Channel width modulates the permeability of DNA origami based nuclear pore mimics.

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.

Integrative spatiotemporal map of nucleocytoplasmic transport.

The Nuclear Pore Complex (NPC) facilitates rapid and selective nucleocytoplasmic transport of molecules as large as ribosomal subunits and viral capsids. It is not clear how key emergent properties of this transport arise from the system components and their interactions. To address this question, we constructed an integrative coarse-grained Brownian dynamics model of transport through a single NPC, followed by coupling it with a kinetic model of Ran-dependent transport in an entire cell. The microscopic model parameters were fitted to reflect experimental data and theoretical information regarding the transport, without making any assumptions about its emergent properties. The resulting reductionist model is validated by reproducing several features of transport not used for its construction, such as the morphology of the central transporter, rates of passive and facilitated diffusion as a function of size and valency, in situ radial distributions of pre-ribosomal subunits, and active transport rates for viral capsids. The model suggests that the NPC functions essentially as a virtual gate whose flexible phenylalanine-glycine (FG) repeat proteins raise an entropy barrier to diffusion through the pore. Importantly, this core functionality is greatly enhanced by several key design features, including 'fuzzy' and transient interactions, multivalency, redundancy in the copy number of FG nucleoporins, exponential coupling of transport kinetics and thermodynamics in accordance with the transition state theory, and coupling to the energy-reliant RanGTP concentration gradient. These design features result in the robust and resilient rate and selectivity of transport for a wide array of cargo ranging from a few kilodaltons to megadaltons in size. By dissecting these features, our model provides a quantitative starting point for rationally modulating the transport system and its artificial mimics.

Mechanism of exportin retention in the cell nucleus.

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 karyopherinß1 (Kapß1 or importinß1), 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 Kapß1 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.

Stable trapping of multiple proteins at physiological conditions using nanoscale chambers with macromolecular gates.

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.

Dynamic molecular mechanism of the nuclear pore complex permeability barrier.

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.

Analysis of Tau/Nucleoporin Interactions by Surface Plasmon Resonance Spectroscopy.

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.

A self-assembling peptidic platform to boost the cellular uptake and nuclear delivery of oligonucleotides.

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.

Multivalent Interactions with Intrinsically Disordered Proteins Probed by Surface Plasmon Resonance.

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.

Phosphorylation but Not Oligomerization Drives the Accumulation of Tau with Nucleoporin Nup98.

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.

Karyopherin enrichment and compensation fortifies the nuclear pore complex against nucleocytoplasmic leakage.

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, importinß1/karyopherinß1 (Kapß1) 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 Kapß1 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.

Surface-modified elastomeric nanofluidic devices for single nanoparticle trapping.

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.

On the asymmetric partitioning of nucleocytoplasmic transport - recent insights and open questions.

Macromolecular cargoes are asymmetrically partitioned in the nucleus or cytoplasm by nucleocytoplasmic transport (NCT). At the center of this activity lies the nuclear pore complex (NPC), through which soluble factors circulate to orchestrate NCT. These include cargo-carrying importin and exportin receptors from the ß-karyopherin (Kapß) family and the small GTPase Ran, which switches between guanosine triphosphate (GTP)- and guanosine diphosphate (GDP)-bound forms to regulate cargo delivery and compartmentalization. Ongoing efforts have shed considerable light on how these soluble factors traverse the NPC permeability barrier to sustain NCT. However, this does not explain how importins and exportins are partitioned in the cytoplasm and nucleus, respectively, nor how a steep RanGTP-RanGDP gradient is maintained across the nuclear envelope. In this Review, we peel away the multiple layers of control that regulate NCT and juxtapose unresolved features against known aspects of NPC function. Finally, we discuss how NPCs might function synergistically with Kapßs, cargoes and Ran to establish the asymmetry of NCT.

Physics of the Nuclear Pore Complex: Theory, Modeling and Experiment.

The hallmark of eukaryotic cells is the nucleus that contains the genome, enclosed by a physical barrier known as the nuclear envelope (NE). On the one hand, this compartmentalization endows the eukaryotic cells with high regulatory complexity and flexibility. On the other hand, it poses a tremendous logistic and energetic problem of transporting millions of molecules per second across the nuclear envelope, to facilitate their biological function in all compartments of the cell. Therefore, eukaryotes have evolved a molecular "nanomachine" known as the Nuclear Pore Complex (NPC). Embedded in the nuclear envelope, NPCs control and regulate all the bi-directional transport between the cell nucleus and the cytoplasm. NPCs combine high molecular specificity of transport with high throughput and speed, and are highly robust with respect to molecular noise and structural perturbations. Remarkably, the functional mechanisms of NPC transport are highly conserved among eukaryotes, from yeast to humans, despite significant differences in the molecular components among various species. The NPC is the largest macromolecular complex in the cell. Yet, despite its significant complexity, it has become clear that its principles of operation can be largely understood based on fundamental physical concepts, as have emerged from a combination of experimental methods of molecular cell biology, biophysics, nanoscience and theoretical and computational modeling. Indeed, many aspects of NPC function can be recapitulated in artificial mimics with a drastically reduced complexity compared to biological pores. We review the current physical understanding of the NPC architecture and function, with the focus on the critical analysis of experimental studies in cells and artificial NPC mimics through the lens of theoretical and computational models. We also discuss the connections between the emerging concepts of NPC operation and other areas of biophysics and bionanotechnology.

Immobilization of arrestin-3 on different biosensor platforms for evaluating GPCR binding.

G protein-coupled receptors (GPCRs) are a large and ubiquitous family of membrane receptors of great pharmacological interest. Cell-based assays are the primary tool for assessing GPCR interactions and activation but their design and intrinsic complexity limit their application. Biosensor-based assays that directly and specifically report GPCR-protein binding (e.g. arrestin or G protein) could provide a good alternative. We present an approach based on the stable immobilization of different arrestin-3 proteins (wild type, and two mutants, mutant X (arrestin-3 I386A) and mutant Y (arrestin-3 R393E)) via histidine tags on NTA(Ni2+)-coated sensors in a defined orientation. Using biolayer interferometry (BLI), surface plasmon resonance (SPR), and quartz crystal microbalance with dissipation (QCM-D), we were able to follow the interaction between the different arrestin-3 proteins and a representative GPCR, jumping spider rhodopsin-1 (JSR1), in a label-free manner in real-time. The interactions were quantified as binding affinity, association and dissociation rate constants. The combination of surface-based biosensing methods indicated that JSR1 showed the strongest binding to arrestin mutant Y. Taken together, this work introduces direct label-free, biosensor-based screening approaches that can be easily adapted for testing interactions of proteins and other compounds with different GPCRs.

Human GBP1 binds LPS to initiate assembly of a caspase-4 activating platform on cytosolic bacteria.

The human non-canonical inflammasome controls caspase-4 activation and gasdermin-D-dependent pyroptosis in response to cytosolic bacterial lipopolysaccharide (LPS). Since LPS binds and oligomerizes caspase-4, the pathway is thought to proceed without dedicated LPS sensors or an activation platform. Here we report that interferon-induced guanylate-binding proteins (GBPs) are required for non-canonical inflammasome activation by cytosolic Salmonella or upon cytosolic delivery of LPS. GBP1 associates with the surface of cytosolic Salmonella seconds after bacterial escape from their vacuole, initiating the recruitment of GBP2-4 to assemble a GBP coat. The GBP coat then promotes the recruitment of caspase-4 to the bacterial surface and caspase activation, in absence of bacteriolysis. Mechanistically, GBP1 binds LPS with high affinity through electrostatic interactions. Our findings indicate that in human epithelial cells GBP1 acts as a cytosolic LPS sensor and assembles a platform for caspase-4 recruitment and activation at LPS-containing membranes as the first step of non-canonical inflammasome signaling.

Organelle-specific targeting of polymersomes into the cell nucleus.

Organelle-specific nanocarriers (NCs) are highly sought after for delivering therapeutic agents into the cell nucleus. This necessitates nucleocytoplasmic transport (NCT) to bypass nuclear pore complexes (NPCs). However, little is known as to how comparably large NCs infiltrate this vital intracellular barrier to enter the nuclear interior. Here, we developed nuclear localization signal (NLS)-conjugated polymersome nanocarriers (NLS-NCs) and studied the NCT mechanism underlying their selective nuclear uptake. Detailed chemical, biophysical, and cellular analyses show that karyopherin receptors are required to authenticate, bind, and escort NLS-NCs through NPCs while Ran guanosine triphosphate (RanGTP) promotes their release from NPCs into the nuclear interior. Ultrastructural analysis by regressive staining transmission electron microscopy further resolves the NLS-NCs on transit in NPCs and inside the nucleus. By elucidating their ability to utilize NCT, these findings demonstrate the efficacy of polymersomes to deliver encapsulated payloads directly into cell nuclei.

Karyopherin enrichment at the nuclear pore complex attenuates Ran permeability.

Ran is a small GTPase whose nucleotide-bound forms cycle through nuclear pore complexes (NPCs) to direct nucleocytoplasmic transport (NCT). Generally, Ran guanosine triphosphate (RanGTP) binds cargo-carrying karyopherin receptors (Kaps) in the nucleus and releases them into the cytoplasm following hydrolysis to Ran guanosine diphosphate (RanGDP). This generates a remarkably steep Ran gradient across the nuclear envelope that sustains compartment-specific cargo delivery and accumulation. However, because NPCs are permeable to small molecules of comparable size, it is unclear how an uncontrolled mixing of RanGTP and RanGDP is prevented. Here, we find that an NPC-enriched pool of karyopherin subunit beta 1 (KPNB1, hereafter referred to as Kapß1) selectively mediates Ran diffusion across the pore but not passive molecules of similar size (e.g. GFP). This is due to RanGTP having a stronger binding interaction with Kapß1 than RanGDP. For this reason, the RanGDP importer, nuclear transport factor 2, facilitates the return of RanGDP into the nucleus following GTP hydrolysis. Accordingly, the enrichment of Kapß1 at NPCs may function as a retention mechanism that preserves the sharp transition of RanGTP and RanGDP in the nucleus and cytoplasm, respectively.

The role of nucleocytoplasmic transport in mechanotransduction.

Cells integrate mechanical and biochemical signals via a process called mechanotransduction to generate essential gene expression patterns in space and time. This is vital for cell migration and proliferation as well as tissue morphogenesis and remodeling. While the force-sensing and force-transducing mechanisms are generally known, it remains unclear how mechanoresponsive transcription factors (TFs) are selectively translocated into the nucleus upon force activation. Such TFs include Yes-Associated Protein (YAP), Myocardin Related Transcription Factors (MRTFs), Hypoxia Induced Factors (HIFs) and others. Here, we discuss how the nucleocytoplasmic transport machinery intersects with mechanoresponsive TFs to facilitate their selective transport through nuclear pore complexes.

Nuclear Pore Membrane Proteins Self-Assemble into Nanopores.

Large multiprotein nanopores remain difficult to reconstitute in vitro, such as, for instance, the nuclear pore complex (NPC) that regulates macromolecular transport between the nucleus and cytoplasm in cells. Here, we report that two NPC pore membrane proteins self-assemble into ∼20 nm diameter nanopores following in vitro reconstitution into lipid bilayers. Pore formation follows from the assembly of Pom121 and Ndc1 oligomers, which arrange into ringlike membrane structures that encircle aqueous, electrically conductive pores. This represents a key step toward reconstituting membrane-embedded NPC mimics for biological studies and biotechnological applications.