# FtsZ Interactions and Biomolecular Condensates as Potential Targets for New Antibiotics
## 5 Reconstruction of Cellular FtsZ Subsystems
Supported lipid bilayers (SLBs), mostly in combination with high-resolution fluorescence microscopy imaging, have been extensively used to characterize the bacterial division machinery and in particular FtsZ. Initial approaches used a fusion of FtsZ to a fluorescent protein and a membrane targeting sequence (FtsZ-YFP-mts), thus overcoming the need of an anchor protein. Reconstitution experiments with these flat bilayers showed the effect of spatial patterning by the MinCDE complex (Figure 1f) on the distribution of FtsZ [104]. SLBs also served as platforms to demonstrate the self-organization of FtsZ polymers into dynamic ring patterns on their own [129] or with FtsA [105]; to show treadmilling of individual FtsZ filaments [105]; to highlight the possible role of ZipA as passive membrane anchor for FtsZ filaments [106]; and, finally, to report the comigration of other divisome proteins with treadmilling FtsZ filaments [107]. Very recently, SLBs were elegantly used as a platform to generate dynamic planar waves of de novo-synthesized MinDE proteins, which spatially regulate FtsZ filament patterns in the presence of de novo-synthesized FtsA [108]. This cell-free expression system allows protein production from a DNA template through the PURE system [130], a mimic of the molecular composition of the bacterial cytoplasm, but which lacks endogenous proteins that might interfere with the reconstituted system. Towards the goal of identifying new antimicrobials, SLBs have been mainly used to characterize the effect of antimicrobial peptides (AMPs) on the membrane, such as formation of lipid fibrils with Temporins B and L [131], or membrane pore formation by the AMP model amhelin [132]. Biosensors can be used for the real-time quantitative characterization of protein-protein and protein-membrane interactions. Biolayer interferometry measures the change in the interferometry signal upon protein interaction after immersion of a biosensor tip, coated with a supported bilayer, into a solution with the protein. This technique has been used, in parallel with coated microbeads, to determine SlmA (Figure 1a) binding to a lipid membrane [103]. These kinds of measurements can be easily employed in the detection of inhibitors of the interaction of SlmA with FtsZ and/or the lipid membrane, potentially interfering with the proper localization of the Z-ring to the midcell division site. The ability to rapidly measure very small volume samples makes this method suitable for compound screening. NanoSPR (surface plasmon resonance) measures the changes in refractive index produced upon binding of a molecule to gold nanorods, and has been used to measure local concentration of the Min proteins (Figure 1f) resulting from their oscillations, i.e., the intrinsic dynamics of the system, using nanorods coated with _E. coli_ lipids [111]. As in the case of nanodiscs, the C-terminal FtsZ peptide has been used to validate assays potentially useful to identify inhibitors of FtsZ-ZipA interactions, based on lipid-coated plasmonic nanosensors with an immobilized soluble version of ZipA [109] (Figure 1b). Finally, plasmonic sensors based on surface-enhanced Raman scattering have been used to detect the interactions between FtsZ and bacterial membrane elements [110]. From this, fast assays to screen for drugs altering the attachment of FtsZ to the membrane could be developed. A further step towards reproducing true cellular macromolecular interactions in vitro consists of reconstruction in biomimetic environments, which reproduce membrane boundary and confinement conditions similar to those found in vivo [55; 56; 57; 58]. This approach allows characterization of a particular cell function in terms of the minimal number of biological components involved in the interaction pattern (including the membrane as an active element), their dynamics, and their localization within the cell-like environment. Importantly, studies of potential inhibitors in crowded environments, although technically more complex, could more reliably assess their effect.