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 or in cells that overexpress protein, the physiological relevance of LLPS is unclear. PERIOD 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. Here we show that when transgene was stably expressed, PER2 formed nuclear phosphorylation-dependent LLPS condensates that recruited other clock proteins. Super-resolution microscopy of endogenous PER2, however, revealed formation of circadian-controlled, rapidly diffusing 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 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 is a key feature of mammalian circadian clock mechanism and the importance of examining endogenous proteins in LLPS and circadian studies.