1. Single-molecule microscopy reveals plasma membrane microdomains created by protein-protein networks that exclude or trap signaling molecules in T cells.
- Author
-
Douglass AD and Vale RD
- Subjects
- Adaptor Proteins, Signal Transducing chemistry, Adaptor Proteins, Signal Transducing metabolism, CD2 Antigens chemistry, CD2 Antigens metabolism, CD2 Antigens physiology, Cell Membrane chemistry, Cell Membrane metabolism, Green Fluorescent Proteins physiology, Humans, Image Processing, Computer-Assisted, Jurkat Cells, Lymphocyte Specific Protein Tyrosine Kinase p56(lck) chemistry, Lymphocyte Specific Protein Tyrosine Kinase p56(lck) metabolism, Membrane Microdomains physiology, Membrane Proteins chemistry, Membrane Proteins metabolism, Phosphoproteins chemistry, Phosphoproteins metabolism, Protein Binding, Signal Transduction physiology, Time Factors, Cell Membrane ultrastructure, Green Fluorescent Proteins chemistry, Membrane Microdomains chemistry, Microscopy, Fluorescence methods, T-Lymphocytes ultrastructure
- Abstract
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.
- Published
- 2005
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