Response of rigor cross-bridges to stretch detected by fluorescence lifetime imaging microscopy of myosin essential light chain in skeletal muscle fibers.
We applied fluorescence lifetime imaging microscopy to map the microenvironment of the myosin essential light chain (ELC) in permeabilized skeletal muscle fibers. Four ELC mutants containing a single cysteine residue at different positions in the C-terminal half of the protein (ELC-127, ELC-142, ELC-160, and ELC-180) were generated by site-directed mutagenesis, labeled with 7-diethylamino-3-((((2-iodoacetamido)ethyl)amino)carbonyl)coumarin, and introduced into permeabilized rabbit psoas fibers. Binding to the myosin heavy chain was associated with a large conformational change in the ELC. When the fibers were moved from relaxation to rigor, the fluorescence lifetime increased for all label positions. However, when 1% stretch was applied to the rigor fibers, the lifetime decreased for ELC-127 and ELC-180 but did not change for ELC-142 and ELC-160. The differential change of fluorescence lifetime demonstrates the shift in position of the C-terminal domain of ELC with respect to the heavy chain and reveals specific locations in the lever arm region sensitive to the mechanical strain propagating from the actin-binding site to the lever arm.
National Heart and Lung Institute, Imperial College London, London SW7 2AZ, United Kingdom. d.ushakov@imperial.ac.uk
Dmitry S
DS
Ushakov
Valentina
V
Caorsi
Delisa
D
Ibanez-Garcia
Hugh B
HB
Manning
Antonios D
AD
Konitsiotis
Timothy G
TG
West
Christopher
C
Dunsby
Paul M
PM
French
Michael A
MA
Ferenczi
D000818:Animals; D001696:Biomechanical Phenomena; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods*; D008958:Models, Molecular; D018485:Muscle Fibers, Skeletal / Q000737:chemistry / Q000378:metabolism*; D009126:Muscle Relaxation; D018995:Myosin Heavy Chains / Q000378:metabolism; D018994:Myosin Light Chains / Q000737:chemistry* / Q000378:metabolism*; D010539:Permeability; D011487:Protein Conformation; D011817:Rabbits
D005456:Fluorescent Dyes; D018994:Myosin Light Chains; D018995:Myosin Heavy Chains