Journal articles: 'Drosophila Indirect Flight Muscles'

1

Fernandes, J., and K. VijayRaghavan. "The development of indirect flight muscle innervation in Drosophila melanogaster." Development 118, no. 1 (May 1, 1993): 215–27. http://dx.doi.org/10.1242/dev.118.1.215.

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We have examined the development of innervation to the indirect flight muscles of Drosophila. During metamorphosis, the larval intersegmental nerve of the mesothorax is remodelled to innervate the dorsal longitudinal muscles and two of the dorsoventral muscles. Another modified larval nerve innervates the remaining dorsoventral muscle. The dorsal longitudinal muscles develop using modified larval muscles as templates while dorsoventral muscles develop without the use of such templates. The development of innervation to the two groups of indirect flight muscles differs in spatial and temporal patterns, which may reflect the different ways in which these muscles develop. The identification of myoblasts associated with thoracic nerves during larval life and the association of migrating myoblasts with nerves during metamorphosis indicate the existence of nerve-muscle interactions during indirect flight muscle development. In addition, the developing pattern of axonal branching suggests a role for the target muscles in respecifying neuromuscular junctions during metamorphosis.

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DeSimone, S., C. Coelho, S. Roy, K. VijayRaghavan, and K. White. "ERECT WING, the Drosophila member of a family of DNA binding proteins is required in imaginal myoblasts for flight muscle development." Development 122, no. 1 (January 1, 1996): 31–39. http://dx.doi.org/10.1242/dev.122.1.31.

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The erect wing locus of the fruit fly Drosophila melanogaster encodes a protein, EWG, that shares extensive homology with the P3A2 DNA binding protein of sea urchin and a recently identified mammalian transcription factor. Loss-of-function erect wing alleles result in embryonic lethality. Viable alleles of erect wing cause severe abnormalities of the indirect flight muscles. We have analyzed the spatial pattern of erect wing expression in the developing indirect flight muscles during postembryonic development. EWG is detected, 10 hours after puparium formation, in myoblasts that will form the indirect flight muscles. The early events of muscle development are normal in ewg mutants. However, a few hours after the onset of erect wing expression in myoblasts, defects are seen in the developing indirect flight muscles which subsequently degenerate. We present results that show that the normal development of the indirect flight muscles requires erect wing expression in the progenitor myoblasts themselves. Finally, we examine the role of target muscles in the arborization of motor axons by studying the developing innervation to the flight muscle in erect wing mutants. Our study demonstrates, for the first time, a role for a regulatory gene expressed in imaginal myoblasts in Drosophila.

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Fernandes, J., M. Bate, and K. Vijayraghavan. "Development of the indirect flight muscles of Drosophila." Development 113, no. 1 (September 1, 1991): 67–77. http://dx.doi.org/10.1242/dev.113.1.67.

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We have followed the pupal development of the indirect flight muscles (IFMs) of Drosophila melanogaster. At the onset of metamorphosis larval muscles start to histolyze, with the exception of a specific set of thoracic muscles. Myoblasts surround these persisting larval muscles and begin the formation of one group of adult indirect flight muscles, the dorsal longitudinal muscles. We show that the other group of indirect flight muscles, the dorsoventral muscles, develops simultaneously but without the use of larval templates. By morphological criteria and by patterns of specific gene expression, our experiments define events in IFM development.

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Oas, Sandy T., Anton L. Bryantsev, and Richard M. Cripps. "Arrest is a regulator of fiber-specific alternative splicing in the indirect flight muscles of Drosophila." Journal of Cell Biology 206, no. 7 (September 22, 2014): 895–908. http://dx.doi.org/10.1083/jcb.201405058.

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Drosophila melanogaster flight muscles are distinct from other skeletal muscles, such as jump muscles, and express several uniquely spliced muscle-associated transcripts. We sought to identify factors mediating splicing differences between the flight and jump muscle fiber types. We found that the ribonucleic acid–binding protein Arrest (Aret) is expressed in flight muscles: in founder cells, Aret accumulates in a novel intranuclear compartment that we termed the Bruno body, and after the onset of muscle differentiation, Aret disperses in the nucleus. Down-regulation of the aret gene led to ultrastructural changes and functional impairment of flight muscles, and transcripts of structural genes expressed in the flight muscles became spliced in a manner characteristic of jump muscles. Aret also potently promoted flight muscle splicing patterns when ectopically expressed in jump muscles or tissue culture cells. Genetically, aret is located downstream of exd (extradenticle), hth (homothorax), and salm (spalt major), transcription factors that control fiber identity. Our observations provide insight into a transcriptional and splicing regulatory network for muscle fiber specification.

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Ghazi, A., S. Anant, and K. Vijay Raghavan. "Apterous mediates development of direct flight muscles autonomously and indirect flight muscles through epidermal cues." Development 127, no. 24 (December 15, 2000): 5309–18. http://dx.doi.org/10.1242/dev.127.24.5309.

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Two physiologically distinct types of muscles, the direct and indirect flight muscles, develop from myoblasts associated with the Drosophila wing disc. We show that the direct flight muscles are specified by the expression of Apterous, a Lim homeodomain protein, in groups of myoblasts. This suggests a mechanism of cell-fate specification by labelling groups of fusion competent myoblasts, in contrast to mechanisms in the embryo, where muscle cell fate is specified by single founder myoblasts. In addition, Apterous is expressed in the developing adult epidermal muscle attachment sites. Here, it functions to regulate the expression of stripe, a gene that is an important element of early patterning of muscle fibres, from the epidermis. Our results, which may have broad implications, suggest novel mechanisms of muscle patterning in the adult, in contrast to embryonic myogenesis.

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Dahl-Halvarsson, Martin, Montse Olive, Malgorzata Pokrzywa, Katarina Ejeskär, Ruth H. Palmer, Anne Elisabeth Uv, and Homa Tajsharghi. "Drosophila model of myosin myopathy rescued by overexpression of a TRIM-protein family member." Proceedings of the National Academy of Sciences 115, no. 28 (June 26, 2018): E6566—E6575. http://dx.doi.org/10.1073/pnas.1800727115.

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Myosin is a molecular motor indispensable for body movement and heart contractility. Apart from pure cardiomyopathy, mutations in MYH7 encoding slow/β-cardiac myosin heavy chain also cause skeletal muscle disease with or without cardiac involvement. Mutations within the α-helical rod domain of MYH7 are mainly associated with Laing distal myopathy. To investigate the mechanisms underlying the pathology of the recurrent causative MYH7 mutation (K1729del), we have developed a Drosophila melanogaster model of Laing distal myopathy by genomic engineering of the Drosophila Mhc locus. Homozygous MhcK1728del animals die during larval/pupal stages, and both homozygous and heterozygous larvae display reduced muscle function. Flies expressing only MhcK1728del in indirect flight and jump muscles, and heterozygous MhcK1728del animals, were flightless, with reduced movement and decreased lifespan. Sarcomeres of MhcK1728del mutant indirect flight muscles and larval body wall muscles were disrupted with clearly disorganized muscle filaments. Homozygous MhcK1728del larvae also demonstrated structural and functional impairments in heart muscle, which were not observed in heterozygous animals, indicating a dose-dependent effect of the mutated allele. The impaired jump and flight ability and the myopathy of indirect flight and leg muscles associated with MhcK1728del were fully suppressed by expression of Abba/Thin, an E3-ligase that is essential for maintaining sarcomere integrity. This model of Laing distal myopathy in Drosophila recapitulates certain morphological phenotypic features seen in Laing distal myopathy patients with the recurrent K1729del mutation. Our observations that Abba/Thin modulates these phenotypes suggest that manipulation of Abba/Thin activity levels may be beneficial in Laing distal myopathy.

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Cripps, R. M., K. D. Becker, M. Mardahl, W. A. Kronert, D. Hodges, and S. I. Bernstein. "Transformation of Drosophila melanogaster with the wild-type myosin heavy-chain gene: rescue of mutant phenotypes and analysis of defects caused by overexpression." Journal of Cell Biology 126, no. 3 (August 1, 1994): 689–99. http://dx.doi.org/10.1083/jcb.126.3.689.

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We have transformed Drosophila melanogaster with a genomic construct containing the entire wild-type myosin heavy-chain gene, Mhc, together with approximately 9 kb of flanking DNA on each side. Three independent lines stably express myosin heavy-chain protein (MHC) at approximately wild-type levels. The MHC produced is functional since it rescues the mutant phenotypes of a number of different Mhc alleles: the amorphic allele Mhc1, the indirect flight muscle and jump muscle-specific amorphic allele Mhc10, and the hypomorphic allele Mhc2. We show that the Mhc2 mutation is due to the insertion of a transposable element in an intron of Mhc. Since a reduction in MHC in the indirect flight muscles alters the myosin/actin protein ratio and results in myofibrillar defects, we determined the effects of an increase in the effective copy number of Mhc. The presence of four copies of Mhc results in overabundance of the protein and a flightless phenotype. Electron microscopy reveals concomitant defects in the indirect flight muscles, with excess thick filaments at the periphery of the myofibrils. Further increases in copy number are lethal. These results demonstrate the usefulness and potential of the transgenic system to study myosin function in Drosophila. They also show that overexpression of wild-type protein in muscle may disrupt the function of not only the indirect flight but also other muscles of the organism.

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Fernandes, J. J., and H. Keshishian. "Nerve-muscle interactions during flight muscle development in Drosophila." Development 125, no. 9 (May 1, 1998): 1769–79. http://dx.doi.org/10.1242/dev.125.9.1769.

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During Drosophila pupal metamorphosis, the motoneurons and muscles differentiate synchronously, providing an opportunity for extensive intercellular regulation during synapse formation. We examined the existence of such interactions by developmentally delaying or permanently eliminating synaptic partners during the formation of indirect flight muscles. When we experimentally delayed muscle development, we found that although adult-specific primary motoneuron branching still occurred, the higher order (synaptic) branching was suspended until the delayed muscle fibers reached a favourable developmental state. In reciprocal experiments we found that denervation caused a decrease in the myoblast pool. Furthermore, the formation of certain muscle fibers (dorsoventral muscles) was specifically blocked. Exceptions were the adult muscles that use larval muscle fibers as myoblast fusion targets (dorsal longitudinal muscles). However, when these muscles were experimentally compelled to develop without their larval precursors, they showed an absolute dependence on the motoneurons for their formation. These data show that the size of the myoblast pool and early events in fiber formation depend on the presence of the nerve, and that, conversely, peripheral arbor development and synaptogenesis is closely synchronized with the developmental state of the muscle.

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9

Chan, W. P., and M. H. Dickinson. "In vivo length oscillations of indirect flight muscles in the fruit fly Drosophila virilis." Journal of Experimental Biology 199, no. 12 (December 1, 1996): 2767–74. http://dx.doi.org/10.1242/jeb.199.12.2767.

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We have used high-speed video microscopy to measure in vivo length oscillations of the indirect flight muscles of the fruit fly Drosophila virilis during tethered flight. The changes in muscle strain were measured by tracking the deformation of the thoracic exoskeleton at the origin and insertion of both the dorsal longitudinal (DLM) and the dorsal ventral (DVM) muscles. The mean peak-to-peak strain amplitudes were found to be 3.5% for the DLMs and 3.3% for the DVMs, although the strain amplitude within individual cycles ranged from 2 to 5%. These values are consistent with the small number of previous measurements of indirect flight muscle strain in other insects, but almost an order of magnitude greater than the strain amplitudes used in most biophysical studies of skinned Drosophila fibers. The results suggest that serial compliance within this sarcomere would need to relieve approximately 70% of the total strain in order for individual crossbridges to remain attached throughout a complete contraction-extension cycle.

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10

Anant, S., S. Roy, and K. Vijay Raghavan. "Twist and Notch negatively regulate adult muscle differentiation in Drosophila." Development 125, no. 8 (April 15, 1998): 1361–69. http://dx.doi.org/10.1242/dev.125.8.1361.

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Twist is required in Drosophila embryogenesis for mesodermal specification and cell-fate choice. We have examined the role of Twist and Notch during adult indirect flight muscle development. Reduction in levels of Twist leads to abnormal myogenesis. Notch reduction causes a similar mutant phenotype and reduces Twist levels. Conversely, persistent expression, in myoblasts, of activated Notch causes continued twist expression and failure of differentiation as assayed by myosin expression. The gain-of-function phenotype of Notch is very similar to that seen upon persistent twist expression. These results point to a relationship between Notch function and twist regulation during indirect flight muscle development and show that decline in Twist levels is a requirement for the differentiation of these muscles, unlike the somatic muscles of the embryo.

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Johnston, J. Spencer, Mary E. Zapalac, and Carl E. Hjelmen. "Flying High—Muscle-Specific Underreplication in Drosophila." Genes 11, no. 3 (February 26, 2020): 246. http://dx.doi.org/10.3390/genes11030246.

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Drosophila underreplicate the DNA of thoracic nuclei, stalling during S phase at a point that is proportional to the total genome size in each species. In polytene tissues, such as the Drosophila salivary glands, all of the nuclei initiate multiple rounds of DNA synthesis and underreplicate. Yet, only half of the nuclei isolated from the thorax stall; the other half do not initiate S phase. Our question was, why half? To address this question, we use flow cytometry to compare underreplication phenotypes between thoracic tissues. When individual thoracic tissues are dissected and the proportion of stalled DNA synthesis is scored in each tissue type, we find that underreplication occurs in the indirect flight muscle, with the majority of underreplicated nuclei in the dorsal longitudinal muscles (DLM). Half of the DNA in the DLM nuclei stall at S phase between the unreplicated G0 and fully replicated G1. The dorsal ventral flight muscle provides the other source of underreplication, and yet, there, the replication stall point is earlier (less DNA replicated), and the endocycle is initiated. The differences in underreplication and ploidy in the indirect flight muscles provide a new tool to study heterochromatin, underreplication and endocycle control.

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12

Chaturvedi, Dhananjay, Sunil Prabhakar, Aman Aggarwal, Krishan B. Atreya, and K. VijayRaghavan. "Adult Drosophila muscle morphometry through microCT reveals dynamics during ageing." Open Biology 9, no. 6 (June 2019): 190087. http://dx.doi.org/10.1098/rsob.190087.

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Indirect flight muscles (IFMs) in adult Drosophila provide the key power stroke for wing beating. They also serve as a valuable model for studying muscle development. An age-dependent decline in Drosophila free flight has been documented, but its relation to gross muscle structure has not yet been explored satisfactorily. Such analyses are impeded by conventional histological preparations and imaging techniques that limit exact morphometry of flight muscles. In this study, we employ microCT scanning on a tissue preparation that retains muscle morphology under homeostatic conditions. Focusing on a subset of IFMs called the dorsal longitudinal muscles (DLMs), we find that DLM volumes increase with age, partially due to the increased separation between myofibrillar fascicles, in a sex-dependent manner. We have uncovered and quantified asymmetry in the size of these muscles on either side of the longitudinal midline. Measurements of this resolution and scale make substantive studies that test the connection between form and function possible. We also demonstrate the application of this method to other insect species making it a valuable tool for histological analysis of insect biodiversity.

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13

Swank, Douglas M. "Mechanical analysis of Drosophila indirect flight and jump muscles." Methods 56, no. 1 (January 2012): 69–77. http://dx.doi.org/10.1016/j.ymeth.2011.10.015.

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14

Vigoreaux, J. O., J. D. Saide, K. Valgeirsdottir, and M. L. Pardue. "Flightin, a novel myofibrillar protein of Drosophila stretch-activated muscles." Journal of Cell Biology 121, no. 3 (May 1, 1993): 587–98. http://dx.doi.org/10.1083/jcb.121.3.587.

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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.

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Hastings, G. A., and C. P. Emerson. "Myosin functional domains encoded by alternative exons are expressed in specific thoracic muscles of Drosophila." Journal of Cell Biology 114, no. 2 (July 15, 1991): 263–76. http://dx.doi.org/10.1083/jcb.114.2.263.

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The Drosophila 36B muscle myosin heavy chain (MHC) gene has five sets of alternatively spliced exons that encode functionally important domains of the MHC protein and provide a combinatorial potential for expression of as many as 480 MHC isoforms. In this study, in situ hybridization analysis has been used to examine the complexity and muscle specificity of MHC isoform expression in the fibrillar indirect flight muscle (IFM), the tubular direct flight muscles (DFM) and tubular tergal depressor of the trochanter muscle (TDT), and the visceral esophageal muscle in the adult thorax. Our results show that alternative splicing of the MHC gene transcripts is precisely regulated in these thoracic muscles, which express three MHC isoforms. Individual thoracic muscles each express transcripts of only one isoform, as detectable by in situ hybridization. An apparently novel fourth MHC isoform, with sequence homology to the rod but not to the head domain of the 36B MHC, is expressed in two direct flight muscles. These findings form a basis for transgenic experiments designed to analyze the muscle-specific functions of MHC domains encoded by alternative exons.

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Cripps, R. M., E. Ball, M. Stark, A. Lawn, and J. C. Sparrow. "Recovery of dominant, autosomal flightless mutants of Drosophila melanogaster and identification of a new gene required for normal muscle structure and function." Genetics 137, no. 1 (May 1, 1994): 151–64. http://dx.doi.org/10.1093/genetics/137.1.151.

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Abstract To identify further mutations affecting muscle function and development in Drosophila melanogaster we recovered 22 autosomal dominant flightless mutations. From these we have isolated eight viable and lethal alleles of the muscle myosin heavy chain gene, and seven viable alleles of the indirect flight muscle (IFM)-specific Act88F actin gene. The Mhc mutations display a variety of phenotypic effects, ranging from reductions in myosin heavy chain content in the indirect flight muscles only, to reductions in the levels of this protein in other muscles. The Act88F mutations range from those which produce no stable actin and have severely abnormal myofibrillar structure, to those which accumulate apparently normal levels of actin in the flight muscles but which still have abnormal myofibrils and fly very poorly. We also recovered two recessive flightless mutants on the third chromosome. The remaining five dominant flightless mutations are all lethal alleles of a gene named lethal(3)Laker. The Laker alleles have been characterized and the gene located in polytene bands 62A10,B1-62B2,4. Laker is a previously unidentified locus which is haplo-insufficient for flight. In addition, adult wild-type heterozygotes and the lethal larval trans-heterozygotes show abnormalities of muscle structure indicating that the Laker gene product is an important component of muscle.

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Maroto, M., J. Arredondo, D. Goulding, R. Marco, B. Bullard, and M. Cervera. "Drosophila paramyosin/miniparamyosin gene products show a large diversity in quantity, localization, and isoform pattern: a possible role in muscle maturation and function." Journal of Cell Biology 134, no. 1 (July 1, 1996): 81–92. http://dx.doi.org/10.1083/jcb.134.1.81.

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The Drosophila paramyosin/miniparamyosin gene expresses two products of different molecular weight transcriptionally regulated from two different promoters. Distinct muscle types also have different relative amounts of myosin, paramyosin, and miniparamyosin, reflecting differences in the organization of their thick filaments. Immunofluorescence and EM data indicate that miniparamyosin is mainly located in the M line and at both ends of the thick filaments in Drosophila indirect flight muscles, while paramyosin is present all along the thick filaments. In the tergal depressor of the trochanter muscle, both proteins are distributed all along the A band. In contrast, in the waterbug, Lethocerus, both paramyosin and miniparamyosin are distributed along the length of the indirect flight and leg muscle thick filaments. Two-dimensional and one-dimensional Western blot analyses have revealed that miniparamyosin has several isoforms, focusing over a very wide pH range, all of which are phosphorylated in vivo. The changes in isoform patterns of miniparamyosin and paramyosin indicate a direct or indirect involvement of these proteins in muscle function and flight. This wide spectrum of potential regulatory characteristics underlines the key importance of paramyosin/miniparamyosin and its complex isoform pattern in the organization of the invertebrate thick filament.

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Jawkar, Saroj, and Upendra Nongthomba. "Indirect flight muscles in Drosophila melanogaster as a tractable model to study muscle development and disease." International Journal of Developmental Biology 64, no. 1-2-3 (2020): 167–73. http://dx.doi.org/10.1387/ijdb.190333un.

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Myogenesis is a complex multifactorial process leading to the formation of the adult muscle. An amalgamation of autonomous processes including myoblast fusion and myofibrillogenesis, as well as non-autonomous processes, such as innervations from neurons and precise connections with attachment sites, are responsible for successful development and function of muscles. In this review, we describe the development of the indirect flight muscles (IFMs) in Drosophila melanogaster, and highlight the use of the IFMs as a model for studying muscle development and disease, based on recent studies on the development and function of IFMs.

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Menke, Stephen D., and Nancy S. Petersen. "Protein synthesis during flight muscle development in normal and wupB indirect flight muscles of Drosophila melanogaster." Insect Biochemistry 19, no. 8 (January 1989): 723–29. http://dx.doi.org/10.1016/0020-1790(89)90052-8.

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Gong, Henry, Weikang Ma, Shaoshuai Chen, Geng Wang, Ramzi Khairallah, and Thomas Irving. "Localization of the Elastic Proteins in the Flight Muscle of Manduca sexta." International Journal of Molecular Sciences 21, no. 15 (July 31, 2020): 5504. http://dx.doi.org/10.3390/ijms21155504.

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The flight muscle of Manduca sexta (DLM1) is an emerging model system for biophysical studies of muscle contraction. Unlike the well-studied indirect flight muscle of Lethocerus and Drosophila, the DLM1 of Manduca is a synchronous muscle, as are the vertebrate cardiac and skeletal muscles. Very little has been published regarding the ultrastructure and protein composition of this muscle. Previous studies have demonstrated that DLM1 express two projectin isoform, two kettin isoforms, and two large Salimus (Sls) isoforms. Such large Sls isoforms have not been observed in the asynchronous flight muscles of Lethocerus and Drosophila. The spatial localization of these proteins was unknown. Here, immuno-localization was used to show that the N-termini of projectin and Salimus are inserted into the Z-band. Projectin spans across the I-band, and the C-terminus is attached to the thick filament in the A-band. The C-terminus of Sls was also located in the A-band. Using confocal microscopy and experimental force-length curves, thin filament lengths were estimated as ~1.5 µm and thick filament lengths were measured as ~2.5 µm. This structural information may help provide an interpretive framework for future studies using this muscle system.

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Li, Jiawei, Hamidreza Rahmani, Fatemeh Abbasi Yeganeh, Hosna Rastegarpouyani, Dianne W. Taylor, Neil B. Wood, Michael J. Previs, Hiroyuki Iwamoto, and Kenneth A. Taylor. "Structure of the Flight Muscle Thick Filament from the Bumble Bee, Bombus ignitus, at 6 Å Resolution." International Journal of Molecular Sciences 24, no. 1 (December 26, 2022): 377. http://dx.doi.org/10.3390/ijms24010377.

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Four insect orders have flight muscles that are both asynchronous and indirect; they are asynchronous in that the wingbeat frequency is decoupled from the frequency of nervous stimulation and indirect in that the muscles attach to the thoracic exoskeleton instead of directly to the wing. Flight muscle thick filaments from two orders, Hemiptera and Diptera, have been imaged at a subnanometer resolution, both of which revealed a myosin tail arrangement referred to as “curved molecular crystalline layers”. Here, we report a thick filament structure from the indirect flight muscles of a third insect order, Hymenoptera, the Asian bumble bee Bombus ignitus. The myosin tails are in general agreement with previous determinations from Lethocerus indicus and Drosophila melanogaster. The Skip 2 region has the same unusual structure as found in Lethocerus indicus thick filaments, an α-helix discontinuity is also seen at Skip 4, but the orientation of the Skip 1 region on the surface of the backbone is less angled with respect to the filament axis than in the other two species. The heads are disordered as in Drosophila, but we observe no non-myosin proteins on the backbone surface that might prohibit the ordering of myosin heads onto the thick filament backbone. There are strong structural similarities among the three species in their non-myosin proteins within the backbone that suggest how one previously unassigned density in Lethocerus might be assigned. Overall, the structure conforms to the previously observed pattern of high similarity in the myosin tail arrangement, but differences in the non-myosin proteins.

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Nongthomba, Upendra, and Nallur B. Ramachandra. "A Direct Screen Identifies New Flight Muscle Mutants on the Drosophila Second Chromosome." Genetics 153, no. 1 (September 1, 1999): 261–74. http://dx.doi.org/10.1093/genetics/153.1.261.

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Abstract An ethyl methanesulfonate mutagenesis of Drosophila melanogaster was undertaken, and >3000 mutagenized second chromosomes were generated. More than 800 homozygous viable lines were established, and adults were screened directly under polarized light for muscle defects. A total of 16 mutant strains in which the indirect flight muscles were reduced in volume or disorganized or were otherwise abnormal were identified. These fell into seven recessive and one semidominant complementation groups. Five of these eight complementation groups, including the semidominant mutation, have been mapped using chromosomal deficiencies and meiotic recombination. Two complementation groups mapped close to the Myosin heavy chain gene, but they are shown to be in different loci. Developmental analysis of three mutations showed that two of these are involved in the early stages of adult myogenesis while the other showed late defects. This is the first report of results from a systematic and direct screen for recessive flight muscle defects. This mutant screen identifies genes affecting the flight muscles, which are distinct from those identified when screening for flightlessness.

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González-Morales, Nicanor, Thomas W. Marsh, Anja Katzemich, Océane Marescal, Yu Shu Xiao, and Frieder Schöck. "Different Evolutionary Trajectories of Two Insect-Specific Paralogous Proteins Involved in Stabilizing Muscle Myofibrils." Genetics 212, no. 3 (May 13, 2019): 743–55. http://dx.doi.org/10.1534/genetics.119.302217.

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Alp/Enigma family members have a unique PDZ domain followed by zero to four LIM domains, and are essential for myofibril assembly across all species analyzed so far. Drosophila melanogaster has three Alp/Enigma family members, Zasp52, Zasp66, and Zasp67. Ortholog search and phylogenetic tree analysis suggest that Zasp genes have a common ancestor, and that Zasp66 and Zasp67 arose by duplication in insects. While Zasp66 has a conserved domain structure across orthologs, Zasp67 domains and lengths are highly variable. In flies, Zasp67 appears to be expressed only in indirect flight muscles, where it colocalizes with Zasp52 at Z-discs. We generated a CRISPR null mutant of Zasp67, which is viable but flightless. We can rescue all phenotypes by re-expressing a Zasp67 transgene at endogenous levels. Zasp67 mutants show extended and broken Z-discs in adult flies, indicating that the protein helps stabilize the highly regular myofibrils of indirect flight muscles. In contrast, a Zasp66 CRISPR null mutant has limited viability, but only mild indirect flight muscle defects illustrating the diverging evolutionary paths these two paralogous genes have taken since they arose by duplication.

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Sandstrom, D. J., and L. L. Restifo. "Epidermal tendon cells require Broad Complex function for correct attachment of the indirect flight muscles in Drosophila melanogaster." Journal of Cell Science 112, no. 22 (November 15, 1999): 4051–65. http://dx.doi.org/10.1242/jcs.112.22.4051.

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Drosophila Broad Complex, a primary response gene in the ecdysone cascade, encodes a family of zinc-finger transcription factors essential for metamorphosis. Broad Complex mutations of the rbp complementation group disrupt attachment of the dorsoventral indirect flight muscles during pupal development. We previously demonstrated that isoform BRC-Z1 mediates the muscle attachment function of rbp(+) and is expressed in both developing muscle fibers and their epidermal attachment sites. We now report two complementary studies to determine the cellular site and mode of action of rbp(+) during maturation of the myotendinous junctions of dorsoventral indirect flight muscles. First, genetic mosaics, produced using the paternal loss method, revealed that the muscle attachment phenotype is determined primarily by the genotype of the dorsal epidermis, with the muscle fiber and the ventral epidermis exerting little or no influence. When the dorsal epidermis was mutant, the vast majority of muscles detached or chose ectopic attachment sites, regardless of the muscle genotype. Conversely, wild-type dorsal epidermis could support attachment of mutant muscles. Second, ultrastructural analysis corroborated and extended these results, revealing defective and delayed differentiation of rbp mutant epidermal tendon cells in the dorsal attachment sites. Tendon cell processes, the stress-bearing links between the epidermis and muscle, were reduced in number and showed delayed appearance of microtubule bundles. In contrast, mutant muscle and ventral epidermis resembled the wild type. In conclusion, BRC-Z1 acts in the dorsal epidermis to ensure differentiation of the myotendinous junction. By analogy with the cell-cell interaction essential for embryonic muscle attachment, we propose that BRC-Z1 regulates one or more components of the epidermal response to a signal from the developing muscle.

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Fernandes, J. J., and H. Keshishian. "Patterning the dorsal longitudinal flight muscles (DLM) of Drosophila: insights from the ablation of larval scaffolds." Development 122, no. 12 (December 1, 1996): 3755–63. http://dx.doi.org/10.1242/dev.122.12.3755.

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The six Dorsal Longitudinal flight Muscles (DLMs) of Drosophila develop from three larval muscles that persist into metamorphosis and serve as scaffolds for the formation of the adult fibers. We have examined the effect of muscle scaffold ablation on the development of DLMs during metamorphosis. Using markers that are specific to muscle and myoblasts we show that in response to the ablation, myoblasts which would normally fuse with the larval muscle, fuse with each other instead, to generate the adult fibers in the appropriate regions of the thorax. The development of these de novo DLMs is delayed and is reflected in the delayed expression of erect wing, a transcription factor thought to control differentiation events associated with myoblast fusion. The newly arising muscles express the appropriate adult-specific Actin isoform (88F), indicating that they have the correct muscle identity. However, there are frequent errors in the number of muscle fibers generated. Ablation of the larval scaffolds for the DLMs has revealed an underlying potential of the DLM myoblasts to initiate de novo myogenesis in a manner that resembles the mode of formation of the Dorso-Ventral Muscles, DVMs, which are the other group of indirect flight muscles. Therefore, it appears that the use of larval scaffolds is a superimposition on a commonly used mechanism of myogenesis in Drosophila. Our results show that the role of the persistent larval muscles in muscle patterning involves the partitioning of DLM myoblasts, and in doing so, they regulate formation of the correct number of DLM fibers.

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Warmke, J., M. Yamakawa, J. Molloy, S. Falkenthal, and D. Maughan. "Myosin light chain-2 mutation affects flight, wing beat frequency, and indirect flight muscle contraction kinetics in Drosophila." Journal of Cell Biology 119, no. 6 (December 15, 1992): 1523–39. http://dx.doi.org/10.1083/jcb.119.6.1523.

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We have used a combination of classical genetic, molecular genetic, histological, biochemical, and biophysical techniques to identify and characterize a null mutation of the myosin light chain-2 (MLC-2) locus of Drosophila melanogaster. Mlc2E38 is a null mutation of the MLC-2 gene resulting from a nonsense mutation at the tenth codon position. Mlc2E38 confers dominant flightless behavior that is associated with reduced wing beat frequency. Mlc2E38 heterozygotes exhibit a 50% reduction of MLC-2 mRNA concentration in adult thoracic musculature, which results in a commensurate reduction of MLC-2 protein in the indirect flight muscles. Indirect flight muscle myofibrils from Mlc2E38 heterozygotes are aberrant, exhibiting myofilaments in disarray at the periphery. Calcium-activated Triton X-100-treated single fiber segments exhibit slower contraction kinetics than wild type. Introduction of a transformed copy of the wild type MLC-2 gene rescues the dominant flightless behavior of Mlc2E38 heterozygotes. Wing beat frequency and single fiber contraction kinetics of a representative rescued line are not significantly different from those of wild type. Together, these results indicate that wild type MLC-2 stoichiometry is required for normal indirect flight muscle assembly and function. Furthermore, these results suggest that the reduced wing beat frequency and possibly the flightless behavior conferred by Mlc2E38 is due in part to slower contraction kinetics of sarcomeric regions devoid or partly deficient in MLC-2.

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Jawkar, Saroj, and Upendra Nongthomba. "CG9650 Regulates the patterning of the indirect flight muscles of Drosophila melanogaster." Mechanisms of Development 145 (July 2017): S99. http://dx.doi.org/10.1016/j.mod.2017.04.256.

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Soanes, Kelly H., and John B. Bell. "Rediscovery and further characterization of the aeroplane (ae) wing posture mutation in Drosophila melanogaster." Genome 42, no. 3 (June 1, 1999): 403–11. http://dx.doi.org/10.1139/g98-143.

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In 1931, Theodore Quelprud characterized a novel spontaneous mutation in Drosophila melanogaster, which was named aeroplane (ae) based on its abnormal wing posture. Although the characterization of the original ae locus was minimal, it is very likely that another allele of this extinct mutation has now been identified. aeroplane-like (ae-l) was isolated as a by-product of a transformation experiment. The apparent wing paralysis is not caused by any obvious abnormalities in the thorax, wing, indirect flight muscles or direct flight muscles. Classical genetic complementation analyses of ae-l with other genes in the region suggest that it represents an allele of a novel locus. Unexpectedly, a molecular examination revealed that the physical lesion identified in the ae-l mutant is exceptionally close to the homeotic gene teashirt (tsh) and, indeed, may represent an unusual allele of teashirt.Key words: aeroplane, teashirt, wing posture, Drosophila, flight.

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O'Donnell, P. T., and S. I. Bernstein. "Molecular and ultrastructural defects in a Drosophila myosin heavy chain mutant: differential effects on muscle function produced by similar thick filament abnormalities." Journal of Cell Biology 107, no. 6 (December 1, 1988): 2601–12. http://dx.doi.org/10.1083/jcb.107.6.2601.

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We have determined the molecular defect of the Drosophila melanogaster myosin heavy chain (MHC) mutation Mhc and the mutation's effect on indirect flight muscle, jump muscle, and larval intersegmental muscle. We show that the Mhc1 mutation is essentially a null allele which results in the dominant-flightless and recessive-lethal phenotypes associated with this mutant (Mogami, K., P. T. O'Donnell, S. I. Bernstein, T. R. F. Wright, C. P. Emerson, Jr. 1986. Proc. Natl. Acad. Sci. USA. 83:1393-1397). The mutation is a 101-bp deletion in the MHC gene which removes most of exon 5 and the intron that precedes it. S1 nuclease mapping indicates that mutant transcripts follow two alternative processing pathways. Both pathways result in the production of mature transcripts with altered reading frames, apparently yielding unstable, truncated MHC proteins. Interestingly, the preferred splicing pathway uses the more distal of two available splice donor sites. We present the first ultrastrutural characterization of a completely MHC-null muscle and show that it lacks any discernable thick filaments. Sarcomeres in these muscles are completely disorganized suggesting that thick filaments play a critical role in sarcomere assembly. To understand why the Mhc1 mutation severely disrupts indirect flight muscle and jump muscle function in heterozygotes, but does not seriously affect the function of other muscle types, we examined the muscle ultrastructure of Mhc1/+ heterozygotes. We find that these organisms have a nearly 50% reduction in the number of thick filaments in indirect flight muscle, jump muscle, and larval intersegmental muscle. In addition, aberrantly shaped thick filaments are common in the jump muscle and larval intersegmental muscle. We suggest that the differential sensitivity of muscle function to the Mhc1 mutation is a consequence of the unique myofilament arrays in each of these muscles. The highly variable myofilament array of larval intersegmental muscle makes its function relatively insensitive to changes in thick filament number and morphology. Conversely, the rigid double hexagonal lattice of the indirect flight muscle, and the organized lattice of the jump muscle cannot be perturbed without interfering with the specialized and evolutionarily more complex functions they perform.

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Moehlman, Andrew T., Gil Kanfer, and Richard J. Youle. "Loss of STING in parkin mutant flies suppresses muscle defects and mitochondria damage." PLOS Genetics 19, no. 7 (July 13, 2023): e1010828. http://dx.doi.org/10.1371/journal.pgen.1010828.

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The early pathogenesis and underlying molecular causes of motor neuron degeneration in Parkinson’s Disease (PD) remains unresolved. In the model organism Drosophila melanogaster, loss of the early-onset PD gene parkin (the ortholog of human PRKN) results in impaired climbing ability, damage to the indirect flight muscles, and mitochondrial fragmentation with swelling. These stressed mitochondria have been proposed to activate innate immune pathways through release of damage associated molecular patterns (DAMPs). Parkin-mediated mitophagy is hypothesized to suppress mitochondrial damage and subsequent activation of the cGAS/STING innate immunity pathway, but the relevance of this interaction in the fly remains unresolved. Using a combination of genetics, immunoassays, and RNA sequencing, we investigated a potential role for STING in the onset of parkin-null phenotypes. Our findings demonstrate that loss of Drosophila STING in flies rescues the thorax muscle defects and the climbing ability of parkin-/- mutants. Loss of STING also suppresses the disrupted mitochondrial morphology in parkin-/- flight muscles, suggesting unexpected feedback of STING on mitochondria integrity or activation of a compensatory mitochondrial pathway. In the animals lacking both parkin and sting, PINK1 is activated and cell death pathways are suppressed. These findings support a unique, non-canonical role for Drosophila STING in the cellular and organismal response to mitochondria stress.

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Bernstein, S. I., C. J. Hansen, K. D. Becker, D. R. Wassenberg, E. S. Roche, J. J. Donady, and C. P. Emerson. "Alternative RNA splicing generates transcripts encoding a thorax-specific isoform of Drosophila melanogaster myosin heavy chain." Molecular and Cellular Biology 6, no. 7 (July 1986): 2511–19. http://dx.doi.org/10.1128/mcb.6.7.2511-2519.1986.

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Genomic and cDNA sequencing studies show that transcripts from the muscle myosin heavy-chain (MHC) gene of Drosophila melanogaster are alternatively spliced, producing RNAs that encode at least two MHC isoforms with different C termini. Transcripts encoding an MHC isoform with 27 unique C-terminal amino acids accumulate during both larval and adult muscle differentiation. Transcripts for the second isoform encode one unique C-terminal amino acid and accumulate almost exclusively in pupal and adult thoracic segments, the location of the indirect flight muscles. The 3' splice acceptor site preceding the thorax-specific exon is unusually purine rich and thus may serve as a thorax-specific splicing signal. We suggest that the alternative C termini of these two MHC isoforms control myofilament assembly and may play a role in generating the distinctive myofilament organizations of flight muscle and other muscle types.

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Bernstein, S. I., C. J. Hansen, K. D. Becker, D. R. Wassenberg, E. S. Roche, J. J. Donady, and C. P. Emerson. "Alternative RNA splicing generates transcripts encoding a thorax-specific isoform of Drosophila melanogaster myosin heavy chain." Molecular and Cellular Biology 6, no. 7 (July 1986): 2511–19. http://dx.doi.org/10.1128/mcb.6.7.2511.

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Genomic and cDNA sequencing studies show that transcripts from the muscle myosin heavy-chain (MHC) gene of Drosophila melanogaster are alternatively spliced, producing RNAs that encode at least two MHC isoforms with different C termini. Transcripts encoding an MHC isoform with 27 unique C-terminal amino acids accumulate during both larval and adult muscle differentiation. Transcripts for the second isoform encode one unique C-terminal amino acid and accumulate almost exclusively in pupal and adult thoracic segments, the location of the indirect flight muscles. The 3' splice acceptor site preceding the thorax-specific exon is unusually purine rich and thus may serve as a thorax-specific splicing signal. We suggest that the alternative C termini of these two MHC isoforms control myofilament assembly and may play a role in generating the distinctive myofilament organizations of flight muscle and other muscle types.

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Nongthomba, Upendra, Mark Cummins, Samantha Clark, Jim O. Vigoreaux, and John C. Sparrow. "Suppression of Muscle Hypercontraction by Mutations in the Myosin Heavy Chain Gene of Drosophila melanogaster." Genetics 164, no. 1 (May 1, 2003): 209–22. http://dx.doi.org/10.1093/genetics/164.1.209.

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Abstract The indirect flight muscles (IFM) of Drosophila melanogaster provide a good genetic system with which to investigate muscle function. Flight muscle contraction is regulated by both stretch and Ca2+-induced thin filament (actin + tropomyosin + troponin complex) activation. Some mutants in troponin-I (TnI) and troponin-T (TnT) genes cause a “hypercontraction” muscle phenotype, suggesting that this condition arises from defects in Ca2+ regulation and actomyosin-generated tension. We have tested the hypothesis that missense mutations of the myosin heavy chain gene, Mhc, which suppress the hypercontraction of the TnI mutant held-up2 (hdp2), do so by reducing actomyosin force production. Here we show that a “headless” Mhc transgenic fly construct that reduces the myosin head concentration in the muscle thick filaments acts as a dose-dependent suppressor of hypercontracting alleles of TnI, TnT, Mhc, and flightin genes. The data suggest that most, if not all, mutants causing hypercontraction require actomyosin-produced forces to do so. Whether all Mhc suppressors act simply by reducing the force production of the thick filament is discussed with respect to current models of myosin function and thin filament activation by the binding of calcium to the troponin complex.

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Chakravorty, Samya, Bertrand C. W. Tanner, Veronica Lee Foelber, Hien Vu, Matthew Rosenthal, Teresa Ruiz, and Jim O. Vigoreaux. "Flightin maintains myofilament lattice organization required for optimal flight power and courtship song quality in Drosophila." Proceedings of the Royal Society B: Biological Sciences 284, no. 1854 (May 3, 2017): 20170431. http://dx.doi.org/10.1098/rspb.2017.0431.

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The indirect flight muscles (IFMs) of Drosophila and other insects with asynchronous flight muscles are characterized by a crystalline myofilament lattice structure. The high-order lattice regularity is considered an adaptation for enhanced power output, but supporting evidence for this claim is lacking. We show that IFMs from transgenic flies expressing flightin with a deletion of its poorly conserved N-terminal domain ( fln ΔN62 ) have reduced inter-thick filament spacing and a less regular lattice. This resulted in a decrease in flight ability by 33% and in skinned fibre oscillatory power output by 57%, but had no effect on wingbeat frequency or frequency of maximum power output, suggesting that the underlying actomyosin kinetics is not affected and that the flight impairment arises from deficits in force transmission. Moreover, we show that fln ΔN62 males produced an abnormal courtship song characterized by a higher sine song frequency and a pulse song with longer pulses and longer inter-pulse intervals (IPIs), the latter implicated in male reproductive success. When presented with a choice, wild-type females chose control males over mutant males in 92% of the competition events. These results demonstrate that flightin N-terminal domain is required for optimal myofilament lattice regularity and IFM activity, enabling powered flight and courtship song production. As the courtship song is subject to female choice, we propose that the low amino acid sequence conservation of the N-terminal domain reflects its role in fine-tuning species-specific courtship songs.

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Barthmaier, Peter, and Eric Fyrberg. "Monitoring Development and Pathology of Drosophila Indirect Flight Muscles Using Green Fluorescent Protein." Developmental Biology 169, no. 2 (June 1995): 770–74. http://dx.doi.org/10.1006/dbio.1995.1186.

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Hiromi, Yasushi, and Yoshiki Hotta. "Actin gene mutations in Drosophila ; heat shock activation in the indirect flight muscles." EMBO Journal 4, no. 7 (July 1985): 1681–87. http://dx.doi.org/10.1002/j.1460-2075.1985.tb03837.x.

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Rao, Deepti S., William A. Kronert, Yiming Guo, Karen H. Hsu, Floyd Sarsoza, and Sanford I. Bernstein. "Reductions in ATPase activity, actin sliding velocity, and myofibril stability yield muscle dysfunction in Drosophila models of myosin-based Freeman–Sheldon syndrome." Molecular Biology of the Cell 30, no. 1 (January 2019): 30–41. http://dx.doi.org/10.1091/mbc.e18-08-0526.

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Using Drosophila melanogaster, we created the first animal models for myosin-based Freeman–Sheldon syndrome (FSS), a dominant form of distal arthrogryposis defined by congenital facial and distal skeletal muscle contractures. Electron microscopy of homozygous mutant indirect flight muscles showed normal (Y583S) or altered (T178I, R672C) myofibril assembly followed by progressive disruption of the myofilament lattice. In contrast, all alleles permitted normal myofibril assembly in the heterozygous state but caused myofibrillar disruption during aging. The severity of myofibril defects in heterozygotes correlated with the level of flight impairment. Thus our Drosophila models mimic the human condition in that FSS mutations are dominant and display varied degrees of phenotypic severity. Molecular modeling indicates that the mutations disrupt communication between the nucleotide-binding site of myosin and its lever arm that drives force production. Each mutant myosin showed reduced in vitro actin sliding velocity, with the two more severe alleles significantly decreasing the catalytic efficiency of actin-activated ATP hydrolysis. The observed reductions in actin motility and catalytic efficiency may serve as the mechanistic basis of the progressive myofibrillar disarray observed in the Drosophila models as well as the prolonged contractile activity responsible for skeletal muscle contractures in FSS patients.

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Reedy, Mary C., Belinda Bullard, and Jim O. Vigoreaux. "Flightin Is Essential for Thick Filament Assembly and Sarcomere Stability in Drosophila Flight Muscles." Journal of Cell Biology 151, no. 7 (December 25, 2000): 1483–500. http://dx.doi.org/10.1083/jcb.151.7.1483.

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Flightin is a multiply phosphorylated, 20-kD myofibrillar protein found in Drosophila indirect flight muscles (IFM). Previous work suggests that flightin plays an essential, as yet undefined, role in normal sarcomere structure and contractile activity. Here we show that flightin is associated with thick filaments where it is likely to interact with the myosin rod. We have created a null mutation for flightin, fln0, that results in loss of flight ability but has no effect on fecundity or viability. Electron microscopy comparing pupa and adult fln0 IFM shows that sarcomeres, and thick and thin filaments in pupal IFM, are 25–30% longer than in wild type. fln0 fibers are abnormally wavy, but sarcomere and myotendon structure in pupa are otherwise normal. Within the first 5 h of adult life and beginning of contractile activity, IFM fibers become disrupted as thick filaments and sarcomeres are variably shortened, and myofibrils are ruptured at the myotendon junction. Unusual empty pockets and granular material interrupt the filament lattice of adult fln0 sarcomeres. Site-specific cleavage of myosin heavy chain occurs during this period. That myosin is cleaved in the absence of flightin is consistent with the immunolocalization of flightin on the thick filament and biochemical and genetic evidence suggesting it is associated with the myosin rod. Our results indicate that flightin is required for the establishment of normal thick filament length during late pupal development and thick filament stability in adult after initiation of contractile activity.

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Chechenova, Maria B., Sara Maes, Sandy T. Oas, Cloyce Nelson, Kaveh G. Kiani, Anton L. Bryantsev, and Richard M. Cripps. "Functional redundancy and nonredundancy between two Troponin C isoforms inDrosophilaadult muscles." Molecular Biology of the Cell 28, no. 6 (March 15, 2017): 760–70. http://dx.doi.org/10.1091/mbc.e16-07-0498.

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We investigated the functional overlap of two muscle Troponin C (TpnC) genes that are expressed in the adult fruit fly, Drosophila melanogaster: TpnC4 is predominantly expressed in the indirect flight muscles (IFMs), whereas TpnC41C is the main isoform in the tergal depressor of the trochanter muscle (TDT; jump muscle). Using CRISPR/Cas9, we created a transgenic line with a homozygous deletion of TpnC41C and compared its phenotype to a line lacking functional TpnC4. We found that the removal of either of these genes leads to expression of the other isoform in both muscle types. The switching between isoforms occurs at the transcriptional level and involves minimal enhancers located upstream of the transcription start points of each gene. Functionally, the two TpnC isoforms were not equal. Although ectopic TpnC4 in TDT muscles was able to maintain jumping ability, TpnC41C in IFMs could not effectively support flying. Simultaneous functional disruption of both TpnC genes resulted in jump-defective and flightless phenotypes of the survivors, as well as abnormal sarcomere organization. These results indicated that TpnC is required for myofibril assembly, and that there is functional specialization among TpnC isoforms in Drosophila.

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Saide, J. D., S. Chin-Bow, J. Hogan-Sheldon, L. Busquets-Turner, J. O. Vigoreaux, K. Valgeirsdottir, and M. L. Pardue. "Characterization of components of Z-bands in the fibrillar flight muscle of Drosophila melanogaster." Journal of Cell Biology 109, no. 5 (November 1, 1989): 2157–67. http://dx.doi.org/10.1083/jcb.109.5.2157.

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Twelve monoclonal antibodies have been raised against proteins in preparations of Z-disks isolated from Drosophila melanogaster flight muscle. The monoclonal antibodies that recognized Z-band components were identified by immunofluorescence microscopy of flight muscle myofibrils. These antibodies have identified three Z-disk antigens on immunoblots of myofibrillar proteins. Monoclonal antibodies alpha:1-4 recognize a 90-100-kD protein which we identify as alpha-actinin on the basis of cross-reactivity with antibodies raised against honeybee and vertebrate alpha-actinins. Monoclonal antibodies P:1-4 bind to the high molecular mass protein, projectin, a component of connecting filaments that link the ends of thick filaments to the Z-band in insect asynchronous flight muscles. The anti-projectin antibodies also stain synchronous muscle, but, surprisingly, the epitopes here are within the A-bands, not between the A- and Z-bands, as in flight muscle. Monoclonal antibodies Z(210):1-4 recognize a 210-kD protein that has not been previously shown to be a Z-band structural component. A fourth antigen, resolved as a doublet (approximately 400/600 kD) on immunoblots of Drosophila fibrillar proteins, is detected by a cross reacting antibody, Z(400):2, raised against a protein in isolated honeybee Z-disks. On Lowicryl sections of asynchronous flight muscle, indirect immunogold staining has localized alpha-actinin and the 210-kD protein throughout the matrix of the Z-band, projectin between the Z- and A-bands, and the 400/600-kD components at the I-band/Z-band junction. Drosophila alpha-actinin, projectin, and the 400/600-kD components share some antigenic determinants with corresponding honeybee proteins, but no honeybee protein interacts with any of the Z(210) antibodies.

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Navarro-Payá, David, Ilona Flis, Michelle A. E. Anderson, Philippa Hawes, Ming Li, Omar S. Akbari, Sanjay Basu, and Luke Alphey. "Targeting female flight for genetic control of mosquitoes." PLOS Neglected Tropical Diseases 14, no. 12 (December 3, 2020): e0008876. http://dx.doi.org/10.1371/journal.pntd.0008876.

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Aedes aegypti Act4 is a paralog of the Drosophila melanogaster indirect flight muscle actin gene Act88F. Act88F has been shown to be haploinsufficient for flight in both males and females (amorphic mutants are dominant). Whereas Act88F is expressed in indirect flight muscles of both males and females, expression of Act4 is substantially female-specific. We therefore used CRISPR/Cas9 and homology directed repair to examine the phenotype of Act4 mutants in two Culicine mosquitoes, Aedes aegypti and Culex quinquefasciatus. A screen for dominant female-flightless mutants in Cx. quinquefasciatus identified one such mutant associated with a six base pair deletion in the CxAct4 coding region. A similar screen in Ae. aegypti identified no dominant mutants. Disruption of the AeAct4 gene by homology-dependent insertion of a fluorescent protein marker cassette gave a recessive female-flightless phenotype in Ae. aegypti. Reproducing the six-base deletion from Cx. quinquefasciatus in Ae. aegypti using oligo-directed mutagenesis generated dominant female-flightless mutants and identified additional dominant female-flightless mutants with other in-frame insertions or deletions. Our data indicate that loss of function mutations in the AeAct4 gene are recessive but that short in-frame deletions produce dominant-negative versions of the AeAct4 protein that interfere with flight muscle function. This makes Act4 an interesting candidate for genetic control methods, particularly population-suppression gene drives targeting female viability/fertility.

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Wang, Yang, Girish C. Melkani, Jennifer A. Suggs, Anju Melkani, William A. Kronert, Anthony Cammarato, and Sanford I. Bernstein. "Expression of the inclusion body myopathy 3 mutation in Drosophila depresses myosin function and stability and recapitulates muscle inclusions and weakness." Molecular Biology of the Cell 23, no. 11 (June 2012): 2057–65. http://dx.doi.org/10.1091/mbc.e12-02-0120.

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Hereditary myosin myopathies are characterized by variable clinical features. Inclusion body myopathy 3 (IBM-3) is an autosomal dominant disease associated with a missense mutation (E706K) in the myosin heavy chain IIa gene. Adult patients experience progressive muscle weakness. Biopsies reveal dystrophic changes, rimmed vacuoles with cytoplasmic inclusions, and focal disorganization of myofilaments. We constructed a transgene encoding E706K myosin and expressed it in Drosophila (E701K) indirect flight and jump muscles to establish a novel homozygous organism with homogeneous populations of fast IBM-3 myosin and muscle fibers. Flight and jump abilities were severely reduced in homozygotes. ATPase and actin sliding velocity of the mutant myosin were depressed >80% compared with wild-type myosin. Light scattering experiments and electron microscopy revealed that mutant myosin heads bear a dramatic propensity to collapse and aggregate. Thus E706K (E701K) myosin appears far more labile than wild-type myosin. Furthermore, mutant fly fibers exhibit ultrastructural hallmarks seen in patients, including cytoplasmic inclusions containing aberrant proteinaceous structures and disorganized muscle filaments. Our Drosophila model reveals the unambiguous consequences of the IBM-3 lesion on fast muscle myosin and fibers. The abnormalities observed in myosin function and muscle ultrastructure likely contribute to muscle weakness observed in our flies and patients.

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Kulke, Michael, Ciprian Neagoe, Bernhard Kolmerer, Ave Minajeva, Horst Hinssen, Belinda Bullard, and Wolfgang A. Linke. "Kettin, a major source of myofibrillar stiffness in Drosophila indirect flight muscle." Journal of Cell Biology 154, no. 5 (September 3, 2001): 1045–58. http://dx.doi.org/10.1083/jcb.200104016.

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Kettin is a high molecular mass protein of insect muscle that in the sarcomeres binds to actin and α-actinin. To investigate kettin's functional role, we combined immunolabeling experiments with mechanical and biochemical studies on indirect flight muscle (IFM) myofibrils of Drosophila melanogaster. Micrographs of stretched IFM sarcomeres labeled with kettin antibodies revealed staining of the Z-disc periphery. After extraction of the kettin-associated actin, the A-band edges were also stained. In contrast, the staining pattern of projectin, another IFM–I-band protein, was not altered by actin removal. Force measurements were performed on single IFM myofibrils to establish the passive length-tension relationship and record passive stiffness. Stiffness decreased within seconds during gelsolin incubation and to a similar degree upon kettin digestion with μ-calpain. Immunoblotting demonstrated the presence of kettin isoforms in normal Drosophila IFM myofibrils and in myofibrils from an actin-null mutant. Dotblot analysis revealed binding of COOH-terminal kettin domains to myosin. We conclude that kettin is attached not only to actin but also to the end of the thick filament. Kettin along with projectin may constitute the elastic filament system of insect IFM and determine the muscle's high stiffness necessary for stretch activation. Possibly, the two proteins modulate myofibrillar stiffness by expressing different size isoforms.

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Miller, Mark S., Panagiotis Lekkas, Joan M. Braddock, Gerrie P. Farman, Bryan A. Ballif, Thomas C. Irving, David W. Maughan, and Jim O. Vigoreaux. "Aging Enhances Indirect Flight Muscle Fiber Performance yet Decreases Flight Ability in Drosophila." Biophysical Journal 95, no. 5 (September 2008): 2391–401. http://dx.doi.org/10.1529/biophysj.108.130005.

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Trujillo, Adriana S., Karen H. Hsu, Meera C. Viswanathan, Anthony Cammarato, and Sanford I. Bernstein. "The R369 Myosin Residue within Loop 4 Is Critical for Actin Binding and Muscle Function in Drosophila." International Journal of Molecular Sciences 23, no. 5 (February 25, 2022): 2533. http://dx.doi.org/10.3390/ijms23052533.

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The myosin molecular motor interacts with actin filaments in an ATP-dependent manner to yield muscle contraction. Myosin heavy chain residue R369 is located within loop 4 at the actin-tropomyosin interface of myosin’s upper 50 kDa subdomain. To probe the importance of R369, we introduced a histidine mutation of that residue into Drosophila myosin and implemented an integrative approach to determine effects at the biochemical, cellular, and whole organism levels. Substituting the similarly charged but bulkier histidine residue reduces maximal actin binding in vitro without affecting myosin ATPase activity. R369H mutants exhibit impaired flight ability that is dominant in heterozygotes and progressive with age in homozygotes. Indirect flight muscle ultrastructure is normal in mutant homozygotes, suggesting that assembly defects or structural deterioration of myofibrils are not causative of reduced flight. Jump ability is also reduced in homozygotes. In contrast to these skeletal muscle defects, R369H mutants show normal heart ultrastructure and function, suggesting that this residue is differentially sensitive to perturbation in different myosin isoforms or muscle types. Overall, our findings indicate that R369 is an actin binding residue that is critical for myosin function in skeletal muscles, and suggest that more severe perturbations at this residue may cause human myopathies through a similar mechanism.

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Prado, A., I. Canal, J. A. Barbas, J. Molloy, and A. Ferrús. "Functional recovery of troponin I in a Drosophila heldup mutant after a second site mutation." Molecular Biology of the Cell 6, no. 11 (November 1995): 1433–41. http://dx.doi.org/10.1091/mbc.6.11.1433.

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To identify proteins that interact in vivo with muscle components we have used a genetic approach based on the isolation of suppressors of mutant alleles of known muscle components. We have applied this system to the case of troponin I (TnI) in Drosophila and its mutant allele heldup2 (hdp2). This mutation causes an alanine to valine substitution at position 116 after a single nucleotide change in a constitutive exon. Among the isolated suppressors, one of them results from a second site mutation at the TnI gene itself. Muscles endowed with TnI mutated at both sites support nearly normal myofibrillar structure, perform notably well in wing beating and flight tests, and isolated muscle fibers produce active force. We show that the structural and functional recovery in this suppressor does not result from a change in the stoichiometric ratio of TnI isoforms. The second site suppression is due to a leucine to phenylalanine change within a heptameric leucine string motif adjacent to the actin binding domain of TnI. These data evidence a structural and functional role for the heptameric leucine string that is most noticeable, if not specific, in the indirect flight muscle.

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K. Vishnudas, Vivek, Shawna S. Guillemette, Panagiotis Lekkas, David W. Maughan, and Jim O. Vigoreaux. "Characterization of the Intracellular Distribution of Adenine Nucleotide Translocase (ANT) in Drosophila Indirect Flight Muscles." CellBio 02, no. 03 (2013): 149–62. http://dx.doi.org/10.4236/cellbio.2013.23017.

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Silva, Rumika, John C. Sparrow, and Michael A. Geeves. "Isolation and kinetic characterisation of myosin and myosin S1 from the Drosophila indirect flight muscles." Journal of Muscle Research and Cell Motility 24, no. 8 (2003): 489–98. http://dx.doi.org/10.1023/b:jure.0000009809.69829.74.

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Standiford, David M., Mary Beth Davis, Weitao Sun, and Charles P. Emerson. "Splice-Junction Elements and Intronic Sequences Regulate Alternative Splicing of the Drosophila Myosin Heavy Chain Gene Transcript." Genetics 147, no. 2 (October 1, 1997): 725–41. http://dx.doi.org/10.1093/genetics/147.2.725.

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Abstract:

The Drosophila muscle myosin heavy chain (Mhc) gene primary transcript contains five alternatively spliced exon groups (exons 3, 7, 9, 11 and 15), each of which contains two to five mutually exclusive members. Individual muscles typically select a specific alternative exon from each group for incorporation into the processed message. We report here on the cis-regulatory mechanisms that direct the processing of alternative exons in Mhc exon 11 in individual muscles using transgenic reporter constructs, RT-PCR and directed mutagenesis. The 6.0-kilobase exon 11 domain is sufficient to direct the correct processing of exon 11 alternatives, demonstrating that the alternative splicing cis-regulatory elements are local to Mhc exon 11. Mutational analysis of Mhc exon 11 reveals that the alternative exon nonconsensus 5′-splice donors are essential for alternative splicing regulation in general, but do not specify alternative exons for inclusion in individual muscles. Rather, we show, through exon substitutions and deletion analyses, that a 360-nucleotide intronic domain precisely directs the normal processing of one exon, Mhc exon 11e, in the indirect flight muscle. These and other data indicate that alternative exons are regulated in appropriate muscles through interactions between intronic alternative splice-specificity elements, nonconsensus exon 11 splice donors and, likely, novel exon-specific alternative splicing factors.

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Miller, Rehae C., Ric Schaaf, David W. Maughan, and Terese R. Tansey. "A non-flight muscle isoform of Drosophila tropomyosin rescues an indirect flight muscle tropomyosin mutant." Journal of Muscle Research and Cell Motility 14, no. 1 (February 1993): 85–98. http://dx.doi.org/10.1007/bf00132183.

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Journal articles on the topic 'Drosophila Indirect Flight Muscles'

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