Time-Domain Fluorescence Lifetime Imaging of cAMP Levels with EPAC-Based FRET Sensors.

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.

Dynamic FRET-FLIM based screening of signal transduction pathways.

Fluorescence Lifetime Imaging (FLIM) is an intrinsically quantitative method to screen for protein-protein interactions and is frequently used to record the outcome of signal transduction events. With new highly sensitive and photon efficient FLIM instrumentation, the technique also becomes attractive to screen, with high temporal resolution, for fast changes in Förster Resonance Energy Transfer (FRET), such as those occurring upon activation of cell signaling. The second messenger cyclic adenosine monophosphate (cAMP) is rapidly formed following activation of certain cell surface receptors. cAMP is subsequently degraded by a set of phosphodiesterases (PDEs) which display cell-type specific expression and may also affect baseline levels of the messenger. To study which specific PDEs contribute most to cAMP regulation, we knocked down individual PDEs and recorded breakdown rates of cAMP levels following transient stimulation in HeLa cells stably expressing the FRET/FLIM sensor, Epac-SH189. Many hundreds of cells were recorded at 5 s intervals for each condition. FLIM time traces were calculated for every cell, and decay kinetics were obtained. cAMP clearance was significantly slower when PDE3A and, to a lesser amount, PDE10A were knocked down, identifying these isoforms as dominant in HeLa cells. However, taking advantage of the quantitative FLIM data, we found that knockdown of individual PDEs has a very limited effect on baseline cAMP levels. By combining photon-efficient FLIM instrumentation with optimized sensors, systematic gene knockdown and an automated open-source analysis pipeline, our study demonstrates that dynamic screening of transient cell signals has become feasible. The quantitative platform described here provides detailed kinetic analysis of cellular signals in individual cells with unprecedented throughput.

cAMP Biosensor Assay Using BacMam Expression System: Studying the Downstream Signaling of LH/hCG Receptor Activation.

Cyclic adenosine monophosphate (cAMP) serves as a second messenger for numerous G-protein-coupled receptors. Changes in cellular cAMP levels reflect the biological activity of various GPCR-specific agents, including protein hormones. cAMP biosensors based on detection of Förster-type resonance energy transfer (FRET) offer unique advantages including the ratiometric nature of measurement, adjustable affinity toward detected molecule, capability of monitoring kinetics of cAMP release, and compatibility with the multi-well format and fluorescence plate reader platforms. In this chapter, we introduce the optimized version of the previously reported method to achieve sufficient and reproducible level of cAMP biosensor protein expression with the means of BacMam transduction system. As a practical challenge, we address the applicability of the designed assay for screening of biological activity of human hormones, including human chorionic gonadotropin (hCG) bearing different posttranslational modifications.