Long, narrow, single-celled tubes are found in many tissues in our body, such as capillaries and nephrons. The excretory canal cell of C. elegans provides a model for investigating the formation and maintenance of narrow tubes. Mutations in a series of exc genes allow the excretory canals to swell into fluid-filled cysts. The position of all these genes has been narrowed, but a few remain to be cloned. We are currently using whole-genome sequencing methods to identify the sequence of
exc-2. Preliminary data suggest that EXC-2 may act upstream of other EXC proteins that effect endosomal recycling. We are characterizing the potential role of EXC proteins in the endosomal transport pathway by monitoring several endosomal compartments via FRAP. Recovery of fluorescence of cytosolic molecules of labeled RAB-5 and RAB-11 are not affected by
exc-2 mutation. A difference was seen, however, with RAB-5 and RAB-11 bound to their target compartments (the early and recycling endosomes, respectively). Puncta carrying marked RAB-5 recovered rapidly after bleaching. Puncta of RAB-11 molecules, exhibited a different behavior. In the wild-type background, bleached puncta recovered fluorescence fully within 180 seconds. When
exc-2 was mutated, however, recovery was essentially halted, with no significant increase in fluorescence visible after 280 seconds. This result suggests that EXC-2 function is required for turnover of RAB-11 on the recycling endosome. We are now analyzing other subcellular markers such as EEA-1, RME-1, GRIP, GLO-1, and RAB-7 in order to gain a broader understanding of the role of EXC-2 and other EXC proteins in the endosomal recycling.