The Recycling GTPase, RAB-10, Regulates Autophagy Flux in Caenorhabditis elegans
Autophagy and endocytosis are two cellular pathways that are vital to cell growth and homeostasis. Autophagy is a dynamic and catabolic process involving the formation of a double-membrane vesicle called the autophagosome, which engulfs long-lived proteins and damaged organelles. Endocytosis involves the uptake of extracellular material into the cell through the formation of intracellular vesicles termed endosomes. Although both endocytosis and autophagy are interconnected processes, the extent to which endocytic proteins and/or compartments contribute to autophagy, and how these endocytic components do so, is still unknown. To improve our understanding of the connections that exist between autophagy and endocytosis, we conducted an RNAi screen for endocytic genes that altered GFP::LGG-1 expression and blocked tissue remodeling in daf-2/IIR mutants. The goal of this screen was to identify new or previously characterized endocytic genes that function in autophagy, further elucidate the mechanism by which these genes control autophagy, and enhance our understanding as to how distantly related processes are interconnected. daf-2 encodes the insulin-like receptor (IIR) and daf-2/IIR loss of function mutants have elevated levels of autophagy, measured by an increase in puncta labeled with the autophagosome reporter, GFP::LGG-1. RNAi-mediated knockdown of autophagy genes in daf-2/IIR mutants results in the formation of GFP::LGG-1-positive aggregates, exemplifying defects in autophagosome formation. Thus, using daf-2/IIR loss of function mutants, we screened for endocytic genes that when depleted by RNAi, altered GFP::LGG-1 expression. One of the genes identified in our screen was the small GTPase, RAB-10, a regulator of endocytic trafficking from yeast to mammals. Our results show that rab-10 loss of function alters the localization pattern of the autophagy reporter, GFP::LGG-1 in wild type and daf-2(e1370) mutant seam cells. We show that the changes in the localization of the GFP::LGG-1 reporter in rab-10 mutants is due to defects in autophagy flux. Supporting defective flux due to rab-10 loss, we find that lysosomal inhibition does not further disrupt GFP::LGG-1 localization in rab-10 mutants. Moreover, autophagosomes fail to fuse with lysosomes in daf-2 mutants treated with rab-10 RNAi, and a loss of rab-10 function results in the accumulation of the autophagy cargo adaptor protein, SQST-1::GFP, in daf-2(e1370) mutants. We determined that RAB-10 is required for the localization of GFP::ATG-9 to punctate structures/foci, which may represent the unique vesicle, called the “ATG-9 reservoir”. Lastly, we found that the GTPase cycling ability of RAB-10 is required for its role in autophagy. Future studies will be needed to determine the exact mechanism by which RAB-10 regulates autophagy flux. In conclusion, our data support a role for rab-10 in promoting autophagy flux and adds to the field of autophagy by reinforcing the importance of RAB GTPases in the regulation of autophagy function.
Biology, Cell Biology, Genetics, Autophagy, Endocytosis, RAB-10, ATG-9, GTPase, Autophagosome, Endosome
City University of New York - Graduate Center
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The Graduate School and University Center of The City University of New York