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.

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