Light Input Pathway of the Neurospora Circadian Clock

脉孢菌昼夜节律钟的光输入途径

• 批准号: 6668052
• 负责人: YI LIU
• 金额: $ 28.86万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6668052
• 关键词: Neurospora; SDS polyacrylamide gel electrophoresis; Sf9 cell line; biological clocks; biological signal transduction; circadian rhythms; circular dichroism; conformation; immunoprecipitation; mass spectrometry; molecular cloning; nonvisual photosensitivity; nuclear magnetic resonance spectroscopy; phosphorylation; photobiology; photochemistry; protein kinase; protein purification; protein structure function; recombinant proteins; transcription factor; yeast two hybrid system

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

描述（由申请人提供）：昼夜节律是内源性细胞过程，可控制几乎所有生物的各种细胞，生理和行为活动。这些生物钟的准确性取决于它们是否可以被环境同步（夹带）。光是将所有昼夜节律从原核生物到哺乳动物的最重要的环境因素，众所周知可以调节许多重要的过程。 Neurospora Crassa是最能理解的昼夜节律系统之一，它为了解分子水平的光夹带机制提供了极好的模型。在Neurospora中，转录因子白领-1（WC-1）和WC-2是昼夜节律反馈回路中对时钟函数必不可少的阳性元素。最近，我们还确定了含有FAD的蛋白质WC-1，是昼夜节律和其他光反应的光感受器。这一发现确立了WC-1为第一个已知的真菌蓝光光感受器。该项目中的实验将解决神经孢子般的昼夜节律系统光输入途径的分子机制。 在特定的目标1中，我们将确定WC-1的感觉如何。我们将使用生化和结构方法来测试WC-1的LOV结构域结合FAD并经历可逆的光循环的假设，并且光引起了WC-1的构象变化。 WC-1在黑暗中被磷酸化，并在光照暴露后变为磷酸化，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磷酸化在光输入和昼夜节律时钟中的作用。我们将使用质谱法来确定WC-1磷酸化位点，并使用遗传和生化方法来了解磷酸化的功能并确定负责的激酶。在特定目标3中，我们将确定介导神经孢子中光输入的新因素。我们将在生化上纯化大型WC蛋白复合物以识别新因素，并将筛选出光输入和昼夜节律时钟中缺陷的新型突变体。这些研究将共同​​帮助我们在遗传，生化和分子方面阐明神经孢子时钟的光输入机制，我们获得的信息应提供与其他生物体中光输入途径相关的基本信息