Abstract
Adenosine triphosphate (ATP) is the central energy carrier of all cells and knowledge on the dynamics of the concentration of ATP ([ATP]) provides important insights into the energetic state of a cell. Several genetically encoded fluorescent nanosensors for ATP were developed, which allow following the cytosolic [ATP] at high spatial and temporal resolution using fluorescence microscopy. However, to calibrate the fluorescent signal to [ATP] has remained challenging. To estimate basal cytosolic [ATP] ([ATP]0) in astrocytes, we here took advantage of two ATP nanosensors of the ATeam-family (ATeam1.03; ATeam1.03YEMK) with different affinities for ATP. Altering [ATP] by external stimuli resulted in characteristic pairs of signal changes of both nanosensors, which depend on [ATP]0. Using this dual nanosensor strategy and epifluorescence microscopy, [ATP]0 was estimated to be around 1.5 mM in primary cultures of cortical astrocytes from mice. Furthermore, in astrocytes in acutely isolated cortical slices from mice expressing both nanosensors after stereotactic injection of AAV-vectors, 2-photon microscopy revealed [ATP]0 of 0.7 mM to 1.3 mM. Finally, the change in [ATP] induced in the cytosol of cultured cortical astrocytes by application of azide, glutamate, and an increased extracellular concentration of K+ were calculated as −0.50 mM, −0.16 mM, and 0.07 mM, respectively. In summary, the dual nanosensor approach adds another option for determining the concentration of [ATP] to the increasing toolbox of fluorescent nanosensors for metabolites. This approach can also be applied to other metabolites when two sensors with different binding properties are available.
Highlights
The metabolism of the brain is realized by a joint effort of all cell types including neurons, glial cells as well as cells constituting the blood vessels
We reasoned that a treatment of cells, which results in a given change in [adenosine triphosphate (ATP)] (d[ATP]), will cause different relative changes of the sensor signal for AT and ATY
At [ATP]0 = 2 mM a change in [ATP] of −0.5 mM will result in dRAT = −38% and dRATY = −17%; while at [ATP]0 = 3 mM the same change in ATP will result in dRAT = −20% and dRATY = −6% (Figure 1B)
Summary
The metabolism of the brain is realized by a joint effort of all cell types including neurons, glial cells as well as cells constituting the blood vessels. Fluorescence nanosensors for metabolites are well suited to follow the concentration of a growing set of metabolites at high spatial and temporal resolution in vitro, in situ, and in vivo (Imamura et al, 2009; Hung et al, 2011; San Martin et al, 2014; Lange et al, 2015; Mächler et al, 2016; Mongeon et al, 2016; Trevisiol et al, 2017; Winkler et al, 2017; Köhler et al, 2018; Barros et al, 2018a; Díaz-García et al, 2019; Zuend et al, 2020) These sensors are proteins composed of a domain which binds the metabolite of interest as well as one or more fluorescent proteins, which change their fluorescent properties upon metabolite binding. Examples of such sensors include sensors for adenosine triphosphate (ATP; e.g., sensors of the ATeam family, Imamura et al, 2009; Queen, Yaginuma et al, 2014; Takaine et al, 2019), the ATP/ADP ratio (Perceval, Berg et al, 2009; PercevalHR, Nguyen et al, 2019), the NADH/NAD+-redox ratio (Peredox, Hung et al, 2011; Sonar, Zhao et al, 2015), NADPH (iNap family, Zhao et al, 2016), glucose (FlipGlu, Fehr et al, 2003; SweetieTS, Díaz-García et al, 2019), lactate (Laconic, San Martín et al, 2013), and pyruvate (Pyronic, San Martín et al, 2014; PyronicSF, Arce-Molina et al, 2020)
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