Internalisation of fluorescein isothiocyanate and fluorescein isothiocyanate-dextran by suspension-cultured plant cells

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Abstract
The uptake of pure non-conjugated fluorescein isothiocyanate (FITC) and of the membraneimpermeant probe FITC—dextran into suspensioncultured carrot cells and protoplasts has been investigated. Commercial samples of a 70K (K=103Mr) FITC—dextran were shown to contain contaminant FITC and/or its degradation products, which were rapidly internalised into the vacuolar system of both cells and protoplasts. However, purified samples of the 70K FITC—dextran were taken up into the vacuoles of cells but not protoplasts after a lh incubation period. This apparent difference in the ability of cells and protoplasts to internalise FITC—dextrans was confirmed using samples of both commercial and purified 20K FITC—dextran as putative endocytotic probes. Both confocal and conventional fluorescence microscopy of FITC—treated cells have shown that FITC was internalised into similar intracellular compartments as was observed in cells treated with three-times purified 70K FITC—dextran. Thus, FITC was a useful fluorophore for rapidly labelling both the putative endocytotic compartments and the pleiomorphic vacuolar system of carrot cells. Kinetic studies indicated that FITC entered the cell by diffusion in the form of the neutral molecule. We have shown that treatment of cells or protoplasts with the drug Probenecid reversibly inhibited the uptake of FITC from the cytoplasm into the vacuole. In addition, the uptake of FITC into isolated vacuoles was enhanced in the presence of Mg-ATP