Optimizing spatial organization of FtsZ rings for large-scale constriction in synthetic cells

Spatially regulated membrane constriction is an important milestone in reconstituting minimal cell division. In giant lipid vesicles, bottom-up approaches have reproduced the assembly, mid-cell positioning, and the initial constriction of an FtsZ-based minimal divisome. However, progressive deformation towards giant vesicle scission by near-equatorial Z rings could so far never be observed. One obvious major limitation has been the scale mismatch, as pure reconstituted FtsZ rings typically exhibit bacterial diameters, too small to constrict typical cell-sized vesicles. Therefore, we explore the potential of other key divisome factors to scale up FtsZ-ring functionality in vitro to match the dimensions required for synthetic cell division. We here focus on cytoFtsN, the cytosolic domain of FtsN, and its effect on FtsZ self-organization. Remarkably, a molar excess of cytoFtsN promotes the formation of large, closed equatorial FtsZ rings on giant vesicle membranes, which are able to constrict to almost full closure. By fluorescence imaging and biochemical analysis, we show that cytoFtsN regulates the spatial organization of the FtsZ network primarily by aligning FtsZ filaments while reducing filament depolymerization. Our findings help to define key requirements in a minimal filament-based system for progressive membrane constriction and thus represent a major step forward towards constructing synthetic cells capable of self-division.