DAY a novel signalling partner of Drosophila melanogaster CRYPTOCHROME

The 24 hour circadian clock controls every facet of an organism's physiology and behaviour. In humans, dysfunction of the clock not only generates temporal disorientation and sleep problems, but is also involved in pathologies such as obesity, mental illness, cardiovascular disease and cancer. The fruitfly Drosophila melanogaster is an ideal model organism for the study of circadian rhythms because the clock mechanism shares the same design and molecular components with mammals. The clock is an endogenous and self-sustained process driven by the interplay of many proteins. Among these there are molecules whose function is to receive and integrate environmental cues, and pass on this information to other clock proteins. The blue-light photopigment CRYPTOCHROME (CRY) is one such molecule, but in some cells, CRY also performs as a core clock protein independent from light. We have been working with CRY for several years and recently have discovered that another protein, we (ironically) call DAY, is able to interact with CRY, but only at night. DAY contains four PDZ domains, which are regions involved in protein-protein interactions. PDZ proteins can bring together membrane bound and cytosolic components to promote certain signalling pathways. Our preliminary work has shown that day mRNA cycles and is present in the lateral brain, most likely in a subset of circadian neurons where CRY functions exclusively as a photopigment. We did not detect the expression of day in the dorsal brain, nor in peripheral cells such as the eyes or the Malpighian tubules, where CRY also provides core clock function, suggesting that DAY may inhibit this role. In this proposal we aim to test this hypothesis as follows. We will characterise the expression of DAY and extend our work on day mRNA. We will provide additional evidence for the interaction between DAY and CRY. We will investigate the molecular, cellular and behavioural phenotypes arising from several combinations of up- and down-regulation of day and cry in central clock neurons and in peripheral tissues. These studies will elucidate the role played by this novel protein involved in the regulation of the circadian clock and will contribute to the understanding of the mode of action of CRY. Moreover, we believe that our work will add some mechanistic information to the catalogue of discrepancies reported for the different classes of central clock neurons.

A large scale yeast-two-hybrid screen has identified the PDZ protein CG15803-PA as producing a highly reliable interaction with Drosophila CRYPTOCHROME (CRY). Interestingly, CRY contains PDZ binding motifs in its C-terminus. We further characterised the interaction and found that CG15803-PA binds to CRY in darkness but not in light, thus we renamed this protein as DARK-ACTIVE-IN YEAST (DAY). Previously we had shown that CRY binds to the clock proteins PERIOD (PER) and TIMELESS (TIM) in a light-dependent manner, although, in Drosophila S2 cells, CRY co-immunoprecipitates with PER and TIM also in the dark. We therefore speculate that, at night, DAY may antagonise the interaction of CRY with other clock proteins, thereby limiting CRY's role to light signalling. It is currently believed that CRY operates exclusively as a photopigment in pacemaker neurons, whereas in peripheral clock cells, or, as we have recently observed, in Dorsal Neurons (a subset of 'pacemaker neurons') CRY additionally functions as a light-independent clock component. Thus, we examined day expression by in situ hybridisation and detected staining in a lateral region of the brain, corresponding to the position of the circadian Lateral Neurons, but not in Dorsal Neurons or in peripheral clocks. We also observed circadian cycling in the abundance of day mRNA. Thus wherever CRY is restricted to a photoreceptor role, DAY is co-expressed, and wherever CRY also plays a clock-related role, DAY is absent, supporting our hypothesis. In this proposal we seek to consolidate and extend these findings and to test our hypothesis on the significance of DAY/CRY interaction. We also propose to investigate the role of day in other behaviours such as geotaxis, following CRY involvement in it, and sexual behaviour, due to the presence of day mRNA in the male accessory glands and to sexual dimorphism in the level of expression in the brain.