Using lanthanide-based resonance energy transfer for in vitro and in vivo studies of biological processes

This review describes key directions in the development of different probes based on complex compounds of lanthanides for in vitro and in vivo researches. The role of microsecond fluorescence of lanthanides for overcoming problems of background fluorescence is considered. The basic classes of synthetic and genetically encoded complex compounds of lanthanides are summarized. Main principles of designing lanthanide-based molecular sensors, including FRET sensors based on lanthanides and colored fluorescent proteins are described. Their applications in bioanalytical research and cellular bioimaging are described. The main principles of cellular bioimaging using lanthanides are formulated, questions of their delivery into cells are considered, and the problem of their potential toxicity for living organisms is discussed. A technique using multiphoton excitation of lanthanides is described.     

Understanding organellar protein folding capacities and assessing their pharmacological modulation by small molecules

Dysfunctional organellar protein quality control machinery leads to protein misfolding associated cardiovascular, neurodegenerative, metabolic and secretory disorders. To understand organellar homeostasis, suitable tools are required which can sense changes in their respective protein folding capacity upon exposure to environmental and pharmacological perturbations. Herein, we have assessed protein folding capacity of cellular organelles using a metastable sensor selectively targeted to cytosol, nucleus, mitochondria, endoplasmic reticulum, golgi and peroxisomes. Microscopy and biochemical data revealed that these sensors report both acute and organelle-specific cellular insults. It also provided insights into contrasting refolding capacities of cellular organelles to recover from proteotoxic challenges. Further, we used these metastable sensors to evaluate pharmacological modulation of organellar protein folding capacity by small molecules. We observed pyrazole based scaffolds increased organellar protein folding capacity through upregulation of chaperones, mainly HSP90 and its co-chaperone HOP which coordinate refolding of misfolded/aggregated species. Overall, our data highlights the potential use of organelle-specific metastable sensors to understand protein folding capacity of sub-cellular compartments and assess pharmacological correction of their proteostasis imbalance. This study also provides additional avenue for use of these organelle-specific metastable sensors in drug discovery programs for identification of novel pharmacophores and drug repositioning of promising scaffolds for protein conformational diseases associated with different cellular organelles.     

Thermodynamic and kinetic characterization of two methyl-accepting chemotaxis heme sensors from Geobacter sulfurreducens reveals the structural origin of their functional difference

The periplasmic sensor domains GSU582 and GSU935 are part of methyl-accepting chemotaxis proteins of the bacterium Geobacter sulfurreducens containing one c-type heme and a PAS-like fold. Their spectroscopic properties were shown previously to share similar spectral features. In both sensors, the heme group is in the high-spin form in the oxidized state and low-spin after reduction and binding of a methionine residue. Therefore, it was proposed that this redox-linked ligand switch might be related to the signal transduction mechanism. We now report the thermodynamic and kinetic characterization of the sensors GSU582 and GSU935 by visible spectroscopy and stopped-flow techniques, at several pH and ionic strength values. Despite their similar spectroscopic features, the midpoint reduction potentials and the rate constants for reduction by dithionite are considerably different in the two sensors. The reduction potentials of both sensors are negative and well framed within the typical anoxic subsurface environments in which Geobacter species predominate. The midpoint reduction potentials of sensor GSU935 are lower than those of GSU582 at all pH and ionic strength values and the same was observed for the reduction rate constants. The origin of the different functional properties of these closely related sensors is rationalized in the terms of the structures. The results suggest that the sensors are designed to function in different working potential ranges, allowing the bacteria to trigger an adequate cellular response in different anoxic subsurface environments. These findings provide an explanation for the co-existence of two similar methyl-accepting chemotaxis proteins in G. sulfurreducens.     

Cell culture models of fatty acid overload: Problems and solutions

High plasma levels of fatty acids occur in a variety of metabolic diseases. Cellular effects of fatty acid overload resulting in negative cellular responses (lipotoxicity) are often studied in vitro, in an attempt to understand mechanisms involved in these diseases. Fatty acids are poorly soluble, and thus usually studied when complexed to albumins such as bovine serum albumin (BSA). The conjugation of fatty acids to albumin requires care pertaining to preparation of the solutions, effective free fatty acid concentrations, use of different fatty acid species, types of BSA, appropriate controls and ensuring cellular fatty acid uptake. This review discusses lipotoxicity models, the potential problems encountered when using these cellular models, as well as practical solutions for difficulties encountered.     

Proteomic analysis of Giardia: Studies from the pre- and post-genomic era

The proteome of Giardia duodenalis has been under study for the last 25 years and has lead to the discovery of valuable information on the biology and variation of the parasite. Proteomic techniques, mainly SDS-PAGE and 2D-PAGE, have been used to investigate protein variation, cellular structure and host parasite interactions. This has allowed for the identification of assemblage and host specific proteins, structural proteins, proteins released by trophozoites upon exposure to host cell monolayers and immunoreactive proteins. These data are important in understanding the pathogenesis of G. duodenalis infections, as well as highlighting potential drug and vaccine targets. There is, however, a large amount of future work needed to fully understand the proteome of this parasite.     

Transient receptor potential channels meet phosphoinositides

Transient receptor potential (TRP) cation channels are unique cellular sensors that are involved in multiple cellular functions, ranging from transduction of sensory signals to the regulation of Ca²⁺ and Mg²⁺ homoeostasis. Malfunctioning of TRP channels is now recognized as the cause of several hereditary and acquired human diseases. At the time of cloning of the first Drosophila TRP channel, a close connection between gating and phosphatidylinositol phosphates (PIPs) was already recognized. In this review, we summarize current knowledge about the mechanisms of interaction between TRP channels and PIPs, and discuss the possible functional implications of TRP–PIP interactions to human physiology and pathophysiology.     

Genome-Wide Investigation and Expression Analyses of WD40 Protein Family in the Model Plant Foxtail Millet (Setaria italica L.)

WD40 proteins play a crucial role in diverse protein-protein interactions by acting as scaffolding molecules and thus assisting in the proper activity of proteins. Hence, systematic characterization and expression profiling of these WD40 genes in foxtail millet would enable us to understand the networks of WD40 proteins and their biological processes and gene functions. In the present study, a genome-wide survey was conducted and 225 potential WD40 genes were identified. Phylogenetic analysis categorized the WD40 proteins into 5 distinct sub-families (I–V). Gene Ontology annotation revealed the biological roles of the WD40 proteins along with its cellular components and molecular functions. In silico comparative mapping with sorghum, maize and rice demonstrated the orthologous relationships and chromosomal rearrangements including duplication, inversion and deletion of WD40 genes. Estimation of synonymous and non-synonymous substitution rates revealed its evolutionary significance in terms of gene-duplication and divergence. Expression profiling against abiotic stresses provided novel insights into specific and/or overlapping expression patterns of SiWD40 genes. Homology modeling enabled three-dimensional structure prediction was performed to understand the molecular functions of WD40 proteins. Although, recent findings had shown the importance of WD40 domains in acting as hubs for cellular networks during many biological processes, it has invited a lesser research attention unlike other common domains. Being a most promiscuous interactors, WD40 domains are versatile in mediating critical cellular functions and hence this genome-wide study especially in the model crop foxtail millet would serve as a blue-print for functional characterization of WD40s in millets and bioenergy grass species. In addition, the present analyses would also assist the research community in choosing the candidate WD40s for comprehensive studies towards crop improvement of millets and biofuel grasses.     

Monitoring cellular redox dynamics using newly developed BRET-based redox sensor proteins

Reactive oxygen species are key factors that strongly affect the cellular redox state and regulate various physiological and cellular phenomena. To monitor changes in the redox state, we previously developed fluorescent redox sensors named Re-Q, the emissions of which are quenched under reduced conditions. However, such fluorescent probes are unsuitable for use in the cells of photosynthetic organisms because they require photoexcitation that may change intracellular conditions and induce autofluorescence, primarily in chlorophylls. In addition, the presence of various chromophore pigments may interfere with fluorescence-based measurements because of their strong absorbance. To overcome these problems, we adopted the bioluminescence resonance energy transfer (BRET) mechanism for the sensor and developed two BRET-based redox sensors by fusing cyan fluorescent protein–based or yellow fluorescent protein–based Re-Q with the luminescent protein Nluc. We named the resulting redox-sensitive BRET-based indicator probes “ROBINc” and “ROBINy.” ROBINc is pH insensitive, which is especially vital for observation in photosynthetic organisms. By using these sensors, we successfully observed dynamic redox changes caused by an anticancer agent in HeLa cells and light/dark-dependent redox changes in the cells of photosynthetic cyanobacterium Synechocystis sp. PCC 6803. Since the newly developed sensors do not require excitation light, they should be especially useful for visualizing intracellular phenomena caused by redox changes in cells containing colored pigments.     

Smad7: not only a regulator, but also a cross-talk mediator of TGF-β signalling

Over the past decade, we have learned that cellular processes, including signalling and metabolism, are highly compartmentalized, and that relevant changes in metabolic state can occur at sub-second timescales. Moreover, we have learned that individual cells in populations, or as part of a tissue, exist in different states. If we want to understand metabolic processes and signalling better, it will be necessary to measure biochemical and biophysical responses of individual cells with high temporal and spatial resolution. Fluorescence imaging has revolutionized all aspects of biology since it has the potential to provide information on the cellular and subcellular distribution of ions and metabolites with sub-second time resolution. In the present review we summarize recent progress in quantifying ions and metabolites in populations of yeast cells as well as in individual yeast cells with the help of quantitative fluorescent indicators, namely FRET metabolite sensors. We discuss the opportunities and potential pitfalls and the controls that help preclude misinterpretation.     

RIG-I family RNA helicases: cytoplasmic sensor for antiviral innate immunity

Viral infection is detected by cellular sensors as foreign nucleic acid and initiates innate antiviral responses, including the activation of type I interferon (IFN) and proinflammatory cytokines. Recent advances in cytoplasmic virus sensors highlight their essential role in the induction of innate immunity. Moreover, it is intriguing to understand how they can discriminate innate RNA from viral foreign RNA. In this mini-review, we focus on these cytoplasmic virus sensors, termed retinoic acid inducible gene-I (RIG-I)-like receptors (RLRs), and discuss their function in the innate immune system.     

Genetically encoded red fluorescent copper(I) sensors for cellular copper(I) imaging

Copper ranks among the most important metal ions in living organism, owing to its key catalytic effect in a range of biochemical processes. Dysregulation of in vivo copper(I) metabolism is extremely toxic and would cause serious diseases in human, such as Wilson’s and Menkes. Thus, it would be highly valuable to have a proper approach to monitor the dynamics of copper(I) in vivo, as it is directly related to the onset of human copper(I)-related diseases. Under these circumstance, developing fluorescent protein based copper(I) sensors is highly demanded. However, these established sensors are mostly based on green or yellow FPs. Fluorescent copper(I) sensors with a spectra in the red range are more desirable due to lower phototoxicity, less auto-fluorescent noise and better penetration of red light. In the present work, we grafted a special red FP into three different location of a copper(I) binding protein, and generate a series of red fluorescent copper(I) sensors. Despite their limited in vivo sensitivity toward copper(I), these sensors are viable for cellular copper(I) imaging. Furthermore, these red fluorescent copper(I) sensors are a good starting point to develop superior copper(I) biosensors capable of imaging copper(I) fluctuations within a truly biologically relevant concentration, and further effort to realize this endeavor is under way.     

Phylogenetic aspects of the sulfate assimilation genes from Thalassiosira pseudonana

Diatoms are unicellular algae responsible for approximately 20 % of global carbon fixation. Their evolution by secondary endocytobiosis resulted in a complex cellular structure and metabolism compared to algae with primary plastids. In the last years the interest on unicellular algae increased. On the one hand assessments suggest that diatom-mediated export production can influence climate change through uptake and sequestration of atmospheric CO₂. On the other hand diatoms are in focus because they are discussed as potential producer of biofuels. To follow the one or other idea it is necessary to investigate the diatoms biochemistry in order to understand the cellular regulatory mechanisms. The sulfur assimilation and methionine synthesis pathways provide S-containing amino acids for the synthesis of proteins and a range of metabolites such as dimethylsulfoniopropionate (DMSP) in order to provide basic metabolic precursors needed for the diatoms metabolism. To obtain an insight into the localization and organization of the sulfur metabolism pathways, the genome of Thalassiosira pseudonana—a model organism for diatom research—might help to understand the fundamental questions on adaptive responses of diatoms to dynamic environmental conditions such as nutrient availability in a broader context.     

A General Workflow for Characterization of Nernstian Dyes and Their Effects on Bacterial Physiology

The electrical membrane potential (V_m) is one of the components of the electrochemical potential of protons across the biological membrane (proton motive force), which powers many vital cellular processes. Because V_m also plays a role in signal transduction, measuring it is of great interest. Over the years, a variety of techniques have been developed for the purpose. In bacteria, given their small size, Nernstian membrane voltage probes are arguably the favorite strategy, and their cytoplasmic accumulation depends on V_m according to the Nernst equation. However, a careful calibration of Nernstian probes that takes into account the tradeoffs between the ease with which the signal from the dye is observed and the dyes’ interactions with cellular physiology is rarely performed. Here, we use a mathematical model to understand such tradeoffs and apply the results to assess the applicability of the Thioflavin T dye as a V_m sensor in Escherichia coli. We identify the conditions in which the dye turns from a V_m probe into an actuator and, based on the model and experimental results, propose a general workflow for the characterization of Nernstian dye candidates.     

Current and emerging methods for probing neuropeptide transmission

Neuropeptides comprise the most diverse category of neurochemicals in the brain, playing critical roles in a wide range of physiological and pathophysiological processes. Monitoring neuropeptides with high spatial and temporal resolution is essential for understanding how peptidergic transmission is regulated throughout the central nervous system. In this review, we provide an overview of current non-optical and optical approaches used to detect neuropeptides, including their design principles, intrinsic properties, and potential limitations. We also highlight the advantages of using G protein‒coupled receptor (GPCR) activation‒based (GRAB) sensors to monitor neuropeptides in vivo with high sensitivity, good specificity, and high spatiotemporal resolution. Finally, we present a promising outlook regarding the development and optimization of new GRAB neuropeptide sensors, as well as their potential applications.     

Light driven hydrogen production in protein based semi-artificial systems

Photobiological hydrogen production has recently attracted interest in terms of being a potential source for an alternative energy carrier. Especially the natural light driven hydrogen metabolism of unicellular green algae appears as an attractive blueprint for a clean and potentially unlimited dihydrogen source. However, the efficiency of in vivo systems is limited by physiological and evolutionary constraints and scientists only begin to understand the regulatory networks influencing cellular hydrogen production. A growing number of projects aim at circumventing these limitations by focusing on semi-artificial systems. They reconstitute parts of the native electron transfer chains in vitro, combining photosystem I as a photoactive element with a proton reducing catalytic element such as hydrogenase enzymes or noble metal nanoparticles. This review summarizes various approaches and discusses limitations that have to be overcome in order to establish economically applicable systems.     

Surface plasmon resonance sensors in cell biology: basics and application

Optical sensors based on the excitation of surface plasmons, referred to as surface plasmon resonance (SPR) sensors, have become a central analytical tool for characterizing and quantifying a wide variety of macromolecular interactions, like receptor–ligand contacts. Besides this classical field of application, in the last 15 years, the development of SPR sensors aiming for the detection and analysis of ligand/cell or host/pathogen interactions, cell/cell contacts, and cellular reactions gained considerable momentum. The number of publications reporting about applications of SPR sensors implementing vital prokaryotic or eukaryotic cells as biorecognition elements for medical diagnostics, environmental monitoring, or biological safety is steadily growing. This review gives a short introduction to the technique of surface plasmon resonance and the parameters that are important for its application in the field of vital cell sensors. Furthermore, the publications concerning the application of such sensors in the analysis of cellular interactions and cellular reactions to extra- and intracellular stimuli are summarized.     

Live monitoring of plant redox and energy physiology with genetically encoded biosensors

Genetically encoded biosensors pave the way for understanding plant redox dynamics and energy metabolism on cellular and subcellular levels.

ADVANCES

• Methodological advances in fluorescent protein-based in vivo biosensing have been instrumental for several paradigm shifts in our understanding of cell physiology, metabolism and signaling.
• An increasing number of genetically encoded biosensors has been used to dissect the dynamics of several distinct redox couples and energy physiology in plants.
• In vivo monitoring using biosensors has pioneered the simultaneous read-out of different physiological parameters in different subcellular locations by parallelized plate reader-based, multiwell fluorimetry, or expression strategies for multiple sensors in parallel.
• Sensing dynamic changes in hydrogen peroxide levels is possible with sensors of the HyPer family, or roGFP fusion variants with a thiol peroxidase.
• Peredox and SoNar family sensors enable direct visualization of NADH/NAD⁺, while iNAP family sensors respond to NADPH concentration in plants.
• Sensor variants with different sensitivity ranges enable use of the most appropriate variant for the specific in vivo environment or experimental scope.