Membrane abscission, the final cut of the last connection between emerging daughter cells, is an indispensable event in the last stage of cell division and in other cellular processes such as endocytosis, virus release or bacterial sporulation. However, its mechanism remains poorly understood, impeding its application as a cell-division machinery for synthetic cells. Here we use fluorescence microscopy and fluorescence recovery after photobleaching measurements to study the in vitro reconstitution of the bacterial protein dynamin A inside liposomes. Upon external reshaping of the liposomes into dumbbells, dynamin A self-assembles at the membrane neck, resulting in membrane hemi-scission and even full scission. Dynamin A proteins constitute a simple one-component division machinery capable of splitting dumbbell-shaped liposomes, marking an important step towards building a synthetic cell.

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The nuclear pore complex (NPC) regulates the selective transport of large biomolecules through the nuclear envelope. As a model system for nuclear transport, we construct NPC mimics by functionalizing the pore walls of freestanding palladium zero-mode waveguides with the FG-nucleoporin Nsp1. This approach enables the measurement of single-molecule translocations through individual pores using optical detection. We probe the selectivity of Nsp1-coated pores by quantitatively comparing the translocation rates of the nuclear transport receptor Kap95 to the inert probe BSA over a wide range of pore sizes from 35 nm to 160 nm. Pores below 55 ± 5 nm show significant selectivity that gradually decreases for larger pores. This finding is corroborated by coarse-grained molecular-dynamics simulations of the Nsp1 mesh within the pore, which suggest that leakage of BSA occurs by diffusion through transient openings within the dynamic mesh. Furthermore, we experimentally observe a modulation of the BSA permeation when varying the concentration of Kap95. The results demonstrate the potential of single-molecule fluorescence measurements on biomimetic NPCs to elucidate the principles of nuclear transport.

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Nuclear pore complexes (NPCs) regulate all molecular transport between the nucleus and the cytoplasm in eukaryotic cells. Intrinsically disordered Phe-Gly nucleoporins (FG-Nups) line the central conduit of NPCs to impart a selective barrier where large proteins are excluded unless bound to a transport receptor (karyopherin; Kap). Here, we assess “Kap-centric” NPC models, which postulate that Kaps participate in establishing the selective barrier. We combine biomimetic nanopores, formed by tethering Nsp1 to the inner wall of a solid-state nanopore, with coarse-grained modeling to show that yeast Kap95 exhibits two populations in Nsp1-coated pores: one population that is transported across the pore in milliseconds, and a second population that is stably assembled within the FG mesh of the pore. Ionic current measurements show a conductance decrease for increasing Kap concentrations and noise data indicate an increase in rigidity of the FG-mesh. Modeling reveals an accumulation of Kap95 near the pore wall, yielding a conductance decrease. We find that Kaps only mildly affect the conformation of the Nsp1 mesh and that, even at high concentrations, Kaps only bind at most 8% of the FG-motifs in the nanopore, indicating that Kap95 occupancy is limited by steric constraints rather than by depletion of available FG-motifs. Our data provide an alternative explanation of the origin of bimodal NPC binding of Kaps, where a stable population of Kaps binds avidly to the NPC periphery, while fast transport proceeds via a central FG-rich channel through lower affinity interactions between Kaps and the cohesive domains of Nsp1. [Figure not available: see fulltext.]

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Inspired by the biological processes of molecular recognition and transportation across membranes, nanopore techniques have evolved in recent decades as ultrasensitive analytical tools for individual molecules. In particular, nanopore-based single-molecule DNA/RNA sequencing has advanced genomic and transcriptomic research due to the portability, lower costs and long reads of these methods. Nanopore applications, however, extend far beyond nucleic acid sequencing. In this Review, we present an overview of the broad applications of nanopores in molecular sensing and sequencing, chemical catalysis and biophysical characterization. We highlight the prospects of applying nanopores for single-protein analysis and sequencing, single-molecule covalent chemistry, clinical sensing applications for single-molecule liquid biopsy, and the use of synthetic biomimetic nanopores as experimental models for natural systems. We suggest that nanopore technologies will continue to be explored to address a number of scientific challenges as control over pore design improves.

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Molecular traffic across lipid membranes is a vital process in cell biology that involves specialized biological pores with a great variety of pore diameters, from fractions of a nanometer to >30 nm. Creating artificial membrane pores covering similar size and complexity will aid the understanding of transmembrane molecular transport in cells, while artificial pores are also a necessary ingredient for synthetic cells. Here, we report the construction of DNA origami nanopores that have an inner diameter as large as 30 nm. We developed methods to successfully insert these ultrawide pores into the lipid membrane of giant unilamellar vesicles (GUVs) by administering the pores concomitantly with vesicle formation in an inverted-emulsion cDICE technique. The reconstituted pores permit the transmembrane diffusion of large macromolecules, such as folded proteins, which demonstrates the formation of large membrane-spanning open pores. The pores are size selective, as dextran molecules with a diameter up to 28 nm can traverse the pores, whereas larger dextran molecules are blocked. By FRAP measurements and modeling of the GFP influx rate, we find that up to hundreds of pores can be functionally reconstituted into a single GUV. Our technique bears great potential for applications across different fields from biomimetics, to synthetic biology, to drug delivery.

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Nuclear Pore Complexes (NPCs) regulate bidirectional transport between the nucleus and the cytoplasm. Intrinsically disordered FG-Nups line the NPC lumen and form a selective barrier, where transport of most proteins is inhibited whereas specific transporter proteins freely pass. The mechanism underlying selective transport through the NPC is still debated. Here, we reconstitute the selective behaviour of the NPC bottom-up by introducing a rationally designed artificial FG-Nup that mimics natural Nups. Using QCM-D, we measure selective binding of the artificial FG-Nup brushes to the transport receptor Kap95 over cytosolic proteins such as BSA. Solid-state nanopores with the artificial FG-Nups lining their inner walls support fast translocation of Kap95 while blocking BSA, thus demonstrating selectivity. Coarse-grained molecular dynamics simulations highlight the formation of a selective meshwork with densities comparable to native NPCs. Our findings show that simple design rules can recapitulate the selective behaviour of native FG-Nups and demonstrate that no specific spacer sequence nor a spatial segregation of different FG-motif types are needed to create selective NPCs.

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At first glance nanopores may appear simple, almost intuitive, to understand given that they are, quite literally, ‘just’ very small pores in a membrane. In fact, one may even wonder why at all we need trained scientists to study such seemingly simple entities. The short answer is that nanopores, as the word suggests, are nanoscale entities and, as such, one can not directly see or experience any of the events that occur down there. The long answer can be found in this thesis. Here, I present and discuss a wide array of nanopores, from biological nanopores like the nuclear pore complex (NPC), to solidstate nanopores, and DNA-origami nanopores. While the central focus of my research is to understand the inner workings of the NPC, a short journey into the world of ion transport in solid-state nanopores is first undertaken, with special emphasis on the random fluctuations of the ion flow within the nanopore, referred to as current noise. Next, I introduce the concept of biomimetic nanopores, where a solid-state nanopore is ‘camouflaged’ by coating its inner surface with purified proteins, resulting in an entity that behaves somewhat like a real NPC. Biomimetic nanopores have enabled us to mimic, study, and gain new insights into how the real NPC works, and bear great potential for further developments and discoveries. @en

Nanopores bear great potential as single-molecule tools for bioanalytical sensing and sequencing, due to their exceptional sensing capabilities, high-throughput, and low cost. The detection principle relies on detecting small differences in the ionic current as biomolecules traverse the nanopore. A major bottleneck for the further progress of this technology is the noise that is present in the ionic current recordings, because it limits the signal-to-noise ratio (SNR) and thereby the effective time resolution of the experiment. Here, we review the main types of noise at low and high frequencies and discuss the underlying physics. Moreover, we compare biological and solid-state nanopores in terms of the SNR, the important figure of merit, by measuring translocations of a short ssDNA through a selected set of nanopores under typical experimental conditions. We find that SiN_x solid-state nanopores provide the highest SNR, due to the large currents at which they can be operated and the relatively low noise at high frequencies. However, the real game-changer for many applications is a controlled slowdown of the translocation speed, which for MspA was shown to increase the SNR > 160-fold. Finally, we discuss practical approaches for lowering the noise for optimal experimental performance and further development of the nanopore technology.

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The performance of solid-state nanopores as promising biosensors is severely hampered by low-frequency 1/f noise in the through-pore ionic current recordings. Here, we develop a model for the 1/f noise in such nanopores, that, unlike previous reports, accounts for contributions from both the pore-cylinder, pore-surface, and access regions. To test our model, we present measurements of the open-pore current noise through solid-state nanopores of different diameters (1-50 nm). To describe the observed trends, it appears essential to include the access resistance in the modeling of the 1/f noise. We attribute a different Hooge constant for the charge carrier fluctuations occurring in the bulk electrolyte and at the pore surface. The model reported here can be used to accurately analyze different contributions to the nanopore low-frequency noise, rendering it a powerful tool for characterizing and comparing different membrane materials in terms of their 1/f noise properties.

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Embedded extrusion bioprinting allows for the generation of complex structures that otherwise cannot be achieved with conventional layer-by-layer deposition from the bottom, by overcoming the limits imposed by gravitational force. By taking advantage of a hydrogel bath, serving as a sacrificial printing environment, it is feasible to extrude a bioink in freeform until the entire structure is deposited and crosslinked. The bioprinted structure can be subsequently released from the supporting hydrogel and used for further applications. Combining this advanced three-dimensional (3D) bioprinting technique with a multimaterial extrusion printhead setup enables the fabrication of complex volumetric structures built from multiple bioinks. The work described in this paper focuses on the optimization of the experimental setup and proposes a workflow to automate the bioprinting process, resulting in a fast and efficient conversion of a virtual 3D model into a physical, extruded structure in freeform using the multimaterial embedded bioprinting system. It is anticipated that further development of this technology will likely lead to widespread applications in areas such as tissue engineering, pharmaceutical testing, and organs-on-chips.

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