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.

Antibacterial activity of ceramide and ceramide analogs against pathogenic Neisseria

“…We next made use of recently synthesized azido-functionalized ceramide analogs 21 , 22 allowing bio-orthogonal click–reactions 18 in order to follow uptake and transport of fluorescently labelled ceramides in N . meningitidis .…”

Acid sphingomyelinase mediates human CD4+ T-cell signaling: potential roles in T-cell responses and diseases

“…Subsequently, ceramides redistribute and accumulate at plasma membrane rapidly to form ceramide-enriched domains and initiate downstream signals. 10 Given the pivotal roles of ASM activity and ceramide redistribution in changing membrane domains and mediating cell signals, 7 we propose broad interactions of ASM with numbers of membrane proteins and receptors. Consequently, through the interactions with those membrane proteins, ASM/ceramide signaling activates relevant signals in T cells and exhibits distinct functions in responses to certain extracellular stimuli.…”

“… 8 , 9 Once generated, ceramide will accumulate at plasma membrane to form ceramide-enriched membrane microcluster, leading to initiation of downstream signals. 10 Through the generation of ceramide, ASM has an important role in regulating cell differentiation, proliferation, and apoptosis. 1 , 11 …”

Nanoscale imaging of bacterial infections by sphingolipid expansion microscopy

“…Ceramide-rich membrane areas promote structural changes within the plasma membrane, which segregate membrane receptors and affect the membrane curvature and vesicle formation, fusion and trafficking. 19,20 We selected ω-N3-C6-ceramide, which is efficiently incorporated into cellular membranes and can be click-labeled with DBCO-functionalized dyes for fluorescence imaging 21,22 , for further functionalization with a primary amino group (Supporting Information and Supplementary Figures S1-S13). Therefore, we synthesized α-NH2-ω-N3-C6-ceramide from (tert-butoxycarbonyl)-Llysine ( Figure 1A).…”