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. cryptochrome | flavoprotein | phototransduction | Drosophila | circadian clock C RYPTOCHROME (CRY) is a highly conserved and evolutionarily ancient flavoprotein expressed widely in prokaryotes and eukaryotes. Light-sensitive Drosophila CRYPTOCHROMEs (dCRYs) exhibit 2 absorbance peaks at 365 nm [ultraviolet (UV) light] and 450 nm (blue light) both in vitro and in cells (1-3). Light activation of dCRY initiates a relatively slow (∼1 h) irreversible process of ubiquitin-mediated degradation of the clock protein TIMELESS (TIM) and dCRY itself, thus resetting the circadian clock (4-8). More recently, dCRY was discovered to mediate rapid membrane depolarization (τ on ∼ 100 ms) and an increased spontaneous action potential firing rate in the lateral ventral neurons (LNvs) in response to blue and UV light (9-12). The mechanisms for light-activated dCRY leading to TIM degradation/clock resetting and membrane depolarization may differ based on timing of the response onset and reversibility/irreversibility following light exposure. For clock resetting, the light activation of dCRY leads to displacement of its short helical C-terminal tail (CTT) allowing TIM binding, which triggers the JETLAG (JET) ubiquitin-ligasemediated targeting of TIM ...

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Nocturnal Anopheles mosquitoes exhibit strong behavioral avoidance to blue-light while diurnal Aedes mosquitoes are behaviorally attracted to blue-light and a wide range of other wavelengths of light. To determine the molecular mechanism of these effects, we expressed light-sensing Anopheles gambiae (AgCRY1) and Aedes aegypti (AeCRY1) Cryptochrome 1 (CRY) genes under a crypGAL4-24 driver line in a mutant Drosophila genetic background lacking native functional CRY, then tested behavioral and electrophysiological effects of mosquito CRY expression relative to positive and negative CRY control conditions. Neither mosquito CRY stops the circadian clock as shown by robust circadian behavioral rhythmicity in constant darkness in flies expressing either AgCRY1 or AeCRY1. AgCRY1 and AeCRY1 both mediate acute increases in large ventral lateral neuronal firing rate evoked by 450 nm blue-light, corresponding to CRY’s peak absorbance in its base state, indicating that both mosquito CRYs are functional, however, AgCRY1 mediates significantly stronger sustained electrophysiological light-evoked depolarization in response to blue-light relative to AeCRY1. In contrast, neither AgCRY1 nor AeCRY1 expression mediates measurable increases in large ventral lateral neuronal firing rates in response to 405 nm violet-light, the peak of the Rhodopsin-7 photoreceptor that is co-expressed in the large lateral ventral neurons. These results are consistent with the known action spectra of type 1 CRYs and lack of response in cry-null controls. AgCRY1 and AeCRY1 expressing flies show behavioral attraction to low intensity blue-light, but AgCRY1 expressing flies show behavioral avoidance to higher intensity blue-light. These results show that nocturnal and diurnal mosquito Cryptochrome 1 proteins mediate differential physiological and behavioral responses to blue-light that are consistent with species-specific mosquito behavior.

Lateral ventral neurons (LNvs) in the fly circadian neural circuit mediate behaviors other than clock resetting, including light-activated acute arousal. Converging sensory inputs often confer functional redundancy. The LNvs have three distinct light input pathways: (1) cell autonomously expressed cryptochrome (CRY), (2) rhodopsin 7 (Rh7), and (3) synaptic inputs from the eyes and other external photoreceptors that express opsins and CRY. We explored the relative photoelectrical and behavioral input contributions of these three photoreceptor systems to determine their functional impact in flies. Patch-clamp electrophysiology measuring light evoked firing frequency (FF) was performed on large LNvs (l-LNvs) in response to UV (365 nm), violet (405 nm), blue (450 nm), or red (635 nm) LED light stimulation, testing controls versus mutants that lack photoreceptor inputs gl60j, cry-null, rh7-null, and double mutant gl60j-cry-null flies. For UV, violet, and blue short wavelength light inputs, all photoreceptor mutants show significantly attenuated action potential FF responses measured in the l-LNv. In contrast, red light FF responses are only significantly attenuated in double mutant gl60j-cry-null flies. We used a light-pulse arousal assay to compare behavioral responses to UV, violet, blue and red light of control and light input mutants, measuring the awakening arousal response of flies during subjective nighttime at two different intensities to capture potential threshold differences (10 and 400 μW/cm2). The light arousal behavioral results are similar to the electrophysiological results, showing significant attenuation of behavioral light responses for mutants compared to control. These results show that the different LNv convergent photoreceptor systems are integrated and together confer functional redundancy for light evoked behavioral arousal.