glo-3, a novel Caenorhabditis elegans gene, is required for lysosome-related organelle biogenesis

Abstract

Gut granules are specialized lysosome-related organelles that act as sites of fat storage in Caenorhabditis elegans intestinal cells. We identified mutations in a gene, glo-3, that functions in the formation of embryonic gut granules. Some glo-3(-) alleles displayed a complete loss of embryonic gut granules, while other glo-3(-) alleles had reduced numbers of gut granules. A subset of glo-3 alleles led to mislocalization of gut granule contents into the intestinal lumen, consistent with a defect in intracellular trafficking. glo-3(-) embryos lacking gut granules developed into adults containing gut granules, indicating that glo-3(+) function may be differentially required during development. We find that glo-3(+) acts in parallel with or downstream of the AP-3 complex and the PGP-2 ABC transporter in gut granule biogenesis. glo-3 encodes a predicted membrane-associated protein that lacks obvious sequence homologs outside of nematodes. glo-3 expression initiates in embryonic intestinal precursors and persists almost exclusively in intestinal cells through adulthood. GLO-3GFP localizes to the gut granule membrane, suggesting it could play a direct role in the trafficking events at the gut granule. smg-1(-) suppression of glo-3(-) nonsense alleles indicates that the C-terminal half of GLO-3, predicted to be present in the cytoplasm, is not necessary for gut granule formation. Our studies identify GLO-3 as a novel player in the formation of lysosome-related organelles.

Figure 1.—

Mutations in glo-3 result in three different embryonic Glo phenotypes. (A and B) Birefringent gut granules (open arrows in B) were present within the intestinal cells of wild-type pretzel-stage embryos. (C and D) glo-3(−) alleles that exhibit a class I phenotype mislocalized birefringent material in the intestinal lumen (solid arrow in D) and did not contain birefringent material within embryonic intestinal cells. (E and F) glo-3(−) alleles that exhibit a class II phenotype mislocalized birefringent material in the intestinal lumen (solid arrow in F) and often contained a few enlarged birefringent granules (open arrow in E) within embryonic intestinal cells. (G and H) glo-3(−) alleles that exhibit a class III phenotype typically contained a reduced number of enlarged birefringent granules within embryonic intestinal cells (open arrows in H) and did not contain birefringent material within the intestinal lumen. Intestinal cells are located between the solid arrowheads in A–H. C. elegans embryos are ∼50 μm in length.

Figure 2.—

Mutations in glo-3 result in two distinct adult Glo phenotypes. Numerous autofluorescent gut granules visualized with a standard FITC filter were present within anterior (A and B) and posterior (G and H) intestinal cells of wild-type animals. glo-3(−) alleles that exhibit a class I phenotype lacked autofluorescent gut granules in anterior intestinal cells (C and D) and displayed substantially reduced numbers of autofluorescent organelles in posterior intestinal cells (I and J). glo-3(−) alleles that exhibit a class II phenotype contained substantially reduced numbers of autofluorescent organelles in both anterior (E and F) and posterior intestinal cells (K and L). The intestinal lumen is marked with a solid arrow in A–L.

Figure 3.—

glo-3(−) adults contain reduced numbers of gut granules. (A–F) In living animals, wild-type gut granules contained autofluorescent material (A, C, and E) that brightly stained with markers for acidity (B), terminal endocytic activity (D), and fat (F). In fixed wild-type adults, gut granules were marked by the PGP-2 protein (M). glo-3(−) adults contained dramatically reduced numbers of autofluorescent gut granules, the majority of which were acidified (H), terminal endocytic compartments (J), that contained fat (L). Nile Red staining of glo-3(−) adults was quite weak and its signal has been increased three to four times that in wild type to aid in its visualization. (O) glo-3(−) adults contained reduced numbers of PGP-2-stained organelles. A few autofluorescent compartments in glo-3(−) adults did not accumulate TRITC-dextran or Nile Red (open arrowheads in J and L). (N and P) Similar numbers of Sudan Black-stained organelles were present in wild-type and glo-3(−) adults. In A–L the intestinal lumen is marked with a solid arrow. Posterior intestinal cells are shown A–P.

Figure 4.—

glo-3(−) embryos lack gut granules but appear to contain other endosomal compartments. Acridine orange stained, acidified compartments present in wild-type 1.5-fold-stage embryos (A) were lacking in glo-3(−) embryos (B) Wild-type embryos displayed fat-containing, Nile Red and BODIPY 493/503-stained compartments (C and E) that were lacking in glo-3(−) embryos (D and F). Wild-type embryos contained anti-FUS-1 and anti-PGP-2 antibody marked compartments (G and I), which were not present in glo-3(−) embryos (H and J). GLO-1∷GFP marked compartments present in wild-type (K) were lacking in glo-3(−) embryos (L). Antibodies that recognize VPS-27 and RAB-5 stained similar organelles in wild-type (M and S) and glo-3(−) (N and T) embryos. Anti-GFP antibodies used to stain embryos expressing RME-1, RAB-5, RAB-7, and LMP-1 fusion proteins showed similar staining patterns in wild-type (O, Q, U, and W) and glo-3(−) embryos (P, R, V, and X), with the exception that enlarged LMP-1∷GFP marked organelles were present in glo-3(−) (open arrows in X). Intestinal cells are located between the solid arrowheads.

Figure 5.—

Predicted structure of the glo-3 gene and encoded protein. (A) The structure of the glo-3 gene and the location and phenotypic class of mutations are shown. (B) The predicted protein sequences and alignment of GLO-3 from C. elegans (Ce), C. briggsae (Cb), and C. remanei (Cr). The location of predicted membrane-spanning domains (TM) identified using TMpred (Hofmann and Stoffel 1993) are shown.

Figure 6.—

glo-3 is expressed in embryonic and adult intestinal cells. Strains carrying an extrachromosomal transgene in which the glo-3 promoter drives the transcription of gfp (glo-3_p∷gfp) resulted in the expression of gfp in intestinal precursors at the E² (A and B) and 1.5-fold stages (C and D). gfp was also expressed in the intestinal cells of L1 larvae (E and F) and adults (G and H). In A and B, the asterisks mark the nuclei of the two gastrulating intestinal precursors. In C–F, intestinal cells are located between the solid arrowheads. The solid arrows mark the intestinal lumen in G and H.

Figure 7.—

GLO-3∷GFP is localized to the gut granule membrane. (A–D) A bean-stage glo-3(zu446) embryo expressing GLO-3∷GFP from the glo-3 promoter showing colocalization of anti-GFP and anti-FUS-1 antibody staining at the same intestinal organelles (white arrows). (F–H) High magnification of the intestine of a GLO-3∷GFP expressing 1.75-fold-stage embryo (white box in E). Birefringent material (pseudocolored red in G and H) is present within GLO-3∷GFP-containing vesicles (white arrows in F–H). Intestinal cells are located between the black arrowheads in A–E.