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

Lena Collenburg, Tim Walter, Anne Burgert, Nora Müller, Jürgen Seibel, Lukasz Japtok, Burkhard Kleuser, Markus Sauer and Sibylle Schneider-Schaulies
J Immunol May 1, 2016, 196 (9) 3951-3962; DOI: https://doi.org/10.4049/jimmunol.1502447
Lena Collenburg
*Institute for Virology and Immunobiology, University of Wuerzburg, D-97078 Wuerzburg, Germany;
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Tim Walter
†Institute of Organic Chemistry, University of Wuerzburg, D-97074 Wuerzburg, Germany;
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Anne Burgert
‡Department of Biotechnology and Biophysics, University of Wuerzburg, D-97074 Wuerzburg, Germany; and
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Nora Müller
*Institute for Virology and Immunobiology, University of Wuerzburg, D-97078 Wuerzburg, Germany;
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Jürgen Seibel
†Institute of Organic Chemistry, University of Wuerzburg, D-97074 Wuerzburg, Germany;
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Lukasz Japtok
§Institute of Nutritional Science, University of Potsdam, D-14558 Nuthetal, Potsdam, Germany
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Burkhard Kleuser
§Institute of Nutritional Science, University of Potsdam, D-14558 Nuthetal, Potsdam, Germany
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Markus Sauer
‡Department of Biotechnology and Biophysics, University of Wuerzburg, D-97074 Wuerzburg, Germany; and
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Sibylle Schneider-Schaulies
*Institute for Virology and Immunobiology, University of Wuerzburg, D-97078 Wuerzburg, Germany;
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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.

Footnotes

  • This work was supported by the Deutsche Forschungsgemeinschaft (Grant RU2123).

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    ASM
    acid SMase
    DC
    dendritic cell
    IS
    immune synapse
    MS
    mass spectrometry
    NBD-C6-cer
    6-([N-(7-nitrobenz-2-oxa-1,3-dimensionaliazol-4-yl)amino]hexanoyl)sphingosine
    N3-C6-cer
    ω-azido-C6-ceramide
    N3-C16-cer
    ω-azido-C16-ceramide
    RT
    room temperature
    SIM
    structured illumination microscopy
    SM
    sphingomyelin
    SMase
    sphingomyelinase.

  • Received November 23, 2015.
  • Accepted March 2, 2016.
  • Copyright © 2016 by The American Association of Immunologists, Inc.
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The Journal of Immunology: 196 (9)
The Journal of Immunology
Vol. 196, Issue 9
1 May 2016
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A Functionalized Sphingolipid Analogue for Studying Redistribution during Activation in Living T Cells
Lena Collenburg, Tim Walter, Anne Burgert, Nora Müller, Jürgen Seibel, Lukasz Japtok, Burkhard Kleuser, Markus Sauer, Sibylle Schneider-Schaulies
The Journal of Immunology May 1, 2016, 196 (9) 3951-3962; DOI: 10.4049/jimmunol.1502447

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A Functionalized Sphingolipid Analogue for Studying Redistribution during Activation in Living T Cells
Lena Collenburg, Tim Walter, Anne Burgert, Nora Müller, Jürgen Seibel, Lukasz Japtok, Burkhard Kleuser, Markus Sauer, Sibylle Schneider-Schaulies
The Journal of Immunology May 1, 2016, 196 (9) 3951-3962; DOI: 10.4049/jimmunol.1502447
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