Regulation of seamless tube development

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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.