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Proteasome function is required for biological timing throughout the twenty-four hour cycle.

van Ooijen G, Dixon LE, Troein C, Millar AJ - Curr. Biol. (2011)

Bottom Line: TOC1 degradation peaks in response to darkness.Targeted protein degradation, unlike transcription and translation, is shown to be essential to sustain TTFL rhythmicity throughout the circadian cycle.Although proteasomal degradation is not necessary for sustained posttranslational oscillations in transcriptionally inactive cells, TTFL and posttranslational oscillators are normally coupled, and proteasome function is crucial to sustain both.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences and Centre for Systems Biology at Edinburgh, University of Edinburgh, The King's Buildings, Mayfield Road, Edinburgh EH9 3JD, UK. gerben.vanooijen@ed.ac.uk

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Proteasomal Inhibition Stops TTFL Rhythmicity Phase-Independently(A) Application of saturating concentrations of MG132 (40 μM, red traces) or vehicle (black traces) to CCA1-LUC cells in constant light.(B) Examples from wedge data of peak phases of individual wells (n ≥ 6) of CCA1-LUC cells subjected to various (0–24) hours of proteasomal inhibition (red wedge, right) or vehicle (gray wedge, left) starting at ZT0 and ending by wash off.(C) Summary of phase shifts (error bars represent SD, n ≥ 6) relative to vehicle-treated controls for all treatment durations (x axis) and starting times. Two black lines represent the expected result, assuming either total resetting by wash off (hypothesis 1) or no effect (hypothesis 2). See also Figure S3.
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fig3: Proteasomal Inhibition Stops TTFL Rhythmicity Phase-Independently(A) Application of saturating concentrations of MG132 (40 μM, red traces) or vehicle (black traces) to CCA1-LUC cells in constant light.(B) Examples from wedge data of peak phases of individual wells (n ≥ 6) of CCA1-LUC cells subjected to various (0–24) hours of proteasomal inhibition (red wedge, right) or vehicle (gray wedge, left) starting at ZT0 and ending by wash off.(C) Summary of phase shifts (error bars represent SD, n ≥ 6) relative to vehicle-treated controls for all treatment durations (x axis) and starting times. Two black lines represent the expected result, assuming either total resetting by wash off (hypothesis 1) or no effect (hypothesis 2). See also Figure S3.

Mentions: Saturating concentrations of MG132 arrested rhythmic behavior of the CCA1-LUC line (Figure 3A). CCA1-LUC cells entrained in LD12:12 cycles were transferred to constant light at dawn (ZT0), and application of MG132 stopped normal oscillatory behavior. After wash off, the cells directly resumed oscillations (Figure S3A), suggesting that treatment was reversible and largely nontoxic. The delay in phase resulting from treatment pulses followed a direct relation with the duration of the treatment (Figure S3B), suggesting that the circadian pacemaker had paused. The period of the reinitiated rhythm after wash off was not substantially affected (Figure S3C), showing that wash off was efficient and that drug concentration was reduced to insignificant levels. The ability of treated cells to re-entrain was demonstrated by reinstating LD12:12 cycles after 48 hr of constant light (Figure S3A). Altogether, this indicates that when drugs are applied at ZT0, the Ostreococus clock is paused by MG132, and cells reset to wash off.


Proteasome function is required for biological timing throughout the twenty-four hour cycle.

van Ooijen G, Dixon LE, Troein C, Millar AJ - Curr. Biol. (2011)

Proteasomal Inhibition Stops TTFL Rhythmicity Phase-Independently(A) Application of saturating concentrations of MG132 (40 μM, red traces) or vehicle (black traces) to CCA1-LUC cells in constant light.(B) Examples from wedge data of peak phases of individual wells (n ≥ 6) of CCA1-LUC cells subjected to various (0–24) hours of proteasomal inhibition (red wedge, right) or vehicle (gray wedge, left) starting at ZT0 and ending by wash off.(C) Summary of phase shifts (error bars represent SD, n ≥ 6) relative to vehicle-treated controls for all treatment durations (x axis) and starting times. Two black lines represent the expected result, assuming either total resetting by wash off (hypothesis 1) or no effect (hypothesis 2). See also Figure S3.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3102177&req=5

fig3: Proteasomal Inhibition Stops TTFL Rhythmicity Phase-Independently(A) Application of saturating concentrations of MG132 (40 μM, red traces) or vehicle (black traces) to CCA1-LUC cells in constant light.(B) Examples from wedge data of peak phases of individual wells (n ≥ 6) of CCA1-LUC cells subjected to various (0–24) hours of proteasomal inhibition (red wedge, right) or vehicle (gray wedge, left) starting at ZT0 and ending by wash off.(C) Summary of phase shifts (error bars represent SD, n ≥ 6) relative to vehicle-treated controls for all treatment durations (x axis) and starting times. Two black lines represent the expected result, assuming either total resetting by wash off (hypothesis 1) or no effect (hypothesis 2). See also Figure S3.
Mentions: Saturating concentrations of MG132 arrested rhythmic behavior of the CCA1-LUC line (Figure 3A). CCA1-LUC cells entrained in LD12:12 cycles were transferred to constant light at dawn (ZT0), and application of MG132 stopped normal oscillatory behavior. After wash off, the cells directly resumed oscillations (Figure S3A), suggesting that treatment was reversible and largely nontoxic. The delay in phase resulting from treatment pulses followed a direct relation with the duration of the treatment (Figure S3B), suggesting that the circadian pacemaker had paused. The period of the reinitiated rhythm after wash off was not substantially affected (Figure S3C), showing that wash off was efficient and that drug concentration was reduced to insignificant levels. The ability of treated cells to re-entrain was demonstrated by reinstating LD12:12 cycles after 48 hr of constant light (Figure S3A). Altogether, this indicates that when drugs are applied at ZT0, the Ostreococus clock is paused by MG132, and cells reset to wash off.

Bottom Line: TOC1 degradation peaks in response to darkness.Targeted protein degradation, unlike transcription and translation, is shown to be essential to sustain TTFL rhythmicity throughout the circadian cycle.Although proteasomal degradation is not necessary for sustained posttranslational oscillations in transcriptionally inactive cells, TTFL and posttranslational oscillators are normally coupled, and proteasome function is crucial to sustain both.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences and Centre for Systems Biology at Edinburgh, University of Edinburgh, The King's Buildings, Mayfield Road, Edinburgh EH9 3JD, UK. gerben.vanooijen@ed.ac.uk

Show MeSH
Related in: MedlinePlus