title: Lipid raft proteins have a random distribution during localized activation of the T-cell receptor.
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 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.
authors.affiliation: MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK.
authors.forename: Oleg O
authors.initials: OO
authors.lastname: Glebov
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Nichols
mesh_terms: D000818:Animals; D019556:COS Cells; D002772:Cholera Toxin; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D006801:Humans; D019169:Jurkat Cells; D008213:Lymphocyte Activation / Q000502:physiology*; D021962:Membrane Microdomains / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D011948:Receptors, Antigen, T-Cell / Q000187:drug effects / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D013601:T-Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D017261:Glycosylphosphatidylinositols; D008565:Membrane Proteins; D011948:Receptors, Antigen, T-Cell; D002772:Cholera Toxin