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In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells
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Mammalian circadian clock proteins form dynamic interacting microbodies distinct from phase separation
Pancheng Xie1,2, Xiaowen Xie1, Congrong Ye1
1Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
Biorxiv : the Preprint Server for Biology
|November 14, 2023
Summary
Physiological relevance of liquid-liquid phase separation (LLPS) in circadian clocks is unclear. Endogenous PERIOD proteins form distinct microbodies, not LLPS condensates, suggesting enzyme-like roles in the mammalian circadian feedback loop.
Area of Science:
- Cell Biology
- Chronobiology
- Biophysics
Background:
- Liquid-liquid phase separation (LLPS) is crucial for biological processes, but its physiological relevance in circadian rhythms remains uncertain.
- Core clock proteins, including PERIOD (PER) proteins, form complexes within the mammalian circadian negative feedback loop.
Approach:
- Investigated PER2 protein behavior using super-resolution microscopy in cells and mouse tissues.
- Differentiated between LLPS condensates formed by transgene overexpression and microbodies formed by endogenous PER2.
- Analyzed interactions between endogenous PER2, BMAL1, and CRY1 under various conditions.
Key Points:
- Overexpressed PER2 forms nuclear, phosphorylation-dependent LLPS condensates.
- Endogenous PER2 forms distinct, rapidly diffusing microbodies regulated by circadian cycles.
- These microbodies are resistant to changes in protein concentration, hexanediol, and phosphorylation status.
- Only a small fraction of endogenous PER2 microbodies transiently interact with BMAL1 and CRY1.
Conclusions:
- Endogenous PER2 microbodies differ significantly from LLPS condensates induced by overexpression.
- The dynamic, transient interactions of core clock proteins suggest an enzyme-like mechanism in circadian regulation.
- Studying endogenous proteins is critical for understanding LLPS and circadian clock mechanisms.
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