Visualizing TCR-induced POLKADOTS formation and NF-κB activation in the D10 T-cell clone and mouse primary effector T cells.
T cells are an immune cell lineage that play a central role in protection against pathogen infection. Antigen, in the form of pathogen-derived peptides, stimulates the T-cell receptor (TCR), leading to activation of the transcription factor, nuclear factor kappa B (NF-κB). The subsequent NF-κB-dependent gene expression program drives expansion and effector differentiation of antigen-specific T cells, leading to the adaptive anti-pathogen immune response. The cell surface TCR transmits activating signals to cytosolic NF-κB by a complex signaling cascade, in which the adapter protein Bcl10 plays a key role. We have previously demonstrated that TCR engagement leads to the formation of cytosolic Bcl10 clusters, called POLKADOTS, that provide a platform for the assembly of the terminal signaling complex that ultimately mediates NF-κB activation. In this chapter, we describe the methods utilized to visualize the formation of TCR-induced POLKADOTS and to study the temporal association between POLKADOTS formation and nuclear translocation of the NF-κB subunit, RelA/p65.
Department of Microbiology and Immunology, Uniformed Services University, Bethesda, MD, 20814, USA.
Suman
S
Paul
Brian C
BC
Schaefer
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D000938:Antigen-Presenting Cells / Q000276:immunology; D000941:Antigens / Q000276:immunology; D000074018:B-Cell CLL-Lymphoma 10 Protein; D002454:Cell Differentiation; D003001:Cloning, Molecular; D004789:Enzyme Activation; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D008213:Lymphocyte Activation / Q000276:immunology; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D016328:NF-kappa B / Q000378:metabolism*; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D015398:Signal Transduction; D016176:T-Lymphocyte Subsets / Q000166:cytology / Q000276:immunology / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D000941:Antigens; D000074018:B-Cell CLL-Lymphoma 10 Protein; C118591:BCL10 protein, human; D016328:NF-kappa B; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins