Distinct mechanisms of Drosophila CRYPTOCHROMEmediated light-evoked membrane depolarization and in vivo clock resetting

Drosophila CRYPTOCHROME (dCRY) mediates electrophysiological depolarization and circadian clock resetting in response to blue or ultraviolet (UV) light. These light evoked biological responses operate at different timescales and possibly through different mechanisms. Whether electron transfer down a conserved chain of tryptophan residues underlies biological responses following dCRY light activation has been controversial. To examine these issues in vivo and in ex vivo whole brain preparations, we generated transgenic flies expressing tryptophan mutant dCRYs in the conserved electron transfer chain, and then measured neuronal electrophysiological phototransduction and behavioral responses to light. Electrophysiological evoked potential analysis shows that dCRY mediates UV and blue light–evoked depolarizations that are long lasting persisting for nearly a minute. Surprisingly, dCRY appears to mediate red light-evoked depolarization in wild-type flies, absent in both cry-null flies, and following acute treatment with flavin-specific inhibitor diphenyleneiodonium (DPI) in wild-type flies. This suggests a previously unsuspected functional signaling role for a neutral semiquinone flavin state (FADH•) for dCRY. The W420 tryptophan residue located closest to the FAD-dCRY interaction site is critical for blue and UV light evoked electrophysiological responses, while other tryptophan residues within electron transfer distance to W420 do not appear to be required for light evoked electrophysiological responses. Mutation of dCRY tryptophan residue W342, more distant from FAD interaction site, mimics the cry-null behavioral light response to constant light exposure. These data indicate that light evoked dCRY electrical depolarization and clock resetting are mediated by distinct mechanisms.