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

生物学中的一个主要问题是行为是如何受到监管的。尽管我们已经在将神经元回路与给定行为联系起来方面取得了进展，但对调节这些回路的基本分子机制的了解相对较少。 昼夜节律是在所有动物中观察到的对行星节律的行为和生理反应，由于关键基因和神经元已经被识别，因此是在分子分辨率上理解行为的极好模型。果蝇是分析神经元活动的理想模式生物，因为昼夜节律神经元不像哺乳动物那样整合到大脑的一个区域。这使得对不同的神经元亚型的询问相对容易，而果蝇可用的广泛的遗传工具便于遗传操作和分析。因此，果蝇的昼夜节律是研究支配行为的基本分子机制的理想系统。 我们已经证明，昼夜节律基因在表达它们的神经元中受到不同的调控，并受到不同的生化调控机制的影响。已知的调节核心昼夜节律蛋白的主要激酶并不是在所有的昼夜节律神经元中都表达，这表明替代机制肯定存在。昼夜节律基因的突变在一个大脑区域产生有害影响，可能对另一个区域没有影响。因此，昼夜节律基因的生化背景对于理解昼夜节律行为是如何调节的至关重要。 我们有三个询问分支：1)使用预测算法和两种不同的无偏质谱分析，识别昼夜节律蛋白质的替代生化机制；2)使用LABL测量分子时钟活动，这是一种能够在体内监控特定神经元时钟的报告系统；3)使用我们开发和适应的最先进的视频系统来分析昼夜节律行为活动。我们独特的方法将蛋白质生物化学、神经元电路和行为联系起来，以揭示调节行为的基本原理。 这种水平的理解最终将使我们能够可靠地预测昼夜节律行为。我们相信，这项工作将广泛地作为证明神经元电路和行为基因通过生化机制联系在一起的原则证据。将调节行为的机制数字化为生化步骤，就像我们在前期工作中已经开始做的那样，将使我们能够可靠地预测苍蝇的行为，作为它们携带的多态的函数。这项工作已经开始挑战生理行为调节方面的既定教条。我开发的LABL技术、我们的生化专业知识和基于视频的行为分析使我的实验室在蛋白质生物化学、神经生物学和行为之间的联系方面取得了重大的新发现。

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.