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

  • Sphingolipids are major components of the plasma membrane. In particular, ceramide serves as an essential building hub for complex sphingolipids, but also as an organizer of membrane domains segregating receptors and signalosomes. Sphingomyelin breakdown as a result of sphingomyelinase activation after ligation of a variety of receptors is the predominant source of ceramides released at the plasma membrane. This especially applies to T lymphocytes where formation of ceramide-enriched membrane microdomains modulates TCR signaling. Because ceramide release and redistribution occur very rapidly in response to receptor ligation, novel tools to further study these processes in living T cells are urgently needed. To meet this demand, we synthesized nontoxic, azido-functionalized ceramides allowing for bio-orthogonal click-reactions to fluorescently label incorporated ceramides, and thus investigate formation of ceramide-enriched domains. Azido-functionalized C-6-ceramides were incorporated into and localized within plasma membraneSphingolipids are major components of the plasma membrane. In particular, ceramide serves as an essential building hub for complex sphingolipids, but also as an organizer of membrane domains segregating receptors and signalosomes. Sphingomyelin breakdown as a result of sphingomyelinase activation after ligation of a variety of receptors is the predominant source of ceramides released at the plasma membrane. This especially applies to T lymphocytes where formation of ceramide-enriched membrane microdomains modulates TCR signaling. Because ceramide release and redistribution occur very rapidly in response to receptor ligation, novel tools to further study these processes in living T cells are urgently needed. To meet this demand, we synthesized nontoxic, azido-functionalized ceramides allowing for bio-orthogonal click-reactions to fluorescently label incorporated ceramides, and thus investigate formation of ceramide-enriched domains. Azido-functionalized C-6-ceramides were incorporated into and localized within plasma membrane microdomains and proximal vesicles in T cells. They segregated into clusters after TCR, and especially CD28 ligation, indicating efficient sorting into plasma membrane domains associated with T cell activation; this was abolished upon sphingomyelinase inhibition. Importantly, T cell activation was not abrogated upon incorporation of the compound, which was efficiently excluded from the immune synapse center as has previously been seen in Ab-based studies using fixed cells. Therefore, the functionalized ceramides are novel, highly potent tools to study the subcellular redistribution of ceramides in the course of T cell activation. Moreover, they will certainly also be generally applicable to studies addressing rapid stimulation-mediated ceramide release in living cells.show moreshow less

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Metadaten
Author:Lena Collenburg, Tim Walter, Anne Burgert, Nora Mueller, Juergen Seibel, Lukasz Japtok, Burkhard KleuserORCiDGND, Markus Sauer, Sibylle Schneider-Schaulies
DOI:https://doi.org/10.4049/jimmunol.1502447
ISSN:0022-1767
ISSN:1550-6606
Pubmed Id:http://www.ncbi.nlm.nih.gov/pubmed?term=27036914
Parent Title (English):The journal of immunology
Publisher:American Assoc. of Immunologists
Place of publication:Bethesda
Document Type:Article
Language:English
Year of first Publication:2016
Year of Completion:2016
Release Date:2020/03/22
Volume:196
Pagenumber:12
First Page:3951
Last Page:3962
Funder:Deutsche Forschungsgemeinschaft [RU2123]
Organizational units:Mathematisch-Naturwissenschaftliche Fakultät / Institut für Ernährungswissenschaft
Peer Review:Referiert