Stephan Nussberger

Controlled insertion of a single protein pore, also referred to as a nanopore, into a free-standing lipid bilayer underpins high-resolution single-channel electrophysiology. Hitherto, conventional bulk reconstitution is stochastic, slow, and prone to multiple insertions. To overcome this, we adapted a hydrogel-confined delivery strategy in which an agarose-filled glass capillary delivers nanopores directly to the bilayer. An optimal window of agarose concentration, aerolysin concentration, and membrane potential raised the single-pore yield to >70% and shortened the waiting time to the first insertion from tens of minutes to a few minutes, without altering native pore conductance. The approach generalizes across the β-barrel toxin aerolysin, the mitochondrial TOM core complex, and a DNA nanopore, making nanopore insertion rapid, controlled, and reproducible.

Bacterial contractile injection systems provide a model for membrane penetration and targeted delivery of molecular cargo through mechanical actuation. Replicating these features in synthetic nanoscale systems remains challenging, particularly with respect to coupling structural organization with dynamic actuation, reversibility and spatiotemporal regulation. Here we report a DNA origami nanosyringe that integrates these capabilities to enable programmable membrane translocation. The DNA origami nanosyringe comprises two ~70-nm DNA origami bundles crosslinked by a ~10-nm gold nanoparticle. One bundle forms a cholesterol-functionalized membrane-anchoring base, whereas the other serves as a DNA fuel-driven sliding needle. After binding to supported lipid bilayers or vesicle membranes, DNA-fuel actuation drives the needle downwards in ~14-nm steps, thereby enabling membrane penetration, while reverse actuation retracts the needle and promotes membrane resealing. We show that this device provides controllable delivery of cargo tethered to the needle tip into lipid-bounded compartments and can regulate biochemical processes within cell-sized environments, including membrane-localized hybridization chain reactions, RNA transcription and catalytic RNA cleavage. These results demonstrate a strategy for constructing dynamic DNA devices that operate at membrane interfaces and coordinate mechanical actuation with biochemical function.

Currently, there is no comprehensive physical model explaining how unfolded polypeptide chains with diverse characteristics are transported into the mitochondria. On a molecular scale, the kinetics of how transit polypeptides approach, are captured by the protein translocation machinery at the outer mitochondrial membrane, and cross the protein translocation pore to enter the intermembrane space remain unclear. This knowledge gap is primarily due to the lack of dynamic single-molecule data on the “protein-conducting channels” involved in mitochondrial protein translocation. In this chapter, we explore the recently resolved sub-nanometer cryo-EM structures, which are a prerequisite for a fundamental understanding of the translocation mechanism, and our existing knowledge of the mitochondrial two-pore outer membrane protein translocation machinery (TOM complex). Particularly intriguing are recent findings from single-molecule TIRF microscopy indicating that the TOM core complex can function as a mechanosensor, with the pores closing upon interaction with nearby membrane structures. We emphasize novel and unexpected correlations between the structural components of the TOM complexes and their dynamic behavior within the membrane environment.

Reconstructing cellular complexity is a central challenge in synthetic biology, with profound implications for understanding life and advancing bio-inspired nanotechnologies. A critical step towards this goal is replicating the dynamic interplay among membrane components and their functions. Here we demonstrate a double-necked synthetic cell microreactor (DCM) that incorporates two dynamic, DNA-based pores in the membrane of a giant unilamellar vesicle. The formation of the DCM leverages a signalling pathway mediated by giant unilamellar vesicle membrane dynamics to coordinate interactions between light-responsive small pores and self-arranged sealable large pores. This system enables sequential, on-demand delivery of molecular reactants with high spatiotemporal precision. Using DCMs, we demonstrate confined biochemical reactions, including a glucose oxidase–myoglobin cascade, cytoskeleton-mimetic actin polymerization and bundling, cell-free Spinach RNA transcription and the synthesis of three-dimensional DNA crystals that extend beyond natural systems. By coupling orchestrated multistep signalling with dynamic control of membrane permeability, the DCM establishes a versatile platform for emulating and expanding the functional complexity of natural cellular systems.

Mitochondria rely on the efficient import of proteins to maintain their functions and regenerate. The translocase of the outer mitochondrial membrane (TOM) complex serves as the primary entry point for the import of mitochondrial proteins. Previous studies have established Tom22 as a multifunctional subunit within the complex and reported mechanosensitive gating-like behavior of the TOM complex. In this study, all-atom molecular dynamics simulations of the TOM core complex reveal large motions of the Tom22 helices that are coupled to global structural rearrangements within the complex, particularly with the α2 helix within the Tom40 pore subunit. Microseconds-long simulations with restraints on the Tom22 helices yield an alternative conformation of the α2 helix that is associated with a reduced ion permeability. The outcome corroborates previous experimental results that reported a reduction in calcium ion flux for transiently stalled TOM complexes. These findings provide a molecular view of a mechanism by which Tom22 modulates the pore architecture of Tom40 and regulates permeability, thus linking the receptor dynamics to the functional control of the mitochondrial protein import.

The shape of biological matter is central to cell function at different length scales and determines how cellular components recognize, interact and respond to one another. However, their shapes are often transient and hard to reprogramme. Here we construct a synthetic cell model composed of signal-responsive DNA nanorafts, biogenic pores and giant unilamellar vesicles (GUVs). We demonstrate that reshaping of DNA rafts at the nanoscale can be coupled to reshaping of GUVs at the microscale. The nanorafts collectively undergo reversible transitions between isotropic and short-range local order on the lipid membrane, programmably remodelling the GUV shape. Assisted by the biogenic pores, during GUV shape recovery the locally ordered DNA rafts perforate the membrane, forming sealable synthetic channels for large cargo transport. Our work outlines a versatile platform for interfacing reconfigurable DNA nanostructures with synthetic cells, expanding the potential of DNA nanotechnology in synthetic biology.

Proteins self-assemble to function in living cells. They may execute essential tasks in the form of monomers, complexes, or supramolecular cages via oligomerization, achieving a sophisticated balance between structural topology and functional dynamics. The modularity and programmability make DNA origami unique in mimicking these key features. Here, we demonstrate three-dimensional reconfigurable DNA origami pincers (DOPs) that multitask on giant unilamellar vesicles (GUVs). By programmably adjusting their pinching angle, the DOPs can dynamically control the degree of GUV remodeling. When oligomerized on the GUV to form origami cages, the DOP units interact with one another and undergo reorganization, resulting in the capture, compartmentalization, and detachment of lipid fragments. This oligomerization process is accompanied with membrane disruptions, enabling the passage of cargo across the membrane. We envisage that interfacing synthetic cells with engineered, multifunctional DNA nanostructures may help to confer customized cellular properties, unleashing the potential of both fields.

To date, there is no general physical model of the mechanism by which unfolded polypeptide chains with different properties are imported into the mitochondria. At the molecular level, it is still unclear how transit polypeptides approach, are captured by the protein translocation machinery in the outer mitochondrial membrane, and how they subsequently cross the entropic barrier of a protein translocation pore to enter the intermembrane space. This deficiency has been due to the lack of detailed structural and dynamic information about the membrane pores. In this review, we focus on the recently determined sub-nanometer cryo-EM structures and our current knowledge of the dynamics of the mitochondrial two-pore outer membrane protein translocation machinery (TOM core complex), which provide a starting point for addressing the above questions. Of particular interest are recent discoveries showing that the TOM core complex can act as a mechanosensor, where the pores close as a result of interaction with membrane-proximal structures. We highlight unusual and new correlations between the structural elements of the TOM complexes and their dynamic behavior in the membrane environment.

Total interference reflection fluorescence (TIRF) microscopy of lipid bilayers is an effective technique for studying the lateral movement and ion channel activity of single integral membrane proteins. Here we describe how to integrate the mitochondrial outer membrane preprotein translocase TOM-CC and its β-barrel protein-conducting channel Tom40 into supported lipid bilayers to identify possible relationships between movement and channel activity. We propose that our approach can be readily applied to membrane protein channels where transient tethering to either membrane-proximal or intramembrane structures is accompanied by a change in channel permeation. Key words TOM β-barrel membrane protein Channels Mitochondria Protein translocation Single molecule TIRF microscopy

The TOM complex is the main entry point for precursor proteins (preproteins) into mitochondria. Preproteins containing targeting sequences are recognized by the TOM complex and imported into mitochondria. We have determined the structure of the TOM core complex from Neurospora crassa by single-particle electron cryomicroscopy at 3.3 Å resolution, showing its interaction with a bound preprotein at 4 Å resolution, and of the TOM holo complex including the Tom20 receptor at 6 to 7 Å resolution. TOM is a transmembrane complex consisting of two β-barrels, three receptor subunits, and three short transmembrane subunits. Tom20 has a transmembrane helix and a receptor domain on the cytoplasmic side. We propose that Tom20 acts as a dynamic gatekeeper, guiding preproteins into the pores of the TOM complex. We analyze the interactions of Tom20 with other TOM subunits, present insights into the structure of the TOM holo complex, and suggest a translocation mechanism.