roth lab presto tango kit  (Addgene inc)

Journal: bioRxiv

Article Title: Functional and phylogenetic analysis of placozoan GPCRs reveal the prebilaterian origin of monoaminergic signalling

doi: 10.1101/2025.04.18.649542

Figure Lengend Snippet: Cluster analysis of major class A GPCR groups from metazoans was obtained using an ad hoc monoamine hidden Markov model based on deorphanized receptors from bilaterians, with a minimum connection cutoff P-value of 1e-28. Each dot represents a GPCR sequence, colour-coded and symbolised according to the key in the top left. Connecting lines between sequences indicate similarity, with P-values noted in the bottom right. Cluster annotations are based on known deorphanised bilaterian class-A GPCRs. The teal squares with black crosses indicate the receptors tested and red crosses indicate receptors deorphanised in Trichoplax adhaerens .

Article Snippet: For this assay, human melatonin A (hMelA; MTNR1A Human) and human melatonin B (hMelB; MTNR1B) receptors were obtained from the Addgene PRESTO-Tango GPCR Kit from the Roth laboratory ( ).

Techniques: Sequencing

Journal: bioRxiv

Article Title: Functional and phylogenetic analysis of placozoan GPCRs reveal the prebilaterian origin of monoaminergic signalling

doi: 10.1101/2025.04.18.649542

Figure Lengend Snippet: (A) Pharmacological assay and pipeline for identifying monoamine-GPCR pairs. (B) Heatmap showing relative luminescence values from two replicates. Colour coding represents luminescence levels as shown in the key. Receptor responses were normalized to the negative control (0%) and five times the negative control (100%) to identify potential signals.

Article Snippet: For this assay, human melatonin A (hMelA; MTNR1A Human) and human melatonin B (hMelB; MTNR1B) receptors were obtained from the Addgene PRESTO-Tango GPCR Kit from the Roth laboratory ( ).

Techniques: Negative Control

Journal: bioRxiv

Article Title: Functional and phylogenetic analysis of placozoan GPCRs reveal the prebilaterian origin of monoaminergic signalling

doi: 10.1101/2025.04.18.649542

Figure Lengend Snippet: Dose-response curves show normalized luminescence plotted against varying ligand concentrations for each monoamine-GPCR pair. The receptor number is indicated above each curve. EC 50 values for the main ligands for each receptor are shown: phenethylamine for Tadh153, tyramine for Tadh165, and tryptamine for Tadh168, Tadh170, and Tadh173. The complete list of EC50 values for all tested compounds is available in Supplementary file 3. B) Synthesis pathway of placozoan monoamine receptor agonists compared to the acetylated versions of the compounds that activate human melatonin receptors . The enzyme AADC that is capable to produce phenethylamine, tyramine and tryptamine is present in placozoans, whereas the enzymes TPH and AANAT are absent in placozoans. Schemes of compounds where drawn using ChemDraw23.1.1

Article Snippet: For this assay, human melatonin A (hMelA; MTNR1A Human) and human melatonin B (hMelB; MTNR1B) receptors were obtained from the Addgene PRESTO-Tango GPCR Kit from the Roth laboratory ( ).

Techniques:

Journal: bioRxiv

Article Title: Functional and phylogenetic analysis of placozoan GPCRs reveal the prebilaterian origin of monoaminergic signalling

doi: 10.1101/2025.04.18.649542

Figure Lengend Snippet: The tree was generated using IQ-TREE2 with the best-fit model LG+G4 and rooted with neuropeptide GPCRs. The tree is partially collapsed for clarity. Node support values (shown as numbers) are based on 1000 replicates. Clades are colour-coded as follows: red for cnidarians, blue for placozoans, orange for bilaterian-specific clades, and green for ctenophores. Black clades represent groups found in both bilaterians and non-bilaterians. Dotted or dashed boxes indicate clades containing receptors that were deorphanized in placozoans. B) Experimental characterisation of human melatonin receptors (hMelA and hMelB) supports the homology relationship of monoamine GPCR between placozoans and humans. Dose-response curves show normalized luminescence plotted against varying ligand concentrations for each monoamine-GPCR pair. The receptor name is indicated above each curve. The table adjacent to the curves lists EC50 values for each compound tested on human receptors. hMelB is activated by tryptamine only at high concentrations.

Article Snippet: For this assay, human melatonin A (hMelA; MTNR1A Human) and human melatonin B (hMelB; MTNR1B) receptors were obtained from the Addgene PRESTO-Tango GPCR Kit from the Roth laboratory ( ).

Techniques: Generated

Journal: bioRxiv

Article Title: In vivo multiplex imaging of dynamic neurochemical networks with designed far-red dopamine sensors

doi: 10.1101/2024.12.22.629999

Figure Lengend Snippet: ( A ) (Left) Schematic diagram illustrating the principle of the far-red dopamine (DA) sensor (top left) and L-Z equilibrium of rhodamine derivatives (bottom left). (Right) Idealized traces depicting the emission spectra of current GFP-and RFP-based sensors, alongside the new far-red and near-infrared (NIR) sensors. ( B ) Optimization of far-red DA sensor variants in response to 100 μM DA application, with stepwise changes in the insertion sites, linker, cpHaloTag and GPCR optimization. The variants in step 1 were screened using the dye JF635, while the variants in steps 2, 3, and 4 were screened using the dye JF646. ( C ) Representative images of HEK293T cells expressing HaloDA1.0 and labeled with JF646, before and after application of 100 μM DA. Scale bar, 20 μm. ( D ) Dose-response curves of HaloDA1.0 and HaloDAmut labeled with JF646 in HEK293T cells; n = 3 wells with 300–500 cells per well. ( E ) One-photon excitation (Ex) and emission (Em) spectra of HaloDA1.0 labeled with JF646 in the presence of 100 μM DA (solid lines) or saline (dashed lines). F.I., fluorescence intensity. ( F ) Maximum ΔF/F 0 (left) and normalized dose-response curves (right) for HaloDA1.0 labeled with the indicated dyes in HEK293T cells; n = 3 wells with 300–500 cells per well for each dye. ( G ) Representative images of cultured rat cortical neurons expressing HaloDA1.0 and labeled with the indicated dyes (top row) and fluorescence response to 100 μM DA (bottom row). Scale bar, 50 μm. ( H ) Dose-response curves (left), maximum ΔF/F 0 (top right), and signal-to-noise ratio (SNR) relative to JF635 (bottom right) for cultured rat cortical neurons expressing HaloDA1.0 and labeled with the indicated dyes; n = 120 regions of interest (ROIs) from 4 coverslips for each dye. ( I ) Normalized ΔF/F 0 (relative to DA) for HaloDA1.0 expressed in cultured neurons and labeled with JF646. SCH, SCH-23390 (D1R antagonist); Etic, eticlopride (D2R antagonist); SKF, SKF-81297 (D1R agonist); Quin, quinpirole (D2R agonist); 5-HT, serotonin; HA, histamine; OA, octopamine; TA, tyramine; ACh, acetylcholine, GABA, γ-aminobutyric acid; Glu, glutamate; L-Dopa, levodopa. All chemicals were applied at 1 μM; n = 3 wells with an average of 50 neurons per well. The inset shows the dose-response curves for DA and norepinephrine (NE); n = 3–4 coverslips with 30 ROIs per coverslip. ( J ) Luciferase complementation assay to measure G protein coupling. Cells expressing miniGs-LgBit alone served as a negative control; n = 3 wells per group. WT, wild-type. ( K ) Tango assay to measure β-arrestin coupling. Non-transfected cells served as a negative control; n = 3 wells per group. ( L ) Schematic diagram depicting the strategy for multiplex imaging (left) and representative images (right) of cultured neurons co-expressing the far-red DA sensor (JF646-labeled HaloDA1.0), the red fluorescent 5-HT sensor (r5-HT1.0), and the green fluorescent NE sensor (NE2m). Scale bar, 50 μm. ( M ) Fluorescence responses of JF646-labeled HaloDA1.0 (magenta), r5-HT1.0 (red), and NE2m (green). Where indicated, DA (1 μM), 5-HT (1 μM), NE (1 μM), yohimbine (YO, 2 μM), RS23597-190 (20 μM), and SCH (10 μM) were applied; n = 40 ROIs from 3 coverslips.

Article Snippet: For the Tango assay, D1R-Tango was cloned from the PRESTO-Tango GPCR Kit (Addgene kit no. 1000000068), and HaloDA1.0-Tango was generated by replacing D1R in D1R-Tango with HaloDA1.0.

Techniques: Expressing, Labeling, Saline, Fluorescence, Cell Culture, Luciferase, Negative Control, Transfection, Multiplex Assay, Imaging