The healthy growth and maintenance of a biological system depends on the precise spatial organization of molecules within the cell through the dissipation of energy. Reaction–diffusion mechanisms can facilitate this organization, as can directional cargo transport orchestrated by motor proteins, by relying on specific protein interactions. However, transport of material through the cell can also be achieved by active processes based on non-specific, purely physical mechanisms, a phenomenon that remains poorly explored. Here, using a combined experimental and theoretical approach, we discover and describe a hidden function of the Escherichia coli MinDE protein system: in addition to forming dynamic patterns, this system accomplishes the directional active transport of functionally unrelated cargo on membranes. Remarkably, this mechanism enables the sorting of diffusive objects according to their effective size, as evidenced using modular DNA origami–streptavidin nanostructures. We show that the diffusive fluxes of MinDE and non-specific cargo couple via density-dependent friction. This non-specific process constitutes a diffusiophoretic mechanism, as yet unknown in a cell biology setting. This nonlinear coupling between diffusive fluxes could represent a generic physical mechanism for establishing intracellular organization.

Upscaling motor protein activity to perform work in man-made devices has long been an ambitious goal in bionanotechnology. The use of hierarchical motor assemblies, as realized in sarcomeres, has so far been complicated by the challenges of arranging sufficiently high numbers of motor proteins with nanoscopic precision. Here, we describe an alternative approach based on actomyosin cortex-like force production, allowing low complexity motor arrangements in a contractile meshwork that can be coated onto soft objects and locally activated by ATP. The design is reminiscent of a motorized exoskeleton actuating protein-based robotic structures from the outside. It readily supports the connection and assembly of micro-three-dimensional printed modules into larger structures, thereby scaling up mechanical work. We provide an analytical model of force production in these systems and demonstrate the design flexibility by three-dimensional printed units performing complex mechanical tasks, such as microhands and microarms that can grasp and wave following light activation.

One of the great challenges of bottom-up synthetic biology is to recreate the cellular geometry and surface functionality required for biological reactions. Of particular interest are lipid membrane interfaces where...

Giant unilamellar phospholipid vesicles are attractive starting points for constructing minimal living cells from the bottom‐up. Their membranes are compatible with many physiologically functional modules and act as selective barriers, while retaining a high morphological flexibility. However, their spherical shape renders them rather inappropriate to study phenomena that are based on distinct cell shape and polarity, such as cell division. Here, a microscale device based on 3D printed protein hydrogel is introduced to induce pH‐stimulated reversible shape changes in trapped vesicles without compromising their free‐standing membranes. Deformations of spheres to at least twice their aspect ratio, but also toward unusual quadratic or triangular shapes can be accomplished. Mechanical force induced by the cages to phase‐separated membrane vesicles can lead to spontaneous shape deformations, from the recurrent formation of dumbbells with curved necks between domains to full budding of membrane domains as separate vesicles. Moreover, shape‐tunable vesicles are particularly desirable when reconstituting geometry‐sensitive protein networks, such as reaction‐diffusion systems. In particular, vesicle shape changes allow to switch between different modes of self‐organized protein oscillations within, and thus, to influence reaction networks directly by external mechanical cues.

as compartmentalization, transport of molecules, metabolism, growth, and ideally, replication through cell division. The latter is one of the hallmarks of life and one of the most intriguing phenomena exhibited by living organisms. [1] Beyond that, synthetic cells could comprise features that biological ones lack, such as an increased stability against environmental conditions, or much simplified programmability for desired new functions to be utilized in (bio)technological applications. [2] One obvious strategy for designing such hybrid protocells includes encapsulating components of the active cell machinery into artificial cell-like compartments. The most prominent methods involve the use of liposomes, which are lipid bilayer compartments that mimic cells with respect to their size and provide basic biochemical functionalities. [3] In these systems, reconstitution of minimal components of cell division has been demonstrated. [3b] A notable example is the MinCDE protein system, which is the spatial regulator of cell division for manyThe integration of active cell machinery with synthetic building blocks is the bridge toward developing synthetic cells with biological functions and beyond. Self-replication is one of the most important tasks of living systems, and various complex machineries exist to execute it. In Escherichia coli, a contractile division ring is positioned to mid-cell by concentration oscillations of self-organizing proteins (MinCDE), where it severs membrane and cell wall. So far, the reconstitution of any cell division machinery has exclusively been tied to liposomes. Here, the reconstitution of a rudimentary bacterial divisome in fully synthetic bicomponent dendrimersomes is shown. By tuning the membrane composition, the interaction of biological machinery with synthetic membranes can be tailored to reproduce its dynamic behavior. This constitutes an important breakthrough in the assembly of synthetic cells with biological elements, as tuning of membrane-divisome interactions is the key to engineering emergent biological behavior from the bottom-up.

In bottom-up synthetic biology, one of the major methodological challenges is to provide reaction spaces that mimic biological systems with regard to topology and surface functionality. Of particular interest are cell- or organelle-shaped membrane compartments, as many protein functions unfold at lipid interfaces. However, shaping artificial cell systems using materials with non-intrusive physicochemical properties, while maintaining flexible lipid interfaces relevant to the reconstituted protein systems, is not straightforward. Herein, we develop micropatterned chambers from CYTOP, a less commonly used polymer with good chemical resistance and a refractive index matching that of water. By forming a self-assembled lipid monolayer on the polymer surface, we dramatically increased the biocompatibility of CYTOP-fabricated systems. The phospholipid interface provides an excellent passivation layer to prevent protein adhesion to the hydrophobic surface, and we succeeded in cell-free protein synthesis inside the chambers. Importantly, the chambers could be sealed after loading by a lipid monolayer, providing a novel platform to study encapsulated systems. We successfully reconstituted pole-to-pole oscillations of the Escherichia coli MinDE system, which responds dramatically to compartment geometry. Furthermore, we present a simplified fabrication of our artificial cell compartments via replica molding, making it a readily accessible technique for standard cleanroom facilities.

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Cell homeostasis requires the maintenance of heterogeneous membrane compositions through both vesicular and non-vesicular transport mechanisms. Yet, our biophysical description of non-vesicular lipid transport remains incomplete. To help fill these gaps, we aimed to identify an accurate reaction coordinate for passive lipid exchange between membranes. Towards this goal, we investigated the elementary steps of lipid exchange, lipid desorption and insertion into a membrane, using molecular simulation. From over 1,000 lipid insertion trajectories of all-atom and coarse-grained lipid models, we discovered a free energy barrier for lipid insertion and identified multiple pathways characterized by splayed lipid intermediates. This barrier appears hidden when only the lipid's displacement normal to the bilayer, which has traditionally been used to describe lipid exchange, is monitored. In contrast, an accurate reaction coordinate measures the breakage and formation of hydrophobic lipid-membrane contacts, which give rise to a barrier for lipid insertion. At the transition state, hydrophobic contacts are just as likely to form as they are to break. Consistent with this fact, membrane distortions and solvent fluctuations, which can both enable and prevent hydrophobic contact formation, are observed in the transition state ensemble. Overall, our results demonstrate that the formation and breakage of hydrophobic contacts is rate limiting for passive lipid exchange and provide a foundation to understand the catalytic function of lipid transfer proteins.