Fluorescence lifetime imaging to detect actomyosin states in mammalian muscle sarcomeres.
We investigated the use of fluorescence lifetime imaging microscopy (FLIM) of a fluorescently labeled ATP analog (3'-O-{N-[3-(7-diethylaminocoumarin-3-carboxamido)propyl]carbamoyl}ATP) to probe in permeabilized muscle fibers the changes in the environment of the nucleotide binding pocket caused by interaction with actin. Spatial averaging of FLIM data of muscle sarcomeres reduces photon noise, permitting detailed analysis of the fluorescence decay profiles. FLIM reveals that the lifetime of the nucleotide, in its ADP form because of the low concentration of nucleotide present, changes depending on whether the nucleotide is free in solution or bound to myosin, and on whether the myosin is bound to actin in an actomyosin complex. Characterization of the fluorescence decays by a multiexponential function allowed us to resolve the lifetimes and amplitudes of each of these populations, namely, the fluorophore bound to myosin, bound to actin, in an actomyosin complex, and free in the filament lattice. This novel application of FLIM to muscle fibers shows that with spatial averaging, detailed information about the nature of nucleotide complexes can be derived.
García
Delisa I
DI
Biological Nanoscience Section, National Heart and Lung Institute, and Photonics Group, Physics Department, Imperial College, London, United Kingdom.
Lanigan
Peter
P
Webb
Martin
M
West
Timothy G
TG
Requejo-Isidro
Jose
J
Auksorius
Egidijus
E
Dunsby
Chris
C
Neil
Mark
M
French
Paul
P
Ferenczi
Michael A
MA
D000205:Actomyosin / Q000737:chemistry* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D055592:Biophysical Phenomena; D001703:Biophysics; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D008856:Microscopy, Fluorescence; D011485:Protein Binding; D016658:Psoas Muscles / Q000737:chemistry / Q000378:metabolism; D011817:Rabbits; D012518:Sarcomeres / Q000737:chemistry* / Q000378:metabolism
D005456:Fluorescent Dyes; D000255:Adenosine Triphosphate; D000205:Actomyosin