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

Starving amoebae of the cellular slime mold Dictyostelium discoideum r eact chemotactically towards the attractant cAMP. In this study, the e ffect of nonhydrolyzable analogs of GTP and GDP on the chemotactic beh avior 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 wi th a fluorescent marker. Stimulation with a cAMP-filled glass capillar y revealed a reduced capacity of loaded cells to migrate towards the c apillary 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 pres ence 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 tr ansient change in the intracellular free calcium concentration ([Ca2+] i), which was detectable in 28% of the cells. Furthermore, large vacuo les 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 neith er filopods nor pseudopods formed. The data suggest that chemotactic m igratory activity involves GTP-dependent processes that participate in the regulation of the Ca2+ homeostasis of the cell and in the regulat ion of membrane traffic that contributes to the directed locomotion.