Ceramide, a central molecule in the sphingolipid biosyn- thesis, plays a critical role as second messenger in cellular signaling that regulates antiproliferative processes, including apoptosis, cell differentiation, and cell cycle arrest in differ-

Sphingolipids are a highly complex class of lipids in terms of structural diversity, metabolism, and cellular functions. While initially seen as inert structural components of eukaryotic cell membranes, there is now substantial evidence that sphingolipids play an important role in signal transduction. (For a recent review on bioactive sphingolipids, see Ref. 1 .) Ceramide, a central molecule in the sphingolipid biosynthesis, plays a critical role as second messenger in cellular signaling that regulates antiproliferative processes, including apoptosis, cell differentiation, and cell cycle arrest in different cell types. Levels of ceramide, a highly bioactive molecule, must be tightly controlled by diverse, coordinated mechanisms, including ceramide degradation, phosphorylation, or sphingolipid metabolism. Glucosylceramide synthase (GCS), also defi ned as ceramide glucosyltransferase (CGT), metabolizes ceramide to glucosylceramide (GlcCer), a glycosylated form of ceramide that does not have antiproliferative activity. GCS plays a crucial role in cell survival after apoptotic stimuli, Abstract Synthesis of glucosylceramide via glucosylceramide synthase (GCS) is a crucial event in higher eukaryotes, both for the production of complex glycosphingolipids and for regulating cellular levels of ceramide, a potent antiproliferative second messenger. In this study, we explored the dependence of the early branching eukaryote Giardia lamblia on GCS activity. Biochemical analyses revealed that the parasite has a GCS located in endoplasmic reticulum (ER) membranes that is active in proliferating and encysting trophozoites. Pharmacological inhibition of GCS induced aberrant cell division, characterized by arrest of cytokinesis, incomplete cleavage furrow formation, and consequent block of replication. Importantly, we showed that increased ceramide levels were responsible for the cytokinesis arrest. In addition, GCS inhibition resulted in prominent ultrastructural abnormalities, including accumulation of cytosolic vesicles, enlarged lysosomes, and clathrin disorganization. Moreover, anterograde traffi cking of the encystations-specifi c protein CWP1 was severely compromised and resulted in inhibition of stage differentiation. Our results reveal novel aspects of lipid metabolism in G. lamblia and specifi cally highlight the vital role of GCS in regulating cell cycle progression, membrane traffi cking events, and stage differentiation in this parasite. In addition, we identifi ed ceramide as a potent bioactive molecule, underscoring the universal conservation of ceramide signaling in eukaryotes. —Štefanić, S., C. Spycher, L. Morf, G. Fabriàs, J. Casas, E. Schraner, P. Wild, A. B. Hehl, and S. Sonda. Glucosylceramide synthesis inhibition affects cell cycle progression, membrane traffi ck-