Abstract
Second messenger molecules in eukaryotic cells relay the signals from activated cell surface receptors to intracellular effector proteins. FRET-based sensors are ideal to visualize and measure the often rapid changes of second messenger concentrations in time and place. Fluorescence Lifetime Imaging (FLIM) is an intrinsically quantitative technique for measuring FRET. Given the recent development of commercially available, sensitive and photon-efficient FLIM instrumentation, it is becoming the method of choice for FRET detection in signaling studies. Here, we describe a detailed protocol for time domain FLIM, using the EPAC-based FRET sensor to measure changes in cellular cAMP levels with high spatiotemporal resolution as an example.
Highlights
Second messengers such as Ca2+, IP3, cGMP, and Cyclic adenosine monophosphate (cAMP) are critical intermediates that relay signals from membrane-bound receptors to intracellular effectors
Intracellular concentrations of cAMP change rapidly when it is synthesized from ATP by a family of adenyl cyclases (ACs) or degraded by phosphodiesterases (PDEs)
We deleted the membrane-binding DEP domain (ΔDEP) of EPAC1, and we introduced point mutations to render EPAC catalytically inactive (CD, T781A, and F782A) so as to prevent unwanted downstream signaling to Rap1 and Rap2 [13, 17]
Summary
Second messengers such as Ca2+, IP3, cGMP, and cAMP are critical intermediates that relay signals from membrane-bound receptors to intracellular effectors. Traditional TCSPC detectors are photon efficient, but have until recently been extremely slow and unable to deliver the speed for biological processes occurring at time scales below tens of seconds Such limitations in speed and experimental complexity were overcome by efforts of several groups, including our laboratory in tight cooperation with Leica Microsystems, and resulted in the development of FLIM instrumentation like the Leica SP8 FALCON [28]. The protocol described here should be applicable to recordings with other intramolecular FRET sensors Note that whereas this protocol focuses on TD-FLIM application to measure cAMP levels in single cells with exceptional quantitative sensitivity, we have previously provided a detailed protocol [31] on the use of FD-FLIM in combination with EPAC-based biosensors and a detailed protocol [32] for cAMP sensing by ratiometric detection of sensitized emission
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