In vitro reconstitution of divisome activation

Abstract

Bacterial cell division is coordinated by the Z-ring, a cytoskeletal structure of treadmilling filaments of FtsZ and their membrane anchors, FtsA and ZipA. For divisome maturation and initiation of constriction, the widely conserved actin-homolog FtsA plays a central role, as it links downstream cell division proteins in the membrane to the Z-ring in the cytoplasm. According to the current model, FtsA initiates cell constriction by switching from an inactive polymeric conformation to an active monomeric form, which then stabilizes the Z-ring and recruits downstream proteins such as FtsN. However, direct biochemical evidence for this mechanism is missing so far. Here, we used biochemical reconstitution experiments in combination with quantitative fluorescence microscopy to study the mechanism of divisome activation in vitro. By comparing the properties of wildtype FtsA and FtsA R286W, a gain-of-function mutant thought to mimic its active state, we found that active FtsA outperforms the wildtype protein in replicating FtsZ treadmilling dynamics, filament stabilization and FtsN recruitment. We could attribute these differences to a faster membrane exchange of FtsA R286W as well as its higher packing density below FtsZ filaments. Using FRET microscopy, we also show that binding of FtsN does not compete with, but promotes FtsA self-interaction. Together, our findings shed new light on the assembly and activation of the bacterial cell division machinery and the mechanism of how FtsA initiates cell constriction.

Min waves without MinC can pattern FtsA-anchored FtsZ filaments on model membranes

“…The difference may be explained by the higher stability of the cytoskeletal structures containing FtsA*, the longer residence time of filaments on the membrane making them less subject to redistribution by MinDE. It has already been reported that FtsZ and FtsA* recruit each other more efficiently to the membrane during filament formation 19 , and that the greater colocalization is mediated by FtsA* filaments having a higher packing density 19,47 . The finding that MinC did not further encourage the redistribution of FtsA*-anchored FtsZ is in agreement with in vivo data showing that FtsA* imparts resistance to high concentrations of MinC, which ordinarily disassembles the Z-ring 44 .…”