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The glucose uptake in skeletal muscle is essential to produce energy through ATP, which is needed by this organ to maintain vital functions. The impairment of glucose uptake compromises the metabolism and function of skeletal muscle and other organs and is a feature of diabetes, obesity, and ageing. There is a need for research to uncover the mechanisms involved in the impairment of glucose uptake in skeletal muscle. In this study, we adapted, developed, optimised, and validated a methodology based on the fluorescence glucose analogue 6-NBDG, combined with a quantitative fluorescence microscopy image analysis, to determine the glucose uptake in two models of skeletal muscle cells: C2C12 myotubes and single fibres isolated from muscle. It was proposed that reactive oxygen and nitrogen species (RONS) and redox homeostasis play an important role in the modulation of intracellular redox signalling pathways associated with glucose uptake. In this study, we prove that the prooxidative intracellular redox environment under oxidative eustress produced by RONS such as hydrogen peroxide and nitric oxide improves glucose uptake in skeletal muscle cells. However, when oxidation is excessive, oxidative distress occurs, and cellular viability is compromised, although there might be an increase in the glucose uptake. Based on the results of this study, the determination of 6-NBDG/glucose uptake in myotubes and skeletal muscle cells is feasible, validated, and will contribute to improve future research.

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It is well-established that cancer and normal cells can be differentiated based on the altered sequence and expression of specific proteins. There are only a few examples, however, showing that cancer and normal cells can be differentiated based on the altered distribution of proteins within intracellular compartments. Here, we review available data on shifts in the intracellular distribution of two proteins, the membrane associated beta-catenin and the actin-binding protein CapG. Both proteins show altered distributions in cancer cells compared to normal cells. These changes are noted (i) in steady state and thus can be visualized by immunohistochemistry-beta-catenin shifts from the plasma membrane to the cell nucleus in cancer cells; and (ii) in the dynamic distribution that can only be revealed using the tools of quantitative live cell microscopy-CapG shuttles faster into the cell nucleus of cancer cells. Both proteins may play a role as prognosticators in gynecologic malignancies: beta-catenin in endometrial cancer and CapG in breast and ovarian cancer. Thus, both proteins may serve as examples of altered intracellular protein distribution in cancer and normal cells.

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The ability to automatically analyze large quantities of image data is a valuable tool for many biochemical assays, as it rapidly provides reliable data. Here, we describe a fast and robust Fiji macro for the analysis of cellular fluorescence microscopy images with single-cell resolution. The macro presented here was validated by successful reconstruction of fluorescent and non-fluorescent cell mixing ratios (for fluorescence fractions ranging between 0 and 100%) and applied to quantify the efficiency of transfection and virus infection inhibition. It performed well compared with manually obtained image quantification data. Its use is not limited to the cases shown here but is applicable for most monolayered cellular assays with nuclei staining. We provide a detailed description of how the macro works and how it is applied to image data. It can be downloaded free of charge and may be used by and modified according to the needs of the user. • Rapid, simple, and reproducible segmentation of eukaryotic cells in confluent cellular assays • Open-source software for use without technical or computational expertise • Single-cell analysis allows identification and quantification of virus infected cell populations and infection inhibition.

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Neurodegenerative disorders (NDs) are diverse age-related conditions also described as "conformational diseases." The hallmark of NDs is the accumulation of disease-specific proteins as toxic misfolded aggregates in some areas of the brain. They lead to the loss of protein homeostasis (proteostasis) that causes neuronal dysfunction and death. A potential therapeutic strategy for NDs is to prevent the accumulation of misfolded proteins by activating the heat shock response (HSR). The HSR maintains proteostasis through the upregulation of heat shock proteins (HSPs), molecular chaperones that recognize misfolded proteins, and either refold them to their functional conformations and/or target them for degradation. However, how to manipulate the expression of HSPs to obtain a therapeutic effect in neurons remains unclear. Furthermore, the regulation of the HSR in neurons is more complex than what we have learned from culturing somatic nonneuronal cells. This chapter describes a method to investigate the induction of HSP70 in primary hippocampal neurons using single-molecule fluorescence in situ hybridization (smFISH). Quantification of smFISH provides the means to analyze neuron-to-neuron variability in the activation of the HSR and enables us to study the transcriptional induction and localization of HSP70 mRNA in primary neurons. This information might be critical to find the druggable steps for developing effective therapies to treat age-related NDs.

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PURPOSE: Transporters at the blood-cerebrospinal fluid (CSF) barrier (BCSFB) play active roles in removing drugs and toxins from the CSF. The goal of this study is to develop a fluorescence microscopy approach to quantitatively study the transepithelial transport processes at the murine BCSFB in real time. METHODS: Choroid plexus (CP) tissues were isolated from mouse lateral ventricles and incubated with anionic (fluorescein-methotrexate, 8-fluorescein-cAMP) or cationic (IDT307) fluorescent probes. The CSF-to-blood transport was imaged and quantified using compartmental segmentation and digital image analysis. Real time images were captured and analyzed to obtain kinetic information and identify the rate-limiting step. The effect of transporter inhibitors was also evaluated. RESULTS: The transport processes of fluorescent probes can be captured and analyzed digitally. The intra- and inter- animal variability were 20.4% and 25.7%, respectively. Real time analysis showed distinct transport kinetics and rate-limiting step for anionic and cationic probes. A CP efflux index was proposed to distinguish between transepithelial flux and intracellular accumulation. Rifampin and MK571 decreased the overall transepithelial transport of anionic probes by more than 90%, indicating a possible involvement of organic anion transporting polypeptides (Oatps) and multidrug resistance-associated proteins (Mrps). CONCLUSIONS: A CP isolation method was described, and a quantitative fluorescence imaging approach was developed to evaluate CSF-to-blood transport in mouse CP. The method is consistent, reproducible, and capable of tracking real time transepithelial transport with temporal and spatial resolution. The approach can be used to evaluate transport mechanisms, assess tissue drug accumulation, and assay potential drug-drug interactions at the BCSFB.

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Lipid nanoparticles have been clinically successful in particular recently within the vaccine field, but better tools are needed to analyze heterogeneities at the single particle level to progress drug delivery designs to the next level. Especially, liposomal nanocarriers are becoming increasingly complex e.g. by employing environmental cues for shedding their protective PEG layer, however a detailed mechanistic understanding of how the dePEGylation varies from liposome-to-liposome is still missing. Here we present the development of a fluorescence microscopy based assay capable of detecting the enzyme mediated dePEGylation of individual liposomes. We employ this methodology to understand how enzyme type-, concentration- and incubation time, in addition to liposome size, affects the dePEGylation at the single particle level.

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Reactive oxygen and nitrogen species (RONS) play an important role in the pathophysiology of skeletal muscle and are involved in the regulation of intracellular signaling pathways, which drive metabolism, regeneration, and adaptation in skeletal muscle. However, the molecular mechanisms underlying these processes are unknown or partially uncovered. We implemented a combination of methodological approaches that are funded for the use of genetically encoded biosensors associated with quantitative fluorescence microscopy imaging to study redox biology in skeletal muscle. Therefore, it was possible to detect and monitor RONS and glutathione redox potential with high specificity and spatio-temporal resolution in two models, isolated skeletal muscle fibers and C2C12 myoblasts/myotubes. Biosensors HyPer3 and roGFP2-Orp1 were examined for the detection of cytosolic hydrogen peroxide; HyPer-mito and HyPer-nuc for the detection of mitochondrial and nuclear hydrogen peroxide; Mito-Grx1-roGFP2 and cyto-Grx1-roGFP2 were used for registration of the glutathione redox potential in mitochondria and cytosol. G-geNOp was proven to detect cytosolic nitric oxide. The fluorescence emitted by the biosensors is affected by pH, and this might have masked the results; therefore, environmental CO2 must be controlled to avoid pH fluctuations. In conclusion, genetically encoded biosensors and quantitative fluorescence microscopy provide a robust methodology to investigate the pathophysiological processes associated with the redox biology of skeletal muscle.

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Elastin-like polypeptides (ELPs) are modular, stimuli-responsive materials that self-assemble into protein-rich microdomains in response to heating. By cloning ELPs to effector proteins, expressed intracellular fusions can even modulate cellular pathways. A critical step in engineering these fusions is to determine and control their intracellular phase transition temperature (Tt). To do so, this Method paper describes a simple live-cell imaging technique to estimate the Tt of non-fluorescent ELP fusion proteins by co-transfection with a fluorescent ELP marker. Intracellular microdomain formation can then be visualized in live cells through the co-assembly of the non-fluorescent and fluorescent ELP fusion proteins. If the two ELP fusions have different Tt, the intracellular ELP mixture phase separates at the temperature corresponding to the fusion with the lower Tt. In addition, co-assembled ELP microdomains often exhibit pronounced differences in size or number, compared to single transfected treatments. These features enable live-cell imaging experiments and image analysis to determine the intracellular Tt of a library of related ELP fusions. As a case study, we employ the recently reported Caveolin1-ELP library (CAV1-ELPs). In addition to providing a detailed protocol, we also report the development of a useful FIJI plugin named SIAL (Simple Image Analysis Library), which contains programs for image randomization and blinding, phenotype scoring, and ROI selection. These tasks are important parts of the protocol detailed here and are also commonly employed in other image analysis workflows.

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Liposomes are the most used drug delivery vehicle and their therapeutic function is closely linked to their lipid composition. Since most liposome characterization is done using bulk techniques, providing only ensemble averages, the lipid composition of all liposomes within the same formulation are typically assumed to be identical. Here we image individual liposomes using confocal microscopy to quantify that liposomal drug delivery formulations, including multiple component mixtures mimicking Doxil, display more than 10-fold variation in their relative lipid composition. Since liposome function is tightly regulated by the physicochemical properties bestowed by the lipid composition, such significant variations could render only a fraction of liposomes therapeutically active. Additionally, we quantified how this degree of compositional inhomogeneity was modulated by liposome preparation method, the saturation state of the membrane lipid, and whether anti-fouling polyethylene glycol (PEG) conjugated lipids were added to the initial lipid mix or inserted after liposome formation. We believe the insights into the factors governing the degree of inhomogeneity offers the possibility for producing more uniform liposomal drug delivery systems, potentially increasing their therapeutic efficacy.

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Caveolae are membrane organelles formed by submicron invaginations in the plasma membrane, and are involved in mechanosensing, cell signaling, and endocytosis. Although implicated broadly in physiology and pathophysiology, better tools are required to elucidate the precise role of caveolar processes through selective activation and inactivation of their trafficking. Our group recently reported that thermally-responsive elastin-like polypeptides (ELPs) can trigger formation of 'genetically engineered protein microdomains (GEPMs)' functionalized with either Clathrin-light chain or the epidermal growth factor receptor. This manuscript is the first report of this strategy to modulate caveolin-1 (CAV1). By attaching different ELP sequences to CAV1, mild heating can be used to self-assemble CAV1-ELP microdomains inside of cells. The temperature of self-assembly can be controlled by tuning the ELP sequence. The formation of CAV1-ELP microdomains internalizes Cholera Toxin Subunit B, a commonly used marker of caveolae mediated endocytosis. CAV1-ELPs also colocalize with Cavin 1, an essential component of functional caveolae biogenesis. With the emerging significance of caveolae in health and disease and the lack of specific probes to rapidly and reversibly affect caveolar function, CAV1-ELP microdomains are a new tool to rapidly probe caveolae associated processes in endocytosis, cell signaling, and mechanosensing.

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Development and homeostasis of multicellular organisms is largely controlled by complex cell-cell signaling networks that rely on specific binding of secreted ligands to cell surface receptors. The Wnt signaling network, as an example, involves multiple ligands and receptors to elicit specific cellular responses. To understand the mechanisms of such a network, ligand-receptor interactions should be characterized quantitatively, ideally in live cells or tissues. Such measurements are possible using fluorescence microscopy yet challenging due to sample movement, low signal-to-background ratio and photobleaching. Here, we present a robust approach based on fluorescence correlation spectroscopy with ultra-high speed axial line scanning, yielding precise equilibrium dissociation coefficients of interactions in the Wnt signaling pathway. Using CRISPR/Cas9 editing to endogenously tag receptors with fluorescent proteins, we demonstrate that the method delivers precise results even with low, near-native amounts of receptors.

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Colocalization aims at characterizing spatial associations between two fluorescently tagged biomolecules by quantifying the co-occurrence and correlation between the two channels acquired in fluorescence microscopy. Colocalization is presented either as the degree of overlap between the two channels or the overlays of the red and green images, with areas of yellow indicating colocalization of the molecules. This problem remains an open issue in diffraction-limited microscopy and raises new challenges with the emergence of superresolution imaging, a microscopic technique awarded by the 2014 Nobel prize in chemistry. We propose GcoPS, for Geo-coPositioning System, an original method that exploits the random sets structure of the tagged molecules to provide an explicit testing procedure. Our simulation study shows that GcoPS unequivocally outperforms the best competitive methods in adverse situations (noise, irregularly shaped fluorescent patterns, and different optical resolutions). GcoPS is also much faster, a decisive advantage to face the huge amount of data in superresolution imaging. We demonstrate the performances of GcoPS on two biological real data sets, obtained by conventional diffraction-limited microscopy technique and by superresolution technique, respectively.

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Visualization of single mRNAs in their native cellular environment provides key information to study gene expression regulation. This fundamental biological question triggered the development of the MS2-MCP (MS2-Capsid Protein) system to tag mRNAs and image their life cycle using widefield fluorescence microscopy. The last two decades have evolved toward improving the qualitative and quantitative characteristics of the MS2-MCP system. Here, we provide a protocol to use the latest versions, MS2V6 and MS2V7, to tag and visualize mRNAs in mammalian cells in culture. The motivation behind engineering MS2V6 and MS2V7 was to overcome a degradation caveat observed in S. cerevisiae with the previous MS2-MCP systems. While for yeast we recommend the use of MS2V6, we found that for live-cell imaging experiments in mammalian cells, the MS2V7 has improved reporter properties.

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Selective transport across the nuclear envelope (NE) is mediated by the nuclear pore complex (NPC), a massive ∼100-MDa assembly composed of multiple copies of ∼30 nuclear pore proteins (Nups). Recent advances have shed light on the composition and structure of NPCs, but approaches that could map their organization in live cells are still lacking. Here, we introduce an in vivo method to perform nuclear radial intensity measurements (NuRIM) using fluorescence microscopy to determine the average position of NE-localized proteins along the nucleocytoplasmic transport axis. We apply NuRIM to study the organization of the NPC and the mobile transport machinery in budding yeast. This reveals a unique snapshot of the intact yeast NPC and identifies distinct steady-state localizations for various NE-associated proteins and nuclear transport factors. We find that the NPC architecture is robust against compositional changes and could also confirm that in contrast to Chlamydomonas reinhardtii, the scaffold Y complex is arranged symmetrically in the yeast NPC. Furthermore, NuRIM was applied to probe the orientation of intrinsically disordered FG-repeat segments, providing insight into their roles in selective NPC permeability and structure.

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The cytoskeleton is key to many essential processes in a plant cell, e.g., growth, division, and defense. Contrary to what "skeleton" implies, the cytoskeleton is highly dynamic, and is able to re-organize itself continuously. The advent of live-cell microscopy and the development of genetically encoded fluorophores enabled detailed observation of the organization and dynamics of the cytoskeleton. Despite the biological importance of the cytoskeletal dynamics, quantitative analyses remain laborious endeavors that only a handful of research teams regularly conduct. With this protocol, we provide a standardized step-by-step guide to analyze the dynamics of microtubules. We provide example data and code for post-processing in Fiji that enables researchers to modify and adapt the routine to their needs. More such tools are needed to quantitatively assess the cytoskeleton and thus to better understand cell biology. © 2019 by John Wiley & Sons, Inc.

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Nanoparticle (NP) interactions with cells and organisms are mediated by a biomolecular adsorption layer, the so-called "protein corona." An in-depth understanding of the corona is a prerequisite to successful and safe application of NPs in biology and medicine. In this work, earlier in situ investigations on small NPs are extended to large polystyrene (PS) NPs of up to 100 nm diameter, using human transferrin (Tf) and human serum albumin (HSA) as model proteins. Direct NP sizing experiments reveal a reversibly bound monolayer protein shell (under saturating conditions) on hydrophilic, carboxyl-functionalized (PS-COOH) NPs, as was earlier observed for much smaller NPs. In contrast, protein binding on hydrophobic, sulfated (PS-OSO3 H) NPs in solvent of low ionic strength is completely irreversible; nevertheless, the thickness of the observed protein corona again corresponds to a protein monolayer. Under conditions of reduced charge repulsion (higher ionic strength), the NPs are colloidally unstable and form large clusters below a certain protein-NP stoichiometric ratio, indicating that the adsorbed proteins induce NP agglomeration. This comprehensive characterization of the persistent protein corona on PS-OSO3 H NPs by nanoparticle sizing and quantitative fluorescence microscopy/nanoscopy reveals mechanistic aspects of molecular interactions occurring during exposure of NPs to biofluids.

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Giardia trophozoites have developed resistance mechanisms to currently available compounds, leading to treatment failures. In this context, the development of new additional agents is mandatory. Sirtuins, which are class III NAD+-dependent histone deacetylases, have been considered important targets for the development of new anti-parasitic drugs. Here, we evaluated the activity of KH-TFMDI, a novel 3-arylideneindolin-2-one-type sirtuin inhibitor, on G. intestinalis trophozoites. This compound decreased the trophozoite growth presenting an IC50 value lower than nicotinamide, a moderately active inhibitor of yeast and human sirtuins. Light and electron microscopy analysis showed the presence of multinucleated cell clusters suggesting that the cytokinesis could be compromised in treated trophozoites. Cell rounding, concomitantly with the folding of the ventro-lateral flange and flagella internalization, was also observed. These cells eventually died by a mechanism which lead to DNA/nuclear damage, formation of multi-lamellar bodies and annexin V binding on the parasite surface. Taken together, these data show that KH-TFMDI has significant effects against G. intestinalis trophozoites proliferation and structural organization and suggest that histone deacetylation pathway should be explored on this protozoon as target for chemotherapy.

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The modulation of expression levels of fluorescent fusion proteins (FFPs) is central for recombinant DNA technologies in modern biology as overexpression of proteins contributes to artifacts in biological experiments. In addition, some microscopy techniques such as fluorescence correlation spectroscopy (FCS) and single-molecule-based techniques are very sensitive to high expression levels of FFPs. To reduce the levels of recombinant protein expression in comparison with the commonly used, very strong CMV promoter, the herpes simplex virus thymidine kinase (TK) gene promoter, and mutants thereof were analyzed. Deletion mutants of the TK promoter were constructed and introduced into the Gateway® system for ectopic expression of enhanced green fluorescent protein (eGFP), monomeric cherry (mCherry), and FFPs containing these FPs. Two promoter constructs, TK2ST and TKTSC, were established, which have optimal low expression levels suitable for FCS studies in U2OS, HeLa CCL2, NIH 3T3, and BALB/c cells. Interestingly, when tested in these four cell lines, promoter constructs having a deletion within TK gene 5'-UTR showed significantly higher protein expression levels than the equivalent constructs lacking this deletion. This suggests that a negative regulatory element is localized within the TK gene 5'-UTR.

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The nuclear pore complex (NPC) is an eightfold symmetrical channel providing selective transport of biomolecules across the nuclear envelope. Each NPC consists of ∼30 different nuclear pore proteins (Nups) all present in multiple copies per NPC. Significant progress has recently been made in the characterization of the vertebrate NPC structure. However, because of the estimated size differences between the vertebrate and yeast NPC, it has been unclear whether the NPC architecture is conserved between species. Here, we have developed a quantitative image analysis pipeline, termed nuclear rim intensity measurement (NuRIM), to precisely determine copy numbers for almost all Nups within native NPCs of budding yeast cells. Our analysis demonstrates that the majority of yeast Nups are present at most in 16 copies per NPC. This reveals a dramatic difference to the stoichiometry determined for the human NPC, suggesting that despite a high degree of individual Nup conservation, the yeast and human NPC architecture is significantly different. Furthermore, using NuRIM, we examined the effects of mutations on NPC stoichiometry. We demonstrate for two paralog pairs of key scaffold Nups, Nup170/Nup157 and Nup192/Nup188, that their altered expression leads to significant changes in the NPC stoichiometry inducing either voids in the NPC structure or substitution of one paralog by the other. Thus, our results not only provide accurate stoichiometry information for the intact yeast NPC but also reveal an intriguing compositional plasticity of the NPC architecture, which may explain how differences in NPC composition could arise in the course of evolution.

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Quantitative fluorescence microscopy techniques are frequently applied to answer fundamental biological questions. Single-molecule RNA imaging methods have enabled the direct observation of the initial steps of the mRNA life cycle in living cells, however, the dynamic mechanisms that regulate mRNA translation are still poorly understood. We have developed an RNA biosensor that can assess the translational state of individual mRNA transcripts with spatiotemporal resolution in living cells. In this chapter, we describe how to perform a TRICK (translating RNA imaging by coat protein knock-off) experiment and specifically focus on a detailed description of our image processing and data analysis procedure.

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