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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., (© 2022. The Author(s).)

The formation of large-scale patterns through molecular self-organization is a basic principle of life. Accordingly, the engineering of protein patterns and gradients is of prime relevance for synthetic biology. As a paradigm for such pattern formation, the bacterial MinDE protein system is based on self-organization of the ATPase MinD and ATPase-activating protein MinE on lipid membranes. Min patterns can be tightly regulated by tuning physical or biochemical parameters. Among the biochemically engineerable modules, MinD's membrane targeting sequence, despite being a key regulating element, has received little attention. Here we attempt to engineer patterns by modulating the membrane affinity of MinD. Unlike the traveling waves or stationary patterns commonly observed in vitro on flat supported membranes, standing-wave oscillations emerge upon elongating MinD's membrane targeting sequence via rationally guided mutagenesis. These patterns are capable of forming gradients and thereby spatially target co-reconstituted downstream proteins, highlighting their functional potential in designing new life-like systems.

The MinDE proteins from E. coli have received great attention as a paradigmatic biological pattern-forming system. Recently, it has surfaced that these proteins do not only generate oscillating concentration gradients driven by ATP hydrolysis, but that they can reversibly deform giant vesicles. In order to explore the potential of Min proteins to actually perform mechanical work, we introduce a new model membrane system, flat vesicle stacks on top of a supported lipid bilayer. MinDE oscillations can repeatedly deform these flat vesicles into tubules and promote progressive membrane spreading through membrane adhesion. Dependent on membrane and buffer compositions, Min oscillations further induce robust bud formation. Altogether, we demonstrate that under specific conditions, MinDE self-organization can result in work performed on biomimetic systems and achieve a straightforward mechanochemical coupling between the MinDE biochemical reaction cycle and membrane transformation., (© 2020 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Modules that switch protein-protein interactions on and off are essential to develop synthetic biology; for example, to construct orthogonal signaling pathways, to control artificial protein structures dynamically, and for protein localization in cells or protocells. In nature, the E. coli MinCDE system couples nucleotide-dependent switching of MinD dimerization to membrane targeting to trigger spatiotemporal pattern formation. Here we present a de novo peptide-based molecular switch that toggles reversibly between monomer and dimer in response to phosphorylation and dephosphorylation. In combination with other modules, we construct fusion proteins that couple switching to lipid-membrane targeting by: (i) tethering a 'cargo' molecule reversibly to a permanent membrane 'anchor'; and (ii) creating a 'membrane-avidity switch' that mimics the MinD system but operates by reversible phosphorylation. These minimal, de novo molecular switches have potential applications for introducing dynamic processes into designed and engineered proteins to augment functions in living cells and add functionality to protocells.

The proteins MinD, MinE and MinC are constitutive for the spatiotemporal organization of cell division in Escherichia coli, in particular, for positioning the division machinery at mid-cell. To achieve this function, the ATPase MinD and the ATPase-activating protein MinE undergo coordinated pole-to-pole oscillations and have thus become a paradigm for protein pattern formation in biology. The exact molecular mechanisms enabling MinDE self-organization, and particularly the role of cooperativity in the membrane binding of MinD, thought to be a key requirement, have remained poorly understood. However, for bottom-up synthetic biology aiming at a de novo design of key cellular features, elucidating these mechanisms is of great relevance. By combining in vitro reconstitution with rationally guided mutagenesis of MinD, we found that when bound to membranes, MinD displays new interfaces for multimerization, which are distinct from the canonical MinD dimerization site. We propose that these additional transient interactions contribute to the local self-enhancement of MinD at the membrane, while their relative lability maintains the structural plasticity required for MinDE wave propagation. This could represent a powerful structural regulation feature not reported so far for self-organizing proteins., Competing Interests: Declaration of Interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Patterns formed by reaction-diffusion mechanisms are crucial for the development or sustenance of most organisms in nature. Patterns include dynamic waves, but are more often found as static distributions, such as animal skin patterns. Yet, a simplistic biological model system to reproduce and quantitatively investigate static reaction-diffusion patterns has been missing so far. Here, we demonstrate that the Escherichia coli Min system, known for its oscillatory behavior between the cell poles, is under certain conditions capable of transitioning to quasi-stationary protein distributions on membranes closely resembling Turing patterns. We systematically titrated both proteins, MinD and MinE, and found that removing all purification tags and linkers from the N-terminus of MinE was critical for static patterns to occur. At small bulk heights, dynamic patterns dominate, such as in rod-shaped microcompartments. We see implications of this work for studying pattern formation in general, but also for creating artificial gradients as downstream cues in synthetic biology applications.

The bacterial Min protein system was encapsulated in giant unilamellar vesicles (GUVs). Using confocal fluorescence microscopy, we identified several distinct modes of spatiotemporal patterns inside spherical GUVs. For osmotically deflated GUVs, the vesicle shape actively changed in concert with the Min oscillations. The periodic relocation of Min proteins from the vesicle lumen to the membrane and back is accompanied by drastic changes in the mechanical properties of the lipid bilayer. In particular, two types of oscillating membrane-shape changes are highlighted: 1) GUVs that repeatedly undergo fission into two connected compartments and fusion of these compartments back into a dumbbell shape and 2) GUVs that show periodic budding and subsequent merging of the buds with the mother vesicle, accompanied by an overall shape change of the vesicle reminiscent of a bouncing ball. These findings demonstrate how reaction-diffusion-based protein self-organization can directly yield visible mechanical effects on membrane compartments, even up to autonomous division, without the need for coupling to cytoskeletal elements., (© 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Living systems employ protein pattern formation to regulate important life processes in space and time. Although pattern-forming protein networks have been identified in various prokaryotes and eukaryotes, their systematic experimental characterization is challenging owing to the complex environment of living cells. In turn, cell-free systems are ideally suited for this goal, as they offer defined molecular environments that can be precisely controlled and manipulated. Towards revealing the molecular basis of protein pattern formation, we outline two complementary approaches: the biochemical reverse engineering of reconstituted networks and the de novo design, or forward engineering, of artificial self-organizing systems. We first illustrate the reverse engineering approach by the example of the Escherichia coli Min system, a model system for protein self-organization based on the reversible and energy-dependent interaction of the ATPase MinD and its activating protein MinE with a lipid membrane. By reconstituting MinE mutants impaired in ATPase stimulation, we demonstrate how large-scale Min protein patterns are modulated by MinE activity and concentration. We then provide a perspective on the de novo design of self-organizing protein networks. Tightly integrated reverse and forward engineering approaches will be key to understanding and engineering the intriguing phenomenon of protein pattern formation.This article is part of the theme issue 'Self-organization in cell biology'., (© 2018 The Author(s).)

Patterns formed by reaction and diffusion are the foundation for many phenomena in biology. However, the experimental study of reaction-diffusion (R-D) systems has so far been dominated by chemical oscillators, for which many tools are available. In this work, we developed a photoswitch for the Min system of Escherichia coli, a versatile biological in vitro R-D system consisting of the antagonistic proteins MinD and MinE. A MinE-derived peptide of 19 amino acids was covalently modified with a photoisomerizable crosslinker based on azobenzene to externally control peptide-mediated depletion of MinD from the membrane. In addition to providing an on-off switch for pattern formation, we achieve frequency-locked resonance with a precise 2D spatial memory, thus allowing new insights into Min protein action on the membrane. Taken together, we provide a tool to study phenomena in pattern formation using biological agents., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The MinDE protein system from E. coli has recently been identified as a minimal biological oscillator, based on two proteins only: The ATPase MinD and the ATPase activating protein MinE. In E. coli, the system works as the molecular ruler to place the divisome at midcell for cell division. Despite its compositional simplicity, the molecular mechanism leading to protein patterns and oscillations is still insufficiently understood. Here we used high-speed atomic force microscopy to analyze the mechanism of MinDE membrane association/dissociation dynamics on isolated membrane patches, down to the level of individual point oscillators. This nanoscale analysis shows that MinD association to and dissociation from the membrane are both highly cooperative but mechanistically different processes. We propose that they represent the two directions of a single allosteric switch leading to MinD filament formation and depolymerization. Association/dissociation are separated by rather long apparently silent periods. The membrane-associated period is characterized by MinD filament multivalent binding, avidity, while the dissociated period is defined by seeding of individual MinD. Analyzing association/dissociation kinetics with varying MinD and MinE concentrations and dependent on membrane patch size allowed us to disentangle the essential dynamic variables of the MinDE oscillation cycle.

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.

Bacterial cell division is arguably one of the most central processes in biology. Despite the identification of many important molecular players, surprisingly little is yet known about the underlying physicochemical mechanisms. However, self-organized protein patterns play key roles during division of Escherichia coli, where division is initiated by the directed localization of FtsZ to the cell middle by an inhibitor gradient arising from pole-to-pole oscillations of MinCDE proteins. In vitro reconstitution studies have established that both the Min system and FtsZ with its membrane adaptor FtsA form dynamic energy-dependent patterns on membranes. Furthermore, recent in vivo and in vitro approaches have shown that Min patterns display rich dynamics in diverse geometries and respond to the progress of cytokinesis., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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.

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., (Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.)

In Escherichia coli, a contractile ring (Z-ring) is formed at midcell before cytokinesis. This ring consists primarily of FtsZ, a tubulin-like GTPase, that assembles into protofilaments similar to those in microtubules but different in their suprastructures. The Min proteins MinC, MinD, and MinE are determinants of Z-ring positioning in E. coli. MinD and MinE oscillate from pole to pole, and genetic and biochemical evidence concludes that MinC positions the Z-ring by coupling its assembly to the oscillations by direct inhibitory interaction. The mechanism of inhibition of FtsZ polymerization and, thus, positioning by MinC, however, is not understood completely. Our in vitro reconstitution experiments suggest that the Z-ring consists of dynamic protofilament bundles in which monomers constantly are exchanged throughout, stochastically creating protofilament ends along the length of the filament. From the coreconstitution of FtsZ with MinCDE, we propose that MinC acts on the filaments in two ways: by increasing the detachment rate of FtsZ-GDP within the filaments and by reducing the attachment rate of FtsZ monomers to filaments by occupying binding sites on the FtsZ filament lattice. Furthermore, our data show that the MinCDE system indeed is sufficient to cause spatial regulation of FtsZ, required for Z-ring positioning.

Unlabelled: Bacterial cell division initiates with the formation of a ring-like structure at the cell center composed of the tubulin homolog FtsZ (the Z-ring), which acts as a scaffold for the assembly of the cell division complex, the divisome. Previous studies have suggested that the divisome is initially composed of FtsZ polymers stabilized by membrane anchors FtsA and ZipA, which then recruit the remaining division proteins. The MinCDE proteins prevent the formation of the Z-ring at poles by oscillating from pole to pole, thereby ensuring that the concentration of the Z-ring inhibitor, MinC, is lowest at the cell center. We show that prior to septum formation, the early-division proteins ZipA, ZapA, and ZapB, along with FtsZ, assemble into complexes that counter-oscillate with respect to MinC, and with the same period. We propose that FtsZ molecules distal from high concentrations of MinC form relatively slowly diffusing filaments that are bound by ZapAB and targeted to the inner membrane by ZipA or FtsA. These complexes may facilitate the early stages of divisome assembly at midcell. As MinC oscillates toward these complexes, FtsZ oligomerization and bundling are inhibited, leading to shorter or monomeric FtsZ complexes, which become less visible by epifluorescence microscopy because of their rapid diffusion. Reconstitution of FtsZ-Min waves on lipid bilayers shows that FtsZ bundles partition away from high concentrations of MinC and that ZapA appears to protect FtsZ from MinC by inhibiting FtsZ turnover., Importance: A big issue in biology for the past 100 years has been that of how a cell finds its middle. In Escherichia coli, over 20 proteins assemble at the cell center at the time of division. We show that the MinCDE proteins, which prevent the formation of septa at the cell pole by inhibiting FtsZ, drive the counter-oscillation of early-cell-division proteins ZapA, ZapB, and ZipA, along with FtsZ. We propose that FtsZ forms filaments at the pole where the MinC concentration is the lowest and acts as a scaffold for binding of ZapA, ZapB, and ZipA: such complexes are disassembled by MinC and reform within the MinC oscillation period before accumulating at the cell center at the time of division. The ability of FtsZ to be targeted to the cell center in the form of oligomers bound by ZipA and ZapAB may facilitate the early stages of divisome assembly.

As a spatial modulator of cytokinesis in Escherichia coli, the Min system cooperates with the nucleoid occlusion mechanism to target the divisome assembly towards mid-cell. Based on a reaction-diffusion mechanism powered by ATP (adenosine triphosphate) hydrolysis, the Min proteins propagate in waves on the cell membrane, resulting in oscillations between the cell poles, thus preventing the formation of the division ring everywhere but in the cell centre. The dynamic behaviour of Min proteins has been successfully reconstructed in vitro on supported lipid bilayers (SLBs), reproducing many of the features observed in the cell. However, there has been a marked discrepancy between the speed of propagation of Min protein waves in vitro, compared with the cellular system. A very plausible explanation is the different mobility of proteins on model membranes, compared with the inner membrane of bacteria. To quantitatively demonstrate how membrane diffusion influences Min wave propagation, we compared Min waves on SLBs with free-standing giant unilamellar vesicles (GUV) membranes which display higher fluidity. Intriguingly, the propagation velocity and wavelength on GUVs are three times higher than those reported on supported bilayers, but the wave period is conserved. This suggests that the shorter spatial period of the patterns in vivo might indeed be primarily explained by lower diffusion coefficients of proteins on the bacterial inner membrane., (© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.)

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., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The components of the bacterial division machinery assemble to form a dynamic ring at mid-cell that drives cytokinesis. The nature of most division proteins and their assembly pathway is known. Our knowledge about the biochemical activities and protein interactions of some key division elements, including those responsible for correct ring positioning, has progressed considerably during the past decade. These developments, together with new imaging and membrane reconstitution technologies, have triggered the 'bottom-up' synthetic approach aiming at reconstructing bacterial division in the test tube, which is required to support conclusions derived from cellular and molecular analysis. Here, we describe recent advances in reconstituting Escherichia coli minimal systems able to reproduce essential functions, such as the initial steps of division (proto-ring assembly) and one of the main positioning mechanisms (Min oscillating system), and discuss future perspectives and experimental challenges., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

In the living cell, proteins are able to organize space much larger than their dimensions. In return, changes of intracellular space can influence biochemical reactions, allowing cells to sense their size and shape. Despite the possibility to reconstitute protein self-organization with only a few purified components, we still lack knowledge of how geometrical boundaries affect spatiotemporal protein patterns. Following a minimal systems approach, we used purified proteins and photolithographically patterned membranes to study the influence of spatial confinement on the self-organization of the Min system, a spatial regulator of bacterial cytokinesis, in vitro. We found that the emerging protein pattern responds even to the lateral, two-dimensional geometry of the membrane such that, as in the three-dimensional cell, Min protein waves travel along the longest axis of the membrane patch. This shows that for spatial sensing the Min system does not need to be enclosed in a three-dimensional compartment. Using a computational model we quantitatively analyzed our experimental findings and identified persistent binding of MinE to the membrane as requirement for the Min system to sense geometry. Our results give insight into the interplay between geometrical confinement and biochemical patterns emerging from a nonlinear reaction-diffusion system.

In Escherichia coli, the pole-to-pole oscillation of the Min proteins directs septum formation to midcell, which is required for symmetric cell division. In vitro, protein waves emerge from the self-organization of MinD, a membrane-binding ATPase, and its activator MinE. For wave propagation, the proteins need to cycle through states of collective membrane binding and unbinding. Although MinD presumably undergoes cooperative membrane attachment, it is unclear how synchronous detachment is coordinated. We used confocal and single-molecule microscopy to elucidate the order of events during Min wave propagation. We propose that protein detachment at the rear of the wave, and the formation of the E-ring, are accomplished by two complementary processes: first, local accumulation of MinE due to rapid rebinding, leading to dynamic instability; and second, a structural change induced by membrane-interaction of MinE in an equimolar MinD-MinE (MinDE) complex, which supports the robustness of pattern formation.

In the bacterium Escherichia coli, the Min proteins oscillate between the cell poles to select the cell center as division site. This dynamic pattern has been proposed to arise by self-organization of these proteins, and several models have suggested a reaction-diffusion type mechanism. Here, we found that the Min proteins spontaneously formed planar surface waves on a flat membrane in vitro. The formation and maintenance of these patterns, which extended for hundreds of micrometers, required adenosine 5'-triphosphate (ATP), and they persisted for hours. We present a reaction-diffusion model of the MinD and MinE dynamics that accounts for our experimental observations and also captures the in vivo oscillations.

Recently, utilization of Machine Learning (ML) has led to astonishing progress in computational protein design, bringing into reach the targeted engineering of proteins for industrial and biomedical applications. However, the design of proteins for emergent functions of core relevance to cells, such as the ability to spatiotemporally self-organize and thereby structure the cellular space, is still extremely challenging. While on the generative side conditional generative models and multi-state design are on the rise, for emergent functions there is a lack of tailored screening methods as typically needed in a protein design project, both computational and experimental. Here we describe a proof-of-principle of how such screening, in silico and in vitro, can be achieved for ML-generated variants of a protein that forms intracellular spatiotemporal patterns. For computational screening we use a structure-based divide-and-conquer approach to find the most promising candidates, while for the subsequent in vitro screening we use synthetic cell-mimics as established by Bottom-Up Synthetic Biology. We then show that the best screened candidate can indeed completely substitute the wildtype gene in Escherichia coli. These results raise great hopes for the next level of synthetic biology, where ML-designed synthetic proteins will be used to engineer cellular functions. Here, the authors introduce a pipeline to screen machine learning generated variants of a protein that forms intracellular spatiotemporal patterns in E. coli, demonstrating the best variants can substitute the wildtype gene. [ABSTRACT FROM AUTHOR]

Molecular self-organziation, also regarded as pattern formation, is crucial for the correct distribution of cellular content. The processes leading to spatiotemporal patterns often involve a multitude of molecules interacting in complex networks, so that only very few cellular pattern-forming systems can be regarded as well understood. Due to its compositional simplicity, the Escherichia coli MinCDE system has, thus, become a paradigm for protein pattern formation. This biological reaction diffusion system spatiotemporally positions the division machinery in E. coli and is closely related to ParA-type ATPases involved in most aspects of spatiotemporal organization in bacteria. The ATPase MinD and the ATPase-activating protein MinE self-organize on the membrane as a reaction matrix. In vivo, these two proteins typically oscillate from pole-to-pole, while in vitro they can form a variety of distinct patterns. MinC is a passenger protein supposedly operating as a downstream cue of the system, coupling it to the division machinery. The MinCDE system has helped to extract not only the principles underlying intracellular patterns, but also how they are shaped by cellular boundaries. Moreover, it serves as a model to investigate how patterns can confer information through specific and non-specific interactions with other molecules. Here, we review how the three Min proteins self-organize to form patterns, their response to geometric boundaries, and how these patterns can in turn induce patterns of other molecules, focusing primarily on experimental approaches and developments. [ABSTRACT FROM AUTHOR]

Abstract: Patterns formed by reaction and diffusion are the foundation for many phenomena in biology. However, the experimental study of reaction–diffusion (R–D) systems has so far been dominated by chemical oscillators, for which many tools are available. In this work, we developed a photoswitch for the Min system of Escherichia coli, a versatile biological in vitro R–D system consisting of the antagonistic proteins MinD and MinE. A MinE‐derived peptide of 19 amino acids was covalently modified with a photoisomerizable crosslinker based on azobenzene to externally control peptide‐mediated depletion of MinD from the membrane. In addition to providing an on–off switch for pattern formation, we achieve frequency‐locked resonance with a precise 2D spatial memory, thus allowing new insights into Min protein action on the membrane. Taken together, we provide a tool to study phenomena in pattern formation using biological agents. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The author argues that synthetic biology and quantitative sciences can help provide new concepts and tools in exploring the origin of life. Particular focus is given to the inherent complexity of living systems. Topics discussed include the lack of fundamental distinction between living and nonliving systems on a molecular level, minimal genome approach in synthetic biology, chemoton theory, and the cell division system of Escherichia coli.

For reconstruction of controlled cell division in a minimal cell model, or protocell, a positioning mechanism that spatially regulates division is indispensable. In Escherichia coli, the Min proteins oscillate from pole to pole to determine the division site by inhibition of the primary divisome protein FtsZ anywhere but in the cell middle. Remarkably, when reconstituted under defined conditions in vitro, the Min proteins self-organize into spatiotemporal patterns in the presence of a lipid membrane and ATP. We review recent progress made in studying the Min system in vitro, particularly focusing on the effects of various physicochemical parameters and boundary conditions on pattern formation. Furthermore, we discuss implications and challenges for utilizing the Min system for division site placement in protocells. [ABSTRACT FROM AUTHOR]

In the framework of synthetic biology, it has become an intriguing question what would be the minimal representation of cell division machinery. Thus, it seems appropriate to compare how cell division is realized in different microorganisms. In particular, the cell division system of Crenarchaeota lacks certain proteins found in most bacteria and Euryarchaeota, such as FtsZ, MreB or the Min system. The Sulfolobaceae family encodes functional homologs of the eukaryotic proteins vacuolar protein sorting 4 (Vps4) and endosomal sorting complex required for transport-III (ESCRT-III). ESCRT-III is essential for several eukaryotic pathways, e.g., budding of intraluminal vesicles, or cytokinesis, whereas Vps4 dissociates the ESCRT-III complex from the membrane. Cell Division A (CdvA) is required for the recruitment of crenarchaeal ESCRT-III proteins to the membrane at mid-cell. The proteins polymerize and form a smaller structure during constriction. Thus, ESCRT-III mediated cell division in Sulfolobus acidocaldarius shows functional analogies to the Z ring observed in prokaryotes like Escherichia coli, which has recently begun to be reconstituted in vitro. In this short perspective, we discuss the possibility of building such an in vitro cell division system on basis of archaeal ESCRT-III. [ABSTRACT FROM AUTHOR]

In Escherichia coli, a contractile ring (Z-ring) is formed at midcell before cytokinesis. This ring consists primarily of FtsZ, a tubulinlike GTPase, that assembles into protofilaments similar to those in microtubules but different in their suprastructures. The Min proteins MinC, MinD, and MinE are determinants of Z-ring positioning in E. coli. MinD and MinE oscillate from pole to pole, and genetic and biochemical evidence concludes that MinC positions the Z-ring by coupling its assembly to the oscillations by direct inhibitory interaction. The mechanism of inhibition of FtsZ polymerization and, thus, positioning by MinC, however, is not understood completely. Our in vitro reconstitution experiments suggest that the Z-ring consists of dynamic protofilament bundles in which monomers constantly are exchanged throughout, stochastically creating protofilament ends along the length of the filament. From the coreconstitution of FtsZ with MinCDE, we propose that MinC acts on the filaments in two ways: by increasing the detachment rate of FtsZ-GDP within the filaments and by reducing the attachment rate of FtsZ monomers to filaments by occupying binding sites on the FtsZ filament lattice. Furthermore, our data show that the MinCDE system indeed is sufficient to cause spatial regulation of FtsZ, required for Z-ring positioning. [ABSTRACT FROM AUTHOR]

The rod-shaped bacterium Escherichia coli selects the cell center as site of division with the help of the proteins MinC, MinD, and MinE. This protein system collectively oscillates between the two cell poles by alternately binding to the membrane in one of the two cell halves. This dynamic behavior, which emerges from the interaction of the ATPase MinD and its activator MinE on the cell membrane, has become a paradigm for protein self-organization. Recently, it has been found that not only the binding of MinD to the membrane, but also interactions of MinE with the membrane contribute to Min-protein self-organization. Here, we show that by accounting for this finding in a computational model, we can comprehensively describe all observed Min-protein patterns in vivo and in vitro. Furthermore, by varying the system's geometry, our computations predict patterns that have not yet been reported. We confirm these predictions experimentally. [ABSTRACT FROM AUTHOR]

Although molecular self-organization and pattern formation are key features of life, only very few pattern-forming biochemical systems have been identi1ed that can be reconstituted and studied vitro under de1ned conditions. A systematic understanding of the underlying mechanisms is often hampered by multiple interactions, conformational 2exibility and other complex features of the pattern forming proteins. Because of its compositional simplicity of only two proteins and a membrane, the MinDE system from Escherichia coli has in the past years been invaluable for deciphering the mechanisms of spatiotemporal self-organization in cells. Here we explored the potential of reducing the complexity of this system even further, by identifying key functional motifs in the effector MinE that could be used to design pattern formation from scratch. In a combined approach of experiment and quantitative modeling, we show that starting from a minimal MinE-MinD interaction motif, pattern formation can be obtained by adding either dimerization or membrane-binding motifs. Moreover, we show that the pathways underlying pattern formation are recruitment-driven cytosolic cycling of MinE and recombination of membrane-bound MinE, and that these differ in their in vivo phenomenology. [ABSTRACT FROM AUTHOR]