Collaborative Research: Biochemical Basis of Cellular Circadian Behavior

通过建立节律行为的遗传基础，生物钟研究在神经科学领域取得了巨大成功。分子钟由正负两部分组成，它们形成了一个反馈回路，构成了大脑节律的基础。然而，“整体大于部分的总和”，我们还不知道少数几个组成部分是如何协调起来产生紧急的日常节奏的。因此，本研究旨在确定核心时钟蛋白的生化特性，使节律行为。为此，该团队开发了一种综合方法，将基于细胞的遗传学和昼夜节律行为的连续监测与生化/生物物理方法相结合。这种方法将使他们不仅可以在试管中孤立地研究时钟蛋白的功能，而且可以在相互作用细胞的振荡行为的背景下研究时钟蛋白。这项工作将揭示时钟的“齿轮和弹簧”是如何相互作用和一起工作的，这些知识将增加我们对大脑如何保持时间的理解，以及蛋白质如何相互作用来调节复杂的大脑功能。PI和他的同事们将使用“从数学到基因再到行为”的平台教授一门讲座/实验课程，并为研究生提供跨学科培训。该团队还将在当地社区教授有关昼夜节律对健康影响的现实世界概念，这与所有背景和年龄的人有关。这些更广泛的影响将加强国家的科学基础设施，提高人们的健康意识。遗传学研究已经确定了形成负反馈机制的几个核心时钟组件，这是昼夜节律行为的基础。已经确定，在核心反馈回路中，BMAL1和CLOCK是两个转录激活因子，而CRY是主要的抑制因子。然而，转录中的负反馈并不一定保证周期性约24小时的重复细胞过程。昼夜节律是由核心时钟因子激活的一种紧急特性，但细胞昼夜节律振荡的生化基础尚未得到很好的理解。该研究小组最近的研究表明，BMAL1和CLOCK结构灵活的c末端在调节与其他时钟因子的动态相互作用中发挥重要作用，从而实现昼夜节律振荡。在拟议的研究中，该团队将采用一种综合方法，将基于细胞的遗传学和动态生物发光分析与生化和生物物理方法相结合，研究它们的c末端如何使用动态相互作用来调节节律幅度和周期长度。通过这种方式，在体外和细胞昼夜节律行为的背景下评估蛋白质功能。通过提供细胞昼夜节律行为的生化基础，本研究将定义昼夜节律转录复合物的分子结构，推进目前对负反馈机制的理解，并进一步实现行为神经生物学的目标。在研究期间，PI和他的同事将为研究生提供跨学科的教育和培训，并向公众介绍昼夜节律对健康的重要影响。

Non-Technical Paragraph By establishing the genetic basis of rhythmic behavior, circadian clock research is a great success story in neuroscience. The molecular clock consists of positive and negative components that form a feedback loop that underlies brain rhythms. However, "the whole is greater than the sum of its parts," and we do not yet know how a handful of components coordinate to give rise to emergent daily rhythms. Therefore, this research aims to identify the biochemical properties of core clock proteins that enable rhythmic behavior. To do this, the team has developed an integrative approach that combines cell-based genetics and continuous monitoring of circadian behavior with biochemical/biophysical methods. This approach will allow them to study the function of clock proteins not only in test tubes in isolation, but also in the context of oscillatory behavior in interacting cells. This work will reveal how the "gears and springs" of the clock interact and function together, and this knowledge will increase our understanding of how the brain keeps time, as well as how proteins interact to regulate complex brain functions. The PI and his colleagues will teach a lecture/lab course that uses a "from math to genes to behavior" platform and provides cross-disciplinary training for graduate students. The team will also teach real world concepts in the local community about the health implications of circadian rhythms, which relates to people of all backgrounds and ages. These broader impacts will strengthen the nation's scientific infrastructure and improve people's health awareness. Technical Paragraph Genetic studies have identified several core clock components that form a negative feedback mechanism, which underlies circadian behavior. It is well established that, in the core feedback loop, BMAL1 and CLOCK are the two transcription activators and CRY serves as the chief repressor. However, negative feedback in transcription does not necessarily warrant recurring cellular processes with a ~24 hr periodicity. The circadian rhythm is an emergent property enabled by the core clock factors, but the biochemical basis of cellular circadian oscillation is not well understood. Recent studies from this research team show that the structurally flexible C-termini of BMAL1 and CLOCK play essential roles in regulating dynamic interactions with other clock factors to enable circadian oscillations. In the proposed research, the team will employ an integrated approach that combines cell-based genetics and kinetic bioluminescence assays with biochemical and biophysical methods to study how their C-termini use dynamic interactions to regulate rhythm amplitude and period length. In this way, protein function is assessed both in vitro and in the context of cellular circadian behavior. By providing a biochemical basis of cellular circadian behavior, this research will define the molecular architecture of the circadian transcription complex, advance current understanding of the negative feedback mechanism, and further the goals of behavioral neurobiology. During the research, the PI and his colleagues will provide cross-disciplinary education and training for graduate students and inform the general public about the important health implications of circadian rhythms.