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

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Abstract
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.