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Past Issue in 2024
Volume: 14, Issue: 22
Biochemistry
Preparation of Protein Lysates Using Biorthogonal Chemical Reporters for Click Reaction and in-Gel Fluorescence Analysis
Bioorthogonal chemical reporters are non-native chemical handles introduced into biomolecules of living systems, typically through metabolic or protein engineering. These functionalities can undergo bioorthogonal reactions, such as copper-catalyzed alkyne-azide cycloaddition (CuAAC), with small-molecule probes, enabling the tagging and visualization of biomolecules. This approach has greatly enhanced our understanding of cellular dynamics, enzyme targeting, and protein post-translational modifications. Herein, we report a protocol for preparing protein lysates for click reaction and in-gel fluorescence analysis, exemplified using alk-16, a terminal alkyne-functionalized stearic acid analog, to investigate proteins with fatty acylation. This protocol provides methods for the fluorescent visualization of chemical reporter–labeled proteomes or individual proteins of interest (POIs).
Measurement of the Activity of Wildtype and Disease-Causing ALPK1 Mutants in Transfected Cells With a 96-Well Format NF-κB/AP-1 Reporter Assay
Alpha-protein kinase 1 (ALPK1) is normally activated by bacterial ADP-heptose as part of the innate immune response, leading to the initiation of downstream signalling events that culminate in the activation of transcription factors such as NF-κB and AP-1. In contrast, disease-causing mutations in ALPK1 that cause ROSAH syndrome or spiradenoma allow ALPK1 to be activated in cells in the absence of bacterial infection (i.e., without ADP-heptose). This protocol describes a semi-quantitative reporter assay based on ALPK1 knockout HEK-Blue cells that measures the activity of transfected wildtype and disease-causing forms of ALPK1 by virtue of their ability to activate the transcription factors NF-κB and AP-1. These cells express a synthetic gene encoding alkaline phosphatase under the control of an NF-κB/AP-1-dependent promoter, and consequently, the activation of ALPK1 leads to the production of alkaline phosphatase, which is secreted into the culture media and can be measured colorimetrically at 645 nm after the addition of a detection reagent.
Quantitative Measurement of the Kinase Activity of Wildtype ALPK1 and Disease-Causing ALPK1 Mutants Using Cell-Free Radiometric Phosphorylation Assays
ALPK1 is an atypical protein kinase that is activated during bacterial infection by ADP-heptose and phosphorylates TIFA to activate a cell signaling pathway. In contrast, specific mutations in ALPK1 allow it to also be activated by endogenous human nucleotide sugars such as UDP-mannose, leading to the phosphorylation of TIFA in the absence of infection. This protocol describes a quantitative, cell-free phosphorylation assay that can directly measure the catalytic activity of wildtype and disease-causing ALPK1 in the presence of different nucleotide sugars. In this method, overexpressed ALPK1 is first immunoprecipitated from the extracts of ALPK1 knockout HEK-Blue cells transfected with plasmids encoding either FLAG-tagged wildtype or mutant ALPK1, and then subjected to a radioactive phosphorylation assay in which the phosphorylation of purified GST-tagged TIFA by ALPK1 is quantified by measuring the incorporation of radioactivity derived from radiolabeled ATP.
Utilizing the Planar Lipid Bilayer Technique to Investigate Drosophila melanogaster dMpv17 Channel Activity
The planar lipid bilayer (PLB) technique represents a highly effective method for the study of membrane protein properties in a controlled environment. The PLB method was employed to investigate the role of mitochondrial inner membrane protein 17 (MPV17), whose mutations are associated with a hepatocerebral form of mitochondrial DNA depletion syndrome (MDS). This protocol presents a comprehensive, step-by-step guide to the assembly and utilization of a PLB system. The procedure comprises the formation of a lipid bilayer over an aperture, the reconstitution of the target protein, and the utilization of electrophysiological recording techniques to monitor channel activity. Furthermore, recommendations are provided for optimizing experimental conditions and overcoming common challenges encountered in PLB experiments. Overall, this protocol highlights the versatility of the PLB technique in advancing our understanding of membrane protein function and its broad application in various fields of research.
Biological Engineering
A Simple Staining Method Using Pyronin Y for Laser Scanning Confocal Microscopy to Evaluate Gelatin Cryogels
This study explores the novel application of pyronin Y for fluorescently labeling extracellular matrices (ECMs) and gelatin cryogels, providing a simple and reliable method for laser scanning confocal microscopy. Pyronin Y exhibited remarkable staining ability of the porous structures of gelatin cryogels, indicating its potential as a reliable tool for evaluating such biomaterials. Confocal imaging of pyronin Y–stained cryogels produced high signal-to-noise ratio images suitable for quantifying pores using Fiji/Image J. Importantly, pyronin Y enabled effective dual-color imaging of cryogel-labeled mesenchymal stem cells, expanding its utility beyond traditional RNA assays. Traditional staining methods like Mason’s trichrome and Sirius Red have limitations in cryogel applications. Pyronin Y emerges as a powerful alternative due to its water solubility, minimal toxicity, and stability. Our results demonstrate pyronin Y’s ability to specifically stain gelatin cryogel's porous structures, surpassing its weak staining of ECMs in 2D. Confocal imaging revealed enduring staining even under rigorous scanning, with no notable photobleaching observed. Furthermore, pyronin Y's combination with Alexa Fluor 647 for dual-color imaging showed promising results, validating its versatility in fluorescence microscopy. In conclusion, this study establishes pyronin Y as a cost-effective and rapid option for fluorescent staining of gelatin cryogels. Its simplicity, efficacy, and compatibility with confocal microscopy make it a valuable tool for characterizing and evaluating gelatin-based biomaterials, contributing significantly to the field of cryogel imaging. The study opens new avenues for dual-color imaging in biomaterial research and tissue engineering, advancing our understanding of cellular interactions within scaffolds.
A Protocol for co-Injecting Cells with Pulverized Fibers for Improved Cell Survival and Engraftment
Adipose tissue is crucial for medical applications such as tissue reconstruction, cosmetic procedures, and correcting soft tissue deformities. Significant advances in the use of adipose tissue have been achieved through Coleman’s studies in fat grafting, which gained widespread acceptance due to its effectiveness and safety. Despite its benefits, adipose tissue grafting faces several limitations, including high absorption rates due to insufficient support or anchorage, replacement by fibrous tissue, migration from the intended site, and loss of the initial desired morphology post-administration. To counteract these constraints, there is a need for improved grafting techniques that enhance the predictability and consistency of outcomes. Biomaterials are extensively used in tissue engineering to support cell adhesion, proliferation, and growth. Both natural and synthetic materials have shown promise in creating suitable microenvironments for adipose tissue regeneration. PLGA, a synthetic copolymer, is particularly notable for its biocompatibility, biodegradability, and tunable mechanical properties. Here, we describe a protocol using milled electrospun poly(lactic-co-glycolic acid) (PLGA) fibers combined with lipoaspirated tissue to create a fibrous slurry for injection. By pulverizing PLGA fiber mats to create fiber fragments with increased pore size and porosity, we can influence key cellular responses and enhance the success of adipose tissue–grafting procedures. This approach improves anchorage and support for adipocytes, thereby increasing cell viability. This method aims to enhance vascularity, perfusion, and volume retention in adipose tissue grafts, which addresses many of the limitations of current approaches to adipose tissue grafting and holds promise for more consistent and successful outcomes.
Biophysics
Small-Molecule Probe for Imaging Oxidative Stress–Induced Carbonylation in Live Cells
Protein carbonylation has been known as the major form of irreversible protein modifications and is also widely used as an indicator of oxidative stress in the biological environment. In the presence of oxidative stress, biological systems tend to produce large amounts of carbonyl moieties; these carbonyl groups do not have particular UV-Vis and fluorescence spectroscopic characteristics that we can differentiate, observe, and detect. Thus, their detection and quantification can only be performed using specific chemical probes. Commercially available fluorescent probes to detect specific carbonylation in biological systems have been used, but their chemical portfolio is still very limited. This protocol outlines the methods and procedures employed to synthesize a probe, (E,Z)-2-(2-(2-hydroxybenzylidene)hydrazonyl)-5-nitrophenol (2Hzin5NP), and assess its impact on carbonylation in human cells. The synthesis involves several steps, including the preparation of its hydrazone compounds mimicking cell carbonyls, 2-Hydrazinyl 5-nitrophenol, (E,Z)-2-(2-ethylidenehydrazonyl)-5-nitrophenol, and the final product (E,Z)-2-(2-(2-hydroxybenzylidene)hydrazonyl)-5-nitrophenol. The evaluation of fluorescence quantum yield and subsequent cell culture experiments are detailed for the investigation of 2Hzin5NP effects on cell proliferation and carbonylation.
Cell Biology
Preparation of Polarity-Marked Microtubules Using a Plus-End Capping DARPin
The eukaryotic cytoskeleton is formed in part by microtubules, which are relatively rigid filaments with inherent structural polarity. One consequence of this polarity is that the two ends of a microtubule have different properties with important consequences for their cellular roles. These differences are often challenging to probe within the crowded environment of the cell. Fluorescence microscopy–based in vitro assays with purified proteins and stabilized microtubules have been used to characterize polarity-dependent and end-specific behaviors. These assays require ways to visualize the polarity of the microtubules, which has previously been achieved either by the addition of fluorescently tagged motor proteins with known directionality or by fluorescently polarity marking the microtubules themselves. However, classical polarity-marking protocols require a particular chemically modified tubulin and generate microtubules with chemically different plus and minus segments. These chemical differences in the segments may affect the behavior of interacting proteins of interest in an undesirable manner. We present here a new protocol that uses a previously characterized, reversibly binding microtubule plus-end capping protein, a designed ankyrin repeat protein (DARPin), to efficiently produce polarity-marked microtubules with different fluorescently labeled, but otherwise biochemically identical, plus- and minus-end segments.
Immunology
Display of Native SARS-CoV-2 Spike on Mammalian Cells to Measure Antibody Affinity and ADCC
The COVID-19 pandemic led to the rapid development of antibody-based therapeutics and vaccines targeting the SARS-CoV-2 spike protein. Several antibodies have been instrumental in protecting vulnerable populations, but their utility was limited by the emergence of spike variants with diminished susceptibility to antibody binding and neutralization. Moreover, these spike variants exhibited reduced neutralization by polyclonal antibodies in vaccinated individuals. Accordingly, the characterization of antibody binding to spike variants is critical to define antibody potency and understand the impact of amino acid changes. A key challenge in this effort is poor spike stability, with most current methods assessing antibody binding using individual domains instead of the intact spike or variants with stabilizing amino acid changes in the ectodomain (e.g., 2P or HexaPro). The use of non-native spike may not accurately predict antibody binding if changes lie within the epitope or alter epitope accessibility by altering spike dynamics. Here, we present methods to characterize antibody affinity for and activity against unmodified SARS-CoV-2 spike protein variants displayed on a mammalian cell membrane that recapitulates the native spike environment on infected cells. These include a flow cytometry–based method to determine the effective antibody binding affinity (KD) and an antibody-dependent cellular cytotoxicity (ADCC) assay to assess Fc-mediated activities. These methods can readily evaluate antibody activity across a panel of spike variants and contribute to our understanding of spike/antibody co-evolution.
Plant Science
Fast and High-Resolution Imaging of Pollinated Stigmatic Cells by Tabletop Scanning Electron Microscopy
In plants, the first interaction between the pollen grain and the epidermal cells of the stigma is crucial for successful reproduction. When the pollen is accepted, it germinates, producing a tube that transports the two sperm cells to the ovules for fertilization. Confocal microscopy has been used to characterize the behavior of stigmatic cells post-pollination [1], but it is time-consuming since it requires the development of a range of fluorescent marker lines. Here, we propose a quick, high-resolution imaging protocol using tabletop scanning electron microscopy. This technique does not require prior sample fixation or fluorescent marker lines. It effectively captures pollen grain behavior from early hydration (a few minutes after pollination) to pollen tube growth within the stigma (1 h after pollination) and is particularly efficient for tracking pollen tube paths.
Optimized Isolation of Lysosome-Related Organelles from Stationary Phase and Iron-Overloaded Chlamydomonas reinhardtii Cells
Lysosome-related organelles (LROs) are a class of heterogeneous subcellular organelles conserved in eukaryotes, performing various functions. An important function of LROs is to mediate phosphorus and metal homeostasis. Chlamydomonas reinhardtii serves as a model organism for investigating metal ion metabolism. Considering that LROs contain polyphosphate and various metal elements, the purification strategy is based on their higher density by fractionating cell lysate through OptiPrep density gradient ultracentrifugation. Here, we optimized a method for purifying LROs from C. reinhardtii cells that have reached stationary phase (sta-LROs) or are overloaded with iron (Fe-LROs). Our protocol provides technical support for further investigations on the biogenesis and function of LROs in C. reinhardtii.
Stem Cell
Multiplex Genome Editing of Human Pluripotent Stem Cells Using Cpf1
Targeted genome editing of human pluripotent stem cells (hPSCs) is critical for basic and translational research and can be achieved with site-specific endonucleases. Cpf1 (CRISPR from Prevotella and Francisella) is a programmable DNA endonuclease with AT-rich PAM sequences. In this protocol, we describe procedures for using a single vector system to deliver Cpf1 and CRISPR RNA (crRNA) for genome editing in hPSCs. This protocol enables indel formation and homologous recombination–mediated precise editing at multiple loci. With the delivery of Cpf1 and a single U6 promoter-driven guide RNA array composed of an AAVS1-targeting and a MAFB-targeting crRNA array, efficient multiplex genome editing at the AAVS1 (knockin) and MAFB (knockout) loci in hPSCs could be achieved in a single experiment. The edited hPSCs expressed pluripotency markers and could differentiate into neurons in vitro. This system also generated INS reporter hPSCs with a 6 kb cassette knockin at the INS locus. The INS reporter cells can differentiate into β-cells that express tdTomato and luciferase, permitting fluorescence-activated cell sorting of hPSC-β-cells. By targeted screening of potential off-target sequences that are most homologous to crRNA sequences, no off-target mutations were detected in any of the tested sequences. This work provides an efficient and flexible system for precise genome editing in mammalian cells including hPSCs with the benefits of less off-target effects.
Preparing Chamber Slides With Pressed Collagen for Live Imaging Monolayers of Primary Human Intestinal Stem Cells
Primary human intestinal stem cells (ISCs) can be cultured and passaged indefinitely as two-dimensional monolayers grown on soft collagen. Culturing ISCs as monolayers enables easy access to the luminal side for chemical treatments and provides a simpler topology for high-resolution imaging compared to cells cultured as three-dimensional organoids. However, the soft collagen required to support primary ISC growth can pose a challenge for live imaging with an inverted microscope, as the collagen creates a steep meniscus when poured into wells. This may lead to uneven growth toward the center of the well, with cells at the edges often extending beyond the working distance of confocal microscopes. We have engineered a 3D-printed collagen mold that enables the preparation of chamber slides with flat, smooth, and reproducible thin collagen layers. These layers are adequate to support ISC growth while being thin enough to optimize live imaging with an inverted microscope. We present methods for constructing the collagen press, preparing chamber slides with pressed collagen, and plating primary human ISCs for growth and analysis.
