Imaging Spatiotemporal Activities of ZAP-70 in Live T Cells Using a FRET-Based Biosensor

Ann Biomed Eng. 2016 Dec;44(12):3510-3521. doi: 10.1007/s10439-016-1683-6. Epub 2016 Jul 6.


The zeta-chain-associated protein kinase 70 kDa (ZAP-70), a member of the spleen tyrosine kinase (Syk) family, plays an essential role in early T cell receptor (TCR) signaling. Defects in ZAP-70 lead to impaired thymocyte development and peripheral T cell activation. To better understand its activation dynamics and regulation, we visualized ZAP-70 activities in single live T cells with a Förster resonance energy transfer (FRET)-based biosensor, which was designed for probing kinase activities of the Syk family. We observed in Jurkat E6.1 T cells rapid and specific FRET changes following anti-CD3 stimulation and subsequent piceatannol inhibition. The initiation of ZAP-70 activation was prompt (within 10 s) and correlates with the accompanied intracellular calcium elevation, as revealed by simultaneous imaging of the biosensor and calcium. Different from the previously reported ZAP-70 activation in the immunological synapse and the opposite pole (anti-synapse), we have observed rapid and sustained ZAP-70 activation only at the synapse with superantigen-pulsed Raji B cells. Furthermore, ZAP-70 signaling was impaired by cholesterol depletion, further supporting the importance of membrane organization in TCR signaling. Together our results provide a direct characterization of the spatiotemporal features of ZAP-70 activity in real time at subcellular levels.

Keywords: Immunological synapse; Membrane microdomains; Syk family kinase; TCR signaling.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Biosensing Techniques / methods*
  • Cholesterol / genetics
  • Cholesterol / metabolism
  • Fluorescence Resonance Energy Transfer / methods*
  • Humans
  • Immunological Synapses / genetics
  • Immunological Synapses / metabolism
  • Jurkat Cells
  • Receptors, Antigen, T-Cell / genetics
  • Receptors, Antigen, T-Cell / metabolism
  • Signal Transduction / physiology*
  • T-Lymphocytes / cytology
  • T-Lymphocytes / metabolism*
  • ZAP-70 Protein-Tyrosine Kinase / genetics
  • ZAP-70 Protein-Tyrosine Kinase / metabolism*


  • Receptors, Antigen, T-Cell
  • Cholesterol
  • ZAP-70 Protein-Tyrosine Kinase
  • ZAP70 protein, human