Fluorescence microscopy view of muscle mechanochemistry
Dmitry Ushakov
Myosin, actin and striated muscle
Actin filament
Muscle actomyosin kinetic cycle
Detached
states
Three Force generating
states
ADP
ATP
Weakly
attached
Pi
Fluorescent Pi binding protein:
Environmentally sensitive coumarin-based dye
Pi binds tightly to the protein and increases coumarin fluorescence
Hirshberg M, Henrick K, Haire LL, Vasisht N, Brune M, Corrie JE, Webb MR. Biochemistry. 1998 37:10381-5.
Skinned fiber
Rigor (zero Ca2+, zero ATP)
Load Ca2+ and NPE-caged ATP
Load labelled sensor
(PBP-MDCC)
Laser flash activation
Mechanical perturbation
ADP or Pi
Dependent
D-Fluor
Force
production
Force and Pi release time courses
Time courses of force, sarcomere length, and phosphate release.
Experiments were at 12C. Fibers were activated from Ca21 rigor using a laser pulse to release ATP from NPE-caged ATP. Laser flash was at time 0, and the release step (0.5% of fiber length) was at 0.4 s (vertical dashed line in all records). Linear fits in panel C to the pre- (0.3–0.4 s) and post- (0.45–0.55 s) step data are shown by the thin lines.
Response to rapid length steps at 12C. (A) Phosphate release. Single exponentials, fitted through zero [Pi] at time 0.4 s, are fitted to the transients; rate constant are 35 s-1 (0.3%L, gray line) and 32 s-1 (0.5%L, black line). (B) Tension recovery records after rapid release steps of 0.3%L (gray) and 0.5%L (black).
Effect of temperature and ADP on cross-bridge dynamics
Calculated distribution of attached cross-bridges after rapid length steps.
(A) Relative occupancy of AM'ADPPi (thin lines) and AM'ADP (thick lines) cross-bridge states during the period of a rapid release step, calculated by the model (Scheme 1). Calculations are shown for 20C with a 0.5%L step (black), 12C with a 0.5%L step (blue), and 12C with a 0.3%L (red).

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