Conformation of ring single-stranded DNA measured by DNA origami structures.

Measuring the mechanical properties of single-stranded DNA (ssDNA) is a complex challenge that has been addressed lately by different methods. We measured the persistence length of ring ssDNA using a combination of a special DNA origami structure, a self-avoiding ring polymer simulation model, and nonparametric estimation statistics. The method overcomes the complexities set forth by previously used methods. We designed the DNA origami nano structures and measured the ring ssDNA polymer conformations using atomic force microscopy. We then calculated their radius of gyration, which was used as a fitting parameter for finding the persistence length. As there is no simple formulation for the radius of gyration distribution, we developed a simulation program consisting of a self-avoiding ring polymer to fit the persistence length to the experimental data. ssDNA naturally forms stem-loops, which should be taken into account in fitting a model to the experimental measurement. To overcome that hurdle, we found the possible loops using minimal energy considerations and used them in our fitting procedure of the persistence length. Due to the statistical nature of the loops formation, we calculated the persistence length for different percentages of loops that are formed. In the range of 25-75% loop formation, we found the persistence length to be 1.9-4.4 nm, and for 50% loop formation we get a persistence length of 2.83 ± 0.63 nm. This estimation narrows the previously known persistence length and provides tools for finding the conformations of ssDNA.

Chromatin Viscoelasticity Measured by Local Dynamic Analysis.

The nucleus in eukaryotic cells is a crowded environment that consists of genetic code along the DNA, together with a condensed solution of proteins, RNA, and other molecules. It is subjected to highly dynamic processes, including cell division, transcription, and DNA repair. In addition, the genome in the nucleus is subjected to external forces applied by the cytoplasmic skeleton and neighboring cells, as well as to internal nuclear forces. These forces oppose the need to maintain the genome order, which may be compensated by the internal nuclear viscoelastic properties that can restrain these forces. The structural and mechanical properties of chromatin inside the nucleus are still not fully clear; however, their importance for the proper functioning of the cells is unquestionable. Different approaches have been developed for this aim, ranging from directly measuring the dynamic and elastic properties of chromatin to studying the interactions of chromatin with the surrounding envelope and nuclear bodies. Although the elasticity of naked DNA in vitro is well characterized, the elasticity of chromatin in live cells is more complex and is still not fully understood. Here, we studied the elastic properties of chromatin by dynamic measurements in live cells, followed by viscoelastic modeling. We measured the trajectories of single chromatin loci, centromeres, and telomeres in live cells and analyzed their dynamics using the Langevin formalism. We assumed that the overall effect of the chromatin network forces can be modeled for each locus by a local harmonic potential and calculated the effective force constant. In addition, we assumed that this harmonic force results from the chromatin network formed by the internal polymer structure together with cross-links formed by the protein complex. We show that lamin A has the greatest effect on chromatin viscoelasticity and that its removal leads to a significant reduction in the local harmonic force.

Chromatin dynamics governed by a set of nuclear structural proteins.

During the past three decades, the study of nuclear and chromatin organization has become of great interest. The organization and dynamics of chromatin are directly responsible for many functions including gene regulation, genome replication, and maintenance. In order to better understand the details of these mechanisms, we need to understand the role of specific proteins that take part in these processes. The genome in the nucleus is organized in different length scales, ranging from the bead-like nucleosomes through topological associated domains up to chromosome territories. The mechanisms that maintain these structures, however, remain to be fully elucidated. Previous works highlighted the significance of lamin A, an important nucleoplasmic protein; however, there are other nuclear structural proteins that are also important for chromatin organization. Studying the organizational aspects of the nucleus is a complex task, and different methods have been developed and adopted for this purpose, including molecular and imaging methods. Here we describe the use of the live-cell imaging method and demonstrate that the dynamics of the nucleus is strongly related to its organizational mechanisms. We labeled different genomic sites in the nucleus and measured the effect of nuclear structural proteins on their dynamics. We studied lamin A, BAF, Emerin, lamin B, CTCF, and Cohesin and discuss how each of them affect chromatin dynamics. Our findings indicate that lamin A and BAF have a significant effect on chromosomes dynamics, while other proteins mildly affect the type of the diffusion while the volume of motion is not affected.

Measuring the Conformation and Persistence Length of Single-Stranded DNA Using a DNA Origami Structure.

Measuring the mechanical properties of single-stranded DNA (ssDNA) is a challenge that has been addressed by different methods lately. The short persistence length, fragile structure, and the appearance of stem loops complicate the measurement, and this leads to a large variability in the measured values. Here we describe an innovative method based on DNA origami for studying the biophysical properties of ssDNA. By synthesizing a DNA origami structure that consists of two rigid rods with an ssDNA segment between them, we developed a method to characterize the effective persistence length of a random-sequence ssDNA while allowing the formation of stem loops. We imaged the structure with an atomic force microscope (AFM); the rigid rods provide a means for the exact identification of the ssDNA ends. This leads to an accurate determination of the end-to-end distance of each ssDNA segment, and by fitting the measured distribution to the ideal chain polymer model we measured an effective persistence length of 1.98 ± 0.72 nm. This method enables one to measure short or long strands of ssDNA, and it can cope with the formation of stem loops that are often formed along ssDNA. We envision that this method can be used for measuring stem loops for determining the effect of repetitive nucleotide sequences and environmental conditions on the mechanical properties of ssDNA and the effect of interacting proteins with ssDNA. We further noted that the method can be extended to nanoprobes for measuring the interactions of specific DNA sequences, because the DNA origami rods (or similar structures) can hold multiple fluorescent probes that can be easily detected.

The role of near-wall drag effects in the dynamics of tethered DNA under shear flow.

We utilized single-molecule tethered particle motion (TPM) tracking, optimized for studying the behavior of short (0.922 µm) dsDNA molecules under shear flow conditions, in the proximity of a wall (surface). These experiments track the individual trajectories through a gold nanobead (40 nm in radius), attached to the loose end of the DNA molecules. Under such circumstances, local interactions with the wall become more pronounced, manifested through hydrodynamic interactions. To elucidate the mechanical mechanism that affects the statistics of the molecular trajectories of the tethered molecules, we estimate the resting diffusion coefficient of our system. Using this value and our measured data, we calculate the orthogonal distance of the extended DNA molecules from the surface. This calculation considers the hydrodynamic drag effect that emerges from the proximity of the molecule to the surface, using the Faxén correction factors. Our finding enables the construction of a scenario according to which the tension along the chain builds up with the applied shear force, driving the loose end of the DNA molecule away from the wall. With the extension from the wall, the characteristic times of the system decrease by three orders of magnitude, while the drag coefficients decay to a plateau value that indicates that the molecule still experiences hydrodynamic effects due to its proximity to the wall.

Genome organization in the nucleus: From dynamic measurements to a functional model.

A biological system is by definition a dynamic environment encompassing kinetic processes that occur at different length scales and time ranges. To explore this type of system, spatial information needs to be acquired at different time scales. This means overcoming significant hurdles, including the need for stable and precise labeling of the required probes and the use of state of the art optical methods. However, to interpret the acquired data, biophysical models that can account for these biological mechanisms need to be developed. The structure and function of a biological system are closely related to its dynamic properties, thus further emphasizing the importance of identifying the rules governing the dynamics that cannot be directly deduced from information on the structure itself. In eukaryotic cells, tens of thousands of genes are packed in the small volume of the nucleus. The genome itself is organized in chromosomes that occupy specific volumes referred to as chromosome territories. This organization is preserved throughout the cell cycle, even though there are no sub-compartments in the nucleus itself. This organization, which is still not fully understood, is crucial for a large number of cellular functions such as gene regulation, DNA breakage repair and error-free cell division. Various techniques are in use today, including imaging, live cell imaging and molecular methods such as chromosome conformation capture (3C) methods to better understand these mechanisms. Live cell imaging methods are becoming well established. These include methods such as Single Particle Tracking (SPT), Continuous Photobleaching (CP), Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Correlation Spectroscopy (FCS) that are currently used for studying proteins, RNA, DNA, gene loci and nuclear bodies. They provide crucial information on its mobility, reorganization, interactions and binding properties. Here we describe how these dynamic methods can be used to gather information on genome organization, its stabilization mechanisms and the proteins that take part in it.

The proteolysis adaptor, NblA, is essential for degradation of the core pigment of the cyanobacterial light-harvesting complex.

The cyanobacterial light-harvesting complex, the phycobilisome, is degraded under nutrient limitation, allowing the cell to adjust light absorbance to its metabolic capacity. This large light-harvesting antenna comprises a core complex of the pigment allophycocyanin, and rod-shaped pigment assemblies emanating from the core. NblA, a low-molecular-weight protein, is essential for degradation of the phycobilisome. NblA mutants exhibit high absorbance of rod pigments under conditions that generally elicit phycobilisome degradation, implicating NblA in degradation of these pigments. However, the vast abundance of rod pigments and the substantial overlap between the absorbance spectra of rod and core pigments has made it difficult to directly associate NblA with proteolysis of the phycobilisome core. Furthermore, lack of allophycocyanin degradation in an NblA mutant may reflect a requirement for rod degradation preceding core degradation, and does not prove direct involvement of NblA in proteolysis of the core pigment. Therefore, in this study, we used a mutant lacking phycocyanin, the rod pigment of Synechococcus elongatusPCC7942, to examine whether NblA is required for allophycocyanin degradation. We demonstrate that NblA is essential for degradation of the core complex of the phycobilisome. Furthermore, fluorescence lifetime imaging microscopy provided in situ evidence for the interaction of NblA with allophycocyanin, and indicated that NblA interacts with allophycocyanin complexes that are associated with the photosynthetic membranes. Based on these data, as well as previous observations indicating interaction of NblA with phycobilisomes attached to the photosynthetic membranes, we suggest a model for sequential phycobilisome disassembly by NblA.

Rapid microscopy measurement of very large spectral images.

The spectral content of a sample provides important information that cannot be detected by the human eye or by using an ordinary RGB camera. The spectrum is typically a fingerprint of the chemical compound, its environmental conditions, phase and geometry. Thus measuring the spectrum at each point of a sample is important for a large range of applications from art preservation through forensics to pathological analysis of a tissue section. To date, however, there is no system that can measure the spectral image of a large sample in a reasonable time. Here we present a novel method for scanning very large spectral images of microscopy samples even if they cannot be viewed in a single field of view of the camera. The system is based on capturing information while the sample is being scanned continuously 'on the fly'. Spectral separation implements Fourier spectroscopy by using an interferometer mounted along the optical axis. High spectral resolution of ~5 nm at 500 nm could be achieved with a diffraction-limited spatial resolution. The acquisition time is fairly high and takes 6-8 minutes for a sample size of 10mm x 10mm measured under a bright-field microscope using a 20X magnification.

Uniform Contraction-Expansion Description of Relative Centromere and Telomere Motion.

Internal organization and dynamics of the eukaryotic nucleus have been at the front of biophysical research in recent years. It is believed that both dynamics and location of chromatin segments are crucial for genetic regulation. Here we study the relative motion between centromeres and telomeres at various distances and at times relevant for genetic activity. Using live-imaging fluorescent microscopy coupled to stochastic analysis of relative trajectories, we find that the interlocus motion is distance-dependent with a varying fractional memory. In addition to short-range constraining, we also observe long-range anisotropic-enhanced parallel diffusion, which contradicts the expectation for classic viscoelastic systems. This motion is linked to uniform expansion and contraction of chromatin in the nucleus, and leads us to define and measure a new (to our knowledge) uniform contraction-expansion diffusion coefficient that enriches the contemporary picture of nuclear behavior. Finally, differences between loci types suggest that different sites along the genome experience distinctive coupling to the nucleoplasm environment at all scales.

The proteolysis adaptor, NblA, initiates protein pigment degradation by interacting with the cyanobacterial light-harvesting complexes.

Degradation of the cyanobacterial protein pigment complexes, the phycobilisomes, is a central acclimation response that controls light energy capture. The small protein, NblA, is essential for proteolysis of these large complexes, which may reach a molecular mass of up to 4 MDa. Interactions of NblA in vitro supported the suggestion that NblA is a proteolysis adaptor that labels the pigment proteins for degradation. The mode of operation of NblA in situ, however, remained unresolved. Particularly, it was unclear whether NblA interacts with phycobilisome proteins while part of the large complex, or alternatively interaction with NblA, necessitates dissociation of pigment subunits from the assembly. Fluorescence intensity profiles demonstrated the preferential presence of NblA::GFP (green fluorescent protein) at the photosynthetic membranes, indicating co-localization with phycobilisomes. Furthermore, fluorescence lifetime imaging microscopy provided in situ evidence for interaction of NblA with phycobilisome protein pigments. Additionally, we demonstrated the role of NblA in vivo as a proteolysis tag based on the rapid degradation of the fusion protein NblA::GFP compared with free GFP. Taken together, these observations demonstrated in vivo the role of NblA as a proteolysis adaptor. Additionally, the interaction of NblA with phycobilisomes indicates that the dissociation of protein pigment subunits from the large complex is not a prerequisite for interaction with this adaptor and, furthermore, implicates NblA in the disassembly of the protein pigment complex. Thus, we suggest that, in the case of proteolysis of the phycobilisome, the adaptor serves a dual function: undermining the complex stability and designating the dissociated pigments for degradation.

Single-allele analysis of transcription kinetics in living mammalian cells.

We generated a system for in vivo visualization and analysis of mammalian mRNA transcriptional kinetics of single alleles in real time, using single-gene integrations. We obtained high-resolution transcription measurements of a single cyclin D1 allele under endogenous or viral promoter control, including quantification of temporal kinetics of transcriptional bursting, promoter firing, nascent mRNA numbers and transcription rates during the cell cycle, and in relation to DNA replication.

Compressive hyperspectral microscopy for cancer detection.

Significance: Hyperspectral microscopy grants the ability to characterize unique properties of tissues based on their spectral fingerprint. The ability to label and measure multiple molecular probes simultaneously provides pathologists and oncologists with a powerful tool to enhance accurate diagnostic and prognostic decisions. As the pathological workload grows, having an objective tool that provides companion diagnostics is of immense importance. Therefore, fast whole-slide spectral imaging systems are of immense importance for automated cancer prognostics that meet current and future needs. Aim: We aim to develop a fast and accurate hyperspectral microscopy system that can be easily integrated with existing microscopes and provide flexibility for optimizing measurement time versus spectral resolution. Approach: The method employs compressive sensing (CS) and a spectrally encoded illumination device integrated into the illumination path of a standard microscope. The spectral encoding is obtained using a compact liquid crystal cell that is operated in a fast mode. It provides time-efficient measurements of the spectral information, is modular and versatile, and can also be used for other applications that require rapid acquisition of hyperspectral images. Results: We demonstrated the acquisition of breast cancer biopsies hyperspectral data of the whole camera area within ∼1 s. This means that a typical 1×1 cm2 biopsy can be measured in ∼10 min. The hyperspectral images with 250 spectral bands are reconstructed from 47 spectrally encoded images in the spectral range of 450 to 700 nm. Conclusions: CS hyperspectral microscopy was successfully demonstrated on a common lab microscope for measuring biopsies stained with the most common stains, such as hematoxylin and eosin. The high spectral resolution demonstrated here in a rather short time indicates the ability to use it further for coping with the highly demanding needs of pathological diagnostics, both for cancer diagnostics and prognostics.

Universal algorithm for identification of fractional Brownian motion. A case of telomere subdiffusion.

We present a systematic statistical analysis of the recently measured individual trajectories of fluorescently labeled telomeres in the nucleus of living human cells. The experiments were performed in the U2OS cancer cell line. We propose an algorithm for identification of the telomere motion. By expanding the previously published data set, we are able to explore the dynamics in six time orders, a task not possible earlier. As a result, we establish a rigorous mathematical characterization of the stochastic process and identify the basic mathematical mechanisms behind the telomere motion. We find that the increments of the motion are stationary, Gaussian, ergodic, and even more chaotic--mixing. Moreover, the obtained memory parameter estimates, as well as the ensemble average mean square displacement reveal subdiffusive behavior at all time spans. All these findings statistically prove a fractional Brownian motion for the telomere trajectories, which is confirmed by a generalized p-variation test. Taking into account the biophysical nature of telomeres as monomers in the chromatin chain, we suggest polymer dynamics as a sufficient framework for their motion with no influence of other models. In addition, these results shed light on other studies of telomere motion and the alternative telomere lengthening mechanism. We hope that identification of these mechanisms will allow the development of a proper physical and biological model for telomere subdynamics. This array of tests can be easily implemented to other data sets to enable quick and accurate analysis of their statistical characteristics.

Cancer detection from stained biopsies using high-speed spectral imaging.

The escalating demand for diagnosing pathological biopsies requires the procedures to be expedited and automated. The existing imaging systems for measuring biopsies only measure color, and even though a lot of effort is invested in deep learning analysis, there are still serious challenges regarding the performance and validity of the data for the intended medical setting. We developed a system that rapidly acquires spectral images from biopsies, followed by spectral classification algorithms. The spectral information is remarkably more informative than the color information, and leads to very high accuracy in identifying cancer cells, as tested on tens of cancer cases. This was improved even more by using artificial intelligence algorithms that required a rather small training set, indicating the high level of information that exists in the spectral images. The most important spectral differences are observed in the nucleus and they are related to aneuploidy in tumor cells. Rapid spectral imaging measurement therefore can bridge the gap in the machine-aided diagnostics of whole biopsies, thus improving patient care, and expediting the treatment procedure.

HU protein induces incoherent DNA persistence length.

HU is a highly conserved protein that is believed to play an important role in the architecture and dynamic compaction of bacterial DNA. Its ability to control DNA bending is crucial for functions such as transcription and replication. The effects of HU on the DNA structure have been studied so far mainly by single molecule methods that require us to apply stretching forces on the DNA and therefore may perturb the DNA-protein interaction. To overcome this hurdle, we study the effect of HU on the DNA structure without applying external forces by using an improved tethered particle motion method. By combining the results with DNA curvature analysis from atomic force microscopy measurements we find that the DNA consists of two different curvature distributions and the measured persistence length is determined by their interplay. As a result, the effective persistence length adopts a bimodal property that depends primarily on the HU concentration. The results can be explained according to a recently suggested model that distinguishes single protein binding from cooperative protein binding.

Novel automated three-dimensional genome scanning based on the nuclear architecture of telomeres.

Telomeres, the end of chromosomes, are organized in a nonoverlapping fashion and form microterritories in nuclei of normal cells. Previous studies have shown that normal and tumor cell nuclei differ in their 3D telomeric organization. The differences include a change in the spatial organization of the telomeres, in telomere numbers and sizes and in the presence of telomeric aggregates. Previous attempts to identify the above parameters of 3D telomere organization were semi-automated. Here we describe the automation of 3D scanning for telomere signatures in interphase nuclei based on three-dimensional fluorescent in situ hybridization (3D-FISH) and, for the first time, define its sensitivity in tumor cell detection. The data were acquired with a high-throughput scanning/acquisition system that allows to measure cells and acquire 3D images of nuclei at high resolution with 40 × or 60 × oil and at a speed of 10,000-15,000 cells h(-1) , depending on the cell density on the slides. The automated scanning, TeloScan, is suitable for large series of samples and sample sizes. We define the sensitivity of this automation for tumor cell detection. The data output includes 3D telomere positions, numbers of telomeric aggregates, telomere numbers, and telomere signal intensities. We were able to detect one aberrant cell in 1,000 normal cells. In conclusions, we are able to detect tumor cells based on 3D architectural profiles of the genome. This new tool could, in the future, assist in patient diagnosis, in the detection of minimal residual disease, in the analysis of treatment response and in treatment decisions.

Translocation frequencies and chromosomal proximities for selected mouse chromosomes in primary B lymphocytes.

Chromosome positions within the nucleus of mammalian cells are nonrandom and it is assumed that chromosomal neighborhoods affect the probability of translocations. Four chromosomes can be involved in c-myc-activating chromosomal translocations in mouse plasmacytoma (PCT): the c-myc gene on mouse chromosome 15 can be juxtaposed to either one of the immunoglobulin (Ig) loci on chromosomes 12 (IgH), 16 (Igλ), or 6 (Igκ). In the BALB/c mouse, the translocation between chromosomes 12 and 15, T(12;15), is most common (90%) while the other two possible translocations, T(6;15) and T(16;15), are much less common (<10%). In contrast, in the BALB/cRb6.15 mouse, T(6;15) is found with the same frequency as T(12;15). We, therefore, examined the distance between chromosomes 15 and 12, 6, and 16 in primary mouse B lymphocytes in order to examine the effect of the chromosome proximity on the translocation frequency. We performed three-dimensional fluorescent in situ hybridization (3D-FISH) with chromosome paints. We acquired three-dimensional image stacks with 90 slices per stack and used constrained iterative deconvolution. The nucleus and chromosomes were segmented from this image stack and the interchromosomal distances were measured. Chromosomes 6 and 15 were found in close proximity in BALB/cRb6.15 mice (82%), whereas they did not share this neighborhood relationship in BALB/c mice. No other chromosome combinations showed such a high percentage of close proximities in either mouse strain. Chromosome positions contribute to translocation frequencies in mouse PCTs. The BALB/cRb6.15 mouse data argue for a proximity relationship of chromosomes that engage in illegitimate recombination. These positions are not, however, the only contributing factor as the T(12;15) translocation preference in BALB/c mice could not be supported by significantly elevated proximity of chromosomes 12 and 15 versus 12 and 16 or 12 and 6. Moreover, while there is a significant increase in T(6;15) in BALB/cRb6.15 mice, T(12;15) still occurs in this mouse strain.

The Dynamics of Lamin a During the Cell Cycle.

Lamin proteins play an essential role in maintaining the nuclear organization and integrity; and lamin A, in particular, plays a major role in the whole volume of the nuclear interior. Although the nucleus is highly organized, it is rather dynamic, it affects crucial nuclear processes and its organization must change as cells progress through the cell cycle. Although many aspects of these changes are already known, the role of lamin A during nuclear assembly and disassembly as well as its underlying mechanisms remains controversial. Here we used live cells imaging and Continuous Photobleaching (CP) method to shed light on the dynamics and mechanisms of lamin A during the cell cycle, combined with imaging flow cytometry measurements, which provides the high-throughput capabilities of flow cytometry with single-cell imaging. As a major analysis tool, we used spatial correlation algorithm for allocating the distribution of lamin A, chromatin and tubulin, as well as their mutual colocalization. Furthermore, we analyzed the distribution of lamin A along the nuclear lamina and in the nucleus interior during the cell cycle. Our results indicate that at the beginning of the cell division that include prophase, metaphase and anaphase, lamin A is distributed throughout the cytoplasm and its concentration in the chromosomal regions is reduced, whereas the spatial correlation between lamin A and tubulin is increased. It implies that lamin A also disassembled in the whole cellular volume. At the telophase and early G1, lamin A is concentrated in the whole volume of the newly formed nuclei of the daughter cells and it assembles to the lamina. We also explored the functional aspects of lamin A during the cell cycle and its binding to the chromatin versus the freely diffusion form. We found that the fraction of the bound proteins of lamin A in the S phase increased, relative to the G1 phase, which means that during replication, the concentration of lamin A on the chromatin increases. All these results shed light on the function of lamin A throughout the cell cycle.

LAP2alpha maintains a mobile and low assembly state of A-type lamins in the nuclear interior.

Lamins form stable filaments at the nuclear periphery in metazoans. Unlike B-type lamins, lamins A and C localize also in the nuclear interior, where they interact with lamin-associated polypeptide 2 alpha (LAP2α). Using antibody labeling, we previously observed a depletion of nucleoplasmic A-type lamins in mouse cells lacking LAP2α. Here, we show that loss of LAP2α actually causes formation of larger, biochemically stable lamin A/C structures in the nuclear interior that are inaccessible to lamin A/C antibodies. While nucleoplasmic lamin A forms from newly expressed pre-lamin A during processing and from soluble mitotic lamins in a LAP2α-independent manner, binding of LAP2α to lamin A/C during interphase inhibits formation of higher order structures, keeping nucleoplasmic lamin A/C in a mobile state independent of lamin A/C S22 phosphorylation. We propose that LAP2α is essential to maintain a mobile lamin A/C pool in the nuclear interior, which is required for proper nuclear functions.

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.