Fusion of myocytes with larval muscle remnants during flight muscle development in the Colorado beetle

The transformation of the slow contracting larval m. obliquus lateralis caudalis II during metamorphosis into the asynchronous indirect flight muscle, m. obliquus lateralis dorsalis, in the Colorado beetle, Leptinotarsa decemlineata, was examined by electron microscopy. Particular attention was paid to the fate of the larval muscle fibres, the origin and behaviour of the myoblasts for flight muscle development and the change of the myofibrillar filament lattice of the larva into that of the adult. In the pre-pupal period, the larval muscles dedifferentiate and fragment. At pupation, the muscle fibres consist of cell fragments containing very few myofibrils. The sarcoplasmic reticulum and the transverse tubular system are greatly reduced. The number of myoblasts developed from satellite cells by mitosis increases considerably. They penetrate the muscle fibre and surround the cell fragments. The new fibres of the flight muscle develop from myocytes fused with the larval fragments. The larval basal lamina, surrounding the cell fragments and myoblasts, is present in pupae up to 1 day old. In pupae about 2.5 days old new myofibrils appear that have the adult filament lattice. The insect muscle transformation and the repair of vertebrate muscle after injury show striking resemblances.     

Direct x-ray observation of a single hexagonal myofilament lattice in native myofibrils of striated muscle

A striated muscle fiber consists of thousands of myofibrils with crystalline hexagonal myofilament lattices. Because the lattices are randomly oriented, the fiber gives rise to an equatorial x-ray diffraction pattern, which is essentially a rotary-averaged "powder diffraction," carrying only information about the distance between the lattice planes. We were able to record an x-ray diffraction pattern from a single myofilament lattice, very likely originating from a single myofibril from the flight muscle of a bumblebee, by orienting the incident x-ray microbeam along the myofibrillar axis (end-on diffraction). The pattern consisted of a number of hexagonally symmetrical diffraction spots whose originating lattice planes were readily identified. This also held true for some of the weak higher order reflections. The spot-like appearance of reflections implies that the lattice order is extremely well maintained for a distance of millimeters, covering up to a thousand of approximately 2.5-microm-long sarcomeres connected in series. The results open the possibility of applying the x-ray microdiffraction technique to study many other micrometer-sized assemblies of functional biomolecules in the cell.     

Evolution of long-range myofibrillar crystallinity in insect flight muscle as examined by X-ray cryomicrodiffraction

Insect flight muscle is known for its crystal-quality regularity of contractile protein arrangement within a sarcomere. We have previously shown by X-ray microdiffraction that the crystal-quality regularity in bumble-bee flight muscle is not confined within a sarcomere, but extends over the entire length of a myofibril (>1000 sarcomeres connected in series). Because of this, the whole myofibril may be regarded as a millimetre-long, natural single protein crystal. Using bright X-ray beams from a synchrotron radiation source, we examined how this long-range crystallinity has evolved among winged insects. We analysed >4600 microdiffraction patterns of quick-frozen myofibrils from 50 insect species, covering all the major winged insect orders. The results show that the occurrence of such long-range crystallinity largely coincides with insect orders with asynchronous muscle operation. However, a few of the more skilled fliers among lower-order insects apparently have developed various degrees of structural regularity, suggesting that the demand for skillful flight has driven the lattice structure towards increased regularity.     

Flight initiations in Drosophila melanogaster are mediated by several distinct motor patterns

We have monitored the patterns of activation of five muscles during flight initiation of Drosophila melanogaster: the tergotrochanteral muscle (a mesothoracic leg extensor), dorsal longitudinal muscles #3, #4 and #6 (wing depressors), and dorsal ventral muscle #Ic (a wing elevator). Stimulation of a pair of large descending interneurons, the giant fibers, activates these muscles in a stereotypic pattern and is thought to evoke escape flight initiation. To investigate the role of the giant fibers in coordinating flight initiation, we have compared the patterns of muscle activation evoked by giant fiber stimulation with those during flight initiations executed voluntarily and evoked by visual and olfactory stimuli. Visually elicited flight initiations exhibit patterns of muscle activation indistinguishable from those evoked by giant fiber stimulation. Olfactory-induced flight initiations exhibit patterns of muscle activation similar to those during voluntary flight initiations. Yet only some benzaldehyde-induced and voluntary flight initiations exhibit patterns of muscle activation similar to those evoked by giant fiber stimulation. These results indicate that visually elicited flight initiations are coordinated by the giant fiber circuit. By contrast, the giant fiber circuit alone cannot account for the patterns of muscle activation observed during the majority of olfactory-induced and voluntary flight initiations.Abbreviations DLM/DLMn dorsal longitudinal muscle/motor neuron - DVM/DVMn dorsal ventral muscle/motor neuron - GF(s) giant fiber interneuron (s) - PSI peripherally synapsing interneuron - TTM/TTMn tergotrochanteral muscle/motor neuron     

The myosin filament superlattice in the flight muscles of flies: A-band lattice optimisation for stretch-activation?

Low-angle X-ray diffraction patterns from relaxed fruitfly (Drosophila) flight muscle recorded on the BioCat beamline at the Argonne Advanced Photon Source (APS) show many features similar to such patterns from the "classic" insect flight muscle in Lethocerus, the giant water bug, but there is a characteristically different pattern of sampling of the myosin filament layer-lines, which indicates the presence of a superlattice of myosin filaments in the Drosophila A-band. We show from analysis of the structure factor for this lattice that the sampling pattern is exactly as expected if adjacent four-stranded myosin filaments, of repeat 116 nm, are axially shifted in the hexagonal A-band lattice by one-third of the 14.5 nm axial spacing between crowns of myosin heads. In addition, electron micrographs of Drosophila and other flies (e.g. the house fly (Musca) and the flesh fly (Sarcophaga)) combined with image processing confirm that the same A-band superlattice occurs in all of these flies; it may be a general property of the Diptera. The different A-band organisation in flies compared with Lethocerus, which operates at a much lower wing beat frequency (approximately 30 Hz) and requires a warm-up period, may be a way of optimising the myosin and actin filament geometry needed both for stretch activation at the higher wing beat frequencies (50 Hz to 1000 Hz) of flies and their need for a rapid escape response.     

Atypical ‘fibrillar’ flight muscle in strepsiptera

The fine structure of the principal and ancillary metathoracic flight muscle fibres in the adult male of a strepsipteran, Elenchus tenuicornis, is described. Power-producing dorsal longitudinal and dorso-ventral flight muscles show features consistent with myoneural asynchrony: myofibrils are large and discrete and are separated by large closely packed mitochondria; the sarcoplasmic reticulum is very reduced but engages with T-system membranes in dyads at the mid-sarcomere H-band level. With respect to other asynchronous insect flight muscles, the fibres of Elenchus are anomalous (i) in the small fibre diameter, (ii) in the variable contour of the myofibrils and (iii) in the absence of tracheolar invagination. The functional significance of these structural features is discussed. Ancillary metathoracic muscles are structurally comparable with other synchronous fibres in possessing an extensive SR compartment. Structural evidence for asynchrony in the flight mechanism of Strepsiptera is considered in the context of the evolution of this mechanism throughout the insect Orders.     

A comparative study of motor patterns during pre-flight warm-up in hawkmoths

Summary Temporal patterns of activation of flight muscles were recorded by means of wires placed extracellularly in thoracic muscles. In the five species of hawkmoths studied, wingstrokes of small amplitude were produced during a preflight warm-up by synchronous contractions of certain groups of muscles which are antagonists in flight. The main depressor muscle, the dorsal longitudinal, was excited in synchrony with some or all of the indirect elevator muscles. Three direct muscles, the subalar, basalar and third axillary muscles, were usually excited out of phase with the dorsal longitudinal muscle. However, details of the motor pattern varied from species to species. During fixed flight phase changes comparable in magnitude to those which occur during the transition from warm-up to flight were observed in Manduca sexta and Smerinthus cerisyi. The results (summarized in Table 2) suggest that a variety of warm-up patterns evolved within the Sphingidae as modifications of a common mechanism generating flight motor patterns.I thank Dr. Harry Lange for assistance in the initial collecting of Manduca sexta and for identifying specimens of this species.     

Ultrastructure of muscle fibres in head and axial muscles of the perch (Perca fluviatilis L.)

Summary White, pink, red and deep red fibres, selected from a head muscle and from axial muscles of the perch, show significant differences in actin filament length, Z line thickness, Z line lattice space, myofibril girth, the percentages volume occupied by T system and terminal cisternae of the SR, and in the degree of T system SR contact per sarcomere. In both muscles the degree of T system SR contact decreases in the order: white, pink, red, deep red, which suggests a decrease of contraction velocity in the same order.The position of the T system (at the Z line or at the AI junction) is related to the actin filament length. The actin filaments in the red fibres are appreciably longer than in the white, which suggests that the sarcomeres of the red fibres have a broader length-tension curve. The Z line thickness is positively correlated with the actin filament length and, in the white and the red fibres, negatively with the degree of sarcomere shortening. Thicker Z lines are suggested to allow greater sarcomere sizes (length or girth).The percentage volume occupied by mitochondria varies independently of the extent of membrane systems.The ultrastructural characteristics of the fibre types are in agreement with the functional roles as reported in literature.     

Myosin heavy chain isoforms regulate muscle function but not myofibril assembly.

Myosin heavy chain (MHC) is the motor protein of muscle thick filaments. Most organisms produce many muscle MHC isoforms with temporally and spatially regulated expression patterns. This suggests that isoforms of MHC have different characteristics necessary for defining specific muscle properties. The single Drosophila muscle Mhc gene yields various isoforms as a result of alternative RNA splicing. To determine whether this multiplicity of MHC isoforms is critical to myofibril assembly and function, we introduced a gene encoding only an embryonic MHC into Drosophila melanogaster. The embryonic transgene acts in a dominant antimorphic manner to disrupt flight muscle function. The transgene was genetically crossed into an MHC null background. Unexpectedly, transformed flies expressing only the embryonic isoform are viable. Adult muscles containing embryonic MHC assemble normally, indicating that the isoform of MHC does not determine the dramatic ultrastructural variation among different muscle types. However, transformed flies are flightless and show reduced jumping and mating ability. Their indirect flight muscle myofibrils progressively deteriorate. Our data show that the proper MHC isoform is critical for specialized muscle function and myofibril stability.     

Mechanism of myofibril growth and proliferation in fish muscle.

The mechanisms of myofibril growth proliferation were investigated in the red and white muscles of fish. In both types of muscle the ratio of lattice filament spacings between the Z disk and M line was found to be greater than that required for perfect transformation of a square into a hexagonal lattice. This mismatch was considered to result in the thin filaments being pulled obliquely instead of at right angles to the Z disk. The angle of pull of the thin filaments was measured in longitudinal sections. The splitting process was found to decrease the degree of pull. Splitting was also observed in transverse sections of the peripheral myofibrils. In both red and white fibres these myofibrils were found to commence splitting when they reached a size of approximately 1-2 mum diameter. Evidence from ultrastructural and autoradiographical studies suggested that growth of the myofibrils within the fibres is centrifugal. The outermost myofibrils appear to be the ones which are being built up and which split. The data indicated that in fish muscle a considerable number of filaments may be added to the daughter regions whilst splitting of the myofibril is still continuing.     

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.     

Anatomy and histochemistry of spread‐wing posture in birds. 4. Eagles soar with fast,not slow muscle fibres

Slow fibres are typically characterized as functioning in avian postural behaviours such as soaring flight and are described for a number of elite soarers such as vultures, pelicans and albatrosses. Golden Eagles and Bald Eagles also display soaring behaviour, and we examined their flight muscles for the presence of slow fibres. Surprisingly, eagles lack a deep layer to the pectoralis found in other soaring species. Additionally, the pectoralis as well as other shoulder muscles had few to no slow muscle fibres. The lack of functionally meaningful numbers of slow muscle fibres in eagle flight muscles indicates that they must rely on fast fibres for posture; these can function in that role due to their high aerobic capacity and also perhaps a ‘tuning’ of muscle contraction frequency to function more efficiently at isometric contractions.     

Protein synthesis during flight muscle development in normal and wupB indirect flight muscles of Drosophila melanogaster

We have looked at protein synthesis in Drosophila pupae during normal and abnormal development of indirect flight muscle. Abnormal development was followed in the dominant flightless mutant wupB isolated by Hotta and Benzer (Genetic Mechanisms of Development, pp. 129–167. Academic Press, New York, 1972). The mutant muscles in adult wupB flies have abnormal morphology and disorganized myofibrils. Protein synthesis in developing muscle was followed on SDS-polyacrylamide gels. During early stages of development (55–60 h) protein synthesis patterns are similar in the mutant and the wild-type. However, at 61 h, the mutant shows a transient increase in synthesis of the 68 and 70 kDa heat shock proteins. This is followed at about 70 h by a divergence of the patterns of synthesis of other proteins seen in the mutant and wild type. These results suggest that induction of heat shock protein synthesis is an early event in abnormal morphogenesis in this mutant.     

"Crystalline" myosin cross-bridge array in relaxed bony fish muscle. Low-angle x-ray diffraction from plaice fin muscle and its interpretation

Detailed structural analysis of muscles normally used to study myosin cross-bridge behavior (e.g., frog sartorius muscle, insect flight muscle) is extremely difficult due to the statistical disorder inherent in their myosin filament arrays. Bony fish muscle is different from all other muscle types in having a myosin filament (A-Band) array with good three-dimensional (crystalline) regularity that is coherent right across each myofibril. Rigorous structure analysis is feasible with fish muscle. We show that low-angle x-ray diffraction patterns from plaice fin muscle contain characteristic vertebrate layer lines at orders of 429 (+/- 0.2) A, that these layer lines are well sampled by row-lines from a simple hexagonal lattice of a-spacing 470 (+/- 2.0) A at rest length and that there are meridional reflections, due to axial perturbations of the basic helix of myosin heads, similar in position to those from frog muscle but differing in relative intensities. Clear trends based on modeling to a resolution of 130 A of the observed intensities in the low angle x-ray diffraction pattern from relaxed plaice fin muscle suggest that: (a) the pattern out to 130 A is more sensitive to the distribution of the two heads than it is to details of the head shape, (b) both heads in one myosin molecule probably tilt axially in the same direction by approximately 20-40 degrees relative to a normal to the thick filament backbone, (c) the center of mass of the heads is at 145 to 160 A radius, and (d) the two heads form a compact structure by lying closely adjacent to each other and almost parallel. Little rotational disorder of the heads can occur. Because of its crystallinity, bony fish muscle provides a uniquely useful structural probe of myosin cross-bridge behavior in other muscle states such as rigor and active contraction.     

Requirements of Kettin, a giant muscle protein highly conserved in overall structure in evolution, for normal muscle function, viability, and flight activity of Drosophila

Kettin is a giant muscle protein originally identified in insect flight muscle Z-discs. Here, we determined the entire nucleotide sequence of Drosophila melanogaster kettin, deduced the amino acid sequence of its protein product (540 kD) along with that of the Caenorhabditis elegans counterpart, and found that the overall primary structure of Kettin has been highly conserved in evolution. The main body of Drosophila Kettin consists of 35 immunoglobulin C2 domains separated by spacers. The central two thirds of spacers are constant in length and share in common two conserved motifs, putative actin binding sites. Neither fibronectin type III nor kinase domains were found. Kettin is present at the Z-disc in several muscle types. Genetic analysis showed that kettin is essential for the formation and maintenance of normal sarcomere structure of muscles and muscle tendons. Accordingly, embryos lacking kettin activity cannot hatch nor can adult flies heterozygous for the kettin mutation fly.     

X-ray diffraction testing for weak-binding crossbridges in relaxed bony fish muscle fibres at low ionic strength

Equatorial X-ray diffraction patterns from single skinned fibres from bony fish muscle (turbot) were obtained with the fibres at 6 degrees C bathed in relaxing solutions of 170 down to 26 mM ionic strength. Diffraction patterns from rigor fibres were also obtained as controls. Unlike fibres from rabbit muscle, which show very clear evidence of substantial crossbridge formation at low ionic strength in what is mechanically a rapid equilibrium ("weak-binding") state (Brenner et al., 1982), diffraction patterns from bony fish fibres showed only a small change in relative peak intensities at low ionic strength (26 mM) compared with normal (170 mM) ionic strength. However, there was a slight ordering of the filament lattice at low ionic strength. The specimen temperature used (about 6 degrees C) was not far from the normal physiological temperature of the fish. Likewise, only a small change was seen by Xu et al. (1987) in patterns from frog fibres at low ionic strength at 2 to 6 degrees C. (Rabbit fibres previously studied, where large changes were seen at temperatures of 5 to 20 degrees C, were about 17 to 32 degrees C below physiological.) The I11/I10 ratio for fish fibres at 26 mM ionic strength was actually lower than that for rabbit even at normal ionic strength. This may be associated with an intrinsic structural difference between these muscles or alternatively with the disordering of the crossbridge helix in rabbit muscle found at low temperature by Wray (1987), and could support the view that rabbit fibres at 5 degrees C and normal ionic strength may already have a significant population of weak-binding crossbridges.     

The Myofibrillar Protein,Projectin, is Highly Conserved Across Insect Evolution Except for Its PEVK Domain

All striated muscles respond to stretch by a delayed increase in tension. This physiological response, known as stretch activation, is, however, predominantly found in vertebrate cardiac muscle and insect asynchronous flight muscles. Stretch activation relies on an elastic third filament system composed of giant proteins known as titin in vertebrates or kettin and projectin in insects. The projectin insect protein functions jointly as a “scaffold and ruler” system during myofibril assembly and as an elastic protein during stretch activation. An evolutionary analysis of the projectin molecule could potentially provide insight into how distinct protein regions may have evolved in response to different evolutionary constraints. We mined candidate genes in representative insect species from Hemiptera to Diptera, from published and novel genome sequence data, and carried out a detailed molecular and phylogenetic analysis. The general domain organization of projectin is highly conserved, as are the protein sequences of its two repeated regions—the immunoglobulin type C and fibronectin type III domains. The conservation in structure and sequence is consistent with the proposed function of projectin as a scaffold and ruler. In contrast, the amino acid sequences of the elastic PEVK domains are noticeably divergent, although their length and overall unusual amino acid makeup are conserved. These patterns suggest that the PEVK region working as an unstructured domain can still maintain its dynamic, and even its three-dimensional, properties, without the need for strict amino acid conservation. Phylogenetic analysis of the projectin proteins also supports a reclassification of the Hymenoptera in relation to Diptera and Coleoptera. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Alterations of crossbridge angle induced by beta, gamma-imido-adenosine-triphosphate. Electron microscope and optical diffraction studies on myofibrillar fragments of abdominal muscles of the crayfish Orconectes limosus.

The molecular basis of muscle contraction is thought to consist of cyclic movements of parts of the myosin molecules (crossbridges). Unitl now different states of the proposed crossbridge cycle could be stablilized and demonstrated by electron microscopy only in the case of highly specialized insect flight muscles. In this paper evidence is presented that it is also possible to induce crossbridge positions corresponding to the rigor [16] and the pseudorelaxed state [3] in non-insect muscles. Homogenization of myofibrils of the abdominal flexors of the crayfish Orconectes limosus in rigor or AMP.PNP-containing solutions brings about two different crossbridge patterns: The formation of crossbridges attached to the actin filaments in a mainly acute (rigor) or in a mainly perpendicular angle (pseudo-relaxed). Optical diffraction patterns taken from electron micrographs of sarcomere fragments are likewise compatible with those taken from sarcomeres of insect flight muscles fixed in comparable conditions [2,3].