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

Jul 5, 2016Current biology : CB

Measuring the Levels and Activity of the Key Body Clock Protein PERIOD2 Inside Cells

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Abstract

A knockin mouse expressing a fluorescent fusion of native PER2 protein (PER2::VENUS) provides new insights into the behavior of circadian proteins in mammals.

PER2::VENUS accurately reflects the circadian functions of wild-type PER2.
The behavior of PER2::VENUS contradicts the Drosophila model, as it does not show circadian gating of nuclear entry.
Fluorescent imaging revealed the first measures of mobility, molecular concentration, and localization of an endogenous circadian protein in mammalian cells.
Mobility and nuclear translocation of PER2 are regulated by casein kinase.
These findings offer new qualitative and quantitative insights into the cellular mechanisms underlying the mammalian circadian clock.

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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.

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

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