Photolysis of caged compound in cilia

Caged cAMP was dissolved in DMSO (Takeuchi and Kurahashi, 2002, 2005, 2008), and caged Ca was dissolved in 10 mM CsOH (Takeuchi et al., 2009, 2013). Both stock solutions were then stored at −20°C in complete darkness for up to 180 d. Before each experiment, the stock was diluted to a Cs solution as described above. After the establishment of the WC recording configuration, caged compounds were introduced to the cell interior by free diffusion.

The whole cilia of an ORC were stimulated with an epifluorescent UV system as previously reported (Takeuchi and Kurahashi, 2002). The UV component from a 100-W xenon lamp was used as the UV source. A computer-regulated magnetic shutter controlled the timing and duration of the UV light. The light intensity was set by a neutral-density wedge filter that was controlled by a pulse motor.

For local uncaging on a single cilium, we used the LSM system as previously reported (Takeuchi and Kurahashi, 2008). An 80-mW UV laser beam (argon laser, λ = 351, 364 nm; Coherent) was used for the photolysis. Throughout the experiments, the UV intensity was regulated by the transmission parameter of an acousto-optic tunable filter device. The region of interest (ROI) function of the LSM system was used for the spatially restricted stimulation as mentioned previously (Takeuchi and Kurahashi, 2008). Colored indicators in figures show the 2D image of light strength constructed by the Gaussian laser beam. The ROI-selected edge corresponds with 50% intensity. Red in the indicator represents 99–100%; orange, 75–99%; yellow, 50–75%; green, 25–50%; and blue, 0.02–25% (Takeuchi and Kurahashi, 2008). The laser beam moves on the x and y axes with the raster scan. During the current recording, the timing and duration of the actual laser irradiation were detected using a UV-photodiode (G6262; Hamamatsu Photonics). Because the sensitivity of the G6262 photodiode to the UV component transmitted via the optical fiber for the UV region was still low, a 488-nm laser was added for detection. This 488-nm laser beam alone did not cause photolysis when examined with cells. For double-pulse stimulation, we continuously used the scan mode and bleach mode.