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: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=684976
• 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.********

生物管构成多细胞生物中许多器官的基本结构。它们在长距离内运输气体，流体和细胞以提供生长和发育所需的基本因素。肾脏，肺部和脉管系统几乎完全由管子组成，它们作为多细胞（上皮）和单细胞（无缝）单位存在。尽管这些管子可以以多种不同的方式发展，但它们都共享了管腔衬里的顶部表面的共同特征。该研究计划的长期目标是使用线虫round虫秀丽隐杆线虫作为模型系统的排泄细胞来理解生物管发展的机制。使用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的试剂的可用性。各种研究表明，内吞运输的关键作用以及肌动蛋白细胞骨架在排泄管扩展和维持膜完整性中的关键作用。然而，它仍然鲜为人知，如何调节这些过程，以确保在管扩展过程中添加协调的膜。我们最近发现，CCM-3蛋白通过小的GTPase CDC-42和内吞回收来调节管道延伸。我们对脑海绵状畸形和CCM-3蛋白的兴趣是由CIHR资助的单独研究分支的主题，我们在其中使用排泄物作为CCM-3活性的读数。从这个试点屏幕中，我们确定了2种中间丝蛋白，3个小的GTPase效应子，2个外囊蛋白和1张形成，它们在消融时引起了管截断。短期目标是描述中间细丝和内吞运输如何调节管道的扩展和膜完整性。因此，我们将首先定义它们的表达模式，并确定它们是否会使用一组荧光标记物影响内吞和/或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是否影响延伸管道所需的细胞器和囊泡的结构。我们还将通过创建双重突变体来定义线性和并行信号通路来进行上学分析。最后，我们将通过对影响管道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。这项工作将揭示有关内吞贩运和中间细丝蛋白如何合作以促进体内无缝管发育的保守机制。********