A study of retrograde transport using Caenorhabditis elegans intestinal cells as a model

Endocytosis mediates the internalization of cell surface proteins and lipids in small vesicles that bud from the plasma membrane and deliver their cargo to endosomes. When cargo proteins reach the endosomes, one important pathway that they may follow is retrograde transport, in which cargos are delivered from endosomes to the Golgi apparatus. The role of the recycling endosome in retrograde transport has not been studied well in the past. We use C. elegans, especially the intestinal cells, as model to study retrograde trafficking, employing a combination of genetics, cell biology, biochemistry and molecular biology tools. We discovered that CDC-42/TOCAs/PARs/WAVE localize to the recycling endosome, and regulate retrograde transport at a step from Recycling endosome to Trans-Golgi Network. When missing functional CDC-42/TOCAs/PARs/WAVE, MIG-14/Wntless is misdirected to the late endosome/lysosome during post-endocytic sorting, and steady-state MIG-14 protein levels are strongly reduced. Neuronal polarity defects in mechanosensory neurons ALM are also observed under these conditions, consistent with a defect in MIG-14-dependent WNT signaling. Interestingly, we also found that mutants lacking retromer components SNX-3, VPS-35, VPS-26, or VPS-29, that aberrantly degrade MIG-14 during retrograde transport, also aberrantly degrade the human Transferrin receptor during early endosome to recycling endosome transport. Furthermore, we tested TGN-38, the worm homolog of a well known retrograde cargo TGN38 that passes through the recycling endosome. When missing functional CDC-42, TGN-38 is mainly trapped at the recycling endosome, while upon loss of functional SNX-3, TGN-38 is trapped at the early endosome. These results suggest CDC-42/TOCAs/PARs/WAVE regulate retrograde transport at a step from Recycling endosome to the Trans-Golgi Network after SNX-3/VPS-35/VPS-26/VPS-29, which regulates early endosome to recycling endosome transport. Lastly, we obtained evidence that CDC-42/TOCAs/PARs/WAVE complex may use clathrin/AP-1 vesicles, and that OCRL-1/CKII may work with CDC-42/TOCAs/PARs/WAVE and regulate retrograde transport from the recycling endosome to Trans-Golgi Network. We hope that our work will shed light on the study of intracellular trafficking, especially retrograde transport.Ph.D.Includes bibliographical referencesby Zhiyong Ba