A multi-functional imaging approach to high-content protein interaction screening

Matthews, D. R. et al. (2012) A multi-functional imaging approach to high-content protein interaction screening. PLoS ONE, 7(4), e33231. (doi: 10.1371/journal.pone.0033231) (PMID:22506000) (PMCID:PMC3323588)

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.

• Item Type: Articles
• Additional Information: The work was supported by Engineering and Physical Sciences Research Councils (EPSRC) grant EP/C546105/1; the King’s College London/University College London Comprehensive Cancer Imaging Centre (CCIC) funded by Cancer Research UK (CRUK) & EPSRC; a CRUK Programme Grant (C133/A/1812) and an endowment fund from Dimbleby Cancer Care to King’s College London.
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Carlin, Dr Leo
• Authors: Matthews, D. R., Fruhwirth, G. O., Weitsman, G., Carlin, L. M., Ofo, E., Keppler, M., Barber, P. R., Tullis, I. D.C., Vojnovic, B., Ng, T., and Ameer-Beg, S. M.
• College/School: College of Medical Veterinary and Life Sciences > School of Cancer Sciences
• Journal Name: PLoS ONE
• Publisher: Public Library of Science
• ISSN: 1932-6203
• ISSN (Online): 1932-6203
• Copyright Holders: Copyright 2012 Matthews et al.
• First Published: First published in PLoS ONE 7(4):e33231
• Publisher Policy: Reproduced under a Creative Commons license