Figure 6. Accumulation of β-catenin at the centrosome after Wnt3a activation.

(a) Frames from time-lapse Video 9 showing YFP-β-catenin accumulation at the centrosome (white arrowheads) and after cell division. Bar = 10 μm. (b) The colocalization (white arrowheads) of YFP-β-catenin (top) or endogenous β-catenin (bottom) with the centrosomal marker pericentrin (red immunofluorescence) in untreated and LiCl-treated cells. Hoechst DNA stain is in blue, and DIC in grey. Boxes show enlarged centrosomal areas. Bar = 10 μm. (c) The relative average intensity of YFP-β-catenin measured in the centrosome (n = 11), membrane, cytoplasm and nucleus (from Figure 2 and 5) of Wnt3a-treated cells. Correlation scores (r) between the nucleus (n), cytoplasm (c), membrane (m) and centrosome (ce) YFP-β-catenin levels are presented at the bottom. (d) The rate of change in YFP-β-catenin levels (ΔI/Δt) accumulation or degradation in the centrosome, membrane, cytoplasm and nucleus over time in Wnt3a- and LiCl-treated cells. (e) Plots of YFP-β-catenin levels in the sub-cellular compartments of individual cells (from Figure 3). Boxes show the correlation scores (r) between the nucleus (n), cytoplasm (c), membrane (m) and centrosome (ce).

DOI: http://dx.doi.org/10.7554/eLife.16748.022

Figure 6—figure supplement 1. Detachment of membranal YFP-β-catenin puncta and movement towards the centrosome.

(a) Frames from Video 10 showing the tracks of several YFP-β-catenin membranal puncta (colored tracks) moving from the membrane region towards the centrosome area (red circle). Time is minutes after addition of Wnt3a. YFP signal is shown in negative greyscale colors. Bar = 10 μm. (b) Maximum time projections of movements of membranal YFP-β-catenin puncta (arrows) towards the centrosome region (circles) in four different cells. The YFP signal is pseudo-colored using the ImageJ ‘Green Fire Blue’ look-up table. Top row are Wnt3a-treated cells. Bottom row are Wnt3a + MG132-treated cells. Bar = 10 μm.

DOI: http://dx.doi.org/10.7554/eLife.16748.023

Figure 6—figure supplement 2. Summary of FRAP measurements of YFP-β-catenin dynamics in subcellular compartments in response to Wnt3a treatment.

(a) Frames showing one pre-bleach frame, the bleach of the YFP-β-catenin in the centrosome of a Wnt3a-treated cell, and frames following the recovery of signal over time. Circle denotes the bleached region. Bar = 10 μm. (b) Averaged data plot of FRAP recovery curves in the cytoplasm (n = 24), nucleus (n = 25), membrane (n = 32) and centrosome (n = 13). Membrane recovery from Figure 5—figure supplement 1 and the import rate to nucleus from Figure 2—figure supplement 1 are also plotted.

DOI: http://dx.doi.org/10.7554/eLife.16748.024