Spectrally and spatially resolved fluorescence lifetime imaging in living cells: TRPV4-microfilament interactions

Time- and space-correlated single photon counting method has been used to demonstrate the interactions of cation channel "transient receptor potential vanilloid 4" (TRPV4) and microfilaments. Living cells co-expressing TRPV4-CFP and actin-YFP, when excited for the donor molecules (CFP) exhibited an emission peak at 527 nm and decrease of the lifetime in the wavelength band 460-490 nm; corresponding to resonance energy transfer to YFP. CFP fluorescence decay was fitted best by a dual mode decay model. Considering the average lifetime of the donor, both in the presence and absence of acceptor yielded an apparent FRET efficiency of similar to 20%. This is rather high placing the minimum distance of chromophores in the two fluorescent proteins in the range of 4 nm. Thus, this study shows for the first time that TRPV4 and actin intimately associate within living cells. The significance of this finding for cell volume regulation is highlighted. (c) 2007 Elsevier Inc. All rights reserved.