Figure 3.

Level control of pmCRY2 using pulse frequency. (a) Experimental TIRF intensity of pmCRY2 (black) as a function of time in the same cell for different activation frequencies (one pulse of 50 ms and 21 μW every 2, 20, and 60 s). The intensity is normalized by the final level of the curve with the period of 2 s. The parameters f and C₀ were obtained from the fitted theoretical values of M_n (green dots) to the experimental curve of period 2 s. These parameters were used to predict the theoretical values of M_n for periods of 20 and 60 s (red dots). (b) Theoretical dependency of the pmCRY2 steady-state value (red) on the period of the activating pulses for f and C₀ extracted in (a). (Black lines) Final level of pmCRY2 for the experimental curves (shown in black) in (a), which correspond theoretically to periods of 26 and 61 s. (c) Experimental values of pmCRY2 over time for a single cell (black). After 5 min, the initial stimulations done every 20 s have been modified to one pulse every 40 s. The experimental curve was fitted with exponentially relaxing functions (red). The normalization is done with regards to the maximal steady value as in (a and b). (d and e) pmCRY2 over time after 2 pulses of blue light spaced out by 25 s and then regular periodic pulses (a period of 25 s after 10 min). (d) Initial 150 s of this experiment (one image every 5 s) with the two calibrating response levels used to extract the parameters f and C₀ (green lines). (e) pmCRY2 over time (black) for the total duration of the experiment with the targeted level (computed using f and C₀ from (d), red line). (f) pmCRY2 over time for a local activation with a constant frequency either with (black) or without (dashed black) a first round of activation at high frequency (six pulses with periodicity of 5 s). In both cases, the steady state is maintained with one pulse every 20 s (50 ms at 9 μW power). To see this figure in color, go online.