Abstract
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.
Highlights
Purinergic signaling regulates a broad range of critical physiological processes such as neuron-glia communication, immune cell responses, and platelet aggregation, and may be a key determinant of disease states such as cancer [1,2,3,4,5,6]
In our initial designs of the sensor, we explored two possible Förster-type resonance energy transfer (FRET)-based architectures that differed in the positioning of the cyan fluorescent protein (CFP)-yellow fluorescent protein (YFP) FRET pair when fused to the ParM protein in order to take advantage of alternative aspects of the adenosine diphosphate (ADP)-dependent structural change
We developed a genetically encoded fluorescent sensor of extracellular ADP called
Summary
Purinergic signaling regulates a broad range of critical physiological processes such as neuron-glia communication, immune cell responses, and platelet aggregation, and may be a key determinant of disease states such as cancer [1,2,3,4,5,6]. We recently developed a genetically-encoded fluorescent sensor of extracellular ATP [13], but there has not been a genetically encoded fluorescent. One major challenge to the development of these purinergic sensors is that sensor of ADP until now. One major challenge to the development of these purinergic sensors is that extracellular nucleotides signal at low concentrations, putatively at hundreds of nanomolar to low extracellular nucleotides signal at low concentrations, putatively at hundreds of nanomolar to low micromolar levels [14]. We take advantage of a bacterial protein that has a high affinity for ADP, micromolar levels [14]. We take advantage of a bacterial protein that has a high affinity for ADP, called ParM, and use it to engineer an extracellular ADP sensor successfully
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