A nonsense mutation within the act88F actin gene disrupts myofibril formation in Drosophila indirect flight muscles

We have investigated the molecular basis of muscle abnormalities in the flightless Drosophila mutant lfm(3)7. This EMS-induced, semi-dominant allele was isolated by Mogami and Hotta (1981) and was shown to disrupt the organization of myofibrils in indirect flight muscles. Here we demonstrate that lfm(3)7 contains a nonsense mutation within codon 355 of the act88F actin gene. A single G greater than A transition converts a tryptophan (TGG) codon to an opal (TGA) terminator, thus deleting the carboxy-terminal 20 amino acids of an actin isoform that accumulates only in thoracic flight muscles. The truncated actin polypeptide is stable, and retains antigenicity to at least two anti-Drosophila actin monoclonal antibodies. We suggest that abnormalities in lfm(3)7 flight muscles result from incorporation of the mutant actin isoform into assembling myofibrils.     

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

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

Alternative exon-encoded regions of Drosophila myosin heavy chain modulate ATPase rates and actin sliding velocity

To investigate the molecular functions of the regions encoded by alternative exons from the single Drosophila myosin heavy chain gene, we made the first kinetic measurements of two muscle myosin isoforms that differ in all alternative regions. Myosin was purified from the indirect flight muscles of wild-type and transgenic flies expressing a major embryonic isoform. The in vitro actin sliding velocity on the flight muscle isoform (6.4 microm x s(-1) at 22 degrees C) is among the fastest reported for a type II myosin and was 9-fold faster than with the embryonic isoform. With smooth muscle tropomyosin bound to actin, the actin sliding velocity on the embryonic isoform increased 6-fold, whereas that on the flight muscle myosin slightly decreased. No difference in the step sizes of Drosophila and rabbit skeletal myosins were found using optical tweezers, suggesting that the slower in vitro velocity with the embryonic isoform is due to altered kinetics. Basal ATPase rates for flight muscle myosin are higher than those of embryonic and rabbit myosin. These differences explain why the embryonic myosin cannot functionally substitute in vivo for the native flight muscle isoform, and demonstrate that one or more of the five myosin heavy chain alternative exons must influence Drosophila myosin kinetics.     

Transformation and rescue of a flightless Drosophila tropomyosin mutant.

In the Drosophila flightless mutant Ifm(3)3, a transposable element inserted into the alternatively spliced fourth exon of the tropomyosin I (TmI) gene prevents proper expression of Ifm-TmI, the tropomyosin isoform found in indirect flight muscle. We have rescued the flightless phenotype of Ifm(3)3 flies using P-element-mediated transformation with a segment of the Drosophila genome containing the wild-type TmI gene plus 2.5 kb of 5' flanking and 2 kb of 3' flanking DNA. The inserted TmI gene is expressed with the proper developmental and tissue specificity, although its level of expression varies among the five transformed lines examined. These conclusions are based on analyses of flight, myofibrillar morphology, and TmI RNA and protein levels. A minimum of two copies of the inserted TmI gene per cell is necessary to restore flight to most of the flies in each line. We also show that the Ifm-TmI isoform is expressed in the leg muscle of wild-type flies and is decreased in Ifm(3)3 leg muscle. Homozygous Ifm(3)3 mutants do not jump. The ability to jump can be restored with a single copy of the wild-type TmI gene per cell.     

Transformation of Drosophila melanogaster with the wild-type myosin heavy-chain gene: rescue of mutant phenotypes and analysis of defects caused by overexpression

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.     

A tropomyosin-2 mutation suppresses a troponin I myopathy in Drosophila

A suppressor mutation, D53, of the held-up(2) allele of the Drosophila melanogaster Troponin I (wupA) gene is described. D53, a missense mutation, S185F, of the tropomyosin-2, Tm2, gene fully suppresses all the phenotypic effects of held-up(2), including the destructive hypercontraction of the indirect flight muscles (IFMs), a lack of jumping, the progressive myopathy of the walking muscles, and reductions in larval crawling and feeding behavior. The suppressor restores normal function of the IFMs, but flight ability decreases with age and correlates with an unusual, progressive structural collapse of the myofibrillar lattice starting at the center. The S185F substitution in Tm2 is close to a troponin T binding site on tropomyosin. Models to explain suppression by D53, derived from current knowledge of the vertebrate troponin-tropomyosin complex structure and functions, are discussed. The effects of S185F are compared with those of two mutations in residues 175 and 180 of human alpha-tropomyosin 1 which cause familial hypertrophic cardiomyopathy (HCM).     

Two Drosophila melanogaster tropomyosin genes: structural and functional aspects.

We compared the structure and function of the two Drosophila melanogaster tropomyosin genes. The most striking structural aspect was their size disparity. Codons 1 through 257 of gene 2 occupied 833 nucleotides and contained only one intron, whereas the corresponding region of gene 1 occupied 17.5 kilobases and was interrupted by eight introns. The intron-exon arrangement of gene 1 reflected evolutionary expansion of tropomyosin via 42- and 49-residue duplications, which are probably actin-binding domains. Functionally, gene 1 was considerably more complex than gene 2; it was active in both muscle and nonmuscle cell lineages, had at least five variable exons, and specified a minimum of five developmentally regulated isoforms. Two of these isoforms, which accumulated only in flight muscles, were unprecedented fusion proteins in which the tropomyosin sequence was joined to a carboxy-terminal proline-rich domain.     

Nucleotide sequence of a cDNA clone encoding a Drosophila muscle tropomyosin II isoform

A cDNA clone was sequenced that contains the entire coding region for the muscle tropomyosin II isoform from Drosophila. The cDNA clone is 1253 nucleotides (nt) long and contains an 88-nt 5'-leader sequence and a 310-nt 3'-untranslated sequence. The muscle tropomyosin II isoform consists of 285 amino acids and is 60% homologous with the previously reported muscle tropomyosin I isoform Drosophila and 55% homologous with rabbit muscle tropomyosin.     

Aberrant splicing of an alternative exon in the Drosophila troponin-T gene affects flight muscle development

During myofibrillogenesis, many muscle structural proteins assemble to form the highly ordered contractile sarcomere. Mutations in these proteins can lead to dysfunctional muscle and various myopathies. We have analyzed the Drosophila melanogaster troponin T (TnT) up1 mutant that specifically affects the indirect flight muscles (IFM) to explore troponin function during myofibrillogenesis. The up1 muscles lack normal sarcomeres and contain "zebra bodies," a phenotypic feature of human nemaline myopathies. We show that the up(1) mutation causes defective splicing of a newly identified alternative TnT exon (10a) that encodes part of the TnT C terminus. This exon is used to generate a TnT isoform specific to the IFM and jump muscles, which during IFM development replaces the exon 10b isoform. Functional differences between the 10a and 10b TnT isoforms may be due to different potential phosphorylation sites, none of which correspond to known phosphorylation sites in human cardiac TnT. The absence of TnT mRNA in up1 IFM reduces mRNA levels of an IFM-specific troponin I (TnI) isoform, but not actin, tropomyosin, or troponin C, suggesting a mechanism controlling expression of TnT and TnI genes may exist that must be examined in the context of human myopathies caused by mutations of these thin filament proteins.     

Protein phosphatase 1beta is required for the maintenance of muscle attachments

Type 1 serine/threonine protein phosphatases (PP1) are important regulators of many cellular and developmental processes, including glycogen metabolism, muscle contraction, and the cell cycle [1] [2] [3] [4] [5]. Drosophila and humans both have multiple genes encoding PP1 isoforms [3] [6] [7]; each has one beta and several alpha isoform genes (alpha(1), alpha(2), alpha(3) in flies, alpha and gamma in humans; mammalian PP1beta is also known as PP1delta). The alpha/beta subtype differences are highly conserved between flies and mammals [6]. Though all these proteins are >85% identical to each other and have indistinguishable activities in vitro, we show here that the Drosophila beta isoform has a distinct biological role. We show that PP1beta9C corresponds to flapwing (flw), previously identified mutants of which are viable but flightless because of defects in indirect flight muscles (IFMs) [8]. We have isolated a new, semi-lethal flw allele that shows a range of defects, especially in muscles, which break away from their attachment sites and degenerate.     

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.     

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

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

Drosophila muscle regulation characterized by electron microscopy and three-dimensional reconstruction of thin filament mutants

Wild-type and mutant thin filaments were isolated directly from "myosinless" Drosophila indirect flight muscles to study the structural basis of muscle regulation genetically. Negatively stained filaments showed tropomyosin with periodically arranged troponin complexes in electron micrographs. Three-dimensional helical reconstruction of wild-type filaments indicated that the positions of tropomyosin on actin in the presence and absence of Ca(2+) were indistinguishable from those in vertebrate striated muscle and consistent with a steric mechanism of regulation by troponin-tropomyosin in Drosophila muscles. Thus, the Drosophila model can be used to study steric regulation. Thin filaments from the Drosophila mutant heldup(2), which possesses a single amino acid conversion in troponin I, were similarly analyzed to assess the Drosophila model genetically. The positions of tropomyosin in the mutant filaments, in both the Ca(2+)-free and the Ca(2+)-induced states, were the same, and identical to that of wild-type filaments in the presence of Ca(2+). Thus, cross-bridge cycling would be expected to proceed uninhibited in these fibers, even in relaxing conditions, and this would account for the dramatic hypercontraction characteristic of these mutant muscles. The interaction of mutant troponin I with Drosophila troponin C is discussed, along with functional differences between troponin C from Drosophila and vertebrates.     

Rediscovery and further characterization of the aeroplane (ae) wing posture mutation in Drosophila melanogaster.

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.