Report |
Address correspondence to Yixian Zheng, Howard Hughes Medical Institute, Dept. of Embryology, Carnegie Institution of Washington, 115 W. University Pkwy., Baltimore, MD 21210. Tel.: (410) 554-1232. Fax: (410) 243-6311. E-mail: zheng{at}ciwemb.edu
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Abstract |
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Key Words: Ran; nuclear; chromatin; nucleotide exchange; fluorescence intensity
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Introduction |
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Although RanGTP gradient appears to exist in vitro, it is not clear whether the gradient also exists in living cells. Most importantly, the mechanism by which RCC1 catalyzes and maintains RanGTP in living cells remains unknown. As a result, little is known about how the cell regulates RanGTP production. Studies of other small GTPases have shown that a plethora of mechanisms are involved in regulating nucleotide exchange catalyzed by GEFs, and understanding these mechanisms have been crucial in deciphering the functions of small GTPases such as Ras, Rho, and Arf in vivo. Clearly, understanding the mechanisms regulating RanGTP production in living cells is essential to understand the Ran system.
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Results and discussion |
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RCC1 is a highly mobile enzyme in interphase and mitosis
To decipher the mechanism of RanGTP gradient production in vivo, it is important to understand how RCC1 interacts with the chromatin in interphase and mitosis. Therefore, we first used FRAP to study the dynamics of RCC1-GFP. A defined region of the 3T3 cell nucleus or condensed mitotic chromosomes was bleached with a laser pulse of 500 ms. The kinetics of recovery, reflecting the mobility of the RCC1-GFP, was measured by sequential imaging (Fig. 2, A and B)
. After photobleaching, 90% of the RCC1 fluorescence signal was recovered within 55 and 10 s, with a half time of
5 and
3 s in interphase and mitotic cells, respectively (Fig. 2 C). The recovery of RCC1-GFP was much faster than that of histone H2B-GFP, which was immobile during our observations (Kimura and Cook, 2001), but slower than the GFP molecule in the nucleus (Fig. 2 D), which freely diffused throughout the nucleus.
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We hope to determine whether and how RCC1 may couple its binding to the chromosomes to the nucleotide exchange on Ran. Although little is known about how RCC1 catalyzes nucleotide exchange on Ran in living cells, the in vitro nucleotide exchange reaction is well documented. Studies using purified RCC1 and Ran showed that RCC1-catalyzed exchange is a multi-step process, involving the formation of ternary and binary complexes of RCC1, Ran, and guanine nucleotides (Klebe et al., 1995a, 1995b; Renault et al., 2001). Because RCC1 destabilizes the binding of nucleotide to Ran, the ternary complex of RCC1Rannucleotide is of low affinity, which dissociates quickly (Klebe et al., 1995a, 1995b; Renault et al., 2001). When there is no free nucleotide, the ternary complex relaxes into the binary complex of RCC1Ran. However, when the free nucleotide concentration is high, the ternary complex dissociates into RCC1 and Rannucleotide (Klebe et al., 1995a, 1995b; Renault et al., 2001). We reasoned that if nucleotide exchange on Ran occurred on the chromatin in vivo, we might find the binary complex on the chromatin.
We used a mutant allele of Ran (RanT24N) to test whether the binary complex of Ran and RCC1 is chromatin bound in the cell. RanT24N has a greatly reduced affinity for nucleotide compared with wild-type Ran; therefore, it forms a stable binary complex with RCC1 due to lack of nucleotide exchange (Dasso et al., 1994; Kornbluth et al., 1994; Klebe et al., 1995a; Lounsbury et al., 1996). If the binary complex is chromosome bound, we should see a clear colocalization of RanT24N and RCC1-GFP on the chromatin in both interphase and mitosis. To test this possibility, we injected rhodamine-labeled (Rh) wild-type RanGDP, Rh-RanGTP, or Rh-RanT24N into interphase nuclei or mitotic cytoplasm of the 3T3 cells expressing RCC1-GFP. Using live imaging, we found that Rh-RanT24N clearly colocalized with RCC1-GFP in interphase nuclei and on mitotic chromosomes (Fig. 4 A). As expected, most Rh-RanGDP is found in the nucleus. Interestingly, in mitosis, Rh-RanGDP was found on condensed chromosomes and in the cytoplasm, resulting in a somewhat diffused distribution (Fig. 4 A). We believe that the localization of Rh-RanGDP to the condensed chromosomes is because RanGDP undergoes nucleotide exchange by forming the binary complex with RCC1, which binds to the condensed chromosomes. Consistent with this idea, we found that although Rh-RanGTP is localized in the interphase nucleus, in mitosis, Rh-RanGTP is diffusely distributed in the mitotic cytosol (Fig. 4 A). Based on these studies, we conclude that the binary complex is chromosome bound in vivo.
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If the above model is correct, excess RanT24N should immobilize RCC1 on the chromatin due to the formation of a stable binary complex that resists nucleotide exchange. On the other hand, excess wild-type RanGDP should only slow down the mobility of RCC1 because of the increased formation of the binary complex that allows nucleotide exchange. Therefore, we used FRAP to probe the mobility of RCC1-GFP in the 3T3 cells after microinjection of purified RanT24N or RanGDP (see Materials and methods). Perturbation by control microinjection into the nucleus had only a small effect on the mobility of RCC1-GFP when compared with cytoplasm-injected or uninjected cells (Fig. 4 D). Similarly, injecting RanGDP at 0.1 mg/ml into the interphase nuclei had no effect on FRAP of RCC1-GFP compared with controls. However, injecting the same amount of RanT24N significantly reduced the mobility of RCC1-GFP (Fig. 4 D).
Next, RanT24N or RanGDP was injected at 10-fold higher concentration (1 mg/ml) into the interphase nuclei. We estimated that injecting at this concentration of Ran could deliver 4 x 106 molecules of Ran into the cells, which is similar to the estimated number of RCC1 molecules in the cell (see Materials and methods). We found that RanGDP injection allowed the recovery of 55% of the RCC1-GFP in the first 10 s, followed by a slow recovery of the fluorescence signal to the level of control injections in the next 110 s (Fig. 4 D). However, RanT24N injection completely blocked the recovery of RCC1-GFP in the bleached spot (Fig. 4, C and D). When the same amount of RanT24N or RanGDP was injected into the mitotic cells, we found that RanT24N completely immobilized RCC1-GFP on the condensed chromosomes, whereas RanGDP only reduced the mobility of RCC1 (Fig. 4, C and E). We also performed FRAP of fluorescently labeled RanGDP or RanT24N that were injected into cells, and found that although RanGDP is mobile, RanT24N is not (unpublished data). These results showed that nucleotide exchange on Ran is required for both RCC1 and Ran to dissociate from the chromatin in both interphase and mitosis. They further suggest that the binary complex of RCC1Ran associates stably with the chromatin in vivo. Successful nucleotide exchange is required for the dissociation of RCC1 from RanGTP, which in turn allows RCC1 and RanGTP to dissociate from the chromatin.
The dissociation of the binary complex of RanRCC1 from the chromatin requires nucleotide exchange
The above in vivo studies revealed that the binary complex of RanRCC1 binds to chromosomes tightly and that nucleotide exchange is required for their dissociation from chromatin. We hope to biochemically confirm these findings by using Xenopus egg extracts that support the formation of chromatin structures from Xenopus sperm in vitro. Sperm were added to the egg extracts supplemented with or without RCC1-GFP in the presence of either RanT24N or RanGDP. We found that the addition of exogenous RanT24N and RanGDP strongly stimulated the binding of both the endogenous RCC1 (Fig. 5
A) and the exogenous RCC1-GFP (Fig. 5 B) to the chromatin assembled from the sperm, which is consistent with the idea that the formation of the binary complex of RCC1Ran enhances the binding of RCC1 to the chromosomes. Next, we used competition assays to determine whether Ran and RCC1 bind to the chromatin tightly in the form of binary complex in the egg extracts. We added Xenopus sperm to the extracts supplemented with Rh-RanT24N, Rh-RanGD, or RCC1-GFP. Excess unlabeled RanT24N, RanGDP, or RCC1 was used as competitors. We found that unlabeled RanGDP and RCC1 readily competed for Rh-RanGDP and RCC1-GFP, respectively. However, unlabeled RanT24N and RCC1 only showed 40 and
20% competition of Rh-RanT24N and RCC1-GFP, even when the competitor concentrations reached 20- and 10-fold excess of the labeled proteins, respectively (Fig. 5, C and D). This suggests that RCC1 and Ran bind stably to the chromatin when they are locked into the RCC1Ran binary complex in the form of RCC1RanT24N.
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We have shown that RCC1 is a highly mobile enzyme, and the binding of RCC1 to the chromatin appears to be subjected to cell cycle regulation. More importantly, as a mobile enzyme, RCC1 couples nucleotide exchange on Ran with chromosome docking to generate RanGTP in vivo. The coupling is established through the stable binding of the RCC1Ran binary complex to chromosomes. Successful nucleotide exchange on the chromatin-bound binary complex dissociates the complex, liberates RCC1, and generates RanGTP on the chromatin. Ran and RCC1 bind to the chromatin via the core histones, which are present in 100-fold molar excess of RCC1 in the cell. Therefore, the chromatin has sufficient capacity to bind to all RCC1Ran binary complexes to support the chromosome-coupling exchange mechanism. Eukaryotic chromosomes are highly dynamic structures that undergo remodeling throughout the cell cycle. A mobile RCC1 and a chromosome-coupled exchange mechanism may be necessary to coordinate the dynamic chromatin reorganization with the production of RanGTP gradient during the cell cycle.
Although the chromosome-coupled nucleotide exchange by RCC1 described in this paper can significantly influence the production of RanGTP gradient, it does not exclude other mechanisms that may also influence RanGTP gradient. For example, the chromosomes were shown to stimulate the GEF activity of RCC1 modestly (Nemergut et al., 2001). This stimulation, in combination with the chromosome-coupled exchange, should further enhance RanGTP production on the chromosomes. In addition, RanGAP1 and RanBP1 present in the cytosol hydrolyze RanGTP into RanGDP, and therefore, should further sharpen the RanGTP gradient across the nuclear envelope in interphase and on the condensed chromosomes in mitosis. Finally, although free RCC1 in cell lysates is active in vitro, the free RCC1 in vivo may be negatively regulated. It will be important to understand the relative contributions of the different mechanisms toward the production of RanGTP gradient.
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Materials and methods |
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Fluorescence microscopy and analysis of salt-extracted RCC1
Immunofluorescence microscopy on cells was performed as described previously (Zhang et al., 2000) using a commercial mAb against RCC1 (MBL International Corporation). Images were obtained with a CCD camera equipped with the MetaMorph® Imaging System (Universal Imaging Corp.). To extract RCC1, nuclei were isolated from cells expressing RCC1-GFP and extracted with increasing salt concentrations as described previously (Ohtsubo et al., 1989). The supernatant and pellet fractions were subjected to SDS-PAGE followed by Western blotting and probing with the anti-RCC1 antibody.
Microscopy of FRAP and FLIP
Cells were plated on a coverslip and mounted onto a glass slide with a depression containing culture medium (DME, 10% FBS, 10 U/ml penicillin/streptomycin, and 25 mM Hepes without phenol red). FRAP and FLIP were performed on a confocal microscope (TCS-SP2; Leica) using the 488-nm laser line of an argon laser at 37 and 23°C with similar results. In FRAP experiments, cells were scanned twice, followed by a single bleach pulse of 500 ms using a spot 1 µm in diameter. Single section images were then collected at 1-s intervals for the first 10 images, followed by 5-s intervals for the next 10 images, and 10-s intervals for the final 10 images with the laser power attenuated to 9% of the bleach intensity. In FLIP experiments, cells were repeatedly imaged and bleached at intervals of 1 s with each bleaching for 250 ms and with imaging identical to those used in FRAP. To determine the relative fluorescence intensity in a region of interest, the fluorescence intensity in the region at each time point was normalized to the change in total fluorescence caused by bleaching and imaging as described previously (Misteli et al., 2000; Phair and Misteli, 2000).
Recombinant protein and microinjection
6his-Ran and 6his-RanT24N were expressed in bacteria and purified using Ni-agarose adopting the methods as described previously (Dasso et al., 1994; Wilde and Zheng, 1999). 6his-RCC1 and 6his-RCC1-GFP were purified using Ni-agarose. Ran and RCC1 were labeled using tetramethyl-rhodamine and fluorescein, respectively. Unlabeled RanGDP or RanT24N in PBS was coinjected with tetramethyl-rhodamine succinididyl ester (Molecular Probes, Inc.) into the nuclei of interphase cells or the cytoplasm of mitotic cells with an InjectMan® microinjection system (Eppendorf) followed by FRAP. Control injections were performed with tetramethyl-rhodamine alone. For localization studies, both proteins were labeled with tetramethyl-rhodamine succinimidyl ester (Molecular Probes, Inc.) and desalted into PBS before injecting into the interphase nucleus or mitotic cytoplasm followed by live imaging and microscopy.
According to our Western blotting analysis (Fig. 1 C), RCC1-GFP is expressed at about the same level as the endogenous RCC1. According to Bischoff and Ponstingl (1995), the number of both endogenous RCC1 and RCC1-GFP expressed in the 3T3 cells should be 106 per cell. Based on our injection condition and the manufacturer's calibration (Eppendorf), we estimated that
0.2 pl of Ran was delivered into the cell per injection. Therefore, at 1 mg/ml of Ran, we delivered
4 x 106 Ran into the cell, whereas at 0.1 mg/ml of Ran, only
0.4 x 106 Ran was injected. If one RanT24N can immobilize one RCC1, injecting Ran at 0.1 mg/ml should lead to partial RCC1 immobilization, whereas injecting Ran at 1 mg/ml should lead to a complete immobilization. Consistent with this prediction, we observed that injecting RanT24N at 0.1 mg/ml lead to partial immobilization of RCC1-GFP, whereas injecting at 1 mg/ml lead to a complete immobilization.
In vitro assays using Xenopus egg extracts
Xenopus egg extracts and sperm were prepared as described previously (Murray and Kirschner, 1989). For Ran competition, sperm was incubated with egg extracts supplemented with 2 µM Rh-RanGDP or Rh-RanT24N in the presence of 040 µM unlabeled RanGDP or RanT24N, respectively, for 40 min at RT. After incubation, the sperm was spun onto coverslips through a glycerol cushion (80 mM Pipes, pH 6.8, 1 mM EGTA, 1 mM MgCl2, and 30% glycerol), fixed with methanol, and stained with DAPI (Wilde and Zheng, 1999). The amount of labeled Ran that remained bound to the sperm chromatin was quantified by taking images using a cooled CCD camera at the same exposure time that is below the saturation limit of the camera. The fluorescence intensity of each sperm was measured, and the background was subtracted from the area next to each sperm using the MetaMorph® software. At least 20 sperm were quantified for each experiment. For RCC1 competition, sperm was incubated with egg extracts supplemented with 0.5 µM RCC1-GFP and 40 µM unlabeled RanGDP or RanT24N in the presence of 05 µM unlabeled RCC1 as competitors.
The following experiments were used to determine whether the binding of RCC1 and Ran to the chromatin is sensitive to free GTP. For assaying RCC1-GFP binding, sperm was added to extracts supplemented with 0.5 µM RCC1-GFP and 40 µM of RanGDP or RanT24N. For assaying Rh-Ran binding, sperm was added to extracts supplemented with 2 µM Rh-RanGDP or Rh-RanT24N. After a 40-min incubation, the sperm was spun onto coverslips through the glycerol cushion without GTP. 250 µl of buffer (10 mM Hepes, pH 7.7, 100 mM KCl, 2 mM MgCl2, 0.1 mM CaCl2, 50 mM sucrose, and 5 mM EGTA) containing 05 mM GTP was added onto the coverslips and incubated for 20 min at RT followed by fixation and DAPI staining. The amount of RCC1-GFP or Rh-Ran that remained on the sperm chromatin were quantified as above. Similar amounts of Rh-Ran and RCC1-GFP remained bound to the chromatin before and after extraction with buffer containing no GTP. This observation suggests that most of Rh-Ran and RCC-GFP remain bound to the chromatin after centrifugation formed the binary complex. To test the stability of the binding of the binary complex to the sperm chromatin in the absence of free GTP, the same experiments as above were performed. After spinning the sperm labeled with RCC1-GFP or Rh-Ran onto coverslips, the sperm was incubated with XB buffer containing 02 µM unlabeled RCC1 and 040 µM unlabeled Ran as competitors for 20 min followed by fixation and DAPI staining. Rh-Ran and RCC1-GFP that remained bound to the sperm chromatin were quantified. The unlabeled RCC1 and Ran competitors were used at the similar ratio as that present in the egg extracts. The concentrations of Ran (20 µM) and RCC1 (1.3 µM) in the egg extract were estimated using quantitative Western blotting with known amounts of purified Ran and RCC1, respectively.
Online supplemental material
Online supplemental material includes kinetic modeling of RCC1 mobility and RanGTP gradient. Figs. S1 and S2 show curve fits of FRAP and FLIP analyses. Fig. S3 shows the simulation of RanGTP concentration profile on the chromatin. Online supplemental material available at http://www.jcb.org/cgi/content/full/jcb.200211004/DC1.
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Footnotes |
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* Abbreviations used in this paper: FLIP, fluorescence loss in photobleaching; GEF, guanine nucleotide exchange factor; RCC1, regulator of chromosome condensation.
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Acknowledgments |
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This work was supported by the Howard Hughes Medical Institute (to Y. Zheng) and by the National Science Foundation (NSE/NIRT 0210718 to D. Wirtz).
Submitted: 1 November 2002
Revised: 17 January 2003
Accepted: 21 January 2003
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References |
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Bilbao-Cortes, D., M. Hetzer, G. Langst, P.B. Becker, and I.W. Mattaj. 2002. Ran binds to chromatin by two distinct mechanisms. Curr. Biol. 12:11511156.[CrossRef][Medline]
Bischoff, F.R., and H. Ponstingl. 1991. Catalysis of guanine nucleotide exchange on Ran by the mitotic regulator RCC1. Nature. 354:8082.[CrossRef][Medline]
Bischoff, F.R., and H. Ponstingl. 1995. Catalysis of guanine nucleotide exchange of Ran by RCC1 and stimulation of hydrolysis of Ran-bound GTP by Ran-GAP1. Methods Enzymol. 257:135144.[Medline]
Dasso, M. 2002. The Ran GTPase: theme and variations. Curr. Biol. 12:R502R508.[CrossRef][Medline]
Dasso, M., S.T. Azuma, T. Ohba, and T. Nishimoto. 1994. A mutant form of the Ran/TC4 protein disrupts nuclear function in Xenopus laevis egg extracts by inhibiting the RCC1 protein, a regulator of chromosome condensation. EMBO J. 13:57325744.[Abstract]
Kalab, P., K. Weis, and R. Heald. 2002. Visualization of a Ran-GTP gradient in interphase and mitotic Xenopus egg extracts. Science. 295:24522456.
Kimura, H., and P.R. Cook. 2001. Kinetics of core histones in living cells: little exchange of H3 and H4 and some rapid exchange of H2B. J. Cell Biol. 153:13411354.
Klebe, C., F.R. Bischoff, H. Ponstingl, and A. Wittinghofer. 1995a. Interaction of the nuclear GTP-binding protein Ran with its regulatory proteins RCC1 and RanGAP1. Biochemistry. 34:639647.[Medline]
Klebe, C., H. Prinz, A. Wittinghofer, and R.S. Goody. 1995b. The kinetic mechanism of Ran-nucleotide exchange catalyzed by RCC1. Biochemistry. 34:1254312552.[Medline]
Kornbluth, S., M. Dasso, and J. Newport. 1994. Evidence for a dual role for TC4 protein in regulating nuclear structure and cell cycle progression. J. Cell Biol. 125:705719.[Abstract]
Lounsbury, K.M., S.A. Richards, K.L. Carey, and I.G. Macara. 1996. Mutations with the Ran/TC4 GTPase. J. Biol. Chem. 271:3283432841.
Mattaj, I., and L. Englmeier. 1998. Nucleocytoplasmic transport: the soluble phase. Annu. Rev. Biochem. 67:265306.[CrossRef][Medline]
Misteli, T., A. Gunjan, R. Hock, B. Michael, and D. Brown. 2000. Dynamic binding of histone H1 to chromatin in living cells. Nature. 408:877881.[CrossRef][Medline]
Murray, A.W., and M.W. Kirschner. 1989. Cyclin synthesis drives the early embryonic cell cycle. Nature. 339:275280.[CrossRef][Medline]
Nemergut, M., C.A. Mizzen, T. Stukenberg, C.D. Allis, and I.G. Macara. 2001. Chromatin docking and exchange activity enhancement of RCC1 by histones H2A and H2B. Science. 292:15401543.
Nishitani, H., M. Ohtsubo, K. Yamashita, H. Iida, J. Pines, H. Yasudo, Y. Shibata, T. Hunter, and T. Nishimoto. 1991. Loss of RCC1, a nuclear DNA-binding protein, uncouples the completion of DNA replication from the activation of cdc2 protein kinase and mitosis. EMBO J. 10:15551564.[Abstract]
Ohtsubo, M., H. Okazaki, and T. Nishimoto. 1989. The RCC1 protein, a regulator for the onset of chromosome condensation locates in the nucleus and binds to DNA. J. Cell Biol. 109:13891397.[Abstract]
Phair, R., and T. Misteli. 2000. High mobility of proteins in the mammalian cell nucleus. Nature. 404:604609.[CrossRef][Medline]
Renault, L., J. Kuhlmann, A. Henkel, and A. Wittinghofer. 2001. Structural basis for guanine nucleotide exchange on Ran by the regulator of chromosome condensation (RCC1). Cell. 105:245255.[CrossRef][Medline]
Seino, H., S. Hisamoto, T. Uzawa, T. Sekiguchi, and T. Nishimoto. 1992. DNA-binding domain of RCC1 protein is not essential for coupling mitosis with DNA replication. J. Cell Sci. 102:393400.[Abstract]
Seki, T., N. Hayashi, and T. Nishimoto. 1996. RCC1 in the Ran pathway. J. Biochem (Tokyo). 120:207214.[Abstract]
Wilde, A., and Y. Zheng. 1999. Stimulation of microtubule aster formation and spindle assembly by the small GTPase Ran. Science. 284:13591362 (see comment).
Zhang, L., T.J. Keating, A. Wilde, G.G. Borisy, and Y. Zheng. 2000. The role of Xgrip210 in gamma tubulin ring complex assembly and centrosome recruitment. J. Cell Biol. 151:15251535.
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