Targeting of MinC to Z-ring

AbstractThe aim of this project was establishment and characterization of an in vitro minimal model system for the bacterial divisome, composed of FtsZ, FtsA and membranes, based on quantitative information about mutual interactions between the components. We initially characterized the binary and self-interactions of individual components, including purified FtsA* and FtsZ-F268C, with and without fluorescent tags, and artificial liposomes, using mainly Microscale thermophoresis (MST) methods. We found that ATP enhances dimerization of FtsA* and its affinity for the membrane. The affinity between FtsA* and monomeric or polymeric FtsZ was measured for the first time. We discovered that while FtsZ dimerization does not change its affinity for FtsA*, the interaction of FtsA* with FtsZ polymers is about 5 fold stronger than with non-polymeric FtsZ, but independent of ATP or ADP binding. Complete characterization of these binary interactions allowed us to approach the construction of the ternary complex of FtsA*, FtsZ, and SOPG LUVs in the presence of GDP and ATP. We analyzed the experimental results using a comprehensive mathematical model for ternary complex formation, which allowed determination of the half-maximal concentrations of the bridging FtsA* for the ternary complex formation (TF50) and inhibition (TI50). This is the first time that a ternary complex of FtsA-FtsZ-membrane was measured in a quantitative experiment. Moreover, these findings extrapolated very well to in vivo conditions. This knowledge can be used to assess the in vivo behavior of FtsZ and FtsA, and offer valuable tools for further investigation of divisome assembly.