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.     

Dense bodies and actin polarity in vertebrate smooth muscle

The arrangement of cytoplasmic dense bodies in vertebrate smooth muscle and their relationship to the thin filaments was studied in cells from rabbit vas deferens and portal vein which were made hyperpermeable (skinned) with saponin and incubated with myosin subfragment 1 (S-1). The dense bodies were obliquely oriented, elongated structures sometimes appearing as chains up to 1.5 microns in length; they were often continuous across the cell for 200 to 300 nm and were interconnected by an oblique network of 10-nm filaments. The arrowheads, formed by S-1 decoration of actins, which inserted into both the sides and ends of dense bodies, always pointed away from the dense body, similar to the polarity of the thin filaments at the Z- bands of skeletal muscle. These results show that the cytoplasmic dense bodies function as anchoring sites for the thin filaments and indicate that the thin filaments, thick filaments, and dense bodies constitute a contractile unit.     

Thin (actin) and thick (myosinlike) filaments in cone contraction in the teleost retina

The long slender retinal cones of fishes shorten in the light and elongate in the dark. Light-induced cone shortening provides a useful model for stuying nonmuscle contraction because it is linear, slow, and repetitive. Cone cells contain both thin (actin) and thick (myosinlike) filaments oriented parallel to the axis of contraction. This study examines the polarities of the cone's thin filaments and the changes in filament distribution which accompany light-induced contraction, in an attempt to elucidate the structural basis for the cone's contractile process. The proximal half of the cone is fixed to its cellular neighbors in the outer nuclear layer while the distal half is free. Thus, all shortening takes place in a necklike region (the myoid) in the distal half of the cone which extends into the space between the neural retina and the pigmented retinal epithelium. Thin filaments are found throughout the length of the cone, whereas thick filaments occur predominantly in the proximal (axon) regions of both light- and dark-adapted cones. Thus, thick filaments are primarily localized outside the region where shortening takes place. Observations from myosin subfragment-1 binding studies suggest that the cone's thin filaments are organized into two opposing sets. In the distal half of the cone (including the myoid), virtually all filaments have proximally directed arrowheads. In the more proximal regions of the axon, many thin filaments have opposite polarity, their arrowheads being distally directed. Near the synaptic proximal end of the light-adapted (contracted) cone, filaments of opposite polarities occur in approximately equal numbers. Thus, in the cone axon there appear to be two overlapping sets of actin filaments whose opposite polarities correspond to the two actin halves of a muscle sarcomere. In elongated, dark-adapted cones, thick filaments are localized throughout the axon region of the cone. In light, thick filaments accumulate towards the proximal end of the cone. These observations are consistent with a "sliding hypothesis" for cone contraction, in which thick myosinlike filaments produce sliding interdigitation of the two sets of oppositely directed actin filaments in the proximal axon region. Thus, the myoid thin filaments would be essentially reeled into the axon region to produce shortening. The mechanism of re-elongation depends on microtubules, as discussed in the companion paper.     

Reorganization of contractile elements in the platelet during clot retraction

Electron microscopic studies have been carried out on human platelets in the clot retraction. In the early stage of clot formation, platelets send out filopodia, in which thin filaments run longitudinally. The thin filaments are often observed to attach to the cell membrane where fibrin strands bind from the extracellular surface. In the later stage of clot formation, thick filaments become observable, mainly in the cell body of the platelets. These thick filaments are arranged to form an ordered array, and thin filaments run parallel to them. The thin filaments often attach to the end of the thick filaments. However, thin filaments are not seen between the arrays of thick filaments. Similar structures are also observed in the cytoskeleton of the contracted platelet. These filaments closely resemble the purified myosin aggregates formed under low ionic strength. Thus, during clot retraction, both actin and myosin in platelets are reorganized into thin and thick filaments, respectively.     

THE ORGANIZATION OF CONTRACTILE FILAMENTS IN A MAMMALIAN SMOOTH MUSCLE

Ordered arrays of thin filaments (65 A diameter) along with other apparently random arrangements of thin and thick filaments (100–200 A diameter) are observed in contracted guinea pig taenia coli rapidly fixed in glutaraldehyde. The thin-filament arrays vary from a few to more than 100 filaments in each array. The arrays are scattered among isolated thin and thick filaments. Some arrays are regular such as hexagonal; other arrays tend to be circular. However, few examples of rosettes with regular arrangements of thin filaments surrounding thick filaments are seen. Optical transforms of electron micrographs of thin-filament arrays give a nearest-neighbor spacing of the thin filaments in agreement with the "actin" filament spacing from x-ray diffraction experiments. Many thick filaments are closely associated with thin-filament arrays. Some thick filaments are hollow circles, although triangular shapes are also found. Thin-filament arrays and thick filaments extend into the cell for distances of at least a micron. Partially relaxed taenia coli shows thin-filament arrays but few thick filaments. The suggestion that thick filaments aggregate prior to contraction and disaggregate during relaxation is promoted by these observations. The results suggest that a sliding filament mechanism operates in smooth muscle as well as in striated muscle.     

LOCALIZATION OF MYOSIN FILAMENTS IN SMOOTH MUSCLE

Thick myosin filaments, in addition to actin filaments, were found in sections of glycerinated chicken gizzard smooth muscle when fixed at a pH below 6.6. The thick filaments were often grouped into bundles and run in the longitudinal axis of the smooth muscle cell. Each thick filament was surrounded by a number of thin filaments, giving the filament arrangement a rosette appearance in cross-section. The exact ratio of thick filaments to thin filaments could not be determined since most arrays were not so regular as those commonly found in striated muscle. Some rosettes had seven or eight thin filaments surrounding a single thick filament. Homogenates of smooth muscle of chicken gizzard also showed both thick and thin filaments when the isolation was carried out at a pH below 6.6, but only thin filaments were found at pH 7.4. No Z or M lines were observed in chicken gizzard muscle containing both thick and thin filaments. The lack of these organizing structures may allow smooth muscle myosin to disaggregate readily at pH 7.4.     

Distribution and polarity of actin in the sensory hair cells of the chinchilla cochlea

Summary The distribution and polarity of actin in sensory hair cells of the chinchilla cochlea has been determined by decoration of actin filaments with myosin sub fragment S₁. Decorated actin filaments of the same polarity were present within the stereocilia above the cuticular plate. However the filaments in the rootlets and the thin filaments projecting laterally from the rootlets into the cuticular plate did not decorate with S₁. Decorated actin filaments were present within the cuticular plate, and near the plasma-membrane filaments of opposite polarity were observed. In the cross-striated region at the base of the cuticular plate of inner hair cells, decorated filaments were present in the dense bands of the cross-striations but the thin filaments perpendicular to the dense bands were not decorated. These results are discussed with respect to the two mechanisms that have been suggested for actin-myosin mediated movement of the stereocilia of inner-ear sensory cells.     

Postmortem changes in the actin-myosin interaction of rabbit skeletal muscle

Postmortem changes in the actin-myosin interaction were studied by determining the amount of thick and thin filaments dissociated by ATP. The amount of separated filaments was very small in myofibrils prepared from muscles in rigor, while it increased markedly during post-rigor storage of muscles. Electron microscopically, separated thick and thin filaments prepared from stored muscles were similar to freshly prepared ones and no signs of proteolytic degradation of either type of filament could be observed. A protein which was released from myofibrils (probably from Z discs) on Ca2+-treatment seemed to be most closely related to the post-rigor dissociation of thick filaments from thin filaments.     

Subsarcomeric distribution of calcium in demembranated fibers of rabbit psoas muscle.

Direct measurements were made of the Ca distribution within sarcomeres of glycerinated rabbit psoas muscle fibers in rigor using electron probe x-ray microanalysis. Both analogue raster analysis and digital x-ray imaging were used to quantitate the Ca distribution along thick and thin filaments as a function of the concentration of free Ca2+. Even when corrected for the estimated contribution of Ca bound to thick filaments, the Ca measured in the region of overlap between thick and thin filaments significantly exceeded the Ca in the I-band at subsaturating concentrations of free Ca2+. At saturating levels of free Ca2+, the excess Ca in the overlap region was diminished but still statistically significant. The data thus suggest that the formation of rigor linkages exerts multiple effects on the binding of Ca2+ to thin filaments in the overlap region by increasing the affinity of troponin C for Ca2+ and possibly by unmasking additional Ca2+ binding sites. The data also show that the cooperativity invested in the thin filaments is insufficient to permit the effects of rigor cross-bridge formation on Ca2+ binding to propagate far along the thin filaments into the I-band.     

Fine Structure of Cardiac Sarcomeres in the Black Widow Spider Latrodectus mactans

Fine structural characteristics of the cardiac muscle and its sarcomere organization in the black widow spider, Latrodectus mactans were examined using transmission electron microscopy. The arrangement of cardiac muscle fibers was quite similar to that of skeletal muscle fibers, but they branched off at the ends and formed multiple connections with adjacent cells. Each cell contained multiple myofibrils and an extensive dyadic sarcotubular system consisting of sarcoplasmic reticulum and T‐tubules. Thin and thick myofilaments were highly organized in regular repetitive arrays and formed contractile sarcomeres. Each repeating band unit of the sarcomere had three apparent striations, but the H‐zone and M‐lines were not prominent. Myofilaments were arranged into distinct sarcomeres defined by adjacent Z‐lines with relatively short lengths of 2.0 μm to 3.3 μm. Cross sections of the A‐band showed hexagon‐like arrangement of thick filaments, but the orbit of thin filaments around each thick filament was different from that seen in other vertebrates. Although each thick filament was surrounded by 12 thin filaments, the filament ratio of thin and thick myofilaments varied from 3:1 to 5:1 because thin filaments were shared by adjacent thick filaments.     

Purification of native myosin filaments from muscle

Analysis of the structure and function of native thick (myosin-containing) filaments of muscle has been hampered in the past by the difficulty of obtaining a pure preparation. We have developed a simple method for purifying native myosin filaments from muscle filament suspensions. The method involves severing thin (actin-containing) filaments into short segments using a Ca(2+)-insensitive fragment of gelsolin, followed by differential centrifugation to purify the thick filaments. By gel electrophoresis, the purified thick filaments show myosin heavy and light chains together with nonmyosin thick filament components. Contamination with actin is below 3.5%. Electron microscopy demonstrates intact thick filaments, with helical cross-bridge order preserved, and essentially complete removal of thin filaments. The method has been developed for striated muscles but can also be used in a modified form to remove contaminating thin filaments from native smooth muscle myofibrils. Such preparations should be useful for thick filament structural and biochemical studies.     

"Twitchin-actin linkage hypothesis" for the catch mechanism in molluscan muscles: evidence that twitchin interacts with myosin, myorod, and paramyosin core and affects properties of actomyosin

"Twitchin-actin linkage hypothesis" for the catch mechanism in molluscan smooth muscles postulates in vivo existence of twitchin links between thin and thick filaments that arise in a phosphorylation-dependent manner [N.S. Shelud'ko, G.G. Matusovskaya, T.V. Permyakova, O.S. Matusovsky, Arch. Biochem. Biophys. 432 (2004) 269-277]. In this paper, we proposed a scheme for a possible catch mechanism involving twitchin links and regulated thin filaments. The experimental evidence in support of the scheme is provided. It was found that twitchin can interact not only with mussel myosin and rabbit F-actin but also with the paramyosin core of thick filaments, myorod, mussel thin filaments, "natural" F-actin from mussel, and skeletal myosin from rabbit. No difference was revealed in binding of twitchin with mussel and rabbit myosin. The capability of twitchin to interact with all thick filament proteins suggests that putative twitchin links can be attached to any site of thick filaments. Addition of twitchin to a mixture of actin and paramyosin filaments, or to a mixture of Ca(2+)-regulated actin and myosin filaments under relaxing conditions caused in both cases similar changes in the optical properties of suspensions, indicating an interaction and aggregation of the filaments. The interaction of actin and myosin filaments in the presence of twitchin under relaxing conditions was not accompanied by an appreciable increase in the MgATPase activity. We suggest that in both cases aggregation of filaments was caused by formation of twitchin links between the filaments. We also demonstrate that native thin filaments from the catch muscle of the mussel Crenomytilus grayanus are Ca(2+)-regulated. Twitchin inhibits the ability of thin filaments to activate myosin MgATPase in the presence of Ca(2+). We suggest that twitchin inhibition of the actin-myosin interaction is due to twitchin-induced switching of the thin filaments to the inactive state.     

Elastic filaments and giant proteins in muscle.

Striated muscle is now known to contain a third major class of filaments, additional to the thick and thin filaments. The presence of such extra filaments has seemed likely for many years, but details of their location, structure, and composition are only now becoming clear. They are composed of massively large proteins and, in contrast to thick and thin filaments, they are elastic.     

Alterations in flightin phosphorylation inDrosophila flight muscles are associated with myofibrillar defects engendered by actin and myosin heavy-chain mutant alleles

Flightin is a 20-kD myofibrillar protein found in the stretch-activated flight muscles ofDrosophila melanogaster. Nine of the eleven isoelectric variants of flightin are generatedin vivo by multiple phosphorylations. The accumulation of these isoelectric variants is affected differently by mutations that eliminate thick filaments or thin filaments. Mutations in the myosin heavy-chain gene that prevent thick filament assembly block accumulation of all flightin variants except N1, the unphosphorylated precursor, which is present at much reduced levels. Mutations in the flight muscle-specific actin gene that block actin synthesis and prevent thin filament assembly disrupt the temporal regulation of flightin phosphorylation, resulting in premature phosphorylation and premature accumulation of flightin phosphovariants. Cellular fractionation of fibers that are devoid of thin filaments show that flightin remains associated with the thick filamentrich cytomatrix. These results suggest that flightin is a structural component of the thick filaments whose regulated phosphorylation is dependent upon the presence of thin filaments.This work was supported by National Science Foundation Grant IBN-9253045.     

Arrangement of filaments and cross-links in the bee flight muscle Z disk by image analysis of oblique sections

Information from oblique thin sections and from three-dimensional reconstructions of tilted, transverse thin sections (Cheng, N., and J. F. Deatherage. 1989. J. Cell Biol. 108:1761-1774) has been combined to determine the three-dimensional structure of the honeybee flight muscle Z disk at 70-A resolution. The overall symmetry and structure of the Z disk and its relationship to the rest of the myofibril have been determined by tracing filaments and connecting elements on electron images of oblique sections which have been enhanced by a local crystallographic averaging technique. In the three-dimensional structure, the connecting density between actin filaments can be described as five compact, crystallographically nonequivalent domains. Features C1 and C2 are located on the transverse twofold rotation axes in the central plane of the Z disk. They are associated with the sides of actin filaments of opposite polarity. Features C3, C4, and C5 are present in two symmetry-related sets which are located on opposite sides of the central plane. C3 and C5 are each associated with two filaments of opposite polarity, interacting with the side of one filament and the end of the other filament. C3 and C5 may be involved in stabilizing actin filament ends inside the Z disk. The location of the threefold symmetric connection C4, relative to the thick filament of the adjacent sarcomere, is determined and its possible relationship to the C filament is considered.     

THE FILAMENT LATTICE OF COCKROACH THORACIC MUSCLE

The fine structure of the tergo-coxal muscle of the cockroach, Leucophaea maderae, has been studied with the electron microscope. This muscle differs from some other types of insect flight muscles inasmuch as the ratio of thin to thick filaments is 4 instead of the characteristic 3. The cockroach flight muscle also differs from the cockroach femoral muscle in thin to thick filament ratios and diameters and in lengths of thick filaments. A comparison of these latter three parameters in a number of vertebrate and invertebrate muscles suggests in general that the diameters and lengths of the thick filaments and thin to thick filament ratios are related.     

Elastic filaments in skeletal muscle revealed by selective removal of thin filaments with plasma gelsolin

Muscle needs an elastic framework to maintain its mechanical stability. Removal of thin filaments in rabbit skeletal muscle with plasma gelsolin has revealed the essential features of elastic filaments. The selective removal of thin filaments was confirmed by staining with phalloidin-rhodamine for fluorescence microscopy, examination of arrowhead formation with myosin subfragment 1 by electron microscopy, and analysis by SDS-PAGE. Thin section electron microscopy revealed the elastic fine filaments (approximately 4 nm in diameter) connecting thick filaments and the Z line. After removal of thin filaments, both rigor stiffness and active tension generation were lost, but the resting tension remained. These observations indicate that the thin filament-free fibers maintain a framework composed of the serial connections of thick filaments, the elastic filaments, and the Z line, which gives passive elasticity to the contractile system of skeletal muscle. The resting tension that remained in the thin filament-free fibers was decreased by mild trypsin treatment. The only protein component that was digested in parallel with the decrease in the resting tension and the disappearance of the elastic filaments was alpha-connectin (also called titin 1), which was transformed from the alpha to the beta form (from titin 1 to 2, respectively). Thus, we conclude that the main protein component of the elastic filaments is alpha-connectin (titin 1).     

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.     

Electron Microscopic Studies of Skeletal and Cardiac Muscle of a Benthic Fish. I. Myofibrillar Structure in Resting and Contracted Muscle

SYNOPSIS. Electron microscopic studies are reported on glycerinatedskeletal and cardiac muscle of a benthic fish, Coryphaenoidesspecies. In white skeletal muscle, the sarcomeres have a restinglength of approximately 1.8 µ, with thick filaments 1.4µ and thin filaments 0.75 µ in length. These dimensionsare somewhat shorter than filament lengths of oilier vertebratemuscles, possibly due to the elfect of volume increase duringassembly of thick and thin filaments at high hydrostatic pressure.During ATP-induced contraction of Coryphaenoides muscle fromsarcomere lengths of 1.8 µ to 1.6 µ, there is acharacteristic interdigitation of thick and thin filaments,with decrease in I band length and no change in length of thickor thin filaments. However, in sarcomeres contracted to lengthsof 1.5 µ. to 1.2 µ, there is a slight shorteningof the A band, apparently due to shortening of thick filaments,that occurs despite the presence of residual I band in the samesarcomeres. There is no obvious crumpling or distortion of thickfilaments during contraction to sarcomere lengths as low as1.0 µ, but filament organization undergoes extensive disarrayat sarcomere lengths approaching 0.7 µ. Although effectsfrom heterogeneity of filament length cannot be excluded withcertainty, the present evidence does suggest that contractionot Coryphaenoides muscle from 1.6 µ to 1.0 µ sarcomerelengih is accompanied by shortening of thick filaments consequentto a structural change within the thick filament core.     

Muscle-like Arrays in a Fibroblast Line

SEVERAL investigators have speculated that the basis for all cellular contractile activity resides in a common molecular mechanism involving an interaction between actin and myosin^1–4. Thin filaments resembling the actin filaments of muscle have indeed been widely observed^3–5 and the recent demonstrations of heavy meromyosin binding to thin filaments^4–6 suggest that these ubiquitous filaments are, in fact, actin. Although muscle-like thick filaments have not been observed in non-muscle cells, myosin thick filaments have been reconstituted from blood platelet preparations¹. To our knowledge, however, no one has presented evidence for the natural occurrence of ordered arrays of thick and thin filaments in non-muscle cells.