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Current Issue in 2024
Volume: 14, Issue: 21
Biochemistry
A Native PAGE Assay for the Biochemical Characterization of G Protein Coupling to GPCRs
G protein-coupled receptors (GPCRs) are a large family of membrane-embedded receptors that have diverse roles in physiology and are major drug targets. GPCRs transduce an agonist binding signal across the membrane to activate intracellular heterotrimeric G proteins. The dynamic nature of the receptors and the complexity of their interactions with agonists and G proteins present significant challenges for biochemical studies. Most biochemical/biophysical methods that have been employed to study GPCR-G protein coupling require purified receptors and are technically difficult. Here, we provide a protocol for a relatively simple and time- and cost-effective membrane protein native PAGE assay, to visualize and biochemically characterize agonist-dependent coupling of detergent-solubilized GPCRs to purified G protein surrogate “mini-G” proteins, which stabilize the receptor in an active state. The assay was developed for our studies of the calcitonin receptor-like receptor, a class B GPCR that mediates the actions of calcitonin gene-related peptide and adrenomedullin peptide agonists. It does not require a purified receptor and it can be used in a screening format with transiently-transfected adherent mammalian cell cultures, to quickly identify detergent-stable complexes amenable to study, or in a quantitative format with membrane preparations, to determine apparent affinities of agonists for the mini-G-coupled receptor and apparent affinities of mini-G proteins for the agonist-occupied receptor. The latter provides a partial measure of agonist efficacy. The method should be applicable to other GPCRs, and has the potential to be adapted to the study of other challenging membrane proteins and their complexes with binding partners.Graphic abstract: Visualizing agonist-dependent mini-G protein coupling and determining apparent binding affinities using the native PAGE assay quantitative formats.
A Simple and Straightforward Approach for Generating Small, Stable, Homogeneous, Unilamellar 1-Palmitoyl 2-Oleoyl Phosphatidylcholine (POPC) Bilayer Vesicles
Various methods have been developed to generate phosphoglyceride liposomes. Approaches resulting in homogeneous populations of unilamellar bilayer vesicles are generally preferred to mimic various cell membrane situations, as well as to optimize aqueous solute trapping efficiency using the least amount of lipid for biotechnological purposes. Most are time-consuming, often tedious, or require specialized equipment, and produce vesicles with limited shelf-life at room temperature or in cold storage. Herein, we describe a straightforward approach that avoids the preceding complications and streamlines the construction of unilamellar bilayer vesicles from 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC)/dihexanoyl phosphatidylcholine (DHPC) bicelle mixtures at room temperature. The resulting vesicles are small (32-36 nm diameter), unilamellar, bilayer vesicles that are homogeneous, stable, and resistant to freeze-thaw alterations.Graphic abstract: Cryo-EM of POPC vesicles formed by dilution of 0.5 q-value POPC/DHPC bicelle mix.
Biophysics
Single Molecular Resolution to Monitor DNA Replication Fork Dynamics upon Stress by DNA Fiber Assay
DNA replication always encounters numerous endogenous and exogenous stresses during the cell cycle. Measuring the cell responses to stress has primarily relied on cell survival and incorporation of radioactive dNTPs, which is limited in resolution. A higher resolution is required to monitor how replication and repair respond to these stresses. This protocol describes a procedure to monitor the length of new synthesized DNA in a single molecular resolution called DNA fiber assay. The fiber assay relies on labeling of nascent DNA with the nucleoside analog 5-Chloro-2'-deoxyuridine (CldU) and 5-Iodo-2'-deoxyuridine (IdU). We can visualize the labeled nascent DNA in single molecular resolution by immunostaining. By measuring labeled DNA length, the assay permits interrogation of replication speed at given conditions, end processing at the reversed fork, and fork restart after repair.
Copper Based Site-directed Spin Labeling of Proteins for Use in Pulsed and Continuous Wave EPR Spectroscopy
Site-directed spin labeling in conjunction with electron paramagnetic resonance (EPR) is an attractive approach to measure residue specific dynamics and point-to-point distance distributions in a biomolecule. Here, we focus on the labeling of proteins with a Cu(II)-nitrilotriacetic acid (NTA) complex, by exploiting two strategically placed histidine residues (called the dHis motif). This labeling strategy has emerged as a means to overcome key limitations of many spin labels. Through utilizing the dHis motif, Cu(II)NTA rigidly binds to a protein without depending on cysteine residues. This protocol outlines three major points: the synthesis of the Cu(II)NTA complex; the measurement of continuous wave and pulsed EPR spectra, to verify a successful synthesis, as well as successful protein labeling; and utilizing Cu(II)NTA labeled proteins, to measure distance constraints and backbone dynamics. In doing so, EPR measurements are less influenced by sidechain motion, which influences the breadth of the measured distance distributions between two spins, as well as the measured residue-specific dynamics. More broadly, such EPR-based distance measurements provide unique structural constraints for integrative structural biophysics and complement traditional biophysical techniques, such as NMR, cryo-EM, FRET, and crystallography.Graphic abstract: Monitoring the success of Cu(II)NTA labeling.
Label-free Quantification of Direct Protein-protein Interactions with Backscattering Interferometry
The functional performance of a cell depends on how macromolecules, in particular proteins, come together in a precise orientation, how they assemble into protein complexes and interact with each other. In order to study protein-protein interactions at a molecular level, a variety of methods to investigate these binding processes yield affinity constants and/or the identification of binding regions. There are several well-established biophysical techniques for biomolecular interaction studies, such as fluorescence spectroscopy and surface plasmon resonance. Although these techniques have been proven to be efficient, they either need labeling or immobilization of one interaction partner. Backscattering interferometry (BSI) is a label-free detection method, which allows label- and immobilization-free interaction analysis under physiologically relevant conditions with high sensitivity and in small volumes. We used BSI to measure the interaction of the neuronal calcium sensor recoverin with its target G protein-coupled receptor kinase 1 (GRK1) as a model system. Increasing concentrations of purified recoverin were mixed with a specific concentration of a GRK1 fusion protein. In this protocol, we provide a full description of the instrumental setup, data acquisition, and evaluation. Equilibrium dissociation constants of recoverin-GRK1 interaction determined by the BSI instrumental setup are in full agreement with affinity constants obtained by different methods as described in the literature.
Cancer Biology
Reconstruction of Human AML Using Functionally and Immunophenotypically Defined Human Haematopoietic Stem and Progenitor Cells as Targeted Populations
Acute myeloid leukaemia (AML) is a highly heterogenous blood cancer, in which the expansion of aberrant myeloid blood cells interferes with the generation and function of normal blood cells. Although key driver mutations and their associated inhibitors have been identified in the last decade, they have not been fully translated into better survival rates for AML patients, which remain dismal. In addition to DNA mutation, studies in mouse models strongly suggest that the cell of origin, where the driver mutation (such as MLL fusions) occurs, emerges as an additional factor that determines the treatment outcome in AML. To investigate its functional relevance in human disease, we have recently reported that AML driven by MLL fusions can transform immunophenotypically and functionally distinctive human hematopoietic stem cells (HSCs) or myeloid progenitors resulting in immunophenotypically indistinguishable human AML. Intriguingly, these cells display differential treatment sensitivities to current treatments, attesting the cell of origin as an important determinant governing treatment outcome for AML. To further facilitate this line of investigation, here we describe a comprehensive disease modelling protocol using human primary haematopoietic cells, which covers all the key steps, from the isolation of immunophenotypically defined human primary haematopoietic stem and progenitor populations, to oncogene transfer via viral transduction, the in vitro liquid culture assay, and finally the xenotransplantation into immunocompromised mice.
Cell Biology
Preparation of Giant Escherichia coli spheroplasts for Electrophysiological Recordings
Prokaryotic ion channels have been instrumental in furthering our understanding of many fundamental aspects of ion channels’ structure and function. However, characterizing the biophysical properties of a prokaryotic ion channel in a native membrane system using patch-clamp electrophysiology is technically challenging. Patch-clamp is regarded as a gold standard technique to study ion channel properties in both native and heterologous expression systems. The presence of a cell wall and the small size of bacterial cells makes it impossible to directly patch clamp using microelectrodes. Here, we describe a method for the preparation of giant E. coli spheroplasts in order to investigate the electrophysiological properties of bacterial cell membranes. Spheroplasts are formed by first inhibiting bacterial cell wall synthesis, followed by enzymatic digestion of the outer cell wall in the presence of a permeabilizing agent. This protocol can be used to characterize the function of any heterologous ion channels or ion transporters expressed in E. coli membranes.
Developmental Biology
A Method to Induce Brown/Beige Adipocyte Differentiation from Murine Preadipocytes
Adipocytes exhibit different morphological and functional characteristics, depending on their anatomical location, developmental origin, and stimulus. While white adipocytes tend to accumulate energy as triglycerides, brown and beige adipocytes tend to direct carbon sources to fuel thermogenesis. White and beige adipocytes originate from common progenitor cells, which are distinct from brown adipocyte precursors. Having a method to study white vs. beige vs. brown adipocyte differentiation may help to unveil the mechanisms driving distinct adipogenic programs. Preadipocytes can be cultured and differentiated in vitro using a combination of compounds to stimulate adipogenesis. Here, we describe and compare protocols designed to stimulate adipocyte differentiation and induce brown/beige-like or white-like characteristics in differentiating adipocytes. The protocols consist in exposing murine preadipocytes to pharmacological stimuli aimed at triggering adipogenesis and inducing (or not) a thermogenic gene expression program. After 8 days of differentiation with a pro-browning cocktail, immortalized preadipocytes isolated from interscapular brown fat (9B cells) or inguinal white fat (9W cells) from the same mouse expressed higher levels of brown/beige adipocyte markers (e.g., Ucp1) and pan-adipocyte differentiation markers (e.g., Pparg, Cebpa and aP2) when compared to the same cells differentiated with a cocktail that lacked brown/beige adipogenic inducers (i.e., rosiglitazone, T3, and indomethacin). Consistent with a higher thermogenic potential of brown vs. beige adipocytes, differentiated 9B cells expressed higher Ucp1 levels than differentiated 9W cells. This simple protocol may help researchers to understand mechanisms of adipogenesis and how adipocytes become thermogenic.
Measurement of Reactive Oxygen and Nitrogen Species in Living Cells Using the Probe 2',7'-Dichlorodihydrofluorescein
Reactive oxygen species and reactive nitrogen species (RONS) are involved in programmed cell death in the context of numerous degenerative and chronic diseases. In particular, the ability of cells to maintain redox homeostasis is necessary for an adaptive cellular response to adverse conditions that can cause damage to proteins and DNA, resulting in apoptosis and genetic mutations. Here, we focus on the 2',7'-dichlorodihydrofluorescein diacetate (DCFH2-DA) assay to detect RONS. Although this fluorescence-based assay is widely utilized due to its high sensitivity to detect changes in cellular redox status that allow measuring alterations in RONS over time, its validity has been a matter of controversy. If correctly carried out, its limitations are understood and results are correctly interpreted, the DCFH2-DA assay is a valuable tool for cell-based studies.
Wounding Zebrafish Larval Epidermis by Laceration
Wound healing is a critical process for maintaining the integrity of tissues, driven in large part by the active migration of cells to cover damaged regions. While the long-term tissue injury response over hours and days has been extensively studied, the rapid early migratory response of cells to injury in vivo is still being uncovered, especially in model systems such as zebrafish larvae, which are ideal for live imaging with high spatiotemporal resolution. Observing these dynamics requires a wounding method that prompts a robust wound response and is compatible with immediate live imaging or other downstream applications. We have developed a procedure for wounding the epidermis in the tailfin of larval zebrafish, which we term “tissue laceration”. In this procedure, the tailfin is impaled with a glass needle that is then dragged through the tissue, which generates a full-thickness wound that elicits a dramatic migratory wound response within seconds from cells up to several hundred micrometers away from the wound. Laceration generates a larger wound response in the first few minutes following wounding compared to other mechanical wounds such as tail transection, and laceration does not require specialized equipment compared to laser wounding methods. This procedure can be used to interrogate the processes by which epidermal cells far away from the wound are able to rapidly detect injury and respond to the wound.
Microbiology
Extraction and Electrophoretic Analysis of Bacterial Lipopolysaccharides and Outer Membrane Proteins
Lipopolysaccharides (LPS) (or lipooligosaccharides [LOS], which lack the O-antigen side chains characteristic of LPS), and outer membrane proteins (OMP) are major cell-surface molecules in the outer membrane (OM) of gram-negative bacteria. The LPS is responsible for causing endotoxic shock in infected hosts and, in conjunction with some OMPs, provides protection to the bacterium against host innate immune defenses and attachment to host cells. Electrophoretic analysis can provide valuable information regarding the size, number, and variability of LPS/LOS and OMP components between bacterial strains and mutants, which aids in understanding the basic biology and virulence factors of a particular species. Furthermore, highly purified extracts are normally not required if only electrophoretic analysis is to be done, and various methods have been established for such procedures. Here, we review ameliorated procedures for fast and convenient extraction of LPS/LOS and protein-enriched outer membranes (PEOM) for optimal electrophoretic resolution. Specifically, we will describe the phenol-based micro-method for LPS/LOS extraction, a differential extraction procedure with sodium lauryl sarcosinate for PEOM, and gel preparation for electrophoretic analysis of LPS/LOS samples in detail.Graphic abstract: Workflow for the preparation and analysis of LPS/LOS and PEOM.
Selection of Vaccinia Virus Recombinants Using CRISPR/Cas9
The engineering of poxvirus genomes is fundamental to primary and applied virology research. Indeed, recombinant poxviruses form the basis for many novel vaccines and virotherapies but producing and purifying these viruses can be arduous. In recent years, CRISPR/Cas9 has become the favoured approach for genome manipulation due to its speed and high success rate. However, recent data suggests poxvirus genomes are not repaired well following Cas9 cleavage. As a result, CRISPR/Cas9 is inefficient as an editing tool, but very effective as a programmable selection agent. Here, we describe protocols for the generation and enrichment of recombinant vaccinia viruses using targeted Cas9 as a selection tool. This novel use of Cas9 is a simple addition to current homologous recombination-based methods that are widespread in the field, facilitating implementation in laboratories already working with poxviruses. This is also the first method that allows for isolation of new vaccinia viruses in less than a fortnight, without the need to incorporate a marker gene or manipulation of large poxvirus genomes in vitro and reactivation with helper viruses. Whilst this protocol describes applications for laboratory strains of vaccinia virus, it should be readily adaptable to other poxviruses.Graphic abstract: Pipeline for Cas9 selection of recombinant poxviruses.
Neuroscience
Delivery of AAV for Expression of Fluorescent Biosensors in Juvenile Mouse Hippocampus
Genetically encoded fluorescent biosensors are versatile tools for studying brain metabolism and function in live tissue. The genetic information for these biosensors can be delivered into the brain by stereotaxic injection of engineered adeno-associated viruses (AAVs), which can selectively target different cell types depending on the capsid serotype and/or the viral promoter. Here, we describe a protocol for intracranial injections of two viral vectors encoding the metabolic biosensor Peredox and the calcium biosensor RCaMP1h. When combined with 2-photon microscopy and fluorescence lifetime imaging, this protocol allows the simultaneous quantitative assessment of changes in the cytosolic NADH/NAD+ ratio and the intracellular Ca2+ levels in individual dentate granule cells from acute hippocampal slices.Graphic abstract: Workflow diagram for biosensor expression in the mouse hippocampus using intracranial injections of adeno-associated viruses.
Assessment of Corticospinal Excitability in Awake Rodents Using EMG-Controlled Intracortical Stimulation
Assessment of corticospinal excitability (CSE) is an essential component of experiments designed to induce or study neuronal plasticity in the motor system. Common examples are paired associative stimulation (PAS), theta-burst stimulation (TBS), intensive motor training, or any methods aimed at potentiating the corticomotor system in the hope of promoting better recovery after neurological insult. To date, rodent models of CSE assessment have mostly been completed under anaesthesia, which greatly affects the level of CSE, as well as the mechanisms of plasticity. Experiments in awake animals are difficult because the ongoing state of behavior affects the excitability of the motor system and complicates the assessment of CSE. To address this issue, we have designed a novel approach for CSE assessment in awake behaving rodents, enabling a reliable measure of evoked motor responses obtained from cortical microstimulation in repeatable conditions of ongoing motor activity. The system relies on chronically implanted intracortical and intramuscular electrodes and a custom-made software control system, enabling the user to require that precise parameters of EMG activity be met before cortical stimulation probes are delivered. This approach could be used for further studies of PAS, TBS or other interventions requiring the assessment of CSE under repeatable conditions. We provide fabrication schematics and a list of materials for the implant, as well as instructions for running a custom-made MATLAB codebase, customizing the PAS protocol, and performing the complete analysis of experimental data. We hope these tools can further facilitate animal research in the field of neuroplasticity and neurorehabilitation.
Plant Science
Non-invasive Imaging of Rice Roots in Non-compacted and Compacted Soil
Roots are the prime organ for nutrient and water uptake and are therefore fundamental to the growth and development of plants. However, physical challenges of a heterogeneous environment and diverse edaphic stresses affect root growth in soil. Compacted soil is a serious global problem, causing inhibition of root elongation, which reduces surface area and impacts resource foraging. Visualisation and quantification of roots in soil is difficult due to this growth substrate’s opaque nature; however, non-destructive imaging technologies are now becoming more widely available to plant and soil scientists working to address this challenge. We have recently developed an integrated approach, combining X-ray Computed Tomography (X-ray CT) and confocal microscopy to image roots grown in compacted soil conditions from a plant to a cellular scale. The method is suited to visualize cellular responses of root tips grown in both non-compacted and compacted soils. This protocol presents a fully integrated workflow, including soil column preparation, creation of compaction conditions, plant growth, imaging, and quantification of root adaptive responses at a cellular scale.
Bacterial Infection and Hypersensitive Response Assays in Arabidopsis-Pseudomonas syringae Pathosystem
Arabidopsis thaliana-Pseudomonas syringae pathosystem has been used as an important model system for studying plant-microbe interactions, leading to many milestones and breakthroughs in the understanding of plant immune system and pathogenesis mechanisms. Bacterial infection and plant disease assessment are key experiments in the studies of plant-pathogen interactions. The hypersensitive response (HR), which is characterized by rapid cell death and tissue collapse after inoculation with a high dose of bacteria, is a hallmark response of plant effector-triggered immunity (ETI), one layer of plant immunity triggered by recognition of pathogen-derived effector proteins. Here, we present a detailed protocol for bacterial disease and hypersensitive response assays applicable to studies of Pseudomonas syringae interaction with various plant species such as Arabidopsis, Nicotiana benthamiana, and tomato.
An in vitro Coupled Assay for PEPC with Control of Bicarbonate Concentration
Phosphoenolpyruvate carboxylase (PEPC) catalyzes a critical step in carbon metabolism in plants and bacteria, the irreversible reaction between bicarbonate and phosphoenolpyruvate to produce the C4 compound oxaloacetate. This enzyme is particularly important in the context of C4 photosynthesis, where it is the initial carbon-fixing enzyme. Many studies have used kinetic approaches to characterize the properties of PEPCs from different species, different post-translational states, and after mutagenesis. Most of these studies have worked at a fixed saturating concentration of bicarbonate. Controlling the concentration of bicarbonate is difficult at low concentrations because of equilibration with atmospheric CO2. We describe here a simple, repeatable, and gas-tight assay system for PEPC that allows bicarbonate concentrations to be controlled above ca. 50 µM.