Glucocorticoids remodel nuclear envelope structure and permeability

Victor Shahin*,{ddagger}, Yvonne Ludwig*, Claudia Schafer, Dessy Nikova and Hans Oberleithner

Institute of Physiology II, University of Münster, Robert-Koch Str. 27b, 48149 Münster, Germany

{ddagger} Author for correspondence (e-mail: shahin{at}uni-muenster.de)

Accepted 7 April 2005


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 Materials and Methods
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The present study describes glucocorticoid induced remodelling of nuclear envelope (NE) structure and permeability. A glucocorticoid analogue, triamcinolone acetonide (TA), is injected into Xenopus laevis oocytes that express an exogeneous glucocorticoid receptor (GR). Electrical, fluorescence and nano-imaging techniques are applied to study the permeability and the structure of the NE at 5 and 60 minutes after injection of TA. A remarkable dilation of nuclear pore complexes (NPCs), a rearrangement of NPC distribution and a significant increase of NE permeability for ions and fluorescent 20 kDa dextran are observed within 5 minutes of TA exposure. At regular distances on local NE patches, NPCs seem to adjoin forming clusters each consisting of several hundred NPCs. Interestingly, at the same time of exposure, hydrophobicity of NPC central channels and NPC-free NE surface increases. The changes in permeability and structure are transient as the NE permeability returns to its initial state within 60 minutes. In conclusion, the NE is a barrier of high plasticity sensitive to hydrophobic molecules. Remodelling of NE structure and permeability is a prerequisite for mediating physiological actions of glucocorticoids.

Key words: Atomic force microscopy, Glucocorticoid receptor, Nuclear envelope, Nuclear pore complex, Triamcinolone acetonide


    Introduction
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 Summary
 Introduction
 Materials and Methods
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The nuclear envelope (NE) is a double membrane composed of at least the following entities: the inner and the outer nuclear membrane, the nuclear pore complexes (NPCs) that join both membranes and the nuclear lamina (Dahl et al., 2004Go; Gruenbaum et al., 2005Go; Holaska et al., 2002Go; Wilson et al., 2001Go). The latter is a fibrous structure that consists of intermediate filament proteins, termed lamins (Gerace and Blobel, 1980Go), which underlie the entire inner surface of the NE linking the NPCs (Aebi et al., 1986Go; Goldberg and Allen, 1992Go; Goldberg and Allen, 1993Go; Hutchison et al., 1994Go; Whytock et al., 1990Go). The NPCs are distributed throughout the entire NE (Aebi et al., 1992Go; Akey and Radermacher, 1993Go; Cronshaw et al., 2002Go; Pante and Aebi, 1996Go; Vasu and Forbes, 2001Go). They represent transport nanomachines that regulate bidirectional nucleocytoplasmic exchange of selected molecules (Fahrenkrog and Aebi, 2003Go; Gerace, 1992Go; Görlich and Mattaj, 1996Go; Oberleithner et al., 2003Go; Rout and Aitchison, 2001Go; Schäfer et al., 2002Go) such as the glucocorticoid receptor (GR). The latter is located in the cytosol. Soon after binding to glucocorticoid hormone the resulting hydrophobic GR-hormone dimer is recognised by a nuclear transport protein, importin-{alpha},ß, that is hydrophobic as well (Ribbeck and Gorlich, 2002Go). In conjunction with importin-{alpha},ß, the hormone-associated GR then traverses the key barrier, the NE, through the NPCs. A single NPC consists of several copies of at least 30 different proteins, termed nucleoporins (Vasu and Forbes, 2001Go), some of which are involved in the transport process (Vasu and Forbes, 2001Go). Various nucleoporins contain hydrophobic phenylalanine-glycine side chains, so-called FG-rich repeats (Vasu and Forbes, 2001Go). The translocation of receptor-cargo complexes through the central channel of the NPC involves binding to these hydrophobic residues. They are believed to determine the selectivity and translocation rate of the NPCs in manner not yet fully understood (Ribbeck and Gorlich, 2002Go). Ribbeck and Gorlich showed that hydrophobic transport cargoes are favoured and thus rapidly translocated through the NPC (Ribbeck and Gorlich, 2002Go). They suggested a transport model, where hydrophobic cargoes cause a reversible collapse of the permeability barrier of NPCs by interaction with the meshwork of the hydrophobic FG-rich repeats within the central channel. This transport model could be generally referred to other hydrophobic transport cargoes such as hormone-associated GRs. Regarding the same model it becomes likely that hydrophobic transport cargoes such as steroid receptors could change not only the permeability but also the conformational state of the NPCs by interaction with the FG-rich repeats. Surprisingly, a change of permeability and structure of the NPCs or the NE, respectively, remains usually unregarded when studying steroid hormone action. We have recently shown structural changes, dilation, of the NPCs in response to the glucocorticoid hormone analogue dexamethasone (Shahin et al., 2004Go). Upon exposure of Chironomus thummi salivary gland cells to the steroid hormone 20-OH ecdysone, changes in nuclear membrane permeability for inorganic ions were observed (Ito and Loewenstein, 1965Go). The latter study indicated that the NE could be involved in the action of steroid hormones. Oberleithner et al. (Oberleithner et al., 1994Go) showed in kidney cells that the NE responds to aldosterone by increasing both, NPCs density and NE permeability within 6 hours of hormone exposure. Using a nano-imaging technique, atomic force microscopy (AFM) (Binnig and Quate, 1986Go), we could visualize some of the endogeneous macromolecules on their route through the NPCs following aldosterone injection into X. laevis oocyte (Schäfer et al., 2002Go). Changes in NPC conformation were paralleled by sharp changes in NE electrical conductivity (Danker et al., 1999Go). In the present study we expressed the exogeneous rat glucocorticoid receptor in X. laevis oocytes and studied the impact of a potent glucocorticoid hormone analogue on the structure and the permeability of the NE and the NPC conformation. We found that NPCs dilate and cluster while the NE barrier becomes leaky at the same time.


    Materials and Methods
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 Materials and Methods
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 References
 
Oocytes injection, isolation of the nuclei and preparation of nuclear envelopes
Oocytes were collected as previously described (Schäfer et al., 2002Go). For the triamcinolone acetonide (TA) experiments, oocytes were injected with 50 nl solution containing TA (TA concentration in the injection fluid: 2 x10–5 mol l–1 in water; TA stock solution dissolved in ethanol: 10–2 mol l–1). Final TA concentration in a single oocyte ranges ~1 µmol l–1 (oocyte volume ~1 µl). Then, nuclei were isolated 5 minutes after injection. Corresponding control experiments were performed by the injection of the respective solvent (ethanol in water). For nuclear isolation, oocytes were transferred into nuclear isolation medium (NIM) (Danker et al., 2001Go). Nuclei were isolated using precision forceps and then cleared from cytoplasmic debris by drawing the nuclei gently into and out of the tip of an Eppendorff pipette. Nuclei were then picked up with a pipette and transferred to a glass coverslip placed under a stereomicroscope. Then, the chromatin was carefully teased out of the nucleus using sharp needles and the nuclear envelope spread onto a Cell-tak (Biosciences, Bedford, MA) coated glass coverslip, with the nucleoplasmic side facing downwards. Subsequently, the flattened NE was rinsed with deionized water and air-dried. All procedures were performed at room temperature (~23°C).



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Fig. 1. Idealized force-distance curve describing a single approach-retract cycle of the AFM tip, which is continuously repeated during surface scanning. The AFM tip is approaching the sample surface (A). The initial contact between the tip and the surface is mediated by the attractive van der Waals forces (contact) that lead to an attraction of the tip toward the surface (B). Hence, the tip applies a constant and default force upon the surface that leads to sample indentation and cantilever deflection (C). Subsequently, the tip tries to retract and to break loose from the surface (D). Various adhesive forces between the sample and the AFM tip, however, hamper tip retraction. These adhesive forces can be taken directly from the force-distance curve (E). The tip withdraws and looses contact to the surface upon overcoming of the adhesive forces (F).

 
Atomic force microscopy
We used a Bioscope AFM (NanoScope IIIa controller, Digital Instruments, Santa Barbara, CA) equipped with an optical microscope. Aluminium coated, V-shaped 290 µm long silicon cantilevers (CSC21\50, Ultra-sharp) with a spring constant of 0.12 N m–1 and pyramidal tips with an estimated tip radius of 10 nm were used. The images were recorded with 512 scan lines per area, at constant force (height mode) in contact mode in air with a scan rate of 3-5 Hz. Images were processed using the Nanoscope III software (Digital Instruments). High-resolution imaging of individual NPCs and associated proteins demanded fine-tuning of the scanning process. This comprised mainly the minimizing of the scanning force between tip and sample to values below 3 nN.

Determination of NPC density, external NPC diameter, NPC opening diameter
About 200 µm2 (eight areas each 5 x5 µm2) per individual NE harvested from individual nuclei of solvent or TA-injected oocytes were imaged with AFM. Data analysis was performed with appropriate software (Nanoscope III software, Digital Instruments, Santa Barbara, CA). In all imaged NE areas we counted the total number of NPCs and calculated the mean NPC density per µm2. As previously described (Shahin et al., 2004Go), profile analysis was performed in order to determine the external NPC diameter and NPC opening diameter. External NPC diameter was obtained by measurement of the NPC `upper rim' diameter. Apparent NPC opening diameter was determined by measurement of the horizontal distance (within the NPC opening) at half-maximal height.

Investigation of hydrophobicity of NPCs and NPC-free NE surface with AFM
Fig. 1 describes the principle of the hydrophobicity measurement using AFM. The AFM uses a diminutive tip (mounted on a soft and bendable cantilever) that scans the sample surface horizontally point by point. Forces that emerge before and after tip contact with the sample surface can be probed. This is done by recording the cantilever deflection as the tip approaches, contacts, and retracts from a surface, then plotting a force curve as a function of the approach-retract travel distance. Thus, this so-called force-distance curve records the adhesive and repulsive forces felt by the tip as it approaches and retracts from a point on the sample surface. Fig. 1 shows how adhesive forces between tip and sample can be directly measured using the force-distance curve. Adhesion forces between the tip and the sample surface develop for numerous reasons. By scanning in air, capillary forces are believed to be the predominant origin of adhesion, which are assumed to be caused by a fluid layer on the sample surface (Jang et al., 2004Go; Sedin and Rowlen, 2000Go). Another reason for an increased adhesion force could be a consequence of interaction between a hydrophobic surface being scanned with a hydrophobic AFM tip. The extent of tip attraction (adhesion force), dependent on surface hydrophobicity, can be directly taken from analysis of the corresponding force-distance curve. To obtain an array of force-distance curves, a hydrophobicity map, over the entire NE area, the so-called force-volume mode can be applied. Using force-volume imaging, each force-distance curve is measured at a unique x-y position in the area, and force-distance curves from an array of x-y points are combined into a three-dimensional array, or `volume' of force data. In so doing, it is possible to obtain information on surface hydrophobicity, point by point on the scanned NE. In the present study, force volume imaging is used to distinguish between the hydrophobicity degree of NPC channel and NPC-free NE surface. A hydrophobic, aluminium coated AFM tip, CSC21 (Ultra-sharp, Mikro Masch, Anfatec, Germany) is used to scan the isolated NEs. Force-volume images of isolated NEs adsorbed onto a glass coverslip were obtained as follows. Contact mode was initially applied to image the NE and to take force curve measurements to provide the general force characteristics of the sample. The so-called microscope's `trigger mode' was set to `relative' and the trigger threshold to 50 nm. Subsequently, a force-volume image was obtained. For this purpose, various parameters were set as follows: Z-scan rate, 15 Hz; FV scan rate 0.11 Hz; number of samples, 64; force per line, 64; samples per line, 64; display mode, retracted. Set-point, Z-scan size and Z-scan start were carried over from force plot settings.

Expression of rat glucocorticoid receptor in X. laevis oocytes
Rat glucocorticoid receptor (rGR) cRNA was transcribed in vitro from pßGR/RN3P (Belikov et al., 2000Go) (kind gift of Ö. Wrange, Karolinska Institute, Stockholm, Sweden), as previously described (Albermann et al., 2004Go), using T3 RNA polymerase (T3 cap-scribe, Roche, Mannheim, Germany) after linearization with restriction endonuclease Acc65I. Stage VI oocytes of Xenopus laevis were manually separated and injected with 10 ng of cRNAGR per oocyte. Subsequently, oocytes were kept in modified Ringer solution at 18°C for 72 hours.

20 kDa dextran permeability of cell nuclei
The underlying principle of exploring passive transport of macromolecules is that a change in nuclear pore complex (NPC) regulatory factors is followed by a change in the passive diffusion rate of fluorescence-labelled dextran. Comparison of diffusion rates under different experimental conditions allows conclusions about passive NPC permeability. The diffusion of FITC (Fluorescein isothiocyanate; Sigma, St Louis, MO) labelled 20 kDa dextran was measured across the nuclear envelope with confocal fluorescence microscopy, as previously described (Enss et al., 2003Go). The light source of the confocal laser-scanning microscope (CLSM Fluoview, Olympus) was an argon/krypton ion laser (Omnichrome®), which generates three excitation wavelengths, 488, 568 and 647 nm. GR expressing oocytes were injected with 50 nl of 2 x10–5 mol l–1 TA or 50 nl solvent. At 5 and 60 minutes after injection, nuclei were isolated and collected in NIM. Subsequently, nuclei were incubated for 15 minutes in NIM containing 5 µmol l–1 FITC labelled dextran and were then placed on Cell-tak in a superfusion chamber to detect the fluorescence of the nuclei.

Nuclear hourglass technique (NHT) and experimental protocol
The technical aspects of the method and its application in isolated cell nuclei have been described in detail previously (Danker et al., 1999Go). In short, the method uses a tapered glass tube, which narrows in its middle part to two thirds of the diameter of the nucleus. A current of up to 1 mA is injected via two massive Ag/AgCl electrodes through either end of the glass tube. The voltage drops across the cell nucleus are measured with two conventional microelectrodes. Since current and voltage are simultaneously measured, the resistance can be continuously calculated. Starting the experiment the nucleus is sucked into the tapered part of the capillary by gentle fluid movement. Thus, the whole current now flows through the accessible parts of the nuclear envelope. The resulting rise in total electrical resistance indicates the nuclear envelope electrical resistance (NEER). To obtain nuclear envelope electrical conductivity (NEEC) reciprocal values of NEER were calculated. In all experimental conditions (solvent and triamcinolone acetonide (TA) experiments), nuclei isolated 5 minutes after injection were brought into the NHT capillary filled with NIM. NEER measurements were started 20 seconds later.

Statistics
Data are presented as the mean±standard error of the mean (s.e.m.). In each experimental series (solvent and triamcinolone acetonide) five nuclei were isolated. n indicates the total number of analysed NPCs or force-curve distances for every time point after injection of solvent or trimacinolone acetonide. Statistical significance of mean values was tested with the unpaired Student's t-test. An asterisk (*) indicates a significant difference of P<0.001 or less. The calculated P values are declared in the respective figure legends.


    Results
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 Materials and Methods
 Results
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 References
 
We inject the glucocorticoid analogue TA into GR-expressing X. laevis oocytes to study induced changes in permeability and structure of the NE. GR accumulates at the chromatin within 5 minutes of TA exposure (Albermann et al., 2004Go). In preliminary experiments using AFM, individual GRs bound to gold-labelled antibodies could be found associated to NPCs under the same conditions (data not shown).

Investigation of NE permeability
TA induced an increase of NE electrical conductivity
The nuclear hourglass technique was developed for measurements of NE electrical resistance (NEER) of X. laevis oocytes (Danker et al., 1999Go). To determine NE electrical conductivity (NEEC), reciprocal values of NEER were calculated. NEEC mirrors NE permeability, which is related to the functional state of the NPCs (Schäfer et al., 2002Go). Fig. 2 describes NEEC changes in response to TA or solvent (ethanol in water, control) at a time scale of 5 minutes upon injection into oocytes. Mean NEEC value of 10 isolated nuclei of non-injected oocytes served as control value that is set to 100% (broken line in the graph of Fig. 2). Solvent injection led to a negligible increase (101.4±1.15%; mean±s.e.m., n=5 nuclei) of NEEC. TA injection, however, significantly increased NEEC (127.4±4.70%; mean±s.e.m., n=5 nuclei).



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Fig. 2. Effect of solvent (ethanol in water) and triamcinolone acetonide (TA) on nuclear envelope electrical conductivity upon 5 minutes of injection into glucocorticoid receptor-expressing X. laevis oocytes. Asterisk (*) indicates a statistically significant difference (unpaired t-test, P<0.001) between the mean data of solvent and TA experiments. The broken line indicates the mean NEEC value of eight non-injected GR-expressing oocytes. The latter mean value was set to 100% (control).

 

TA induced a transient increase of NE permeability for 20 kDa FITC-dextran
We investigated the impact of TA and solvent injection on the permeability of the NE for fluorescence labelled intermediate-sized macromolecules, 20 kDa FITC-dextran. NE permeability for FITC-dextran, nuclear fluorescence respectively, was measured in response to TA and solvent at a time scale of 5 and 60 minutes after injection. Nuclear fluorescence of the injected oocytes was compared with the nuclear fluorescence of non-injected oocytes (control, n=10 oocytes nuclei). Non-injected oocytes revealed a modest level of nuclear fluorescence that was set to 100% (control nuclear fluorescence). A change in nuclear fluorescence of injected oocytes was compared with control nuclear fluorescence and determined as proportions (%). As seen in Fig. 3, no remarkable change (98.9±3.8%; mean±s.e.m., n=15 nuclei) of nuclear fluorescence was detectable 5 minutes after solvent injection. By contrast, a significant increase (124.2±5.5%; mean±s.e.m., n=16 nuclei) of nuclear fluorescence was observed 5 minutes upon TA injection. In addition, the increase of NE permeability for 20 kDa dextran was transient: 60 minutes after TA injection, the detected fluorescence from stimulated oocytes decreased to its initial value (103.9±0.8%; mean±s.e.m., n=10 nuclei).



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Fig. 3. (A) Representative confocal fluorescence images of nuclear envelope permeability for 20 kDa FITC-dextran, nuclear fluorescence respectively, in response to triamcinolone acetonide (TA) and solvent at a time scale of 5 minutes after injection into glucocorticoid receptors-expressing X. laevis oocytes. (B) Change of nuclear envelope permeability for 20 kDa FITC-dextran, nuclear fluorescence respectively, in response to triamcinolone acetonide (TA) and solvent at a time scale of 5 and 60 minutes after injection of TA into glucocorticoid receptor-expressing X. laevis oocytes. Asterisk (*) indicates a statistically significant difference (unpaired t-test, P<0.0009) between the mean data of solvent and TA experiments.

 

Investigation of NE structure with AFM
TA induced a dilation and a rearrangement of NPCs distribution on the NE surface
Fig. 4 displays representative AFM images of the cytoplasmic face of the NE 5 minutes after solvent or TA injection into oocytes. Upon solvent injection, no remarkable changes of the conformational state and the distribution of the NPCs in the NE were detected compared with non-injected oocytes (data not shown here). Only one NPC pattern covers the entire surface of the NE. NPCs appear as smooth rings non-randomly distributed throughout the entire NE surface. Uneven NPC-free areas, so-called NPC-free NE surface, were commonly visible. TA injection, however, led to remarkable changes of NPC conformational states and to dramatic rearrangement of NPC distribution in the NE. Two NPC patterns (I and II) equally covered the entire surface of the NE. Mean NPC density in pattern I decreased from 30.1 (solvent condition) to 21.7 per µm2. In pattern II, however, mean NPC density increased from 30.1 to 39.2 per µm2. NPC-free NE surface was commonly detectable in pattern I but barely in pattern II, where consistently, several hundred NPCs adjoin forming clusters. Following NPC density reduction in pattern I mean distances between adjacent NPCs in pattern I increased from 97.73±4.06 nm (solvent conditions) to 114.97±6.64 nm. In pattern II, however, following the NPC density increase compared with solvent, mean distance between adjacent NPCs considerably decreased to 39.21±1.01 nm. In both patterns I and II, the external NPC diameter and the apparent NPC opening dilated conspicuously. Table 1 summarizes the observations mentioned above.



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Fig. 4. AFM images of the cytoplasmic nuclear envelope surface of glucocorticoid receptor (GR)-expressing X. laevis oocytes 5 minutes after solvent (ethanol in water, at the left) or triamcinolone acetonide (at the right) injection.

 

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Table 1. Effect of TA on the NE and NPCs

 

Estimation of NPC opening and NE surface hydrophobicity using AFM
TA induced an increase in hydrophobicity of the NPC opening and the NPC-free NE surface
Fig. 5 describes an assessement of the hydrophobicity degree of NPC opening and NPC-free NE surface 5 minutes upon injection of either solvent or TA into X. laevis oocytes. The assessement of hydrophobicity was determined by measurement of the adhesion forces between the AFM tip and the scanned NE area, using a hydrophobic, aluminium coated AFM tip, CSC21 (Ultrasharp, Mikro Masch, Anfatec, Germany) as described in methods. To obtain an array of force-distance curves, a hydrophobicity map, over the entire NE area being scanned, the so-called force-volume mode is applied. To characterize the adhesion forces on both, a hydrophobic and a hydrophilic surface, respectively, silicon or poly-l-lysine coated glass coverslips were scanned with the AFM tip CSC21. Mean adhesion force on the silicon surface ranged 3.79±0.04 nN; (number of analysed force-distance curves=30). Mean adhesion force on poly-l-lysine surface, however, was clearly less and ranged 0.62±0.01 nN; (number of analysed force-distance curves=30). These data indicate that the applied AFM tip CSC21 is rather hydrophobic, thus much more attracted on the hydrophobic silicon surface being scanned. After characterization of the AFM tip surface properties, similar adhesion force measurements were carried out on both, the NPC opening and the NPC-free NE surface using the same tip. At 5 minutes after solvent injection, mean adhesion force of NPC opening ranged 0.27±0.00 nN and of NPC-free NE surface 0.60±0.02. Within 5 minutes, TA injection led to an increase of both forces by ~200% and ~132%, respectively. Table 2 summarizes the above mentioned adhesion force measurements.



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Fig. 5. Assessement of the hydrophobicity degree of NPC opening and NPC-free NE surface 5 minutes upon injection of either solvent or TA into X. laevis oocytes. The assessment of hydrophobicity was determined by measurement of the adhesion forces between the AFM tip and the scanned NE area, using a hydrophobic, aluminium coated AFM tip, CSC21 (Ultra-sharp, Mikro Masch, Anfatec, Germany). To obtain an array of force-distance curves, a hydrophobicity map, over the entire NE area being scanned, the so-called force-volume mode is applied. The white spikes in the upper force volume image result from loss of contact between the AFM tip and the sample. Nuclear envelope (NE), nuclear pore complexes (NPCs), triamcinolone acetonide (TA).

 

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Table 2. Effect of TA on NPC opening and surface hydrophobicity

 


    Discussion
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 Summary
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 Materials and Methods
 Results
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 References
 
The present study describes remarkable changes of permeability and structure of the NE following exposure to the glucocorticoid analogue TA.

Glucocorticoids affect hydrophobicity of the NPC opening, lead to its dilation, causing a leakiness of the NE barrier
Proposed model
The observed dilation of NPC opening might become explainable by regarding the following works and various others cited therein. Ribbeck and Gorlich (Ribbeck and Gorlich, 2002Go) postulated the following model: abundant FG-rich nucleoporins inside the NPC central channel are proposed to interact mutually via their hydrophobic repeats to form a flexible meshwork of nucleoporins; nuclear transport receptors (e.g. importin-{alpha},ß) overcome this barrier very rapidly because they are more hydrophobic than the average soluble proteins and interact directly with the hydrophobic FG nucleoporins repeats.

Hydrophobic molecules, such as aliphatic alcohols, are believed to induce a dissociation of structural and FG-rich nucleoporins leading to a disruption of the permeability barrier of wild type NPCs (Shulga and Goldfarb, 2003Go). Interestingly, the effectiveness of the alcohols is roughly proportional to their relative hydrophobicity.

Like the aliphatic alcohols mentioned above, also TA is a hydrophobic molecule. Upon TA-binding, the formed complex, consisting of TA-GR and the hydrophobic nuclear transport receptor (importin-{alpha},ß), would become even more hydrophobic and soluble in the flexible FG nucleoporin meshwork inside the NPC opening. This might lead to molecular interactions between FG-rich nucleoporins and the complex being translocated. Such interactions could be exerted not only by the nuclear transport receptor to which the TA-bound GR is associated, but also by TA itself. This assumption is confirmed in Fig. 5, which indicates a doubling in hydrophobicity of the NPC opening upon TA exposure. TA itself or in conjunction with the also hydrophobic nuclear transport receptor seems to accumulate within the NPC opening leading to a significant increase in hydrophobicity there. Such an accumulation would cause a dissociation of FG-rich nucleoporins in the NPC opening, affecting the structure of the nucleoporin meshwork. As a result of nucleoporins dissociation the dilation state or the diameter of the apparent NPC opening, respectively, would increase. Taken together, the extent of NPC opening dilation might depend on the hydrophobicity of the molecules to be translocated.

Physiological relevance
Dilation of NPC opening was accompanied by a parallel and significant increase of NE permeability for small ions and intermediate-sized macromolecules (20 kDa FITC-dextran). It seems likely that the increase of NE permeability is a consequence of the dilation of the opening of the NPC, the only transport pathway between the cytosol and the nucleus. We assume that the glucocorticoid-induced increase of NE permeability is of crucial physiological relevance. As steroid hormones, glucocorticoids trigger gene expression that requires nuclear translocation of not only the glucocorticoid-GR-complex but also of various universal transcription factors (macromolecules). Thus, increase of NE permeability for macromolecules (as shown for 20 kDa dextran), could be a prerequisite for mediating gene expression. Also the increase of NE permeability for ions seems to be important with respect to the `ion hypothesis of gene activation' that was postulated more than 40 years ago by Kroeger (Kroeger, 1966Go) and later confirmed in our laboratory (Wünsch et al., 1993Go). A change of intracellular ion composition, in particular a change in free Ca2+ (Hardingham et al., 1997Go; Schäfer et al., 2003Go) and H+ (Oberleithner et al., 1993Go; Wünsch et al., 1993Go), seems necessary for steroid-induced gene activation (Mazzanti et al., 2001Go).

Glucocorticoids interact with structures of the NE surface, initiate a lateral movement of NPCs and a rearrangement of their distribution
Proposed model
The observed TA-induced rearrangement of NPC distribution and the formation of NPC clusters indicate that NPCs are capable of undergoing a lateral movement within the NE plane. The lateral movement of NPCs into clusters may result from dissociation of nucleoporins that normally restrict NPC dynamics within the NE plane. The nucleoporins POM121 and Nup153 were shown to be involved in NPC clustering in apoptosis (Beckman et al., 2004Go; Kihlmark et al., 2001Go). POM121 is one of two [besides gp210 (Courvalin et al., 1990Go; Greber et al., 1990Go; Wozniak and Blobel, 1992Go)] integral membrane proteins that are known to tether the vertebrate NPC to the inner and outer membrane of NE. We cannot rule out the possibility that these nucleoporins could be involved in mediating a lateral NPC movement. However, it seems unlikely that NPC movement could be mediated exclusively by nucleoporins. After all, NPCs are held in place by various barrier components including the nuclear lamina and particularly both membranes of the NE. The NPCs are densely surrounded by a hydrophobic layer composed of membrane lipids, which restrict their lateral movement. In other words, NPCs should be able reversibly to displace these membrane lipids to undergo a lateral movement in the NE membranes. Alternatively, a change of the composition of NE membrane lipids could be induced. Such a change could be induced by physiological stimulation, for example with glucocorticoids, as indicated in Fig. 5. In the latter figure it becomes very probable that TA could have interacted with still unknown structures, probably lipids, of the NPC-free NE surface. Furthermore, these structures could be membrane components that restrict the lateral movement of protein complexes such as NPCs. In fact, in cellular membranes, the movement of lipids and proteins is believed to be regulated by lipid microdomains, so-called lipid-rafts (Henderson et al., 2004Go; Simons and Ikonen, 1997Go). The latter are believed to be involved in various other processes like cellular signalling and protein trafficking (Simons and Toomre, 2000Go). They were shown to mediate rapid physiological actions of glucocorticoids analogues (Van Laethem et al., 2003Go). It is conceivable that the glucocorticoid TA could also have interacted with such rafts. Possibly as a consequence of this interaction, hydrophobicity of the NE surface is affected, a lateral movement of NPCs is enabled and NPCs clusters are formed.

Physiological relevance
The non-random distribution of NPCs on the NE was supposed to reflect the periodic organization of the subjacent genome (Blobel, 1985Go): expanded (transcribable) and compacted domains alternate with each other. Various studies involved a role of NPCs in both, transcription and silencing of genes (Casolari et al., 2004Go; Feuerbach et al., 2002Go; Galy et al., 2000Go). With respect to these studies and citations therein, the following assumption is conceivable: non-random distributed, transcribable chromatin domains would be subjacent to NPC clusters while repressive chromatin domains could be subjacent to NE regions devoid of NPCs clusters; consequently, a spread of transcriptional activation could be prevented. However, why should NPCs form clusters? Transcribable genes would physically interact with a subset of NPCs simultaneously; a spatial approximation of NPCs, following lateral movement, would become very convenient or even necessary, to enable such an interaction. The association between NPC clusters and subjacent active genes could occur via intranuclear channels (Strouboulis and Wolffe, 1996Go). The latter are interconnecting open channels, which have been observed radiating from the nuclear interior towards the NPCs (Berezney et al., 1995Go). Through this intranuclear pathway, transport substrates could be driven to the NPC bidirectionally. However, whether all NPCs possess such channels is unclear. Twenty six years ago, three models of association between NPCs and intranuclear channels were postulated (Schatten and Thoman, 1978Go). One of these models indicated that a single intranuclear channel might be able to bifurcate. We would like to speculate that a given intranuclear channel might branch out to service numerous NPCs simultaneously. It could build a `collecting duct'. This duct would drive activated GRs to their target genes and/or newly-synthesized mRNA to a given NPC in the cluster. Thus, again a spatial approximation of numerous NPCs or NPC clustering would become necessary to use the full capacity of intranuclear channels.

In conclusion, the NE is a multicomponent structure of high plasticity. It is not only a transport barrier but also a key mediator of physiological processes, e.g. steroid hormone action.


    Acknowledgments
 
The excellent technical assistance of Barbara Windoffer is gratefully acknowledged. The study was supported by the `Innovative Medizinische Forschung' (SH-110315) and the `Volkswagenstiftung' (BD-151103).


    Footnotes
 
* These authors contributed equally to this work Back


    References
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

Aebi, U., Cohn, J., Buhle, L. and Gerace, L. (1986). The nuclear lamina is a meshwork of intermediate-type filaments. Nature 323, 560-564.[CrossRef][Medline]

Aebi, U., Pante, N. and Jarnik, M. (1992). Structure and function of the nuclear pore complex, a supramolecular machine mediating molecular trafficking across the nuclear envelope. Verh. Dtsch. Zool. Ges. 85, 285-296.

Akey, C. W. and Radermacher, M. (1993). Architecture of the Xenopus nuclear pore complex revealed by three-dimensional cryo-electron microscopy. J. Cell Biol. 122, 1-19.[Abstract]

Albermann, L., Shahin, V., Ludwig, Y., Schafer, C., Schillers, H. and Oberleithner, H. (2004). Evidence for importin alpha independent nuclear translocation of glucocorticoid receptors in Xenopus laevis oocytes. Cell Physiol. Biochem. 14, 343-350.[CrossRef][Medline]

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