Fig. 1: Quantitative nanoscale imaging of major protein families by PharmacoSTORM.
From: PharmacoSTORM nanoscale pharmacology reveals cariprazine binding on Islands of Calleja granule cells
a General workflow of the development of PharmacoSTORM probes, demonstrated by the example of a CB1R ligand. (I) The docking pose of the fluorescently tagged CB1R agonist (fluo-cannabinoid, yellow) in the CB1R (gray) (PDB:5XRA). The hydrogen bonds between the protein residues (red) and the probe are represented as yellow dashed lines. (II) In vitro cAMP assay for the functional characterization of fluo-cannabinoid activity on CB1R (n = 5, logEC50 = −7.48 nM, Emax = 36.1%). Reference compound was WIN55,212-2 (n = 4, logEC50 = −8.03 nM, Emax = 51.5%). (III) Confocal microscopic image of HEK 293 cells expressing GFP-CB1R, labeled with 100 nM fluo-cannabinoid; (IV) Comparison of confocal and STORM images depicting fluo-cannabinoid binding to CB1Rs on the same plasma membrane segment. b, e, h Surface representations of three PharmacoSTORM probes, bound to their respective protein targets that represent three major protein families. b GPCR:CB1R, (PDB:5XRA, gray) with fluo-cannabinoid (yellow); e enzyme: MAGL (PDB:6BQ0, gray) with DH-463 (yellow); h ion channel: α7-nAchR (PDB:4HQP, gray) with fluorescent α-bungarotoxin (yellow). c, f, i Competitive ligand-binding measurements by dual direct STORM imaging demonstrate the high specificity of PharmacoSTORM probes. Target proteins were visualized simultaneously by pharmacoprobe- and antibody-based labeling. c 100 nM fluo-cannabinoid and anti-GFP immunostaining for GFP-CB1R in the absence (vehicle) or presence of CB1R antagonist rimonabant (Rim, 10 µM). f 100 nM DH-463 and anti-GFP immunostaining for GFP-MAGL pretreated either with vehicle or the MAGL inhibitor JZL184 (JZL, 10 µM). i 1 ng/µl Alexa647-α-bungarotoxin and anti-HA immunostaining for α7-nAchR-HA pretreated either with vehicle or the α7-nAchR antagonist methyllycaconitine (MLA, 10 µM). d, g, j Scatter dot plots display the ratio of PharmacoSTORM and ImmunoSTORM localization points (LPs) in the absence or presence of the unlabeled competitive ligands. Data are normalized to average vehicle (Veh) pretreatment. Two-tailed Mann–Whitney tests were performed. d n = 5, P = 0.0079; g n = 4, P = 0.0286; j n = 4, P = 0.0286. Data are presented as mean ± SEM, n values indicate biologically independent experiments in all panels.
