[
Development & Evolution Meeting,
2008]
We have isolated a mutant strain with a greater than two-fold increase in fat accumulation compared to wild type. Genetic epistasis experiments indicate that the defect is not caused by a reduction in the activity of the insulin/insulin-like or TGF-beta signaling pathways but rather from a defect downstream of or in parallel to these pathways. We made extracts of fats from the mutants and analyzed them by gas chromatography. This analysis revealed that the fatty acids C16:1n9 and C18:1n9 were present at disproportionately higher levels than in wild type but that the fatty acid distribution was otherwise similar to that in wild type. Thus the mutant causes a global increase in fat accumulation as well as changes in the distribution of specific fatty acids. We have performed RNAi with a panel of 150 clones that have been shown to reduce fat in a wild-type background (1) and found 15 genes that can reduce fat in the background of the mutant. At the same time, we have performed QRT-PCR with 120 genes implicated in the fatty acid synthesis, beta-oxidation or other aspects of fat metabolism (2) and identified 25 genes whose expression is altered in the mutant. The expression and RNAi data are consistent with a model in which the increase in fat in the mutant is caused by increased expression of CPT-5, a carnitine palmitoyl transferase, and reduced expression of F52B11.2 and F08F8.1, which encode a phosphomannomutase and a novel protein respectively.
[
International Worm Meeting,
2015]
The contributions of non-proteinaceous molecules (e.g. lipids and sugars) to polarity and tubulogenesis are poorly understood. In an unbiased tubulogenesis screen, we previously identified a requirement of lipid-biosynthetic enzymes for C. elegans intestinal polarity. A follow-on tiered lipid-biosynthetic-pathway-screen approach* allowed us to identify glycosphingolipids (GSLs), presumed membrane raft components, as the synthesized compounds mediating the enzyme's function. The analysis procured in vivo evidence for GSLs' role in apical sorting and supported aspects of the controversial lipid-raft theory on polarity. Similarly controversial is a proposed requirement for sugars, specifically N-glycans, for polarity via apical sorting. To systematically investigate an in vivo requirement for sugars in polarity, we here use this biosynthetic-pathway-screen approach to query carbohydrate biosynthesis.In an initial broad tier-I screen we examined apical marker displacement subsequent to RNAi with each of 209 molecules predicted to affect synthesis, transfer, modification and transport of nucleotide-sugars, N-glycans, O-glycans, glycosaminoglycans and GPI-anchors. Only two enzyme knockdowns affected polarity: F52B11.2, a phosphomannomutase homolog, catalyzing activated nucleotide-sugar synthesis; and BUS-8, an alpha-1,3-mannosyltransferase homolog, needed for lipid-linked oligosaccharide N-, but not O-glycosylation. The follow-on tightened tier-II RNAi screen was thus targeted towards N-glycosylation and detected phenocopy when depleting 7 more genes: F09E5.2, a BUS-8 paralog; four oligosaccharyltransferase-complex components and a dolichol-phosphate-mannosyl-transferase-complex- and GDP-mannose-pyrophosphorylase subunit each. These screens thus identify an in vivo requirement for N-glycan biosynthesis in apical polarity. To examine whether N-glycan diversification was required for function, we next knocked down 104 N-glycan-derivative-specific lectins. This tier III screen, combined with triple mutant analyses of critical enzymes, failed to detect any such requirement. In contrast, an RNAi screen of N-glycosylated proteins revealed that this class of molecules is enriched for proteins required for apical marker placement and intestinal lumen morphogenesis. This class of molecules contains lumen-associated mucins as well as most secreted ECM components, raising the possibility that these molecules, via their sugar-containing portions, affect trafficking pathways for apical polarity. *Zhang et al., 2015, Nature Protocol, 10, 681-700.