Label-free impedance measurements to unravel biomolecular interactions involved in G protein-coupled receptor signaling.

G protein-coupled receptors (GPCRs) are among the most heavily addressed drug targets in medicinal chemistry and pharmacology. The screening for new agonists or antagonists has been largely based on genetically engineered cells overexpressing the receptor to study binding of ligands directly or via intracellular signaling events downstream of receptor activation. These approaches are often invasive in nature, need to be conducted as endpoint assays, require isotope- or fluorophore-labeling and significant genetic manipulation. In contrast to that, non-invasive and label-free impedance measurements are capable of monitoring ligand-receptor interactions in target cells with endogenous receptor expression in real time. The cells expressing the receptor are grown on planar gold-film electrodes that are integrated into regular cell culture dishes. This article will highlight several impedance-based assay formats to characterize biomolecular interactions between ligands and their GPCRs in vitro, comprising agonist and antagonist characterization, dose-response relationships, receptor desensitization, and signal transduction profiling.

Impedance analysis of adherent cells after in situ electroporation-mediated delivery of bioactive proteins, DNA and nanoparticles in µL-volumes.

Specific intracellular manipulation of animal cells is a persistent goal in experimental cell biology. Such manipulations allow precise and targeted interference with signaling cascades, metabolic pathways, or bi-molecular interactions for subsequent tracking of functional consequences. However, most biomolecules capable of molecular recognition are membrane impermeable. The ability to introduce these molecules into the cytoplasm and then to apply appropriate readouts to monitor the corresponding cell response could prove to be an important research tool. This study describes such an experimental approach combining in situ electroporation (ISE) as a means to efficiently deliver biomolecules to the cytoplasm with an impedance-based, time-resolved analysis of cell status using electric cell-substrate impedance sensing (ECIS). In this approach, gold-film electrodes, deposited on the bottom of regular culture dishes, are used for both electroporation and monitoring. The design of the electrode layout and measurement chamber allows working with sample volumes as small as 10 µL. A miniaturized setup for combined electroporation and impedance sensing (µISE-ECIS) was applied to load different adherent cells with bioactive macromolecules including enzymes, antibodies, nucleic acids and quantum dot nanoparticles. The cell response after loading the cytoplasm with RNase A or cytochrome c (in the presence or absence of caspase inhibitors) was tracked by non-invasive impedance readings in real-time.

Expression profiles of proton-sensing G-protein coupled receptors in common skin tumors.

The proton-sensing GPCRs (pH-GPCRs) GPR4 (GPR19), TDAG8 (GPR65, T-cell death associated gene 8), OGR1 (GPR68, ovarian cancer GPCR1), and G2A (GPR132, G2 accumulation protein) are involved in sensing and transducing changes in extracellular pH (pHe). Extracellular acidification is a central hallmark of solid cancer. pH-GPCR function has been associated with cancer cell proliferation, adhesion, migration and metastasis, as well as with modulation of the immune system. Little is known about the expression levels and role of pH-GPCRs in skin cancer. To better understand the functions of pH-GPCRs in skin cancer in vivo, we examined the expression-profiles of GPR4, TDAG8, OGR1 and G2A in four common skin tumors, i.e. squamous cell carcinoma (SCC), malignant melanoma (MM), compound nevus cell nevi (NCN), basal cell carcinoma (BCC). We performed immunohistochemistry and immunofluorescence staining on paraffin-embedded tissue samples acquired from patients suffering from SCC, MM, NCN or BCC. We show the expression of pH-GPCRs in four common skin cancers. Different expression patterns in the investigated skin cancer types indicate that the different pH-GPCRs may have distinct functions in tumor progression and serve as novel therapeutic targets.

pH sensing in skin tumors: Methods to study the involvement of GPCRs, acid-sensing ion channels and transient receptor potential vanilloid channels.

Solid tumors exhibit an inversed pH gradient with increased intracellular pH (pHi ) and decreased extracellular pH (pHe ). This inside-out pH gradient is generated via sodium/hydrogen antiporter 1, vacuolar-type H + ATPases, monocarboxylate transporters, (bi)carbonate (co)transporters and carboanhydrases. Our knowledge on how pHe -signals are sensed and what the respective receptors induce inside cells is scarce. Some pH-sensitive receptors (GPR4, GPR65/TDAG8, GPR68/OGR1, GPR132/G2A, possibly GPR31 and GPR151) and ion channels (acid-sensing ion channels ASICs, transient receptor potential vanilloid receptors TRPVs) transduce signals inside cells. As little is known on the expression and function of these pH sensors, we used immunostainings to study tissue samples from common and rare skin cancers. Our current and future work is directed towards investigating the impact of all the pH-sensing receptors in different skin tumors using cell culture techniques with selective knockdown/knockout (siRNA/CRISPR-Cas9). To study cell migration and proliferation, novel impedance-based wound healing assays have been developed and are used. The field of pH sensing in tumors and wounds holds great promise for the development of pH-targeting therapies, either against pH regulators or sensors to inhibit cell proliferation and migration.

Stepwise Dosing Protocol for Increased Throughput in Label-Free Impedance-Based GPCR Assays.

Label-free impedance-based assays are increasingly used to non-invasively study ligand-induced GPCR activation in cell culture experiments. The approach provides real-time cell monitoring with a device-dependent time resolution down to several tens of milliseconds and it is highly automated. However, when sample numbers get high (e.g., dose-response studies for various different ligands), the cost for the disposable electrode arrays as well as the available time resolution for sequential well-by-well recordings may become limiting. Therefore, we here present a serial agonist addition protocol which has the potential to significantly increase the output of label-free GPCR assays. Using the serial agonist addition protocol, a GPCR agonist is added sequentially in increasing concentrations to a single cell layer while continuously monitoring the sample's impedance (agonist mode). With this serial approach, it is now possible to establish a full dose-response curve for a GPCR agonist from just one single cell layer. The serial agonist addition protocol is applicable to different GPCR coupling types, Gq Gi/0 or Gs and it is compatible with recombinant and endogenous expression levels of the receptor under study. Receptor blocking by GPCR antagonists is assessable as well (antagonist mode).

Cytocompatibility of Mats Prepared from Different Electrospun Polymer Nanofibers.

Mats of cytocompatible polymer fibers are needed as scaffolds in tissue engineering or as wound healing supports. Most recently, they have emerged as matrix-material to allow for in situ chemo- and biosensing inside intact tissue fragments or surrogates. Electrospinning of such fibers from polymer solutions provides extended options to control the structural and functional properties of the resulting fiber mats. We have prepared electrospun polymeric fiber mats from poly(lactic acid) (PLA), polystyrene (PS), and poly(vinyl pyrrolidone) (PVP) with two different fiber densities. Mats and individual fibers were characterized with respect to their dimensions, morphology, and their compatibility with human keratinocytes (HaCaT) selected as a biological model. Microscopic inspection revealed that HaCaT cells were viable on mats from all three polymers with only a negligible fraction of dead cells, similar to planar control surfaces. Growth in the presence of the fiber mats did not alter cellular metabolism (ATP, redox state) and did not induce significant production of cytokines (interleukin-6 (IL-6); monocyte chemoattractant protein-1 (MCP-1)). However, we did observe that fiber density changed the overall topography of the resulting mats and led to differences in the establishment of continuous cell sheets. In conclusion, the findings support the suitability of electrospun polymeric fiber mats made from PLA, PS, or PVP as potential biocompatible matrices for future two-dimensional (2D) or three-dimensional (3D) sensing of vital parameters from tissue in health and disease.

Mitochondria control store-operated Ca2+ entry through Na+ and redox signals.

Mitochondria exert important control over plasma membrane (PM) Orai1 channels mediating store-operated Ca2+ entry (SOCE). Although the sensing of endoplasmic reticulum (ER) Ca2+ stores by STIM proteins and coupling to Orai1 channels is well understood, how mitochondria communicate with Orai1 channels to regulate SOCE activation remains elusive. Here, we reveal that SOCE is accompanied by a rise in cytosolic Na+ that is critical in activating the mitochondrial Na+/Ca2+ exchanger (NCLX) causing enhanced mitochondrial Na+ uptake and Ca2+ efflux. Omission of extracellular Na+ prevents the cytosolic Na+ rise, inhibits NCLX activity, and impairs SOCE and Orai1 channel current. We show further that SOCE activates a mitochondrial redox transient which is dependent on NCLX and is required for preventing Orai1 inactivation through oxidation of a critical cysteine (Cys195) in the third transmembrane helix of Orai1. We show that mitochondrial targeting of catalase is sufficient to rescue redox transients, SOCE, and Orai1 currents in NCLX-deficient cells. Our findings identify a hitherto unknown NCLX-mediated pathway that coordinates Na+ and Ca2+ signals to effect mitochondrial redox control over SOCE.

Calcium Signaling Is Dispensable for Receptor Regulation of Endothelial Barrier Function.

Endothelial barrier function is tightly regulated by plasma membrane receptors and is crucial for tissue fluid homeostasis; its dysfunction causes disease, including sepsis and inflammation. The ubiquitous activation of Ca2+ signaling upon phospholipase C-coupled receptor ligation leads quite naturally to the assumption that Ca2+ signaling is required for receptor-regulated endothelial barrier function. This widespread hypothesis draws analogy from smooth muscle and proposes the requirement of G protein-coupled receptor (GPCR)-generated Ca2+ signaling in activating the endothelial contractile apparatus and generating interendothelial gaps. Notwithstanding endothelia being non-excitable in nature, the hypothesis of Ca2+-induced endothelial contraction has been invoked to explain actions of GPCR agonists that either disrupt or stabilize endothelial barrier function. Here, we challenge this correlative hypothesis by showing a lack of causal link between GPCR-generated Ca2+ signaling and changes in human microvascular endothelial barrier function. We used three endogenous GPCR agonists: thrombin and histamine, which disrupt endothelial barrier function, and sphingosine-1-phosphate, which stabilizes barrier function. The qualitatively different effects of these three agonists on endothelial barrier function occur independently of Ca2+ entry through the ubiquitous store-operated Ca2+ entry channel Orai1, global Ca2+ entry across the plasma membrane, and Ca2+ release from internal stores. However, disruption of endothelial barrier function by thrombin and histamine requires the Ca2+ sensor stromal interacting molecule-1 (STIM1), whereas sphingosine-1-phosphate-mediated enhancement of endothelial barrier function occurs independently of STIM1. We conclude that although STIM1 is required for GPCR-mediated disruption of barrier function, a causal link between GPCR-induced cytoplasmic Ca2+ increases and acute changes in barrier function is missing. Thus, the cytosolic Ca2+-induced endothelial contraction is a cum hoc fallacy that should be abandoned.

Leukotriene-C4 synthase, a critical enzyme in the activation of store-independent Orai1/Orai3 channels, is required for neointimal hyperplasia.

Leukotriene-C4 synthase (LTC4S) generates LTC4 from arachidonic acid metabolism. LTC4 is a proinflammatory factor that acts on plasma membrane cysteinyl leukotriene receptors. Recently, however, we showed that LTC4 was also a cytosolic second messenger that activated store-independent LTC4-regulated Ca(2+) (LRC) channels encoded by Orai1/Orai3 heteromultimers in vascular smooth muscle cells (VSMCs). We showed that Orai3 and LRC currents were up-regulated in medial and neointimal VSMCs after vascular injury and that Orai3 knockdown inhibited LRC currents and neointimal hyperplasia. However, the role of LTC4S in neointima formation remains unknown. Here we show that LTC4S knockdown inhibited LRC currents in VSMCs. We performed in vivo experiments where rat left carotid arteries were injured using balloon angioplasty to cause neointimal hyperplasia. Neointima formation was associated with up-regulation of LTC4S protein expression in VSMCs. Inhibition of LTC4S expression in injured carotids by lentiviral particles encoding shRNA inhibited neointima formation and inward and outward vessel remodeling. LRC current activation did not cause nuclear factor for activated T cells (NFAT) nuclear translocation in VSMCs. Surprisingly, knockdown of either LTC4S or Orai3 yielded more robust and sustained Akt1 and Akt2 phosphorylation on Ser-473/Ser-474 upon serum stimulation. LTC4S and Orai3 knockdown inhibited VSMC migration in vitro with no effect on proliferation. Akt activity was suppressed in neointimal and medial VSMCs from injured vessels at 2 weeks postinjury but was restored when the up-regulation of either LTC4S or Orai3 was prevented by shRNA. We conclude that LTC4S and Orai3 altered Akt signaling to promote VSMC migration and neointima formation.

Impedance analysis of GPCR-mediated changes in endothelial barrier function: overview and fundamental considerations for stable and reproducible measurements.

The past 20 years has seen significant growth in using impedance-based assays to understand the molecular underpinning of endothelial and epithelial barrier function in response to physiological agonists and pharmacological and toxicological compounds. Most studies on barrier function use G protein-coupled receptor (GPCR) agonists which couple to fast and transient changes in barrier properties. The power of impedance-based techniques such as electric cell-substrate impedance sensing (ECIS) resides in its ability to detect minute changes in cell layer integrity label-free and in real-time ranging from seconds to days. We provide a comprehensive overview of the biophysical principles, applications, and recent developments in impedance-based methodologies. Despite extensive application of impedance analysis in endothelial barrier research, little attention has been paid to data analysis and critical experimental variables, which are both essential for signal stability and reproducibility. We describe the rationale behind common ECIS data presentation and interpretation and illustrate practical guidelines to improve signal intensity by adapting technical parameters such as electrode layout, monitoring frequency, or parameter (resistance versus impedance magnitude). Moreover, we discuss the impact of experimental parameters, including cell source, liquid handling, and agonist preparation on signal intensity and kinetics. Our discussions are supported by experimental data obtained from human microvascular endothelial cells challenged with three GPCR agonists, thrombin, histamine, and sphingosine-1-phosphate.

Emerging roles of Orai3 in pathophysiology.

Calcium (Ca(2+)) is a ubiquitous second messenger that regulates a plethora of physiological functions. Deregulation of calcium homeostasis has been reported in a wide variety of pathological conditions including cardiovascular disorders, cancer and neurodegenerative diseases. One of the most ubiquitous pathways involved in regulated Ca(2+) influx into cells is the store-operated Ca(2+) entry (SOCE) pathway. In 2006, Orai1 was identified as the channel protein that mediates SOCE in immune cells. Orai1 has two mammalian homologs, Orai2 and Orai3. Although Orai1 has been the most widely studied Orai isoform, Orai3 has recently received significant attention. Under native conditions, Orai3 was demonstrated to be an important component of store-independent arachidonate-regulated Ca(2+) (ARC) entry in HEK293 cells, and more recently of a store-independent leukotrieneC4-regulated Ca(2+) (LRC) entry pathway in vascular smooth muscle cells. Recent studies have shown upregulation of Orai3 in estrogen receptor-expressing breast cancers and a critical role for Orai3 in breast cancer development in immune-compromised mice. Orai3 upregulation was also shown to contribute to vascular smooth muscle remodeling and neointimal hyperplasia caused by vascular injury. Furthermore, Orai3 has been shown to contribute to proliferation of effector T-lymphocytes under oxidative stress. In this review, we will discuss the role of Orai3 in reported pathophysiological conditions and will contribute ideas on the potential role of Orai3 in native Ca(2+) signaling pathways and human disease.

What role for store-operated Ca²âº entry in muscle?

Store-operated Ca²âº entry (SOCE) is a receptor-regulated Ca²âº entry pathway that is both ubiquitous and evolutionarily conserved. SOCE is activated by depletion of intracellular Ca²âº stores through receptor-mediated production of inositol 1,4,5-trisphosphate (IP3). The depletion of endoplasmic reticulum (ER) Ca²âº is sensed by stromal interaction molecule 1 (STIM1). On store depletion, STIM1 aggregates and moves to areas where the ER comes close to the plasma membrane (PM; within 25 nm) to interact with Orai1 channels and activate Ca²âº entry. Ca²âº entry through store-operated Ca²âº (SOC) channels, originally thought to mediate the replenishment of Ca²âº stores, participate in active downstream signaling by coupling to the activation of enzymes and transcription factors that control a wide variety of long-term cell functions such as proliferation, growth, and migration. SOCE has also been proposed to contribute to short-term cellular responses such as muscle contractility. While there are significant STIM1/Orai1 protein levels and SOCE activity in adult skeletal muscle, the precise role of SOCE in skeletal muscle contractility is not clear. The dependence on SOCE during cardiac and smooth muscle contractility is even less certain. Here, we will hypothesize on the contribution of SOCE in muscle and its potential role in contractility and signaling.

Ultra-small, highly stable, and membrane-impermeable fluorescent nanosensors for oxygen.

We report on the preparation of ultra-small fluorescent nanosensors for oxygen via a one-pot approach. The nanoparticles have a hydrophobic core capable of firmly hosting hydrophobic luminescent oxygen probes. Their surface is composed of a dense and long-chain poly(ethylene glycol) shell, which renders them cell-membrane impermeable but yet highly sensitive to oxygen, and also highly stable in aqueous solutions and cell culture media. These features make them potentially suitable for sensing oxygen in extracellular fluids such as blood, interstitial and brain fluid, in (micro) bioreactors and micro- or nanoscale fluidic devices. Four kinds of nanosensors are presented, whose excitation spectra cover a wide spectral range (395-630 nm), thus matching many common laser lines, and with emission maxima ranging from 565 to 800 nm, thereby minimizing interference from background luminescence of biomatter. The unquenched lifetimes are on the order of 5.8-234 µs, which-in turn-enables lifetime imaging and additional background separation via time-gated methods.

Ultra-small, highly stable, and sensitive dual nanosensors for imaging intracellular oxygen and pH in cytosol.

We report on the first dual nanosensors for imaging of pH values and oxygen partial pressure in cells. The sensors have a unique nanostructure in that a soft core structure is rigidized with a silane reagent, while poly(ethylene glycol) chains form an outer shell. Lipophilic oxygen-sensitive probes and reference dyes are encapsulated inside the hydrophobic core, while a pH-sensitive probe is covalently attached to the poly(ethylene glycol) end-group on the shell. The core/shell structure renders the nanosensors well dispersed and highly stable in various kinds of aqueous media. Their average size is 12 nm, and they respond to both pH and oxygen in the physiological range. They do not pass cell membranes, but can be internalized into the cellular cytosol by electroporation, upon which they enable sensing and imaging of pH values and oxygen with high spatial resolution. The nanosensor strategy shown here is expected to be applicable to the development of various other kinds of multiple nanosensors for in vivo studies.

Impedance analysis of adherent cells after in situ electroporation: non-invasive monitoring during intracellular manipulations.

In this study adherent animal cells were grown to confluence on circular gold-film electrodes of 250 µm diameter that had been deposited on the surface of a regular culture dish. The impedance of the cell-covered electrode was measured at designated frequencies to monitor the behavior of the cells with time. This approach is referred to as electric cell-substrate impedance sensing or short ECIS in the literature. The gold-film electrodes were also used to deliver well-defined AC voltage pulses of several volts amplitude and several hundred milliseconds duration to the adherent cells in order to achieve reversible membrane electroporation (in situ electroporation=ISE). Electroporation-assisted introduction of membrane impermeable molecules into the cytoplasm was studied by using FITC-labeled dextran molecules of different molecular weights. Probes as big as 2MDa were successfully loaded into the cells residing on the electrode surface. Time-resolved impedance measurements before and immediately after the electroporation pulse revealed the kinetics of membrane resealing as well as subsequent changes in cell morphology. Cells recovered from the electroporation pulse within less than 90 min. When membrane-impermeable, bioactive compounds like N(3)(-) or bleomycin were introduced into the cells by in situ electroporation, concomitant ECIS readings sensitively reported on the associated response of the cells to these toxins as a function of time (ISE-ECIS).