Regulation of seamless tube development

无缝管发展监管

• 批准号: RGPIN-2016-06638
• 负责人: Derry, William
• 金额: $ 2.4万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=616428
• 关键词: Regulation; seamless; tube; development

Biological tubes make up fundamental structures of many organs in multicellular organisms. They function to transport gases, fluids and cells over long distances to supply essential factors necessary for growth and development. Kidneys, lungs and vasculature are comprised almost entirely of tubes, and these exist as both multicellular (epithelial) and unicellular (seamless) units. Although these tubes can develop in many different ways they all share the common feature of an apical surface lining the lumen. The long-term goals of this research program are to understand the mechanisms by which biological tubes develop using the excretory cell of the nematode roundworm Caenorhabditis elegans as a model system. The key advantages of using the worm are its powerful genetics, in vivo imaging, and availability of reagents for the excretory system. Various studies have shown critical roles for endocytic trafficking and the actin cytoskeleton in excretory canal extension and maintenance of membrane integrity. However, it remains poorly understood how these processes are regulated to ensure coordinated membrane addition during tube extension. We recently found that the CCM-3 protein regulates canal extension through the small GTPase CDC-42 and endocytic recycling. Our interests in cerebral cavernous malformations and the CCM-3 protein is the subject of a separate branch of research funded by CIHR, where we use the excretory canal as a readout for CCM-3 activity. To understand the basic mechanisms governing excretory canal development we surveyed a list of cytoskeletal and trafficking genes using RNA interference (RNAi) to knock down their expression. From this pilot screen we identified 2 intermediate filament proteins, 3 small GTPase effectors, 2 exocyst proteins, and 1 formin that caused canal truncations when ablated. The short-term goals are to delineate how intermediate filaments and endocytic trafficking regulate tube extension and membrane integrity. Therefore, we will first define their expression patterns and determine if they affect the localization and/or expression of endocytic and cytoskeletal proteins using a panel of fluorescent markers. Using electron microscopy we will determine if these proteins affect the structures of organelles and vesicles required to extend canals. We will also perform epistasis analysis by creating double mutants to define linear and parallel signalling pathways. Finally, we will delineate upstream and downstream components of these pathways by performing a genome-wide RNAi screen for genes that affect canal extension and membrane integrity. This work will uncover conserved mechanisms governing how endocytic trafficking and intermediate filament proteins collaborate to promote seamless tube development in vivo.

生物管构成了多细胞生物中许多器官的基本结构。它们的功能是长距离运输气体、液体和细胞，以提供生长和发育所需的必需因子。肾脏、肺和脉管系统几乎全部由管组成，它们以多细胞（上皮）和单细胞（无缝）单位存在。尽管这些管可以以多种不同的方式发育，但它们都有一个共同特征，即内衬的顶端表面。该研究计划的长期目标是利用线虫秀丽隐杆线虫的排泄细胞作为模型系统来了解生物管发育的机制。使用该蠕虫的主要优点是其强大的遗传学、体内成像以及排泄系统试剂的可用性。各种研究表明内吞运输和肌动蛋白细胞骨架在排泄道延伸和膜完整性维持中发挥着关键作用。然而，人们对如何调节这些过程以确保在管延伸过程中协调膜添加仍然知之甚少。我们最近发现 CCM-3 蛋白通过小 GTP 酶 CDC-42 和内吞再循环来调节根管延伸。我们对脑海绵状血管瘤和 CCM-3 蛋白的兴趣是 CIHR 资助的一个独立研究分支的主题，其中我们使用排泄道作为 CCM-3 活性的读数。 为了了解控制排泄道发育的基本机制，我们调查了一系列细胞骨架和运输基因，使用 RNA 干扰 (RNAi) 来降低其表达。从这个试点筛选中，我们鉴定了 2 种中间丝蛋白、3 种小 GTP 酶效应子、2 种胞外囊蛋白和 1 种在消融时导致根管截断的福尔明。短期目标是描述中间丝和内吞运输如何调节管延伸和膜完整性。因此，我们将首先定义它们的表达模式，并使用一组荧光标记确定它们是否影响内吞蛋白和细胞骨架蛋白的定位和/或表达。使用电子显微镜，我们将确定这些蛋白质是否影响延伸根管所需的细胞器和囊泡的结构。我们还将通过创建双突变体来定义线性和平行信号通路来进行上位分析。最后，我们将通过对影响根管延伸和膜完整性的基因进行全基因组 RNAi 筛选来描绘这些途径的上游和下游组件。这项工作将揭示控制内吞运输和中间丝蛋白如何协作促进体内无缝管发育的保守机制。