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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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Disruption of APC2 self-association leads to defects in destructosome assembly in live Drosophila S2 cells. In all cases, Axin is GFP tagged and APC2 is mCherry tagged. Dotted lines indicate cell boundaries. (A) When expressed alone in S2 cells, Axin-GFP oligomers can be visualized as cytoplasmic puncta (1), and mCh-APC2-FL (2) is primarily cortical. When coexpressed, mCh-APC2-FL colocalizes in cytoplasmic puncta with Axin-GFP (3). Removal of APC2's Axin interaction domains (APC2-∆SAMP) disrupts this colocalization (4). ASAD mutants (both ΔASAD and ASADPro) colocalize with Axin-GFP, but cells coexpressing these proteins exhibit defects in puncta assembly and morphology (5, 6). (B) Quantification of puncta size in S2 cells expressing Axin alone and coexpressing Axin and APC2 in cells sorted into three expression level categories (high, medium, and low) by FACS using Axin-GFP. Images were taken under the same imaging conditions, and puncta size was determined using Imaris. Puncta were then divided into three classes based on area (micrometers squared). Coexpression with APC2-FL is associated with fewer, larger puncta at all three expression levels (see puncta/cell ratios). Coexpression with the ASAD mutants showed increase in the number of small puncta only in the low category. Owing to the disrupted puncta morphology in ASAD mutants, we were only able to assess the puncta size in this category. Two-tailed z test demonstrates significant differences between different groups. Scale bar, 10 μm.
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Figure 3: Disruption of APC2 self-association leads to defects in destructosome assembly in live Drosophila S2 cells. In all cases, Axin is GFP tagged and APC2 is mCherry tagged. Dotted lines indicate cell boundaries. (A) When expressed alone in S2 cells, Axin-GFP oligomers can be visualized as cytoplasmic puncta (1), and mCh-APC2-FL (2) is primarily cortical. When coexpressed, mCh-APC2-FL colocalizes in cytoplasmic puncta with Axin-GFP (3). Removal of APC2's Axin interaction domains (APC2-∆SAMP) disrupts this colocalization (4). ASAD mutants (both ΔASAD and ASADPro) colocalize with Axin-GFP, but cells coexpressing these proteins exhibit defects in puncta assembly and morphology (5, 6). (B) Quantification of puncta size in S2 cells expressing Axin alone and coexpressing Axin and APC2 in cells sorted into three expression level categories (high, medium, and low) by FACS using Axin-GFP. Images were taken under the same imaging conditions, and puncta size was determined using Imaris. Puncta were then divided into three classes based on area (micrometers squared). Coexpression with APC2-FL is associated with fewer, larger puncta at all three expression levels (see puncta/cell ratios). Coexpression with the ASAD mutants showed increase in the number of small puncta only in the low category. Owing to the disrupted puncta morphology in ASAD mutants, we were only able to assess the puncta size in this category. Two-tailed z test demonstrates significant differences between different groups. Scale bar, 10 μm.

Mentions: The destructosome is typically visualized as cytoplasmic Axin puncta that are observed both endogenously and when Axin is overexpressed in cell culture and intact tissues (Fagotto et al., 1999; Schwarz-Romond et al., 2007b; Faux et al., 2008; Fiedler et al., 2011). Overexpressed Axin tagged with green fluorescent protein (GFP), FLAG, red fluorescent protein, myc, or hemagglutinin (HA) localizes to cytoplasmic puncta in a variety of vertebrate and fly cultured cells, including S2, SW480, HeLa, MDCK, and Cos-7. Overexpressed Axin has been shown to rescue β-cat destruction in colorectal cancer cell lines (Behrens et al., 1998; Hart et al., 1998; Nakamura et al., 1998; Roberts et al., 2011), and in Drosophila embryos, cytoplasmic Axin-GFP puncta become cortical when cells activate the Wnt pathway, suggesting that these overexpression puncta are responsive to Wnt pathway activation (Mendoza-Topaz et al., 2011). Axin can self-associate via its C-terminal DIX domain (also called DAX), which is essential for its function in β-cat destruction and for its ability to form puncta (Schwarz-Romond et al., 2007a, b). It was recently shown that APC is essential for destructosome assembly, as in the absence of APC, Axin failed to form functional destructosomes (Mendoza-Topaz et al., 2011). Like Axin, we predicted that APC2 contributes to the formation of the destructosome through its ability to self-associate and form larger macromolecular assemblages (Kunttas-Tatli et al., 2012). To test this hypothesis, we coexpressed Axin and APC2 in Drosophila S2 cells. When expressed alone, both Axin-GFP (Figure 3A1) and Axin-HA (Supplemental Figure S2A) formed cytoplasmic puncta, albeit smaller in the case of Axin-HA. Thus the GFP tag may have a slight effect on puncta size. On the other hand, mCh-APC2-FL localized primarily to the cell cortex (Figure 3A2; Zhou et al., 2011). When coexpressed with Axin-GFP, mCh-APC2-FL redistributed and localized primarily in the cytoplasmic Axin puncta (Figure 3A3). Deletion of the Axin-binding SAMP repeats from APC2 (APC2-∆SAMP) restored cortical localization of APC2 (Figure 3A4), indicating that the primary mechanism for APC2's incorporation into Axin puncta is its direct association with Axin (Roberts et al., 2011).


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)

Disruption of APC2 self-association leads to defects in destructosome assembly in live Drosophila S2 cells. In all cases, Axin is GFP tagged and APC2 is mCherry tagged. Dotted lines indicate cell boundaries. (A) When expressed alone in S2 cells, Axin-GFP oligomers can be visualized as cytoplasmic puncta (1), and mCh-APC2-FL (2) is primarily cortical. When coexpressed, mCh-APC2-FL colocalizes in cytoplasmic puncta with Axin-GFP (3). Removal of APC2's Axin interaction domains (APC2-∆SAMP) disrupts this colocalization (4). ASAD mutants (both ΔASAD and ASADPro) colocalize with Axin-GFP, but cells coexpressing these proteins exhibit defects in puncta assembly and morphology (5, 6). (B) Quantification of puncta size in S2 cells expressing Axin alone and coexpressing Axin and APC2 in cells sorted into three expression level categories (high, medium, and low) by FACS using Axin-GFP. Images were taken under the same imaging conditions, and puncta size was determined using Imaris. Puncta were then divided into three classes based on area (micrometers squared). Coexpression with APC2-FL is associated with fewer, larger puncta at all three expression levels (see puncta/cell ratios). Coexpression with the ASAD mutants showed increase in the number of small puncta only in the low category. Owing to the disrupted puncta morphology in ASAD mutants, we were only able to assess the puncta size in this category. Two-tailed z test demonstrates significant differences between different groups. Scale bar, 10 μm.
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Figure 3: Disruption of APC2 self-association leads to defects in destructosome assembly in live Drosophila S2 cells. In all cases, Axin is GFP tagged and APC2 is mCherry tagged. Dotted lines indicate cell boundaries. (A) When expressed alone in S2 cells, Axin-GFP oligomers can be visualized as cytoplasmic puncta (1), and mCh-APC2-FL (2) is primarily cortical. When coexpressed, mCh-APC2-FL colocalizes in cytoplasmic puncta with Axin-GFP (3). Removal of APC2's Axin interaction domains (APC2-∆SAMP) disrupts this colocalization (4). ASAD mutants (both ΔASAD and ASADPro) colocalize with Axin-GFP, but cells coexpressing these proteins exhibit defects in puncta assembly and morphology (5, 6). (B) Quantification of puncta size in S2 cells expressing Axin alone and coexpressing Axin and APC2 in cells sorted into three expression level categories (high, medium, and low) by FACS using Axin-GFP. Images were taken under the same imaging conditions, and puncta size was determined using Imaris. Puncta were then divided into three classes based on area (micrometers squared). Coexpression with APC2-FL is associated with fewer, larger puncta at all three expression levels (see puncta/cell ratios). Coexpression with the ASAD mutants showed increase in the number of small puncta only in the low category. Owing to the disrupted puncta morphology in ASAD mutants, we were only able to assess the puncta size in this category. Two-tailed z test demonstrates significant differences between different groups. Scale bar, 10 μm.
Mentions: The destructosome is typically visualized as cytoplasmic Axin puncta that are observed both endogenously and when Axin is overexpressed in cell culture and intact tissues (Fagotto et al., 1999; Schwarz-Romond et al., 2007b; Faux et al., 2008; Fiedler et al., 2011). Overexpressed Axin tagged with green fluorescent protein (GFP), FLAG, red fluorescent protein, myc, or hemagglutinin (HA) localizes to cytoplasmic puncta in a variety of vertebrate and fly cultured cells, including S2, SW480, HeLa, MDCK, and Cos-7. Overexpressed Axin has been shown to rescue β-cat destruction in colorectal cancer cell lines (Behrens et al., 1998; Hart et al., 1998; Nakamura et al., 1998; Roberts et al., 2011), and in Drosophila embryos, cytoplasmic Axin-GFP puncta become cortical when cells activate the Wnt pathway, suggesting that these overexpression puncta are responsive to Wnt pathway activation (Mendoza-Topaz et al., 2011). Axin can self-associate via its C-terminal DIX domain (also called DAX), which is essential for its function in β-cat destruction and for its ability to form puncta (Schwarz-Romond et al., 2007a, b). It was recently shown that APC is essential for destructosome assembly, as in the absence of APC, Axin failed to form functional destructosomes (Mendoza-Topaz et al., 2011). Like Axin, we predicted that APC2 contributes to the formation of the destructosome through its ability to self-associate and form larger macromolecular assemblages (Kunttas-Tatli et al., 2012). To test this hypothesis, we coexpressed Axin and APC2 in Drosophila S2 cells. When expressed alone, both Axin-GFP (Figure 3A1) and Axin-HA (Supplemental Figure S2A) formed cytoplasmic puncta, albeit smaller in the case of Axin-HA. Thus the GFP tag may have a slight effect on puncta size. On the other hand, mCh-APC2-FL localized primarily to the cell cortex (Figure 3A2; Zhou et al., 2011). When coexpressed with Axin-GFP, mCh-APC2-FL redistributed and localized primarily in the cytoplasmic Axin puncta (Figure 3A3). Deletion of the Axin-binding SAMP repeats from APC2 (APC2-∆SAMP) restored cortical localization of APC2 (Figure 3A4), indicating that the primary mechanism for APC2's incorporation into Axin puncta is its direct association with Axin (Roberts et al., 2011).

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