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First published online 30 August 2006
doi: 10.1242/dev.02575

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Suppression of the cup-5 mucolipidosis type IV-related lysosomal dysfunction by the inactivation of an ABC transporter in C. elegans

Department of Molecular and Cellular Biology, Life Sciences South Room 531, University of Arizona, Tucson, AZ 85721, USA.

View larger version (25K): [in a new window]   Fig. 1. Suppression of cup-5(zu223) by mrp-4(cd8). (A) Percentage of laid embryos of the indicated genotypes that hatched. (B) Percentage of hatched embryos of the indicated genotypes that developed into adults. (C) Percentage of adults of the indicated genotypes that were fertile. Normal media (white bars), media that included 14 mM methylpyruvate (mPvy; gray bars). (D) Sequence of mrp-4 showing the position of the SL1 trans-splice site, the translation initiation codon and open reading frame of the first exon (uppercase), and the sequence change in the cd8 mutant allele. The complete sequence of MRP-4 is available from GenBank (Accession Number NP_509658).

View larger version (35K): [in a new window]   Fig. 2. mrp-4(cd8) suppresses the receptor degradation defect and the developmental defect of cup-5(zu223). Confocal micrographs of `1.5-fold' embryos of the indicated genotypes immunostained for the detection of RME-2, LIN-12 or IFB-2. Images of embryos stained with the same antibody were taken with the same exposure and magnification. All strains also carried the array. The outlines of the embryos are drawn. Scale bar: 10 µm.

View larger version (67K): [in a new window]   Fig. 3. mrp-4(cd8) suppresses yolk degradation and lipid accumulation defects of cup-5(zu223). (A) Confocal micrographs of `comma' stage embryos laid by hermaphrodites carrying the YP170::GFP transgene (green in merge) and grown on plates containing LysoTracker Red (red in merge). All images of the same marker were taken with the same exposure and at the same magnification. Arrows indicate staining of intestinal cells. (B) Quantitation of the surface area of the YP170::GFP/LysoTracker Red granules in intestinal cells shown in A. One pixel is 0.01 µm2. (C) Confocal micrographs of `1.5-fold' stage embryos laid by the indicated hermaphrodites carrying the GFP::LGG-1 transgene. Long arrows indicate staining of intestinal cells. This transgene also carries a marker that expresses GFP in pharyngeal cells (short arrows). All images were taken with the same exposure and at the same magnification. (D) Confocal micrographs of wild-type, mrp-4(cd8), cup-5(zu223) or mrp-4(cd8); cup-5(zu223) `comma' stage embryos stained using the TUNEL assay to detect DNA-strand breaks. The average number of TUNEL-staining bodies in each strain is indicated at the top of each panel. (E) Confocal micrographs of `1.5 fold' stage embryos laid by hermaphrodites carrying the YP170::GFP transgene (green in merge) and stained for Nile Red (red in merge). All images of the same marker were taken with the same exposure and at the same magnification. Arrows indicate staining of intestinal cells. (F) Quantitation of the intensity per surface area of the Nile Red granules in intestinal cells shown in E. Scale bars: 10 µm in all images. The alleles in all panels are mrp-4(cd8) and cup-5(zu223).

View larger version (48K): [in a new window]   Fig. 4. Functional complementation of mrp-4(cd8) and localization of MRP-4::GFP. (A) Confocal micrographs of `1.5-fold' stage embryo or adult hermaphrodites expressing a transcriptional fusion of the mrp-4 promoter to GFP. (B) Percent of embryos that hatch and percent of hatched embryos that develop to adults in the indicated strains. (C) Confocal micrographs of `1.5-fold' stage embryos laid by hermaphrodites carrying the MRP-4::GFP transgene (green in merge) and grown on plates containing LysoTracker Red (red in merge). All images of the same marker were taken with the same exposure and at the same magnification. (D) Quantitation of the surface area of the LysoTracker Red granules in intestinal cells shown in C. Wild-type and cup-5(zu223) values are included for comparison. One pixel is 0.01 µm2. (E) Confocal micrographs of `1.5 fold' stage embryos laid by hermaphrodites carrying the MRP-4::GFP transgene (green in merge) and stained with Nile Red (red in merge). All images of the same marker were taken with the same exposure and at the same magnification. (F) Quantitation of the intensity per surface area of the Nile Red granules in intestinal cells shown in E. Wild type and cup-5(zu223) values are included for comparison. The alleles in all panels are mrp-4(cd8) and cup-5(zu223). Scale bar: 10 µm in C,D. Large arrows indicate staining of intestinal cells. The MRP-4::GFP transgene also carries a marker that expresses GFP in pharyngeal cells (small arrows).

View larger version (13K): [in a new window]   Fig. 5. Suppression of other mutations by mrp-4(cd8). (A) Percentage of laid embryos of the indicated genotypes that hatched. ced-9 embryos that hatched arrested development as L1 larvae. All of the hatched embryos from other strains developed into adults. (B) Percentage of laid embryos of the indicated genotypes that hatched after control or rme-2 RNAi. Alleles used are ced-9(n1950n2161), rme-2((b1008), cpl-1(ok360), cup-5(zu223) and mrp-4(cd8).