Single-Molecule Imaging of Nuclear Transport

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the last decade. The investigation of molecular interactions with high temporal and spatial resolutions deep within cells has remained challenging due to the inherently weak signals arising from individual molecules. Recent works by Yang et al. demonstrated that narrow-field epifluorescence microscopy allows visualization of nucleocytoplasmic transport at the single molecule level. By the single molecule approach, important kinetics, such as nuclear transport time and efficiency, for signal-dependent and independent cargo molecules have been obtained. Here we described a protocol for the methodological approach with an improved spatiotemporal resolution of 0.4 ms and 12 nm. The improved resolution enabled us to capture transient active transport and passive diffusion events through the nuclear pore complexes (NPC) in semi-intact cells. We expect this method to be used in elucidating other binding and trafficking events within cells.Download video file.^{(133M, mp4)}     

Single molecule studies of nucleocytoplasmic transport

Molecular traffic between the cytoplasm and the nucleoplasm of eukaryotic cells is mediated by nuclear pore complexes (NPCs). Hundreds, if not thousands, of molecules interact with and transit through each NPC every second. The pore is blocked by a permeability barrier, which consists of a network of intrinsically unfolded polypeptides containing thousands of phenylalanine-glycine (FG) repeat motifs. This FG-network rejects larger molecules and admits smaller molecules or cargos bound to nuclear transport receptors (NTRs). For a cargo transport complex, minimally consisting of a cargo molecule plus an NTR, access to the permeability barrier is provided by interactions between the NTR and the FG repeat motifs. Numerous models have been postulated to explain the controlled accessibility and the transport characteristics of the FG-network, but the amorphous, flexible nature of this structure has hindered characterization. A relatively recent development is the ability to monitor the real-time movement of single molecules through individual NPCs via single molecule fluorescence (SMF) microscopy. A major advantage of this approach is that it can be used to continuously monitor a series of specific molecular interactions in an active pore with millisecond time resolution, which therefore allows one to distinguish between kinetic and thermodynamic control. Novel insights and prospects for the future are outlined in this review. This article is part of a Special Issue entitled: Regulation of Signaling and Cellular Fate through Modulation of Nuclear Protein Import.     

Nuclear transport of single molecules: dwell times at the nuclear pore complex

The mechanism by which macromolecules are selectively translocated through the nuclear pore complex (NPC) is still essentially unresolved. Single molecule methods can provide unique information on topographic properties and kinetic processes of asynchronous supramolecular assemblies with excellent spatial and time resolution. Here, single-molecule far-field fluorescence microscopy was applied to the NPC of permeabilized cells. The nucleoporin Nup358 could be localized at a distance of 70 nm from POM121-GFP along the NPC axis. Binding sites of NTF2, the transport receptor of RanGDP, were observed in cytoplasmic filaments and central framework, but not nucleoplasmic filaments of the NPC. The dwell times of NTF2 and transportin 1 at their NPC binding sites were 5.8 +/- 0.2 and 7.1 +/- 0.2 ms, respectively. Notably, the dwell times of these receptors were reduced upon binding to a specific transport substrate, suggesting that translocation is accelerated for loaded receptor molecules. Together with the known transport rates, our data suggest that nucleocytoplasmic transport occurs via multiple parallel pathways within single NPCs.     

Widefield deconvolution epifluorescence microscopy combined with fluorescence in situ hybridization reveals the spatial arrangement of bacteria in sponge tissue

Widefield deconvolution epifluorescence microscopy (WDEM) combined with fluorescence in situ hybridization (FISH) was performed to identify and characterize single bacterial cells within sections of the mediterranean sponge Chondrosia reniformis. Sponges were embedded in paraffin wax or plastic prior to the preparation of thin sections, in situ hybridization and microscopy. Serial digital images generated by widefield epifluorescence microscopy were visualized using an exhaustive photon reassignment deconvolution algorithm and three-dimensional rendering software. Computer processing of series of images taken at different focal planes with the deconvolution technique provided deblurred three-dimensional images with high optical resolution on a submicron scale. Results from the deconvolution enhanced widefield microscopy were compared with conventional epifluorescent microscopical images. By the application of the deconvolution algorithm on digital image data obtained with widefield epifluorescence microscopy after FISH, the occurrence and spatial arrangement of Desulfovibrionaceae closely associated with micropores of Chondrosia reniformis could be visualized.     

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.     

Nuclear side conformational changes in the nuclear pore complex following calcium release from the nuclear membrane

Changes in nuclear pore complex (NPC) structure are studied following treatments modifying the cisternal calcium levels located between the two lipid bilayers that together form the nuclear envelope. Since the NPC forms the only known passageway across the nuclear envelope, it plays a central role in nucleocytoplasmic transport. Understanding the origin of conformational changes that may affect this trafficking or modify cargo interactions with the NPC is, therefore, necessary to completely understand the function of these complex molecules. In previous studies on the cytoplasmic side of the nuclear envelope, a central mass was observed in the pore of the NPC and its location was shown to be sensitive to the cisternal calcium levels. Here we report atomic force microscopy (AFM) measurements on the nuclear side of the envelope, which also reveal a cisternal calcium dependence in the conformational state of the NPC. These measurements, made at the single nuclear pore level, reveal a displacement of the central mass towards the nuclear side of the membrane following treatments with adenophostin A, a specific agonist of calcium channels (inositol 1,4,5-trisphosphate (IP(3)) receptors) located in the nuclear envelope. We further demonstrate that these conformational changes are observed in nuclear pores lacking the basket structure while samples prepared in the presence of protease inhibitors retain baskets and block AFM measurements of the channel. While these measurements are unable to distinguish whether the central mass is cargo or an integral component of the NPC, its dose-dependent displacement with cisternal calcium levels does suggest links to transport or to changes in cargo interactions with the NPC. Taken together with previous measurements done on the cytoplasmic side of the nuclear envelope, these studies argue against a piston-like displacement of the central mass and instead suggest a more complicated mechanism. One possibility involves a concerted collapse of the NPC rings towards one another following cisternal calcium release, thus leading to the apparent emergence of the central mass from each side of the NPC.     

Detection and characterization of single biomolecules at surfaces

The investigation of bio-molecules has entered a new age since the development of methodologies capable of studies at the level of single molecules. In biology, most molecules show a complex dynamical behavior, with individual motions and transitions between different states, occurring as highly correlated in space and time within an arrangement of various elements. In order to resolve such dynamical changes in ensemble average techniques, one would have to synchronize all molecules, which is hard to achieve and might interfere with important system properties. Single molecule studies, in contrast, do not require pretreatment of the system and resume, therefore, much less invasive methodologies. Here, we review recent employments for the investigation of bio-molecules on surfaces, in which the high local and temporal resolution of two complementary techniques, atomic force microscopy and single molecule fluorescence microscopy, is used to address single molecules. Novel methodologies for the characterization of biologically relevant parameters, functions and dynamical aspects of individual molecules are described.     

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM)

Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and sensitivity in cells because the resolution of conventional light microscopy is diffraction-limited to ~250 nm. By combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM), PPIs can be visualized in cells with single molecule sensitivity and nanometer spatial resolution. BiFC is a commonly used technique for visualizing PPIs with fluorescence contrast, which involves splitting of a fluorescent protein into two non-fluorescent fragments. PALM is a recent superresolution microscopy technique for imaging biological samples at the nanometer and single molecule scales, which uses phototransformable fluorescent probes such as photoactivatable fluorescent proteins (PA-FPs). BiFC-PALM was demonstrated by splitting PAmCherry1, a PA-FP compatible with PALM, for its monomeric nature, good single molecule brightness, high contrast ratio, and utility for stoichiometry measurements. When split between amino acids 159 and 160, PAmCherry1 can be made into a BiFC probe that reconstitutes efficiently at 37 °C with high specificity to PPIs and low non-specific reconstitution. Ras-Raf interaction is used as an example to show how BiFC-PALM helps to probe interactions at the nanometer scale and with single molecule resolution. Their diffusion can also be tracked in live cells using single molecule tracking (smt-) PALM. In this protocol, factors to consider when designing the fusion proteins for BiFC-PALM are discussed, sample preparation, image acquisition, and data analysis steps are explained, and a few exemplary results are showcased. Providing high spatial resolution, specificity, and sensitivity, BiFC-PALM is a useful tool for studying PPIs in intact biological samples.     

Visualization of cargo concentration by COPII minimal machinery in a planar lipid membrane

Selective protein export from the endoplasmic reticulum is mediated by COPII vesicles. Here, we investigated the dynamics of fluorescently labelled cargo and non‐cargo proteins during COPII vesicle formation using single‐molecule microscopy combined with an artificial planar lipid bilayer. Single‐molecule analysis showed that the Sar1p–Sec23/24p‐cargo complex, but not the Sar1p–Sec23/24p complex, undergoes partial dimerization before Sec13/31p recruitment. On addition of a complete COPII mixture, cargo molecules start to assemble into fluorescent spots and clusters followed by vesicle release from the planar membrane. We show that continuous GTPase cycles of Sar1p facilitate cargo concentration into COPII vesicle buds, and at the same time, non‐cargo proteins are excluded from cargo clusters. We propose that the minimal set of COPII components is required not only to concentrate cargo molecules, but also to mediate exclusion of non‐cargo proteins from the COPII vesicles.     

The nuclear basket mediates perinuclear mRNA scanning in budding yeast

After synthesis and transit through the nucleus, messenger RNAs (mRNAs) are exported to the cytoplasm through the nuclear pore complex (NPC). At the NPC, messenger ribonucleoproteins (mRNPs) first encounter the nuclear basket where mRNP rearrangements are thought to allow access to the transport channel. Here, we use single mRNA resolution live cell microscopy and subdiffraction particle tracking to follow individual mRNAs on their path toward the cytoplasm. We show that when reaching the nuclear periphery, RNAs are not immediately exported but scan along the nuclear periphery, likely to find a nuclear pore allowing export. Deletion or mutation of the nuclear basket proteins MLP1/2 or the mRNA binding protein Nab2 changes the scanning behavior of mRNPs at the nuclear periphery, shortens residency time at nuclear pores, and results in frequent release of mRNAs back into the nucleoplasm. These observations suggest a role for the nuclear basket in providing an interaction platform that keeps RNAs at the periphery, possibly to allow mRNP rearrangements before export.     

Towards reconciling structure and function in the nuclear pore complex

The spatial separation between the cytoplasm and the cell nucleus necessitates the continuous exchange of macromolecular cargo across the double-membraned nuclear envelope. Being the only passageway in and out of the nucleus, the nuclear pore complex (NPC) has the principal function of regulating the high throughput of nucleocytoplasmic transport in a highly selective manner so as to maintain cellular order and function. Here, we present a retrospective review of the evidence that has led to the current understanding of both NPC structure and function. Looking towards the future, we contemplate on how various outstanding effects and nanoscopic characteristics ought to be addressed, with the goal of reconciling structure and function into a single unified picture of the NPC.     

Lighting up the nuclear pore complex

It is generally accepted that transport through the nuclear pore complex (NPC) involves an abundance of phenylalanine-glycine rich protein domains (FG-domains) that serve as docking sites for soluble nuclear transport receptors (NTRs) and their cargo complexes. But the precise mechanism of translocation through the NPC allowing for high speed and selectivity is still vividly debated. To ultimately decipher the underlying gating mechanism it is indispensable to shed more light on the molecular arrangement of FG-domains and the distribution of NTR-binding sites within the central channel of the NPC. In this review we revisit current transport models, summarize recent results regarding translocation through the NPC obtained by super-resolution microscopy and finally discuss the status and potential of optical methods in the analysis of the NPC.     

An integrated instrumental setup for the combination of atomic force microscopy with optical spectroscopy

In recent years, the study of single biomolecules using fluorescence microscopy and atomic force microscopy (AFM) techniques has resulted in a plethora of new information regarding the physics underlying these complex biological systems. It is especially advantageous to be able to measure the optical, topographical, and mechanical properties of single molecules simultaneously. Here an AFM is used that is especially designed for integration with an inverted optical microscope and that has a near-infrared light source (850 nm) to eliminate interference between the optical experiment and the AFM operation. The Tip Assisted Optics (TAO) system consists of an additional 100 x 100-microm(2) X-Y scanner for the sample, which can be independently and simultaneously used with the AFM scanner. This allows the offset to be removed between the confocal optical image obtained with the sample scanner and the simultaneously acquired AFM topography image. The tip can be positioned exactly into the optical focus while the user can still navigate within the AFM image for imaging or manipulation of the sample. Thus the tip-enhancement effect can be maximized and it becomes possible to perform single molecule manipulation experiments within the focus of a confocal optical image. Here this is applied to simultaneous measurement of single quantum dot fluorescence and topography with high spatial resolution.     

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.     

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.     

Random Walk of Processive, Quantum Dot-Labeled Myosin Va Molecules within the Actin Cortex of COS-7 Cells

Myosin Va (myoVa) is an actin-based intracellular cargo transporter. In vitro experiments have established that a single myoVa moves processively along actin tracks, but less is known about how this motor operates within cells. Here we track the movement of a quantum dot (Qdot)-labeled myoVa HMM in COS-7 cells using total internal reflectance fluorescence microscopy. This labeling approach is unique in that it allows myoVa, instead of its cargo, to be tracked. Single-particle analysis showed short periods (≤0.5 s) of ATP-sensitive linear motion. The mean velocity of these trajectories was 604 nm/s and independent of the number of myoVa molecules attached to the Qdot. With high time (16.6 ms) and spatial (15 nm) resolution imaging, Qdot-labeled myoVa moved with sequential 75 nm steps per head, at a rate of 16 s⁻¹, similarly to myoVa in vitro. Monte Carlo modeling suggests that the random nature of the trajectories represents processive myoVa motors undergoing a random walk through the dense and randomly oriented cortical actin network.     

Multiscale dynamics in nucleocytoplasmic transport

The nuclear pore complex (NPC) has long been viewed as a point-like entry and exit channel between the nucleus and the cytoplasm. New data support a different view whereby the complex displays distinct spatial dynamics of variable duration ranging from milliseconds to events spanning the entire cell cycle. Discrete interaction sites outside the central channel become apparent, and transport regulation at these sites seems to be of greater importance than currently thought. Nuclear pore components are highly active outside the NPC or impact the fate of cargo transport away from the nuclear pore. The NPC is a highly dynamic, crowded environment-constantly loaded with cargo while providing selectivity based on unfolded proteins. Taken together, this comprises a new paradigm in how we view import/export dynamics and emphasizes the multiscale nature of NPC-mediated cellular transport.     

Assembly of anthrax toxin pore: Lethal‐factor complexes into lipid nanodiscs

We have devised a procedure to incorporate the anthrax protective antigen (PA) pore complexed with the N‐terminal domain of anthrax lethal factor (LF_N) into lipid nanodiscs and analyzed the resulting complexes by negative‐stain electron microscopy. Insertion into nanodiscs was performed without relying on primary and secondary detergent screens. The preparations were relatively pure, and the percentage of PA pore inserted into nanodiscs on EM grids was high (～43%). Three‐dimensional analysis of negatively stained single particles revealed the LF_N‐PA nanodisc complex mirroring the previous unliganded PA pore nanodisc structure, but with additional protein density consistent with multiple bound LF_N molecules on the PA cap region. The assembly procedure will facilitate collection of higher resolution cryo‐EM LF_N‐PA nanodisc structures and use of advanced automated particle selection methods.     

Single nuclear pores visualized by confocal microscopy and image processing.

How nuclear pore complexes, mediating the transport of nucleic acids, proteins, and metabolites between cell nucleus and cytoplasm, are arranged in the nuclear envelope is essentially unknown. Here we describe a method combining high-resolution confocal imaging with image processing and pattern recognition to visualize single nuclear pore complexes (120 nm diameter), determine their relative positions with nanometer accuracy, and analyze their distribution in situ. The method was tested by means of a model system in which the very same sample areas could be imaged by confocal and electron microscopy. It was thus found that single fluorescent beads of 105 nm nominal diameter could be localized with a lateral accuracy of <20 nm and an axial accuracy of approximately 20 nm. The method was applied to digitonin-permeabilized 3T3 cells, whose nuclear pore complexes were fluorescently labeled with the anti-nucleoporin antibody mAb414. Stacks of optical sections were generated by confocal imaging at high resolution. Herein the nuclear pore complexes appeared as bright diffraction-limited spots whose centers were localized by fitting them by three-dimensional gaussians. The nearest-neighbor distribution function and the pair correlation function were calculated and found to agree well with those of randomly distributed hard cylinders of 138 +/- 17 nm diameter, but not with those of randomly distributed points or nonrandomly distributed cylinders. This was supported by a cluster analysis. Implications for the direct observation of the transport of single particles and molecules through individual nuclear pore complexes are discussed.