Abstract
The T cell receptor (TCR) to NF-κB signaling pathway plays a critical role in regulation of proliferation and effector T cell differentiation and function. In naïve T cells, data suggest that most or all key cytoplasmic NF-κB signaling occurs in a TCR-proximal manner at the immunological synapse (IS). However, the subcellular organization of cytoplasmic NF-κB-activating complexes in effector T cells is more complex, involving signaling molecules and regulatory mechanisms beyond those operative in naïve cells. Additionally, in effector T cells, much signaling occurs at cytoplasmic locations distant from the IS. Visualization of these cytoplasmic signaling complexes has provided key insights into the complex and dynamic regulation of NF-κB signal transduction in effector T cells. In this chapter, we provide in-depth protocols for activating and preparing effector T cells for fluorescence imaging, as well as a discussion of the effective application of distinct imaging methodologies, including confocal and super-resolution microscopy and imaging flow cytometry.
Key words
- Signal transduction
- T lymphocyte
- T cell receptor
- POLKADOTS
- Bcl10
- NF-κB
- Confocal microscopy
- Super-resolution microscopy
- Imaging flow cytometry
- Retroviruses
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Acknowledgments
Supported by a grant from the US National Institutes of Health (GM109887-01 to BCS), an Irvington postdoctoral fellowship (to MT) from the Cancer Research Institute, and predoctoral fellowships (to SP) from the American Heart Association (10PRE3150039) and the Henry M. Jackson Foundation for the Advancement of Military Medicine. The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Department of Defense, the Uniformed Services University of the Health Sciences, or any other agency of the US government.
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Traver, M.K., Paul, S., Schaefer, B.C. (2017). T Cell Receptor Activation of NF-κB in Effector T Cells: Visualizing Signaling Events Within and Beyond the Cytoplasmic Domain of the Immunological Synapse. In: Baldari, C., Dustin, M. (eds) The Immune Synapse. Methods in Molecular Biology, vol 1584. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6881-7_8
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DOI: https://doi.org/10.1007/978-1-4939-6881-7_8
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