Molecular Anchoring of Resilin in the Elastic Cuticle of Insects

为了运动和身体灵活，昆虫的角质层由刚性和柔性单元组成。弹性角质层的一个重要组成部分是弹性蛋白，这是一种类似橡胶的蛋白质基质，于上世纪60年代被发现，存在于各种昆虫物种中。含有弹性蛋白的角质层的一个中心特征是交联弹性蛋白单体（前弹性蛋白）的二酪氨酸的发生，从而说明了角质层的柔韧性和弹性。在宏观水平上，二酪氨酸的自荧光已被准用于间接检测弹性蛋白。相比之下，含有树脂的角质层的分子组织学组织还没有被研究过。事实上，我们甚至不清楚弹性蛋白是由一种还是几种蛋白质组成的。此外，尽管Resilin的机械性能已被深入研究，但其对昆虫适应性，性能和行为的意义尚未探索。在这个项目中，我建议使用果蝇黑腹果蝇来研究弹性蛋白的这些分子组织学和生物学方面。我们用两种主要的策略来解决这个问题，同时采用一种进化的方法。1)利用含有亲和启动子和3-prime gfp标签的前弹性蛋白基因基因组构建体，直接监测弹性蛋白在活蝇中的完整表达模式。在共聚焦显微镜下，Resilin-GFP信号将与二酪氨酸的自荧光相关联。我们将详细描述表达弹性蛋白的细胞。通过透射电镜实验确定弹性蛋白在角质层内的锚定作用。2)应用RNAi和Crispr/Cas9技术，在有机体和超微结构水平上对Resilin功能减少或消除的果蝇进行详细的表征。初步结果强调了这一成功的努力。正如预期的那样，弹性蛋白在机翼铰链中表达。一致地，RNAi或crispr诱导的促弹性蛋白下调会引起翅膀姿态失败。为了解决非单一蛋白弹性蛋白基质的可能性，我们还将在遗传和组织学实验中研究Cpr56F和Muc91这两种可能的前弹性蛋白冗余蛋白的功能。侧翼进化方法涉及到人类害虫臭虫，也就是众所周知的臭虫。在这种昆虫中，鉴定前弹性蛋白基因是困难的，因为六个候选基因存在于Cimex基因组中。利用经典的原位杂交实验结合RNAi技术，我们将确定那些负责Cimex角质层不同区域弹性的基因。该子项目将揭示昆虫中弹性蛋白和含弹性蛋白角质层数量的进化问题。总之，这个项目将大大有助于我们从分子-组织学到生物-有机体水平对昆虫中弹性蛋白功能的理解。

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.