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First published online March 22, 2006
doi: 10.1242/10.1242/jcs.02864

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Nucleo-cytoplasmic distribution of ß-catenin is regulated by retention

Nikolaus-Fiebiger-Center for Molecular Medicine, University of Erlangen-Nürnberg, Glückstr. 6, 91054 Erlangen, Germany

View larger version (35K): [in a new window]   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.

View larger version (24K): [in a new window]   Fig. 2. TCF4 slows down ß-catenin in the nucleus and decelerates its nucleo-cytoplasmic shuttling. (A) Live cell images of mYFP-ß-cateninS45A and co-expressed TCF4-mCFP in HEK293T cells. Bar, 10 µm. (B) Compartment bleach analysis of ß-catenin shuttling in the absence or presence of co-expressed TCF4-mCFP. (C) Nuclear point bleach analysis of mYFP-ß-catenin mobility in the absence or presence of TCF4-mCFP. TCF4-mYFP mobility was determined in a separate experiment. The cartoons in B and C designate compartment bleaches and point bleaches, respectively.

View larger version (28K): [in a new window]   Fig. 3. BCL9 slows down ß-catenin nucleo-cytoplasmic shuttling. (A) Live cell images of mYFP-ß-cateninS45A and co-transfected mCFP-BCL9v in the absence (left panels) or presence (right panels) of mRFP-Pygopus2. Bar, 10 µm. (B) Compartment bleach analysis of mYFP-ß-catenin shuttling in the absence or presence of mCFP-BCL9v and mRFP-Pygopus2. mYFP-BCL9v shuttling was analysed in a separate experiment. (C) Nuclear point bleach analysis of mYFP-ß-catenin in the absence or presence of mCFP-BCL9v and mRFP-Pygopus2. The mobilities of mYFP-BCL9v and mYFP-Pygopus were determined in separate experiments. The cartoons in B and C designate compartment bleaches and point bleaches, respectively.

View larger version (27K): [in a new window]   Fig. 4. APC slows down ß-catenin in the cytoplasm and slightly decelerates ß-catenin nucleo-cytoplasmic shuttling. (A) Live cell images of mYFP-ß-cateninS45A and co-expressed mCFP-APC. The tip of a protrusion where APC and ß-catenin co-accumulate is indicated by an arrow. Bar, 10 µm. (B) Compartment bleach analysis of mYFP-ß-cateninS45A shuttling in the absence or presence of mCFP-APC. (C) Cytoplasmic point bleach analysis of mYFP-ß-cateninS45A in the absence or presence of mCFP-APC. mYFP-APC mobility was determined in a separate experiment. Note the low mobility of APC and ß-cateninS45A at the tips of cellular protrusions (tips) as compared to the cell body (cb). The cartoons in B and C designate compartment bleaches and point bleaches, respectively.

View larger version (29K): [in a new window]   Fig. 5. Axin and axin2 slow down ß-catenin in the cytoplasm and do not accelerate ß-catenin nucleo-cytoplasmic shuttling. (A) Live cell images of mYFP-ß-cateninS45A (upper panels) and co-expressed mCFP-tagged axin, axin2 or axinC (lower panels). Bar, 10 µm. (B) Compartment bleach analysis of mYFP-ß-catenin in the absence or presence of axin, axin2 or axinC. (C) Cytoplasmic point bleach analysis of mYFP-ß-catenin in the absence or presence of axin, axin2 or axinC. The mobilities of mYFP-tagged axin, axin2 and axinC were determined in separate experiments. The cartoons in B and C designate compartment bleaches and point bleaches, respectively.

View larger version (38K): [in a new window]   Fig. 6. APC and axin mediate ß-catenin cytoplasmic accumulation by a CRM-1 independent mechanism. (A) Immunofluorescence staining of endogenous ß-catenin (first two panels) and live cell images of HEK293T cells expressing mYFP-ß-cateninS45A or mYFP-ß-cateninNES in the presence of the indicated interactors (other panels) before and after 1 hour of leptomycin B treatment. Bars, 10 µm. (B) Compartment bleach analysis of mYFP-ß-catenin shuttling before and after 1 hour of leptomycin B treatment. The cartoon designates compartment bleaches.

View larger version (30K): [in a new window]   Fig. 7. Wnt signalling does not influence ß-catenin nucleo-cytoplasmic shuttling. (A) Immunofluorescence staining of endogenous ß-catenin (upper panels) in HEK293T cells in the absence or presence of mRFP-tagged Wnt-3a or dominant active LRP6 (LRP6da). Lower panels depict the fluorescence of the RFP-tagged activators in the same microscopic field as in the upper panels. Bar, 10 µm. (B) Western blot of hypotonic lysates of HEK293T cells transfected with mYFP-ß-catenin and the indicated activators, stained for ß-catenin (top) and ß-actin (bottom). (C) TOPglow reporter assay with mYFP-ß-catenin and indicated interactors. Fold changes were determined by calculating ratios of relative luciferase activities of TOP- to FOP samples and normalizing to the mYFP control. Error bars indicate standard deviations. (D) Compartment bleach analysis of mYFP-ß-catenin in the absence or presence of dominant active LRP6 or Wnt3a. The cartoon designates compartment bleaches.

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