Expression and function of the Drosophila ACT88F actin isoform is not restricted to the indirect flight muscles.

Most higher eukaryotic genomes contain multiple actin genes, yet the sequence differences between isoforms are few. In Drosophila melanogaster it was previously established that one of the six actin genes, Act88F, is expressed only in the indirect flight muscles (IFMs). These muscles are highly specialised for oscillatory contractions to power flight. The implication was that this isoform had tissue-specific properties. In this paper we show using two reporter constructs expressing either beta-galactosidase, Act88F-lacZ, or the green fluorescent protein, Act88F-GFP, that the Act88F promoter is active in a small number of other muscles, including leg (femoral) and uterine muscles. However, the levels of Act88F driven non-IFM expression are much less than in the IFMs. We have confirmed endogenous Act88F gene expression in these other muscles by in situ hybridisation studies. Using null and antimorphic mutants to show decreased walking ability and delayed/reduced oviposition we demonstrated that Act88F expression is functionally important in multiple muscle groups. Since the mutant effects are mild, this supports the expectation that other actin genes are also expressed in these muscles. The Act88F-GFP promoter-reporter also detects Act88F-driven expression in the bristle-forming cells in the pupal wings. The implications of these results for the functions and developmental expression of the Drosophila ACT88F isoform are discussed.

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).

Drosophila ACT88F indirect flight muscle-specific actin is not N-terminally acetylated: a mutation in N-terminal processing affects actin function.

Many eukaryotic proteins are co and post-translationally modified at their N termini by removal of one or two amino acid residues and N(alpha)-acetylation. Actins show two different forms of N-terminal processing dependent on their N-terminal sequence. In class II actins, which include muscle actins, the common primary sequence of Met-Cys-Asp-actin is processed to acetyl-Asp-actin. The functional significance of this in vivo is unknown. We have studied the indirect flight muscle-specific actin, ACT88F, of Drosophila melanogaster. Our results show that ACT88F is N-terminally processed in vivo as a class II actin by removal of the first two amino acid residues (Met and Cys), but that uniquely the N terminus is not acetylated. In addition we show that ACT88F is methylated, probably at His73. Flies carrying the mod(-) mutation fail to complete post-translational processing of ACT88F. We propose that the mod gene product is normally responsible for removing N-acetyl-cysteine from actin. The biological significance of this process is demonstrated by observations that retention of the N-acetyl-cysteine in ACT88F affects the flight muscle function of mod(-) flies. This suggests that the extreme N terminus affects actomyosin interactions in vivo, a proposal we have examined by in vitro motility assays of ACT88F F-actin from mod(-) flies. The mod(-) actin only moves in the presence of methylcellulose, a viscosity-enhancing agent, where it moves at velocities slightly, but significantly, reduced compared to wild-type. These data confirm that N-acetyl-cysteine at the N terminus affects actomyosin interactions, probably by reducing formation of the initial actomyosin collision complex, a process known to involve the actin N terminus.

A direct screen identifies new flight muscle mutants on the Drosophila second chromosome.

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.