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Volume: 16, Issue: 10
Biochemistry
PEPTERGENT: A Peptide-Based Reagent for Detergent-Free Extraction of Membrane Proteins and Purification of Membrane Proteomes
Peptergent is a novel class of amphipathic peptides that enables detergent-free extraction of membrane proteins (MPs) from lipid bilayers. This reagent self-assembles around hydrophobic transmembrane regions, forming stable, water-soluble complexes that can be isolated directly from biological membranes. Peptergent therefore bypasses the limitations imposed by traditional detergents, which often destabilize protein assemblies. Since detergents are completely avoided, MPs are directly amenable to structural and mass spectrometry (MS) analysis, thereby addressing their persistent underrepresentation in proteomic datasets and improving their accessibility in drug-screening strategies. We present here a streamlined protocol for MPs extraction with the Peptergent PDET-1, followed by exchange into His-tagged Peptidiscs for Ni-NTA-based affinity purification. The method encompasses membrane isolation, peptide preparation, protein extraction, clarification, and MPs exchange from Peptergents to Peptidiscs. This workflow yields an enriched membrane proteome compatible with downstream LC-MS/MS analysis for improved identification of multi-pass MPs.
Bioinformatics and Computational Biology
A Step-by-Step GUI-Based Protocol for Molecular Dating Analysis Using PhyloSuite v2
In current genomic research, molecular dating is challenged by both imperfect substitution modeling and analysis efficiency, as genome-scale datasets often exhibit substantial rate heterogeneity and complex patterns of sequence evolution, which can make divergence-time estimation sensitive to modeling assumptions and computational settings. Meanwhile, commonly used molecular dating workflows remain operationally demanding; preparing correctly formatted inputs, implementing model settings, configuring fossil calibrations, and performing basic diagnostics and visualization frequently require multiple tools and extensive manual steps, resulting in high hands-on time and avoidable operational errors. To facilitate the practical implementation of molecular dating analyses and lower the operational barrier for users, this protocol describes a GUI-based workflow in PhyloSuite v2 for molecular dating analysis. Using a dataset of fish nuclear genomes as an example, the tutorial covers multi-format data import, visual configuration of fossil calibrations, automatic selection and implementation of substitution models, automation of complex analytical procedures, and assessment of Markov chain Monte Carlo (MCMC) convergence, along with data visualization. Through this protocol, users can quickly master the full workflow—from input preparation and molecular dating to MCMC sample statistical assessment and timetree visualization—thus significantly enhancing the efficiency of molecular dating analysis and result verification.
Biophysics
Using Single-Particle Fluorescence Microscopy to Quantify Substrate Binding of Peptidoglycan-Modification Enzymes
Peptidoglycan (PG), a network of glycan strands crosslinked by short peptides, is an essential and bacterial-specific structure that determines cell shape and protects cells from lysis. Understanding how bacteria assemble, maintain, and modify their PG not only addresses fundamental questions in cell biology but also provides a basis for developing strategies to treat bacterial infections. Although several in vitro methods, such as zymography, Remazol Brilliant Blue (RBB) assay, and LC-MS analyses, are available to quantify the activities of PG-modification enzymes, these approaches are not readily applicable in vivo. Here, we describe a single-particle tracking photo-activated localization microscopy (sptPALM)-based method to quantify the binding of enzymes to PG in vivo, which serves as a proxy for their enzymatic activities. Because the PG meshwork is relatively immobile, fluorescently tagged enzymes that transiently or stably bind it exhibit reduced mobility, reflected by lower diffusion coefficients. This approach provides sensitive, quantitative, and real-time insights into enzyme behavior in vivo under diverse physiological conditions or genetic backgrounds. The protocol is particularly valuable for investigating PG-modification enzymes that are essential or functionally redundant, which are often difficult to analyze using traditional genetic methods.
Cell Biology
Quantification of Spatial Patterns of Microtubule Transport by Kinesin-1 Head and Tail
The conventional kinesin-1 is a plus-end-directed microtubule-dependent motor protein with distinct motor head, stalk, and tail domains. Along with the motor head, which binds and walks along microtubules in an adenosine 5’-triphosphate (ATP) dependent manner, kinesin also contains a C-terminal microtubule binding tail. Motor-driven collective motility is well characterized using in vitro gliding assays, which show uninterrupted, smooth trajectories of transport. However, gliding assays driven by the full-length Drosophila kinesin-1 with both head and tail resulted in the emergence of spontaneous spatial microtubule patterns and stop-and-go motion. This was reproduced by an equimolar ratio of the active head and passive tail. Here, we describe the detailed protocol to reconstitute these microtubule gliding assays using multiple motor types: the full-length kinesin-1, the motor head or tail, mixtures of both head and tail, and a rigor mutant of the kinesin. We provide details of the approach taken to acquire the image time-series, to then quantify the spatial patterns that result from these motor combinations. Our approach provides a framework to systematically characterize the spatiotemporal effects of molecular motor-driven collective microtubule transport.
Isolation and Biophysical Characterization of Extracellular Vesicles Released by Myocytes
Extracellular vesicles (EVs) are lipid bilayer–enclosed vesicles released by diverse cell types and found in various body fluids. Because their composition and cargo dynamically respond to physiological and environmental cues, EVs hold promise both as biomarkers and as carriers for therapeutic delivery. Skeletal muscle functions as an endocrine organ, secreting myokines and EVs that modulate a wide range of cellular processes. The murine C2C12 cell line is a widely used in vitro model for investigating muscle biology. Here, we describe a protocol for isolating EVs from differentiated C2C12 myocytes. The isolated EVs are characterized and validated using western blotting, transmission electron microscopy (TEM), and dynamic light scattering (DLS) analysis. This workflow provides a robust platform for studying the molecular composition and functional roles of muscle-derived EVs.
Immunology
A Versatile In Vitro Quantitative Assay for Macrophage Efferocytosis in Diverse Research Applications
Macrophage efferocytosis is a previously unrecognized key pathogenic event, engulfing apoptotic targets, preventing inflammation and necrosis, and maintaining immune homeostasis. The phagocytic function can be disrupted by harmful factors and toxic substances. This protocol describes a versatile visualized in vitro method that can be used for the detection of general efferocytosis. This method is applicable to a wide range of research scenarios. As a representative application, it can be used to evaluate macrophage efferocytosis dysfunction in diseases linked to harmful exposures, including atherosclerosis, chronic inflammation, and malignant tumors. Among them, the detection of the effects of oxidized low-density lipoprotein (ox-LDL) and arsenite on macrophage efferocytosis capacity is an exemplary application of this protocol. Primary macrophages collected from mice were labeled with a cell-tracking dye and exposed to ox-LDL or arsenite, then co-cultured with apoptotic thymocytes or hepatocytes (labeled with another cell-tracking dye) for 2 h at a ratio of 5:1. Macrophage efferocytosis was visualized using a laser confocal microscope. The results indicate that arsenite impaired macrophage efferocytosis, leading to insufficient clearance of apoptotic thymocytes or hepatocytes. This method can be extended to subsequent studies, including those involving different types of phagocytes, apoptotic cell models, and research related to exposure to various factors.
Microbiology
An In Vitro A-431 Epithelial Cell Infection Model for Studying Fungal Pathogenicity and Immune Responses Associated With Vulvovaginal Candidiasis
Vulvovaginal candidiasis (VVC), also known as vaginal thrush, is an infection of the vulvovaginal mucosa caused by fungi of the Candida genus. Particularly for patients suffering from recurrent infection, the disease has a significant impact on their quality of life. The still unknown aspects of disease pathogenesis, as well as factors driving the development of infections and recurrence, represent a challenge for both clinical practitioners and patients. Mouse models and patient studies have suggested important roles of the microbiome, deployment of fungal pathogenicity mechanisms in the vagina, and dysregulated immune responses for VVC pathology. Dissecting their individual contributions can reveal specific processes associated with infection and may inspire novel therapeutic strategies. Epithelial in vitro infection models have been playing a key role in dissecting a crucial interaction during VVC, the invasion and infection of the vaginal mucosa. They have been instrumental in characterizing candidalysin as a fungal toxin that damages epithelial cells and elicits initial inflammatory responses to catalyze downstream inflammation. Moreover, they have also revealed potential protective immune pathways. Such a standardized epithelial cell infection model offers high versatility and compatibility with different downstream assays to link epithelial responses with other processes during VVC. This protocol describes a general A-431 vulvovaginal epithelial cell–Candida infection model in detail and provides several adaptations, such as live-cell imaging and mRNA silencing, as well as possible follow-up readouts, like the quantification of cytokine release, cytotoxicity, and neutrophil recruitment to study diverse processes relevant to VVC research.
Plasmid Curing of Pseudoalteromonas haloplanktis TAC125 Using Homologous Recombination and PTasRNA Gene Silencing
Pseudoalteromonas haloplanktis TAC125 is a psychrophilic marine bacterium widely used to study cold adaptation and increasingly exploited as a non-conventional platform for biotechnological applications. The strain harbors the endogenous megaplasmid pMEGA (64.7 kb), whose presence may limit its exploitation as a cell factory, making its elimination advantageous to strain engineering. Traditional plasmid-curing approaches based on chemical and physical agents are often inefficient and unsuitable for stable endogenous replicons, such as pMEGA. Here, we describe a targeted protocol for pMEGA curing in P. haloplanktis TAC125 that combines homologous recombination with paired-termini antisense RNA (PTasRNA) gene silencing. First, a selectable marker cassette is inserted into pMEGA by homologous recombination using a suicide vector, enabling selective discrimination between plasmid-positive and plasmid-cured bacteria. Next, PTasRNA gene silencing technology is applied to target a gene essential for the replication of pMEGA, thereby transiently interfering with its replication and promoting its loss. This approach provides a specific method to cure a highly stable endogenous megaplasmid in a psychrophilic non-conventional bacterium, enabling improved functional studies and strain optimization, establishing a broadly applicable framework for targeted curing across diverse bacterial systems.
Neuroscience
Chemoenzymatic Labeling Method for Detection of O-GlcNAcylated α-Synuclein Proteins by Western Blot
α-Synuclein (α-syn) aggregation has emerged as a key pathogenetic feature in several neurodegenerative disorders. The α-syn protein has various conformational strains, each with unique structural features that influence their cytotoxicity, propagation, and neuroinflammation. A post-translational modification known as O-GlcNAcylation has been found to influence the toxicity of α-syn and its propensity to aggregate. Difficulties in detecting and quantifying this modification are a major challenge to understanding its roles among the conformational forms of α-syn. We now describe a protocol for detecting O-GlcNAcylated α-syn that combines a click chemistry labeling approach and western blotting. This chemoenzymatic method involves the transfer of azido-modified galactose (GalNAz) from UDP-GalNAz to O-GlcNAcylated proteins, enabling their further functionalization with alkyne-containing polyethylene glycol of defined molecular weight. This protocol facilitates the determination of the glycosylation status of varying conformations of α-syn and their stoichiometric ratios.
Plant Science
3D Reconstruction of Mature Arabidopsis Ovules Using FIB-SEM to Study Filiform Apparatus Morphology
Volume electron microscopy based on serial sectioning allows for three-dimensional (3D) visualization and analysis of the internal structures of tissues, cells, and organelles. One such technique, focused ion beam (FIB) scanning electron microscopy (SEM), has the advantages of nanoscale sectioning and high z-resolution, but the disadvantage of limited volume processing. Because of this limitation, targeting localized objects by FIB-SEM is difficult. Here, we developed a FIB-SEM observation workflow that enables the analysis of the filiform apparatus of synergid cells enclosed in the Arabidopsis ovule. In this protocol, plant samples are stained, embedded, trimmed, and carbon-coated while maintaining their orientation within the tissue. Then, sequential observations are performed using Cut & See function of FIB-SEM, followed by image processing for 3D reconstruction. Utilization of multi-scanning and image cropping from high-resolution data helps to identify localized targets within plant tissue. The filiform apparatus, which is an invaginated cell wall structure of the synergid cells, shows distinct contrast in each image, allowing for segmentation using brightness-based binarization. Such segmentation avoids the need to manually trace complex structures and facilitates 3D reconstruction by volume electron microscopy.
Analysis of Cauline Leaf Development in Arabidopsis thaliana Using Time-Lapse Confocal Microscopy
Understanding cellular growth dynamics in plants requires precise, long-term imaging of developing tissues. Cauline leaves are produced during the transition from vegetative to reproductive development and provide a useful system for studying how laminar organs diversify in form and function. While other laminar organs, such as rosette leaves and sepals, have been extensively studied, early cauline leaf development remains technically challenging to capture due to their concealed position, curved morphology, and the presence of dense trichomes. Here, we provide a complete pipeline for the dissection, confocal imaging, 2.5D segmentation, and image analysis of initiating cauline leaves in Arabidopsis thaliana. This method enables reproducible, high-resolution imaging of cauline leaves, supporting robust quantitative analysis of growth across developmental stages at cellular scale resolution.
Biolayer Interferometry (BLI) to Quantify RALF1–Pectin Interactions
Cellular function relies on a network of precisely regulated interactions among macromolecules such as proteins, peptides, carbohydrates, and nucleic acids. These molecular interactions regulate vital processes, including signaling, structural organization, and developmental patterning. Biolayer interferometry (BLI) is a label-free optical biosensing technique that enables real-time quantification of such interactions. This protocol describes how to use BLI to assess the binding affinity between a biotinylated plant peptide hormone (RALF1) and cell wall–derived oligogalacturonides (OG25–50) on the Octet RED96 platform. Streptavidin-coated biosensors are employed to immobilize the ligand, while analyte binding is monitored through wavelength shifts in the reflected light. The protocol includes detailed steps for sensor preparation, assay setup, software configuration, and kinetic data analysis. While optimized for plant peptide–matrix interactions, the method is broadly adaptable to other macromolecular systems across biological disciplines.
Correction
