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

Cadra, Sophie; Gucciardi, Alexia; Valignat, Marie-Pierre; Théodoly, Olivier; Vacaflores, Aldo; Houtman, Jon C. D.; Lellouch, Annemarie C.

doi:10.1016/j.bbrc.2015.02.117

Cited by 14 publications

(20 citation statements)

References 24 publications

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“…All primers were custom made by Microsynth (Balgach, Switzerland). pcDNA3.1 + ROZA XL and pcDNA3.1 + ROZA XL YF (27) were a kind gift of Annemarie Lellouch (Addgene plasmids #64194 and #64195).…”

Section: Methodsmentioning

confidence: 99%

CCR7 Is Recruited to the Immunological Synapse, Acts as Co-stimulatory Molecule and Drives LFA-1 Clustering for Efficient T Cell Adhesion Through ZAP70

et al. 2019

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The chemokine receptor CCR7 guides T cells and dendritic cells to and within lymph nodes to launch the onset of adaptive immunity. Here, we demonstrate that CCR7 in addition acts as a potent co-stimulatory molecule in T cell activation. We found that antigen recognition and engagement of the TCR results in CCR7 accumulation at the immunological synapse where CCR7 and the TCR co-localize within sub-synaptic vesicles. We demonstrate that CCR7 triggering alone is sufficient to recruit and activate ZAP70, a critical kinase for T cell activation, through Src kinase, whereas TCR CCR7 co-stimulation results in increased and prolonged ZAP70 kinase activity. Finally, we show that ZAP70, acting as adapter molecule, is critical for CCR7-mediated inside-out signaling to integrins, thereby modulating LFA-1 valency regulation to promote cell adhesion, a key step in immunological synapse formation and efficient T cell activation.

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Section: Methodsmentioning

confidence: 99%

CCR7 Is Recruited to the Immunological Synapse, Acts as Co-stimulatory Molecule and Drives LFA-1 Clustering for Efficient T Cell Adhesion Through ZAP70

et al. 2019

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“…They could determine the activity of ZAP-70 during TCR-mediated signalling and interestingly found the activation not only at the TCR-APC interface pole (synapse), but also at the opposite side (anti-synapse). This biosensor was recently further developed and improved by Cadra et al [ 146 ].…”

Section: Types Of Fret Biosensors and Their Practical Applicationsmentioning

confidence: 99%

Fluorescent Proteins as Genetically Encoded FRET Biosensors in Life Sciences

Hochreiter

Garcia

Schmid

2015

Sensors

166

121

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Fluorescence- or Förster resonance energy transfer (FRET) is a measurable physical energy transfer phenomenon between appropriate chromophores, when they are in sufficient proximity, usually within 10 nm. This feature has made them incredibly useful tools for many biomedical studies on molecular interactions. Furthermore, this principle is increasingly exploited for the design of biosensors, where two chromophores are linked with a sensory domain controlling their distance and thus the degree of FRET. The versatility of these FRET-biosensors made it possible to assess a vast amount of biological variables in a fast and standardized manner, allowing not only high-throughput studies but also sub-cellular measurements of biological processes. In this review, we aim at giving an overview over the recent advances in genetically encoded, fluorescent-protein based FRET-biosensors, as these represent the largest and most vividly growing group of FRET-based sensors. For easy understanding, we are grouping them into four categories, depending on their molecular mechanism. These are based on: (a) cleavage; (b) conformational-change; (c) mechanical force and (d) changes in the micro-environment. We also address the many issues and considerations that come with the development of FRET-based biosensors, as well as the possibilities that are available to measure them.

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“…31,34,38 FRET biosensors for ZAP-70 were reported with its detection of ZAP-70 kinase activity at both the immunological synapse and the opposing pole of synapse. 8,29 Based on the same principle, we have developed and validated a biosensor specific for Syk family kinases and observed Syk activation through FcγRIIA and PDGF receptor signaling. 40 Since Syk and ZAP-70 are differentially expressed in immune cells (e.g.…”

Section: Introductionmentioning

confidence: 99%

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

Xue

Sun

et al. 2016

Ann Biomed Eng

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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 sec) 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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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

Cited by 14 publications

References 24 publications

CCR7 Is Recruited to the Immunological Synapse, Acts as Co-stimulatory Molecule and Drives LFA-1 Clustering for Efficient T Cell Adhesion Through ZAP70

CCR7 Is Recruited to the Immunological Synapse, Acts as Co-stimulatory Molecule and Drives LFA-1 Clustering for Efficient T Cell Adhesion Through ZAP70

Fluorescent Proteins as Genetically Encoded FRET Biosensors in Life Sciences

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

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