Localization and Interaction of Circadian Clock and TOR Pathway Components in Neurospora Crassa

Circadian (daily) rhythmicity is an attribute of almost all eukaryotic cells and some prokaryotes. Our lab utilizes the filamentous fungus Neurospora crassa as a model organism to study the molecular basis of rhythmicity. Models for the endogenous oscillators that regulate circadian rhythms in eukaryotes are primarily based on a small number of ‘‘clock genes’’ operating as transcription/translation feedback loops (TTFL). Still, rhythms can be observed, when TTFLs are nonoperating. Understanding the mechanism of rhythmicity operating outside of TTFLs is the key unresolved problem in circadian biology. Our lab has identified two genes in Neurospora crassa required for this TTFL-less rhythmicity, vta, and gtr2. Both are components of the TOR (Target of Rapamycin) nutrient-sensing pathway preserved in eukaryotes. Coimmunoprecipitation and mass spectrometry found TOR pathway components, including GTR2 (homologous to yeast Gtr2 and RAG C/D in mammals) as binding partners of VTA (homologous to yeast EGO1 and mammalian LAMTOR 1). In this thesis I report reciprocal co-IP with GTR2 confirming VTA as a binding partner. I also report that the expression of GTR2 protein is rhythmic, and VTA is required for GTR2 rhythmicity. FRQ protein, central to the clock TTFL, is rhythmic in the presence of GTR2 but dampened in the absence of GTR2. I also report my research regarding the subcellular localization of VTA and GTR2 in different nutritional states and the presence and absence of TOR pathway inhibitors. Kog1/Raptor is a regulator for TOR1 activity in yeast and mammals, and I report the localization of KOG1 in Neurospora crassa. My data indicate that GTR2-RFP localizes in filamentous structures identified as vacuolar membranes but is cytoplasmic in the absence of VTA. KOG1-GFP looks similar to GTR2-RFP. In the presence of arginine, KOG-1 is localized around the vacuole. Starvation using no glucose media looks similar to the vta ko condition in the GTR2 strain. In KOG-1GFP, there is the presence of KOG1 P bodies around the edges of the vacuole. The latter effect is similar to the yeast homolog kog-1 protein behavior in the absence of glucose. In the presence of TOR pathway inhibitors, Torin I and Torin II, there is an abnormal structural morphology in both cases. It is indicated that there is a destruction of the vacuolar structure and internal hyphal network in inhibitors that moves the fluorescence signal to the outer plasma membrane. These results support the connection between the mutual components of the clock and TOR pathway and the essential interaction to maintain the proper function of the signaling pathway and the circadian clock. As we have previously reported, the TTFL cannot explain the whole circadian clock mechanism. An independent oscillator may be linked to the TTFL to help maintain its function. This link is identified as the TOR pathway, a significant and nutrient-sensitive pathway conserved in eukaryotes. The circadian clock is also conserved in almost all organisms. Our results established a network between the control of metabolism through the TOR pathway and the circadian clock.