High-resolution multicolor imaging of dynamic signaling complexes in T cells stimulated by planar substrates.
The dynamic visualization of developing immunological synapses has been hindered by the difficulty of imaging the contact between the T cell and the antigen-presenting cell (APC). Here, we describe a technique in which T cell responses are constrained to a planar stimulatory substrate. This approach, when used in conjunction with immunofluorescent staining procedures or fluorescent protein tags, greatly facilitates detection of the dynamic molecular rearrangements that accompany the formation of contacts and the initiation of signal transduction through the T cell receptor (TCR). Using this method, we have observed signaling complexes of dynamically varying compositions that possess distinct fates.
Bunnell
Stephen C
SC
Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4255, USA. sbunnell@helix.nih.gov
Barr
Valarie A
VA
Fuller
Claudette L
CL
Samelson
Lawrence E
LE
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D000938:Antigen-Presenting Cells / Q000502:physiology; D001426:Bacterial Proteins / Q000096:biosynthesis; D017252:CD3 Complex; D015194:Diffusion Chambers, Culture; D019084:Fluorescent Antibody Technique, Indirect; D049452:Green Fluorescent Proteins; D006801:Humans; D007202:Indicators and Reagents; D019169:Jurkat Cells; D008164:Luminescent Proteins / Q000096:biosynthesis; D008565:Membrane Proteins / Q000276:immunology / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D018715:Microscopy, Video; D011948:Receptors, Antigen, T-Cell / Q000276:immunology / Q000378:metabolism; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000276:immunology / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D015398:Signal Transduction / Q000276:immunology*; D013601:T-Lymphocytes / Q000502:physiology*; D016707:Tissue Fixation; D014162:Transfection; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D017252:CD3 Complex; C115335:CD3E protein, human; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D008565:Membrane Proteins; D011948:Receptors, Antigen, T-Cell; D016693:Receptors, Antigen, T-Cell, alpha-beta; D011993:Recombinant Fusion Proteins; C054979:antigen T cell receptor, zeta chain; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins