Drosophila DBT lacking protein kinase activity produces long-period and arrhythmic circadian behavioral and molecular rhythms

A mutation (K38R) which specifically eliminates kinase activity was created in the Drosophila melanogaster ckI gene (doubletime [dbt]). In vitro, DBT protein carrying the K38R mutation (DBT^K/R) interacted with Period protein (PER) but lacked kinase activity. In cell culture and in flies, DBT^K/R antagonized the phosphorylation and degradation of PER, and it damped the oscillation of PER in vivo. Overexpression of short-period, long-period, or wild-type DBT in flies produced the same circadian periods produced by the corresponding alleles of the endogenous gene. These mutations therefore dictate an altered “set point” for period length that is not altered by overexpression. Overexpression of the DBT^K/R produced effects proportional to the titration of endogenous DBT, with long circadian periods at lower expression levels and arrhythmicity at higher levels. This first analysis of adult flies with a virtual lack of DBT activity demonstrates that DBT's kinase activity is necessary for normal circadian rhythms and that a general reduction of DBT kinase activity does not produce short periods.     

Drosophila and vertebrate casein kinase Idelta exhibits evolutionary conservation of circadian function

Mutations lowering the kinase activity of Drosophila Doubletime (DBT) and vertebrate casein kinase I/δ (CKI/δ) produce long-period, short-period, and arrhythmic circadian rhythms. Since most ckI short-period mutants have been isolated in mammals, while the long-period mutants have been found mostly in Drosophila, lowered kinase activity may have opposite consequences in flies and vertebrates, because of differences between the kinases or their circadian mechanisms. However, the results of this article establish that the Drosophila dbt mutations have similar effects on period (PER) protein phosphorylation by the fly and vertebrate enzymes in vitro and that Drosophila DBT has an inhibitory C-terminal domain and exhibits autophosphorylation, as does vertebrate CKI/δ. Moreover, expression of either Drosophila DBT or the vertebrate CKIδ kinase carrying the Drosophila dbt^S or vertebrate tau mutations in all circadian cells leads to short-period circadian rhythms. By contrast, vertebrate CKIδ carrying the dbt^L mutation does not lengthen circadian rhythms, while Drosophila DBT^L does. Different effects of the dbt^S and tau mutations on the oscillations of PER phosphorylation suggest that the mutations shorten the circadian period differently. The results demonstrate a high degree of evolutionary conservation of fly and vertebrate CKIδ and of the functions affected by their period-shortening mutations.     

Effects of Different PER Translational Kinetics on the Dynamics of a Core Circadian Clock Model

Living beings display self-sustained daily rhythms in multiple biological processes, which persist in the absence of external cues since they are generated by endogenous circadian clocks. The period (per) gene is a central player within the core molecular mechanism for keeping circadian time in most animals. Recently, the modulation PER translation has been reported, both in mammals and flies, suggesting that translational regulation of clock components is important for the proper clock gene expression and molecular clock performance. Because translational regulation ultimately implies changes in the kinetics of translation and, therefore, in the circadian clock dynamics, we sought to study how and to what extent the molecular clock dynamics is affected by the kinetics of PER translation. With this objective, we used a minimal mathematical model of the molecular circadian clock to qualitatively characterize the dynamical changes derived from kinetically different PER translational mechanisms. We found that the emergence of self-sustained oscillations with characteristic period, amplitude, and phase lag (time delays) between per mRNA and protein expression depends on the kinetic parameters related to PER translation. Interestingly, under certain conditions, a PER translation mechanism with saturable kinetics introduces longer time delays than a mechanism ruled by a first-order kinetics. In addition, the kinetic laws of PER translation significantly changed the sensitivity of our model to parameters related to the synthesis and degradation of per mRNA and PER degradation. Lastly, we found a set of parameters, with realistic values, for which our model reproduces some experimental results reported recently for Drosophila melanogaster and we present some predictions derived from our analysis.     

PER-TIM interactions with the photoreceptor cryptochrome mediate circadian temperature responses in Drosophila

Drosophila cryptochrome (CRY) is a key circadian photoreceptor that interacts with the period and timeless proteins (PER and TIM) in a light-dependent manner. We show here that a heat pulse also mediates this interaction, and heat-induced phase shifts are severely reduced in the cryptochrome loss-of-function mutant cry^b. The period mutant per^L manifests a comparable CRY dependence and dramatically enhanced temperature sensitivity of biochemical interactions and behavioral phase shifting. Remarkably, CRY is also critical for most of the abnormal temperature compensation of per^L flies, because a per^L; cry^b strain manifests nearly normal temperature compensation. Finally, light and temperature act together to affect rhythms in wild-type flies. The results indicate a role for CRY in circadian temperature as well as light regulation and suggest that these two features of the external 24-h cycle normally act together to dictate circadian phase.     

Relationships between the Circadian System and Alzheimer's Disease-Like Symptoms in Drosophila

Circadian clocks coordinate physiological, neurological, and behavioral functions into circa 24 hour rhythms, and the molecular mechanisms underlying circadian clock oscillations are conserved from Drosophila to humans. Clock oscillations and clock-controlled rhythms are known to dampen during aging; additionally, genetic or environmental clock disruption leads to accelerated aging and increased susceptibility to age-related pathologies. Neurodegenerative diseases, such as Alzheimer''s disease (AD), are associated with a decay of circadian rhythms, but it is not clear whether circadian disruption accelerates neuronal and motor decline associated with these diseases. To address this question, we utilized transgenic Drosophila expressing various Amyloid-β (Aβ) peptides, which are prone to form aggregates characteristic of AD pathology in humans. We compared development of AD-like symptoms in adult flies expressing Aβ peptides in the wild type background and in flies with clocks disrupted via a null mutation in the clock gene period (per⁰¹). No significant differences were observed in longevity, climbing ability and brain neurodegeneration levels between control and clock-deficient flies, suggesting that loss of clock function does not exacerbate pathogenicity caused by human-derived Aβ peptides in flies. However, AD-like pathologies affected the circadian system in aging flies. We report that rest/activity rhythms were impaired in an age-dependent manner. Flies expressing the highly pathogenic arctic Aβ peptide showed a dramatic degradation of these rhythms in tune with their reduced longevity and impaired climbing ability. At the same time, the central pacemaker remained intact in these flies providing evidence that expression of Aβ peptides causes rhythm degradation downstream from the central clock mechanism.     

In Vivo Role of Phosphorylation of Cryptochrome 2 in the Mouse Circadian Clock

The circadian clock is finely regulated by posttranslational modifications of clock components. Mouse CRY2, a critical player in the mammalian clock, is phosphorylated at Ser557 for proteasome-mediated degradation, but its in vivo role in circadian organization was not revealed. Here, we generated CRY2(S557A) mutant mice, in which Ser557 phosphorylation is specifically abolished. The mutation lengthened free-running periods of the behavioral rhythms and PER2::LUC bioluminescence rhythms of cultured liver. In livers from mutant mice, the nuclear CRY2 level was elevated, with enhanced PER2 nuclear occupancy and suppression of E-box-regulated genes. Thus, Ser557 phosphorylation-dependent regulation of CRY2 is essential for proper clock oscillation in vivo.     

PER-TIM Interactions with the Photoreceptor Cryptochrome Mediate Circadian Temperature Responses in Drosophila

Drosophila cryptochrome (CRY) is a key circadian photoreceptor that interacts with the period and timeless proteins (PER and TIM) in a light-dependent manner. We show here that a heat pulse also mediates this interaction, and heat-induced phase shifts are severely reduced in the cryptochrome loss-of-function mutant cry^b. The period mutant per^L manifests a comparable CRY dependence and dramatically enhanced temperature sensitivity of biochemical interactions and behavioral phase shifting. Remarkably, CRY is also critical for most of the abnormal temperature compensation of per^L flies, because a per^L; cry^b strain manifests nearly normal temperature compensation. Finally, light and temperature act together to affect rhythms in wild-type flies. The results indicate a role for CRY in circadian temperature as well as light regulation and suggest that these two features of the external 24-h cycle normally act together to dictate circadian phase.