Reversible membrane deformations by straight DNA origami filaments.

Membrane-active cytoskeletal elements, such as FtsZ, septin or actin, form filamentous polymers able to induce and stabilize curvature on cellular membranes. In order to emulate the characteristic dynamic self-assembly properties of cytoskeletal subunits in vitro, biomimetic synthetic scaffolds were here developed using DNA origami. In contrast to our earlier work with pre-curved scaffolds, we specifically assessed the potential of origami mimicking straight filaments, such as actin and microtubules, by origami presenting cholesteryl anchors for membrane binding and additional blunt end stacking interactions for controllable polymerization into linear filaments. By assessing the interaction of our DNA nanostructures with model membranes using fluorescence microscopy, we show that filaments can be formed, upon increasing MgCl2 in solution, for structures displaying blunt ends; and can subsequently depolymerize, by decreasing the concentration of MgCl2. Distinctive spike-like membrane protrusions were generated on giant unilamellar vesicles at high membrane-bound filament densities, and the presence of such deformations was reversible and shown to correlate with the MgCl2-triggered polymerization of DNA origami subunits into filamentous aggregates. In the end, our approach reveals the formation of membrane-bound filaments as a minimal requirement for membrane shaping by straight cytoskeletal-like objects

Shaping Giant Membrane Vesicles in 3D-Printed Protein Hydrogel Cages

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

DNA Nanostructures on Membranes as Tools for Synthetic Biology

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

Membrane-coated 3D architectures for bottom-up synthetic biology dagger

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 many protein functions take place. However, cellular 3D geometries are often complex, and custom-shaping stable lipid membranes on relevant spatial scales in the micrometer range has been hard to accomplish reproducibly. Here, we use two-photon direct laser writing to 3D print microenvironments with length scales relevant to cellular processes and reactions. We formed lipid bilayers on the surfaces of these printed structures, and we evaluated multiple combinatorial scenarios, where physiologically relevant membrane compositions were generated on several different polymer surfaces. Functional dynamic protein systems were reconstituted in vitro and their self-organization was observed in response to the 3D geometry. This method proves very useful to template biological membranes with an additional spatial dimension, and thus allows a better understanding of protein function in relation to the complex morphology of cells and organelles.We also thank the Biochemistry Core Facility of the Max Planck Institute of Biochemistry for assistance with protein purification, and the Imaging Core Facility of the same institution for assistance on the 4D image visualisation

Non-Equilibrium Large-Scale Membrane Transformations Driven by MinDE Biochemical Reaction Cycles

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.We thank MPI‐B Core Facility for assistance in protein purification

Membrane-Mediated Self-Organization of Rod-Like DNA Origami on Supported Lipid Bilayers

Organization of elongated particles into ordered phases on 2D surfaces and interfaces has been extensively studied during the last decades both theoretically and experimentally. For mutually repulsive particles on solid nondeformable substrates, the process is controlled only by the aspect ratio and the surface density of the adsorbed particles. The local elastic response of soft substrates to particle adhesion can drastically change the collective behavior of adsorbed rod-like particles resulting in their self-organization via substrate-mediated interparticle attraction. Here, high-speed atomic force microscopy is used to study the organization of DNA origami particles on locally responsive supported lipid bilayers (SLBs) in comparison with that on nondeformable solid mica surfaces. At high surface coverage, the aspect ratio-dependent anisotropic phases expected for densely packed particles are observed. At intermediate and low surface densities, however, a drastically different phenomenology is observed: surprisingly strong surface-mediated interparticle attraction of DNA origami particles is found on SLBs resulting in their self-organization compared to their purely repulsive interaction on a mica surface. The formation of organized aggregates of elongated DNA origami particles on SLBs is explained by exceptionally strong nanoparticle adhesion to the membrane that responds with a local deformation in spite of the presence of the solid support

Features of MOG required for recognition by patients with MOG antibody-associated disorders

Antibodies to myelin oligodendrocyte glycoprotein (MOG-Abs) define a distinct disease entity. Here we aimed to understand essential structural features of MOG required for recognition by autoantibodies from patients. We produced the N-terminal part of MOG in a conformationally correct form; this domain was insufficient to identify patients with MOG-Abs by ELISA even after site-directed binding. This was neither due to a lack of lipid embedding nor to a missing putative epitope at the C-terminus, which we confirmed to be an intracellular domain. When MOG was displayed on transfected cells, patients with MOG-Abs recognized full-length MOG much better than its N-terminal part with the first hydrophobic domain (P < 0.0001). Even antibodies affinity-purified with the extracellular part of MOG recognized full-length MOG better than the extracellular part of MOG after transfection. The second hydrophobic domain of MOG enhanced the recognition of the extracellular part of MOG by antibodies from patients as seen with truncated variants of MOG. We confirmed the pivotal role of the second hydrophobic domain by fusing the intracellular part of MOG from the evolutionary distant opossum to the human extracellular part; the chimeric construct restored the antibody binding completely. Further, we found that in contrast to 8-18C5, MOG-Abs from patients bound preferentially as F(ab')(2) rather than Fab. It was previously found that bivalent binding of human IgG1, the prominent isotype of MOG-Abs, requires that its target antigen is displayed at a distance of 13-16 nm. We found that, upon transfection, molecules of MOG did not interact so closely to induce a Forster resonance energy transfer signal, indicating that they are more than 6 nm apart. We propose that the intracellular part of MOG holds the monomers apart at a suitable distance for bivalent binding; this could explain why a cell-based assay is needed to identify MOG-Abs. Our finding that MOG-Abs from most patients require bivalent binding has implications for understanding the pathogenesis of MOG-Ab associated disorders. Since bivalently bound antibodies have been reported to only poorly bind C1q, we speculate that the pathogenicity of MOG-Abs is mostly mediated by other mechanisms than complement activation. Therefore, therapeutic inhibition of complement activation should be less efficient in MOG-Ab associated disorders than in patients with antibodies to aquaporin-4

Revolving around constriction by ESCRT-III

The endosomal sorting complex required for transport (ESCRT)-III is critical for membrane abscission; however, the mechanism underlying ESCRT-III-mediated membrane constriction remains elusive. A study of the dynamic assembly and disassembly of the ESCRT-III machinery in vitro and in vivo now suggests that the turnover of the observed spiralling filaments is critical for membrane abscission during cytokinesis

Molecular Mechanism of Autophagic Membrane-Scaffold Assembly and Disassembly

Autophagy is a catabolic pathway that sequesters undesired cellular material into autophagosomes for delivery to lysosomes for degradation. A key step in the pathway is the covalent conjugation of the ubiquitin-related protein Atg8 to phosphatidylethanolamine (Atg8-PE) in autophagic membranes by a complex consisting of Atg16 and the Atg12Atg5 conjugate. Atg8 controls the expansion of autophagic precursor membranes, but the underlying mechanism remains unclear. Here, we reconstitute Atg8 conjugation on giant unilamellar vesicles and supported lipid bilayers. We found that Atg8-PE associates with Atg12-Atg5-Atg16 into a membrane scaffold. By contrast, scaffold formation is counteracted by the mitochondrial cargo adaptor Atg32 through competition with Atg12-Atg5 for Atg8 binding. Atg4, previously known to recycle Atg8 from membranes, disassembles the scaffold. Importantly, mutants of Atg12 and Atg16 deficient in scaffold formation in vitro impair autophagy in vivo. This suggests that autophagic scaffolds are critical for phagophore biogenesis and thus autophagy