Single-molecule microscopy reveals plasma membrane microdomains created by protein-protein networks that exclude or trap signaling molecules in T cells.
Membrane subdomains have been implicated in T cell signaling, although their properties and mechanisms of formation remain controversial. Here, we have used single-molecule and scanning confocal imaging to characterize the behavior of GFP-tagged signaling proteins in Jurkat T cells. We show that the coreceptor CD2, the adaptor protein LAT, and tyrosine kinase Lck cocluster in discrete microdomains in the plasma membrane of signaling T cells. These microdomains require protein-protein interactions mediated through phosphorylation of LAT and are not maintained by interactions with actin or lipid rafts. Using a two color imaging approach that allows tracking of single molecules relative to the CD2/LAT/Lck clusters, we demonstrate that these microdomains exclude and limit the free diffusion of molecules in the membrane but also can trap and immobilize specific proteins. Our data suggest that diffusional trapping through protein-protein interactions creates microdomains that concentrate or exclude cell surface proteins to facilitate T cell signaling.
Douglass
Adam D
AD
The Howard Hughes Medical Institute and Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, California 94107, USA.
Vale
Ronald D
RD
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000378:metabolism; D018801:CD2 Antigens / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure*; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000502:physiology; D006801:Humans; D007091:Image Processing, Computer-Assisted; D019169:Jurkat Cells; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000737:chemistry / Q000378:metabolism; D021962:Membrane Microdomains / Q000737:chemistry* / Q000502:physiology; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods*; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D015398:Signal Transduction / Q000502:physiology; D013601:T-Lymphocytes / Q000648:ultrastructure*; D013997:Time Factors
D048868:Adaptor Proteins, Signal Transducing; D018801:CD2 Antigens; C110810:LAT protein, human; D008565:Membrane Proteins; D010750:Phosphoproteins; D049452:Green Fluorescent Proteins; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck)