Genetic Analysis of Grinder Formation in Caenorhabditis Elegans: Regulation by RAB-6.2 and Its GTPase Activating Protein EAT-17




Anselmo, Sarah Straud

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The C. elegans grinder is an intricately designed, macromolecular structure located in the terminal bulb of the pharynx. It acts as the teeth of C. elegans, crushing bacteria before they are passed to the intestine. The grinder is a specialized cuticular structure and is shed and rebuilt at each larval molt. While we have a fairly decent view regarding the mechanics of the grinder, we know surprisingly little about its composition or how it is formed. The nematode grinder has been studied for over 100 years, but no one has yet described the molecular events controlling grinder formation and/or grinder function.
To understand how the grinder is formed, I have focused on cloning and characterizing eat-17. eat-17 mutants have rudimentary, malformed grinders: the grinder plates are smaller than normal, disorganized in structure, and often improperly arranged. I found that eat-17 encodes an ~825 amino acid Rab GTPase activating protein with a series of C-terminal coiled-coil domains. Its closest human homolog is Evi5, a putative oncogene whose function is not currently understood. Rabs are key regulators of vesicle transport, and cycle between active, GTP bound and inactive, GDP bound states. GTPase activating proteins (GAPs) catalyze the hydrolysis of GTP, allowing Rabs to be extracted from membranes and recycled for additional rounds of signaling. I found that the GAP activity of EAT-17 is important for its function: 21% (16/76) of mutants expressing a wild-type version of eat-17 are rescued for defects in grinder formation, while only 2.9% (3/113) of worms expressing catalytically inactive versions of EAT-17 are rescued. I performed RNAi against the 27 putative Rabs in the C. elegans genome and found that rab-6.2 RNAi causes grinder defects similar to those seen in eat-17 mutants. GFP reporters show that both EAT-17 and RAB-6.2 are expressed in terminal bulb muscle, the site of grinder secretion. By yeast two-hybrid, I have demonstrated a direct interaction between RAB-6.2 and EAT-17. These data suggest that EAT-17 and RAB-6.2 work together in regulating grinder formation. Genetic interaction studies suggest that RAB-6.1 may play a role in a similar cellular process.

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