Several lines of evidence support the idea that increases in intracellular free calcium concentration ([Ca2+]i) regulate the chromosome motion. To directly test this we have iontophoretically injected Ca2+ or related signaling agents into Tradescantia stamen hair cells during anaphase and measured their effect on chromosome motion and on the Ca2+ levels. Ca2+ at (+)1 nA for 10 s (~ 1 μM) causes a transient (20 s) twofold increase in the rate of chromosome motion, while at higher levels it slows or completely stops motion. Ca2+ buffers, EGTA, and 5,5'-dibromo-1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, which transiently suppress the ion level, also momentarily stop motion. Injection of K+, Cl-, or Mg2+, as controls, have no effect on motion. The injection of GTPγS, and to a lesser extent GTP, enhances motion similarly to a low level of Ca2+. However, inositol 1,4,5-triphosphate, ATPγS, ATP, and GDPβS have no effect. Measurement of the [Ca2+](i) with indo-1 reveals that the direct injections of Ca2+ produce the expected increases. GTPγS, on the other hand, causes only a small [Ca2+](i) rise, which by itself is insufficient to increase the rate of chromosome motion. Further studies reveal that any negative ion injection, presumably through hyperpolarization of the membrane potential, generates a similar small pulse of Ca2+, yet these agents have no effect on motion. Two major conclusions from these studies are as follows. (a) Increased [Ca2+](i) can enhance the rate of motion, if administered in a narrow physiological window around 1 μM; concentrations above 1 μM or below the physiological resting level will slow or stop chromosomes. (b) GTPγS enhances motion by a mechanism that does not cause a sustained uniform rise of [Ca2+](i) in the spindle; this effect may be mediated through very localized [Ca2+](i) changes or Ca2+-independent effectors.
