Biochemistry

The human pathogenic yeast Candida albicans can attach to epithelial cells or indwelling medical devices to form biofilms. These microbial communities are highly problematic in the clinic as they reduce both sensitivity to antifungal drugs and detection of fungi by the immune system. Amyloid structures are highly organized quaternary structures that play a critical role in biofilm establishment by allowing fungal cells to adhere to each other. Thus, fungal amyloids are exciting targets to develop new antifungal strategies. Thioflavin T is a specific fluorescent dye widely used to study amyloid properties of target proteins in vitro (spectrophotometry) and in vivo (epifluorescence/confocal microscopy). Notably, thioflavin T has been used to demonstrate the ability of Als5, a C. albicans adhesin, to form an amyloid fiber upon adhesion. We have developed a pipeline that allows us to study amyloid properties of target proteins using thioflavin T staining in vitro and in vivo, as well as in intact fungal biofilms. In brief, we used thioflavin T to sequentially stain (i) amyloid peptides, (ii) recombinant proteins, (iii) fungal cells treated or not with amyloid peptides, (iv) fungal amyloids enriched by cell fractionation, and (v) intact biofilms of C. albicans. Contrary to other methods, our pipeline gives a complete picture of the amyloid behavior of target proteins, from in vitro analysis to intact fungal biofilms. Using this pipeline will allow an assessment of the relevance of the in vitro results in cells and the impact of amyloids on the development and/or maintenance of fungal biofilm.

Key features

• Study of amyloid properties of fungal proteins.

• Visualization of the subcellular localization of fungal amyloid material using epifluorescence or confocal microscopy.

• Unraveling of the amyloid properties of target proteins and their physiological meaning for biofilm formation.

• Observation of the presence of amyloid structures with live-cell imaging on intact fungal biofilm using confocal microscopy.

Graphical overview

Biomolecular condensates are membrane-less assemblies of proteins and nucleic acids formed through liquid–liquid phase separation (LLPS). These assemblies are known to temporally and spatially regulate numerous biological activities and cellular processes in plants and animals. In vitro phase separation assay using recombinant proteins represents one of the standard ways to examine the properties of proteins undergoing LLPS. Here, we present a detailed protocol to investigate in vitro LLPS using in vitro expressed and purified recombinant proteins.

Nitrate (NO₃^–) is an essential element and nutrient for plants and animals. Despite extensive studies on the regulation of nitrate uptake and downstream responses in various cells, our knowledge of the distribution of nitrogen forms in different root cell types and their cellular compartments is still limited. Previous physiological models have relied on in vitro biochemistry and metabolite level analysis, which limits the ability to differentiate between cell types and compartments. Here, to address this, we report a nuclear-localized, genetically encoded fluorescent biosensor, which we named nlsNitraMeter3.0, for the quantitative visualization of nitrate concentration and distribution at the cellular level in Arabidopsis thaliana. This biosensor was specifically designed for nitrate measurements, not nitrite. Through genetic engineering to create and select sensors using yeast, Xenopus oocyte, and Arabidopsis expression systems, we developed a reversible and highly specific nitrate sensor. This method, combined with fluorescence imaging systems such as confocal microscopy, allows for the understanding and monitoring of nitrate transporter activity in plant root cells in a minimally invasive manner. Furthermore, this approach enables the functional analysis of nitrate transporters and the measurement of nitrate distribution in plants, providing a valuable tool for plant biology research. In summary, we provide a protocol for sensor development and a biosensor that can be used to monitor nitrate levels in plants.

Key features

• This protocol builds upon the concept of FRET biosensors for in vivo visualization of spatiotemporal nitrate levels at a cellular resolution.

• Nitrate levels can be quantified utilizing the biosensor in conjunction with either a plate reader or a fluorescence microscope.

Reconstitution of membrane proteins into large unilamellar vesicles is an essential approach for their functional analysis under chemically defined conditions. The orientation of the protein in the liposomal membrane after reconstitution depends on many parameters, and its assessment is important prior to functional measurements. Common approaches for determining the orientation of a membrane-inserted protein are based on limited proteolytic digest, impermeable labeling reagents for specific amino acids, or membrane-impermeable quenchers for fluorescent proteins. Here, we describe a simple site-specific fluorescent assay based on self-labeling enzyme tags to determine the orientation of membrane proteins after reconstitution, exemplified on a reconstituted SNAP-tag plant H⁺-ATPase. This versatile method should benefit the optimization of reconstitution conditions and the analysis of many types of membrane proteins.

Graphical abstract:

Immunofluorescence is a technique to visualize the localization of specific molecule targets within cells using the specificity of antibodies. Here, we describe a protocol to detect two different protein components in a cell simultaneously. Antibody concentrations to be used vary from cell to cell and should be optimized for different cell types. In this protocol, we perform co-immunofluorescence of mitochondrial ribosomal protein L7/L12 (MRPL12) and nuclear factor erythroid 2-related factor 2 (Nrf2), a potential transcription factor of MRPL12, in HK-2 cells, as an example. Taking advantage of the diverse set of antibodies raised in different species, we are able to analyze the colocalization and expression of these proteins.

Understanding the folding pathway of any protein is of utmost importance for deciphering the folding problems under adverse conditions. We can obtain important information about the folding pathway by monitoring the folding of any protein from its unfolded state. It is usually very difficult to monitor the folding process in real time as the process is generally very fast, and we need a suitable read out. In this protocol, we have solved this issue by using a protein that is non-fluorescent in its unfolded state but fluoresces in its native state after folding. The kinetics of refolding can be monitored by following the increase in fluorescence in real time. Previously, this was generally achieved by either monitoring a protein’s enzymatic activity or measuring the tryptophan fluorescence, where the signal output depends on well-described enzymatic activity or the frequency of tryptophan residues present in the proteins, respectively. Here, we describe a simple and real-time assay to monitor the refolding of sGFP, a recently described slow-folding mutant of yeGFP (yeast enhanced GFP). We unfold this protein using chemical denaturant and refold in a suitable buffer, monitoring the increase in fluorescence over time. GFP is fluorescent only when correctly folded; thus, using this technique, we can measure the true rate of protein refolding by following the increase in fluorescence over time. Therefore, sGFP can be used as an ideal model to study the in vitro protein folding process. Accordingly, the effects of different conditions and molecules on the protein folding pathway can be efficiently studied using sGFP as a model protein.

Graphical abstract:

Schematic of the steps involved in the sGFP refolding pathway. Native sGFP is unfolded by chemical denaturation using 6 M GuHCl at 25°C for 1 hour and then refolded in refolding buffer by 100-fold dilution.

The production of reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress are tightly linked. The generation of ROS can be both the cause and a consequence of ER stress pathways, and an increasing number of human diseases are characterized by tissue atrophy in response to ER stress and oxidative injury. For the assessment of modulators of ER luminal ROS generation and for mechanistic studies, methods to monitor changes in ER reduction-oxidation (redox) states in a time-resolved and organelle-specific manner are needed. This has been greatly facilitated by the development of genetically encoded fluorescent probes, which can be targeted to different subcellular locations by specific amino acid extensions. One of these probes is the yellow fluorescent protein-based redox biosensor, HyPer. Here, we provide a protocol for the time-resolved monitoring of the oxidizing milieu in the ER of adherent mammalian cells using the ratiometric sensor, HyPerER, which is specifically targeted to the ER lumen.

Immunofluorescence is a reliable method for identifying specific proteins in neuronal and glial cell populations of the hypothalamus. Several immunofluorescence protocols are available to detect protein markers and neuropeptides in the hypothalamus; however, published methods may vary in subtle details that can potentially impact the final outcome of the procedure. Here, we provide a detailed protocol suitable for thin cryostat sections, which has been successful for specific antibodies directed against key markers of hypothalamic neurons and glial cells. We include every detail concerning brain tissue collection, processing, sectioning, and labeling with optimal dilutions of antibodies with the aim of reducing non-specific background. Our background-optimized immunostaining protocol has been routinely used in the lab and allows efficient detection of specific neuropeptides, glial cells, and markers of inflammation and endoplasmic reticulum stress in the hypothalamus.

The intracellular interferon regulatory factor 5 (IRF5) dimerization assay is a technique designed to measure molecular interaction(s) with endogenous IRF5. Here, we present two methods that detect endogenous IRF5 homodimerization and interaction of endogenous IR5 with cell penetrating peptide (CPP) inhibitors. Briefly, to detect endogenous IRF5 dimers, THP-1 cells are incubated in the presence or absence of the IRF5-targeted CPP (IRF5-CPP) inhibitor for 30 min then the cells are stimulated with R848 for 1 h. Cell lysates are separated by native-polyacrylamide gel electrophoresis (PAGE) and IRF5 dimers are detected by immunoblotting with IRF5 antibodies. To detect endogenous interactions between IRF5 and FITC-labeled IRF5-CPP, an in-cell fluorescence resonance energy transfer (FRET) assay is used. In this assay, THP-1 cells are left untreated or treated with FITC-IRF5-CPP conjugated inhibitors for 1 h. Next, cells are fixed, permeabilized, and stained with anti-IRF5 and TRITC-conjugated secondary antibodies. Transfer of fluorescence can be measured and calculated as FRET units. These methods provide rapid and accurate assays to detect IRF5 molecular interactions.

Microtubules (MT) are the most rigid component of the cytoskeleton. Nevertheless, they often appear highly curved in the cellular context and the mechanisms governing their overall shape are poorly understood. Currently, in vitro microtubule analysis relies primarily on electron microscopy for its high resolution and Total Internal Reflection Fluorescence (TIRF) microscopy for its ability to image live fluorescently-labelled microtubules and associated proteins. For three-dimensional analyses of microtubules with micrometer curvatures, we have developed an assay in which MTs are polymerized in vitro from MT seeds adhered to a glass slide in a manner similar to conventional TIRF microscopy protocols. Free fluorescent molecules are removed and the MTs are fixed by perfusion. The MTs can then be observed using a confocal microscope with an Airyscan module for higher resolution. This protocol allows the imaging of microtubules that have retained their original three-dimensional shape and is compatible with high-resolution immunofluorescence detection.