Method of cell handling affects leakiness of cell surface labeling and detection of intracellular keratins.
Keratins are a subgroup of cytoskeletal intermediate filament proteins found in most epithelial cells. Some reports have suggested that keratins may be found on the cell surface as well as their well-accepted cytoskeletal location. A major part of the evidence in the interpretation of cell surface expression of keratins is cell surface radioiodination. Here we show that lactoperoxidase-catalyzed iodination of colonic and breast tissue culture cells results in radiolabeling of the keratins when cells are manipulated. No labeling of keratins is detected when cells are labeled directly on the tissue culture dish. A similar result was obtained when intact cells were biotinylated using water-soluble sulfo-NHS-biotin. Partitioning of the keratins to a soluble and an insoluble pool after "cell surface" 125I-labeling showed that both pools became iodinated. Indirect immunofluorescence showed that binding of a panel of anti-keratin antibodies to intact epithelial cells occurs only on the cells that are more adherent, which are the cells that require longer manipulation to remove from the tissue culture dish. Taken together, our results indicate that the reported expression of cell surface keratins in some cells likely reflects intracellular keratins. In addition, the method of epithelial cell handling can dramatically alter the leakiness of cell surface iodination techniques.
Riopel
C L
CL
Department of Medicine, Palo Alto VA Medical Center, California 94304.
Butt
I
I
Omary
M B
MB
D000230:Adenocarcinoma / Q000737:chemistry* / Q000473:pathology* / Q000648:ultrastructure; D000911:Antibodies, Monoclonal / Q000032:analysis / Q000276:immunology; D001710:Biotin; D001943:Breast Neoplasms / Q000737:chemistry* / Q000473:pathology* / Q000648:ultrastructure; D002462:Cell Membrane / Q000737:chemistry / Q000502:physiology / Q000648:ultrastructure; D002463:Cell Membrane Permeability / Q000502:physiology*; D003110:Colonic Neoplasms / Q000737:chemistry* / Q000473:pathology* / Q000648:ultrastructure; D003599:Cytoskeleton / Q000737:chemistry / Q000502:physiology / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D006801:Humans; D007382:Intermediate Filaments / Q000737:chemistry / Q000502:physiology / Q000648:ultrastructure; D007457:Iodine Radioisotopes; D007633:Keratins / Q000032:analysis* / Q000276:immunology; D008722:Methods; D011233:Precipitin Tests; D013048:Specimen Handling / Q000379:methods*; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D007457:Iodine Radioisotopes; D007633:Keratins; D001710:Biotin