Fourth-Generation Epac-Based FRET Sensors for cAMP Feature Exceptional Brightness, Photostability and Dynamic Range: Characterization of Dedicated Sensors for FLIM, for Ratiometry and with High Affinity

Abstract

Epac-based FRET sensors have been widely used for the detection of cAMP concentrations in living cells. Originally developed by us as well as others, we have since then reported several important optimizations that make these sensors favourite among many cell biologists. We here report cloning and characterization of our fourth generation of cAMP sensors, which feature outstanding photostability, dynamic range and signal-to-noise ratio. The design is based on mTurquoise2, currently the brightest and most bleaching-resistant donor, and a new acceptor cassette that consists of a tandem of two cp173Venus fluorophores. We also report variants with a single point mutation, Q270E, in the Epac moiety, which decreases the dissociation constant of cAMP from 9.5 to 4 μM, and thus increases the affinity ~ 2.5-fold. Finally, we also prepared and characterized dedicated variants with non-emitting (dark) acceptors for single-wavelength FLIM acquisition that display an exceptional near-doubling of fluorescence lifetime upon saturation of cAMP levels. We believe this generation of cAMP outperforms all other sensors and therefore recommend these sensors for all future studies.

Quantitative Single-Cell Analysis of Signaling Pathways Activated Immediately Downstream of Histamine Receptor Subtypes

“…FRET reporters can be developed to measure every step in the GPCR signaling cascade. FRET biosensors are available to measure ligand binding to the GPCR (Stoddart et al, 2015), GPCR activation (Vilardaga et al, 2003), GPCR and G-protein interaction (Hein et al, 2005;Stumpf and Hoffmann, 2016), G-protein activation (Janetopoulos et al, 2001;Adjobo-Hermans et al, 2011), Ca 21 release (Nagai et al, 2004), cAMP production (Klarenbeek et al, 2015), and activation of downstream effectors such as protein kinase C (Verbeek et al, 2008), RhoA (van Unen et al, 2015a), and inositol 1,4,5-trisphosphate (Gulyás et al, 2015). The preferred option is to use FRET biosensors that report on the specific activation of one of the heterotrimeric G-protein subfamilies directly stimulated by a GPCR.…”

Dysregulation of Neuronal Ca 2+ Channel Linked to Heightened Sympathetic Phenotype in Prohypertensive States

“…For intracellular measurements of cAMP and PKA activity, cells were infected 0 -24 h after culture with the FRET-based cAMP sensors Epac-H30 (Ponsioen et al, 2004), Epac-H187 (Klarenbeek et al, 2015) or the PKA sensor AKAR4 (Depry et al, 2011; Lefkimmiatis et al, 2013) for 24 h. FRET sensor-expressing neurons were imaged 3-4 d after isolation on an inverted Nikon microscope. This was connected to an OptoLED fluores-cence imaging system (Cairn Research ) equipped with a 40ϫ oilimmersion objective, a CoolSnap HQ2 digital CCD camera (Photometics), and a beam-splitter (DV2; Photometrics), which included the emission filters for CFP and YFP acquisition (dichroic mirror 505DCXR).…”

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

“…The third exemplar FLIM FRET experiment concerns a genetically expressed FRET biosensor for Exchange protein activated by cyclic adenosine monophosphate (cAMP (EPAC)), which is a Rap-1 guanine exchange factor that binds specifically to cAMP. This EPAC FRET biosensor (EPAC-S H188 ) utilizes mTurquoise2 fluorescent protein (mTq2FP) as the donor fluorophore and incorporates two Venus-FP to implement a composite acceptor 46 . cAMP is a ubiquitous second messenger and is involved in a plethora of intracellular processes throughout many different organisms.…”