Figure 1.
Cryptochromes and DNA Photolyases
(A) Evolutionary relatedness and domain structure of plant and animal cryptochromes. N-terminal region is highly conserved and binds the same light-absorbing cofactors: a folate at the N-terminal region and a catalytic flavin chromophore at the C-terminal region. Plant cryptochromes are most related to Type I CPD photolyases, whereas animal cryptochromes are most related to 6–4 photolyases.
(B) The photocycle of plant cryptochromes. In the dark, the flavin chromophore is in its oxidized redox state. Blue light induces conversion to a meta-stable semiquinone redox state that is the activated signaling state. Green light causes further reduction to the fully reduced redox state of flavin, which is inactive in signaling. In the dark, fully reduced flavin reoxidizes to the fully oxidized form and can be reactivated by blue light. The photocycle of plant cryptochromes is different from DNA photolyases, in which only the fully reduced redox state is catalytically active.
Figure 2.
Redox State of Animal Cryptochromes and Photoreduction In Vivo
(A) Time course of Dmcry protein degradation in irradiated flies. Irradiation of flies and quantitation of signal was performed as described in Materials and Methods.
(B) Action spectrum of Dmcry activity in living flies. Y-axis is a measure of activity (irradiance expressed as time to induce 50% Dmcry degradation) at indicated wavelengths. Data was obtained from plots of time courses of Dmcry degradation in Figure S1.
(C) In vivo difference spectrum of Dmcry flavin photoconversion. Dmcry-overexpressing insect cells were irradiated with blue light (445 ± 10 nm, 25 μmol m−2 s−1), and excitation spectra were taken at the indicated intervals by monitoring emission at 525 nm in a fluorescence spectrophotometer. The successive plots were obtained by subtraction of spectra from the (dark) baseline spectrum taken before light treatment (see Figure S2). Peak excitation efficiency at 450 nm decreased as a function of time, consistent with photoreduction of oxidized flavin in vivo.
(D) In vivo difference spectrum of Hscry1 flavin photoconversion in vivo. Full-length Hscry1 were irradiated with blue light (445 ± 10 nm, 25 μmol m−2 s−1), and excitation spectra were taken at the indicated intervals by monitoring emission at 525 nm in a fluorescence spectrophotometer. The successive plots were obtained by subtraction of spectra from the (dark) baseline spectrum taken before light treatment (see Figure S2).
Figure 3.
Disruption of the Putative Electron Transfer Chain Alters Photoresponse of Dmcry at Low Photon Fluence Rates
(A) Insect cells expressing high levels of either wild-type Dmcry or tryptophan to phenylalanine mutants were irradiated in white light (150 μmol m−2 sec−1), and excitation spectra taken at the indicated intervals to determine levels of remaining oxidized flavins. Plant cryptochromes (wild-type Atcry1, and W400F and W324F mutants) are included for comparison.
(B) Same as in (A) except irradiation was performed at reduced blue-light intensity (450 nm, 10 μmol m−2 sec−1).
Figure 4.
Animal Cryptochrome Photocycle Includes Light-Dependent Radical Accumulation In Vivo
(A) X-band continuous-wave (cw)-EPR frozen-solution spectra of intact Sf21 cells expressing Dmcry and Hscry1. A–C, Sf21 insect cells expressing Dmcry after different blue-light illumination times: A, 0 min; B, 3 min; and C, 6 min; D–F, Sf21 insect cells expressing hscry1 after different blue-light illumination times: D, 0 min; E, 6 min; and F, 15 min. Moreover, two Dmcry mutants (W397F and W342F) were recorded under dark and after blue-light illumination conditions: H, 0 min; I, 12 min for W397F; and J, 0 min; K, 12 min for W342F. For more experimental details, see [17,18].
(B) Comparison of X-band–pulsed (Davies) ENDOR spectra for illuminated Dmcry in intact Sf21 insect cells and purified protein. A, cells (sample C in Figure 4A). B, purified protein. The magnified range above 20 MHz (inset trace B) shows the absence of the characteristic ENDOR signal from the N(5)H proton for neutral flavin radicals [54], clearly identifying the radical as anionic flavin species.
Figure 5.
Green Light Alters Cryptochrome Photoconversion In Vivo That Correlates with Function
(A) Living flies dark adapted for 3 d were irradiated with either blue (B) (445 ± 10 nm, 10 μmol m−2 s−1) light or bichromatically with (445 ± 10 nm, 10 μmol m−2 s−1) plus green (G) (550 ± 25 nm, 50 μmol m−2 s−1) light (B+G). Western blot with Dmcry antibody was as in Figure 2; equivalent load was verified both by Bradford assay and by staining of total proteins on gels. Three independent trials were performed with similar reduced response to B+G. a.u., arbitrary units.
(B) Dmcry photoreduction in living insect cell culture was monitored under conditions of bichromatic irradiation. Peak excitation efficiency at 450 nm was determined in duplicate samples over the indicated time course as above (Figure 1C and 1D). Samples were treated with either B at 25 μmol m−2 s−1 or bichromatically with B+G (550 ± 25 nm, 150 μmol m−2 s−1) light over the same interval. Deduced 450-nm peak intensities were plotted over time. No difference was observed in uninfected control cells under conditions of B or B+G irradiation.
Figure 6.
Light Alters Biochemical Properties of Hscry1 in an In Vivo Context
Transgenic flies expressing full-length Hscry1 of two independently transformed lines (A: w;31.2A and B: w;26.9A) were assayed for protein expression either in the dark or after the indicated times of irradiation at 150 μmol m−2 s−1 white light.