Abstract
BackgroundThe trafficking of the adenomatous polyposis coli (APC) tumour suppressor protein in mammalian cells is a perennially controversial topic. Immunostaining evidence for an actin-associated APC localisation at intercellular junctions has been previously presented, though live imaging of mammalian junctional APC has not been documented.ResultsUsing live imaging of transfected COS-7 cells we observed intercellular junction-associated pools of GFP-APC in addition to previously documented microtubule-associated GFP-APC and a variety of minor localisations. Although both microtubule and junction-associated populations could co-exist within individual cells, they differed in their subcellular location, dynamic behaviour and sensitivity to cytoskeletal poisons. GFP-APC deletion mutant analysis indicated that a protein truncated immediately after the APC armadillo repeat domain retained the ability to localise to adhesive membranes in transfected cells. Supporting this, we also observed junctional APC immunostaining in cultures of human colorectal cancer cell line that express truncated forms of APC.ConclusionOur data indicate that APC can be found in two spatially separate populations at the cell periphery and these populations can co-exist in the same cell. The first localisation is highly dynamic and associated with microtubules near free edges and in cell vertices, while the second is comparatively static and is closely associated with actin at sites of cell-cell contact. Our imaging confirms that human GFP-APC possesses many of the localisations and behaviours previously seen by live imaging of Xenopus GFP-APC. However, we report the novel finding that GFP-APC puncta can remain associated with the ends of shrinking microtubules. Deletion analysis indicated that the N-terminal region of the APC protein mediated its junctional localisation, consistent with our observation that truncated APC proteins in colon cancer cell lines are still capable of localising to the cell cortex. This may have implications for the development of colorectal cancer.
Highlights
The trafficking of the adenomatous polyposis coli (APC) tumour suppressor protein in mammalian cells is a perennially controversial topic
In this study we investigated the intracellular distribution of APC using live imaging of human GFP-APC fusion proteins in mammalian cells
In this study we show that APC is capable to localising to both microtubules and to junctions within the cell, depending on cellular context
Summary
The trafficking of the adenomatous polyposis coli (APC) tumour suppressor protein in mammalian cells is a perennially controversial topic. Two mammalian APC distributions have previously been described as populations found at peripheral cellular sites. The first of these to be identified and widely accepted consists of APC clusters that localize to specific cortical sites in a microtubule-dependent manner [3,4,5]. Support for this distribution has been presented in studies examining the behavior of Xenopus APC-GFP fusion proteins in living cells [6]. Restoration of expression of fulllength APC in a colorectal cancer cell line has been shown to promote cell-cell adhesion [8] while in Drosophila the APC homologue E-APC has been shown to associate with and play a role in maintaining the integrity of epithelial cell junctions, a localization mediated by its armadillo repeats [9]
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