Hydrostatic pressure studies of native and synthetic thick filaments: II. Native thick filaments from rabbit skeletal muscle

Native thick filaments isolated from freshly prepared rabbit psoas muscle were found to be resistant to pressure-induced dissociation. With increasing pressure application and release, a bimodal distribution of filament lengths was observed. The shorter filament length is associated with filament breakage at the center of the bare zone, while the longer length is associated with relatively intact filaments. Intact filaments and filament halves decrease in length by no more than 20% after exposure to and release of 14,000 psi. Bimodal distributions were not observed in equivalent experiments performed on filaments isolated from muscle glycerinated and stored at -20 degrees C for 6 months. Instead, filament dissociation proceeds linearly as a function of increasing pressure. Filaments prepared from muscle glycerinated and stored for 2 and 4 months exhibited pressure-induced behavior intermediate between the filaments prepared from fresh muscle and filaments prepared from muscle stored for 6 months. Since there appears to be no difference in the protein profiles of the various muscle samples, it is possible that stabilization of the native thick filament against hydrostatic pressure arises from trapped ions that are leached out over time.     

The use of cryomethods for research on the sarcomere ultrastructure of rabbit skeletal muscles

The ultrastructure of sarcomeres of glycerinated rabbit psoas muscle was studied using freeze-fracture-etching, freeze-drying and optical diffraction techniques in comparison with the investigation of this muscle by plastic sections and negative staining methods. In frozen and dried myofibrils isolated from the above muscle the stripes of minor proteins location in A- and I-disks were clearly seen. The pivot structure in thick filaments was revealed in longitudinal fractures of the muscle. The ordered arrangement of myosin heads (crossbridges) associated with actin filaments was preserved in frozen longitudinal fractures as evidenced by optical diffraction. Freeze etching technique allowed to revealed some details of Z-line structure: alpha-actinin bridges connecting the ends of actin filaments of neighbouring sarcomeres and to preserve the lateral struts between actin filaments in I-disks.     

Intermediate filaments in smooth muscle from pregnant and non-pregnant human uterus.

The intermediate filament proteins desmin and vimentin from pregnant and non-pregnant uterine muscle and smooth-muscle cells in culture were analysed using SDS/PAGE. The desmin content in uterine muscle increases dramatically during pregnancy, whereas vimentin remains unchanged or changes very little. When muscle cells are kept in culture, a considerable increase in vimentin content is observed as compared with vimentin in freshly isolated non-pregnant uterine tissue. Our results strengthen the view that vimentin and desmin filaments have independent function and turnover, and point to a predominantly structural role for desmin filaments.     

Rigor crossbridges are double-headed in fast muscle from crayfish

The structure of rigor crossbridges was examined by comparing rigor crossbridges in fast muscle fibers from glycerol-extracted abdominal flexor muscle of crayfish with those in "natively decorated" thin filaments from the same muscle. Natively decorated thin filaments were obtained by dissociating the backbone of the myosin filaments of rigor myofibrils in 0.6 M KCl. Intact fibers were freeze-fractured, deep-etched, and rotary shadowed; isolated filaments were either negatively stained or freeze dried and rotary shadowed. The crossbridges on the natively decorated actin maintain the original spacing and the disposition in chevrons and double chevrons for several hours, indicating that no rearrangement of the actomyosin interactions occurs. Thus the crossbridges of the natively decorated filaments were formed within the geometrical constraints of the intact myofibril. The majority of crossbridges in the intact muscle have a triangular shape indicative of double-headed crossbridge. The triangular shape is maintained in the isolated filaments and negative staining resolves two heads in a single crossbridge. In the isolated filaments, crossbridges are attached at uniform acute angles. Unlike those in insect flight muscle (Taylor et al., 1984), lead and rear elements of the double chevron may be both double-headed. Deep-etched images reveal a twisted arrangement of subfilaments in the backbone of the thick filament.     

Changes in thick filament length in Limulus striated muscle

Here we describe the change in thick filament length in striated muscle of Limulus, the horseshoe crab. Long thick filaments (4.0 microns) are isolated from living, unstimulated Limulus striated muscle while those isolated from either electrically or K+-stimulated fibers are significantly shorter (3.1 microns) (P less than 0.001). Filaments isolated from muscle glycerinated at long sarcomere lengths are long (4.4 microns) while those isolated from muscle glycerinated at short sarcomere lengths are short (2.9 microns) and the difference is significant (P less than 0.001). Thin filaments are 2.4 microns in length. The shortening of thick filaments is related to the wide range of sarcomere lengths exhibited by Limulus telson striated muscle.     

Polarity of myofilaments in molluscan smooth muscle

Summary Myofilaments were isolated by gently homogenizing smooth muscle cells isolated from the pedal retractor muscle (PRM) of Mytilus edulis, and observed by electron microscopy. The thick filaments isolated in the presence of ATP (10–20 mM) had projections of myosin heads except near their centre (central bare zone). After extraction of myosin, the paramyosin core of the thick filaments showed a Bear-Selby net or a striated pattern with a main periodicity of 14.5 nm. Both the Bear-Selby net and the striated patterns had a polarity that reversed at the centre of the filament where the patterns were obscured. The thin filaments were attached to dense bodies. Decoration of the thin filaments with heavy meromyosin showed that they have opposite polarity on opposing sides of the dense body. The results indicate that the thick filaments are bipolar and also that the dense bodies are functionally analogous to the Z-disk of the striated muscle.     

Electron microscopic and optical diffraction analysis of the structure of scorpion muscle thick filaments

We rapidly and gently isolated thick filaments from scorpion tail muscle by a modification of the technique previously described for isolating Limulus thick filaments. Images of negatively stained filaments appeared to be highly periodic, with a well-preserved myosin cross-bridge array. Optical diffraction patterns of the electron micrograph images were detailed and similar to optical diffraction patterns from Limulus and tarantula thick filaments. Analysis of the optical diffraction patterns and computed Fourier transforms, together with the appearance of the filaments in the micrographs, suggested a model for the filaments in which the myosin cross-bridges were arranged on four helical strands with 12 cross-bridges per turn of each strand, thus giving the observed repeat every third cross-bridge level. Comparison of the scorpion thick filaments with those isolated from the closely related chelicerate arthropods, Limulus and tarantula, revealed that they were remarkably similar in appearance and helical symmetry but different in diameter.     

Elastic filaments in situ in cardiac muscle: deep-etch replica analysis in combination with selective removal of actin and myosin filaments

To clarify the full picture of the connectin (titin) filament network in situ, we selectively removed actin and myosin filaments from cardiac muscle fibers by gelsolin and potassium acetate treatment, respectively, and observed the residual elastic filament network by deep-etch replica electron microscopy. In the A bands, elastic filaments of uniform diameter (6-7 nm) projecting from the M line ran parallel, and extended into the I bands. At the junction line in the I bands, which may correspond to the N2 line in skeletal muscle, individual elastic filaments branched into two or more thinner strands, which repeatedly joined and branched to reach the Z line. Considering that cardiac muscle lacks nebulin, it is very likely that these elastic filaments were composed predominantly of connectin molecules; indeed, anti-connectin monoclonal antibody specifically stained these elastic filaments. Further, striations of approximately 4 nm, characteristic of isolated connectin molecules, were also observed in the elastic filaments. Taking recent analyses of the structure of isolated connectin molecules into consideration, we concluded that individual connectin molecules stretched between the M and Z lines and that each elastic filament consisted of laterally-associated connectin molecules. Close comparison of these images with the replica images of intact and S1-decorated sarcomeres led us to conclude that, in intact sarcomeres, the elastic filaments were laterally associated with myosin and actin filaments in the A and I bands, respectively. Interestingly, it was shown that the elastic property of connectin filaments was not restricted by their lateral association with actin filaments in intact sarcomeres. Finally, we have proposed a new structural model of the cardiac muscle sarcomere that includes connectin filaments.     

The active cross-bridge motions of isolated thick filaments from myosin-regulated muscles detected by quasi-elastic light scattering.

Intensity fluctuation spectroscopy has been used successfully as a probe that can detect an increase in high-frequency internal motions of isolated thick filaments of Limulus muscle upon the addition of calcium ions. We have attributed such motions to cross-bridge motion instead of to an increase in the flexibility of the filament backbone. Here we show that after cleavage of the S-1 and then the S-2 moieties with papain, cross-linking the myosin heads to the filament backbone, or heat denaturation (42 degrees C, 10 min), the increase in the high frequency internal motions in the thick filaments no longer occurs. Congo Red, which has been shown to induce shortening of isolated myofibrils, also increases the high-frequency motions of the isolated filaments. Furthermore, the increase is suppressed by treating the filaments with a myosin ATPase inhibitor such as vanadate ions (10 mM) or by replacing ATP with either an equimolar CrADP or the nonhydrolyzable ATP analogue beta, gamma-imido-adenine-5'-triphosphate (AMP-PNP). Calcium ions have a similar effect on isolated thick filaments from scallop muscle, where the myosin is known to be regulatory. Calcium ions have no such effect on thick filaments isolated from frog muscle, which is believed not to be regulated by calcium binding to myosin. These results confirm our earlier supposition that the additional high frequency internal motions of the thick filaments isolated from striated muscle of Limulus are related to the energy dependent, active cross-bridge motions.     

DIRECT ISOLATION OF NATIVE THIN FILAMENTS FROM EMBRYONIC MUSCLE CELLS*

A method is presented for the release of “native” thin filaments from 13-day old embryonic chick muscle without tryptic digestion or desoxycholate (DOC) solubilization of Z bands. The isolated filaments were 50–60 Å in diameter, of variable length, and formed “arrowhead-like” complexes with heavy meromyosin (HMM). In addition, the filaments interacted with purified myosin to form actomyosin as effectively as action extracted from an acetone powder of muscle. The Mg⁺⁺-dependent ATPase activity and extent of superprecipitation of the synthetic actomyosin required a low concentration of Ca⁺⁺, strongly suggesting the presence of troponin and tropomyosin on the thin filaments isolated from muscle at this stage of embryogenesis. The native thin filaments were more sensitive to trypsin than synthetic F-actin prepared from an acetone powder based on measurements of flow birefrengence, viscosity and the ability to activate myosin ATPase.     

The dystrophin complex forms a mechanically strong link between the sarcolemma and costameric actin

The absence of dystrophin complex leads to disorganization of the force-transmitting costameric cytoskeleton and disruption of sarcolemmal membrane integrity in skeletal muscle. However, it has not been determined whether the dystrophin complex can form a mechanically strong bond with any costameric protein. We performed confocal immunofluorescence analysis of isolated sarcolemma that were mechanically peeled from skeletal fibers of mouse hindlimb muscle. A population of gamma-actin filaments was stably associated with sarcolemma isolated from normal muscle and displayed a costameric pattern that precisely overlapped with dystrophin. However, costameric actin was absent from all sarcolemma isolated from dystrophin-deficient mdx mouse muscle even though it was localized to costameres in situ. Vinculin, alpha-actinin, beta-dystroglycan and utrophin were all retained on mdx sarcolemma, indicating that the loss of costameric actin was not due to generalized membrane instability. Our data demonstrate that the dystrophin complex forms a mechanically strong link between the sarcolemma and the costameric cytoskeleton through interaction with gamma-actin filaments. Destabilization of costameric actin filaments may also be an important precursor to the costamere disarray observed in dystrophin-deficient muscle. Finally, these methods will be broadly useful in assessing the mechanical integrity of the membrane cytoskeleton in dystrophic animal models lacking other costameric proteins.     

Mammalian cardiac muscle thick filaments: their periodicity and interactions with actin

Cardiac muscle has been extensively studied, but little information is available on the detailed macromolecular structure of its thick filament. To elucidate the structure of these filaments I have developed a procedure to isolate the cardiac thick filaments for study by electron microscopy and computer image analysis. This procedure uses chemical skinning with Triton X-100 to avoid contraction of the muscle that occurs using the procedures previously developed for isolation of skeletal muscle thick filaments. The negatively stained isolated filaments appear highly periodic, with a helical repeat every third cross-bridge level (43 nm). Computed Fourier transforms of the filaments show a strong set of layer lines corresponding to a 43-nm near-helical repeat out to the 6th layer line. Additional meridional reflections extend to at least the 12th layer line in averaged transforms of the filaments. The highly periodic structure of the filaments clearly suggests that the weakness of the layer lines in x-ray diffraction patterns of heart muscle is not due to an inherently more disordered cross-bridge arrangement. In addition, the isolated thick filaments are unusual in their strong tendency to remain bound to actin by anti-rigor oriented cross-bridges (state II or state III cross-bridges) under relaxing conditions.     

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.     

Isolation and composition of thick filaments from rabbit skeletal muscle

A method has been developed for the isolation of thick filaments from rabbit skeletal muscle. We found that the thick filaments of this muscle are readily dispersed in the presence of a relaxing medium if the M and Z-line structures are first extracted in a low-salt solvent system. Thick filaments were separated from thin filaments by zone sedimentation in a 10% to 30% glycerol density gradient. The isolated filaments are homogeneous in length (1.5 to 1.6 μm) and retain the physical characteristics of these structures observed in sectioned muscle. Gel electrophoresis of thick filaments in the presence of sodium dodecyl sulfate showed a band of C-protein as well as bands with mobilities characteristic of the heavy and light chains of myosin. No other protein species was detected in these experiments. Thus our results provide evidence against the presence of a special protein component which would serve as the core of the skeletal thick filament structure. From the relative stain density of bands, the molar ratio of C-protein to myosin was estimated to be 1 to 5.8.     

Isolation of F-actin filaments. Comparison of F-actin filament preparations from normal and dystrophic mouse muscle.

To investigate the dystrophic influence on the characteristics of actin, a method for the isolation of F-actin filaments from the skeletal muscle of small sizes, i.e., less than 0.5 g, was devised. In this method, minced muscle was treated with collagenase and hyaluronidase, and the isolated filaments were washed with adenosine triphosphate (ATP). Upon examination in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the ATP-washed filaments showed a protein component identical in mobility to actin in untreated myofibrils or to that prepared by the conventional method. Electron microscopic appearances of the filaments were similar to those of F-actin filaments described in the literature. The dimensions of the filaments were 0.5--2.5 micrometer in length and 60--70 A in diameter. The ability to activate the Mg-adenosine triphosphatase or myosin was found to be Ca2+ independent. In all aspects of the above characteristics, the filaments from leg muscles of 129/Re dydy dystrophic mice and their litter mates were observed to be identical.     

Microfilaments and tropomyosin of cultured mammalian cells: isolation and characterization

Microfilaments were isolated from cultured mammalian cells, utilizing procedures similar to those for isolation of "native" thin filaments from muscle. Isolated microfilaments from rat embryo, baby hamster kidney (BHK- 21), and Swiss mouse 3T3 cells appeared structurally similar to muscle thin filaments, exhibiting long, 6 nm Diam profiles with a beaded, helical substructure. An arrowhead pattern was observed after reaction of isolated microfilaments with rabbit skeletal muscle myosin subfragment 1. Under appropriate conditions, isolated microfilaments will aggregate into a form that resembles microfilament bundles seen in situ cultured cells. Isolated microfilaments represent a complex of proteins including actin. Some of these components have been tentatively identified, based on coelectrophoresis with purified proteins, as myosin, tropomyosin, and a high molecular weight actin-binding protein. The tropomyosin components of isolated microfilaments were unexpected; polypeptides comigrated on SDS-polyacrylamide gels with both muscle and nonmuscle types of tropomyosin. In order to identify more specifically these subunits, we isolated and partially characterized tropomyosin from three cell types. BHK-21 cell tropomyosin was similar to other nonmuscle tropomyosins, as judged by several criteria. However, tropomyosin isolated from rate embryo and 3T3 cells contained subunits that comigrated with both skeletal muscle and nonmuscle types of myosin, whereas the BHK cell protein consistently contained a minor muscle-like subunit. The array of tropomyosin subunits present in a cell culture was reflected in the polypeptide chain pattern seen on SDS-polyacrylamide gels of microfilaments isolated from that culture. These studies provide a starting point for correlating changes in the ultrastructural organization of microfilaments with alterations in their protein composition.     

The alteration of myosin isoform compartmentation in specific mutants of Caenorhabditis elegans

Myosin isoforms A and B are located at the surface of the central and polar regions, respectively, of thick filaments in body muscle cells of Caenorhabditis elegans, whereas paramyosin and a distinct core structure comprise the backbones of these filaments. Thick filaments and related structures were isolated from nematode mutants that have altered thick filament protein compositions. These mutant filaments and their complexes with specific antibodies were studied by electron microscopy to determine the distribution of the two myosins. The compartmentation of the two myosin isoforms in body wall muscle thick filaments depends not only upon the intrinsic properties of the myosins but their interactions with other components such as paramyosin and their relative quantities determined by synthesis.     

Two kinds of thick filament in smooth muscle cells in the adductor of a clam, Chlamys nobilis

The ultrastructure of the opaque portion of the adductor muscle in the pecten Chlamys nobilis was investigated. The opaque portion was composed of smooth muscle cells that contained thin and thick filaments. The thick filaments were classified into two kinds, thinner and thicker, according to the statistical analysis of diameters. They were also classified as being shorter and longer, when isolated native filaments were examined. The thick filaments may consequently be classified into two kinds: thinner and shorter filaments, and thicker and longer ones. The thinner and shorter filaments were about 26.5 nm in diameter and 7.5 mum in length, and the thicker and longer ones were about 42.0 nm in diameter and 13.0 mum in length, respectively. A regular periodicity was apparent on the surface of the core after removal of myosin molecules from its surface. The periodicity seemed similar for the two kinds of thick filament.     

Role of vimentin in smooth muscle force development

Vimentin intermediate filaments undergo spatial reorganization in cultured smooth muscle cells in response to contractile activation; however, the role of vimentin in the physiological properties of smooth muscle has not been well elucidated. Tracheal smooth muscle strips were loaded with antisense oligonucleotides (ODNs) against vimentin and then cultured for 2 days to allow for protein degradation. Treatment with vimentin antisense, but not sense, ODNs suppressed vimentin protein expression; neither vimentin antisense nor sense ODNs affected protein levels of desmin and actin. Force development in response to ACh stimulation or KCl depolarization was lower in vimentin-deficient tissues than in vimentin sense ODN- or non-ODN-treated muscle strips. Passive tension was also depressed in vimentin-depleted muscle tissues. Vimentin downregulation did not attenuate increases in myosin light chain (MLC) phosphorylation in response to contractile stimulation or basal MLC phosphorylation. In vimentin sense ODN-treated or non-ODN-treated smooth muscle strips, the desmosomal protein plakoglobin was primarily localized in the cell periphery. The membrane-associated localization of plakoglobin was reduced in vimentin-depleted muscle tissues. These studies suggest that vimentin filaments play an important role in mediating active force development and passive tension, which are not regulated by MLC phosphorylation. Vimentin downregulation impairs the structural organization of desmosomes, which may be associated with the decrease in force development. intermediate filaments; cytoskeleton; contraction; desmin     

THE FINE STRUCTURE OF THE SHELL MUSCLE OF PATELLID PROSOBRANCH LIMPETS

The structure of the shell muscle of eleven species of patellidlimpet is described from light and transmission electron microscopestudies. Although the muscle has many structural characteristicstypical of molluscan smooth muscle, it also has a number ofunusual features. At the electron microscope level two myofibretypes are distinguishable. Type I cells, present in all species,contain conventional contractile apparatus in the form of thickand thin filaments. Thick filaments contain paramyosin and varyin diameter between 20—180 nm. An axial striation witha repeat of 14.2 nm is calculated from optically diffractedmicrographs of isolated thick filaments. Transverse sectionsof thick filaments reveal bands from which the transverse repeatof the paramyosin crystal lattice is calculated. Type II myofibres,which are present in five species, contain a novel arrangementof thin filaments with electron-dense regions at intervals of80–150 nm. The striated thin filaments are similar inappearance to the microfilament bundles and stress-fibres ofnon-muscle cells. They also have similarities to the leptomericorganelles of some vertebrate muscle tissues. Associated withthe muscle is an unusually large amount of collagen which hasa periodicity of 62 nm calculated from optical diffraction patternsof isolated collagen fibrils. (Received 3 July 1989; accepted 12 October 1989)