ACaenorhabditis elegansmodel of orotic aciduria reveals enlarged lysosome‐related organelles in embryos lackingumps‐1function

Abstract

Gut granules are cell type‐specific lysosome‐related organelles found within the intestinal cells of Caenorhabditis elegans. To investigate the regulation of lysosome‐related organelle size, we screened for C. elegans mutants with substantially enlarged gut granules, identifying alleles of the vacuolar‐type H+‐ATPase and uridine‐5′‐monophosphate synthase (UMPS)‐1. UMPS‐1 catalyzes the conversion of orotic acid to UMP; this comprises the two terminal steps in de novo pyrimidine biosynthesis. Mutations in the orthologous human gene UMPS result in the rare genetic disease orotic aciduria. The umps‐1(−) mutation promoted the enlargement of gut granules to 250 times their normal size, whereas other endolysosomal organelles were not similarly affected. UMPS‐1::green fluorescent protein was expressed in embryonic and adult intestinal cells, where it was cytoplasmically localized and not obviously associated with gut granules. Whereas the umps‐1(−) mutant is viable, combination of umps‐1(−) with mutations disrupting gut granule biogenesis resulted in synthetic lethality. The effects of mutations in pyr‐1, which encodes the enzyme catalyzing the first three steps of de novo pyrimidine biosynthesis, did not phenotypically resemble those of umps‐1(−); instead, the synthetic lethality and enlargement of gut granules exhibited by the umps‐1(−) mutant was suppressed by pyr‐1(−). In a search for factors that mediate the enlargement of gut granules in the umps‐1(−) mutant, we identified WHT‐2, an ABCG transporter previously implicated in gut granule function. Our data suggest that umps‐1(−) leads to enlargement of gut granules through a build‐up of orotic acid. WHT‐2 possibly facilitates the increase in gut granule size of the umps‐1(−) mutant by transporting orotic acid into the gut granule and promoting osmotically induced swelling of the compartment.

C. elegans BLOC-1 Functions in Trafficking to Lysosome-Related Gut Granules

“…glo-2(zu455) did not dramatically alter the appearance or distribution of RAB-5 containing early endosomes (Figure 2W–Z), RAB-7 containing late endosomes/lysosomes (Figure 2S–V), or RME-1::GFP containing recycling endosomes (Figure 2A′–B′). Conventional lysosomes in intestinal cells contained the soluble glucosylceramidase F11E6.1::mCherry and the cathepsin protease CPR-6::mCherry [33] and their formation and morphology was not obviously altered by glo-2(zu455) (Figure 2K–N). In C. elegans embryos, alterations in trafficking to lysosomes compromise their degradative properties, which dramatically alters the number and morphology of yolk platelets normally consumed during embryogenesis [63], [64], [65], [66], [67].…”

“…cdf-2 encodes a zinc transporter homologous to mammalian ZnT2/ZnT3 [70]. A functional CDF-2::GFP fusion protein localized to gut granules marked with PGP-2 (Figure 9A–C) [33], [70]. Conventional lysosomes containing F11E6.1::mCherry were positioned near the apical surface of embryonic intestinal cells and lacked CDF-2::GFP (Figure 9 G–I).…”

“…The numbers of birefringent granules were approximated and the localization was determined relative to the position of the lumen seen by DIC microscopy. Embryos were stained with the vital stains LysoSensor Green (Invitrogen) and Nile Red (Sigma, St. Louis, MI, USA) as described [4], [33], and visualized with Zeiss 38 Ex: BP470/40 Em: BP525/50 and Zeiss 45 Ex: BP560/40 Em: BP630/75 filters, respectively. GFP and mCherry protein fusions were detected with these same filters in living embryos.…”

SLC30A10: A novel manganese transporter

“…CDF-1 and CDF-2 are known Zn transporters. 24,25 TTM-1 and TOC-1 were predicted to regulate Zn homeostasis, while Y105E8A.3 has an unknown function. First, we established a Mn dose-dependent lethality curve of larva stage 4 (L4) worms in a RNAi sensitive strain GR1373 [eri-1(mg366)] (Fig.…”

Genetics of Lipid-Storage Management inCaenorhabditis elegansEmbryos

“…In C. elegans, yolk seems to be only partially degraded during embryogenesis, as evidenced by the presence of yolk particles in larval intestines Schierenberg 1992, 1996;Grant and Hirsh 1999). Further, lipid-containing droplets have been partially characterized in the C. elegans embryo (Levitte et al 2010;Ehmke et al 2014). In this study, we used C. elegans to identify genes that function in the processing of lipid-containing droplets, especially in the embryo.…”