Research ResourceTechniques

cAMPr: A single-wavelength fluorescent sensor for cyclic AMP

See allHide authors and affiliations

Sci. Signal.  06 Mar 2018:
Vol. 11, Issue 520, eaah3738
DOI: 10.1126/scisignal.aah3738
  • Fig. 1 Design and selection of a cpGFP-based cAMP sensor.

    (A) Schematic and putative mechanism of cAMPr. PKA-C (orange) is the full-length catalytic subunit of protein kinase A, cpGFP (green) is circularly permuted GFP, and PKA-R 91-244 (blue) contains amino acid residues 91 to 244 of the regulatory subunit of PKA. The binding of cyclic adenosine monophosphate (cAMP; yellow) to the PKA-R peptide triggers a conformational change that releases PKA-C from PKA-R and increases fluorescence. (B) Flowchart of linker library generation and screening as a fusion protein with the signal peptide from TorA in Escherichia coli (gray). PG is a two–amino acid linker inserted between PKA-C (C, orange) and cpGFP (G, green). AC is a two–amino acid linker inserted between cpGFP and PKA-R amino acids 91 to 244 (R, blue) to generate C-G-R. Three different linker libraries were made with four random amino acids (X)4 inserted between PG and cpGFP (left), between PG and cpGFP and between cpGFP and AC (middle), or between cpGFP and AC (right). The most strongly fluorescent bacteria were selected by flow cytometry analysis. The chosen bacteria were then grown before plasmid DNA was extracted.

  • Fig. 2 Initial testing and characterization of cAMPr in ES cells.

    (A) Overview of the screening process in embryonic stem (ES) cells. Forskolin was added to ES cells expressing the cAMP sensor variants, and those colonies that exhibited an increase in fluorescence were selected and sequenced. The sequences of the linkers of the forskolin-responsive clones are shown. (B) Left: Response of ES cell colonies expressing cAMPr to 40 μM forskolin (red; n = 15 colonies) or dimethyl sulfoxide (DMSO) vehicle (blue; n = 16 colonies) from two independent experiments. The gray box indicates the time of drug addition. Light red and blue shading represents the SEM. Data are normalized to the fluorescence at time zero. Right: Images show representative single ES colonies before (Pre; left) and after (Post; right) the application of 40 μM forskolin (top) or DMSO (bottom). P was calculated using a standard unpaired two-sided t test. (C) Response of cAMPr in digitonin-permeabilized ES cells to 1 mM cAMP or 1 mM cGMP. Left: Graph shows the average of at least 13 colonies from two independent experiments, and the data were plotted as described in (B). Right: Bar graph showing the maximum fold changes in fluorescence plotted for 1 mM cAMP (red) and 1 mM cGMP (Blue). (D) Response of ES cell colonies expressing cAMPr (left) or cAMP Difference Detector in situ (cADDis) (right) to the indicated concentrations of forskolin. Cells expressing cADDis that were exposed to DMSO are shown in black. Data are from 14 to 20 colonies from two independent experiments for all treatments. Data are plotted as described in (B). (E) Maximum average (max avg) fold change in fluorescence in response to the indicated concentrations of forskolin in ES cells expressing cAMPr (left) or cADDis (right) normalized to the maximum response to 40 μM forskolin using data from (D). Two-sample unpaired t tests for cAMPr showed P < 0.05 for each value plotted compared to the next highest concentration (40 μM versus 10 μM, P < 0.05; 10 μM versus 1 μM, P < 0.001; 1 μM versus 100 nM, P < 0.001; 100 nM versus 10 nM, P < 10−5). For cADDis, two-sample unpaired t tests only showed statistical significant differences for 1 μM versus 100 nM when compared to the next highest value (40 μM versus 10 μM, P = 0.7; 10 μM versus 1 μM, P = 0.5; 1 μM versus 100 nM, P < 10−8, 100 nM versus 10 nM, P = 0.10; 10 nM versus DMSO, P = 0.44).

  • Fig. 3 cAMPr kinetics and two-photon compatibility in ES cells in a flow chamber.

    (A) Response of cAMPr-expressing ES cells to DMSO (blue) and 40 μM forskolin (red) applied for the indicated periods to cells in a flow chamber. Data are plotted as the fold change in fluorescence normalized to that at time zero. Graph shows an average of 10 colonies from a single experiment imaged by single-photon excitation. (B) Response of cAMPr-expressing ES cells to sequential exposure to forskolin. Forskolin (40 μM; red) was applied to cAMPr-expressing ES cells in a flow chamber from t = 120 to 240 s and from t = 660 to 800 s. The graph shows an average of 15 colonies from a single experiment using single-photon excitation, and the data are plotted as described in (A). (C) Response of cAMPr-expressing ES cells to 40 μM forskolin (red) in a flow chamber and imaged by two-photon microscopy. Graph shows an average of 15 colonies from a single experiment, and the data are plotted as described in (A).

  • Fig. 4 cAMPr responses in ES cells induced to differentiate into neurons.

    (A) Schematic of the protocols used to induce ES cells to differentiate into neurons. Doxycycline was added to induce cAMPr expression. (B) Left: Response of retinoic acid (RA)–differentiated neurons expressing cAMPr to either 40 μM forskolin (red) or DMSO (blue). Data are from 14 and 18 neurons, respectively, from two independent experiments. Data are plotted as described in Fig. 2B. Right: Images of individual representative RA-differentiated neurons expressing cAMPr showing fluorescence before (Pre) and after (Post) the addition of 40 μM forskolin (top) or DMSO (bottom). (C) Left: Response of Smoothened agonist (SAG)–differentiated neurons expressing cAMPr to 10 μM forskolin, 1 mM dopamine, or 100 μM γ-aminobutyric acid (GABA). Data are from 18 to 20 neurons per treatment, with two replicates per treatment, from two independent experiments. Data are plotted as described in Fig. 2B. Right: Representative images of a typical SAG-differentiated neuron expressing cAMPr before (Pre) and after (Post) the addition of 1 mM dopamine. The same neuron is shown in grayscale (top) and pseudocolor (bottom). (D) Responses of SAG-differentiated neurons expressing either cAMPr (left) or cADDis (right) to the indicated concentrations of dopamine. Fluorescence was quantified from 15 to 20 neurons for each dopamine concentration from two independent experiments. Two-sample unpaired t tests showed a statistical significance of P < 0.05 for all dopamine concentrations in neurons expressing cAMPr except for 1 mM versus 100 μM when compared to the next highest value (1 mM versus 100 μM, P = 0.90; 100 μM versus 10 μM, P < 0.05; 10 μM versus 1 μM, P < 10−8; 1 μM versus 100 nM, P < 0.01). For cADDis-expressing neurons, only 10 μM versus 1 μM was statistically significantly different (1 mM versus 100 μM, P = 0.07; 100 μM versus 10 μM, P = 0.90; 10 μM versus 1 μM, P < 10−7; 1 μM versus 100 nM, P = 0.75).

  • Fig. 5 Testing cAMPr in Drosophila pacemaker neurons.

    (A) Top: Diagram showing an adult Drosophila brain and the approximate location of adult lateral ventral circadian pacemaker neurons (LNvs) expressing cAMPr in Pdf-Gal4/UAS-cAMPr; Pdf-Gal4/+ adult flies (green). Bottom: Response of LNvs expressing cAMPr to either 40 μM forskolin (red) or DMSO (blue). The data are an average of 12 LNvs from three brains. (B) Top: Diagram showing an adult Drosophila brain and the approximate location of adult LNvs expressing Epac-camps in Pdf-Gal4/UAS-Epac-camps 50A; Pdf-Gal4/+ adult flies (light blue). Bottom: Response of LNvs expressing Epac-camps to either 40 μM forskolin (red; n = 5 neurons from two brains) or DMSO (blue; n = 8 neurons from three brains). (C) Diagram illustrating communication between larval LNv and DN (dorsal neuron) clock neurons. cAMPr (green) was expressed in all nine clock neurons per hemisphere using tim(UAS)-Gal4 (five LNvs and four DNs). The four pigment-dispersing factor (PDF)–expressing LNvs were also programmed to express the mammalian purinergic receptor P2X2 (orange circle). Adenosine 5′-triphosphate (ATP) stimulates the four LNvs to fire and release the neuropeptide PDF (black dots), which bind to and activate the PDF receptor (PDFR; black circles) present on all larval clock neurons. Flies of the following genotypes were tested using two-photon imaging: tim(UAS)-Gal4/UAS-cAMPr; Pdf-LexA, LexAOP-P2X2/+ (Pdf > P2X2); tim(UAS)-Gal4/UAS-cAMPr; +/TM6B] (Control). (D) Left: Images of cAMPr responses in larval DNs before (Pre-ATP) and after (Post-ATP) the application of ATP. Images are in grayscale (top) and pseudocolor (bottom). DNs were from larvae in which P2X2 was expressed only in LNvs, and cAMPr was expressed in all clock neurons. Right: Quantification of the cAMPr responses in larval DNs. Red: DNs with P2X2 in LNvs and cAMPr in all clock neurons. Data are from four neurons from three brains. Blue: DNs from control larvae with no Pdf-LexA or LexA-P2X2 transgenes. Data are from 12 neurons from three brains. ATP (2.5 mM) was added during the time represented by the gray box. Data are plotted as described in Fig. 2B. (E) Left: Images of cAMPr responses in larval LNvs before (Pre-ATP) and after (Post-ATP) the application of ATP. Images are in grayscale (top) and pseudocolor (bottom). LNvs were from larvae in which P2X2 was expressed in LNvs and cAMPr was expressed in all clock neurons. Right: Quantification of cAMPr responses in DNs. Red: LNvs from larvae with P2X2 in LNvs and cAMPr in all clock neurons. Data are from 22 neurons from three brains. Blue: LNvs from control larvae with no Pdf-LexA or LexA-P2X2 transgenes. Data are from 23 neurons from three brains. ATP (2.5 mM) was added during the time represented by the gray box. Data are plotted as described in Fig. 2B.

  • Fig. 6 Simultaneous measurement of Ca2+ and cAMP in whole brains.

    (A) Images of PDF-expressing LNv cell bodies or projections before (0 s) or during (150 and 300 s) the treatment of larval brains with carbachol. Top: cAMPr fluorescence in green and pseudocolor to measure intracellular cAMP. Middle: RCaMP1h in red and pseudocolor to measure intracellular Ca2+. Bottom: Merged cAMPr (green) and RCaMP1h (red) fluorescence images to simultaneously show cAMP and Ca2+. (B) RCaMP1h fluorescence from 12 PDF-expressing LNv cell bodies and projections from four brains. Shading indicates SEM. (C) cAMPr fluorescence from 12 PDF-expressing LNv cell bodies and projections from four brains. Shading indicates SEM. (D) Data replotted from (B) and (C) to compare the dynamics of changes in cAMPr and RCaMP1h fluorescence in PDF-expressing LNv cell bodies. (E) Data replotted from (B) and (C) to compare the dynamics of changes in cAMPr and RCaMP1h fluorescence in PDF-expressing LNv projections.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/520/eaah3738/DC1

    Fig. S1. Comparison of cAMPr and C-G-R in ES cells.

    Fig. S2. cAMPr excitation and emission spectra in ES cells.

    Fig. S3. Responses of RA- and SAG-differentiated neurons to neurotransmitters.

    Fig. S4. ATP-treated larval clock neurons respond to forskolin.

    Fig. S5. Two-photon imaging of cAMPr and RCaMP1h in PDF-expressing LNvs.

    Table S1. Locomotor activity rhythms of adult flies in constant darkness.

    Table S2. Primers used for cloning.

    Movie S1. Time lapse of ES cells expressing cAMPr exposed to forskolin.

    Movie S2. Time lapse of ES cells expressing cAMPr exposed to DMSO.

    Movie S3. Time lapse of RA-differentiated neurons exposed to forskolin.

    Movie S4. Time lapse of SAG-differentiated neurons exposed to forskolin.

    Movie S5. Time lapse of SAG-differentiated neurons exposed to dopamine.

    Movie S6. Time lapse of RA-differentiated neurons exposed to dopamine.

    Movie S7. Time lapse of the response of DNs expressing cAMPr to activation of LNvs by ATP shown in pseudocolor.

    Movie S8. Time lapse of the response of LNvs expressing cAMPr to activation by ATP shown in pseudocolor.

    Movie S9. Time lapse of the response of LNvs expressing cAMPr and RCaMP1h to carbachol.

  • Supplementary Materials for:

    cAMPr: A single-wavelength fluorescent sensor for cyclic AMP

    Christopher R. Hackley, Esteban O. Mazzoni, Justin Blau*

    *Corresponding author. Email: justin.blau{at}nyu.edu

    This PDF file includes:

    • Fig. S1. Comparison of cAMPr and C-G-R in ES cells.
    • Fig. S2. cAMPr excitation and emission spectra in ES cells.
    • Fig. S3. Responses of RA- and SAG-differentiated neurons to neurotransmitters.
    • Fig. S4. ATP-treated larval clock neurons respond to forskolin.
    • Fig. S5. Two-photon imaging of cAMPr and RCaMP1h in PDF-expressing LNvs.
    • Table S1. Locomotor activity rhythms of adult flies in constant darkness.
    • Table S2. Primers used for cloning.
    • Legends for movies S1 to S9

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.avi format). Time lapse of ES cells expressing cAMPr exposed to forskolin.
    • Movie S2 (.avi format). Time lapse of ES cells expressing cAMPr exposed to DMSO.
    • Movie S3 (.avi format). Time lapse of RA-differentiated neurons exposed to forskolin.
    • Movie S4 (.mov format). Time lapse of SAG-differentiated neurons exposed to forskolin.
    • Movie S5 (.avi format). Time lapse of SAG-differentiated neurons exposed to dopamine.
    • Movie S6 (.avi format). Time lapse of RA-differentiated neurons exposed to dopamine.
    • Movie S7 (.avi format). Time lapse of the response of DNs expressing cAMPr to activation of LNvs by ATP shown in pseudocolor.
    • Movie S8 (.avi format). Time lapse of the response of LNvs expressing cAMPr to activation by ATP shown in pseudocolor.
    • Movie S9 (.avi format). Time lapse of the response of LNvs expressing cAMPr and RCaMP1h to carbachol.

    © 2018 American Association for the Advancement of Science

Navigate This Article