Microenvironments created by liquid-liquid phase transition control the dynamic distribution of bacterial division FtsZ protein

Abstract

The influence of membrane-free microcompartments resulting from crowding-induced liquid/liquid phase separation (LLPS) on the dynamic spatial organization of FtsZ, the main component of the bacterial division machinery, has been studied using several LLPS systems. The GTP-dependent assembly cycle of FtsZ is thought to be crucial for the formation of the septal ring, which is highly regulated in time and space. We found that FtsZ accumulates in one of the phases and/or at the interface, depending on the system composition and on the oligomerization state of the protein. These results were observed both in bulk LLPS and in lipid-stabilized, phase-separated aqueous microdroplets. The visualization of the droplets revealed that both the location and structural arrangement of FtsZ filaments is determined by the nature of the LLPS. Relocation upon depolymerization of the dynamic filaments suggests the protein may shift among microenvironments in response to changes in its association state. The existence of these dynamic compartments driven by phase transitions can alter the local composition and reactivity of FtsZ during its life cycle acting as a nonspecific modulating factor of cell function.

Bacterial FtsZ protein forms phase‐separated condensates with its nucleoid‐associated inhibitor SlmA

“…This is likely because of the accumulation of the two proteins and the SBS in this phase (Appendix Fig S11A–D). As in the single crowder solutions, none or very few condensates were observed in this LLPS system for SlmA with or without the SBS (Appendix Fig S11B and C), and for FtsZ either alone or in the presence of only SlmA (Appendix Fig S12) or only SBS (Appendix Fig S11E). Control experiments showed that these findings were highly reproducible, irrespectively of the fluorescent dyes used and of which element of the condensate was labeled (Appendix Figs S10 and S12).…”

“…Appendix Figs S13C, S3 and S12), likely because of the unspecific charge effects of the crowder DNA and/or unspecific binding of SlmA to these highly concentrated sequences. The simultaneous presence of SlmA, FtsZ, and DNA was determinant to achieve significant condensation in this LLPS system as no condensates were formed by FtsZ on its own , and a limited number were found for SlmA with or without SBS (Appendix Fig S13B). Like in the crowded systems previously described (see above), FtsZ·SlmA·SBS condensates obtained at high concentrations of the unspecific DNA were dynamic, as they captured FtsZ freshly added to the sample (Fig EV3B).…”

“…To determine the effect that microenvironments as those found in the cell might have on FtsZÁSlmAÁSBS condensation, similar experiments to the ones described above were carried out in PEG/dextran LLPS systems, in which unassembled FtsZ distributes asymmetrically, partitioning preferentially into the dextran-rich phase [36]. Confocal microscopy images of an emulsion of PEG/dextran containing FtsZ and SlmAÁSBS showed abundant condensates of similar size as the ones previously found in homogeneous crowders (see above), in which the two division proteins and SBS colocalized ( Fig EV2A, Appendix Fig S10A-C).…”

“…Thus, in the presence of high concentrations of homogeneous or heterogeneous crowders, whether inert polymers, DNA, or proteins, the tendency of FtsZ to assemble into higher-order fibers was found to be significantly enhanced [17,34,35]. Besides, the reconstitution of FtsZ in crowded liquid-liquid phase separation (LLPS) systems mimicking microenvironments and compartmentalization, in bulk or encapsulated in microdroplets, demonstrated uneven distribution of FtsZ among preexisting phases and/or interfaces, which could be reversibly modulated by the association state of FtsZ [36,37]. These studies suggest that phase separation that may occur in the cytoplasm could affect the organization and reactivity of FtsZ, but phase separation of FtsZ itself was not observed.…”

Specific Spatial Localization of Actin and DNA in a Water/Water Microdroplet: Self‐Emergence of a Cell‐Like Structure

“…Recently, it has been suggested that the crowded cellular environment plays a fundamental role in the construction of subcellular organelles and granules, as well as in the cellular morphology, that is, liquid droplets (cytoplasmic bodies) consisting of RNA and proteins, assembly of the bacterial cytoskeleton protein FtsZ, and cytoskeletal networks . Interestingly, it is becoming clear that these systems lack specific regulatory factors, which suggests that subcellular structures are generated spontaneously under nonspecific environmental factors in a self‐organized manner under the crowded conditions of the cytoplasm.…”