1 Department of Anatomy and Cell Biology, and
2 Department of Biochemistry and Molecular Biology, University of Florida, College of Medicine, Gainesville, FL 32610, USA
*Author for correspondence (e-mail: feldherr{at}anatomy.med.ufl.edu)
Accepted September 12, 2001
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SUMMARY |
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Key words: Nuclear pore complex, Nuclear transport, p53
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INTRODUCTION |
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The functional size of the nuclear pores (or more specifically the central transport channel) is variable, and dependent on the physiological state of the cells (Feldherr and Akin, 1995a). By using colloidal gold particles coated with proteins containing classical NLSs (signals that use the importin /ß pathway) as substrates for signal-mediated transport, it was determined that the upper limit for nuclear import in normal proliferating BALB/c 3T3 cells is approximately 250 Å. In quiescent populations, this value can decrease by up to 100 Å (Feldherr and Akin, 1991), whereas functional pore size increases by about 40 Å in SV40-transformed BALB/c 3T3 cell lines (Feldherr et al., 1992). Changes of this magnitude could significantly alter the transport rates of large mRNP particles and ribosomal subunits.
It was found that the increase in nuclear import in SV40-transformed cells was caused by large T antigen (Feldherr et al., 1992). This conclusion was based on the finding that the microinjection of purified large T antigen resulted in a significant increase in the dimensions of gold particles able to enter the nucleoplasm, comparable with that observed in transformed cells. Experiments using a series of large T mutants suggested that the transport increase is dependent on p53 binding (Feldherr et al., 1994). In the same study, supporting evidence that sequestering p53 alters transport capacity was obtained by transfecting cells with mutant p53, which forms oligomers with wild-type p53, thereby reducing its effective concentration, and also by microinjecting anti-p53 antibodies. Both of these procedures resulted in a significant increase in functional pore size. Subsequently, it was determined that 100,000 g cytosolic extracts from SV40-transformed cells increased functional pore size when microinjected into non-transformed BALB/c 3T3 cells (Feldherr and Akin, 1995b). This activity could be blocked by treating the extracts with a variety of kinase inhibitors, including specific PKC inhibitors. Taken together, the above studies imply that large T antigen initiates a series of events that result in a PKC-dependent increase in nuclear transport, and that p53 is an intermediate in these events.
To further test the above scenario, which implicates p53 as a factor in modulating nuclear transport, we investigated the effects of cycloheximide, and the p53 inhibitor pifithrin- on nuclear import. Since p53 is a short-lived protein, inhibiting polypeptide synthesis should cause a rapid depletion of endogenous p53, and an increase in functional pore size. The present study shows that this is the case. A 3 hour treatment with cycloheximide resulted in a significant increase in the nuclear import of large gold particles. In contrast to the results obtained with large substrates, cycloheximide had no significant effect on either the transport rates of small substrates (BSA-NLS conjugates), or the passive diffusion of proteins through the pores. The latter results show that the changes in pore size are coupled to events that occur during signal-mediated transport. Incubation of the cells in pifithrin-
also increased the functional size of the pores.
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MATERIALS AND METHODS |
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Cells that were injected with colloidal gold, and analyzed by electron microscopy, were cultured on gridded ACLAR coverslips (Allied Corp., Morristown, NJ) as described (Feldherr and Akin, 1990). Fluorescent analysis was performed on cells grown on glass coverslips.
Inhibitors
Stock solutions of MG-132 (10 nM), and pifithrin- (10 mg/ml) were prepared in dimethyl sulfoxide. Stock solutions of cycloheximide (10 mg/ml) were made up in ion-free water. Cycloheximide was obtained from Sigma (St Louis, MO); the other inhibitors were purchased from Calbiochem (San Diego, CA).
Nuclear transport substrates
Colloidal gold particles, ranging in diameter from 80 to 360 Å were prepared by reducing gold chloride with sodium citrate (Frens, 1973). The coating protein, BSA-NLS, was prepared by conjugating bovine serum albumin (Sigma) with a synthetic peptide, CGGGPKKKRKVGG, which contains the SV40 large T NLS (underlined). The peptide was synthesized by the University of Florida protein core facility. The conjugation step was performed using the cross-linker m-maleimidobenzoyl-N-hydroxysuccinimide ester (Pierce, Rockford, IL), as reported (Lanford et al., 1986). It was estimated by SDS-PAGE that each BSA molecule was conjugated with an average of 8 peptides. The gold particles were coated with BSA-NLS as outlined previously (Dworetzky et al., 1988). The protein coat increases overall particle diameter by approximately 30 Å.
To prepare fluorescent BSA-NLS, BSA was first labeled with fluorescein isothiocyanate (Sigma) as previously described (Feldherr and Akin, 1999), and subsequently conjugated with NLS peptide as above. Fluorescein isothiocyanate was also used to label BSA, and ovalbumin (Sigma) for diffusion studies; 260 kDa dextran, labeled with FITC, was purchased from Sigma.
Prior to microinjection, all transport substrates were dialyzed against intracellular medium, 117 mM KCl, 10 mM NaCl, 6 mM K2HPO4, and 4 mM KH2PO4 (pH 7.0).
Microinjection
Microinjections were performed at 37°C in a CO2-enriched atmosphere using an inverted Nikon Diaphot microscope, a Narishige hydraulic micromanipulator, and a continuous-flow injection system (Feldherr and Akin, 1990). The micropipets had tip diameters of approximately 0.5-0.7 µm.
EM and fluorescent analysis
Details of the EM procedures have been reported previously (Feldherr and Akin, 1990). Briefly, the cells were fixed in 1% glutaraldehyde, postfixed in 1% OsO4 and analyzed using a JEOL 100CX electron microscope.
The nuclear import rates of fluorescent-labeled transport substrates were determined using an intensified Hamamatsu CCD camera, and a MetaMorph imaging system (Universal Imaging System, West Chester, PA). At the specified times after cytoplasmic injection, images of the cells were collected using a Nikon PlanApo 60x oil immersion lens, and the N/C ratios were determined by measuring fluorescence in equal areas of cytoplasm and nucleoplasm. To avoid UV damage to the cells, exposure at each time point was less than 1 second and a number 16 neutral density filter was used.
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RESULTS |
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The cycloheximide effect is initiated by a short lived protein and requires at least one additional factor
To study the effect of protein turnover on cycloheximide-dependent increases in transport capacity, cells were incubated for 3 hours in 50 µg/ml cycloheximide alone, or in cycloheximide plus 10 nM MG-132, a proteasome inhibitor. The control cells were not pretreated. Nuclear transport was analyzed using colloidal gold, as described above. As can be seen in Table 2A, the increase in transport capacity that resulted from cycloheximide treatment was blocked by inhibiting proteasome activity. These results indicate that the effect of cycloheximide on pore size is due to the depletion of a short-lived protein; thus, if turnover is prevented by inactivating proteasomes, there is no significant change in transport capacity.
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Cycloheximide has no effect on the import of small NLS-substrates or passive diffusion
The intracellular distributions of FITC-BSA-NLS, 1 and 60 minutes after injection into control cells and cells treated with 50 µg/ml cycloheximide for 3 hours, are shown in Fig. 1A (left). The N/C fluorescence ratios were quantified at the time points indicated, and are plotted in Fig. 1B. There was no significant difference in nuclear import kinetics in the control versus the experimental cells, demonstrating that cycloheximide had no effect on the signal-mediated transport of a smaller substrate (approximately 70 Å in diameter), which is appreciably below the exclusion limit of the nuclear pores. Similarly, a 3 hour treatment with cycloheximide had no significant effect on the passive diffusion of either FITC-labeled ovalbumin or BSA (Fig. 1A, right; Fig. 1B). The high N/C ratios obtained after 1 minute in the diffusion experiments were due to background fluorescence from overlapping and underlying cytoplasm. This was demonstrated by injecting FITC-labeled, 260 kDa dextran, which is too large to diffuse across the envelope, but still had an initial N/C ratio of approximately 0.6.
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Pifithrin- has the same effect on pore size as cycloheximide
As a further test for the possible involvement of p53 in the regulation of transport capacity, experiments were performed using the inhibitor pifithrin- (Komarov et al., 1999). The nuclear uptake of colloidal gold was examined in control fibroblasts, and cells that were incubated in 30 µM pifithrin-
for 3 hours. The results (Table 3) show that exposure to pifithrin-
significantly increased functional pore size (both the percentage of large particles that entered the nucleus and the N/C ratio) to approximately the same degree as cycloheximide treatment. However, the same pifithrin-
treatment (30 µM for 3 hours) had no significant effect on the nuclear import rate of FITC-BSA-NLS, compared with controls (Fig. 2).
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DISCUSSION |
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Although conclusive evidence is not available, a possible candidate for the initiating factor is the short-lived tumor suppressor, p53. As discussed in the Introduction, antibodies to p53 as well as transfection with mutant forms of p53 cause a functional increase in pore diameter, similar in magnitude to those reported here. The results obtained with pifithrin-, which blocks p53-dependent transcription activation, provide further support for the involvement of p53 and indicate that a factor downstream of p53 is required to enhance transport.
Our previous results (see Introduction) that extracts from SV40-transformed BALB/c 3T3 cells can increase functional pore size when microinjected into non-transformed cells, and that the increase can be blocked by PKC inhibitors, raises the possibility that cycloheximide-dependent transport effects might also require kinase activity. Unfortunately, experiments designed to test this hypothesis were inconclusive. Cells were simultaneously treated with cycloheximide and various kinase inhibitors, a procedure that prevented an increase in the transport of large gold particles; however, 3 hour exposures to kinase inhibitors alone also altered transport capacity, making it difficult to evaluate their specific effects on cycloheximide activity.
It is currently believed that signal-mediated translocation through the nuclear pore complex occurs by sequential steps of adsorption and desorption of the receptor-substrate complex to FG repeat nucleoporins that are associated with the central transport channel (Bayliss et al., 2000). However, the mechanism that restricts the passive diffusion of molecules approximately 25 Å in diameter and above (Paine et al., 1975), but allows NLS-containing substrates as large as 250 Å in diameter to rapidly enter the nucleus, remains controversial. Theoretically, this could be accomplished in several ways. One possibility is a mechanical gating system, which could involve either two separate gates, located at either end of the transport channel (Akey, 1990; Kiseleva et al., 2000), or a single gate, positioned in the middle of the channel (Feldherr and Akin, 1997). Activation of a gating system by the receptor-substrate complex could allow selective translocation of large, signal-containing substrates, while restricting diffusion of molecules that are not targeted for exchange. Rout et al. introduced the concept of a virtual gate (Rout et al., 2000). This model is based on a detailed study of yeast nuclear pore complexes, which indicated that both ends of the transport channel are encircled by filamentous FG-containing nucleoporins. It was suggested that these filamentous proteins undergo active Brownian movement and present a formidable barrier to the diffusion of large molecules that lack FG-binding domains; however, substrates that contain FG-binding sequences (e.g. transport receptors) would be transiently retained, thus facilitating diffusion through the central channel. Most recently, a selective phase model has been proposed for exchange through the pores (Ribbeck and Gorlich, 2001). According to this model, the central transport channel is occupied by a low density meshwork of FG-repeat nucleoporins. It is suggested that this meshwork would function as a semi-liquid phase, in which transport complexes containing FG-binding sites could partition and, thus, translocate across the pores. The finding, in this investigation, that changes in functional channel size are not accompanied by overall changes in the physical characteristics of the transport channel (as determined from diffusion studies), does not appear to distinguish among the above possibilities. However, it does imply that, as a result of cycloheximide treatment, there is an increased capacity of the gate to dilate, whether it be mechanical or virtual. Presumably, this would involve conformational changes of the nucleoporins, which are initiated by their interaction with the receptor-substrate complex. It is also possible that the degree of dilation is regulated by the phosphorylation state of the nucleoporins.
Although all models of the pore complex incorporate a centrally located pathway for the translocation of macromolecules, the specific nature of the pathway is still in question. Several investigators have proposed that transport occurs through a cylindrical transporter element. In the model developed by Akey and Radermacher, for example, the transporter element is represented as a hourglass-shaped structure with minimum and maximum outer diameters of 320 and 420 Å, respectively (Akey and Radermacher, 1993). The wall, at its thinnest point, is approximately 75-90 Å thick. Alternatively, it is possible that the central pathway is simply an open channel through the pore complex, rather than a distinct structural element (Stoffler et al., 1999). We have calculated that channel diameters of approximately 310 Å and 370 Å in normal and cycloheximide-treated cells, respectively, would be necessary to account for the observed transport of large gold particles. It would appear that the open channel model would be more compatible with these size requirements than a more restricted pathway associated with a transporter element.
Future investigations directed toward understanding the overall significance, and the molecular events that regulate the functional dimensions of the central transport channel should include the following: (1) establishing whether variations in pore size can occur in all cells, or are restricted to specific cell types; (2) identifying the factor(s) downstream of p53 that activates the subsequent events responsible for increasing pore size (possible candidates for this factor include cyclin G, p21 and mdm2); and (3) determining whether cycloheximide and pifithrin- treatments are accompanied by changes in the phosphorylation patterns of nucleoporins. This data might help identify specific pore components that are directly involved in modulating pore size.
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ACKNOWLEDGMENTS |
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