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: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=710335
• 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.

在多细胞生物体中，生物管构成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基本因素。肾、肺和脉管系统几乎完全由管组成，并且这些管以多细胞（上皮）和单细胞（无缝）单位存在。虽然这些管可以以许多不同的方式发展，但它们都具有共同的特征，即顶端表面衬着管腔。这项研究计划的长期目标是了解生物管的发展机制，使用线虫的排泄细胞作为模型系统。使用蠕虫的主要优势是其强大的遗传学，体内成像和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管延伸和维持膜完整性中起着关键作用。然而，人们仍然不太了解如何调节这些过程，以确保在管延伸过程中协调的膜添加。我们最近发现CCM-3蛋白通过小的GTdR CDC-42和内吞再循环来调节运河的延伸。我们对脑海绵状畸形和CCM-3蛋白的兴趣是CIHR资助的一个独立的分支研究的主题，我们使用排泄管作为CCM-3活性的读数。 为了了解控制排泄管发育的基本机制，我们调查了一系列细胞骨架和运输基因，使用RNA干扰（RNAi）来敲低它们的表达。从这个试点筛选，我们确定了2个中间丝蛋白，3个小的GTdR效应，2个外囊蛋白，和1个在消融时引起管截断的GTdR。短期目标是描绘中间丝和内吞运输如何调节管的延伸和膜的完整性。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物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会影响细胞器和囊泡的结构，需要延长运河。我们还将通过创建双突变体来定义线性和平行信号传导途径来进行上位性分析。最后，我们将通过对影响运河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成分。这项工作将揭示保守的机制如何内吞运输和中间丝蛋白合作，以促进无缝管的发展在体内。