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 ∼20-kDa (182 amino acids) protein that is expressed exclusively in the IFM (33). Flightin binds the light meromyosin region of myosin, ∼2/3 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 Drosophilaand 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 important roles in maintaining muscle integrity. Flightin is required for normal thick filament assembly and for establishing or maintaining in vivo filament length and flexural rigidity (8, 26). Thick filaments assembled in the absence of flightin are, on average, >30% longer and are 30–45% more flexible than normal filaments (8). Thick filaments lacking flightin are also more prone to in vivo fragmentation, indicating that flightin is essential for their structural integrity (26). Notably, genetic ablation of flightin expression results in complete loss of flight due to structurally and mechanically compromised flight muscles (13, 26). Sarcomere degradation and fiber hypercontraction are often extreme in the absence of flightin, suggesting that flightin fulfills a crucial role in maintaining normal myofilament lattice integrity of the IFM. Thus, flightin's influence spans from the sarcomere to the fiber, both of which are routinely disrupted by contractile forces when flightin is absent or present in reduced amounts (13, 18, 23,26). The remarkable and distinct phenotypes manifested by flightin mutants (3, 8, 13, 23, 26, 31) and mutants that affect flightin expression (13, 18, 23) raise important questions about the functional roles and molecular properties of this unique protein. However, predicting flightin sequences that may fulfill important functional roles is difficult because flightin's amino acid sequence is not similar to any known protein domains. Therefore, we are left with the alternative approach of identifying flightin homologues and making predictions about protein function based on regions of sequence conservation across many species. A comparison of flightin sequences from 12 Drosophila species reveal a tripartite organization (Fig. 1). The three distinct regions appear to be under different evolutionary constraints, raising the possibility that these regions define separate functional domains. In this study, we focus on the COOH-terminal region (amino acids 137 to 182) whose sequence shows an intermediate conservation profile, compared with the poorly conserved NH2-terminal region and the highly conserved midregion. High conservation suggests that the midregion contains critical sequences for flightin's interaction with the filament lattice, and we expect that genetic manipulation of this region would produce a phenotype similar to the flightin null. In contrast, the COOH-terminal region shows strong patterns of conservation only among closely related species (e.g., D. persimilis and D. pseudoobscura), suggesting that its function may be taxon-specific. To investigate the structural and functional influences of flightin's COOH terminus, we generated a new mutant allele by removing 44 COOH-terminal amino acids (Fig. 2). We find that the COOH-terminal truncated flightin associates with thick filaments but that the absence of the COOH terminus reduces myofilament lattice order and sarcomere regularity. Structural changes in IFM fibers with the COOH-terminal truncated flightin lead to decreased cross-bridge binding and reduced power output, which results in flies that are unable to beat their wings.