Roles of CUP-5, the Caenorhabditis elegans orthologue of human TRPML1, in lysosome and gut granule biogenesis

Abstract

Background: CUP-5 is a Transient Receptor Potential protein in C. elegans that is the orthologue of mammalian TRPML1. Loss of TRPML1 results in the lysosomal storage disorder Mucolipidosis type IV. Loss of CUP-5 results in embryonic lethality and the accumulation of enlarged yolk granules in developing intestinal cells. The embryonic lethality of cup-5 mutants is rescued by mutations in mrp-4, which is required for gut granule differentiation. Gut granules are intestine-specific lysosome-related organelles that accumulate birefringent material. This link between CUP-5 and gut granules led us to determine the roles of CUP-5 in lysosome and gut granule biogenesis in developing intestinal cells.

Results: We show that CUP-5 protein localizes to lysosomes, but not to gut granules, in developing intestinal cells. Loss of CUP-5 results in defects in endo-lysosomal transport in developing intestinal cells of C. elegans embryos. This ultimately leads to the appearance of enlarged terminal vacuoles that show defective lysosomal degradation and that have lysosomal and endosomal markers. In contrast, gut granule biogenesis is normal in the absence of CUP-5. Furthermore, loss of CUP-5 does not result in inappropriate fusion or mixing of content between lysosomes and gut granules.

Conclusions: Using an in vivo model of MLIV, we show that there is a defect in lysosomal transport/biogenesis that is earlier than the presumed function of TRPML1 in terminal lysosomes. Our results indicate that CUP-5 is required for the biogenesis of lysosomes but not of gut granules. Thus, cellular phenotypes in Mucolipidosis type IV are likely not due to defects in lysosome-related organelle biogenesis, but due to progressive defects in lysosomal transport that lead to severe lysosomal dysfunction.

Figure 1

Late endocytic and lysosome-related organelles in developing intestinal cells of wild type embryos. A) Epifluorescence images of a wild type embryo that expresses YP170::GFP and stained with Nile Red. B) Confocal images of a wild type embryo that expresses YP170::GFP and immunostained to detect PGP-2. C) Epifluorescence images of a wild type embryo that expresses YP170::GFP and LMP-1::TagRFP(S158T). D) Confocal images of a wild type embryo that expresses YP170::GFP and immunostained to detect PGP-2. Bottom panels are magnified images of the regions indicated in the top panels. Scale bars in whole embryo images represent 10 μm; scale bars in magnified images represent 2 μm.

Figure 2

CUP-5 protein localization in developing intestinal cells. A) Percent lethality of WT, cup-5(zu223), cup-5(zu223); Pelt-2::gfp::cup-5 and cup-5(zu223); Pelt-2:: mCherry::cup-5 embryos. Error bars represent the 95% confidence intervals for significance. B) Epifluorescence images of a wild type embryo that expresses LMP-1::GFP and mCherry::CUP-5. C) Confocal images of a wild type embryo that expresses GFP::CUP-5 and LMP-1::TagRFP(S158T). D) Epifluorescence images of a wild type embryo that expresses YP170::GFP and mCherry::CUP-5. E) Confocal images of a wild type embryo that expresses GFP::CUP-5 and immunostained to detect PGP-2. Bottom panels are magnified images of the regions indicated in the top panels. Scale bars in whole embryo images represent 10 μm; scale bars in magnified images represent 2 μm.

Figure 3

Late endocytic and lysosome-related organelles in developing intestinal cells of cup-5 mutant embryos. A) Confocal images of a cup-5(zu223) embryo that expresses YP170::GFP and stained with Nile Red. B) Confocal images of a cup-5(zu223) embryo that expresses YP170::GFP and immunostained to detect PGP-2. C) Epifluorescence images of a cup-5(zu223) embryo that expresses YP170::GFP and LMP-1::TagRFP(S158T). Small arrows point to organelles containing only YP170::GFP; large arrowheads point to organelles containing both YP170::GFP and LMP-1::TagRFP(S158T). D) Confocal images of a cup-5(zu223) embryo that expresses LMP-1::GFP and immunostained to detect PGP-2. Bottom panels are magnified images of the regions indicated in the top panels. Scale bars in whole embryo images represent 10 μm; scale bars in magnified images represent 2 μm.

Figure 4

cup-5 mutant effects on sizes and intensities of markers of late endocytic and lysosome-related organelles in developing intestinal cells. Images of wild type and cup-5(zu223) embryos, as well as quantification of sizes and intensities per unit area of compartments with YP170::GFP (A), LMP-1::GFP (B), PGP-2 (C), and UV (D) signals. Images for the same marker were taken using the same microscopy parameters. Scale bars represent 10 μm. Error bars represent the 95% confidence intervals for significance, and asterisks represent statistical significance at p < 0.05, compared to WT. In cup-5(zu223) embryos, the difference in areas of YP170::GFP organelles (A) with or without LMP-1::TagRFP(S158T) is also statistically significant.

Figure 5

Model of CUP-5 functions in developing intestinal cells. Drawings of intestinal cells showing the markers used in this study in wild type and cup-5 mutant embryos. Organelles are not drawn to actual scale.