Limits...
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.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.Dishevelled is almost entirely cytosolic, but does not significantly cofractionate with the 20S complex.

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

Apical membrane localization of APC in normal mouse colon. Immunolocalization of APC (red) and β-catenin (green) in tissue sections of normal mouse colon. (a and b) Apical membrane staining of epithelial cells in the upper portion of colonic crypts which face the lumen of the digestive tract in normal mouse colon (arrows). (c) Low APC immunoreactivity in epithelial cells towards the base of the crypts. Additional APC staining in nonepithelial cells in the lamina propria (arrowheads) and throughout the submucosa and muscularis layers. (d) APC staining is effectively blocked with the specific neutralizing peptide against which the antibody was raised. There is no β-catenin staining, because normal mouse IgG was substituted for β-catenin primary antibody in control sections. Note residual immunoreactivity of secondary anti–mouse IgG antibody with cells in the lamina propria (green).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196003&req=5

Figure 6: Apical membrane localization of APC in normal mouse colon. Immunolocalization of APC (red) and β-catenin (green) in tissue sections of normal mouse colon. (a and b) Apical membrane staining of epithelial cells in the upper portion of colonic crypts which face the lumen of the digestive tract in normal mouse colon (arrows). (c) Low APC immunoreactivity in epithelial cells towards the base of the crypts. Additional APC staining in nonepithelial cells in the lamina propria (arrowheads) and throughout the submucosa and muscularis layers. (d) APC staining is effectively blocked with the specific neutralizing peptide against which the antibody was raised. There is no β-catenin staining, because normal mouse IgG was substituted for β-catenin primary antibody in control sections. Note residual immunoreactivity of secondary anti–mouse IgG antibody with cells in the lamina propria (green).

Mentions: To determine where APC localizes in situ, we examined samples of normal mouse colon by immunofluorescence microscopy. APC was enriched at the apical membrane of epithelial cells predominantly in upper regions of colonic crypts towards the lumen of the digestive tract (Fig. 6, a and b). In addition, some diffuse cytoplasmic staining was detected. In contrast, β-catenin was seen mainly at the lateral borders of colonocytes and no obvious colocalization with APC was found. Immunoreactivity towards the APC protein in epithelial cells decreased substantially towards the base of the crypts (Fig. 6 c). Additionally, APC was detected in nonepithelial cells in the lamina propria and throughout the submucosa and muscularis layers. APC staining was effectively blocked with the competing peptide against which the anti-APC antibody was raised (Fig. 6 d).


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 membrane localization of APC in normal mouse colon. Immunolocalization of APC (red) and β-catenin (green) in tissue sections of normal mouse colon. (a and b) Apical membrane staining of epithelial cells in the upper portion of colonic crypts which face the lumen of the digestive tract in normal mouse colon (arrows). (c) Low APC immunoreactivity in epithelial cells towards the base of the crypts. Additional APC staining in nonepithelial cells in the lamina propria (arrowheads) and throughout the submucosa and muscularis layers. (d) APC staining is effectively blocked with the specific neutralizing peptide against which the antibody was raised. There is no β-catenin staining, because normal mouse IgG was substituted for β-catenin primary antibody in control sections. Note residual immunoreactivity of secondary anti–mouse IgG antibody with cells in the lamina propria (green).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 6: Apical membrane localization of APC in normal mouse colon. Immunolocalization of APC (red) and β-catenin (green) in tissue sections of normal mouse colon. (a and b) Apical membrane staining of epithelial cells in the upper portion of colonic crypts which face the lumen of the digestive tract in normal mouse colon (arrows). (c) Low APC immunoreactivity in epithelial cells towards the base of the crypts. Additional APC staining in nonepithelial cells in the lamina propria (arrowheads) and throughout the submucosa and muscularis layers. (d) APC staining is effectively blocked with the specific neutralizing peptide against which the antibody was raised. There is no β-catenin staining, because normal mouse IgG was substituted for β-catenin primary antibody in control sections. Note residual immunoreactivity of secondary anti–mouse IgG antibody with cells in the lamina propria (green).
Mentions: To determine where APC localizes in situ, we examined samples of normal mouse colon by immunofluorescence microscopy. APC was enriched at the apical membrane of epithelial cells predominantly in upper regions of colonic crypts towards the lumen of the digestive tract (Fig. 6, a and b). In addition, some diffuse cytoplasmic staining was detected. In contrast, β-catenin was seen mainly at the lateral borders of colonocytes and no obvious colocalization with APC was found. Immunoreactivity towards the APC protein in epithelial cells decreased substantially towards the base of the crypts (Fig. 6 c). Additionally, APC was detected in nonepithelial cells in the lamina propria and throughout the submucosa and muscularis layers. APC staining was effectively blocked with the competing peptide against which the anti-APC antibody was raised (Fig. 6 d).

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.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.Dishevelled is almost entirely cytosolic, but does not significantly cofractionate with the 20S complex.

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