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

Abstract

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 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 the flavin-specific inhibitor diphenyleneiodonium 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 the dCRY tryptophan residue W342, more distant from the 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.

Keywords: Drosophila; circadian clock; cryptochrome; flavoprotein; phototransduction.