Reconstitution of TCR signaling using supported lipid bilayers

Xiaolei Su, Jonathon A. Ditlev, Michael K. Rosen, Ronald D. Vale

Research output: Chapter in Book/Report/Conference proceedingChapter

8 Scopus citations

Abstract

Biochemical reconstitution has served as an important tool for understanding the mechanisms of many cellular processes including DNA replication, transcription, translation, vesicle trafficking, and ubiquitin-mediated proteolysis. Here, we demonstrate that biochemical reconstitution can be applied to studying a complex signaling pathway involving as many as 12 proteins or protein complexes acting at the surface of model membranes. We show that a temporal sequence of events in activated T cells beginning with phosphorylation of the T cell receptor and culminating in the activation of actin polymerization can be replicated in vitro. Our reconstitution demonstrates the sufficiency of these proteins in producing many of the complex behaviors observed during T cell activation. The ability to manipulate all of the components, measure reaction rates, and observe molecular behaviors, including at single molecule resolution, has enabled us to gain insight into some of the important biochemical features of this signaling pathway such as microcluster formation. The same system could be adapted to study other membrane-proximal signaling pathways, including growth factor receptors, death receptors, and Eph receptors.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages65-76
Number of pages12
DOIs
StatePublished - 2017

Publication series

NameMethods in Molecular Biology
Volume1584
ISSN (Print)1064-3745

Keywords

  • Actin
  • LAT
  • Microcluster
  • Multivalency
  • Phase separation
  • Reconstitution
  • Supported lipid bilayer
  • TCR

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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  • Cite this

    Su, X., Ditlev, J. A., Rosen, M. K., & Vale, R. D. (2017). Reconstitution of TCR signaling using supported lipid bilayers. In Methods in Molecular Biology (pp. 65-76). (Methods in Molecular Biology; Vol. 1584). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-6881-7_5