# FtsZ Interactions and Biomolecular Condensates as Potential Targets for New Antibiotics
## 5 Reconstruction of Cellular FtsZ Subsystems
Intracellular crowdingis known to greatly affect the reactivity of molecules and enhance their associated states through volume exclusion, in turn modulated by other nonspecific attractive and repulsive effects [133; 134; 135]. It is therefore probable that functional complexes resembling their physiological counterparts form in vitro only under those conditions, which would contribute valuable information on a drug candidate's mechanism of action and dose-response as opposed to assays conducted in diluted conditions. A plethora of different approximations exists that, although not yet extensively used for identifying inhibitors, hold great promise as platforms for screening and characterization of compounds that inhibit bacterial cell division (Figure 3, Table 1). Among them, microdroplets (inverted micelles) and liposomes, particularly giant unilamellar vesicles (GUVs), have been employed most often because of their membrane fluidity and size range similar to bacterial cells. These sizes are ideal for the characterization of mesoscale cytoskeletal systems, allowing visualization of polymer networks formed by FtsZ (below), or the bacterial actin homolog MreB [136]. Similar systems have been used to monitor polymers of eukaryotic tubulin [137], actin [138], and myosin [139] with an obvious application for drug testing. In a notable use of liposomes to address whether FtsZ polymers can exert a constrictive force on the cytoplasmic membrane, chimeras of FtsZ fused to both YFP and an artificial membrane targeting sequence (mts, mentioned above) were externally added to multilamellar liposomes. These chimeras exerted different forces on the liposome surface depending on the position of the mts sequence, with either concave depressions and tubulation (FtsZ-YFP-mts) or convex bulges (mts-FtsZ-YFP) [140]. Similar FtsZ chimeras were added to tubular lipids to characterize the arrangement of FtsZ filaments from _E. coli_ and _Mycobacterium tuberculosis_ on their surface [141]. The de novo-synthesized MinDE system (Figure 1f), mentioned as part of the SLB reconstructions, has been shown to oscillate between membrane and lumen and produce autonomous deformation of liposomes [108]. The same cell-free gene expression system was employed in liposomes and lipid bilayers to reconstitute FtsZ-FtsA ring-like structures that were able to construct the liposomes [113]. Reconstitution of FtsZ and a ZipA (Figure 1b) variant into permeable GUVs resulted in membrane shrinkage modulated by dynamics of FtsZ polymers, which were externally triggered by diffusion of GTP and magnesium through membrane pores. Vesicle collapse was inhibited by addition of the FtsZ C-terminal peptide, which interfered with FtsZ-ZipA interaction [114]. Deformation of the membrane was also achieved by de novo synthesis of FtsZ and ZipA in GUVs, but not with the FtsZ-mts chimera [115]. Finally, lipid vesicles produced by manual emulsion were deformed into rod-shaped containers by trapping them in microfluidic devices, allowing visualization studies of FtsZ-mts filament arrangement in bacterial cell-like geometries [142]. Cell-like systems with precisely controlled composition, such as microdroplets generated by microfluidics [112], provide avenues for quantitative analysis of proteins in a crowded environment and confined by a lipid boundary. This technology allowed the measurement of self-organizing behavior of FtsZ polymers [117] and its modulation in a system that mimics a compartmentalized cytoplasm [118]. The use of traps in microfluidics chips provided a way to assess the effects of microdroplet shape and size on the arrangement of FtsZ polymer networks in the presence of ZipA (Figure 1b) [116]. Microdroplets containing FtsZ could also be transformed off-chip into permeable GUVs by an adapted droplet transfer method [118]. This strategy resulted in a lipid bilayer more closely resembling the membrane, where protein polymerization could be triggered from the outside.