Mammalian circadian clock proteins form dynamic interacting microbodies distinct from phase separation

Liquid-liquid phase separation (LLPS) underlies diverse biological processes. Because most LLPS studies were performed in vitro using recombinant proteins or in cells that overexpress protein, the physiological relevance of LLPS for endogenous protein is often unclear. PERIOD, the intrinsically diso...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2023-12, Vol.120 (52), p.e2318274120
Main Authors: Xie, Pancheng, Xie, Xiaowen, Ye, Congrong, Dean, Kevin M, Laothamatas, Isara, Taufique, S K Tahajjul, Takahashi, Joseph, Yamazaki, Shin, Xu, Ying, Liu, Yi
Format: Article
Language:English
Subjects:
Animal tissues
Animals
ARNTL Transcription Factors - genetics
ARNTL Transcription Factors - metabolism
Biological activity
Biological clocks
Biological Sciences
BMAL1 protein
Circadian Clocks - genetics
Circadian rhythm
Circadian Rhythm - genetics
Circadian rhythms
CLOCK Proteins - genetics
CLOCK Proteins - metabolism
Condensates
Control theory
Cryptochromes
Feedback
Feedback loops
Liquid phases
Mammals
Mammals - metabolism
Mice
Microbodies
Microbodies - metabolism
Negative feedback
Period 2 protein
Period Circadian Proteins - genetics
Period Circadian Proteins - metabolism
Phase Separation
Phosphorylation
Proteins
Transgenes
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Summary:Liquid-liquid phase separation (LLPS) underlies diverse biological processes. Because most LLPS studies were performed in vitro using recombinant proteins or in cells that overexpress protein, the physiological relevance of LLPS for endogenous protein is often unclear. PERIOD, the intrinsically disordered domain-rich proteins, are central mammalian circadian clock components and interact with other clock proteins in the core circadian negative feedback loop. Different core clock proteins were previously shown to form large complexes. Circadian clock studies often rely on experiments that overexpress clock proteins. Here, we show that when Per2 transgene was stably expressed in cells, PER2 protein formed nuclear phosphorylation-dependent slow-moving LLPS condensates that recruited other clock proteins. Super-resolution microscopy of endogenous PER2, however, revealed formation of circadian-controlled, rapidly diffusing nuclear microbodies that were resistant to protein concentration changes, hexanediol treatment, and loss of phosphorylation, indicating that they are distinct from the LLPS condensates caused by protein overexpression. Surprisingly, only a small fraction of endogenous PER2 microbodies transiently interact with endogenous BMAL1 and CRY1, a conclusion that was confirmed in cells and in mice tissues, suggesting an enzyme-like mechanism in the circadian negative feedback process. Together, these results demonstrate that the dynamic interactions of core clock proteins are a key feature of mammalian circadian clock mechanism and the importance of examining endogenous proteins in LLPS and circadian clock studies.
ISSN:0027-8424
1091-6490
1091-6490
DOI:10.1073/pnas.2318274120