Structure of the cross-striated adductor muscle of the scallop

The structure of the cross-striated adductor muscle of the scallop has been studied by electron microscopy and X-ray diffraction using living relaxed, glycerol-extracted (rigor), fixed and dried muscles. The thick filaments are arranged in a hexagonal lattice whose size varies with sarcomere length so as to maintain a constant lattice volume. In the overlap region there are approximately 12 thin filaments about each thick filament and these are arranged in a partially disordered lattice similar to that found in other invertebrate muscles, giving a thin-to-thick filament ratio in this region of 6:1.The thin filaments, which contain actin and tropomyosin, are about 1 μm long and the actin subunits are arranged on a helix of pitch 2 × 38.5 nm. The thick filaments, which contain myosin and paramyosin, are about 1.76 μm long and have a backbone diameter of about 21 nm. We propose that these filaments have a core of paramyosin about 6 nm in diameter, around which the myosin molecules pack. In living relaxed muscle, the projecting myosin heads are symmetrically arranged. The data are consistent with a six-stranded helix, each strand having a pitch of 290 nm. The projections along the strands each correspond to the heads of one or two myosin molecules and occur at alternating intervals of 13 and 16 nm. In rigor muscle these projections move away from the backbone and attach to the thin filaments.In both living and dried muscle, alternate planes of thick filaments are staggered longitudinally relative to each other by about 7.2 nm. This gives rise to a body-centred orthorhombic lattice with a unit cell twice the volume of the basic filament lattice.     

Three-dimensional structure of the vertebrate muscle A-band: II. The myosin filament superlattice

The three-dimensional arrangement of the myosin filaments in the A-band of frog sartorius muscle was studied using electron micrographs of very thin and accurately cut transverse sections through the bare region (on each side of the M-band) where the thick filament shafts are roughly triangular in shape. It was found that the orientations of these triangular profiles are arranged to give a superlattice of the same size and shape as that proposed by Huxley & Brown (1967) on the basis of X-ray diffraction evidence, but the contents of the superlattice may not be as they suggested. The results from detailed image analysis strongly suggest that myosin filaments (which have been shown to have 3-fold rotational symmetry, Luther, 1978; Luther, Munro and Squire, unpublished results) are arranged with one of two orientations which are 60 ° (or 180 °) apart. This arrangement of filaments with 3-fold symmetry is not that predicted for a superlattice with the symmetry suggested by Huxley & Brown.Two rules define the way in which the orientations of neighbouring filaments are defined. Rule (1): no three mutually adjacent filaments in the hexagonal array of filaments in the A-band can all have identical orientations; and rule (2): no three successive filaments along a 10$\text{1}\text{?}$ row in the filament array can have identical orientations. These two no-three-alike rules are sufficient to describe the observed arrangement of filament profiles in the frog bare region (except for some minor violations discussed in the text), and they lead automatically to the generation of the required superlattice. The A-band structure in fish muscle is different; there is no superlattice and the triangular bare region profiles have only one orientation. The frog superlattice and fish simple lattice are explained directly in terms of different interactions between the M-bridges in the M-bands of these muscles. The observed structures require that the myosin filament symmetry at the centre of the M-band is that of the dihedral point group 32. The two possible forms of interaction between filaments with this symmetry (apart from a completely random structure) give rise to the observed A-band lattices in frog and fish muscles. The 3-fold rotational symmetry of the myosin filaments required to explain the observed micrographs also requires that the myosin crossbridge arrangements around the actin filaments in frog and fish muscles will be different. It is suggested that the structure in the frog A-band (and in the A-bands of other higher vertebrates) has evolved from that in fish to improve the distribution of crossbridges around the aotin filaments. The X-ray diffraction evidence of Huxley & Brown (1967) will be accounted for in terms of the proposed A-band structure in a further paper in this series.     

Structural changes in thin filaments of crab striated muscle.

Low angle X-ray diffraction patterns were recorded from crab leg muscle in living resting state and in rigor (glycerol-extracted). Both resting and rigor patterns showed a series of layer-lines arising from a helical arrangement of actin subunits in the thin filaments. In the resting state, the crossover repeat of the long-pitch actin helices was 36.6 nm, and the symmetry of the genetic actin helix was an intermediate between $\text{26}\text{12}$ and $\text{28}\text{13}$. When the muscle went into rigor, the crossover repeat changed to 38.3 nm and the helical symmetry to $\text{28}\text{13}$.In the living resting pattern, six other reflections were observed on the meridian and in the near-meridional region. These were indexed as orders of 2 × 38.2 nm and could be assigned to troponin molecules; the spacings and the intensity distributions of these reflections could be explained by the model proposed by Ohtsuki (1974) for the arrangement of troponin molecules in the thin filaments.The muscle in rigor gave meridional and near-meridional reflections at orders of 2 × 38.3 nm. These were identified as the same series of reflections as was assigned to troponin in the living resting pattern, but were more intense and could be seen up to higher orders. We consider that the myosin heads attached to the thin filament at regular intervals along its axis also contribute to these reflections in the rigor pattern.     

3D Structure of fish muscle myosin filaments

Myosin filaments isolated from goldfish (Carassius auratus) muscle under relaxing conditions and viewed in negative stain by electron microscopy have been subjected to 3D helical reconstruction to provide details of the myosin head arrangement in relaxed muscle. Previous X-ray diffraction studies of fish muscle (plaice) myosin filaments have suggested that the heads project a long way from the filament surface rather than lying down flat and that heads in a single myosin molecule tend to interact with each other rather than with heads from adjacent molecules. Evidence has also been presented that the head tilt is away from the M-band. Here we seek to confirm these conclusions using a totally independent method. By using 3D helical reconstruction of isolated myosin filaments the known perturbation of the head array in vertebrate muscles was inevitably averaged out. The 3D reconstruction was therefore compared with the X-ray model after it too had been helically averaged. The resulting images showed the same characteristic features: heads projecting out from the filament backbone to high radius and the motor domains at higher radius and further away from the M-band than the light-chain-binding neck domains (lever arms) of the heads.     

Head-Head Interactions of Resting Myosin Crossbridges in Intact Frog Skeletal Muscles,Revealed by Synchrotron X-Ray Fiber Diffraction

The intensities of the myosin-based layer lines in the x-ray diffraction patterns from live resting frog skeletal muscles with full thick-thin filament overlap from which partial lattice sampling effects had been removed were analyzed to elucidate the configurations of myosin crossbridges around the thick filament backbone to nanometer resolution. The repeat of myosin binding protein C (C-protein) molecules on the thick filaments was determined to be 45.33 nm, slightly longer than that of myosin crossbridges. With the inclusion of structural information for C-proteins and a pre-powerstroke head shape, modeling in terms of a mixed population of regular and perturbed regions of myosin crown repeats along the filament revealed that the myosin filament had azimuthal perturbations of crossbridges in addition to axial perturbations in the perturbed region, producing pseudo-six-fold rotational symmetry in the structure projected down the filament axis. Myosin crossbridges had a different organization about the filament axis in each of the regular and perturbed regions. In the regular region that lacks C-proteins, there were inter-molecular interactions between the myosin heads in axially adjacent crown levels. In the perturbed region that contains C-proteins, in addition to inter-molecular interactions between the myosin heads in the closest adjacent crown levels, there were also intra-molecular interactions between the paired heads on the same crown level. Common features of the interactions in both regions were interactions between a portion of the 50-kDa-domain and part of the converter domain of the myosin heads, similar to those found in the phosphorylation-regulated invertebrate myosin. These interactions are primarily electrostatic and the converter domain is responsible for the head-head interactions. Thus multiple head-head interactions of myosin crossbridges also characterize the switched-off state and have an important role in the regulation or other functions of myosin in thin filament-regulated muscles as well as in the thick filament-regulated muscles.     

Three-dimensional reconstruction of tarantula myosin filaments suggests how phosphorylation may regulate myosin activity

Muscle contraction involves the interaction of the myosin heads of the thick filaments with actin subunits of the thin filaments. Relaxation occurs when this interaction is blocked by molecular switches on these filaments. In many muscles, myosin-linked regulation involves phosphorylation of the myosin regulatory light chains (RLCs). Electron microscopy of vertebrate smooth muscle myosin molecules (regulated by phosphorylation) has provided insight into the relaxed structure, revealing that myosin is switched off by intramolecular interactions between its two heads, the free head and the blocked head. Three-dimensional reconstruction of frozen-hydrated specimens revealed that this asymmetric head interaction is also present in native thick filaments of tarantula striated muscle. Our goal in this study was to elucidate the structural features of the tarantula filament involved in phosphorylation-based regulation. A new reconstruction revealed intra- and intermolecular myosin interactions in addition to those seen previously. To help interpret the interactions, we sequenced the tarantula RLC and fitted an atomic model of the myosin head that included the predicted RLC atomic structure and an S2 (subfragment 2) crystal structure to the reconstruction. The fitting suggests one intramolecular interaction, between the cardiomyopathy loop of the free head and its own S2, and two intermolecular interactions, between the cardiac loop of the free head and the essential light chain of the blocked head and between the Leu305-Gln327 interaction loop of the free head and the N-terminal fragment of the RLC of the blocked head. These interactions, added to those previously described, would help switch off the thick filament. Molecular dynamics simulations suggest how phosphorylation could increase the helical content of the RLC N-terminus, weakening these interactions, thus releasing both heads and activating the thick filament.     

3D structure of relaxed fish muscle myosin filaments by single particle analysis

To understand the structural changes involved in the force-producing myosin cross-bridge cycle in vertebrate muscle it is necessary to know the arrangement and conformation of the myosin heads at the start of the cycle (i.e. the relaxed state). Myosin filaments isolated from goldfish muscle under relaxing conditions and viewed in negative stain by electron microscopy (EM) were divided into segments and subjected to three-dimensional (3D) single particle analysis without imposing helical symmetry. This allowed the known systematic departure from helicity characteristic of vertebrate striated muscle myosin filaments to be preserved and visualised. The resulting 3D reconstruction reveals details to about 55 A resolution of the myosin head density distribution in the three non-equivalent head 'crowns' in the 429 A myosin filament repeat. The analysis maintained the well-documented axial perturbations of the myosin head crowns and revealed substantial azimuthal perturbations between crowns with relatively little radial perturbation. Azimuthal rotations between crowns were approximately 60 degrees , 60 degrees and 0 degrees , rather than the regular 40 degrees characteristic of an unperturbed helix. The new density map correlates quite well with the head conformations analysed in other EM studies and in the relaxed fish muscle myosin filament structure modelled from X-ray fibre diffraction data. The reconstruction provides information on the polarity of the myosin head array in the A-band, important in understanding the geometry of the myosin head interaction with actin during the cross-bridge cycle, and supports a number of conclusions previously inferred by other methods. The observed azimuthal head perturbations are consistent with the X-ray modelling results from intact muscle, indicating that the observed perturbations are an intrinsic property of the myosin filaments and are not induced by the proximity of actin filaments in the muscle A-band lattice. Comparison of the axial density profile derived in this study with the axial density profile of the X-ray model of the fish myosin filaments which was restricted to contributions from the myosin heads allows the identification of a non-myosin density peak associated with the azimuthally perturbed head crown which can be interpreted as a possible location for C-protein or X-protein (MyBP-C or -X). This position for C-protein is also consistent with the C-zone interference function deduced from previous analysis of the meridional X-ray pattern from frog muscle. It appears that, along with other functions, C-(X-) protein may have the role of slewing the heads of one crown so that they do not clash with the neighbouring actin filaments, but are readily available to interact with actin when the muscle is activated.     

Interacting-heads motif explains the X-ray diffraction pattern of relaxed vertebrate skeletal muscle

Electron microscopy (EM) shows that myosin heads in thick filaments isolated from striated muscles interact with each other and with the myosin tail under relaxing conditions. This “interacting-heads motif” (IHM) is highly conserved across the animal kingdom and is thought to be the basis of the super-relaxed state. However, a recent X-ray modeling study concludes, contrary to expectation, that the IHM is not present in relaxed intact muscle. We propose that this conclusion results from modeling with a thick filament 3D reconstruction in which the myosin heads have radially collapsed onto the thick filament backbone, not from absence of the IHM. Such radial collapse, by about 3–4 nm, is well established in EM studies of negatively stained myosin filaments, on which the reconstruction was based. We have tested this idea by carrying out similar X-ray modeling and determining the effect of the radial position of the heads on the goodness of fit to the X-ray pattern. We find that, when the IHM is modeled into a thick filament at a radius 3–4 nm greater than that modeled in the recent study, there is good agreement with the X-ray pattern. When the original (collapsed) radial position is used, the fit is poor, in agreement with that study. We show that modeling of the low-angle region of the X-ray pattern is relatively insensitive to the conformation of the myosin heads but very sensitive to their radial distance from the filament axis. We conclude that the IHM is sufficient to explain the X-ray diffraction pattern of intact muscle when placed at the appropriate radius.     

Assembly of thick filaments and myofibrils occurs in the absence of the myosin head

We investigated the importance of the myosin head in thick filament formation and myofibrillogenesis by generating transgenic Drosophila lines expressing either an embryonic or an adult isoform of the myosin rod in their indirect flight muscles. The headless myosin molecules retain the regulatory light-chain binding site, the alpha-helical rod and the C-terminal tailpiece. Both isoforms of headless myosin co-assemble with endogenous full-length myosin in wild-type muscle cells. However, rod polypeptides interfere with muscle function and cause a flightless phenotype. Electron microscopy demonstrates that this results from an antimorphic effect upon myofibril assembly. Thick filaments assemble when the myosin rod is expressed in mutant indirect flight muscles where no full-length myosin heavy chain is produced. These filaments show the characteristic hollow cross-section observed in wild type. The headless thick filaments can assemble with thin filaments into hexagonally packed arrays resembling normal myofibrils. However, thick filament length as well as sarcomere length and myofibril shape are abnormal. Therefore, thick filament assembly and many aspects of myofibrillogenesis are independent of the myosin head and these processes are regulated by the myosin rod and tailpiece. However, interaction of the myosin head with other myofibrillar components is necessary for defining filament length and myofibril dimensions.     

Blebbistatin stabilizes the helical order of myosin filaments by promoting the switch 2 closed state

Blebbistatin is a small-molecule, high-affinity, noncompetitive inhibitor of myosin II. We have used negative staining electron microscopy to study the effects of blebbistatin on the organization of the myosin heads on muscle thick filaments. Loss of ADP and Pi from the heads causes thick filaments to lose their helical ordering. In the presence of 100 μM blebbistatin, disordering was at least 10 times slower. In the M·ADP state, myosin heads are also disordered. When blebbistatin was added to M·ADP thick filaments, helical ordering was restored. However, blebbistatin did not improve the order of thick filaments lacking bound nucleotide. Addition of calcium to relaxed muscle homogenates induced thick-thin filament interaction and filament sliding. In the presence of blebbistatin, filament interaction was inhibited. These structural observations support the conclusion, based on biochemical studies, that blebbistatin inhibits myosin ATPase and actin interaction by stabilizing the closed switch 2 structure of the myosin head. These properties make blebbistatin a useful tool in structural and functional studies of cell motility and muscle contraction.     

The equatorial x-ray diffraction patterns of crustacean striated muscles

The equatorial X-ray diffraction patterns from the striated muscles of the crab and crayfish showed a series of reflections arising from the hexagonal myofilament lattice. Some of the reflections in the region of $\text{1}\text{14}\text{to}\text{1}\text{11}\text{nm}{}^{\mathrm{?1}}$ had intensities comparable to those of the 1,0 and 1,1 reflections. This can be accounted for by certain myofilament lattice models in which the thick filaments are hollow.     

Probing Muscle Myosin Motor Action: X-Ray (M3 and M6) Interference Measurements Report Motor Domain Not Lever Arm Movement

The key question in understanding how force and movement are produced in muscle concerns the nature of the cyclic interaction of myosin molecules with actin filaments. The lever arm of the globular head of each myosin molecule is thought in some way to swing axially on the actin-attached motor domain, thus propelling the actin filament past the myosin filament. Recent X-ray diffraction studies of vertebrate muscle, especially those involving the analysis of interference effects between myosin head arrays in the two halves of the thick filaments, have been claimed to prove that the lever arm moves at the same time as the sliding of actin and myosin filaments in response to muscle length or force steps. It was suggested that the sliding of myosin and actin filaments, the level of force produced and the lever arm angle are all directly coupled and that other models of lever arm movement will not fit the X-ray data. Here, we show that, in addition to interference across the A-band, which must be occurring, the observed meridional M3 and M6 X-ray intensity changes can all be explained very well by the changing diffraction effects during filament sliding caused by heads stereospecifically attached to actin moving axially relative to a population of detached or non-stereospecifically attached heads that remain fixed in position relative to the myosin filament backbone. Crucially, and contrary to previous interpretations, the X-ray interference results provide little direct information about the position of the myosin head lever arm; they are, in fact, reporting relative motor domain movements. The implications of the new interpretation are briefly assessed.     

Refined structure of bony fish muscle myosin filaments from low-angle X-ray diffraction data

Application of X-ray diffraction methods to the elucidation of the arrangement of the myosin heads on myosin filaments in resting muscles is made simpler when the muscles themselves are well ordered in 3D. Bony fish muscle for the vertebrates and insect flight muscle for the invertebrates are the muscles of choice for this analysis. The rich, well-sampled, low-angle X-ray diffraction pattern from bony fish muscle has previously been modelled with an R-factor of 3.4% between observed and calculated transforms on the assumption that the two heads in one myosin molecule have the same shape. However, recent evidence from other kinds of analysis of other muscles has shown that this assumption may not be valid. There is evidence that the motor domain of one head in each pair may interact with the neck region of the second head. This possibility has been tested directly in the present analysis which extends the X-ray modelling of fish muscle myosin filaments by permitting independent shape changes of the two heads in one molecule. The new model, with a computed R-factor of 1.19% against 56 independent reflections, shows that in fish muscle also there is a marked asymmetry in the organisation of each head pair.     

The effect of fixative tonicity on the myosin filament lattice volume of frog muscle fixed following exposure to normal or hypertonic Ringer

Synopsis Frog sartorius muscles have been fixed sequentially with acrolein and osmium tetroxide dissolved in vehicles of various tonicities, and the myosin filament spacings and sarcomere lengths measured with the electron microscope. From these dimensions the myosin unit-cell volume has been calculated and compared with X-ray diffraction data to determine the effect of fixation. In muscles soaked in normal Ringer and afterwards fixed using normal Ringer as a vehicle for the fixation agents, the unitcell volume undergoes a 10.4% reduction during the preparative procedure. Muscles soaked in hypertonic Ringer undergo a similar reduction in volume during fixation, provided hypertonic Ringer is used as the vehicle; if they are fixed in normal Ringer, the lattice swells during fixation, even if the change to the normal tonicity vehicle occurs after acrolein fixation. If blocks suitable for embedding are cut from the muscles before, rather than after, osmium fixation, more complex changes in intracellular dimensions may occur, including artefactual swelling of the T-system. It is concluded that fixation of tissues exposed to modifications of normal physiological solutions should be performed using the same modified solutions as fixative vehicles.     

A molecular model of phosphorylation-based activation and potentiation of tarantula muscle thick filaments

Myosin filaments from many muscles are activated by phosphorylation of their regulatory light chains (RLCs). To elucidate the structural mechanism of activation, we have studied RLC phosphorylation in tarantula thick filaments, whose high-resolution structure is known. In the relaxed state, tarantula RLCs are ∼ 50% non-phosphorylated and 50% mono-phosphorylated, while on activation, mono-phosphorylation increases, and some RLCs become bi-phosphorylated. Mass spectrometry shows that relaxed-state mono-phosphorylation occurs on Ser35, while Ca²⁺-activated phosphorylation is on Ser45, both located near the RLC N-terminus. The sequences around these serines suggest that they are the targets for protein kinase C and myosin light chain kinase (MLCK), respectively. The atomic model of the tarantula filament shows that the two myosin heads (“free” and “blocked”) are in different environments, with only the free head serines readily accessible to kinases. Thus, protein kinase C Ser35 mono-phosphorylation in relaxed filaments would occur only on the free heads. Structural considerations suggest that these heads are less strongly bound to the filament backbone and may oscillate occasionally between attached and detached states (“swaying” heads). These heads would be available for immediate actin interaction upon Ca²⁺ activation of the thin filaments. Once MLCK becomes activated, it phosphorylates free heads on Ser45. These heads become fully mobile, exposing blocked head Ser45 to MLCK. This would release the blocked heads, allowing their interaction with actin. On this model, twitch force would be produced by rapid interaction of swaying free heads with activated thin filaments, while prolonged exposure to Ca²⁺ on tetanus would recruit new MLCK-activated heads, resulting in force potentiation.     

DAAM Is Required for Thin Filament Formation and Sarcomerogenesis during Muscle Development in Drosophila

During muscle development, myosin and actin containing filaments assemble into the highly organized sarcomeric structure critical for muscle function. Although sarcomerogenesis clearly involves the de novo formation of actin filaments, this process remained poorly understood. Here we show that mouse and Drosophila members of the DAAM formin family are sarcomere-associated actin assembly factors enriched at the Z-disc and M-band. Analysis of dDAAM mutants revealed a pivotal role in myofibrillogenesis of larval somatic muscles, indirect flight muscles and the heart. We found that loss of dDAAM function results in multiple defects in sarcomere development including thin and thick filament disorganization, Z-disc and M-band formation, and a near complete absence of the myofibrillar lattice. Collectively, our data suggest that dDAAM is required for the initial assembly of thin filaments, and subsequently it promotes filament elongation by assembling short actin polymers that anneal to the pointed end of the growing filaments, and by antagonizing the capping protein Tropomodulin.     

Multiple structures of thick filaments in resting cardiac muscle and their influence on cross-bridge interactions.

Based on two criteria, the tightness of packing of myosin rods within the backbone of the filament and the degree of order of the myosin heads, thick filaments isolated from a control group of rat hearts had three different structures. Two of the structures of thick filaments had ordered myosin heads and were distinguishable from each other by the difference in tightness of packing of the myosin rods. Depending on the packing, their structure has been called loose or tight. The third structure had narrow shafts and disordered myosin heads extending at different angles from the backbone. This structure has been called disordered. After phosphorylation of myosin-binding protein C (MyBP-C) with protein kinase A (PKA), almost all thick filaments exhibited the loose structure. Transitions from one structure to another in quiescent muscles were produced by changing the concentration of extracellular Ca. The probability of interaction between isolated thick and thin filaments in control, PKA-treated preparations, and preparations exposed to different Ca concentrations was estimated by electron microscopy. Interactions were more frequent with phosphorylated thick filaments having the loose structure than with either the tight or disordered structure. In view of the presence of MgATP and the absence of Ca, the interaction between the myosin heads and the thin filaments was most likely the weak attachment that precedes the force-generating steps in the cross-bridge cycle. These results suggest that phosphorylation of MyBP-C in cardiac thick filaments increases the probability of cross-bridges forming weak attachments to thin filaments in the absence of activation. This mechanism may modulate the number of cross-bridges generating force during activation.     

Millisecond time-resolved changes occurring in Ca2+-regulated myosin filaments upon relaxation

Contraction of many muscles is activated in part by the binding of Ca²⁺ to, or phosphorylation of, the myosin heads on the surface of the thick filaments. In relaxed muscle, the myosin heads are helically ordered and undergo minimal interaction with actin. On Ca²⁺ binding or phosphorylation, the head array becomes disordered, reflecting breakage of the head-head and other interactions that underlie the ordered structure. Loosening of the heads from the filament surface enables them to interact with actin filaments, bringing about contraction. On relaxation, the heads return to their ordered positions on the filament backbone. In scallop striated adductor muscle, the disordering that takes place on Ca²⁺ binding occurs on the millisecond time scale, suggesting that it is a key element of muscle activation. Here we have studied the reverse process. Using time-resolved negative staining electron microscopy, we show that the rate of reordering on removal of Ca²⁺ also occurs on the same physiological time scale. Direct observation of images together with analysis of their Fourier transforms shows that activated heads regain their axial ordering within 20 ms and become ordered in their final helical positions within 50 ms. This rapid reordering suggests that reformation of the ordered structure, and the head-head and other interactions that underlie it, is a critical element of the relaxation process.     

Human platelet myosin. II. In vitro assembly and structure of myosin filaments

We have used electron microscopy and solubility measurements to investigate the assembly and structure of purified human platelet myosin and myosin rod into filaments. In buffers with ionic strengths of less than 0.3 M, platelet myosin forms filaments which are remarkable for their small size, being only 320 nm long and 10-11 nm wide in the center of the bare zone. The dimensions of these filaments are not affected greatly by variation of the pH between 7 and 8, variation of the ionic strength between 0.05 and 0.2 M, the presence or absence of 1 mM Mg++ or ATP, or variation of the myosin concentration between 0.05 and 0.7 mg/ml. In 1 mM Ca++ and at pH 6.5 the filaments grow slightly larger. More than 90% of purified platelet myosin molecules assemble into filaments in 0.1 M KC1 at pH 7. Purified preparations of the tail fragment of platelet myosin also form filaments. These filaments are slightly larger than myosin filaments formed under the same conditions, indicating that the size of the myosin filaments may be influenced by some interaction between the head and tail portions of myosin molecules. Calculations based on the size and shape of the myosin filaments, the dimensions of the myosin molecule and analysis of the bare zone reveal that the synthetic platelet myosin filaments consists of 28 myosin molecules arranged in a bipolar array with the heads of two myosin molecules projecting from the backbone of the filament at 14-15 nm intervals. The heads appear to be loosely attached to the backbone by a flexible portion of the myosin tail. Given the concentration of myosin in platelets and the number of myosin molecules per filament, very few of these thin myosin filaments should be present in a thin section of a platelet, even if all of the myosin molecules are aggregated into filaments.     

Direct observation of molecular motility by light microscopy

We used video-fluorescence microscopy to directly observe the sliding movement of single fluorescently labeled actin filaments along myosin fixed on a glass surface. Single actin filaments labeled with phalloidin-tetramethyl-rhodamine, which stabilizes the filament structure of actin, could be seen very clearly and continuously for at least 60 min in 02-free solution, and the sensitivity was high enough to see very short actin filaments less than 40 nm long that contained less than eight dye molecules. The actin filaments were observed to move along double-headed and, similarly, single-headed myosin filaments on which the density of the heads varied widely in the presence of ATP, showing that the cooperative interaction between the two heads of the myosin molecule is not essential to produce the sliding movement. The velocity of actin filament independent of filament length (greater than 1 micron) was almost unchanged until the density of myosin heads along the thick filament was decreased from six heads/14.3 nm to 1 head/34 nm. This result suggests that five to ten heads are sufficient to support the maximum sliding velocity of actin filaments (5 micron/s) under unloaded conditions. In order for five to ten myosin heads to achieve the observed maximum velocity, the sliding distance of actin filaments during one ATP cycle must be more than 60 nm.