Light Input Pathway of the Neurospora Circadian Clock

描述(申请人提供)：生物钟是一种内源性细胞过程，它控制着几乎所有生物体中各种各样的细胞、生理和行为活动。这些生物钟的精确度取决于它们能否被环境同步(携带)。从原核生物到哺乳动物，光是影响所有生物钟的最重要的环境因素，并被认为调节着许多重要的过程。粗糙脉孢菌是最被人们理解的生物钟系统之一，它为在分子水平上理解光携带机制提供了一个很好的模型。在脉孢子虫中，转录因子白领-1(Wc-1)和Wc-2是生物钟功能所必需的昼夜反馈环中的正元件。最近，我们还发现Wc-1，一种含有FAD的蛋白，是生物钟和其他光反应的光感受器。这一发现确立了WC-1是第一个已知的真菌蓝光感受器。本项目中的实验将研究脉孢子虫昼夜节律系统的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。我们将使用生化和结构方法来验证WC-1的LOV结构域与FAD结合并经历可逆光循环，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，光照后过度磷酸化，磷酸化事件可能调节其稳定性和活性。因此，在特定的目标2中，我们将确定WC-1磷酸化在光输入和生物钟中的作用。我们将使用质谱仪来确定WC-1的磷酸化位点，并使用遗传学和生化的方法来了解磷酸化的功能，并确定负责的激酶(S)。在特定的目标3中，我们将确定在脉孢子虫中调节光输入的新因素。我们将对大的WC蛋白复合体进行生化纯化，以确定新的因子，并筛选具有光输入和生物钟缺陷的新突变体。总之，这些研究将帮助我们从遗传、生化和分子方面阐明脉孢子虫生物钟的光输入机制，我们获得的信息应该提供与其他生物的光输入途径相关的基本信息。

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