ROZA-XL, an improved FRET based biosensor with an increased dynamic range for visualizing Zeta Associated Protein 70 kD (ZAP-70) tyrosine kinase activity in live T cells
ROZA-XL, an improved FRET based biosensor with an increased dynamic range for visualizing Zeta Associated Protein 70 kD (ZAP-70) tyrosine kinase activity in live T cells
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DOI：
10.1016/j.bbrc.2015.02.117
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发表时间：
2015-04-10
影响因子：
3.1
通讯作者：
Lellouch, Annemarie C.
中科院分区：
文献类型：
--
作者：
Cadra, Sophie;Gucciardi, Alexia;Lellouch, Annemarie C.
基因编码FRET为基础的生物传感器允许一个可视化的空间和时间进化的特定酶活性的活细胞。我们之前曾报道过一种基于FRET的生物传感器的创建，该传感器专门针对ζ相关蛋白- 70kd (ZAP-70) (Randriamampita等人，2008)，一种Syk家族蛋白酪氨酸激酶。ZAP-70对早期T细胞受体（TCR）信号事件、T淋巴细胞发育至关重要，也与整合素介导的T淋巴细胞迁移有关。为了便于研究免疫突触形成或细胞迁移等动态现象中ZAP-70激酶活性，我们设计并制备了第二代ZAP-70特异性生物传感器。在这里，我们描述了一种名为ROZA-XL的新型生物传感器，其动态范围比其前身大3-4倍，并且在Jurkat人类T细胞系中表达时具有强大的基线FRET值。我们证明，这种生物传感器的强大行为允许在单个细胞水平上快速分析TCR介导的ZAP-70激酶活性，如简单的终点分析所示，其中表达ROZA-XL的细胞被允许与刺激性抗cd3epsilon涂覆的盖层相互作用。(C) 2015爱思唯尔公司版权所有。
Genetically encoded FRET based biosensors allow one to visualize the spatial and temporal evolution of specific enzyme activities in live cells. We have previously reported the creation of a FRET based biosensor specific for Zeta-Associated Protein -70 kD (ZAP-70) (Randriamampita et al., 2008), a Syk family protein tyrosine kinase. ZAP-70 is essential for early T cell receptor (TCR) signaling events, T lymphocyte development and has also been implicated in integrin mediated T lymphocyte migration. In order to facilitate the study of ZAP-70 kinase activity during dynamic phenomena such as immunological synapse formation or cell migration, we have designed and prepared a second generation of ZAP-70 specific biosensors. Here we describe a novel biosensor named ROZA-XL, that displays a 3-4 times greater dynamic range than its predecessor and possesses a robust baseline FRET value when expressed in the Jurkat human T cell line. We demonstrate that the robust behavior of this biosensor allows for rapid analysis of TCR mediated of ZAP-70 kinase activity at a single cell level, as shown in a simple end point assay in which ROZA-XL expressing cells are allowed to interact with stimulatory anti-CD3epsilon coated coverslips. (C) 2015 Elsevier Inc. All rights reserved.