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Self-association of the APC tumor suppressor is required for the assembly, stability, and activity of the Wnt signaling destruction complex.

Kunttas-Tatli E, Roberts DM, McCartney BM - Mol. Biol. Cell (2014)

Bottom Line: The destruction complex forms macromolecular particles we termed the destructosome.Here we show that a novel N-terminal coil, the APC self-association domain (ASAD), found in vertebrate and invertebrate APCs, directly mediates self-association of Drosophila APC2 and plays an essential role in the assembly and stability of the destructosome that regulates β-cat degradation in Drosophila and human cells.These results suggest that APC proteins are required not only for the activity of the destructosome, but also for the assembly and stability of this macromolecular machine.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213.

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APC2 self-association stabilizes the destructosome. (A–C) Plots of ΔF/F for each condition. Similar-sized Axin-GFP puncta were selected, and the recovery of individual bleached spots is shown in unique colors for each condition. Black lines are regression lines. Regression analysis indicates that the relationship between time and fluorescence varies by condition. ANOVA for regression lines, p < 0.0001; Tukey–Kramer HSD posthoc test for each pair, p < 0.05. (D) To compare the rate of recovery, we calculated the slope of the regression line for each individual sample and compared the means of these slopes for each condition. Means are plotted with SEM whiskers; Tukey–Kramer HSD posthoc test for each pair, p < 0.05. (E) To compare the difference in mobile and immobile fractions at the end of the experiment, time-zero normalized degree of recovery at our last time point (384.12 s) for the three conditions was determined. Means are plotted with SEM whiskers; Tukey–Kramer HSD posthoc test for each pair, p < 0.05. (F) We observed a significant difference in the number of puncta merging and separating events (a measure of puncta dynamics) between Axin-GFP/APC2-ΔASAD puncta compared with Axin-GFP or Axin-GFP/APC2-FL puncta. This behavior is rarely observed in Axin-GFP and Axin-GFP/APC2-FL puncta. Scale bar, 2 μm. Student's t test, p < 0.001 between the mutant and either of the other conditions. (G) Axin-GFP/APC2-ΔASAD puncta are highly dynamic. The white arrow tracks the merging of two puncta. Time stamp in minutes:seconds.
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Figure 5: APC2 self-association stabilizes the destructosome. (A–C) Plots of ΔF/F for each condition. Similar-sized Axin-GFP puncta were selected, and the recovery of individual bleached spots is shown in unique colors for each condition. Black lines are regression lines. Regression analysis indicates that the relationship between time and fluorescence varies by condition. ANOVA for regression lines, p < 0.0001; Tukey–Kramer HSD posthoc test for each pair, p < 0.05. (D) To compare the rate of recovery, we calculated the slope of the regression line for each individual sample and compared the means of these slopes for each condition. Means are plotted with SEM whiskers; Tukey–Kramer HSD posthoc test for each pair, p < 0.05. (E) To compare the difference in mobile and immobile fractions at the end of the experiment, time-zero normalized degree of recovery at our last time point (384.12 s) for the three conditions was determined. Means are plotted with SEM whiskers; Tukey–Kramer HSD posthoc test for each pair, p < 0.05. (F) We observed a significant difference in the number of puncta merging and separating events (a measure of puncta dynamics) between Axin-GFP/APC2-ΔASAD puncta compared with Axin-GFP or Axin-GFP/APC2-FL puncta. This behavior is rarely observed in Axin-GFP and Axin-GFP/APC2-FL puncta. Scale bar, 2 μm. Student's t test, p < 0.001 between the mutant and either of the other conditions. (G) Axin-GFP/APC2-ΔASAD puncta are highly dynamic. The white arrow tracks the merging of two puncta. Time stamp in minutes:seconds.

Mentions: For these FRAP experiments, we chose similarly sized puncta for each condition and kept the bleached area constant. For the ∆ASAD mutant, where the puncta can be interconnected at higher expression levels, we chose cells with relatively low expression, for which we could see isolated, individual puncta. To compare the Axin-GFP fluorescence recovery among the three conditions, we first normalized for the starting postbleach fluorescence by calculating ∆F/F for each time point in each condition and plotted this over time (Figure 5, A–C). To compare the rates of recovery, we compared the mean slope of the regression lines for each condition (Figure 5D). Axin-GFP/APC2-∆ASAD puncta displayed a significantly greater slope than either Axin-GFP alone or Axin-GFP/APC2-FL puncta. Conversely, Axin-GFP/APC2-FL puncta exhibited a significantly reduced slope compared with the other conditions (Figure 5D). Axin-alone puncta exhibited the greatest variation in rate of recovery; some puncta displayed Axin-GFP/APC2-FL–like properties, whereas others exhibited Axin-GFP/APC2-∆ASAD–like properties (Figure 5A). Cells expressing Axin-GFP alone express significantly more Axin than the low level of endogenous APC2 in these cells (Zhou and McCartney, unpublished data). This suggests that at a high Axin:APC2 ratio, Axin turnover rates are not well controlled and fluctuate as a consequence. When the Axin:APC2 ratio is closer to 1, as in the case of Axin-GFP/APC2-FL puncta, Axin-GFP is stabilized, and the overall rates of recovery decrease significantly (Figure 5D). On the other hand, disrupting APC2 self-association appears to drive Axin-GFP toward the opposite end of its dynamic spectrum (Figure 5D).


Self-association of the APC tumor suppressor is required for the assembly, stability, and activity of the Wnt signaling destruction complex.

Kunttas-Tatli E, Roberts DM, McCartney BM - Mol. Biol. Cell (2014)

APC2 self-association stabilizes the destructosome. (A–C) Plots of ΔF/F for each condition. Similar-sized Axin-GFP puncta were selected, and the recovery of individual bleached spots is shown in unique colors for each condition. Black lines are regression lines. Regression analysis indicates that the relationship between time and fluorescence varies by condition. ANOVA for regression lines, p < 0.0001; Tukey–Kramer HSD posthoc test for each pair, p < 0.05. (D) To compare the rate of recovery, we calculated the slope of the regression line for each individual sample and compared the means of these slopes for each condition. Means are plotted with SEM whiskers; Tukey–Kramer HSD posthoc test for each pair, p < 0.05. (E) To compare the difference in mobile and immobile fractions at the end of the experiment, time-zero normalized degree of recovery at our last time point (384.12 s) for the three conditions was determined. Means are plotted with SEM whiskers; Tukey–Kramer HSD posthoc test for each pair, p < 0.05. (F) We observed a significant difference in the number of puncta merging and separating events (a measure of puncta dynamics) between Axin-GFP/APC2-ΔASAD puncta compared with Axin-GFP or Axin-GFP/APC2-FL puncta. This behavior is rarely observed in Axin-GFP and Axin-GFP/APC2-FL puncta. Scale bar, 2 μm. Student's t test, p < 0.001 between the mutant and either of the other conditions. (G) Axin-GFP/APC2-ΔASAD puncta are highly dynamic. The white arrow tracks the merging of two puncta. Time stamp in minutes:seconds.
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Related In: Results  -  Collection

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Figure 5: APC2 self-association stabilizes the destructosome. (A–C) Plots of ΔF/F for each condition. Similar-sized Axin-GFP puncta were selected, and the recovery of individual bleached spots is shown in unique colors for each condition. Black lines are regression lines. Regression analysis indicates that the relationship between time and fluorescence varies by condition. ANOVA for regression lines, p < 0.0001; Tukey–Kramer HSD posthoc test for each pair, p < 0.05. (D) To compare the rate of recovery, we calculated the slope of the regression line for each individual sample and compared the means of these slopes for each condition. Means are plotted with SEM whiskers; Tukey–Kramer HSD posthoc test for each pair, p < 0.05. (E) To compare the difference in mobile and immobile fractions at the end of the experiment, time-zero normalized degree of recovery at our last time point (384.12 s) for the three conditions was determined. Means are plotted with SEM whiskers; Tukey–Kramer HSD posthoc test for each pair, p < 0.05. (F) We observed a significant difference in the number of puncta merging and separating events (a measure of puncta dynamics) between Axin-GFP/APC2-ΔASAD puncta compared with Axin-GFP or Axin-GFP/APC2-FL puncta. This behavior is rarely observed in Axin-GFP and Axin-GFP/APC2-FL puncta. Scale bar, 2 μm. Student's t test, p < 0.001 between the mutant and either of the other conditions. (G) Axin-GFP/APC2-ΔASAD puncta are highly dynamic. The white arrow tracks the merging of two puncta. Time stamp in minutes:seconds.
Mentions: For these FRAP experiments, we chose similarly sized puncta for each condition and kept the bleached area constant. For the ∆ASAD mutant, where the puncta can be interconnected at higher expression levels, we chose cells with relatively low expression, for which we could see isolated, individual puncta. To compare the Axin-GFP fluorescence recovery among the three conditions, we first normalized for the starting postbleach fluorescence by calculating ∆F/F for each time point in each condition and plotted this over time (Figure 5, A–C). To compare the rates of recovery, we compared the mean slope of the regression lines for each condition (Figure 5D). Axin-GFP/APC2-∆ASAD puncta displayed a significantly greater slope than either Axin-GFP alone or Axin-GFP/APC2-FL puncta. Conversely, Axin-GFP/APC2-FL puncta exhibited a significantly reduced slope compared with the other conditions (Figure 5D). Axin-alone puncta exhibited the greatest variation in rate of recovery; some puncta displayed Axin-GFP/APC2-FL–like properties, whereas others exhibited Axin-GFP/APC2-∆ASAD–like properties (Figure 5A). Cells expressing Axin-GFP alone express significantly more Axin than the low level of endogenous APC2 in these cells (Zhou and McCartney, unpublished data). This suggests that at a high Axin:APC2 ratio, Axin turnover rates are not well controlled and fluctuate as a consequence. When the Axin:APC2 ratio is closer to 1, as in the case of Axin-GFP/APC2-FL puncta, Axin-GFP is stabilized, and the overall rates of recovery decrease significantly (Figure 5D). On the other hand, disrupting APC2 self-association appears to drive Axin-GFP toward the opposite end of its dynamic spectrum (Figure 5D).

Bottom Line: The destruction complex forms macromolecular particles we termed the destructosome.Here we show that a novel N-terminal coil, the APC self-association domain (ASAD), found in vertebrate and invertebrate APCs, directly mediates self-association of Drosophila APC2 and plays an essential role in the assembly and stability of the destructosome that regulates β-cat degradation in Drosophila and human cells.These results suggest that APC proteins are required not only for the activity of the destructosome, but also for the assembly and stability of this macromolecular machine.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213.

Show MeSH
Related in: MedlinePlus