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Proceedings Paper

Dynamic imaging of protein-protein interactions by MP-FLIM
Author(s): Simon M. Ameer-Beg; Marion Peter; Melanie D Keppler; Soren Prag; Paul R. Barber; Tony C. Ng; Borivoj Vojnovic
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Paper Abstract

The spatio-temporal localization of molecular interactions within cells in situ is of great importance in elucidating the key mechanisms in regulation of fundamental process within the cell. Measurements of such near-field localization of protein complexes may be achieved by the detection of fluorescence (or Forster) resonance energy transfer (FRET) between protein-conjugated fluorophores. We demonstrate the applicability of time-correlated single photon counting multiphoton microscopy to the spatio-temporal localization of protein-protein interactions in live and fixed cell populations. Intramolecular interactions between protein hetero-dimers are investigated using green fluorescent protein variants. We present an improved monomeric form of the red fluorescent protein, mRFP1, as the acceptor in biological fluorescence resonance energy transfer (FRET) experiments using the enhanced green fluorescent protein as donor. We find particular advantage in using this fluorophore pair for quantitative measurements of FRET. The technique was exploited to demonstrate a novel receptor-kinase interaction between the chemokine receptor (CXCR4) and protein kinase C (PKC) α in carcinoma cells for both live and fixed cell experiments.

Paper Details

Date Published: 30 March 2005
PDF: 10 pages
Proc. SPIE 5700, Multiphoton Microscopy in the Biomedical Sciences V, (30 March 2005); doi: 10.1117/12.589202
Show Author Affiliations
Simon M. Ameer-Beg, Gray Cancer Institute, Mount Vernon Hospital (United Kingdom)
Marion Peter, King's College London, Guy's Medical School (United Kingdom)
Melanie D Keppler, King's College London, Guy's Medical School (United Kingdom)
Soren Prag, King's College London, Guy's Medical School (United Kingdom)
Paul R. Barber, Gray Cancer Institute, Mount Vernon Hospital (United Kingdom)
Tony C. Ng, King's College London, Guy's Medical School (United Kingdom)
Borivoj Vojnovic, Gray Cancer Institute, Mount Vernon Hospital (United Kingdom)


Published in SPIE Proceedings Vol. 5700:
Multiphoton Microscopy in the Biomedical Sciences V
Ammasi Periasamy; Peter T. C. So, Editor(s)

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