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Reconstitution of TCR Signaling Using Supported Lipid Bilayers

  • Xiaolei Su
  • Jonathon A. Ditlev
  • Michael K. Rosen
  • Ronald D. ValeEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1584)

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.

Key words

TCR Microcluster Reconstitution Supported lipid bilayer Multivalency Phase separation Actin LAT 

Notes

Acknowledgment

We thank Marcus Taylor and Enfu Hui for their assistance in developing the reconstitution assays. This work was supported by grants from the HCIA program of HHMI, the NIH (R01-GM56322 to M.K.R.), and Welch Foundation (I-1544 to M.K.R.). X.S. was supported by CRI Irvington postdoctoral fellowship. J.A.D. was supported by NRSA F32 award 5-F32-DK101188.

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Xiaolei Su
    • 1
    • 2
  • Jonathon A. Ditlev
    • 1
    • 3
  • Michael K. Rosen
    • 1
    • 3
  • Ronald D. Vale
    • 1
    • 2
    Email author
  1. 1.Marine Biological LaboratoryThe HHMI Summer InstituteWoods HoleUSA
  2. 2.Department of Cellular and Molecular Pharmacology and Howard Hughes Medical InstituteUniversity of CaliforniaSan FranciscoUSA
  3. 3.Department of Biophysics and Howard Hughes Medical InstituteUniversity of Texas Southwestern Medical CenterDallasUSA

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