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Lipid raft proteins have a random distribution during localized activation of the T-cell receptor

Nature Cell Biology volume 6, pages 238–243 (2004)Cite this article

Abstract

The extent to which lipid raft proteins are organized in functional clusters within the plasma membrane is central to the debate on structure and function of rafts1,2,3. Glycosylphosphatidylinositol (GPI)-linked proteins are characteristic components of biochemically defined rafts1,4,5. Several studies report a function for rafts in T-cell stimulation6,7,8, but it is unclear whether molecules involved in T-cell receptor (TCR) signalling are recruited to (or excluded from) T-cell synapses through asymmetric distribution of raft microdomains or through specific protein–protein interactions9,10. Here we used FRET analysis11 in live cells to determine whether GPI-linked proteins are clustered in the plasma membrane of unstimulated cells, and at regions where TCR signalling has been activated using antibody-coated beads. Multiple criteria suggested that FRET between different GPI-linked fluorescent proteins in COS-7 or unstimulated Jurkat T-cells is generated by a random, un-clustered distribution. Stimulation of TCR signalling in Jurkat cells resulted in localized increases in fluorescence of GPI-linked fluorescent proteins and cholera toxin B-subunit (CTB)12. However, measurements of FRET and ratio imaging showed that there was no detectable clustering and no overall enrichment of GPI-linked proteins or CTB in these regions.

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Figure 1: GPI-anchored proteins have a predominantly random distribution.
Figure 2: Fluorescence from lipid raft markers during TCR activation.
Figure 3: FRET ratio imaging of GPI-linked proteins.
Figure 4: Ratio imaging of cells labelled with raft and non-raft markers.

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Acknowledgements

Thanks to T. Zimmermann, P. Keller, A. Kenworthy, L. Samelson, T. Harder and K. Simons for DNA constructs, and S. Munro, H. Pelham, K. Schmidt and A. Kenworthy for critical reading of the manuscript.

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  1. MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK

    Oleg O. Glebov & Benjamin J. Nichols

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  1. Oleg O. Glebov
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Correspondence to Benjamin J. Nichols.

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Glebov, O., Nichols, B. Lipid raft proteins have a random distribution during localized activation of the T-cell receptor. Nat Cell Biol 6, 238–243 (2004). https://doi.org/10.1038/ncb1103

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