PHOTODYNAMIC KILLING OF Dictyostelium discoideum AMOEBAE MEDIATED BY 4',5'-DIIODOFLUORESCEIN ISOTHIOCYANATE DEXTRAN. A STRATEGY FOR THE ISOLATION OF THERMOCONDITIONAL ENDOCYTOSIS MUTANTS

4′, 5′-Diiodofluorescein dextran has been synthesized by iodination of fluorescein dextran and it was used as a sensitizer in photodynamic killing of amoebae of the cellular slime mold Dictyostelium discoideum. Amoebae from axenic strain AX2 incubated in the presence of 4′,5′-diiodofluorescein dextran efficiently internalized the molecule by fluid-phase pinocytosis. Subsequently, illumination of cells showed dose- and time-dependent loss of viability. The tertiary amine, 2,4- diaza[2,2,2]bicyclooctane, significantly decreased phototoxicity, whereas the presence of D₂O increased phototoxicity. These effects were fully consistent with a role for excited-state singlet oxygen generated inside the endosomal compartments in the mechanism of photoinjury. A strategy was developed to enrich for temperature-sensitive Dictyostelium endocytosis mutants in a nitrosoguanidine mutagenized population. Incubation with 4′,5′-diiodofluorescein dextran at 27°C to load endosomal compartments was followed by light treatment to kill parent-type cells selectively. In contrast, mutants unable to internalize the fluid-phase marker would survive the treatment. Six temperature-sensitive clones were selected that grew normally at 21°C but did not grow at 27°C. They all showed markedly decreased fluid-phase pinocytosis capacity at nonpermissive temperature as compared to their activity at 21 °C. These mutant strains should be useful in studies designed to characterize endocytosis mechanisms in Dictyostelium.