O-GlcNAcylation of CEP44 Promotes Its Droplet Formation and Regulates Its Localization.

Area of Science:

• Cell Biology
• Biochemistry
• Molecular Biology

Background:

• The centrosome is crucial for cell division and organization.
• Centrosomal protein 44 (CEP44) plays key roles in centriole duplication and spindle integrity.
• Liquid-liquid phase separation (LLPS) is a mechanism for forming cellular condensates, but its role in CEP44 function was unknown.

Purpose of the Study:

• To investigate whether CEP44 undergoes liquid-liquid phase separation (LLPS).
• To determine the role of O-GlcNAcylation in regulating CEP44's phase separation properties.
• To explore how post-translational modifications affect CEP44's structural dynamics and localization.

Main Methods:

• Identification of intrinsically disordered regions (IDRs) in CEP44.
• In vitro and in vivo droplet formation assays to assess LLPS.
• Immunoblotting to detect CEP44 O-GlcNAcylation and interaction with O-GlcNAc transferase (OGT).
• Immunostaining to visualize CEP44 droplets and assess the effect of O-GlcNAcylation on droplet fusion.

Main Results:

• CEP44 possesses intrinsically disordered regions and forms liquid droplets in vitro and in vivo, confirming LLPS capability.
• CEP44 undergoes O-GlcNAcylation, indicating interaction with OGT.
• O-GlcNAcylation significantly promotes CEP44 droplet fusion.
• Computational analysis suggests potential interplay between O-GlcNAcylation and phosphorylation in modulating CEP44 structure.

Conclusions:

• CEP44 exhibits liquid-liquid phase separation (LLPS) properties.
• O-GlcNAcylation is a critical regulator of CEP44 droplet fusion and potentially influences its subcellular localization.
• These findings provide new insights into the molecular mechanisms governing centrosome assembly and function.