Trafficking of Acetyl‐C16‐Ceramide‐NBD with Long‐Term Stability and No Cytotoxicity into the Golgi Complex

Abstract

The Golgi complex plays a prominent role in the modification and sorting of lipids and proteins, and is a highly dynamic organelle that is dispersed and rearranged before and after mitosis. Several reagents including 4-nitrobenzo-2-oxa-1,3-diazole-labeled C6-ceramide (NBD-C6-ceramide, a ceramide having an NBD-bound C6-N-acyl chain) and Golgi-specific proteins that emit fluorescence are used as Golgi markers. In the present study, we synthesized a new ceramide analog, acetyl-C16-ceramide-NBD (a ceramide having an acetylated C-1 hydroxyl group, C16-N-acyl chain, and NBD-bound C15-sphingosine), and showed that it preferentially accumulated in the Golgi complex without cytotoxicity for over 24 h. Pathways for cellular uptake and interorganelle trafficking of acetyl-C16-ceramide-NBD were investigated. Acetyl-C16-ceramide-NBD was transported to the Golgi complex via ceramide transport proteins. In contrast to NBD-C6-ceramide, acetyl-C16-ceramide-NBD was resistant to ceramide metabolic enzymes such as sphingomyelin synthase and glucosylceramide synthase. Because of its weaker cytotoxicity and resistance to ceramide metabolic enzymes, the localization of the Golgi complex could be observed in acetyl-C16-ceramide-NBD-labeled cells before and after mitosis. Lipids including sphingolipids play a structural role in the cellular membranes, and regulate physiological and pathological conditions in cells and tissues and in vivo by themselves. The backbone structure of sphingolipids including ceramide is sphingosine, and sphingosine-1-phosphate (S1P, the phosphate ester of the C-1 position of sphingosine) is an important signaling molecule (1,2). Ceramide, which has an N-acyl chain at the C-2 position of sphingosine, is a central point from where various sphingolipids are synthesized and interconverted. The hydroxyl (OH) † These authors contributed equally to this work.group at the C-1 position of ceramide is modified by various enzymes: phosphorylation by ceramide kinase and glucosylation by glucosylceramide (GlcCer) synthase produce ceramide-1-phosphate (C1P) and GlcCer, respectively. Sphingomyelin (SM) synthase transfers a phosphocholine head group from phosphatidylcholine to the OH group at the C-1 position of ceramide and produces SM.Many studies previously reported the usefulness of fluorescence-labeled ceramides and ceramide-related molecules in the study of uptake systems and the intracellular traffic of molecules. 4-Nitrobenzo-2-oxa-1, 476 www.traffic.dk

A Functionalized Sphingolipid Analogue for Studying Redistribution during Activation in Living T Cells

“…6). In line with previous observations made with other cell types (35), NBD-C 6 -cer also proved to be toxic when added to living Jurkat T cells even after 10 min of application, which was not the case for our compounds added at the same concentration (Fig. 2).…”

“…As revealed for NBD-C 6 -cer, which directly reaches the Golgi in a variety of cells and is mainly converted into NBDglucosylceramide, NBD-SM, NBD-ceramide-1-phosphate, and NBD-capronic acid within 2 h, sphingolipid analogues serve as substrate for metabolizing enzymes (35). Because they are minimally modified analogues, alkyne lipids also proved to be reliable substrates for metabolizing enzymes (15); and though we did not address this issue directly, it is more than likely that N 3 -C 6 -cer is also further metabolized.…”

Effects of Glycerophospholipids on Ceramide Kinase Activity: Cardiolipin-Affected Cellular Formation of Ceramide-1-phosphate

“…NBD-labeled ceramide metabolites including NBD-C1P were separated using TLC, and the levels of metabolites were determined as described previously. 14,26,27) Absolute values (pmol/well) of ceramide metabolites in HepG2 cells were similar to those in cells previously reported, 14,26,27) and the relative levels of NBD-sphingomyelin, NBD-glucosylceramide, and NBD-C1P at 30 min after labeling with NBD-ceramide were 5-10 (% of NBD-ceramide), 3-6, and 0.05-0.1%, respectively. Since the ceramide metabolites showed a wide range of values and the absolute values varied depending on experiments, the data in Figs.…”

Sphingolipid metabolism, transport, and functions in plants: Recent progress and future perspectives

“…In plants, polyglycosylated GIPCs in the tobacco PM have been shown to cluster around the outer layer of the PM fraction using the immune colloidal gold technique ( Cacas et al., 2016 ). Fluorophore-labeled sphingolipids are widely used for lipid imaging in mammalian cells ( Makiyama et al., 2015 ). Fluorescence correlation spectroscopy (FCS) with a stimulated emission depletion (STED) microscope (STED-FCS) can reveal lipid dynamics within the PM ( Eggeling et al., 2009 ).…”