Fig. 1. ß-catenin shuttles efficiently between nucleus and cytoplasm and is mobile in both compartments. (A) Time-lapse microscopy of HEK293T cells transfected with mYFP-ß-catenin during compartment bleach experiments. After one initial image, the entire cytoplasm (top) or the entire nucleus (bottom) was bleached and fluorescence recovery was monitored. Bars, 10 µm. (B) Mean recovery curves from mYFP-ß-catenin-expressing cells bleached in the nucleus or in the cytoplasm. The ratio of the fluorescence in the bleached compartment to that of the whole cell prior to photobleaching was set at 100%. The identical recovery kinetics in the nucleus and cytoplasm indicate that ß-catenin is shuttling at an equilibrium. The cartoons in B-E designate compartment bleaches. (C) Compartment bleach analysis of cells expressing moderate amounts of mYFP-ß-catenin as used for all further analyses or five times higher levels. Inset: average total cell fluorescence in both groups. (D) Compartment bleach analysis comparing the shuttling kinetics of mYFP-ß-catenin or mYFP-ß-cateninS45A (120 kDa) and passively diffusing controls mYFP (30kD) and a YFP-CFP fusion protein (60 kDa). (E) Compartment bleach analysis of ß-catenin deletion mutants and ß-catenin fusion proteins with an IB NES alone or a MAPKK consensus NES in combination with the SV40 NLS that are actively shuttled by the exportin and the importin/exportin system, respectively. (F,G) Point bleach analysis of mYFP-ß-catenin mobility in the cytoplasm (F) or the nucleus (G). A small area within one compartment (indicated by a circle) was bleached and the recovery relative to the whole compartment was monitored. Cartoons in F and G designate cytoplasmic or nuclear point bleaches. Total monitoring time was 45 seconds compared to 15 minutes in the compartment bleach analyses, due to much higher mobility of ß-catenin within than between the compartments.