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Curr Biol. 2016 Jul 25;26(14):1880-6. doi: 10.1016/j.cub.2016.05.018. Epub 2016 Jun 30.

Visualizing and Quantifying Intracellular Behavior and Abundance of the Core Circadian Clock Protein PERIOD2.

Author information

1
Neurobiology Division, Medical Research Council (MRC) Laboratory of Molecular Biology (LMB), Francis Crick Avenue, Cambridge CB2 0QH, UK.
2
Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, UK.
3
Neurobiology Division, Medical Research Council (MRC) Laboratory of Molecular Biology (LMB), Francis Crick Avenue, Cambridge CB2 0QH, UK. Electronic address: mha@mrc-lmb.cam.ac.uk.
4
Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, UK. Electronic address: andrew.loudon@manchester.ac.uk.

Abstract

Transcriptional-translational feedback loops (TTFLs) are a conserved molecular motif of circadian clocks. The principal clock in mammals is the suprachiasmatic nucleus (SCN) of the hypothalamus. In SCN neurons, auto-regulatory feedback on core clock genes Period (Per) and Cryptochrome (Cry) following nuclear entry of their protein products is the basis of circadian oscillation [1, 2]. In Drosophila clock neurons, the movement of dPer into the nucleus is subject to a circadian gate that generates a delay in the TTFL, and this delay is thought to be critical for oscillation [3, 4]. Analysis of the Drosophila clock has strongly influenced models of the mammalian clock, and such models typically infer complex spatiotemporal, intracellular behaviors of mammalian clock proteins. There are, however, no direct measures of the intracellular behavior of endogenous circadian proteins to support this: dynamic analyses have been limited and often have no circadian dimension [5-7]. We therefore generated a knockin mouse expressing a fluorescent fusion of native PER2 protein (PER2::VENUS) for live imaging. PER2::VENUS recapitulates the circadian functions of wild-type PER2 and, importantly, the behavior of PER2::VENUS runs counter to the Drosophila model: it does not exhibit circadian gating of nuclear entry. Using fluorescent imaging of PER2::VENUS, we acquired the first measures of mobility, molecular concentration, and localization of an endogenous circadian protein in individual mammalian cells, and we showed how the mobility and nuclear translocation of PER2 are regulated by casein kinase. These results provide new qualitative and quantitative insights into the cellular mechanism of the mammalian circadian clock.

PMID:
27374340
PMCID:
PMC4963210
DOI:
10.1016/j.cub.2016.05.018
[Indexed for MEDLINE]
Free PMC Article

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