Activatable bioluminescence to monitor circadian clock mechanisms in specific Drosophila neurons in vivo

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One of the major questions in biology is how behaviour is regulated. Although we have made progress in linking a neuronal circuit to a given behaviour, an understanding of the fundamental molecular mechanisms regulating these circuits has been comparatively lacking. Circadian rhythms are behavioural and physiological responses to planetary rhythms observed in all animals, and serve as an excellent model for understanding behaviour at molecular resolution because the key genes and neurons have been identified. Drosophila melanogaster is an ideal model organism for analyzing neuronal activity because the circadian neurons are not consolidated into one area of the brain as they are in mammals. This allows interrogation of distinct neuronal sub types with relative ease, while the wide array of genetic tools available in Drosophila facilitate genetic manipulation and analysis. Therefore, circadian rhythms in Drosophila is an ideal system to study the fundamental molecular mechanisms that govern behaviour. We have shown that circadian genes are regulated differently in the neurons in which they are expressed and are subject to different biochemical regulatory mechanisms. The principle kinases known to regulate core circadian proteins are not expressed in all circadian neurons, suggesting that substitute mechanisms must exist. A mutation in a circadian gene that has a deleterious effect in one brain region may have no effect in another. Therefore the biochemical context of a circadian gene is critical to understanding how circadian behaviour is regulated. We have three branches of interrogation: 1) identifying alternative biochemical mechanisms of circadian proteins, using prediction algorithms and two different unbiased mass spectrometry analyses; 2) measuring molecular clock activity using LABL, a reporter system that enables monitoring the clock in specific neurons, in vivo; 3) analysis of circadian behavioural activity using state-of-the-art video systems that we have developed and adapted. Our unique approach will link protein biochemistry, neuronal circuitry and behaviour to reveal the fundamental principles that regulate behaviour. This level of understanding will ultimately allow us to reliably predict circadian behaviour. We believe that this work will broadly serve as proof of principle in demonstrating that neuronal circuitry and behavioural genes are linked through biochemical mechanisms . Digitizing mechanisms that regulate behaviour into biochemical steps, as we have begun to do in our preliminary work will allow us to reliably predict the behaviour of flies as a function of the polymorphisms that they carry. This work has already begun to challenge established dogma in circadian behaviour regulation. The LABL technology I developed, our biochemical expertise and our video-based behavioural analysis uniquely position my lab to make significant new discoveries in the links between protein biochemistry, neurobiology and behaviour.