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. 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 ...

Tuning flavin environment to detect and control light-induced conformational switching in Drosophila cryptochrome

“…However, not all dCRY functionality depends on interaction with the core clock machinery 66 – 69 . In particular, dCRY can directly modulate neuron firing frequency in response to light and this property also correlates with flavin photoreduction 68 , 69 . The role that CTT release plays in neuronal firing frequency is currently unknown, but reversibility of the photocycle may be relevant for these activities.…”

“…The DEER experiments also indicate that flavin loss is not an obligatory step of the photocycle and dCRY can repeatedly release the CTT during cycles of light activation. It is notable that the ability of dCRY to modulate neuronal firing frequency has a small red-light dependence 68 . As the NSQ would be the only flavin species to absorb at these wavelengths, the red-light effect may involve the conversion of a small pool of inactive NSQ to active fully reduced FADH − , which, like the ASQ, would carry a negative charge.…”

Weekend Light Shifts Evoke Persistent Drosophila Circadian Neural Network Desynchrony

“…Furthermore, LL rapidly induces circadian behavioral rhythmicity ( Pittendrigh and Daan, 1976 ; Baik et al, 2018 , 2020 ). Circadian rhythmicity under an LL entrainment is preserved in mutant flies lacking functional CRY ( Emery et al, 2000 ; Dolezelova et al, 2007 ; Baik et al, 2019 ). In close agreement with well characterized LL in vivo behavioral responses, PER-cycling rapidly dampens throughout the circadian circuit in response to LL exposure in imaged whole brains, thus providing another line of validation for the whole-brain imaging method.…”

“…Light entrainment in flies lacking single light input channels (CRY or external opsin expressing photoreceptors or Rh7) yields very discernable but slower behavioral light circadian phase shift responses ( Helfrich-Förster et al, 2001 ; Ni et al, 2017 ), indicating these input channels are largely functionally redundant (although there are measurable differences between the number of transient days needed for reentrainment at different light intensities and morning vs evening reentrainment depending on which light input channel is missing). Electrophysiological light responses can be recorded in circadian neurons using light stimulus parameters that are optimized for opsin activation in eyes but are insufficient in duration (and perhaps amplitude) for CRY activation ( Li et al, 2018 ; see Baik et al, 2019 for detailed light parameters for CRY activation). As expected, we find that overall circuit entrainment by direct light is comparable whether the compound eye is present or is completely removed.…”

Parallel Synaptic Acetylcholine Signals Facilitate Large Monopolar Cell Repolarization and Modulate Visual Behavior inDrosophila

“…Image-forming vision initiates with light activation of rods and cones in the vertebrate retina or eight photoreceptors (R1-R8) in the Drosophila retina, which send information about the color, contrast, and motion of objects to brain. In addition, Melanopsin in the vertebrate intrinsically photosensitive retinal ganglion cells or the flavoprotein Cryptochrome and Rhodopsin 7 in Drosophila pacemaker neurons generate non-image-forming vision to synchronize the biological clocks with the external light/dark cycle (Berson et al, 2002;Hattar et al, 2002;Ni et al, 2017;Baik et al, 2019). In both vertebrates and invertebrates, absorption of light photons in image-forming photoreceptors causes a conformational change of the light receptor rhodopsin, which subsequently activates the phototransduction cascade.…”