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.

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