Kindlins are essential coactivators, with talin, of the cell surface receptors integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin β-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3's role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.
21 Related JoVE Articles!
Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays
Institutions: University of Toronto, University of Toronto, University of Regina.
Phenotypes are determined by a complex series of physical (e.g.
protein-protein) and functional (e.g.
gene-gene or genetic) interactions (GI)1
. While physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships2
. Large-scale GI maps have been reported for eukaryotic organisms like yeast3-7
, but GI information remains sparse for prokaryotes8
, which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods9, 10
Here, we present the key steps required to perform quantitative E. coli
Synthetic Genetic Array (eSGA) screening procedure on a genome-scale9
, using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format.
Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g.
the 'Keio' collection11
) and essential gene hypomorphic mutations (i.e.
alleles conferring reduced protein expression, stability, or activity9, 12, 13
) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system14
. GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected9
. Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process2
. Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e.
slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex2
as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred9
Genetics, Issue 69, Molecular Biology, Medicine, Biochemistry, Microbiology, Aggravating, alleviating, conjugation, double mutant, Escherichia coli, genetic interaction, Gram-negative bacteria, homologous recombination, network, synthetic lethality or sickness, suppression
Detection of Toxin Translocation into the Host Cytosol by Surface Plasmon Resonance
Institutions: University of Central Florida.
AB toxins consist of an enzymatic A subunit and a cell-binding B subunit1
. These toxins are secreted into the extracellular milieu, but they act upon targets within the eukaryotic cytosol. Some AB toxins travel by vesicle carriers from the cell surface to the endoplasmic reticulum (ER) before entering the cytosol2-4
. In the ER, the catalytic A chain dissociates from the rest of the toxin and moves through a protein-conducting channel to reach its cytosolic target5
. The translocated, cytosolic A chain is difficult to detect because toxin trafficking to the ER is an extremely inefficient process: most internalized toxin is routed to the lysosomes for degradation, so only a small fraction of surface-bound toxin reaches the Golgi apparatus and ER6-12
To monitor toxin translocation from the ER to the cytosol in cultured cells, we combined a subcellular fractionation protocol with the highly sensitive detection method of surface plasmon resonance (SPR)13-15
. The plasma membrane of toxin-treated cells is selectively permeabilized with digitonin, allowing collection of a cytosolic fraction which is subsequently perfused over an SPR sensor coated with an anti-toxin A chain antibody. The antibody-coated sensor can capture and detect pg/mL quantities of cytosolic toxin. With this protocol, it is possible to follow the kinetics of toxin entry into the cytosol and to characterize inhibitory effects on the translocation event. The concentration of cytosolic toxin can also be calculated from a standard curve generated with known quantities of A chain standards that have been perfused over the sensor. Our method represents a rapid, sensitive, and quantitative detection system that does not require radiolabeling or other modifications to the target toxin.
Immunology, Issue 59, Surface plasmon resonance, AB toxin, translocation, endoplasmic reticulum, cell culture, cholera toxin, pertussis toxin
Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag
Institutions: Royal Institute of Technology.
Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization1
. To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used2
. The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions3
. The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies1,2
Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding4
, were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A5
, a small, bispecific molecule with affinity for both HSA and the novel target was identified6
The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli
with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix.
This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination1,7
Molecular Biology, Issue 59, Affinity chromatography, albumin-binding domain, human serum albumin, Z-domain
Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids
Institutions: Washington University, Washington University, Washington University.
Microbes have complex metabolic pathways that can be investigated using biochemistry and functional genomics methods. One important technique to examine cell central metabolism and discover new enzymes is 13
C-assisted metabolism analysis 1. This technique is based on isotopic labeling, whereby microbes are fed with a 13
C labeled substrates. By tracing the atom transition paths between metabolites in the biochemical network, we can determine functional pathways and discover new enzymes.
As a complementary method to transcriptomics and proteomics, approaches for isotopomer-assisted analysis of metabolic pathways contain three major steps 2
, we grow cells with 13
C labeled substrates. In this step, the composition of the medium and the selection of labeled substrates are two key factors. To avoid measurement noises from non-labeled carbon in nutrient supplements, a minimal medium with a sole carbon source is required. Further, the choice of a labeled substrate is based on how effectively it will elucidate the pathway being analyzed. Because novel enzymes often involve different reaction stereochemistry or intermediate products, in general, singly labeled carbon substrates are more informative for detection of novel pathways than uniformly labeled ones for detection of novel pathways3, 4
, we analyze amino acid labeling patterns using GC-MS. Amino acids are abundant in protein and thus can be obtained from biomass hydrolysis. Amino acids can be derivatized by N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (TBDMS) before GC separation. TBDMS derivatized amino acids can be fragmented by MS and result in different arrays of fragments. Based on the mass to charge (m/z) ratio of fragmented and unfragmented amino acids, we can deduce the possible labeled patterns of the central metabolites that are precursors of the amino acids. Third
, we trace 13C carbon transitions in the proposed pathways and, based on the isotopomer data, confirm whether these pathways are active 2
. Measurement of amino acids provides isotopic labeling information about eight crucial precursor metabolites in the central metabolism. These metabolic key nodes can reflect the functions of associated central pathways.
C-assisted metabolism analysis via proteinogenic amino acids can be widely used for functional characterization of poorly-characterized microbial metabolism1
. In this protocol, we will use Cyanothece
51142 as the model strain to demonstrate the use of labeled carbon substrates for discovering new enzymatic functions.
Molecular Biology, Issue 59, GC-MS, novel pathway, metabolism, labeling, phototrophic microorganism
Assaying the Kinase Activity of LRRK2 in vitro
Institutions: UCL Institute of Neurology.
Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a GTPase domain (termed ROC, for Ras of Complex proteins) and a kinase domain1
. The discovery in 2004 of mutations in LRRK2 that cause Parkinson's disease (PD) resulted in LRRK2 being the focus of a huge volume of research into its normal function and how the protein goes awry in the disease state2,3
. Initial investigations into the function of LRRK2 focused on its enzymatic activities4-6
. Although a clear picture has yet to emerge of a consistent alteration in these due to mutations, data from a number of groups has highlighted the importance of the kinase activity of LRRK2 in cell death linked to mutations7,8
. Recent publications have reported inhibitors targeting the kinase activity of LRRK2, providing a key experimental tool9-11
. In light of these data, it is likely that the enzymatic properties of LRRK2 afford us an important window into the biology of this protein, although whether they are potential drug targets for Parkinson's is open to debate.
A number of different approaches have been used to assay the kinase activity of LRRK2. Initially, assays were carried out using epitope tagged protein overexpressed in mammalian cell lines and immunoprecipitated, with the assays carried out using this protein immobilised on agarose beads4,5,7
. Subsequently, purified recombinant fragments of LRRK2 in solution have also been used, for example a GST tagged fragment purified from insect cells containing residues 970 to 2527 of LRRK212
. Recently, Daniëls et al.
reported the isolation of full length LRRK2 in solution from human embryonic kidney cells, however this protein is not widely available13
. In contrast, the GST fusion truncated form of LRRK2 is commercially available (from Invitrogen, see table 1 for details), and provides a convenient tool for demonstrating an assay for LRRK2 kinase activity. Several different outputs for LRRK2 kinase activity have been reported. Autophosphorylation of LRRK2 itself, phosphorylation of Myelin Basic Protein (MBP) as a generic kinase substrate and phosphorylation of an artificial substrate - dubbed LRRKtide, based upon phosphorylation of threonine 558 in Moesin - have all been used, as have a series of putative physiological substrates including α-synuclein, Moesin and 4-EBP14-17
. The status of these proteins as substrates for LRRK2 remains unclear, and as such the protocol described below will focus on using MBP as a generic substrate, noting the utility of this system to assay LRRK2 kinase activity directed against a range of potential substrates.
Molecular Biology, Issue 59, Kinase, LRRK2, Parkinson's disease
Associated Chromosome Trap for Identifying Long-range DNA Interactions
Institutions: Stanford University School of Medicine.
Genetic information encoded by DNA is organized in a complex and highly regulated chromatin structure. Each chromosome occupies a specific territory, that may change according to stage of development or cell cycle. Gene expression can occur in specialized transcriptional factories where chromatin segments may loop out from various chromosome territories, leading to co-localization of DNA segments which may exist on different chromosomes or far apart on the same chromosome. The Associated Chromosome Trap (ACT) assay provides an effective methodology to identify these long-range DNA associations in an unbiased fashion by extending and modifying the chromosome conformation capture technique. The ACT assay makes it possible for us to investigate mechanisms of transcriptional regulation in trans, and can help explain the relationship of nuclear architecture to gene expression in normal physiology and during disease states.
Molecular Biology, Issue 50, Associated chromosomal Trap, DNA long-range interaction, nuclear architecture, gene regulation
High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli
Institutions: California Institute of Technology, California Institute of Technology.
Cellulase enzymes (endoglucanases, cellobiohydrolases, and β-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1
. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2
. Of particular interest are fungal cellulases3-8
, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli
the catalytic domains of endoglucanases from H. jecorina3,9
, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli
expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10
and Phanerochaete chrysosporium11-12
We present a method for high throughput screening of fungal endoglucanase activity in E. coli
. (Fig 1
) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (“halos”) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness.
Traditional bacterial whole cell CMC/CR activity assays13
involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14
for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes
, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus
and Penicillium decumbens
(shown in Figure 2
), suggesting that this protocol is applicable to enzymes from a range of organisms.
Molecular Biology, Issue 54, cellulase, endoglucanase, CMC, Congo Red
Nanomechanics of Drug-target Interactions and Antibacterial Resistance Detection
Institutions: University College London.
The cantilever sensor, which acts as a transducer of reactions between model bacterial cell wall matrix immobilized on its surface and antibiotic drugs in solution, has shown considerable potential in biochemical sensing applications with unprecedented sensitivity and specificity1-5
. The drug-target interactions generate surface stress, causing the cantilever to bend, and the signal can be analyzed optically when it is illuminated by a laser. The change in surface stress measured with nano-scale precision allows disruptions of the biomechanics of model bacterial cell wall targets to be tracked in real time. Despite offering considerable advantages, multiple cantilever sensor arrays have never been applied in quantifying drug-target binding interactions.
Here, we report on the use of silicon multiple cantilever arrays coated with alkanethiol self-assembled monolayers mimicking bacterial cell wall matrix to quantitatively study antibiotic binding interactions. To understand the impact of vancomycin on the mechanics of bacterial cell wall structures1,6,7
. We developed a new model1
which proposes that cantilever bending can be described by two independent factors; i) namely a chemical factor, which is given by a classical Langmuir adsorption isotherm, from which we calculate the thermodynamic equilibrium dissociation constant (Kd
) and ii) a geometrical factor, essentially a measure of how bacterial peptide receptors are distributed on the cantilever surface. The surface distribution of peptide receptors (p
) is used to investigate the dependence of geometry and ligand loading. It is shown that a threshold value of p ~
10% is critical to sensing applications. Below which there is no detectable bending signal while above this value, the bending signal increases almost linearly, revealing that stress is a product of a local chemical binding factor and a geometrical factor combined by the mechanical connectivity of reacted regions and provides a new paradigm for design of powerful agents to combat superbug infections.
Immunology, Issue 80, Engineering, Technology, Diagnostic Techniques and Procedures, Early Diagnosis, Bacterial Infections and Mycoses, Lipids, Amino Acids, Peptides, and Proteins, Chemical Actions and Uses, Diagnosis, Therapeutics, Surface stress, vancomycin, mucopeptides, cantilever sensor
Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate
Institutions: KU Leuven and Leuven Institute for Neuroscience and Disease (LIND).
Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, a Ras of complex proteins domain (ROC), a C-terminal of ROC domain (COR), and leucine-rich and ankyrin-like repeats at the N-terminus. The precise cellular roles of LRRK1 and LRRK2 have yet to be elucidated, however LRRK1 has been implicated in tyrosine kinase receptor signaling1,2
, while LRRK2 is implicated in the pathogenesis of Parkinson's disease3,4
. In this report, we present a protocol to label the LRRK1 and LRRK2 proteins in cells with 32
P orthophosphate, thereby providing a means to measure the overall phosphorylation levels of these 2 proteins in cells. In brief, affinity tagged LRRK proteins are expressed in HEK293T cells which are exposed to medium containing 32
P-orthophosphate. The 32
P-orthophosphate is assimilated by the cells after only a few hours of incubation and all molecules in the cell containing phosphates are thereby radioactively labeled. Via the affinity tag (3xflag) the LRRK proteins are isolated from other cellular components by immunoprecipitation. Immunoprecipitates are then separated via SDS-PAGE, blotted to PVDF membranes and analysis of the incorporated phosphates is performed by autoradiography (32
P signal) and western detection (protein signal) of the proteins on the blots. The protocol can readily be adapted to monitor phosphorylation of any other protein that can be expressed in cells and isolated by immunoprecipitation.
Cellular Biology, Issue 79, biology (general), biochemistry, bioengineering (general), LRRK1, LRRK2, metabolic labeling, 32P orthophosphate, immunoprecipitation, autoradiography
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
Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry
Institutions: University of Exeter.
A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most of these require access to specialist equipment, and often require a degree of expertise to effectively establish reliable experiments and analyze data. Differential scanning fluorimetry (DSF) is being increasingly used as a robust method for initial screening of proteins for interacting small molecules, either for identifying physiological partners or for hit discovery. This technique has the advantage that it requires only a PCR machine suitable for quantitative PCR, and so suitable instrumentation is available in most institutions; an excellent range of protocols are already available; and there are strong precedents in the literature for multiple uses of the method. Past work has proposed several means of calculating dissociation constants from DSF data, but these are mathematically demanding. Here, we demonstrate a method for estimating dissociation constants from a moderate amount of DSF experimental data. These data can typically be collected and analyzed within a single day. We demonstrate how different models can be used to fit data collected from simple binding events, and where cooperative binding or independent binding sites are present. Finally, we present an example of data analysis in a case where standard models do not apply. These methods are illustrated with data collected on commercially available control proteins, and two proteins from our research program. Overall, our method provides a straightforward way for researchers to rapidly gain further insight into protein-ligand interactions using DSF.
Biophysics, Issue 91, differential scanning fluorimetry, dissociation constant, protein-ligand interactions, StepOne, cooperativity, WcbI.
Specificity Analysis of Protein Lysine Methyltransferases Using SPOT Peptide Arrays
Institutions: Stuttgart University.
Lysine methylation is an emerging post-translation modification and it has been identified on several histone and non-histone proteins, where it plays crucial roles in cell development and many diseases. Approximately 5,000 lysine methylation sites were identified on different proteins, which are set by few dozens of protein lysine methyltransferases. This suggests that each PKMT methylates multiple proteins, however till now only one or two substrates have been identified for several of these enzymes. To approach this problem, we have introduced peptide array based substrate specificity analyses of PKMTs. Peptide arrays are powerful tools to characterize the specificity of PKMTs because methylation of several substrates with different sequences can be tested on one array. We synthesized peptide arrays on cellulose membrane using an Intavis SPOT synthesizer and analyzed the specificity of various PKMTs. Based on the results, for several of these enzymes, novel substrates could be identified. For example, for NSD1 by employing peptide arrays, we showed that it methylates K44 of H4 instead of the reported H4K20 and in addition H1.5K168 is the highly preferred substrate over the previously known H3K36. Hence, peptide arrays are powerful tools to biochemically characterize the PKMTs.
Biochemistry, Issue 93, Peptide arrays, solid phase peptide synthesis, SPOT synthesis, protein lysine methyltransferases, substrate specificity profile analysis, lysine methylation
Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions
Institutions: University of Toronto, University of Toronto, University of Toronto.
The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae
as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub
) and the N-terminal half of the ubiquitin moiety (Nub
). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub
which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub
moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.
Cellular Biology, Issue 36, protein-protein interaction, membrane, split-ubiquitin, yeast, library screening, Y2H, yeast two-hybrid, MYTH
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
Using Coculture to Detect Chemically Mediated Interspecies Interactions
Institutions: University of North Carolina at Chapel Hill .
In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by secreting metabolites. These metabolites have the potential to modulate the physiology and differentiation of their microbial neighbors and are likely important factors in the establishment and maintenance of complex microbial communities. We have developed a fluorescence-based coculture screen to identify such chemically mediated microbial interactions. The screen involves combining a fluorescent transcriptional reporter strain with environmental microbes on solid media and allowing the colonies to grow in coculture. The fluorescent transcriptional reporter is designed so that the chosen bacterial strain fluoresces when it is expressing a particular phenotype of interest (i.e.
biofilm formation, sporulation, virulence factor production, etc
.) Screening is performed under growth conditions where this phenotype is not
expressed (and therefore the reporter strain is typically nonfluorescent). When an environmental microbe secretes a metabolite that activates this phenotype, it diffuses through the agar and activates the fluorescent reporter construct. This allows the inducing-metabolite-producing microbe to be detected: they are the nonfluorescent colonies most proximal to the fluorescent colonies. Thus, this screen allows the identification of environmental microbes that produce diffusible metabolites that activate a particular physiological response in a reporter strain. This publication discusses how to: a) select appropriate coculture screening conditions, b) prepare the reporter and environmental microbes for screening, c) perform the coculture screen, d) isolate putative inducing organisms, and e) confirm their activity in a secondary screen. We developed this method to screen for soil organisms that activate biofilm matrix-production in Bacillus subtilis
; however, we also discuss considerations for applying this approach to other genetically tractable bacteria.
Microbiology, Issue 80, High-Throughput Screening Assays, Genes, Reporter, Microbial Interactions, Soil Microbiology, Coculture, microbial interactions, screen, fluorescent transcriptional reporters, Bacillus subtilis
Microwave-assisted Functionalization of Poly(ethylene glycol) and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation
Institutions: University of Rochester, University of Rochester, University of Rochester Medical Center.
One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large variety of PEG molecular weights and configurations (arm number, arm length, and branching pattern) affords researchers tight control over resulting hydrogel structures and properties, including Young’s modulus and mesh size. This video will illustrate a rapid, efficient, solvent-free, microwave-assisted method to methacrylate PEG precursors into poly(ethylene glycol) dimethacrylate (PEGDM). This synthetic method provides much-needed starting materials for applications in drug delivery and regenerative medicine. The demonstrated method is superior to traditional methacrylation methods as it is significantly faster and simpler, as well as more economical and environmentally friendly, using smaller amounts of reagents and solvents. We will also demonstrate an adaptation of this technique for on-resin methacrylamide functionalization of peptides. This on-resin method allows the N-terminus of peptides to be functionalized with methacrylamide groups prior to deprotection and cleavage from resin. This allows for selective addition of methacrylamide groups to the N-termini of the peptides while amino acids with reactive side groups (e.g.
primary amine of lysine, primary alcohol of serine, secondary alcohols of threonine, and phenol of tyrosine) remain protected, preventing functionalization at multiple sites. This article will detail common analytical methods (proton Nuclear Magnetic Resonance spectroscopy (;
H-NMR) and Matrix Assisted Laser Desorption Ionization Time of Flight mass spectrometry (MALDI-ToF)) to assess the efficiency of the functionalizations. Common pitfalls and suggested troubleshooting methods will be addressed, as will modifications of the technique which can be used to further tune macromer functionality and resulting hydrogel physical and chemical properties. Use of synthesized products for the formation of hydrogels for drug delivery and cell-material interaction studies will be demonstrated, with particular attention paid to modifying hydrogel composition to affect mesh size, controlling hydrogel stiffness and drug release.
Chemistry, Issue 80, Poly(ethylene glycol), peptides, polymerization, polymers, methacrylation, peptide functionalization, 1H-NMR, MALDI-ToF, hydrogels, macromer synthesis
Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy
Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the enzymes onto the noncatalytic scaffold protein is directed by interactions among a family of related receptor-ligand pairs comprising interacting cohesin and dockerin modules. The extremely strong binding between cohesin and dockerin modules results in dissociation constants in the low picomolar to nanomolar range, which may hamper accurate off-rate measurements with conventional bulk methods. Single-molecule force spectroscopy (SMFS) with the atomic force microscope measures the response of individual biomolecules to force, and in contrast to other single-molecule manipulation methods (i.e.
optical tweezers), is optimal for studying high-affinity receptor-ligand interactions because of its ability to probe the high-force regime (>120 pN). Here we present our complete protocol for studying cellulosomal protein assemblies at the single-molecule level. Using a protein topology derived from the native cellulosome, we worked with enzyme-dockerin and carbohydrate binding module-cohesin (CBM-cohesin) fusion proteins, each with an accessible free thiol group at an engineered cysteine residue. We present our site-specific surface immobilization protocol, along with our measurement and data analysis procedure for obtaining detailed binding parameters for the high-affinity complex. We demonstrate how to quantify single subdomain unfolding forces, complex rupture forces, kinetic off-rates, and potential widths of the binding well. The successful application of these methods in characterizing the cohesin-dockerin interaction responsible for assembly of multidomain cellulolytic complexes is further described.
Bioengineering, Issue 82, biophysics, protein unfolding, atomic force microscopy, surface immobilization
The Structure of Skilled Forelimb Reaching in the Rat: A Movement Rating Scale
Institutions: University of Lethbridge.
Skilled reaching for food is an evolutionary ancient act and is displayed by many animal species, including those in the sister clades of rodents and primates. The video describes a test situation that allows filming of repeated acts of reaching for food by the rat that has been mildly food deprived. A rat is trained to reach through a slot in a holding box for food pellet that it grasps and then places in its mouth for eating. Reaching is accomplished in the main by proximally driven movements of the limb but distal limb movements are used for pronating the paw, grasping the food, and releasing the food into the mouth. Each reach is divided into at least 10 movements of the forelimb and the reaching act is facilitated by postural adjustments. Each of the movements is described and examples of the movements are given from a number of viewing perspectives. By rating each movement element on a 3-point scale, the reach can be quantified. A number of studies have demonstrated that the movement elements are altered by motor system damage, including damage to the motor cortex, basal ganglia, brainstem, and spinal cord. The movements are also altered in neurological conditions that can be modeled in the rat, including Parkinson's disease and Huntington's disease. Thus, the rating scale is useful for quantifying motor impairments and the effectiveness of neural restoration and rehabilitation. Because the reaching act for the rat is very similar to that displayed by humans and nonhuman primates, the scale can be used for comparative purposes. from a number of viewing perspectives. By rating each movement element on a 3-point scale, the reach can be quantified. A number of studies have demonstrated that the movement elements are altered by motor system damage, including damage to the motor cortex, basal ganglia, brainstem, and spinal cord. The movements are also altered in neurological conditions that can be modeled in the rat, including Parkinson's disease and Huntington's disease. Thus, the rating scale is useful for quantifying motor impairments and the effectiveness of neural restoration and rehabilitation.
Experiments on animals were performed in accordance with the guidelines and regulations set forth by the University of Lethbridge Animal Care Committee in accordance with the regulations of the Canadian Council on Animal Care.
Neuroscience, Issue 18, rat skilled reaching, rat reaching scale, rat, rat movement element rating scale, reaching elements
Reaggregate Thymus Cultures
Institutions: University of Birmingham .
Stromal cells within lymphoid tissues are organized into three-dimensional structures that provide a scaffold that is thought to control the migration and development of haemopoeitic cells. Importantly, the maintenance of this three-dimensional organization appears to be critical for normal stromal cell function, with two-dimensional monolayer cultures often being shown to be capable of supporting only individual fragments of lymphoid tissue function. In the thymus, complex networks of cortical and medullary epithelial cells act as a framework that controls the recruitment, proliferation, differentiation and survival of lymphoid progenitors as they undergo the multi-stage process of intrathymic T-cell development. Understanding the functional role of individual stromal compartments in the thymus is essential in determining how the thymus imposes self/non-self discrimination. Here we describe a technique in which we exploit the plasticity of fetal tissues to re-associate into intact three-dimensional structures in vitro
, following their enzymatic disaggregation. The dissociation of fetal thymus lobes into heterogeneous cellular mixtures, followed by their separation into individual cellular components, is then combined with the in vitro
re-association of these desired cell types into three-dimensional reaggregate structures at defined ratios, thereby providing an opportunity to investigate particular aspects of T-cell development under defined cellular conditions. (This article is based on work first reported Methods in Molecular Biology 2007, Vol. 380 pages 185-196).
Immunology, Issue 18, Springer Protocols, Thymus, 2-dGuo, Thymus Organ Cultures, Immune Tolerance, Positive and Negative Selection, Lymphoid Development
A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse
Institutions: Columbia University.
Alzheimer's disease (AD) is a progressive neurodegenerative disease that is pathologically characterized by extracellular deposition of β-amyloid peptide (Aβ) and intraneuronal accumulation of hyperphosphorylated tau protein. Because cerebrospinal fluid (CSF) is in direct contact with the extracellular space of the brain, it provides a reflection of the biochemical changes in the brain in response to pathological processes. CSF from AD patients shows a decrease in the 42 amino-acid form of Aβ (Aβ42), and increases in total tau and hyperphosphorylated tau, though the mechanisms responsible for these changes are still not fully understood. Transgenic (Tg) mouse models of AD provide an excellent opportunity to investigate how and why Aβ or tau levels in CSF change as the disease progresses. Here, we demonstrate a refined cisterna magna puncture technique for CSF sampling from the mouse. This extremely gentle sampling technique allows serial CSF samples to be obtained from the same mouse at 2-3 month intervals which greatly minimizes the confounding effect of between-mouse variability in Aβ or tau levels, making it possible to detect subtle alterations over time. In combination with Aβ and tau ELISA, this technique will be useful for studies designed to investigate the relationship between the levels of CSF Aβ42 and tau, and their metabolism in the brain in AD mouse models. Studies in Tg mice could provide important validation as to the potential of CSF Aβ or tau levels to be used as biological markers for monitoring disease progression, and to monitor the effect of therapeutic interventions. As the mice can be sacrificed and the brains can be examined for biochemical or histological changes, the mechanisms underlying the CSF changes can be better assessed. These data are likely to be informative for interpretation of human AD CSF changes.
Neuroscience, Issue 21, Cerebrospinal fluid, Alzheimer's disease, Transgenic mouse, β-amyloid, tau
Interview: HIV-1 Proviral DNA Excision Using an Evolved Recombinase
Institutions: Heinrich-Pette-Institute for Experimental Virology and Immunology, University of Hamburg.
HIV-1 integrates into the host chromosome of infected cells and persists as a provirus flanked by long terminal repeats. Current treatment strategies primarily target virus enzymes or virus-cell fusion, suppressing the viral life cycle without eradicating the infection. Since the integrated provirus is not targeted by these approaches, new resistant strains of HIV-1 may emerge. Here, we report that the engineered recombinase Tre (see Molecular evolution of the Tre recombinase , Buchholz, F., Max Planck Institute for Cell Biology and Genetics, Dresden) efficiently excises integrated HIV-1 proviral DNA from the genome of infected cells. We produced loxLTR containing viral pseudotypes and infected HeLa cells to examine whether Tre recombinase can excise the provirus from the genome of HIV-1 infected human cells. A virus particle-releasing cell line was cloned and transfected with a plasmid expressing Tre or with a parental control vector. Recombinase activity and virus production were monitored. All assays demonstrated the efficient deletion of the provirus from infected cells without visible cytotoxic effects. These results serve as proof of principle that it is possible to evolve a recombinase to specifically target an HIV-1 LTR and that this recombinase is capable of excising the HIV-1 provirus from the genome of HIV-1-infected human cells.
Before an engineered recombinase could enter the therapeutic arena, however, significant obstacles need to be overcome. Among the most critical issues, that we face, are an efficient and safe delivery to targeted cells and the absence of side effects.
Medicine, Issue 16, HIV, Cell Biology, Recombinase, provirus, HeLa Cells