Characterization of Liquid-Liquid Phase Separation of Companion of Cellulose Synthases under Stress Mimicking Conditions

Companion of Cellulose Synthase (CC), has previously been shown to sustain cellulose synthesis during salt stress. However, the role of CCs in this process is not well-understood. In this study, we show that the intrinsically disordered N-terminal domain of Arabidopsis CC1 (CC1NTD) can undergo liquid-liquid phase separation (LLPS) under defined reducing conditions in the presence of trimethylamine N-oxide (TMAO), a naturally occurring plant stress osmolyte. The observed biomolecular condensates are enriched in CC1NTD and display liquid-like behavior, including spherical morphology, fusion, and dynamic exchange with the surrounding solution. We further show that TMAO compacts CC1NTD without inducing folding, that CC1NTD condensates recruit tubulin and accelerate microtubule polymerization while retaining dynamic properties. Additionally, we also studied the intrinsically disordered N-terminal domain of CC2, a closely related paralog to CC1. We found that CC2NTD remains disordered but does not phase separate independently under the same experimental conditions. However, it does partition into CC1NTD condensates. Together, these results define a condition-dependent condensate state for CC1NTD and link that state to tubulin-related function. Finally, we propose a novel mechanism for the formation and maintenance of stress-associated cellulose synthase compartments driven by LLPS of the disordered regions of CSC and accessory proteins.