Nematode excretory canals are thin hollow tubules that run the length of the animal, and are built from a single cell. Extension and maintenance of these tubules requires concomitant growth of the basal outer surface of the canals and of the apical luminal surface. In C. elegans, loss-of-function mutations in any of a series of exc genes cause the canal lumen to swell into large fluid-filled cysts. These cysts form as a result of mechanical failure of the actin-based cytoskeleton lining the canal apical surface.
Several exc mutations (
exc-1,
exc-9, and
exc-2) show genetic interactions with
exc-5, which encodes a guanine exchange factor homologous to the mammalian FGD proteins that activate CDC42. We have examined constructs expressing subcellular organelle markers (including EEA-1, CHC-1, RAB-5, RAB-7, RAB-11, GRIP, and GLO-1, CDC-42, and the CDC-42-binding domain of WSP-1) within the excretory canals, and examined their placement in wild type,
exc-5 mutant, and
exc-5-overexpressing strains. In
exc-5 mutants, early endosomes (marked by EEA-1) accumulate greatly in areas of the canals adjacent to and surrounding areas where large cysts formed. Most notably, we saw these accumulations of labeled EEA-1 in areas of the canal prior to the formation of cysts. Endocytic vesicles (labeled with RAB-5) also accumulated to some extent in these mutants. Recycling endosomes (marked by RME-1), conversely, were strikingly depleted in areas of cyst formation. In animals overexpressing
exc-5, the RME-1 marker accumulated, while EEA-1 expression was lowered. We conclude that EXC-5 regulates trafficking of early endosomes necessary to recycle proteins that maintain the apical cytoskeleton. Mammalian FGD4 was reported by others to be necessary to maintain Schwann cells to maintain the myelin sheath to prevent the neuropathy Charcot-Marie-Tooth Syndrome Type 4H. Our results imply that FGD proteins may be needed to maintain efficient recycling of proteins to maintain the structure of these single-celled tubes as well.
We are grateful for marker constructs and advice supplied by Barth Grant, John White, Brian Ackley, Erik Lundquist, and Monica Driscoll, as well as support from NINDS R03-NS067323 and the Inez Jay Fund of the University of Kansas.