MinCDE exploits the dynamic nature of FtsZ filaments for its spatial regulation

Abstract

In Escherichia coli, a contractile ring (Z-ring) is formed at midcell before cytokinesis. This ring consists primarily of FtsZ, a tubulin-like GTPase, that assembles into protofilaments similar to those in microtubules but different in their suprastructures. The Min proteins MinC, MinD, and MinE are determinants of Z-ring positioning in E. coli. MinD and MinE oscillate from pole to pole, and genetic and biochemical evidence concludes that MinC positions the Z-ring by coupling its assembly to the oscillations by direct inhibitory interaction. The mechanism of inhibition of FtsZ polymerization and, thus, positioning by MinC, however, is not understood completely. Our in vitro reconstitution experiments suggest that the Z-ring consists of dynamic protofilament bundles in which monomers constantly are exchanged throughout, stochastically creating protofilament ends along the length of the filament. From the coreconstitution of FtsZ with MinCDE, we propose that MinC acts on the filaments in two ways: by increasing the detachment rate of FtsZ-GDP within the filaments and by reducing the attachment rate of FtsZ monomers to filaments by occupying binding sites on the FtsZ filament lattice. Furthermore, our data show that the MinCDE system indeed is sufficient to cause spatial regulation of FtsZ, required for Z-ring positioning.

Keywords: bacterial cytoskeleton; cell division; depolymerization; self-organization.

Fig. 1.

Polymerization and depolymerization of an FtsZ network. (A) Polymerization of 0.1 µM FtsZ on a supported lipid bilayer, with frames taken every 10 s. (B) Growth characteristics of the FtsZ network. A higher concentration of FtsZ results in an increased net length of filaments (increased area coverage). (C) Increased concentrations of FtsZ result in increased area coverage/net length of FtsZ filaments. (D) Depolymerization of FtsZ filaments is qualitatively the reverse of assembly. (E) FRAP of an FtsZ filament network assembled at 0.2 µM. The spatial pattern before and after fluorescence recovery remains unchanged. (F) Kymograph of FRAP along a filament, showing uniform recovery at optical resolution limits. (G) Fluorescence recovery curves after photobleaching of FtsZ-YFP-MTS with GTP and GMPCPP. (H) A temporal overlay of two-color TIRF imaging shows NZ interacting throughout the length of the filament bundle. The overlay image shows FtsZ in blue, NZ in green, and colocalization in white pixels. (I) Schematic representing a polar FtsZ filament and a filament bundle. Scale bar: A and D, 1 µm; C and E, 10 µm; I, 5 µm.

Fig. 2.

Residence time distributions and interactions of MinC with FtsZ. Single-molecule residence time distributions of (A) FtsZ-Cy5, (B) NZ, and (C) EGFP-MinC dimer on FtsZ filament bundles. (D) FtsZ bundles can be depolymerized stepwise by addition of MinC, and the MinC-induced depolymerization rates are increased in the presence of MinD. (E) Twenty-nanometer resolution localization of EGFP-MinC on an FtsZ bundle network. (F) Montage (Left) and kymograph (Right) of a depolymerizing FtsZ filament, showing uniform loss of intensity along the length. (G) Filaments showing fragmentation events while being depolymerized by MinC. (H) Depolymerization of FtsZ polymers upon addition of MinC. Scale bar: E, F, and H, 5 µm.

Fig. 3.

MinC and NZ disassemble FtsZ polymers with different dynamics. (A) Depolymerization dynamics of FtsZ by MinC fit to the model described in this paper. FtsZ was assembled at a concentration of 0.8 µM. Fit parameters are summarized in Fig. S2B. (B) FCCS studies of MinC and NZ interaction with FtsZ in the solution above the FtsZ network; for details, see the main text and SI Appendix, Text S1. (C) Montage showing depolymerization of FtsZ filaments on addition of NZ. The characteristics are similar to those of MinC-induced depolymerization: an overall decrease in filament intensity as well as breakage of the filaments. (D) Depolymerization dynamics of FtsZ bundles assembled at a concentration of 0.8 µM by NZ. The fits are described in SI Appendix, Text S3; Eq. S5 was used for fitting. (E) Initial rates of depolymerization of FtsZ with MinC, NZ, and MinD + MinC. (F) The experimental dependence of the rates k_a and k_d on MinC concentration. The experimental values are in blue and the fit is in red. Scale bars: C, 10 µm.

Fig. 4.

Schematic showing the FtsZ filament bundles and their interaction with NZ and MinC. FtsZ bundles assemble by (i) longitudinal annealing and (ii) lateral interactions. In steady state, the FtsZ bundles constantly exchange subunits with the solution as a result of GTP hydrolysis. This results in stochastic exposing of the (−) ends. About 50% of the subunits in the bundle lattice are bound to GDP. NZ binds to the (−) ends of the filaments. NZ can sequester monomers in the solution (iii), and it can cap the filament (−) ends in the bundles (iv). MinC also binds to the (−) end of the FtsZ filaments through the N-terminal, capping and preventing annealing (v). MinC also interacts with the C-terminal of FtsZ through its C-terminal. MinC can bind to exposed (−) ends in the filaments caused by a leaving FtsZ subunit. Once bound to FtsZ, MinC remains bound until the subunit leaves, frustrating lateral interactions with incoming FtsZ subunits or FtsZ fragments (vi). The net action of MinC also results in a release of FtsZ-GDP subunits trapped in the filament lattice, resulting in a concentration-dependent increase in the rate of depolymerization.

Fig. 5.

Spatial regulation of FtsZ by MinCDE waves. (A) FtsZ is depolymerized by MinCDE waves. In the absence of MinC, FtsZ is not spatially regulated, Concentrations used were 1 µM MinD, 1.5 µM MinE, 0.5 µM MinC, and 2 µM FtsZ. MinE was doped with 20 mol% MinE-Cy5 and FtsZ with 50 mol% FtsZ-YFP-MTS. (B) The filaments show a reaction-dominant recovery on photobleaching (note the sharp boundaries throughout the recovery) as on supported lipid bilayers. (C) Montage showing depolymerization and repolymerization cycles at a fixed spot in the sample with time. The white arrow points in the direction of the Min waves. Scale bars: A–C, 25 µm.