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 approximately 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.
Ramadass
Radhan
R
Kinematic Cell Research Group, Institute for Cell Biology and Neuroscience, JW Goethe University, Max-von-Laue-Strasse 9, D-60438 Frankfurt/Main, Germany. ramadass@bio.uni-frankfurt.de
Becker
Daniel
D
Jendrach
Marina
M
Bereiter-Hahn
Jürgen
J
D008841:Actin Cytoskeleton / Q000378:metabolism*; D000199:Actins / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods; D050916:TRPV Cation Channels / Q000378:metabolism*; D014162:Transfection
D000199:Actins; C485182:Cyan Fluorescent Protein; D050916:TRPV Cation Channels; C416986:TRPV4 protein, human; D049452:Green Fluorescent Proteins