| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
* Nano-Optics Group, National Competence Center for Research in Nanoscale Science, Institute of Physics; Biographics Laboratory 3R and Department of Molecular Pharmacy; and M. E. Müller Institute, Biozentrum, University of Basel, CH-4056 Basel, Switzerland
Correspondence: Address reprint requests to Bert Hecht, Nano-Optics Group, National Competence Center for Research in Nanoscale Science, Institute of Physics, University of Basel, Klingelbergstrasse 82, CH-4056 Basel, Switzerland. Fax: 41-61-267-3795; E-Mail: bert.hecht{at}unibas.ch.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONEXPERIMENTALRESULTS AND DISCUSSIONCONCLUSIONACKNOWLEDGEMENTSREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTALRESULTS AND DISCUSSIONCONCLUSIONACKNOWLEDGEMENTSREFERENCES |
|---|
). Therefore, the study of virus-nucleus interactions is of importance for the basic understanding of virology and cell biology as well as for important technological applications, like gene therapy vector systems.
Hepatitis B virus (HBV) is a major human infectious pathogen that causes acute and chronic hepatitis and eventually hepatocellular carcinoma. Its capsid exhibits icosahedral symmetry and is built of 180 or 240 copies of a single, virally encoded core protein (2). The mechanism by which HBV cores are targeted and transported into the nucleus without disassembly has been of great interest, since its capsid diameter (
35 nm) is close to the physical diameter (45 nm) of the NPC central pore (3–5). Previous bulk transport studies suggest that binding to and transport through the NPC is mediated by transport receptors called importins. The association with importin a has been shown to take place at the COOH-terminus of the HBV core protein, which harbors nuclear localization signal (NLS)-like amino acid sequences (6,7). However, cryoelectron microscopy has shown that the COOH-terminus is not surface exposed in capsids (2,7).
Single-particle-tracking experiments have recently gained substantial attention for their ability to trace specific interactions in living cells. Experiments with single particles have distinct advantages compared to ensemble experiments because they can: i), unravel dynamic processes at the molecular level taking place on various timescales without synchronizing the experiment; and ii), reveal the presence of subensembles in complex, heterogeneous systems (8–10). This is because, by definition, single-particle experiments avoid ensemble averaging. For example, properties of lipid membranes supported on solid substrates (11,12) or in living cells (13–17) have been investigated by single-molecule diffusion studies in the past. Also, viral infection pathways have been studied using single-molecule-labeled viruses to follow the entry of viruses into cells and their transport to the nucleus in real time (18). Furthermore, a study on nuclear trafficking of viral genes by single-particle tracking (19) showed heterogeneous interactions between the viral genes and NPCs with a large range of dissociation rate constants. Recently, single-molecule studies of the nuclear transport of a model protein substrate (20) and of nuclear transport receptors (21) have been performed.
Here, we apply single-molecule fluorescence techniques to investigate the interaction of single HBV core capsids with individual NPCs to study the initial steps of capsid import in detail. As a model system we have selected a recombinant HBV core capsid assembled from a 149-amino-acid version of its core capsid protein (22). The core capsid protein was COOH-terminally truncated to lack the NLS-like sequence and the phosphorylation sites. To render the capsids fluorescent, green fluorescent protein (GFP) was inserted into the immunodominant loop (amino acids 78–83) of the capsid protein. FCS and wide-field fluorescence microscopy are applied to study the interaction of single capsids with individual NPCs. In contrast to previous studies (20,21), we were able to suppress to a large extent the frequent transient nonspecific encounters of the capsids with the NE by single-capsid FCS and two-color colocalization of the NPCs and capsids. Thus, long-lasting specific binding events of single capsids to individual NPCs could be isolated. Our investigations reveal the capsids' capability to interact specifically with NPCs even in the absence of the COOH-terminal domain that is generally thought to harbor an NLS. This finding, in turn, suggests the occurrence of a direct HBV core capsid-NPC association that is not mediated by importins.
|
EXPERIMENTAL |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTALRESULTS AND DISCUSSIONCONCLUSIONACKNOWLEDGEMENTSREFERENCES |
|---|
). In brief, the sequence encoding the GFP was inserted in a parental PET15-derived plasmid containing the truncated HBV core protein (amino acids 1–149) in the immunodominant loop (amino acids 238–243). The HBV-GFP construct was overexpressed by transforming into Escherichia coli BL21 cells and purified using a Ni-loaded chelating sepharose column (GE Healthcare, München, Germany). This recombinant construct was chosen because it efficiently self-assembles into icosahedral capsids in the test tube (22). The resulting capsids are of two size classes, T = 3 (180 subunits) and T = 4 (240 subunits) (2), thus yielding fluorescent capsids tagged with either 180 or 240 copies of GFP. Capsid formation and size of the assembled particles were controlled by negative stain electron microscopy.
Cell culturing and immunocytochemistry
HeLa cells were cultured on glass coverslips in Dulbecco's Modified Eagle Medium supplemented with 10% fetal calf serum overnight. On the day of the experiment, the cells were permeabilized with digitonin (40 µg/ml). NPCs were labeled in transport buffer (20 mM Hepes, pH 7.3, 110 mM potassium acetate, 5 mM sodium acetate, 2 mM magnesium acetate, 1 mM EGTA, 2 mM dithiothreitol, 2% bovine serum albumin and proteases) with a monoclonal antibody, mAb414 (BABCO, Berkeley, CA), that recognizes the FG-repeat domain of the nucleoporins (23). The primary antibody was fluorescently labeled with a secondary Texas-Red-conjugated monoclonal antibody.
Single-molecule experiments
The HeLa cell cultures labeled with mAb414 were employed immediately for confocal or wide-field microscopy. The cells were kept at room temperature throughout the measurement. Directly before the experiment, the transport buffer was exchanged by a transport mixture containing the transport buffer, a HeLa cell cytosolic extract, ATP, creatine phosphokinase, and creatine phosphatase. HBV core capsids tagged with GFP were added at a concentration of 10–9 M, low enough for single-particle detection.
Microscopy methods
Confocal and wide-field microscopy setups
An epifluorescence microscope was arranged to perform both confocal and wide-field microscopy (Fig. 1). Two laser beams, one at 532 nm (ps-pulsed (pulse width 8 ps, repetition rate 80 MHz), frequency-doubled Nd:Van laser GE-100, Time-Bandwidth Products, Zurich, Switzerland), and one at 488 nm (Ar+-laser, SpectraPhysics, Mountain View, CA), were coupled into the back aperture of an oil immersion microscope objective (Nikon, Tokyo, Japan; x100, 1.45 numerical aperture) using a suitable color beam splitter (505dclp, Chroma Technology, Brattleboro, VT). For wide-field microscopy, the illumination spot was widened to a diameter of 10 µm using a lens. The resulting fluorescent emission was collected by the same objective lens and passed through holographic notch filters before reaching a flipping mirror that enabled the switching of the detection pathway between a charge-coupled device camera (EMCCD, IXon, Andor, Ireland) and single-photon counting modules (SPCM-AQR-13, Perkin Elmer, Fremont, CA). After flipping the mirror the fluorescence is divided into a red and green part by a dichroic beamsplitter (545dclp, Chroma Technology) and filtered by respective band-pass filters (HQ510/20 and D605/50, Chroma Technology).
|
10 µm in diameter was illuminated, resulting in an excitation intensity of 0.9–1.3 kW/cm2. Series of single-molecule fluorescence images recorded by wide-field microscopy were analyzed using the software Igor Pro (WaveMetrics, Lake Oswego, OR). Within each image showing fluorescent NPCs, close encounters of NPCs with HBV core capsids, being possible candidates for binding events, were preselected if their peak-to-peak distance was <2 pixels (220 nm). The HBV core capsid image series with nearby NPCs were then analyzed to track down binding events. To determine the position, width, amplitude, and local background of spots representing either single NPCs or single HBV core capsids bound to the NE, an asymmetric two-dimensional Gaussian was fitted to a 7 x 7 pixel subregion of the images that contained the selected spot using the Levenberg-Marquardt algorithm. From the spatial positions of a capsid appearing in subsequent images, trajectories were obtained by standard procedures (11,24). In addition to the numerical values of the fit parameters, the nonlinear fit routine also provides estimates for the uncertainty of the parameters. Fits that resulted in positional errors relative to the spot size of >35% were discarded from further analysis. Spots and trajectories of HBV core capsids residing within a radius of 110 nm around a given NPC in the ventral NE were scored for colocalization. This distance criterion is based on the physical size of an NPC (5,25,26) including the length of the cytoplasmic filaments.
|
= 532 nm) showing the spatial positions of individual red fluorescent NPCs (see Fig. 2 a). Then a single, well-isolated NPC was positioned within the confocal volume and the excitation was switched to blue (1–2 µW, = 488 nm). Time traces revealing fluorescence intensity fluctuations in the green (GFP) channel due to diffusing GFP-tagged HBV core capsids were recorded for 200 s from that confocal volume. Autocorrelation functions of such fluorescence time traces were calculated offline. Rare specific binding events which presumably have longer durations than diffusive events are expected to be difficult to detect in such an analysis. This is due to the fact that a major fraction of the fluorescence fluctuation detected in the confocal volume stems from capsids that do enter the confocal volume but do not specifically interact with the NPC (see Fig. 4). To capture the occasional specific binding of an HBV core capsid to an NPC, the time traces were also analyzed by determining the length of the observed fluorescence bursts. To identify a burst as an event originating from the capsids, the average fluorescence intensity of the corresponding trace plus three times its standard deviation was taken as the threshold for the start of that burst. A burst was assumed to last until the signal fell under the average fluorescence intensity plus twice its standard deviation. By plotting histograms of burst durations, we are able to distinguish between long, specific and short, nonspecific events.
|
7 x 7 µm were acquired before and after recording a time series of a single NPC. Individual NPCs exhibited hardly any movement over the timescale of our experiment, consistent with a previous finding (27) and as expected for permeabilized cells. To monitor nonspecific binding of the GFP-tagged HBV core capsids to the nuclear membrane, the confocal volume was positioned at spots along the ventral area of the NE where no labeled NPCs were present. The cytoplasmic diffusion of HBV core capsids was monitored by focusing into the cytoplasm away from the nucleus.
Simulation of FCS measurements
To aid the interpretation of the FCS data, we performed Monte Carlo simulations employing NPCSim, developed for this investigation. Initially, HBV core capsid particles are distributed randomly in a 3 x 3 x 3 µm3 box. The box has a structure that constitutes a random distribution of obstacles to represent cytoplasm-containing components such as organelles, protein, and RNA with which the capsids may interact (see Fig. 6, insets). The energy of a capsid configuration is determined by its interaction with obstacles. The latter are represented by square-well potentials of depth V and width r ("valley" model) (28). The parameters V and r are assigned to each obstacle separately using a random number generator. Periodic boundary conditions are assigned to the x-z- and y-z-plane; reflective boundary conditions were implemented for the x-y-plane representing the NE where an NPC could be inserted. An NPC is treated as a single, separate obstacle modeled as a square-well potential with variable diameter and depth. During each time interval, 
t, the particles move in space by a distance, x, which is computed from a normal distribution representing random diffusion:
 | (1) |
x if it fulfills the Boltzmann criterion, i.e., 
, that forces the system in the canonical ensemble at a constant temperature. Here, E is the energy difference between the previous and actual capsid position and 
is the probability that the trial move is accepted. If a random number is smaller than , the move is performed, but if it is larger than , the move is rejected and the capsid remains in its actual position.
|
 | (2) |
We used a diffusion constant D that was obtained from FCS studies in the cytoplasm. Unknown parameters such as the density of capsids and obstacles, the maximum capsids-obstacles interaction, and the maximum depth of the square-well potential of the obstacles representing the cytoplasm were also adjusted in such a way as to obtain an event-length distribution similar to that obtained from the control experiments.
|
RESULTS AND DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTALRESULTS AND DISCUSSIONCONCLUSIONACKNOWLEDGEMENTSREFERENCES |
|---|
,4). Control experiments using unlabeled NPCs yielded results indistinguishable from Fig. 3 b.
|
250 nm and 750 nm, respectively, only a few fluorescence bursts are expected to be events that reflect capsids-NPC interaction. To distinguish those bursts from those of the freely diffusing capsids, as references, we have also recorded fluorescence time traces i) in the cytoplasm away from the nucleus, and ii) at the NE with no NPC present (see insets of Fig. 5, a and c). To discriminate the few specific binding events among a vast number of nonspecific diffusion events, the durations of individual bursts were determined. Histograms of burst durations are shown in Fig. 5, a–c, corresponding to different sites in digitonin-permeabilized HeLa cells. Fluorescence bursts from HBV core capsids can be due to i) ‘free’ diffusion through the confocal volume, ii) collision or interaction with the NE, or iii) interaction with an NPC. On average, the fluorescence bursts are longer for freely diffusing capsids in the cytoplasm (Fig. 5 c) than for capsids diffusing close to the NE (Fig. 5 a). This is due to a significant reduction in the effective cytoplasmic volume available to the capsids in the presence of the NE (see Fig. 4). However, if the NE residing in the confocal volume contains an NPC, very long-lasting fluorescence bursts start to noticeably contribute above the background of ‘free’ diffusion events as shown in Fig. 5 b. Such longer lasting fluorescence bursts (Fig. 5 b, inset) are clearly distinguishable in the time traces from the events detected in the absence of an NPC either with (Fig. 5 a, inset) or without (Fig. 5 c, inset) the NE. Fitting an exponential curve to the histogram of burst width recorded in the cytoplasm without the NE (Fig. 5 c) yields a typical burst duration of 
. If the NE is in the confocal volume but there is no NPC present (Fig. 5 a), the fit yields a faster decay with a time constant 
, as expected. If an NPC is present in the confocal volume, the character of the histogram of burst width changes. Now a second decay rate, 
obtained by employing a sum of two exponential curves, is necessary to obtain a satisfactory fit, as shown in Fig. 5 b. The second exponential decay characterizes the longer specific capsid-NPC binding events.
|
where 
is an anomalous diffusion coefficient and 
is an anomalous diffusion exponent characterizing the dependence of the mean-square displacement of the particle on time. For fluorescence time traces recorded in the cytoplasm away from the nucleus, 
and 
were determined (Fig. 5 c, inset), whereas for fluorescence time traces recorded at the NE without NPC, 
and 
were obtained (Fig. 5 a, inset). The evidently faster diffusion indicated by 
found for capsids residing in a confocal volume placed near the NE is again consistent with the reduced effective cytoplasmic volume mentioned above. The degree of anomalous diffusion represented by is similar for both cases. The autocorrelation function obtained from the experiments with an NPC yields a significant increase in the degree of anomalous diffusion, 
. This suggests that the presence of an NPC in the confocal volume creates additional interaction with the capsids. The anomalous diffusion coefficient obtained from the fit is reduced to 
compared to the case without NPC at the NE. Although the actual values from the fits do not describe the effect of the presence of an NPC quantitatively since the autocorrelation functions average all the bursts detected as fluorescence intensity fluctuations, the effect on (namely on the dependence of the mean-square displacement of the capsids on time) indeed indicates a specific interaction between NPCs and capsids. A simulation of the experimental system was performed to investigate the effect of the presence of the NE and an NPC in the confocal volume. A simplified system to mimic the confocal volume in the experiment as described in Materials and Methods was used to produce fluorescence time traces. Histograms of burst lengths obtained for the three situations: a), at the NE but without an NPC, b), at the NE with an NPC, and c), in the cytoplasm, are shown in Fig. 6. The observed behavior is consistent with the experiments.
Capsid-NPC interaction visualized by wide-field microscopy
We have performed wide-field microscopy to follow the movement of individual capsids in the vicinity of NPCs and to directly visualize their interaction with an NPC by dual-color colocalization. From series of HBV core capsid fluorescent images recorded at a rate of 50 frames/s, the duration of binding events was obtained for events colocalizing with an NPC and at positions without NPCs at the ventral plane of the NE. The histograms of such binding event lengths at the NE are shown in Fig. 7, a and b. Capsid binding events were determined as follows: a continuous series of capsid images were pooled out, during which the peak-to-peak distance of a capsid and an NPC remained within a range corresponding to the physical geometry of an NPC including the length of its cytoplasmic filaments. The binding events, on the other hand, were considered as nonspecific if they occurred without any labeled NPC in the vicinity. Fitting of exponentials to the histograms of binding event durations indicates a longer average duration of binding events at the position of NPCs (Fig. 7 b, 
) compared to the case where no NPC is present (Fig. 7 a, 
).
|
. To this end the positions of capsids within each trace have been used to calculate an average distance and angle as well as the respective standard deviations. Since the NPC is rotationally symmetric we disregard the absolute angular coordinate and project all angles to = 0. The resulting probability density distribution shows two major contributions: 1), a confined peak both in radial (±9 nm) and angular (±2°) directions 44 nm from the central axis, and 2), another peak that is much weaker and shows a large spread both in radial (±16 nm) and angular (±22°) directions 93 nm from the central axis. Comparison of this probability density distribution to the dimension of an NPC (Fig. 8 b) shows that the first type of binding events coincides with the ring moiety of the NPCs and the second is consistent with interactions of capsids with the cytoplasmic filaments. The correlation between the length and the position of binding events and the nature of capsid movements at the NPCs must be further investigated, for instance, for any relevance to distinction between interactions that lead to an import from interactions that fail to lead to an import.
|
|
CONCLUSION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTALRESULTS AND DISCUSSIONCONCLUSIONACKNOWLEDGEMENTSREFERENCES |
|---|
|
ACKNOWLEDGEMENTS |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTALRESULTS AND DISCUSSIONCONCLUSIONACKNOWLEDGEMENTSREFERENCES |
|---|
We also gratefully acknowledge financial support from the Swiss National Science Foundation via the National Center of Competence in Research in Nanoscale Science, a research grant (B.F.), and a research professorship (B.H.) as well as from the M. E. Müller Foundation (U.A.), and the Kanton Basel Stadt (B.F.).
Submitted on April 24, 2006; accepted for publication July 12, 2006.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTALRESULTS AND DISCUSSIONCONCLUSIONACKNOWLEDGEMENTSREFERENCES |
|---|
2. Crowther, R. A., N. A. Kiselev, B. Bottcher, J. A. Berriman, G. P. Borisova, V. Ose, and P. Pumpens. 1994. Three-dimensional structure of hepatitis B virus core particles determined by electron cryomicroscopy. Cell. 77:943–950.[CrossRef][Medline]
3. Panté, N., and M. Kann. 2002. Nuclear pore complex is able to transport macromolecules with diameters of 39 nm. Mol. Biol. Cell. 13:425–434.
4. Rabe, B., A. Vlachou, N. Panté, A. Helenius, and M. Kann. 2003. Nuclear import of hepatitis B virus capsids and release of the viral genome. Proc. Natl. Acad. Sci. USA. 100:9849–9854.
5. Stoffler, D., B. Feja, B. Fahrenkrog, J. Walz, D. Typke, and U. Aebi. 2003. Cryo-electron tomography provides novel insights into nuclear pore architecture: implications for nucleocytoplasmic transport. J. Mol. Biol. 328:119–130.[CrossRef][Medline]
6. Kann, M., B. Sodeik, A. Vlachou, W. H. Gerlich, and A. Helenius. 1999. Phosphorylation-dependent binding of hepatitis B virus core particles to the nuclear pore complex. J. Cell Biol. 145:45–55.
7. Steven, A. C., J. F. Conway, N. Cheng, N. R. Watts, D. M. Belnap, A. Harris, S. J. Stahl, and P. T. Wingfield. 2005. Structure, assembly, and antigenicity of hepatitis B virus capsid proteins. Adv. Virus Res. 64:125–164.[Medline]
8. Basché, T., W. E. Moerner, M. Orrit, and U. P. Wild, editors. 1999. Single-Molecule Optical Detection, Imaging and Spectroscopy. VCH, Weinheim, Germany.
9. Rigler, R., M. Orrit, and T. Basché, editors. 2001. Single Molecule Spectroscopy. Springer Series in Chemical Physics. Springer, Berlin.
10. Special issue. 1999. Frontiers in chemistry: single molecules. Science. 283:1667.[CrossRef]
11. Schmidt, Th., G. J. Schütz, W. Baumgartner, H. J. Gruber, and H. Schindler. 1996. Imaging of single molecule diffusion. Proc. Natl. Acad. Sci. USA. 93:2926–2929.
12. Schütz, G. J., G. Kada, V. P. Pastushenko, and H. Schindler. 2000. Properties of lipid microdomains in a muscle cell membrane visualized by single molecule microscopy. EMBO J. 19:892–901.[CrossRef][Medline]
13. Lommerse, P. H. M., G. A. Blab, L. Cognet, G. S. Harms, B. E. Snaar-Jagalska, H. P. Spaink, and Th. Schmidt. 2004. Single-molecule imaging of the H-Ras membrane-anchor reveals domains in the cytoplasmic leaflet of the cell membrane. Biophys. J. 86:609–616.
14. Sako, Y., S. Minoguchi, and T. Yanagida. 2000. Single-molecule imaging of EGFR signalling on the surface of living cells. Nat. Cell Biol. 2:168–172.[CrossRef][Medline]
15. Sako, Y., and T. Yanagida. 2003. Single-molecule visualization in cell biology. Nat. Rev. Mol. Cell Biol. 4:SS1–SS5.
16. Schütz, G. J., J. Hesse, G. Freudenthaler, V. Ph. Pastushenko, H.-G. Knaus, B. Pragl, and H. Schindler. 2000. 3D mapping of individual ion channels on living cells. Single Mol. 1:153–157.[CrossRef]
17. Vrljic, M., S. Y. Nishimura, S. Brasselet, W. E. Moerner, and H. M. McConnell. 2002. Translational diffusion of individual class II MHC membrane proteins. Biophys. J. 83:2681–2692.
18. Seisenberger, G., M. U. Ried, Th. Endreß, H. Büning, M. Hallek, and C. Braüchle. 2001. Real-time single-molecule imaging of the infection pathway of an adeno-associated virus. Science. 294:1929–1932.
19. Babcock, H. P., C. Chen, and X. Zhuang. 2004. Using single-particle tracking to study nuclear trafficking of viral genes. Biophys. J. 87:2749–2758.
20. Yang, W., J. Gelles, and S. M. Musser. 2004. Imaging of single-molecule translocation through nuclear pore complexes. Proc. Natl. Acad. Sci. USA. 101:12887–12892.
21. Kubitscheck, U., D. Grünwald, A. Hoekstra, D. Rohleder, T. Kues, J. P. Siebrasse, and R. Peters. 2005. Nuclear transport of single molecules: dwell times at the nuclear pore complex. J. Cell Biol. 168:233–243.
22. Kratz, P. A., B. Böttcher, and M. Nassal. 1999. Native display of complete foreign protein domains on the surface of hepatitis B virus capsids. Proc. Natl. Acad. Sci. USA. 96:1915–1920.
23. Davis, N. L., G. Wertz, M. Schubert, and R. A. Lazzarini. 1985. Role of the nucleocapsid protein in regulating vesicular stomatitis virus RNA synthesis. Heinz Arnheitera. Cell. 41:259–267.[CrossRef][Medline]
24. Kusumi, A., Y. Sako, and M. Yamamoto. 1993. Confined lateral diffusion of membrane receptors as studied by single particle tracking (nanovid microscopy). Effects of calcium-induced differentiation in cultured epithelial cells. Biophys. J. 65:2021–2040.
25. Beck, M., F. Förster, M. Ecke, J. M. Plitzko, F. Melchior, G. Gerisch, W. Baumeister, and O. Medalia. 2004. Nuclear pore complex structure and dynamics revealed by cryoelectron tomography. Science. 306:1387–1390.
26. Fahrenkrog, B., E. C. Hurt, U. Aebi, and N. Panté. 1998. Molecular architecture of the yeast nuclear pore complex: localization of Nsp1p subcomplexes. J. Cell Biol. 143:577–588.
27. Daigle, N., J. Beaudouin, L. Hartnell, G. Imreh, E. Hallberg, J. Lippincott-Schwartz, and J. Ellenberg. 2001. Nuclear pore complexes form immobile networks and have a very low turnover in live mammalian cells. J. Cell Biol. 154:71–84.
28. Saxton, M. J. 1996. Anomalous diffusion due to binding: a Monte Carlo study. Biophys. J. 70:1250–1262.
29. Fahrenkrog, B., J. Köser, and U. Aebi. 2004. The nuclear pore complex: a jack of all trades? Trends Biochem. Sci. 29:175–182.[CrossRef][Medline]
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
, (b) 
, and (c) 
. Sketches of the simulated structure for each situation are shown as insets.
