The Fluorescence Lifetime of a Single Actin-bound Fluorophore During Contraction of Skeletal Muscle

Abstract

During interaction of actin with myosin, cross-bridges impart cyclical impulses to thin filaments. A cross-bridge spends part of cycle time strongly attached to actin (ts) during which it generates force, and remaining time (td) detached from thin filaments. The environment of a binding site on actin is different when a cross-bridge is attached and detached from thin filaments. Here we report, for the first time, measurements of the environment of a single actin binding site in rigor, relaxed and during isometric contraction of skeletal muscle. The environment was monitored by tracking the fluorescent lifetime (τ) of single Alexa488-phalloidin molecule bound to actin. The fluorescent lifetime is the averaged rate of decay of fluorescent species from the excited state. It depends on a variety of environmental factors. Lifetime of a single phalloidin molecule located at the center of the Overlap-band was measured every 50 msec during 60 sec of rigor, relaxation and contraction of muscle. The lifetime of rigor muscle was large when a cross-bridge was bound to actin, low when it was dissociated from it and intermediate during contraction. The “duty cycle” of a cross-bridge (Ψ) - defined as the fraction of the total cross-bridge cycle that myosin spends attached to actin in a force generating state [Ψ=ts/(ts+td)] was calculated from lifetime as 60%.