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.     

Isolation of 88F Actin Mutants ofDrosophila melanogasterand Possible Alterations in the Mutant Actin Structures

The 88F actin (act88F) gene ofDrosophila melanogasterencodes an actin isoform that is expressed exclusively in the indirect flight muscle. In order to isolate a large number of act88F mutants, an efficient screening method was used to obtain dominant flightless mutants. Genetic analyses revealed that 25 mutations were located near or at the act88F locus. From each mutant strain, the DNA fragments including the coding region of the act88F gene were asymmetrically amplified by the polymerase chain reaction method, and the amplified fragments were directly sequenced. Eighteen of them were found to have point mutations within their coding regions. Of these, 13 were novel alleles of this gene. We have characterised these mutations in detail. First, their flight abilities were tested after introducing two normal alleles of this gene. Second, two-dimensional gel electrophoresis was used to examine actin isoforms and whole thorax proteins. Third, morphological anomalies of indirect flight muscle fibres and myofibrils were examined with an optical microscope. On the basis of these phenotypes and the known atomic structure of actin, possible alterations in the structure of actin brought about by these mutations are discussed.     

Actomyosin kinetics and in vitro motility of wild-type Drosophila actin and the effects of two mutations in the Act88F gene.

Two missense mutations of the flight muscle-specific actin gene of Drosophila melanogaster, Act88F, assemble into normally structured myofibrils but affect the flight ability of flies and the mechanical kinetics of isolated muscle fibers. We describe the isolation of actin from different homozygous Act88F strains, including wild-type, an Act88F null mutant (KM88), and two Act88F single point mutations (E316K and G368E), their biochemical interactions with rabbit myosin subfragment 1 (S1), and behavior with rabbit myosin and heavy meromyosin in in vitro motility assays. The rabbit and wild-type Drosophila actins have different association rate constants with S1 (2.64 and 1.77 microM-1 s-1, respectively) and in vitro motilities (2.51, 1.60 microns s-1) clearly demonstrating an isoform-specific difference. The G368E mutation shows a reduced affinity for rabbit S1 compared with the wild type (increasing from 0.11 to 0.17 microM) and a reduced velocity in vitro (reduced by 19%). The E316K mutant actin has no change in affinity for myosin S1 or in vitro motility with heavy meromyosin but does have a reduced in vitro motility (15%) with myosin. These results are discussed with respect to the recently published atomic models for the actomyosin structure and our findings that G368E fibers show a reduced rate constant for delayed tension development and increased fiber stiffness. We interpret these results as possibly caused either by effects on A1 myosin light chain binding or conformational changes within the subdomain 1 of actin, which contains the myosin binding site. E316K is discussed with respect to its likely position within the tropomyosin binding site of actin.     

Two missense alleles of the Drosophila melanogaster act88F actin gene are strongly antimorphic but only weakly induce synthesis of heat shock proteins.

We have characterized two extant mutations of the flight muscle-specific act88F actin gene of Drosophila melanogaster. Both defective alleles were recovered from flightless mutants isolated previously (K. Mogami and Y. Hotta, Mol. Gen. Genet. 183:409-417, 1981). By directly sequencing the mutant alleles, we demonstrated that in act88FIfm(3)2 a single G-C to A-T transition converted arginine-28 to cysteine and that in act88FIfm(3)4 a single A-T to T-A transversion changed isoleucine-76 to phenylalanine. We showed that the actins encoded by either allele were strongly antimorphic. Mutant alleles effectively disrupted myofibril structure and function in the flight muscles of strains having the diploid complement of wild-type act88F genes. However, unlike antimorphic actins encoded by three previously characterized act88F alleles, neither that encoded by act88FIfm(3)2 nor that encoded by act88FIfm(3)4 was a strong inducer of heat shock protein synthesis.     

Differential Epitope Tagging of Actin in Transformed Drosophila Produces Distinct Effects on Myofibril Assembly and Function of the Indirect Flight Muscle

We have tested the impact of tags on the structure and function of indirect flight muscle (IFM)-specific Act88F actin by transforming mutant Drosophila melanogaster, which do not express endogenous actin in their IFMs, with tagged Act88F constructs. Epitope tagging is often the method of choice to monitor the fate of a protein when a specific antibody is not available. Studies addressing the functional significance of the closely related actin isoforms rely almost exclusively on tagged exogenous actin, because only few antibodies exist that can discriminate between isoforms. Thereby it is widely presumed that the tag does not significantly interfere with protein function. However, in most studies the tagged actin is expressed in a background of endogenous actin and, as a rule, represents only a minor fraction of the total actin. The Act88F gene encodes the only Drosophila actin isoform exclusively expressed in the highly ordered IFM. Null mutations in this gene do not affect viability, but phenotypic effects in transformants can be directly attributed to the transgene. Transgenic flies that express Act88F with either a 6x histidine tag or an 11-residue peptide derived from vesicular stomatitis virus G protein at the C terminus were flightless. Overall, the ultrastructure of the IFM resembled that of the Act88F null mutant, and only low amounts of C-terminally tagged actins were found. In contrast, expression of N-terminally tagged Act88F at amounts comparable with that of wild-type flies yielded fairly normal-looking myofibrils and partially reconstituted flight ability in the transformants. Our findings suggest that the N terminus of actin is less sensitive to modifications than the C terminus, because it can be tagged and still polymerize into functional thin filaments.     

Variable N-terminal regions of muscle myosin heavy chain modulate ATPase rate and actin sliding velocity

We integratively assessed the function of alternative versions of a region near the N terminus of Drosophila muscle myosin heavy chain (encoded by exon 3a or 3b). We exchanged the alternative exon 3 regions between an embryonic isoform and the indirect flight muscle isoform. Each chimeric myosin was expressed in Drosophila indirect flight muscle, in the absence of other myosin isoforms, allowing for purified protein analysis and whole organism locomotory studies. The flight muscle isoform generates higher in vitro actin sliding velocity and solution ATPase rates than the embryonic isoform. Exchanging the embryonic exon 3 region into the flight muscle isoform decreased ATPase rates to embryonic levels but did not affect actin sliding velocity or flight muscle ultrastructure. Interestingly, this swap only slightly impaired flight ability. Exchanging the flight muscle-specific exon 3 region into the embryonic isoform increased actin sliding velocity 3-fold and improved indirect flight muscle ultrastructure integrity but failed to rescue the flightless phenotype of flies expressing embryonic myosin. These results suggest that the two structural versions of the exon 3 domain independently influence the kinetics of at least two steps of the actomyosin cross-bridge cycle.     

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.     

Assembly of thick filaments and myofibrils occurs in the absence of the myosin head

We investigated the importance of the myosin head in thick filament formation and myofibrillogenesis by generating transgenic Drosophila lines expressing either an embryonic or an adult isoform of the myosin rod in their indirect flight muscles. The headless myosin molecules retain the regulatory light-chain binding site, the alpha-helical rod and the C-terminal tailpiece. Both isoforms of headless myosin co-assemble with endogenous full-length myosin in wild-type muscle cells. However, rod polypeptides interfere with muscle function and cause a flightless phenotype. Electron microscopy demonstrates that this results from an antimorphic effect upon myofibril assembly. Thick filaments assemble when the myosin rod is expressed in mutant indirect flight muscles where no full-length myosin heavy chain is produced. These filaments show the characteristic hollow cross-section observed in wild type. The headless thick filaments can assemble with thin filaments into hexagonally packed arrays resembling normal myofibrils. However, thick filament length as well as sarcomere length and myofibril shape are abnormal. Therefore, thick filament assembly and many aspects of myofibrillogenesis are independent of the myosin head and these processes are regulated by the myosin rod and tailpiece. However, interaction of the myosin head with other myofibrillar components is necessary for defining filament length and myofibril dimensions.     

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.     

Actin residue glu(93) is identified as an amino acid affecting myosin binding.

Many mutants have been described that affect the function of the actin encoded by the Drosophila melanogaster indirect flight muscle-specific actin gene, Act88F. We describe the development of procedures for purification of this actin from the other isoforms expressed in the fly as well as in vitro motility, single molecule force/displacement measurements, and stop-flow solution kinetic studies of the wild-type actin and that of the E93K mutation of the Act88F gene. We show that this mutation affects in vitro motility of F-actin, in both the presence and absence of methylcellulose, and the ability of the ACT88F actin to bind the S1 fragment of rabbit skeletal myosin. However, optical tweezer measurements of the actomyosin working stroke and the force transmitted from the rabbit heavy meromyosin to and through F-actin are unchanged by the mutation. These results support the proposal (Holmes, K. C. (1995) Biophys J. 68, (suppl.) 2-7) that actin residue Glu(93) is part of the secondary myosin binding site and suggest that myosin binding occurs first at the primary myosin binding site and then at the secondary site.     

Actin with tumor-related mutation is antimorphic in Drosophila muscle: two distinct modes of myofibrillar disruption by antimorphic actins

A mutant beta-actin with an amino acid substitution from Gly-245 to Asp has been shown to be related to tumorigenic transformation of a human fibroblast cell line (Leavitt, J. et al. (1987) Mol. Cell. Biol. 7, 2467-2476). To examine the effects of this mutation, we artificially introduced the same amino acid change into the Act88F actin gene of Drosophila melanogaster. The gene (Act88FGD245) was inserted in the Drosophila genome to make transgenic adult flies which synthesize the mutant actin in the indirect flight muscles. The mutant actin was found to be antimorphic with regard to flight and also to cause myofibrillar disruption in transformants even in the presence of two normal alleles. It was initially incorporated into myofibrils and later induced their degeneration from center to periphery. This mode of myofibrillar disruption is distinct from that of previously reported Act88F mutations, where defects are found only in the peripheral region of myofibrils. This indicates that actin functions are altered differently in the two classes of antimorphic mutations.     

Suppression of muscle hypercontraction by mutations in the myosin heavy chain gene of Drosophila melanogaster

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 Ca(2+)-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 Ca(2+) 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-up(2) (hdp(2)), 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.     

Kinetic analysis of Drosophila muscle myosin isoforms suggests a novel mode of mechanochemical coupling

The molecular mechanism of myosin function was addressed by measuring transient kinetic parameters of naturally occurring and chimeric Drosophila muscle myosin isoforms. We assessed the native embryonic isoform, the native indirect flight muscle isoform, and two chimeric isoforms containing converter domains exchanged between the indirect flight muscle and embryonic isoforms. Myosin was purified from the indirect flight muscles of transgenic flies, and S1 was produced by alpha-chymotryptic digestion. Previous studies in vertebrate and scallop myosins have shown a correlation between actin filament velocity in motility assays and cross-bridge detachment rate, specifically the rate of ADP release. In contrast, our study showed no correlation between the detachment rate and actin filament velocity in Drosophila myosin isoforms and further that the converter domain does not significantly influence the biochemical kinetics governing the detachment of myosin from actin. We suggest that evolutionary pressure on a single muscle myosin gene may maintain a fast detachment rate in all isoforms. As a result, the attachment rate and completion of the power stroke or the equilibrium between actin.myosin.ADP states may define actin filament velocity for these myosin isoforms.     

Actin gene mutations in Drosophila; heat shock activation in the indirect flight muscles

We have identified four mutations affecting the actin III isoform in the indirect flight muscles (IFM) of Drosophila. One mutation does not produce any protein product, and three direct the synthesis of electrophoretic variants of actin. Complementation tests and recombination mapping indicate that all mutations are alleles of an actin gene at chromosomal band 88F (act88F gene). The effect of these mutations is restricted to the IFM. We conclude that the act88F gene is expressed only in the IFM to encode actin III, which is its major isoform. In two of the actin mutants, heat shock proteins are constitutively expressed in the IFM. Genetic evidence strongly suggest that this anomaly is primarily caused by the mutations in the act88F structural gene.     

Functions of the myosin ATP and actin binding sites are required for C. elegans thick filament assembly

We have determined the positions and sequences of 31 dominant mutations affecting a C. elegans muscle myosin heavy chain gene. These mutations alter thick filament structure in heterozygotes by interfering with the ability of wild-type myosin to assemble into stable thick filaments. These assembly-disruptive mutations are missense alleles affecting the globular head of myosin. The most strongly dominant alleles alter highly conserved residues of the myosin ATP binding site, indicating that functions of the myosin ATPase are important for thick filament assembly. Other alleles alter the site at which myosin binds actin.     

The flightless Drosophila mutant raised has two distinct genetic lesions affecting accumulation of myofibrillar proteins in flight muscles

We have used a combination of histological, molecular, and genetic techniques to investigate the flightless Drosophila mutant raised. Electron microscopy of indirect flight muscles of raised homozygotes confirms that they are grossly abnormal, lacking thin filaments and Z discs. These defects correspond to aberrant protein accumulation in thoraces, where several myofibrillar components are reduced or absent. Utilizing the germ-line transformation technique we demonstrate that one genetic lesion associated with the raised phenotype resides within the act88F actin gene, which, as a result, fails to specify normal mRNA accumulation during thoracic muscle differentiation. We also provide evidence for a distinct second genetic lesion, which apparently eliminates proper posttranslational modification of two myofibrillar proteins, one of which is actin.     

Heat shock gene activation by mutant actin is independent of myofibril degeneration in Drosophila muscle

Artificially mutagenized Drosophila Act88F actin genes with triple and double mutations were expressed in the indirect flight muscles of transgenic flies. The triple mutant actin, GD245T (Gly-36----Glu, Glu-83----Asp, and Gly-245----Asp), induced heat shock protein (hsp) synthesis without affecting flight ability. On the other hand, the double mutation, GD245D (Gly-36----Glu and Glu-83----Asp), disrupted myofibrils but induced little hsp synthesis. These results demonstrate that myofibril degeneration is not the primary cause of the anomalous heat shock gene activation by mutant actins.