Abstract
Permeable vesicles containing the proto-ring anchoring ZipA protein shrink when FtsZ, the main cell division protein, polymerizes in the presence of GTP. Shrinkage, resembling the constriction of the cytoplasmic membrane, occurs at ZipA densities higher than those found in the cell and is modulated by the dynamics of the FtsZ polymer. In vivo, an excess of ZipA generates multilayered membrane inclusions within the cytoplasm and causes the loss of the membrane function as a permeability barrier. Overproduction of ZipA at levels that block septation is accompanied by the displacement of FtsZ and two additional division proteins, FtsA and FtsN, from potential septation sites to clusters that colocalize with ZipA near the membrane. The results show that elementary constriction events mediated by defined elements involved in cell division can be evidenced both in bacteria and in vesicles.
Highlights
Before constriction ZipA anchors FtsZ to the E. coli inner membrane as part of the cell division proto-ring
Escherichia coli Strains and Growth Conditions—Bacterial strains used in this study were MC1061 [17, 18] for in vivo studies of His-ZipA overproduction, BL21(DE3) for gene overexpression and protein purification, and VIP205 to deplete the amount of FtsZ by removing 30 M isopropyl 1-thio--D-galactopyranoside from the culture, or restore it by its readdition [19]
Encapsulated FtsZ Polymerizes Inside Permeable Giant Unilamellar Vesicles (GUVs) upon Addition of GTP and Magnesium—Giant vesicles made from egg yolk phosphatidylcholine were produced at high yield by an optimized droplet transfer protocol [30] under physiological potassium concentrations (100 mM) and in the presence of inert macromolecules at high concentration (50 mg/ml Ficoll 70) to mimic natural crowding conditions and to help to visualize FtsZ polymers inside the vesicle [28]
Summary
Before constriction ZipA anchors FtsZ to the E. coli inner membrane as part of the cell division proto-ring. Results: Dynamic FtsZ polymers shrink ZipA-containing vesicles whereas excess of ZipA invaginates the E. coli membrane destroying the permeability barrier. Permeable vesicles containing the proto-ring anchoring ZipA protein shrink when FtsZ, the main cell division protein, polymerizes in the presence of GTP. In this work we have used phosphatidylcholine giant unilamellar vesicles containing FtsZ in their lumen and ZipA inserted into the inner surface of the lipid phase These vesicles have been made permeable by integration of the pore-forming protein ␣-hemolysin allowing us to control the polymerization of FtsZ from outside the vesicle upon the addition of GTP and magnesium. The mechanical properties of the complexes formed upon interaction of FtsZ polymers with the ZipA-containing vesicles have been compared with the morphological and biological changes observed in living cells in which ZipA has been overproduced
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