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Past Issue in 2024
Volume: 14, Issue: 5
Cell Biology
Real-Time Autophagic Flux Measurements in Live Cells Using a Novel Fluorescent Marker DAPRed
Autophagy is a conserved homeostatic mechanism involved in cellular homeostasis and many disease processes. Although it was first described in yeast cells undergoing starvation, we have learned over the years that autophagy gets activated in many stress conditions and during development and aging in mammalian cells. Understanding the fundamental mechanisms underlying autophagy effects can bring us closer to better insights into the pathogenesis of many disease conditions (e.g., cardiac muscle necrosis, Alzheimer’s disease, and chronic lung injury). Due to the complex and dynamic nature of the autophagic processes, many different techniques (e.g., western blotting, fluorescent labeling, and genetic modifications of key autophagy proteins) have been developed to delineate autophagy effects. Although these methods are valid, they are not well suited for the assessment of time-dependent autophagy kinetics. Here, we describe a novel approach: the use of DAPRed for autophagic flux measurement via live cell imaging, utilizing A549 cells, that can visualize and quantify autophagic flux in real time in single live cells. This approach is relatively straightforward in comparison to other experimental procedures and should be applicable to any in vitro cell/tissue models.Key features• Allows real-time qualitative imaging of autophagic flux at single-cell level.• Primary cells and cell lines can also be utilized with this technique.• Use of confocal microscopy allows visualization of autophagy without disturbing cellular functions.
Monitoring Intestinal Organoid–Derived Monolayer Barrier Functions with Electric Cell–Substrate Impedance Sensing (ECIS)
The measurement of transepithelial electrical resistance across confluent cell monolayer systems is the most commonly used technique to study intestinal barrier development and integrity. Electric cell substrate impedance sensing (ECIS) is a real-time, label-free, impedance-based method used to study various cell behaviors such as cell growth, viability, migration, and barrier function in vitro. So far, the ECIS technology has exclusively been performed on cell lines. Organoids, however, are cultured from tissue-specific stem cells, which better recapitulate cell functions and the heterogeneity of the parent tissue than cell lines and are therefore more physiologically relevant for research and modeling of human diseases. In this protocol paper, we demonstrate that ECIS technology can be successfully applied on 2D monolayers generated from patient-derived intestinal organoids.Key features• We present a protocol that allows the assessment of various cell functions, such as proliferation and barrier formation, with ECIS on organoid-derived monolayers.• The protocol facilitates intestinal barrier research on patient tissue-derived organoids, providing a valuable tool for disease modeling.
Microbiology
Bacterial Pathogen-mediated Suppression of Host Trafficking to Lysosomes: Fluorescence Microscopy-based DQ-Red BSA Analysis
Intracellular bacterial pathogens have evolved to be adept at manipulating host cellular function for the benefit of the pathogen, often by means of secreted virulence factors that target host pathways for modulation. The lysosomal pathway is an essential cellular response pathway to intracellular pathogens and, as such, represents a common target for bacterial-mediated evasion. Here, we describe a method to quantitatively assess bacterial pathogen–mediated suppression of host cell trafficking to lysosomes, using Salmonella enterica serovar Typhimurium infection of epithelial cells as a model. This live-cell imaging assay involves the use of a BODIPY TR-X conjugate of BSA (DQ-Red BSA) that traffics to and fluoresces in functional lysosomes. This method can be adapted to study infection with a broad array of pathogens in diverse host cell types. It is capable of being applied to identify secreted virulence factors responsible for a phenotype of interest as well as domains within the bacterial protein that are important for mediating the phenotype. Collectively, these tools can provide invaluable insight into the mechanisms of pathogenesis of a diverse array of pathogenic bacteria, with the potential to uncover virulence factors that may be suitable targets for therapeutic intervention.Key features• Infection-based analysis of bacterial-mediated suppression of host trafficking to lysosomes, using Salmonella enterica serovar Typhimurium infection of human epithelial cells as a model.• Live microscopy–based analysis allows for the visualization of individually infected host cells and is amenable to phenotype quantification.• Assay can be adapted to a broad array of pathogens and diverse host cell types.• Assay can identify virulence factors mediating a phenotype and protein domains that mediate a phenotype.
Mobilization of Plasmids from Bacteria into Diatoms by Conjugation Technique
Diatoms serve as a source for a variety of compounds with particularbiotechnological interest. Therefore, redirecting the flow to a specific pathwayrequires the elucidation of the gene’s specific function. The mostcommonly used method in diatoms is biolistic transformation, which is a veryexpensive and time-consuming method. The use of episomes that are maintained asclosed circles at a copy number equivalent to native chromosomes has become auseful genetic system for protein expression that avoids multiple insertions,position-specific effects on expression, and potential knockout of non-targetedgenes. These episomes can be introduced from bacteria into diatoms viaconjugation. Here, we describe a detailed protocol for gene expression thatincludes 1) the gateway cloning strategy and 2) the conjugation protocol for themobilization of plasmids from bacteria to diatoms.
An Optimized P. berghei Liver Stage–HepG2 Infection Model for Simultaneous Quantitative Bioimaging of Host and Parasite Nascent Proteomes
The Plasmodium parasites that cause malaria undergo an obligate, asymptomatic developmental stage in the host liver before initiating the symptomatic blood-stage infection. The parasite liver stage is a key intervention point for antimalarial chemoprophylaxis: successful targeting of liver-stage parasites prevents disease development in individuals and can help to reduce parasite transmission in populations, as the gametocyte forms that transmit infection to mosquitos are exclusively found in the blood stage. Antimalarial drugs that can target multiple parasite stages are thus highly desirable, and one emerging cellular target for such multistage active compounds is the process of protein synthesis or translation. Quantitative study of liver stage translation, and thus mechanistic evaluation of translation inhibitors against liver stage parasites, is not amenable to the methods allowing quantification of asexual blood stage translation, such as radiolabeled amino acid incorporation or lysate-based translation of reporter transcripts. Here, we present a method using o-propargyl puromycin (OPP) labeling of host and parasite nascent proteomes in the P. berghei-HepG2 infection model, followed by automated confocal image acquisition and computational separation of P. berghei vs. H. sapiens nascent proteome signals to allow simultaneous readout of the effects of translation inhibitors on both host and parasite. This protocol details our HepG2 cell culture and infected monolayer handling optimized for microscopy, our OPP labeling workflow, and our approach to automated confocal imaging, image processing, and data analysis.Key features• Uses the o-propargyl puromycin labeling technique developed by Liu et al. to quantitatively analyze protein synthesis in Plasmodium berghei liver-stage parasites in actively translating hepatoma cells.• This quantitative approach should be adaptable for other puromycin-sensitive intracellular pathogens residing in actively translating host cells.• The P. berghei–infected HepG2 recovery and reseeding protocol presented here is of use in applications beyond nascent proteome labeling and quantification.Graphical overview
Analysis of Cleavage Activity of Dengue Virus Protease by Co-transfections
The genome of the dengue virus codes for a single polypeptide that yields three structural and seven non-structural (NS) proteins upon post-translational modifications. Among them, NS protein-3 (NS3) possesses protease activity, involved in the processing of the self-polypeptide and in the cleavage of host proteins. Identification and analysis of such host proteins as substrates of this protease facilitate the development of specific drugs. In vitro cleavage analysis has been applied, which requires homogeneously purified components. However, the expression and purification of both S3 and erythroid differentiation regulatory factor 1 (EDRF1) are difficult and unsuccessful on many occasions. EDRF1 was identified as an interacting protein of dengue virus protease (NS3). The amino acid sequence analysis indicates the presence of NS3 cleavage sites in this protein. As EDRF1 is a high-molecular-weight (~138 kDa) protein, it is difficult to express and purify the complete protein. In this protocol, we clone the domain of the EDRF1 protein (C-terminal end) containing the cleavage site and the NS3 into two different eukaryotic expression vectors containing different tags. These recombinant vectors are co-transfected into mammalian cells. The cell lysate is subjected to SDS-PAGE followed by western blotting with anti-tag antibodies. Data suggest the disappearance of the EDRF1 band in the lane co-transfected along with NS3 protease but present in the lane transfected with only EDRF1, suggesting EDRF1 as a novel substrate of NS3 protease. This protocol is useful in identifying the substrates of viral-encoded proteases using ex vivo conditions. Further, this protocol can be used to screen anti-protease molecules.Key features• This protocol requires the cloning of protease and substrate into two different eukaryotic expression vectors with different tags.• Involves the transfection and co-transfection of both the above recombinant vectors individually and together.• Involves western blotting of the same PVDF membrane containing total proteins of the cell lysate with two different antibodies.• Does not require purified proteins for the analysis of cleavage of any suspected substrate by the protease.Graphical overview
Neuroscience
Simple Rescue of Opaque Tissue Previously Cleared by iDISCO
Recent advancements in tissue-clearing techniques and volumetric imaging have greatly facilitated visualization and quantification of biomolecules, organelles, and cells in intact organs or even entire organisms. Generally, there are two types of clearing methods: hydrophobic and hydrophilic (i.e., clearing with organic or aqueous solvents, respectively). The popular iDISCO approach and its modifications are hydrophobic methods that involve dehydration, delipidation, decolorization (optional), decalcification (optional), and refractive-index (RI) matching steps. Cleared samples are often stored for a relatively long period of time and imaged repeatedly. However, cleared tissues can become opaque over time, which prevents accurate reimaging. We reasoned that the resurgent haziness is likely due to rehydration, residual lipids, and uneven RI deep inside those tissue samples. For rescue, we have developed a simple procedure based on iDISCO. Beginning with a methanol dehydration, samples are delipidated using dichloromethane, followed by RI matching with dibenzyl ether (DBE). This simple method effectively re-clears mouse brains that have turned opaque during months of storage, allowing the user to effectively image immunolabeled samples over longer periods of time.Key features• This simple protocol rescues previously cleared tissue that has turned opaque.• The method does not cause detectable loss of immunofluorescence from previously stained samples.Graphical overview
Stem Cell
Immunofluorescent Staining Assay of 3D Cell Culture of Colonoids Isolated from Mice Colon
Here, we describe immunofluorescent (IF) staining assay of 3D cell culture colonoids isolated from mice colon as described previously. Primary cultures developed from isolated colonic stem cells are called colonoids. Immunofluorescence can be used to analyze the distribution of proteins, glycans, and small molecules—both biological and non-biological ones. Four-day-old colonoid cell cultures grown on Lab-Tek 8-well plate are fixed by paraformaldehyde. Fixed colonoids are then subjected to antigen retrieval and blocking followed by incubation with primary antibody. A corresponding secondary antibody tagged with desired fluorescence is used to visualize primary antibody–marked protein. Counter staining to stain actin filaments and nucleus to assess cell structure and DNA in nucleus is performed by choosing the other two contrasting fluorescences. IF staining of colonoids can be utilized to visualize molecular markers of cell behavior. This technique can be used for translation research by isolating colonoids from colitis patients’ colons, monitoring the biomarkers, and customizing their treatments.Key features• Analysis of molecular markers of cell behavior.Protocol to visualize proteins in 3D cell culture.• This protocol requires colonoids isolated from mice colon grown on matrigel support.• Protocol requires at least eight days to complete.Graphical overview