Fig. 1: Reconstitution of FtsZ or MinCDE systems inside lipid vesicles under macromolecular crowding conditions.

a Left: Schematic illustration of the molecular mechanism of the minimal bacterial division system. FtsZ polymerizes upon GTP hydrolysis and binds the membrane through FtsA to assemble into the FtsZ-ring. The dynamic MinCDE system self-organizes into oscillatory waves inhibiting FtsZ polymerization at cell poles. Right: Our experimental setup by which the minimal division system is reconstituted inside lipid vesicles using two different approaches: the purified components or the cell-free expression of the system. In both cases, we observe a minimal FtsZ-ring at the equatorial plane of the vesicle. b Top: schematic representation of FtsZ-Venus-mts. Bottom: 3D max projection of encapsulated 2 µM of FtsZ-Venus-mts (green) in the absence (left) or presence (right) of macromolecular crowding using 100 g/L Dextran70 and 1 mM GTP. Scale bars: 15 µm. c Frequency of FtsZ structures formed inside lipid vesicles. FtsZ structures were differentiated into three categories: Bundles assembling a mesh, FtsZ dots, or luminal localization (n = 295, 230, 212, 242, 226, 218, 262 for no additives, 25, 50, 100 g/L Dextran70, 25, 50, 100 g/L Ficoll70, respectively). We could not observe the assembly of FtsZ-rings regardless of crowding conditions. d Left: Representative confocal image of purified MinCDE proteins inside lipid vesicles in the presence of macromolecular crowding (50 g/L Dextran70). Capital alphabet letters correspond to the kymographs on the right side (0.5 µM mScarlet-I-MinC, 3 µM MinD, and 3 µM MinE). Scale bar: 15 µm. Right: Kymographs of different MinCDE wave patterns captured by mScarlet-I-MinC fluorescence. The Kymograph is a 2D representation of a 1D object along with elapsed time. In this case, the fluorescence of mScarlet-I-MinC on the membrane is represented over time. The Y axis represents the signal at the membrane of the circumference (however, circumference was straightened to present the 1D information) while the X axis represents the time. Different intensity patterns observed in the Kymographs can be related with the Min oscillation modes on the membrane. e Frequencies of vesicles containing variations on the MinCDE dynamics (absence of waves, pole-to-pole oscillations, traveling waves, or pulsing) at different macromolecular crowding concentrations (0–100 g/L) using Dextran70 or Ficoll70 (0.5 µM mScarlet-I-MinC, 3 µM MinD, and 3 µM MinE) (n = 374, 227, 181, 161, 297, 181, 298, 303 for no additives, 25, 50, 100 g/L Dextran70, mScarlet-I-MinC^G10D in 100 g/L Dextran70, 25, 50, 100 g/L Ficoll70, respectively). Images were collected after 10 min of vesicle preparation for 1–2 h.