Effect of constant light and circadian entrainment of perS flies: evidence for light-mediated delay of the negative feedback loop in Drosophila.

Abstract

Light is the dominant environmental cue that provides temporal information to circadian pacemakers. In Drosophila melanogaster some period gene mutants have altered free‐running circadian periods but entrain to 24 h light‐dark cycles. To address the mechanism of light entrainment in Drosophila, we examined the effects of constant light on the period gene (per) and timeless gene (tim) products in wild‐type and perS flies. The results indicate that light affects three features of the PER‐TIM program: PER and TIM phosphorylation, PER and TIM accumulation, and per and tim RNA cycling. A post‐transcriptional effect on the PER‐TIM complex is the likely primary clock target, which then delays the subsequent decrease in per and tim RNA levels. This is consistent with a negative feedback loop, in which the PER‐TIM complex contributes to the decrease in per and tim RNA levels, presumably at the transcriptional level. There are enhanced constant light effects on the perS mutant, which further support negative feedback as well as support its importance to entrainment of these flies to a 24 h cycle, far from their intrinsic period of 19 h. The perS mutant leads to a truncated protein accumulation phase and a subsequent premature perS RNA increase. A standard 24 h light‐dark cycle delays the negative feedback circuit and extends the RNA and protein profiles, compensating for the accelerated RNA increase and restoring the rhythms to wild‐type‐like periodicity.

PDF and cAMP enhance PER stability in Drosophila clock neurons

“…This was a prime candidate in part because Neurospora PKA phosphorylates and stabilizes the important transcriptional repressor FREQUENCY (FRQ) (22), which functions in Neurospora rhythms similarly to Drosophila PER. To assess the role of PKA in PER stability regulation, we combined FSK with the specific PKA inhibitor PKI (14)(15)(16)(17)(18)(19)(20)(21)(22). The results show that PKI addition significantly reduces the effect of FSK (Fig.…”

“…They were incubated in AHL for 6 h, corresponding to an in vivo time of ZT4 or 4 h after lights on. PER levels at ZT4 are much lower than their peak levels at ZT22 (15,17). As there is rather little per mRNA and therefore probably rather little PER synthesis between ZT22 and ZT4 (27), it is likely that the normal decrease in PER levels observed in vivo between ZT22 and ZT4 is due to PER degradation.…”

“…To avoid the well-known and important effect of light on clock protein stability (17,24,25), circadian-blind cry 01 flies were used in these experiments; cry 01 flies manifest normal PER cycling within PDF neurons (23,26). Flies were collected at Zeitgeber time 22 (ZT22) (10 h after lights off and 2 h before lights on) when PER is at or near peak levels (15,17), and the brains were immediately dissected. They were incubated in AHL for 6 h, corresponding to an in vivo time of ZT4 or 4 h after lights on.…”

“…per S flies have a very short circadian period (∼19 h vs. ∼24 h for wild-type flies) (13), and PERS has a markedly shorter half-life than PER (14)(15)(16)(17)(18). This suggests that the function of PDF and enhanced cAMP levels is to slow the pace of PER degradation in PDFR-expressing clock neurons; the intrinsically fast degradation of PERS can bypass this regulatory step.…”

Synchronization of theDrosophilaCircadian Clock by Temperature Cycles

“…In LL and constant temperatures, molecular and behavioral rhythmicity breaks down (Konopka et al 1989;Marrus et al 1996), presumably because of the lightinduced and Cry-mediated degradation of Tim (see, e.g., Stanewsky et al 1998;Ceriani et al 1999;Busza et al 2004). Nevertheless, robust behavioral synchronization and oscillations of per and tim gene products are observed in LL and temperature cycles (Glaser and Stanewsky 2005;Matsumoto et al 1998;Miyasako et al 2007;Yoshii et al 2002Yoshii et al , 2005.…”