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

Area of Science:

• Molecular Biology
• Chronobiology
• Biophysics

Background:

• Liquid-liquid phase separation (LLPS) is crucial for biological processes, but its role with endogenous proteins is often unclear.
• Core circadian clock proteins, like PERIOD (PER), are intrinsically disordered and form complexes, yet studies often use overexpression systems.
• Understanding LLPS of endogenous circadian proteins is vital for deciphering the molecular mechanisms of the circadian clock.

Purpose of the Study:

• To investigate the physiological relevance of LLPS for endogenous circadian clock proteins.
• To differentiate between LLPS condensates formed by overexpressed proteins and structures formed by endogenous proteins.
• To elucidate the dynamic interactions of core clock proteins in the mammalian circadian negative feedback loop.

Main Methods:

• Stable expression of Per2 transgene in cells to observe LLPS condensates.
• Super-resolution microscopy to visualize endogenous PER2 within nuclear microbodies.
• Analysis of microbody characteristics (diffusion, resistance to treatments) and interactions with other clock proteins (BMAL1, CRY1) in cells and mouse tissues.

Main Results:

• Overexpressed PER2 formed slow-moving, phosphorylation-dependent LLPS condensates.
• Endogenous PER2 formed rapidly diffusing nuclear microbodies, distinct from LLPS condensates and resistant to various treatments.
• Only a small fraction of endogenous PER2 microbodies transiently interacted with BMAL1 and CRY1, suggesting an enzyme-like mechanism.

Conclusions:

• Mammalian circadian clock mechanisms rely on the dynamic interactions of core clock proteins, not solely on large LLPS condensates.
• Overexpression studies may not accurately reflect the behavior of endogenous circadian proteins.
• Investigating endogenous proteins is crucial for understanding LLPS and circadian clock function.