Kinetics of pinocytosis studied by flow cytometry

Kinetics of pinocytosis of FITC-Dextran (mw 70 000) was analysed in mouse L-cells using flow cytometry with a fluorescence-activated cell sorter (FACS). In each experiment information on 2 X 10(4) individual, living cells was obtained. The population of L-cells thus analysed was shown to be rather homogeneous with respect to cell size, and the peak of the FITC-fluorescence curve--a histogram of the accumulated signals from L-cells exposed to FITC-Dextran--was shown to be representative of the average size of the L-cells. It was found that the cellular accumulation of FITC-Dextran was proportional to the tracer. However, the rate of accumulation decreased with increasing time of incubation (up to 3 h). When cells were pulse-labeled for 10 min at 37 degrees C, rinsed carefully with ice-cold PBS, and reincubated at 37 degrees C for various periods of time, about 50% of the initial cellular FITC-fluorescence disappeared within approximately 15 min of reincubation, whereafter no measurable decrease in cellular fluorescence was seen. In addition, a rapid increase of intact FITC-Dextran molecules in the reincubation medium occurred within the first 15 to 30 min of reincubation, whereafter no further increase took place. Thus, a large portion of previously endocytosed FITC-Dextran becomes rapidly exocytosed, while the rest becomes sequestered within a compartment from where little or no exocytosis occurs. The deviation from linearity in accumulation related to time, and the distinct biphasic kinetics of exocytosis are taken to support a generalized two-compartment model for endocytosis and membrane recycling, the two compartments being endocytic vacuoles (endosomes) and (secondary) lysosomes, respectively.