Regulatory Light Chain Phosphorylation and N-Terminal Extension Increase Cross-Bridge Binding and Power Output in Drosophila at In Vivo Myofilament Lattice Spacing

Abstract

The N-terminal extension and phosphorylation of the myosin regulatory light chain (RLC) independently improve Drosophila melanogaster flight performance. Here we examine the functional and structural role of the RLC in chemically skinned fibers at various thick and thin filament lattice spacings from four transgenic Drosophila lines: rescued null or control (Dmlc2(+)), truncated N-terminal extension (Dmlc2(Δ2-46)), disrupted myosin light chain kinase phosphorylation sites (Dmlc2(S66A,S67A)), and dual mutant (Dmlc2(Δ2-46; S66A,S67A)). The N-terminal extension truncation and phosphorylation sites disruption mutations decreased oscillatory power output and the frequency of maximum power output in maximally Ca(2+)-activated fibers compressed to near in vivo inter-thick filament spacing, with the phosphorylation sites disruption mutation having a larger affect. The diminished power output parameters with the N-terminal extension truncation and phosphorylation sites disruption mutations were due to the reduction of the number of strongly-bound cross-bridges and rate of myosin force production, with the larger parameter reductions in the phosphorylation sites disruption mutation additionally related to reduced myosin attachment time. The phosphorylation and N-terminal extension-dependent boost in cross-bridge kinetics corroborates previous structural data, which indicate these RLC attributes play a complementary role in moving and orienting myosin heads toward actin target sites, thereby increasing fiber and whole fly power generation.

Constitutive phosphorylation of cardiac myosin regulatory light chain prevents development of hypertrophic cardiomyopathy in mice

“…On the other hand, RLC phosphorylation was proposed to modify the mechanical properties of the myosin lever arm and its ability to control the step size of cardiac myosin and advancing the longer 8-nm step frequency compared with nonphosphorylated myosin (25). An RLC phosphorylation-elicited longer myosin working stroke would then lead to increased contractile force and power (26). We believe that this mechanism may underlie the pseudophosphorylation-induced prevention of the development of the D166V-HCM phenotype in transgenic S15D-D166V mice.…”

In vitro and in vivo single myosin step-sizes in striated muscle

“…Changes to the phosphorylation level of Ser15 in ventricular RLC in the cardiac muscle affect power output in model organisms (Miller et al 2011; Scruggs and Solaro 2011). We investigated if RLC phosphorylation affects the unitary step-size/step-frequency using the Qdot assay.…”

The effect of work cycle frequency on the potentiation of dynamic force in mouse fast twitch skeletal muscle

“…Sweeney and colleagues showed that this disruption was due to charge repulsion between the myosin head and the thick filament, as the addition of a negative charge to the RLC mimicked this effect of phosphorylation (Sweeney et al, 1994). In terms of myosin function, the lateral displacement and/or increased mobility of the myosin head relative to the thick filament may facilitate formation of the actomyosin complex and the strongly bound, force-generating cross-bridge state in a variety of species (Levine et al, 1996;Levine et al, 1998;Miller et al, 2011;Sweeney et al, 1994). In support of this idea, Yang and colleagues showed that RLC phosphorylation effects on isometric force were mimicked by myofilament lattice spacing compression.…”