Dynamic imaging of protein-protein interactions by MP-FLIM

Simon M. Ameer-Beg; Marion Peter; Melanie D Keppler; Soren Prag; Paul R. Barber; Tony C. Ng; Borivoj Vojnovic

doi:10.1117/12.589202

30 March 2005 Dynamic imaging of protein-protein interactions by MP-FLIM

Simon M. Ameer-Beg, Marion Peter, Melanie D Keppler, Soren Prag, Paul R. Barber, Tony C. Ng, Borivoj Vojnovic

Proceedings Volume 5700, Multiphoton Microscopy in the Biomedical Sciences V; (2005) https://doi.org/10.1117/12.589202
Event: SPIE BiOS, 2005, San Jose, CA, United States

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.

Citation Download Citation

Simon M. Ameer-Beg, Marion Peter, Melanie D Keppler, Soren Prag, Paul R. Barber, Tony C. Ng, and Borivoj Vojnovic "Dynamic imaging of protein-protein interactions by MP-FLIM", Proc. SPIE 5700, Multiphoton Microscopy in the Biomedical Sciences V, (30 March 2005); https://doi.org/10.1117/12.589202

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