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Fig. 4. Only APC in the 10S complex binds to microtubules, binding to ß-catenin and microtubules are inversely related functions of APC. (A) Fractions corresponding to the 10S and 23S complexes from control HeLa cells and HeLa cells treated with SB216763 for 12 hours as indicated were incubated with control buffers (no MTs) or taxol stabilised microtubules (+ MTs). Microtubules and associated proteins (Pel) were separated from unbound material (Sup) by sedimentation through a glycerol cushion and subjected to PAGE. The top part of each gel was transferred to nitrocellulose, followed by immunoblotting with antibodies against APC and ß-catenin. The bottom part of each gel was stained with Coomassie blue to show the distribution of tubulin. Upon addition of polymerised tubulin only APC in the 10S complex was detected bound to microtubules. ß-Catenin was not recovered bound to microtubules. (B) Total cell lysates from HeLa control cells or SB-treated HeLa cells, or DLD-1 and HCT 116 cells were incubated with control buffer (no MTs) or taxol stablised microtubules (+ MTs). Microtubules and associated proteins were recovered by centrifugation through a glycerol cushion and probed with antibodies against APC. APC co-sedimented specifically with microtubules in HeLa cells, only slightly in DLD-1 cells and extremely poorly in HCT 116 cells. (C) SW480 cells were co-transfected with GFP or GFP-tagged APC constructs and a TOP reporter plasmid carrying a TCF-specific promoter, or the FOP plasmid, a control plasmid carrying a mutated promoter, for 48 hours. The APC constructs encoded the following proteins: full length APC, APC lacking the direct microtubule binding site (residues 2168-2451) (APCdeltaMT), APC4, a fragment lacking the N-terminal 1000 amino acids, or APC4 lacking the direct microtubule binding site (APC4deltaMT), M-APC (residues 1014-2038) that have been described in detail (Zumbrunn et al., 2001), or GFP. Luciferase activity was measured in FOP- and TOP-transfected cells and the TOP:FOP ratio was calculated to control for transfection efficiency and background. Luciferase activity was significantly reduced by all APC proteins compared with GFP alone. However, APC that lacks the direct microtubule binding site was more efficient in producing this effect, regardless of whether the N-terminal domain was present or not. Similar results were observed after 24 hours of transfection. Data represent triplicate readings.
