Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. Alterations of this program can lead to the growth of damaged cells, a hallmark of cancer development, or to premature cell death such as seen in neurodegenerative diseases. Much of what is known concerning the molecular basis for translational control has been obtained from polysome analysis using a density gradient fractionation system. This technique relies on ultracentrifugation of cytoplasmic extracts on a linear sucrose gradient. Once the spin is completed, the system allows fractionation and quantification of centrifuged zones corresponding to different translating ribosomes populations, thus resulting in a polysome profile. Changes in the polysome profile are indicative of changes or defects in translation initiation that occur in response to various types of stress. This technique also allows to assess the role of specific proteins on translation initiation, and to measure translational activity of specific mRNAs. Here we describe our protocol to perform polysome profiles in order to assess translation initiation of eukaryotic cells and tissues under either normal or stress growth conditions.
21 Related JoVE Articles!
Visualization of G3BP Stress Granules Dynamics in Live Primary Cells
Institutions: Institut de Génétique Moléculaire de Montpellier, CNRS UMR 5535.
SGs can be visualized in cells by immunostaining of specific protein components or polyA+ mRNAs. SGs are highly dynamic and the study of their assembly and fate is important to understand the cellular response to stress. The deficiency in key factors of SGs like G3BP (RasGAP SH3 domain Binding Protein) leads to developmental defects in mice and alterations of the Central Nervous System. To study the dynamics of SGs in cells from an organism, one can culture primary cells and follow the localization of a transfected tagged component of SGs. We describe time-lapse experiment to observe G3BP1-containing SGs in Mouse Embryonic Fibroblasts (MEFs). This technique can also be used to study G3BP-containing SGs in live neurons, which is crucial as it was recently shown that these SGs are formed at the onset of neurodegenerative diseases like Alzheimer's disease. This approach can be adapted to any other cellular body and granule protein component, and performed with transgenic animals, allowing the live study of granules dynamics for example in the absence of a specific factor of these granules.
Cellular Biology, Issue 87, Stress granule (SG), G3BP, primary cells, neurons
Bimolecular Fluorescence Complementation
Institutions: University of Illinois at Chicago.
Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex.
Conventional methods such as immunoprecipitation do not provide information on the spatial localization of complexes. In contrast, BiFC monitors the interaction and subcellular compartmentalization of protein complexes. In this method, a fluororescent protein is split into amino- and carboxy-terminal non-fluorescent fragments which are then fused to two proteins of interest. Interaction of the proteins results in reconstitution of the fluorophore (Figure 1)1,2
. A limitation of BiFC is that once the fragmented fluorophore is reconstituted the complex is irreversible3
. This limitation is advantageous in detecting transient or weak interactions, but precludes a kinetic analysis of complex dynamics. An additional caveat is that the reconstituted flourophore requires 30min to mature and fluoresce, again precluding the observation of real time interactions4
. BiFC is a specific example of the protein fragment complementation assay (PCA) which employs reporter proteins such as green fluorescent protein variants (BiFC), dihydrofolate reductase, b-lactamase, and luciferase to measure protein:protein interactions5,6
. Alternative methods to study protein:protein interactions in cells include fluorescence co-localization and Förster resonance energy transfer (FRET)7
. For co-localization, two proteins are individually tagged either directly with a fluorophore or by indirect immunofluorescence. However, this approach leads to high background of non-interacting proteins making it difficult to interpret co-localization data. In addition, due to the limits of resolution of confocal microscopy, two proteins may appear co-localized without necessarily interacting. With BiFC, fluorescence is only observed when the two proteins of interest interact. FRET is another excellent method for studying protein:protein interactions, but can be technically challenging. FRET experiments require the donor and acceptor to be of similar brightness and stoichiometry in the cell. In addition, one must account for bleed through of the donor into the acceptor channel and vice versa. Unlike FRET, BiFC has little background fluorescence, little post processing of image data, does not require high overexpression, and can detect weak or transient interactions. Bioluminescence resonance energy transfer (BRET) is a method similar to FRET except the donor is an enzyme (e.g. luciferase) that catalyzes a substrate to become bioluminescent thereby exciting an acceptor. BRET lacks the technical problems of bleed through and high background fluorescence but lacks the ability to provide spatial information due to the lack of substrate localization to specific compartments8
. Overall, BiFC is an excellent method for visualizing subcellular localization of protein complexes to gain insight into compartmentalized signaling.
Cellular Biology, Issue 50, Fluorescence, imaging, compartmentalized signaling, subcellular localization, signal transduction
Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses
Institutions: University of Alabama Huntsville, Stanford University .
Recently, structural and biochemical studies have detailed many of the molecular events that occur in the ribosome during inhibition of protein synthesis by antibiotics and during nascent polypeptide synthesis. Some of these antibiotics, and regulatory nascent polypeptides mostly in the form of peptidyl-tRNAs, inhibit either peptide bond formation or translation termination1-7
. These inhibitory events can stop the movement of the ribosome, a phenomenon termed "translational arrest". Translation arrest induced by either an antibiotic or a nascent polypeptide has been shown to regulate the expression of genes involved in diverse cellular functions such as cell growth, antibiotic resistance, protein translocation and cell metabolism8-13
. Knowledge of how antibiotics and regulatory nascent polypeptides alter ribosome function is essential if we are to understand the complete role of the ribosome in translation, in every organism.
Here, we describe a simple methodology that can be used to purify, exclusively, for analysis, those ribosomes translating a specific mRNA and containing a specific peptidyl-tRNA14
. This procedure is based on selective isolation of translating ribosomes bound to a biotin-labeled mRNA. These translational complexes are separated from other ribosomes in the same mixture, using streptavidin paramagnetic beads (SMB) and a magnetic field (MF). Biotin-labeled mRNAs are synthesized by run-off transcription assays using as templates PCR-generated DNA fragments that contain T7 transcriptional promoters. T7 RNA polymerase incorporates biotin-16-UMP from biotin-UTP; under our conditions approximately ten biotin-16-UMP molecules are incorporated in a 600 nt mRNA with a 25% UMP content. These biotin-labeled mRNAs are then isolated, and used in in vitro
translation assays performed with release factor 2 (RF2)-depleted cell-free extracts obtained from Escherichia coli
strains containing wild type or mutant ribosomes. Ribosomes translating the biotin-labeled mRNA sequences are stalled at the stop codon region, due to the absence of the RF2 protein, which normally accomplishes translation termination. Stalled ribosomes containing the newly synthesized peptidyl-tRNA are isolated and removed from the translation reactions using SMB and an MF. These beads only bind biotin-containing messages.
The isolated, translational complexes, can be used to analyze the structural and functional features of wild type or mutant ribosomal components, or peptidyl-tRNA sequences, as well as determining ribosome interaction with antibiotics or other molecular factors 1,14-16
. To examine the function of these isolated ribosome complexes, peptidyl-transferase assays can be performed in the presence of the antibiotic puromycin1
. To study structural changes in translational complexes, well established procedures can be used, such as i) crosslinking to specific amino acids14
and/or ii) alkylation protection assays1,14,17
Molecular Biology, Issue 48, Ribosome stalling, ribosome isolation, peptidyl-tRNA, in vitro translation, RNA chemical modification, puromycin, antibiotics.
Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides
Institutions: Cleveland State University.
Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5
. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli
protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA
. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAG
P-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9
. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7
. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo
in E. coli
cells and in vitro
in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11
. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro
Molecular Biology, Issue 64, Ribosome, nascent polypeptides, co-translational protein folding, translational arrest, in vitro translation
Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA
Institutions: Cleveland State University.
The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1
. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1
. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2
. Codon bias is organism as well as tissue specific2,3
. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4
. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8
. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9
. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo
) protein folding1,7,10,11
. To understand the process of protein folding in vivo
, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8
. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro
translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro
transcribed mRNA is used for in vitro
translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.
Genetics, Issue 65, Molecular Biology, Ribosome, Nascent polypeptide, Co-translational protein folding, Synonymous codon usage, gene regulation
Preparation of Segmented Microtubules to Study Motions Driven by the Disassembling Microtubule Ends
Institutions: Russian Academy of Sciences, Federal Research Center of Pediatric Hematology, Oncology and Immunology, Moscow, Russia, University of Pennsylvania.
Microtubule depolymerization can provide force to transport different protein complexes and protein-coated beads in vitro
. The underlying mechanisms are thought to play a vital role in the microtubule-dependent chromosome motions during cell division, but the relevant proteins and their exact roles are ill-defined. Thus, there is a growing need to develop assays with which to study such motility in vitro
using purified components and defined biochemical milieu. Microtubules, however, are inherently unstable polymers; their switching between growth and shortening is stochastic and difficult to control. The protocols we describe here take advantage of the segmented microtubules that are made with the photoablatable stabilizing caps. Depolymerization of such segmented microtubules can be triggered with high temporal and spatial resolution, thereby assisting studies of motility at the disassembling microtubule ends. This technique can be used to carry out a quantitative analysis of the number of molecules in the fluorescently-labeled protein complexes, which move processively with dynamic microtubule ends. To optimize a signal-to-noise ratio in this and other quantitative fluorescent assays, coverslips should be treated to reduce nonspecific absorption of soluble fluorescently-labeled proteins. Detailed protocols are provided to take into account the unevenness of fluorescent illumination, and determine the intensity of a single fluorophore using equidistant Gaussian fit. Finally, we describe the use of segmented microtubules to study microtubule-dependent motions of the protein-coated microbeads, providing insights into the ability of different motor and nonmotor proteins to couple microtubule depolymerization to processive cargo motion.
Basic Protocol, Issue 85, microscopy flow chamber, single-molecule fluorescence, laser trap, microtubule-binding protein, microtubule-dependent motor, microtubule tip-tracking
Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons
Institutions: University of Pennsylvania, Integrated DNA Technologies, Inc..
The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3’-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.
Genetics, Issue 90, RNA, imaging, single molecule, fluorescence, living cell
Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species
Institutions: Uppsala University, Swedish University of Agricultural Sciences.
The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of tissue specific expression is limited because of problems in dissecting individual tissues. Expression data needs to be confirmed and complemented with expression patterns using e.g. in situ
hybridization, a technique used to localize cell specific mRNA expression. The in situ
hybridization method is laborious, time-consuming and often requires extensive optimization depending on species and tissue. In situ
experiments are relatively more difficult to perform in woody species such as the conifer Norway spruce (Picea abies
). Here we present a modified DIG in situ
hybridization protocol, which is fast and applicable on a wide range of plant species including P. abies
. With just a few adjustments, including altered RNase treatment and proteinase K concentration, we could use the protocol to study tissue specific expression of homologous genes in male reproductive organs of one gymnosperm and two angiosperm species; P. abies, Arabidopsis thaliana
and Brassica napus
. The protocol worked equally well for the species and genes studied. AtAP3
were observed in second and third whorl floral organs in A. thaliana
and B. napus
and DAL13 in microsporophylls of male cones from P. abies
. For P. abies
the proteinase K concentration, used to permeablize the tissues, had to be increased to 3 g/ml instead of 1 g/ml, possibly due to more compact tissues and higher levels of phenolics and polysaccharides. For all species the RNase treatment was removed due to reduced signal strength without a corresponding increase in specificity. By comparing tissue specific expression patterns of homologous genes from both flowering plants and a coniferous tree we demonstrate that the DIG in situ
protocol presented here, with only minute adjustments, can be applied to a wide range of plant species. Hence, the protocol avoids both extensive species specific optimization and the laborious use of radioactively labeled probes in favor of DIG labeled probes. We have chosen to illustrate the technically demanding steps of the protocol in our film.
Anna Karlgren and Jenny Carlsson contributed equally to this study.
Corresponding authors: Anna Karlgren at Anna.Karlgren@ebc.uu.se and Jens F. Sundström at Jens.Sundstrom@vbsg.slu.se
Plant Biology, Issue 26, RNA, expression analysis, Norway spruce, Arabidopsis, rapeseed, conifers
Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis
Institutions: Purdue University.
embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene expression in these tissues. Thus, to analyze cell-specific gene expression profiles from Drosophila
tissues, it may be necessary to isolate specific cell types with high purity and at sufficient yields for downstream applications such as transcriptional profiling and chromatin immunoprecipitation. However, the irregular cellular morphology in tissues such as the central nervous system, coupled with the rare population of specific cell types in these tissues, can pose challenges for traditional methods of cell isolation such as laser microdissection and fluorescence-activated cell sorting (FACS). Here, an alternative approach to characterizing cell-specific gene expression profiles using affinity-based isolation of tagged nuclei, rather than whole cells, is described. Nuclei in the specific cell type of interest are genetically labeled with a nuclear envelope-localized EGFP tag using the Gal4/UAS binary expression system. These EGFP-tagged nuclei can be isolated using antibodies against GFP that are coupled to magnetic beads. The approach described in this protocol enables consistent isolation of nuclei from specific cell types in the Drosophila
larval central nervous system at high purity and at sufficient levels for expression analysis, even when these cell types comprise less than 2% of the total cell population in the tissue. This approach can be used to isolate nuclei from a wide variety of Drosophila
embryonic and larval cell types using specific Gal4 drivers, and may be useful for isolating nuclei from cell types that are not suitable for FACS or laser microdissection.
Biochemistry, Issue 85, Gene Expression, nuclei isolation, Drosophila, KASH, GFP, cell-type specific
Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction
Institutions: Georgia Health Sciences University.
The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid.
We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.
Cellular Biology, Issue 50, ceramide, phosphatidylserine, lipid-protein interaction, atypical PKC
Using Reverse Genetics to Manipulate the NSs Gene of the Rift Valley Fever Virus MP-12 Strain to Improve Vaccine Safety and Efficacy
Institutions: University of Texas Medical Branch.
Rift Valley fever virus (RVFV), which causes hemorrhagic fever, neurological disorders or blindness in humans, and a high rate abortion and fetal malformation in ruminants1
, has been classified as a HHS/USDA overlap select agent and a risk group 3 pathogen. It belongs to the genus Phlebovirus
in the family Bunyaviridae
and is one of the most virulent members of this family. Several reverse genetics systems for the RVFV MP-12 vaccine strain2,3
as well as wild-type RVFV strains 4-6
, including ZH548 and ZH501, have been developed since 2006. The MP-12 strain (which is a risk group 2 pathogen and a non-select agent) is highly attenuated by several mutations in its M- and L-segments, but still carries virulent S-segment RNA3
, which encodes a functional virulence factor, NSs. The rMP12-C13type (C13type) carrying 69% in-frame deletion of NSs ORF lacks all the known NSs functions, while it replicates as efficient as does MP-12 in VeroE6 cells lacking type-I IFN. NSs induces a shut-off of host transcription including interferon (IFN)-beta mRNA7,8
and promotes degradation of double-stranded RNA-dependent protein kinase (PKR) at the post-translational level.9,10
IFN-beta is transcriptionally upregulated by interferon regulatory factor 3 (IRF-3), NF-kB and activator protein-1 (AP-1), and the binding of IFN-beta to IFN-alpha/beta receptor (IFNAR) stimulates the transcription of IFN-alpha genes or other interferon stimulated genes (ISGs)11
, which induces host antiviral activities, whereas host transcription suppression including IFN-beta gene by NSs prevents the gene upregulations of those ISGs in response to viral replication although IRF-3, NF-kB and activator protein-1 (AP-1) can be activated by RVFV7. . Thus, NSs is an excellent target to further attenuate MP-12, and to enhance host innate immune responses by abolishing the IFN-beta suppression function. Here, we describe a protocol for generating a recombinant MP-12 encoding mutated NSs, and provide an example of a screening method to identify NSs mutants lacking the function to suppress IFN-beta mRNA synthesis. In addition to its essential role in innate immunity, type-I IFN is important for the maturation of dendritic cells and the induction of an adaptive immune response12-14
. Thus, NSs mutants inducing type-I IFN are further attenuated, but at the same time are more efficient at stimulating host immune responses than wild-type MP-12, which makes them ideal candidates for vaccination approaches.
Immunology, Issue 57, Rift Valley fever virus, reverse genetics, NSs, MP-12, vaccine development
Isolation of mRNAs Associated with Yeast Mitochondria to Study Mechanisms of Localized Translation
Institutions: Technion - Israel Institute of Technology.
Most of mitochondrial proteins are encoded in the nucleus and need to be imported into the organelle. Import may occur while the protein is synthesized near the mitochondria. Support for this possibility is derived from recent studies, in which many mRNAs encoding mitochondrial proteins were shown to be localized to the mitochondria vicinity. Together with earlier demonstrations of ribosomes’ association with the outer membrane, these results suggest a localized translation process. Such localized translation may improve import efficiency, provide unique regulation sites and minimize cases of ectopic expression. Diverse methods have been used to characterize the factors and elements that mediate localized translation. Standard among these is subcellular fractionation by differential centrifugation. This protocol has the advantage of isolation of mRNAs, ribosomes and proteins in a single procedure. These can then be characterized by various molecular and biochemical methods. Furthermore, transcriptomics and proteomics methods can be applied to the resulting material, thereby allow genome-wide insights. The utilization of yeast as a model organism for such studies has the advantages of speed, costs and simplicity. Furthermore, the advanced genetic tools and available deletion strains facilitate verification of candidate factors.
Biochemistry, Issue 85, mitochondria, mRNA localization, Yeast, S. cerevisiae, microarray, localized translation, biochemical fractionation
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
Polysome Fractionation and Analysis of Mammalian Translatomes on a Genome-wide Scale
Institutions: McGill University, Karolinska Institutet, McGill University.
mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. Accordingly, dysregulation of mRNA translation is considered to play a major role in a variety of pathological states including cancer. Ribosomes also host chaperones, which facilitate folding of nascent polypeptides, thereby modulating function and stability of newly synthesized polypeptides. In addition, emerging data indicate that ribosomes serve as a platform for a repertoire of signaling molecules, which are implicated in a variety of post-translational modifications of newly synthesized polypeptides as they emerge from the ribosome, and/or components of translational machinery. Herein, a well-established method of ribosome fractionation using sucrose density gradient centrifugation is described. In conjunction with the in-house developed “anota” algorithm this method allows direct determination of differential translation of individual mRNAs on a genome-wide scale. Moreover, this versatile protocol can be used for a variety of biochemical studies aiming to dissect the function of ribosome-associated protein complexes, including those that play a central role in folding and degradation of newly synthesized polypeptides.
Biochemistry, Issue 87, Cells, Eukaryota, Nutritional and Metabolic Diseases, Neoplasms, Metabolic Phenomena, Cell Physiological Phenomena, mRNA translation, ribosomes,
protein synthesis, genome-wide analysis, translatome, mTOR, eIF4E, 4E-BP1
High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry
Institutions: Medical College of Wisconsin, Stanford University School of Medicine, Medical College of Wisconsin, Hong Kong University, Johns Hopkins University School of Medicine, Medical College of Wisconsin.
There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle “in a dish” for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.
Cellular Biology, Issue 91, human induced pluripotent stem cell, flow cytometry, directed differentiation, cardiomyocyte, IRX4, TNNI3, TNNT2, MCL2v, MLC2a
Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer
Institutions: Max Planck Institute for Psycholinguistics, Donders Institute for Brain, Cognition and Behaviour.
Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis
luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.
Cellular Biology, Issue 87, Protein-protein interactions, Bioluminescence Resonance Energy Transfer, Live cell, Transfection, Luciferase, Yellow Fluorescent Protein, Mutations
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
Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport
Institutions: VECT-HORUS SAS, CNRS, NICN UMR 7259.
The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro
model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm·cm2
on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 ± 0.11 x 10-3
cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro
BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.
Medicine, Issue 88, rat brain endothelial cells (RBEC), mouse, spinal cord, tight junction (TJ), receptor-mediated transport (RMT), low density lipoprotein (LDL), LDLR, transferrin, TfR, P-glycoprotein (P-gp), transendothelial electrical resistance (TEER),
Identification of Post-translational Modifications of Plant Protein Complexes
Institutions: University of Warwick, Norwich Research Park, The Australian National University.
Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via
the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.
Plant Biology, Issue 84, plant-microbe interactions, protein complex purification, mass spectrometry, protein phosphorylation, Prf, Pto, AvrPto, AvrPtoB
Interview: Protein Folding and Studies of Neurodegenerative Diseases
Institutions: MIT - Massachusetts Institute of Technology.
In this interview, Dr. Lindquist describes relationships between protein folding, prion diseases and neurodegenerative disorders. The problem of the protein folding is at the core of the modern biology. In addition to their traditional biochemical functions, proteins can mediate transfer of biological information and therefore can be considered a genetic material. This recently discovered function of proteins has important implications for studies of human disorders. Dr. Lindquist also describes current experimental approaches to investigate the mechanism of neurodegenerative diseases based on genetic studies in model organisms.
Neuroscience, issue 17, protein folding, brain, neuron, prion, neurodegenerative disease, yeast, screen, Translational Research
Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells
Institutions: University of Toronto.
In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e.
"free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ
hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.
Cellular Biology, Issue 70, Biochemistry, Genetics, Molecular Biology, Genomics, mRNA localization, RNA, digitonin extraction, cell fractionation, endoplasmic reticulum, secretion, microscopy, imaging, fluorescent in situ hybridization, FISH, cell biology