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Detection of Osmotic Shock-Induced Extracellular Nucleotide Release with a Genetically Encoded Fluorescent Sensor of ADP and ATP.
Trull, Keelan J; Miller, Piper; Tat, Kiet; Varney, S Ashley; Conley, Jason M; Tantama, Mathew.
Afiliación
  • Trull KJ; Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, IN 47907, USA.
  • Miller P; Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, IN 47907, USA.
  • Tat K; Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, IN 47907, USA.
  • Varney SA; Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, IN 47907, USA.
  • Conley JM; Department of Pediatrics, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
  • Tantama M; Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, IN 47907, USA. mt4@wellesley.edu.
Sensors (Basel) ; 19(15)2019 Jul 24.
Article en En | MEDLINE | ID: mdl-31344821
Purinergic signals, such as extracellular adenosine triphosphate (ATP) and adenosine diphosphate (ADP), mediate intercellular communication and stress responses throughout mammalian tissues, but the dynamics of their release and clearance are still not well understood. Although physiochemical methods provide important insight into physiology, genetically encoded optical sensors have proven particularly powerful in the quantification of signaling in live specimens. Indeed, genetically encoded luminescent and fluorescent sensors provide new insights into ATP-mediated purinergic signaling. However, new tools to detect extracellular ADP are still required. To this end, in this study, we use protein engineering to generate a new genetically encoded sensor that employs a high-affinity bacterial ADP-binding protein and reports a change in occupancy with a change in the Förster-type resonance energy transfer (FRET) between cyan and yellow fluorescent proteins. We characterize the sensor in both protein solution studies, as well as live-cell microscopy. This new sensor responds to nanomolar and micromolar concentrations of ADP and ATP in solution, respectively, and in principle it is the first fully-genetically encoded sensor with sufficiently high affinity for ADP to detect low levels of extracellular ADP. Furthermore, we demonstrate that tethering the sensor to the cell surface enables the detection of physiologically relevant nucleotide release induced by hypoosmotic shock as a model of tissue edema. Thus, we provide a new tool to study purinergic signaling that can be used across genetically tractable model systems.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Técnicas Biosensibles / Adenosina Difosfato / Adenosina Trifosfato / Edema Tipo de estudio: Diagnostic_studies / Prognostic_studies Límite: Humans Idioma: En Revista: Sensors (Basel) Año: 2019 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Suiza

Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Técnicas Biosensibles / Adenosina Difosfato / Adenosina Trifosfato / Edema Tipo de estudio: Diagnostic_studies / Prognostic_studies Límite: Humans Idioma: En Revista: Sensors (Basel) Año: 2019 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Suiza