Different Evolutionary Trajectories of Two Insect-Specific Paralogous Proteins Involved in Stabilizing Muscle Myofibrils

Abstract

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. <i>Drosophila melanogaster</i> has three Alp/Enigma family members, Zasp52, Zasp66, and Zasp67. Ortholog search and phylogenetic tree analysis suggest that <i>Zasp</i> genes have a common ancestor, and that <i>Zasp66</i> and <i>Zasp67</i> 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 <i>Zasp67</i>, which is viable but flightless. We can rescue all phenotypes by re-expressing a <i>Zasp67</i> transgene at endogenous levels. <i>Zasp67</i> 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 <i>Zasp66</i> 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.