Molecular Anchoring of Resilin in the Elastic Cuticle of Insects

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To allow locomotion and body flexibility, the insect cuticle is arranged of stiff and flexible units. A prominent component of the flexible and elastic cuticle is Resilin, a rubber-like protein matrix discovered in the 60s of last century and found in various insect species. A central feature of Resilin-containing cuticles is the incidence of di-tyrosines that cross-link Resilin monomers (Pro-Resilin), thereby accounting for the flexibility and elasticity of the cuticle. Auto-fluorescence of di-tyrosines has quasi been used as an indirect detection of Resilin at the macroscopic level. By contrast, the molecular-histological organisation of Resilin-containing cuticles has not been studied at all. Indeed, it is even not clear whether Resilin is composed of one or several proteins. Moreover, although the mechanical properties of Resilin have been intensively investigated, its significance for insect fitness, performance and behaviour is unexplored.In this project, I propose to use the fruit fly Drosophila melanogaster to study these molecular-histological and biological aspects of Resilin. We address this issue with two major strategies flanked by an evolutionary approach. 1) A genomic construct with the pro-resilin gene including its own promoter and a 3-prime gfp tag will be used to monitor directly the complete expression pattern of Resilin in live flies. The Resilin-GFP signal will be correlated with the auto-fluorescence of di-tyrosines by confocal microscopy. Cells expressing Resilin will be characterised in detail. Anchoring of Resilin within the cuticle will be determined by transmission electron microscopic experiments. 2) Applying RNAi and Crispr/Cas9 techniques, flies with reduced or eliminated Resilin function will be characterised in detail at the organismic and ultrastructural levels. Preliminary results underline this successful endeavour. Resilin is, as expected, expressed among others in wing hinges. Consistently, the RNAi or Crispr-induced down-regulation of pro-resilin provoke a wing posture failure. Addressing the possibility of a non-single-protein Resilin matrix, we will also investigate the function of Cpr56F and Muc91, two potential Pro-Resilin-redundant proteins in gentic and histological experiments. The flanking evolutionary approach involves the human pest Cimex lectularius, known as the bedbug. In this insect, identification of the pro-resilin gene is difficult as six candidate genes are present in the Cimex genome. Using classical in situ hybridisation experiments combined with the RNAi technique we will identify those genes that are responsible for elasticity of different regions of the Cimex cuticle. This sub-project will shed light on the problem of the evolution of the number of Resilin proteins and Resilin-containing cuticles in insects.Together, this project will substantially contribute to our understanding of Resilin function in insects, from the molecular-histological to the biological-organismic level.