Figure 5. R2 and B regulate APC2 dynamics in the destruction complex, and regulate βcat removal from the destruction complex.
(A) APC2 mutants. (B–F) SW480 cells transfected with Axin-RFP and indicated GFP-APC2 constructs (B) FRAP assay. Deleting either R2 or B slows APC2's turnover in Axin puncta. (C–E) Axin-RFP, GFP-APC2 constructs, βcat (inset = boxes). (C) βcat is essentially undetectable in APC2:Axin puncta (arrows). (D) βcat accumulates in APC2ΔR2:Axin puncta (arrows). (E) βcat strongly accumulates in APC2ΔB:Axin puncta (arrows). (F) Deletion of R2 or B impair the ability of APC2 to aid Axin in reducing βcat fluorescent intensity.
Figure 5—figure supplement 1. Colocalization of APC2's Arm repeat domain with Axin is controlled by R2 and B and APC2 without R2 or region B still stabilizes Axin complexes.
(A) Diagram of APC2 constructs. (B) GFP-APC2 and Axin-RFP colocalize with one another in SW480 cells and reduce βcat levels (inset = box in B). (C) Deleting APC2's SAMPs reduces colocalization with Axin (inset = box in C). (D) Deleting R2 from APC2ΔSAMPs enhances colocalization with Axin (inset = box in D). (E) APC2ΔSAMPs lacking B colocalizes strongly with the Axin (inset = box in E). (F) Puncta area of APC2ΔR2 + Axin and APC2ΔB + Axin is similar to wildtype APC2 + Axin and differs from puncta formed by Axin alone. Analysis of confocal images, total of n = 30 cells. Student's t-test was used. (G) Wildtype APC2, APC2∆R2 and APC2∆B all reduce Axin puncta number. Quantitation of puncta number of confocal images used in (F). (H) Comparison of puncta volumes using SIM images reveals that APC2ΔR2 + Axin and APC2ΔB + Axin form puncta that are in between those formed by Axin alone and those formed by APC2 + Axin puncta. Volumes across puncta population. Axin (n = 3), APC2 + Axin (n = 11), APC2ΔR2 + Axin (n = 6), and APC2ΔB + Axin (n = 9). ANOVA-Bonferroni was used. (I,J) APC2ΔR2 + Axin and APC2ΔB + Axin puncta maintain complex internal structure. Closeups of puncta of SIM high resolution images of APC2ΔB + Axin (I) and APC2ΔR2 + Axin expressed in SW480 cells. (K) Deleting R2 or B does not significantly diminish the ability of APC2 to stabilize Axin in the destruction complex.