Fig. 4. Miranda moves three-dimensionally in the cytoplasm by passive diffusion, but shows a spatially limited and slower movement at the cortex. (A-E) FRAP experiments in living embryos co-expressing Miranda-GFP (green) and Histone2A-mRFP (red) or PON-GFP. White circles indicate selectively bleached regions. (A,B) Prebleach and postbleach images of prophase NBs are shown. Cytoplasmic Miranda-GFP could not be bleached on the apical (A) or basal (B) side of the cell by using the same bleaching intensity that was used to eliminate signal from cortical Miranda-GFP (E), indicative of three-dimensional diffusion. (C) Cytoplasmic Miranda-GFP and freely diffusing eGFP showed similar kinetics. Both have a significantly shorter recovery time than Miranda at the basal cortex (E). (D) Miranda-GFP at prophase and pro/metaphase. Miranda-GFP was repeatedly bleached at high laser intensity at pro/metaphase to remove signal from the NB (white circle). Subsequently, no basal crescent was detected in metaphase (white brackets), suggesting that ubiquitously localized Miranda at pro/metaphase is required to form the basal crescent at metaphase. (E) Miranda-GFP signal bleached at the basal cortical crescent (white circle) recovered at a slower rate than did cytoplasmic Miranda, suggesting that Miranda does not diffuse freely at the cortex. (F) Quantification of the relative fluorescent intensity over time shows that cortical Miranda moves slower (t_1/2=6.76±0.67 seconds) than cytoplasmic Miranda (t_1/2<1.5 seconds), but at similar rate to cortical PON-GFP (t_1/2=6.78±0.43 seconds). The recovery rates of Miranda and PON at the cortex are still fast, indicating that the two proteins show dynamic association with the cortex.