Article |
Address correspondence to Xueliang Zhu, Institute of Biochemistry and Cell Biology, 320 Yue Yang Rd., Shanghai 200031, China. Tel.: 86-21-54921406. Fax: 86-21-54921011. email: xlzhu{at}sibs.ac.cn
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Abstract |
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Key Words: NudE; organelle; motility; time-lapse microscopy; RNA interference
The online version of this article includes supplemental material.
Abbreviations used in this paper: DHC, dynein heavy chain; DIC, dynein intermediate chain; ERGIC, ER-to-Golgi intermediate compartment; hPL, human placental lactogen; MT, microtubule; RNAi, RNA interference; SiRNA, small interference RNA; Tet, tetracycline.
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Introduction |
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Cytoplasmic dynein, a microtubule (MT)-based and minus enddirected motor, is a large complex composed of two dynein heavy chains (DHCs) of 550 kD, three to four dynein intermediate chains (DICs) of 74 kD, four light intermediate chains of
55 kD, and light chains of 822 kD (Hirokawa, 1998; Karki and Holzbaur, 1999). It exerts multiple functions, from the movement of chromosomes, formation and maintenance of the mitotic spindle during mitosis (Karki and Holzbaur, 1999; Brunet and Vernos, 2001) to centripetal transit and juxta-centrosomal distributions of membranous organelles, including the Golgi apparatus, ERGIC, endosomes, and lysosomes (Hirokawa, 1998; Karki and Holzbaur, 1999). Subtle mutations in DHC can specifically cause motor neuron degeneration diseases correlated with defects in the axonal retrograde transport and abnormal migration (Hafezparast et al., 2003). It is believed that cytoplasmic dynein anchors to its target sites through interaction with dynactin, another multisubunit complex (Holleran et al., 1998). However, how its motor activity is regulated is not fully understood.
In Aspergillus nidulans, even distribution of fungal nuclei along hypha requires a group of nuclear distribution factors. Among them are dynein subunits (e.g., NudA and NudG), NudE, and NudF, the fungal orthologue of mammalian Lis1 (Xiang et al., 1994, 1995; Willins et al., 1997; Efimov and Morris, 2000). NudE and NudF appear to be regulators of dynein. Lis1 is a WD-40 repeat protein whose complete loss results in early embryonic lethality in mice (Hirotsune et al., 1998). Moreover, human heterozygotes of Lis1 mutations suffer from type I lissencephaly, a severe congenital disease with smooth brain surfaces and disorganized cortical layering of the central nervous system due to neuronal migration defects (Hirotsune et al., 1998; Wynshaw-Boris and Gambello, 2001; Gupta et al., 2002). NudE is also conserved in eukaryotes, with two isoforms in mammals, NudE and Nudel (for NudE-like). Both proteins partially localize to the centrosome and are likely to have similar functions (Feng et al., 2000; Niethammer et al., 2000; Sasaki et al., 2000; Yan et al., 2003). Lis1, NudE/Nudel, and dynein interact with each other directly (Feng et al., 2000; Niethammer et al., 2000; Sasaki et al., 2000; Tai et al., 2002). Nudel is also a substrate of Cdk5/p35, a brain-specific kinase critical for neuronal migration, which is suggestive of a linker between the Cdk5 and dynein pathways (Niethammer et al., 2000; Sasaki et al., 2000). 14-3-3, a member in the ubiquitous phosphoserine/threonine-binding protein 14-3-3 family, associates with Cdk5/p35-phosphorylated Nudel and protects the latter from dephosphorylation (Toyo-oka et al., 2003). Haploinsufficiencies of both Lis1 and 14-3-3
are implicated in neuronal migration defects more severe than lack of Lis1 alone (Toyo-oka et al., 2003). These lines of evidence suggest that Lis1 and NudE/Nudel function in neuronal migration through an evolutionarily conserved dynein pathway (Wynshaw-Boris and Gambello, 2001; Gupta et al., 2002).
We recently showed that Nudel is functionally involved in dynein-mediated poleward transport of kinetochore proteins, a process contributing to inactivation of the spindle checkpoint (Howell et al., 2001) in mitosis (Yan et al., 2003; Yang et al., 2003). Nudel and NudE are phosphorylated in M phase by Cdc2 and probably Erk1/2 (Yan et al., 2003). Nevertheless, the significance of Nudeldynein interaction has not been tested. Whether or not Nudel functions in membrane traffic also remains unknown.
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Results |
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Because Nudel homodimerizes through its NH2-terminal coiled coil domain in yeast two-hybrid assays (Sasaki et al., 2000), we examined if NudelN20 retained this property. As shown in Fig. 1 C, both FLAG-Nudel (lane 4) and NudelN20 (lane 5) pulled down GFP-NudelP2N, a mutant containing the NH2-terminal 201 amino acids, indicating that NudelN20 was still able to homodimerize. Thus, its phenotypes would be mainly attributed to loss of Lis1 association.
Involvement of Nudel in membrane trafficking
Cytoplasmic dynein is essential for proper positioning of many membranous organelles in the vicinity of centrosomes in a MT-dependent way. Disruption of dynein activity by antibody microinjection, targeted disruption, or overexpression of dynamitin to displace dynein from its cargoes leads to dispersion/fragmentation of the Golgi cisternae, lysosomes, endosomes, and ERGIC throughout the cytoplasm due to impairment of minus enddirected motions (Burkhardt et al., 1997; Harada et al., 1998). Thus, we examined whether mutant Nudel affected distributions of the membrane organelles in CV1 cells.
We initially examined the cis-Golgi cisternae decorated by anti-GM130 antibody (Lowe et al., 1998). Its perinuclear clustering was not affected in the majority of FLAG-Nudel expressors (Fig. 2 A, 1; and Fig. 2 B). In contrast, severe fragmentation and dispersion of the Golgi apparatus were observed upon expression of the mutant defective in binding to either Lis1 or DHC (Fig. 2 A, 2; Fig. 2 B; and not depicted). The extent of Golgi fragmentation strongly correlated with expression levels, with the DHC-binding defective mutant as the most potent because the threshold to cause Golgi fragmentation was relatively low and the cisternae were frequently scattered widely in the cytoplasm (Fig. 2 A, 2; and not depicted). Considering a background of 3% in surrounding untransfected cells, NudelN20/C36 had little effect (Fig. 2 A, 3; and Fig. 2 B). Similar effects were observed for lysosomes (Fig. 2, B and C), endosomes formed by transferrin receptor-mediated uptake (Fig. 2, B and D) or by fluid phase uptake of Texas redlabeled dextran (not depicted).
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Detailed analysis was performed manually. In the 2-min windows of monitoring, 113 lysosomes exhibited directional motions in 10 GFP-Nudel expressors (average 11.3 lysosomes per cell), whereas 71 were found in 15 cells expressing the mutant lacking DHC binding (average 4.7 per cell). In the mutant expressors, all the lysosomes had total run lengths (track lengths) shorter than 16 µm (minimal 1.4 µm, maximal 15.5 µm; Fig. 5 E). In contrast, 25.7% lysosomes with directional motions traveled longer than 16 µm (minimal 2.7 µm, maximal 84.9 µm) in GFP-Nudel expressors (Fig. 5 E). Such a difference was mainly due to lack of minus enddirected movement because the average inward motion events in a mutant expressor were 6.1-fold lower than in a wild-type expressor, compared with only 1.9-fold decrease in the average outward motion events per cell (Fig. 5 F and Videos 1 and 2). Consistently, the velocities of inward motions in the mutant expressors were lower than 1.2 µm/s, whereas 20.0% of those in wild-type expressors moved faster than that (maximal 2.7 µm/s; Fig. S1 A, available at http://www.jcb.org/cgi/content/full/jcb.200308058/DC1). Conversely, although lysosomes in wild-type expressors tended to move faster outwardly (Fig. S1 B), the difference was not as significant compared with that for inward motions. Similarly, the difference in the duration time of continual movements was also less apparent for either moving directions (Fig. S1, C and D). Together, the major defects in the mutant expressors were considerable reductions in both the frequencies and velocities of the minus enddirected motions.
Reduction of protein secretion by mutant Nudel
What would be the collective outcomes when both organizations and traffic of multiple membrane organelles were compromised by mutant Nudel? To partially address this question, we examined the effect on protein secretion using human placental lactogen (hPL; Walker et al., 1991) tagged with GFP at the COOH terminus as a marker. Secretion of hPL-GFP was confirmed through its detection in the medium (Fig. 6 A). Microscopic examinations indicated that the protein in the medium was not a result of cell death (unpublished data). For comparable results, we used the tetracycline (Tet)-responsive promotor (Gossen and Bujard, 1992) to control the expression of FLAG-Nudel and mutants (Fig. 6 B). Immunofluorescence staining indicated that over 90% of FLAG-positive cells coexpressed hPL-GFP in cultures lacking Tet, whereas only a few FLAG-positive cells were noticed in cultures with Tet (unpublished data).
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Dispersion of the Golgi cisternae upon silencing of Nudel expression
To further corroborate a role of Nudel in membrane traffic, we knocked down its expression through RNA interference (RNAi). Liposome transfection of small interference RNA (SiRNA) for Nudel into HEK293T cells with pEGFP-F to express a membrane-associated GFP marker resistant to methanol fixation (Jiang and Hunter, 1998) resulted in extensive cell death. In a typical set of experiments, when 10 random areas were examined 3 d after transfection, only 11.8% transfectants survived compared with those transfected with pEGFP-F alone. Immunoblotting indicated a 37% decrease of Nudel level compared with control cells (Fig. 7 A), which was informative but might not reflect the real situation because most cells with insufficient Nudel might be inviable and excluded from the sample. In fact, a more dramatic repression was achieved when exogenous Nudel was examined. In the presence of the SiRNA, GFP-Nudel level was only 2.3% of the control (Fig. 7 B, lanes 1 and 2; and Fig. 7 C, 16). In comparison, the SiRNA resulted in a 45% reduction for GFP-NudE (Fig. 7 B, lanes 3 and 4; and Fig. 7 C, 79), which shares a 53% identity with Nudel (Yan et al., 2003), with little effects on ß-actin, GFP, and red fluorescence protein DsRed (Fig. 7, AC). Moreover, despite the low levels of GFP-Nudel in the presence of the SiRNA, cell death was not obvious. Approximately 87% of transfectants survived, probably due to maintenance of critical Nudel levels by the trace amount of GFP-Nudel (Fig. 7 B, lane 2). Together, we attributed the cell death phenotype to repressed Nudel expression but not to toxicity of the SiRNA preparations. Therefore, the Nudel SiRNA was highly efficient and specific. Interestingly, expression of GFP-NudE in the presence of Nudel SiRNA (Fig. 7 C, 79) also prevented cells from extensive death. 72% of transfectants survived.
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Discussion |
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Several lines of evidence collectively indicate that Nudel functions in endomembrane flux through cytoplasmic dynein. First, a mutant incapable of direct interaction with DHC (Fig. 1; Sasaki et al., 2000) causes dispersions of the aforementioned organelles whose perinuclear distributions require dynein-mediated transport (Figs. 24). The phenotypes closely resemble those when dynein functions are inactivated by other means (Burkhardt et al., 1997; Harada et al., 1998; Valetti et al., 1999). In fact, the phenotypes for WGA-positive vesicles caused by overexpression of either dynamitin or the DHC-binding defective Nudel mutant are basically indistinguishable (Fig. 3). Moreover, selective disruption of Nudel's interaction with Lis1, another dynein partner, also leads to similar but slightly milder phenotypes (Fig. 1 and not depicted). However, organelles such as the ER and COPI-coated vesicles, whose maintenance and traffic do not entirely depend on dynein (Lippincott-Schwartz et al., 1995; Burkhardt et al., 1997), are not significantly affected by these mutants.
Second, time-lapse microscopy reveals clear defects in minus enddirected motions of lysosomes in living cells expressing the DHC-binding defective Nudel mutant (Fig. 5). Both the frequencies and velocities of inward trafficking are considerably reduced compared with those in the wild-type expressors. Despite similar tendencies for outward motilities, the differences are not as significant. We also confirmed that the radial MT arrays were not disrupted in transfectants (unpublished data). Therefore, dispersions of the membrane organelles and the lack of inward motions were not due to loss of MT focus at the MT-organizing center.
Finally, fragmentation of the Golgi apparatus after knocking down Nudel expression by RNAi further indicates requirement of Nudel for dynein function (Fig. 7). Moreover, the extensive cell death after repression of endogenous Nudel by RNAi and the rescue by exogenous protein also highlight its importance, like Lis1 (Hirotsune et al., 1998), for cell survival. Such properties may at least partially reflect critical roles of cytoplasmic dynein in trafficking, mitosis, and cell migration. In addition, several previous works suggest NudE as a functional paralogue of Nudel (Feng et al., 2000; Sasaki et al., 2000; Yan et al., 2003). Consistently, inhibition of NudE by RNAi also exhibited similar cell death and conferred the Golgi fragmentation in 59.2 ± 6.9% HEK293T cells (unpublished data). Prevention of Nudel SiRNA-induced cell death by overexpressing GFP-NudE also suggests a complementary effect. Therefore, Nudel, NudE, and Lis1 serve as regulators for a variety of dynein functions.
Three factors, the motorcargo association, motorMT interaction, and motor activity, dictate cargo motilities directed by MT-based motors. Neither the Lis1- nor the DHC-binding defective mutants attenuate the membrane associations of dynactin/dynein (unpublished data). In the time-lapse experiments, the similar durations for inward motions in the wild-type Nudel, or the mutant expressors (Fig. S1), suggest roughly intact dyneincargo and dyneinMT interactions. Consistently, in mitotic cells, blocking the dynein-mediated transport of kinetochore proteins to spindle poles by these mutants does not disrupt localizations of dynein and its cargo proteins on the spindle, also suggesting undisrupted dyneincargo and dyneinMT interactions (Yan et al., 2003; Yang et al., 2003; unpublished data). Together, the reduced velocities and frequencies of the inward lysosome trafficking (Fig. 5) are mainly attributed to impaired dynein motor activity by the DHC-binding defective mutant. In contrast, kinesin activity appears fairly maintained. The moderately decreased outward motilities (Fig. 5 F and Fig. S1) may be due to improbability for peripheral lysosomes to further move outwardly without preceding inward traffic. Therefore, it is concluded that the Nudel mutants mainly impair dynein motor activity by selectively abolishing either the NudelLis1 or the NudelDHC interaction (Fig.7 E). Moreover, the double mutant lacking interaction with both Lis1 and DHC was basically null, indicating that the remaining regions of Nudel, e.g., the dimerization domain (Fig. 1; Sasaki et al., 2000), have little effect in regulating dynein motor (Fig. 7 E). In comparison with Nudel, phenotypes by dynamitin overexpression are due to dissociation of dynein from the dynactin complex, and thus from its cargos (Burkhardt et al., 1997). Nudel also functionally differs from the Rab family of small GTPase, which regulates dynein-mediated membrane traffic by recruiting the dyneindynactin complex to appropriate membrane organelles (Jordens et al., 2001; Matanis et al., 2002).
Inhibition of hPL-GFP secretion by mutant Nudel (Fig. 6) may be delineated as a collective result of multiple defects. On the one hand because MT disassembly by nocodazole does not abolish protein secretion but only diminishes the secretion rate of a similar protein marker (Wacker et al., 1997); MT-based motors appear just as important for the efficiencies of membrane trafficking. Similarly, lack of retrograde transport and perinuclear distributions of many organelles in the secretory pathway through inactivation of dynein may contribute to the reduced rates of protein secretion. On the other hand, because many factors need to be recycled back to the previous organelles during membrane trafficking (Lippincott-Schwartz et al., 1989; Mallet and Maxfield, 1999; Matanis et al., 2002), interruption of such processes by impairing dynein activity may also indirectly affect secretion.
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Materials and methods |
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Cell culture and transfection
Monkey kidney CV1 and human embryonic kidney HEK293T cells were grown in DME (GIBCO BRL) supplemented with 10% calf serum (Sijiqing Company) in an atmosphere containing 5% CO2. Transfection was performed using calcium phosphate method unless otherwise indicated. Generally, HEK293T was used for biochemical studies due to high transfection efficiencies of up to 80%, whereas CV1 was used for cytological experiments because it is a normal cell line with larger cell sizes and better attachment to coverslips to allow easier examinations on vesicle distributions. Both cell lines express Nudel (Fig. 7; Yan et al., 2003).
Antibodies and staining reagents
Anti-FLAG M2, anti-DIC, anti-ßCOP, anti-tubulin, and anti
-tubulin mAbs, TRITC-WGA, and Texas redlabeled Con A were purchased from Sigma-Aldrich. Antidynamitin mAb was obtained from BD Biosciences. Polyclonal anti-DHC and anti-Lis1 antibodies were obtained from Santa Cruz Biotechnology, Inc. Lysotracker red (provided by M. Zhao, Chinese Academy of Sciences, Shanghai, China), Texas redconjugated transferrin, and dextran were from Molecular Probes. Secondary antibodies labeled with FITC, TRITC, or Cy5 were purchased from Pierce Chemical Co. or Rockland. Antibodies to ERGIC53, GM130, or Nudel were provided by H.-P. Hauri (University of Basel, Basel, Switzerland), M. Lowe (University of Manchester, Manchester, UK), and L.-H. Tsai (Harvard Medical School, Boston, MA), respectively.
Coimmunoprecipitation and immunoblotting
Transfected HEK293T cells were collected and washed twice with PBS. Coimmunoprecipitation using anti-FLAG M2 affinity resin (Sigma-Aldrich) was performed as described previously (Yan et al., 2003). Blots were developed in Renaissance ECL reagent (NEN Life Science Products) and exposed to X-ray films (Kodak). Band intensities were quantitated using Adobe Photoshop 6.0 (Yan et al., 2003). Data were presented as the mean ± SD.
Uptake of endocytic tracers
For tracing receptor-mediated endocytosis, CV1 cells were incubated with 20 µg/ml of Texas red transferrin or with dextran for 30 min at 37°C before immediate fixation with 4% PFA at 37°C for 15 min (Kauppi et al., 2002). To analyze the endocytic traffic of WGA-binding sites in plasma membranes, CV1 cells were incubated with 10 µg/ml TRITC-conjugated WGA in PBS for 30 min at 4°C, rinsed, and cultured in serum-free medium at 37°C (Raub et al., 1990). Samples were fixed at 0 and 60 min with PFA.
Fluorescence staining and microscopy
Cells grown on sterile coverslips were fixed in cold methanol or 4% PFA before indirect immunofluorescence staining using appropriate antibodies. Nuclear DNA was stained with DAPI. The cis-Golgi cisternae, trans-Golgi cisternae, or ER was decorated with anti-GM130 antibody (Lowe et al., 1998), TRITC-WGA (Virtanen et al., 1980), or Texas redlabeled Con A (Virtanen et al., 1980). The ERGIC was labeled with anti-ERGIC53 mAb (Hauri et al., 2000), whereas COPI vesicles were labeled with anti-ßCOP mAb. Lysosomes were stained with LysoTracker red (Molecular Probes) at a concentration of 50 nM in living cells followed by three times of wash before time-lapse microscopy or fixation in 4% PFA. Cells with clearly dispersed organelles were scored in a blind fashion independently by three researchers with results averaged. Percentages of affected cells were presented as the mean ± SD from two to three independent experiments. Images were captured using a cooled CCD SPOT II (Diagnostic Instruments) on a microscope (model BX51; Olympus) with UPlanApo 100x/1.35 Oil Iris and UPlanFL 60x/1.25 Oil Iris objectives, except that those stained with cy5-conjugated secondary antibodies (Fig. 3 D) were captured using a CCD camera (model DC350F; Leica) on a microscope (model DM5000B; Leica). Grayscale images (1,315 x 1,033 pixels) were recorded and converted to color using Confocal Assistant 4.02 when required. Illustrations were organized using Photoshop. Sometimes, nonlinear adjustment was applied to DAPI staining for optimal indication of nuclear positions.
Time-lapse microscopy was performed at 37°C using a LUMPlanFL 60x/0.9 water immersion objective. Images were recorded at 2-s intervals including exposure time for 2 min and processed using an NIH image program and Quick-time v5.02. Vesicle movement was defined as "directional" when the direction was fixed in three or more consecutive frames with the run length (track length) between two adjacent frames longer than 0.7 µm (equivalent to 4 pixels in images), otherwise it was considered "random." The directions were further classified as "inward" or "outward" for movement toward or away from the nucleus, or "other" if difficult to define. For track display, coordinates of individual vesicles in each frame were measured using the image program, stored in Microsoft Excel, and converted to tracks using SigmaPlot. The tracks were superimposed onto an outline of the cell drawn according to its differential interference contrast image. The run length of each directional motion was the track length from the start position to the next pause position. The velocity for each directional movement was an average obtained by dividing the run length by the duration time. The total run length of a vesicle was defined as the sum of all its run lengths regardless of direction and succession.
Secretion assays
HEK293T cells were cotransfected with pCD-hPL-GFP, pUHDF-Nudel, or a mutant and p15-1 at a molar ratio of 1:4:4 for 24 h in the presence of 1 µg/ml Tet (Gossen and Bujard, 1992). After three times of gentle wash, cells were split equally into two 60-mm dishes to guarantee minimal variations in cell numbers and transfection efficiencies. One plate was cultured in the presence of Tet, whereas the other was cultured without Tet. A glass coverslip was placed in each dish. After an additional 12 h of incubation, old culture media were removed and 2 ml of corresponding fresh media was added. 30 µl of each medium was collected at 0, 12, and 24 h. At the end of the experiment, cells were collected for immunoblotting. The coverslips were fixed in methanol and stained with anti-FLAG mAb to evaluate the quality of induction and cotransfection efficiencies. For quantitation, hPL-GFP intensities in each pair of samples at 24 h were normalized to corresponding BSA levels. Relative secretion levels were calculated by setting the level in the Tet+ sample as 100. Results were averaged from two experiments and presented as the mean ± SD.
SiRNA preparation and transfection
Nudel and NudE SiRNA were prepared using Genesilencer kit following the manufacturer's protocols (Gene Therapy Systems). In brief, the first 500 bp of Nudel or NudE cDNA from the start codon was transcribed in vitro into double stranded RNA using T7 RNA polymerase followed by cleavage with Dicer enzyme (Myers et al., 2003) for 24 h and column purification. Transfection of SiRNA was performed in 12-well plates with Lipofectamine2000 (Life Technologies) as described by the manufacturer for adherent cell lines. 0.1 µg pGFP-Nudel or pGFP-NudE, 0.4 µg DsRed plasmid, and 0.12 µg SiRNA formulated into liposomes were applied per well. Cells were processed 48 h after transfection for immunoblotting and 48, 72, and 96 h after transfection for fluorescence microscopy. To repress the endogenous protein, 0.10.3 µg SiRNA and 0.1 µg pEGFP-F (CLONTECH Laboratories, Inc.; Jiang and Hunter, 1998) were transfected per well. Cells were processed 72 h after transfection.
Online supplemental material
Quicktime movies (six frames per second) show lysosome motilities in typical CV1 cells expressing GFP-Nudel (Video 1, frame 1) or GFP-NudelC36 (Video 2, frame 1). The tracks of five lysosomes with the longest run lengths in each cell are displayed in the last frame. Red tracks refer to motions with net outward displacements. Representative frames in Videos 1 and 2 are displayed in Fig. 5 (C and D) to show movement of typical vesicles. Detailed analysis is shown in Fig. 5 (E and F) and Fig. S1. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200308058/DC1.
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Acknowledgments |
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This work was supported by grants 30025021, 30330330, and 39970160 from the National Science Foundation of China, grant KSCX2-2-02 from the Chinese Academy of Sciences, and grant 2002CB713802 (The National Key Basic Research and Development Plan) from the Ministry of Science and Technology of China.
Submitted: 11 August 2003
Accepted: 14 January 2004
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