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Apical membrane localization of the adenomatous polyposis coli tumor suppressor protein and subcellular distribution of the beta-catenin destruction complex in polarized epithelial cells.

Reinacher-Schick A, Gumbiner BM - J. Cell Biol. (2001)

Bottom Line: Reports on the subcellular localization of APC in various cell systems have differed significantly and have been consistent with an association with a cytosolic complex, with microtubules, with the nucleus, or with the cortical actin cytoskeleton.Dishevelled is almost entirely cytosolic, but does not significantly cofractionate with the 20S complex.The disproportionate amount of APC in the apical membrane and the lack of other destruction complex components in the 60S fraction of APC raise questions about whether these pools of APC take part in the degradation of beta-catenin, or alternatively, whether they could be involved in other functions of the protein that still must be determined.

View Article: PubMed Central - PubMed

Affiliation: Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.

ABSTRACT
The adenomatous polyposis coli (APC) protein is implicated in the majority of hereditary and sporadic colon cancers. APC is known to function as a tumor suppressor through downregulation of beta-catenin as part of a high molecular weight complex known as the beta-catenin destruction complex. The molecular composition of the intact complex and its site of action in the cell are still not well understood. Reports on the subcellular localization of APC in various cell systems have differed significantly and have been consistent with an association with a cytosolic complex, with microtubules, with the nucleus, or with the cortical actin cytoskeleton. To better understand the role of APC and the destruction complex in colorectal cancer, we have begun to characterize and isolate these complexes from confluent polarized human colon epithelial cell monolayers and other epithelial cell types. Subcellular fractionation and immunofluorescence microscopy reveal that a predominant fraction of APC associates tightly with the apical plasma membrane in a variety of epithelial cell types. This apical membrane association is not dependent on the mutational status of either APC or beta-catenin. An additional pool of APC is cytosolic and fractionates into two distinct high molecular weight complexes, 20S and 60S in size. Only the 20S fraction contains an appreciable portion of the cellular axin and small but detectable amounts of glycogen synthase kinase 3beta and beta-catenin. Therefore, it is likely to correspond to the previously characterized beta-catenin destruction complex. Dishevelled is almost entirely cytosolic, but does not significantly cofractionate with the 20S complex. The disproportionate amount of APC in the apical membrane and the lack of other destruction complex components in the 60S fraction of APC raise questions about whether these pools of APC take part in the degradation of beta-catenin, or alternatively, whether they could be involved in other functions of the protein that still must be determined.

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Apical localization of APC in the HCT116 colon carcinoma cell line. Immunolocalization of endogenous APC (red) and β-catenin (green) in HCT116 cells. (a and b) Negative control for APC and β-catenin staining, respectively. Normal mouse (a) or normal rabbit IgG (b) was substituted for primary antibodies before incubation with respective secondary antibodies. (c) Peptide competition (+ peptide apical). Anti–β-catenin and anti-APC antibodies were preincubated with an excess of the neutralizing peptide against which the anti-APC antibody was raised. A transverse apical section is shown. (d–f) Images of successive sections of fully confluent, polarized HCT116 cells using confocal microscopy: basal (d), intermediate (e), and apical (f) sections. (g) Corresponding perpendicular section (z-axis).
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Figure 3: Apical localization of APC in the HCT116 colon carcinoma cell line. Immunolocalization of endogenous APC (red) and β-catenin (green) in HCT116 cells. (a and b) Negative control for APC and β-catenin staining, respectively. Normal mouse (a) or normal rabbit IgG (b) was substituted for primary antibodies before incubation with respective secondary antibodies. (c) Peptide competition (+ peptide apical). Anti–β-catenin and anti-APC antibodies were preincubated with an excess of the neutralizing peptide against which the anti-APC antibody was raised. A transverse apical section is shown. (d–f) Images of successive sections of fully confluent, polarized HCT116 cells using confocal microscopy: basal (d), intermediate (e), and apical (f) sections. (g) Corresponding perpendicular section (z-axis).

Mentions: To determine where the membrane-associated fraction of APC localizes in HCT116 cells, we used indirect immunofluorescence microscopy with the polyclonal rabbit IgG antibody N-15 raised against an NH2-terminal peptide of human APC. Immunolocalization was carried out on fully confluent, polarized epithelial cell monolayers. Confocal microscopic analysis of immunostained HCT116 colon carcinoma cells showed that APC predominantly localized to the apical plasma membrane and to a very small degree to the apicolateral cell border (Fig. 3, d–g). APC and β-catenin, for the most part, did not colocalize. β-Catenin staining was mostly seen laterally with very little overlap of the two proteins detectable at the apicolateral cell borders (Fig. 3 g). Essentially no APC was seen in more basal sections of the cell (Fig. 3 d). APC staining was most pronounced in apical sections of the cells (Fig. 3 f). The apical localization of APC is best observed in the z-section of analyzed monolayers (Fig. 3 g). For controls using normal mouse or rabbit IgG, no specific staining was detected (Fig. 3, a and b). Furthermore, apical staining of APC was effectively blocked by preincubation of the antibodies with the neutralizing peptide against which the anti-APC antibody was raised (Fig. 3 c).


Apical membrane localization of the adenomatous polyposis coli tumor suppressor protein and subcellular distribution of the beta-catenin destruction complex in polarized epithelial cells.

Reinacher-Schick A, Gumbiner BM - J. Cell Biol. (2001)

Apical localization of APC in the HCT116 colon carcinoma cell line. Immunolocalization of endogenous APC (red) and β-catenin (green) in HCT116 cells. (a and b) Negative control for APC and β-catenin staining, respectively. Normal mouse (a) or normal rabbit IgG (b) was substituted for primary antibodies before incubation with respective secondary antibodies. (c) Peptide competition (+ peptide apical). Anti–β-catenin and anti-APC antibodies were preincubated with an excess of the neutralizing peptide against which the anti-APC antibody was raised. A transverse apical section is shown. (d–f) Images of successive sections of fully confluent, polarized HCT116 cells using confocal microscopy: basal (d), intermediate (e), and apical (f) sections. (g) Corresponding perpendicular section (z-axis).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196003&req=5

Figure 3: Apical localization of APC in the HCT116 colon carcinoma cell line. Immunolocalization of endogenous APC (red) and β-catenin (green) in HCT116 cells. (a and b) Negative control for APC and β-catenin staining, respectively. Normal mouse (a) or normal rabbit IgG (b) was substituted for primary antibodies before incubation with respective secondary antibodies. (c) Peptide competition (+ peptide apical). Anti–β-catenin and anti-APC antibodies were preincubated with an excess of the neutralizing peptide against which the anti-APC antibody was raised. A transverse apical section is shown. (d–f) Images of successive sections of fully confluent, polarized HCT116 cells using confocal microscopy: basal (d), intermediate (e), and apical (f) sections. (g) Corresponding perpendicular section (z-axis).
Mentions: To determine where the membrane-associated fraction of APC localizes in HCT116 cells, we used indirect immunofluorescence microscopy with the polyclonal rabbit IgG antibody N-15 raised against an NH2-terminal peptide of human APC. Immunolocalization was carried out on fully confluent, polarized epithelial cell monolayers. Confocal microscopic analysis of immunostained HCT116 colon carcinoma cells showed that APC predominantly localized to the apical plasma membrane and to a very small degree to the apicolateral cell border (Fig. 3, d–g). APC and β-catenin, for the most part, did not colocalize. β-Catenin staining was mostly seen laterally with very little overlap of the two proteins detectable at the apicolateral cell borders (Fig. 3 g). Essentially no APC was seen in more basal sections of the cell (Fig. 3 d). APC staining was most pronounced in apical sections of the cells (Fig. 3 f). The apical localization of APC is best observed in the z-section of analyzed monolayers (Fig. 3 g). For controls using normal mouse or rabbit IgG, no specific staining was detected (Fig. 3, a and b). Furthermore, apical staining of APC was effectively blocked by preincubation of the antibodies with the neutralizing peptide against which the anti-APC antibody was raised (Fig. 3 c).

Bottom Line: Reports on the subcellular localization of APC in various cell systems have differed significantly and have been consistent with an association with a cytosolic complex, with microtubules, with the nucleus, or with the cortical actin cytoskeleton.Dishevelled is almost entirely cytosolic, but does not significantly cofractionate with the 20S complex.The disproportionate amount of APC in the apical membrane and the lack of other destruction complex components in the 60S fraction of APC raise questions about whether these pools of APC take part in the degradation of beta-catenin, or alternatively, whether they could be involved in other functions of the protein that still must be determined.

View Article: PubMed Central - PubMed

Affiliation: Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.

ABSTRACT
The adenomatous polyposis coli (APC) protein is implicated in the majority of hereditary and sporadic colon cancers. APC is known to function as a tumor suppressor through downregulation of beta-catenin as part of a high molecular weight complex known as the beta-catenin destruction complex. The molecular composition of the intact complex and its site of action in the cell are still not well understood. Reports on the subcellular localization of APC in various cell systems have differed significantly and have been consistent with an association with a cytosolic complex, with microtubules, with the nucleus, or with the cortical actin cytoskeleton. To better understand the role of APC and the destruction complex in colorectal cancer, we have begun to characterize and isolate these complexes from confluent polarized human colon epithelial cell monolayers and other epithelial cell types. Subcellular fractionation and immunofluorescence microscopy reveal that a predominant fraction of APC associates tightly with the apical plasma membrane in a variety of epithelial cell types. This apical membrane association is not dependent on the mutational status of either APC or beta-catenin. An additional pool of APC is cytosolic and fractionates into two distinct high molecular weight complexes, 20S and 60S in size. Only the 20S fraction contains an appreciable portion of the cellular axin and small but detectable amounts of glycogen synthase kinase 3beta and beta-catenin. Therefore, it is likely to correspond to the previously characterized beta-catenin destruction complex. Dishevelled is almost entirely cytosolic, but does not significantly cofractionate with the 20S complex. The disproportionate amount of APC in the apical membrane and the lack of other destruction complex components in the 60S fraction of APC raise questions about whether these pools of APC take part in the degradation of beta-catenin, or alternatively, whether they could be involved in other functions of the protein that still must be determined.

Show MeSH
Related in: MedlinePlus