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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