Study of the mechanism of septum localization during bacterial cell division

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

Mid-cell localization of the cell division septum in bacteria such as E. coli is controlled by a set of proteins, MinC, MinD, MinE, and FtsZ. FtsZ is a tubulin homologue and the first component of the dynamic septum assembly machinery to polymerize in the presence of GTP on the inner membrane at the mid-cell as the cell prepares for division. FtsZ polymerization is confined to mid-cell by the action of the three Min proteins. MinC is an inhibitor of FtsZ polymerization when localized on the membrane, but on its own, it does not bind to membrane and needs the help of MinD, an ATP-dependent membrane binding protein. MinC binds to the ATP-activated MinD dimers bound to the membrane, and these two proteins generally co-localize on the membrane. MinE also interacts with MinD dimers on the membrane, and when present at high densities on the membrane, it can displace MinC from MinD. More importantly, MinE helps membrane binding of MinD-ATP and also activates MinD ATPase, and hence MinE controls both membrane association and dissociation dynamics of MinD. In vivo imaging studies have demonstrated a cell pole-to-cell pole oscillating pattern self-organization by MinD and MinE proteins, resulting in a time-averaged concentration minimum of MinD, and hence MinC protein on the membrane at the mid-cell. This earlier discovery strongly indicated the system is a prototypical example of the Reaction-Diffusion Turing pattern-self-organization system. Above observation explained why FtsZ polymerization is restricted to mid-cell. However, a detailed molecular mechanism of this bio-patterning reaction system remains enigmatic. This project aims to investigate the biochemical and biophysical principles of this dynamic molecular pattern self-organization system by combining a variety of techniques, including a reconstituted cell-free reaction system we have established that recapitulates aspects of in vivo system dynamics. Techniques and instruments have been developed to study these oscillation systems in vitro by using a sensitive fluorescence microscope system. By using fluorescence-labeled MinD and MinE proteins, assembly and disassembly of these proteins on a supported lipid bilayer on the slide glass surface are monitored under a variety of experimental conditions. We successfully reconstituted a variety of modes of self-organized dynamic pattern formation by MinD and MinE proteins on the membrane surface in the presence of ATP. We also study the kinetic parameters of the biochemical reaction steps involved in the reaction. Recent progress allowed us to propose the first comprehensive detailed molecular mechanistic model for this reaction system that is supported by a large body of experimental observations we have accumulated. Further mechanistic details of the dynamic pattern organization are currently studied combining biochemical, biophysical and mathematical approaches. Currently, in collaboration with scientists in LCP/NIDDK, conformational dynamics of MinE protein by itself, and its transient membrane interaction dynamics that plays critical roles in the control of MinD-membrane interaction dynamics is investigated using NMR techniques. In addition, we are currently investigating the nature of MinD-MinE protein complexes that form on the membrane as transient reaction intermediates that control biochemical activities of MinD protein bound to the membrane in the oscillation phase specific manner in order to further refine our molecular mechanistic model. This study is in part aimed at advancement of our knowledge at molecular details on how a set of protein molecules could orchestrate a spatial control of cellular events that occur at a much larger length-scale than the individual protein molecules by reaction-diffusion principles without assembling protein filaments that spans the distance. Large number of spatial pattern self-organization systems in biology are expected to involve conceptually similar mechanistic principles, and this study is hoped to contribute to further advance our understanding of more complex systems that operate involving related principles.