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

Abstract

The centrosomal protein of 44 kDa (CEP44) is essential for centriole duplication, centrosome cohesion, and spindle integrity. It localizes to the proximal end of centrioles and associates with spindle microtubules. Liquid-liquid phase separation (LLPS) is a process by which biomolecules undergo demixing into distinct liquid-like phases, facilitating the formation of cellular condensates such as the centrosome. However, whether CEP44 possesses LLPS properties remains unclear. In this study, we identified intrinsically disordered regions (IDRs) within CEP44, and droplet formation assays confirmed its capacity to form liquid droplets in vivo and in vitro. Immunoblotting detected O-GlcNAcylation of CEP44, indicating its interaction with O-GlcNAc transferase (OGT). Subsequent immunostaining demonstrated that O-GlcNAcylation promotes CEP44 droplet fusion. Post-translational modification prediction analysis suggested a potential interplay between O-GlcNAcylation and phosphorylation that may modulate the structural dynamics of CEP44. Overall, our findings reveal the LLPS capability of CEP44 and underscore the critical role of O-GlcNAcylation in regulating CEP44 droplet fusion and potentially influencing its subcellular localization.