Will we be ever able to produce living matter artificially? Despite our increasingly precise understanding of the details of life, its fundamental principles still lie in the dark. Armed with today's technology and knowledge about living systems, it is high time for us to re-address this persistent challenge in understanding nature. Graphics: Monika Krause, MPIB.

Werden wir je in der Lage sein, lebende Systeme künstlich zu erzeugen? Trotz unseres immer präziseren Verständnisses verschiedenster Aspekte des Lebens sind seine fundamentalen Prinzipien noch immer nicht wirklich verstanden. Angesichts modernster Technologien und eines immensen Wissenszuwachses in den Biowissenschaften über die letzten Jahrzehnte ist es an der Zeit, sich dieser notorischen Herausforderung im Verständnis der Natur erneut zu widmen. Graphik: Monika Krause, MPIB.

Ceramide is a pro-apoptotic sphingolipid with unique physical characteristics. Often viewed as a second messenger, its generation can modulate the structure of lipid rafts. We prepared three photoswitchable ceramides, ACes, which contain an azobenzene photoswitch allowing for optical control over the N-acyl chain. Using combined atomic force and confocal fluorescence microscopy, we demonstrate that the ACes enable reversible switching of lipid domains in raft-mimicking supported lipid bilayers (SLBs). In the trans-configuration, the ACes localize into the liquid-ordered (Lo) phase. Photoisomerization to the cis-form triggers a fluidification of the Lo domains, as liquid-disordered (Ld) “lakes” are formed within the rafts. Photoisomerization back to the trans-state with blue light stimulates a rigidification inside the Ld phase, as the formation of small Lo domains. These changes can be repeated over multiple cycles, enabling a dynamic spatiotemporal control of the lipid raft structure with light.

We introduce a simple experimental system to study dynamics of needle-like nanoobjects in two dimensions (2D) as a function of their surface density close to the isotropic–nematic transition. Using fluorescence correlation spectroscopy, we find that translational and rotational diffusion of rigid DNA origami nanoneedles bound to freestanding lipid membranes is strongly suppressed upon an increase in the surface particle density. Our experimental observations show a good agreement with results of Monte Carlo simulations of Brownian hard needles in 2D.

The Escherichia coli Min proteins select the cell middle for division by oscillating between the cell poles where they inhibit the divisome protein FtsZ. Reconstitution of Min proteins on a lipid membrane in vitro yields their self-organization into surface waves. In biomimetic compartments, pole-to-pole oscillations can be obtained which direct FtsZ to the middle. This establishes bottom-up synthetic biology as a promising approach to reconstitute complex dynamics and spatial cues in vitro.

Supported lipid bilayers (SLBs) are broadly used as minimal membrane models and commonly produced by vesicle fusion (VF) on solid supports. Despite its advantages, VF does not allow the controlled formation of bilayers that mimic the leaflet asymmetry in lipid composition normally found in biological systems. Here we present a simple, quick, and versatile method to produce SLBs with a desired asymmetric lipid composition which is stable for ca. 4 h. We apply methyl-β-cyclodextrin mediated lipid exchange to SLBs formed by VF to enrich the upper leaflet of the bilayer with sphingomyelin. The bilayer asymmetry is assessed by fluorescence correlation spectroscopy, measuring the lipid mobility separately in each leaflet. To check the compatibility of the method with the most common protein reconstitution approaches, we report the production of asymmetric SLBs (aSLBs) in the presence of a glycosylphosphatidylinositol-anchored protein, reconstituted in the bilayer both, via direct protein insertion, and via proteoliposomes fusion. We finally apply aSLBs to study phase separation and transbilayer lipid movement of raft-mimicking lipid mixtures. The observed differences in terms of phase separation in symmetric and asymmetric SLBs with the same overall lipid composition provide further experimental evidence that the transversal lipid distribution affects the overall lipid miscibility and allow to temporally investigate leaflet mixing.

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.

Glycosaminoglycans (GAGs) are important constituents of extracellular matrices (ECMs). As charged polymers, they do most likely influence lipid and protein dynamics in the outer leaflet of plasma membranes. In this study, we investigated their specific effect, depending on concentration, on lipid diffusion in model membranes. In our assay, GAGs are simply attached electrostatically to supported phospholipid (DOPC) bilayers doped with small amounts of cationic lipid (DOTAP) at physiological pH. Lipid dynamics are characterized via the diffusion of fluorescent lipid analogs (DiD/DiO), determined by fluorescence correlation spectroscopy (FCS). We find that diffusion of DiD is significantly affected by the attachment of GAG. Quite surprisingly, short chains (≤10 disaccharide units) of hyaluronic acid (unsulfated GAG) on the membrane surface affect the DiD diffusion coefficients stronger than medium or long chains (≥100 disaccharide units). In particular, short chains of hyaluronic acids at micromolar concentrations display a 2-fold decrease of the diffusion coefficients compared to the situation without GAG. At nanomolar concentrations of hyaluronic acid of both short and long chains, DiD diffusion remains unaltered. In contrast, sulfated GAGs, such as heparan sulfate (HS) and heparin, affect the lipid diffusion already at sub-micromolar concentrations, albeit not as strongly, with a less than 1.5 fold reduction of the diffusion coefficient. Chondroitin sulfate, another class of sulfated GAGs, did not impose any effect on DiD diffusion in the supported phospholipid bilayer at the concentrations studied. We also investigated desulfated heparin, to explore the role of sulfation and to compare its effect with HA. It is observed that heparin derivatives with lower degrees of sulfation have little effect on the lipid diffusion. Altogether, our results suggest that the presence of certain carbohydrate polymers in the ECM does have a noticeable effect on lipid dynamics in biological membranes.

Fluorescence correlation spectroscopy (FCS) measurements are widely used for determination of diffusion coefficients of lipids and proteins in biological membranes. In recent years, several variants of FCS have been introduced. However, a comprehensive comparison of these methods on identical systems has so far been lacking. In addition, there exist no consistent values of already determined diffusion coefficients for well-known or widely used membrane systems. This study aims to contribute to a better comparability of FCS experiments on membranes by determining the absolute diffusion coefficient of the fluorescent lipid analog 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine (DiD) in giant unilamellar vesicles (GUVs) made of dioleoylphosphatidylcholine (DOPC), which can in future studies be used as a reference value. For this purpose, five FCS variants, employing different calibration methods, were compared. Potential error sources for each particular FCS method and strategies to avoid them are discussed. The obtained absolute diffusion coefficients for DiD in DOPC were in good agreement for all investigated FCS variants. An average diffusion coefficient of D = 10.0 ± 0.4 μm2 s–1 at 23.5 ± 1.5 °C was obtained. The independent confirmation with different methods indicates that this value can be safely used for calibration purposes. Moreover, the comparability of the methods also in the case of slow diffusion was verified by measuring diffusion coefficients of DiD in GUVs consisting of DOPC and cholesterol.

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 the context of minimal systems design, there are two areas in which the reductionist approach has been particularly successful: studies of molecular motors on cytoskeletal filaments, and of protein–lipid interactions in model membranes. However, a minimal cortex, that is, the interface between membrane and cytoskeleton, has just begun to be functionally reconstituted. A key property of living cells is their ability to change their shape in response to extracellular and intracellular stimuli. Although studied in live cells since decades, the mutual dependence between cytoskeleton and membrane dynamics in these large-scale transformations is still poorly understood. Here we report on inspiring recent in vitro work in this direction, and the promises it holds for a better understanding of key cellular processes.Graphical abstractDownload high-res image (278KB)Download full-size imageHighlights► We discuss recent advances in studying membrane–cytoskeleton interactions. ► Membrane deformation and actin structure formation through reconstituted actin networks on lipid bilayers. ► Building minimal cells — methods of actin encapsulation in liposomes. ► Reconstitution of biomimetic actin cortices.

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.

Recently, a new and versatile assay to determine the partitioning coefficient KP as a measure for the affinity of peripheral membrane proteins for lipid bilayers was presented in the research article entitled, “Introducing a fluorescence-based standard to quantify protein partitioning into membranes” [1]. Here, the well-characterized binding of hexahistidine-tag (His6) to NTA(Ni) was utilized. Complementarily, this data article reports the average diffusion coefficient D of His6-tagged enhanced green fluorescent protein (eGFP-His6) and the fluorescent lipid analog ATTO‐647N‐DOPE in giant unilamellar vesicles (GUVs) containing different amounts of NTA(Ni) lipids. In addition, dissociation constants Kd of the NTA(Ni)/eGFP-His6 system are reported. Further, a conversion between Kd and KP is provided.

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.

Bacterial cell division is driven by an FtsZ ring in which the FtsZ protein localizes at mid-cell and recruits other proteins, forming a divisome. In Escherichia coli, the first molecular assembly of the divisome, the proto-ring, is formed by the association of FtsZ polymers to the cytoplasmic membrane through the membrane-tethering FtsA and ZipA proteins. The MinCDE system plays a major role in the site selection of the division ring because these proteins oscillate from pole to pole in such a way that the concentration of the FtsZ-ring inhibitor, MinC, is minimal at the cell center, thus favoring FtsZ assembly in this region. We show that MinCDE drives the formation of waves of FtsZ polymers associated to bilayers by ZipA, which propagate as antiphase patterns with respect to those of Min as revealed by confocal fluorescence microscopy. The emergence of these FtsZ waves results from the displacement of FtsZ polymers from the vicinity of the membrane by MinCD, which efficiently competes with ZipA for the C-terminal region of FtsZ, a central hub for multiple interactions that are essential for division. The coupling between FtsZ polymers and Min is enhanced at higher surface densities of ZipA or in the presence of crowding agents that favor the accumulation of FtsZ polymers near the membrane. The association of FtsZ polymers to the membrane modifies the response of FtsZ to Min, and comigrating Min-FtsZ waves are observed when FtsZ is free in solution and not attached to the membrane by ZipA. Taken together, our findings show that the dynamic Min patterns modulate the spatial distribution of FtsZ polymers in controlled minimal membranes. We propose that ZipA plays an important role in mid-cell recruitment of FtsZ orchestrated by MinCDE.

Over the last decade, functionally designed DNA nanostructures applied to lipid membranes prompted important achievements in the fields of biophysics and synthetic biology. Taking advantage of the universal rules for self-assembly of complementary oligonucleotides, DNA has proven to be an extremely versatile biocompatible building material on the nanoscale. The possibility to chemically integrate functional groups into oligonucleotides, most notably with lipophilic anchors, enabled a widespread usage of DNA as a viable alternative to proteins with respect to functional activity on membranes. As described throughout this review, hybrid DNA-lipid nanostructures can mediate events such as vesicle docking and fusion, or selective partitioning of molecules into phase-separated membranes. Moreover, the major benefit of DNA structural constructs, such as DNA tiles and DNA origami, is the reproducibility and simplicity of their design. DNA nanotechnology can produce functional structures with subnanometer precision and allow for a tight control over their biochemical functionality, e.g., interaction partners. DNA-based membrane nanopores and origami structures able to assemble into two-dimensional networks on top of lipid bilayers are recent examples of the manifold of complex devices that can be achieved. In this review, we will shortly present some of the potentially most relevant avenues and accomplishments of membrane-anchored DNA nanostructures for investigating, engineering, and mimicking lipid membrane-related biophysical processes.

Diffusion of lipids and proteins within the cell membrane is essential for numerous membrane-dependent processes including signaling and molecular interactions. It is assumed that the membrane-associated cytoskeleton modulates lateral diffusion. Here, we use a minimal actin cortex to directly study proposed effects of an actin meshwork on the diffusion in a well-defined system. The lateral diffusion of a lipid and a protein probe at varying densities of membrane-bound actin was characterized by fluorescence correlation spectroscopy (FCS). A clear correlation of actin density and reduction in mobility was observed for both the lipid and the protein probe. At high actin densities, the effect on the protein probe was ∼3.5-fold stronger compared to the lipid. Moreover, addition of myosin filaments, which contract the actin mesh, allowed switching between fast and slow diffusion in the minimal system. Spot variation FCS was in accordance with a model of fast microscopic diffusion and slower macroscopic diffusion. Complementing Monte Carlo simulations support the analysis of the experimental FCS data. Our results suggest a stronger interaction of the actin mesh with the larger protein probe compared to the lipid. This might point toward a mechanism where cortical actin controls membrane diffusion in a strong size-dependent manner.

We study the effect of a minimal cytoskeletal network formed on the surface of giant unilamellar vesicles by the prokaryotic tubulin homolog, FtsZ, on phase separation in freestanding lipid membranes. FtsZ has been modified to interact with the membrane through a membrane targeting sequence from the prokaryotic protein MinD. FtsZ with the attached membrane targeting sequence efficiently forms a highly interconnected network on membranes with a concentration-dependent mesh size, much similar to the eukaryotic cytoskeletal network underlying the plasma membrane. Using giant unilamellar vesicles formed from a quaternary lipid mixture, we demonstrate that the artificial membrane-associated cytoskeleton, on the one hand, suppresses large-scale phase separation below the phase transition temperature, and, on the other hand, preserves phase separation above the transition temperature. Our experimental observations support the ideas put forward in our previous simulation study: In particular, the picket fence effect on phase separation may explain why micrometer-scale membrane domains are observed in isolated, cytoskeleton-free giant plasma membrane vesicles, but not in intact cell membranes. The experimentally observed suppression of large-scale phase separation much below the transition temperatures also serves as an argument in favor of the cryoprotective role of the cytoskeleton.

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.