title: Visualizing and Quantifying Intracellular Behavior and Abundance of the Core Circadian Clock Protein PERIOD2.
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.
authors.affiliation: Neurobiology Division, Medical Research Council (MRC) Laboratory of Molecular Biology (LMB), Francis Crick Avenue, Cambridge CB2 0QH, UK.
authors.forename: Nicola J
authors.initials: NJ
authors.lastname: Smyllie
authors.affiliation: Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, UK.
authors.forename: Violetta
authors.initials: V
authors.lastname: Pilorz
authors.affiliation: Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, UK.
authors.forename: James
authors.initials: J
authors.lastname: Boyd
authors.affiliation: Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, UK.
authors.forename: Qing-Jun
authors.initials: QJ
authors.lastname: Meng
authors.affiliation: Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, UK.
authors.forename: Ben
authors.initials: B
authors.lastname: Saer
authors.affiliation: Neurobiology Division, Medical Research Council (MRC) Laboratory of Molecular Biology (LMB), Francis Crick Avenue, Cambridge CB2 0QH, UK.
authors.forename: Johanna E
authors.initials: JE
authors.lastname: Chesham
authors.affiliation: Neurobiology Division, Medical Research Council (MRC) Laboratory of Molecular Biology (LMB), Francis Crick Avenue, Cambridge CB2 0QH, UK.
authors.forename: Elizabeth S
authors.initials: ES
authors.lastname: Maywood
authors.affiliation: Neurobiology Division, Medical Research Council (MRC) Laboratory of Molecular Biology (LMB), Francis Crick Avenue, Cambridge CB2 0QH, UK.
authors.forename: Toke P
authors.initials: TP
authors.lastname: Krogager
authors.affiliation: Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, UK.
authors.forename: David G
authors.initials: DG
authors.lastname: Spiller
authors.affiliation: Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, UK.
authors.forename: Raymond
authors.initials: R
authors.lastname: Boot-Handford
authors.affiliation: Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, UK.
authors.forename: Michael R H
authors.initials: MR
authors.lastname: White
authors.affiliation: 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.
authors.forename: Michael H
authors.initials: MH
authors.lastname: Hastings
authors.affiliation: Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, UK. Electronic address: andrew.loudon@manchester.ac.uk.
authors.forename: Andrew S I
authors.initials: AS
authors.lastname: Loudon
mesh_terms: D000818:Animals; D057906:Circadian Clocks / Q000235:genetics*; D051379:Mice / Q000235:genetics*; D056950:Period Circadian Proteins / Q000235:genetics* / Q000378:metabolism; D013493:Suprachiasmatic Nucleus / Q000378:metabolism*
chemical_list: C513312:Per2 protein, mouse; D056950:Period Circadian Proteins