Alp/Enigma family members have a unique PDZ domain followed by zero to four LIM domains, and are essential for myofibril assembly across all species analyzed so far. Drosophila melanogaster has three Alp/Enigma family members, Zasp52, Zasp66, and Zasp67. Ortholog search and phylogenetic tree analysis suggest that Zasp genes have a common ancestor, and that Zasp66 and Zasp67 arose by duplication in insects. While Zasp66 has a conserved domain structure across orthologs, Zasp67 domains and lengths are highly variable. In flies, Zasp67 appears to be expressed only in indirect flight muscles, where it colocalizes with Zasp52 at Z-discs. We generated a CRISPR null mutant of Zasp67, which is viable but flightless. We can rescue all phenotypes by re-expressing a Zasp67 transgene at endogenous levels. Zasp67 mutants show extended and broken Z-discs in adult flies, indicating that the protein helps stabilize the highly regular myofibrils of indirect flight muscles. In contrast, a Zasp66 CRISPR null mutant has limited viability, but only mild indirect flight muscle defects illustrating the diverging evolutionary paths these two paralogous genes have taken since they arose by duplication.
Keywords: Alp/Enigma family; Drosophila melanogaster Zasp67; LIM; PDZ; Zasp52; Zasp66; indirect flight muscle; myofibril assembly.
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