cellular Biology, Developmental Biology, Anatomy, Physiology, Genetics, Flow Cytometry, Cell Cycle, DNA Replication, Metamorphosis, Biological, drosophila, Gal4/UAS, insect metamorphosis, animal model
Metabolic Labeling of Newly Transcribed RNA for High Resolution Gene Expression Profiling of RNA Synthesis, Processing and Decay in Cell Culture
Institutions: Max von Pettenkofer Institute, University of Cambridge, Ludwig-Maximilians-University Munich.
The development of whole-transcriptome microarrays and next-generation sequencing has revolutionized our understanding of the complexity of cellular gene expression. Along with a better understanding of the involved molecular mechanisms, precise measurements of the underlying kinetics have become increasingly important. Here, these powerful methodologies face major limitations due to intrinsic properties of the template samples they study, i.e.
total cellular RNA. In many cases changes in total cellular RNA occur either too slowly or too quickly to represent the underlying molecular events and their kinetics with sufficient resolution. In addition, the contribution of alterations in RNA synthesis, processing, and decay are not readily differentiated.
We recently developed high-resolution gene expression profiling to overcome these limitations. Our approach is based on metabolic labeling of newly transcribed RNA with 4-thiouridine (thus also referred to as 4sU-tagging) followed by rigorous purification of newly transcribed RNA using thiol-specific biotinylation and streptavidin-coated magnetic beads. It is applicable to a broad range of organisms including vertebrates, Drosophila
, and yeast. We successfully applied 4sU-tagging to study real-time kinetics of transcription factor activities, provide precise measurements of RNA half-lives, and obtain novel insights into the kinetics of RNA processing. Finally, computational modeling can be employed to generate an integrated, comprehensive analysis of the underlying molecular mechanisms.
Genetics, Issue 78, Cellular Biology, Molecular Biology, Microbiology, Biochemistry, Eukaryota, Investigative Techniques, Biological Phenomena, Gene expression profiling, RNA synthesis, RNA processing, RNA decay, 4-thiouridine, 4sU-tagging, microarray analysis, RNA-seq, RNA, DNA, PCR, sequencing
Visualizing Single-molecule DNA Replication with Fluorescence Microscopy
Institutions: Harvard Medical School.
We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.
Cellular Biology, Issue 32, single-molecule, fluorescence, DNA replication, biophysics
Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis
Institutions: University of Michigan-Ann Arbor.
Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution of existing proteins, and the assembly of new protein complexes can be stimulated by a variety of DNA lesions and mismatched DNA base pairs. Fluorescence microscopy has been used as a powerful experimental tool for visualizing and quantifying these and other responses to DNA lesions and to monitor DNA replication status within the complex subcellular architecture of a living cell. Translational fusions between fluorescent reporter proteins and components of the DNA replication and repair machinery have been used to determine the cues that target DNA repair proteins to their cognate lesions in vivo
and to understand how these proteins are organized within bacterial cells. In addition, transcriptional and translational fusions linked to DNA damage inducible promoters have revealed which cells within a population have activated genotoxic stress responses. In this review, we provide a detailed protocol for using fluorescence microscopy to image the assembly of DNA repair and DNA replication complexes in single bacterial cells. In particular, this work focuses on imaging mismatch repair proteins, homologous recombination, DNA replication and an SOS-inducible protein in Bacillus subtilis
. All of the procedures described here are easily amenable for imaging protein complexes in a variety of bacterial species.
Microbiology, Issue 36, mismatch repair, DNA repair, microscopy, DNA replication, Bacillus subtilis, GFP, SOS, FM4-64, fluorescence microscopy
Direct Restart of a Replication Fork Stalled by a Head-On RNA Polymerase
Institutions: Rockefeller University.
studies suggest that replication forks are arrested due to encounters with head-on transcription complexes. Yet, the fate of the replisome and RNA polymerase (RNAP) following a head-on collision is unknown. Here, we find that the E. coli
replisome stalls upon collision with a head-on transcription complex, but instead of collapsing, the replication fork remains highly stable and eventually resumes elongation after displacing the RNAP from DNA. We also find that the transcription-repair coupling factor, Mfd, promotes direct restart of the fork following the collision by facilitating displacement of the RNAP. These findings demonstrate the intrinsic stability of the replication apparatus and a novel role for the transcription-coupled repair pathway in promoting replication past a RNAP block.
Cellular Biology, Issue 38, replication, transcription, transcription-coupled repair, replisome, RNA polymerase, collision
In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy
Institutions: University of Wisconsin - Milwaukee, University of Wisconsin - Milwaukee.
The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial applications as well as increases basic fundamental biological knowledge. Förster (Fluorescence) Resonance Energy Transfer (FRET) between a donor molecule in an electronically excited state and a nearby acceptor molecule has been frequently utilized for studies of protein-protein interactions in living cells. The proteins of interest are tagged with two different types of fluorescent probes and expressed in biological cells. The fluorescent probes are then excited, typically using laser light, and the spectral properties of the fluorescence emission emanating from the fluorescent probes is collected and analyzed. Information regarding the degree of the protein interactions is embedded in the spectral emission data. Typically, the cell must be scanned a number of times in order to accumulate enough spectral information to accurately quantify the extent of the protein interactions for each region of interest within the cell. However, the molecular composition of these regions may change during the course of the acquisition process, limiting the spatial determination of the quantitative values of the apparent FRET efficiencies to an average over entire cells. By means of a spectrally resolved two-photon microscope, we are able to obtain a full set of spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level spectral data, a map of FRET efficiencies throughout the cell is calculated. By applying a simple theory of FRET in oligomeric complexes to the experimentally obtained distribution of FRET efficiencies throughout the cell, a single spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. Here we describe the procedure of preparing biological cells (the yeast Saccharomyces cerevisiae
) expressing membrane receptors (sterile 2 α-factor receptors) tagged with two different types of fluorescent probes. Furthermore, we illustrate critical factors involved in collecting fluorescence data using the spectrally resolved two-photon microscopy imaging system. The use of this protocol may be extended to study any type of protein which can be expressed in a living cell with a fluorescent marker attached to it.
Cellular Biology, Issue 47, Forster (Fluorescence) Resonance Energy Transfer (FRET), protein-protein interactions, protein complex, in vivo determinations, spectral resolution, two-photon microscopy, G protein-coupled receptor (GPCR), sterile 2 alpha-factor protein (Ste2p)
A Protocol for Computer-Based Protein Structure and Function Prediction
Institutions: University of Michigan , University of Kansas.
Genome sequencing projects have ciphered millions of protein sequence, which require knowledge of their structure and function to improve the understanding of their biological role. Although experimental methods can provide detailed information for a small fraction of these proteins, computational modeling is needed for the majority of protein molecules which are experimentally uncharacterized. The I-TASSER server is an on-line workbench for high-resolution modeling of protein structure and function. Given a protein sequence, a typical output from the I-TASSER server includes secondary structure prediction, predicted solvent accessibility of each residue, homologous template proteins detected by threading and structure alignments, up to five full-length tertiary structural models, and structure-based functional annotations for enzyme classification, Gene Ontology terms and protein-ligand binding sites. All the predictions are tagged with a confidence score which tells how accurate the predictions are without knowing the experimental data. To facilitate the special requests of end users, the server provides channels to accept user-specified inter-residue distance and contact maps to interactively change the I-TASSER modeling; it also allows users to specify any proteins as template, or to exclude any template proteins during the structure assembly simulations. The structural information could be collected by the users based on experimental evidences or biological insights with the purpose of improving the quality of I-TASSER predictions. The server was evaluated as the best programs for protein structure and function predictions in the recent community-wide CASP experiments. There are currently >20,000 registered scientists from over 100 countries who are using the on-line I-TASSER server.
Biochemistry, Issue 57, On-line server, I-TASSER, protein structure prediction, function prediction
MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression
Institutions: Wayne State University , Wayne State University .
We have developed 3D coculture models, which we term MAME (m
rchitecture and m
ngineering), and used them for live-cell imaging in real-time of cell:cell interactions. Our overall goal was to develop models that recapitulate the architecture of preinvasive breast lesions to study their progression to an invasive phenotype. Specifically, we developed models to analyze interactions among pre-malignant breast epithelial cell variants and other cell types of the tumor microenvironment that have been implicated in enhancing or reducing the progression of preinvasive breast epithelial cells to invasive ductal carcinomas. Other cell types studied to date are myoepithelial cells, fibroblasts, macrophages and blood and lymphatic microvascular endothelial cells. In addition to the MAME models, which are designed to recapitulate the cellular interactions within the breast during cancer progression, we have developed comparable models for the progression of prostate cancers.
Here we illustrate the procedures for establishing the 3D cocultures along with the use of live-cell imaging and a functional proteolysis assay to follow the transition of cocultures of breast ductal carcinoma in situ
(DCIS) cells and fibroblasts to an invasive phenotype over time, in this case over twenty-three days in culture. The MAME cocultures consist of multiple layers. Fibroblasts are embedded in the bottom layer of type I collagen. On that is placed a layer of reconstituted basement membrane (rBM) on which DCIS cells are seeded. A final top layer of 2% rBM is included and replenished with every change of media. To image proteolysis associated with the progression to an invasive phenotype, we use dye-quenched (DQ) fluorescent matrix proteins (DQ-collagen I mixed with the layer of collagen I and DQ-collagen IV mixed with the middle layer of rBM) and observe live cultures using confocal microscopy. Optical sections are captured, processed and reconstructed in 3D with Volocity visualization software. Over the course of 23 days in MAME cocultures, the DCIS cells proliferate and coalesce into large invasive structures. Fibroblasts migrate and become incorporated into these invasive structures. Fluorescent proteolytic fragments of the collagens are found in association with the surface of DCIS structures, intracellularly, and also dispersed throughout the surrounding matrix. Drugs that target proteolytic, chemokine/cytokine and kinase pathways or modifications in the cellular composition of the cocultures can reduce the invasiveness, suggesting that MAME models can be used as preclinical screens for novel therapeutic approaches.
Medicine, Issue 60, Immunology, Breast, cancer, extracellular matrix, invasion, proteolysis, tumor microenvironment
Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies
Institutions: University of California, Los Angeles .
In the biological sciences there have been technological advances that catapult the discipline into golden ages of discovery. For example, the field of microbiology was transformed with the advent of Anton van Leeuwenhoek's microscope, which allowed scientists to visualize prokaryotes for the first time. The development of the polymerase chain reaction (PCR) is one of those innovations that changed the course of molecular science with its impact spanning countless subdisciplines in biology. The theoretical process was outlined by Keppe and coworkers in 1971; however, it was another 14 years until the complete PCR procedure was described and experimentally applied by Kary Mullis while at Cetus Corporation in 1985. Automation and refinement of this technique progressed with the introduction of a thermal stable DNA polymerase from the bacterium Thermus aquaticus
, consequently the name Taq
PCR is a powerful amplification technique that can generate an ample supply of a specific segment of DNA (i.e., an amplicon) from only a small amount of starting material (i.e., DNA template or target sequence). While straightforward and generally trouble-free, there are pitfalls that complicate the reaction producing spurious results. When PCR fails it can lead to many non-specific DNA products of varying sizes that appear as a ladder or smear of bands on agarose gels. Sometimes no products form at all. Another potential problem occurs when mutations are unintentionally introduced in the amplicons, resulting in a heterogeneous population of PCR products. PCR failures can become frustrating unless patience and careful troubleshooting are employed to sort out and solve the problem(s). This protocol outlines the basic principles of PCR, provides a methodology that will result in amplification of most target sequences, and presents strategies for optimizing a reaction. By following this PCR guide, students should be able to:
● Set up reactions and thermal cycling conditions for a conventional PCR experiment
● Understand the function of various reaction components and their overall effect on a PCR experiment
● Design and optimize a PCR experiment for any DNA template
● Troubleshoot failed PCR experiments
Basic Protocols, Issue 63, PCR, optimization, primer design, melting temperature, Tm, troubleshooting, additives, enhancers, template DNA quantification, thermal cycler, molecular biology, genetics
Super-resolution Imaging of the Bacterial Division Machinery
Institutions: The Johns Hopkins University School of Medicine.
Bacterial cell division requires the coordinated assembly of more than ten essential proteins at midcell1,2
. Central to this process is the formation of a ring-like suprastructure (Z-ring) by the FtsZ protein at the division plan3,4
. The Z-ring consists of multiple single-stranded FtsZ protofilaments, and understanding the arrangement of the protofilaments inside the Z-ring will provide insight into the mechanism of Z-ring assembly and its function as a force generator5,6
. This information has remained elusive due to current limitations in conventional fluorescence microscopy and electron microscopy. Conventional fluorescence microscopy is unable to provide a high-resolution image of the Z-ring due to the diffraction limit of light (~200 nm). Electron cryotomographic imaging has detected scattered FtsZ protofilaments in small C. crescentus
, but is difficult to apply to larger cells such as E. coli
or B. subtilis
. Here we describe the application of a super-resolution fluorescence microscopy method, Photoactivated Localization Microscopy (PALM), to quantitatively characterize the structural organization of the E. coli
PALM imaging offers both high spatial resolution (~35 nm) and specific labeling to enable unambiguous identification of target proteins. We labeled FtsZ with the photoactivatable fluorescent protein mEos2, which switches from green fluorescence (excitation = 488 nm) to red fluorescence (excitation = 561 nm) upon activation at 405 nm9
. During a PALM experiment, single FtsZ-mEos2 molecules are stochastically activated and the corresponding centroid positions of the single molecules are determined with <20 nm precision. A super-resolution image of the Z-ring is then reconstructed by superimposing the centroid positions of all detected FtsZ-mEos2 molecules.
Using this method, we found that the Z-ring has a fixed width of ~100 nm and is composed of a loose bundle of FtsZ protofilaments that overlap with each other in three dimensions. These data provide a springboard for further investigations of the cell cycle dependent changes of the Z-ring10
and can be applied to other proteins of interest.
Biophysics, Issue 71, Cellular Biology, Microbiology, Molecular Biology, Structural Biology, Chemistry, Physics, super-resolution imaging, PALM, FtsZ, mEos2, cell division, cytokinesis, divisome
Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica
Institutions: Case Western Reserve University , Case Western Reserve University , Case Western Reserve University .
In animals with large identified neurons (e.g.
mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4
. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5
. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.
This extracellular technique has advantages. First, extracellular electrodes can stimulate and record neurons through the sheath5
, so it does not need to be removed. Thus, neurons will be healthier in extracellular experiments than in intracellular ones. Second, if ganglia are rotated by appropriate pinning of the sheath, extracellular electrodes can access neurons on both sides of the ganglion, which makes it easier and more efficient to identify multiple neurons in the same preparation. Third, extracellular electrodes do not need to penetrate cells, and thus can be easily moved back and forth among neurons, causing less damage to them. This is especially useful when one tries to record multiple neurons during repeating motor patterns that may only persist for minutes. Fourth, extracellular electrodes are more flexible than intracellular ones during muscle movements. Intracellular electrodes may pull out and damage neurons during muscle contractions. In contrast, since extracellular electrodes are gently pressed onto the sheath above neurons, they usually stay above the same neuron during muscle contractions, and thus can be used in more intact preparations.
To uniquely identify motor neurons for a motor pool (in particular, the I1/I3 muscle in Aplysia
) using extracellular electrodes, one can use features that do not require intracellular measurements as criteria: soma size and location, axonal projection, and muscle innervation4,6,7
. For the particular motor pool used to illustrate the technique, we recorded from buccal nerves 2 and 3 to measure axonal projections, and measured the contraction forces of the I1/I3 muscle to determine the pattern of muscle innervation for the individual motor neurons.
We demonstrate the complete process of first identifying motor neurons using muscle innervation, then characterizing their timing during motor patterns, creating a simplified diagnostic method for rapid identification. The simplified and more rapid diagnostic method is superior for more intact preparations, e.g.
in the suspended buccal mass preparation8
or in vivo9
. This process can also be applied in other motor pools10,11,12
or in other animal systems2,3,13,14
Neuroscience, Issue 73, Physiology, Biomedical Engineering, Anatomy, Behavior, Neurobiology, Animal, Neurosciences, Neurophysiology, Electrophysiology, Aplysia, Aplysia californica, California sea slug, invertebrate, feeding, buccal mass, ganglia, motor neurons, neurons, extracellular stimulation and recordings, extracellular electrodes, animal model
Concentration Determination of Nucleic Acids and Proteins Using the Micro-volume Bio-spec Nano Spectrophotometer
Institutions: Scientific Instruments.
Nucleic Acid quantitation procedures have advanced significantly in the last three decades. More and more, molecular biologists require consistent small-volume analysis of nucleic acid samples for their experiments. The BioSpec-nano provides a potential solution to the problems of inaccurate, non-reproducible results, inherent in current DNA quantitation methods, via specialized optics and a sensitive PDA detector. The BioSpec-nano also has automated functionality such that mounting, measurement, and cleaning are done by the instrument, thereby eliminating tedious, repetitive, and inconsistent placement of the fiber optic element and manual cleaning.
In this study, data is presented on the quantification of DNA and protein, as well as on measurement reproducibility and accuracy. Automated sample contact and rapid scanning allows measurement in three seconds, resulting in excellent throughput. Data analysis is carried out using the built-in features of the software. The formula used for calculating DNA concentration is:
Sample Concentration = DF · (OD260-OD320)· NACF (1)
Where DF = sample dilution factor and NACF = nucleic acid concentration factor.
The Nucleic Acid concentration factor is set in accordance with the analyte selected1
Protein concentration results can be expressed as μg/ mL or as moles/L by entering e280 and molecular weight values respectively. When residue values for Tyr, Trp and Cysteine (S-S bond) are entered in the e280Calc tab, the extinction coefficient values are calculated as e280 = 5500 x (Trp residues) + 1490 x (Tyr residues) + 125 x (cysteine S-S bond). The e280 value is used by the software for concentration calculation.
In addition to concentration determination of nucleic acids and protein, the BioSpec-nano can be used as an ultra micro-volume spectrophotometer for many other analytes or as a standard spectrophotometer using 5 mm pathlength cells.
Molecular Biology, Issue 48, Nucleic acid quantitation, protein quantitation, micro-volume analysis, label quantitation
Principles of Site-Specific Recombinase (SSR) Technology
Institutions: Max Plank Institute for Molecular Cell Biology and Genetics, Dresden.
Site-specific recombinase (SSR) technology allows the manipulation of gene structure to explore gene function and has become an integral tool of molecular biology. Site-specific recombinases are proteins that bind to distinct DNA target sequences. The Cre/lox system was first described in bacteriophages during the 1980's. Cre recombinase is a Type I topoisomerase that catalyzes site-specific recombination of DNA between two loxP (locus of X-over P1) sites. The Cre/lox system does not require any cofactors. LoxP sequences contain distinct binding sites for Cre recombinases that surround a directional core sequence where recombination and rearrangement takes place. When cells contain loxP sites and express the Cre recombinase, a recombination event occurs. Double-stranded DNA is cut at both loxP sites by the Cre recombinase, rearranged, and ligated ("scissors and glue"). Products of the recombination event depend on the relative orientation of the asymmetric sequences.
SSR technology is frequently used as a tool to explore gene function. Here the gene of interest is flanked with Cre target sites loxP ("floxed"). Animals are then crossed with animals expressing the Cre recombinase under the control of a tissue-specific promoter. In tissues that express the Cre recombinase it binds to target sequences and excises the floxed gene. Controlled gene deletion allows the investigation of gene function in specific tissues and at distinct time points. Analysis of gene function employing SSR technology --- conditional mutagenesis -- has significant advantages over traditional knock-outs where gene deletion is frequently lethal.
Cellular Biology, Issue 15, Molecular Biology, Site-Specific Recombinase, Cre recombinase, Cre/lox system, transgenic animals, transgenic technology
Laser Capture Microdissection of Mammalian Tissue
Institutions: University of California, Irvine (UCI).
Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or formalin-fixed, paraffin-embedded tissue sections. LMD techniques rely on a thermo labile membrane placed either on top of, or underneath, the tissue section. In one method, focused laser energy is used to melt the membrane onto the underlying cells, which can then be lifted out of the tissue section. In the other, the laser energy vaporizes the foil along a path "drawn" on the tissue, allowing the selected cells to fall into a collection device. Each technique allows the selection of cells with a minimum resolution of several microns. DNA, RNA, protein, and lipid samples may be isolated and analyzed from micro-dissected samples. In this video, we demonstrate the use of the Leica AS-LMD laser microdissection instrument in seven segments, including an introduction to the principles of LMD, initializing the instrument for use, general considerations for sample preparation, mounting the specimen and setting up capture tubes, aligning the microscope, adjusting the capture controls, and capturing tissue specimens. Laser-capture micro-dissection enables the investigator to isolate samples of pure cell populations as small as a few cell-equivalents. This allows the analysis of cells of interest that are free of neighboring contaminants, which may confound experimental results.
Issue 8, Basic Protocols, Laser Capture Microdissection, Microdissection Techniques, Leica
Purifying Plasmid DNA from Bacterial Colonies Using the Qiagen Miniprep Kit
Institutions: University of California, Irvine (UCI).
Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial culture is a quick way for clone verification or DNA isolation, followed by further enzymatic reactions (polymerase chain reaction and restriction enzyme digestion). Here, we video-recorded the general procedures of miniprep through the QIAGEN's QIAprep 8 Miniprep Kit, aiming to introducing this highly efficient technique to the general beginners for molecular biology techniques. The whole procedure is based on alkaline lysis of E. coli cells followed by adsorption of DNA onto silica in the presence of high salt. It consists of three steps: 1) preparation and clearing of a bacterial lysate, 2) adsorption of DNA onto the QIAprep membrane, 3) washing and elution of plasmid DNA. All steps are performed without the use of phenol, chloroform, CsCl, ethidium bromide, and without alcohol precipitation. It usually takes less than 2 hours to finish the entire procedure.
Issue 6, Basic Protocols, plasmid, DNA, purification, Qiagen