Isolation by fluorescence-activated cell sorting of Chinese hamster ovary cell lines with pleiotropic, temperature-conditional defects in receptor recycling.
We have isolated several Chinese hamster ovary cell lines with temperature-sensitive defects in the recycling of receptors after endocytosis. These cell lines were selected using fluorescence-activated cell sorting for retention of a pulse of labeled transferrin after a chase in the presence of unlabeled transferrin. One of these cell lines, TfT1.11, was selected for further characterization. In TfT1.11 the trapping of transferrin within the cells is paralleled by a loss of cell surface transferrin receptors. Within 4 h after the shift from 33 to 41 degrees C the surface binding of transferrin is reduced to 18% of parental cells at 41 degrees C. The trapping of transferrin and the loss of transferrin receptor from the cell surface are caused by a temperature-conditional 5.5-fold decrease in the initial rate of transferrin recycling. TfT1.11 cells also rapidly lose 89% of their ability to take up alpha 2-macroglobulin after the temperature shift to 41 degrees C. These data indicate that the TfT1.11 cell line has a pleiotropic defect in receptor recycling.
Cain
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CC
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213.
Wilson
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Murphy
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D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004705:Endocytosis; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D007700:Kinetics; D009154:Mutation; D010053:Ovary / Q000166:cytology / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D013696:Temperature; D014168:Transferrin / Q000378:metabolism; D000511:alpha-Macroglobulins / Q000378:metabolism
D011956:Receptors, Cell Surface; D014168:Transferrin; D000511:alpha-Macroglobulins; D000255:Adenosine Triphosphate