Fluorescent fusion proteins have revolutionized examination of proteins in living cells. Still, studies using these proteins are met with criticism because proteins are modified and ectopically expressed, in contrast to immunofluorescence studies. However, introducing immunoreagents inside cells can cause protein extraction or relocalization, not reflecting the in vivo situation. Here we discuss pitfalls of immunofluorescence labeling that often receive little attention and argue that immunostaining experiments in dead, permeabilized cells should be complemented with live-cell imaging when scrutinizing protein localization.
Injection of tumor cells in mice more than 30 years ago resulted in the discovery of an epithelial antigen, later defined as a cell adhesion molecule (EpCAM). Although EpCAM has since evoked significant interest as a target in cancer therapy, mechanistic insights on the functions of this glycoprotein have been emerging only very recently. This may have been caused by the multitude of functions attributed to the glycoprotein, its localization at different subcellular sites and complex posttranslational modifications. Here, we review how EpCAM modifies cell-cell contact adhesion strength and tissue plasticity, and how it regulates cell proliferation and differentiation. Major knowledge derived from human diseases will be highlighted: Mutant EpCAM that is absent from the cell surface leads to fatal intestinal abnormalities (congenital tufting enteropathy). EpCAM-mediated cell proliferation in cancer may result from signaling (i) via regulated intramembrane proteolysis and/or (ii) the localization and association with binding partners in specialized membrane microdomains. New insight in EpCAM signaling will help to develop optimized cancer therapies and open new avenues in the field of regenerative medicine.
EpCAM [epithelial cell adhesion molecule; CD326 (cluster of differentiation 326)] is highly expressed on epithelium-derived tumours and can play a role in cell proliferation. Recently, RIP (regulated intramembrane proteolysis) has been implicated as the trigger for EpCAM-mediated proliferative signalling. However, RIP does not explain all EpCAM-derived protein fragments. To shed light on how proteolytic cleavage is involved in EpCAM signalling, we characterized the protein biochemically using antibodies binding to three different EpCAM domains. Using a newly generated anti-EpCAM antibody, we find that EpCAM can be cleaved at multiple positions within its ectodomain in addition to described peptides, revealing that EpCAM is processed via distinct proteolytic pathways. Here, we report on four new peptides, but also discuss the previously described cleavage products to provide a comprehensive picture of EpCAM cleavage at multiple positions. The complex regulation of EpCAM might not only result in the absence of full-length EpCAM, but the newly formed EpCAM-derived proteins may have their own signalling properties.
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