Kinetics of histone endocytosis in Chinese hamster ovary cells. A flow cytofluorometric analysis.

The endocytosis of histones by cultured cells was examined by flow cytofluorometry. Monolayer cultures of Chinese hamster ovary cells were incubated with fluorescein-labeled histone for various periods of time and then trypsinized to remove surface-bound protein. Internalization followed first order kinetics with a half-time of 45 min, and was linear in histone concentration up to 80 microgram/ml. Since fluorescein fluorescence decreases with decreasing pH, the fluorescence of labeled histone contained in lysosomes was expected to be decreased relative to its fluorescence at neutral pH. This was demonstrated by using chloroquine to increase the lysosomal pH of intact cells. The fluorescence of labeled histones incorporated into cells increased when those cells were incubated with 50 microM chloroquine and remeasured. This provides a method for measuring the kinetics of entry of a fluorescent probe into lysosomes. Internalization into lysosomes began almost immediately upon addition of histone, but stable nonlysosomal fluorescence appeared only after a lag of 1 h. Using suspension cultures, the short term binding and internalization kinetics were also measured. In pulse-chase experiments, lysosomal fluorescence decreased with a half-time of 30 min, but nonlysosomal fluorescence decreased with a half-time of almost 12 h, probably as a result of cell division. These results demonstrate the usefulness of flow cytometry for the quantitation and characterization of endocytosis in cultured cells.