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

SummaryTranscriptional-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

Search space comparison of genes and proteins for the comparison of EvsO differentially expressed genes/proteins in SILAC and GEMA.

<p>Panel <b>A</b>. Comparison of search spaces containing all genes/proteins quantified in SILAC and GEMA analyses. Panel <b>B</b>. Numbers of differentially expressed genes/proteins in the EvsO comparison according to method (SILAC fold change >1.3, p-value <0.01; GEMA fold change >1.5, BH-p-value <0.01). Red numbers indicate up-regulations while green indicates down-regulations. The brown number indicates genes/proteins found down-regulated in SILAC while up-regulated in GEMA. In both GEMA and SILAC, 7% of the genes/proteins in the respective search spaces were found to be regulated in the EvsO comparison.</p

Circadian actin dynamics drive rhythmic fibroblast mobilization during wound healing

The circadian clock in fibroblasts determines the efficiency of wound healing through rhythmic regulation of actin cytoskeletal dynamics.</jats:p

Labeling and identifying cell-specific proteomes in the mouse brain.

We develop an approach to tag proteomes synthesized by specific cell types in dissociated cortex, brain slices, and the brains of live mice. By viral-mediated expression of an orthogonal pyrrolysyl-tRNA synthetase-tRNAXXX pair in a cell type of interest and providing a non-canonical amino acid with a chemical handle, we selectively label neuronal or glial proteomes. The method enables the identification of proteins from spatially and genetically defined regions of the brain.Michael J Fox Foundation (MJFF) (unknown), Parkinson's UK (None), Cure Parkinson's Trust (unknown

HepG2-SF cell proliferation during FA incubation.

<p>Proliferation of HepG2-SF cells in supplemented medium (100 µM FFA) was followed during six days of incubation and measured on day 0, 2, 4 and 6 by CyQuant cell proliferation assay, where fluorescence (y-axis) is a measure of cell numbers. HepG2-SF cell proliferation is differentially affected by EA, OA and SA. EA supplemented cells appear to be compromised on their growth rate when compared to <i>Control</i>, OA and SA supplemented cells. * marks measurements significantly differing from controls using the unpaired t-test, two-tailed with a 95% confidence interval.</p

IPA categories and functions specifically perturbed by EA as compared to OA, comparing GEMA and SILAC data.

<p><b>Panel </b><b>A</b> displays perturbed IPA categories. The y-axis reports the significances of the perturbation and is plotted as minus log of the BH multiple testing corrected p-value calculated by IPA based on the specific data set. <b>Panel </b><b>B</b> displays the 24 significantly perturbed functions found in the top three IPA categories for both SILAC and GEMA. For both panels, the dotted line is the threshold BH-p-value 0.01, with orange bars representing the GEMA data and the blue bars representing the SILAC data.</p

The FA composition of HepG2-SF phospholipids analyzed by gas chromatography.

<p>The composition of phospholipids from HepG2-SF cells incubated 6 days in 100 µM oleic, elaidic, or stearic acid supplemented medium were determined. Panel A and Panel B are the high and low abundant FAs present in the PLs, respectively. Each bar represents the average of three biological replicas. The y-axis is the percent of total FA methyl esters measured. The x-axis indicates the different PL FAs measured as their methyl esters. Color coding corresponds to the different supplemented FFAs. Panel C shows a heatmap representation of the distribution of FAs in PLs of the HepG2-SF cells after supplementation. The FA composition of HepG2-SF phospholipids change depending on the different supplemented fatty acids, <i>Stearic</i> and <i>Oleic</i> profiles are alike whereas the <i>Elaidic</i> FA PL profile differs from all the other groups. * marks measurements significantly differing from controls using the unpaired t-test, two-tailed with a 95% confidence interval.</p

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

SummaryTranscriptional-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