Efficient nuclear delivery of DNA remains a significant challenge for non-viral vectors, limiting their broader application in gene therapy. A key intracellular barrier is represented by the nuclear envelope, where the nuclear pore complex (NPC) restricts passive diffusion of macromolecules, and prevents more than 97% of delivered cytosolic DNA from entering the nucleus. Our study addresses this major limitation with a targeted delivery strategy leveraging nuclear localization signaling (NLS) peptides to enhance nuclear import of plasmid DNA (pDNA). NLS peptides were non-covalently complexed with pDNA and co-encapsulated in lipid nanoparticles (LNPs). Using fluorescently labeled LNPs and pDNA, the intracellular trafficking behavior from cellular uptake to endosomal escape and nuclear entry was characterized with and without the co-encapsulation of the NLS peptide. pDNA-NLS-functionalized LNPs achieved a 2.5-fold increase in transfection efficiency compared to non-complexed controls in vitro. Mechanistic studies indicate that cytosolic importins recognize the co-delivered NLS peptides, facilitating active transport of the released pDNA through the nuclear pore complex. In vivo studies in zebrafish larvae and wildtype mice are consistent with an NLS-dependent enhancement in gene expression, displaying a 10-fold increase after 72 h in mice. These findings demonstrate that LNPs can be combined with NLS peptides to promote non-viral DNA delivery to the nucleus by endogenous nuclear import pathways.

Due to its pivotal role as a regulator of nucleocytoplasmic transport, the structure and dynamic gating mechanism of the nuclear pore complex (NPC) is a subject of immense interest. Here, we report key recent advancements discussed at the Selective Transport Control in Biological and Biomimetic Nanopores meeting (Monte Verità, Switzerland, 2024) that gathered NPC experts from a range of disciplines. Novel insights were reported from cutting-edge super-resolution techniques that enable the direct interrogation of the NPC’s dynamic central transporter; computational models that unravel the mechanisms of the selective barrier; and synthetic NPC mimics as valuable in vitro models for delineating NPC permeability and transport dynamics. Altogether, three major insights were highlighted: (i) the presence of dynamically organised nuclear transport pathways within the NPC, (ii) the role of nuclear transport receptors that enrich and reinforce the NPC’s selective permeability barrier, and (iii) the ability of DNA origami nanostructures to mimic aspects of the NPC with unprecedented precision. Overall, the advancements marked a convergence in our understanding of NPC function by unraveling its dynamic gating mechanism at the nanoscale.

ILC is a distinct and aggressive subtype of breast cancer (BC) characterized by a unique single-file cell growth pattern and a propensity for diffuse metastasis, including leptomeningeal and peritoneal involvement. A hallmark of this disease is the loss of E-cadherin, a critical adhesion molecule, which disrupts cell-cell junctions and alters the mechanical properties of both cancer cells and their surrounding microenvironment. These changes contribute to the infiltrative behavior and metastatic potential of the disease. Understanding the mechanobiology of ILC offers promising avenues for targeted therapeutic interventions. Methods: Breast tissue (BT) samples from a blinded, single-center prospective study at the University of Basel’s Breast Clinic were analyzed. Nanomechanical properties were evaluated using the AFM-based Automated and Reliable Tissue Diagnostics (ARTIDIS) system on BT of patients (pts) with clinically indicated biopsy. All provided informed consent, with clinical data collected for up to 10 years(y). A total of 588 fresh BT samples from 545 pts suspected of BC were included, analyzed via the ARTIDISNET platform. Results: This study included 128 invasive BC patients (112 IDC, 16 ILC). Median ages were 62.8y (IDC) and 76.4y (ILC). Tumor characteristics were similar, but Ki-67 <10% was more common in ILC (56.3% vs. 27.7%), while >30% was higher in IDC (25% vs. 12.5%). Grade 3 tumors were also more frequent in IDC (32.1% vs. 18.8%). Nodal involvement was 37.5% in IDC and 12.5% in ILC. Tumor stages were similar, though stage II was more common in ILC (50% vs. 37.5%). Luminal A/B subtypes were 30.4%/45.5% (IDC) and 56.3%/31.3% (ILC). HER2+ was 15.1% (IDC) and 12.6% (ILC). Stiffness, adhesion, and dissipation profiles were significantly different between ILC and IDC, indicating that each cancer type has distinct mechanical properties. ILC exhibited higher stiffness overall, while IDC showed more consistent stiffness across samples. Additionally, ILC cells had lower dissipation compared to IDC, suggesting that ILC may have a stiffer tumor environment, possibly due to variations in tissue structure or extracellular matrix composition. When analyzing ILC subtypes (classical vs. variants), classical ILC showed a wider stiffness distribution, likely because its cells are more scattered. In contrast, ILC variants had a narrower stiffness range, indicating more clustered cell growth. Conclusion: This study provides a groundbreaking nanomechanical characterization of invasive lobular carcinoma (ILC) using ARTIDIS technology. It distinguishes ILC from invasive ductal carcinoma (IDC) based on unique stiffness and dissipation signatures while identifying aggressive ILC subtypes, surpassing current clinical technologies. These insights promise improved diagnostic precision and personalized therapies for ILC. The ANGEL trial, enrolling over 2,700 patients globally, is underway to further validate these findings. Marko Loparic, Carolina Ortiz Velez, Reiner Oropesa Nuñez, Sara Nizzero, Tobias Appenzeller, Leonie Briner, Rosemarie Burian, Christian Raez, Simone Muenst-Soysal, Tatjana Vlaijnic, Ellen Obermann, Sophie Dellas, Serafino Forte, Zlatko Marušić, Roderick Lim, Ahmed Jizawi, Philipp Oertle, Viola Heinzelmann-Schwarz, Marija Plodinec. Unraveling the nanomechanical characteristics of invasive lobular carcinoma (ILC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB352.

Surface acoustic wave (SAW)-driven acoustic tweezers have been widely explored for high-resolution ultrasonic sample manipulation. Among these, hybrid acoustic tweezers comprising a reusable SAW chip and a disposable glass or silicon microfluidic chip as a superstrate offer advantages such as reduced experimental costs and minimized cross-contamination between experiments. However, maximizing the acoustic pressure within the microfluidic channel requires efficient acoustic coupling between the SAW and the microfluidic structure. In this work, we investigate the frequency-dependent characteristics of acoustophoresis of 50 MHz hybrid acoustic tweezers composed of a SAW chip and a silicon microfluidic chip. We elucidate the role of the bulk acoustic wave resonances in the silicon substrate in facilitating the formation of acoustic standing waves within the microfluidic channel. Experimental results demonstrate the generation of acoustic pressures up to 2.1 ± 0.5 MPa inside the channel. The fabricated device was successfully used to probe the transient viscoelastic deformation of HEK293T cells and to trap motile cells Tetrahymena. These findings highlight the potential of the propose hybrid acoustic tweezers as a platform for acoutsto-mechanical testing of soft matter and biological samples.

Nuclear pore complexes (NPCs) enable rapid, selective, and robust nucleocytoplasmic transport. To explain how transport emerges from the system components and their interactions, we used experimental data and theoretical information to construct an integrative Brownian dynamics model of transport through an NPC, coupled to a kinetic model of transport in the cell. The model recapitulates key aspects of transport for a wide range of molecular cargoes, including preribosomes and viral capsids. Our model quantifies how flexible phenylalanine-glycine (FG) repeat proteins create an entropic barrier to passive diffusion and how this barrier is selectively lowered in facilitated diffusion by the many transient interactions of nuclear transport receptors with the FG repeats. Selective transport is enhanced by "fuzzy" multivalent interactions, redundant FG repeat mass, coupling to the energy-dependent RanGTP concentration gradient, and exponential dependence of transport kinetics on the transport barrier. Our model will facilitate rational modulation of the NPC and its artificial mimics.

Bacteria respond to environmental stimuli and attacks from competing organisms. Pseudomonas aeruginosa assembles the type VI secretion system (H1-T6SS) to precisely retaliate against aggressive competing bacteria. However, we lack an understanding of how the H1-T6SS assembly dynamically responds to nanomechanical forces. To address this, we analyzed live cells using correlative atomic force microscopy (AFM) and fluorescence microscopy. We show that indentation forces above 7 nanonewtons trigger local, repeated and targeted H1-T6SS assemblies within seconds of impact by the AFM tip. Analysis of the corresponding AFM force curves shows that a breach of a single layer of the cell envelope is necessary and sufficient for triggering H1-T6SS assembly. Accordingly, polymyxin B nonapeptide, which damages the outer membrane, also triggers H1-T6SS assembly. This suggests that P. aeruginosa has evolved a danger-sensing mechanism that enables rapid and precise deployment of its antibacterial H1-T6SS in response to breaches in the outer membrane.

Protocells offer a versatile material for dissecting cellular processes and developing simplified biomimetic systems by combining biological components with synthetic ones. However, a gap exists between the integrity and complex functionality of native organelles such as nuclei, and bottom-up strategies reducing cellular functions within a synthetic environment. Here, this gap is bridged by incorporating native nuclei into polymeric giant unilamellar vesicles (pGUVs) using double-emulsion microfluidics. It is shown that the nuclei retain their morphology and nuclear envelope integrity, facilitating the import of co-encapsulated peptide-based multicompartment micelles (MCMs) via nuclear localization signals (NLS). Importantly, it is demonstrated that the nuclear import machinery remains functional inside the protocells, and by enriching the GUV interior with nuclear import-promoting factors, the delivery efficiency of NLS-MCMs significantly increases. The findings reveal that nucleated protocells preserve nuclear function and integrity for extended periods, providing a new platform for studying nuclear processes in a simplified, yet biologically relevant, environment. This approach opens avenues for creating advanced biohybrid materials, offering opportunities to investigate organelle behavior and their interactions with cellular components in greater detail. The findings establish a foundation for high-throughput applications in synthetic biology and contribute valuable insights into sustaining complex cellular functions in engineered systems.

Exportin receptors are concentrated in the nucleus to transport essential cargoes out of it. A mislocalization of exportins to the cytoplasm is linked to disease. Hence, it is important to understand how their containment within the nucleus is regulated. Here, we have studied the nuclear efflux of exportin2 (cellular apoptosis susceptibility protein or CAS) that delivers karyopherinα (Kapα or importinα), the cargo adaptor for karyopherinβ1 (Kapβ1 or importinβ1), to the cytoplasm in a Ran guanosine triphosphate (RanGTP)-mediated manner. We show that the N-terminus of CAS attenuates the interaction of RanGTPase activating protein 1 (RanGAP1) with RanGTP to slow GTP hydrolysis, which suppresses CAS nuclear exit at nuclear pore complexes (NPCs). Strikingly, a single phosphomimetic mutation (T18D) at the CAS N-terminus is sufficient to abolish its nuclear retention and coincides with metastatic cellular behavior. Furthermore, downregulating Kapβ1 disrupts CAS nuclear retention, which highlights the balance between their respective functions that is essential for maintaining the Kapα transport cycle. Therefore, NPCs play a functional role in selectively partitioning exportins in the cell nucleus.

Nucleoporins (nups) in the central channel of nuclear pore complexes (NPCs) form a selective barrier that suppresses the diffusion of most macromolecules while enabling rapid transport of nuclear transport receptors (NTRs) with bound cargos. The complex molecular interactions between nups and NTRs have been thought to underlie the gatekeeping function of the NPC. Recent studies have shown considerable variation in NPC diameter but how altering NPC diameter might impact the selective barrier properties remains unclear. Here, we build DNA nanopores with programmable diameters and nup arrangement to mimic NPCs of different diameters. We use hepatitis B virus (HBV) capsids as a model for large-size cargos. We find that Nup62 proteins form a dynamic cross-channel meshwork impermeable to HBV capsids when grafted on the interior of 60-nm wide nanopores but not in 79-nm pores, where Nup62 cluster locally. Furthermore, importing substantially changes the dynamics of Nup62 assemblies and facilitates the passage of HBV capsids through NPC mimics containing Nup62 and Nup153. Our study shows the transport channel width is critical to the permeability of nup barriers and underscores the role of NTRs in dynamically remodeling nup assemblies and mediating the nuclear entry of viruses.

Nuclear pore complexes (NPCs) mediate nucleocytoplasmic transport of specific macromolecules while impeding the exchange of unsolicited material. However, key aspects of this gating mechanism remain controversial. To address this issue, we determined the nanoscopic behavior of the permeability barrier directly within yeast S. cerevisiae NPCs at transport-relevant timescales. We show that the large intrinsically disordered domains of phenylalanine-glycine repeat nucleoporins (FG Nups) exhibit highly dynamic fluctuations to create transient voids in the permeability barrier that continuously shape-shift and reseal, resembling a radial polymer brush. Together with cargo-carrying transport factors the FG domains form a feature called the central plug, which is also highly dynamic. Remarkably, NPC mutants with longer FG domains show interweaving meshwork-like behavior that attenuates nucleocytoplasmic transport in vivo. Importantly, the bona fide nanoscale NPC behaviors and morphologies are not recapitulated by in vitro FG domain hydrogels. NPCs also exclude self-assembling FG domain condensates in vivo, thereby indicating that the permeability barrier is not generated by a self-assembling phase condensate, but rather is largely a polymer brush, organized by the NPC scaffold, whose dynamic gating selectivity is strongly enhanced by the presence of transport factors.

The possibility to detect and analyze single or few biological molecules is very important for understanding interactions and reaction mechanisms. Ideally, the molecules should be confined to a nanoscale volume so that the observation time by optical methods can be extended. However, it has proven difficult to develop reliable, non-invasive trapping techniques for biomolecules under physiological conditions. Here we present a platform for long-term tether-free (solution phase) trapping of proteins without exposing them to any field gradient forces. We show that a responsive polymer brush can make solid state nanopores switch between a fully open and a fully closed state with respect to proteins, while always allowing the passage of solvent, ions and small molecules. This makes it possible to trap a very high number of proteins (500-1000) inside nanoscale chambers as small as one attoliter, reaching concentrations up to 60 gL-1. Our method is fully compatible with parallelization by imaging arrays of nanochambers. Additionally, we show that enzymatic cascade reactions can be performed with multiple native enzymes under full nanoscale confinement and steady supply of reactants. This platform will greatly extend the possibilities to optically analyze interactions involving multiple proteins, such as the dynamics of oligomerization events.

Multivalent interactions underpin associations between intrinsically disordered proteins (IDPs) and their binding partners. This is a subject of considerable interest and governs how nuclear transport receptors (NTRs) orchestrate the nucleocytoplasmic transport (NCT) of signal-specific cargoes through nuclear pore complexes (NPCs) in eukaryotic cells. Specifically, IDPs termed phenylalanine-glycine nucleoporins (FG Nups) exert multivalent interactions with NTRs to facilitate their transport selectivity and speed through the NPC. Here, we document the use of surface plasmon resonance (SPR) to quantify the affinity and kinetics of NTR-FG Nup binding as a function of FG Nup surface density. Moreover, we describe an in situ method that measures conformational height changes that occur in a FG Nup layer following NTR-binding. Protocols by which the as-obtained SPR results are treated with respect to mass transport limitations are further described. Overall, the SPR methodology described here can be applied to studying multivalent interactions and the role of avidity in diverse biological and biointerfacial systems.

Tau, a soluble and predominantly neuronal protein, is best known for its microtubule (MT)-binding function in the cytosol, where it decisively contributes to stability as well as modulation of MT dynamics. In Alzheimer’s disease and other tauopathies, Tau is altered into forming intracellular neurofibrillary tangles; additionally, also a mislocalization from the cytosol to the nucleus has been observed where interactions of Tau with the nucleus become possible. Using surface plasmon resonance (SPR), it was recently shown that Tau can directly interact with certain nucleoporins (e.g., Nup98), components of the nuclear pore complex (NPC). The NPC constitutes large regulated pores in the nuclear envelope that facilitate the bidirectional exchange of proteins, nucleic acids, and other biomolecules between the inner section of the nucleus and the cytosol, the nucleocytoplasmic transport. The mechanism of Tau/Nup interactions is as yet unknown, and a systematic interaction analysis of Tau with different Nups can be of high value to decipher the molecular binding mechanism of Tau to Nups. SPR is a useful tool to analyze binding affinities and kinetic parameters in a label-free environment. While one interaction partner is immobilized on a sensor chip, the second is supplied within a constant flow of buffer. Binding of mobile molecules to immobilized ones changes the refractive index of the medium close to the sensor surface with the signal being proportional to the bound mass. In this chapter, we describe the application of the SPR technique for the investigation of Tau binding to nucleoporins.

Nuclear pore complexes (NPCs) discriminate nonspecific macromolecules from importin and exportin receptors, collectively termed "karyopherins" (Kaps), that mediate nucleocytoplasmic transport. This selective barrier function is attributed to the behavior of intrinsically disordered phenylalanine-glycine nucleoporins (FG Nups) that guard the NPC channel. However, NPCs in vivo are typically enriched with different Kaps, and how they impact the NPC barrier remains unknown. Here, we show that two major Kaps, importinβ1/karyopherinβ1 (Kapβ1) and exportin 1/chromosomal maintenance 1 (CRM1), are required to fortify NPC barrier function in vivo. Their enrichment at the NPC is sustained by promiscuous binding interactions with the FG Nups, which enable CRM1 to compensate for the loss of Kapβ1 as a means to maintain NPC barrier function. However, such a compensatory mechanism is constrained by the cellular abundances and different binding kinetics for each respective Kap, as evidenced for importin-5. Consequently, we find that NPC malfunction and nucleocytoplasmic leakage result from poor Kap enrichment.

We study the rotation characteristics and the mechanism to produce the acoustic torque for polymer thin-film acoustic rotors levitated in ambient air in a 40 kHz ultrasound standing wave. It was found that the polymer thin-film disk can be efficiently rotated by attaching short blade around, of which shape can control the direction of the rotation. We further demonstrated that miniaturized acoustic rotors down to 25 µm in diameter can be rotated in liquid by in-plane standing wave produced by 2–10 MHz ultrasound. Our study will pave the way for further optimization of acoustic rotors as sample holders for experiments that demand sample rotation and the exploration of micron-scale acoustic rotors in liquid.

Tau is a neuronal protein that stabilizes axonal microtubules (MTs) in the central nervous system. In Alzheimer's disease (AD) and other tauopathies, phosphorylated Tau accumulates in intracellular aggregates, a pathological hallmark of these diseases. However, the chronological order of pathological changes in Tau prior to its cytosolic aggregation remains unresolved. These include its phosphorylation and detachment from MTs, mislocalization into the somatodendritic compartment, and oligomerization in the cytosol. Recently, we showed that Tau can interact with phenylalanine-glycine (FG)-rich nucleoporins (Nups), including Nup98, that form a diffusion barrier inside nuclear pore complexes (NPCs), leading to defects in nucleocytoplasmic transport. Here, we used surface plasmon resonance (SPR) and bio-layer interferometry (BLI) to investigate the molecular details of Tau:Nup98 interactions and determined how Tau phosphorylation and oligomerization impact the interactions. Importantly, phosphorylation, but not acetylation, strongly facilitates the accumulation of Tau with Nup98. Oligomerization, however, seems to inhibit Tau:Nup98 interactions, suggesting that Tau-FG Nup interactions occur prior to oligomerization. Overall, these results provide fundamental insights into the molecular mechanisms of Tau-FG Nup interactions within NPCs, which might explain how stress-and disease-associated posttranslational modifications (PTMs) may lead to Tau-induced nucleocytoplasmic transport (NCT) failure. Intervention strategies that could rescue Tau-induced NCT failure in AD and tauopathies will be further discussed.

The design of non-viral vectors that efficiently deliver genetic materials into cells, in particular to the nucleus, remains a major challenge in gene therapy and vaccine development. To tackle the problems associated with cellular uptake and nuclear targeting, here we introduce a delivery platform based on the self-assembly of an amphiphilic peptide carrying an N-terminal KRKR sequence that functions as a nuclear localization signal (NLS). By means of a single-step self-assembly process, the amphiphilic peptides afford the generation of NLS-functionalized multicompartment micellar nanostructures that can embed various oligonucleotides between their individual compartments. Detailed physicochemical, cellular and ultrastructural analyses demonstrated that integrating an NLS in the hydrophilic domain of the peptide along with tuning its hydrophobic domain led to self-assembled DNA-loaded multicompartment micelles (MCMs) with enhanced cellular uptake and nuclear translocation. We showed that the nuclear targeting ensued via the NLS interaction with the nuclear transport receptors of the karyopherin family. Importantly, we observed that the treatment of MCF-7 cells with NLS-MCMs loaded with anti-BCL2 antisense oligonucleotides resulted in up to 86% knockdown of BCL2, an inhibitor of apoptosis that is overexpressed in more than half of all human cancers. We envision that this platform can be used to efficiently entrap and deliver diverse genetic payloads to the nucleus and find applications in basic research and biomedicine.

Our work focuses on the development of simpler and effective production of nanofluidic devices for high-throughput charged single nanoparticle trapping in an aqueous environment. Single nanoparticle confinement using electrostatic trapping has been an effective approach to study the fundamental properties of charged molecules under a controlled aqueous environment. Conventionally, geometry-induced electrostatic trapping devices are fabricated using SiOx-based substrates and comprise nanochannels imbedded with nanoindentations such as nanopockets, nanoslits and nanogrids. These geometry-induced electrostatic trapping devices can only trap negatively charged particles, and therefore, to trap positively charged particles, modification of the device surface is required. However, the surface modification process of a nanofluidic device is cumbersome and time consuming. Therefore, here, we present a novel approach for the development of surface-modified geometry-induced electrostatic trapping devices that reduces the surface modification time from nearly 5 days to just a few hours. We utilized polydimethylsiloxane for the development of a surface-modified geometry-induced electrostatic trapping device. To demonstrate the device efficiency and success of the surface modification procedure, a comparison study between a PDMS-based geometry-induced electrostatic trapping device and the surface-modified polydimethylsiloxane-based device was performed. The device surface was modified with two layers of polyelectrolytes (1: poly(ethyleneimine) and 2: poly(styrenesulfonate)), which led to an overall negatively charged surface. Our experiments revealed the presence of a homogeneous surface charge density inside the fluidic devices and equivalent trapping strengths for the surface-modified and native polydimethylsiloxane-based geometry-induced electrostatic trapping devices. This work paves the way towards broader use of geometry-induced electrostatic trapping devices in the fields of biosensing, disease diagnosis, molecular analysis, fluid quality control and pathogen detection.