Frank A. Pepe

The myosin filament: I. Structural organization from antibody staining observed in electron microscopy

Abstract A model for the myosin filament is presented. The model is based on: (1) the staining pattern observed with anti-myosin in electron microscopy and its relation to the M-line and pseudo-H-zone; (2) the presence of six radially distributed bridges between the filaments in the M-line; (3) the triangular profiles seen in cross-sections of the thick filament through the pseudo-H-zone. The most significant features of the model are that (1) the packing of the myosin molecules along the filament is altered by the tapered ends and the overlap of molecules in the pseudo-H-zone, (2) the myosin filaments are ordered in the A-band in positions restricted by the ability to form M-bridges between the filaments and (3) the approximately 430-A repeat period generally seen in the A-band can be accounted for solely on the basis of superposition of the cross-bridges. The model of the myosin filament has the following important structural characteristics: (1) myosin molecules are aggregated in parallel rows; (2) in the pseudo-H-zone the myosin molecules are aggregated tail (L-meromyosin) to tail and everywhere else head to tail; (3) in the M-line region there is tail to tail abutment of myosin molecules in the same row; (4) between the pseudo-H-zone and the tapered ends, there is an overlap of one period (approx. 430 A) in the myosin molecules along each row; (5) there are 12 rows in a cross-section through the M-line region and 18 rows in a cross-section between the pseudo-H-zone and the tapered ends; (6) the myosin molecules are packed so that a triangular profile is obtained in a cross-section through the pseudo-H-zone. Nine rows are on the surface and three centrally located; (7) these 12 rows make up three sets of four rows. In each set there are two pairs. There is a stagger of one-third of a period between the rows of a pair and a stagger of one period between pairs.

The myosin filament. III. C-protein.

Antibody labeling for C-protein observed in fluorescence microscopy shows that C-protein is located only in the middle one-third of each half of the A-band. In electron microscopy the labeling shows up as stripes spaced 430apart. The restricted location of C-protein to these stripes cannot be determined by electron microscopy alone. The earlier assumption that the stripes reflect the axial repeat of the myosin in the myosin filament ( Pepe, 1967 a ) is valid, since C-protein binds to the filaments with the repeat of the underlying molecules ( Pepe et al. , 1975 ; Rome et al. , 1973 ; Pepe, 1972 ). The labeling pattern for C-protein occurs in the same position as part of the labeling pattern for myosin. This explains why similar anti-myosin labeling patterns are observed in fluorescence microscopy regardless of the presence or absence of contaminating antibody to C-protein. In electron microscopy the labeling patterns are clearly different. This can be explained by the difference in detectability of antibody protein in fluorescence and electron microscopy. The antibody labeling experiments show that proteins other than C-protein are also present on the myosin filaments. Although antibody to these other proteins is detectable by antibody labeling, it is not detectable by immuno-diffusion against even the initial crude myosin extract.

The distribution of protein antigens in striated myofibrils

Abstract The method of localizing specific protein antigens by antibody staining has been applied to skeletal muscle. Some progress has been made in determining the distributions of myosin, actin, and tropomyosin within the sarcomere, by using fluorescein-labelled antibodies and the light microscope. In addition, work has been begun on the use of the electron microscope for visualizing antibody staining. The fundamental problem of determining the immunochemical specificity of such staining procedures has been considered, and, in the case of myosin, attacked experimentally. This has led to new studies on the splitting of myosin by trypsin, on the properties of the four major fragments derived from myosin by brief digestion, and on the differential localization of these parts of myosin within the A-band region of the sarcomere. It is concluded that the L-1 portion of myosin, which according to Cohen and Szent-Gyorgyi is largely, α-helical in structure, lies in the lateral edges of the A-band of the relaxed sarcomere. The other parts of the myosin complex appear to lie in the more central region of the A-band, but detailed mapping and immunochemical identification of these parts remains to be done. Although the evidence at hand is not sufficient to permit a definite conclusion regarding the configuration of the myosin molecule in the relaxed sarcomere, it is suggested that the molecule in its native state may be greatly extended.

The myosin filament: II. Interaction between myosin and actin filaments observed using antibody staining in fluorescent and electron microscopy

Abstract Using absorption techniques and fluorescent antimyosin staining, it is possible to identify three antigenically specific regions on the myosin molecule. The availability of these regions for antibody staining in different parts of the A-band reflects differences in the organization of myosin molecules in these parts. These results confirm and extend previous observations. The relationship of these results to the model of the myosin filament presented in the accompanying paper provides information concerning the interaction between the myosin and the actin filaments. The three antigenically distinct regions of the myosin molecule correspond to the two fragments produced by the action of trypsin (heavy meromyosin and light meromyosin) and the region of the molecule sensitive to trypsin digestion. The availability of these antigenic sites is as follows: (1) heavy meromyosin antigenic sites are available in the A-band only where no overlap of thin and thick filaments occurs. Everywhere else these sites are interacting with the actin filaments. (2) Trypsin-sensitive antigenic sites are only available in the middle one-third of each half of the A-band. In this region the myosin molecules in the filaments are precisely packed, leading to stable aggregation. On interaction with actin the myosin molecules are not able to bend out of the filament easily so that light meromyosin sites never become available for staining. Thus severe distortions are imposed on the hinge (trypsin-sensitive) portion of the cross-bridge, exposing sites otherwise unavailable for staining. (3) Light meromyosin antigenic sites are available only at the lateral edges of the A-band. In this region the packing of the molecules is affected by the taper of the filament and the molecules are able to bend out of the filament more easily on interaction with actin. Little or no distortion is imposed on the hinge region and no trypsin-sensitive sites are available for staining.

Purification of uterine myosin and synthetic filament formation

Abstract Rabbit uterine myosin was purified by DEAE-Sephadex column chromatography. The purified myosin was shown to be free from contamination with actin or other impurities by sodium dodecyl sulfate gel electrophoresis. It was also shown to have two light chains with molecular weights of 22,000 and 17,000. The C protein normally found in crude, rabbit skeletal muscle myosin was not found in crude, rabbit uterine myosin. On reducing the ionic strength of a solution of rabbit uterine myosin, the myosin molecules first aggregated to form short, tapered bipolar filaments with bare zones. These were observed in the presence or absence of 10 m m -magnesium. These filaments ranged in length from 0.3 μm to 0.6 μm. On reaching 0.1 m -KCl, and only if 10 m m -magnesium was present, the filaments grew in length by linear overlap of the tapered bipolar filaments and obliteration of the bare zone regions. These filaments ranged in length from 0.7 μm to 1.2 μm.

The myosin filament: VI. Myosin content

Abstract 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: IX. Determination of subfilament positions by computer processing of electron micrographs

Abstract Computer image processing of electron micrographs has been employed to delineate the position of thick filament subunits in transverse sections of extensively crosslinked vertebrate skeletal muscle. Both back projection and rotational averaging methods indicate the presence of 12 subunits arranged on an approximately hexagonal lattice similar to that proposed by Pepe (1967). The spacing between subunits and the myosin content of the thick filament indicate that these subunits probably contain more than one myosin molecule and are most likely dimers.

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.

Axial packing in light meromyosin paracrystals

Abstract The positions of ends of molecules have been correlated with the striation pattern in negatively stained paracrystals of light meromyosin, and the pattern of deposition of C-protein on paracrystals has been examined both in negative stain and in section. The data show that in paracrystals of papain LMM ‡ , molecules may be related by overlaps of 16 or 44 nm; in paracrystals of chymotryptic LMM, molecules may overlap by multiples of 14 nm. The polarity by which overlapping molecules are related may be deduced for those molecules which bind C-protein. In paracrystals of papain LMM, molecules may overlap by 16 nm head-to-head or by 44 nm head-to-tail; in paracrystals of chymotryptic LMM, the head-to-head and head-to-tail overlaps are 14 and 42 nm. The binding of C-protein at 42 nm intervals to paracrystals whose staining pattern shows an undifferentiated 14 nm periodicity indicates that some feature of molecular packing must repeat at 42 nm intervals. The observation of C-protein bound at the edges of paracrystals suggests that the C-protein binding site is near one end of the LMM molecule.

Analysis of Antibody Staining Patterns Obtained with Striated Myofibrils in Fluorescence Microscopy and Electron Microscopy

Publisher Summary Antibody staining methods have been used in both fluorescence and electron microscopy to identify the presence and distribution of protein in the myofibril. For fluorescence microscopy, the antibody is tagged with fluorescein and its localization in the myofibril is determined by observing the fluorescence of the conjugate. In electron microscopy an initial attempt is made to use mercury as a tag to identify the localization of the antibody. However, the greatest progress has been made using unlabeled antibody. In the myofibril, using both fluorescence and electron microscopy, it has been possible to obtain information concerning the molecular organization and interactions of some of the myofibrillar proteins being localized. The details of the antibody staining patterns obtained with myofibrils have been interpreted as either contradicting the sliding filament model for muscle contraction or agreeing with it. The myofibrillar proteins which have been studied are myosin, actin, tropomyosin, and troponin. Of these, the most work has been done on myosin. In general glycerinated fibrils have been used for staining. The chapter discusses the use of antibodies for the study of the striated myofibril and general considerations for the interpretation of antibody staining patterns. Analysis of antibody staining patterns in terms of distribution, organization, and interaction of protein molecules in the myofibril is presented in the chapter.