Method of cell handling affects leakiness of cell surface labeling and detection of intracellular keratins

TY - JOUR

T1 - Method of cell handling affects leakiness of cell surface labeling and detection of intracellular keratins

AU - Riopel, Carrie L.

AU - Butt, Isha

AU - Omary, M. Bishr

PY - 1993

Y1 - 1993

N2 - 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. © 1993 Wiley‐Liss, Inc.

AB - 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. © 1993 Wiley‐Liss, Inc.

KW - cell surface iodination

KW - cell surface keratins

KW - intermediate filaments

KW - keratins

UR - http://www.scopus.com/inward/record.url?scp=0027172198&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0027172198&partnerID=8YFLogxK

U2 - 10.1002/cm.970260108

DO - 10.1002/cm.970260108

M3 - Article

C2 - 7693356

SN - 0886-1544

VL - 26

SP - 77

EP - 87

JO - Cell Motility and the Cytoskeleton

JF - Cell Motility and the Cytoskeleton

IS - 1

ER -