Search Results (89)

Adenosine triphosphate (ATP) is readily released into the extracellular space as an autocrine and paracrine purinergic signaling molecule. We originally reported a genetically encoded, fluorescent protein-based Förster Resonance Energy Transfer (FRET) biosensor that can detect micromolar levels of extracellular ATP. Through mutagenesis of the ATP binding site and optimization of cell-surface display, here we report the development of a second-generation biosensor called ECATS2 with greater than three-fold higher affinity for extracellular ATP. We found that the tether length between the FRET biosensor and the cell surface anchor is critical to optimization of its performance. Furthermore, we demonstrate that the improved sensor can detect extracellular ATP release upon hypoosmotic stress in cultured astrocytes. This new sensor contributes an improved tool for the ratiometric detection of extracellular ATP dynamics and purinergic signaling. Full article

Genetically encoded fluorescent protein (FP)-based biosensors have revolutionized cell biology research by enabling real-time monitoring of molecular activities in live cells with exceptional spatial and temporal resolution. Multiplexed biosensing advances this capability by allowing the simultaneous tracking of multiple signaling pathways to uncover network interactions and dynamic coordination. However, challenges in spectral overlap limit broader implementation. Innovative strategies have been devised to address these challenges, including spectral separation through FP palette expansion and novel biosensor designs, temporal differentiation using photochromic or reversibly switching FPs, and spatial segregation of biosensors to specific subcellular regions or through cell barcoding techniques. Combining multiplexed biosensors with artificial intelligence-driven analysis holds great potential for uncovering cellular decision-making processes. Continued innovation in this field will deepen our understanding of molecular networks in cells, with implications for both fundamental biology and therapeutic development. Full article

iPSCs and their derivatives are used to investigate the molecular genetic mechanisms of human diseases, to identify therapeutic targets, and to screen for small molecules. Combining technologies for generating patient-specific iPSC lines and genome editing allows us to create cell models with unique characteristics. We obtained and characterized three iPSC lines by reprogramming peripheral blood mononuclear cells of a patient with Huntington’s disease (HD) using episomal vectors encoding Yamanaka factors. iPSC lines expressed pluripotency marker genes, had normal karyotypes and were capable of differentiating into all three germ layers. The obtained iPSC lines are useful for modeling disease progression in vitro and studying pathological mechanisms of HD, such as ER stress. A transgene of genetically encoded biosensor XBP1-TagRFP was introduced into the iPSCs to visualize ER stress state of cells. The study demonstrated that iPSC-derived medium spiny neurons develop ER stress, though the IRE1-mediated pathway does not seem to be involved in the process. Full article

Metabolism is a tightly controlled, but plastic network of pathways that allow cells to grow and maintain homeostasis. As a normal cell transforms into a malignant cancer cell and proliferates to establish a tumor, it utilizes a variety of metabolic pathways that support growth, proliferation, and survival. Cancer cells alter metabolic pathways in different contexts, leading to complex metabolic heterogeneity within a tumor. There is an unmet need to characterize how cancer cells alter how they use resources from the environment to evolve, spread to other sites of the body, and survive current standard-of-care therapies. We review key techniques and methods that are currently used to study cancer metabolism and provide drawbacks and considerations in using one over another. The goal of this review is to provide a methods’ guide to study different aspects of cell and tissue metabolism, how they can be applied to cancer, and discuss future perspectives on advancements in these areas. Full article

γ-Secretase has primarily been studied in neurons, whereas increasing evidence highlights its importance in microglia. Previous research has shown that the pharmacological inhibition of γ-secretase impairs microglial phagocytic activity. In this study, we used a genetically encoded Förster resonance energy transfer (FRET)-based biosensor to record γ-secretase activity, aiming to determine if naturally occurring cell-by-cell variations in endogenous γ-secretase activity are associated with phagocytic activity. Using the Notch1 N100 Y-T biosensor, we found that the regulation of endogenous γ-secretase activity varies among individual BV-2 microglial cells. Our multiplexed time-lapse imaging revealed that the phagocytosis of E. coli bioparticles was impaired in cells with lower γ-secretase activity compared to those with higher activity. Complementary biochemical analysis, utilizing Zymosan bioparticles and fluorescence-activated cell sorting (FACS), further demonstrated that cells with reduced phagocytic activity exhibited decreased endogenous γ-secretase activity. Collectively, our confirmatory study supports previous findings that microglial phagocytic activity is closely linked to γ-secretase and emphasizes the essential role of γ-secretase in microglia. Full article

Background: Tauopathy has been identified as a prevalent causative agent of neurodegenerative diseases, including frontotemporal dementia with parkinsonism-17 (FTDP-17). This rare hereditary neurodegenerative condition is characterised by the manifestation of parkinsonism and behavioural changes. The majority of cases of FTDP-17 are associated with mutations in the MAPT gene, which encodes the tau protein. MAPT mutations lead to disruption of the balance between 3R and 4R tau forms, which causes destabilisation of microtubules and impairment of cellular organelle functions, particularly mitochondrial dysfunction. The development of model systems and tools for studying the molecular, genetic, and biochemical mechanisms underlying FTDP-17 and testing therapies at the cellular level is an urgent necessity. Methods: In this study, we generated transgenic lines of induced pluripotent stem cells (iPSCs) from a patient carrying the pathogenic mutation c.2013T > G (rs63750756, p.N279K) of MAPT and a healthy donor. A doxycycline-controlled transgene of the genetically encoded biosensor MitoTimer was integrated into the AAVS1 locus of these cells. The MitoTimer biosensor allows for lifetime monitoring of the turnover of mitochondria in neuronal cells derived from directed iPSC differentiation. The fact that transcription of the transgene can be induced by doxycycline provides additional possibilities for pulse labelling of newly formed mitochondria. Results: Transgenic iPSC lines provide a unique tool to study the molecular and genetic mechanisms of FTDP-17 caused by the presence of the c.2013T > G (p.N279K) mutation, as well as to test potential drugs in vitro. Full article

Multiple antibiotic resistance regulators (MarRs) control the transcription of genes in the mar operon of Escherichia coli in the presence of salicylic acid (SA). The interaction with SA induces conformational changes in the MarR released from the promoter of the mar operon, turning on transcription. We constructed an SA-specific E. coli cell-based biosensor by fusing the promoter of the mar operon (P_marO) and the gene that encodes an enhanced green fluorescent protein (egfp). Because SA and aspirin are structurally similar, a biosensor for monitoring aspirin can be obtained by genetically engineering MarR to be aspirin (ASP)-responsive. To shift the selectivity of MarR toward ASP, we changed the residues around the ligand-binding sites by site-directed mutagenesis. We examined the effects of genetic engineering on MarR by introducing MarRs with P_marO-egfp into E. coli. Among the tested mutants, MarR T72A improved the ASP responses by approximately 3 times compared to the wild-type MarR, while still showing an SA response. Although the MarR T72A biosensor exhibited mutual interference between SA and ASP, it accurately determined the ASP concentration in spiked water and medicine samples with over 90% accuracy. While the ASP biosensors still require improvement, our results provide valuable insights for developing E. coli cell-based biosensors for ASP and transcription factor-based biosensors in general. Full article

Malate is a key intermediate in the citric acid cycle, an enzymatic cascade that is central to cellular energy metabolism and that has been applied to make biofuel cells. To enable real-time sensing of malate levels, we have engineered a genetically encoded, protein-based fluorescent biosensor called Malon specifically responsive to malate by performing structure-based mutagenesis of the Cache-binding domain of the Citron GFP-based biosensor. Malon demonstrates high specificity and fluorescence activation in response to malate, and has been applied to monitor enzymatic reactions in vitro. Furthermore, we successfully incorporated Malon into redox polymer hydrogels and bacterial cells, enabling analysis of malate levels in these materials and living systems. These results show the potential for fluorescent biosensors in enzymatic cascade monitoring within biomaterials and present Malon as a novel tool for bioelectronic devices. Full article

For rapid and convenient detection of living endothelial cells (ECs) specifically without immunostaining, we developed a biosensor based on turn-on fluorescent protein, named LV-EcpG. It includes a high-affinity peptide E12P obtained through phage display technology for specifically recognizing ECs and a turn-on EGFP fused with two linker peptides. The “on-off” switching mechanism of this genetically encoded fluorescent protein-based biosensor (FPB) ensured that fluorescence signals were activated only when binding with ECs, thus enabling these FPB characters for direct, visual, and non-invasive detection of ECs. Its specificity and multicolor imaging capability established LV-EcpG as a powerful tool for live EC research, with significant potential for diagnosing and treating cardiovascular diseases and tumor angiogenesis. Full article

Microbial alkane degradation pathways provide biological routes for converting these hydrocarbons into higher-value products. We recently reported the functional expression of a methyl-alkylsuccinate synthase (Mas) system in Escherichia coli, allowing for the heterologous anaerobic activation of short-chain alkanes. However, the enzymatic activation of methane via natural or engineered alkylsuccinate synthases has yet to be reported. To address this, we employed high-throughput screening to engineer the itaconate (IA)-responsive regulatory protein ItcR (WT-ItcR) from Yersinia pseudotuberculosis to instead respond to methylsuccinate (MS, the product of methane addition to fumarate), resulting in genetically encoded biosensors for MS. Here, we describe ItcR variants that, when regulating fluorescent protein expression in E. coli, show increased sensitivity, improved overall response, and enhanced specificity toward exogenously added MS relative to the wild-type repressor. Structural modeling and analysis of the ItcR ligand binding pocket provide insights into the altered molecular recognition. In addition to serving as biosensors for screening alkylsuccinate synthases capable of methane activation, MS-responsive ItcR variants also establish a framework for the directed evolution of other molecular reporters, targeting longer-chain alkylsuccinate products or other succinate derivatives. Full article

Zebrafish larvae have emerged as a valuable model for studying heart physiology and pathophysiology, as well as for drug discovery, in part thanks to its transparency, which simplifies microscopy. However, in fluorescence-based optical mapping, the beating of the heart results in motion artifacts. Two approaches have been employed to eliminate heart motion during calcium or voltage mapping in zebrafish larvae: the knockdown of cardiac troponin T2A and the use of myosin inhibitors. However, these methods disrupt the mechano-electric and mechano-mechanic coupling mechanisms. We have used ratiometric genetically encoded biosensors to image calcium in the beating heart of intact zebrafish larvae because ratiometric quantification corrects for motion artifacts. In this study, we found that halting heart motion by genetic means (injection of tnnt2a morpholino) or chemical tools (incubation with para-aminoblebbistatin) leads to bradycardia, and increases calcium levels and the size of the calcium transients, likely by abolishing a feedback mechanism that connects contraction with calcium regulation. These outcomes were not influenced by the calcium-binding domain of the gene-encoded biosensors employed, as biosensors with a modified troponin C (Twitch-4), calmodulin (mCyRFP1-GCaMP6f), or the photoprotein aequorin (GFP-aequorin) all yielded similar results. Cardiac contraction appears to be an important regulator of systolic and diastolic Ca²⁺ levels, and of the heart rate. Full article

C-terminal Src kinase (CSK) is the major inhibitory kinase for Src family kinases (SFKs) through the phosphorylation of their C-tail tyrosine sites, and it regulates various types of cellular activity in association with SFK function. As a cytoplasmic protein, CSK needs be recruited to the plasma membrane to regulate SFKs’ activity. The regulatory mechanism behind CSK activity and its subcellular localization remains largely unclear. In this work, we developed a genetically encoded biosensor based on fluorescence resonance energy transfer (FRET) to visualize the CSK activity in live cells. The biosensor, with an optimized substrate peptide, confirmed the crucial Arg¹⁰⁷ site in the CSK SH2 domain and displayed sensitivity and specificity to CSK activity, while showing minor responses to co-transfected Src and Fyn. FRET measurements showed that CSK had a relatively mild level of kinase activity in comparison to Src and Fyn in rat airway smooth muscle cells. The biosensor tagged with different submembrane-targeting signals detected CSK activity at both non-lipid raft and lipid raft microregions, while it showed a higher FRET level at non-lipid ones. Co-transfected receptor-type protein tyrosine phosphatase alpha (PTPα) had an inhibitory effect on the CSK FRET response. The biosensor did not detect obvious changes in CSK activity between metastatic cancer cells and normal ones. In conclusion, a novel FRET biosensor was generated to monitor CSK activity and demonstrated CSK activity existing in both non-lipid and lipid raft membrane microregions, being more present at non-lipid ones. Full article

Endoplasmic reticulum (ER) stress is involved in the pathogenesis of many human diseases, such as cancer, type 2 diabetes, kidney disease, atherosclerosis and neurodegenerative diseases, in particular Parkinson’s disease (PD). Since there is currently no treatment for PD, a better understanding of the molecular mechanisms underlying its pathogenesis, including the mechanisms of the switch from adaptation in the form of unfolded protein response (UPR) to apoptosis under ER stress conditions, may help in the search for treatment methods. Genetically encoded biosensors based on fluorescent proteins are suitable tools that facilitate the study of living cells and visualization of molecular events in real time. The combination of technologies to generate patient-specific iPSC lines and genetically encoded biosensors allows the creation of cell models with new properties. Using CRISPR-Cas9-mediated homologous recombination at the AAVS1 locus of iPSC with the genetic variant p.N370S (rs76763715) in the GBA1 gene, we created a cell model designed to study the activation conditions of the IRE1-XBP1 cascade of the UPR system. The cell lines obtained have a doxycycline-dependent expression of the genetically encoded biosensor XBP1-TagRFP, possess all the properties of human pluripotent cells, and can be used to test physical conditions and chemical compounds that affect the development of ER stress, the functioning of the UPR system, and in particular, the IRE1-XBP1 cascade. Full article

Engineered microorganisms such as the probiotic strain Escherichia coli Nissle 1917 (EcN) offer a strategy to sense and modulate the concentration of metabolites or therapeutics in the gastrointestinal tract. Here, we present an approach to regulate the production of the depression-associated metabolite gamma-aminobutyric acid (GABA) in EcN using genetic circuits that implement negative feedback. We engineered EcN to produce GABA by overexpressing glutamate decarboxylase and applied an intracellular GABA biosensor to identify growth conditions that improve GABA biosynthesis. We next employed characterized genetically encoded NOT gates to construct genetic circuits with layered feedback to control the rate of GABA biosynthesis and the concentration of GABA produced. Looking ahead, this approach may be utilized to design feedback control of microbial metabolite biosynthesis to achieve designable smart microbes that act as living therapeutics. Full article

Neurotransmitters (NTs) are endogenous low-molecular-weight chemical compounds that transmit synaptic signals in the central nervous system. These NTs play a crucial role in facilitating signal communication, motor control, and processes related to memory and learning. Abnormalities in the levels of NTs lead to chronic mental health disorders and heart diseases. Therefore, detecting imbalances in the levels of NTs is important for diagnosing early stages of diseases associated with NTs. Sensing technologies detect NTs rapidly, specifically, and selectively, overcoming the limitations of conventional diagnostic methods. In this review, we focus on the fluorescence-based biosensors that use nanomaterials such as metal clusters, carbon dots, and quantum dots. Additionally, we review biomaterial-based, including aptamer- and enzyme-based, and genetically encoded biosensors. Furthermore, we elaborate on the fluorescence mechanisms, including fluorescence resonance energy transfer, photon-induced electron transfer, intramolecular charge transfer, and excited-state intramolecular proton transfer, in the context of their applications for the detection of NTs. We also discuss the significance of NTs in human physiological functions, address the current challenges in designing fluorescence-based biosensors for the detection of NTs, and explore their future development. Full article