Nuclear accumulation of plasmid DNA can be enhanced by non-selective gating of the nuclear pore

One of the major obstacles in non-viral gene transfer is the nuclear membrane. Attempts to improve the transport of DNA to the nucleus through the use of nuclear localization signals or importin-β have achieved limited success. It has been proposed that the nuclear pore complexes (NPCs) through which nucleocytoplasmic transport occurs are filled with a hydrophobic phase through which hydrophobic importins can dissolve. Therefore, considering the hydrophobic nature of the NPC channel, we evaluated whether a non-selective gating of nuclear pores by trans-cyclohexane-1,2-diol (TCHD), an amphipathic alcohol that reversibly collapses the permeability barrier of the NPCs, could be obtained and used as an alternative method to facilitate nuclear entry of plasmid DNA. Our data demonstrate for the first time that TCHD makes the nucleus permeable for both high molecular weight dextrans and plasmid DNA (pDNA) at non-toxic concentrations. Furthermore, in line with these observations, TCHD enhanced the transfection efficacy of both naked DNA and lipoplexes. In conclusion, based on the proposed structure of NPCs we succeeded to temporarily open the NPCs for macromolecules as large as pDNAs and demonstrated that this can significantly enhance non-viral gene delivery.     

Nup62‐mediated nuclear import of p63 in squamous cell carcinoma

Nuclear pore complexes (NPCs) are multiprotein channels that bridge the nucleus with the cytoplasm and regulate all nucleo‐cytoplasmic traffic. NPCs are built by the repetition of ~30 different proteins known as nucleoporins (Nups). Accumulating evidence has revealed a diversity in NPC composition that is critical for cell‐specific functionality and fate determination. A new report by Hazawa et al 1 now identifies the central transport channel nucleoporin Nup62 as a novel regulator of cell proliferation and differentiation in squamous cell carcinoma (SCC), via modulation of p63 nucleo‐cytoplasmic transport. These findings provide further evidence on how alterations in NPC composition might be utilized to determine cell fate.     

Steroids dilate nuclear pores imaged with atomic force microscopy

Macromolecules that act in the cell nucleus must overcome the nuclear envelope (NE). This barrier between cytosol and the nucleus is perforated by nuclear pore complexes (NPCs) that serve as translocation machineries. We visualized the translocation process at the NE surface, applying a nanotechnical approach using atomic force microscopy (AFM). In order to initiate protein targeting to NPCs, dexamethasone (dex) was injected into Xenopus laevis oocytes. Dex is a synthetic steroid of great therapeutic relevance that specifically binds to glucocorticoid receptors and thus triggers an intracellular signal cascade involving the cell nucleus. Ninety and 180 sec after dex injection cell nuclei were isolated, the NEs spread on glass and scanned with AFM. With single molecule resolution we observed that dex initiated proteins (DIPs) first bind to NPC-free areas of the outer nuclear membrane. This causes NPCs to dilate. Then, in a second step, DIPs attach directly to NPCs and enter the dilated central channels. DIPs accumulation and NPC conformational changes were blocked by RU486, a specific glucocorticoid receptor antagonist. In conclusion, dex exposure induces NPC dilation. NPCs change conformation already prior to transport. The NPC dilation signal is most likely transmitted through NPC associated filaments or yet unknown structures in the NE outer membrane. NPC dilation could have significant impact on nuclear targeting of therapeutic macromolecules.     

The dynamics of karyopherin-mediated nuclear transport.

The regulated exchange of proteins and nucleic acids between the nucleus and cytoplasm demands a complex interplay between nuclear pore complexes (NPCs), which provide conduits in the nuclear envelope, and mobile transport receptors (or karyopherins, also known as importins/exportins) that bind and mediate the translocation of cargoes through the NPCs. Biochemical characterization of individual karyopherins has led to the identification of many of their cargoes and to the elucidation of the mechanisms by which they mediate transport. Likewise, the characterization of numerous NPC-associated components, in combination with structural studies of NPCs, have begun to address the possible mechanisms that drive nucleocytoplasmic transport, and the role that different nucleoporins play in the transport process. Some recent studies indicate that several NPC-associated factors, previously thought to be stable components of the NPC, dynamically interact with both nuclear and cytoplasmic aspects of the NPC. The mobility of these components challenges our conventional view of the NPC as the stationary phase of transport. These components and their potiential roles in nucleo-cytoplasmic transport are discussed.     

The C-terminal domain of Nup93 is essential for assembly of the structural backbone of nuclear pore complexes

Nuclear pore complexes (NPCs) are large macromolecular assemblies that control all transport across the nuclear envelope. They are formed by about 30 nucleoporins (Nups), which can be roughly categorized into those forming the structural skeleton of the pore and those creating the central channel and thus providing the transport and gating properties of the NPC. Here we show that the conserved nucleoporin Nup93 is essential for NPC assembly and connects both portions of the NPC. Although the C-terminal domain of the protein is necessary and sufficient for the assembly of a minimal structural backbone, full-length Nup93 is required for the additional recruitment of the Nup62 complex and the establishment of transport-competent NPCs.     

Evidence for Ca2+- and ATP-sensitive peripheral channels in nuclear pore complexes.

In eukaryotic cells the nuclear envelope (NE) serves as a functional barrier between cytosol and nucleoplasm perforated by nuclear pore complexes (NPCs). Both active and passive transport of ions and macromolecules are thought to be mediated by the centrally located large NPC channel. However, 3-dimensional imaging of NPCs based on electron microscopy indicates the existence of additional small channels of unknown function located in the NPC periphery. By means of the recently developed nuclear hourglass technique that measures NE electrical conductance, we evaluated passive electrically driven transport through NPCs. In isolated Xenopus laevis oocyte nuclei, we varied ambient Ca2+ and ATP in the cytosolic solution and/or chelated Ca2+ in the perinuclear stores in order to assess the role of Ca2+ in regulating passive ion transport. We noticed that NE electrical conductance is large under conditions where macromolecule permeability is known to be low. In addition, atomic force microscopy applied to native NPCs detects multiple small pores in the NPC periphery consistent with channel openings. Peripheral pores were detectable only in the presence of ATP. We conclude that NPC transport of ions and macromolecules occurs through different routes. We present a model in which NE ion flux does not occur through the central NPC channel but rather through Ca2+- and ATP-activated peripheral channels of individual NPCs.     

Charge as a Selection Criterion for Translocation through the Nuclear Pore Complex

Nuclear pore complexes (NPCs) are highly selective filters that control the exchange of material between nucleus and cytoplasm. The principles that govern selective filtering by NPCs are not fully understood. Previous studies find that cellular proteins capable of fast translocation through NPCs (transport receptors) are characterized by a high proportion of hydrophobic surface regions. Our analysis finds that transport receptors and their complexes are also highly negatively charged. Moreover, NPC components that constitute the permeability barrier are positively charged. We estimate that electrostatic interactions between a transport receptor and the NPC result in an energy gain of several k _B T, which would enable significantly increased translocation rates of transport receptors relative to other cellular proteins. We suggest that negative charge is an essential criterion for selective passage through the NPC.     

A Highlights from MBoC Selection: Three-dimensional superresolution fluorescence microscopy maps the variable molecular architecture of the nuclear pore complex

Nuclear pore complexes (NPCs) are large macromolecular machines that mediate the traffic between the nucleus and the cytoplasm. In vertebrates, each NPC consists of ∼1000 proteins, termed nucleoporins, and has a mass of more than 100 MDa. While a pseudo-atomic static model of the central scaffold of the NPC has recently been assembled by integrating data from isolated proteins and complexes, many structural components still remain elusive due to the enormous size and flexibility of the NPC. Here, we explored the power of three-dimensional (3D) superresolution microscopy combined with computational classification and averaging to explore the 3D structure of the NPC in single human cells. We show that this approach can build the first integrated 3D structural map containing both central as well as peripheral NPC subunits with molecular specificity and nanoscale resolution. Our unbiased classification of more than 10,000 individual NPCs indicates that the nuclear ring and the nuclear basket can adopt different conformations. Our approach opens up the exciting possibility to relate different structural states of the NPC to function in situ.     

Characterisation of the passive permeability barrier of nuclear pore complexes

Nuclear pore complexes (NPCs) restrict uncontrolled nucleocytoplasmic fluxes of inert macromolecules but permit facilitated translocation of nuclear transport receptors and their cargo complexes. We probed the passive barrier of NPCs and observed sieve‐like properties with a dominating mesh or channel radius of 2.6 nm, which is narrower than proposed earlier. A small fraction of diffusion channels has a wider opening, explaining the very slow passage of larger molecules. The observed dominant passive diameter approximates the distance of adjacent hydrophobic clusters of FG repeats, supporting the model that the barrier is made of FG repeat domains cross‐linked with a spacing of an FG repeat unit length. Wheat germ agglutinin and the dominant‐negative importin β^45‐462 fragment were previously regarded as selective inhibitors of facilitated NPC passage. We now observed that they do not distinguish between the passive and the facilitated mode. Instead, their inhibitory effect correlates with the size of the NPC‐passing molecule. They have little effect on small species, inhibit the passage of green fluorescent protein‐sized objects >10‐fold and virtually block the translocation of larger ones. This suggests that passive and facilitated NPC passage proceed through one and the same permeability barrier.     

Calcium-mediated structural changes of native nuclear pore complexes monitored by time-lapse atomic force microscopy

Nuclear pore complexes (NPCs) are large macromolecular assemblies embedded in the double membrane nuclear envelope. They are the major gateways mediating transport of ions, small molecules, proteins, RNAs, and ribonucleoprotein particles in and out of the nucleus in interphase cells. Understanding structural changes at the level of individual pores will be a prerequisite to eventually correlate the molecular architecture of the NPC with its distinct functional states during nucleocytoplasmic transport. Toward this goal, we have employed time-lapse atomic force microscopy of native NPCs kept in buffer, and recorded calcium-mediated structural changes such as the opening (i.e. +Ca2+) and closing (i.e. -Ca2+) of individual nuclear baskets. Most likely, this structural change of the nuclear basket involves its distal ring which may act as an iris-like diaphragm. In order to directly correlate distinct structural features with corresponding functional states and dynamic aspects, we also addressed the question of whether the "central plug" or "transporter" actually represents a calcium-sensitive component of the NPC involved in mediating nucleocytoplasmic transport. Our data indicate that in the absence of ATP, cytoplasmic plugging/unplugging of the NPC is insensitive to calcium.     

Nuclear pore complex is able to transport macromolecules with diameters of about 39 nm

Bidirectional transport of macromolecules between the nucleus and the cytoplasm occurs through the nuclear pore complexes (NPCs) by a signal-mediated mechanism that is directed by targeting signals (NLSs) residing on the transported molecules or "cargoes." Nuclear transport starts after interaction of the targeting signal with soluble cellular receptors. After the formation of the cargo-receptor complex in the cytosol, this complex crosses the NPC. Herein, we use gold particles of various sizes coated with cargo-receptor complexes to determine precisely how large macromolecules crossing the NPC by the signal-mediated transport mechanism could be. We found that cargo-receptor-gold complexes with diameter close to 39 nm could be translocated by the NPC. This implies that macromolecules much larger than the assumed functional NPC diameter of 26 nm can be transported into the karyoplasm. The physiological relevance of this finding was supported by the observation that intact nucleocapsids of human hepatitis B virus with diameters of 32 and 36 nm are able to cross the nuclear pore without disassembly.     

Structural Analysis of a Metazoan Nuclear Pore Complex Reveals a Fused Concentric Ring Architecture

The sole gateway for molecular exchange between the cytoplasm and the nucleus is the nuclear pore complex (NPC). This large supramolecular assembly mediates transport of cargo into and out of the nucleus and fuse the inner and outer nuclear membranes to form an aqueous translocation channel. The NPC is composed of eight proteinaceous asymmetric units forming a pseudo-8-fold symmetric passage. Due to its shear size, complexity, and plastic nature, dissecting the high-resolution three-dimensional structure of the NPC in its hydrated state is a formidable challenge. Toward this goal, we applied cryo-electron tomography to spread nuclear envelopes from Xenopus oocytes. To compensate for perturbations of the 8-fold symmetry of individual NPCs, we performed symmetry-independent asymmetric unit averaging of three-dimensional tomographic NPC volumes to eventually yield a refined model at 6.4 nm resolution. This approach revealed novel structural features, particularly in the spoke-ring complex and luminal domains. Fused concentric ring architecture of the spoke-ring complex was found along the translocation channel. Additionally, a comparison of the refined Xenopus model to that of its Dictyostelium homologue yielded similar pore diameters at the level of the three canonical rings, although the Xenopus NPC was found to be 30% taller than the Dictyostelium pore. This discrepancy is attributed primarily to the relatively low homology and different organization of some nucleoporins in the Dictyostelium genome as compared to that of vertebrates. Nevertheless, the experimental conditions impose a preferred axial orientation of the NPCs within spread Xenopus oocyte nuclear envelopes. This may at least in part explain the increased height of the reconstructed vertebrate NPCs compared to those obtained from tomographic reconstruction of intact Dictyostelium nuclei.     

A pathway separate from the central channel through the nuclear pore complex for inorganic ions and small macromolecules

Nuclear pore complexes (NPCs) are supramolecular nanomachines that mediate the exchange of macromolecules and inorganic ions between the nucleus and the cytosol. Although there is no doubt that large cargo is transported through the centrally located channel, the route of ions and small molecules remains debatable. We thus tested the hypothesis that there are two separate pathways by imaging NPCs using atomic force microscopy, NPC electrical conductivity measurements, and macromolecule permeability assays. Our data indicate a spatial separation between the active transport of macromolecules through the central channel and the passive transport of ions and small macromolecules through the pore periphery.     

Conformational changes of the in situ nuclear pore complex.

By bridging the double membrane separating the cell nucleus and cytoplasm, nuclear pore complexes (NPCs) are crucial pathways for the exchange of ions, proteins, and RNA between these two cellular compartments. A structure in the central lumen of the NPC, called the nuclear transport protein, central granule, or nuclear plug, appeared to gate diffusion of intermediate-sized molecules (10-40 kDa) across the nuclear membranes. Visualization of the NPC required drying and fixation of the specimen for electron and atomic force microscopy (AFM), a requirement that has raised doubts about the physiological relevance of the observation. Here we present AFM images of the outer nuclear membranes and NPCs of Xenopus laevis oocytes under more physiological conditions. Measured under a variety of Ca2+ depletion conditions, the central granule appeared to occupy and occlude the lumen of the pore in >80% of NPCs compared to <10% in controls. In a few instances images were obtained of the same NPCs as the solution was changed from control saline to store depletion conditions, and finally to store repletion conditions. We conclude that the central lumen of the nuclear pore complex undergoes a conformational change in response to depletion of nuclear cisternal Ca2+ levels.     

USING ATOMIC FORCE MICROSCOPY TO INVESTIGATE PATCH-CLAMPED NUCLEAR MEMBRANE

Nuclear patch clamp is an emerging research field that aims to disclose the electrical phenomena underlying macromolecular transport across the nuclear envelope (NE), its properties as an ion barrier and its function as an intracellular calcium store. The authors combined the patch clamp technique with atomic force microscopy (AFM) to investigate the structure—function relationship of NE. In principle, patch clamp currents, recorded from the NE can indicate the activity of the nuclear pore complexes (NPCs) and/or of ion channels in the two biomembranes that compose the NE. However, the role of the NPCs is still unclear because the observed NE current in patch clamp experiments is lower than expected from the known density of the NPCs. Therefore, AFM was applied to link patch clamp currents to structure. The membrane patch was excised from the nuclear envelope and, after electrical evaluation, transferred from the patch pipette to a substrate. We could identify the native nuclear membrane patches with AFM at a lateral and a vertical resolution of 3nm and 0.1nm, respectively. It was shown that complete NE together with NPCs can be excised from the nucleus after their functional identification in patch clamp experiments. However, we also show that membranes of the endoplasmic reticulum can contaminate the tip of the patch pipette during nuclear patch clamp experiments. This possibility must be considered carefully in nuclear patch clamp experiments.     

Large cargo transport by nuclear pores: implications for the spatial organization of FG‐nucleoporins

Nuclear pore complexes (NPCs) mediate cargo traffic between the nucleus and the cytoplasm of eukaryotic cells. Nuclear transport receptors (NTRs) carry cargos through NPCs by transiently binding to phenylalanine‐glycine (FG) repeats on intrinsically disordered polypeptides decorating the NPCs. Major impediments to understand the transport mechanism are the thousands of FG binding sites on each NPC, whose spatial distribution is unknown, and multiple binding sites per NTR, which leads to multivalent interactions. Using single molecule fluorescence microscopy, we show that multiple NTR molecules are required for efficient transport of a large cargo, while a single NTR promotes binding to the NPC but not transport. Particle trajectories and theoretical modelling reveal a crucial role for multivalent NTR interactions with the FG network and indicate a non‐uniform FG repeat distribution. A quantitative model is developed wherein the cytoplasmic side of the pore is characterized by a low effective concentration of free FG repeats and a weak FG‐NTR affinity, and the centrally located dense permeability barrier is overcome by multivalent interactions, which provide the affinity necessary to permeate the barrier.     

Dynamic nuclear pore complexes: life on the edge

The exchange of molecules between the nucleus and cytoplasm is mediated through nuclear pore complexes (NPCs) embedded in the nuclear envelope. Altering the interactions between transport receptors and their cargo has been shown to be a major regulatory mechanism to control traffic through NPCs. New evidence now suggests that NPC proteins play active roles in translocation, and that transport is also controlled by dynamic changes in NPC composition and architecture. This view of ever-changing NPCs necessitates the re-evaluation of current models of nuclear transport and how this process is regulated.