In vitro actin filament sliding velocities produced by mixtures of different types of myosin

Abstract

Using in vitro motility assays, we examined the sliding velocity of actin filaments generated by pairwise mixings of six different types of actively cycling myosins. In isolation, the six myosins translocated actin filaments at differing velocities. We found that only small proportions of a more slowly translating myosin type could significantly inhibit the sliding velocity generated by a myosin type that translocated filaments rapidly. In other experiments, the addition of noncycling, unphosphorylated smooth and nonmuscle myosin to actively translating myosin also inhibited the rapid sliding velocity, but to a significantly reduced extent. The data were analyzed in terms of a model derived from the original working cross-bridge model of A.F. Huxley. We found that the inhibition of rapidly translating myosins by slowly cycling was primarily dependent upon only a single parameter, the cross-bridge detachment rate at the end of the working powerstroke. In contrast, the inhibition induced by the presence of noncycling, unphosphorylated myosins required a change in another parameter, the transition rate from the weakly attached actomyosin state to the strongly attached state at the beginning of the cross-bridge power stroke.

Myofibrillar remodelling in cardiac hypertrophy, heart failure and cardiomyopathies

“…Approximately 35% of fHCM cases are attributed to β-MHC, 20% to cMyBP-C, 15% to TnT, less than 5% to α-TM and fewer cases to TnI, VLC 1 , VLC 2 , alpha-actin, titin and α-MHC gene mutations (12,178,179). The exact mechanism by which the mutations induce hypertrophic transformation is not fully understood, but many mutations appear to impart a diverse array of functional defects (force generation or transmission), including reduced myosin ATPase activity, actin-myosin interaction, cross-bridge kinetics, myocyte contractility and Ca 2+ sensitivity (23,52,(180)(181)(182). Therefore, HCM seems to be a reactive adaptation to the underlying disturbance of the contractile apparatus, which is caused mostly by missense mutations or deletions in any of the proteins.…”

Mutations in Human Nonmuscle Myosin IIA Found in Patients with May-Hegglin Anomaly and Fechtner Syndrome Result in Impaired Enzymatic Function

“…nation, 10 nm/s, should be detectable in our assay and was not observed. Cuda et al (19) and Harris et al (18) demonstrated that when two actively cycling myosins with different inherent rates of motility are mixed in the motility assay, the more slowly cycling myosin dominates the velocity.…”

“…Fourth, it is possible the N93K mutation does not allow the HMM to fully adopt the active state upon phosphorylation, and thus the molecule mostly remains in an inhibited state that is able to interact weakly with actin filaments but cannot support their movement. The behavior of noncyclying unphosphorylated smooth and nonmuscle myosins when mixed with an actively cycling myosin has also been studied and modeled (18,19). The unphosphorylated noncycling myosins impose a load on the actively cycling myosins but do so in a non-dominant manner.…”

Phosphorylation-dependent Regulation Is Absent in a Nonmuscle Heavy Meromyosin Construct with One Complete Head and One Head Lacking the Motor Domain

“…To explain this, they suggested that there was an equilibrium between a fully off-state and fully on-state that was shifted by phosphorylation. Myosin molecules in the off-state such as unphosphorylated smooth or nonmuscle HMM, which are themselves incapable of moving actin filaments, have been shown to be able to exert a drag on activated cycling myosins in this same type of assay (29,30). Thus, Sweeney et al (16) rationalized that the completely turned off molecules in their preparation would act to prevent the movement of the fully on molecules in the same preparation and thus slow the observed sliding velocity.…”