Biochemical and functional analysis of daywake, a novel wake-sleep regulator in drosophila melanogaster
Creators: name: Villegas, Gabriel
Publisher: name: No Publisher Supplied
Year: 2020

description: D. melanogaster is an extremely powerful genetic animal model to study why and how we sleep. Like humans, the majority of sleep in D. melanogaster occurs during the night. However, similar to many other day-active animals, D. melanogaster exhibits a midday sleep or “siesta”, most frequently associated with limiting exposure to the hot midday sun. Recent evidence indicates a strong genetic component influencing siesta behavior in humans. While nighttime and daytime sleep serve different functions, the role and mechanisms for siesta are little understood.Initial studies from our laboratory showed that midday siesta in D. melanogaster is down-regulated by the cold-enhanced splicing of the 3’ terminal intron (termed dmpi8) in the period (per) gene, a key component of the circadian (~24 h) clock. More recent results revealed that dmpi8 splicing influences midday siesta in-trans via up-regulating the expression of a slightly overlapping gene termed daywake (dyw) that functions in an anti-siesta capacity. Overall, the main idea from prior work is that dyw mainly functions in thermal-adaptation whereby its increased levels on cool days reduces siesta levels by promoting midday wakefulness on days where the threat from heat exposure is diminished.The dyw gene encodes a juvenile hormone binding protein (JHBP), which are known to be secreted into the hemolymph as part of signaling cascades. In this thesis I raised anti-DYW antibodies and used them to characterize the DYW protein at the biochemical and cellular levels in both flies and cultured Drosophila cells. DYW is found in key circadian cells in the brain, the pigment-dispersing factor (PDF)-expressing neurons, consistent with earlier work showing that manipulating dyw levels in these cells modulates midday siesta. DYW levels in the head are responsive to temperature and differ in male and female adult flies. There are two major isoforms of DYW in adult flies that appear to reflect post-translational differences. Indeed, studies in cultured Drosophila cells indicate that DYW stability and/or maturation is heavily dependent on a glycosylation event at a single residue position. Further, this work shows that DYW is secreted and that this secretion is reliant on the presence of an N-terminal signal peptide comprised of the first 25 residues in DYW. My studies suggest a model whereby DYW functions as a JHBP that is produced in the brain followed by cleavage and entry into the circulatory system where it interacts with target tissues to regulate sleep-wake behavior.In related work, CRISPR technology was used to generate dyw knock-out (dyw-KO) flies. Remarkably, although dyw-KO flies exhibit increased midday sleep compared to wildtype controls as expected, when challenged with malnourishment we observe major differences in the regulation of nighttime sleep between dyw-KO flies and controls. This finding suggests that with prolonged hunger, dyw mainly stimulates wake during the night. Presumably, this helps in foraging for food but shifts this activity towards the night when the threat from sun exposure is eliminated. Together the studies presented in this thesis substantially increase our understanding of how dyw functions. It is proposed that dyw has a more broad adaptive role in integrating multiple survival threats (e.g., heat, hunger) to optimize day-night levels of wake-sleep behavior to best meet current environmental challenges. Because dyw is part of a larger family of lipid-binding carriers found in animals, the work might reveal further insights into the interplay between sleep and metabolism, key to human well-being and linked to many diseases. In addition to my work on dyw, this thesis also includes some key results from my earlier studies aimed at biochemically characterizing the PER circadian clock protein.