Fluorescent Receptor Research Articles

Efficient fluorescent recognition of ATP/GTP by a water-soluble bisquinolinium pyridine-2,6-dicarboxamide compound. Crystal structures, spectroscopic studies and interaction mode with DNA.

The new dicationic pyridine-2,6-dicarboxamide-based compound 1 bearing two N-alkylquinolinium units was synthesized, structurally determined by single-crystal X-ray diffraction, and studied in-depth as a fluorescent receptor for nucleotides and inorganic phosphorylated anions in pure water. The addition of nucleotides to 1 at pH = 7.0 quenches its blue emission with a selective affinity towards adenosine 5'-triphosphate (ATP) and guanosine 5'-tripohosphate (GTP) over other nucleotides such CTP, UTP, ADP, AMP, dicarboxylates and inorganic anions. On the basis of the spectroscopic tools (1H, 31P NMR, UV-vis, fluorescence), MS measurements and DFT calculations, receptor 1 binds ATP with high affinity (log K = 5.04) through the simultaneous formation of strong hydrogen bonds and π-π interactions between the adenosine fragment and quinolinium ring with binding energy calculated in 8.7 kcal mol-1. High affinity for ATP/GTP is attributed to the high acidity of amides and preorganized rigid structure of 1. Receptor 1 is an order of magnitude more selective for ATP than GTP. An efficient photoinduced electron transfer quenching mechanism with simultaneous receptor-ATP complexation in both the excited and ground states is proposed. Additionally, multiple spectroscopic studies and molecular dynamics simulations showed that 1 can intercalate into DNA base pairs.

Fluorescent Nanozeolite Receptors for the Highly Selective and Sensitive Detection of Neurotransmitters in Water and Biofluids (Adv. Mater. 49/2021)

Detection of Neurotransmitters So far, only modestly performing synthetic binders have been accessible for small bioactive molecules such as serotonin and dopamine. In article number 2104614, Luisa De Cola, Frank Biedermann, and co-workers introduce zeolite-based artificial receptors (ZARs), which are microporous inorganic–organic hybrid materials applicable for the optical-based detection of serotonin and dopamine in urine at physiological relevant concentrations. These ZARs outperform protein receptors with respect to response times, robustness and scalability.

Improved Discrimination of Tumors with Low and Heterogeneous EGFR Expression in Fluorescence-Guided Surgery Through Paired-Agent Protocols

PurposeThe goal of fluorescence-guided surgery (FGS) in oncology is to improve the surgical therapeutic index by enhancing contrast between cancerous and healthy tissues. However, optimal discrimination between these tissues is complicated by the nonspecific uptake and retention of molecular targeted agents and the variance of fluorescence signal. Paired-agent imaging (PAI) employs co-administration of an untargeted imaging agent with a molecular targeted agent, providing a normalization factor to minimize nonspecific and varied signals. The resulting measured binding potential is quantitative and equivalent to in vivo immunohistochemistry of the target protein. This study demonstrates that PAI improves the accuracy of tumor-to-healthy tissue discrimination compared to single-agent imaging for in vivo FGS.ProceduresPAI using a fluorescent anti-epidermal growth factor receptor (EGFR) affibody molecule (ABY-029, eIND 122,681) with untargeted IRDye 700DX carboxylate was compared to ABY-029 alone in an oral squamous cell carcinoma xenograft mouse model at 3 h after dye administration (n = 30).ResultsPAI significantly enhanced tumor discrimination, as compared to ABY-029 alone in low EGFR-expressing tumors and highly heterogeneous populations including multiple cell lines with varying expression (diagnostic accuracy: 0.908 vs. 0.854 and 0.908 vs. 0.822; and ROC curve AUC: 0.963 vs. 0.909 and 0.957 vs. 0.909, respectively) indicating a potential for universal FGS image thresholds to determine surgical margins. In addition, PAI achieved significantly higher diagnostic ability than ABY-029 alone 0.25–5-h post injection and exhibited a stronger correlation to EGFR expression heterogeneity.ConclusionThe quantitative receptor delineation of PAI promises to improve the surgical therapeutic index of cancer resection in a clinically relevant timeline.

Fluorescent Nanozeolite Receptors for the Highly Selective and Sensitive Detection of Neurotransmitters in Water and Biofluids.

The design and preparation of synthetic binders (SBs) applicable for small biomolecule sensing in aqueous media remains very challenging. SBs designed by the lock-and-key principle can be selective for their target analyte but usually show an insufficient binding strength in water. In contrast, SBs based on symmetric macrocycles with a hydrophobic cavity can display high binding affinities but generally suffer from indiscriminate binding of many analytes. Herein, a completely new and modular receptor design strategy based on microporous hybrid materials is presented yielding zeolite-based artificial receptors (ZARs) which reversibly bind the neurotransmitters serotonin and dopamine with unprecedented affinity and selectivity even in saline biofluids. ZARs are thought to uniquely exploit both the non-classical hydrophobic effect and direct non-covalent recognition motifs, which is supported by in-depth photophysical, and calorimetric experiments combined with full atomistic modeling. ZARs are thermally and chemically robust and can be readily prepared at gram scales. Their applicability for the label-free monitoring of important enzymatic reactions, for (two-photon) fluorescence imaging, and for high-throughput diagnostics in biofluids is demonstrated. This study showcases that artificial receptor based on microporous hybrid materials can overcome standing limitations of synthetic chemosensors, paving the way towards personalized diagnostics and metabolomics.

Isophthalamide bearing 1-pyrenylethynyl group as a highly fluorescent hydrogen bond motif for anion receptors

Isophthalamide-based receptor 2 bearing 1-pyrenylethynyl group as a fluorophore was prepared for a ratiometric fluorescence sensor for anions. Receptor 2 showed small hypsochromic shift by UV–vis titrations and ratiometric fluorescence changes upon the addition of anions, in particular biologically important anions such as AcO−, H2PO4−, and Cl− with high association constants. The high quantum yield of receptor 2 (ΦF = 0.88) implies that 5-(1-pyrenyl)isophthaloyl amide spacer is a versatile component for various fluorescence receptors.

MP40-05 c-MET RECEPTOR-TARGETED FLUORESCENCE ON THE ROAD TO IMAGE-GUIDED SURGERY IN PENILE SQUAMOUS CELL CARCINOMA PATIENTS

You have accessJournal of UrologyPenile & Testicular Cancer I (MP40)1 Sep 2021MP40-05 c-MET RECEPTOR-TARGETED FLUORESCENCE ON THE ROAD TO IMAGE-GUIDED SURGERY IN PENILE SQUAMOUS CELL CARCINOMA PATIENTS Hielke de Vries, Elise Bekers, Matthias van Oosterom, Baris Karakullukcu, Henk van der Poel, Fijs van Leeuwen, Tessa Buckle, and Oscar Brouwer Hielke de VriesHielke de Vries More articles by this author , Elise BekersElise Bekers More articles by this author , Matthias van OosteromMatthias van Oosterom More articles by this author , Baris KarakullukcuBaris Karakullukcu More articles by this author , Henk van der PoelHenk van der Poel More articles by this author , Fijs van LeeuwenFijs van Leeuwen More articles by this author , Tessa BuckleTessa Buckle More articles by this author , and Oscar BrouwerOscar Brouwer More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002055.05AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: In penile squamous cell carcinoma (pSCC), primary surgery aims to obtain oncologically safe margins while minimizing mutilation. Surgical guidance provided by receptor-specific tracers could potentially improve margin detection and reduce unnecessary excision of healthy tissue. Here, we present the first results of a prospective feasibility study for real-time intraoperative visualization of pSCC using a fluorescent c-MET receptor targeting tracer (EMI-137). METHODS: EMI-137 tracer performance was initially assessed ex vivo (N=10) via incubation of freshly excised pSCC in a solution containing EMI-137 (500 nM). The in vivo potential of c-MET targeting and intraoperative tumour visualization was assessed after intravenous administration of EMI-137 in five pSCC patients scheduled for surgical resection using a Cy5 fluorescence camera. Fluorescence imaging results were related to standard pathological tumour evaluation and c-MET immunohistochemistry. Three of the five in vivo patients also underwent a sentinel node resection after local administration of the hybrid tracer ICG-99mTc-nanocolloid, which could be imaged using a near-infrared fluorescence camera. RESULTS: No tracer-related adverse events were encountered. Both ex vivo and in vivo, EMI-137 enabled c-MET based tumour visualization in all patients. Histopathological analyses showed that all pSCC's expressed c-MET, with expression levels of ≥70% in 14/15 patients. Moreover, the highest c-MET expression levels were seen on the outside rim of the tumours, and a visual correlation was found between c-MET expression and fluorescence signal intensity. No complications were encountered when combining primary tumour targeting with lymphatic mapping. As such, simultaneous use of Cy5 and ICG in the same patient proved to be feasible. CONCLUSIONS: Fluorescence imaging of c-MET receptor-expressing pSCC tumours after intravenous injection of EMI-137 was shown to be feasible and can be combined with fluorescence-based lymphatic mapping. This combination is unique and paves the way towards further development of this surgical guidance approach. Source of Funding: This research was financially supported by a Netherlands Organization for Scientific Research TTW-VICI grant (Grant No. TTW 16141). EMI-137 was provided by Edinburgh Molecular Imaging (EMI), KARL STORZ provided the Cy5 fluorescence camera, and the FIS-00 near-infrared fluorescence camera was made available by Hamamatsu photonics © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e716-e716 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Hielke de Vries More articles by this author Elise Bekers More articles by this author Matthias van Oosterom More articles by this author Baris Karakullukcu More articles by this author Henk van der Poel More articles by this author Fijs van Leeuwen More articles by this author Tessa Buckle More articles by this author Oscar Brouwer More articles by this author Expand All Advertisement Loading ...

Preclinical Evaluation of a Fluorescent Probe Targeting Receptor CDCP1 for Identification of Ovarian Cancer.

Optimal cytoreduction for ovarian cancer is often challenging because of aggressive tumor biology and advanced stage. It is a critical issue since the extent of residual disease after surgery is the key predictor of ovarian cancer patient survival. For a limited number of cancers, fluorescence-guided surgery has emerged as an effective aid for tumor delineation and effective cytoreduction. The intravenously administered fluorescent agent, most commonly indocyanine green (ICG), accumulates preferentially in tumors, which are visualized under a fluorescent light source to aid surgery. Insufficient tumor specificity has limited the broad application of these agents in surgical oncology including for ovarian cancer. In this study, we developed a novel tumor-selective fluorescent agent by chemically linking ICG to mouse monoclonal antibody 10D7 that specifically recognizes an ovarian cancer-enriched cell surface receptor, CUB-domain-containing protein 1 (CDCP1). 10D7ICG has high affinity for purified recombinant CDCP1 and CDCP1 that is located on the surface of ovarian cancer cells in vitro and in vivo. Our results show that intravenously administered 10D7ICG accumulates preferentially in ovarian cancer, permitting visualization of xenograft tumors in mice. The data suggest CDCP1 as a rational target for tumor-specific fluorescence-guided surgery for ovarian cancer.

Red fluorescent nanoprobe based on Ag@Au nanoparticles and graphene quantum dots for H2O2 determination and living cell imaging.

A sensitive and turn-on fluorescence nanoprobe based on core-shell Ag@Au nanoparticles (Ag@AuNPs) as a fluorescence receptor and red emissive graphene quantum dots (GQDs) as a donor was fabricated. They were conjugated together through π-π stacking between the GQDs and single-strand DNA modified at the Ag@AuNPs surface. The absorption spectrum of the receptorsignificantly overlapped with the donor emission spectrum, leading to a strong Förster resonance energy transfer (FRET) and thus a dramatic quenching. The sensing mechanism relies on fluorescence recovery following DNA cleavage by •OH produced from Fenton-like reaction between the peroxidase-like Ag nanocore and H2O2. The red emissive feature (Ex/Em, 520nm/560nm) provides low background in physiological samples. The •OH production, great spectrum overlapping, and red emission together contributes to good sensitivity and living cell imaging capability. The fluorescence assay (intensity at 560nm) achieves a low detection limit of 0.49μM H2O2 and a wide linear range from 5 to 200μM, superior to most of the reported fluorescent probes. The RSD value for 100μM H2O2 was 1.4%. Thenanoprobe exhibits excellent anti-interferences and shows low cytotoxicity. The recovery of 100μM standard H2O2 in a cancer cell lysate was 85.8%. Most satisfactorily, it can realize monitoring and imaging H2O2 in living cells. This study not only presents a sensitive H2O2 probe but also provides a platform for detecting other types of reactive oxygen species.

Two novel red-emitting fluorescent chemosensors for trace water determination with high sensitivity and large Stokes shift

Water plays a crucial role in human daily life and industrial production. However, the real-time detection technology for trace water still needed to be developed. In this paper, two red-emitting fluorescent receptors (HDBF and NDBF) for the recognition of water were designed and synthesized based on the β-diketone difluoroboron complex and natural product vanillin. The two fluorescent probes showed excellent optical properties such as large Stokes shifts (227 nm and 286 nm), long emission wavelengths (627 nm and 686 nm), and high fluorescence quantum yields (11.28% and 6.15%). The limits of detection of HDBF and NDBF for water were determined as low as 0.016% and 0.018%, respectively. Their detecting mechanism towards water was confirmed as the hydrogen-bonding interaction by 1H NMR titration analysis. Furthermore, the two fluorescent sensors were successfully utilized in probing trace water in 1,4-dioxane by utilizing wet cotton swab as carrier, which suggested their practical application value for trace water determination.

Abstract 1154: Site-specific gene editing with ABBIE for T-cell therapy

Abstract Therapeutic T-cell engineering using viral vectors has been extensively explored as the most promising therapeutic purpose. However, virus-mediated gene delivery carries many challenges and limitations, such as random insertion of transgene into chromosome, posing risk of oncogenic transformation of the host cells, and complexity in the manufacturing process.We are developing a gene-editing tool, ABBIE (A Binding-Based Integrase Enzyme) which combines the specificity of Cas9 with the non-destructive DNA integration conferred by HIV integrase. Unlike endonucleases, HIV integrase can efficiently insert LTR-flanked DNA without causing dsDNA breaks. In our system, by use of fusing protein of enzymatically dead Cas9 (Cas9DN) to integrase of HIV-1 or its mutants, we were able to perform the gene knock-out (KO) and knock-in (KI) in one step at the target genome loci. For proof of concept, we generated the ABBIE proteins and donor DNA containing fluorescent gene with LTR sequence and tested the ABBIE mediated knock-in with conversion of fluorescent proteins. The integration of fluorescent donor DNA into genome was observed in the 293 T-cells transfected with both WT and Mut ABBIE proteins. Higher integration rate was observed in the cells expressing Mut ABBIE protein. Further, we applied this strategy to deliver the donor fluorescent DNA or chimeric antigen receptors (CARs) via electroporation into the HPRT1 locus for specific insertion using primary human T cells. We observed an average of ~30% and ~16% knock-in efficiency as measured by flow cytometry of GFP+ and CD34+ expression, respectively. The site-specific transgene integration was tested by treating the cells with 6- thioguanine (6-TG). We will further optimize the electroporation condition to maximize editing efficiency. This novel therapeutic toolkit will provide a modular platform for inserting therapeutic genes safely and efficiently into the target genome, further accelerate the offshore immunotherapy development. Citation Format: Jihyun Park, Xiaohong Wang, Leonardo Mirandola, Maurizio Chiriva-Internati. Site-specific gene editing with ABBIE for T-cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1154.

A Vibration-Induced-Emission-Based Fluorescent Chemosensor for the Selective and Visual Recognition of Glucose.

The development of chemosensors to detect analytes in biologically relevant solutions is a challenging task. We report the synthesis of a fluorescent receptor that combines vibration‐induced emission (VIE) and dynamic covalent chemistry for the detection of glucose in aqueous media. We show that the bis‐2‐(N‐methylaminomethyl)phenylboronic acid‐decorated N,N′‐diphenyl‐dihydrodibenzo[a,c]phenazine (DPAC) receptor 1 can detect glucose and discriminate between closely related monosaccharides including those commonly found in blood. Preliminary studies suggest monosaccharides bind to the DPAC‐receptor with a 1:1 stoichiometry to produce pseudomacrocyclic complexes, which in turn leads to distinct optical changes in the fluorescent emission of the receptor for each host. Moreover, the complexation‐induced change in emission can be detected visually and quantified in a ratiometric way. Our results highlight the potential of VIE‐type receptors for the quantitative determination of saccharides in biological samples.

A Vibration‐Induced‐Emission‐Based Fluorescent Chemosensor for the Selective and Visual Recognition of Glucose

AbstractThe development of chemosensors to detect analytes in biologically relevant solutions is a challenging task. We report the synthesis of a fluorescent receptor that combines vibration‐induced emission (VIE) and dynamic covalent chemistry for the detection of glucose in aqueous media. We show that the bis‐2‐(N‐methylaminomethyl)phenylboronic acid‐decorated N,N′‐diphenyl‐dihydrodibenzo[a,c]phenazine (DPAC) receptor 1 can detect glucose and discriminate between closely related monosaccharides including those commonly found in blood. Preliminary studies suggest monosaccharides bind to the DPAC‐receptor with a 1:1 stoichiometry to produce pseudomacrocyclic complexes, which in turn leads to distinct optical changes in the fluorescent emission of the receptor for each host. Moreover, the complexation‐induced change in emission can be detected visually and quantified in a ratiometric way. Our results highlight the potential of VIE‐type receptors for the quantitative determination of saccharides in biological samples.

Effects of the herbicide Irgarol 1051 on the transcriptome of hermatypic coral Acropora tenuis and its symbiotic dinoflagellates

Coral reefs face multiple threats, including climate change, agricultural runoff, shipping activities, coastal development, and chemical pollutants. Irgarol 1051, a PSII herbicide, has been used as an antifouling booster since the previously used antibiofouling agent tributyltin (TBT) was banned worldwide. Although the mechanisms through which elevated temperatures cause coral bleaching have been reported, it remains unclear how PSII herbicides cause bleaching. Thus, in this study, we investigated the transcriptomes of Acropora tenuis and its symbiotic dinoflagellates by RNA-sequencing (RNA-Seq) to elucidate the molecular mechanisms underlying Irgarol-induced bleaching. Coral exposure to 10 μg/L Irgarol for 7 d affected coral body colour, specifically by an increase in their red, green, and blue (RGB) values; however, no such effect was observed in corals exposed to 1 μg/L Irgarol. RNA-Seq revealed the differentially expressed genes (DEGs) in corals and symbiotic dinoflagellates following Irgarol exposure. Coral DEGs encoded green fluorescent protein, blue-light-sensing photoreceptor (cryptochrome), chromoprotein, caspase 8, and nuclear receptors; DEGs in symbiotic dinoflagellates encoded light-harvesting proteins, photosystem II proteins, and heat shock proteins (i.e. HSP70 and HSP90), and ubiquitin. Bioinformatic analyses revealed that both Irgarol treatments disrupted various gene ontology terms, pathways, and protein interaction networks; these are different in corals (e.g. oxidative phosphorylation, metabolic pathway, transforming growth factor-β signalling pathway, adherens junction, and apoptosis) and symbiotic dinoflagellates (e.g. protein processing in endoplasmic reticulum, carbon fixation in photosynthetic organisms, metabolic pathway, and photosynthesis). Our data suggest that Irgarol disrupts the expression of various coral genes, thereby affecting various gene ontology terms, pathways, and protein interaction networks. Our study provides new insights into the potential molecular mechanisms underlying the bleaching effect of PSII herbicides, such as Irgarol, on corals and symbiotic dinoflagellates.

A dual-response naphthalene-armed calix[4]arene based fluorescence receptor for Zr(IV) and Fe(II) via Ligand to metal charge transfer

Abstract A new and dual-response calix[4]arene based fluorescence sensor; 5, 11, 17, 23-tetra t-butyl-25, 26, 27, 28 tetracarboxylate naphthalene calix [4]arene (C4N) has been designed and developed for the recognition of Zr4+ and Fe2+. The fairly linear range of C4N for Zr4+ and Fe2+ is 5−205 nM and 5–210 nM respectively. This sensor exhibits favourable selectivity towards Zr4+ and Fe2+ with the detection limit of 1.98 nM and 4.05 nM respectively via ligand to metal charge transfer (LMCT). This interesting supramolecular architecture has been exploited by UV–vis spectroscopy, voltammetry, electrospray ionization mass spectroscopy (ESI-MS), powder x-ray diffraction (PXRD) study and fourier transform infrared spectroscopy (FT-IR). The binding constant of C4N with Zr4+ and Fe2+ is calculated to be 8.139 × 108 M−1 and 8.430 × 108 M−1 respectively. The quantum mechanical study of C4N, C4N: Zr4+ and C4N: Fe2+ has been implemented using Gaussian 09 W software. Hybrid Becke’s three parameters exchange functional (B3LYP) level using standard STO-3 G basis set is used to optimize geometry of C4N and its composite metal cations. To strengthen the experimental florescence study, highest occupied molecular orbital-lowest unoccupied molecular orbital (HOMO-LUMO) energy band gap for C4N and C4N with Fe2+ and Zr4+ is also carried out, which reveals the enhancement and quenching effect in the luminous study. The molecular docking study has performed different protein receptors of metal-phosphodiesterase proteins (4MCW, 4MDZ, 4ME4, 6NVP and 6NVO) with C4N using HEX software.

Fluorescence modulation of naphthalene containing salicyl hydrazide-based receptor through aggregation-induced emission enhancement approach: Dual detection of lanthanum and cyanide ions in semi-aqueous medium.

The sensing activity of naphthalene containing salicyl hydrazide-based fluorescence receptor has been improved through aggregation-induced enhanced emission mechanism approach in semi-aqueous medium. The receptor has been found to be selective toward La3+ with approximately 70-fold fluorescence enhancement due to a combined effect of keto-enol tautomerism inhibition and chelation enhanced fluorescence with a detection limit of 3.91 × 10-6 M. In addition, the receptor is also able to sense CN- with a detection limit of 3.55 × 10-6 M via deprotonation effect, justifying its multiple analyte sensing behaviour. Hence, the current analytical methodology improves the sensing activity of the probe and also provides a greener alternative for La3+ and CN- detection.

Highly selective fluorescence detection of Cu2+ based on Schiff base functionalized periodic mesoporous organosilicas.

A novel copper ion sensing periodic mesoporous organosilica (SCN-PMO) was obtained by incorporating a Schiff base-based fluorescent receptor into the pore walls of mesoporous silica, which exhibited a well ordered mesoporous structure and excellent optical properties demonstrated by various characterization results. SCN-PMO possessed high selectivity and sensitivity towards Cu2+ based on its specific fluorescence response. The detection limit of SCN-PMO could be as low as 6.7 × 10-7 M. Due to protection of the silica network, SCN chromophores in PMOs exhibited higher photostability and the resulting material possessed great repeatability. Additionally, the fluorescence changes of SCN-PMO towards copper ions in vivo (zebrafish) showed that SCN-PMO has potential application as a nanoprobe in biological fields.

Design and property investigation on a five-interaction-based fluorescent anion receptor clip

A molecular clip combining a doubly substituted fluorescent anion–π donor probe and two flexible arms bearing H-bond motifs constitutes a new generation of anion receptors. Five simultaneous non-covalent interactions are highlighted by theoretical complexation studies with five different anions. A large range of analytical techniques (electrospray-tandem mass spectrometry, NMR, UV-visible, steady-state and time-resolved fluorescence) were deployed to evaluate the stoichiometry and association constants with the selected anions. The photophysical and anion–π donor properties of the tetrazine ring allowed fine characterization of the binding properties of the ligand. Based on previously published results, an anti-cooperativity effect in non-covalent interactions was demonstrated.

Dye-functionalized phosphate-binding macrocycles: from nucleotide to G-quadruplex recognition and “turn-on” fluorescence sensing

A PBM-dye architecture to design “turn-on” fluorescent receptors for G-quadruplexes of DNA is presented, which involves the connection of phosphate binding macrocycles (PBM) with dyes.

Extrasynaptic acetylcholine signaling through a muscarinic receptor regulates cell migration

Acetylcholine (ACh) promotes various cell migrations in vitro, but there are few investigations into this nonsynaptic role of ACh signaling in vivo. Here we investigate the function of a muscarinic receptor on an epithelial cell migration in Caenorhabditis elegans We show that the migratory gonad leader cell, the linker cell (LC), uses an M1/M3/M5-like muscarinic ACh receptor GAR-3 to receive extrasynaptic ACh signaling from cholinergic neurons for its migration. Either the loss of the GAR-3 receptor in the LC or the inhibition of ACh release from cholinergic neurons resulted in migratory path defects. The overactivation of the GAR-3 muscarinic receptor caused the LC to reverse its orientation through its downstream effectors Gαq/egl-30, PLCβ/egl-8, and TRIO/unc-73 This reversal response only occurred in the fourth larval stage, which corresponds to the developmental time when the GAR-3::yellow fluorescent protein receptor in the membrane relocalizes from a uniform to an asymmetric distribution. These findings suggest a role for the GAR-3 muscarinic receptor in determining the direction of LC migration.

Membrane Permeability and Its Activation Energies in Dependence on Analyte, Lipid, and Phase Type Obtained by the Fluorescent Artificial Receptor Membrane Assay.

Time-resolved monitoring of the permeability of analytes is of utmost importance in membrane research. Existing methods are restricted to single-point determinations or flat synthetic membranes, limiting access to biologically relevant kinetic parameters (permeation rate constant, permeation coefficients). We now use the recently introduced fluorescent artificial receptor membrane assay (FARMA) as a method to monitor, in real time, the permeation of indole derivatives through liposomal membranes of different lipid compositions. This method is based on the liposomal encapsulation of a chemosensing ensemble or "fluorescent artificial receptor", consisting of 2,7-dimethyldiazapyrenium as a fluorescent dye and cucurbit[8]uril as the macrocyclic receptor, that responds to the complexation of a permeating aromatic analyte by fluorescence quenching. FARMA does not require a fluorescent labeling of the analytes and allows access to permeability coefficients in the range from 10-8 to 10-4 cm s-1. The effect of temperature on the permeation rate of a series of indole derivatives across the phospholipid membranes was studied. The activation energies for permeation through POPC/POPS phospholipid membranes were in the range of 28-96 kJ mol-1. To study the effect of different lipid phases on the membrane permeability, we performed experiments with DPPC/DOPS vesicles, which showed a phase transition from a gel phase to a liquid-crystalline phase, where the activation energies for the permeation process were expected to show a dramatic change. Accordingly, for the permeation of the indole derivatives into the DPPC/DOPS liposomes, discontinuities were observed in the Arrhenius plots, from which the permeation activation energies for the distinct phases could be determined, for example, for tryptamine 245 kJ mol-1 in the gel phase and 47 kJ mol-1 in the liquid-crystalline phase.