Chromosomal association of Ran during meiotic and mitotic divisions

The function of the small GTPase Ran was first studied in mutants, where its absence led to many defects such as in DNA replication, nuclear envelope structure, chromatin structure and cell cycle progression (reviewed inSazer, 1996;Moore, 2001). A cell-free system led to the recognition of the importance of Ran in nuclear transport,and Ran now has a well established, central role in that process (e.g.Mattaj and Englmeier, 1998;Gorlich and Kutay, 1999). Recently, work with another cell-free system has implicated Ran in M phase processes; this work has been extensively reviewed (e.g.Sazer and Dasso, 2000;Kahana and Cleveland, 2001;Dasso, 2001). In retrospect,the multiple functions of Ran in interphase and M phase are consistent with the many defects observed in the first mutant studies.

The M phase studies have made use of the cell-free extract system from Xenopus eggs, where addition of sperm chromatin or DNA coated beads induces the formation of a bipolar microtubule spindle centered on the added DNA. The initial experiments indicated that generation of Ran-GTP was necessary for assembly of the spindle (Wilde et al., 1999;Ohba et al., 1999;Kalab et al., 1999;Carazo-Salas et al., 1999). Importins α and β have been identified as the target of Ran-GTP; it appears that Ran-GTP displaces microtubule polymerization promoters such as TPX2 or NuMA from importin (Gruss et al.,2001; Nachury et al.,2001; Wiese et al.,2001). A Ran-GTP gradient near the chromosomes in Xenopusegg extracts has been visualized (Kalab et al., 2002). In addition to the work on spindle assembly, it has also been shown that Ran has an essential role in nuclear envelope reformation after mitosis (Zhang and Clarke,2000; Hetzer et al.,2000; Zhang and Clarke,2001). There remain unresolved issues about Ran's role in M phase;for instance, verification in living cells of the roles for Ran identified in extracts is required, and has begun to be addressed(Bamba et al., 2002), and to what degree Ran is involved in all eucaryotic cell meioses and mitoses needs to be resolved.

Light microscopic imaging seems likely to be useful for learning more about Ran and its interactions. For many proteins, the amount of fluorescent analogue required for imaging is in excess of the endogenous protein concentration, which may perturb the natural pathways. Because Ran is one of the most abundant proteins in the cell(Bischoff and Ponstingl, 1991),there should be less danger of disrupting the cell in this way. Another advantage is that Ran's biochemical properties have been well characterized. A major factor in this progress is that Ran is soluble and can be readily produced as a functional recombinant protein (unlike all other small GTPases,Ran has no lipid modifications). Lastly, there is only one isoform of Ran, one known exchange factor and only a few GAPs (GTPase-activating proteins)(Macara et al., 2000), all of which make it simpler to try to interpret observations in living cells. To further investigate Ran during M phase, we have observed fluorescent Ran localization and dynamics in several types of living cells.

Methods for handling Xenopus eggs were as described elsewhere(Terasaki et al., 2001). In brief, immature Xenopus oocytes were matured in culture by exposure to 1 μg/ml progesterone; the oocytes became arrested at meiosis II metaphase ∼3 hours after white spot formation(Gallo et al., 1995). For immunofluorescence, eggs were fixed in -70°C methanol for 1.5 hours,warmed at room temperature for 30 minutes and then hydrated in 2:1 methanol:PBS (20 minutes), 1:2 methanol:PBS (20 minutes), then PBS. Eggs were incubated in 2.5 μg/ml anti-Ran monoclonal antibody (R32620; Transduction Labs; San Diego, CA) for 1.5 hours followed by 1:50 rhodamine goat anti-mouse(ICN; Costa Mesa, CA) for 1 hour. For DNA double-labeling, eggs were incubated in 10 μg/ml Hoechst dye 33342 (Sigma Chemical Co, St. Louis, MO) for 10 minutes. The eggs were imaged with a Zeiss LSM 410 confocal microscope with UV and visible optics. Hoechst-stained chromosomes can be seen easily in living eggs by wide field fluorescence microscopy(Gallo et al., 1995), but with other fluorescent labels where the contrast is not as high, the spindle region of living or fixed eggs is imaged better by confocal microscopy.

For live cell imaging, Rh-Ran was injected into immature oocytes at a final concentration of 1-2 μM. The endogenous concentration in Xenopusoocytes has not been determined. In HeLa cells, Ran is 0.36% of total protein(Bischoff and Ponstingl, 1991);if the total protein concentration is 100 mg/ml in cells, this corresponds to 14 μM (Ran MW 25,000). After maturation, the egg was imaged using an MRC 600 confocal microscope (Bio-Rad; Cambridge, MA) with a krypton argon laser,coupled to an upright microscope with a 40×Plan neofluar N.A. 1.3 objective lens (Zeiss; Thornwood, NY). For photobleaching, a macro was written in the BioRad SOM software to control the scan and image acquisition while the neutral density and laser power levels were switched manually. Quantification was done using public domain NIH Image program (available at http://rsb.info.nih.gov/nih-image/).

For imaging anaphase of meiosis II, frog eggs were activated by puncturing the egg surface with a microneedle. The eggs were in an open sided chamber made of silicon rubber (Ronsil, thickness 0.03 inches; North American Reiss,Corp, Blackstone, VA). The needle puncture was done on an upright microscope using a 10× objective lens, then the egg was transferred to a dissecting scope, where a #0 thickness cover slip was placed on top of the egg and maneuvered so that the white spot was positioned straight up. The white spot was located under a 40× oil immersion lens on the confocal microscope,and then scanning was begun to image the calcium wave. Sometimes the wave had already passed, and sometimes it failed to be initiated [later experiments suggested that Ins(1,4,5)P₃ injection is more reliable]. With the confocal microscope set to continuous scanning, images were collected on an optical memory disk recorder (Panasonic TQ303F, Secaucus, NJ) with a custom-made trigger circuit.

Fully grown, immature mouse oocytes were collected from the ovaries of PMSG-primed NSA (CF-1) mice (Harlan Sprague-Dawley) in a minimal essential medium (MEM), then washed into MEM without dibutryl cAMP to induce maturation. Matured eggs were microinjected as described previously(Mehlmann and Kline, 1994) and were examined within 30 minutes of injection while being maintained at 37°C on the confocal microscope stage. Rh-Ran was injected in a 3% volume injection, with a resulting final concentration of 2.5 μM.

Starfish (Asterina miniata) were obtained from Bodega Bay, CA. Methods for obtaining and handling the gametes were described previously(Jaffe et al., 1993). Mercury-loaded pipettes were used for quantitative microinjection; detailed description of this method and equipment used is available at http://egg.uchc.edu/injection/. Embryos were kept in an 18°C incubator until imaged.

Mitotic NRK cells in an injection chamber on a heated microscope stage were pressure injected (<10% volume injection) with a 5:1 mix of Alexa 488 Ran(20 mg/ml) and 70 kDa Rh dextran (2 mg/ml). They were imaged with a Zeiss Planapo 1.4 numerical aperture objective lens using a Zeiss 510 confocal microscope.

Tetramethylrhodamine or Alexa 488 was conjugated to the single exposed cysteine (C112) of the three cysteine residues of human Ran(Ribbeck et al., 1998). Both Rh-Ran and Alexa 488-Ran (3 μM) stimulated import of NPC-M9 substrate into digitonin-permeabilized cells as much as wild-type Ran. In this assay, many GTPase cycles are required for import to occur, so if either hydrolysis or exchange on RCC1 were impaired, there would be no import. GTP hydrolysis by both fluorescent conjugates was stimulated by RanGAP with identical kinetics to wild-type Ran. The labeling efficiency of both was greater than 92% in comparison with absorption spectra with tetramethylrhodamine or Alexa 488, so this activity cannot be due to the presence of an active unlabeled fraction. We concluded that Rh-Ran and Alexa 488-Ran are as active as the wild-type unlabeled protein.

GFP-Ran was isolated in a visual screen of a cDNA library concatenated with GFP (VLP55) (Rolls et al.,1999). It consists of an N-terminal GFP followed by a 22 amino-acid linker region (GGGLDPRVRSDGRGDASGRNAA) and the entire coding sequence for human Ran. GFP-Ran was inserted into the pSP64-S expression vector by standard PCR methods resulting in a consensus echinoderm kozak sequence (aattcaaa). RNA was prepared in vitro using the SP6 mMESSAGE mMACHINE kit (Ambion; Austin, TX). Owing to difficulties in producing GFP-Ran for biochemical characterization, its activity was not tested in vitro.

The experimentally observed exponential recovery does not necessarily mean that there is only one type of interaction of Ran with chromosomes. For instance, if there is a second interaction with much larger k_off, there would be a fast recovery for this interaction,which would be complete by the time of the first experimental time point. Also, if the number of binding sites is very different, then the less-abundant site will make only a small contribution to the recovery curve.

The last part of Equation 3 can be used for estimating K+[R] from experiments with different amounts of [R^*] by multiplying the experimental [R^*B]/[R^*] by [R^*], which puts the right hand side into the form of the Michaelis-Menten equation with parameters V_m=B_t and K_m=K+[R]. The analysis is made somewhat more complex because the endogenous Ran concentration is not known and it is not known whether free Ran or complexed Ran is binding to the chromosomes. When α is equal to the fraction of the total nuclear Ran(either endogenous or fluorescent), which is available for binding, the expression for K_m is (K/α)+[R].

Xenopus eggs are arrested in metaphase of meiosis II. The meiotic spindle is near the surface in a ∼20 μm diameter pigment-free region in the central region of the pigmented half of the egg. The spindle axis is oriented perpendicular to the cell surface, so that the chromosomes are arranged more or less in a single plane parallel to and about 20 μm from the egg surface. The chromosomes were labeled with Hoechst dye and Ran was localized by immunofluorescence using a mouse monoclonal antibody originally raised against human Ran that specifically recognizes Xenopus Ran(Zhang et al., 1999). Ran fluorescence colocalized with the chromosomes(Fig. 1A).

For imaging Ran localization and dynamics in living eggs, we used a rhodamine-labeled Ran (Rh-Ran). The activity of Rh-Ran was tested in GTPase assays in vitro and behaved identically to unmodified Ran (see Materials and Methods). Microinjected Rh-Ran accumulated at the chromosomes(Fig. 1B). A GFP chimera of Ran was isolated in a visual screen of a cDNA library concatenated with GFP[termed VLP55; (Rolls et al.,1999)]. GFP-Ran, expressed by mRNA injection, also accumulated at the chromosomes (data not shown).

Mature mouse oocytes are arrested in metaphase of meiosis II. As in Xenopus eggs, microinjected Rh-Ran localized to the mouse egg chromosomes (Fig. 1C)(Verlhac et al., 2000).

The meiosis II metaphase arrest in Xenopus eggs was released by raising intracellular calcium. The egg surface was punctured with a microneedle (prick activation); a calcium wave begins at this point and takes several minutes to cross the egg. Rh-Ran was co-injected with calcium green dextran in order to determine exactly when the calcium wave rose in the spindle region. The Rh-Ran labeled chromosomes began anaphase movements between 12-16 minutes after the calcium rose in the spindle region(n=3) (Fig. 2). The timing corresponds well with anaphase onset in cell-free extracts stimulated by addition of calcium (Murray,1991; Desai et al.,1998). Experiments with Rh-tubulin and calcium green dextran gave the same results (data not shown). Rh-Ran remained associated with the chromosomes at approximately the same brightness. In later stages, Rh-Ran gradually became incorporated into the newly forming nuclear envelope(Fig. 2).

In order to image Ran throughout meiosis and mitosis, we used starfish oocytes and embryos; they are smaller than frog eggs (∼180 μm versus∼1.3 mm), are optically clear, and are easier to maintain on the microscope stage than mouse eggs. Immature starfish oocytes are arrested in late G2 of meiosis I and possess a large nucleus. Microinjected Rh-Ran, as well as GFP-Ran expressed by mRNA injection, became localized to the nuclear envelope. During maturation induced by 1-methyladenine, fluorescence at the nuclear envelope disappeared with nuclear envelope breakdown, whereas the fluorescent globules migrated to the animal pole(Fig. 3A). In oocytes co-injected with Rh-tubulin to label the microtubules, the fluorescent Ran became part of the meiotic spindle and separated at the time of polar body formation (Fig. 3B). This corresponds exactly with the known distribution and behavior of chromosomes in starfish oocytes (Shirai et al.,1990; Ookata et al.,1992).

Fluorescently labeled Ran was also observed during mitosis in developing embryos. The fluorescent nucleotide Oregon Green dUTP is incorporated into newly synthesized DNA (Carroll et al.,1999) and was used in double labeling experiments in embryonic blastomeres. There was a close correspondence of the Oregon Green dUTP-labeled chromosomes and Rh-Ran throughout the entire mitotic phase(Fig. 3C). In the mitotic cells as well, microtubules and Ran localized as expected(Fig. 3D).

When fluorescent Ran was microinjected into mitotic mammalian cells, it was found to be distributed throughout the mitotic spindle region, with no apparent accumulation on the chromosomes(Fig. 4). However, fluorescent dextran was excluded from the chromosomal regions. When a mixture of 70 kDa Rh-dextran and fluorescent Ran was injected into mitotic NRK (normal rat kidney) cells, the fluorescent dextran was excluded from the region of the chromosomes while the fluorescent Ran was not excluded(Fig. 4). If Ran were not interacting with chromosomes, it should have the same distribution as the dextran. Thus, in mitotic mammalian cultured cells, Ran appears to be associated with chromosomes, although to a lesser degree than in Xenopus, mouse or starfish eggs.

Rh-Ran in a small region of chromosomes was photobleached with intense laser excitation, and the redistribution of the remaining fluorescence was monitored. In meiosis-I-metaphase-arrested Xenopus eggs, Rh-Ran fluorescence redistributed to photobleached chromosomal regions with a 50%recovery time of 11.7±4.0 second (s.d.; n=8 eggs)(Fig. 5A,B). In blastula-stage starfish embryos, a bleached area of metaphase-chromosome-associated Rh-Ran recovered to 50% by 4.7±1.7 seconds (s.d.; n=5 cells)(Fig. 5C,D). Owing to the changing shape and position of anaphase chromosomes, we could not determine whether the exchange rates change after the metaphase-anaphase transition.

The chromosomal association of Ran was examined more quantitatively in immature starfish oocytes, where the nucleus is intact but the chromosomes are partially condensed.

The photobleached chromosomal Ran recovery was found to be described very well with an exponential rate constant ∼0.06 second^-1,corresponding to a 50% recovery time of ∼11 seconds(Fig. 6A). The experimentally observed exponential recovery is consistent with chromosomal Ran undergoing one major interaction, of the simple type described above, and also allows one to equate the exponential rate constant to the k_off. Had the experimentally observed recovery been non-exponential, it would not be possible to draw these conclusions.

For simple binding interactions, a standard procedure is to measure the bound ligand as a function of total ligand concentration; this analysis yields the dissociation constant K_d and the concentration of binding sites. Four different amounts of Rh-Ran were injected into starfish oocytes and allowed to come to equilibrium, then measurements of the brightest part of the chromosome (typically a 1-2 μm diameter region of interest) along with a neighboring nuclear region were made. In oocytes injected with 10 kDa rhodamine dextran and Alexa 488-Ran, the fluorescent dextran was just as bright in the nucleus as in regions of the chromosomal Rh-Ran labeling(Fig. 6B), leading to the conclusion that the soluble Ran can diffuse into the regions of the chromosomes. All of the chromosomes are present in a small space in mitotic cells (Fig. 4C), so it seems reasonable that the scattered individual and more extended chromosomes in immature oocytes do not exclude soluble molecules. We therefore subtracted nuclear from chromosomal fluorescence to get the value for bound Ran. As with the FRAP recovery, the relationship between bound Ran and added Ran appears to be characteristic of a single type of binding site(Fig. 6C). Fitting the curve yields a value of 30 μM for the concentration of Ran-binding sites within the space occupied by the chromosomes.

To determine the K_d, it is necessary to know the amount of free ligand that is available for binding. However, the endogenous Ran concentration in starfish oocytes is unknown. Furthermore, the fractionα of the nuclear Ran that is available for binding to chromosomes is unknown. Much of nuclear Ran is probably bound to proteins such as import/export factors while a smaller amount exists as free Ran-GTP and Ran-GDP. It is not known which of these is the chromosome-binding unit. These uncertainties do not affect the above conclusions about binding to a single type of site, or the concentration of the sites, nor do they affect the FRAP determination of k_off. Also, even with these uncertainties, it is possible to estimate the sum (K_d/α) +[R], where [R] is the total endogenous nuclear Ran and α is the fraction of the total nuclear Ran that is available for binding to chromosomes (see Materials and Methods). The data suggested that the sum of(K_d/α) + [R] was 9-10 μM. Estimates of nuclear Ran concentration in other cell types are in the range of 8-10 μM. With these values, K_d<1 μM and is smaller for smaller α.

As the recent evidence for Ran involvement in M phase is based on work in Xenopus egg extracts, we investigated Ran's distribution and dynamics in intact Xenopus eggs. Ran was localized by immunofluorescence to chromosomes of Xenopus meiosis-II-arrested eggs. Fluorescently tagged Ran localized to the chromosomes in the Xenopus eggs, as well as in starfish oocytes and embryos and mouse eggs. Anaphase was observed in artificially activated Xenopus eggs; to our knowledge, this is the first time that this has been imaged. In this system, it is necessary to image the spindle in the vertical orientation, so that the anaphase movements do not occur in one plane of focus. In starfish, the entire meiotic process and also the early mitotic divisions can be imaged with a more favorable orientation. In both Xenopus and starfish, Ran appeared to remain associated with the chromosomes to the same degree throughout M phase. In mammalian cultured cells, Ran was present throughout the spindle region. However, the condensed chromosomes exclude soluble molecules from the space they occupy. When this is taken into account, it is evident that Ran is associated with chromosomes in mammalian cultured cells, although to a lesser degree than in the other cells we observed.

An earlier report found no association of Ran with chromosomes in fixed or digitonin-permeabilized mitotic mammalian cells in culture(Ren et al., 1993). However,formaldehyde was used to fix the cells for immunofluorescence in that study,whereas we find that methanol fixation preserves the chromosomal distribution. Also, there was no energy source in the digitonin-permeabilization medium. Stably associated molecules may remain bound under these conditions, but our data indicate a dynamic association of Ran with chromosomes, and it would not be expected that Ran would remain bound to the chromosomes under these conditions.

Our findings are more in agreement with recent in vivo and in vitro work. Ran was localized by immunofluorescence at kinetochores of chromosomes in C. elegans (Bamba et al.,2002). Transient association of Ran with condensed chromatin added to Xenopus extracts was seen(Zhang et al., 1999), and Ran binding to nucleoplasmin-decondensed sperm chromatin has been demonstrated(Bilbao-Cortes et al.,2002).

It is not known what constitutes the Ran chromosomal binding unit. Ran, in its GTP or GDP form, may bind by itself, or may bind to another protein(s),which then binds to chromosomes (see further discussion below). Even with these uncertainties, photobleaching and binding experiments indicate that the association of the Ran-binding unit follows mass action kinetics to a single type of binding site of relatively high concentration within the space occupied by the chromosomes (∼30 μM) and that the k_off for binding is ∼0.06 second^-1. Because we are using a fluorescently tagged human Ran in starfish oocytes, the values for endogenous Ran may be different.

As for the chromosomal binding site for Ran, one possibility is that it is located on the protein RCC1. This protein is thought to be continuously associated with the chromosomes(Carazo-Salas et al., 1999),and since it is the only known GDP/GTP exchange factor for Ran, there must be a steady-state concentration of Ran bound to RCC1 owing to the turnover through RCC1. From in vitro studies of purified proteins, three kinetic steps are involved in the GDP/GTP exchange that occurs on RCC1; the slowest k_off, and therefore the likely rate-determining step, is ∼20 second^-1 (Klebe et al.,1995b). This is much greater than the k_off value of 0.06 second^-1 measured for Ran association with starfish chromosomes. The discrepancy suggests that most of the chromosomal Ran is involved in an interaction with another protein and that the steady-state concentration of Ran bound to RCC1 is a small fraction of the chromosomal Ran. In support of this, a significant amount of Ran can still bind to sperm chromatin depleted of RCC1 (Bilbao-Cortes et al., 2002). Another possibility is that RCC1 is regulated during mitosis by modification or association with other proteins and that its kinetic properties measured in vitro are different from those in vivo.

Recent ideas regarding Ran's role in spindle assembly had predicted a Ran-GTP gradient at the chromosomes; this gradient was recently demonstrated(Kalab et al., 2002). However,there have been no predictions of a high concentration of Ran at the chromosomes. One possibility is that the chromosomal interactions that we observe keep Ran-GTP near the chromosomes. An analogy may be made with the sarcoplasmic reticulum in striated muscle. Calcium is stored in the sarcoplasmic reticulum lumen, but the calcium-binding protein calsequestrin is only located in the terminal cisternae, near the calcium release sites(Franzini-Armstrong and Protasi,1997). In a similar way, Ran associations with chromosomes may serve to concentrate Ran-GTP in the area of the cell where it will function. This is supported by the finding that the chromatin-bound Ran increases membrane vesicle association with chromatin in an in vitro assay for nuclear envelope reformation (Bilbao-Cortes et al.,2002).

We thank Linda Runft, Jason Kirk, Susan Krueger, Nathalie Daigle, Joel Beaudoin and Virginie Hachet for help with experiments, Evelyn Houliston for advice on immunofluorescence and Alicia Smith, Petr Kalab, Ann Cowan, Iain Mattaj and Ian Macara for useful discussions. We also thank Andrew Giusti,Laurinda Jaffe, Jim Galbraith and Tom Rapoport for advice and/or critical reading of the manuscript. Supported by grants to M.T. from the Patrick and Catherine Donaghue Foundation, the Human Frontier Science Program, and the NIH(RO1-GM60389). M.M.R. was a Howard Hughes Predoctoral Fellow in the Biological Sciences. B.S. was supported by National Institutes of Health grant 1P41RR13186-01A1 for the National Resource for Cell Analysis and Modeling.