Genetics of the Drosophila flight muscle myofibril: a window into the biology of complex systems.

This essay reviews the long tradition of experimental genetics of the Drosophila indirect flight muscles (IFM). It discusses how genetics can operate in tandem with multidisciplinary approaches to provide a description, in molecular terms, of the functional properties of the muscle myofibril. In particular, studies at the interface of genetics and proteomics address protein function at the cellular scale and offer an outstanding platform with which to elucidate how the myofibril works. Two generalizations can be enunciated from the studies reviewed. First, the study of mutant IFM proteomes provides insight into how proteins are functionally organized in the myofibril. Second, IFM mutants can give rise to structural and contractile defects that are unrelated, a reflection of the dual function that myofibrillar proteins play as fundamental components of the sarcomeric framework and biochemical "parts" of the contractile "engine".

Role of the elastic protein projectin in stretch activation and work output of Drosophila flight muscles.

We examine how the stretch activation response of the Drosophila indirect flight muscles (IFM) is affected by the projectin mutation bentDominant. IFM from flies heterozygous for this mutation (bentD/+) produce approximately 85% full length projectin and approximately 15% truncated projectin lacking the kinase domain and more C-terminal sequences. Passive stiffness and power output of mutant fibers is similar to that of wild-type (+/+) fibers, but the amplitude of the stretch activation response (delayed tension rise) was significantly reduced. Measurement of actomyosin kinetics by sinusoidal analysis revealed that the apparent rate constant of the delayed tension rise (2 pi b) increased in proportion to the decrease in amplitude, accounting for the near wild-type levels of power output and nearly normal flight ability. These results suggest that projectin plays a crucial role in stretch activation, possibly through its protein kinase activity, by modulating crossbridge recruitment and kinetics.

The effect of removing the N-terminal extension of the Drosophila myosin regulatory light chain upon flight ability and the contractile dynamics of indirect flight muscle.

The Drosophila myosin regulatory light chain (DMLC2) is homologous to MLC2s of vertebrate organisms, except for the presence of a unique 46-amino acid N-terminal extension. To study the role of the DMLC2 N-terminal extension in Drosophila flight muscle, we constructed a truncated form of the Dmlc2 gene lacking amino acids 2-46 (Dmlc2(Delta2-46)). The mutant gene was expressed in vivo, with no wild-type Dmlc2 gene expression, via P-element-mediated germline transformation. Expression of the truncated DMLC2 rescues the recessive lethality and dominant flightless phenotype of the Dmlc2 null, with no discernible effect on indirect flight muscle (IFM) sarcomere assembly. Homozygous Dmlc2(Delta2-46) flies have reduced IFM dynamic stiffness and elastic modulus at the frequency of maximum power output. The viscous modulus, a measure of the fly's ability to perform oscillatory work, was not significantly affected in Dmlc2(Delta2-46) IFM. In vivo flight performance measurements of Dmlc2(Delta2-46) flies using a visual closed-loop flight arena show deficits in maximum metabolic power (P(*)(CO(2))), mechanical power (P(*)(mech)), and flight force. However, mutant flies were capable of generating flight force levels comparable to body weight, thus enabling them to fly, albeit with diminished performance. The reduction in elastic modulus in Dmlc2(Delta2-46) skinned fibers is consistent with the N-terminal extension being a link between the thick and thin filaments that is parallel to the cross-bridges. Removal of this parallel link causes an unfavorable shift in the resonant properties of the flight system, thus leading to attenuated flight performance.

Flightin is essential for thick filament assembly and sarcomere stability in Drosophila flight muscles.

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, fln(0), that results in loss of flight ability but has no effect on fecundity or viability. Electron microscopy comparing pupa and adult fln(0) IFM shows that sarcomeres, and thick and thin filaments in pupal IFM, are 25-30% longer than in wild type. fln(0) 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 fln(0) 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.

The Drosophila projectin mutant, bentD, has reduced stretch activation and altered indirect flight muscle kinetics.

Projectin is a ca. 900 kDa protein that is a member of the titin protein superfamily. In skeletal muscle titins are involved in the longitudinal reinforcement of the sarcomere by connecting the Z-band to the M-line. In insect indirect flight muscle (IFM), projectin is believed to form the connecting filaments that link the Z-band to the thick filaments and is responsible for the high relaxed stiffness found in this muscle type. The Drosophila mutant bentD (btD) has been shown to have a breakpoint close to the carboxy-terminal kinase domain of the projectin sequence. Homozygotes for btD are embryonic lethal but heterozygotes (btD/+) are viable. Here we show that btD/+ flies have normal flight ability and a slightly elevated wing beat frequency (btD/+ 223+/-13 Hz; +/+ 203+/-5 Hz, mean +/- SD; P < 0.01). Electron microscopy of btD/+ IFM show normal ultrastructure but skinned fiber mechanics show reduced stretch activation and oscillatory work. Although btD/+ IFM power output was at wild-type levels, maximum power was achieved at a higher frequency of applied length perturbation (btD/+ 151+/-6 Hz; +/+ 102+/-14 Hz; P < 0.01). Results were interpreted in the context of a viscoelastic model of the sarcomere and indicate altered cross-bridge kinetics of the power-producing step. These results show that the btD mutation reduces oscillatory work in a way consistent with the proposed role of the connecting filaments in the stretch activation response of IFM.

A genetic deficiency that spans the flightin gene of Drosophila melanogaster affects the ultrastructure and function of the flight muscles.

We have developed a reverse-genetic approach to study the function of flightin, a unique protein of the flight muscle myofibril of Drosophila melanogaster. We describe the generation and characterization of Df(3L)fln1, a lethal genetic deficiency in the 76BE region of the third chromosome which deletes several genes, including the gene for flightin. We show that heterozygous flies harboring the Df(3L)fln1 mutation exhibit both impaired flight and ultrastructural defects in their flight muscle myofibrils. We found that the mutation does not interfere with assembly of the myofibril but leads to disorganization of peripheral myofilaments in adult myofibrils. Most myofibrils, nevertheless, retain an intact core that represents approximately 80 % of the normal lattice diameter. Mechanical analysis of single skinned flight muscle fibers demonstrates that the mutation has no significant effect on net power output but increases the frequency at which maximum power is delivered to the wings, potentially reducing the overall performance of the flight system. The results suggest that flightin is an indispensable part of the flight muscle contractile mechanism.

Phosphorylation-dependent power output of transgenic flies: an integrated study.

We examine how the structure and function of indirect flight muscle (IFM) and the entire flight system of Drosophila melanogaster are affected by phosphorylation of the myosin regulatory light chain (MLC2). This integrated study uses site-directed mutagenesis to examine the relationship between removal of the myosin light chain kinase (MLCK) phosphorylation site, in vivo function of the flight system (flight tests, wing kinematics, metabolism, power output), isolated IFM fiber mechanics, MLC2 isoform pattern, and sarcomeric ultrastructure. The MLC2 mutants exhibit graded impairment of flight ability that correlates with a reduction in both IFM and flight system power output and a reduction in the constitutive level of MLC2 phosphorylation. The MLC2 mutants have wild-type IFM sarcomere and cross-bridge structures, ruling out obvious changes in the ultrastructure as the cause of the reduced performance. We describe a viscoelastic model of cross-bridge dynamics based on sinusoidal length perturbation analysis (Nyquist plots) of skinned IFM fibers. The sinusoidal analysis suggests the high power output of Drosophila IFM required for flight results from a phosphorylation-dependent recruitment of power-generating cross-bridges rather than a change in kinetics of the power generating step. The reduction in cross-bridge number appears to affect the way mutant flies generate flight forces of sufficient magnitude to keep them airborne. In two MLC2 mutant strains that exhibit a reduced IFM power output, flies appear to compensate by lowering wingbeat frequency and by elevating wingstroke amplitude (and presumably muscle strain). This behavioral alteration is not seen in another mutant strain in which the power output and estimated number of recruited cross-bridges is similar to that of wild type.

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.

Multiple isoelectric variants of flightin in Drosophila stretch-activated muscles are generated by temporally regulated phosphorylations.

Drosophila stretch-activated flight muscles contain flightin, a novel myofibrillar protein that interacts with myosin filaments. We have identified eleven flightin isoelectric variants that can be subdivided into phosphorylated and non-phosphorylated subclasses. Flight muscles of late pupal stage P15, at which time myofibrillogenesis has been completed but the muscle has yet to be used, contain primarily non-phosphorylated variants. A dramatic increase in flightin phosphorylation occurs subsequent to eclosion. As the young adult matures, increasingly phosphorylated variants are generated following a precise ontogenetic progression. Adults 5-6 h old and older contain the entire set of flightin isoelectric variants. All nine phosphovariants remain metabolically active throughout adult life as evidenced by their ability to incorporate radioactive phosphate in older adults. Our results suggest the possibility that all nine phosphorylated variants originate from a single precursor by sequential phosphorylation. Phosphorylation of flightin may thus serve both structural and regulatory functional roles.

Alterations in flightin phosphorylation in Drosophila flight muscles are associated with myofibrillar defects engendered by actin and myosin heavy-chain mutant alleles.

Flightin is a 20-kD myofibrillar protein found in the stretch-activated flight muscles of Drosophila melanogaster. Nine of the eleven isoelectric variants of flightin are generated in vivo by multiple phosphorylations. The accumulation of these isoelectric variants is affected differently by mutations that eliminate thick filaments or thin filaments. Mutations in the myosin heavy-chain gene that prevent thick filament assembly block accumulation of all flightin variants except N1, the unphosphorylated precursor, which is present at much reduced levels. Mutations in the flight muscle-specific actin gene that block actin synthesis and prevent thin filament assembly disrupt the temporal regulation of flightin phosphorylation, resulting in premature phosphorylation and premature accumulation of flightin phosphovariants. Cellular fractionation of fibers that are devoid of thin filaments show that flightin remains associated with the thick filament-rich cytomatrix. These results suggest that flightin is a structural component of the thick filaments whose regulated phosphorylation is dependent upon the presence of thin filaments.

The muscle Z band: lessons in stress management.

Two of the most characteristic features of striated muscle are (i) its ability to contract and generate tension when activated and (ii) its ability to return to its original length and form after contraction or stretching ceases. These two properties are to a large extent the primary manifestations of separate sets of filament systems: contractile actin and myosin filaments and viscoelastic titin and intermediate filaments. Z bands function as a common link that mechanically integrates contractile and elastic elements and as such they play a fundamental role in transmission of active and passive forces. Differences in Z band structure have been described for distinct classes of muscle and fibre types. The diversity in Z band architecture has been built around its phylogenetically conserved role as an actin-anchoring structure. Novel proteins are likely to account for structural and functional differences seen across the phyla.

Flightin, a novel myofibrillar protein of Drosophila stretch-activated muscles.

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.

Structurally different Drosophila striated muscles utilize distinct variants of Z-band-associated proteins.

Monoclonal antibodies raised against four proteins from insect asynchronous flight muscle have been used to characterize the cross-reacting proteins in synchronous muscle of Drosophila melanogaster. Two proteins, alpha-actinin and Z(400/600), are found at the Z-band of every muscle examined. A larger variant of alpha-actinin is specific for the perforated Z-bands of supercontractile muscle. A third Z-band protein, Z(210), has a very limited distribution. It is found only in the asynchronous muscle and in the large cells of the jump muscle (tergal depressor of the trochanter). The absence of Z(210) from the anterior four small cells of the jump muscle demonstrates that cells within the same muscle do not have identical Z-band composition. The fourth protein, projectin, greater than 600 kDa polypeptide component of the connecting filaments in asynchronous muscle, is also detected in all synchronous muscles studied. Surprisingly, projectin is detected in the region of the thick filaments in synchronous muscles, rather than between the thick filaments and the Z-band, as in asynchronous muscles. Despite their different locations, the projectins of synchronous and asynchronous muscles are very similar, but not identical, as judged by SDS-PAGE and by peptide mapping. Projectin shows immunological cross-reactivity with twitchin, a nematode giant protein that is a component of the body wall A-band and shares similarities with vertebrate titin.

Characterization of components of Z-bands in the fibrillar flight muscle of Drosophila melanogaster.

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.

Alternative 5C actin transcripts are localized in different patterns during Drosophila embryogenesis.

The Drosophila actin gene located at cytogenetic position 5C forms at least 9 and perhaps as many as 15 different transcripts with the use of alternative transcriptional start points, differential splicing, and different regions of cleavage/polyadenylation. Each transcript contains one of two alternative 5' exons. We have subcloned unique recombinant DNA probes specific for each separate 5' exon and for three polyadenylation regions into vectors containing T3 and T7 promoters. Single stranded, tritium-labeled RNA probes were generated in vitro from these constructs. These probes have been hybridized in situ to RNA transcripts present in tissue sections from Drosophila embryos. The results of these experiments indicate that transcripts homologous to the two separate 5' exon-specific probes accumulate in strikingly different patterns during Drosophila development. Thus the incorporation of a particular 5' exon into a transcript is correlated with tissue-specific localization of that transcript. In contrast, probes for each of the three polyadenylation regions do not detect any tissue-specific localization of transcripts.

Stage-specific selection of alternative transcriptional initiation sites from the 5C actin gene of Drosophila melanogaster.

The transcription unit of the 5C actin gene exhibits a complex organization that is unique among the six actin genes of Drosophila melanogaster. Three different mRNA size classes showing distinct patterns of accumulation throughout development are detected on Northern blots. We have determined the structure of the various 5C actin transcripts by exon mapping using strand-specific RNA probes, primer extension analysis, and DNA sequences analysis of both cDNA and genomic clones. All the transcripts share a single protein-coding nucleotide sequence but are heterogeneous in the 5' and 3' untranslated regions. The 5' untranslated region of each transcript consists of either one of two small exons (exon 1 and exon 2) which are alternatively spliced to a single acceptor site 8 bp upstream from the translation initiation codon in exon 3. Results from primer extension analysis suggest that transcription can initiate from either exon 1 or exon 2, and also from a third site within exon 2. We detect an increase in the relative abundance of exon 1-containing transcripts at larval and pupal stages, as well as a change in the proportion of transcripts that initiate at either of the two exon 2 sites. Five polyadenylation sites have been found within three termination/processing regions that define the three size classes of polyadenylated transcripts. The results of our experiments indicate the existence in vivo of all possible combinations of 5' exon with 3' polyadenylation site. However, particular combinations of 5' initiation site and 3' polyadenylation site are preferred at certain developmental stages.