Douglass and Vale combined confocal and total internal reflection microscopy to investigate clustering into membrane microdomains of plasma membrane proteins involved in T cell signaling and concluded that these depended on protein-protein interactions and not lipid rafts. T cell activation by antigen-presenting cells stimulates formation of the immunological synapse; however, the mechanisms underlying formation of this signaling complex remain poorly understood. Douglass and Vale visualized signaling proteins and their mutants that are or are not associated with lipid rafts and found that, when expressed in Jurkat T cells, the mobility of single green fluorescent protein (GFP)-labeled molecules was heterogeneous and did not correlate with association with lipid rafts. Stimulation with antibodies to the T cell receptor decreased the mobility of the raft-associated adaptor protein LAT, as well as that of a nonraft-associated LAT mutant. Stimulation promoted clustering of the nonraft-associated coreceptor CD2 and its colocalization with LAT and the raft-associated tyrosine kinase Lck but not with a nonphosphorylated raft-associated LAT mutant that does not recruit SH2-containing proteins. CD2 clustering was attenuated in a cell line expressing little LAT; it was rescued by expression of LAT but not of the nonphosphorylated mutant. The location of signaling clusters containing CD2, LAT, and Lck was static, whereas individual proteins moved in and out of the cluster. Dual color imaging of the movement of single molecules labeled with GFP relative to clusters containing CD2 labeled with monomeric red fluorescent protein (mRFP) indicated that the mobility of LAT and Lck was sharply reduced inside clusters. Thus, the authors conclude that formation of the signaling clusters, and the immobilization of proteins therein, was independent of association with lipid rafts but depended on protein-protein interactions involving LAT.
A. D. Douglass, R. D. Vale, Single-molecule microscopy reveals plasma membrane microdomains created by protein-protein networks that exclude or trap signaling molecules in T cells. Cell 121, 937-950 (2005). [PubMed]