In vitro FRET imaging of cAMP signaling
The PDE4DIP NM_001002811 transcript was cloned into an mCherry-tagged
plasmid and a point mutation, resulting in an alanine to threonine
substitution at amino acid 123, was induced using site directed
mutagenesis and verified by sequencing. Human embryonic kidney (HEK)
cell line 293T was transfected with the PDE4DIP mutant and control
plasmids using the Lipofectamine transfection kit. Transfection
efficiency was verified by percentage m-cherry expression on fluorescent
microscopy (>50%). The cells were then co-transfected with
the FRET sensor plasmids using the Lipofectamine kit and incubated on
35mm microwell dishes with 14mm glass coverslip for 3 days to achieve
60-70% confluency.
Intracellular cAMP imaging was done using FRET sensors and confocal
microscopy at baseline, and at 4 minutes after stimulation with 1uM
isoproterenol (cAMP activator). Further imaging was done 4 minutes after
stimulation with 25 uM of Forskolin (strong cAMP activator) under live
confocal microscopy. A fourth generation Epac-based FRET sensor
(mTurquoise2Δ-Epac (CD, ΔDEP, Q270E)-tdcp173Venus (Epac-SH187)) was used
as previously described (Klarenbeek, Goedhart, van Batenburg,
Groenewald, & Jalink, 2015). This sensor consists of a full-length
cAMP-binding Rap-1 activating protein Epac, which is sandwiched between
the donor and acceptor fluorescent proteins with larger conformational
change, and FRET change compared to sensors with partial Epac. Fourth
generation of cAMP sensors have superior photo-stability, dynamic range
and signal-to-noise ratios. The design is based on mTurquoise2 as a
bleaching-resistant donor, and a tandem of two cp173Venus fluorophores
as acceptors.
FRET was quantified using 3 cube ratiometric imaging with analysis in
MATLAB using previously developed custom software (A. Kumar et al.,
2016). Images were acquired on an inverted Nikon Eclipse Ti widefield
microscope equipped with a cooled charged-coupled device Cool SNAP HQ2
camera using a 20x 0.75 NA objective at 37°C. Three sequential images
were acquired with the following filter combinations: donor
(mTurquoise2) channel with 460/20 (excitation filter-ex), T455lp
(dichroic mirror-di) and 500/22 (emission filter-em); FRET channel with
460/20 (ex), T455lp (di) and 435/30 (em); and acceptor (Venus) channel
with 492/18 (ex), T515lp (di) and 535/30 (em) filter combinations. For
analysis, donor leakage was determined from 293T cells transfected with
mTurquoise2 and acceptor cross excitation was obtained from
Vinculin-Venus transfected cells. All three FRET images (mTurquoise2,
Venus, FRET) were background subtracted and filtered by three-point
smoothening. FRET maps and pixel-wise FRET index was calculated as:
FRET index = [FRET channel – x(Donor channel) – y(Acceptor
channel)]/[Acceptor channel]
Where x is the leakage co-efficient and y is the cross-excitation
fraction. Masks for each cell were generated by thresholding mCherry
positive cells using the mTurquoise images. Mean FRET index per cell was
calculated for each field under each treatment condition.