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

• Published in: Current biology Vol. 26; no. 14; pp. 1880 - 1886
• Main Authors: Smyllie, Nicola J., Pilorz, Violetta, Boyd, James, Meng, Qing-Jun, Saer, Ben, Chesham, Johanna E., Maywood, Elizabeth S., Krogager, Toke P., Spiller, David G., Boot-Handford, Raymond, White, Michael R.H., Hastings, Michael H., Loudon, Andrew S.I.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 25.07.2016; Cell Press
• Subjects: Animals; Circadian Clocks - genetics; Mice - genetics; Period Circadian Proteins - genetics; Period Circadian Proteins - metabolism; Suprachiasmatic Nucleus - metabolism
• ISSN: 0960-9822; 1879-0445
• DOI: 10.1016/j.cub.2016.05.018

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. [Display omitted] •Reporter mouse is used for real-time fluorescent imaging of mammalian clock protein PER2•In contrast to Drosophila, localization of PER2 is not subject to circadian gating•Circadian abundance, mobility, and intracellular dynamics of native PER2 are quantified•Casein kinase1 controls nucleocytoplasmic mobility of PER2 alongside circadian period Smyllie et al. use a fluorescent reporter mouse to image the circadian dynamics of PER2, a key component of the circadian clock in the suprachiasmatic nucleus and fibroblasts. They reveal marked divergence of the mechanisms of mouse and fly clock cells, and they provide quantitative data to support reappraisal of current models of the mammalian clock.