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.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

Localization of APC and microtubules in subconfluent epithelial cells. Immunolocalization of APC (red) and β-tubulin (green) in subconfluent HCT116 (a–c) and MDCK cells (d–e). (a) Localization of APC at the tips of cell processes containing microtubules is detected, albeit infrequently (<5% of all microtubule-containing protrusions). (b–f) APC is present over much of the cell surface and enriched all along the edges of the cell body and cell protrusions. (f) Higher magnification view of e.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196003&req=5

Figure 7: Localization of APC and microtubules in subconfluent epithelial cells. Immunolocalization of APC (red) and β-tubulin (green) in subconfluent HCT116 (a–c) and MDCK cells (d–e). (a) Localization of APC at the tips of cell processes containing microtubules is detected, albeit infrequently (<5% of all microtubule-containing protrusions). (b–f) APC is present over much of the cell surface and enriched all along the edges of the cell body and cell protrusions. (f) Higher magnification view of e.

Mentions: Since a lot of attention has been given to the association of APC with microtubules, we also performed double immunostaining for APC and β-tubulin. In fully confluent cell monolayers, no apparent colocalization of APC and microtubules was observed (data not shown). In subconfluent HCT116 cells (Fig. 7, a–c) and subconfluent MDCK cells (Fig. 7, d–f), APC was found over large regions of the cell surface and perhaps in the cytoplasm. We did occasionally detect APC at the tips of cell protrusions where microtubules project, as reported previously (Nathke et al. 1996); however, these were very rare (<5% of identified microtubule-containing protrusions) (Fig. 7 a). Much more commonly observed was APC distributed all along the surface of the cell protrusions (Fig. 7, b–f). Staining patterns for HCT116 and MDCK cells were very similar.


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)

Localization of APC and microtubules in subconfluent epithelial cells. Immunolocalization of APC (red) and β-tubulin (green) in subconfluent HCT116 (a–c) and MDCK cells (d–e). (a) Localization of APC at the tips of cell processes containing microtubules is detected, albeit infrequently (<5% of all microtubule-containing protrusions). (b–f) APC is present over much of the cell surface and enriched all along the edges of the cell body and cell protrusions. (f) Higher magnification view of e.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: Localization of APC and microtubules in subconfluent epithelial cells. Immunolocalization of APC (red) and β-tubulin (green) in subconfluent HCT116 (a–c) and MDCK cells (d–e). (a) Localization of APC at the tips of cell processes containing microtubules is detected, albeit infrequently (<5% of all microtubule-containing protrusions). (b–f) APC is present over much of the cell surface and enriched all along the edges of the cell body and cell protrusions. (f) Higher magnification view of e.
Mentions: Since a lot of attention has been given to the association of APC with microtubules, we also performed double immunostaining for APC and β-tubulin. In fully confluent cell monolayers, no apparent colocalization of APC and microtubules was observed (data not shown). In subconfluent HCT116 cells (Fig. 7, a–c) and subconfluent MDCK cells (Fig. 7, d–f), APC was found over large regions of the cell surface and perhaps in the cytoplasm. We did occasionally detect APC at the tips of cell protrusions where microtubules project, as reported previously (Nathke et al. 1996); however, these were very rare (<5% of identified microtubule-containing protrusions) (Fig. 7 a). Much more commonly observed was APC distributed all along the surface of the cell protrusions (Fig. 7, b–f). Staining patterns for HCT116 and MDCK cells were very similar.

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