Light Input Pathway of the Neurospora Circadian Clock

描述（由申请人提供）：昼夜节律钟是控制几乎所有生物体中各种细胞、生理和行为活动的内源性细胞过程。这些生物钟的准确性取决于它们是否能被环境同步（携带）。光是影响从原核生物到哺乳动物所有生物钟的最重要的环境因素，并且已知调节许多重要过程。粗糙脉孢菌是人们最了解的生物钟系统之一，为在分子水平上理解光夹带机制提供了一个很好的模型。在脉孢菌中，转录因子白色COLLAR-1（WC-1）和WC-2是生物钟功能所必需的昼夜节律反馈回路中的正元件。最近，我们还确定了WC-1，一个FAD蛋白，作为光受体的生物钟和其他光反应。这一发现确立了WC- 1作为第一个已知的真菌蓝光感光体。本项目的实验将探讨脉孢菌昼夜节律系统光输入途径的分子机制。 在具体目标1中，我们将确定WC-1如何感知光线。我们将使用生物化学和结构的方法来测试WC-1的LOV结构域结合FAD并经历可逆的光循环，以及光诱导WC-1的构象变化的假设。WC-1在黑暗中被磷酸化，在光照后变得过度磷酸化，并且磷酸化事件可以调节其稳定性和活性。因此，在特定目标2中，我们将确定WC-1磷酸化在光输入和昼夜节律钟中的作用。我们将使用质谱法来确定WC-1磷酸化位点，并使用遗传和生物化学方法来了解磷酸化的功能，并确定负责的激酶。在具体目标3中，我们将确定新的因素，介导光输入脉孢菌。我们将生化纯化大WC蛋白复合物，以确定新的因素，我们将筛选新的突变体与缺陷的光输入和昼夜节律钟。总之，这些研究将有助于我们从遗传、生化和分子方面阐明脉孢菌生物钟的光输入机制，我们获得的信息将为其他生物体的光输入途径提供相关的基础信息

DESCRIPTION (provided by applicant): Circadian clocks are endogenous cellular processes that control a wide variety of cellular, physiological, and behavioral activities in almost all organisms. The accuracy of these biological clocks is dependent on whether they can be synchronized (entrained) by the environment. Light is the most important environmental factor that entrains all circadian clocks from prokaryotes to mammals, and is known to regulate many important processes. Neurospora crassa, one of the best understood circadian clock systems, offers an excellent model for understanding the light entrainment mechanism at the molecular level. In Neurospora, the transcription factors WHITE COLLAR-1 (WC-1) and WC-2 are the positive elements in the circadian feedback loops that are essential for the clock function. Recently, we also identified WC-1, a FAD-containing protein, as the photoreceptor for circadian clock and other light responses. This finding established WC- 1 as the first known fungal blue light photoreceptor. The experiments in this project will address the molecular mechanisms of the light input pathway of the Neurospora circadian system. In Specific Aim 1, we will determine how WC-1 senses light. We will use biochemical and structural approaches to test the hypotheses that the LOV domain of WC-1 binds FAD and undergoes reversible photocycles, and that light induces conformational changes of WC-1. WC-1 is phosphorylated in the dark and becomes hyperphosphorylated after light exposure, and the phosphorylation events may regulate its stability and activity. Therefore, in Specific Aim 2, we will determine the role of WC-1 phosphorylation in the light input and circadian clock. We will use mass spectrometry to determine WC-1 phosphorylation sites, and use genetic and biochemical approaches to understand the functions of phosphorylation and to identify the kinase(s) responsible. In Specific Aim 3, we will identify novel factors that mediate light input in Neurospora. We will biochemically purify the large WC protein complex to identify novel factors, and we will screen for novel mutants with defects in light input and circadian clock. Together, these studies will help us to elucidate the light input mechanism of the Neurospora clock in genetic, biochemical, and molecular terms, and the information we obtain should provide fundamental information relevant to the light input pathway in other organisms