Giovanni M. Lesa and Esther Marza One of the central challenges in biology is to understand the precise function and regulation of the huge variety of complex lipids found in animal cells. Many lines of evidence suggest that glycosphingolipids (GSLs), complex glycosylated lipids derived from ceramide, participate in several key cellular events leading to neuronal development, cell growth and proliferation, apoptosis and cell migration. Although the precise function of GSLs is unclear, it has been proposed that GSLs form microdomains, or rafts, that would provide the structural environment for interactions between ligands, plasma membrane receptors, and downstream molecules. This would improve or allow signalling. However, it has been difficult to prove that these microdomains are functionally relevant or whether they exist. To clarify whether GSLs exert essential functions in C. elegans cells, we inactivated ceramide glucosyl transferase (CGT), an enzyme needed for GSL biosynthesis. Analysis of the complete genome sequence revealed that C. elegans has three CGTs:
cgt-1 (T06C12.10),
cgt-2 (F20B4.6), and
cgt-3 (F59G1.1). Inactivation of each single cgt gene does not cause any visible phenotype. However, double mutant animals in which two CGTs,
cgt-1 and
cgt-3, are both knocked out, stop growing at the L1 larval stage, undergo progressive paralysis, and eventually die.. We carried out knockdown experiments at different time points and found that cgt activity is required embryonically or within the L1 larval stage. We used constructs expressing fully functional CGT-1::GFP and CGT-3::GFP proteins to determine the localization of CGT-1 and CGT-3. We found that the pharyngeal intestinal valve and a cell/small group of cells in the tail (probably the intestinal-rectal valve) are the only tissues that express both cgts, suggesting that these epithelial tissues might be the focus of the lethal cgt phenotype. To test whether this is the case, we used a variety of promoters to drive expression of
cgt-1 and
cgt-3 in specific tissues. We could rescue the growth arrest phenotype associated with cgt inactivation by only expressing
cgt-1 or
cgt-3 in the pharyngeal intestinal valve and in the intestinal-rectal valve. Because expression in these tissues could also rescue the lethality associated with inactivation of all 3 cgts, we conclude that cgts, and thus also GSLs, are dispensable in most C. elegans cells.