Live Cell Monitoring Research Articles

Single-shot quantitative differential phase contrast microscope using a single calcite beam displacer

This paper presents the development of a single-shot, partially spatially coherent quantitative differential phase contrast microscopy (Q-DPCM) system. The optical scheme comprises a polarizer, lenses, calcite beam displacer, and analyzer, which can be seamlessly integrated to an existing bright-field microscopy system, transforming it into a Q-DPCM system. It utilizes a partially spatially coherent light source, enabling single-shot quantitative differential phase recovery of the specimens with high spatial phase sensitivity. It generates highly sensitive quantitative differential phase images of the specimens along one direction, like a gradient light interference microscopy (GLIM) system, using only a single interferogram. First, we validated the differential phase measurement capability of the system through experiments on polystyrene spheres (diameter 5.2 µm) and HeLa cells. Next, the system is utilized to generate quantitative phase maps of human red blood cells using two orthogonal differential interferograms recorded at two orientations of the calcite beam displacer. Further, the Q-DPCM system is implemented for 1-h time-lapse live cell monitoring, revealing the dynamics of intracellular granules such as nucleolus and lipids in U2OS cells.

Enhanced kinase translocation reporters for simultaneous real-time measurement of PKA, ERK, and Ca2.

Kinase translocation reporters (KTRs) are powerful tools for single-cell measurement of time-integrated kinase activity but suffer from restricted dynamic range and limited sensitivity, particularly in neurons. To address these limitations, we developed enhanced KTRs (eKTRs) for protein kinase A (PKA) and extracellular signal-regulated kinase (ERK) that display high sensitivity, rapid response kinetics, broad dynamic range, cell type-specific tuning, and an ability to detect PKA and ERK activity in primary sensory neurons. Moreover, co-expression of optically separable eKTRs for PKA and ERK revealed the kinetics of expected and unexpected crosstalk between PKA, ERK, protein kinase C, and calcium signaling pathways, demonstrating the utility of eKTRs for live cell monitoring of diverse and interacting signaling pathways. These results open the door to improved live-cell and in vivo measurements of key signaling pathways in neurons, while at the same time demonstrating the importance of KTR size and NLS strength to KTR dynamics.

Lighting up arginine metabolism reveals its functional diversity in physiology and pathology.

Arginine is one of the most metabolically versatile amino acids and plays pivotal roles in diverse biological and pathological processes; however, sensitive tracking of arginine dynamics in situ remains technically challenging. Here, we engineer high-performance fluorescent biosensors, denoted sensitive to arginine (STAR), to illuminate arginine metabolism in cells, mice, and clinical samples. Utilizing STAR, we demonstrate the effects of different amino acids in regulating intra- and extracellular arginine levels. STAR enabled live-cell monitoring of arginine fluctuations during macrophage activation, phagocytosis, efferocytosis, and senescence and revealed cellular senescence depending on arginine availability. Moreover, a simple and fast assay based on STAR revealed that serum arginine levels tended to increase with age, and the elevated serum arginine level is a potential indicator for discriminating the progression and severity of vitiligo. Collectively, our study provides important insights into the metabolic and functional roles of arginine, as well as its potential in diagnostic and therapeutic applications.

Obtaining source material for cellular agriculture

Cellular Agriculture (CellAg) is an attractive concept for innovative technology with the intent to provide food and nutrition complementary to existing supply streams. The past decade has seen considerable progress in the field with advancement of cellular technology that delivers the initial building blocks for meaningful implementation. The availability of natural cell-based material that can serve as nutrient-filled source for human consumption at low cost is a critical challenge for the emerging cellular agriculture industry. Therefore, here the isolation of bovine myofibroblasts of the Black Angus breed has been pursued and accomplished together with its characterisation by using RNA sequencing and proteomics through western blotting.To transition CellAg from a concept to a game changing technology for the industry, multiple challenges need to be overcome. The field requires powerful initial material, i.e., dedicated cells that can proliferate and differentiate robustly at scale. The methodology described allows for the production of healthy cells, which have been unequivocally characterized as clonal representatives of a stable myofibroblast cell line using transcriptomics and proteomics validation. Stringent and rigorous live cell monitoring of a nascent cell line derived from healthy muscle tissue allowed for stable cell growth.In this research article, a simple and precise methodology is presented for creating a bovine myofibroblast cell line (Bov.mia). Our work puts forward a low-tech use of materials and expertise that is devoid of transgenic approaches, thus creating a reliable approach for lab-scale research.

Next-generation 3D tumor modeling: A microfluidic platform with biocompatible red carbon dots for live cell imaging in co-cultured elongated spheroid tumor model

Co-culture spheroids mimic tumor architecture more accurately than traditional 2D cell cultures, but non-invasive, long-term tracking of live cells within these 3D models remains a challenge. This study addresses this critical need by developing a novel approach for live cell imaging in U-87/HUF co-culture spheroids. We introduce water-soluble, biocompatible red carbon dots (R-CDs) exhibiting exceptional stability and brightness (21% quantum yield) specifically designed for imaging within these 3D models. Furthermore, we designed a microfluidic chip with ellipsoid-shaped microwells to efficiently generate two distinct co-culture spheroid types: direct mixing and core-shell. R-CDs enabled non-invasive tracking of U-87 cancer cell location within these 3D models demonstrating their efficacy for long-term monitoring of live cells in cancer research. This R-CD and microfluidic technology has the potential to accelerate cancer drug discovery by enabling live cell studies in 3D tumor models.

Live Cell Monitoring of Phosphodiesterase Inhibition by Sulfonylurea Drugs.

Sulfonylureas (SUs) are a class of antidiabetic drugs widely used in the management of diabetes mellitus type 2. They promote insulin secretion by inhibiting the ATP-sensitive potassium channel in pancreatic β-cells. Recently, the exchange protein directly activated by cAMP (Epac) was identified as a new class of target proteins of SUs that might contribute to their antidiabetic effect, through the activation of the Ras-like guanosine triphosphatase Rap1, which has been controversially discussed. We used human embryonic kidney (HEK) 293 cells expressing genetic constructs of various Förster resonance energy transfer (FRET)-based biosensors containing different versions of Epac1 and Epac2 isoforms, alone or fused to different phosphodiesterases (PDEs), to monitor SU-induced conformational changes in Epac or direct PDE inhibition in real time. We show that SUs can both induce conformational changes in the Epac2 protein but not in Epac1, and directly inhibit the PDE3 and PDE4 families, thereby increasing cAMP levels in the direct vicinity of these PDEs. Furthermore, we demonstrate that the binding site of SUs in Epac2 is distinct from that of cAMP and is located between the amino acids E443 and E460. Using biochemical assays, we could also show that tolbutamide can inhibit PDE activity through an allosteric mechanism. Therefore, the cAMP-elevating capacity due to allosteric PDE inhibition in addition to direct Epac activation may contribute to the therapeutic effects of SU drugs.

Recent Developments in Small-Molecule Fluorescent Probes for Cellular Senescence

Cellular senescence is a recently emerged research topic in modern biology. Often described as a double-edged sword, it encompasses numerous essential biological processes, including beneficial effects such as wound healing and embryonic development, as well as detrimental contributions to chronic inflammation and tumor development. Consequently, there is an increasing need to unravel the intricate networks of senescence and develop reliable detection methods to distinguish it from related phenomena. To address these challenges, a variety of detection methods have been developed. In particular, small-molecule fluorescent probes offer distinct advantages such as suitability for real-time live cell monitoring and in vivo imaging, superior tunable properties, and versatile applications. In this review, we explored recent advancements in the development of small-molecule fluorescent probes toward monitoring cellular senescence by targeting various senescence-related biological phenomena. These phenomena include the upregulation of senescence-associated enzymes, perturbation of the subcellular environment, and increased endogenous ROS levels. Moreover, multi-senescence biomarker-targeting approaches are also discussed to improve their sensitivities and specificities for the detection of cellular senescence. With recent advances in senescence probe development, current challenges in this field are also discussed to facilitate further progress.

A dual-mode, image-enhanced, miniaturized microscopy system for incubator-compatible monitoring of live cells

Imaging live cells under stable culture conditions is essential to investigate cell physiological activities and proliferation. To achieve this goal, typically, a specialized incubation chamber that creates desired culture conditions needs to be incorporated into a microscopy system to perform cell monitoring. However, such imaging systems are generally large and costly, hampering their wide applications. Recent advances in the field of miniaturized microscopy systems have enabled incubator cell monitoring, providing a hospitable environment for live cells. Although these systems are more cost-effective, they are usually limited in imaging modalities and spatial temporal resolution. Here, we present a dual-mode, image-enhanced, miniaturized microscopy system (termed MiniCube) for direct monitoring of live cells inside incubators. MiniCube enables both bright field imaging and fluorescence imaging with single-cell spatial resolution and sub-second temporal resolution. Moreover, this system can also perform cell monitoring inside the incubator with tunable time scales ranging from a few seconds to days. Meanwhile, automatic cell segmentation and image enhancement are realized by the proposed data analysis pipeline of this system, and the signal-to-noise ratio (SNR) of acquired data is significantly improved using a deep learning based image denoising algorithm. Image data can be acquired with 5 times lower light exposure while maintaining comparable SNR. The versatility of this miniaturized microscopy system lends itself to various applications in biology studies, providing a practical platform and method for studying live cell dynamics within the incubator.

Monitoring of three-dimensional live-cell cultures using a multimode, multiscale imaging system combining confocal fluorescence microscopy and optical coherence microscopy

Live-cell monitoring involves long-term observation and analysis of living cells in tissue cultures, which develop cells or tissues in an artificial environment and are essential for tissue engineering. Fluorescence microscopy (FM) is widely used as a monitoring tool owing to its powerful ability to visualize protein distribution within cells and tissues, thereby providing information on their functions in biological processes. To reduce the photobleaching effect, which is a major limiting factor in FM, FM is generally combined with other imaging modalities, such as phase contrast and differential interference contrast microscopy, which are nondestructive to fluorochromes, to selectively use fluorescence signals only in specific areas of interest within a sample. However, these methods are restricted to thin samples and cannot produce depth-resolved images. Here, we propose a method to determine three-dimensional (3D) positions in volumetric samples for application in high-resolution 3D fluorescence imaging using a multimode and multiscale imaging system that combines optical coherence microscopy (OCM) and line confocal FM (LC–FM). We also demonstrate the benefits of multimodal imaging in 3D cell culture monitoring. To evaluate the performance of the proposed system and method, we rapidly and accurately located the regions of interest in 3D tissue culture models, such as tumor spheroids and microvascular bed, and instantaneously acquired volumetric high-resolution fluorescence images. We also used an integrated system to monitor tumor spheroids mixed with extracellular matrix (ECM) over five days of the culture process, and the FM–OCM integrated technique successfully imaged the overall 3D structures, such as the distribution and boundaries of the spheroids and ECM as well as stained tumor cells. This complementary information is useful for understanding the relationship between cellular behaviors, such as the proliferation and migration of tumor cells, and microenvironments, such as the ECM.

A Bioorthogonal Dual Fluorogenic Probe for the Live-Cell Monitoring of Nutrient Uptake by Mammalian Cells.

Cells rely heavily on the uptake of exogenous nutrients for survival, growth, and differentiation. Yet quantifying the uptake of small molecule nutrients at the single cell level is difficult. Here we present a new approach to studying the nutrient uptake in live single cells using Inverse Electron-Demand Diels Alder (IEDDA) chemistry. We have modified carboxyfluorescein-diacetate-succinimidyl esters (CFSE)-a quenched fluorophore that can covalently react with proteins and is only turned on in the cytosol of a cell following esterase activity-with a tetrazine. This tetrazine serves as a second quencher for the pendant fluorophore. Upon reaction with nutrients modified with an electron-rich or strained dienophile in an IEDDA reaction, this quenching group is destroyed, thereby enabling the probe to fluoresce. This has allowed us to monitor the uptake of a variety of dienophile-containing nutrients in live primary immune cell populations using flow cytometry and live-cell microscopy.

Pooled multicolour tagging for visualizing subcellular protein dynamics

Imaging-based methods are widely used for studying the subcellular localization of proteins in living cells. While routine for individual proteins, global monitoring of protein dynamics following perturbation typically relies on arrayed panels of fluorescently tagged cell lines, limiting throughput and scalability. Here, we describe a strategy that combines high-throughput microscopy, computer vision and machine learning to detect perturbation-induced changes in multicolour tagged visual proteomics cell (vpCell) pools. We use genome-wide and cancer-focused intron-targeting sgRNA libraries to generate vpCell pools and a large, arrayed collection of clones each expressing two different endogenously tagged fluorescent proteins. Individual clones can be identified in vpCell pools by image analysis using the localization patterns and expression level of the tagged proteins as visual barcodes, enabling simultaneous live-cell monitoring of large sets of proteins. To demonstrate broad applicability and scale, we test the effects of antiproliferative compounds on a pool with cancer-related proteins, on which we identify widespread protein localization changes and new inhibitors of the nuclear import/export machinery. The time-resolved characterization of changes in subcellular localization and abundance of proteins upon perturbation in a pooled format highlights the power of the vpCell approach for drug discovery and mechanism-of-action studies.

Live Cell Monitoring of Separase Activity, a Key Enzymatic Reaction for Chromosome Segregation, with Chimeric FRET-Based Molecular Sensor upon Cell Cycle Progression.

Separase is a key cysteine protease in the separation of sister chromatids through the digestion of the cohesin ring that inhibits chromosome segregation as a trigger of the metaphase-anaphase transition in eukaryotes. Its activity is highly regulated by binding with securin and cyclinB-CDK1 complex. These bindings prevent the proteolytic activity of separase until the onset of anaphase. Chromosome missegregation and aneuploidy are frequently observed in malignancies. However, there are some difficulties in biochemical examinations due to the instability of separase in vitro and the fact that few spatiotemporal resolution approaches exist for monitoring live separase activity throughout mitotic processes. Here, we have developed FRET-based molecular sensors, including GFP variants, with separase-cleavable sequences as donors and covalently attached fluorescent dyes as acceptor molecules. These are applicable to conventional live cell imaging and flow cytometric analysis because of efficient live cell uptake. We investigated the performance of equivalent molecular sensors, either localized or not localized inside the nucleus under cell cycle control, using flow cytometry. Synchronized cell cycle progression rendered significant separase activity detections in both molecular sensors. We obtained consistent outcomes with localized molecular sensor introduction and cell cycle control by fluorescent microscopic observations. We thus established live cell separase activity monitoring systems that can be used specifically or statistically, which could lead to the elucidation of separase properties in detail.

Abstract 1644: Exploring the role of a TERT intronic tandem repeat in regulating cell proliferative responses to environmental factors and cancer risk

Abstract The chr 5p15.33 genomic locus encoding telomerase reverse transcriptase (TERT) is structurally complex, with several highly polymorphic intronic variable number tandem repeats (VNTRs). A germline VNTR located in TERT intron 6 (VNTR6-1, 38 bp repeat unit) has previously been associated with TERT alternative splicing but its potential role in regulating cellular functions and disease risk is unclear. To functionally characterize VNTR6-1, we deleted this region (1-2 kb) using CRISPR/Cas9 in UMUC3 (bladder cancer cell line). Cell count growth tracking of parental and VNTR6-1 knockout (VNTR6-1 KO) cells via automated live-cell monitoring revealed different relative growth rates under serum starvation vs. normal conditions, suggesting that VNTR6-1 might be sensitive to growth factors present in culture media. Evaluation of growth in media with full vs. charcoal-stripped (CS) serum, showed significant differences, with higher cell counts in parental vs. VNTR6-1 KO in CS-serum (P=4.1e-3), parental vs. parental in full (P=1.57e-4) or CS-serum (P=4.76e-2), but not in parental vs. VNTR6-1 KO in full serum (P=0.378). After 5-6 days of growth, cells in CS-serum also rapidly began outgrowing cells in full serum. We observed that VNTR6-1 KO dampened the outgrowth response in cells lacking growth factors in CS-serum. Proliferation-specific growth assays tracking the dilution of an incorporated dye in cells by flow cytometry further showed that VNTR6-1 KO leads to more pronounced proliferation differences due to the presence or absence of serum-supplied factors. Overall, cell growth patterns appear to be sensitive to serum in interaction with VNTR6-1, suggesting a potential VNTR6-1-dependent response to extracellular signaling. In human populations, we have identified two VNTR6-1 groups: Short (24-27 copies) and Long (40.5-66.5 copies). In the PLCO dataset (n=100,445 cancer cases and controls), logistic regression showed that VNTR6-1-Long group was associated with a decreased risk in male-prevalent cancers such as bladder and prostate, but an increased risk in female-prevalent cancers such as breast, endometrial and ovarian. These results support a link between the TERT region, and potentially VNTR6-1, with hormone signaling and cancer risk. Motif analysis within VNTR6-1 predicted binding sites for several transcription factors, including those associated with development and cell growth. Together, these results suggest that VNTR6-1 may be involved in regulating cell growth, potentially by facilitating responses to extracellular signaling pathways. Additionally, given the location of VNTR6-1 within a multi-cancer GWAS region, our findings provide functional context for the role of this polymorphic repeat in differential risk and outcomes in certain cancers. Citation Format: Michelle Ho, Oscar Florez-Vargas, Maxwell Hogshead, Brenen Papenberg, Chia Han Lee, Chi Zhang, Kaitlin Forsythe, Wusheng Yan, Raj Chari, Ludmila Prokunina-Olsson. Exploring the role of a TERT intronic tandem repeat in regulating cell proliferative responses to environmental factors and cancer risk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1644.

Monitoring redox stress in human airway epithelial cells exposed to woodsmoke at an air–liquid interface

Wildland fires contribute significantly to the ambient air pollution burden worldwide, causing a range of adverse health effects in exposed populations. The toxicity of woodsmoke, a complex mixture of gases, volatile organic compounds, and particulate matter, is commonly studied in vitro using isolated exposures of conventionally cultured lung cells to either resuspended particulate matter or organic solvent extracts of smoke, leading to incomplete toxicity evaluations. This study aimed to improve our understanding of the effects of woodsmoke inhalation by building an advanced in vitro exposure system that emulates human exposure of the airway epithelium. We report the development and characterization of an innovative system that permits live-cell monitoring of the intracellular redox status of differentiated primary human bronchial epithelial cells cultured at an air–liquid interface (pHBEC-ALI) as they are exposed to unfractionated woodsmoke generated in a tube furnace in real time. pHBEC-ALI exposed to freshly generated woodsmoke showed oxidative changes that were dose-dependent and reversible, and not attributable to carbon monoxide exposure. These findings show the utility of this novel system for studying the molecular initiating events underlying woodsmoke-induced toxicity in a physiologically relevant in vitro model, and its potential to provide biological plausibility for risk assessment and public health measures.

A Pipeline for Dynamic Analysis of Mitochondrial Content in Developing T Cells: Bridging the Gap Between High-Throughput Flow Cytometry and Single-Cell Microscopy Analysis.

Analyzing the dynamics of mitochondrial content in developing T cells is crucial for understanding the metabolic state during Tcell development. However, monitoring mitochondrial content in real-time needs a balance of cell viability and image resolution. In this chapter, we present experimental protocols for measuring mitochondrial content in developing T cells using three modalities: bulk analysis via flow cytometry, volumetric imaging in laser scanning confocal microscopy, and dynamic live-cell monitoring in spinning disc confocal microscopy. Next, we provide an image segmentation and centroid tracking-based analysis pipeline for automated quantification of a large number of microscopy images. These protocols together offer comprehensive approaches to investigate mitochondrial dynamics in developing T cells, enabling a deeper understanding of their metabolic processes.

Real-time monitoring of glucose metabolism and effects of metformin on HepG2 cells using 13C in-cell NMR spectroscopy

Metformin is currently a strong candidate antitumor agent for multiple cancers, and has the potential to inhibit cancer cell viability, growth, and proliferation. Metabolic reprogramming is a critical feature of cancer cells. However, the effects of metformin which targets glucose metabolism on HepG2 cancer cells remain unclear. In this study, to explore the effects of metformin on glucose metabolism in HepG2 cells, we conducted real-time metabolomic monitoring of live HepG2 cells treated with metformin using 13C in-cell NMR spectroscopy. Metabolic tracing with U–13C6-glucose revealed that metformin significantly increased the production of 13C-G3P and 13C-glycerol, which were reported to attenuate liver cancer development, but decreased the production of potential oncogenesis-supportive metabolites, including 13C-lactate, 13C-alanine, 13C-glycine, and 13C-glutamate. Moreover, the expression levels of enzymes associated with the measured metabolites were carried out. The results showed that the levels of ALT1, MCT4, GPD2 and MPC1 were greatly reduced, which were consistent with the changes of measured metabolites in 13C in-cell NMR spectroscopy. Overall, our approach directly provides fundamental insights into the effects of metformin on glucose metabolism in live HepG2 cells, and highlights the potential mechanism of metformin, including the increase in production of G3P and glycerol derived from glucose, as well as the inhibition of glucose incorporation into lactate, alanine, glutamate, and glycine.

Создание генетически кодируемого светоиндуцируемого сенсора для визуализации ядрышка

Nucleolus plays a vital role in enhancing rRNA production and maintaining ribosome biogenesis in tumor cells, making the nucleolus a desirable target for genetic and oncological research. The most convenient method for nucleolus monitoring is fluorescent microscopy, combining high efficiency and accessibility. Nevertheless, currently available fluorescent visualization methods are unsuitable for live-cell monitoring of nucleolus because they require continuous labeling. To address this issue, we have developed a genetically encoded Light-Activated Nucleolus Sensing (LANS) system for real-time nucleolar visualization. The combination of eMags domains and reader domain of DPF3 protein, responsible respectively for the light-induced dimerization and targeting the nucleolus, allowed LANS system to efficiently target nucleolus in several cancer cell lines without affecting cell morphology. This system makes it possible to increase the representation of the LANS2 sample in the nucleolus by 1.5 times relative to the fluorescence intensity values obtained before irradiation of the nucleolus. LANS holds the potential to accelerate the search for new drugs and enhance the primary screening of drug compounds in in vivo models.

Fibrinogen Fragment X Mediates Endothelial Barrier Disruption via Suppression of VE-Cadherin

IntroductionMajor traumatic injury is associated with early hemorrhage-related and late-stage deaths due to multiple organ failure (MOF). While improvements to hemostatic resuscitation have significantly reduced hemorrhage-related deaths, the incidence of MOF among trauma patients remains high. Dysregulation of vascular endothelial cell (EC) barrier function is a central mechanism in the development of MOF; however, the mechanistic triggers remain unknown. Accelerated fibrinolysis occurs in a majority of trauma patients, resulting in high circulating levels of fibrin(ogen) degradation products, such as fragment X. To date, the relationship between fragment X and EC dysregulation and barrier disruption is unknown. The goal of this study was to determine the effects of fragment X on EC barrier integrity and expression of paracellular junctional proteins that regulate barrier function. MethodsHuman lung microvascular endothelial cells (HLMVECs) were treated with increasing concentrations of fragment X (1, 10, and 100 μg/mL), and barrier function was monitored using the xCELLigence live-cell monitoring system. Quantitative PCR (qPCR) was performed to measure changes in EC expression of 84 genes. Immunofluorescent (IF) cytostaining was performed to validate qPCR findings. ResultsFragment X treatment significantly increased endothelial permeability over time (P < 0.05). There was also a significant reduction in VE-cadherin mRNA expression in fragment X-treated HLMVECs compared to control (P = 0.01), which was confirmed by IF staining. ConclusionsFragment X may induce EC hyperpermeability by reducing VE-cadherin expression. This suggests that a targeted approach to disrupting EC-fragment X interactions could mitigate EC barrier disruption, organ edema, and MOF associated with major trauma.

THU596 Heteromerization Of The Angiotensin II Type 1 Receptor And The Bradykinin Type 2 Receptor

Abstract Disclosure: E.K. Johnstone: Grant Recipient; Self; Australian Research Council. K.D. Pfleger: Grant Recipient; Self; Australian Research Council. Stock Owner; Self; Dimerix Bioscience Pty Ltd. Background: Receptor heteromerization is the phenomenon whereby two different receptors form a functional complex that attains unique pharmacological properties. Consequently, receptor heteromers can be considered novel drug targets, with the potential to achieve greater therapeutic selectivity and specificity. In a landmark study, the first receptor heteromer to be associated with a disease was the heteromer that forms between the angiotensin II type 1 (AT1) receptor and the bradykinin type 2 (B2) receptor (1). The AT1-B2 heteromer reportedly led to increased angiotensin II signalling, which was implicated in the angiotensin II hypersensitivity involved in pre-eclampsia. However, the validity of this study was questioned by a collaboration of several groups who were unable to find any evidence for a physical or a function interaction between the two receptors (2). As a consequence of these conflicting studies, the existence of the AT1-B2 receptor heteromer has remained controversial. Aim: Investigation of evidence for heteromerization of the AT1 receptor and the B2 receptor. Methods: AT1 receptor and B2 receptor pharmacology and potential heteromerization was investigated in HEK293FT cells using various bioluminescence resonance energy transfer (BRET)-proximity based assays, as well as Dimerix’s Receptor-Heteromer Investigation Technology (Receptor-HIT) (3). Receptor-HIT enables the investigation of receptor heteromers through their specific ligand-induced proximity to interacting proteins. BRET assays, including in the Receptor-HIT configuration, allow real time, live cell monitoring of proximity between biomolecules of interest. Results: Using the Receptor-HIT assay, we confirmed the existence of the AT1-B2 receptor heteromer in HEK293FT cells. We found that the heteromer was able to recruit various GPCR interacting proteins, such as G proteins and arrestin in a bradykinin-dependent manner. Additionally, the heteromer also internalised and trafficked through the cell upon treatment with bradykinin. The close proximity of the two receptors was confirmed using a variation of the Receptor-HIT assay, which monitors receptor-ligand binding rather than protein-protein interactions. Discussion: Our study supports the existence of the AT1-B2 receptor heteromer and identifies various pharmacological properties of the complex. Further studies are required to relate these molecular properties to physiological consequences of heteromerization. (1)AbdAlla et al. Nat Med2001, 7 (9), 1003 1009.(2)Hansen et al. J Biol Chem2009;284(3):1831 1839.(3)Johnstone et al. Biochem Soc T. 2021;49:1555–65. Presentation: Thursday, June 15, 2023

Cover Picture: Chemical probes based on GOLD: The discovery and elucidation of mechanism (BKCS 8/2023) Larissa Miasiro Ciaramicoli, Young‐Tae Chang

Fluorescent probes are extensively used for detection and monitoring of live cells, in particular, due to its ability to distinguish different cell types. Therefore, gating‐oriented live‐cell distinction (GOLD) mechanism describes how the unique composition of cell membrane transporters controls the influx and efflux of dyes to create a distinct staining pathway. Herein, a series of techniques are detailed for elucidation of known and unknown target transporters related to the GOLD mechanism. More details are available in the article by Larissa Miasiro Ciaramicoli and Young‐Tae Chang.image