Allosteric regulation by cooperative conformational changes of actin filaments drives mutually exclusive binding with cofilin and myosin

Heavy meromyosin (HMM) of myosin II and cofilin each binds to actin filaments cooperatively and forms clusters along the filaments, but it is unknown whether the two cooperative bindings are correlated and what physiological roles they have. Fluorescence microscopy demonstrated that HMM-GFP and cofilin-mCherry each bound cooperatively to different parts of actin filaments when they were added simultaneously in 0.2 μM ATP, indicating that the two cooperative bindings are mutually exclusive. In 0.1 mM ATP, the motor domain of myosin (S1) strongly inhibited the formation of cofilin clusters along actin filaments. Under this condition, most actin protomers were unoccupied by S1 at any given moment, suggesting that transiently bound S1 alters the structure of actin filaments cooperatively and/or persistently to inhibit cofilin binding. Consistently, cosedimentation experiments using copolymers of actin and actin-S1 fusion protein demonstrated that the fusion protein affects the neighboring actin protomers, reducing their affinity for cofilin. In reciprocal experiments, cofilin-actin fusion protein reduced the affinity of neighboring actin protomers for S1. Thus, allosteric regulation by cooperative conformational changes of actin filaments contributes to mutually exclusive cooperative binding of myosin II and cofilin to actin filaments, and presumably to the differential localization of both proteins in cells

Stretching Actin Filaments within Cells Enhances their Affinity for the Myosin II Motor Domain

To test the hypothesis that the myosin II motor domain (S1) preferentially binds to specific subsets of actin filaments in vivo, we expressed GFP-fused S1 with mutations that enhanced its affinity for actin in Dictyostelium cells. Consistent with the hypothesis, the GFP-S1 mutants were localized along specific portions of the cell cortex. Comparison with rhodamine-phalloidin staining in fixed cells demonstrated that the GFP-S1 probes preferentially bound to actin filaments in the rear cortex and cleavage furrows, where actin filaments are stretched by interaction with endogenous myosin II filaments. The GFP-S1 probes were similarly enriched in the cortex stretched passively by traction forces in the absence of myosin II or by external forces using a microcapillary. The preferential binding of GFP-S1 mutants to stretched actin filaments did not depend on cortexillin I or PTEN, two proteins previously implicated in the recruitment of myosin II filaments to stretched cortex. These results suggested that it is the stretching of the actin filaments itself that increases their affinity for the myosin II motor domain. In contrast, the GFP-fused myosin I motor domain did not localize to stretched actin filaments, which suggests different preferences of the motor domains for different structures of actin filaments play a role in distinct intracellular localizations of myosin I and II. We propose a scheme in which the stretching of actin filaments, the preferential binding of myosin II filaments to stretched actin filaments, and myosin II-dependent contraction form a positive feedback loop that contributes to the stabilization of cell polarity and to the responsiveness of the cells to external mechanical stimuli

Identification of functional differences between recombinant human α and β cardiac myosin motors

The myosin isoform composition of the heart is dynamic in health and disease and has been shown to affect contractile velocity and force generation. While different mammalian species express different proportions of α and β myosin heavy chain, healthy human heart ventricles express these isoforms in a ratio of about 1:9 (α:β) while failing human ventricles express no detectable α-myosin. We report here fast-kinetic analysis of recombinant human α and β myosin heavy chain motor domains. This represents the first such analysis of any human muscle myosin motor and the first of α-myosin from any species. Our findings reveal substantial isoform differences in individual kinetic parameters, overall contractile character, and predicted cycle times. For these parameters, α-subfragment 1 (S1) is far more similar to adult fast skeletal muscle myosin isoforms than to the slow β isoform despite 91% sequence identity between the motor domains of α- and β-myosin. Among the features that differentiate α- from β-S1: the ATP hydrolysis step of α-S1 is ~ten-fold faster than β-S1, α-S1 exhibits ~five-fold weaker actin affinity than β-S1, and actin·α-S1 exhibits rapid ADP release, which is >ten-fold faster than ADP release for β-S1. Overall, the cycle times are ten-fold faster for α-S1 but the portion of time each myosin spends tightly bound to actin (the duty ratio) is similar. Sequence analysis points to regions that might underlie the basis for this finding

Heavy and light roles: myosin in the morphogenesis of the heart

Myosin is an essential component of cardiac muscle, from the onset of cardiogenesis through to the adult heart. Although traditionally known for its role in energy transduction and force development, recent studies suggest that both myosin heavy-chain and myosin lightchain proteins are required for a correctly formed heart. Myosins are structural proteins that are not only expressed from early stages of heart development, but when mutated in humans they may give rise to congenital heart defects. This review will discuss the roles of myosin, specifically with regards to the developing heart. The expression of each myosin protein will be described, and the effects that altering expression has on the heart in embryogenesis in different animal models will be discussed. The human molecular genetics of the myosins will also be reviewed

Functional characterization of vertebrate nonmuscle myosin IIB isoforms using Dictyostelium chimeric myosin II.

The alternatively spliced isoform of nonmuscle myosin II heavy chain B (MHC-IIB) with an insert of 21 amino acids in the actin-binding surface loop (loop 2), MHC-IIB(B2), is expressed specifically in the central nervous system of vertebrates. To examine the role of the B2 insert in the motor activity of the myosin II molecule, we expressed chimeric myosin heavy chain molecules using the Dictyostelium myosin II heavy chain as the backbone. We replaced the Dictyostelium native loop 2 with either the noninserted form of loop 2 from human MHC-IIB or the B2-inserted form of loop 2 from human MHC-IIB(B2). The transformant Dictyostelium cells expressing only the B2-inserted chimeric myosin formed unusual fruiting bodies. We then assessed the function of chimeric proteins, using an in vitro motility assay and by measuring ATPase activities and binding to F-actin. We demonstrate that the insertion of the B2 sequence reduces the motor activity of Dictyostelium myosin II, with reduction of the maximal actin-activated ATPase activity and a decrease in the affinity for actin. In addition, we demonstrate that the native loop 2 sequence of Dictyostelium myosin II is required for the regulation of the actin-activated ATPase activity by phosphorylation of the regulatory light chain

Cooperativity between two heads of dictyostelium myosin II in in vitro motility and ATP hydrolysis.

To elucidate the significance of the two-headed structure of myosin II, we have engineered and characterized recombinant single-headed myosin II. A tail segment of a myosin II heavy chain fused with a His-tag was expressed in wild-type Dictyostelium cells. Single-headed myosin, which consists of a full length myosin heavy chain and a tagged tail, was isolated on the basis of the affinities for Nickel agarose and actin. Actin sliding velocity by the single-headed myosin was about half of the two-headed, whereas the minimum density of the heads to support continuous movement was twofold higher. Actin-activated MgATPase activity of the single-headed myosin in solution in the presence of 24 microM actin was less than half of the two headed. This decrease is primarily because of fourfold-elevated Kapp for actin and secondary to 40% lower Vmax. These results suggest that the two heads of a Dictyostelium myosin II molecule act cooperatively on an actin filament. We propose a mechanism by which two heads move actin efficiently based on the cooperativity