Timing of chitin/chitosan matrix assembly in insects

昆虫中甲壳素/壳聚糖基质组装的时间

• 批准号: 525893614
• 负责人: Professor Dr. Stanislav N. Gorb
• 金额: --
• 依托单位: Arbeitsgruppe Funktionelle Morphologie und Biomechanik
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/525893614?language=en
• 关键词: Timing; chitin; chitosan; matrix; assembly

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.

N-乙酰氨基葡萄糖多糖几丁质及其脱乙酰衍生物壳聚糖是昆虫角质层中主要的细胞外支架。在不同的身体部位和阶段，特定的角质层蛋白组与几丁质/壳聚糖相互作用，形成结晶、无定形或复合结构，这些结构定义了角质层的区域特异性材料和物理特性。由几丁质/壳聚糖和相关蛋白质组成的基质在时间和空间上的组装，包括其对机械和生物功能的影响，尚未被探索。根据 CodeXi 序言，我们认为几丁质和壳聚糖 (FA) 之间的组织特异性比率以及脱乙酰化 (PA) 模式由角质层蛋白根据在体内的位置来解释。在这个联合项目中，我们使用果蝇的腿和可选的翅膀作为模型角质层系统来解决这些问题。在遗传和分子水平上，我们探索了两种几丁质/壳聚糖结合蛋白 Pro-Resilin 和 Cpr56F 的功能，重点关注转子（运动所需）和跗骨（粘附所需）。首先，我们将监测荧光标记版本的 Pro-Resilin 和 Cpr56F 的时间和空间分布，这些版本是我们应用 Crispr/Cas9 基因编辑协议生成的。同时，我们将检查几丁质脱乙酰酶 Vermiform (Verm) 和 Serpentine (Serp) 以及正确 Pro-Resilin 定位所需的 C 型凝集素 Schlaff (Slf) 的标记版本的表达。使用真菌壳聚糖酶和壳聚糖结合蛋白，我们绘制了腿节中壳聚糖的分布图。基因编辑将用于交换各自的启动子和几丁质/壳聚糖结合域，以完善 Pro-Resilin 和 Cpr56F 对角质层功能的时间和空间影响的研究。随后，在扩展的组织学、生物力学和行为论文中对转基因果蝇的角质层特性进行了表征，并最终与昆虫角质层中特定的局部几丁质/壳聚糖代码联系起来。这项工作将作为甲壳素/壳聚糖基质在生活世界中产生、解释和使用的分子机制的一个完整的例子。