Defects in the Drosophila myosin rod permit sarcomere assembly but cause flight muscle degeneration.

We have determined the molecular and ultrastructural defects associated with three homozygous-viable myosin heavy chain mutations of Drosophila melanogaster. These mutations cause a dominant flightless phenotype but allow relatively normal assembly of indirect flight muscle myofibrils. As adults age, the contents of the indirect flight muscle myofibers are pulled to one end of the thorax. This apparently results from myofibril "hyper-contraction", and leads to sarcomere rupture and random myofilament orientation. All three mutations cause single amino acid changes in the light meromyosin region of the myosin rod. Two change the same glutamic acid to a lysine residue and the third affects an amino acid five residues away, substituting histidine for arginine. Both affected residues are conserved in muscle myosins, cytoplasmic myosins and paramyosins. The mutations are associated with age-dependent, site-specific degradation of myosin heavy chain and failure to accumulate phosphorylated forms of flightin, an indirect flight muscle-specific protein previously localized to the thick filament. Given the repeating nature of the hydrophobic and charged amino acid residues of the myosin rod and the near-normal assembly of myofibrils in the indirect flight muscle of these mutants, it is remarkable that single amino acid changes in the rod cause such severe defects. It is also interesting that these severe defects are not apparent in other muscles. These phenomena likely arise from the highly organized nature and rigorous performance requirements of indirect flight muscle, and perhaps from the interaction of myosin with flightin, a protein specific to this muscle type.

A charge change in an evolutionarily-conserved region of the myosin globular head prevents myosin and thick filament accumulation in Drosophila.

We have determined the molecular lesion in Mhc9, a homozygous-viable mutant of the Drosophila muscle myosin heavy chain gene. This mutation is in an adult-specific alternative exon (exon 9a) which encodes a portion of the myosin head that is highly conserved among both cytoplasmic and muscle myosins of all organisms. The mutation results in a charge change in the evolutionarily invariant amino acid residue 482. The phenotype of the homozygous mutant is identical to that of an organism having a stop codon within alternative exon 9a, i.e. lack of thick filaments in the indirect flight muscles and a greatly reduced number of thick filaments in the small cells of the jump muscles. This phenotype correlates with the known expression pattern of exon 9a. Genetic, biochemical and ultrastructural analyses show that the failure to accumulate thick filaments in the mutant is not a result of aberrant interactions with thin filaments and that the mutant myosin heavy chain does not poison assembly of wild-type thick filaments. Our results, in conjunction with recent structural and mutant studies by others, indicate that residue 482 is important for generating ATPase activity and for myosin stability in muscle.

Analysis of Drosophila paramyosin: identification of a novel isoform which is restricted to a subset of adult muscles.

In this report we show that Drosophila melanogaster muscles contain the standard form of the thick filament protein paramyosin, as well as a novel paramyosin isoform, which we call miniparamyosin. We have isolated Drosophila paramyosin using previously established methods. This protein is approximately 105 kD and cross-reacts with polyclonal antibodies made against Caenorhabditis elegans or Heliocopris dilloni paramyosin. The Heliocopris antibody also cross-reacts with a approximately 55-kD protein which may be miniparamyosin. We have cloned and sequenced cDNA's encoding both Drosophila isoforms. Standard paramyosin has short nonhelical regions at each terminus flanking the expected alpha-helical heptad repeat seen in other paramyosins and in myosin heavy chains. The COOH-terminal 363 amino acids are identical in standard and miniparamyosin. However, the smaller isoform has 114 residues at the NH2 terminus that are unique as compared to the current protein sequence data base. The paramyosin gene is located at chromosome position 66E1. It appears to use two promoters to generate mRNA's that have either of two different 5' coding sequences joined to common 3' exons. Each protein isoform is encoded by two transcripts that differ only in the usage of polyadenylation signals. This results in four size classes of paramyosin mRNA which are expressed in a developmentally regulated pattern consistent with that observed for other muscle-specific RNA's in Drosophila. In situ hybridization to Drosophila tissue sections shows that standard paramyosin is expressed in all larval and adult muscle tissues whereas miniparamyosin is restricted to a subset of the adult musculature. Thus miniparamyosin is a novel muscle-specific protein that likely plays a role in thick filament structure or function in some adult muscles of Drosophila.

Alternative myosin hinge regions are utilized in a tissue-specific fashion that correlates with muscle contraction speed.

By comparing the structure of wild-type and mutant muscle myosin heavy chain (MHC) genes of Drosophila melanogaster, we have identified the defect in the homozygous-viable, flightless mutant Mhc10. The mutation is within the 3' splice acceptor of an alternative exon (exon 15a) that encodes the central region of the MHC hinge. The splice acceptor defect prevents the accumulation of mRNAs containing exon 15a, whereas transcripts with a divergent copy of this exon (exon 15b) are unaffected by the mutation. In situ hybridization and Northern blot analysis of wild-type organisms reveals that exon 15b is used in larval MHCs, whereas exons 15a and/or 15b are used in adult tissues. Because Mhc10 mutants fail to accumulate transcripts encoding MHC protein with hinge region a, analysis of their muscle-specific reduction in thick filament number serves as a sensitive assay system for determining the pattern of accumulation of MHCs with alternative hinge regions. Electron microscopic comparisons of various muscles from wild-type and Mhc10 adults reveals that those that contract rapidly or develop high levels of tension utilize only hinge region a, those that contract at moderate rates accumulate MHCs of both types, and those that are slowly contracting have MHCs with hinge region b. The presence of alternative hinge-coding exons and their highly tissue-specific usage suggests that this portion of the MHC molecule is important to the isoform-specific properties of MHC that lead to the different physiological and ultrastructural characteristics of various Drosophila muscle types. The absence of other alternative exons in the rod-coding region, aside from those shown previously to encode alternative carboxyl termini, demonstrates that the bulk of the myosin rod is not involved in the generation of isoform-specific properties of the MHC molecule.

Ultrastructural and molecular analyses of homozygous-viable Drosophila melanogaster muscle mutants indicate there is a complex pattern of myosin heavy-chain isoform distribution.

We describe the ultrastructural and initial molecular characterization of four homozygous-viable, dominant-flightless mutants of Drosophila melanogaster. Genetic mapping indicates that these mutations are inseparable from the known muscle myosin heavy-chain (MHC) allele Mhc1, and each mutation results in a muscle-specific reduction in MHC protein accumulation. The indirect flight muscles (IFMs) of each of these homozygous mutants fail to accumulate MHC, lack thick filaments, and do not display normal cylindrical myofibrils. As opposed to the null phenotype observed in the IFM, normal amounts of MHC accumulate in the leg muscles of three of these mutants, whereas the fourth mutant shows a 45% reduction in leg muscle MHC. The ultrastructure of the tergal depressor of the trochanter muscle TDT, or jump muscle) is normal in one mutant, completely lacks thick filaments in a second mutant, and displays a reduction of thick filaments in two mutants. The thick filament reduction in this latter class of mutants is limited to the four smaller anterior cells of the TDT, indicating that the TDT is a mixed fiber-type muscle. Because all isoforms of muscle MHC are encoded by alternative splicing of transcripts from a single gene, our results suggest that there is a complex pattern of MHC isoform accumulation in Drosophila. The phenotypes of the homozygous-viable mutants provide evidence for the differential localization of MHC isoforms in different muscles, within the same muscle, and even within a single muscle cell. The mutant characteristics also suggest that the use of some alternative exons is shared among the IFM, TDT, and additional muscles whereas the use of others is unique to the IFM.

Molecular and ultrastructural defects in a Drosophila myosin heavy chain mutant: differential effects on muscle function produced by similar thick filament abnormalities.

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.

Analysis of the 5' end of the Drosophila muscle myosin heavy chain gene. Alternatively spliced transcripts initiate at a single site and intron locations are conserved compared to myosin genes of other organisms.

We have localized the transcription start site of the Drosophila melanogaster muscle myosin heavy chain (MHC) gene and find that all forms of the alternatively spliced MHC mRNA initiate at the same location. Therefore the alternative inclusion/exclusion of the 3' penultimate exon in transcripts from this gene (Bernstein, S.I., Hansen, C.J., Becker, K.D., Wassenberg, D.R., II, Roche, E.S., Donady, J.J., and Emerson, C. P., Jr. (1986) Mol. Cell. Biol. 6, 2511-2519; Rozek, C.E., and Davidson, N. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 2128-2134) does not result from the use of different 5' transcription initiation sites. This gene is the first invertebrate MHC gene shown to have TATA and CAAT box consensus sequences and a noncoding 5' exon, properties that are shared with some vertebrate and invertebrate contractile protein genes. The intron that splits the 5' noncoding region of the Drosophila MHC gene contains no major conserved elements relative to other Drosophila contractile protein genes. The introns within the coding region near the 5' end of the Drosophila MHC gene are located at the same sites as nematode and vertebrate MHC gene introns, indicating that these MHC genes are derived from a common ancestral sequence. The putative ATP binding domain encoded in the fourth exon of the Drosophila MHC gene is highly conserved relative to vertebrate, invertebrate, and non-muscle MHC genes suggesting that each of these myosins bind ATP by the same mechanism. Two divergent copies of the third exon are present within the 5' region of the Drosophila MHC gene, suggesting that alternative splicing produces MHC isoforms with different globular head regions.

Mutations of the Drosophila myosin heavy-chain gene: effects on transcription, myosin accumulation, and muscle function.

Mutations of the myosin heavy-chain (MHC) gene of Drosophila melanogaster were identified among a group of dominant flightless and recessive lethal mutants (map position 2-52, 36A8-B1,2). One mutation is a 0.1-kilobase deletion in the 5' region of the MHC gene and reduces MHC protein in the leg and thoracic muscles of heterozygotes to levels found in 36AC haploids. Three mutations are insertions of 8-to 10-kilobase DNA elements within the MHC gene and produce truncated MHC transcripts. Heterozygotes of these insertional mutations possess levels of MHC intermediate between those of haploids and diploids. An additional mutation has no gross alteration of the MHC gene or its RNA transcripts. Although leg and larval muscles function normally in each mutant heterozygote, indirect flight muscles are defective and possess disorganized myofibrils. Homozygous mutants die during embryonic or larval development and display abnormal muscle function prior to death. These findings provide direct genetic evidence that the MHC gene at 36B (2L) is essential for both larval and adult muscle development and function. The results are consistent with the previous molecular evidence that Drosophila, unlike other organisms, has only a single muscle MHC gene per haploid genome. Quantitative expression of both copies of the MHC gene is required for function of indirect flight muscle, whereas expression of a single MHC gene is sufficient for function of larval muscles and adult tubular muscles.