Abstract
T cells form an immunological synapse (IS) that sustains and regulates signals for cell stimulation. Enriched in the IS is the Src family kinase Lck. Conversely, the membrane phosphatase CD45, which activates Src family kinases, is excluded, and this is necessary to avoid quenching of T cell receptor phosphosignals. Data suggest that this arrangement occurs by an enrichment of cholesterol-dependent rafts in the IS. However, the role of rafts in structuring the IS remains unclear. To address this question, we used fluorescence resonance energy transfer (FRET) to interrogate the nanoscopic structure of the IS. The FRET probes consisted of membrane-anchored fluorescent proteins with distinct affinities for rafts. Both the raft and nonraft probes exhibited clustering in the IS. However, co-clustering of raft donor-acceptor pairs was 10-fold greater than co-clustering of raft-nonraft pairs. We measured the effect of disrupting rafts in the IS on CD45 localization and Lck regulation by treating stimulated T cells with filipin. The filipin specifically disrupted co-clustering of the raft FRET pairs in the IS and allowed targeting of CD45 to the IS and dephosphorylation of the regulatory tyrosine of Lck. Clustering of the raft probes was also sensitive to latrunctulin B, which disrupts actin filaments. Strikingly, enriching the cortical cytoskeleton using jasplakinolide maintained raft probe co-clustering, CD45 exclusion, and Lck regulation in the IS following the addition of filipin. These data show the actin cytoskeleton maintains a membrane raft environment in the IS that promotes Lck regulation by excluding CD45.
Highlights
immunological synapse (IS) has multiple roles in T cell activation, including sustaining and regulating signals from the TCR [2,3,4,5,6,7]
The IS has a distinct architecture, in many cases represented by a central supramolecular activation complex (c-SMAC) that is surrounded by a peripheral supramolecular activation complex (p-SMAC) [8, 9]
Localization of Membrane-anchored Fluorescent Proteins to the T Cell IS—We illustrate in Fig. 1A the membraneanchored fluorescent proteins (FPs) that we used for labeling the raft and nonraft fractions of the plasma membrane
Summary
Much of the uncertainty regarding the mechanisms that define IS macrostructure, such as CD45 exclusion, relate to a poor understanding of its plasma membrane nanostructure This is especially a shortcoming in models relating to rafts because most occur as nanoscale domains [20, 24, 40]. Conditions that disrupted DRM-associated probe clustering resulted in deregulation of Lck by dephosphorylation of its Tyr505 These data suggest that rafts are structured by actin filaments that attach to the plasma membrane, producing domains that function in Lck regulation [43]. We report findings that show exclusion of CD45 from the IS that occurs by structuring of rafts at the cell interface We show that these properties are necessary for maintaining regulation of Lck in the IS
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