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) A scheme of hyBRET-Epac, a cAMP sensor. (G) MDCK cells expressing hyBRET-Epac were imaged to generate Turquoise/YPet ratio images representing cAMP concentration, [cAMP], in pseudocolor. The image of Turquoise represents the cell density, which is seeded at 1.2 × 105 cells cm-2. The time 0 was set as just before cAMP production. (H) 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.