Exoskeletal cuticle proteins enable Drosophila locomotion

Exo- and Endoskeleton function enables muscle-mediated locomotion in animals. In mammals, the defective protein matrix of bones found in systematic skeletal disorders such as osteoporosis causes fractures and severe skeletal deformations under high muscle tension. We identified an analogous mechanism for integrating muscle-mediated tension into the apical extracellular matrix (aECM) of the invertebrate body wall exoskeleton. Obstructor chitin-binding proteins, the chitin deacetylases, Chitinases, and the matrix-protecting proteins Knickkopf and Retroactive are epidermally expressed during late embryogenesis. Their control of forming epidermal chitinous structures protects the exoskeletal aECM from collapsing when embryos start moving and hatch as larvae. In a larval locomotion assay we tested the function of these cuticle related genes. Gene mutations and knockdowns caused changes in normal movement behavior and lower the speed of larvae. Moreover, we found that the transmembrane Zona Pellucida domain protein Piopio provides the adhesion between the epidermal apical membrane and the overlaying chitinous aECM in a matriptase-dependent manner. A failure of Piopio and chitin-associated proteins leads to exoskeletal deformations and detachment from the epidermal membrane, destabilizing muscle forces and impairing larval mobility. Our data identifies a protein network that transforms the chitinous aECM into a stable exoskeleton that directly resists muscle impact at epidermal tendon cells, thereby serving locomotion. Demonstrating the importance of these proteins in producing aECM as a three-dimensional cuticular scaffold for exoskeletal function opens up opportunities for the development of biomimetic applications of synthetic materials. STATEMENT OF SIGNIFICANCE: Chitin-based materials include hydrogels, microcapsules, membranous films, sponges, tubes, and various porous structures. In nature, chitin structures form cuticles, which serves as the exoskeleton of arthropods. Using Drosophila melanogaster, we have performed systematic analyses to identify the proteins and enzymes that organize chitin polymers in 3D structures of the cuticle exoskeleton. Three-dimensional laser-scanning and ultrastructural electron microscopy revealed deformations of the cuticle structure, lack of cellular cuticle adhesion, and overall changes in the flexibility of the chitin-based material, leading to insufficient function of the exoskeleton. Components such as the identified proteins and enzymes, which play a unique role in the organization of the chitin fibers and the formation of the exoskeleton, offer suitable materials for tissue engineering for biomimetic applications.