• Figure 9.
    Download figureOpen in new tabFigure 9. Comparing the kinetics of CCND1 transcriptional activation to the dynamics of β-catenin nuclear accumulation rate of change following Wnt signaling in living cells.

    (a) Plots of the average transcriptional activation kinetics of CCND1-MS2 (red) following Wnt3a activation, compared to the plot of rate of change in β-catenin nuclear accumulation (green). (b) Scheme of the dynamic changes occurring in the studied cell system following Wnt signaling. Top - from left to right: Levels of β-catenin (yellow) in the nucleus are normally low but after the addition of Wnt3a to the medium a significant and rapid increase in the nucleus is observed, peaking after 2–3 hr. β-catenin levels later decline in the nucleus and cytoplasm due to degradation. While this is the average behavior in the population (e.g. cells 1 and 2), when examining individual cells, different dynamics such as multiple pulsations (e.g. cell 3) and rapid initial accumulation (e.g. cell 4) are observed. β-catenin levels increase simultaneously at the membrane and at the centrosome. Bottom- β-catenin induces cyclin D1 transcriptional activity (green dot), and modulation of the transcriptional reaction can be observed as the gene reaches higher levels of activity, for longer periods of time. The rate of change in β-catenin accumulation (blue curve, top), rather than the actual levels of β-catenin in the nucleus, correlate with the kinetics of transcriptional activation.

    DOI: http://dx.doi.org/10.7554/eLife.16748.033

  • Video 1. Transcriptional activation of CCND1 in response to Wnt3a.

    HEK293 CCND1-MS2 cells stably expressing MS2-GFP (green) were treated with Wnt3a. The transcribed CCND1 mRNA on the active gene is seen as a bright green dot. The fluorescent signal on the active genes was enhanced using ImageJ 'Spot Enhancing Filter 2D' in order to clearly present the active sites in the movie. Cells were imaged every 15 min for 6 hr.

    DOI: http://dx.doi.org/10.7554/eLife.16748.005

  • Video 2. YFP-β-catenin dynamics at steady state and after Wnt3a activation.

    HEK293 CCND1-MS2 cells stably expressing YFP-β-catenin were treated with Wnt3a (top) and showed nuclear and cytoplasmic accumulation of YFP-β-catenin, followed by slow egress. No change in YFP-β-catenin levels was seen in mock-treated cells (bottom). Right – The YFP signal is pseudo-colored using ImageJ ‘Royal’ look-up table to show YFP-β-catenin levels. Cells were imaged every 15 min for 510 min.

    DOI: http://dx.doi.org/10.7554/eLife.16748.008

  • Video 3. YFP-β-catenin dynamics in individual cells.

    HEK293 CCND1-MS2 cells stably expressing YFP-β-catenin were treated with Wnt3a, and the dynamics of the protein were observed in individual cells. The YFP signal is pseudo-colored using ImageJ ‘Green Fire Blue’ look-up table to show YFP-β-catenin levels. Cells were imaged every 15 min for 825 min.

    DOI: http://dx.doi.org/10.7554/eLife.16748.012

  • Video 4. YFP-β-catenin dynamics in response to LiCl.

    HEK293 CCND1-MS2 cells stably expressing YFP-β-catenin were treated with LiCl and increased accumulation of the protein was observed. The YFP signal is pseudo-colored using ImageJ ‘Green Fire Blue’ look-up table to show YFP-β-catenin levels. Cells were imaged every 15 min for 825 min.

    DOI: http://dx.doi.org/10.7554/eLife.16748.013

  • Video 5. YFP-β-catenin dynamics following Wnt3a activation during cell division.

    HEK293 CCND1-MS2 cells stably expressing YFP-β-catenin were treated with Wnt3a, and the dynamics of the protein in the nucleus were followed over time. Two cells that undergo mitosis were observed in the field. The levels of the protein in the daughter cells formed from the upper cell were low (also note the appearance and division of the centrosome detected via YFP-β-catenin). In comparison, in the bottom mitotic cell, one daughter cell accumulated high YFP-β-catenin levels very rapidly, while the other responded slowly and had very low levels. The YFP signal is pseudo-colored using ImageJ ‘Fire’ look-up table to show YFP-β-catenin levels. Cells were imaged every 15 min for 225 min.

    DOI: http://dx.doi.org/10.7554/eLife.16748.016

  • Video 6. YFP-β-catenin dynamics following Wnt3a activation during the cell cycle.

    HEK293 CCND1-MS2 cells stably expressing YFP-β-catenin (yellow) and the Fucci markers for G1 (red) and G2 (cyan), were treated with Wnt3a, and the dynamics of the protein were followed over time. Cells were imaged every 15 min for 1065 min.

    DOI: http://dx.doi.org/10.7554/eLife.16748.017

  • Video 7. YFP-β-catenin dynamics following Wnt3a activation during cell division.

    HEK293 CCND1-MS2 cells stably expressing YFP-β-catenin (yellow) and the Fucci markers for G1 (red) and G2 (cyan), were treated with Wnt3a, and the dynamics of the protein were followed over time in four cells that undergo mitosis. Cells were imaged every 15 min for 705 min.

    DOI: http://dx.doi.org/10.7554/eLife.16748.018

  • Video 8. YFP-β-catenin dynamics at the cell membrane following Wnt3a activation.

    HEK293 CCND1-MS2 cells stably expressing YFP-β-catenin were treated with Wnt3a, and the dynamics of the protein at the membrane were followed over time, and were similar to the nucleus and cytoplasm accumulation. The YFP signal is pseudo-colored using ImageJ ‘Green Fire Blue’ look-up table to show YFP-β-catenin levels. Cells were imaged every 15 min for 1065 min.

    DOI: http://dx.doi.org/10.7554/eLife.16748.021

  • Video 9. YFP-β-catenin accumulation at the centrosome following Wnt3a activation.

    HEK293 CCND1-MS2 cells stably expressing YFP-β-catenin were treated with Wnt3a, and the dynamics of the protein at the centrosome were observed in parallel to the accumulation in the nucleus and cytoplasm. The separation of the centrosome in a cell during division can be seen after the 960 time point. Cells were imaged every 15 min for 1005 min.

    DOI: http://dx.doi.org/10.7554/eLife.16748.025

  • Video 10. YFP-β-catenin puncta move from the membrane to the centrosome.

    HEK293 CCND1-MS2 cells stably expressing YFP-β-catenin were treated with Wnt3a. At the 300 min time point, a series of YFP-β-catenin puncta can be tracked (track colors) moving from the membrane to the centrosome. An inverted presentation of the movie shows the movie puncta (black dots). Cell was imaged every 15 min for 1005 min.

    DOI: http://dx.doi.org/10.7554/eLife.16748.026

  • Video 11. YFP-β-catenin puncta move from the membrane to the centrosome.

    HEK293 CCND1-MS2 cells stably expressing YFP-β-catenin were treated with Wnt3a and MG132. At the 165 min time point, a series of YFP-β-catenin puncta can be tracked (track colors) moving from the membrane to the centrosome. The YFP signal is pseudo-colored using ImageJ ‘Green Fire Blue’ look-up table to show YFP-β-catenin levels. Cells were imaged every 15 min for 1065 min.

    DOI: http://dx.doi.org/10.7554/eLife.16748.027

  • Video 12. Prolonged activation of CCND1 after Wnt activation.

    HEK293 CCND1-MS2 cells stably expressing MS2-GFP (green) were treated with Wnt3a. CCND1 mRNA transcription could be detected 15 min after Wnt3a (green dot, transcription site) and continued for 4 hr. Cells were imaged every 15 min for 270 min.

    DOI: http://dx.doi.org/10.7554/eLife.16748.030

  • Supplementary file 1.Statistical analysis performed in this study.

    (a) The statistical significance p values (t test) at each time point for the percentage of cells showing an active CCND1-MS2 gene (refers to Figure 1b) between control and Wnt3a-treated cells. (b-f) Mann-Whitney test for comparison between two independent FRAP/FLIP experiments. A statistical comparison between all datasets of two individual FRAP/FLIP experiments are depicted in each plot and are illustrated as a single red circle which marks the p-value (y axis) for all intensity values measured for each time point (x axis). The top and bottom dotted lines indicate where p-value equals 0.05. (b) Statistically significant difference between the FRAP dynamics of YFP-β-catenin in the nucleus under Wnt3a treatment versus overexpression of YFP-β-catenin that enters the nucleus without signal, and (c) between the FRAP and (d) FLIP import and export dynamics (refers to Figure 2—figure supplement 1). (e) No statistically significant difference between YFP-β-catenin at the cell membrane between mock-treated and Wnt3a-treated cells (refers to Figure 5—figure supplement 1). (f) No statistically significant difference between YFP-β-catenin at the cell membrane between mock-treated and LiCl-treated cells (refers to Figure 5—figure supplement 1).

    DOI: http://dx.doi.org/10.7554/eLife.16748.034

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