THE FINE STRUCTURE OF THE VENTRAL INTERSEGMENTAL ABDOMINAL MUSCLES OF THE INSECT RHODNIUS PROLIXUS DURING THE MOLTING CYCLE : I. Muscle Structure at Molting

Rhodnius prolixus, a South American insect, molts five times in its development to an adult after emerging from the egg. Each molting cycle is triggered with a blood-meal. The ventral intersegmental abdominal muscles of Rhodnius develop during each molting cycle and are functional at molting. The fine structure of these fully developed muscles from fourth stage larval insects is studied. They have the characteristic structure of slow muscles. They have multiple motor nerve endings, and the myofibrils are poorly defined in cross-section. Longitudinal sections show long sarcomeres (8–10 µ), irregular Z-lines, and no apparent H zones. No M line is seen. Transverse sections through the A-band region show that each hexagonally arranged thick filament is surrounded by 12 thin filaments. Two thin filaments are shared by two neighboring thick filaments. The ratio of thin to thick filaments is 6:1. This structure is related to that found in vertebrate skeletal muscle and insect flight muscle.     

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.     

Thin filaments are not of uniform length in rat skeletal muscle

The variation in thin filament length was investigated in slow and fast muscle from adult and neonatal rats. Soleus (slow) muscle from adult, 3- , 7-, and 9-d-old rats, and extensor digitorum longus (EDL; fast) muscle from adult rats were serially cross-sectioned. The number of thin filaments per 0.06 microns2 (TF#) was counted for individual myofibrils followed from the H zone of one sarcomere, through the I-Z-I region, to the H zone of an adjacent sarcomere TF# was pooled by distance from the Z band or AI junction. In both adult muscles, thin filament length varied from 0.18 to 1.20 microns, with approximately 25% of the thin filaments less than 0.7 microns in length. In 7- and 9- d soleus, thin filament length ranged from 0.18 to 1.08 microns; except for the longest (0.18 to 1.20 microns) filaments, the distribution of thin filament lengths was similar to that in adult muscle. In 3-d soleus, thin filament length was more uniform, with less than 5% of the filaments shorter than 0.7 microns. In all neonatal muscles, there were approximately 15% fewer thin filaments per unit area as compared to adult muscles. We conclude: (a) In rat skeletal muscle, thin filaments are not of uniform length, ranging in length from 0.18 to 1.20 microns. (b) There may be two stages of thin filament assembly in neonatal muscle: between 3 and 7 d when short thin filaments may be preferentially or synthesized or inserted near the Z-band, and between 9 d and adult when thin filaments of all lengths may be synthesized or inserted into the myofibril.     

Paramyosin content and thick filament structure in insect muscles

Summary The myosin filaments of the flight muscles of the locust Locusta migratoria, the cockchafer Melolontha melolontha and the femur muscles of L. migratoria have solid centers. Those of the flight muscles of the housefly Musca domestica and Drosophila melanogaster are tubular. Electron micrographs of myofibrils of the fleshfly Phormia terrae-novae contain both filament types within one sarcomere and suggest the existence of 4 cross-bridges per crown.Estimates of the ratios of myosin to paramyosin and of myosin to actin on sodium dodecyl sulphate-polyacrylamide gels yielded paramyosin contents of 9% of the thick filament mass for the solid and 2.6% for the tubular filaments (3.8% for P. terrae-novae). Based on the myosinactin ratios up to 6 myosin dimers per crown could be calculated.The molar ratio of actin to arthrin on SDS gels was found to be 3.37 for native and extracted myofibrils of flight muscles from P. terrae-novae. Arthrin is also present in isolated actin filaments suggesting that it is localized in or on the thin filaments. If we assume that it is constituent part of the helices of the thin filaments the number of myosin dimers per crown can be diminished to 4.5, considerably closer to the values obtained by evaluation of electron micrographs.Dedicated to Prof. Dr. Bernhard Rensch on his 85^th birthday     

Electron microscopy and three-dimensional reconstruction of native thin filaments reveal species-specific differences in regulatory strand densities

Throughout the animal kingdom striated muscle contraction is regulated by the thin filament troponin-tropomyosin complex. Homologous regulatory components are shared among vertebrate and arthropod muscles; however, unique protein extensions and/or components characterize the latter. The Troponin T (TnT) isoforms of Drosophila indirect flight and tarantula femur muscle for example contain distinct C-terminal extensions and are ∼20% larger overall than their vertebrate counterpart. Using electron microscopy and three-dimensional helical reconstruction of native Drosophila, tarantula and frog muscle thin filaments we have identified species-specific differences in tropomyosin regulatory strand densities. The strands on the arthropod thin filaments were significantly larger in diameter than those from vertebrates, although not significantly different from each other. These findings reflect differences in the regulatory troponin-tropomyosin complex, which are likely due to the larger TnT molecules aligning and extending along much of the tropomyosin strands’ length. Such an arrangement potentially alters the physical properties of the regulatory strands and may help establish contractile characteristics unique to certain arthropod muscles.     

The Regulation of Catch in Molluscan Muscle

Molluscan catch muscles are smooth muscles. As with mammalian smooth muscles, there is no transverse ordering of filaments or dense bodies. In contrast to mammalian smooth muscles, two size ranges of filaments are present. The thick filaments are long as well as large in diameter and contain paramyosin. The thin filaments contain actin and appear to run into and join the dense bodies. Vesicles are present which may be part of a sarcoplasmic reticulum. Neural activation of contraction in Mytilus muscle is similar to that observed in mammalian smooth muscles, and in some respects to frog striated muscle. The relaxing nerves, which reduce catch, are unique to catch muscles. 5-Hydroxytryptamine, which appears to mediate relaxation, specifically blocks catch tension but increases the ability of the muscle to fire spikes. It is speculated that Mytilus muscle actomyosin is activated by a Ca⁺⁺-releasing mechanism, and that 5-hydroxytryptamine may reduce catch and increase excitability by influencing the rate of removal of intracellular free Ca⁺⁺.     

Flightin, a novel myofibrillar protein of Drosophila stretch-activated muscles

The indirect flight muscles of Drosophila are adapted for rapid oscillatory movements which depend on properties of the contractile apparatus itself. Flight muscles are stretch activated and the frequency of contraction in these muscles is independent of the rate of nerve impulses. Little is known about the molecular basis of these adaptations. We now report a novel protein that is found only in flight muscles and has, therefore, been named flightin. Although we detect only one gene (in polytene region 76D) for flightin, this protein has several isoforms (relative gel mobilities, 27-30 kD; pIs, 4.6-6.0). These isoforms appear to be created by posttranslational modifications. A subset of these isoforms is absent in newly emerged adults but appears when the adult develops the ability to fly. In intact muscles flightin is associated with the A band of the sarcomere, where evidence suggests it interacts with the myosin filaments. Computer database searches do not reveal extensive similarity to any known protein. However, the NH2-terminal 12 residues show similarity to the NH2- terminal sequence of actin, a region that interacts with myosin. These features suggest a role for flightin in the regulation of contraction, possibly by modulating actin-myosin interaction.     

Muscular contraction and cytoplasmic streaming: A new general hypothesis

Several experiments point out that some crossbridges remain attached to the thin filaments at rest. It is assumed, in this paper, that these cross-bridges exert mechanical tractions on the thin filaments, directed from the thin to the thick filaments. When contraction is triggered off, a conformational change of the attached crossbridges is induced by the chemical energy released from ATP splitting. This conformational change leads to the reduction of the mechanical tensions. The electrostatic repulsive forces between the filaments become therefore automatically preponderant. This phenomenon induces a sideways expansion of the filament lattice and, taking into account the elasticity of muscle, a contraction in the direction of the filaments. This model accounts for the most important physiological and thermodynamical properties of muscle (tension-length curves, responses to quick stretch and quick release, Fenn effect, Hill's relation, behaviour of skinned fibres). It is directly applicable to all kinds of muscles and to cytoplasmic streaming, provided only actin, but not necessarily myosin, filaments are present in the cell.     

Structure of multinucleated smooth muscle cells of the ascidian Halocynthia roretzi

Summary Cells isolated from ascidian smooth muscle were about 1.5–2 mm in length. Each contained 20–40 nucle in proportion to cell length. The cytoplasm was characterized by the presence of an enormous quantity of glycogen particles, tubular elements of sarcoplasmic reticulum coupled to the cell membrane, and conspicuous contractile elements. Thick and thin filaments had diameters of about 14–16 nm and 6–7 nm, respectively. The population density of the thick filaments was much higher (mean 270/m² filament area) than in vertebrate smooth muscles. The ratio of thick to thin filaments was about 16. All the thick filaments were surrounded by a single row of 5–9 thin filaments forming a rosette, and cross-bridges with periodicities of 14.5 and 29 nm were found between them. The contractile apparatus consisted of numerous myofibrils which were arranged nearly along the cell axis and were separated from each other by a network of 10-nm filaments. The myofibrils further consisted of many irregularly arranged sarcomerelike structures, each of which was comprised of a small group of thick and thin filaments with attached dense bodies.     

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)     

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.     

Muscle abnormalities in Drosophila melanogaster heldup mutants are caused by missing or aberrant troponin-I isoforms

We have investigated the molecular bases of muscle abnormalities in four Drosophila melanogaster heldup mutants. We find that the heldup gene encodes troponin-I, one of the principal regulatory proteins associated with skeletal muscle thin filaments. heldup3, heldup4, and heldup5 mutants, all of which have grossly abnormal flight muscle myofibrils, lack mRNAs encoding one or more troponin-I isoforms. In contrast, heldup2, an especially interesting mutant wherein flight muscles are atrophic, synthesizes the complete mRNA complement. By sequencing mutant troponin-I cDNAs we demonstrate that the molecular basis for muscle degeneration in heldup2 is conversion of an invariant alanine residue to valine. We finally show that degeneration of heldup2 thin filament/Z-disc networks can be prevented by eliminating thick filaments from flight muscles using a null allele of the sarcomeric myosin heavy chain gene. This latter observation suggests that actomyosin interactions exacerbate the structural or functional defect resulting from the troponin-I mutation.     

Muscle anatomy is a primary determinant of muscle relaxation dynamics in the lobster (<Emphasis Type="Italic">Panulirus interruptus</Emphasis>) stomatogastric system

We stained sarcomere thin filaments with fluorescently labeled phalloidin, measured sarcomere and muscle length, and calculated sarcomere number in pyloric and gastric mill muscles. A wide range of sarcomere lengths (3.25–12.29 μm), muscle lengths (5.9–21.1 mm), and sarcomere numbers (648–3,036) were observed. Sarcomere number differences occurred both because of changes in sarcomere length and muscle length, and sarcomere and muscle length varied independently. This independence, the wide range of sarcomere numbers present, and the muscles being all ‘slow’, graded muscles allowed us to use these data to test Huxley and Neidergerke’s (1954) hypothesis that muscle dynamics depend on sarcomere number. The time constants of exponential fits to contraction relaxations were used to measure muscle dynamics, and comparison of theoretical predictions and experimental results quantitatively confirm the predicted dependence. The differing dynamics of the various pyloric muscles are likely functionally important, and the dependence of muscle dynamics on sarcomere number implies that sarcomere number is likely closely regulated in these muscles. The stomatogastric system may thus be an excellent model system for studying the mechanisms regulating muscle sarcomere number.     

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.     

Mutating the Converter-Relay Interface of Drosophila Myosin Perturbs ATPase Activity, Actin Motility, Myofibril Stability and Flight Ability

We used an integrative approach to probe the significance of the interaction between the relay loop and converter domain of the myosin molecular motor from Drosophila melanogaster indirect flight muscle. During the myosin mechanochemical cycle, ATP-induced twisting of the relay loop is hypothesized to reposition the converter, resulting in cocking of the contiguous lever arm into the pre-power stroke configuration. The subsequent movement of the lever arm through its power stroke generates muscle contraction by causing myosin heads to pull on actin filaments. We generated a transgenic line expressing myosin with a mutation in the converter domain (R759E) at a site of relay loop interaction. Molecular modeling suggests that the interface between the relay loop and converter domain of R759E myosin would be significantly disrupted during the mechanochemical cycle. The mutation depressed calcium as well as basal and actin-activated MgATPase (V_max) by ∼ 60% compared to wild-type myosin, but there is no change in apparent actin affinity (K_m). While ATP or AMP-PNP (adenylyl-imidodiphosphate) binding to wild-type myosin subfragment-1 enhanced tryptophan fluorescence by ∼ 15% or ∼ 8%, respectively, enhancement does not occur in the mutant. This suggests that the mutation reduces lever arm movement. The mutation decreases in vitro motility of actin filaments by ∼ 35%. Mutant pupal indirect flight muscles display normal myofibril assembly, myofibril shape, and double-hexagonal arrangement of thick and thin filaments. Two-day-old fibers have occasional “cracking” of the crystal-like array of myofilaments. Fibers from 1-week-old adults show more severe cracking and frayed myofibrils with some disruption of the myofilament lattice. Flight ability is reduced in 2-day-old flies compared to wild-type controls, with no upward mobility but some horizontal flight. In 1-week-old adults, flight capability is lost. Thus, altered myosin function permits myofibril assembly, but results in a progressive disruption of the myofilament lattice and flight ability. We conclude that R759 in the myosin converter domain is essential for normal ATPase activity, in vitro motility and locomotion. Our results provide the first mutational evidence that intramolecular signaling between the relay loop and converter domain is critical for myosin function both in vitro and in muscle.     

The ultrastructure of the striated muscles of Derocheilocaris typica (Mystacocarida,Crustacea)

The striated muscles of Derocheilocaris typica consist of mononucleated cells, each containing one filament bundle. Large muscles consist of two or more cells adjacent to each other. The mitochondria line up along the filament bundle on one side. The nucleus is situated in the mitochondrial row and has a small cytoplasmic area around it filled with glycogen. The sarcomeres are between 3 and 6 μm long. The Z-line and H band are present. Six thin filaments surround one thick filament. All muscles belong to the phasic type. The tubular system emanates from the ends of the muscle cell and penetrates the whole cell. The tubules are formed as cisterns, which also open at the cell membrane at the level of the I bands. They have sarcoplasmic cisterns on both sides forming a continuous triad system. Partially transformed epidermal cells mediate muscle insertions on the cuticle. Tendons are formed with the transformed epidermal cells being supplemented by fibroblasts forming collagen fibers. Dorsal and ventral abdominal muscles are innervated from the dorso-lateral nerve arising from the nerve chain. Each muscle cell receives one axon, which forms one synapse on the mitochondrial-free side of the muscles. Axons form terminal spines, which make axo-axonal synapses.     

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.     

X-ray diffraction evidence for the extensibility of actin and myosin filaments during muscle contraction.

To clarify the extensibility of thin actin and thick myosin filaments in muscle, we examined the spacings of actin and myosin filament-based reflections in x-ray diffraction patterns at high resolution during isometric contraction of frog skeletal muscles and steady lengthening of the active muscles using synchrotron radiation as an intense x-ray source and a storage phosphor plate as a high sensitivity, high resolution area detector. Spacing of the actin meridional reflection at approximately 1/2.7 nm-1, which corresponds to the axial rise per actin subunit in the thin filament, increased about 0.25% during isometric contraction of muscles at full overlap length of thick and thin filaments. The changes in muscles stretched to approximately half overlap of the filaments, when they were scaled linearly up to the full isometric tension, gave an increase of approximately 0.3%. Conversely, the spacing decreased by approximately 0.1% upon activation of muscles at nonoverlap length. Slow stretching of a contracting muscle increased tension and increased this spacing over the isometric contraction value. Scaled up to a 100% tension increase, this corresponds to a approximately 0.26% additional change, consistent with that of the initial isometric contraction. Taken together, the extensibility of the actin filament amounts to 3-4 nm of elongation when a muscle switches from relaxation to maximum isometric contraction. Axial spacings of the layer-line reflections at approximately 1/5.1 nm-1 and approximately 1/5.9 nm-1 corresponding to the pitches of the right- and left-handed genetic helices of the actin filament, showed similar changes to that of the meridional reflection during isometric contraction of muscles at full overlap. The spacing changes of these reflections, which also depend on the mechanical load on the muscle, indicate that elongation is accompanied by slight changes of the actin helical structure possibly because of the axial force exerted by the actomyosin cross-bridges. Additional small spacing changes of the myosin meridional reflections during length changes applied to contracting muscles represented an increase of approximately 0.26% (scaled up to a 100% tension increase) in the myosin periodicity, suggesting that such spacing changes correspond to a tension-related extension of the myosin filaments. Elongation of the myosin filament backbone amounts to approximately 2.1 nm per half sarcomere. The results indicate that a large part (approximately 70%) of the sarcomere compliance of an active muscle is caused by the extensibility of the actin and myosin filaments; 42% of the compliance resides in the actin filaments, and 27% of it is in the myosin filaments.     

Myofilament proteins in the synchronous flight muscles of Manduca sexta show both similarities and differences to Drosophila melanogaster

Insect flight muscles have been classified as either synchronous or asynchronous based on the coupling between excitation and contraction. In the moth Manduca sexta, the flight muscles are synchronous and do not display stretch activation, which is a property of asynchronous muscles. We annotated the M. sexta genes encoding the major myofibrillar proteins and analyzed their isoform pattern and expression. Comparison with the homologous genes in Drosophila melanogaster indicates both difference and similarities. For proteins such as myosin heavy chain, tropomyosin, and troponin I the availability and number of potential variants generated by alternative spicing is mostly conserved between the two insects. The exon usage associated with flight muscles indicates that some exon sets are similarly used in the two insects, whereas others diverge. For actin the number of individual genes is different and there is no evidence for a flight muscle specific isoform. In contrast for troponin C, the number of genes is similar, as well as the isoform composition in flight muscles despite the different calcium regulation. Both troponin I and tropomyosin can include COOH-terminal hydrophobic extensions similar to tropomyosinH and troponinH found in D. melanogaster and the honeybee respectively.