Journal of Muscle Research and Cell Motility 15, 607-616 (1994) Multiple isoelectric variants of flightin in Drosophila stretch-activated muscles are generated by temporally regulated phosphorylations JIM O. VIGOREAUX* AND LOUISE M. PERRY • Department of Zoology, University of VermonL Burlington, VT 05405, USA Received 25 April 1994; revised 23 May 1994; accepted 24 May 1994 Summary 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 edosion. 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. Introduction Dipterans such as Drosophila melanogaster are capable of beating their wings at rates approaching 200 Hz. The muscles responsible for these very rapid wing movements are the indirect flight muscles (IFM) which occupy a large percentage of the thoracic volume. Numerous studies over several decades have shown that the physiological properties of insect IFM share many features in common with other muscle types (Tregear, 1977). Like all other muscles, calcium is needed for activation of the IFM, but unlike other muscle types, calcium activation results in low tension and low ATPase activity. Maximal activation occurs only after a slight length extension of approximately 3-8% over rest length (Pringle, 1978). Stretch results in a delayed increase in tension which, in the living insect, initiates oscillatory contractions (Thorson & White, 1969). This phenomenon, known as stretch-activation, is present in all muscles; however, only in insect fibrillar flight muscle and mammalian heart muscle is its effect large enough (both in amplitude and duration) to be functionally significant (Pringle, 1978; Steiger, 1971; *To whom correspondence should be addressed. 0142-4319 © 1994 Chapman & Hall Abbott & Steiger, 1977). The IFM is also classified as asynchronous since the frequency of wing beats is not synchronized with the frequency of nerve impulses (Pringle, 1978). The ability of skinned IFM fibres to contract and do work at constant Ca 2+ concentration (independent of nerve impulses) suggests that com- ponents within the fibre itself are responsible for stretch- activation (Jewell & Ruegg, 1966). The presence of different myofibrillar protein isoelectric variants and/or novel protein components in the IFM could explain, at least in part, the distinct mechanics of this muscle type. In recent years novel insect myofibrillar proteins have been identified in the IFM and implicated in stretch- activation. Two of the better characterized components are arthrin, a stable ubiquinated actin (Ball et al., 1987), and troponin H, a modified tropomyosin (Bullard et al., 1988). Their functions, however, remain to be elucidated. More recently, we have identified flightin, an IFM-specific protein that, unlike arthrin and troponin H, does not appear to be related to any known muscle protein (Vigoreaux et al., 1993). This novel myofibrillar protein may thus perform a critical role in defining the mechanical properties of stretch-activated muscles.