Analyses of dynamic properties of the MinD cytoskeleton

Abstract

In Escherichia coli, placement of the cell division site at midcell is governed by pole-to-pole oscillation of the Min proteins. The Min system contains three proteins in which MinD belongs to a superfamily of the Walker-type ATPases and possesses intriguing cytoskeleton properties. Accumulating evidence indicates that pole-to-pole oscillation of the Min proteins underlies cycles of assembly and disassembly of the MinD protein filaments on the membrane that are regulated by MinE and nucleotide-binding and hydrolysis. Our goal is to obtain better understanding on the cytoskeleton dynamics of MinD both in vivo and in vitro. We first analyze the ability of Gfp-MinD diffusion on membrane sites using the fluorescence recovery after photobleaching (FRAP) technique. We observed directional recovery of Gfp-MinD fluorescence on the membrane, indicating possible exchange of protein molecules within underlay MinD protein filaments. On the other hand, in order to reconstitute an in vitro environment for studying MinD dynamics and MinD-membrane interactions, we again used the FRAP technique to characterize the supported lipid bilayers prepared from E. coli lipid extracts. Fluorescently-labeled MinD and other components of the Min system are applied to the supported lipid bilayers for microscopy studies. Fluorescently-labeled MinD in the presence of ATP appears capable of forming short rod-like structures. The observation is consistent with the fact that self-assembly of MinD protein filaments requires ATP. In summary, the Min system serves as a model system for studying cytoskeleton dynamics that is coupled to fundamental cellular functions. Moreover, such dynamic properties may be common across different types of cytoskeletal proteins.