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Figure 1 | BMC Research Notes

Figure 1

From: Improved molecular toolkit for cAMP studies in live cells

Figure 1

Characteristics of mCherry-PACα. (A) PACα and mCherry have distinct excitation spectra, predicting independent excitation. (B, C) Sequences of merged panels of transmitted light images, mCherry fluorescence images (red), and 488 nm PACα excitation (blue). The transmission image is omitted during the 488 nm excitation because of saturation of the image (6 min, 2nd and 4th rows). Both bath application of 10 μM forskolin (1st row) and one-minute photoactivation of PACα (2nd row) induce arrest of circumferential movements of circus cells' cytoplasm. In contrast, excitation of mCherry alone (3rd row) or blue light illumination of cells lacking PACα (4th row) does not affect circus cells' movements. Arrows indicate the direction of the circus movement in the first image of each row and each time the direction switches. Cells were split in 4 quadrants and were scored as moving when a blob of cytoplasm was seen in all the quadrants during the 4 minutes following stimulation. ≥ 13 cells were scored for each condition. (D, E) mCherry-PACα-injected embryos twitch when illuminated with blue light to excite PACα (2nd row), but not when exposed to green light to excite mCherry (1st and 3rd row). Dashed white line marks the initial position of the head of the embryo (-300 ms). Dashed magenta line indicates the head position during (0 ms) and following (+300 ms) the twitch. Embryos that do not express mCherry-PACα are insensitive to both blue and green light. Images were extracted from the movie shown in Additional File 2. Images were chosen 300 ms before (-300 ms), during (0 ms) and 300 ms after (+300 ms) the embryo twitches under blue illumination (BP 470/40 filter). Embryos were immobile under green illumination (BP 560/40 filter), and the 600 ms portions of the recording was chosen randomly (1st and 3rd row). Embryos were scored as responsive when they were not completely immobile during the illumination period. ≥ 35 embryos were scored for each condition. Error bars, sem. *** p < 0.001. C, Chi Square test. E, ANOVA.

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