# FtsZ Interactions and Biomolecular Condensates as Potential Targets for New Antibiotics
## 5 Reconstruction of Cellular FtsZ Subsystems  
Reconstruction of selected subsets of proteins within the FtsZ interaction network in synthetic platforms that display different features of the cellular environment, relevant for the function of this protein, may be a suitable strategy for the identification of antimicrobial candidates. Not only can these approaches shed further light onto mechanisms used by molecules identified through assays in dilute solutions or in cells, but also the measured effects of these molecules would more closely resemble those occurring in vivo, potentially facilitating the selection of candidates. One of the key elements to be considered in such studies is the cytoplasmic membrane, which plays an active part in divisome function, as most essential divisome proteins interact directly with it. Some divisome proteins anchor FtsZ polymers to the membrane (FtsA, ZipA; Figure 1b), some directly interact with the membrane surface at some point while exerting their function (MinDE, SlmA, MatP; Figure 1a,e,f), and others are integral membrane proteins (FtsK, FtsEX, the FtsQBL complex, and the FtsWI septum synthesis enzymes). Consequently, it is important to include membranes in order to evaluate more accurately the interactions of FtsZ and to study protein-membrane interactions that regulate cell division. The different available approaches start with protein-free lipid bilayers, to which proteins are externally added (Figure 3, Table 1). For a detailed review of membrane transformation by molecular assemblies, see [57]. Membrane proteins can be stabilized in solution by reconstruction in phospholipid bilayer nanodiscs, which are small portions of a lipid bilayer surrounded by two copies of an amphipatic scaffold protein [100]. Nanodiscs enable the analysis of membrane protein interactions by the many techniques available to study soluble proteins, in a more native-like environment than when using detergents. Nanodisc technology has been applied to the analysis of interactions of full-length (including the transmembrane region) ZipA (Figure 1b) with FtsZ oligomers and with GTP-induced FtsZ polymers [37]. The ZipA-interacting C-terminal tail of FtsZ was used in that study to prove the utility of analytical ultracentrifugation and FCS assays developed to find compounds that interfere with the FtsZ-ZipA interactions. Reconstruction of membrane protein interactions have also been conducted using microbeads of various materials, such as silica or polystyrene, and of uniform size, coated with a lipid bilayer or monolayer. Such lipid-coated microbeads have been used to characterize the binding of FtsZ oligomers and polymers to a His-tagged ZipA variant anchored to the lipid membrane, as well as the influence of receptor density on this binding, by measurement of the fluorescence signal corresponding to the amount of unbound protein in the supernatant after sedimentation of the microbeads [101]. The same procedure was used earlier to quantify the interaction of FtsA (Figure 1b) with membranes of different compositions, including the _E. coli_ cytoplasmic membrane [102]. The affinity of FtsA to membranes was highly dependent on lipid composition, indicating that this approach could serve as a basis for the characterization of the effects of compounds on membrane remodeling [125] and/or FtsZ anchoring, both modulated by FtsA during cytokinesis. Experiments with coated microbeads also provided evidence that SlmA (Figure 1a) can interact with lipids and, only upon FtsZ interaction, recruit its targeted specific DNA sequences to these lipids [103]. Although more laborious than other types of experiments, this procedure is highly reproducible and suitable for the screening of compounds that affect these interactions, as tested by proof-of-concept experiments with molecular inhibitors of the FtsZ-ZipA interaction [128].