Single molecule imaging reveals the concerted release of myosin from regulated thin filaments

Abstract

Regulated thin filaments (RTFs) tightly control striated muscle contraction through calcium binding to troponin, which in turn shifts the position of tropomyosin on actin to expose myosin binding sites. The binding of the first myosin holds tropomyosin in a position such that more myosin binding sites on actin are available, resulting in cooperative activation. Troponin and tropomyosin also act to turn off the thin filament; however, this is antagonized by the high local concentration of myosin, questioning how the thin filament relaxes. To provide molecular details of deactivation we use the RTF tightrope assay, in which single RTFs are suspended between pedestals above a microscope coverslip surface. Single molecule imaging of GFP tagged myosin-S1 (S1-GFP) is used to follow the activation of RTF tightropes. In sub-maximal activation conditions, S1-GFP molecules bind forming metastable clusters, from which release and rebinding of S1-GFP leads to prolonged activation in these regions. Because the RTFs are not fully active we are able to directly observe deactivation in real time. Using a Reversible Jump Markov Chain Monte Carlo model we are able to dynamically assess the fate of active regions. This analysis reveals that myosin binding occurs in a stochastic stepwise fashion; however, an unexpectedly large probability of multiple simultaneous detachments is observed. This suggests that deactivation of the thin filament is a coordinated, active process.