Molecular Anchoring of Resilin in the Elastic Cuticle of Insects

昆虫弹性角质层中节肢弹性蛋白的分子锚定

• 批准号: 395400575
• 负责人: Privatdozent Dr. Bernard Moussian
• 金额: --
• 依托单位: Abteilung Genetik der Tiere
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/395400575?language=en
• 关键词: Molecular; Anchoring; Resilin; Elastic; Cuticle

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.

为了允许运动和身体的灵活性，昆虫的角质层是由刚性和柔性单元组成的。弹性角质层的主要成分是Resilin，它是上个世纪60年代发现的一种橡胶样蛋白质基质，存在于各种昆虫物种中。含有树脂弹性蛋白的角质层的中心特征是发生交联树脂弹性蛋白单体（Pro-Resilin）的二酪氨酸，从而解释角质层的柔韧性和弹性。二酪氨酸的自发荧光已准用作在宏观水平上间接检测Resilin。相比之下，含有弹性蛋白的铜的分子组织学组织还没有被研究。事实上，甚至不清楚Resilin是由一种还是几种蛋白质组成。此外，虽然Resilin的机械性能已被深入研究，其意义的昆虫健身，性能和behaviors.In这个项目中，我建议使用果蝇研究这些分子组织学和生物学方面的Resilin。我们解决这个问题的两个主要战略两侧的进化方法。1)具有包括其自身启动子和3-prime gfp标签的节枝弹性蛋白原基因的基因组构建体将用于直接监测节枝弹性蛋白在活蝇中的完整表达模式。通过共聚焦显微镜将Resilin-GFP信号与双酪氨酸的自发荧光相关联。将详细表征表达Resilin的细胞。将通过透射电子显微镜实验确定表皮内的Resilin的包封。2)应用RNAi和Crispr/Cas9技术，将在器官和超微结构水平上详细表征具有减少或消除的Resilin功能的果蝇。初步结果表明这一努力是成功的。如预期的那样，Resilin在机翼铰链中表达。因此，RNAi或Crispr诱导的节枝弹性蛋白原下调引起翅姿势失败。为了解决非单一蛋白质Resilin基质的可能性，我们还将研究Cpr 56 F和Muc 91的功能，这两种潜在的Pro-Resilin冗余蛋白质在遗传学和组织学实验中。侧翼进化方法涉及人类害虫Cimex lectularius，即臭虫。在这种昆虫中，由于Cimex基因组中存在六个候选基因，所以鉴定前节枝弹性蛋白基因是困难的。使用经典的原位杂交实验结合RNAi技术，我们将确定这些基因是负责不同地区的Cimex角质层的弹性。该子项目将揭示昆虫中Resilin蛋白和含Resilin的cucuminase数量的进化问题，共同为我们从分子组织学到生物器官水平理解昆虫中Resilin的功能做出贡献。