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

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Abstract

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.

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Acknowledgments

This work was supported by NIH Grant R01AI044902 (to C.Z.), NIH HL098472, HL109142, HL121365 (Y.W.). We thank Dr. Arthur Weiss for kindly sharing the Jurkat P116 cell line expressing kinase-inactive ZAP-70.

Author information

Author notes

  1. Xue Xiang

    Present address: UnionPay Smart Co., Ltd, Shanghai, China

  2. Jie Sun

    Present address: Center for Cell Engineering, Memorial Sloan Kettering Cancer Center, New York, USA

  3. Jianhua Wu

    Present address: School of Bioscience, South China University of Technology, Guangzhou, China

  4. Yingxiao Wang

    Present address: Department of Bioengineering, University of California, San Diego, La Jolla, CA, USA

Authors and Affiliations

  1. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA, USA

    Kaitao Li, Xue Xiang & Cheng Zhu

  2. School of Life Sciences, SUN YAT-SEN University, Guangzhou, China

    Xue Xiang & Jianhua Wu

  3. Department of Bioengineering and Beckman Institute for Advanced Science and Technology, University of Illinois, Urbana-Champaign, Urbana, IL, USA

    Jie Sun & Yingxiao Wang

  4. Centre d’Immunologie de Marseille-Luminy, Aix Marseille Université UM2, Inserm, U1104, CNRS UMR7280, 13288, Marseille, France

    Hai-Tao He

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  1. Kaitao Li
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Correspondence to Cheng Zhu.

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Associate Editor Sriram Neelamegham oversaw the review of this article.

Kaitao Li and Xue Xiang are the co-first authors.

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Li, K., Xiang, X., Sun, J. et al. Imaging Spatiotemporal Activities of ZAP-70 in Live T Cells Using a FRET-Based Biosensor. Ann Biomed Eng 44, 3510–3521 (2016). https://doi.org/10.1007/s10439-016-1683-6

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