title: A Functionalized Sphingolipid Analogue for Studying Redistribution during Activation in Living T Cells.
abstract: 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 C6-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.
authors.affiliation: Institute for Virology and Immunobiology, University of Wuerzburg, D-97078 Wuerzburg, Germany;
authors.forename: Lena
authors.initials: L
authors.lastname: Collenburg
authors.affiliation: Institute of Organic Chemistry, University of Wuerzburg, D-97074 Wuerzburg, Germany;
authors.forename: Tim
authors.identifier: 0000-0002-5961-8927
authors.initials: T
authors.lastname: Walter
authors.affiliation: Department of Biotechnology and Biophysics, University of Wuerzburg, D-97074 Wuerzburg, Germany; and.
authors.forename: Anne
authors.initials: A
authors.lastname: Burgert
authors.affiliation: Institute for Virology and Immunobiology, University of Wuerzburg, D-97078 Wuerzburg, Germany;
authors.forename: Nora
authors.identifier: 0000-0002-9512-1555
authors.initials: N
authors.lastname: Müller
authors.affiliation: Institute of Organic Chemistry, University of Wuerzburg, D-97074 Wuerzburg, Germany;
authors.forename: Jürgen
authors.identifier: 0000-0002-8036-4853
authors.initials: J
authors.lastname: Seibel
authors.affiliation: Institute of Nutritional Science, University of Potsdam, D-14558 Nuthetal, Potsdam, Germany.
authors.forename: Lukasz
authors.initials: L
authors.lastname: Japtok
authors.affiliation: Institute of Nutritional Science, University of Potsdam, D-14558 Nuthetal, Potsdam, Germany.
authors.forename: Burkhard
authors.identifier: 0000-0002-1888-9595
authors.initials: B
authors.lastname: Kleuser
authors.affiliation: Department of Biotechnology and Biophysics, University of Wuerzburg, D-97074 Wuerzburg, Germany; and.
authors.forename: Markus
authors.initials: M
authors.lastname: Sauer
authors.affiliation: Institute for Virology and Immunobiology, University of Wuerzburg, D-97078 Wuerzburg, Germany; s-s-s@vim.uni-wuerzburg.de.
authors.forename: Sibylle
authors.initials: S
authors.lastname: Schneider-Schaulies
mesh_terms: D001386:Azides / Q000737:chemistry / Q000378:metabolism*; D002478:Cells, Cultured; D006801:Humans; D008213:Lymphocyte Activation; D021962:Membrane Microdomains / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods*; D021381:Protein Transport; D011940:Receptor Aggregation; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D015398:Signal Transduction; D013107:Sphingolipids / Q000737:chemistry / Q000378:metabolism*; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism*
chemical_list: D001386:Azides; D011948:Receptors, Antigen, T-Cell; D013107:Sphingolipids