Uncovering the essential roles of glutamate carboxypeptidase 2 orthologs in Caenorhabditis elegans.

Human glutamate carboxypeptidase 2 (GCP2) from the M28B metalloprotease group is an important target for therapy in neurological disorders and an established tumor marker. However, its physiological functions remain unclear. To better understand general roles, we used the model organism Caenorhabditis elegans to genetically manipulate its three existing orthologous genes and evaluate the impact on worm physiology. The results of gene knockout studies showed that C. elegans GCP2 orthologs affect the pharyngeal physiology, reproduction, and structural integrity of the organism. Promoter-driven GFP expression revealed distinct localization for each of the three gene paralogs, with gcp-2.1 being most abundant in muscles, intestine, and pharyngeal interneurons, gcp-2.2 restricted to the phasmid neurons, and gcp-2.3 located in the excretory cell. The present study provides new insight into the unique phenotypic effects of GCP2 gene knockouts in C. elegans, and the specific tissue localizations. We believe that elucidation of particular roles in a non-mammalian organism can help to explain important questions linked to physiology of this protease group and in extension to human GCP2 involvement in pathophysiological processes.

Human myotubularin-related protein 9 regulates ER-to-Golgi trafficking and modulates WNT3A secretion.

Regulation of phosphatidylinositol phosphates plays a crucial role in signal transduction, membrane trafficking or autophagy. Members of the myotubularin family of lipid phosphatases contribute to phosphoinositide metabolism by counteracting the activity of phosphoinositide kinases. The mechanisms determining their subcellular localization and targeting to specific membrane compartments are still poorly understood. We show here that the inactive phosphatase MTMR9 localizes to the intermediate compartment and to the Golgi apparatus and is able to recruit its active phosphatase partners MTMR6 and MTMR8 to these locations. Furthermore, MTMR8 and MTMR9 co-localize with the small GTPase RAB1A and regulate its localization. Loss of MTMR9 expression compromises the integrity of the Golgi apparatus and results in altered distribution of RAB1A and actin nucleation-promoting factor WHAMM. Loss or overexpression of MTMR9 leads to decreased rate of protein secretion. We demonstrate that secretion of physiologically relevant cargo exemplified by the WNT3A protein is affected after perturbation of MTMR9 levels.

Inhibition of late endosomal maturation restores Wnt secretion in Caenorhabditis elegans vps-29 retromer mutants.

Secretion of Wnt proteins is mediated by the Wnt sorting receptor Wls, which transports Wnt from the Golgi to the cell surface for release. To maintain efficient Wnt secretion, Wls is recycled back to the trans-Golgi network (TGN) through a retromer dependent endosome to TGN retrieval pathway. It has recently been shown that this is mediated by an alternative retromer pathway in which the sorting nexin SNX3 interacts with the cargo-selective subcomplex of the retromer to sort Wls into a retrieval pathway that is morphologically distinct from the classical SNX-BAR dependent retromer pathway. Here, we investigated how sorting of Wls between the two different retromer pathways is specified. We found that when the function of the cargo-selective subcomplex of the retromer is partially disrupted, Wnt secretion can be restored by interfering with the maturation of late endosomes to lysosomes. This leads to an accumulation of Wls in late endosomes and facilitates the retrieval of Wls through a SNX-BAR dependent retromer pathway. Our results are consistent with a model in which spatial separation of the SNX3 and SNX-BAR retromer complexes along the endosomal maturation pathway as well as cargo-specific mechanisms contribute to the selective retrieval of Wls through the SNX3 retromer pathway.

Retromer dependent recycling of the Wnt secretion factor Wls is dispensable for stem cell maintenance in the mammalian intestinal epithelium.

In C. elegans and Drosophila, retromer mediated retrograde transport of Wntless (Wls) from endosomes to the trans-Golgi network (TGN) is required for Wnt secretion. When this retrograde transport pathway is blocked, Wls is missorted to lysosomes and degraded, resulting in reduced Wnt secretion and various Wnt related phenotypes. In the mammalian intestine, Wnt signaling is essential to maintain stem cells. This prompted us to ask if retromer mediated Wls recycling is also important for Wnt signaling and stem cell maintenance in this system. To answer this question, we generated a conditional Vps35 (fl) allele. As Vps35 is an essential subunit of the retromer complex, this genetic tool allowed us to inducibly interfere with retromer function in the intestinal epithelium. Using a pan-intestinal epithelial Cre line (Villin-CreERT2), we did not observe defects in crypt or villus morphology after deletion of Vps35 from the intestinal epithelium. Wnt secreted from the mesenchyme of the intestine may compensate for a reduction in epithelial Wnt secretion. To exclude the effect of the mesenchyme, we generated intestinal organoid cultures. Loss of Vps35 in intestinal organoids did not affect the overall morphology of the organoids. We were able to culture Vps35 (∆/∆) organoids for many passages without Wnt supplementation in the growth medium. However, Wls protein levels were reduced and we observed a subtle growth defect in the Vps35 (∆/∆) organoids. These results confirm the role of retromer in the retrograde trafficking of Wls in the intestine, but show that retromer mediated Wls recycling is not essential to maintain Wnt signaling or stem cell proliferation in the intestinal epithelium.