Fig. 4. Reconstitution of minimal division ring placement system by co-expression of five essential components.

a Schematic illustration of co-expression systems of FtsA, FtsZ-G55-Venus-Q56, mCherry-MinC, MinD, and MinE. b 3D max projection of FtsZ-ring formation and Min protein localization at the pole of the vesicles after 100 min of co-expression at 37 °C (1 nM ftsAopt, 3 nM ftsZopt-G55-Venus-Q56, 2 nM mCherry-MinC, and 1 nM minDEoperon templates). mCherry-MinC and FtsZ-G55-Venus-Q56 are indicated in magenta and green, respectively. Scale bar: 20 μm. c Enlarged images of FtsZ-structure and Min wave patterns inside lipid vesicles after 3 h of cell-free expression. Pole-to-pole oscillations and static patterns stabilize the FtsZ-ring structures, and in both cases, FtsZ-ring can constrict vesicles. Experimental condition is the same as Fig. 4b. Scale bars: 10 μm. d Time-lapse images of the formation of the FtsZ-ring structure and constriction of the vesicle driven by the pole-to-pole oscillations of MinCDE. Top panels: 3D max projection of the merged image of mCherry-MinC (magenta) and FtsZ-G55-Venus-Q56 (green). The isotropic FtsZ mesh is condensed into a ring structure after the emergence of pole-to-pole oscillations, which later constricts the vesicle membrane. Bottom panels: Differential interference contrast (DIC) images of the equatorial plane of the vesicle. Red lines indicate aspect ratios of the vesicle before and after constriction by FtsZ-ring, yellow triangles indicate necks of the constriction by FtsZ-ring. Experimental condition is the same as Fig. 4b and the recording started after 50 min of cell-free expression. Scale bar: 10 μm. e Percentage of the ring structure and deformed vesicles among different vesicle sizes after 2–3 h co-expression of 5 essential genes (at the same template ratios as Fig. 4b) at 37 °C. Proportions of both FtsZ-ring structures and deformation of the vesicles increase along vesicle diameter, while deformation never appears under 12 μm in a diameter. n = 40 for each diameter range. f Scatter plot of the aspect ratios of deformed vesicles against vesicle diameter. The correlation coefficient shows moderate correlation (Pearson’s r = −0.40), suggesting greater effective force generation by FtsZ-rings within larger vesicles. Gray and blue dots (with respective background colors) indicate non-deformed vesicles (aspect ratio > 0.95) and deformed vesicles (aspect ratio < 0.95), respectively. n = 79 from three independent biological replicates. Experimental condition is the same as shown in Fig. 4e. Source data are provided as a Source Data file.