Towards a mechanistic description of PopZ self-assembly from monomers to micron-sized condensates.

Abstract

We use a combination of in vitro techniques to characterize the formation of phase separated PopZ condensates. The pole-organizing protein Z (PopZ) phase separates into microdomains at the poles of the bacterium Caulobacter crescentus and modulates asymmetric division by selectively sequestering regulatory reaction pathways. PopZ is conserved across α-proteobacteria and plays a key role in cytosol organization. Using mass-photometry, we found that PopZ self-assembles into a distinct distribution of highly stable oligomers in a concentration-dependent manner. Strikingly, PopZ forms trimers and hexamers but seems to only grow beyond hexamers by the addition of more hexamers. Mutations in its oligomerization domain affect the oligomeric distribution as well as the fluidity and saturation concentration of the resulting condensate. PopZ phase separation requires divalent cations, even above its saturation concentration. This allows us to study PopZ at identical concentrations before and after condensation. To characterize the identity of the relevant oligomers and the interparticle interactions that drive phase separation, we study PopZ across three length scales. On the 0.1-1 nanomolar scale, we employ single-molecule FRET to monitor conformational changes within the structured OD and between the OD and the adjacent IDR upon oligomerization and condensation. On the 1-100 nanomolar scale, we use small angle x-ray scattering (SAXS) to monitor the size and shape of PopZ oligomers and microfilaments. Finally, on the micron scale, we study condensates' size, internal and exchange dynamics, and material properties using differential interference contrast and fluorescence microscopies. Combined, our work provides a mechanistic description of PopZ assembly from monomers to micron-sized condensates.