A Multi-Functional Imaging Approach to High-Content Protein Interaction Screening

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 bas...

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Published in:	PloS one Vol. 7; no. 4; p. e33231
Main Authors:	Matthews, Daniel R., Fruhwirth, Gilbert O., Weitsman, Gregory, Carlin, Leo M., Ofo, Enyinnaya, Keppler, Melanie, Barber, Paul R., Tullis, Iain D. C., Vojnovic, Borivoj, Ng, Tony, Ameer-Beg, Simon M.
Format:	Journal Article
Language:	English
Published:	United States Public Library of Science 10.04.2012 Public Library of Science (PLoS)
Subjects:	Amino acids Analytical chemistry Anisotropy Assaying Biology Biophysics Biosensing Techniques - instrumentation Biosensing Techniques - methods Biosensors Cancer Cdc42 protein Cell Line, Tumor Chemokines CXCR4 protein Data analysis Detection equipment Energy transfer Fluorescence Fluorescence Polarization - instrumentation Fluorescence Polarization - methods Fluorescence resonance energy transfer Fluorescence Resonance Energy Transfer - instrumentation Fluorescence Resonance Energy Transfer - methods Green Fluorescent Proteins - chemistry Humans Image processing Information processing Instrumentation Internalization Lifetime Luminescent Proteins - chemistry Medical imaging Microscopy Oncology Photons Physics Protein interaction Protein Interaction Domains and Motifs Protein-protein interactions Proteins Proteins - chemistry Receptors, CXCR4 - chemistry Recipes Red Fluorescent Protein Scaling Screening Sensitivity and Specificity Small Molecule Libraries - chemistry Studies Time correlation functions United Kingdom > UK United States > US
ISSN:	1932-6203, 1932-6203
Online Access:	Get full text
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Summary:	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.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 14 content type line 23 Conceived and designed the experiments: SMA TN DRM. Performed the experiments: DRM GOF GW LMC EO. Analyzed the data: DRM SMA. Contributed reagents/materials/analysis tools: MK PB IT BV. Wrote the paper: DRM SMA. Current address: Centre for Molecular & Cellular Biology of Inflammation, King’s College London, London, United Kingdom
ISSN:	1932-6203 1932-6203
DOI:	10.1371/journal.pone.0033231