Intracellular protein gradients are significant determinants of spatial organization. However, little is known about how protein patterns are established, and how their positional information directs downstream processes. We have accomplished the reconstitution of a protein concentration gradient that directs the assembly of the cell division machinery in E.coli from the bottom-up. Reconstituting self-organized oscillations of MinCDE proteins in membrane-clad soft-polymer compartments, we demonstrate that distinct time-averaged protein concentration gradients are established. Our minimal system allows to study complex organizational principles, such as spatial control of division site placement by intracellular protein gradients, under simplified conditions. In particular, we demonstrate that FtsZ, which marks the cell division site in many bacteria, can be targeted to the middle of a cell-like compartment. Moreover, we show that compartment geometry plays a major role in Min gradient establishment, and provide evidence for a geometry-mediated mechanism to partition Min proteins during bacterial development.DOI: http://dx.doi.org/10.7554/eLife.03949.001

Cell division in bacteria is highly regulated in time and space. The use of micrometer-sized sample volumes and model membranes allows the pole-to-pole oscillations of spatial regulators for bacterial cell division to be reconstituted in a synthetic minimal system.

The E. coli MinDE oscillator is a paradigm for protein self-organization and gradient formation. Previously, we reconstituted Min protein wave patterns on flat membranes as well as gradient-forming pole-to-pole oscillations in cell-shaped PDMS microcompartments. These oscillations appeared to require direct membrane interaction of the ATPase activating protein MinE. However, it remained unclear how exactly Min protein dynamics are regulated by MinE membrane binding. Here, we dissect the role of MinE’s membrane targeting sequence (MTS) by reconstituting various MinE mutants in 2D and 3D geometries. We demonstrate that the MTS defines the lower limit of the concentration-dependent wavelength of Min protein patterns while restraining MinE’s ability to stimulate MinD’s ATPase activity. Strikingly, a markedly reduced length scale—obtainable even by single mutations—is associated with a rich variety of multistable dynamic modes in cell-shaped compartments. This dramatic remodeling in response to biochemical changes reveals a remarkable trade-off between robustness and versatility of the Min oscillator.

The Min proteins from E.coli position the bacterial cell‐division machinery through pole‐to‐pole oscillations. In vitro, Min protein self‐organization can be reconstituted in the presence of a lipid membrane as a catalytic surface. However, Min dynamics have so far not been reconstituted in fully membrane‐enclosed volumes. Microdroplets interfaced by lipid monolayers were employed as a simple 3D mimic of cellular compartments to reconstitute Min protein oscillations. We demonstrate that lipid monolayers are sufficient to fulfil the catalytic role of the membrane and thus represent a facile platform to investigate Min protein regulated dynamics of the cell‐division protein FtsZ‐mts. In particular, we show that droplet containers reveal distinct Min oscillation modes, and reveal a dependence of FtsZ‐mts structures on compartment size. Finally, co‐reconstitution of Min proteins and FtsZ‐mts in droplets yields antagonistic localization, thus demonstrating that droplets indeed support the analysis of complex bacterial self‐organization in confined volumes.

a b s t r a c t Self-organization of proteins into large-scale structures is of pivotal importance for the organization of cells. The Min protein system of the bacterium Escherichia coli is a prime example of how pattern formation occurs via reaction-diffusion. We have previously demonstrated how Min protein patterns are influenced by compartment geometry. Here we probe the influence of membrane surface topology, as an additional regulatory element. Using microstructured membrane-clad soft polymer substrates, Min protein patterns can be aligned. We demonstrate that Min pattern alignment starts early during pattern formation and show that macroscopic millimeter-sized areas of protein patterns of well-defined orientation can be generated.

The Min proteins from E.coli position the bacterial cell-division machinery through pole-to-pole oscillations. In vitro,M in protein self-organization can be reconstituted in the presence of al ipid membrane as ac atalytic surface. However,M in dynamics have so far not been reconstituted in fully membrane-enclosed volumes.M icrodroplets interfaced by lipid monolayers were employed as as imple 3D mimic of cellular compartments to reconstitute Min protein oscillations. We demonstrate that lipid monolayers are sufficient to fulfil the catalytic role of the membrane and thus represent af acile platform to investigate Min protein regulated dynamics of the cell-division protein FtsZ-mts.I np articular,w es howt hat droplet containers reveal distinct Min oscillation modes,a nd reveal ad ependence of FtsZ-mts structures on compartment size. Finally,co-reconstitution of Min proteins and FtsZ-mts in droplets yields antagonistic localization, thus demonstrating that droplets indeed support the analysis of complex bacterial self-organization in confined volumes.The self-organization of proteins into large-scale structures and patterns is fundamental to all living systems,a nd is required to regulate complex cellular processes such as protein localization and cell division. As triking example of protein self-organization is the Min protein system (comprising the proteins MinC,M inD,a nd MinE) of the bacterium Escherichia coli, [1] which oscillates between the cell poles. These pole-to-pole oscillations generate atime-averaged nonhomogeneous concentration gradient with am inimum at the mid-cell plane,t he future cell division site. [2][3][4][5][6][7] Intriguingly, only am inimal set of components (the two membrane interacting proteins MinD and MinE, al ipid membrane, and ATP) has been shown to establish Min oscillations. [8][9][10][11][12] Theprotein MinC follows the oscillating MinDE patterns by binding to MinD,a nd it directly inhibits the assembly of the cell-division protein FtsZ at the poles. [3,7,[13][14][15] In this way,the main division initiator FtsZ, which assembles into aring-like structure (Z ring), is directed to the middle of the cell. [4] To study Min oscillations under well-defined conditions, cell-free systems are being developed that provide ahigh level of control over physicochemical parameters.P reviously,w e have reconstituted the pole-to-pole oscillations of the Min proteins,a sw ell as their ability to spatially direct FtsZ-mts (FtsZ fused to am embrane-targeting sequence) [16] to the middle of am icrofabricated compartment. [12,17] However, although the cell membrane of E. colii sac losed compartment, Min oscillations have thus far only been successfully reconstituted in volumes not fully enclosed by membranes, but open at least at one site. [12,18,19] Moreover,a lthough membrane-attached FtsZ rings have been observed in vesicles [16,20,21] and FtsZ bundles have been characterized in the lumen of droplets, [22] we are just beginning to understand how systems parameters,s uch as compartment ge...

Even simple cells like bacteria have precisely regulated cellular anatomies, which allow them to grow, divide and to respond to internal or external cues with high fidelity. How spatial and temporal intracellular organization in prokaryotic cells is achieved and maintained on the basis of locally interacting proteins still remains largely a mystery. Bulk biochemical assays with purified components and in vivo experiments help us to approach key cellular processes from two opposite ends, in terms of minimal and maximal complexity. However, to understand how cellular phenomena emerge, that are more than the sum of their parts, we have to assemble cellular subsystems step by step from the bottom up. Here, we review recent in vitro reconstitution experiments with proteins of the bacterial cell division machinery and illustrate how they help to shed light on fundamental cellular mechanisms that constitute spatiotemporal order and regulate cell division.

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