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First published online 13 May 2008
doi: 10.1242/jcs.029470

Journal of Cell Science 121, 1916-1925 (2008)
Published by The Company of Biologists 2008
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Novel self-association of the APC molecule affects APC clusters and cell migration

Zhuoyu Li, Karin Kroboth, Ian P. Newton and Inke S. Näthke*

Division of Cell and Developmental Biology, School of Life Sciences, University of Dundee, WTB/MSI Complex, Dow Street, Dundee, DD1 5EH, UK


Figure 1
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  		Fig. 1. In vitro binding between N-APC fragments and full-length C-APC. (A) Schematic of full-length APC (aa 1-2843), and the position and exact residues contained in the fragments that were used in this study. (B) Autoradiogram of His-tagged N1, N2 and N3 APC fragments that were generated using an in vitro translation system in the presence of 35S-methionine. (C) Coomassie gel and immunoblot of purified His-tagged C-APC, C3 and N3 using an anti-His-tag antibody. (D) Purified C-APC was incubated with in vitro translation reaction products for N1, N2 and N3. C-APC was immunoprecipitated with a polyclonal anti-C-APC antibody and bound N-APC fragments were detected by autoradiography. For control samples (Ctrl), the purified C-APC protein was omitted. To confirm that equal amounts of C-APC were used in each case, the same samples were immunoblotted with a monoclonal anti-C-APC antibody.

 

Figure 2
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  		Fig. 2. Binding of C-APC fragments to different endogenous N-APC fragments. (A,B) GFP-tagged C-APC or GFP-tagged C1, C2, C3 or C3m fragments were transiently expressed in SW480 (A) or Colo320 (B) cells. Please note that the GFP tag increased the molecular weight of these proteins so that they appear larger than in Fig. 1. Lysates from transfected cells were blotted with a monoclonal anti-GFP antibody (upper panel). The endogenous N-APC was precipitated with a monoclonal anti-N-APC antibody and the bound C-APC fragments were detected using an anti-GFP antibody (middle panel). Cells transfected with GFP alone acted as controls. (C) Purified C3 (C3–His-tag) was immobilised using a polyclonal anti-C-APC antibody. Individual N-APC fragments labelled with 35S-methionine were prepared by in vitro translation and added to the immobilised C3 fragments. Bound N-APC fragments were detected using autoradiography.

 

Figure 3
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  		Fig. 3. Binding of N3 to itself is regulated by phosphorylation. (A) Equal amounts of proteins from cell lysates of SW480 or Colo320 cells transfected with GFP-N3 or GFP alone were prepared and immunoprecipitated with a monoclonal anti-N-APC antibody. Total lysates (left panels) and proteins bound to the immunoprecipitated material (right panels) were probed with an anti-GFP antibody. Only the longer N-APC in SW480 cells bound to GFP-N3. (B) Endogenous N-APC was immunoprecipitated from SW480 cells and treated with {lambda}-phosphatase ({lambda}-ppase; +) or control buffer (–). Purified His-tagged C-APC, C3 or N3 were added and bound proteins were detected using a monoclonal anti-His antibody. Only the binding of N3 was significantly inhibited when N-APC was dephosphorylated. In addition, the precipitated material was probed with a monoclonal anti-N-APC antibody to confirm that equal amounts of N-APC were recovered in each case (upper panel) and also to illustrate that treatment with {lambda}-phosphatase increased the electrophoretic mobility of the N-APC in these cells, confirming that the N-terminal third of APC is normally phosphorylated in cells. (C) Immunoprecipitated N-APC from SW480 cells was incubated with purified His-tagged C-APC, C3 or N3 in the presence (+) or absence (–) of GST-tagged 14-3-3 protein. Bound C-APC, C3 and N3 were detected with a monoclonal anti-His antibody, and the presence of 14-3-3 was confirmed with a polyclonal anti-GST antibody (lower panel). (D) Immunoprecipitated endogenous APC from SW480 or Colo320 cells was incubated with purified GST–14-3-3 in the presence (+) or absence (–) of {lambda}-phosphatase. Only the N-APC from SW480 cells bound 14-3-3 proteins strongly and this binding required phosphorylation of N-APC, consistent with the idea that the N3 region (absent in the APC in Colo320 cells) is required for this interaction. (E) Immunoprecipitated endogenous APC from SW480 cells was treated with {lambda}-phosphatase or control buffer before purified GST–14-3-3{zeta} was added to the beads. After washing, purified N3 was added to each sample. Bound proteins were detected with the indicated antibodies. The control sample did not include the cell lysate. Both phosphorylation and the binding of 14-3-3 enhance the ability of N3 to bind N-APC.

 

Figure 4
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  		Fig. 4. APC-cluster formation in MDCK cells is inhibited by expression of N3 or by the inhibition of 14-3-3 proteins. (A) Representative images of the different types of endogenous APC (a-c) or GFP-APC protein (a'-c') clusters used to categorise cells: (a) cluster, (b) dispersed cluster, (c) dispersed. APC is green; tubulin is shown in red. (B) APC-cluster formation in cells was scored using a total of ~100 cells randomly selected from three independent experiments. (a) The localisation of endogenous APC in MDCK cells was scored in cells transiently transfected with N3, C3 or GFP. Expression of N3 caused a marked decrease in the number of cells with clustered APC and led to more cells with dispersed APC, whereas C3 had a more moderate effect. (b) The localisation of GFP-tagged full-length APC in MDCK cells was compared in cells co-transfected with GST or with GST-N3. Again, the presence of N3 led to a decrease in clustering of GFP-APC. Similar data was obtained in PTK2 cells (supplementary material Fig. S4). (c) The localisation of endogenous APC was scored in cells treated with a phospho-peptide (inhibitor) or the non-phospho version of the same peptide (control). Inhibition of 14-3-3 caused a reduction in the number of cells with clustered APC. (d) Clusters formed by GFP-tagged APC lacking N3 (GFP-APC{Delta}N3) and GFP-APC were scored as above. GFP-APC{Delta}N3 forms tighter clusters compared with GFP-APC. Similar data was obtained in PTK2 cells (supplementary material Fig. S4). (C) Images of live cells expressing GFP-APC together with mCherry-N3 (a) or mCherry-C3 (b) reveal enrichment of N3 in APC clusters. C3 is not enriched in the clusters, but is also not excluded from them.

 

Figure 5
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  		Fig. 5. MDCK cells stably expressing N3 migrate more slowly. (A) MDCK cells stably expressing GFP or GFP-N3 were imaged at the time of induction of a scratch into a monolayer (shaded blue) and 8 hours later (shaded grey). Cells expressing N3 migrated more slowly. (B) The migration of MDCK cells stably expressing N3 or GFP was measured over 8 hours and was significantly reduced compared with GFP-expressing control cells. Data from four independent experiments using two different clones are shown. Asterisk indicates statistical significance (P<0.001). (C) Lysates from MDCK cells stably expressing GFP-N3 or GFP corresponding to equal amounts of protein were blotted with anti-GFP antibodies to show the relative expression levels of these proteins.

 

Figure 6
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  		Fig. 6. Clusters formed by APC{Delta}N3 are less dynamic. (A-C) Images of clusters containing GFP-APC (A), GFP-APC{Delta}N3 (B) or GFP-APC{Delta}C3 (C) were recorded every 100 seconds to monitor shape and intensity changes over time. The summed intensity in each cluster was recorded and two examples of different types of clusters at 0, 300, 600 and 900 seconds are shown. Please note that these images show projections of the total summed intensity, not maximum intensity projections. This makes their appearance more fuzzy, but allows the changes in total intensity to be quantitated and visualised accurately. The appearance of clusters formed by GFP-APC changed noticeably more than of those formed by GFP-APC{Delta}N3, whereas changes by those formed by GFP-APC{Delta}C3 were intermediate. (D) Total fluorescent intensity in selected clusters was measured and recorded over time (seconds). The intensity in each cluster relative to that at the start is plotted for each time point to show that clusters formed by GFP-APC{Delta}N3 are less dynamics than those formed by GFP-APC or GFP-APC{Delta}C3.

 

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