Mid-cell localization of the cell division septum in bacteria such as E. coli is controlled by a set of proteins including MinC, MinD, MinE, and FtsZ. FtsZ is the first structural component of the septum to polymerize on the inner membrane at the mid-cell when the cell starts to divide. FtsZ polymerization is limited to mid-cell by the action of the three Min proteins. MinC is an inhibitor of FtsZ polymerization, but on its own, it does not exhibit specific membrane localization. Instead, it binds to MinD, which is an ATP-dependent membrane binding protein and the two proteins co-localize on the membrane. MinE interacts with MinD and thought to control MinD ATPase activity and hence its membrane association dissociation dynamics. In vivo imaging studies have demonstrated oscillating pattern formation of these two proteins, resulting in the minimum concentration of MinD, hence MinC at the mid-cell region when averaged over time. This observation explained why FtsZ polymerization is restricted to mid-cell. However, detailed molecular mechanism of this bio-patterning reaction system is still poorly understood, due in part to the absence of suitable cell free reaction system to study this pattern formation reaction in detail. This project aims to investigate the biochemical and biophysical mechanism of the dynamic aspects of this reaction system by combining a variety of techniques. ? ? Techniques and instruments have been developed to study these reactions at the single molecule detection level by using a sensitive fluorescence microscope/CCD camera system. Using GFP-tagged and fluorescent dye coupled MinD and MinE proteins, assembly and disassembly of these proteins on lipid bilayer that mimics bacterial inner membrane that is immobilized on a slide glass surface was monitored under a variety of reaction conditions. We learned that: MinD, in the presence of ATP associates with membrane with rapid on- and off-rates. Limited polymerization of MinD could be observed on the membrane. Kinetic analysis of the dynamics, as well as the influence of MinE on MinD membrane association, has been investigated. ? ? The reaction system studied here is an example of biomolecular patterning reaction, and the experimental techniques developed here will be exploited for the parallel studies of mechanistically related reaction systems.
