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

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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 rafts. Glycosylphosphatidylinositol (GPI)-linked proteins are characteristic components of biochemically defined rafts. Several studies report a function for rafts in T-cell stimulation, 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 interactions. Here we used FRET analysis 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). 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.