Timing of chitin/chitosan matrix assembly in insects

N-乙酰氨基葡萄糖多糖、甲壳素及其脱乙酰化的壳聚糖是昆虫角质层的主要胞外支架。在不同的身体部位和阶段，特定的角质层蛋白与甲壳素/壳聚糖相互作用，形成结晶、无定形或复合结构，定义特定区域的角质层材料和物理属性。甲壳素/壳聚糖及其相关蛋白质组成的基质在时间和空间上的组装，包括它对机械和生物功能的影响，还没有被探索。根据CodeXI的前言，我们认为甲壳素和壳聚糖(FA)之间的组织特异性比率(FA)和脱乙酰基(PA)模式是由角质层蛋白解释的，这取决于角质层蛋白在体内的位置。在这个联合项目中，我们使用果蝇的腿和可选的翅膀作为角质层系统的模型来解决这些问题。在基因和分子水平上，我们探讨了两个甲壳素/壳聚糖结合蛋白Pro-resilin和Cpr56F的功能，重点是大转子(运动所需)和踝关节(粘连所需)。首先，我们将监测我们已经应用Crispr/Cas9的基因编辑协议生成的Pro-resilin和Cpr56F的荧光标记版本的时间和空间分布。同时，我们将研究几丁质脱乙酰酶的标记版本的表达，包括VERM和SERP，以及正确定位Pro-resilin所需的C型凝集素Schlaff(SLF)。使用真菌壳聚糖酶和壳聚糖结合蛋白，我们绘制了壳聚糖在腿部节段的分布。基因编辑将用于交换各自的启动子和甲壳素/壳聚糖结合结构域，以完善Pro-resilin和Cpr56F对角质层功能的时空影响的研究。转基因果蝇的角质层特性随后在广泛的组织学、生物力学和行为学文章中得到了表征，并最终与昆虫角质层中特定的和局部的甲壳素/壳聚糖代码联系在一起。这项工作将作为甲壳素/壳聚糖基质在生物世界中产生、解释和使用的分子机制的一个完整的例子。

The N-acetyl-glucosamine polysaccharide chitin and its deacetylated derivative chitosan are major extracellular scaffolds in the cuticle of insects. In different body parts and stages, specific sets of cuticle proteins interact with chitin/chitosan forming a crystalline, amorphic or composite structures that define the region-specific material and physical properties of the cuticle. The assembly of the matrix composed of chitin/chitosan and the associated proteins in time and space including its impact on the mechanical and biological function is unexplored. In line with the CodeXi preamble, we reckon that the tissue-specific ratio between chitin and chitosan (FA) and pattern of deacetylation (PA) are interpreted by cuticle proteins depending on the position in the body. In this joint project, we address these problems using the leg and optionally the wing of the fruit fly Drosophila melanogaster as a model cuticle system. At the genetic and molecular level, we explore the functions of the two chitin/chitosan-binding proteins Pro-Resilin and Cpr56F focusing on the trochanter (needed for locomotion) and the tarsus (needed for adhesion). To begin with, we will monitor the temporal and spatial distribution of fluorescently tagged versions of Pro-Resilin and Cpr56F that we have already generated applying the gene editing protocol of Crispr/Cas9. In parallel, we will examine the expression of tagged versions of the chitin deacetylases Vermiform (Verm) and Serpentine (Serp) as well as the C-type lectin Schlaff (Slf) that is needed for correct Pro-Resilin localization. Using fungal chitosanases and chitosan-binding proteins, we map chitosan distribution in the leg segments. Gene editing will be applied to swap respective promotors and chitin/chitosan-binding domains in order to refine the study of temporal and spatial impact of Pro-Resilin and Cpr56F on cuticle function. The cuticle properties of the genetically modified flies are subsequently characterised in extended histological, biomechanical and behavioural essays, and eventually linked to the specific and local chitin/chitosan code in the insect cuticle. This work will serve as an integral example for the molecular mechanisms of generation, interpretation, and use of a chitin/chitosan matrix in the living world.