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doi: 10.1242/10.1242/jcs.00136

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Chromosomal association of Ran during meiotic and mitotic divisions

¹ Department of Physiology, University of Connecticut Health Center, Farmington, CT 06032, USA
² Marine Biological Laboratory, Woods Hole, MA 02543, USA
³ Department of Cell Biology and Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02115, USA
⁴ European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany

View larger version (73K): [in a new window]   Fig. 1. Ran associates with the chromosomes. (A) In meiosis-II-metaphase-arrested Xenopus eggs, double-labeling of Ran by immunofluorescence and chromosomes by Hoechst dye shows colocalization of Ran and chromosomes. (B) Microinjected Rh-Ran is associated with the chromosomes in Xenopus eggs. (C) Microinjected Rh-Ran is associated with chromosomes in mature mouse eggs. The dark circular region in the egg is the oil drop from the injection. Bars, 10 µm.

View larger version (47K): [in a new window]   Fig. 2. Ran localization in activated Xenopus eggs. Xenopus oocytes were co-injected with calcium green dextran (15 µM final concentration) and Rh-Ran (1.2 µM) then were matured. (A) The matured eggs were prick activated to initiate a calcium wave that releases the meiosis II arrest. The calcium green image shown was taken a few seconds after the calcium wave had passed through the spindle region. The calcium wave is progressing from right to left. The bright circular region is the location of the meiotic spindle; it is bright because of the lack of volume-excluding yolk platelets. (B) Rh-Ran in the meiotic spindle of the egg in the preceding panel was imaged at high magnification. Since the spindle is oriented perpendicularly to the cell surface, it was necessary to take z series sequences to document anaphase movements. z series with a step of 3.1 µm were taken at approximately 2 minute intervals (each series of 14 images took 35 seconds to obtain) and were made into stereo pairs. Timing of the pairs shown is indicated on the figure. Anaphase movements appear to begin between 13 and 15 minutes. Nuclear envelopes reform around individual chromosomes after they have separated (Lemaitre et al., 1998); these should later fuse to form a single nucleus but this was not imaged. See stereo movie http://terasaki.uchc.edu/ran/anaphase.mov. Bars, 10 µm.

View larger version (129K): [in a new window]   Fig. 3. Ran localizes to the chromosomes during meiosis and mitosis in starfish oocytes and embryos. (A) GFP-Ran was expressed in immature starfish oocytes by injection of mRNA. Oocytes were imaged before and during maturation; time elapsed after addition of the maturation hormone 1-methyladenine is indicated. In immature oocytes, GFP-Ran is located at the nuclear envelope. In addition, it is accumulated at globules inside the nuclear envelope. These globules move to the animal pole during maturation. Bar, 25 µm. (B) Double-labeling of Ran and microtubules during meiosis I. GFP-Ran RNA and rhodamine tubulin (5 µM final concentration in cell) were co-injected; after expression of GFP-Ran, the oocyte was matured and imaged. The light/dark border running diagonally is the surface of the oocyte near the animal pole seen from the side, and the sequence shows the extrusion of the first polar body. The location of GFP-Ran with respect to the meiotic spindle is consistent with association with the chromosomes. Bar, 10 µm. (C) Double-labeling of Ran and chromosomes during mitosis. Oocytes were co-injected with Rh-Ran (1.4 µM final concentration in the cell) and Oregon Green 488-5-dUTP (5 µM final concentration in cell), which becomes incorporated into newly synthesized DNA. The oocytes were matured, fertilized and allowed to develop into blastulae, where mitotic divisions were imaged. Rh-Ran appears to colocalize with the DNA label through all stages of mitosis. See movie http://terasaki.uchc.edu/ran/randutp.mov. (D) Double-labeling of Ran and microtubules during mitosis. Oocytes were co-injected with GFP-Ran mRNA and rhodamine tubulin as in (B), then matured, fertilized and allowed to develop into blastulae. Bar, 10 µm.

View larger version (106K): [in a new window]   Fig. 4. Association of Ran with chromosomes in a cultured mammalian cell. A mitotic NRK cell was injected with Alexa 488 Ran and 70 kDa Rh dextran. (A) The position of the chromosomes is seen in a scanning transmission light image. (B) 70 kDa Rh dextran is excluded from the region of the chromosomes. (C) Fluorescent Ran is present throughout the spindle region, and in particular, in the region of the chromosomes. Since a non-interacting molecule should have a similar distribution to the fluorescent dextran, this is evidence for a Ran association with chromosomes. Bar, 10 µm.

View larger version (75K): [in a new window]   Fig. 5. Dynamics of Rh-Ran. (A) Microinjected Rh-Ran in a Xenopus egg was photobleached in the rectangular region shown in the upper left panel, and the recovery was followed in images taken every 4.7 seconds. The photobleach consisted of eight consecutive slow scans lasting 24.8 seconds in the outlined rectangular region, with 750x the light intensity as for imaging. As seen in the first post-bleach image, the chromosomes and cytosol became dimmer outside as well as within the bleach zone. This is likely to be a consequence of exchange of cytosolic Rh-Ran with chromosomal Rh-Ran. Bar, 10 µm. (B) Time course of fluorescence recovery of one of the chromosomes within the bleached region. (C) Rh-Ran was microinjected into immature starfish oocytes (final concentration of 1-2 µM). The oocytes were matured, fertilized and allowed to develop to the blastula stage (128-256 cells), where mitotic cells were imaged. Rh-Ran associated with metaphase chromosomes was photobleached in a similar manner as described for Xenopus eggs, except the `normal' scan speed of the BioRad confocal microscope was used. The bleach period was eight consecutive scans lasting 8.5 seconds, and images were obtained afterwards every 2.5 seconds; timing of the images shown are indicated on the figure. Bar, 10 µm. (D) Fluorescence recovery of a photobleached region of the metaphase chromosomes. As in Xenopus, the parts of the chromosomes that were not irradiated were dimmer immediately after photobleaching. In contrast to Xenopus (Fig. 5B), the fluorescence does not return to the original value. The likely explanation is that the bleaching protocol depletes a very small fraction of the total Ran in frog oocytes but depletes a large fraction in the much smaller starfish blastomeres.

View larger version (29K): [in a new window]   Fig. 6. Steady-state Ran association with chromosomes in immature starfish oocytes. (A) Recovery from photobleaching. Fluorescence from the brightest part of a chromosome was measured and is plotted by open squares whereas the nuclear fluorescence is denoted by filled triangles. The 0 second time point corresponds to the first post-bleach image where the nuclear and chromosomal fluorescence are set to 0 (symbols not shown at this time point because they would overlap). The fluorescence recovery at subsequent time points is equal to the increase in fluorescence relative to the fluorescence in the first post-bleach image. The nuclear fluorescence has already recovered by the second post-bleach image, and clearly recovers faster than the chromosomal fluorescence. If the chromosomal recovery is not limited by diffusion of soluble Ran, and if the interaction follows mass action kinetics with a single type of binding site, the chromosomal recovery should be exponential. The theory line shows an exponential recovery with rate constant 0.06 second-1. (B) Oocytes were co-injected with Alexa 488 Ran (7 µM final concentration) and 10 kDa rhodamine dextran (100 µg/ml) and were imaged separately with either the 488 nm line or the 568 nm line in the confocal microscope. The nucleus takes up most of the image, with the cytoplasm in the upper left corner of each image. The images show that the dextran is not excluded from the region of the chromosomes, probably because the chromosomes are not fully condensed in the meiosis-I-prophase-arrested oocytes. In other experiments, oocytes were injected with 10 kDa dextran alone, and z series sections failed to detect signs of volume exclusion. The lack of exclusion indicates that the nuclear fluorescence should be subtracted from the chromosomal fluorescence to get a more accurate value for the bound Ran. Bar, 10 µm. (C) Binding curve for chromosomal Ran for different amounts of injected fluorescent Ran. The theory line shows the predicted values for the concentration of binding sites in the chromosomal space of 30 µM; the close correspondence supports the idea that the interaction is with a single type of binding site.

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