Introduction of calcium buffers into the cytosol of Dictyostelium discoideum amoebae alters cell morphology and inhibits chemotaxis

Abstract

Differentiating Dictyostelium discoideum amoebae respond chemotactically towards the attractant cAMP. To test whether chemotaxis requires the establishment of a spatial gradient of the cytosolic calcium concentration ([Ca2+]i) we scrape-loaded calcium chelating agents with different affinities for Ca2+ into the cytosol of the cells. The buffers were 1,2-bis(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA) and its derivatives. Parameters analyzed were general cell morphology and the capability to protrude pseudopods and to migrate towards a cAMP-filled capillary. The chelators dose- and time-dependently inhibited spreading of the amoebae on the substrate. Both oriented pseudopod formation and locomotion of the cells were reduced. This effect was overcome by extracellular Ca2+, but not Mg2+. The effects of BAPTA derivatives were compared to the inhibition by BAPTA. A dose-response curve was obtained; 5,5'-difluoro-BAPTA was the most potent analogue. We conclude that a [Ca2+]i-gradient is necessary for orientation and locomotion. Chemotaxis experiments performed in the presence of extracellular EGTA revealed that liberation of Ca2+ from intracellular stores is sufficient for pseudopod formation; yet under physiological conditions influx of extracellular Ca2+ is also used to establish the gradient.