Anal. Chem. 2005, 77, 2147-2156
Permeability of the Nuclear Envelope at Isolated Xenopus Oocyte Nuclei Studied by Scanning Electrochemical Microscopy Jidong Guo and Shigeru Amemiya*
Department of Chemistry, University of Pittsburgh, 219 Parkman Avenue, Pittsburgh, Pennsylvania 15260
In interphase eukaryotic cells, molecular transport between the cytoplasm and the nucleus is mediated by the nuclear pore complex (NPC), which perforates the doublemembraned nuclear envelope (NE). Local permeability of the NE at large intact nuclei (∼400 µm in diameter) isolated from Xenopus laevis oocytes was studied by scanning electrochemical microscopy (SECM). Steadystate tip current versus tip-nucleus distance curves (approach curves) were measured with 10- and 2-µmdiameter Pt disk microelectrodes at the nuclei in isotonic buffer solutions containing redox-active molecules. The approach curves in the normalized form are independent of the tip diameter, indicating diffusion-limited membrane transport of the redox molecules. SECM chronoamperometry demonstrated that a decrease in the steady-state tip current at short tip-nucleus distances is due to smaller diffusion coefficients and concentrations of the redox molecules in the nucleus than those in the buffer solution. The experimental approach curves fit very well with theoretical ones for freely permeable membranes, yielding the NE permeability to the molecules that is at least 2 orders of magnitude larger than permeability of bilayer lipid membranes and cell membranes. This result indicates that passive transport of the redox molecules across the NE is facilitated by open NPC pores. The flux of the redox molecules sustainable by a single NPC channel (>9.8 × 106 molecules per NPC per second) and the diameter of the channel pore (>15 nm) were estimated from the SECM data by assuming the NE as an array of nanometer-sized NPC pores. The effects of the redox molecules on the nucleus and the NPC function were examined by studying signal-mediated nuclear import of rhodamine-labeled bovine serum albumin with and without nuclear localization signals by fluorescence microscopy. In interphase eukaryotic cells, the nucleus is spatially separated from the cytoplasm by the double membrane of the nuclear envelope (NE).1 Many species of electrolytes, small molecules, proteins, and RNAs are transported through the nuclear pore * To whom correspondence should be addressed. E-mail:
[email protected]. Fax: 412-624-5259. (1) Alberts, B.; Johnson, A.; Lewis, J.; Raff, M.; Roberts, K.; Walter, P. Molecular Biology of the Cell, 4th ed.; Garland Science: New York, 2002; pp 669-676. 10.1021/ac048370j CCC: $30.25 Published on Web 02/24/2005
© 2005 American Chemical Society
complex (NPC), which perforates the NE.2-6 The NPC is a large complex (estimated mass of 60-120 MDa) composed of 30 or more distinct proteins termed nucleoporins.7 The NPC serves as the sole gate for nucleocytoplasmic transport, which is based on two different mechanisms, i.e., passive and facilitated transport. The molecules that are smaller than 10-40 kDa can passively diffuse through the NPC pore. The NPC also mediates transport of larger proteins and RNAs with nuclear transport signals. For example, a macromolecule with a nuclear localization signal peptide is recognized by a heterodimer of importin R and β in the cytoplasm and imported into the nucleus, where importin R (∼55 kDa) binds the signal peptide on the macromolecule and importin β (∼100 kDa) facilitates translocation of the importinsubstrate complex through the NPC. Therefore, the permeable complexes are much larger than the molecules that are passively permeable through the NPC and can be as large as up to 39 nm in diameter.8 During the past four decades, physiological and dynamic states of the NPC channel were studied by electrophysiological methods.9 Electrical resistance of the NE was measured with glass micropipet electrodes penetrated into the intact nucleus.10,11 In contrast to cell membranes, the NE resistance was found to be very low, suggesting that most NPCs are open under physiological conditions. The resistance thus measured, however, corresponds to the whole NE embedding thousands of NPC channels. Therefore, dynamic information about each channel’s state cannot be obtained. Also, the total resistance of large nuclei of amphibian oocytes is too low to be measured accurately with this method while the nuclei have been widely used for evaluation of nuclear transport and for studies of NPC structure and function. To overcome these limitations, permeability of a small area of the NE was studied by the patch clamp technique. Current recording with a “gigaseal” was successfully achieved to find the ion (2) Wente, S. R. Science 2000, 288, 1374-1377. (3) Macara, I. G. Microbiol. Mol. Biol. Rev. 2001, 65, 570-594. (4) Weis, K. Cell 2003, 112, 441-451. (5) Bednenko, J.; Cingolani, G.; Gerace, L. Traffic 2003, 4, 127-135. (6) Rout, M. P.; Aitchison, J. D.; Magnasco, M. O.; Chait, B. T. Trends Cell Biol. 2003, 13, 622-628. (7) Fahrenkrog, B.; Aebi, U. Nat. Rev. Mol. Cell Biol. 2003, 4, 757-766. (8) Pante, N.; Kann, M. Mol. Biol. Cell 2002, 13, 425-434. (9) Mazzanti, M.; Bustamante, J. O.; Oberleithner, H. Physiol. Rev. 2001, 81, 1-19. (10) Kanno, Y.; Loewenstein, W. R. Exp. Cell Res. 1963, 31, 149-166. (11) Loewenstein, W. R.; Kanno, Y. J. Gen. Physol. 1963, 46, 1123-1140.
Analytical Chemistry, Vol. 77, No. 7, April 1, 2005 2147
channels in the NE other than the NPC.12,13 The small patch area, however, contains many NPCs (7-50 NPCs/µm2) so that most NPCs must be closed during patch clamp experiments with the gigaseal. This conclusion is inconsistent with efficient passive transport at the NE as observed by the resistance measurements and also by the other methods,14 suggesting that the closed state is not likely to be a physiological state of the NPC. More recently, Oberleithner and co-workers developed the novel technique that is called the nuclear hourglass technique.15 When the nucleus is sucked into the narrow part of a tapered glass tube filled with an electrolyte solution, tube resistance increases, which corresponds to the resistance of the nucleus. The method was used to determine the resistance of the large intact nucleus isolated from Xenopus laevis oocytes. The low resistance thus obtained (456 ( 13 Ω) indicates high ionic permeability of the NPC, supporting the conclusion that most NPCs are open. In contrast to the patch clamp technique, however, the hourglass technique does not offer high spatial resolution to study dynamics of a small number of the NPCs. Scanning electrochemical microscopy (SECM) was established as a noninvasive method to study highly permeable membranes with high spatial resolution.16 In a SECM membrane experiment,17 an ultramicroelectrode is brought to the surface of a bilayer lipid membrane (BLM) formed between two aqueous solutions containing redox-active molecules. These molecules are electrolyzed at the electrode to be depleted locally in the gap between the tip and the membrane. The concentration gradient in the gap causes flux of the molecules across the membrane, which is detected at the electrode. By studying the steady-state tip current versus tipmembrane distance curve (approach curve), the membrane permeability can be determined without any tip-membrane contact. Also, an external application of membrane potential or current is not necessary to induce the molecular flux for the permeability measurement. In addition to its noninvasive principle, the membrane flux is localized at the small area of the NE under the tip so that fast membrane transport can be studied with high spatial resolution. Bard and co-workers recently used this approach to study more complicated transport processes at BLMs such as facilitated I- transport18 and gramicidin-mediated K+ transport.19 The technique was also applied for permeability study of a single, living algal protoplast.20 While these SECM membrane transport studies were carried out under steady-state conditions, Unwin and co-workers combined chronoamperometric techniques with approach curve measurements to study partitioning processes of redox-active molecules across unmodified or lipid monolayer(12) Mazzanti, M.; Defelice, L. J.; Cohen, J.; Malter, H. Nature 1990, 343, 764767. (13) Mak, D. O. D.; Foskett, J. K. J. Biol. Chem. 1994, 269, 29375-29378. (14) Peters, R. Biochim. Biophys. Acta 1986, 864, 305-359. (15) Danker, T.; Schillers, H.; Storck, J.; Shahin, V.; Kra¨mer, B.; Wilhelmi, M.; Oberleithner, H. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 13530-13535. (16) Bath, B. D.; White, H. S.; Scott, E. R. In Scanning Electrochemical Microscopy; Bard, A. J., Mirkin, M. V., Eds.; Marcel Dekker: New York, 2001; pp 343395. (17) Yamada, H.; Matsue, T.; Uchida, I. Biochem. Biophys. Res. Commun. 1991, 180, 1330-1334. (18) Tsionsky, M.; Zhou, J.; Amemiya, S.; Fan, F.-R. F.; Bard, A. J.; Dryfe, R. A. W. Anal. Chem. 1999, 71, 4300-4305. (19) Amemiya, S.; Bard, A. J. Anal. Chem. 2000, 72, 4940-4948. (20) Yasukawa, T.; Uchida, I.; Matsue, T. Biochim. Biophys. Acta 1998, 1369, 152-158.
2148 Analytical Chemistry, Vol. 77, No. 7, April 1, 2005
Figure 1. Scheme of SECM experiments at the intact nucleus (not to scale). X is a redox molecule.
modified interfaces between liquid/liquid and air/liquid phases.21,22 Importantly, SECM-based chronoamperometry at an electrode positioned at a side of the interface allows determinations of the diffusion coefficient and concentration of the redox molecules at the opposite side of the interface without contact from the electrode. These two parameters are necessary to quantitatively interpret steady-state SECM approach curves. Here we report on application of SECM for study of the NE permeability at the intact nucleus isolated from X. laevis oocytes (Figure 1). SECM is suitable for study of the highly permeable NE because it offers a high mass-transfer rate for diffusion of a redox molecule in the gap between the tip and the NE, which, with a disk-shaped probe, can be in the range of D/a ) 0.1-0.02 cm/s for the disk radius, a, of 1-5 µm and the diffusion coefficient of the molecule, D, of 10-5 cm2/s. Also, the spatial resolution of SECM with a few micrometer-diameter probe is not only much better than that of the glass pipet and hourglass techniques but also comparable to that of the patch clamp technique. Recently, single artificial nanopores were detected using nanometer-sized SECM probes.23,24 Also, in the SECM experiments, all electrodes are positioned outside the nucleus so that the NPCs are not damaged by physical contact,25 by surface tension change,26 or electrophoretically plugged with macromolecules.27 SECM experiments with metal electrodes, however, require use of redox-active molecules, which do not exist under physiological conditions. Therefore, the effects of the redox molecules on the nucleus and the NPC function were examined by studying signal-mediated nuclear import of rhodamine-labeled bovine serum albumin (BSA) with and without nuclear localization signals by fluorescence microscopy. MODEL Figure 1 illustrates the SECM approach curve and chronoamperometry experiments at the nucleus. Initially, a redox-active (21) Barker, A. L.; Macpherson, J. V.; Slevin, C. J.; Unwin, P. R. J. Phys. Chem. B 1998, 102, 1586-1598. (22) Barker, A. L.; Unwin, P. R. J. Phys. Chem. B 2001, 105, 12019-12031. (23) Macpherson, J. V.; Jones, C. E.; Barker, A. L.; Unwin, P. R. Anal. Chem. 2002, 74, 1841-1848. (24) Lee, S.; Zhang, Y.; White, H. S.; Harrell, C. C.; Martin, C. R. Anal. Chem. 2004, 76, 6108-6115. (25) Danker, T.; Mazzanti, M.; Tonini, R.; Rakowska, A.; Oberleithner, H. Cell Biol. Int. 1997, 21, 747-757. (26) Stoffler, D.; Feja, B.; Fahrenkrog, B.; Walz, J.; Typke, D.; Aebi, U. J. Mol. Biol. 2003, 328, 119-130. (27) Danker, T.; Shahin, V.; Schlune, A.; Schafer, C.; Oberleithner, H. J. Membr. Biol. 2001, 184, 91-99.
[
molecule, X, is partitioned across the NE between the outer buffer solution and the nucleus k1
X (outer solution) y\ z X (nucleus) k
(1)
2
where k1 and k2 are the first-order heterogeneous rate constants of the forward and backward reactions, respectively. The equilibrium concentrations of the redox molecule in the outer solution and in the nucleus, c01 and c02, respectively, are related to the partitioning equilibrium constant, Ke, as
Ke ) c02/c01
(2)
]
∂c1(r,z,t) ∂z
D1
z)d
) k2c2(r,d,t) - k1c1(r,d,t)
and for the nucleus side of the membrane as
[
]
∂c2(r,z,t) ∂z
D2
z)d
) k2c2(r,d,t) - k1c1(r,d,t)
The equilibrium constant can be also expressed by the rate constants as
The other boundary conditions are
Ke ) k1/k2
c1(r,0,t) ) 0
(3)
[
]
∂c1(r,z,t) ∂2c1(r,z,t) 1 ∂c1(r,z,t) ∂2c1(r,z,t) ) D1 + + ∂t r ∂r ∂r2 ∂z2
[
]
[
]
∂c1(r,z,t) ∂z
z)0
01.5 × 10-1 >7.1 × 10-1 >6.9 × 10-1 >7.3 × 10-1
BLMb 5.0 ×
10-3
cell membranec 5.0 × 10-3
0.6 × 10-3d 4.0 × 10-3e
0.7 cm/s. Only experiments with a 10-µm-diameter probe were successful for the anionic ferrocenes so that their transfer rate constants are >0.15 cm/s. Importantly, these rate constants are at least 2 orders of magnitudes larger than those at BLMs17 and cell membranes20 (also in Table 2). This result indicates that even ferrocene 2154 Analytical Chemistry, Vol. 77, No. 7, April 1, 2005
Figure 9. Schematic diagrams to illustrate (A) an electrode covered with the NE and the diffusion layer when the NPC pore is (B) large and (C) small in comparison with the channel-channel distance. The arrows indicate diffusion of redox molecules to the NE. The dotted lines illustrate their isoconcentration profiles at the NPCs.
molecules with relatively high hydrophobicity are transferred much faster across the NE than across hydrophobic BLMs and cell membranes. Therefore, we conclude that the redox molecules that can be detected with 2- and 10-µm-diameter SECM probes are transported through the NPC rather than across the double membrane of the NE. Estimations of Single-Channel Flux and Pore Diameter. While the approach curves were analyzed by assuming a laterally homogeneous membrane, the transport pathway is localized at the NPCs. Further information about the transport process can be obtained by considering the NE as an array of nanometersized NPC pores. The flux of redox molecules through a single NPC channel was estimated as the resulting redox current at the tip. With the 2-µm electrode, the theoretical approach curve that fits with the experimental curve for FcCH2OH in Figure 8 gives a current value of 249 pA at the zero tip-membrane distance. Assuming the channel density of 50 channels/µm,2,15 157 channels are embedded in the small patch of the NE (3.14 µm2) that covers the 2-µm-diameter disk electrode at the zero distance (Figure 9A). The average current at a single NPC is obtained as 249 pA/157 NPCs ) 1.56 pA/NPC at 1 mM FcCH2OH, which corresponds to the flux of 9.8 × 106 molecules per NPC per second (Note that the current and flux are proportional to the FcCH2OH concentration.). Moreover, the flux sustainable by a single NPC should be larger than this value because (1) the flux is limited by diffusion of the redox molecules from the bulk solution to the NE rather than by their translocation through the NPC, and (2) the current density at disk electrodes is nonuniform, i.e., the disk edge is geometrically more accessible to the diffusing molecules,39 so that the NPCs near the edge needs to sustain the flux that is larger than the average value. The large single-channel flux is due to a large pore size, which can also be estimated from SECM approach curves. At the zero electrode-membrane distance (Figure 9A), a steady-state diffusion layer of the redox molecules builds up from the electrode surface into the nucleus through the freely permeable NE. In this case, (39) Bard, A. J.; Faulkner, L. R. Electrochemical Methods: Fundamentals and Applications, 2nd ed.; John Wiley & Sons: New York, 2001.
Figure 10. Fluorescence microscopic images of the intact nuclei incubated in the transport media; the isotonic MIB solution based on 1.5% PVP containing (A) 0.5 µM BSA labeled with tetramethylrhodamine isocyanate, (B) 0.5 µM sulforhodamine-labeled BSA with nuclear localization signal peptides, 0.5 µM importin R2, 0.5 µM importin β1, and energy mix, and (C) 0.5 µM signal-attached fluorescent BSA. The top and bottom images are those of the nuclei incubated in the transport media without and with 1 mM FcCH2OH, respectively. Fluorescence microscopic images of the intact nuclei that were pretreated in the MIB solution without PVP and incubated in the transport media containing 0.5 µM signalattached fluorescent BSA and (D) no other factor, or (E) 1 µM importin R2, 1 µM importin β1, and energy mix.
the pore diameter must be large enough in comparison with the channel-channel distance so that the diffusion layer at each NPC pore overlaps, resulting in an overall diffusion layer with a thickness that is several times that of the electrode radius (Figure 9A and B). Therefore, the total flux of the redox molecules across the membrane in contact with the electrode is limited by the overall diffusion layer. However, if the pore size is very small, the diffusion layer at each NPC is localized without overlap (Figure 9C). Here, the total flux through the membrane is limited by the total number of the NPCs on the NE patch, i.e., by membrane transport, which is inconsistent with the experimental result. For estimation of the pore diameter, the diffusion problem of the NPC array was treated as that of a microelectrode array (or a partially blocked electrode).40-42 For example, it was shown that there is significant overlap between the steady-state diffusion layers at a pair of hemispherical microelectrodes when the center-center distance is less than 10 times of the electrode diameter.41 By applying this simple criterion for the random array of the NPCs with the channel-channel distance of ∼150 nm,43 the diffusion layer at each NPC overlaps when the channel diameter is larger than 15 nm. This value is in the range of the previously estimated values (10-40 nm)7,43 while the central pore of the NPC is not disk-shaped; the pore has a length of ∼90 nm, is narrowest (40-50 nm in diameter) at the middle, and widens to ∼70 nm toward the cytoplasmic and nucleoplasmic periphery.7 Nuclear Import of Rhodamine-Labeled BSA with and without Nuclear Localization Signals. While SECM is a unique, noninvasive method to provide quantitative information about partitioning processes of the redox molecules at the nucleus, use of such nonphysiological substances raises a concern about their (40) Amatore, C.; Save´ant, J. M.; Tessier, D. J. Electroanal. Chem. 1983, 147, 39-51. (41) Alfred, L. C. R.; Oldham, K. B. J. Electroanal. Chem. 1995, 396, 257-263. (42) Beriet, C.; Ferrigno, R.; Girault, H. H. J. Electroanal. Chem. 2000, 486, 56-64. (43) Keminer, O.; Peters, R. Biophys. J. 1999, 77, 217-228.
effects on the nucleus sample. Indeed, the nucleus became opaque in buffer solutions containing Ru(NH3)63+. This result suggests some interactions between Ru(NH3)63+ and the nucleoplasm while any effect of the other redox molecules on the nucleus was not apparent under an optical microscope. Therefore, effects of the redox molecules on the nucleus and the NPC function were further examined by studying nuclear import of rhodamine-labeled BSA with and without nuclear localization signals by fluorescent microscopy.33 BSA (67 kDa) is much larger than the molecules that are passively permeable through the NPC. Incubation of the intact nucleus in transport media containing rhodamine-labeled BSA resulted in negligible fluorescence in the nucleus (Figure 10A top). This result confirms that the isolated nucleus is not leaky for this size of macromolecule. Similar results were obtained when the transport media also contained any redox molecule listed in Tables 1 and 2 (Figure 10A bottom with FcCH2OH). On the other hand, rhodamine-labeled BSA with nuclear localization signals was imported into the nucleus by a signal-mediated transport pathway, where importin R2 recognizes the signal peptides on the BSA molecule and importin β1 facilitates the translocation of the importin-BSA complex through the NPC. After incubation in the isotonic transport media containing signal-attached fluorescent BSA, importin R2, importin β1, and energy mix, the nucleus became highly fluorescent (Figure 10B top). Similar results were obtained by incubating the nucleus in the transport media also containing any of the redox molecules (Figure 10B bottom with FcCH2OH). While the NPCs on the isolated nucleus are known to serve as a selective gate for signal-mediated macromolecular transport,33 these results confirm that the NPCs keep the primary function even in the presence of the redox molecules. The nucleus also became fluorescent, while it was significantly less intense, after incubation in isotonic transport media containing signal-attached fluorescent BSA but no importin or energy mix (Figure 10C). This result indicates that the nucleus provides Analytical Chemistry, Vol. 77, No. 7, April 1, 2005
2155
importin R and β so that the signal-mediated transport can be facilitated without externally providing these proteins. It was reported that soluble proteins are gradually lost only to 50% of the original level from the Xenopus oocyte nucleus in the isotonic MIB buffer solution with PVP while the proteins are quickly lost almost to the 10% level in the buffer solution without PVP.38 To confirm that the nucleus serves as a reservoir of importin R and β, the nucleus was pretreated in a buffer solution without PVP for 1 h and then incubated in the isotonic transport media containing 1.5% PVP and signal-attached fluorescent BSA. After such a pretreatment, only a small amount of signal-attached fluorescent BSA was imported into the nucleus (Figure 10D). With externally added importins, however, the pretreated nucleus became much more fluorescent. With 2 times higher concentrations of importins, the nucleus became as fluorescent as that of the nucleus without pretreatment (Figure 10E). It should be noted that the fluorescence intensity of the nucleus incubated with signal-attached fluorescent BSA was similar to or less than that of the incubating solution. This result indicates that, in contrast to in vivo, the signal-attached fluorescent BSA was equilibrated between inside and outside of the nucleus and was not concentrated into the nucleus. Also, signal-attached BSA can be exported from the nucleus into the isotonic MIB solution, causing a slight fluorescence around the nucleus as shown in Figure 10B, C, and E. This result indicates that BSA was not dissociated from the importin complex by competitive binding of RanGTP to importin β in the nucleus so that the stable BSAimportin complex with a long half-life (∼1 h) lasted in the nucleus to be exported.44 These phenomena, however, were observed both with and without the redox molecules. CONCLUSIONS SECM was used to study permeability of the NE at the intact nucleus isolated from X. laevis oocytes. The approach curve and chronoamperometric data demonstrated that the NE is so permeable to the redox molecules that the overall membrane transport (44) Gilchrist, D.; Mykytka, B.; Rexach, M. J. Biol. Chem. 2002, 277, 1816118172. (45) Duffy, N. W.; Harper, J.; Ramani, P.; Ranatunge-Bandarage, R.; Robinson, B. H.; Simpson, J. J. Organomet. Chem. 1998, 564, 125-131.
2156
Analytical Chemistry, Vol. 77, No. 7, April 1, 2005
is limited by diffusion of the redox molecules from the bulk nucleus to the NE under the SECM experimental conditions. This conclusion is qualitatively consistent with that obtained using the glass pipet and hourglass techniques, supporting the conclusion that most NPCs on the nucleus are open. In contrast to these electrophysiological techniques, however, the membrane transport across the NE and the mass transfer in the nucleus can be studied quantitatively and separately by SECM using the same intact nucleus. Therefore, such a control experiment with the NEremoved nucleus is not required to correct the contribution of the nucleoplasm to the measured permeability of the whole intact nucleus.15 SECM offers a spatial resolution as high as the patch clamp technique, allowing permeability measurement at a small area of the NE (∼3 µm2). The results as obtained by these techniques, however, are very different. While the closed state of the NPCs as observed under patch clamp experiments is not likely to be their physiological state, SECM demonstrated that the small area of the NE can still sustain the large flux of the redox molecules, indicating highly efficient passive transport through the NPCs. While no significant effect of the redox molecules on the signalmediated macromolecular import through the NPC was observed in our fluorescent study, all possible effects on the physiological properties of the NPC cannot be excluded. Nevertheless, the SECM data are still consistent with the structural information about the NPC, i.e, the large pore diameter and the high density. ACKNOWLEDGMENT We thank the Research Corporation and National Science Foundation (DBI-0242561) for financial support. This work was also supported by the University of Pittsburgh. We also thank Prof. R. A. Frizzell, Department of Cell Biology and Physiology, University of Pittsburgh for gifts of Xenopus oocytes. We appreciate the assistance of Tracy L. Wazenegger, Gregg P. Kotchey, and Shawn P. O′Leary in preliminary experiments. Received for review January 12, 2005. AC048370J
November
3,
2004.
Accepted
