Comparison of G-protein coupled receptor desensitization-related beta-arrestin redistribution using confocal and non-confocal imaging.
High Content Screening (HCS), a combination of fluorescence microscopic imaging and automated image analysis, has become a frequently applied tool to study test compound effects in cellular disease-modelling systems. In this work, we compared a confocal and a non-confocal cellular HCS system, the IN Cell Analyzers(1) 3,000 and 1,000, respectively. As a cellular model system we used the Transfluor technology in the 384-well microtiter plate (MTP) format. The Transfluor HCS assay for G-protein coupled receptor (GPCR) activation is based on the recruitment of a green fluorescent protein-labelled arrestin (ArrGFP) from the cytosol to the plasma membrane. We investigated two GPCRs, the wild-type (wt) beta2 adrenergic receptor (beta2AR) and the beta2AR-enhanced (E), a C-terminally mutated receptor with a higher affinity to arrestin. Upon agonist stimulation, the beta2AR-wt induced the redistribution of ArrGFP to coated pits, the beta2AR-E maintained the interaction with ArrGFP down to the formation of endocytic vesicles. Our findings reveal that the assay is feasible on both instruments, with sufficiently robust Z' statistics. Improved Z' statistics, though, are achieved with the confocal system, particularly in case of weak signals. Moreover, throughput is dramatically higher for the IN Cell Analyzer 3,000. We conclude that, depending on the needs for throughput and assay biology, either instrument may fulfil a successful role in the drug discovery process. Confocal optics, however, provide a better basis for the detection of smaller subcellular structures with lower fluorescence intensity.
Haasen
Dorothea
D
Boehringer Ingelheim Pharma GmbH & Co. KG, Department of Integrated Lead Discovery, Birkendorfer Str. 65, D-88397 Biberach, Germany.
Wolff
Michael
M
Valler
Martin J
MJ
Heilker
Ralf
R
D058666:Adrenergic beta-2 Receptor Agonists; D058672:Adrenergic beta-2 Receptor Antagonists; D019390:Arrestins / Q000737:chemistry*; D002462:Cell Membrane / Q000737:chemistry; D002478:Cells, Cultured; D003600:Cytosol / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000295:instrumentation / Q000379:methods*; D007545:Isoproterenol / Q000494:pharmacology; D018613:Microscopy, Confocal / Q000295:instrumentation / Q000379:methods; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods; D008954:Models, Biological; D011433:Propranolol / Q000494:pharmacology; D018343:Receptors, Adrenergic, beta-2 / Q000737:chemistry*; D018035:Receptors, Odorant / Q000819:agonists / Q000037:antagonists & inhibitors / Q000737:chemistry*; D012680:Sensitivity and Specificity; D013329:Structure-Activity Relationship; D000071557:beta-Arrestins
D058666:Adrenergic beta-2 Receptor Agonists; D058672:Adrenergic beta-2 Receptor Antagonists; D019390:Arrestins; D018343:Receptors, Adrenergic, beta-2; D018035:Receptors, Odorant; D000071557:beta-Arrestins; D049452:Green Fluorescent Proteins; C077170:G-protein coupled receptor OR3; D011433:Propranolol; D007545:Isoproterenol