Figure 3

Restricted mobility of GFP-Dnmt1S in the cytoplasm of mouse preimplantation embryos. A) The GFP-Dnmt1S expression construct was microinjected in 1-cell stage embryos and a portion of the cytoplasm of one blastomere was bleached at the 2-cell stage. The upper row shows bleaching of a living embryo while the lower row shows a fixed control. Note that only regions immediately adjacent to the bleached area show decreased fluorescence. A very sharp bleaching boundary in fixed controls shows that such decrease is not due to poor sharpness of the bleaching beam, but to diffusion of GFP-Dnmt1S from adjacent sites. B) Salt extraction of endogenous Dnmt1S from 2-cell embryos. Soluble (S) and insoluble (P) fractions were analysed by immunoblotting with an anti-Dnmt1 antibody. C) Localisation dependent mobility of Dnmt1S in 1-cell embryos. A square bleached area (indicated) including a small fraction of the male pronucleus (outlined) was produced in a 1-cell embryo microinjected with the GFP-Dnmt1S construct. After bleaching no fluorescence remained in the entire pronucleus, while in the cytoplasm fluorescence was depleted only within and in proximity of the bleached area, indicating that the mobility of GFP-Dnmt1S is specifically restricted in the cytoplasm. Insets on the right show magnifications of the bleached area at the indicated time points. A) and C) show optical sections obtained by confocal microscopy (scale bars = 20 μm).