Phosphate and ADP Differently Inhibit Coordinated Smooth Muscle Myosin Groups

Abstract

Actin filaments propelled in vitro by groups of skeletal muscle myosin motors exhibit distinct phases of active sliding or arrest, whose occurrence depends on actin length (L) within a range of up to 1.0 μm. Smooth muscle myosin filaments are exponentially distributed with ≈150 nm average length in vivo--suggesting relevance of the L-dependence of myosin group kinetics. Here, we found L-dependent actin arrest and sliding in in vitro motility assays of smooth muscle myosin. We perturbed individual myosin kinetics with varying, physiological concentrations of phosphate (Pi, release associated with main power stroke) and adenosine diphosphate (ADP, release associated with minor mechanical step). Adenosine triphosphate was kept constant at physiological concentration. Increasing [Pi] lowered the fraction of time for which actin was actively sliding, reflected in reduced average sliding velocity (ν) and motile fraction (fmot, fraction of time that filaments are moving); increasing [ADP] increased the fraction of time actively sliding and reduced the velocity while sliding, reflected in reduced ν and increased fmot. We introduced specific Pi and ADP effects on individual myosin kinetics into our recently developed mathematical model of actin propulsion by myosin groups. Simulations matched our experimental observations and described the inhibition of myosin group kinetics. At low [Pi] and [ADP], actin arrest and sliding were reflected by two distinct chemical states of the myosin group. Upon [Pi] increase, the probability of the active state decreased; upon [ADP] increase, the probability of the active state increased, but the active state became increasingly similar to the arrested state.

Spreading of perturbations in myosin group kinetics along actin filaments

“…(C) Three example time courses showing instantaneous sliding velocities ("frame-to-frame velocities," V f 2f ) for actin filaments of different length ðLÞ. As previous work has shown, actin sliding exhibits L-dependent, bistable switching (8,10); the shortest actin filaments (diffraction-limited, L K 0.3 μm) are permanently arrested; slightly longer actin filaments exhibit bistable switching between sliding and arrest; for L ≥ 1 μm, actin slides continuously. (D) Previous work explained how the mechanical coupling of individual myosin motors via the actin filament results in 2 emergent states in myosin groups, which underlie the actin stop-and-go motion (8,10).…”

“…Myosin binding is oriented with respect to the filament sliding direction. (B) Individual myosin binding sites go through unidirectional kinetic cycles consisting of 3 main states (8,10): unoccupied (Free), pre-power stroke (Pre), and post-power stroke (Post). The transitions occur by load-independent myosin attachment and load-dependent mechanochemical transitions.…”

“…Specifically, the group kinetic states corresponding to actin arrest or sliding are molecularly characterized by persistent binding at all myosin binding sites or a balanced mixture of bound and unbound binding sites, respectively. Furthermore, it was shown that experimentally induced changes in single myosin mechanochemistry proliferate into altered actin sliding patterns (9,10). Last, statistical distributions of actin sliding velocities for different lengths of filaments can be extracted using automated actin tracking software (9).…”

“…Motility Assay, Video Recording, and Analysis of Actin Sliding. Buffers, proteins, and flow-through chambers were prepared including several steps for the removal of catalytically defective myosin as previously described (8,10). Concentrations of myosin and filamin were controlled by dilution in the initial myosin perfusion buffer and the final motility buffer, respectively.…”

“…The mechanical work levels W ‡ and W eq can be calculated by applying the local mechanical equilibrium condition with and without the additional strain for the protein of interest to reach its transition state, respectively. The resulting rates were included in an extended Gillespie algorithm, to simulate actin sliding, which was sampled at time intervals Δt = 0.33 s as described previously (8,10). Details are described in SI Appendix.…”