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Melanopsin-dependent photo-perturbation reveals desynchronization underlying the singularity of mammalian circadian clocks


Singularity behaviour in circadian clocks1,2 — the loss of robust circadian rhythms following exposure to a stimulus such as a pulse of bright light — is one of the fundamental but mysterious properties of clocks. To quantitatively perturb and accurately measure the dynamics of cellular clocks3,4, we synthetically produced photo-responsiveness within mammalian cells by exogenously introducing the photoreceptor melanopsin5,6,7,8 and continuously monitoring the effect of photo-perturbation on the state of cellular clocks. Here we report that a critical light pulse drives cellular clocks into singularity behaviour. Our theoretical analysis consistently predicts and subsequent single-cell level observation directly proves that desynchronization of individual cellular clocks underlies singularity behaviour. Our theoretical framework also explains why singularity behaviours have been experimentally observed in various organisms, and it suggests that desynchronization is a plausible mechanism for the observable singularity of circadian clocks. Importantly, these in vitro and in silico findings are further supported by in vivo observations that desynchronization underlies the multicell-level amplitude decrease in the rat suprachiasmatic nucleus induced by critical light pulses.

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Figure 1: Synthetic implementation of photo-responsiveness within mammalian clock cells.
Figure 2: Phase shift of mammalian clocks induced by photo-perturbation.
Figure 3: Amplitude changes of mammalian clocks induced by photo-perturbation.
Figure 4: Desynchronization underlying the singularity behaviour of mammalian clocks.
Figure 5: Desynchronization underlying the multicell-level amplitude decrease in the rat suprachiasmatic nucleus (SCN) induced by the critical light pulses.


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This research was supported by intramural Grant-in-Aid from the Center for Developmental Biology (CDB), Director's Fund from CDB (H.R.U.), KAKENHI (Grant-in-Aid for Scientific Research) on Priority Areas 'Systems Genomics' from the Ministry of Education, Culture, Sports, Science and Technology of Japan (H.R.U. and H.U.), Grant-in-Aid for Scientific Research on 'Development of Basic Technology to Control Biological Systems Using Chemical Compounds' from the New Energy and Industrial Technology Development Organization (NEDO) of Japan (H.R.U.) and Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists 17-4169 (T.J.K). We thank for Wataru Kishimoto for technical supports on high-throughput monitoring system, Rikuhiro G. Yamada for support for data analysis, Maki Ukai-Tadenuma for technical support, Yoichi Minami for discussion for in vivo experiments, and Douglas Sipp Michael Royle, Danny Forger for critical comments on the manuscript. We also thank John J. Tyson for his suggestion regarding Winfree's original idea of desynchronization.

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K.Y. and H.R.U. developed the concept of synthetic implementation of photo-responsiveness within NIH3T3 cells by using melanopsin. T.K. designed a high-throughput monitoring device. H.U. constructed all materials used in this work and performed high-throughput real-time luciferase reporter assays, single-cell real-time bioluminescence imaging and pharmacological assays. T.J.K. developed the theory, data-analysis methods, and the image-analysis software used in this work, analysed high-throughput, single-cell real-time bioluminescence, in situ hybridization, and locomotor activity data, and performed single-cell real-time bioluminescence imaging. M.N. and M.S. performed behaviour analysis of rats. M.N., K.M. and Y.S. performed in situ hybridization and analysed the data. H.U., T.J.K. and H.R.U wrote the manuscript. All authors discussed the results and commented on the manuscript text.

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Correspondence to Hiroki R. Ueda.

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Ukai, H., Kobayashi, T., Nagano, M. et al. Melanopsin-dependent photo-perturbation reveals desynchronization underlying the singularity of mammalian circadian clocks. Nat Cell Biol 9, 1327–1334 (2007).

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