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
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