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 ...
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.
Wild-type and mutant thin filaments were isolated directly from "myosinless" Drosophila indirect flight muscles to study the structural basis of muscle regulation genetically. Negatively stained filaments showed tropomyosin with periodically arranged troponin complexes in electron micrographs. Three-dimensional helical reconstruction of wild-type filaments indicated that the positions of tropomyosin on actin in the presence and absence of Ca(2+) were indistinguishable from those in vertebrate striated muscle and consistent with a steric mechanism of regulation by troponin-tropomyosin in Drosophila muscles. Thus, the Drosophila model can be used to study steric regulation. Thin filaments from the Drosophila mutant heldup(2), which possesses a single amino acid conversion in troponin I, were similarly analyzed to assess the Drosophila model genetically. The positions of tropomyosin in the mutant filaments, in both the Ca(2+)-free and the Ca(2+)-induced states, were the same, and identical to that of wild-type filaments in the presence of Ca(2+). Thus, cross-bridge cycling would be expected to proceed uninhibited in these fibers, even in relaxing conditions, and this would account for the dramatic hypercontraction characteristic of these mutant muscles. The interaction of mutant troponin I with Drosophila troponin C is discussed, along with functional differences between troponin C from Drosophila and vertebrates.
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