title: A multi-functional imaging approach to high-content protein interaction screening.
abstract: Functional imaging can provide a level of quantification that is not possible in what might be termed traditional high-content screening. This is due to the fact that the current state-of-the-art high-content screening systems take the approach of scaling-up single cell assays, and are therefore based on essentially pictorial measures as assay indicators. Such phenotypic analyses have become extremely sophisticated, advancing screening enormously, but this approach can still be somewhat subjective. We describe the development, and validation, of a prototype high-content screening platform that combines steady-state fluorescence anisotropy imaging with fluorescence lifetime imaging (FLIM). This functional approach allows objective, quantitative screening of small molecule libraries in protein-protein interaction assays. We discuss the development of the instrumentation, the process by which information on fluorescence resonance energy transfer (FRET) can be extracted from wide-field, acceptor fluorescence anisotropy imaging and cross-checking of this modality using lifetime imaging by time-correlated single-photon counting. Imaging of cells expressing protein constructs where eGFP and mRFP1 are linked with amino-acid chains of various lengths (7, 19 and 32 amino acids) shows the two methodologies to be highly correlated. We validate our approach using a small-scale inhibitor screen of a Cdc42 FRET biosensor probe expressed in epidermoid cancer cells (A431) in a 96 microwell-plate format. We also show that acceptor fluorescence anisotropy can be used to measure variations in hetero-FRET in protein-protein interactions. We demonstrate this using a screen of inhibitors of internalization of the transmembrane receptor, CXCR4. These assays enable us to demonstrate all the capabilities of the instrument, image processing and analytical techniques that have been developed. Direct correlation between acceptor anisotropy and donor FLIM is observed for FRET assays, providing an opportunity to rapidly screen proteins, interacting on the nano-meter scale, using wide-field imaging.
authors.affiliation: Division of Cancer Studies, Randall Division of Cell and Molecular Biophysics, King's College London, London, United Kingdom.
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Matthews
authors.forename: Gilbert O
authors.initials: GO
authors.lastname: Fruhwirth
authors.forename: Gregory
authors.initials: G
authors.lastname: Weitsman
authors.forename: Leo M
authors.initials: LM
authors.lastname: Carlin
authors.forename: Enyinnaya
authors.initials: E
authors.lastname: Ofo
authors.forename: Melanie
authors.initials: M
authors.lastname: Keppler
authors.forename: Paul R
authors.initials: PR
authors.lastname: Barber
authors.forename: Iain D C
authors.initials: ID
authors.lastname: Tullis
authors.forename: Borivoj
authors.initials: B
authors.lastname: Vojnovic
authors.forename: Tony
authors.initials: T
authors.lastname: Ng
authors.forename: Simon M
authors.initials: SM
authors.lastname: Ameer-Beg
mesh_terms: D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods*; D045744:Cell Line, Tumor; D005454:Fluorescence Polarization / Q000295:instrumentation* / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation* / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry; D017785:Photons; D054730:Protein Interaction Domains and Motifs; D011506:Proteins / Q000737:chemistry*; D019718:Receptors, CXCR4 / Q000737:chemistry; D012680:Sensitivity and Specificity; D054852:Small Molecule Libraries / Q000737:chemistry; D000097573:Red Fluorescent Protein
chemical_list: D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D011506:Proteins; D019718:Receptors, CXCR4; D054852:Small Molecule Libraries; D000097573:Red Fluorescent Protein; C527276:CXCR4 protein, human; C485184:enhanced green fluorescent protein