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Solid State Physics Laboratory, ETH Zürich, 8093 Zürich, Switzerland
Correspondence: Address reprint requests to Klaus Ensslin, Solid State Physics Laboratory, ETH Zürich, 8093 Zürich, Switzerland. E-mail: ensslin{at}phys.ethz.ch.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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The importin ß transport complex is the best characterized nuclear transport pathway of eukaryotes. In this transport pathway specific recognition of the NPC is mediated by importin ß whereas cargo binding is relegated to an adaptor molecule known as importin 
. It is thus the union of cargo-bound importin with importin ß that forms the intact transport complex, which then targets to the NPC for subsequent translocation via the interactions of importin ß. In particular, importin ß has affinity for nuclear pore proteins, or nucleoporins, containing Phenylalanine (F) and Glycine (G) repeating motifs (Radu et al., 1995
; Stewart, 2000). The transport complex remains irreversibly bound to peripheral regions of the NPC until it becomes associated with Ran-GTP, a Ras related GTPase (Görlich and Kutay, 1999). It is the binding of Ran-GTP within the nuclear compartment that causes importin ß to dissociate from peripheral components of the NPC, thus effectively terminating the transport process. By contrast, the interactions of importin ß with luminal nucleoporins need to be highly transient in order for transport through the lumen of the NPC to be rapid. This mechanistic disparity might indicate that the FG-repeats of peripheral and luminal filamentous NPC proteins differ in their intrinsic importin ß binding affinity. Indeed, the finding that replacing the isoleucine residue at position 178 with an aspartic acid residue greatly reduces the binding of importin ß to FG-repeats of peripheral filamentous nucleoporins (Bayliss et al., 2000), while at the same time supporting efficient transport through the NPC (U. Kutay, unpublished results), may testify to such a mechanistic scenario. Interestingly, a recent report has shown that the filamentous nucleoporins of the cytoplasmic compartment may be, in fact, dispensable for translocation of importin ß through the NPC (Walther et al., 2002). It thus may be that more peripheral filamentous FG-nucleoporins act to concentrate cargo at the mouth of the NPC via high-affinity interactions, whereas luminal FG-nucleoporins subserve rapid translocation of cargo via lower-affinity interactions (cf. Rout et al., 2000).
In a previous study we examined the effect of binding of mutant importin ß proteins on NPC topology (Jäggi et al., 2003). We found that the extent of accumulation of bound mutant transport receptors over the luminal domain of the NPC as visualized using scanning force microscopy (SFM) agreed well with the relative binding affinity of the mutant constructs for the NPC as determined in biochemical measurements. The most dramatic case was that of the 45-462 importin ß mutant, which due to the truncation of its amino terminal end is unable to interact with Ran proteins and hence binds irreversibly to the NPC (Kutay et al., 1997). Furthermore, truncation of the carboxyl terminal of this mutant impedes its interaction with importin and hence its association with nuclear localization sequence-containing cargo. Initial biochemical studies conducted with the 45-462 mutant receptor demonstrated that it accumulated at the NPC and blocked all receptor-mediated transport through the nuclear envelope, although the exact mechanism of block remained elusive.
Transport receptor binding to the NPC is not necessarily synonymous with the translocation process. The translocation of cargo through the NPC involves the combination of several independent processes, namely transport receptor binding, the actual translocation of cargo through the NPC, the transport of RanGDP into the nucleus, nucleotide exchange of RanGDP to RanGTP within the nucleus, and finally the association of RanGTP with importin ß. The binding of the 45-462 mutant receptor to the NPC thus only represents the unique binding kinetics of importin ß to FG-repeats of the NPC. In this report, we investigate the binding characteristics of importin 45-462 as a function of incubation time and initial free transport receptor concentration.
The nuclear pore complex is a 125-MDa macromolecular complex exhibiting an octagonal, radially symmetrical structure that has been the focus of intense structural characterization on both the electron microscopic (Akey, 1995; Stoffler et al., 1999; Panté and Aebi, 1996) and scanning force microscopic levels (Stoffler et al., 1999; Nevo et al., 2000; Danker and Oberleithner, 2000). Conformational changes in the central channel region of the NPC have been previously visualized by SFM measurements under conditions of calcium store depletion (Perez-Terzic et al., 1996; Moore-Nichols et al., 2002; Jäggi et al., 2003). Specifically, depleting the perinuclear calcium stores by a variety of biochemical means causes the appearance of a molecular structure within the lumen of the NPC commonly referred to as the central translocator plug, central granule, or central channel complex. Since its initial discovery (Akey, 1990; Akey and Radermacher, 1993), the nature of the central translocator plug has been a matter of much controversy. There are those that explain its existence as cargo caught in transit (Stoffler et al., 1999). On the other hand, the fact that the central plug can be shown to extrude in real time in isolated patches lacking both cytoplasmic and nucleoplasmic constituents argues against this interpretation (Wang and Clapham, 1999) and has led to the notion that the central translocator plug is an integral part of the NPC. We have previously shown that the topological characteristics of the central translocator plug are clearly distinct from those obtained under conditions where a variety of transport receptors are bound to the NPC (Jäggi et al., 2003). In this report we further demonstrate that the surface characteristics of the central translocator plug are distinct from those of the rest of the NPC or bound transport receptors. Therefore, if the topographic characteristics of a transport receptor bound to the NPC can be equated to that of cargo undergoing transport, then the central plug is most likely not cargo caught in transit or, alternatively, represents cargo of unique structural characteristics.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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). Tapping mode AFM was employed to minimize lateral forces exerted on the sample. Typical cantilever amplitudes were 10 nm, and setpoints used for height feedback operation were larger than 90% of the free amplitude. Scan speed was adjusted to 
2 µm/s.
Data analysis
Changes in NPC topology were investigated by taking cross-sectional profiles through single NPCs as previously described (Jäggi et al., 2003). Briefly, to minimize artifacts induced by thermal drift the profile measurements were taken along the fast scan direction of the SFM. The NPC topology was characterized by measurements of upper rim diameter (d) and central pore depth (z) as indicated in the inset of Fig. 1 B. Measuring pore diameter at the upper rim, rather than at the outer circumference of the NPC, eliminated any uncertainties associated with limited tip asperity. Thus, diameter measurements were independent of the tip radius and aspect ratio. Measured data sets for depth and diameter were well described by single Gaussian distributions. In all cases diameter and depth data are given as mean values ± SD.
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, 2003). Briefly, oocytes were stored (18°C) in Modified Barth's Solution consisting of (in mM); 1 KCl, 0.82 MgSO4, 0.41 CaCl2, 0.33 Ca(NO3)2, 2.4 NaHCO3, 88 NaCl, and 10 HEPES, pH 7.4. Nuclear envelopes were typically prepared for scanning on the day of oocyte isolation. Nevertheless, analogous results could also be obtained on nuclear envelopes from oocytes stored in Modified Barth's Solution for up to 3 days. After dissection nuclei were immediately transferred into a low salt buffer consisting of (in mM): 1 KCl, 0.5 MgCl2, and 15 Tris. Nuclei were washed several times in cold (10°C) low salt buffer to remove any adherent cellular material and debri. After cleaning, nuclei were then transferred to Mock Intracellular Buffer consisting of (in mM); 90 KCl, 10 NaCl, 2 MgCl2, 0.75 CaCl2, 1.1 EGTA, and 15 Tris, pH 7.32, in which they were incubated in indicated experimental conditions. After incubation in importin ß 45-462 mutant protein for the indicated times and concentrations or the incubation in 10 mM EGTA for 10 min the nuclei were fixed in 2% formaldehyde and 1% glutaraldehyde at 4°C overnight. In preparation for scanning, nuclei were placed intact onto plastic tissue culture dishes, partially dried, and rehydrated in distilled water. Since the nuclei were imaged intact it was clear that imaging was performed on the outer (cytosolic), rather than on the inner (nucleoplasmic), nuclear envelope.
Molecular biology
The generation, expression, and purification of the mutant importin ß fragment, 45-462, was described previously (Kutay et al., 1997). Nuclear envelopes were incubated with mutant fragment for the indicated times and concentrations before overnight fixation.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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). The time dependence of 45-462 binding is shown in Fig. 1. Since the binding of the 45-462 mutant is an irreversible process the accumulation of bound protein over the central channel region indicates the saturation of available binding sites for importin ß. In fact, very brief exposures to the 45-462 mutant protein were necessary in order to be able to clearly resolve the central channel depression of the NPC using SFM before its occupancy with bound protein. Thus, at very short exposure times to the 45-462 mutant protein the NPCs were virtually indistinguishable from those from control samples. After only 10 s of exposure to 45-462 fragment the filling of the central channel region was apparent. Saturation of 45-462 binding was apparent shortly after only 2 min of incubation. In an earlier report Ribbeck and Görlich (2001) demonstrated that transportin import into the nuclear compartment was apparent within 5 s and reached saturation after 2 min. The mechanisms for transportin translocation and 45-462 binding, although obviously related, differ on a phenomenological level, especially at early time points when translocation through the NPC might represent a nonlinear phase of the filling of the central channel region. Nevertheless, within these boundary conditions we conclude that the temporal characteristics of transportin import into intact nuclei are strikingly similar to our data examining the irreversible binding of importin ß 45-462.
Dose-dependent binding
The previous time course was conducted with a set concentration of 45-462 protein of 1 µM. We next examined the concentration dependence of 45-462 mutant receptors binding to the NPC after a fixed incubation period of 2 min. Fig. 2 shows that the accumulation of mutant protein was apparent in the nanomolar range and saturated in the micromolar range. Again, these values are similar to those of Ribbeck and Görlich (2001) for transportin import into the nuclear compartment. Due to the irreversible nature of the binding of the 45-462 mutant to the NPC, values of rate constants cannot be calculated.
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; Stoffler et al., 1999; Moore-Nichols, et al., 2002; Jäggi et al., 2003). There is some debate as to whether it is cargo caught in transit (Stoffler et al., 1999) or represents an integral part of the structure. For a more detailed discussion of the origin of the central translocator plug see Moore-Nichols et al. (2002). In this context we would like to report on experimental findings that might shed some light on this issue. Fig. 3 shows consecutive scans performed on a single EGTA-treated NPC clearly exhibiting a central translocator plug protruding from the lumen of the NPC (Fig. 3 A). Interestingly, the volume of the translocator decreased with repeated scanning (3 A, b and c), as if portions of the translocator structure were being swept away by the scanning tip. Furthermore, the translocator surface exhibited a grainy and irregular appearance much like that often attributed to instabilities in height feedback operation. By contrast, the surrounding NPC body and the nuclear envelope surface were consistently imaged at high resolution. This is illustrated in greater detail in the profiles taken across the diameter of the same NPC (Fig. 3 B). The amplitude of the noise level in the height measurement increases as the cantilever scans across the surface of the central translocator (arrow), which is increasingly evident as more of the plug is scratched away. Furthermore, such surface characteristics were never observed in scans performed under identical AFM operation conditions examining the binding of importin ß mutants on the NPC surface. On the basis of structural properties and variations in surface characteristics we are thus able to distinguish binding of transport molecules, represented by importin ß 45-462, from the occurrence of the central granule.
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Attractive forces between tip and sample can lead to cantilever instabilities and irregular height feedback if the force gradient becomes larger than the cantilever's force constant. This effect, commonly referred to as snap-in/snap-to-contact, is most frequently encountered in air due to the large capillary forces arising from the thin water film covering a sample surface. In our experiments, however, both tip and sample are completely immersed in liquids. Therefore, capillary forces are eliminated and the observed snap-in effect must therefore arise from attractive interactions between the cantilever and the central plug surface. We propose that these attractive forces are hydrophobic rather than electrostatic in nature.
In aqueous environments surface charges are strongly screened by soluble counterions, an effect attributed to the formation of a diffuse electrical double layer (Israelachvili, 1991; Müller et al., 1999). In our experiments the concentration of protein surface charges is greatly diminished through the use of formaldehyde/glutaraldehyde fixatives. Attractive electrostatic interactions are thus expected to be negligible.
Hydrophobic interactions, on the other hand, may be sufficiently strong to cause snap-in effects. Interactions between hydrophobic surfaces in aqueous liquids are always attractive (see Israelachvili, 1991, for a detailed description of hydrophobicity and hydrophobic interactions). The silicon nitride SFM tips used in our measurements were used without prior cleaning in oxygen plasma or under ultraviolet illumination, rendering them hydrophobic as determined from contact angle measurements (Knapp and Stemmer, 1999). Indeed, similar scanning instabilities are often observed between SFM tips and membranes or proteins that have been damaged, thereby exposing their hydrophobic interiors. It is thus relevant that the NPC rim and surrounding nuclear envelope was consistently imaged at higher resolution, possibly indicating that the central granule surface is more hydrophobic in nature. Importantly, the snap-to-contact effect becomes increasingly evident as the scanning tip exposes more granular material.
Relevance to previous work
The ability of transport receptors to interact with the NPC is largely determined by hydrophobic interactions (Radu et al., 1995; Stewart, 2000). These interactions are, on the one hand, mediated by the FG-repeats of nucleoporins that line the translocation pathway through the NPC and, on the other hand, by hydrophobic pockets expressed on the surface of importin ß. Ribbeck and Görlich (2002) proposed that through such an arrangement transport receptor-bound cargo is able to specifically interact and thus effectively partition into the hydrophobic phase established by interconnected FG-repeats within the lumen of the NPC. While this model is still being actively discussed in the field they were able to show that small amphipathic alcohols that presumably interfere with these intraluminal hydrophobic interactions are able to disrupt the permeability barrier normally imposed by the NPC. In a previous report we showed that these small polar alcohols cause the NPC to dilate and proposed that such amphipathic alcohols, by way of disrupting the associations between luminal FG-repeats, relieve stress within the NPC structure (Jäggi et al., 2003). In further support of this interpretation Bickel and Bruinsma (2002) have recently conducted a physical analysis of the properties that would be required for cargo to partition through a confined reversible gel in a poor solvent, such as the selective hydrophobic phase proposed to fill the lumen of the NPC by Ribbeck and Görlich (2002). In the analysis put forth by Bickel and Bruinsma (2002) they proposed that intraluminal tension would intrinsically arise from the thermal fluctuations of the connections between FG-repeats comprising the reversible gel within the NPC lumen. Disrupting these connections could feasibly account for the NPC dilation observed by us (Jäggi et al., 2003) and explain the previously described breakdown in NPC specificity observed with these same small polar alcohols (Ribbeck and Görlich, 2002). Finally, the hydrophobic nature of the central translocator plug may imply that it originates from the luminal FG-repeats. The appearance of the central channel plug might thus represent the extrusion of the luminal FG-repeat matrix in response to inwardly directed pressure within the lumen of the NPC possibly (cf. Bickel and Bruinsma, 2002).
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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), thereby giving validity to our experimental approach. Furthermore, using this same technique we were able to show that the central translocator plug can be modified by repeated scanning with a hydrophopic tip, leaving any other domain of the NPC or nuclear envelope intact. The unique hydrophobic nature of the central translocator plug may arise from FG-repeats forming part of the NPC lumen. This result may indicate that the transport receptor binding sites (FG-repeats) expressed on the cytoplasmic rim of the NPC may be distinct in hydrophobic character and possibly binding capacity. We hence give two important examples where SFM was clearly able to provide quantitative measurements of related, although functionally distinct, transport phenomena. |
ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Financial support from ETH Zürich is acknowledged.
Submitted on March 20, 2003; accepted for publication August 11, 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Akey, C. W. 1990. Visualization of transport-related configurations of the nuclear pore transporter. Biophys. J. 58:341–355.
Akey, C. W., and M. Radermacher. 1993. Architecture of the Xenopus nuclear pore complex revealed by three-dimensional cryo-electron microscopy. J. Cell Biol. 122:1–19.
Bayliss, R., T. Littlewood, and M. Stewart. 2000. Structural basis for the interaction between FxFG nucleoporin repeats and importin-beta in nuclear trafficking. Cell. 102:99–108.[Medline]
Bickel, T., and R. Bruinsma. 2002. The nuclear pore complex mystery and anomalous diffusion in reversible gels. Biophys. J. 83:3079–3087.
Danker, T., and H. Oberleithner. 2000. Nuclear pore function viewed with atomic force microscopy. Pflügers Arch. – Eur. J. Physiol. 439:671–681.
Görlich, D., and U. Kutay. 1999. Transport between the cell nucleus and the cytoplasm. Annu. Rev. Cell Dev. Biol. 15:607–660.[Medline]
Israelachvili, J. 1991. Intermolecular and Surface Forces, 2nd ed. Academic Press, New York.
Jäggi, R. D., A. Franco-Obregón, P. Mühlhäusser, F. Thomas, U. Kutay, and K. Ensslin. 2003. Modulation of nuclear pore topology by transport modifiers. Biophys. J. 84:665–670.
Jäggi, R. D., A. Franco-Obregón, P. Studerus, and K. Ensslin. 2001. Detailed analysis of forces influencing lateral resolution for Q-control and tapping mode. Appl. Phys. Lett. 79:135–137.
Knapp, H. F., and A. Stemmer. 1999. Preparation, comparison, and performance of hydrophobic AFM tips. Surface and Interface Analysis. 27:324–331.
Kutay, U., E. Izaurralde, F. R. Bischoff, I. W. Mattaj, and D. Görlich. 1997. Dominant-negative mutants of importin-beta block multiple pathways of import and export through the nuclear pore complex. EMBO J. 16:1153–1163.[Medline]
Moore-Nichols, D., A. Arnott, and R. C. Dunn. 2002. Regulation of nuclear pore complex conformation by IP3 receptor activation. Biophys. J. 83:1421–1428.
Müller, D., D. Fotiadis, S. Scheuring, S. A. Müller, and A. Engel. 1999. Electrostatically balanced subnanometer imaging of biological specimens by atomic force microscope. Biophys. J. 76:1101–1111.
Nevo, R., P. Markiewicz, R. Kapon, M. Elbaum, and Z. Reich. 2000. High-resolution imaging of the nuclar pore complex by AC scanning force microscopy. Single Molecules. 1:109–114.
Panté, N., and U. Aebi. 1996. Molecular dissection of the nuclear pore complex. Crit. Rev. Biochem. Mol. Biol. 31:153–199.[Medline]
Perez-Terzic, C., J. Pyle, M. Jaconi, L. Stehno-Bittel, and D. E. Clapham. 1996. Conformational states of the nuclear pore complex induced by depletion of nuclear Ca2+ stores. Science. 273:1875–1877.
Radu, A., M. S. Moore, and G. Blobel. 1995. The peptide repeat domain of nucleoporin Nup98 functions as a docking site in transport across the nuclear pore complex. Cell. 81:215–222.[Medline]
Ribbeck, K., and D. Görlich. 2002. The permeability barrier of nuclear pore complexes appears to operate via hydrophobic exclusion. EMBO J. 21:2664–2671.[Medline]
Ribbeck, K., and D. Görlich. 2001. Kinetic analysis of translocation through nuclear pore complexes. EMBO J. 20:1320–1330.[Medline]
Rout, M. P., J. D. Aitchison, A. Suprapto, K. Hjertaas, Y. Zhao, and T. Chait. 2000. The yeast nuclear pore complex: composition, architecture, and transport mechanism. J. Cell Biol. 148:635–651.
Stewart, M. 2000. Insights into the molecular mechanism of nuclear trafficking using nuclear transport factor 2 (NTF2). Cell Struct. Funct. 25:217–225.[Medline]
Stoffler, D., B. Fahrenkrog, and U. Aebi. 1999. The nuclear pore complex: from molecular architecture to functional dynamics. Curr. Opin. Cell Biol. 11:391–401.[Medline]
Stoffler, D., K. N. Goldie, B. Feja, and U. Aebi. 1999. Calcium-mediated structural changes of native nuclear pore complexes monitored by time-lapse atomic force microscopy. Mol. Biol. 287:741–752.
Walther, T. C., H. S. Pickersgill, V. C. Cordes, M. W. Goldberg, T. D. Allen, I. W. Mattaj, and F. Fornerod. 2002. The cytoplasmic filaments of the nuclear pore complex are dispensable for selective nuclear protein import. J. Cell Biol. 158:63–77.
Wang, H., and D. E. Clapham. 1999. Conformational changes of the in situ nuclear pore complex. Biophys. J. 77:241–247.
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