The tumor suppressor p53 is subject to both nuclear import and export, and both are fast, energy-dependent and lectin-inhibited.

Human p53 was expressed in E. coli, purified, labeled with fluorescein iodoacetamide (IAF) and characterized for sequence-specific DNA binding and epitope disposition. Injected into the cytoplasm or nuclei of 3T3 cells IAF-p53 was imported into or exported from nuclei within minutes. Import was inhibited by coinjection of the lectin wheat germ agglutinine (WGA). In contrast, the peptide-protein conjugate NLS-HSA carrying the nuclear localization sequence (NLS) of the SV40 T antigen was only imported but not exported. 3T3 polykaryons were injected with IAF-p53 and photo-bleached by Scanning Microphotolysis in such a manner that only a single nucleus per polykaryon remained non-bleached. IAF-p53 was found to migrate rapidly (halftime 10 min) from non-bleached into bleached nuclei, while NLS-HSA did not. In digitonin permeabilized cells IAF-p53 was imported into nuclei. When removed from the medium after nuclear accumulation IAF-p53 was exported from the nuclei. Nuclear import and export of IAF-p53 both were rapid (halftimes of a few minutes, 22 C) and strongly inhibited by WGA or incubation on ice. NLS-HSA was only imported but not exported. We conclude that the nucleocytoplasmic transport of p53, in contrast to that of NLS-HSA, is bidirectional and that transport in both directions is carrier mediated and energy dependent. These results suggest that p53 contains nuclear export signals (NES) in addition to import signals (NLS) and thus open new views on the potential regulation of p53 cellular fractions.

The cutaneous microfibrillar apparatus contains latent transforming growth factor-beta binding protein-1 (LTBP-1) and is a repository for latent TGF-beta1.

The transforming growth factors-beta1 and beta2 (TGF-beta) stimulate synthesis of extracellular matrix proteins in vitro and appear upregulated in fibrotic conditions, in scar formation, and in wound healing. The extracellular matrix in turn might also act as a scavenger or repository for TGF-beta. We therefore studied the in situ distribution of latent TGF binding protein-1 (LTBP-1) and latent TGF-beta1 on extracellular matrix elements of normal human skin and skin regenerating from cultured keratinocyte autografts. We localized both LTBP-1 and latent TGF-beta1 to fibrillin-containing (elastic) microfibrils. Both LTBP-1 and latent TGF-beta1 were already present during the earliest stages of the de novo formation of the microfibrillar apparatus, i.e., on fusiform, randomly oriented microfibrils that later coalesced to form the typical candelabra-like structures in the papillary dermis. We show herewith that LTBP-1 exerts a dual role as a component of fibrillin-microfibrils of the skin and in targeting latent TGF-beta1 to the cutaneous microfibrillar apparatus. Thus, this major connective tissue structure does not only serve as a force bearing element and scaffold for elastin deposition in the dermis, but also as an important repository for latent TGF-beta in the skin.

Human dermal fibroblasts express prohormone convertases 1 and 2 and produce proopiomelanocortin-derived peptides.

In the last few years it has become apparent that the skin is a locoregional source for several proopiomelanocortin-derived peptides including alpha-melanocyte-stimulating hormone, adrenocorticotropin, and beta-endorphin. The enzymes that regulate expression of these neuropeptides are the prohormone convertases 1 and 2. In this study we demonstrate, by reverse transcriptase polymerase chain reaction and Western immunoblotting, that cultured human dermal fibroblasts express prohormone convertases 1 and 2 as well as 7B2, which is an essential cofactor for enzymatic activity of prohormone convertase 2. Immunofluorescence studies revealed prohormone convertase 1 to be mainly expressed in the perinuclear region in vesicular structures resembling the trans-Golgi network, whereas prohormone convertase 2 was found in the trans-Golgi network as well as in vesicular structures diffusely distributed in the peripheral cytoplasm. Expression of both enzymes was also confirmed in fibroblasts of normal adult human skin by immunohistochemistry using antibodies against prohormone convertases 1 and 2 and vimentin. To assess the relevance of prohormone convertase 1 and 2 expression in human dermal fibroblasts, we studied the expression of proopiomelanocortin and proopiomelanocortin-derived peptides. Proopiomelanocortin expression was detected by reverse transcriptase polymerase chain reaction and Western immunoblotting. Alpha-melanocyte-stimulating hormone, adrenocorticotropin, and beta-endorphin were mainly located in vesicular structures as demonstrated by immunofluorescence. Production of these peptides was confirmed by radioimmunoassay, immunoradiometric assay, or enzyme immunoassay. Among several stimuli tested, interleukin-1 was found to upregulate production of alpha-melanocyte-stimulating hormone in human dermal fibroblasts. In summary, we have shown that human dermal fibroblasts express the enzymatic machinery for proopiomelanocortin processing and make proopiomelanocortin, alpha-melanocyte-stimulating hormone, adrenocorticotropin, and beta-endorphin. Production of proopiomelanocortin peptides by human dermal fibroblasts may be relevant for fibroblast functions such as collagen degradation and/or regulation of dermal immune responses.

Transglutaminase activity in the eye: cross-linking in epithelia and connective tissue structures.

PURPOSE: To assess the distribution of transglutaminase (TGase) activity in ocular tissues and the target structures for cross-linking. METHODS: Cryosections from human and cynomolgus monkey eyes were incubated with the biotinylated amine donor substrate cadaverine (biotC), which was subsequently visualized with streptavidin-peroxidase. Confocal laser scanning was used to colocalize biotC and fibrillin, a major component of elastic microfibrils and the zonular fibers in particular. Cryosections and isolated bovine zonules were treated with purified TGase 2 and biotC. The distribution of different TGases (1, 2, 3, and factor XIII) was confirmed immunohistochemically. RESULTS: Virtually all ocular tissues showed TGase activity with a remarkable preponderance for the ciliary body, zonular fibers, and blood vessel walls. Confocal laser scanning revealed fibrillin-containing microfibrils as a major target for TGase activity, in particular the ciliary zonules. Corneal epithelium and basement membrane showed a TGase cross-linking pattern similar to skin. Treatment of cryosections and isolated bovine zonular fibers with purified TGase 2 led to additional incorporation of biotC into extracellular matrix, particularly zonular fibers. The immunohistochemically predominant TGase 2 was associated with epithelia and particularly with connective tissue fibers. TGase 1 was restricted to the corneal epithelium, whereas factor XIII was found to be associated only with blood vessels. TGase 3 was absent. CONCLUSIONS: TGase 2 appears to be an important cross-linker and thus stabilizer of ocular connective tissue. In particular, the zonular fibers are a major target for TGase 2. This is of relevance in hereditary microfibrillopathies such as Marfan syndrome, which exhibits distinct ocular manifestations such as elongated bulbus, retinal detachment, and subluxation of the lens. Purified or recombinant TGase might be of therapeutic use in the future.

Characterization of a polyclonal antibody raised against the human melanocortin-1 receptor.

We have generated a polyclonal antibody raised against a synthetic peptide corresponding to the amino acids 2-18 of the extracellular, N-terminal domain of the human melanocortin-1 receptor (MC-1R). Specificity of the affinity-purified anti-MC-1R antibody was confirmed by dot blot analysis with the antigenic peptide. The antibody detected MC-1R antigenicity on the surface of normal human melanocytes and WM35 melanoma cells, as shown by FACS and immunofluorescence analysis. The antibody was suitable for immunoperoxidase staining of deparaffinized skin sections, revealing prominent MC-1R staining of a cutaneous melanoma as opposed to undiseased skin in which normal melanocytes were only occasionally immunoreactive. Distinct adnexal structures in normal skin also displayed MC-1R immunostaining. Specificity of the MC-1R immunoreactivity in each technique was confirmed by preabsorption with the immunogenic peptide, omission, or substitution of the primary antibody with preimmune serum. Our results provide a baseline for future studies on MC-1R expression in diseased human skin.

High-resolution near-field optical imaging of single nuclear pore complexes under physiological conditions.

Scanning near-field optical microscopy (SNOM) circumvents the diffraction limit of conventional light microscopy and is able to achieve optical resolutions substantially below 100 nm. However, in the field of cell biology SNOM has been rarely applied, probably because previous techniques for sample-distance control are less sensitive in liquid than in air. Recently we developed a distance control based on a tuning fork in tapping mode, which is also well-suited for imaging in solution. Here we show that this approach can be used to visualize single membrane protein complexes kept in physiological media throughout. Nuclear envelopes were isolated from Xenopus laevis oocytes at conditions shown recently to conserve the transport functions of the nuclear pore complex (NPC). Isolated nuclear envelopes were fluorescently labeled by antibodies against specific proteins of the NPC (NUP153 and p62) and imaged at a resolution of approximately 60 nm. The lateral distribution of epitopes within the supramolecular NPC could be inferred from an analysis of the intensity distribution of the fluorescence spots. The different number densities of p62- and NUP153-labeled NPCs are determined and discussed. Thus we show that SNOM opens up new possibilities for directly visualizing the transport of single particles through single NPCs and other transporters.

Scanning microphotolysis: three-dimensional diffusion measurement and optical single-transporter recording.

Scanning microphotolysis (SCAMP) is a combination of fluorescence microphotolysis and confocal laser scanning microscopy. A laser scanning microscope is equipped with an optical switch able to modulate the power or/and wavelength of the laser beam in less than a microsecond while a dedicated computer program is employed to precisely coordinate scanning process and laser beam modulation. By these means it becomes possible to vary the power or/and wavelength of the laser beam during scanning at a precision of one resolution element. Patterns of almost arbitrary design can be written into the object by photolysis, e.g., photobleaching or photoactivation. The dissipation of the photolysis pattern by diffusion or other types of molecular transport can be followed at confocal resolution and used to characterize the transport process. SCAMP can be employed in conjunction with single-photon or multiphoton excitation. Furthermore, it can be easily installed on virtually any confocal laser scanning microscope. We summarize at first the conceptual and practical basis of SCAMP. Then, two novel applications are discussed: (i) measurements of translational diffusion coefficients in truly three-dimensional systems at diffraction-limited resolution, and (ii) optical recording of single transporters in membrane patches.

Osmotically evoked shrinking of guard-cell protoplasts causes vesicular retrieval of plasma membrane into the cytoplasm.

The dye FM1-43 was used alone or in combination with measurements of the membrane capacitance (Cm) to monitor membrane changes in protoplasts from Vicia faba L. guard cells. Confocal images of protoplasts incubated with FM1-43 (10 microM) at constant ambient osmotic pressure (pi omicron) revealed in confocal images a slow internalisation of FM1-43-labelled membrane into the cytoplasm. As a result of this process the relative fluorescence intensity of the cell interior (fFM,i) increased with reference to the total fluorescence (fFM,t) by 7.4 x 10(-4) min(-1). This steady internalisation of dye suggests the occurrence of constitutive endocytosis under constant osmotic pressure. Steady internalisation of FM1-43 labelled membrane caused a prominent staining of a ring-like structure located beneath the plasma membrane. Abrupt elevation of pi omicron by 200 mosmol kg(-1) caused, over the first minutes of incubation, a rapid internalisation of FM1-43 fluorescence into the cytoplasm concomitant with a decrease in cell perimeter. Within the first 5 min the cell perimeter decreased by 7.9%. Over the same time fFM,i/fFM,t increased by 0.13, reflecting internalisation of fluorescent label into the cytoplasm. Combined measurements of Cm and total fluorescence of a protoplast (fFM,p) showed that an increase in pi omicron evoked a decrease in Cm but no change in fFM,p. This means that surface contraction of the protoplast is due to retrieval of excess membrane from the plasma membrane and internalisation into the cytoplasm. Further inspection of confocal images revealed that protoplast shrinking was only occasionally associated with internalisation of giant vesicles (median diameter 2.7 microm) with FM 1-43-labelled membrane. But, in all cases, osmotic contraction was correlated with a diffuse distribution of FM1-43 label throughout the cytoplasm. From this, we conclude that endocytosis of small vesicles into the cytoplasm is the obligatory process by which cells accommodate an osmotically driven decrease in membrane surface area.

Visualization and tracking of single protein molecules in the cell nucleus.

A recently developed laser fluorescence videomicroscopy method was used to determine for the first time the intranuclear trajectories of single protein molecules. Using the recombinant Escherichia coli beta-galactosidase protein P4K, labeled with an average of 4.6 ALEXA 488 chromophores per tetramer, single P4K molecules could be localized and tracked in the nuclei of permeabilized 3T3 cells at a spatial accuracy of approximately 30 nm and a time resolution of 18 ms. Our previous photobleaching measurements indicated that P4K had two fractions inside the nucleus, a larger mobile and a smaller immobile fraction. The present study supported this observation but revealed a much larger variety of mobility classes. Thus, a fraction of P4K molecules appeared to be truly immobile while another fraction was mobile but confined to very small areas. In addition, a large fraction of the P4K molecules appeared to be mobile and to move over extended distances by diffusion. However, a quantitative analysis showed that at least two subpopulations were present differing widely in diffusion coefficients. Importantly, both the diffusion coefficients and the fractions of these subpopulations were time-dependent. Our results suggest that proteins can move inside the nucleus over extended distances by diffusion. However, intranuclear protein diffusion is severely restricted, most likely by multiple association-dissociation events and/or impermeable obstacles.

Lateral diffusion measurement at high spatial resolution by scanning microphotolysis in a confocal microscope.

Fluorescence photobleaching methods have been widely used to study diffusion processes in the plasma membrane of single living cells and other membrane systems. Here we describe the application of a new photobleaching technique, scanning microphotolysis. Employing a recently developed extension module to a commercial confocal microscope, an intensive laser beam was switched on and off during scanning according to a user definable image mask. Thereby the location, geometry, and number of photolysed spots could be chosen arbitrarily, their size ranging from tens of micrometers down to the diffraction limit. Therewith we bleached circular areas on the surface of single living 3T3 cells labeled with the fluorescent lipid analog NBD-HPC. Subsequently, the fluorescence recovery process was observed using the attenuated laser beam for excitation. This yielded image stacks representing snapshots of the spatial distribution of fluorescent molecules. From these we computed the radial distribution functions of the photobleached dye molecules. The variance of these distributions is linearly related to the diffusion constant, time, and the mobile fraction of the diffusing species. Furthermore, we compared directly the theoretically expected and measured distribution functions, and could thus determine the diffusion coefficient from each single image. The results of these two new evaluation methods (D = 0.3 +/- 0.1 micron 2/s) agreed well with the outcome of conventional fluorescence recovery measurements. We show that by scanning microphotolysis information on dynamical processes such as diffusion of lipids or proteins can be acquired at the superior spatial resolution of a confocal laser scanning microscope.

Scanning microphotolysis: a new photobleaching technique based on fast intensity modulation of a scanned laser beam and confocal imaging.

The fluorescence photobleaching method has been widely used to study molecular transport in single living cells and other microsystems while confocal microscopy has opened new avenues to high-resolution, three-dimensional imaging. A new technique, scanning microphotolysis (Scamp), combines the potential of photobleaching, beam scanning and confocal imaging. A confocal scanning laser microscope was equipped with a sufficiently powerful laser and a novel device, the 'Scamper'. This consisted essentially of a filter changer, an acousto-optical modulator (AOM) and a computer. The computer was programmed to activate the AOM during scanning according to a freely defined image mask. As a result, almost any desired pattern could be bleached ('written') into fluorescent samples at high definition and then imaged ('read') at non-bleaching conditions, employing full confocal resolution. Furthermore, molecular transport could be followed by imaging the dissipation of bleach patterns. Experiments with living cells concerning dynamic processes in cytoskeletal filaments and the lateral mobility of membrane lipids suggest a wide range of potential biological applications. Thus, Scamp offers new possibilities for the optical manipulation and analysis of both technical and biological microsystems.

Imaging and tracking of single GFP molecules in solution.

Visualization and tracking of single fluorescent molecules is a recent development in optical microscopy holding great promise for the study of cell biological processes. However, all experimental strategies realized so far confined the observation to extremely thin interfacial layers. The detection and characterization of single molecules in three-dimensionally extended systems such as living cells has yet to be accomplished. We show, here, for the first time that single protein molecules can be visualized and tracked in three-dimensional (3D) samples at room temperature. Using a wide-field fluorescence microscope equipped with an Ar(+)-laser and a low-light-level CCD camera, single molecules of the green fluorescent protein (GFP) were detected in gels and viscous solutions at depths of up to approximately 10 microm from the interface. A time resolution of 5 ms was achieved by a high-speed framing mode. The two-dimensional localization accuracy was determined to be approximately 30 nm. The number of photons emitted by single GFP molecules before photodestruction was found to be < or = 4 * 10(5). Freely diffusing GFP molecules could be tracked over up to nine images acquired at a frame rate of approximately 80 Hz. From the trajectories, the diffusion coefficients of single GFP molecules were derived and found to agree well with expectation and microphotolysis measurements. Our results imply that the visualization and tracking of single molecules in living cells is possible.

Line-scanning microphotolysis for diffraction-limited measurements of lateral diffusion.

Fluorescence microphotolysis was combined with confocal laser-scanning microscopy to yield a method, herein referred to as line-scanning microphotolysis (LINESCAMP), for the measurement of molecular transport at a lateral resolution of approximately 0.34 microns and a temporal resolution of approximately 0.5 ms. A confocal microscope was operated in the line scan mode, while the laser beam power could be switched during scanning between low monitoring and high photolysing levels in less then a microsecond. The number and location of line segments to be photolysed could be freely determined. The length of the photolysed segments could be also chosen and was only limited by diffraction. Together with instrumentation a new, completely general, theoretical framework for the evaluation of diffusion measurements was developed. Based on the numerical simulation of diffusion processes employing a modified Crank-Nicholson scheme, the theory could be applied to any photobleaching geometry and profile as the initial condition and took into account the convolution with the microscope point spread function. With small diffraction-limited areas, the method yielded accurate values for diffusion coefficients in the range between approximately 10(-4) and 1 micron2 s-1. A first application of the method to the diffusion of a fluorescently labeled tracer inside the cell nucleus showed the potential of the method for the study of complex biological systems.

Calcium pump kinetics determined in single erythrocyte ghosts by microphotolysis and confocal imaging.

The activity of the plasma membrane calcium pump was measured in single cells. Human red blood cell ghosts were loaded with a fluorescent calcium indicator and either caged calcium and ATP (protocol A) or caged ATP and calcium (protocol B). In a suitably modified laser scanning microscope either calcium or ATP were released by a short UV light pulse. The time-dependent fluorescence intensity of the calcium indicator was then followed in single ghosts by repetitive confocal imaging. The fluorescence intensity was converted into calcium concentration, which in turn was used to derive the kinetic parameters of the calcium pump, the Michaelis-Menten constant Km, and the maximal transport rate vmax. Km and vmax values derived in this manner were 24 +/- 14 microM and 1.0 +/- 0.6 microM/(ghost s) for protocol A, and 4 +/- 3 microM and 1.0 +/- 0.6 microM/(ghost s) for protocol B, respectively. The difference between A and B is presumably caused by calmodulin, which is inactive in the experiments with protocol A. The possibilities to extend the new method to living nucleus-containing cells transiently transfected with mutants of the plasma membrane calcium pump are discussed.

Mapping of nucleoporins to the center of the nuclear pore complex by post-embedding immunogold electron microscopy.

Ultrathin sections of Lowicryl K4M embedded cultured 3T3 cells, human keratinocytes and mouse/rat liver tissue were incubated with polyspecific primary antibodies against p62 and other nucleoporins followed by 10 nm gold labeled secondary antibodies. By quantitatively evaluating both cross sections and tangential sections of the NPC, we found that irrespective of the cell type antibodies predominantly bound within a radius of 25 nm around the central axis of the nuclear pore complex (NPC). Superposition of a current structural model of the NPC with the nucleoporin distribution observed by us showed that nucleoporins mapped predominatly to the controversely discussed 'central granule'. Our experimental approach was verified by mapping gp210, another nuclear pore protein, at or very close to the NPC in the perinuclear cisterna thus establishing a distribution pattern completely different from that of the nucleoporins.

Two-photon scanning microphotolysis for three-dimensional data storage and biological transport measurements.

Scanning microphotolysis is a method that permits the user to select, within the scanning field of a confocal microscope, areas of arbitrary geometry for photobleaching or photoactivation. Two-photon absorption, by contrast, confers on laser scanning microscopy a true spatial selectivity by restricting excitation to very small focal volumes. In the present study the two methods were combined by complementing a laser scanning microscope with both a fast programmable optical switch and a titan sapphire laser. The efficiency and accuracy of fluorescence photobleaching induced by two-photon absorption were determined using fluorescein-containing polyacrylamide gels. At optimal conditions a single scan was sufficient to reduce the gel fluorescence by approximately 40%. Under these conditions the spatial accuracy of photobleaching was 0.5 +/- 0.1 micron in the lateral (x.y) and 3.5 +/- 0.5 micron in the axial (z) direction, without deconvolution accounting for the optical resolution. Deconvolution improved the accuracy values by approximately 30%. The method was applied to write complex three-dimensional patterns into thick gels by successively scanning many closely spaced layers, each according to an individual image mask. Membrane transport was studied in a model tissue consisting of human erythrocyte ghosts carrying large transmembrane pores and packed into three-dimensional arrays. Upon equilibration with a fluorescent transport substrate single ghosts could be selectively photobleached and the influx of fresh transport substrate be monitored. The results suggest that two-photon scanning microphotolysis provides new possibilities for the optical analysis and manipulation of both technical and biological microsystems.

Optical single-channel analysis of the aerolysin pore in erythrocyte membranes.

Scanning microphotolysis (Scamp), a recently developed photobleaching technique, was used to analyze the transport of two small organic anions and one inorganic cation through single pores formed in human erythrocyte membranes by the channel-forming toxin aerolysin secreted by Aeromonas species. The transport rate constants of erythrocyte ghosts carrying a single aerolysin pore were determined to be (1.83 +/- 0.43) x 10(-3) s-1 for Lucifer yellow, (0.33 +/- 0.10) x 10(-3) s-1 for carboxyfluorescein, and (8.20 +/- 2.30) x 10(-3) s-1 for Ca2+. The radius of the aerolysin pore was derived from the rate constants to be 19-23 A, taking steric hindrance and viscous drag into account. The size of the Ca2+ rate constant implies that at physiological extracellular Ca2+ concentrations (> 1 mM) the intracellular Ca2+ concentration would be elevated to the critical level of > 1 microM in much less than a second after formation of a single aerolysin pore in the plasma membrane. Thus changes in the levels of Ca2+ or other critical intracellular components may be more likely to cause cell death than osmotic imbalance.

Localization of analyte molecules in MALDI preparations by confocal laser scanning microscopy.

In this study, the incorporation of Texas Red-labeled avidin into crystals of 2,5-dihydroxybenzoic acid (2,5-DHB) and 2,6-DHB (used as matrixes for matrix-assisted laser desorption/ionization (MALDI)) was investigated by fluorescence spectrophotometry and confocal laser scanning microscopy (CLSM). The analyte distribution in crystals, grown slowly under controlled conditions, was compared to the analyte localization in different standard preparations (dried-droplet and thin-layer preparation). Texas Red turned out to be a useful fluorescence label in the acidic environments of typical matrixes. Earlier results by absorption spectrophotometry could be confirmed by fluorescence measurements; 2,5-DHB incorporates the analyte proportionally, while 2,6-DHB excludes the protein from its crystal lattice. It is found that the analyte distribution can be analyzed well in both single crystals and standard preparation, by CLSM using Texas Red-labeled analytes. The present study allows for a conclusive and consistent interpretation of analyte incorporation into MALDI preparations.

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.