Katja Zieske

Reconstitution of self-organizing protein gradients as spatial cues in cell-free systems

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

Reconstitution of Pole-to-Pole Oscillations of Min Proteins in Microengineered Polydimethylsiloxane Compartments

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.

Surface topology assisted alignment of Min protein waves

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.

Protein Patterns and Oscillations on Lipid Monolayers and in Microdroplets

Abstract 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.

Large-scale modulation of reconstituted Min protein patterns and gradients by defined mutations in MinE’s membrane targeting sequence

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.

Reconstituting geometry-modulated protein patterns in membrane compartments.

The MinCDE protein system from Escherichia coli has become one of the most striking paradigms of protein self-organization and biological pattern formation. The whole set of Min proteins is functionally active to position the divisome machinery by inhibiting Z ring assembly away from mid-cell. This is accomplished by an oscillation behavior between the cell poles, induced by the reaction between the two antagonistic proteins MinD and MinE, which has long caught the attention of quantitative biologists. Technical advances in fluorescence microscopy and molecular biology have allowed us in the past years to reconstitute this MinDE self-organization in cell-free environments on model membranes. We verified the compositional simplicity of protein systems principally required for biological pattern formation, and subjected the mechanism to quantitative biophysical analysis on a single-molecule level. On flat extended membranes, MinD and MinE self-organized into parallel propagating waves. Moreover, employing microsystems technology to construct membrane-clad soft polymer compartments mimicking the shape of native E. coli cells has further enabled us to faithfully reproduce Min protein oscillations. We further investigated the response of this self-organizing molecular system to three-dimensional compartment geometry. We could show that Min protein patterns depend strongly on the size and shape of the compartment, and the oscillation axis can only be preserved within a certain length interval and narrow width of the compartment. This renders the Min system a perfectly adapted oscillator to the bacterial cell geometry.

Reconstitution of Protein Dynamics Involved in Bacterial Cell Division

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.