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, fln0, that results in loss of flight ability but has no effect on fecundity or viability. Electron microscopy comparing pupa and adult fln0 IFM shows that sarcomeres, and thick and thin filaments in pupal IFM, are 25–30% longer than in wild type. fln0 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 fln0 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.

Abstract. 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-hand 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 c~:1-4 recognize a 90-100-kD protein which we identify as o~-actinin on the basis of cross-reactivity with antibodies raised against honeybee and vertebrate ot-actinins. Monoclonal antibodies P:l-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 (,o400/600 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 ot-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 ol-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.T HE Z-band is an electron-dense structural component of striated muscle. It serves as an attachment site for thin filaments and transmits tension between neighboring sarcomeres during contraction. Electron micrographs of both vertebrate muscle and insect fibrillar muscle show Z-bands with a highly ordered, almost crystalline, appearance in cross section (for reviews see 1, 4, 12, 40, 42). Several Z-band proteins have been identified from both vertebrate and insect species (2, 3, 6, 7, 18, 20-22, 25, 29, 30, 33-38); however, the manner in which these proteins are organized within the Z-band lattice is poorly understood. Moreover, the developmental programs that lead to the early organization of the Z-band are only beginning to be clarified.The study of insect Z-bands has been carried out primarily on the flight muscles of the honeybee (Apis) and the giant water bug (Lethocerus). These insects are particularly favorable for biochemical studies of muscle because their size and the predominance of the flight muscle permit isolation of reasonable amounts of homogeneous muscle tissue. The much smaller size of Drosophila presents obstacles for biochemical analyses but facilitates the genetic analyses that are proving to be another useful approach to the study of muscle structure ...

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.

Abstract. 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; pls, 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.T HE indirect flight muscles (IFM) ~ of Drosophila melanogaster are a group of thoracic fibers whose oscillatory contractions power wing beats at high frequencies, far greater than the firing rate of motor nerves. With steady neural input intracellular calcium levels remain high in the IFM, and the muscle responds to slight changes in length with delayed changes in tension (35,51). This property, called stretch activation, allows the IFM to drive the mechanically resonant wing/thorax system at its very high natural frequency. Since skinned fibers also do oscillatory work in the presence of calcium and ATE it is assumed that components of the myofibril itself are responsible for this adaptation (18).The mechanism underlying stretch activation is not known. Wray (64) has suggested that a slight longitudinal displacement of thick and thin filaments increases the number of cross-bridges recruited by optimally aligning the matching actin and myosin filament periodicities. Recently, however, Squire (45) has challenged this hypothesis on the basis of detailed analysis of filament organization. Alternative proposals are that longitudinal displacement of filaments might influence tension development by changing the angle of attachment of cross-bridges bound to actin (45, 51, 52), J. Vigoreaux's present address is Department of Zoology, University of Vermont, Burlington, VT 05405-0086.

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.

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We investigated the effects of aging on Drosophila melanogaster indirect flight muscle from the whole organism to the actomyosin cross-bridge. Median-aged (49-day-old) flies were flight impaired, had normal myofilament number and packing, barely longer sarcomeres, and slight mitochondrial deterioration compared with young (3-day-old) flies. Old (56-day-old) flies were unable to beat their wings, had deteriorated ultrastructure with severe mitochondrial damage, and their skinned fibers failed to activate with calcium. Small-amplitude sinusoidal length perturbation analysis showed median-aged indirect flight muscle fibers developed greater than twice the isometric force and power output of young fibers, yet cross-bridge kinetics were similar. Large increases in elastic and viscous moduli amplitude under active, passive, and rigor conditions suggest that median-aged fibers become stiffer longitudinally. Small-angle x-ray diffraction indicates that myosin heads move increasingly toward the thin filament with age, accounting for the increased transverse stiffness via cross-bridge formation. We propose that the observed protein composition changes in the connecting filaments, which anchor the thick filaments to the Z-disk, produce compensatory increases in longitudinal stiffness, isometric tension, power and actomyosin interaction in aging indirect flight muscle. We also speculate that a lack of MgATP due to damaged mitochondria accounts for the decreased flight performance.

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".