Functional characterization of the molecular timer of the circadian clock of Neurospora crassa.
Circadian rhythms are ~24 hr oscillations present in essentially every mammalian cell. Daily changes in the environment (e.g. light, food, temperature) have likely been the main drivers for the evolution of circadian clocks. Such clocks not only need to be robust towards external and internal fluctuations, but also adaptive to environmental cues (Zeitgebers, e.g. metabolic state, light, temperature, etc.). While the means by which the molecular clockwork (essentially a gene-regulatory network) can be synchronized to external Zeitgebers are overwhelmingly manifold, the molecular mechanisms are often little understood. Both, the generation of self- sustained oscillations ("limit cycles") as well as a rapid response to Zeitgebers, likely require nonlinear kinetics, such as switch-like behavior of transcriptional regulation. Here, we propose [as a first aim] to investigate experimentally and by mathematical modelling the effect of redox signals on an essential molecular switch within the circadian oscillator (i.e. the interaction of the key clock proteins PER2 and CRY1). Specifically, we will e.g. (i) perform fluorescence resonance energy transfer (FRET) experiments using our newly generated knock-in cell lines to characterize this interaction at the single cell level; (ii) use proximity labelling experiments to identify potential components of a redox relay; (iii) characterize the oxidation state of endogenous CRY1 using appro- priate mass spectrometry techniques; (iv) use mathematical modelling to combine our novel redox oscillator model with the canonical gene regulatory clock model. As a next conceptual step, we will study the role and mechanisms of various other important Zeitgebers beyond redox, thereby likely revealing key principles of “network switching”. Specifically, we propose [as a second aim] to experimentally and theoretically investigate network switches of single cell circadian oscillators in response to Zeitgeber stimuli. To this end, we will e.g. (i) explore how Zeitgebers switch the various nodes of the circadian network using systematic single cell fluorescence microscopy with our newly generated knock-in cells; (ii) extract critical oscillator properties (e.g. relaxation rates) from single cell kinetic data to mathematically reconstruct the circadian network; (iii) chemically control specific network nodes to study necessity and sufficiency of specific nodes for network switching. To- gether, we anticipate obtaining a quantitative description in space and time of the mechanisms how a circadian oscillator network rapidly responds to environmental signals – a defining principle of circadian clocks.