In vitro reconstitution of the initial stages of the bacterial cell division machinery

Abstract

Fission of many prokaryotes as well as some eukaryotic organelles depends on the self-assembly of the FtsZ protein into a membrane-associated ring structure early in the division process. Different components of the machinery are then sequentially recruited. Although the assembly order has been established, the molecular interactions and the understanding of the force-generating mechanism of this dividing machinery have remained elusive. It is desirable to develop simple reconstituted systems that attempt to reproduce, at least partially, some of the stages of the process. High-resolution studies of Escherichia coli FtsZ filaments' structure and dynamics on mica have allowed the identification of relevant interactions between filaments that suggest a mechanism by which the polymers could generate force on the membrane. Reconstituting the membrane-anchoring protein ZipA on E. coli lipid membrane on surfaces is now providing information on how the membrane attachment regulates FtsZ polymer dynamics and indicates the important role played by the lipid composition of the membrane.

Fig. 1

Topographic images of FtsZ filaments adsorbed on mica. a Polymers formed in solution in the presence of 10 mM GTP and adsorbed for a few minutes on the mica arranged as a densely packed layer of filaments on the surface. b Filaments adsorbed at a lower density rearranged as spirals on the surface

Fig. 2

Modeling of short filaments. a Protein filaments are considered as beads on a string kept together by a spring constant κ and a preferential angle θ₀. b An image of one of the filaments analyzed to obtain the distribution of angles between monomers shown in c

Fig. 3

Modeling of long filaments. a Images of the same filaments taken at different times. Shorter filaments on the left lengthen to form the spirals shown at the right. c The two drawings are taken from the Langevin dynamic simulations run using the potential shown in b (Eq. 2) to explore the equilibrium configurations adopted by extended filaments diffusing on a two-dimensional surface

Fig. 4

Orientation of native ZipA on a planar lipid bilayer. a Formation of the first supported membrane on a mica surface. b and c The step in which the protein–lipid–detergent solubilized complex is incubated in the presence of BioBeads to remove excess detergent. The histidines in the amino terminal end of the ZipA bind to the first bilayer and orient the protein, while removal of the detergent allows for the formation of a second bilayer in which the protein is incorporated. d The surface that can then be exposed to the FtsZ protein solution and analyzed with the AFM tip

Fig. 5

a A mica surface (darker region) partially covered by a lipid bilayer. b The same surface after incubation with the protein. FtsZ filaments adsorb to the bare mica but not to the lipid surface. c The surface of E. coli lipids with ZipA after being exposed to FtsZ in the presence of GTP. d The same region 15 min later, after the formation of FtsZ filament bundles