Single-headed Myosin Research Articles

Tomographic three-dimensional reconstruction of insect flight muscle partially relaxed by AMPPNP and ethylene glycol.

Rigor insect flight muscle (IFM) can be relaxed without ATP by increasing ethylene glycol concentration in the presence of adenosine 5'-[beta'gamma- imido]triphosphate (AMPPNP). Fibers poised at a critical glycol concentration retain rigor stiffness but support no sustained tension ("glycol-stiff state"). This suggests that many crossbridges are weakly attached to actin, possibly at the beginning of the power stroke. Unaveraged three-dimensional tomograms of "glycol-stiff" sarcomeres show crossbridges large enough to contain only a single myosin head, originating from dense collars every 14.5 nm. Crossbridges with an average 90 degrees axial angle contact actin midway between troponin subunits, which identifies the actin azimuth in each 38.7-nm period, in the same region as the actin target zone of the 45 degrees angled rigor lead bridges. These 90 degrees "target zone" bridges originate from the thick filament and approach actin at azimuthal angles similar to rigor lead bridges. Another class of glycol-PNP crossbridge binds outside the rigor actin target zone. These "nontarget zone" bridges display irregular forms and vary widely in axial and azimuthal attachment angles. Fitting the acto-myosin subfragment 1 atomic structure into the tomogram reveals that 90 degrees target zone bridges share with rigor a similar contact interface with actin, while nontarget crossbridges have variable contact interfaces. This suggests that target zone bridges interact specifically with actin, while nontarget zone bridges may not. Target zone bridges constitute only approximately 25% of the myosin heads, implying that both specific and nonspecific attachments contribute to the high stiffness. The 90 degrees target zone bridges may represent a preforce attachment that produces force by rotation of the motor domain over actin, possibly independent of the regulatory domain movements.

Brush border myosin-I structure and ADP-dependent conformational changes revealed by cryoelectron microscopy and image analysis.

Brush border myosin-I (BBM-I) is a single-headed myosin found in the microvilli of intestinal epithelial cells, where it forms lateral bridges connecting the core bundle of actin filaments to the plasma membrane. Extending previous observations (Jontes, J.D., E.M. Wilson-Kubalek, and R.A. Milligan. 1995. Nature [Lond.]. 378:751-753), we have used cryoelectron microscopy and helical image analysis to generate three-dimensional (3D) maps of actin filaments decorated with BBM-I in both the presence and absence of 1 mM MgADP. In the improved 3D maps, we are able to see the entire light chain-binding domain, containing density for all three calmodulin light chains. This has enabled us to model a high resolution structure of BBM-I using the crystal structures of the chicken skeletal muscle myosin catalytic domain and essential light chain. Thus, we are able to directly measure the full magnitude of the ADP-dependent tail swing. The approximately 31 degrees swing corresponds to approximately 63 A at the end of the rigid light chain-binding domain. Comparison of the behavior of BBM-I with skeletal and smooth muscle subfragments-1 suggests that there are substantial differences in the structure and energetics of the biochemical transitions in the actomyosin ATPase cycle.

Myosin head interactions in Ca2+-activated skinned rabbit skeletal muscle fibers.

Interactions between the two myosin heads were studied in skinned rabbit slow-twitch muscle fibers activated in the presence of vanadate (Vi), a phosphate analog. The strong complex between Vi, MgADP, and myosin trapped the myosin in an inactivated myosin x MgADP x Vi state. Electron paramagnetic resonance spectroscopy was used to quantitate the fraction of myosin heads trapped in the presence of a spin labeled analog of ATP (SLATP). Force was found to depend directly on the fraction of untrapped heads. At high [Vi] (low force), most untrapped heads would have a trapped partner. The equivalence of force with the proportion of untrapped heads shows that the isometric force produced by a single untrapped myosin head on a molecule with a trapped partner is equivalent to that produced by either head of a myosin molecule with neither head trapped. The actin-activated MgATPase activities of one-headed and two-headed skeletal myosin species were inhibited similarly by Vi, suggesting that trapping one head did not preclude trapping its partner. These data indicate that the two skeletal muscle myosin heads can function without interacting during maximal Ca2+-activated force generation.

Single Molecule Imaging of Fluorophores and Enzymatic Reactions Achieved by Objective-Type Total Internal Reflection Fluorescence Microscopy

Imaging of single fluorescent molecules has been achieved in a relatively simple manner using objective-type total internal reflection fluorescence microscopy (TIRFM). Switching from epi-fluorescence microscopy to objective-type TIRFM was achieved by translation of a single mirror in the system. Clear images of single molecules of an orange fluorescent dye, Cy3, were obtained with a fluorescence-to-background ratio of 12, using a conventional high aperture objective (PlanApo, 100 ×, NA 1.4) with ordinary coverslips and immersion oil. This method allowed visualization of single molecules under scanning probe microscopes. Taking advantage of the technique of single molecule imaging, individual ATP turnovers have been visualized with a fluorescent ATP analogue, Cy3-ATP, using a simple experimental strategy. Clear on/off signals were obtained that correspond to the association and dissociation of single Cy3-ATP/ADP molecules with a single myosin head molecule. This method will allow a variety of single-molecular assays of biomolecular functions to be performed using fluorescently labeled substrates, ligands, messengers, and biologically active molecules. Thus, the present technique provides a simple yet powerful and universal tool for researchers to probe the events of single molecules.

Force-dependent and force-independent heat production in single slow- and fast-twitch muscle fibres from Xenopus laevis.

1. The origin of labile heat production, i.e. a heat component which rapidly decays after the onset of stimulation, and of stable (maintenance) heat production was investigated in intact single fast-twitch (type 1) and slow-twitch (type 3) iliofibularis muscle fibres from Xenopus laevis, at 20 degrees C, by varying stimulation frequency and by varying sarcomere length and the concentration of 2,3-butanedione 2-monoxime (BDM) added. 2. The labile heat produced consisted of a force-independent and a force-dependent part. The average parvalbumin (PA) content found in type 1 fibre bundles (0.84 +/- 0.08 mM; mean +/- S.E.M.; n = 5) and in type 3 fibre bundles (0.12 +/- 0.02 mM; n = 5) indicates that the force-independent labile heat is explained by Ca(2+)-Mg2+ exchange on PA, and amounts to a molar enthalpy change of -78 kJ (molPA)-1. 3. Force-dependent labile heat during fused contractions was similar to the calculated heat production resulting from the formation of force-generating cross-bridges, assuming an enthalpy change associated with cross-bridge formation of -30 kJ mol-1. 4. Activation heat, i.e. the part of the total stable heat that is not related to the contractile apparatus, and of which the calcium sequestration by the sarcoplasmic reticulum is the most important contributor, determined by varying sarcomere length or BDM concentration, was identical. For fused contractions the fraction activation heat of the stable maintenance rate of heat production was 34 +/- 4% (mean +/- S.E.M.; n = 13) in type 1 fibres, and 52 +/- 4% (n = 15) in type 3 fibres. In unfused contractions this was 48 +/- 5% (n = 13) in type 1 fibres, and 35 +/- 2% (n = 11) in type 3 fibres. 5. From the force-dependent stable rate of heat production the economy of cross-bridge cycling, expressed as the force-time integral for a single myosin head per ATP molecule hydrolysed, was calculated. It followed that cross-bridge interaction in type 3 fibres is more economical than in type 1 fibres, and that fused contractions are more economical than unfused contractions.

Single-headed Scallop Myosin and Regulation

Single-headed scallop myosin (shM) was prepared by papain digestion of filamentous scallop myosin and purified by hydrophobic interaction chromatography. The shM preparation consisted of equimolar amounts of polypeptides corresponding to an intact heavy chain, rod chain, essential light chain, and regulatory light chain. In electron micrographs the shape of shM showed the presence of a single head domain to which a normal looking rod was attached. Myosin and shM bound Ca2+ with association constants of 5 x 10(6) and 11 x 10(6) M-1, respectively. The ATPase activity of shM was activated about 3-fold by Ca2+. Both heads of myosin and shM had comparable ATPase activities in the presence of Ca2+. The activation of the ATPase activity of single-headed scallop myosin by Ca2+ paralleled closely the Ca2+ binding, in sharp contrast to the activation of intact myosin by Ca2+, which is highly cooperative. Single turnover experiments of myosin with radioactive ATP gave a half-life for the ATPase cycle of approximately 3 min in the presence of EGTA, whereas that of single-headed myosin was shorter than approximately 30 s, which was the resolution time of these measurements. The results suggest that the presence of two heads, as well as the attachment of the head to the coiled coil rod, contribute to the regulation of scallop myosin by Ca2+.

Proposed Modification of the Huxley—Simmons Model for Myosin Head Motion along an Actin Filament

A model is proposed for myosin head motion along an actin filament which accommodates recent experimental data. The model includes three attached states of a myosin head and is thus similar to the classical Huxley & Simmons (1971) model, but differs in that an explicit expression is given for the spatial distribution of potential energy wells for the myosin head. Our model also differs from the classical model, in that it assumes that the proportion of myosin heads attached to actin filament is constant and independent of shortening velocity, as suggested by X-ray diffraction data. Furthermore, it posits that the crossbridge is string-like rather than spring like. This modified model fits well to the experimental data in the following respects. (1) The calculated tension dependence of muscle stiffness agrees with the observation by Ford et al.(1985 J. Physiol. 361,131–150). (2) A myosin head under low load can move as far as 60 nm along an actin filament during one ATP hydrolysis cycle in muscle, in agreement with the results by Yanagida et al.(1985 Nature 316,366–369) and others. (3) The model predicts that such movements consist of a series of elementary steps of 11 nm. (4) A single myosin head hardly moves after the first step of 11 nm under the condition of in vitroexperiment carried out by Finer et al.(1994 Nature 368,113–119), in agreement with their observation. (5) The calculated energy liberation rate reproduces the characteristics of Hill's equation. (6) The “double-hyperbolic force-velocity relation” reported by Edman (1988 J. Physiol. 404,301–321) can be understood in terms of a potential barrier against movement of a potential well in which a myosin head is trapped.

Structure and Function of the 10 S Conformation of Smooth Muscle Myosin

Smooth myosin regulatory light chain (RLC) was exchanged with RLC labeled with benzophenone-4-iodoacetamide at Cys-108. Irradiation under conditions that favor the folded (10 S) conformation resulted in 10 S cross-linked myosin that could not unfold. Purified 10 S cross-linked myosin was cross-linked between the RLC of one head to light meromyosin between leucine 1554 and glutamate 1583, adjacent to a predicted noncoiled region, approximately 60 nm from the tip of the tail. At high ionic strength without actin, product release from one-half of the heads was slow (like 10 S) whereas the other half were activated. This suggests that tail binding to the RLC carboxyl-terminal domain stabilizes ionic interactions important to slow nucleotide release. With actin, product release from both (un)phosphorylated 10 S cross-linked myosin was from one slow population similar to unphosphorylated filaments. 10 S cross-linked myosin weakly bound actin (dissociation constant > 500 microM) and did not move actin in vitro. Single-headed myosin did not fold or trap nucleotide. These and other data suggest that "trapping" occurs only with both heads and the tail binds to a newly formed site, which includes the RLC carboxyl-terminal domain, once trapping has occurred.

Myosin Minireview Series

Myosin Minireview Series

Effect of inorganic phosphate and ADP on the myofilament sliding induced by laser flash photolysis of caged ATP

Using the technique of laser flash photolysis of caged ATP, we have suggested that, under nearly isometric conditions, the unitary distance of myofilament sliding per ATP molecule (myosin head powerstroke) is about 10 nm. To give further information about the mechanism of myofilament sliding, we studied the effect of inorganic phosphate (P i) and ADP on the photoreleased ATP-induced shortening of single glycerinated muscle fibers under very small external loads. Both the velocity and the distance of the myofilament sliding induced by 150 μM ATP increased by P i (20 mM), and decreased by ADP (0.4 mM). On the other hand, P i and ADP showed no significant effect on the myofilament sliding induced by 100 and 75 μM ATP. The potentiating effect of P i on the myofilament sliding with 150 μM ATP can be explained as being due to the increase in population of AM · ADP · P i with corresponding decrease in population of AM · ADP, and also the increase in population of M · ADP · P i and M · ATP. Meanwhile, the inhibitory effect of ADP can be simply accounted for to be due to an accumulation of AM · ADP that already finished their force generating process. The ineffectiveness of P i and ADP on the myofilament sliding with 100 and 75 μM ATP is consistent with the view that it is caused by almost synchronized single myosin head powerstrokes.

Protein-protein interactions in the rigor actomyosin complex.

Since it has not been possible to crystallize the actomyosin complex, the x-ray structures of the individual proteins together with data obtained by fiber diffraction and electron microscopy have been used to build detailed models of filamentous actin (f-actin) and the actomyosin rigor complex. In the f-actin model, a single monomer uses 10 surface loops and two alpha-helices to make sometimes complicated interactions with its four neighbors. In the myosin molecule, both the essential and regulatory light chains show considerable structural homology to calmodulin. General principles are evident in their mode of attachment to the target alpha-helix of the myosin heavy chain. The essential light chain also makes contacts with other parts of the heavy chain and with the regulatory light chain. The actomyosin rigor interface is extensive, involving interaction of a single myosin head with regions on two adjacent actin monomers. A number of hydrophobic residues on the apposing faces of actin and myosin contribute to the main binding site. This site is flanked on three sides by charged myosin surface loops that form predominantly ionic interactions with adjacent regions of actin. Hydrogen bonding is likely to play a significant role in actin-actin and actin-myosin interactions since many of the contacts involve loops. The model building approach used with actomyosin is applicable to other multicomponent assemblies of biological interest and is a powerful method for revealing molecular interactions and providing insights into the mode of action of the assemblies.

Movement and force produced by a single myosin head.

Muscle contraction is driven by the cyclical interaction of myosin with actin, coupled to the breakdown of ATP. Studies of the interaction of filamentous myosin and of a double-headed proteolytic fragment, heavy meromyosin (HMM), with actin have demonstrated discrete mechanical events, arising from stochastic interaction of single myosin molecules with actin. Here we show, using an optical-tweezers transducer, that a single myosin subfragment-1 (S1), which is a single myosin head, can act as an independent generator of force and movement. Our analysis accounts for the broad distribution of displacement amplitudes observed, and indicates that the underlying movement (working stroke) produced by a single acto-S1 interaction is approximately 4 nm, considerably shorter than previous estimates but consistent with structural data. We measure the average force generated by S1 or HMM to be at least 1.7 pN under isometric conditions.

Single-headed myosin II acts as a dominant negative mutation in Dictyostelium.

Conventional myosin II is an essential protein for cytokinesis, capping of cell surface receptors, and development of Dictyostelium cells. Myosin II also plays an important role in the polarization and movement of cells. All conventional myosins are double-headed molecules but the significance of this structure is not understood since single-headed myosin II can produce movement and force in vitro. We found that expression of the tail portion of myosin II in Dictyostelium led to the formation of single-headed myosin II in vivo. The resultant cells contain an approximately equal ratio of double- and single-headed myosin II molecules. Surprisingly, these cells were completely blocked in cytokinesis and capping of concanavalin A receptors although development into fruiting bodies was not impaired. We found that this phenotype is not due to defects in myosin light chain phosphorylation. These results show that single-headed myosin II cannot function properly in vivo and that it acts as a dominant negative mutation for myosin II function. These results suggest the possibility that cooperativity of myosin II heads is critical for force production in vivo.

Two Heads Are Required for Phosphorylation-dependent Regulation of Smooth Muscle Myosin

Recent structural evidence (Rayment, I., Holden, H. M., Whittaker, M., Yohn, C. B., Lorenz, M., Holmes, K. C., and Milligan, R. A. (1993) Science 261, 58-65) suggests that the two heads of skeletal muscle myosin interact when the protein is bound to filamentous actin. Direct chemical cross-linking experiments show that the two heads of smooth muscle myosin interact in the presence of filamentous actin and the absence of ATP (Onishi, H., Maita, T., Matsuda, G., and Fujiwara, K. (1992) Biochemistry 31, 1201-1210). Head-head interactions may be important in the mechanism of phosphorylation-dependent regulation of smooth muscle myosin. To explore the structural elements essential for phosphorylation-dependent regulation, we purified a proteolytic fragment of chicken gizzard myosin containing only one head attached to an intact tail. This molecule contained a partially digested regulatory light chain, which was replaced with exogenously added intact light chain in either the thiophosphorylated or the unphosphorylated state. Control experiments showed that this replacement was nearly quantitative and did not alter the actin-activated ATPase of this myosin. Electron micrographs confirmed that the single-headed preparation contained an intact form of single-headed myosin. The unphosphorylated single-headed myosin hydrolyzed ATP rapidly and moved actin filaments in an in vitro motility assay. Phosphorylation had minimal effects upon these properties. Therefore, we conclude that phosphorylation-dependent regulation in this myosin requires two heads. These findings may have important implications in studies of other regulated motor proteins that contain two motor domains.

Isolation of Myosin from Dictyostelium Cytoskeletons

Dictyostelium has several isoforms of "unconventional" single-headed myosins that do not assemble into filaments (myosin I). In contrast, there is only one form of conventional myosin (myosin II) that self-assembles into bipolar thick filaments, and molecular genetic studies have shown that this myosin is an essential motor protein for several cell functions, including cytokinesis. Myosin II is phosphorylated on both the heavy chain and light chain, and these modifications regulate assembly and ATPase activity. A large fraction of the myosin II remains associated with structures that are resistant to extraction with nonionic detergents (the Triton cytoskeleton). These cytoskeletons retain the ability to contract in an ATP-dependent manner and can easily be isolated by low-speed centrifugation. A method for the rapid purification of myosin II from cytoskeletons is described. This method is simple and avoids the need for sonication, ultracentrifugation of large volumes, and overnight dialysis. The subunit composition, assembly properties, and enzyme activity are similar to those reported for myosin prepared from soluble extracts.

Myosin II distribution in neurons is consistent with a role in growth cone motility but not synaptic vesicle mobilization

We have generated a polyclonal antibody against myosin II from a neuronally derived cell line in order to assess potential roles for myosin II in growth cone movement and synaptic transmission. The distribution of neuronal myosin II, in isolated cells as well as in tissues of the adult rat brain and spinal cord, was examined at the light microscopic and ultrastructural levels. In isolated neuroblastoma cells and dorsal root ganglion neurons, myosin II was found at the leading edge of growth cones, within neuritic processes and cell soma, and adjacent to the plasma membrane. The subcellular distribution of myosin II overlapped significantly with that of both actin and single-headed myosin I. These results implicate both myosin I and myosin II as molecular motors required for neurite elongation and growth cone motility. An exclusive postsynaptic distribution of myosin II in neurons of the mature central nervous system suggests that myosin II cannot play a role in the mobilization of synaptic vesicles, but could participate in synaptic plasticity.

An approach to reconstituting motility of single myosin molecules

Over the last five years, the value of in vitro motility assays as probes of the mechanical properties of the actin-myosin interaction has been amply demonstrated. Motility assays in which single fluorescent actin filaments are observed moving over surfaces coated with myosin or its soluble fragments are now used in many laboratories. They have been applied to a wide range of problems including the study of structure-function relationships in the myosin molecule and measurement of fundamental properties of the myosin head. However, one limitation of these assays has been uncertainty over the number of myosin heads interacting with each sliding filament, that frustrates attempts to determine properties of individual heads. In order to address this limitation, we have modified the conditions of the actin sliding filament assay to reduce the number of heads interacting with each filament. Our goal is to establish an assay in which the motor function of a single myosin head can be characterized from the movement of a single actin filament.

Ca2+ stimulates the Mg2(+)-ATPase activity of brush border myosin I with three or four calmodulin light chains but inhibits with less than two bound.

Brush border myosin I from chicken intestinal microvilli is a membrane-associated, single-headed myosin composed of a 119-kDa heavy chain and several calmodulin light chains. We first describe in detail a new procedure for the rapid purification of brush border myosin I in greater than 99% purity with a yield of 40%, significantly higher than for previous methods. The subunit stoichiometry was determined to be 4 calmodulin light chains/myosin I heavy chain by amino acid compositional analysis of the separated subunits. We have studied the effects of Ca2+ and temperature on dissociation of calmodulin from myosin I and on myosin I Mg2(+)-ATPase and contractile activities. At 30 degrees C the actin-activable ATPase activity is stimulated 2-fold at 10-700 microM Ca2+. Dissociation of 1 calmodulin occurs at 25-50 microM Ca2+, but this has no effect on actin activation. The contractile activity of myosin I, expressed as superprecipitation, is greatly enhanced by Ca2+ under conditions in which 1 calmodulin is dissociated. This calmodulin is thus not essential for actin activation or superprecipitation. Myosin I was found to be highly temperature-sensitive, with an increase to 37 degrees C resulting in dissociation of 1 calmodulin at below 10(-7) M Ca2+ and an additional 1.5 calmodulins at 1-10 microM Ca2+. A complete loss of actin activation accompanies the Ca2(+)-induced calmodulin dissociation at 37 degrees C. Our conclusion is that physiological levels of Ca2+ can either stimulate or inhibit the mechanoenzyme activities of brush border myosin I in vitro, with the mode of regulation determined by the number of associated calmodulin light chains.

Three-dimensional image reconstruction of insect flight muscle. II. The rigor actin layer.

The averaged structure of rigor cross-bridges in insect flight muscle is further revealed by three-dimensional reconstruction from 25-nm sections containing a single layer of thin filaments. These exhibit two thin filament orientations that differ by 60 degrees from each other and from myac layer filaments. Data from multiple tilt views (to +/- 60 degrees) was supplemented by data from thick sections (equivalent to 90 degrees tilts). In combination with the reconstruction from the myac layer (Taylor et al., 1989), the entire unit cell is reconstructed, giving the most complete view of in situ cross-bridges yet obtained. All our reconstructions show two classes of averaged rigor cross-bridges. Lead bridges have a triangular shape with leading edge angled at approximately 45 degrees and trailing edge angled at approximately 90 degrees to the filament axis. We propose that the lead bridge contains two myosin heads of differing conformation bound along one strand of F-actin. The lead bridge is associated with a region of the thin filament that is apparently untwisted. We suggest that the untwisting may reflect the distribution of strain between myosin and actin resulting from two-headed, single filament binding in the lead bridge. Rear bridges are oriented at approximately 90 degrees to the filament axis, and are smaller and more cylindrical, suggesting that they consist of single myosin heads. The rear bridge is associated with a region of apparently normal thin filament twist. We propose that differing myosin head angles and conformations consistently observed in rigor embody different stages of the power stroke which have been trapped by a temporal sequence of rigor cross-bridge formation under the constraints of the intact filament lattice.

Binding of myosin I to membrane lipids.

The single-headed myosins called myosin-I were first isolated from the protozoan Acanthamoeba and subsequently identified in other cells. We previously reported evidence that myosin-I is responsible for the movement of membranes, extracted from Acanthamoeba, along actin filaments in vitro. Here we show for the first time that myosin-I can bind directly to NaOH-extracted membranes isolated from Acanthamoeba and to vesicles of pure lipids with an affinity sufficient for extensive binding in the cell. Membrane-bound myosin-I may provide a mechanism for many cellular movements previously thought to involve filamentous myosin-II.