COOH-terminal truncation of flightin decreases myofilament lattice organization, cross-bridge binding, and power output in Drosophila indirect flight muscle

Abstract

insects is characterized by a near crystalline myofilament lattice structure that likely evolved to achieve high power output. In Drosophila IFM, the myosin rod binding protein flightin plays a crucial role in thick filament organization and sarcomere integrity. Here we investigate the extent to which the COOH terminus of flightin contributes to IFM structure and mechanical performance using transgenic Drosophila expressing a truncated flightin lacking the 44 COOH-terminal amino acids (fln ⌬C44 ). Electron microscopy and Xray diffraction measurements show decreased myofilament lattice order in the fln ⌬C44 line compared with control, a transgenic flightinnull rescued line (fln ϩ ). fln ⌬C44 fibers produced roughly 1/3 the oscillatory work and power of fln ϩ , with reduced frequencies of maximum work (123 Hz vs. 154 Hz) and power (139 Hz vs. 187 Hz) output, indicating slower myosin cycling kinetics. These reductions in work and power stem from a slower rate of cross-bridge recruitment and decreased cross-bridge binding in fln ⌬C44 fibers, although the mean duration of cross-bridge attachment was not different between both lines. The decreases in lattice order and myosin kinetics resulted in fln ⌬C44 flies being unable to beat their wings. These results indicate that the COOH terminus of flightin is necessary for normal myofilament lattice organization, thereby facilitating the cross-bridge binding required to achieve high power output for flight. fiber mechanics; cross-bridge kinetics; thick filaments IN MUSCLE, THE THICK AND THIN filament lattice provides the structural and mechanical foundation for transmitting contractile forces throughout the cell. The highly ordered indirect flight muscle (IFM) of Drosophila melanogaster is an attractive model system to study the relationship between lattice structure and muscle function, because its in vivo lattice organization can be measured via X-ray diffraction in living flies (15) and its function can be measured from the whole fly to the molecule (14,20,30). In addition, the means for producing genetic alterations of specific proteins in D. melanogaster are well established, permitting precise manipulation of thick and thin filament proteins. In this study, we combine these approaches to define the role of flightin, specifically the COOH terminus, in lattice organization and its effects on cross-bridge cycling kinetics and overall muscle performance.In Drosophila, flightin is a ϳ20-kDa (182 amino acids) protein that is expressed exclusively in the IFM (33). Flightin binds the light meromyosin region of myosin, ϳ2/3 of the way down the rod, because substituting aspartic acid 1554 for lysine abolishes flightin's interaction in vitro (1) and accumulation in vivo (18). Immunolocalization studies in Drosophila and Lethocerus IFM indicate that flightin is associated with the thick filament backbone (25,26), consistent with studies that show flightin is absent in IFM lacking thick filaments (29).Studies using Drosophila mutants demonstrate that flightin plays several importa...

Flightin maintains myofilament lattice organization required for optimal flight power and courtship song quality inDrosophila

“…Contrary to deletion of the N-terminal domain, deletion of the C-terminal domain affects thick filament length but not stiffness ( persistence length [28]) nor passive skinned fibre stiffness (elastic modulus [22]). These results reveal a clear demarcation of functional roles between the N-terminal and the C-terminal domains and confirm that the N-terminal domain has a prominent role in maintaining thick filament stiffness and myofilament lattice geometry.…”

“…A comparison of the results of this study with those that examined the properties of fln DC44 [22] provides important insight into the structure-function relation in flightin. Upon skinning, flightin remained associated with fln DN62 and fln DC44 muscle fibres, indicating that neither the N-terminal domain nor the C-terminal domain is required for myosin binding [18].…”

“…Preparation of muscle fibres from 2-3 days-old flies, solutions used, mechanical measurements and curve fitting were carried out as in previous studies [22,26] with some modifications as described in electronic supplementary material, Method 9.…”

“…The structural, physiological and flight phenotypes are fully rescued by a wild-type flightin transgene [21], further evidence of the strict flightin requirement for IFM function. By contrast, a truncated flightin transgene lacking the COOH-terminal 44 amino acids ( fln DC44 ) only partially rescues the structural and mechanical defects evident in fln 0 IFMs and is unable to rescue flightlessness [22].…”

Intrinsic disorder and multiple phosphorylations constrain the evolution of the flightin N-terminal region

“…Characterization of flightin mutants in Drosophila melanogaster has revealed how flightin function manifests across levels of biological hierarchy by contributing to length determination and flexural rigidity of thick filaments, sarcomere organization, myofibril integrity, fiber contractile mechanics, and flight behavior [9, 12–18]. Phylogenetic and protein sequence analyses revealed that flightin is endemic to Pancrustacea (Hexapoda + Crustacea) and led to the identification of WYR, a novel ~52 amino acid protein domain characterized by strictly conserved tryptophan (W) and arginine (R) sites and a high content of tyrosines (Y) [19].…”