Spatiotemporal patterning regulation in artificial cells by synthesizing a bacterial spatiotemporal regulator using a defined protein expression system.

Constructing a minimal machinery for autonomous self-division of synthetic cells is a major goal of bottom-up synthetic biology. One paradigm has been the E. coli divisome, with the MinCDE protein system guiding assembly and positioning of a presumably contractile ring based on FtsZ and its membrane adaptor FtsA. Here, we demonstrate the full in vitro reconstitution of this machinery consisting of five proteins within lipid vesicles, allowing to observe the following sequence of events in real time: 1) Assembly of an isotropic filamentous FtsZ network, 2) its condensation into a ring-like structure, along with pole-to-pole mode selection of Min oscillations resulting in equatorial positioning, and 3) onset of ring constriction, deforming the vesicles from spherical shape. Besides demonstrating these essential features, we highlight the importance of decisive experimental factors, such as macromolecular crowding. Our results provide an exceptional showcase of the emergence of cell division in a minimal system, and may represent a step towards developing a synthetic cell.

The Min system, a system that determines the bacterial cell division plane, uses changes in the localization of proteins (a Min wave) that emerges by reaction-diffusion coupling. Although previous studies have shown that space sizes and boundaries modulate the shape and speed of Min waves, their effects on wave emergence were still elusive. Here, by using a microsized fully confined space to mimic live cells, we revealed that confinement changes the conditions for the emergence of Min waves. In the microsized space, an increased surface-to-volume ratio changed the localization efficiency of proteins on membranes, and therefore, suppression of the localization change was necessary for the stable generation of Min waves. Furthermore, we showed that the cell-sized space strictly limits parameters for wave emergence because confinement inhibits both the instability and excitability of the system. These results show that confinement of reaction-diffusion systems has the potential to control spatiotemporal patterns in live cells.

23Min system, which determines the cell division plane of bacteria, uses the localization 24 change of protein (Min wave) emerged by a reaction-diffusion coupling. Although 25 previous studies have shown that cell-sized space and boundaries modulate shape and 26 speed of Min waves, its effects on Min wave emergence was still elusive. Here, by using a 27 fully confined microsized space as a mimic of live cells, we revealed that confinement 28 changes conditions for Min wave emergence. In the microsized space, an increase of 29 surface-to-volume ratio changed the localization efficiency of proteins on membranes, and 30 therefore, suppression of the localization change was necessary to produce stable Min 31 wave generations. Furthermore, we showed that the cell-sized space more strictly limits 32 parameters for wave emergence because confinement inhibits instability and excitability 33 of the system. These results illuminate that confinement of reaction-diffusion systems 34 works as a controller of spatiotemporal patterns in live cells. 35 36 37 38 39 52 53 To date, Min wave is the only biological RD system reconstituted in vitro. Reconstitution 54 of Min wave was firstly shown by spotting a mixture of MinD, MinE, and ATP on 2-55 demensinal (2D) planar membranes comprising E. coli polar lipid extract in open 56 geometry(7). The following studies based on a 2D planar system have clarified the 57 mechanisms of wave generation and the characteristics of Min waves (8-13). In vitro 58 studies have demonstrated that external environments such as boundary shape, protein 59 concentration, and lipids alter patterns, velocities, wavelengths and shapes of Min waves 60 (9)(10)(11)(12)(13)(14). In particular, boundary shapes prepared by PDMS chambers significantly change 61 the behavior of Min waves with studies showing that a rod-shape is important in terms of 62 inducing the pole-to-pole oscillation found in living cells (10, 12, 13). 63 64 Due to the importance of Min waves for initiating division at a precise location, the 65 timing, conditions, and regulation of their emergence should be investigated. However, 66 despite the critical conditions for Min wave emergence including environmental effects 67 having been surveyed in open space, the effect of confinement in cell-sized space, which 68 is one of the most remarkable features of living cells, has been poorly addressed. Although 69 some studies have reported reconstitution of Min waves in fully confined cell-sized spaces 70 (12, 13, 15), lipid conditions were modified or spaces were closed after observing wave 71 generation. The necessity of these treatments suggests that cell-sized space affects the 72 condition for Min wave emergence. 73 74 Recent studies have unveiled that confinement inside cell-sized space alters both behaviors 75 of biochemical reactions and molecular diffusions (16-18). Because RD waves appear 76 only in limited parameter ranges (19, 20), encapsulation inside cell-sized space should 77 shift the condition for Min wave emergence such as...

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