title: 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.
authors.affiliation: Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260-8675, Japan.
authors.forename: Tomohiko
authors.initials: T
authors.lastname: Makiyama
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Nakamura
authors.forename: Nobuo
authors.initials: N
authors.lastname: Nagasaka
authors.forename: Hisahiro
authors.initials: H
authors.lastname: Yamashita
authors.forename: Takuya
authors.initials: T
authors.lastname: Honda
authors.forename: Naoto
authors.initials: N
authors.lastname: Yamaguchi
authors.forename: Atsushi
authors.initials: A
authors.lastname: Nishida
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Murayama
mesh_terms: D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D001692:Biological Transport; D016466:CHO Cells; D002352:Carrier Proteins / Q000378:metabolism; D018929:Cell Culture Techniques; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002518:Ceramides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D003412:Cricetulus; D005456:Fluorescent Dyes; D006056:Golgi Apparatus / Q000187:drug effects* / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D008938:Mitosis / Q000187:drug effects; D015394:Molecular Structure
chemical_list: D002352:Carrier Proteins; D002518:Ceramides; D005456:Fluorescent Dyes; C000604916:acetyl-C16-ceramide-NBD; D016718:Arachidonic Acid; D009327:4-Chloro-7-nitrobenzofurazan
keywords: CERT; CHO cells; Golgi complex; acetyl-C16-ceramide-NBD; cytotoxicity