Cygnets'. Intracellular Guanosine 3',5'-
Cyclic Monophosphate Sensing
in Primary Cells Using Fluorescence
Guanosine 3',5'-cyclic monophosphate (cGMP) is a
key player in the regulation of various physiological
processes, including smooth muscle tone, neuronal
excitability, epithelial electrolyte transport, phototransduction
in the retina, and cell adhesion
(Eigenthaler et al.
, 1999; Kaupp and Seifert, 2002;
Lincoln et al.
, 2001; Schlossmann et al.
, 2003). Although
the cGMP second messenger pathway has been
steadily gaining recognition for its role in intracellular
signaling, cGMP remains the least well understood
member of the cyclic nucleotide family due to the following
peculiarities of the cGMP signal transduction
system. (1) The control of cGMP levels is complex, with
formation of cGMP occurring through two different
forms of guanylyl cyclases (Russwurm and Koesling,
2002; Wedel and Garbers, 2001) and degradation by a
number of cGMP-specific phosphodiesterases (PDEs)
(Rybalkin et al.
, 2003). (2) The intracellular actions of
cGMP are mediated by cGMP-dependent protein
kinases (PKGs) (Hofmann et al.
, 2000) and by several
types of cyclic nucleotide-activated ion channels
(Kaupp and Seifert, 2002). (3) The enzymes modulating
cGMP levels are expressed differentially throughout
mammalian tissues. Furthermore, a tightly
controlled equilibrium of synthesis and breakdown
generates highly flexible intracellular cGMP transients,
contributing to the experimental and conceptual obstacles
posed by the multiplicity in mechanisms of cGMP
signaling and the difficulty of studying cGMP in
broken cell preparations.
Genetically encoded indicators based on the insertion
of conformationally sensitive domains between
two mutants of green fluorescent protein (GFP) that
participate in fluorescence resonance energy transfer
(FRET) have proved to be powerful tools for observing
the dynamics of intracellular signaling molecules
noninvasively. Examples of such indicators include
those that detect the second messengers Ca2+
(Miyawaki et al.
, 1997) and cAMP (Zaccolo and
Pozzan, 2002). For the construction of our cGMP indicators,
which we have named cygnets (cyclic GMP
indicators using energy transfer), we chose PKG as
the central cGMP sensor because it binds cGMP with
high affinity, undergoes a conformational change in
response to cGMP, and is not restricted to membranes
(Pfeifer et al.
, 1999; Ruth et al.
, 1991; Zhao et al.
We have demonstrated the validity of this intramolecular
FRET approach and could show that cygnets (i)
are exclusively selective for cGMP, (ii) allow detection
of intracellular cGMP in single living cells, (iii) are
fully reversible to monitor fast spatial and temporal
cGMP changes, and (iv) are minimally invasive when
analyzing intracellular cGMP signaling events (Honda et al.
, 2001; Sawyer et al.
, 2003). This article provides
a detailed methodology for the use of our latest
cGMP indicator version, Cygnet-2.1, in primary cell
II. MATERIALS AND
The RFL-6 (CCL-192) established cell line is from
American Type Culture Collection. The FuGENE 6
transfection reagent is from Roche (1814443). Cygnet-
2.1 DNA (pcDNA3.1(-)-Cygnet-2.1) is constructed as
described previously (Honda et al.
Dulbecco's modification of Eagle's medium
(DMEM, with 4.5g/liter glucose and L
with sodium pyruvate; MT 10-013-CM), Ham's F-12
medium (MT 10-080-CM), fetal bovine serum (FBS, MT
35-010-CV), trypsin EDTA (MT 25-052-CI), amphotericin
B (MT 30-003-CI), and gentamycin sulfate
(30-005-CR) are from cellgro by Mediatech, Inc. Bovine
serum albumin (BSA, A7638) and potassium phosphate
, P-3786) and monobasic
, P-0662)] are from Sigma-Aldrich. Elastase
(100617) is from ICN Biomedicals. Collagenase
(199152) is from Worthington Biochemical Corporation.
Penicillin / streptomycin / neomycin (15640-055) is
from Gibco-BRL/Invitrogen. Collagen (354231), BD
Falcon polystyrene 35 × 10-mm (353001) and 60 × 15-
mm (353004) cell culture dishes are from BD Biosciences.
Benzamidine hydrochloride (105240250) and
EDTA (118430010) are from Fisher Scientific. The extracellular
solution used for microscopic imaging consists
of Hank's balanced salt solution (HBSS; cellgro by
Mediatech, MT 21-020-CV) with 20mM
(cellgro by Mediatech, MT 25-060-CI) and 2g/liter
glucose (Sigma-Aldrich, G-7021).
cGMP (40732-48-7; reconstituted in distilled H2
and fluorescence grade 8-(4-chlorophenylthio)guanosine-
3', 5'-cyclic monophosphate [8-pCPT-cGMP;
51239-26-0; reconstituted in dimethyl sulfoxide
(DMSO)] are from BIOLOG Life Science Institute.
The following chemicals have been used to modulate
intracellular cGMP levels: Nitric oxide donors
S-nitrosoglutathione (GSNO; Calbiochem 487920,
protect from light and reconstitute in cold distilled
O free of divalent cations other than Ca2+
; stock solution
is stable for 2h at 4°C), N
[NOC-22, spermine NONOate; Calbiochem 567703;
reconstitute in 0.1 N
NaOH, ≥pH 10; t½
of NO release
= 230min in phosphate-buffered saline (PBS), pH 7.4,
22°C] diethylamine NONOate (DEA NONOate;
Calbiochem 292500, reconstitute in distilled H2
of NO release - 16 min in PBS, pH 7.4, 22°C), and (±)- S
-acetylpenicillamine (SNAP; Calbiochem
487910; protect from light and reconstitute in DMSO;
tl/2 of NO release = 10 h) are reconstituted to 100× the final concentration of 100µM
prior to use and stored
on ice until needed.
Atrial [ANP (3-28), rat; 14-5-44A] and brain [BNP (1-
32), Rat; 14-5-11A] natriuretic peptides are from American
Peptide Company. The C-type natriuretic peptide
[CNP (6-22), human and porcine; 05-23-0310] is from
Calbiochem. Peptides are reconstituted in distilled
O to 100 µM
stock solutions and stored at -20°C for
several months until their use at a final concentration
of 0.1-1 µM
The phosphodiesterase inhibitors 3-isobutyl-1-
methylxanthine (IBMX; 410957; nonspecific inhibitor
of cAMP and cGMP phosphodiesterases, final concentration
), vinpocetine (677500; PDE I inhibitor;
final concentration 0.2 mM
isobutyl-l-methylxanthine (MM-IBMX; 454202; PDE
I inhibitor; final concentration 40µM
(2-hydroxy-3-nonyl)adenine, HCl (EHNA; 324630;
PDE II inhibitor; final concentration 8 µM
in distilled H2
bipyridine)-5-carbonitrile (Milrinone; 475840; PDE
III inhibitor; final concentration 3 µM
(Rolipram; 557330; PDE IV inhibitor; final concentration
), and 1,4-dihydro-5-(2-propoxyphenyl)-
684500; PDE V inhibitor; final concentration 4.5µM
are from Calbiochem. After reconstitution in DMSO
(unless otherwise noted), stock solutions are stored
at -20°C and used within 2 months. Sildenafil is a
generous gift from Pfizer.
The fluorescence spectrometer F-4500 is from
Hitachi. Quartz fluorescence cells (14-385-918A) for
spectrophotometers and Dithiothreitol (DTT, 16568-
0050) are from Fisher Scientific. Dithiothreitol (DTT,
A light-duty portable punch size XX (130010001)
outfitted with a 0.5-in. round dye (Type O) is from
Roper Whitney of Rockford, Inc. A Sylgard 184 silicone
elastomer kit is from Dow Corning Corporation.
Coverslips (22 × 22 mm, 1 thickness; 12-544-10) are
acquired from Fisher Scientific.
An inverted Nikon Diaphot 200 microscope
equipped with a Nikon Fluor 40/1.30 oil Ph4DL objective
(Part 140010) is outfitted with an ORCA ER cooled
charge-coupled device camera (Hamamatsu). Three
filter wheels, one each for excitation, emission, and
neutral density filters, and a shutter at the excitation
filter wheel are controlled by Lambda 10-2 optical filter
changers from Sutter Instruments. A lambda LS xenon
arc lamp and power supply are also obtained from
Sutter Instruments. The Cameleons 2 filter set (71007a)
purchased from Chroma Technologies for dual emission consists of a D440/20x excitation filter, a 455DCLP
dichroic, and two emission filters (D485/40m and
D535/30m). Neutral density filters (0.1, 0.3, 0.5, 1, 2, 3)
are also obtained from Chroma. Image acquisition is
controlled by a computer loaded with Metamorph
and Metafluor 4.64 software from Universal Imaging
(Media, PA). A stage adaptor to hold 35-mm imaging
dishes was constructed at the Instrumentation and
Model Facility at the University of Vermont.
A. Cygnet Expression and Purification
Express recombinant Cygnet-2.1 protein in
Spodoptera frugiperda (Sf9) cells using the Bac-to-Bac
baculovirus system (GIBCO/BRL) and purify using
cAMP-agarose as described earlier (Honda et al.
B. In Vitro cGMP Titration
Perform cGMP titrations of Cygnet-2.1 by adding
of protein to a quartz fluorescence cell with
buffer (50 mM
, pH 6.8, 10 mM DTT, 10 mM
EDTA) for a final volume of
500 µl. Excite samples in a fluorescence spectrometer at
432nm and monitor emission intensities from 450
to 550nm. Plat the ratios of the 475 to 525 emission
intensities against the concentration of cGMP added to
the sample to generate a titration curve.
C. Cell Culture
The following cell types have been used successfully
to express cygnets and monitor intracellular
cGMP levels. Details on their preparation and handling
are as follow.
1. Rat Fetal Lung Fibroblast Cells (RFL-6)
Cells should be cultured according to the supplier.
Briefly, grow RFL-6 cells in Ham's F12 medium supplemented
with 20% fetal bovine serum at 37°C, 5%
. Subculture every 5-6 days with 0.25% trypsin at
a ratio of 1:4 and plate on glass-bottom dishes for
imaging. RFL-6 cells should not used beyond passage
number eight. Routinely verify cGMP responses with
cygnet-transfected RFL-6 cells, an established cell line
known to respond with high cGMP levels upon stimulation
(Ishii et al.
2. Rat Aortic Smooth Muscle Cells (RASMC)
Preparation and Culture of RASMC
- Euthanize mature female or male
Sprague-Dawley rat (250-350g) by a lethal dose of
pentobarbital sodium and exsanguination and remove
- Clean aorta of fat and connective tissue and slice
into 1- to 2-mm rings with a sterile scalpel.
- With a scalpel, score a 60-mm cell culture dish to
create a grid of three horizontal and three vertical
lines, and embed each ring where two grooves
- To prevent rings from detaching from the bottom
of the dish, carefully add DMEM containing 10% FBS,
50µg/ml gentamicin, and 2.5µg/ml amphoteracin B
and incubate at 37°C in humidified 5% CO2. Replenish
media every 2-3 days and smooth muscle cells will
proliferate from the aortic explants within 1 week.
- When cells reach confluency between gridlines,
remove artery sections and rinse plate with trypsin.
Add 1 ml fresh trypsin and aspirate off, leaving only a
small amount in the dish. Incubate at 37°C until cells
have detached, less than 5 min.
- Resuspend cells in 2-3 ml supplemented media
and transfer approximately 300 µl of cell suspension to
glass portion only of glass-bottom imaging dishes.
- When cells have adhered to the coverslip,
aspirate media and replace with 2ml fresh media in
entire dish. These cells are henceforth referred to as
passage one (P1) cells. Use only P1 cells in imaging
Alternatively, RASMC can be dissociated from the
aorta using an isolation protocol adapted from
Cornwell and Lincoln (1989) and Smith and Brock
D. Preparation of Imaging Dishes
- After extracting aorta, place in isolation media
consisting of DMEM, 20mM HEPES, 1 mg/ml BSA,
5µg/ml amphoteracin B, and 50µg/ml gentamicin.
- Clean aorta of fat and connective tissue and incubate
in isolation media supplemented with 1 mg/ml
elastase and 130 units/ml collagenase for 8min at
- Rinse aorta to remove endothelial and other
nonadherent cells, and remove tunicae adventitia as an
- Mince the medial layer of the aorta and digest
pieces in isolation media supplemented with 200
units/ml collagenase for 1 to 2h or until single cells
- Wash cells twice in isolation media and culture
in DMEM, 10% FCS, and 50µg/ml gentamicin.
E. Transfection of Cygnet-2.1
- Prepare glass-bottomed dishes for fluorescence
imaging by punching 0.5-in. holes in the bottoms of
35-mm cell culture dishes with an industrial punch.
- Mix 1 part Sylgard 184 curing agent with 10 parts
Sylgard 184 silicone elastomer (w/w) carefully to
avoid introducing air bubbles.
- Pipette a thin line of Sylgard around the hole on the
bottom of the dish. Place a coverslip over the ring
of Sylgard and tamp down with a cotton-tipped
applicator to seal.
- Allow Sylgard to cure overnight.
- Sterilize dishes in a sterile hood under ultraviolet
light for at least 30min.
- Dishes may be treated with 0.1 mg/ml collagen to
promote cell adherence to coverslips.
F. Data Acquisition and Analysis
Dual-Emission Imaging Protocol Using Metafluor
- Grow primary RASMC to 50-60% confluency on 35-
mm glass-bottomed dishes.
- For each 35-mm dish, pipette 3µl FuGENE 6
directly into 200µL serum-free DMEM in a plastic
1.5-ml microfuge tube, minimizing the contact that
FuGENE 6 has with the wall of the tube.
- Add 1 µg highly purified pcDNA3.1(-)-Cygnet-2.1
to the DMEM-FuGENE 6. Close the tube and mix
- Allow DNA/lipid complexes to form at room temperature
for 15 min.
- Without removing media from 35-mm dishes, add
transfection mixture dropwise using a pipette. Distribute
transfection mixture around dish by gently
shaking dish side to side.
- Incubate RASMC at 37°C for 24 h and RFL-6 cells 48
h before imaging. Culture media may be replaced
after 6 h of transfection, if desired. However, leaving
cells in FuGENE until the time of imaging does not
- Open Metafluor software from Universal
Imaging Corporation and select "New" on the toolbar
to begin a new experiment.
- Select configure/configure acquisition and define
wavelengths 1 and 2 as ECFP and EYFP, respectively,
and the excitation wavelength for both as 440nm. The
emission wavelengths for ECFP and EYFP must be
defined as 480 and 535nm, respectively. Chose
file/save protocol file to save these parameters. When
starting Metafluor from now on, begin by selecting
"Protocol" and then open a new experiment.
- Check the "Save Images" box in the control
panel. As long as the box remains checked, every
image acquired from now on will be saved as part of
the file you are now prompted to name. Note: Each
experiment is saved as an .inf file, which is composed
of a .tif file of every image acquired during the
- To locate transfected cells, manually select
the 440-nm excitation filter and open the shutter (if
applicable) in the illumination control box found in the
- Focus the transfected cell(s) of interest through
the camera by selecting "Focus" on the control panel
and the focusing screen will appear. Choose "Start
Focusing" and your image will appear. Bring the
image displayed on the monitor into focus and then
click on "Stop Focusing." Selecting "Close" will bring
you back to the experimental menu.
- Select "Acquire One" on the toolbar to get an
idea of what your image will look like. Cygnettransfected
cells should be visible in both 475 and 535
emission channels (Figs. 1A and 1B). Cells can be
pseudocolored during at a later period during data
analysis to correlate color hue with 475/535 emission
ratio (Figs. 1C and 1D). If the cell is too bright (saturated
pixels appear black) or too dim, the exposure
time may need to be altered in the configure acquisition
- To start the experiment, press "Acquire" on the
control panel menu and images will be captured at
intervals until "Pause" is pressed.
- To set the time lapse between image acquisitions
to 10s, select "Timelapse" on the control panel menu
and enter the desired time.
- Define regions on your image in which to
monitor intensity values by selecting "Regions" on the
toolbar. Select the circle tool and place a circle on a
dark area of the image for background measurements.
Then trace several regions (shape and number depending
on cell) on the cell(s) of which you would like to
monitor the fluorescence. Select "Close" to return to
the experimental menu.
Note: The defined regions appear on one of the
images, but fluorescence intensity data are collected
from both wavelength channels in the same exact position
defined on the cell. Graph 1 and Graph 2 plot the
wavelength intensity value and ratio of (wavelength
1)/(wavelength 2) intensity values, respectively,
- As the program acquires images and plots data
collected from the regions that you have defined,
monitor the trace in Graph 2, the plot of the
ECFP/EYFP ratio. Once you have established a stable
baseline, pharmacological agents can be added and the event markers used to note when things were added
(select "Events" on the toolbar to define and mark
events). At the end of an experiment, a cell-permeable
cGMP analog can be applied to verify indicator fidelity
- To end the experiment, select "Close" on the
|FIGURE 1 Cygnet-2.1 expression indicates cytosolic localization and nuclear exclusion in cultured rat
aortic smooth muscle cells (top) as shown by the fluorescence images of (A) ECFP (480nm emission) and
(B) EYFPcitrine (535 nm emission). Pseudocolor representations of the 480- to 535-nm FRET ratio at resting
(C) and elevated (D) cGMP levels were elicited with 50 µM 8-pCPT-cGMP.
|FIGURE 2 (A) Cygnet-2.1 expressed in an individual RASM cell
exhibits a 30% EYFP/citrine ratio change in response to a saturating
dose of the membrane-permeable cGMP analog 8-CPT-cGMP. (B) In
vitro Cygnet-2.1 titration with cGMP reveals a maximum FRET ratio
change of 45%. Saturating concentrations of the analog 8-CPT-cGMP
can only generate a 30% change. (C) A RASM cell expressing Cygnet- 2.1 was permeabilized with 50µM digitonin in an extracellular solution
containing 8 µM cGMP. The indicator responded to the cGMP
influx with a FRET ratio change of 47%.
Making a Movie of a Pseudocolored Cell
- To collect data, select "Open" from the toolbar
and select an experiment.
- Define regions for background and on the cell(s)
- To subtract the background (i.e., make the
intensity of background equal to zero), select run
experiment/reference images. In the pull-down menu,
choose to subtract the average intensity of a region and
indicate which number refers to the region defining
the background. Check the box on the lower left-hand
corner to "Subtract Background" and close the menu.
- Check "Log Data" on the control panel to save
the intensity and ratio values of the wavelengths associated
with each region.
- Press "Forward" on the control panel to run
through the experiment. As each image appears, the
intensity and ratio values for each region on each
wavelength channel (ECFP and EYFP) are saved. To
make certain that the regions stay in the approximate
same place on the cell, their placement may need
adjustment. The log file that has been generated can be
opened in a spreadsheet program and time plotted
against the ECFP/EYFP intensity ratio.
- To save individual images, select the desired
image by playing through the experiment using the
"Forward" button or the slider bar and then click on
the image so that the window is highlighted. Under
utilities/save as an 8-bit image, select to save the
image as a .TIF file.
G. Example of cGMP Sensing in Primary Rat
Aortic Smooth Muscle Cells
- To create a movie of a pseudocolored cell, define
regions on the background and cell(s) and subtract
background as described previously.
- Play through the experiment and determine the
minimum and maximum ratio values the cell displays
by referencing Graph 2.
- Select configure/image display control and
choose Ratio 1 (ECFP/EYFP) from the top pull-down
menu. From the lower pull-down menus choose intensity
modulated display (IMD). In the "Minimum" and
"Maximum" boxes, enter the approximate minimum
and maximum ratio values determined from Graph 2.
Setting the ratio values slightly inside the actual
minimum and maximum values often enhances the
visual effect as seen in the Ratio 1 box.
- After optimizing the color changes the cell
undergoes throughout the experiment, check the
"Save Ratio" box on the control panel and forward
through the entire experiment. The images displayed
in the Ratio 1 box have now been saved as .TIF files.
- Exit Metafluor and open Metamorph.
- Select file/build stack/numbered names and
select the first image. When prompted, select the last
image. A "stack" of .TIF files has now been created.
- Select stack/make movie, and a movie is generated
from the compiled stack, which can be saved as
an .AVI file under the stack menu.
- A representative movie corresponding to the
trace shown in Fig. 3 is published as supplemental
data on the Cell Biology website.
Note: .AVI files generated with Metamorph are very
large (40-100MB) but can be compressed down to a
few megabytes (50× or more) using a program such as
At approximately 50-60% confluency, passage one
cells were transfected with FuGENE 6 using a 3:1 ratio
of FuGENE reagent to pcDNA3.1-Cygnet-2.1 DNA.
Cells were imaged 24h posttransfection at 25°C using
Hank's balanced salt solution with 20 mM
7.35) and glucose (2g/liter) as the extracellular solution
and 500-ms exposures at 10-s intervals.
Cygnet-2.1 expressed in both rat aortic smooth
muscle cells (Figs. 1A and 1B) and RFL-6 cells (Honda et al.
, 2001) demonstrated cytosolic localization and
nuclear exclusion when ECFP and citrine emissions
were viewed individually. Pseudocoloring of the
cell (Figs. 2C and 2D) correlates the ratio of the
ECFP/citrine emissions to a color scale, with lower
and higher ratios and cGMP levels represented by blue
and red, respectively. A saturating dose of the cell
membrane-permeable cGMP analog 8-pCPT-cGMP
changed the pseudocoloring from blue to red (Fig. 1D)
and correlates to a 30% increase in FRET ratio (Fig. 2A).
However, in vitro
results with purified Cygnet-2.1
showed that cGMP consistently causes a 40-50% ratio change. To analyze this apparent discrepancy, Cygnet-
2.1 purified from Sf9 cells was titrated with cGMP
and 8-CPT-cGMP (Fig. 2B). The purified indicator's
maximum ratio change attained with cGMP was
approximately 45%, while the analog still only elicited
a maximal ratio change of 30%. In fact, in cells permeabilized
digitonin in the presence of saturating
concentrations of cGMP, cygnets demonstrated
a 47% FRET ratio change as expected from the in vitro
results (Fig. 2C).
Activating the particulate guanylate cyclase with 1
brain natriuretic peptide produced a FRET ratio
change of 28% (Fig. 3A). Atrial natriuretic peptide produced
a similar response, and both ANP- and BNPinduced
cGMP accumulation could be washed out to
near baseline (data not shown). Analysis of the
individual emission intensities of ECFP and citrine
demonstrates the mechanism of our cGMP indicator:
cGMP generated in response to the activation of
guanylate cyclase binds to the indicator and triggers
a conformational change in the receptor. Subsequent
alterations in the relative spatial arrangement of the
two fluorophores result in a loss of energy transfer
from ECFP to citrine and an increase in ECFP emission
and a decrease in that of citrine (Fig. 3B).
|FIGURE 3 (A) Stimulation of the particulate guanylate cyclase
(pGC) NPR-A with the natriuretic peptide BNP produces a FRET
ratio change of approximately 28%. (B) Analysis of the individual
emission intensities of ECFP and citrine during pGC activation
demonstrates an increase in ECFP and a decrease in citrine
Experiments acquiring images at 10-s intervals can
run for greater than 30min. We have accumulated as
many as 200 exposures before applying a stimulus and
have observed no significant loss in indicator sensitivity.
It should be noted that cells have the tendency to
react quite differently to the same stimulus.
- Transfection efficiency and cygnet expression
may be low in some cells, particularly in primary
cultures. Optimal expression may require alternative
transfection reagents and optimization of transfection
conditions, depending on the cell type.
- Because intracellular cGMP fluctuations can
potentially be buffered by the expression of the
indicator, it is important to compare the total
cGMP-binding capacity of untransfected and cygnettransfected
cells. This can be accomplished by the use
of antibodies to PKG to determine if total PKG
immunoreactivity, and therefore cGMP-binding capacity,
is elevated by the expression of cygnets.
- Because excessive illumination of cygnets may
result in photobleaching of GFP mutants, fluorophore
excitation should occur at the minimum intensity,
frequency, and duration possible. The emission profiles
of ECFP and citrine should be monitored to ascertain
that changes in FRET are not due to aberrant
behavior of the individual fluorophores due to photobleaching
or other phenomena such as pH-induced
This work was supported by NSF Grant MCB-9983097
(WRGD) and the Totman Medical Research Trust.
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