TY - JOUR A1 - Collenburg, Lena A1 - Walter, Tim A1 - Burgert, Anne A1 - Mueller, Nora A1 - Seibel, Juergen A1 - Japtok, Lukasz A1 - Kleuser, Burkhard A1 - Sauer, Markus A1 - Schneider-Schaulies, Sibylle T1 - A Functionalized Sphingolipid Analogue for Studying Redistribution during Activation in Living T Cells JF - The journal of immunology N2 - 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 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. Y1 - 2016 U6 - https://doi.org/10.4049/jimmunol.1502447 SN - 0022-1767 SN - 1550-6606 VL - 196 SP - 3951 EP - 3962 PB - American Assoc. of Immunologists CY - Bethesda ER -