Regulation of seamless tube development

生物管构成了多细胞生物体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基本因子。肾、肺和血管系统几乎完全由管子组成，这些管子既以多细胞(上皮)单位存在，也以单细胞(无缝)单位存在。尽管这些管子可以以许多不同的方式发育，但它们都有一个共同的特征，即管腔内衬着根尖表面。这项研究计划的长期目标是以线虫线虫的排泄物细胞为模型系统，了解生物管发育的机制。使用蠕虫的主要优势是它强大的遗传学、活体成像以及排泄系统试剂的可用性。各种研究表明，内吞转运和肌动蛋白细胞骨架在排泄道延伸和维持细胞膜完整性方面起着关键作用。然而，人们仍然不清楚这些过程是如何调节的，以确保在试管延伸过程中协调膜添加。我们最近发现CCM-3蛋白通过小的GTP酶CDC-42和内吞循环来调节管道的延伸。我们对大脑海绵状畸形和CCM-3蛋白的兴趣是由CIHR资助的另一项研究的主题，在该研究中，我们使用排泄管作为CCM-3活性的读数。*为了了解排泄管发育的基本机制，我们使用RNA干扰(RNAi)调查了一系列细胞骨架和运输基因，以抑制它们的表达。从这个初步筛选中，我们鉴定了2个中间丝蛋白、3个小的GTP酶效应子、2个胞外蛋白和1个福尔明，它们在消融时会导致管道截断。短期目标是描绘中间细丝和内吞运输如何调节管子延伸和膜的完整性。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来确定它们是否影响内细胞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管道所需的细胞器和囊泡的结构。我们还将通过创建双突变来定义线性和平行的信号通路来执行上位性分析。最后，我们将通过进行全基因组范围的RNAi筛查来描述这些通路的上游和下游部分，以寻找影响椎管延伸和膜完整性的基因。这项工作将揭示保守的机制，控制细胞内转运和中间丝蛋白如何合作，促进体内无缝管的发育。

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.********