T cell signal regulation by the actin cytoskeleton.
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.
Chichili
Gurunadh R
GR
Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA.
Westmuckett
Andrew D
AD
Rodgers
William
W
D000199:Actins / Q000378:metabolism*; D000970:Antineoplastic Agents / Q000494:pharmacology; D002784:Cholesterol / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism*; D047630:Depsipeptides / Q000494:pharmacology; D005372:Filipin / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D054992:Immunological Synapses / Q000187:drug effects / Q000378:metabolism*; D019169:Jurkat Cells; D017493:Leukocyte Common Antigens / Q000378:metabolism*; D008213:Lymphocyte Activation; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000378:metabolism*; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism*; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism; D013601:T-Lymphocytes / Q000187:drug effects / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism
D000199:Actins; D000970:Antineoplastic Agents; D047630:Depsipeptides; D011948:Receptors, Antigen, T-Cell; C057531:jasplakinolide; D014443:Tyrosine; D005372:Filipin; D002784:Cholesterol; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D017493:Leukocyte Common Antigens