Dynamics of Regulated Thin Filament Motility

Abstract

The cooperative activation of regulated thin filaments (RTFs) is important for efficient calcium handling in striated muscle. Although myosin binding to an RTF has been proposed as a mechanism for cooperative activation, we have recently shown that myosin binding constitutively rather than cooperatively activates RTFs; in other words the binding of a single myosin head fully activates the regulatory unit of an RTF. The model predicts a well-defined relationship between the calcium concentration at half-maximal activation (pCa50) and all factor that influences actin-myosin attachment, katt, or detachment, kdet, kinetics. To test this model, we use an in vitro motility assay to determine the effects of altering katt and kdet on the calcium dependence of RTF sliding velocities, V. The model accurately describes our observed changes in pCa50 when katt and kdet are modulated with changes in [ATP] and [ADP], upon addition of inhibitors, and when myosin densities are varied. The model further predicts that partially activated V result from the RTF switching between fully active and inactive states rather than the RTF maintaining a partial level of activation. To test this hypothesis, we are using an in vitro motility assay to measure the dynamics of speckled fluorescent actin filaments with high spatial accuracy and temporal resolution. Our model and data suggest that calcium sensitivity of striated muscle is altered by changes in actin-myosin attachment and detachment kinetics.