The myosin filament: VII. Changes in internal structure along the length of the filament

Improved fixation procedures have enabled substructure to be observed by electron microscopy in transverse sections of vertebrate skeletal muscle thick filaments as thin as 140 nm. Optical diffraction combined with digital autocorrelation analysis, focal series and tilting experiments have confirmed the presence of a regular substructure having a repeat near 4 nm and shown that it is highly unlikely to be an artifact associated with the electron microscope imaging system. The results obtained strongly suggest that the thick filament is constructed from a bundle of rod-like subfilaments arranged parallel to the thick filament axis to within less than a degree. This cannot easily be reconciled with the general theory of thick filament structure proposed by Squire (1973), but it is consistent with the model proposed by Pepe, 1966, Pepe, 1967. Optical diffraction of 140 nm thick serial transverse sections has also suggested a structural change along the length of the filament that is manifest by a variation in the proportion of filaments showing strong diffraction maxima in one, two or three directions.     

Periodically arranged interactions within the myosin filament backbone revealed by mechanical unzipping

Numerous types of biological motion are driven by myosin thick filaments. Although the exact structure of the filament backbone is not known, it has long been hypothesized that periodically arranged charged regions along the myosin tail are the main contributors to filament stability. Here we provide a direct experimental test of this model by mechanically pulling apart synthetic myosin thick filaments. We find that unzipping is accompanied by broad force peaks periodically spaced at 4-, 14- and 43-nm intervals. This spacing correlates with the repeat distance of highly charged regions along the myosin tail. Lowering ionic strength does not change force-peak periodicity but increases the forces necessary for unzipping. The force peaks are partially reversible, indicating that the interactions are rapidly re-established upon mechanical relaxation. Thus, the zipping together of myosin tails via consecutive formation of periodically spaced bonds may be the underlying mechanism of spontaneous thick filament formation.     

General model of myosin filament structure. 3. Molecular packing arrangements in myosin filaments

Following the original proposals about myosin filament structure put forward as part of a general myosin filament model (Squire, 1971, 1972) it is here shown what the most likely molecular packing arrangements within the backbones of certain myosin filaments would be assuming that the model is correct. That this is so is already indicated by recently published experimental results which have confirmed several predictions of the model (Bullard and Reedy, 1972; Reedy et al., 1972; Tregear and Squire, 1973).The starting point in the analysis of the myosin packing arrangements is the model for the myosin ribbons in vertebrate smooth muscle proposed by Small &; Squire (1972). It is shown that there is only one reasonable type of packing arrangement for the rod portions of the myosin molecules which will account for the known structure of the ribbons and which is consistent with the known properties of myosin molecules. The dominant interactions in this packing scheme are between parallel myosin molecules which are related by axial shifts of 430 Å and 720 Å. In this analysis the myosin rods are treated as uniform rods of electron density and only the general features of two-strand coiled-coil molecules are considered.Since the general myosin filament model is based on the assumption that the structures of different types of myosin filament must be closely related, the packing scheme derived for the myosin ribbons is used to deduce the structures of the main parts (excluding the bare zones) of the myosin filaments in a variety of muscles. It is shown in each case that there is only one packing scheme consistent with all the available data on these filaments and that in each filament type exactly the same interactions between myosin rods are involved. In other words the myosin-myosin interactions involved in filament formation are specific, they involve molecular shifts of either 430 Å or 720 Å, and are virtually identical in all the different myosin filaments which have been considered. Apart from the myosin ribbons, these are the filaments in vertebrate skeletal muscle, insect flight muscle and certain molluscan muscles.In the case of the thick filaments in vertebrate skeletal muscle the form of the myosin packing arrangement in the bare zone is considered and a packing scheme proposed which involves antiparallel overlaps between myosin rods of 1300 Å and 430 Å. It is shown that this scheme readily explains the triangular profiles of the myosin filaments in the bare zone (Pepe, 1967, 1971) and many other observations on the form of these myosin filaments.Finally it is shown that the cores of several different myosin filaments, assuming they contain protein, may consist of different arrangements of one or other of two types of core subfilament.     

The myosin filament. VI. Myosin content.

There has been some disagreement about the number of myosin molecules in vertebrate skeletal myosin filaments calculated from the myosin to actin weight ratio determined by quantitative sodium dodecyl sulfate/polyacrylamide gel electrophoresis (Tregear &; Squire, 1973; Potter, 1974; Morimoto &; Harrington, 1974). In this work it was found that (1) thoroughly washed fibrils are required to obtain the true value for the myosin to actin weight ratio. (2) Neither actin nor myosin is extracted preferentially during the required washing procedure. (3) There are four myosin molecules per 14.3 nm interval along the myosin filament or about 400 myosin molecules per filament.From published estimates of the number of molecules of C-protein per myosin filament (Offer et al., 1973; Morimoto &; Harrington, 1974) and the findings in this work, we conclude that there are four molecules of C-protein at each of the 14 C-protein binding positions along the filament, i.e. one C-protein molecule for each of the four myosin molecules contributing to the cross-bridges at each position.     

The myosin filament: XI. Filament assembly

The critical parameters required for the assembly of myosin filaments with a length distribution comparable to that for native myosin filaments were examined. It was found that: Two steps are required in the dilution of a myosin solution from 0.6M KCl to 0.15M KCl. In Step I the KCl concentration is reduced from 0.6 to 0.3M KCl and in Step II from 0.3 to 0.15M KCl. The rate of change of KCl required for Step I is different than that required for Step II. Increasing the total time of dilution in either Step I or II alone leads to an increase in length and a broadening of the length distribution. In Step I assembly of myosin molecules into nonsedimentable units occurs. These may be the basic units from which the filaments are assembled in Step II. Rapid dilution in Step I alone has no effect on the length distribution obtained at 0.15M KCl, but rapid dilution in Step II alone leads to short filaments (about 0.6 micron). Increasing the time of dilution in Step II alone to 3 hrs or 6 hrs gives a bimodal distribution in lengths with one peak at about 0.8 micron and the other at about 2.2 microns. The length distribution obtained at 0.15M KCl is not critically dependent on information contained in the portion of the filament previously assembled in Step II, but is critically dependent on the rate of change of KCl concentration during the assembly of the rest of the filament.     

Intermediate filament structure.

In the past year, several new developments concerning the structure of intermediate filament proteins and their assembly into intact intermediate filaments have been made: the coiled-coil structure of a rod domain has been elucidated; the basis of the chain interaction and its role in intermediate filament assembly has been specified; the organization of nearest-neighbour molecules in keratin intermediate filaments has been determined; and the glycine loop structures of the terminal domains of epidermal keratin chains have been defined. In addition, mutations in intermediate filament chains that promote pathology have been reported for the first time.     

The myosin filament. XIII. The sensitivity of LMM assembly to Mg.ATP

An LMM fragment (Mr 62,000) of myosin has been prepared which has aggregation properties that are sensitive to the presence of Mg.ATP. Aggregation of the LMM by reducing the ionic strength in the presence of 1 mM Mg.ATP produces non-periodic aggregates which gradually rearrange to paracrystals with a 43 nm axial repeat pattern. This fragment includes the C-terminal end of the myosin rod starting at residue 1376. Therefore, at least one of the Mg.ATP binding sites responsible for this effect is located somewhere along this region of the myosin rod. Although assembly of the rod fragment of myosin into paracrystals does not show sensitivity to Mg.ATP, assembly of intact myosin molecules to form filaments does show sensitivity to Mg.ATP. For myosin filaments, assembly initially gives a broad distribution around a mean length of 1.5 microns, which sharpens around the mean length with time. The rearrangement of the LMM rods and intact myosin molecules both induced by the presence of Mg.ATP are probably related. These findings highlight the complexity of the cooperative interactions between different portions of the myosin molecule that are involved in determining the assembly properties of the intact molecule.     

The myosin filament. X. Observation of nine subfilaments in transverse sections

The molecular packing of the subfilaments in muscle thick filaments has been investigated by electron microscopy. Thin (80-100 nm) transverse sections of vertebrate skeletal muscle were cut, and 129 electron microscope images of thick filaments from 15 different areas including seven to ten images in each area were analyzed by computer image processing. The transverse sections were limited to the portion of the filaments between the bare zone and the C-protein bearing region. Of the 129 images, six were discarded because they were structurally disrupted, 17 did not show evidence for the presence of subfilaments from the autocorrelation function, and four did not show evidence for three-fold rotational symmetry from the power spectrum. The remaining 102 filaments all showed evidence for three-fold rotational symmetry, consistent with other available evidence (Pepe, 1982). From the analysis of these images by rotational filtering, we have found that the vertebrate skeletal myosin filament is made up of nine subfilaments and that the image appears to have trigonal symmetry. Of these subfilaments, six are arranged with a center-to-center spacing of about 4 nm and the other three on the surface of the filament are distorted from this arrangement. Three additional densities, which together with the other nine, correspond to the pattern of 12 densities previously observed in more highly selected images (Stewart et al., 1981; Pepe and Drucker, 1972) were observed in 5% of the images. Another pattern of nine subfilaments peripherally arranged around the circumference of the filament was observed occasionally. This latter image may represent the organization of the subfilaments in the bare zone region of the filament, resulting from sampling of individual filaments displaced longitudinally relative to the other filaments in the A-band.     

Modulation of myosin filament organization by C-protein family members.

We have analyzed the interactions between two types of sarcomeric proteins: myosin heavy chain (MyHC) and members of an abundant thick filament-associated protein family (myosin-binding protein; MyBP). Previous work has demonstrated that when MyHC is transiently transfected into mammalian nonmuscle COS cells, the expressed protein forms spindle-shaped structures consisting of bundles of myosin thick filaments. Co-expression of MyHC and MyBP-C or -H modulates the MyHC structures, resulting in dramatically longer cables consisting of myosin and MyBP encircling the nucleus. Immunoelectron microscopy indicates that these cable structures are more uniform in diameter than the spindle structures consisting solely of MyHC, and that the myosin filaments are compacted in the presence of MyBP. Deletion analysis of MyBP-H indicates that cable formation is dependent on the carboxy terminal 24 amino acids. Neither the MyHC spindles nor the MyHC/MyBP cables associate with the endogenous actin cytoskeleton of the COS cell. While there is no apparent co-localization between these structures and the microtubule network, colchicine treatment of the cells promotes the formation of longer assemblages, suggesting that cytoskeletal architecture may physically impede or regulate polymer formation/extension. The data presented here contribute to a greater understanding of the interactions between the MyBP family and MyHC, and provide additional evidence for functional homology between MyBP-C and MyBP-H.     

The mammalian cardiac muscle thick filament: backbone contributions to meridional reflections

Information about the structure of the vertebrate striated muscle thick filament backbone is important for understanding the arrangement of both the rod portion of the myosin molecule and the accessory proteins associated with the backbone region of the filament. Although models of the backbone have been proposed, direct data on the structure of the backbone is limited. In this study, we provide evidence that electron micrographs of isolated negatively stained cardiac thick filaments contain significant information about the filament backbone. Computed Fourier transforms from isolated cardiac thick filaments show meridional (or near meridional) reflections on the 10th and 11th layer lines that are particularly strong. Comparison of Fourier filtrations of the filaments that exclude, or include, these reflections, provide evidence that these reflections originate at least in part from a series of striations on the backbone at a approximately 4 nm spacing. The striations are likely to result either from the packing of the myosin rods, or from proteins such as titin associated with the filament backbone.     

Effects of actin filament cross-linking and filament length on actin- myosin interaction

We have used two actin-binding proteins of the intestinal brush border, TW 260/240 and villin, to examine the effects of filament cross-linking and filament length on myosin-actin interactions. TW 260/240 is a nonerythroid spectrin that is a potent cross-linker of actin filaments. In the presence of this cross-linker we observed a concentration- dependent enhancement of skeletal muscle actomyosin ATPase activity (150-560% of control; maximum enhancement at a 1:70-80 TW 260/240:actin molar ratio). TW 260/240 did not cause a similar enhancement of either acto-heavy meromyosin (HMM) ATPase or acto-myosin subfragment-one (S1) ATPase. Villin, a Ca2+-dependent filament capping and severing protein of the intestinal microvillus, was used to generate populations of actin filaments of various lengths from less than 20 nm to 2.0 microns; (villin:actin ratios of 1:2 to 1:4,000). The effect of filament length on actomyosin ATPase was biphasic. At villin:actin molar ratios of 1:2- 25 actin-activated myosin ATPase activity was inhibited to 20-80% of control values, with maximum inhibition observed at the highest villin:actin ratio. The ATPase activities of acto-HMM and acto-S1 were also inhibited at these short filament lengths. At intermediate filament lengths generated at villin:actin ratios of 1:40-400 (average lengths 0.26-1.1 micron) an enhancement of actomyosin ATPase was observed (130-260% of controls), with a maximum enhancement at average filament lengths of 0.5 micron. The levels of actomyosin ATPase fell off to control values at low concentrations of villin where filament length distributions were almost those of controls. Unlike intact myosin, the actin-activated ATPase of neither HMM nor S1 showed an enhancement at these intermediate actin filament lengths.     

Intermediate filament structure: 2. Molecular interactions in the filament

Molecules of intermediate filament (IF) proteins contain a central rod domain in which the two constituent chains have a predominantly α-helical conformation and are coiled around one another to form segments of two-strand rope. Possible interactions between the two long segments, termed 1B and 2 were investigated by a technique successfully employed in studies of the modes of association of collagen molecules by Miller and coworkers. Prominent maxima were found in all of the six possible modes of association between the rod domain segments in individual IF proteins and certain maxima were found to be common to all IF. The surface lattice of the IF from α-keratin has been determined and possible bonding arrangements between the rod-domain segments are catalogued. A systematic search was carried out for combinations of interaction maxima which were consistent with the dimensions of the surface lattice. By the further application of stereochemical constraints, models for the topological arrangement of the rod-domain segments on the surface lattice were derived and these are illustrated and discussed.     

Intermediate filament structure

In a previous communication (Biosci. Rep. 3, 517–525, 1993) we described quantitative X-ray diffraction studies of -keratin which were shown to be consistent with the presence of finite arrays of repeating units, successive arrays being set down at axial intervals of 470 Å. In addition the axial interval between repeating units in an array was shown to be 197.9 Å. It was suggested that this could most readily be explained by supposing that a surfacelattice was present which contained a dislocation along a helical path with unit heighth = 470 Å and unit twist |t| = 49.1° . The number of repeating units was shown to be in the range 7–9. With 7 repeats the mismatch of the lattice along the dislocation is small and this choice was used to develop a detailed model for the filament. Subsequent studies of molecular interactions have shown however that the coiled-coil rope segments in the rod domain of the molecule are most probably oriented parallel to the dislocation, and so minimization of lattice mismatch may be less important than originally supposed. In the present communication it is shown that the choice of 8, rather than 7, for the number of repeating units yields a model which is more compatible with estimates of the linear density and also provides the basis for a general model for polymorphism in intermediate filament lattices.