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

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. Single-headed myosin produces approximately half the displacement and force (approximately 6 nm; 0.7 pN) of double-headed myosin (approximately 10 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.

A minimal motor domain from chicken skeletal muscle myosin.

The myosin head (S1) consists of a wide, globular region that contains the actin- and nucleotide-binding sites and an alpha-helical, extended region that is stabilized by the presence of two classes of light chains. The essential light chain abuts the globular domain, whereas the regulatory light chain lies near the head-rod junction of myosin. Removal of the essential light chain by a mild denaturant exposes the underlying heavy chain to proteolysis by chymotrypsin. The cleaved fragment, or "motor domain" (MD), migrates as a single band on SDS-polyacrylamide gel electrophoresis, with a slightly greater mobility than S1 prepared by papain or chymotrypsin. Three-dimensional image analysis of actin filaments decorated with MD reveals a structure similar to S1, but shorter by an amount consistent with the absence of a light chain-binding domain. The actin-activated MgATPase activity of MD is similar to that of S1 in Vmax and Km. But the ability of MD to move actin filaments in a motility assay is considerably reduced relative to S1. We conclude that the globular, active site region of the myosin head is a stable, independently folded domain with intrinsic motor activity, but the coupling efficiency between ATP hydrolysis and movement declines markedly as the light chain binding region is truncated.

The essential light chain is required for full force production by skeletal muscle myosin.

Myosin, a molecular motor that is responsible for muscle contraction, is composed of two heavy chains each with two light chains. The crystal structure of subfragment 1 indicates that both the regulatory light chains (RLCs) and the essential light chains (ELCs) stabilize an extended alpha-helical segment of the heavy chain. It has recently been shown in a motility assay that removal of either light chain markedly reduces actin filament sliding velocity without a significant loss in actin-activated ATPase activity. Here we demonstrate by single actin filament force measurements that RLC removal has little effect on isometric force, whereas ELC removal reduces isometric force by over 50%. These data are interpreted with a simple mechanical model where subfragment 1 behaves as a torque motor whose leyer arm length is sensitive to light-chain removal. Although the effect of removing RLCs fits within the confines of this model, altered crossbridge kinetics, as reflected in a reduced unloaded duty cycle, probably contributes to the reduced velocity and force production of ELC-deficient myosins.

Coupling of ATPase activity and motility in smooth muscle myosin is mediated by the regulatory light chain.

Smooth muscle myosin acts as a molecular motor only if the regulatory light chain (RLC) is phosphorylated. This subunit can be removed from myosin by a novel method involving the use of trifluoperazine. The motility of RLC-deficient myosin is very slow, but native properties are restored when RLC is rebound. Truncating 6 residues from the COOH terminus of the RLC had no effect on phosphorylated myosin's motor properties, while removal of the last 12 residues reduced velocity by approximately 30%. Very slow movement was observed once 26 residues were deleted, or with myosin containing only the COOH-terminal RLC domain. These two mutants thus mimicked the behavior of RLC-deficient myosin, with the important difference that the mutant myosins were monodisperse when assayed by sedimentation velocity and electron microscopy. The decreased motility therefore cannot be caused by aggregation. A common feature of RLC-deficient myosin and the mutant myosins that moved actin slowly was an increased myosin ATPase compared with dephosphorylated myosin, and a lower actin-activated ATPase than obtained with phosphorylated myosin. These results suggest that the COOH-terminal portion of an intact RLC is involved in interactions that regulate myosin's "on-off" switch, both in terms of completely inhibiting and completely activating the molecule.

Function of skeletal muscle myosin heavy and light chain isoforms by an in vitro motility assay.

The functional significance of the large number of myosin isoforms in skeletal muscles is poorly understood. Myosin molecules that have the same heavy chain, but differ in their essential or alkali light chains, have the same actin-activated ATPase activity. Similarly, the many heavy chain isoforms that appear during the course of muscle development do not show any significant differences in enzymatic activity. By means of an in vitro motility assay, we have measured the analogue of unloaded shortening velocity for myosin isoforms in a reconstituted actomyosin system. We find that both light and heavy chain isoforms translocate actin filaments at distinct velocities. These results support the hypothesis that myosin isoforms are the primary determinant for the range of shortening velocities adopted by a muscle in response to changing functional demands.

Skeletal muscle myosin light chains are essential for physiological speeds of shortening.

In muscle each myosin head contains a regulatory light chain (LC2) that is wrapped around the head/rod junction, and an alkali light chain that is distal to LC2 (ref. 1). The role of these light chains in vertebrate skeletal muscle myosin has remained obscure. Here we prepare heavy chains that are free of both light chains in order to determine by a motility assay whether the light chains are necessary for movement. We find that removal of light chains from myosin reduces the velocity of actin filaments from 8.8 microns s-1 to 0.8 microns s-1 without significantly decreasing the ATPase activity. Reconstitution of myosin with LC2 or alkali light chain increases filament velocity to intermediate rates, and readdition of both classes of light chains fully restores the original sliding velocity. We conclude that even though the light chains are not essential for enzymatic activity, light-chain/heavy-chain interactions play an important part in the conversion of chemical energy into movement.

Neonatal and adult myosin heavy chains form homodimers during avian skeletal muscle development.

Myosin isoforms contribute to the heterogeneity and adaptability of skeletal muscle fibers. Besides the well-characterized slow and fast muscle myosins, there are those isoforms that appear transiently during the course of muscle development. At a stage of development when two different myosins are coexpressed, the possibility arises for the existence of heterodimers, molecules containing two different heavy chains, or homodimers, molecules with two identical heavy chains. The question of whether neonatal and adult myosin isoforms can associate to form a stable heterodimer was addressed by using stage-specific monoclonal antibodies in conjunction with immunological and electron microscopic techniques. We find that independent of the ratio of adult to neonatal myosin, depending on the age of the animal, the myosin heavy chains form predominantly homodimeric molecules. The small amount of hybrid species present suggests that either the rod portion of the two heavy chain isoforms differs too much in sequence to form a stable alpha-helical coiled coil, or that the biosynthesis of the heavy chains precludes the formation of heterodimeric molecules.

Myosin subunit interactions. Localization of the alkali light chains.

Myosin homodimers, molecules containing either the A1 or the A2 light chain, do not exchange their light chains under conditions approximating physiological temperature and ionic strength. Myosin heterodimers, molecules containing both A1 and A2 light chains, are therefore formed at the time of synthesis rather than by a labile subunit exchange. Antibodies specific for the amino-terminal region of the alkali light chains were used to localize these subunits in myosin by immunoelectron microscopy. The close proximity of the alkali light chain to the 5,5'-dithiobis-(2-nitrobenzoic acid) light chain in the "neck" region of the myosin head is consistent with the finding that the 5,5'-dithiobis-(2-nitrobenzoic acid) light chain influences subunit interactions between the alkali light chain and heavy chain in vertebrate skeletal muscle myosin.

Myosin isozymes in avian skeletal muscles. I. Sequential expression of myosin isozymes in developing chicken pectoralis muscles.

Myosin has been purified from chicken pectoralis muscle at various stages of development, from 10 days' incubation to approximately 10 months after hatching. Embryonic myosin from the earliest stage showed a high level of ATPase activity, similar to that obtained for adult pectoralis myosin. Two-dimensional peptide mapping of partial chymotryptic digests showed, however, that is heavy chain is quite different from that of adult fast myosin. The immunological crossreactivity observed between embryonic myosin and adult fast (pectoralis) myosin is therefore due to shared antigenic determinants rather than the presence of any adult isoforms. In an accompanying paper we will show that embryonic myosin at 10 days' incubation is not a single species, but consists of at least two heavy chain isozymes. The minor fraction binds slow light chains preferentially, and appears to be largely responsible for the observed crossreactivity with slow (ALD) myosin. None of the embryonic myosins is equivalent to the adult forms. Prior to hatching, LC3f is present only in very small amounts (less than 5%), and the adult light chain pattern, containing LC1f and LC3f in equimolar amounts, is not generated until after one week post-hatching. At about that time a new heavy chain population is detected, different from either the embryonic heavy chain or the adult heavy chain. The adult heavy chain peptide pattern appears from about three weeks' post-hatching, but a map indistinguishable from that of adult myosin is not observed until about 26 weeks. None of the observed differences in peptide maps can be related to different strains of chicken; pectoralis myosin from adult White Rock gave an identical map to that from White Leghorn. Unexpectedly, posterior latissimus dorsi (PLD) myosin from White Leghorn appears to be different from pectoralis myosin from the same strain, despite the histochemical and immunocytochemical similarity of the two muscles. We conclude that myosin polymorphism is widespread in muscle tissue, and that the expression of myosin isozymes and their subunits is under developmental regulation.

Myosin isozymes in avian skeletal muscles. II. Fractionation of myosin isozymes from adult and embryonic chicken pectoralis muscle by immuno-affinity chromatography.

Chicken pectoralis consists primarily of large white fibres, which react exclusively with antibodies prepared against adult fast myosin. There is, however, a small region of uniformly red fibres which responds to antibodies against adult slow myosin as well as adult fast myosin. The myosin extracted from this red region is also heterogeneous as shown by the presence of both slow and fast light chains. By means of immunoadsorbents, it has been possible to separate the 'red myosin' into a 'fast' component and a 'slow' component. These two fractions have been characterized with respect to their light and heavy chain content by one-dimensional and two-dimensional gel electrophoresis. The myosin heavy chain was reduced to the smaller fragments required for electrophoresis by proteolytic degradation. We conclude from the electrophoretic patterns that the 'fast' and 'slow' myosin components from the pectoralis red region closely resemble the myosin from the white region of the pectoralis and the myosin from the slow anterior latissimus dorsi (ALD) muscle. The demonstration of a 'slow myosin' in adult pectoralis muscle raises the possibility that the crossreactivity of embryonic pectoralis myosin with anti-slow (ALD) myosin antibodies might be due to the presence of such slow components in embryonic chicken muscle. Direct isolation of a slow component from embryonic pectoralis was achieved by immunoadsorption, as described for adult mixed muscle myosin. Analysis of the subunit composition by gel electrophoresis shows an enrichment in adult-type slow light chains, but the heavy chain pattern is quite distinct from that of adult slow heavy chain. These studies suggest that several myosin isozymes exist in embryonic chicken pectoralis, but that none is identical to those myosins found in the different fibres of the adult pectoralis muscle.

Mechanical properties and myosin light chain composition of skinned muscle fibres from adult and new-born rabbits.

1. The maximum velocity of shortening, Vmax, and stiffness were measured in skinned single fibre segments from psoas and soleus muscles of adult rabbits and psoas muscles of new-born rabbits, and the myosin light chain composition was also determined in the same segments used in the mechanical studies. 2. Vmax was obtained at 15 degrees C during maximal activation at pCa 5.49 using a method involving measurement of the time required to take up various amounts of slack imposed on the segments. Stiffness was measured during activation at 10 degrees C by application of length steps complete in 0.6 msec. The myosin light chain composition of the segments was then determined by SDS-polyacrylamide gel electrophoresis. 3. Only fast type light chains were found to be present in the psoas fibre segments, though the relative amounts of myosin LC1f, LC2f and LC3f in these segments was somewhat variable. In most instances, the sum of the amounts of LC1f and LC3f present was equivalent to the amount of LC2f. Only slow type light chains were found in the soleus segments and the sum of the amounts of LC1as and LC1bs was about equal to the amount of LC2s. 4. The results indicate that there are no consistent relationships between Vmax, tension development or stiffness and LC1f/LC2f in the segments from adult and new-born psoas muscles, or between these mechanical parameters and LC1as/LC2s or LC1bs/LC2s in the adult soleus segments. However, the psoas segments, which had light chains of the fast type, had Vmax values that were consistently higher than those of the soleus segments, which had light chains of the slow type. 5. The stiffness values obtained in each of the three kinds of muscle were similar, suggesting that cross-bridge stiffness is similar in rabbit skeletal muscles of different type and age. Moreover, the results indicate that the amount of end compliance introduced by the connections to the fibre segments has a marked influence on the stiffness that is measured.