Fig. 1 Distinct signaling hubs localized at the opposite cell poles in C. crescentus. (Image by SIAT) 

As a well-established model for studying bacterial ACD, Caulobacter crescentus produces a motile swarmer cell and a sessile stalked cell during each cell cycle. In the pre-division cell stage, the polarization of new-pole signaling proteins by the scaffold PodJ coordinates to modulate the phosphorylation levels of a set of downstream signaling proteins, thus determining the cell fate of C. crescentus. 

The researchers found that phase separation played an essential role in the C. crescentus PodJ-signaling hub assembly. PodJ formed biomolecular condensates both in vitro and in vivo. Either the coiled-coil 4–6 region or the intrinsically disordered region in PodJ could mediate PodJ phase separation. In addition, both of the phase separation-related domains were found to be involved in recruiting client signaling proteins, indicating that phase separation of PodJ functionally contributes to forming the PodJ–client complexes. 

Moreover, a negative regulation of PodJ phase separation by the old-cell-pole scaffold protein SpmX was observed. SpmX inhibited PodJ condensate formation and promoted PodJ condensate aging in vitro. In living cells, SpmX was found to impede the cell-pole accumulation of PodJ and client recruitment, suggesting it may be involved in the new-to-old cell-pole remodeling. 

"The results revealed that phase separation modulates the assembly and dynamics of scaffold-signaling hubs in C. crescentus," said Prof. ZHAO Wei, corresponding author of the study. "It may serve as a general biophysical approach for assembling scaffold-signaling complexes and regulating ACD. Similar methods could be employed for rational engineering of artificial organelles and other membraneless biocatalytic compartments."  

Fig. 2 Schematic illustration of C. crescentus cells with membraneless biomolecular condensates. (Image by SIAT)