INTRACELLULAR GUANOSINE-5'-O-(3-THIOTRIPHOSPHATE) BLOCKS CHEMOTACTIC MOTILITY OF DICTYOSTELIUM-DISCOIDEUM AMEBAS

Starving amoebae of the cellular slime mold Dictyostelium discoideum react chemotactically towards the attractant cAMP. In this study, the effect of nonhydrolyzable analogs of GTP and GDP on the chemotactic behavior was analyzed with light microscopic techniques. Guanosine-5'-0-(2-thiotriphosphate) (GTP-gammaS) or guanosine-5'-0-(2-thiodiphosphate) (GDPbetaS) was scrape-loaded into the cytoplasm of cells, together with a fluorescent marker. Stimulation with a cAMP-filled glass capillary revealed a reduced capacity of loaded cells to migrate towards the capillary tip. Most cells still protruded filopods in the direction of the capillary tip, but full extension of pseudopods was inhibited in a dose-dependent and reversible manner. This indicates that in the presence of the analogs, chemotactic sensing still occurs, and that a more distal step of the cascade of events leading to the formation of the pseudopod is impaired. In cells loaded with the analogs together with the calcium indicator fura-2, stimulation with 10 muM cAMP led to a transient change in the intracellular free calcium concentration ([Ca2+]i), which was detectable in 28% of the cells. Furthermore, large vacuoles were found containing high amounts of calcium. On the other hand, clamping of [Ca2+]i at low levels with 1,2-bis(2-aminophenoxy) ethane N,N,N',N'-tetraacetic acid (BAPTA) also inhibited motility, with neither filopods nor pseudopods formed. The data suggest that chemotactic migratory activity involves GTP-dependent processes that participate in the regulation of the Ca2+ homeostasis of the cell and in the regulation of membrane traffic that contributes to the directed locomotion.