Two heads of myosin are better than one for generating force and motion

Abstract

Several classes of the myosin superfamily are distinguished by their ''double-headed'' structure, where each head is a molecular motor capable of hydrolyzing ATP and interacting with actin to generate force and motion. The functional significance of this dimeric structure, however, has eluded investigators since its discovery in the late 1960s. Using an optical-trap transducer, we have measured the unitary displacement and force produced by double-headed and single-headed smooth-and skeletal-muscle myosins. Singleheaded myosin produces approximately half the displacement and force (Ϸ6 nm; 0.7 pN) of double-headed myosin (Ϸ10 nm; 1.4 pN) during a unitary interaction with actin. These data suggest that muscle myosins require both heads to generate maximal force and motion.Muscle shortening is driven by a cyclical interaction between the contractile proteins myosin and actin. During this cycle the dimeric molecular motor, myosin, transduces chemical energy into mechanical work. Although the contractile system has been investigated extensively, the details of this process and the functional significance of myosin's ''double-headed'' structure have remained unclear since the dimeric structure was discovered in the late 1960s (ref. 1; see refs. 2 and 3 for review). Based on the available biochemical and mechanical data (4-8), early models assumed that the two heads of myosin act independently (9, 10). Enzymatic assays clearly showed that the actin-activated ATPase activity per head was the same for proteolytically prepared myosin-head fragments (subfragment-1, S1) and for single-and double-headed heavy meromyosin (HMM; ref. 4). An even more compelling example of independent head action was observed when reconstituted actomyosin threads were used. In these experiments, ensembles of single-headed myosin generated half the force of native myosin (5). More recently, by using an optical trap, single S1 heads were shown to move actin and produce force, suggesting that a dimeric structure is not necessary for the production of work in vitro (11). Additional single-molecule experiments have shown that in synthetic thick filaments, single-and double-headed myosins produce similar unitary step displacements (12).However, evidence suggesting a functional difference between single-and double-headed myosin species came from measurements of the binding of S1 and HMM to actin in the absence of nucleotide (6, 7). These data showed a smaller association constant for HMM than would be expected if the heads bound independently, implying that the two heads are sterically constrained from simultaneously interacting with actin. More recent kinetic studies comparing S1 and HMM suggest that the two heads may be coordinated in their transition from the weakly bound to the strongly bound state (13).Because the functional significance of myosin's two-headed structure remains ambiguous, we have performed a singlemolecule mechanical comparison between single-and doubleheaded smooth-and skeletal-muscle myosins. Using an optical-trap assay ...

Kinetic Characterization of Nonmuscle Myosin IIB at the Single Molecule Level

“…Using optical tweezers, Tyska et al (50) showed that in case of smooth and skeletal myosins the dwell times of the singleand double-headed molecules were very similar to each other in both unloaded and loaded conditions, which is consistent with our findings for NMIIB HMM. These authors also demonstrated that the stroke sizes of the double-headed molecule smooth and skeletal muscle myosins were approximately double that of the single-headed fragment.…”

Phosphorylation of a Single Head of Smooth Muscle Myosin Activates the Whole Molecule

“…9). This suggestion is supported by laser trap studies on single-headed and heterodimeric molecules which indicated that one head performs all of the work on the actin filament (24,37,38). Single-headed binding is also consistent with the very low duty cycle which myosin II exhibits under unloaded conditions (4% for both smooth and skeletal in the motility assay) (39).…”

Mouse Macrophages Completely Lacking Rho Subfamily GTPases (RhoA, RhoB, and RhoC) Have Severe Lamellipodial Retraction Defects, but Robust Chemotactic Navigation and Altered Motility

“…Ultimately, cell shape changes and cell motility require, in addition to local volume changes (10), the spatial coordination of two ATP-dependent events (11)(12)(13): F-actin polymerization and motor protein-driven actomyosin contraction. Activated Rho subfamily Rho GTPases act via Rho kinases (ROCK1 and ROCK2; Rho-associated coiled-coil-containing protein kinases) to induce phosphorylation of myosin II regulatory light chains, which promotes actomyosin contraction.…”