Figure 4. APC2’s Arm rpts and SAMPs each are required to stabilize APC2:Axin complexes.
(A) APC2 mutants. (B and C) FRAP analyses, SW480 cells. (B) APC2 needs both the Arm rpts and SAMPs to robustly associate with Axin puncta. Student's t-test. (C) Axin stabilization by APC2 is abolished when either APC2's Arm rpts or SAMPs are deleted. (D,K) SIM super-resolution images, SW480 cells expressing indicated constructs. (D–F) GFP-APC2ΔArm and Axin-RFP. (E–F) Close-ups, X–Y slice. Axin structure resembles complexes formed by Axin alone. (G and H) Axin-RFP puncta and APC2:Axin puncta for comparison. (I–K) GFP-APC2ΔSAMPs and Axin-RFP. (J,K) Close-ups. Axin does not form a complex internal structure when APC2 ΔSAMPs is expressed. (L) Puncta volume in SIM images of Axin (n = 3 cells), APC2 + Axin (n = 11), APC2ΔArm + Axin(n = 9), and APC2ΔSAMPs + Axin (n = 5) expressing cells. Deleting either the Arm rpts or the SAMPs inhibits APC2's ability to enhance puncta volume. ANOVA-Bonferroni. (M) Puncta area, confocal images. Area differences are consistent with volumes in (L). Student's t-test. (N) Puncta number, confocal images. Deleting Arm rpts or SAMPs in APC2 fails to decrease number of APC2:Axin puncta as does wildtype APC2. (O) APC mutants lacking the Arm rpts or SAMP motif show decreased ability to reduce βcat levels in SW480 cells. Quantification, total cell βcat fluorescent intensity normalized to untransfected cells.