title: Imaging Spatiotemporal Activities of ZAP-70 in Live T Cells Using a FRET-Based Biosensor.
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.
authors.affiliation: Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA, USA.
authors.forename: Kaitao
authors.initials: K
authors.lastname: Li
authors.affiliation: Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA, USA.|School of Life Sciences, SUN YAT-SEN University, Guangzhou, China.|UnionPay Smart Co., Ltd, Shanghai, China.
authors.forename: Xue
authors.initials: X
authors.lastname: Xiang
authors.affiliation: Department of Bioengineering and Beckman Institute for Advanced Science and Technology, University of Illinois, Urbana-Champaign, Urbana, IL, USA.|Center for Cell Engineering, Memorial Sloan Kettering Cancer Center, New York, USA.
authors.forename: Jie
authors.initials: J
authors.lastname: Sun
authors.affiliation: Centre d'Immunologie de Marseille-Luminy, Aix Marseille Université UM2, Inserm, U1104, CNRS UMR7280, 13288, Marseille, France.
authors.forename: Hai-Tao
authors.initials: HT
authors.lastname: He
authors.affiliation: School of Life Sciences, SUN YAT-SEN University, Guangzhou, China.|School of Bioscience, South China University of Technology, Guangzhou, China.
authors.forename: Jianhua
authors.initials: J
authors.lastname: Wu
authors.affiliation: Department of Bioengineering and Beckman Institute for Advanced Science and Technology, University of Illinois, Urbana-Champaign, Urbana, IL, USA.|Department of Bioengineering, University of California, San Diego, La Jolla, CA, USA.
authors.forename: Yingxiao
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA, USA. cheng.zhu@bme.gatech.edu.
authors.forename: Cheng
authors.initials: C
authors.lastname: Zhu
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D002784:Cholesterol / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D054992:Immunological Synapses / Q000235:genetics / Q000378:metabolism; D019169:Jurkat Cells; D011948:Receptors, Antigen, T-Cell / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D013601:T-Lymphocytes / Q000166:cytology / Q000378:metabolism*; D051746:ZAP-70 Protein-Tyrosine Kinase / Q000235:genetics / Q000378:metabolism*
chemical_list: D011948:Receptors, Antigen, T-Cell; D002784:Cholesterol; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human
keywords: Immunological synapse; Membrane microdomains; Syk family kinase; TCR signaling