Disruption of Cryptochrome partially restores circadian rhythmicity to the arrhythmic period mutant of Drosophila

Abstract

The Drosophila melanogaster circadian clock is generated by interlocked feedback loops, and null mutations in core genes such as period and timeless generate behavioral arrhythmicity in constant darkness. In light-dark cycles, the elevation in locomotor activity that usually anticipates the light on or off signals is severely compromised in these mutants. Light transduction pathways mediated by the rhodopsins and the dedicated circadian blue light photoreceptor cryptochrome are also critical in providing the circadian clock with entraining light signals from the environment. The cry b mutation reduces the light sensitivity of the fly's clock, yet locomotor activity rhythms in constant darkness or light-dark cycles are relatively normal, because the rhodopsins compensate for the lack of cryptochrome function. Remarkably, when we combined a period-null mutation with cry b , circadian rhythmicity in locomotor behavior in light-dark cycles, as measured by a number of different criteria, was restored. This effect was significantly reduced in timeless-null mutant backgrounds. Circadian rhythmicity in constant darkness was not restored, and TIM protein did not exhibit oscillations in level or localize to the nuclei of brain neurons known to be essential for circadian locomotor activity. Therefore, we have uncovered residual rhythmicity in the absence of period gene function that may be mediated by a previously undescribed period-independent role for timeless in the Drosophila circadian pacemaker. Although we do not yet have a molecular correlate for these apparently iconoclastic observations, we provide a systems explanation for these results based on differential sensitivities of subsets of circadian pacemaker neurons to light.anticipation ͉ phase shift ͉ oscillator ͉ hourglass ͉ timeless T he PERIOD (PER) and TIMELESS (TIM) proteins are core components of the negative feedback loop that drives circadian oscillations in Drosophila. TIM stabilizes PER, and the PER and TIM proteins cooperate in nuclear entry and retention in key pacemaker cells at night (1). TIM degrades rapidly in response to light, releasing PER to repress per and tim transcription (1). D. melanogaster individuals carrying the null mutation per 01 display arrhythmic locomotor activity under constant darkness (DD), whereas in light-dark (LD) cycles, their locomotor activity is elevated under illumination and reduced in darkness, reflecting the ''masking'' effect of light (2, 3). Another mutation in the gene encoding the blue-light photoreceptor cryptochrome, cry b , gives attenuated light responses in a circadian context (4-7), but normal circadian locomotor behavior in 24-h LD cycles. As part of another study of seasonal behavioral responses to temperature and light effected via changes in per 3Ј splicing, we generated a per 01 ; cry b double mutant (8). We noted that a modest cycle in wild-type per 3Ј splicing in LD cycles at 29°C was exaggerated in per 01 and cry b mutants, yet in the double mutant, the amplitude of the splicing reverted back to ap...

Circadian Rhythm Gene Regulation in the Housefly Musca domestica

“…It is interesting to note that in Musca at ZT24 the l-LNvs are the only group of putative clock cells to show cytoplasmic PER and nuclearcytoplasmic TIM and that both types of reagents give much weaker signals. This might suggest that this neuronal cluster might have a special function in the circadian network, as suggested for Drosophila (Collins et al 2005). Furthermore, in Drosophila the l-LNvs are the only group of neurons where, under particular environmental conditions, for example, in constant conditions, PER and TIM nuclear accumulation can be decoupled (Yang and Sehgal 2001;Shafer et al 2002;Rieger et al 2006).…”

“…Furthermore, in Drosophila the l-LNvs are the only group of neurons where, under particular environmental conditions, for example, in constant conditions, PER and TIM nuclear accumulation can be decoupled (Yang and Sehgal 2001;Shafer et al 2002;Rieger et al 2006). These cells possibly represent a strategic point in the neuronal network of Diptera where a physiological response to a combination of environmental variables might be amplified for entrainment (Collins et al 2005). As for the more nuclear subcellular distribution of MdTIM in the l-LNvs of Musca compared to MdPER, this could reflect a more prominent role for MdTIM as a negative regulator in the housefly, as has been suggested for A. pernyii (Chang et al 2003).…”

The hormonal and circadian basis for insect photoperiodic timing

“…However, in the case of per 01 , there is considerable evidence for a residual circadian oscillator that might be underlying the photoperiodic response in the mutant, providing a convenient escape clause [180][181][182]. If this is the case, then the tim 01 results from Drosophila (and other species)…”

Even a stopped clock tells the right time twice a day: circadian timekeeping in Drosophila

“…However, this is confounded by the observation of anticipatory evening activity in per 01 ; cry b double mutant flies that should lack a functional clock in all clock cells [79]. Anticipation is TIMdependent, as both per 01 ; tim 01 ; cry b triple mutants and tim 01 ; cry b double mutants showed no anticipation.…”

“…to generate rhythmic evening activity in LD. However, TIM does not enter the nucleus of either the M or E cells in per 01 ; cry b mutants, making it unclear exactly how much clock function is sufficient to drive LD rhythmicity [79]. …”