Radial spread of PKA activation, RSPA, in MDCK cells

(A) A scheme of Booster-PKA, a PKA sensor. (B) Booster-PKA-expressing MDCK cells in a confluent condition were observed every 15 seconds under a fluorescence microscope (Video 1). The image of mKOκ represents the cell density, which is seeded at 2.4 × 105 cells cm-2.mKate2 and mKOκ images were acquired to generate mKate2/mKOκ ratio images representing PKA activity in pseudocolor. The time 0 is set the just before initiation of PKA activation. The radius of RSPA as determined in (C) was plotted as a function of time. (C) Procedure to call RSPA positive. The original ratio images were binarized with the threshold value 1.3 of mKate2/mKOκ ratio. The fitted radius of RSPA, r, was defined as the radius of a circle with the same area. When r is greater than 15 µm, it is counted as RSPA. The detailed procedure is in the method section. (D, E) MDCK cells expressing Booster-PKA were seeded at the indicated density and analyzed. Representative images in indicated cell densities are shown in pseudocolor (D). Each color in panel E represents an individual experiment. Red lines indicate average values. (F) MDCK cells expressing Booster-PKA in the presence of the inhibitors were imaged and analyzed for the RSPA frequency. Reagents are as follows: DMSO, 0.1% v/v DMSO; PKAi, 20 μM H89; Apyrase, 10 unit mL−1; EP2i, 10 μM PF-04418948; EP4i, 1 μM ONO-AE3-208; COXi, 10 μM indomethacin. The frequency of RSPA was analyzed 20–80 min after the treatment. Each dot represents an individual experiment. Red lines indicate their average value. p values were calculated between the labeled sample and the DMSO-treated sample.

RSPA in the basal layer of mouse auricular epidermis

(A) A scheme of AKAR3EV, a PKA sensor. (B) Transgenic mice expressing AKAR3EV were observed under a two-photon excitation microscope. Shown are an ECFP image and a YPet/ECFP ratio image representing the cell density and PKA activity in pseudocolor, respectively. (C) Three mice were administrated a COXi, 30 mg/kg flurbiprofen intraperitoneally. The frequency of RSPA in pre-treatment was quantitated more than 40 min before the injection. Similarly, the frequency of a post-COX inhibitor treatment was analyzed 15–58, 15–97, and 15–63 min. Each dotted line represents an individual mouse experiment. (D) Magnified views of a RSPA in vivo. Shown are an ECFP image and a YPet/ECFP ratio image representing the cell density and PKA activity in pseudocolor, respectively. The radius was determined as in Fig. 1C with the detection limit of 6.4 µm. (E) The properties of RSPA are compared between in vivo and in vitro. Data from three independent experiments are shown for each condition. The data of MDCK is from Fig. 1E, seeded at the 1.2 × 105 cells cm−2. Cell density and frequency of RSPA data are values per experiment. Peak radius data are pooled from three independent experiments for each condition. Red lines indicate average values.

RSPA induction by calcium transients

(A) MDCK cells expressing GCaMP6s and Booster-PKA were observed for calcium transients and RSPA. Calcium transients were represented by the fluorescence of GCaMP normalized to the basal level (F/F0). RSPA is analyzed as in Fig. 1C. (B) The frequency of RSPA was analyzed 20–80 min after 30 μM BAPTA-AM treatment. Data from three or four independent experiments. The DMSO control data set is from Fig. 1F. (C) Maximum projection images of the ratio over 10 min. An arrow or arrowheads represent calcium transients with or without RSPA, respectively. Shown is a part of Video 3. (D) MDCK cells expressing GCaMP6s were seeded at the indicated density. Calcium transients showing F/F0 to be greater than three were counted in an indicated cell density. (E) Schematic representation of RSPA induction using Gq-DREADD. MDCK cells expressing Gq-DREADD served as producer cells, while MDCK cells expressing Booster-PKA were employed as reporter cells. (F, G) MDCK cells expressing Gq-DREADD with GCaMP6s or Booster-PKA were mixed and plated, treated with 1 μM CNO, and imaged. Blue circled cells are producer cells, expressing Gq-DREADD. The FRET ratio, the value of mKate2/mKOκ, in each pixel is shown in pseudocolor as indicated. The time 0 was set as just before CNO addition. In panel I, cells showing an F/F0 value greater than 4 were analyzed for their RSPA as Fig. 1C. Red lines indicate their average value.

A switch-like response of RSPA to calcium transients

(A) Schematic representation of RSPA induction using OptoSTIM1. MDCK cells expressing OptoSTIM1 and R-GECO1 were employed as the producer cells. The Booster-PKA-expressing MDCK cells, deficient in COX-1 and COX-2 (COX-DKO), were employed as the reporter cells. (B) The producer cells were stimulated by a flashlight during imaging. Blue circled cells in the mKOκ image are the producer cells. The FRET ratio, the value of mKate2/mKOκ, in each pixel is shown in pseudocolor as indicated. The time 0 was set as just before blue light irradiation. The detection limit for the RSPA radius was 26 µm, as shown in the shaded area. (C) Flashlight illumination was repeated with increasing LED power. (D) Vertically aligned dots (left) are the results from an individual producer cell. The right panel shows the relationship between R-GECO fluorescence intensity and the radius of RSPA.

Effect of cell density on PGE2 sensitivity and the transcriptome.

(A) MDCK cells expressing Gq-DREADD and GCaMP6s were employed as the parental producer cells. Meanwhile, MDCK cells expressing Booster-PKA were employed as the parental reporter cells. The genes knocked out by CRIPSR/Cas9 system are depicted in the figure. Analysis was performed as in Fig. 3G. Each producer cell exhibiting F/F0 values greater than 3 was analyzed for the occurrence of RSPA. Data from two independent experiments was summed up. (B) Inter- and intra-cellular pathway of RSPA. (C) COX-DKO MDCK cells expressing Booster-PKA were plated at the indicated cell density, treated with increasing concentrations of PGE2, and analyzed for PKA activity. The mKate2/mKOκ ratio representing PKA activity was calculated and plotted against PGE2 concentration. The average intensity of the whole view field of mKate2 or mKOκ, at 20 to 30 min after the addition of PGE2, was applied to calculate the mKate2/mKOκ ratio. Three or four independent experiments were performed. (D) COX-DKO MDCK cells were seeded at the indicated cell densities and subjected to RNA-Seq analysis. FPKM values of other genes are in Figure S2. N.D. represents Not Detected.

Requirement of ERK activation for RSPA.

(A) MDCK cells expressing EKAREV and Booster-PKA were observed for ERK and PKA activities every 5 min and 1 min, respectively. The mKate2/mKOκ ratio image represents PKA activity in pseudocolor. The YPet/ECFP ratio image represents ERK activity in IMD mode. RSPA is indicated by white circles. (B) The ERK activities within 10 µm from the center of RSPA and within 10 µm from randomly set positions with a random number table generated by Python are plotted in the left panel. Each colored dot represents an average value of an independent experiment. The right scattered plot shows the relationship between ERK activity and the size of RSPA. (C) The correlation between ERK activation and RSPA is shown. This is a part of Video 4. (D) Cross-correlation analysis of PKA and ERK activities. The average and S. D. values from 67 samples are shown in the black lines and blue shades, respectively. (E) Temporal cross-correlations between RSPA and ERK activation rate. The black line indicates the average temporal cross-correlation coefficients with S.D. (F) MDCK cells expressing Booster-PKA were imaged in the presence of the following reagents: 0.1% v/v DMSO, 50 ng mL−1 EGF, 1 μM PD0325901 (MEKi), and 1 μM AG1478 (EGFRi). The frequency of RSPA was analyzed 20–80 min after the treatment. Each dot represents an individual experiment. Red lines indicate their average value. The control data set is from Fig. 1F. p values were calculated between the labeled sample and the DMSO-treated sample. (G, H) Similar to Fig. 2. Transgenic mice expressing AKAR3EV were observed under a two-photon excitation microscope and administrated a MEKi, 5 mg/kg PD0325901 intravenously. Shown are an ECFP image and a YPet/ECFP ratio image representing the cell density and PKA activity in pseudocolor, respectively. The images of PKA activity were projected over 30 min. The frequency of RSPA in pre-treatment was quantitated more than 120 min before the injection. Similarly, the frequency of RSPA in a post-treatment was analyzed 15–90, 15–125, 15-85, and 15–115 min. Each dotted line represents an individual mouse experiment.

Models for PGE2 secretion.

The frequency of calcium transients is cell density-dependent manner. While the ERK activation wave is there in both conditions. Because both calcium transient and ERK activation are required for RSPA, the probability for PGE2 secretion is regulated as “AND gate”.