Single Nuclear Pore Complexes Research Articles

Visualizing nuclear pore complex plasticity with Pan-Expansion Microscopy.

Cell-type specific and environmentally-responsive plasticity in nuclear pore complex (NPC) composition and structure is an emerging area of investigation, but its molecular underpinnings remain ill defined. To understand the cause and consequence of NPC plasticity requires technologies to visualize differences within individual NPCs across the thousands in a given nucleus. We evaluate the utility of Pan Expansion Microscopy (Pan-ExM), which enables 16-20 fold isotropic cell enlargement while preserving the proteome, to reveal NPC plasticity. NPCs are robustly identified by deep learning-facilitated segmentation as tripartite structures corresponding to the nucleoplasmic ring, inner ring with central transport channel, and cytoplasmic ring, as confirmed by immunostaining. We demonstrate a range of NPC diameters with a bias for dilated NPCs at the basal nuclear surface, often in local clusters. These diameter biases are eliminated by disrupting linker of nucleoskeleton and cytoskeleton (LINC) complex-dependent connections between the nuclear envelope (NE) and the cytoskeleton, supporting that they reflect local variations in NE tension. Pan-ExM further reveals that the transmembrane nucleoporin/nup POM121 resides specifically at the nuclear ring in multiple model cell lines, surprising given the expectation that it would be a component of the inner ring like other transmembrane nups. Remarkably, however, POM121 shifts from the nuclear ring to the inner ring specifically in aged induced pluripotent stem cell derived neurons (iPSNs) from a patient with C9orf72 amyotrophic lateral sclerosis (ALS). Thus, Pan-ExM allows the visualization of changes in NPC architecture that may underlie early steps in an ALS pathomechanism. Taken together, Pan-ExM is a powerful and accessible tool to visualize NPC plasticity in physiological and pathological contexts at single NPC resolution.

Nuclear basket proteins regulate the distribution and mobility of nuclear pore complexes in budding yeast.

Nuclear pore complexes (NPCs) mediate all traffic between the nucleus and the cytoplasm and are among the most stable protein assemblies in cells. Budding yeast cells carry two variants of NPCs which differ in the presence or absence of the nuclear basket proteins Mlp1, Mlp2, and Pml39. The binding of these basket proteins occurs very late in NPC assembly and Mlp-positive NPCs are excluded from the region of the nuclear envelope that borders the nucleolus. Here, we use recombination-induced tag exchange to investigate the stability of all the NPC subcomplexes within individual NPCs. We show that the nuclear basket proteins Mlp1, Mlp2, and Pml39 remain stably associated with NPCs through multiple cell-division cycles, and that Mlp1/2 are responsible for the exclusion of NPCs from the nucleolar territory. In addition, we demonstrate that binding of the FG-nucleoporins Nup1 and Nup2 depletes also Mlp-negative NPCs from this region by an independent pathway. We develop a method for single NPC tracking in budding yeast and observe that NPCs exhibit increased mobility in the absence of nuclear basket components. Our data suggest that the distribution of NPCs on the nucleus is governed by multiple interaction of nuclear basket proteins with the nuclear interior.

Nanoscale Quantitative Imaging of Single Nuclear Pore Complexes by Scanning Electrochemical Microscopy.

The nuclear pore complex (NPC) is a proteinaceous nanopore that solely and selectively regulates the molecular transport between the cytoplasm and nucleus of a eukaryotic cell. The ∼50 nm-diameter pore of the NPC perforates the double-membrane nuclear envelope to mediate both passive and facilitated molecular transport, thereby playing paramount biological and biomedical roles. Herein, we visualize single NPCs by scanning electrochemical microscopy (SECM). The high spatial resolution is accomplished by employing ∼25 nm-diameter ion-selective nanopipets to monitor the passive transport of tetrabutylammonium at individual NPCs. SECM images are quantitatively analyzed by employing the finite element method to confirm that this work represents the highest-resolution nanoscale SECM imaging of biological samples. Significantly, we apply the powerful imaging technique to address the long-debated origin of the central plug of the NPC. Nanoscale SECM imaging demonstrates that unplugged NPCs are more permeable to the small probe ion than are plugged NPCs. This result supports the hypothesis that the central plug is not an intrinsic transporter, but is an impermeable macromolecule, e.g., a ribonucleoprotein, trapped in the nanopore. Moreover, this result also supports the transport mechanism where the NPC is divided into the central pathway for RNA export and the peripheral pathway for protein import to efficiently mediate the bidirectional traffic.

Improving STED microscopy with SUPPOSe: enhancing resolution from a single-image

Here we apply the SUPPOSe algorithm on images acquired using Stimulated Emission Depletion (STED) microscopy with the aim of improving the resolution limit achieved. We processed images of the nuclear pore complex (NPC) from cell lines in which the Nup96 nucleoporin was endogenously labeled. This reference protein forms a ring whose diameter is ∼107 nm with 8 corners ∼42 nm apart from each other. The stereotypic arrangement of proteins in the NPC has been used as reference structures to characterize the performance of a variety of microscopy techniques. STED microscopy images resolve the ring arrangement but not the eightfold symmetry of the NPC. After applying the SUPPOSe algorithm to the STED images, we were able to solve the octagonal structure of the NPC. After processing 562 single NPC, the average radius of the NPC was found to be R = 54.2 ± 2.9 nm, being consistent with the theoretical distances of this structure. To verify that the solutions obtained are compatible with a NPC-type geometry, we rotate the solutions to optimally fit an eightfold-symmetric pattern and we count the number of corners that contain at least one localization. Fitting a probabilistic model to the histogram of the number of bright corners gives an effective labeling efficiency of 31%, which is in agreement with the values reported in for other cell lines and ligands used in Single Molecule Localization microscopy, showing that SUPPOSe can reliably retrieve sub-resolution, nanoscale objects from single acquisitions even in noisy conditions.

The magnified view: from ancient trinkets to single nuclear pore complexes.

A journey from the earliest known use of lenses and magnifying glasses in ancient times, through the development of microscopes and towards modern electron microscopy techniques. The evolving technology and improved microscopes enabled the discovery of intracellular organelles, the nucleus and nuclear pore complexes (NPCs). Current advances have led to composite three-dimensional models showing NPC structure in unprecedented detail but relying on the averaging of many images. A complementary approach is field emission scanning electron microscopy providing topographic surface images that are easily and intuitively interpreted by our brain. Recent advances in this technique have made it possible to expose nuclei from human cells and to focus on individual NPCs and their architectural features.

Nuclear export of pre-60S particles through the nuclear pore complex.

The nuclear pore complex (NPC) is the bidirectional gate that mediates the exchange of macromolecules or their assemblies between nucleus and cytoplasm1-3. The assembly intermediates of the ribosomal subunits, pre-60S and pre-40S particles, are among the largest cargoes of the NPC and the export of these gigantic ribonucleoproteins requires numerous export factors4,5. Here we report the cryo-electron microscopy structure of native pre-60S particles trapped in the channel of yeast NPCs. In addition to known assembly factors, multiple factors with export functions are also included in the structure. These factors in general bind to either the flexible regions or subunit interface of the pre-60S particle, and virtually form many anchor sites for NPC binding. Through interactions with phenylalanine-glycine (FG)repeats from various nucleoporins of NPC, these factors collectively facilitate the passage of the pre-60S particle through the central FG repeat network of the NPC. Moreover, in silico analysis of the axial and radial distribution of pre-60S particles within the NPC shows that a single NPC can take up to four pre-60S particles simultaneously, and pre-60S particles are enriched in theinner ring regions close to the wall of the NPC with the solvent-exposed surface facing the centre of the nuclear pore. Our data suggest a translocation model for the export of pre-60S particles through the NPC.

Direct Visualization of Single Nuclear Pore Complex Proteins Using Genetically‐Encoded Probes for DNA‐PAINT

AbstractThe nuclear pore complex (NPC) is one of the largest and most complex protein assemblies in the cell and, among other functions, serves as the gatekeeper of nucleocytoplasmic transport. Unraveling its molecular architecture and functioning has been an active research topic for decades with recent cryogenic electron microscopy and super‐resolution studies advancing our understanding of the architecture of the NPC complex. However, the specific and direct visualization of single copies of NPC proteins is thus far elusive. Herein, we combine genetically‐encoded self‐labeling enzymes such as SNAP‐tag and HaloTag with DNA‐PAINT microscopy. We resolve single copies of nucleoporins in the human Y‐complex in three dimensions with a precision of circa 3 nm, enabling studies of multicomponent complexes on the level of single proteins in cells using optical fluorescence microscopy.

Direct Visualization of Single Nuclear Pore Complex Proteins Using Genetically-Encoded Probes for DNA-PAINT.

The nuclear pore complex (NPC) is one of the largest and most complex protein assemblies in the cell and, among other functions, serves as the gatekeeper of nucleocytoplasmic transport. Unraveling its molecular architecture and functioning has been an active research topic for decades with recent cryogenic electron microscopy and super‐resolution studies advancing our understanding of the architecture of the NPC complex. However, the specific and direct visualization of single copies of NPC proteins is thus far elusive. Herein, we combine genetically‐encoded self‐labeling enzymes such as SNAP‐tag and HaloTag with DNA‐PAINT microscopy. We resolve single copies of nucleoporins in the human Y‐complex in three dimensions with a precision of circa 3 nm, enabling studies of multicomponent complexes on the level of single proteins in cells using optical fluorescence microscopy.

Imaging within single NPCs reveals NXF1's role in mRNA export on the cytoplasmic side of the pore.

Translocation of mRNA through the nuclear pore complex (NPC) requires interactions with different NPC regions. To determine the interactions that are crucial for effective mRNA export in living cells, we examined mRNA export within individual pores by applying various types of mRNA export blocks that stalled mRNPs at different stages of transition. Focusing on the major mRNA export factor NXF1, we found that initial mRNP binding to the NPC did not require NXF1 in the NPC, whereas release into the cytoplasm did. NXF1 localization in the NPC did not require RNA or RNA binding. Superresolution microscopy showed that NXF1 consistently occupied positions on the cytoplasmic side of the NPC. Interactions with specific nucleoporins were pinpointed using FLIM-FRET for measuring protein-protein interactions inside single NPCs, showing that Dbp5 helicase activity of mRNA release is conserved in yeast and humans. Altogether, we find that specific interactions on the cytoplasmic side of the NPC are fundamental for the directional flow of mRNA export.

The mode of mitosis is dramatically modified by deletion of a single nuclear pore complex gene in Aspergillus nidulans

Nuclear pore complex (NPC) proteins (Nups) play multiple roles during mitosis. In this study we expand these roles and reveal that in Aspergillus nidulans, compromising the core Nup84-120 subcomplex of the NPC modifies the mitotic behavior of the nuclear envelope (NE). In wildtype cells, the NE undergoes simultaneous double pinching events to separate daughter nuclei during mitotic exit, whereas in Nup84-120 complex mutants, only one restriction of the NE is observed. Investigating the basis for this modified behavior of the NE in Nup deleted cells uncovered previously unrealized roles for core Nups in mitotic exit. During wildtype anaphase, the NE surrounds the two separating daughter DNA masses which typically flank the central nucleolus, to form three distinct nuclear compartments. In contrast, deletion of core Nups frequently results in early nucleolar eviction from the mitotic nucleus, in turn causing an uncharacteristic dumbbell-shaped NE morphology of anaphase nuclei with a nuclear membrane bridge connecting the two forming G1 nuclei. Importantly, the absence of the nucleolus between the separating daughter nuclei during anaphase delays chromosome segregation and progression into G1 as nuclei remain connected by chromatin bridges. Proteins localizing to late segregating chromosome arms are observed between forming daughter nuclei, and the mitotic spindle fails to resolve in a timely manner. These chromatin bridges are occupied by the Aurora kinase until nuclei have fully separated, suggesting involvement of Aurora in monitoring mitotic spindle and nuclear membrane resolution during mitotic exit. Our findings thus reveal a novel requirement for core Nups in mediating nucleolar positioning during mitosis, which dictates the pattern of NE fissions during karyokinesis and facilitates normal chromosome segregation. The findings additionally demonstrate that the mode of mitosis can be dramatically modified by deletion of a single NPC gene and reveals surprising fluidity in mitotic mechanisms.

A user-interactive algorithm quantifying nuclear pore complex distribution within the nuclear lamina network in single molecular localization microscopic image.

For decades, components of the mammalian nuclear envelope (NE), such as the nuclear lamina and nuclear pore complexes (NPCs), have been largely resistant to quantitative cell biological analysis using conventional fluorescence microscopy. This is in part due to their sub diffraction limit dimensions. Super-resolution microscopy, a major advancement in cell biology research, has now made possible the acquisition of images in which nuclear lamin networks and single NPCs can be resolved in intact mammalian somatic cells. In particular, single molecule localization microscopy is able to generate data sets that are accurate enough to allow detailed quantitative analysis. Here we describe an algorithm that will identify the centroid of single NPCs and will determine their localization relative to the distribution of lamin protein filaments. Using this algorithm, a percentage of NPCs localized within the nuclear lamin network was accurately calculated, that could be compared between cells expressing different lamin complements. With modifications tweaked according to user specified sample images, this algorithm serves as a semi-automatic and fast computational tool to quantify and compare the localization and distribution of two or more cellular components at the nanometre scale.

(Invited) High-Resolution Scanning Electrochemical Microscopy of Biological and Artificial Nanopores

In this presentation, we discuss applications of scanning electrochemical microscopy (SECM) to high-resolution imaging of biological and artificial nanopores. SECM imaging enables us to obtain unprecedented information about the kinetics, pathways, and mechanism of molecular transport through single nanopores. We characterize nanopipet electrodes as nanoscale SECM probes by transmission electron microscopy to verify their small diameters of ~30 nm. The resultant high spatial resolution of our SECM instrument is quantitatively demonstrated by imaging ion transport through the single nanopores of a thin solid silicon membrane. The nanoscale SECM approach thus established is applied to image a biological nanopore, the nuclear pore complex (NPC), in aqueous solution, which eliminates artifacts that are caused when the NPC is imaged by electron microscopy in vacuum and atomic force microscopy in air. Our SECM study of single NPCs is significant, because it will led to a greater mechanistic understanding of molecular transport through the NPC, which plays crucial roles in the gene expression regulation and gene delivery and is linked to many human diseases and therapeutics of genetic disorders.

Characterization of nuclear pore complex components in fission yeast Schizosaccharomyces pombe

The nuclear pore complex (NPC) is an enormous proteinaceous complex composed of multiple copies of about 30 different proteins called nucleoporins. In this study, we analyzed the composition of the NPC in the model organism Schizosaccharomyces pombe using strains in which individual nucleoporins were tagged with GFP. We identified 31 proteins as nucleoporins by their localization to the nuclear periphery. Gene disruption analysis in previous studies coupled with gene disruption analysis in the present study indicates that 15 of these nucleoporins are essential for vegetative cell growth and the other 16 nucleoporins are non-essential. Among the 16 non-essential nucleoporins, 11 are required for normal progression through meiosis and their disruption caused abnormal spore formation or poor spore viability. Based on fluorescence measurements of GFP-fused nucleoporins, we estimated the composition of the NPC in S. pombe and found that the organization of the S. pombe NPC is largely similar to that of other organisms; a single NPC was estimated as being 45.8–47.8 MDa in size. We also used fluorescence measurements of single NPCs and quantitative western blotting to analyze the composition of the Nup107-Nup160 subcomplex, which plays an indispensable role in NPC organization and function. Our analysis revealed low amounts of Nup107 and Nup131 and high amounts of Nup132 in the Nup107-Nup160 subcomplex, suggesting that the composition of this complex in S. pombe may differ from that in S. cerevisiae and humans. Comparative analysis of NPCs in various organisms will lead to a comprehensive understanding of the functional architecture of the NPC.

Charge Effects on Nucleocytoplamsic Transport Studied by Single-Molecule Microscopy

In eukaryotic cells, the transport of genetic material and proteins between nucleus and cytoplasm is mediated by the nuclear pore complexes (NPCs) embedded in the double-membrane nuclear envelope. A highly selective barrier formed by phenylalanine-glycine (FG)-nucleoporin (Nup) with net positive charge in the NPC allows for passive diffusion of signal-independent small molecules (< 40 kDa) and transportin-facilitated translocation of signal-dependent large molecules (up to 50 MDa). Previously it was suggested that the positively charged FG-barrier would inhibit the transport of positively charged transiting molecules. However, the fundamental inhibition mechanism and the detailed pathways for both transport modes remain poorly understood. Here, we employ an innovative single-molecule technique, single-point edge-excitation subdiffraction (SPEED) microscopy, to track fluorescent single molecules with different charges transiting through single native NPCs. We have obtained the transport kinetics and the 3D transport pathways for both passive diffusion of variously charged GFPs (27 kDa) and facilitated translocation of transportin-cargo complexes with different charges. Our results indicate that (i): the positively charged selective barrier in the NPC possesses an axial central conduit for passive diffusion of small molecules with different charges, and allows for transportin-facilitated translocation of differently charged cargos via the peripheral regions around the central conduit; and (ii) the positively charged environment in the NPC significantly affects the transport pathway, transport time and efficiency for both passive and facilitated transport.

Super-Resolution by Feedback Imaging: Mechanisms of Translocation through the Nuclear Pore Complex

Nuclear pore complexes (NPCs) are the gateways for nucleocytoplasmic exchange. Because single molecules undergo rapid transport, it is challenging to follow their motion in live cells. Hence fundamental questions remain in regard to the nanomechanical basis of selective gating of molecules through the NPC in live cells.We set out to address these issues by a combination of fluorescence correlation spectroscopy (FCS) and real-time tracking of the center of mass of single NPCs in live cells. The center of mass tracking allows us to create an “Einstein trap” in which the thermal motion of the entire pore is compensated so that we observe the shuttling of single molecules in the reference frame of the pore. Using this setup we demonstrate that the transport of Karyopherin-β1 (Kapβ1) receptor is regulated to produce a characteristic narrow correlation time within the NPC, which is the signature of directed-motion events. We also show that the back and forth components of Kapβ1 transport are coupled by energy-consuming processes. Analogously, the dynamics of nucleoporin-153 (Nup153) at the nanoscale is characterized by a similar correlation pattern between two separate positions within the NPC. By means of the pair correlation function (pCF) analysis we separate the two components of Nup153 exchange: a fast collapse into compact conformations (cytoplasm-to-nucleus) and a slightly slower release into extended conformations (nucleus-to-cytoplasm). We demonstrate that this signature activity is directly linked to the functional import of classical transport receptors and cargoes.Thus, we propose that the selective gating through intact NPCs may be powered by spring-like molecular engines As far as we know, this is the first time that a selective gating model is proposed on the basis of experimental evidences obtained in live, unperturbed cells.Work supported by NIH-P41 P41-RRO3155,P50-GM076516.

Capturing directed molecular motion in the nuclear pore complex of live cells

Nuclear pore complexes (NPCs) are gateways for nucleocytoplasmic exchange. Intrinsically disordered nucleoporins (Nups) form a selective filter inside the NPC, taking a central role in the vital nucleocytoplasmic transport mechanism. How such intricate meshwork relates to function and gives rise to a transport mechanism is still unclear. Here we set out to tackle this issue in intact cells by an established combination of fluorescence correlation spectroscopy and real-time tracking of the center of mass of single NPCs. We find the dynamics of nucleoporin Nup153 to be regulated so as to produce rapid, discrete exchange between two separate positions within the NPC. A similar behavior is also observed for both karyopherinβ1 transport-receptor and cargoes destined to nuclear import. Thus, we argue that directed Nup-mediated molecular motion may represent an intrinsic feature of the overall selective gating through intact NPCs.

Molecular Springs in the Nuclear Pore Complex of Live Cells

Nuclear pore complexes (NPCs) are the gateways for nucleocytoplasmic exchange. Measurements of molecular transport through NPCs may provide valuable information to unravel the mechanism of communication between the nucleus and the cytoplasm. Unfortunately, because single molecules undergo very rapid transport, it is challenging to follow their motion in live cells. We set out to address the nanomechanical basis of pore function in intact cells by a combination of fluorescence correlation spectroscopy (FCS) and real-time tracking of the center of mass of single NPCs.

Fluorescence Correlation Spectroscopy of Intact Nuclear Pore Complexes

No methods proposed thus far have the sensitivity to measure the transport of single molecules through single nuclear pore complexes (NPCs) in intact cells. Here we demonstrate that fluorescence correlation spectroscopy (FCS) combined with real-time tracking of the center of mass of single NPCs in live, unperturbed cells allows us to detect the transport of single molecules in a reference system of a pore with high temporal (millisecond) and spatial (limited by diffraction) resolution. We find that the transport of the classical receptor karyopherin-β1 (Kapβ1) is regulated so as to produce a peculiar distribution of characteristic times at the NPC. This regulation, which is spatially restricted to the pore, depends on the properties and metabolic energy of Kapβ1. As such, this method provides a powerful tool for studying nucleocytoplasmic shuttling at the nanometer scale under physiological conditions.

Single Molecule Studies of Nuclear Transport

The translocation of single biomolecules through nanopores is one of the most fundamental processes of life. For example, the import of histones into the cell nucleus via the nuclear pore complex (NPC) is crucial for DNA condensation (1,2). We aim to gain a deeper understanding of NPC translocation on a single molecule level by combining optical tweezers with patch clamp techniques. This gives us the unique possibility to analyze the interaction forces between a biological macromolecule and the NPC while measuring the relevant time scales by electrophysiological recordings. We present preliminary results on the characterisation of the properties of NPCs concerning mode and duration of macromolecular translocation. Nuclei extracted from the syntactical coconut cocos nucifera are one model system. We demonstrate the selectivity of the NPC using an import signal tagged with GFP. We determine the conductance of a single nuclear pore to be approximately 490pS. Our results allow for estimation of the nuclear pore density to be between one and four per μm2. From observations of the ionic current across the pores, we are able to observe gating and to derive the translocation time of cargo across single NPCs . As a technical development towards nuclear pore force spectroscopy we introduce optical fiber illumination for real-time tracking of optically trapped particles. We demonstrate video-based fluctuation analysis of a single colloid at 10,000 Hz in order to study the dynamics of an attached biological macromolecule with microsecond time resolution (3-5).

Tracking Single Nuclear Pore Complexes in Live Cells: Importin β Transport and Beyond

The molecular details of transport through nuclear pore complexes (NPCs) have been well characterized, including the key role of Importin Beta (ImpB) receptor. However, the overall system behavior in intact cells is difficult to analyze because of its highly dynamic nature. Contrary to the common single particle tracking (SPT) approach that tracks an isolated particle as it moves through the pore, we set out to track the center of mass of the entire single NPC. While we track the pore, single molecules pass through it, and their location and dynamics are analyzed by fluorescence correlation spectroscopy (FCS). By this unconventional approach we find that ImpB transport is regulated so as to produce a characteristic bump in the autocorrelation function at the NPC. This regulation is spatially restricted to the pore, dependent on ImpB properties, pore structure, and metabolic energy. Combined to simulations our results suggest that ImpB movement within the pore is likely to be directed instead of unbiased, and that the back and forth components of its shuttling are coupled in time. We use our results to discriminate between existing NPC functional models, and identify key features that must be essential for transport in the intact pore.