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Eukaryotic cells have been constantly challenged throughout their evolution by pathogens, mechanical stresses, or toxic compounds that induce plasma membrane (PM) or endolysosomal membrane damage. The survival of the wounded cells depends on damage detection and repair machineries that are evolutionary conserved between protozoan, plants, and animals. We use the social amoeba Dictyostelium discoideum as a model system to study bacteria, mechanical or sterile membrane damage that allows us to identify and monitor factors involved in PM, endolysosomal damage response (ELDR), and endolysosomal homeostasis. Importantly, the sterile damage techniques presented here homogenously affect cell populations, which allows to phenotype mutant strains and quantify various aspects of cell fitness using live cell microscopy. This is instrumental to functionally assess genes involved in the repair of damaged plasma membrane or intracellular compartments and the degradation of extensively damaged compartments. Here, we describe how to inflict sterile PM or endolysosomal membrane damage, how to monitor the cell-intrinsic response to damage, and how to proxy proton leakage from damaged acidic compartments and quantify cell viability.

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PIN proteins establish the auxin concentration gradient, which coordinates plant growth. PIN1-4 and 7 localized at the plasma membrane (PM) and facilitate polar auxin transport while the endoplasmic reticulum (ER) localized PIN5 and PIN8 maintain the intracellular auxin homeostasis. Although an antagonistic activity of PIN5 and PIN8 proteins in regulating the intracellular auxin homeostasis and other developmental events have been reported, the membrane topology of these proteins, which might be a basis for their antagonistic function, is poorly understood. In this study we optimized digitonin based PM-permeabilizing protocols coupled with immunocytochemistry labeling to map the membrane topology of PIN5 and PIN8 in Arabidopsis thaliana root cells. Our results indicate that, except for the similarities in the orientation of the N-terminus, PIN5 and PIN8 have an opposite orientation of the central hydrophilic loop and the C-terminus, as well as an unequal number of transmembrane domains (TMDs). PIN8 has ten TMDs with groups of five alpha-helices separated by the central hydrophilic loop (HL) residing in the ER lumen, and its N- and C-terminals are positioned in the cytoplasm. However, the topology of PIN5 comprises nine TMDs. Its N-terminal end and the central HL face the cytoplasm while its C-terminus resides in the ER lumen. Overall, this study shows that PIN5 and PIN8 proteins have a divergent membrane topology while introducing a toolkit of methods for studying membrane topology of integral proteins including those localized at the ER membrane.

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We report the development of a 384-well formatted NanoBRET assay to characterize molecular glues of 14-3-3/client interactions in living cells. The seven isoforms of 14-3-3 are dimeric hub proteins with diverse roles including transcription factor regulation and signal transduction. 14-3-3 interacts with hundreds of client proteins to regulate their function and is therefore an ideal therapeutic target when client selectivity can be achieved. We have developed the NanoBRET system for three 14-3-3σ client proteins CRAF, TAZ, and estrogen receptor α (ERα), which represent three specific binding modes. We have measured stabilization of 14-3-3σ/client complexes by molecular glues with EC50 values between 100 nM and 1 µM in cells, which align with the EC50 values calculated by fluorescence anisotropy in vitro. Developing this NanoBRET system for the hub protein 14-3-3σ allows for a streamlined approach, bypassing multiple optimization steps in the assay development process for other 14-3-3σ clients. The NanoBRET system allows for an assessment of PPI stabilization in a more physiologically relevant, cell-based environment using full-length proteins. The method is applicable to diverse protein-protein interactions (PPIs) and offers a robust platform to explore libraries of compounds for both PPI stabilizers and inhibitors.

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Immunocytochemistry combined with confocal or superresolution microscopy allows us to observe molecular localization and intracellular structures. However, it is challenging to analyze individual neurons in brain tissue, where neurons are densely packed. In contrast, we can easily observe structures such as the axonal growth cone and dendritic spines in dissociated individual neurons. Thus, the immunocytochemistry of primary cultured neurons is often used because it reflects the in vivo condition at least in part. Here, we describe a method for indirect fluorescence immunocytochemistry of primary cultured neurons from the embryonic cerebral cortex. This involves multiple steps including fixation, permeabilization, and antibody reaction, and in particular, we introduce an optimized protocol for permeabilization to enable the precise localization of target molecules.

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In this work, iron oxide (Fe3O4) magnetic nanoparticles (MNPs) and graphene oxide (GO) nanosheets were prepared via the co-precipitation technique and the Modified Hummer method. Fe3O4 MNPs and GO nanosheets were combined to prepare Fe3O4/GO nanocomposite and subsequently conjugated with Digitonin (DIG) in order to obtain a dual-targeted delivery system based on DIG/Fe3O4/GO nanocomposite. SEM images reveal the presence of Fe3O4 MNPs at a scale of 100 nm, exhibiting dispersion between the GO nanosheets. Aggregation of the DIG/Fe3O4/GO nanocomposite was observed at various size scales. The XRD structural analysis confirms the crystal structure of the prepared samples. The Fe3O4 MNPs demonstrated the main XRD-diffracted peaks. Also, GO nanosheets exhibit crystalline characteristics on the (001) and (002) planes. The predominant peaks observed in the DIG/GO/Fe3O4 nanocomposite are attributed to the crystal phases of Fe3O4 MNPs. The FT-IR vibrational modes observed in the GO/DIG/Fe3O4 nanocomposite indicate the presence of crosslinking between GO nanosheet layers and the Fe3O4 MNPs. The antioxidant activity of the prepared samples was measured and the DIG/GO/Fe3O4 nanocomposite demonstrated a significantly high antioxidant activity in both 2-diphenyl-1-picrylhydrazyl (DPPH·) and 2,2-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS·+) tests.

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Insect-transmitted trypanosomatid parasite infections cause life-threatening neglected tropical diseases (NTDs), including African sleeping sickness, Chagas disease and leishmaniasis. In these parasites, glycosomes are unique organelles that are essential for the parasite survival. Proper biogenesis of glycosomes is crucial to ensure correct compartmentation of the glycosomal metabolism. Genetic or chemical disruption of the glycosome biogenesis leads to a mislocalization of the glycosomal enzymes into the cytosol, which results in toxicity to the parasites. Here, we describe a detailed protocol for biochemical fractionation of Trypanosoma brucei parasites to detect mislocalization of glycosomal proteins to the cytosol. This approach utilizes increasing concentrations of digitonin that first permeabilizes the plasma membrane, followed by permeabilization of other organelles, depending on their cholesterol content. Fractionated samples can be further analyzed using immunoblotting for specific marker proteins or quantified by the specific enzyme activities.

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Migraine is a highly prevalent neurological disease affecting circa 1 billion patients worldwide with severe incapacitating symptoms, which significantly diminishes the quality of life. As self-medication practice, oral administration of triptans is the most common option, despite its relatively slow therapeutic onset and low drug bioavailability. To overcome these issues, here we present, to the best of our knowledge, the first study on the possibility of oral transmucosal delivery of one of the safest triptans, namely eletriptan hydrobromide (EB). Based on a comprehensive set of in vitro and ex vivo experiments, we highlight the conditions required for oral transmucosal delivery, potentially giving rise to similar, or even higher, drug plasma concentrations expected from conventional oral administration. With histology and tissue integrity studies, we conclude that EB neither induces morphological changes nor impairs the integrity of the mucosal barrier following 4 h of exposure. On a cellular level, EB is internalized in human oral keratinocytes within the first 5 min without inducing toxicity at the relevant concentrations for transmucosal delivery. Considering that the pKa of EB falls within the physiologically range, we systematically investigated the effect of pH on both solubility and transmucosal permeation. When the pH is increased from 6.8 to 10.4, the drug solubility decreases drastically from 14.7 to 0.07 mg/mL. At pH 6.8, EB gave rise to the highest drug flux and total permeated amount across mucosa, while at pH 10.4 EB shows greater permeability coefficient and thus higher ratio of permeated drug versus applied drug. Permeation experiments with model membranes confirmed the pH dependent permeation profile of EB. The distribution of EB in different cellular compartments of keratinocytes is pH dependent. In brief, high drug ionization leads to higher association with the cell membrane, suggesting ionic interactions between EB and the phospholipid head groups. Moreover, we show that the chemical permeation enhancer DMSO can be used to enhance the drug permeation significantly (i.e., 12 to 36-fold increase). Taken together, this study presents important findings on transmucosal delivery of eletriptan via the oral cavity and paves the way for clinical investigations for a fast and safe migraine treatment.

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The bioluminescent luciferin-luciferase reaction is based on the oxidation of D-luciferin by oxygen in the presence of ATP and magnesium ions, catalyzed by firefly luciferase. The possibilities of using this reaction to study the influence of external effectors of a physical and chemical nature (temperature exposure, additions of drugs, membrane-active compounds, etc.) on living cells (prokaryotes and eukaryotes) are considered. Examples of the use of test systems based on living cells producing thermostable firefly luciferase for monitoring cellular homeostasis are given. The study of the kinetics of changes in the concentration of ATP and luciferase inside and outside cells made it possible to determine in dynamics the metabolic activity, cytotoxicity, and survival of cells under conditions of cellular stress, to study the processes of ATP synthesis/hydrolysis, and to evaluate the effectiveness of lytic agents in changing the permeability of the cell membrane.

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The Na+/K+-ATPase α1 subunit (ATP1A1) is a potential target for hepatic carcinoma (HCC) treatment, which plays a key role in Na+/K+ exchange, metabolism, signal transduction, etc. In vivo, we found that Panax notoginseng saponins (PNS) could inhibit tumor growth and significantly downregulate the expression and phosphorylation of ATP1A1/AKT/ERK in tumor-bearing mice. Our study aims to explore the potential effects of PNS on the regulation of ATP1A1 and the possible mechanisms of antitumor activity. The effects of PNS on HepG2 cell viability, migration, and apoptosis were examined in vitro. Fluorescence, Western blot, and RT-PCR analyses were used to examine the protein and gene expression. Further analysis was assessed with a Na+/K+-ATPase inhibitor (digitonin) and sorafenib in vitro. We found that the ATP1A1 expression was markedly higher in HepG2 cells than in L02 cells and PNS exhibited a dose-dependent effect on the expression of ATP1A and the regulation of AKT/ERK signaling pathways. Digitonin did not affect the expression of ATP1A1 but attenuated the effects of PNS on the regulation of ATP1A1/AKT/ERK signaling pathways and enhanced the antitumor effect of PNS by promoting nuclear fragmentation. Taken together, PNS inhibited the proliferation of HepG2 cells via downregulation of ATP1A1 and signal transduction. Our findings will aid a data basis for the clinical use of PNS.

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The varied applications of nanotechnology have paved way for several breakthroughs in the realm of biomedical technology. In this challenging era when illness multiplies, timely and accurate disease diagnosis is very important. Thus, well founded novel approaches matter very much in areas like disease diagnosis and monitoring. Nanomedicine has tremendous implications in the given context. An elevated cholesterol concentration in blood is risky and is associated with cardiovascular diseases (CVD). CVD remains the No. 1 global cause of death and hence there is an urge to understand cholesterol level and take preventive measures. Highly fluorescent graphene quantum dots (GQs) are well known for their biocompatibility, non toxicity and aqueous solubility. Here in we report an easy and sensitive non enzymatic based cholesterol detection using digitonin conjugated graphene quantum dots (GDG). Selectivity studies and the cholesterol detection in human blood serum suggests the probe to be reliable and selective for blood cholesterol monitoring. Digitonin conjugated fluorescent graphene quantumdots, an efficient probe for cholesterol sensing.

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The BlueNative page (BNGE) gel has been the reference technique for studying the electron transport chain organization since it was established 20 years ago. Although the migration of supercomplexes has been demonstrated being real, there are still several concerns about its ability to reveal genuine interactions between respiratory complexes. Moreover, the use of different solubilization conditions generates conflicting interpretations. Here, we thoroughly compare the impact of different digitonin concentrations on the liquid dispersions' physical properties and correlate with the respiratory complexes' migration pattern and supercomplexes. Our results demonstrate that digitonin concentration generates liquid dispersions with specific size and variability critical to distinguish between a real association of complexes from being trapped in the same micelle.

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Saponins are a diverse group of secondary plant metabolites, some of which display hemolytic toxicity due to plasma membrane permeabilization. This feature is employed in biological applications for transferring hydrophilic molecules through cell membranes. Widely used commercial saponins include digitonin and saponins from soap tree bark, both of which constitute complex mixtures of little definition. We assessed the permeabilization power of pure saponins towards cellular membranes in an effort to detect novel properties and to improve existing applications. In a respirometric assay, we characterized half-maximal permeabilization of the plasma membrane for different metabolites, of the mitochondrial outer membrane for cytochrome C and the full solubilization of mitochondrial inner membrane protein complexes. Beyond the complete list as repository for the field, we highlight several findings with direct applicability. First, we identified and validated α-chaconine as alternative permeabilization agent in respirometric assays of cultured cells and isolated synaptosomes, superior to digitonin in its tolerability for mitochondria. Second, we identified glycyrrhizic acid to form exceptionally small pores impermeable for adenosine diphosphate. Third, in a concentration dependent manner, tomatine proved to be able to selectively permeabilize the mitochondrial outer, but not inner membrane, allowing for novel states in which to determine cytochrome C oxidase activity. In summary, we provide a list of the permeabilization properties of 18 pure saponins. The identification of two saponins, namely tomatine and chaconine, with direct usability in improved or novel cell biological applications within this small subgroup demonstrates the tremendous potential for further functional screening of pure saponins.

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The present paper discusses the use of monolayers of lipid mixtures mimicking the composition of biological membranes of bacteria, erythrocyte and yeast in the context of the anti-bacterial, hemolytic and anti-fungal activity of saponins. Saponins are plant-produced glycosidic biosurfactants with either steroidal or triterpenoidal aglycone. In the present study we used digitonin as a representative steroidal saponin (extracted from Digitalis purpurea) and a mixture of triterpenoid saponins from Quillaja saponaria Molina. The effect of saponins was studied first on monolayers consisting of single lipids characteristic for the given type of biological membrane, and then - on model mixed lipid monolayers. Finally, the monolayers were formed from total lipid extracts of natural cell membranes (E. coli and S. cerevisiae) to verify the results obtained in the simplified models. The effect of saponins on monolayers was studied by a combination of surface pressure relaxation, infrared reflection - absorption spectroscopy (IRRAS) and fluorescence microscopy. In line with expectations, sterols (cholesterol and ergosterol) play a major role in the saponin-lipid interactions in monolayers, which may explain especially the hemolytic and antifungal properties of saponins. In contrast, bacterial membranes are devoid of sterols, although the presence of similar compounds may be responsible for their affinity to saponins. Nevertheless, the effect of saponins on bacterial models is less pronounced than for the erythrocyte or fungal ones.

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Currently, SAMHD1 is the only known dNTPase in human cells. It also suppresses the replication of both retroviruses and retroelements. SAMHD1 contains a classic nuclear localization sequence (NLS) and resides in the nucleus in live or fixed cells. It has been reported that alteration or removal of NLS does not affect the dNTPase or the antiviral activity of SAMHD1. However, it was unclear whether the nuclear localization was involved in SAMHD1-mediated suppression against retroelements such as long interspersed element type 1 (LINE-1 or L1). In this study, we reported that SAMHD1 is a nucleocytoplasmic shuttling protein. Digitonin-based cytoplasm/nucleus fractionation tests suggested that SAMHD1 is capable of being exported from the nucleus, which was confirmed by introducing exogenous exportin Xpo1 in live cells. Interestingly, altering the protein's subcellular localization by mutating or removing NLS significantly enhances SAMHD1's potency in L1 suppression. Further tests with SAMHD1 mutants indicated that nucleocytoplasmic shuttling is important for SAMHD1-mediated L1 suppression. Finally, we demonstrated that the cytoplasmic distribution of SAMHD1 leads to an enhanced depletion of L1 ORF2p. Taken together, our data have revealed SAMHD1 as a nucleocytoplasmic shuttling protein, and associated such a new feature of SAMHD1 with its potency against L1 retrotransposition, which provides more insights to the understanding of SAMHD1 and its role in L1 suppression.

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Pollen tubes require functional mitochondria in order to achieve fast and sustained growth. In addition, cell wall expansion requires a calcium gradient in the tube apex formed by a dedicated array of calcium pumps and channels. Most studies have traditionally focused on the molecular aspects of calcium interactions and transport across the pollen tube plasmalemma. However, calcium transients across mitochondrial membranes from pollen tubes are beginning to be studied. Here, we report the presence of a ruthenium red-sensitive mitochondrial calcium uniporter-like activity in tobacco pollen tubes with functional oxidative phosphorylation. The present study provides a framework to measure in situ specifics of mitochondrial transport and respiration in pollen tubes from different plants. The relevance of a mitochondrial calcium uniporter for pollen tube growth is discussed.

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Every organ in the body is capable of synthesizing cholesterol de novo but at rates that vary with a constellation of factors. A significant proportion of the hydrogen atoms present in cholesterol that is synthesized in the body are derived from water. Thus, although water ordinarily makes up the bulk of body mass, the acute enrichment of the body water pool with a sufficiently large amount of tritiated water over a short interval of time (usually 1 h) yields measurable rates of incorporation of the labeled water into newly generated cholesterol and also fatty acids. Such data can provide a quantitative measure of how specific genetic, dietary, and pharmacological manipulations impact not just the rate of cholesterol synthesis in particular organs but also rates of whole-body cholesterol production and turnover.

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Many Gram-negative bacterial pathogens use type III secretion systems to export proteins that act directly on the host and aid in the infectious process. Extracellular bacteria primarily rely upon the type III secretion system to insert or inject effector proteins into the cytosol of their host cell in order to perturb intracellular signaling events and aid in pathogenesis. Intracellular bacteria can also depend on the T3SS translocation of effector proteins from vacuolar compartments into the vacuolar membrane or host cell cytosol where they can modulate intracellular trafficking and/or signaling pathways necessary for their growth and survival. Biochemical fractionation of infected cells in vitro enables detection of these events, making it possible to identify relevant protein-protein interactions, characterize phenotypes of mutant strains and understand how these effector proteins impact host cells. In this chapter we provide methods for the analysis of translocated effector proteins using biochemical and mechanical fractionation procedures.

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In general, cultivation and purification of intracellular pathogenic rickettsiae represents a risk for laboratory personnel due to exposure to highly infectious aerosol or accidental inoculation during these procedures. In this study, we describe an alternative, effective and time saving technique for rickettsial purification using digitonin to release intracellular bacteria from host cell without physical disruption. No significant differences were noted in yield and infectivity between digitonin treated rickettsiae and rickettsiae purified by sonication. This is the first report of using digitonin in purification of pathogenic rickettsiae and this approach might be effective for other intracellular pathogenic bacteria.

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Nuclei can be relatively easily extracted from homogenized liver due to the softness of the tissue and crudely separated from other cellular organelles by low-speed centrifugation due to the comparatively large size of nuclei. However, further purification is complicated by nuclear envelope continuity with the endoplasmic reticulum, invaginations containing mitochondria, and connections to the cytoskeleton. Subsequent purification to nuclear envelopes is additionally confounded by connections of inner nuclear membrane proteins to chromatin. For these reasons, it is necessary to confirm proteomic identification of nuclear envelope proteins by testing targeting of individual proteins. The proteomic identification of nuclear envelope fractions is affected by the tendencies of transmembrane proteins to have extreme isoelectric points, strongly hydrophobic peptides, posttranslational modifications, and a propensity to aggregate, thus making proteolysis inefficient. To circumvent these problems, we have developed a MudPIT approach that uses multiple extractions and sequential proteolysis to increase identifications. Here we describe methods for isolating nuclear envelopes, determining their proteome by MudPIT, and confirming their targeting to the nuclear periphery by microscopy.

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The high concentration of cholesterol in the plasma membrane relative to the endomembranes of eukaryotic cells allows the selective permeabilization of the plasma membrane with the glycoside digitonin leaving the intracellular membrane bound organelles intact. In this chapter, we describe the basic method to use digitonin permeabilized cells to reconstitute the transport of proteins containing nuclear localization signals into the nucleus. The assay requires only a target cell line that can be permeabilized with digitonin, a source of soluble transport factors, typically provided by the cytosol fraction of cultured cells, and a cargo protein of interest. No other specialized equipment is required other than a fluorescence microscope. The assay can be used to identify transport factors required to transport specific proteins, to study the regulation of protein transport, or to study nuclear protein transport under different conditions.

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